Research Collection

Doctoral Thesis

Engineering inorganic nanomaterials for the capturing, storageand release of biomolecules

Author(s): Zlateski, Vladimir

Publication Date: 2016

Permanent Link: https://doi.org/10.3929/ethz-a-010750035

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ETH Library

DISS. ETH No. 23852

ENGINEERING INORGANIC NANOMATERIALS FOR THE CAPTURING, STORAGE

AND RELEASE OF BIOMOLECULES

A thesis submitted to attain the degree of

DOCTOR OF SCIENCES of ETH ZURICH

(Dr. sc. ETH Zurich)

presented by

VLADIMIR ZLATESKI

MSc Molecular Life Sciences, Jacobs University

born on 12.05.1987

citizen of Macedonia

accepted on the recommendation of

Prof. Dr. Wendelin J. Stark, examiner

Prof. Dr. Javier Pérez-Ramírez, co-examiner

Dr. Robert N. Grass, co-examiner

2016

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3

Acknowledgments

The PhD as such has been a very exciting journey with some ups and downs, a journey where

one has to be persistent, show some strong will when things don`t work, fight through and

finally enjoy the moments of success as much as one can because there aren`t many of them.

As a PhD candidate I have acquired a substantial amount of knowledge and skills, a powerful

add-on to both my professional and private lives. It wouldn`t have been possible to complete

such a life-chapter without the help and support of many people around me. I would like to

take a moment to thank the people responsible for my success and well-being.

First and foremost, I would like to express my profound gratitude to Prof. Wendelin Stark for

giving me the opportunity to join the Functional Materials Laboratory (FML). His impressive

way of conducting applied research (thinking out of the box), inspirational talks and business

knowledge and experience are some of the things one could learn from. Wendelin was ready

to show tolerance and understanding when things were not going as smooth and I thank him

for that.

My special thanks go to my direct supervisor, Dr. Robert Grass, mainly for his constant

availability and big scientific help in terms of many valuable discussions and numerous given

advises. Robert is a very smart and knowledgeable scientist from whom I acquired a whole

new way of perceiving science and conducting research. He was my mentor who guided me

through the whole PhD and I am utterly grateful for that.

I kindly acknowledge Prof. Javier Pérez-Ramírez for accepting to co-examine my dissertation

and I thank him for the help with my scientific work.

Big big thanks to the whole research group for the great time and support both inside and

outside of the lab. I would like to start with Michela Puddu. We started together, were part of

the Mag(net)icFun network, traveled around for workshops and shared the workload of filling

out paperwork for the European project. I thank her for the support and nice time in the lab

and as a friend outside of the lab. The same is valid for Gediminas Mikutis. In addition to our

professional discussions about research, I enjoyed the social events we attended together and

especially the weekend trips. I would like to express my sincere gratitude towards all current

and former FML PhD members with whom I had the chance to work together: Antoine, Alex,

Chälli, Jonas, Carlos, Elia, Mario, Sam, Tino, Dirk, Lukas, Philipp, Michael L., Corinne,

Nicholas, Weida, Xavier, Michele, Konstantin, Nadine, Mirjam, Renzo, Roland, Fabian,

Michael R., Schumi, Daniela, Nora, Aline, Norman and Stephanie. Those people contributed

4

to the positive spirit of the group and it`s my greatest pleasure to have shared moments with

all of them. Rafi, Balder, Adi, Adi Z. and Elias deserve an acknowledgement for the nice time

we had together during their stay in the group and beyond.

Special thanks go to my girlfriend Natanja Fleckenstein. Her love, support and appreciation

helped me prevail even in the most stressful times. A wellness weekend was always a good

idea. In addition, I would like to thank the big circle of friends which was surrounding me in

the past 4 years, helping me cheer up and showing support and understanding in difficult

moments. I am also very fortunate to have met the nice people and remote colleagues from the

European project network.

Olivera Evrova, who is a good friend and a fellow scientist, deserves a special

acknowledgment because without her this PhD wouldn`t have happened.

I am mostly grateful to my beloved parents Mirjana and Radoslav who spent substantial

amounts of time and effort trying to raise me in the best way possible. Their unconditional

love and belief in me throughout the past 29 years was of crucial importance to both my

personal and career developments. My brother Aleksandar is also acknowledged for his help

and support.

This work has been financially supported by the EU-ITN network Mag(net)icFun (PITN-GA-

2012-290248), which is kindly acknowledged.

5

Table of contents

Acknowledgments 3 

Zusammenfassung 8 

Summary 10 

1  Biomolecules and nanomaterials: how the interactions turn into applications 12 

1.1  Nanotechnology – a whole new world at the bottom 13 

1.2  Bionanotechnology is where biology inspires nanotechnology 14 

1.2.1  Bionanostructures 1: Mimicking natural biomolecules interactions 16 

1.2.2  Bionanostructures 2: Inorganic particles-biomolecules interactions exist in

nature 17 

1.3  Towards applications of biomolecule-nanomaterial hybrids 18 

1.3.1  Biomolecules – a template in nanomaterials fabrication 18 

1.3.2  Biomolecule-functionalized nanomaterials – main interactions 19 

1.3.2.1  DNA-nanomaterial conjugates  20 

1.3.2.2  Protein-nanomaterial conjugates  21 

1.4  Selected applications of biomolecule-nanomaterial conjugates 24 

1.4.1  Nanomaterials in biocatalysis 24 

1.4.2  Nanomaterials in DNA enrichment 26 

2  Efficient Magnetic Recycling of Covalently Attached Enzymes on Carbon-Coated

Metallic Nanomagnets 29 

2.1  Introduction 30 

2.2  Experimental section 31 

2.2.1  Particles activation for bioconjugation 31 

2.2.2  Enzyme immobilization 32 

2.2.3  Enzymatic activity assays 32 

2.2.4  Enzymatic activity calculation 33 

2.2.5  Protein concentration measurement 33 

2.2.6  Large-scale experiment 34 

2.2.7  Analytics 34 

2.2.8  Desorption experiment 35 

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2.2.9  Cobalt leaching experiment 35 

2.2.10  Magnetic separation with a small magnet 36 

2.3  Results and discussion 36 

2.4  Conclusion 44 

2.5  Contribution of the authors 45 

3  Immobilizing and de-immobilizing enzymes on mesoporous silica 46 

3.1  Introduction 47 

3.2  Experimental section 48 

3.2.1  MCF mesoporous silica synthesis 48 

3.2.2  Mercury intrusion 49 

3.2.3  Nitrogen sorption 49 

3.2.4  Small-angle X-ray scattering (SAXS) 49 

3.2.5  TEM and SEM analysis 50 

3.2.6  β-glucosidase immobilization, entrapment and release 50 

3.2.7  Enzymatic activity assays 51 

3.2.8  Enzymatic activity calculation 51 

3.2.9  Protein concentration measurement 52 

3.2.10  Thermal stability test 52 

3.2.11  Fluoride buffer influence on enzymatic activity 52 

3.3  Results and discussion 52 

3.4  Conclusion 59 

3.5  Contribution of the authors 59 

4  Selective ssDNA enrichment by magnetic up-concentration using glass

microarray chemistry 60 

4.1  Introduction 61 

4.2  Experimental section 62 

4.2.1  Diazonium chemistry (Fe/C-OH) 62 

4.2.2  Silica coating (Fe/C-SiO2) 62 

4.2.3  APTES functionalization (Fe/C-SiO2-NH2) 63 

4.2.4  ssDNA binding (Fe/C-SiO2-ssDNA) 63 

4.2.5  Second strand hybridization/melting experiments 64 

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4.2.6  FTIR spectroscopy 65 

4.2.7  Nitrogen sorption experiment 65 

4.2.8  STEM analysis 66 

4.2.9  X-ray diffraction (XRD) 66 

4.2.10  C, H, N elemental analysis 66 

4.3  Results and discussion 66 

4.4  Conclusion 74 

5  Conclusion and outlook 75 

Appendix 79 

A.1  Supporting information to Chapter 2 80 

A.2  Supporting information to Chapter 3 84 

References 86 

8

Zusammenfassung

Partikel-Hybridsysteme aus Biomolekülen und anorganischen Materialien sind aufgrund ihrer

Anwesenheit in der Natur gut bekannt. Sie werden von optimierten biologischen Prozessen,

die sich auf der Nanoskala befinden, geregelt. Die Nanotechnologie als wissenschaftliche

Disziplin hat viel vom erweiterten Verständnis der schwachen chemischen Wechselwirkungen

und der kombinierten Eigenschaften der biomolekularen/anorganischen Hybridmaterialien

profitiert. Dies hat während der letzten Jahrzehnte zur Entwicklung von neuartigen,

einzigartigen und intelligenten Hybridmaterialien geführt. Diese Materialien sind für

biologische und auch für nichtbiologische Anwendungen vielversprechend. Die vorliegende

Doktorarbeit setzt sich mit den Fortschritten in der Entwicklung und Anwendung von

biomolekularen/anorganischen Partikel-Hybridsysteme (vor allem Proteine und Nukleinsären

in Kombination mit anorganischen Materialien) in verschiedenen Disziplinen auseinander.

Kapitel 1 fasst die Wechselwirkungen zwischen Biomolekülen und anorganischen Partikeln

zusammen, mit dem Fokus auf Nanomaterialien. Dieses Kapital gibt einen Überblick über die

Nanotechnologie und über das neue Feld der Nanobiotechnologie. Hier sehen wir auf welche

Art und Weise die Biomoleküle und die anorganischen Materialien zusammenwirken können

und wie daraus einen Vorteil erzielt werden kann. Es wird aufgezeigt, wie das Verständnis

dieser Wechselwirkungen von Forschern angewendet wurde, um eine Vielfalt von

Hybridmaterialien zu erschaffen, welche für biologische und nichtbiologische Anwendungen

nützlich sind. Zum Schluss wird ausführlich über die Anwendungen dieser

biomolekularen/anorganischen Partikel-Hybridsysteme in den Bereichen Biokatalyse und

DNA-Anreicherung gesprochen.

Kapitel 2 konzentriert sich auf die Anwendung von Hybridmaterialien bestehend aus einer

Kombination von Enzymen und magnetischen Nanopartikeln in der Biokatalyse. Im Streben

nach robusten und wiederbenutztbaren Biokatalysatoren für die industrielle synthetische

Chemie, nimmt die Bedeutung von Nanotechnologie stetig zu. In letzter Zeit wurden vermehrt

Biomoleküle, insbesondere Enzyme, auf verschiedene Nanomaterialien immobilisiert.

Kohlenstoff-beschichtete magnetische Nanopartikel haben sich aufgrund ihrer grossen

Oberfläche, der hohen magnetische Sättigung und der bekannten Chemie als ein

vielversprechender Enzymträger erwiesen. Hier wird gezeigt, wie kohlenstoffbeschichtete

Kobaltnanopartikel chemisch funktionalisiert werden können um Enzyme auf die Oberfläche

zu immobilisieren. Die Enzym/Kobaltpartikel Konjugate konnten sowohl im Kleinansatz

(Milliliter) als auch in grösseren Pilotreaktionen (mehrere Liter) rezykliert werden.

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In Kapitel 3 wird ein neuartiges Vorgehen gezeigt, mit dem Enzyme auf mesoporöse Silikate

immobilisiert, eingeschlossen, inaktiviert und danach beliebig wieder in aktivem Zustand

freigesetzt werden können. Beta-Glukosidase wurde als Modellenzym auf mesozellulären

Silika-Schaumstoff immobilisiert. Zusätzlich wurde das Enzym durch das Ausfällen von

Silika in den Kanälen des Materials eingeschlossen. Das Enzym war im eingeschlossenen

Zustand nicht reaktiv und zeigte eine grosse Thermostabilität. Nach einem milden Silika-

Auflösungsschritt durch die Verwendung eines fluoridhaltigen Puffers wies das Enzym

wieder den grössten Teil der ursprünglichen Aktivität auf. Dieses Prinzip zur Enzymlagerung

in Silika kann neben Beta-Glukosidase auch auf weitere Enzyme angewendet werden.

Kapitel 4 beschreibt die Verwendung von neuartigem Silikat-beschichteten Eisen-Kohlenstoff

Verbundwerkstoff für die Anreicherung von spezifischen DNA Sequenzen. Dank des

magnetischen Kernes können die Partikel sehr schnell abgetrennt werden und die intrinsisch

nicht-DNA bindende Silikatoberfläche ermöglicht eine einfache chemische

Funktionalisierungen mit Hilfe von Silanchemie. Das im Labor hergestellte Material wurde

chemisch funktionalisiert um einzelsträngige DNA-Moleküle auf die Silikaoberfläche

kovalent binden zu können. Das mit einzelsträngiger DNA geladene Material wurde dafür

verwendet um auf selektive Art und Weise die komplementäre Sequenz aus einer Mischung

von verschiedenen DNA-Sequenzen aufzukonzentrieren. Hier wird somit eine einfache,

schnelle und zuverlässige Methode erarbeitet, welche die selektive Bindung,

Aufkonzentration, Aufreinigung und zum Schluss das Detektion der zu analysierenden DNA-

Sequenz ermöglicht.

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Summary

Biomolecule/inorganic particle hybrid systems are well known for their existence in living

organisms. They are governed by nanoscale bioprocesses that have been optimized for years.

Nanotechnology is the field that largely profited from the understanding of those weak

interactions and from combining the chemical and physical properties of both entities into a

single unit. Throughout the last decades this has led to the development of novel, unique and

smart hybrid materials which hold a great promise for both biological and non-biological

applications. In the present thesis recent advances in the design and application of

biomolecule/inorganic particle hybrids (mainly proteins and nucleic acids as biomolecules) in

different disciplines are reported.

Chapter 1 gives an overview of the interactions between biomolecules and inorganic materials

with a focus on nanomaterials. It gives a brief introduction about the field of nanotechnology

and the emerging field of bionanotechnology. In this chapter we see how biomolecules and

inorganic materials interact in nature to their mutual benefit. We see how scientists exploited

the understanding of these interactions for the purpose of creating a variety of hybrid

materials and use them in many biological and non-biological applications. Lastly, we talk

more in detail about the applications of such biomolecule-inorganic material hybrids in

biocatalysis and DNA enrichment.

Chapter 2 focuses on the use of enzyme/magnetic nanoparticle hybrids in biocatalysis. In the

pursuit of robust and reusable biocatalysts for industrial synthetic chemistry,

nanobiotechnology is currently taking a significant part. Recently, enzymes have been

immobilized on different nanoscaffold supports. Carbon-coated metallic nanoparticles were

found to be a practically useful support for enzyme immobilization due to their large surface

area, high magnetic saturation, and familiar surface chemistry. Carbon-coated cobalt

nanoparticles were chemically functionalized, activated for bioconjugation and subsequently

used in enzyme immobilization. The enzyme-support conjugates could be recycled on a

millilitre to litre scale.

In Chapter 3, we talk about a novel approach to immobilize and then release enzymes from

mesoporous silicates. Beta glucosidase was immobilised as a model enzyme within

mesocellular foam (MCF) at a high loading. The enzyme was further entrapped within the

material by precipitating additional silica within the channels. Although unreactive while

entrapped, in this state the enzyme was highly stable towards heat treatments. Upon release

from the matrix by a mild silica dissolution step involving a fluoride comprising buffer, the

11

enzyme regained most of its original activity. The principle can be adapted to many

previously developed mesoporous silica/enzyme biocomposites.

Chapter 4 describes the use of novel silica-coated iron-carbon composites in DNA

enrichment. The magnetic iron core allows a fast separation whereas the silica surface has an

anti-fouling character and could be easily functionalized simply by silane chemistry. The in-

house produced material was further functionalized for DNA binding and single-stranded

DNA sequences were covalently bound to the silica surface. The ssDNA-loaded material was

used to selectively fish out the complementary oligonucleotide from a DNA mixture, from

different volumes and at different concentrations. An easy, fast and reliable procedure to bind

and release a target ssDNA and subsequently detect and quantify it with standard in-house

equipment was shown.

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13

1.1 Nanotechnology – a whole new world at the bottom

“I would like to describe a field, in which little has been done, but in which an enormous

amount can be done in principle. Furthermore, a point that is most important is that it would

have an enormous number of technical applications.”

Richard P. Feynman, 1959

Back in 1959, the renowned American theoretical physicist Richard Feynman gave a lecture

of a ground-breaking character at the American Chemical Society (ACS) meeting at Caltech.1

He made people aware of the technological importance to go small; however his lecture was

forgotten shortly after it took place. It`s only 30 years later when people re-discovered his

talk, after the field of nanotechnology was already becoming a game-changer in the field of

science and technology. With the sentence: “There is plenty of room at the bottom”, Feynman

deservedly found his place in the history of nanotechnology and has a worldwide reputation

as being one of the pioneers of the field. Feynman foresaw the need to go small, where whole

new worlds were waiting to be discovered.

But what in fact is nanotechnology? Although one can still argue the details of the most

representative definition, the following three points reflect the main idea and distinguish the

field from other initiatives. Nanoscience and technology is a field that focuses on: 1) the

development of methods and surface analytical tools for building structures and materials,

typically on the below 100 nanometer scale, 2) the identification of the chemical and physical

consequences of miniaturization, and 3) the use of such properties in the development of

novel and functional materials and devices.2 The field of nanotechnology aided by the novel

tool developments is characterized by having a broad and multidisciplinary character,

including all of the basic scientific fields: physics, chemistry, biology, medicine and materials

science & engineering.

Dealing with the matter on the nanoscale is usually accompanied by properties that are

significantly different from that of the bulk materials. By adjusting the size, composition and

shape of the materials on the sub-100 nanometer length scale, the optical, electrical,

mechanical, magnetic, and chemical properties can be manipulated which lead to the

development of many different classes of nanomaterials. In general the nanomaterials are

classified into groups according to their dimensions. 0D category includes those

nanomaterials wherein all of their dimensions belong within the nanoscale (100 nm ≤), for

ex. the nanoparticles; 1D category contains nanomaterials having one dimension outside the

14

nanoscale (nanowires for ex.); 2D category contains nanomaterials having two dimensions not

confined in the nanoscale (thin films for example); 3D category contains nanomaterials with

no single dimension in the nanoscale. The reason why they are called nanomaterials lies in the

fact that they either possess some sort of nanocrystalline structure or involve the presence of

features at the nanoscale. The most typical nanomaterials are given in Table 1.1.

The big window of opportunities is responsible for the high importance of the nanomaterials

in many technological areas, among which: electronics,3, 4 sensors,5, 6 catalysis7 and

medicine.8

Table 1.1 Typical nanomaterials9

Nanomaterials Size (approx.) Materials

(a) Nanocrystals (quantum dots) diameter 1–10 nm Metals, semiconductors, magnetic

materials

Other nanoparticles diameter 1–100 nm Ceramic oxides

(b) Nanowires diameter 1–100 nm Metals, semiconductors, oxides,

sulfides, nitrides

Nanotubes diameter 1–100 nm Carbon, layered metal

chalcogenides

(c) 2-Dimensional arrays (of nano

particles)

several nm2–mm2 Metals, semiconductors, magnetic

materials

Surfaces (thin films) thickness 1–1000 nm Various materials

(d) 3-Dimensional structures Several nm in all three

dimensions

semiconductors, magnetic

materials

1.2 Bionanotechnology is where biology inspires nanotechnology

The growing maturity of nanotechnology has led to the establishment of more focused

subdisciplines including especially that of bionanotechnology.10 This field can more easily be

defined as the intersection of nanotechnology and biology. Nano-biological hybrid materials

are considered “value-added” in that they are capable of far more than each individual

component alone (Figure 1.1). The biological world can either provide the inspiration or the

end goal. Biosystems are governed by nanoscale processes that have been optimized over

millions of years.11 What nature in fact does in living systems is rearranging matter with the

help of “weak” molecular interactions, such as: van der Waals forces, hydrogen bonds,

electrostatic dipoles etc. in order biological processes to be efficiently accomplished.12

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1.2.1 Bionanostructures 1: Mimicking natural biomolecules interactions

Because of their nano-size and the special molecular structure, the nucleic acids (DNA and

RNA) are in fact convenient and logical building blocks for assembling new nanomaterials.13,

14 DNA, the hereditary molecule of the living organisms, is composed of two strands, held

together by hydrogen bonds between the base pairs (adenine binds thymine, and cytosine

binds guanine); RNA is more often found in nature as a single-stranded molecule, and

intramolecular base pairing (guanine with cytosine and adenine with uracil) can occur. Thanks

to the well understood structural features of the DNA (controlled hybridization and melting of

the complementary bases) we are able to synthetically construct more complex structural units

in order to achieve specific shapes and geometries of the final products. Some well-known

examples include the pioneering work and DNA construction strategy of Seeman to build

branched DNA scaffolds with the help of the “sticky ends” (short single-stranded extensions

protruding from the double helix).13, 15 Other examples include DNA origami, where a long

scaffold strand is folded by hundreds of short auxiliary strands into a complex shape,16 as well

as other two and three dimensional structures such as DNA lattices and crystals.17-19 RNA

nanotechnology is a field still in its infancy compared to DNA nanotechnology. Nevertheless,

work on RNA manipulation showing successful of 2D and 3D structure construction has been

done.20, 21

Proteins are large biomolecules (few nm in size) consisting of one or more chains of amino

acid residues. The unique nature of the protein folding (usually in a 3D structure) has attracted

a lot of attention in the world of nanotechnology. The understanding of the 3D structure of the

proteins has proven to be extremely valuable from scientific and engineering prospective.

Similar to nucleic acids people started assembling protein structures with different shapes and

complexity, a nice overview given by Yeates.22 Linear assemblies have been designed using

peptide-based systems. Ghadiri and co-workers synthesized cyclic polypeptides containing

alternating D- and L-amino acids which then crystalize into hollow tubular structures upon

protonation.23 β-sheet proteins, and amyloid proteins in particular, have served as useful

guides in designing self-assembling proteins.24 A combinatorial library of rationally

designed polypeptides (β-sheets) was constructed where polypeptides unexpectedly self-

assembled into fibrils.25 When each subunit reacts with other 3 or more instead of 2 we obtain

ordered architectures by polyvalent design instead of the plain linear ones. The Dotan group

17

for example based their recent crystal design on the lectin concanavalin which is a tetrameric

protein that binds a carbohydrate molecule in each of its four binding sites.26

Although relevant applications have begun to emerge, scientists so far mainly focused on

controlling and reshaping the matter at a molecular level. Issues like high-cost of production

and error rate of assembly still limit their expansion into the applied world. The combination

of biomolecules with other micro- or nanoscale materials (usually inorganic materials) to

form hybrids, proved to be more mature for real life applications. In the following paragraphs,

we are going to read more about the interaction of biomolecules with nanomaterials and its

impact on modern science.

1.2.2 Bionanostructures 2: Inorganic particles-biomolecules interactions exist in nature

Until now we have talked about the interactions of biomolecules among themselves. In fact,

interactions between biomolecules and inorganic materials exist in nature and are well known

in the scientific world. Biominerals are inorganic materials which are assembled inside living

organisms in order to harden and stiffen existing tissues. They are produced in a process

known as biomineralization where biomolecules are usually involved as facilitators. In

biological organisms, organic molecules appear to exert a remarkable level of control over the

nucleation, composition (principal and trace ions) and crystallographic phase of

minerals.27 As mentioned before, profound understanding of the biomolecule-inorganic

material interactions not only helps in deciphering how nature works but more importantly in

designing novel materials and processing technologies for different fields. A few selected

areas where the biomolecule-nanomaterial hybrids have made a high impact are: bio-imaging,

implant integration and food and drug handling.28, 29 In order to make things concise and

easier to organize we are going to write separate sub-chapters explaining DNA and proteins

interactions with inorganic materials and their applications.

Looking more in detail in the interaction between inorganic materials and biomolecules in

nature, it is known that peptides and proteins are involved in most, if not all, stages of

biomineral formation, from transport, to nucleation and growth. This affinity binding, also

known as molecular recognition, arises from a single or multiple of the non-covalent

interactions such as hydrogen bonding, electrostatic interactions, hydrophobic interactions

etc.30 One famous example of such organic-inorganic interactions in nature is the red abalone

shells which are made of calcium carbonate but are nevertheless incredibly tough. In this case

intercalated protein interacts with the crystalline calcium carbonate plates.31 Another example

18

are magnetotactic bacteria. Magnetosomes are cellular structures characteristic for the

magnetotactic bacteria which contain single domains iron oxide particles and proteins

embedded in the membrane.32 The transmembrane proteins found in the magnetosome

membrane have been identified to associate with the growth of magnetite crystals.33, 34 Many

small proteins have been found tightly bound to bacterial magnetite but not inside the crystals.

The generation of hierarchically ordered silica structures in the presence of proteins is yet

another important example of biomineralization.35, 36 Diatoms, a group of unicellular algae

sheathed in a silicified external cell wall known as the frustule, are a perfect example. In the

case of diatoms, the current state of knowledge is that complex patterned macroscopic

structures are built up from nanometre sized amorphous silica particles in the presence of

proteins and/or polyamines. Silicifying polypeptides known as silaffins were originally

isolated as proteinaceous components of the frustule.37 The 19-mer synthetic peptide known

as R5 has been used widely in studies of peptide-driven silica formation.38

1.3 Towards applications of biomolecule-nanomaterial hybrids

The initial understanding of the biomolecule-inorganic material interactions present in nature

further motivated scientists to deepen and broaden up the scope towards real-life applications.

Such nanostructures can already be produced readily in large quantities from a whole range of

materials that belong to the group of metals, metal oxides or semiconductor materials. The

contribution of biomolecules to the field of nanotechnology spans all the way from the

synthesis of nanomaterials to the final applications of the biomolecule-functionalized

nanostructures.

1.3.1 Biomolecules – a template in nanomaterials fabrication

The importance of biomolecules in nanomaterials synthesis should not be neglected. There are

two main ways to produce nanomaterials: a) a top-down approach that relies on expensive

high-precision equipment to chop-down large pieces into smaller, which makes it costly and

b) a bottom-up approach by which the nanomaterial self-assembles from its precursors in a

liquid medium. The bottom-up approach is cheaper but less controlled in size and shape.

Those challenges can be addressed by the use of nanostructured templates for guiding the

assembly of nanoscopic building blocks. DNA-templated nanofabrication is still a developing

discipline which is divided in three main groups: 1) fabrication of nanomaterials directly on

DNA, 2) assembly of nano/meso-scale materials with DNA and 3) use of DNA templates in

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20

Nanomaterials are often functionalized with biomolecules and up to now many biological

molecules have been immobilized on polymer matrices and inorganic supports through a

variety of techniques that include non-covalent interactions (for ex. physical adsorption or

encapsulation) and covalent coupling. The modified supports with biomolecules (here we will

limit ourselves to DNA and proteins) are then used in numerous biotechnological

applications.

1.3.2.1 DNA-nanomaterial conjugates

DNA-nanomaterial conjugates can be obtained by simply letting the DNA strand adsorb on

the surface or via formation of a stable covalent bond (Figure 1.2). Nucleic acids are often

attached to particles through noncovalent electrostatic interactions. DNA and RNA, being

negatively charged, adsorb quickly onto positively charged nanoparticle surface. To this aim,

nanoparticles are functionalized with positively charged groups or polymers otherwise

repulsion takes place. The electrostatically driven adsorption of negatively charged DNA on

positively charged Cd2+ rich CdS NPs has been studied in detail. Calf thymus DNA was

adsorbed on CdS NPs where it was shown that the binding is entropically driven, largely by

release of counterions from the interface.47 The high binding affinity of DNA onto Cd2+-rich

CdS NPs was also shown to quench fluorescence opening up the possibility to use the system

in the chemical sensing field.48 In addition, it was found that “kinked” oligonucleotides,

which exhibit a curvature of ≈3 nm from crystallographic and biochemical experiments, bind

more tightly and faster to NPs that display curvatures of similar dimensions relative to straight

or bent DNA.49 Although unexpected from the surface charges (both DNA and silica carry

negative charges at neutral pH), DNA also adsorbs on silica materials in the presence of

chaotropic salts. The adsorption is based on electrostatic interactions, dehydration of DNA

and silica surface, and hydrogen bond formation.50, 51 The adsorption of nucleic acids on silica

surfaces in the presence of chaotropic agents is regularly employed in the solid phase

purification of nucleic acids using silica beads or gels. Adsorption (shell formation) of

oligonucleotides on gold nanoparticles with displacement of citrate ions was also

demonstrated.52

Covalent linkage has become very popular due to the stability of the bond and the variety of

functional groups available to modify the oligonucleotide of choice. In the case of gold

particles, a well-established way to couple DNA is by utilizing the strong Au-S bonds, where

the DNA of choice is modified with alkylthiol groups at either 3`or 5`53, 54 or with ten

successive thiophosphate thymine residues linked to the 5`end.55 The thiols spontaneously

21

bind to a gold surface, allowing for DNA coupling. Thiolated DNA oligonucleotides have

been covalently attached to propylmaleimid functionalized gold nanoparticles.56 Alkyne-

modified DNA has been covalently coupled to azide-terminated gold57 and superparamagnetic

nanoparticles58 through copper-catalyzed click chemistry. Phosphorothioate oligonucleotides

were covalently bound to the neutral CdS but also Cd2+ rich and S rich NPs.59 On thiol-

functionalized silica nanoparticles disulfide-modified DNA oligonucleotides have been

immobilized via a thiol/disulfide exchange reaction, in which the disulfide on the 5′ end of the

oligonucleotide reacts with the thiol functional group of the MPTS (3-

mercaptopropyltrimethoxysilane) layer on the silica nanoparticles.60 Silica particles were also

used as a support in DNA binding where alkyne-azide cycloaddition reaction was employed

between the dibenzyl cyclooctyne (DBCO) group containing oligonucleotide and the azide-

functionalized silica surface.61 Another widely used category of nanoparticles are magnetic

nanoparticles. Successful biotinylated ssDNA attachment to the streptavidin-immobilized iron

oxide NPs has been shown. The original iron oxide surface was first functionalized with

amino groups to which streptavidin was covalently attached by carbodiimide activation and

peptide coupling.62 Other research groups also used the highly specific streptavidin-biotin

binding to attach oligonucleotides to nanomaterials.63

Mainly for the purpose of intracellular delivery, DNA was encapsulated in an array of

materials. Recently the ability of natural phospholipids to self-assemble was mimicked by

amphiphilic copolymers. A fully scalable and extrusion-free method was developed to rapidly

and reproducibly prepare stabilized plasmid lipid particles (SPLP) for non-viral, systemic

gene therapy. The particles were prepared instantaneously by mixing lipids dissolved in

ethanol with an aqueous solution of DNA in a controlled, stepwise manner.64 DNA was

also encapsulated within poly(2-(methacryloyloxy)ethyl phosphorylcholine)–poly(2-

(diisopropylamino)ethyl methacrylate) (PMPC–PDPA) copolymer vesicles at neutral pH,

whereas lowering the solution pH leads to the formation of DNA–copolymer complexes.65

These two copolymer nanostructures ensure protection of plasmid DNA. Protection from

degradation and the potential to be applied to electronic devices used in biosensors lead to the

development of a way to encapsulate DNA in carbon nanotubes using a plasma ion irradiation

method in electrolyte solutions with DNA.66

1.3.2.2 Protein-nanomaterial conjugates

Protein-nanoparticle conjugates are also very popular in nanotechnology. Three main binding

strategies that apply here are: adsorption, covalent binding and encapsulation (entrapment).

22

Some of the major driving forces behind protein adsorption include: surface energy,

intermolecular forces, hydrophobicity, and ionic or electrostatic interaction. Talking about

electrostatic interactions, NPs stabilized by anionic ligands such as carboxylic acid derivatives

(citrate, tartrate, lipoic acid) are used to bind positively charged proteins and cationic ligands

serve the purpose of binding to negatively charged proteins. The overall protein charge is

dependent on its isoelectric point (pI) and the pH of the reaction solution, meaning that by

changing the solution pH one can switch between adsorption and desorption. One of the

famous nano-supports used in protein adsorption via electrostatic interactions are the gold

nano-structures. Shenton and co-workers immobilized positively charged antibodies on gold

and silver NPs produced by citrate reduction. This helped them to form 2D and 3D assemblies

of NPs when the corresponding antigen was added. Such assemblies are promising in the field

of biosensors.67 Another example is the gold nanoplates. Coated with anionic phospholipids

produced by photoreduction of NaAuCl4 in the presence of dimyristoyl-L-alpha-phosphatidyl-

dl-glycerol, they were used to bind positively charged proteins.68 Besides the gold, silica

nano-supports are also widely utilized in the field of protein adsorption. The size and

curvature influence of the negatively charged silica nanoparticles on a protein (Human

carbonic anhydrase I) adsorption was investigated by Lundqvist and co-workers.69 The

electrostatic deposition of biomolecules, particularly proteins or enzymes, can also be

extended to multilayer-level assemblies.70 Caruso and co-workers extended this in the area of

layer-by-layer assemblies of biomolecules on nanoparticles. In one of his works he deposited

protein/electrolyte layers on polystyrene (PS) latex particles in a way that they were

oppositely charged to one another, thereby facilitating growth of the films through

electrostatic interactions.71 Apart from the electrostatic interactions some proteins bind to

surfaces via hydrophobic interactions. One example is the lipase which is a robust enzyme

with a hydrophobic surface. Hydrophobic binding of lipases by adsorption has proven

successful due to the enzymes affinity for water/oil interfaces. One widely used example in

biocatalysis is the Candida Antarctica Lipase B (CALB). The enzyme was successfully

immobilized via hydrophobic interaction on a variety of polystyrene resins by Chen and co-

workers.72 Lee and co-workers immobilized lipases on SDS-modified magnetite NPs with the

aim to recycle the enzyme multiple times.73

Covalent binding of proteins (especially enzymes) to surfaces is also very common. The

strength of the chemical bond is crucial in overcoming stability and inactivation problems.

Low-molecular bifunctional linkers, which have anchor groups for their attachment to NP

surfaces and functional groups for their further covalent coupling to the target biomolecules

23

were extensively used in the generation of covalent-tethered conjugates of biomolecules with

various NPs.74 The available amino, carboxylic acid or thiol (not a lot on average) groups on

the surface could be directly involved in many different coupling reactions. Gold

nanoparticles can be easily and efficiently functionalized with thiolated molecules and amino

or carboxylic acid groups, which in turn, are conjugated with the protein of interest mainly for

the application in biosensors. Glucose oxidase was covalently attached to a gold nanoparticles

monolayer-modified electrode for the purpose of glucose biosensor formation.75 The surface

of the gold nanoparticles was functionalized with cystamine exposing an array of available

amino groups which would further react with the aldehyde groups of the periodate-oxidized

glucose oxidase via the well-known Schiff base reaction. Scientists also demonstrated a

feasible approach in multilayer assembly of glucose oxidase/gold nanoparticles on the Au

electrode surface using cysteamine as cross-linker.76 Magnetic nanoparticles (MNPs) are

another support famous for covalent immobilization of proteins. The same enzyme glucose

oxidase was covalently immobilized on amino-functionalized iron oxide nanoparticles and

was found to be stable over a wide range of pH and temperature conditions.77 Candida rugosa

lipase was covalently immobilized to magnetic nanoparticles (γ-Fe2O3) by a carbo-diimide

linkage and was shown to maintain significant activity after one month of storage.78

Cholesterol oxidase is another enzyme that was immobilized on MNPs (Fe3O4) via carbo-

diimide linkage for the use in sensors in clinical applications. Diagnostic and therapeutic

applications in cancer and infectious disease as well as uses in gene and drug

delivery have also been found for silica (SiO2) NPs-protein conjugates.79 Silica NPs, doped

with a dye (FITC) during the particle production process, found their application in bio-

imaging. The silanol groups on the surface were used to couple the TAT-regulatory protein

via N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) coupling chemistry.80 10 amino

acids, also known as the HIV-TAT peptide, are responsible for translocation through the

plasma membrane. Primary or secondary antibodies were covalently immobilized onto

the silica NP surface in order to selectively and efficiently bind various cancer cells.80, 81

Beyond adsorption and covalent attachment, the encapsulation of proteins, such as

entrapment within a porous matrix, is a useful technique for biotechnological and biomedical

applications for ex. in biosensing, affinity columns, biocatalysis and therapeutics.82 Scientists

introduced a novel method for fabricating nano- and microscale polyaniline particles

containing an entrapped oxidoreductase enzyme for the use in biosensing applications.83

Another recent development has been the ingenious use of an emulsion containing an aqueous

solution of polyethyleneimine (PEI) to encapsulate Trametes versicolor laccase.84 Surface

24

area, average pore size tenability and chemical inertness are some of the factors that may

promote the usage of silica matrices in protein entrapment. Very few research works

published so far report on silica entrapment of biomolecules such as enzymes and antibodies.

In one of their works, Luckarift and co-workers managed to develop a method to encapsulate

lysozyme in amorphous silica and titania.85 This rapid one-pot precipitation was found to be

induced by the lysozyme itself at room temperature. Having this property in mind scientists

adsorbed lysozyme on gold surfaces and then let it initiate silica formation. The enzyme was

entrapped in the layer surrounding the gold nanoparticles.86

1.4 Selected applications of biomolecule-nanomaterial conjugates

1.4.1 Nanomaterials in biocatalysis

Biocatalysis is the catalysis in living systems. In the biocatalytic reactions biocatalysts also

known as enzymes are engaged in the chemical transformations of organic compounds. The

main role of enzymes is to increase the rate of chemical reactions with a very high specificity

and selectivity. Enzymes mainly function in aqueous reaction media. Having the chance to

eliminate many of the steps present in the organic synthesis of a chemical compound as well

as to avoid the harsh reaction conditions (for ex. organic solvents) especially in industrial

large-scale plants, scientists found enzymes to be an attractive alternative. However, since

enzymes are naturally designed for the confined cellular environments in living organisms,

maintaining structural stability and activity over time became an issue when used under

operational conditions in industry. The high enzyme production cost and the separation of the

biocatalyst from the end product after reaction are other two issues that should be taken into

account. A common approach is to phase-separate the enzyme from the reaction mix by

immobilization. In this way the enzyme stability is increased and re-usability of the

biocatalyst by separating the solid phase is possible. Since the large-scale use of immobilized

enzymes, which started more than 50 years ago, substantial efforts have been made to

optimize the carrier material for better catalytic efficiency and stability. Materials of different

shape, size and composition have been used as solid supports. The surface area to volume

ratio, high loading and the mass transfer resistance are among the most important factors that

influence catalytic efficiency. In addition to those factors, nanoscale biocatalyst systems

exhibit unique behaviours that distinguish them from traditional immobilized systems. The

Brownian motion of nanoparticles, confining effect of nanopores and self-assembling

behavio

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26

term stability and robust re-use of the enzyme compared to the physisorbed one.78 Magnetic

Fe3O4 nanoparticles treated with (3-aminopropyl)triethoxysilane were used as the

immobilization platform for lipase, where the enzyme was covalently bound to the amino-

functionalized magnetic nanoparticles by using glutaraldehyde as a coupling reagent.92 This

approach resulted in very high enzymatic activity retention. Other industrially valuable

enzymes like α-CT (alpha-chymotrypsin)93 glucose oxidase94 and cholesterol oxidase62 were

successfully bound on magnetic nanoparticles.

Mesoporous materials have also been widely used to host enzymes by adsorbing them in the

pores or even by forming a stable covalent bond between the enzyme and the inner surface of

the pore. Adsorption of enzymes on pre-fabricated porous inorganic supports, such as

mesoporous silicates (MPS), is currently one of the most attractive enzyme immobilization

methods due to the offered simplicity, support stability and large surface area. Peroxidases

have been widely utilized in applications for the decomposition of pollutants, such as lignin or

dioxins. Takanashi and co-workers managed to produce a mesoporous silica matrix with a

pore size just big enough to fit horseradish peroxidase.91 The retained enzymatic activity was

high and low leaching of the enzyme was reported. Scientists then started using all the know-

how in pore fine-tuning in order to find the most suitable dimensions for enzyme binding.

Wang and co-workers went one step further and managed to additionally stabilize α-CT by a

multipoint covalent attachment of the enzyme to the newly processed highly-ordered sol-gel

glass.95 The result was improved stability and retained high activity after binding.

1.4.2 Nanomaterials in DNA enrichment

The field of DNA enrichment received a lot of attention after the breakthroughs in DNA

sequencing technologies.96 Since targeted analysis limited to a certain genomic region would

substantially reduce time and cost, scientists started using DNA microarrays as platforms of

choice in order to hybridize the target DNA sequence, elute and detect it.97, 98 The core

principle behind microarrays is hybridization between two DNA strands, the property of

complementary nucleic acid single strands to specifically pair with each other by forming

hydrogen bonds between the complementary nucleotide base pairs (Figure 1.4 A).

Hybridization is one of the three main ways employed in DNA enrichment. Other support-

free methods like molecular inversion probes (MIPs) and PCR amplification are also used.99

DNA hybridization in fact could be: a) attachment to a solid surface like in the case of DNA

microarrays, meaning solid-based and b) solution-based hybridization, where particles

equipped with the complementary DNA sequence were dispersed in solution (Figure 1.4 B).

Solution

microar

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28

DNA binding to charged surfaces for the purpose of intracellular delivery and protection

against external conditions was demonstrated by Kneuer and co-workers.100 Silica

nanoparticles were synthesized and then functionalized with cationic functional groups which

allowed for plasmid DNA binding due to the electrostatic interactions between the charged

surface and the DNA. The same strategy based on amino-modified functionalized silica

nanoparticles but a novel approach was later shown by He and co-workers.101 Once again,

functionalized silica nanoparticles displaying a positive surface charge at neutral pH due to

the presence of amino groups were used in binding and enrichment of plasmid DNA.

However those examples show only the idea of enrichment but do not follow the single

stranded DNA (ssDNA) hybridization concept as in the case of the microarrays.

In the last two approaches charge-to-charge interactions are exploited. In a recent study

however, Song et al. managed to bind a ssDNA sequence to core-shell magnetic graphitic

nanocapsules (MGNs) by adsorbing it on the graphitic shell via π -stacking interactions

between the nucleotide bases at the end of the sequence and the MGN outer graphitic

shells.102 A decrease in quenching demonstrated the hybridization of the complementary

sequence and the 5-fold up-concentration, achieved simply with the aid of a magnet, led to

even higher increase in fluorescence. The use of magnetic nanoparticles in genomic DNA

enrichment was once again shown by Gnirke and co-workers.103 They used pre-designed

biotinylated RNA “baits” to bind to complementary sequences from a “pond” of DNA

fragments and then enrich them by letting them bind to streptavidin-coated magnetic particles.

They have developed a hybrid-selection method for enriching specific subsets of a genome

that is flexible, scalable and efficient but also independant of the DNA sequencing equipment.

This work is important in the later developments of the solution-based hybridization concept.

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30

2.1 Introduction

Recent advances in biotechnology have opened the way for the widespread application of

biocatalysis in industrial organic synthesis by offering biodegradable catalysts (enzymes) with

a high stereo-, chemo- and regio-selectivity as well as mild operating conditions.104 Enzyme

separation from product after reaction, efficient recovery and reuse of costly enzymes

especially in industrial (large-scale) processes have led to the development of enzyme

immobilization via adsorption, entrapment, cross-linking and covalent linkage on solid

supports.105 Throughout the years, substantial research has been completed in order to

optimize the carrier material for having high surface area/volume ratio, enzyme loading and

substrate diffusion.87 The most important property of enzyme carriers is their mechanical and

chemical stability over a vast range of pH values.104 The latest achievements in

nanotechnology show a possibility to develop a revolutionary class of biocatalyst

nanostructured materials (nanoparticles, nanofibers, mesoporous materials and single enzyme

nanoparticles) for enzyme immobilization. When enzymatic carriers are in the sub-

micrometer scale one limitation is the difficulty of catalyst separation from solution.

Magnetically driven separations are much easier and faster than cross-flow filtration and

centrifugation. Magnetic nanoparticles with good stability (core/shell particles) and high

magnetic saturation (Ms) are of great interest in biocatalysis as enzymatic carriers because

they combine the advantages of easy and fast separation with high dispersion and

reactivity.106 Iron oxide nanoparticles (maghemite, γ-Fe2O3, or magnetite, Fe3O4), with a

magnetic saturation of 30-85 (emu/g), have shown to be a possible platform for enzyme

immobilization.78, 93, 94, 107-112 The Ms of the iron oxide nanoparticles however leads to a slow

separation from liquid media (min to h) even after applying strong magnetic fields.113, 114

Further, the iron oxide materials have a limited chemical stability and product leaching115, 116

as well as loss of immobilized enzyme is a problem when scaling the idea of magnetic

separation to larger volumes. Carbon coated metal nanoparticles (Co/C and Fe3C/C), which

combine the beneficial magnetic properties of the core (high magnetic saturation of >158

emu/g) and the possibility of covalent surface chemistry, have been reported in literature

(average size of 30 nm)117 and are commercially available. The advantages of the carbon

coated metal nanomagnets compared to iron oxides (e.g. SPIONS) have previously been

reported for applications in semi-heterogeneous catalysis118, 119, water120 and blood113, 121

purification. For application in biocatalysis, the possibility of linking the enzyme to the

support via a reliable organic chemistry based procedure is especially attractive as it promises

good catalyst recycling and low proteinous by-products in the final product.

31

In this study, we report a successful covalent immobilization of three widely used enzymes

on the surface of carbon coated metallic nanoparticles: β-glucosidase (β-Glu) from almonds

(E.C. 3.2.1.21), α-chymotrypsin (α-CT) from bovine pancreas (E.C. 3.4.21.1) and Candida

Antarctica Lipase B (CALB) (E.C. 3.1.1.3). We show that these enzymes retain a large

fraction of their activity when immobilized on the nanosupport, can be easily separated (s to

min) from reaction media and reused with consistent results (Figure 1a). This was shown not

only on a small (mL) but also on larger scale utilizing a 20 liter glass reactor modified with a

magnetic filter.

2.2 Experimental section

2.2.1 Particles activation for bioconjugation

The first step is diazonium chemistry. The Co/C nanoparticles (5 g, TurboBeads, Zurich) were

suspended in dH2O (250 mL) by the use of an ultrasonic bath (Sonorex RK 106, Bandelin) for

15 min. A cooled (ice bath) solution of 2-(4-aminophenyl)ethanol (1.5 g, 11 mmol, Sigma-

Aldrich) in 50 mL dH2O and concentrated HCl (5 ml, Sigma-Aldrich) was prepared and

added to the particle suspension followed by another ultrasonic bath cycle of 30 min. In the

first 5 min a cool solution of sodium nitrite (2 g, 29 mmol, Fluka) in 15 ml deionized H2O was

added drop-wise to the final suspension. The Co/C-Ph-EtOH nanoparticles were recovered

from the reaction mixture with the aid of a neodymium based magnet (Q-30-30-15-N,

Webcraft GmbH, side length 30 mm) and washed 4x with water, 4x with ethanol, 2x with

ethylacetate and 4x with acetone. Each washing step consisted of suspending the particles in

the solvent, 4 minutes ultrasonication and retracting the particles from the solvent by the aid

of the magnet. After washing the particles were dried in a vacuum oven at 60° C.

DSC-activation is the second step. Under N2 flow the Co/C-Ph-EtOH nano-particles produced

via diazonium chemistry (300 mg) were added to a previously evacuated Schlenk flask

together with N, N’-Disuccinimidyl carbonate (200 mg, 0.78 mmol, Fluka) and 4-

Dimethylaminopyridine (50 mg, 0.41 mmol, Acros) by quickly opening and closing the

septum. Dimethylformamide (30 mL, dry) was then injected through the septum and the

reaction mixture was suspended by the use of an ultrasonic bath (20 minutes) and shaken

overnight on an orbital shaker (VXR basic, IKA). The nanoparticles were recovered from the

reaction mixture with the aid of a magnet and washed 2x with dimethylformamide (DMF) and

32

2x with acetone. Each washing step consisted of suspending the particles in the solvent, 4

minutes ultrasonication and retracting the particles from the solvent by the aid of the magnet.

2.2.2 Enzyme immobilization

The DSC-activated particles (300 mg) immediately after washing were suspended in acetone

(300 mL) out of which 50 mL were taken for analysis. The remaining particles (250 mg) were

recovered by magnet, the acetone was discarded and respective enzyme solution (80 mL) was

added. The enzyme solutions contained either lyophilized powder in the cases of β-Glu from

almonds (50 mg, Sigma-Aldrich) and α-chymotrypsin from bovine pancreas (25 mg, Sigma-

Aldrich) or enzyme in liquid storage solution in the case of the Candida Antarctica lipase B

(250 µL, Almac Sciences) dissolved in 80 mL of dH2O. The final suspensions were

ultrasonicated for 5 min and left shaking for 5 hours. The particles were then washed 5x with

dH2O. Each washing step consisted of suspending the particles in water, 1 minute

ultrasonication and retracting the particles from the solution by the aid of a magnet. The

immobilized enzymes were stored at +4 °C in dH2O containing 0.02 % NaN3 (Brunschwig).

Apart from the covalently immobilized enzyme, adsorption was also performed. The

procedure is the same as for the covalent immobilization with a difference in the enzymatic

support (non-functionalized Co/C instead of the DSC-activated particles were used).

2.2.3 Enzymatic activity assays

α-chymotrypsin protocol: The increase in absorbance (production of N-benzoyl-L-tyrosine)

over time at a wavelength of 256 nm was measured on a spectrophotometer (Nanodrop 2000c,

Thermo scientific) in a quartz cuvette at 25 °C. The assay mixture of the free enzyme

contained: Tris/HCl buffer pH 7.8 (1.42 mL, 80 mM, Fluka), N-benzoyl-L-tyrosine ethyl ester

substrate (1.4 mL, 1.18 mM, Sigma-Aldrich), CaCl2 x 2 H2O (80 µL, 2 M, Fluka) and either

HCl (100 µL, 1 mM, Sigma) in case of the blank or enzyme solution (100 µL) in case of the

sample to a final volume of 3 mL. Immediately after the enzyme addition 1.5 mL of the

mixture were transferred to a cuvette and absorbance was monitored each minute for 8

minutes. The assay mixture of the immobilized enzyme contained five times the components

of the free enzyme mixture to a final volume of 15 mL. Reaction started with the addition of

the immobilized enzyme suspension (500 µL) and was measured every second minute for 14

minutes. Each time a sample was taken out (1.5 mL) the particles were retracted with the aid

of a magnetic separator and the clear solution was measured in a cuvette.

33

β-glucosidase protocol: The increase in absorbance (production of p-nitrophenol) over time at

a wavelength of 405 nm was measured on a micro-titer plate reader (Infinite f200 Tecan) in a

transparent flat bottom 96-well plate (TPP) at 25 °C. The assay mixture of both free and

immobilized enzyme contained 4-nitrophenyl β-D-glucopyranoside (11 mg, 0.037 mmol,

Sigma-Aldrich) dissolved in PBS buffer (1.404 mL, Gibco, adjusted to pH 6.5 with 0.1 M

HCl) to which enzyme solution was added (48 µL). Samples (242 µL) were added to a

stopping NaHCO3 (Fluka) buffer solution (62 µL, 0.1 M, pH 11) and transferred to the

microplate for measurement. In the case of the immobilized enzyme, particles were first

retracted with the aid of a magnetic separator and the clear supernatant was put into the

stopping buffer. Samples were taken every minute for 4 minutes in total.

CALB protocol (tributyrin hydrolysis assay): Lipase catalysed hydrolysis of tributyrin (Sigma-

Aldrich) was followed titrametrically with the aid of a pH meter (Metler Toledo) in a thermo-

regulated (28 °C) reaction vessel. The vessel contained potassium phosphate buffer pH 7.0

(60 mL, 5 mM) and tributyrin (5 mL) to which enzyme was added (100 µL of each free and

immobilized suspension). Reaction took place for 2 hours and a burette titration with NaOH

(0.1 M) followed to get to pH 7.

2.2.4 Enzymatic activity calculation

The specific enzymatic activities (U/mg) of both free and immobilized -Glu and -CT were

calculated with the given formula: Specific Activity =(∆A x Vt x Df)/( x l x Vs x C) where

∆A=(∆A Test - ∆A Blank)/min. at the desired wavelength (256 or 405 nm); Vt=total volume

of the reaction mixture; Df=dilution factor; =extinction coefficient; l=path length;

Vs=volume of the sample; C=protein concentration.

The extinction coefficient in the case of the p-nitrophenol (β-Glu assay) was calculated under

our assay conditions; =13394.43 (M-1 x cm-1) and in the case of the N-benzoyl-L-tyrosine (α-

CT assay) is 0.964 (mM-1 x cm-1) (Sigma-Aldrich). The path length is l=1 cm.

The specific enzymatic activity (U/mg) of both free and immobilized CALB was estimated

according to the titrant consumption over time. One enzymatic unit (U) corresponds to a

consumption rate of 1 µmol NaOH per minute. When divided by the total enzyme mass (mg)

in the reaction mix we obtain the specific enzymatic activity.

2.2.5 Protein concentration measurement

The amount of protein immobilized on the particles was estimated from the C, H, N

percentage mass increase after immobilization obtained by elemental microanalysis

34

measurement (Vario Micro Cube, Elementar) and from knowing the elemental content of the

enzymes (% N and % C).

2.2.6 Large-scale experiment

The large scale experiments of the covalently immobilized -Glu were performed in a 20 L

reactor tank (Büchi Glas Uster) equipped with a stirrer, flow pump, flow meter and modified

with a magnetic filter. The substrate solutions (2x) were prepared by dissolving 4-nitrophenyl

β-D-glucopyranoside (2 g, 6.64 mmol) in PBS buffer pH 6.5 (15 L). After the tank was filled

with substrate solution the enzyme-particle conjugates were added (600 mg) and the reaction

was left to take place (18 min.) under continuous stirring. The particles were separated with

an aid of a small magnet placed on the wall of the reaction vessel (15 min.), the yellow

product was discarded and the second substrate solution was added. The magnet was

removed, particles suspended and the reaction was left to take place once again (18 min.). The

solution was then pumped through the built-in magnetic filter.

2.2.7 Analytics

FTIR spectroscopy: Samples were prepared in pure KBr (Sigma-Aldrich) (5wt% particles)

and measured by a Tensor 27 Spectrometer (Bruker Optics, equipped with a diffuse

reflectance accessory, DiffusIR™, Pike technologies).

SEM/STEM analysis: The increase in mass of the nanoparticle-enzyme conjugates was shown

by elemental microanalysis. Samples were prepared in gelatin capsules and VSM hysteresis

data was obtained (MicroMag 3900 VSM) and their morphology was investigated by means

of scanning electron microscopy (SEM) and scanning transmission electron microscopy

(STEM) (FEI Nova NanoSEM 450 and FEI Magellan 400 FEG ). For SEM, the samples were

sputter-coated with a 3–4 nm platinum layer and pictured at 5 kV. For STEM, the NPs were

loaded onto copper/carbon grids and the microscope was operated at 30 kV.

XPS analysis: Sample pre-treatment: a spatula tip of the powder was put on top of double side

sticky C-tape. No further sample treatment was performed. XPS analysis was performed using

a PhI5000 VersaProbe spectrometer (ULVAC-PHI, INC.) equipped with a 180° spherical

capacitor energy analyzer and a multi-channel detection system with 16 channels. Spectra

were acquired at a base pressure of 5 10-8 Pa using a focused scanning monochromatic Al-Ka

source (1486.6 eV) with a spot size of 200 μm. The instrument was run in the FAT analyzer

mode with electrons emitted at 45° to the surface normal. Pass energy used for survey scans

was 187.85 eV and 46.95 eV for detail spectra. Charge neutralization utilizing both a cool

35

cathode electron flood source (1.2 eV) and very low energy Ar+–ions (10 eV) was applied

throughout the analysis.

Peptide analysis: 50 L sample (27.4 pmol/L) from both native and immobilized -Glu in

water with 50 L buffer (10 mM Tris/2 mM CaCl2, pH 8.2) plus 5 L trypsin (100 ng/L in

10 mM HCl) were incubated overnight at 37 °C. Supernatant was collected and the beads

were extracted with 50 L 0.1% TFA/50% acetonitrile. All supernatants were combined,

dried, dissolved in 100 L 0.1% formic acid and transferred to autosampler vials for

LC/MS/MS. The instrument used is a Q Exactive Hybrid Quadrupole-Orbitrap Mass

Spectrometer (Thermo Fisher Scientific inc.) equipped with a nanoAcquity UPLC System

(Waters Corp.). 5 L of each sample were injected for analysis. Database searches were

performed by using the Mascot (NCBI_nr, all species) search program.

2.2.8 Desorption experiment

Solutions of acetonitrile were prepared by mixing acetonitrile (HPLC grade, Sigma-Aldrich)

with water to obtain the following concentrations: 10, 30, 50, 70 and 90 %. 20 mg of adsorbed

protein particles were dispersed in 20 mL of the acetonitrile solutions by ultrasonication for 3

min and then shaken at 600 rpm for 20 min. The solvent was discarded with the aid of a

magnet and particles were dried in a vacuum oven overnight. Sodium dodecyl sulfate (SDS)

was prepared by dissolving 1 g of SDS powder (Fisher Scientific UK, electrophoresis grade)

into 20 mL dH20 and cooking it in the microwave for couple of seconds until it dissolves

completely. 10 mg of Co/C--Glu (adsorbed) were dispersed in 2 mL of the 5% SDS by

ultrasonicating for 3 min and then incubating in a thermomixer (Eppendorf, comfort) for 10

min at 95 °C. Five washing cycles were performed with water where the particles were

retracted by magnet each time. Particles were dried in a vacuum oven. Phenol solution was

prepared by dissolving 1 g of phenol powder (Sigma-Aldrich, puriss) into 20 dH2O. 10 mg of

Co/C--Glu (adsorbed) were dispersed in 2 mL of the 5% phenol solution by ultrasonicating

for 3 min and shaking at 600 rpm for 20 min. The solvent was discarded with the aid of a

magnet; particles were washed 5 times with water and dried in a vacuum oven overnight.

2.2.9 Cobalt leaching experiment

Atomic absorption spectroscopy analysis (Varian, Spectraa 220 FS) was performed to

determine Co concentration from solution. 1 mg of the β-Glu immobilized Co/C nanoparticles

were incubated for 30 min in 20 mL of the enzymatic reaction buffer (PBS pH 6.5), particles

36

were retracted with the aid of a magnet and the clear supernatant was analyzed. Standard

curve was prepared by using Co standard for AAS (Fluka).

2.2.10 Magnetic separation with a small magnet

Two dispersions both containing dispersed nanoparticles in water either Co/C or Dynabeads

at a concentration of 1 mg/mL were prepared in a final volume of 20 mL. A small cubical

neodymium based permanent magnet with a side length of 1.1 cm was placed between the

vials. Images were taken at different time points. The separation speed was correlated to

increase in relative transmission through the vial.

2.3 Results and discussion

Prior to covalently attaching the enzymes to the surface of the magnetic beads, enzymes were

merely adsorbed to the hydrophobic surface (garphene-like carbon) of the beads (Figure

2.1b). The immobilization of proteins on hydrophobic surfaces is a common technique in

protein immobilization and is best known from the preparation of ELISA (enzyme-linked

immunosorbent assay) plates.122 Indeed, when the particles were immersed in a glucosidase

solution they were rapidly covered with a monolayer of the enzyme (30 mg enzyme per 1 g

particles). After washing away any non-bound material, the enzyme adsorbed onto the particle

surface retained about 50% of its original activity (see Scheme A1.1 in the Appendix for the

enzyme immobilization methodology). However, during several reuse cycles the activity of

the particles decreased rapidly (Figure 2.1e). The following experiment was conducted to

elucidate why the activity of the particles decreased: enzyme loaded particles were added to a

substrate solution and the reaction was followed by measuring the product formed (UV-VIS)

for 3 minutes, when the particles were removed by magnetic attraction. Any product

formation after removal of the particles could then be ascribed to the non-removed enzyme

(i.e. non-bound enzyme). We found this method to be most sensitive and it quickly showed

the degree of enzyme loss from the particle surface (Figure 2.1d, dotted line).

Several methods were tried to chemically desorb previously adsorbed -Glu on the Co/C. The

methods included single washings of the Co/C--Glu (adsorbed) with aqueous solutions of

acetonitrile (10, 30, 50, 70, and 90%), phenol (5%) or SDS (5%). Elemental microanalysis

was utilized to follow the course of any protein desorption, as this is a very precise and

Figure

homoge

recover

further w

covalen

surface

DSC, D

(nitroph

magneti

experim

f) Comp

immobil

2.1 a) Th

eneously mi

red from th

work-up; b)

nt attachmen

for the imm

DMAP, dry D

henol, optic

ic particle

ments of the

parison betw

lized enzym

he biocataly

ixed with t

he product

) Methods f

nt (to the rig

mobilization

DMF, RT, 2

cal absorptio

es with ad

same produ

ween the en

mes.

yst immobi

the substra

by magneti

for enzyme i

ght); c) Che

n of enzyme

24 h, iii) enz

on at 405 n

dsorbed an

uct; activity

nzymatic ac

ilized onto

ate during

ic force aft

immobilizat

emical funct

es, E=enzym

zyme, H2O,

nm) after rep

nd chemic

y in both ca

ctivities and

the partic

the reactio

fter reaction

tion employ

tionalizatio

me: i) HCl,

RT, 5h; d)

epeated add

cally attach

ases is relati

d protein lo

cle surface

on mixture.

n and direc

yed: adsorpt

n and activa

NaNO2, H

Tracking of

dition and m

hed -Glu

ive to the on

oading of th

(center) is

It can be

ctly reused

tion (to the

ation of the

H2O, RT, 30

f product fo

magnetic rem

u. e) Recy

ne of the fir

he three ch

37

s quasi-

rapidly

without

left) and

e particle

0 min, ii)

ormation

moval of

yclability

rst cycle.

emically

38

Table 2.1 C, H, N weight change of the adsorbed Co/C–β–Glu after being washed with

different solvents relative to the non-washed adsorbed enzyme mass % determined by

elemental microanalysis measurement (Co/ C–β–Glu (adsorbed) - C: 8.8 %, H: 0.41 %, N:

0.74 %)

Sample C (%) H (%) N (%)

Acetonitrile (10%) + 0.3 + 0.04 + 0.01

Acetonitrile (30%) + 0.2 + 0.01 + 0.01

Acetonitrile (50%) + 0.2 + 0.03 + 0.01

Acetonitrile (70%) + 0.1 + 0.04 + 0

Acetonitrile (90%) - 0.2 + 0 - 0.01

Phenol in H2O (5%) + 0.05 + 0.06 + 0

SDS in H20 (5%) + 1.15 + 0.25 - 0.01

quantitative measurement technique. In all described cases with our washing strategy no

substantial amount of protein could be eluted in a short time (20 min of shaking at 600 rpm,

See Table 2.1). In literature there is already data on carbon nanotubes (CNTs) and their

interaction with proteins. Although geometrically different both CNTs and our Co/C

nanoparticles share the same surface structure, which is a graphene-like layer. There are four

types of forces that can contribute to the non-covalent binding of proteins on carbon: Van der

Waals interactions (π-π stacking), hydrophobic interactions, amphiphilicity and weaker but

still existing electrostatic interactions.123 These forces are very strong to even hold most of the

enzyme molecules attached to such a surface without the need of a covalent binding.124

However in order to achieve a 100 % effective biocatalytic switch we had to be sure that not

even a single molecule desorbs from the particles surface during prolonged enzymatic action

and following prolonged product storage, which was not the case with the adsorbed β-Glu

(Figure 2.1d). Slow (course of hours/days) desorption of enzyme molecules from a surface

can happen due to the transient complex turning where due to structural changes of the

biomolecules some enzymes which are adsorbed loosely are squeezed out spontaneously and

released in time.125, 126 This is consistent with the loss of enzymatic activity after prolonged

storage of the enzyme adsorbed to the magnetic substrate (Figure 2.2). Further, repetitive

retractions with the aid of a magnet could also have a mechanical impact releasing free

enzyme in solution after multiple particle collisions.

Figure

period a

retained

after 22

immobil

To redu

attached

Diazoni

rather in

introduc

Disucci

of the

microan

the NH

which th

at 1650

proteins

yielded

confirm

case of

metallic

Co/C-D

is slight

O comp

2.2 Enzym

at +4 °C. Th

d after one

2 days of

lized enzym

uce the en

d to the surf

ium chemis

nert carbon

ction of an

nimidyl car

immobiliz

nalysis (See

S-carbonate

hen disappe

and 1540 c

s.128 To qua

a protein lo

med by X-ra

Co/C-Ph-E

c state. Co,

DSC activate

tly higher th

ponents or a

matic activit

The covalent

month whe

storage. T

mes measure

zyme loss

face of the p

stry is one

n surfaces a

n alcohol g

rbonate (DS

zation were

e Appendix,

e (asymmet

ears after pr

cm-1, corresp

antify the am

oading of 4.

ay photoele

tOH particl

C, O and N

ed particles

han expecte

an incomple

ty changes

tly attached

ereas the ad

The enzyma

ed immediat

to an abso

particles by

of the few

and its app

group on th

SC) activati

e followed

, Table A1.

tric stretch

rotein additi

ponding to

mount of bo

.5 wt% (Fig

ectron spect

les Co, C an

N could be c

where Co i

ed (5). This

ete DSC fu

of the imm

d enzyme (so

dsorbed enz

atic activiti

telly after im

olute minim

diazonium

chemistrie

plication wi

he particle

ion and pep

by FTIR

1). The IR p

of the NH

ion. The fin

amide 1 an

ound enzym

gure 2.1f). T

troscopy an

nd O could

clearly dete

is in a meta

indicates a

unctionalizat

mobilized β-

olid line, squ

zyme (dash

ies are rela

mmobilizati

mum, the a

and carbam

es known fo

ith 2-4(-am

surface wh

ptide immob

R spectrosc

peak at 174

S-carbonyl)

nal particles

d 2 vibratio

me, CHN mic

The NHS-es

nalysis (Tab

be detected

ected as exp

llic state. T

an additiona

tion. Simila

Glu over a

uares) show

ed line, cir

ative to th

on (100 %).

active prote

mate chemis

or the funct

inophenyl)e

hich is com

bilization. T

opy (Figur

45 cm-1 refle

)127 during

displayed I

ons and bend

croanalysis

ster formati

ble A1.3 in

d but no N.

ected from

he O to N r

l O source

ar to the ads

a prolonged

wed 76 % of

rcles) showe

he activities

.

ein was co

stry (Figure

ctionalizatio

ethanol ena

mpatible wi

The individu

re 2.3) an

ects the pre

the activati

IR absorptio

ds characte

was condu

ion was add

n Appendix)

Co was ma

the structu

ratio was 6.

such as Co

dsorbed enzy

39

d storage

f activity

ed 23 %

s of the

ovalently

2.1c).

on of the

ables the

ith N,N-

ual steps

nd CHN

esence of

ion step,

on peaks

ristic for

ucted and

ditionally

). In the

ainly in a

re of the

9, which

-O or C-

yme, the

40

Figure

immobil

chemica

activity

adsorbe

magneti

be perfo

the supe

catalyst

by a sec

The reli

where n

retractin

decrease

prolong

the cova

To give

analysis

the Co/C

immobi

in the M

bound v

that the

2.3 Fourie

lization (fro

ally attache

under the

ed on the pa

ic particles.

ormed. As i

ernatant 3 m

t to the sam

cond magne

iable nature

new substra

ng the partic

e in activi

ged, repeate

alent immob

e an insight

s from prote

C magnetic

ilized enzym

MS spectrum

via this sequ

e most ab

r transform

om bottom t

d β-Glu had

same cond

article surfa

. Therefore,

it can be se

min after th

me substrate

etic separati

e of the bond

ate solutions

cles by mag

ity (ca 5%

d use. Also

bilization (F

t into the b

eolytic dige

c beads was

me, the sequ

m of the free

uence ((K)E

undant pep

m IR (FTIR)

to top).

d an initial

ditions (Fig

ace, the chem

, a fully ma

een from th

he magnetic

solution, th

on step.

d was also s

s (8 times in

gnet and dis

% per run)

o the mainta

Figure 2.2).

binding on

sts of β-Glu

performed

uence at ~10

e sample. F

EDIDAVFR

ptide in th

) spectra of

activity of ~

gure 2.1f).

mically atta

agnetic activ

he figure the

c particle re

he reaction c

supported b

n a row) w

scarding the

can be at

ained activi

the protein

u both free i

(Figure A1

000 m/z wa

rom this da

R(A)). Furth

he covalent

f the consec

~50% when

However, i

ached enzym

vity switch

ere is no in

emoval. Aft

could be tur

by the reusab

were added t

e last produ

ttributed to

ity after sto

n level, LC/

in solution a

1.2). In the M

as poorly co

ata it is assu

her support

tly bound

cutive steps

n compared

in compari

me complet

(Figure 2.1

crease in pr

er addition

rned on aga

bility exper

to the same

uct solution.

enzyme d

orage is add

/MS/MS pe

and covalen

MS spectru

vered, whic

umed that th

ting this hyp

enzyme d

s leading to

d to the free

son to the

tely stayed

1d, solid lin

roduct form

of a new m

ain, to be tu

riment (Figu

catalyst, ea

. The slight

deactivation

ditional evid

eptide ident

ntly immobi

um of the co

ch was well

he enzyme i

pothesis is

digest (~17

o enzyme

enzyme

enzyme

with the

ne) could

mation in

magnetic

urned off

ure 2.1e)

ach time

t gradual

n during

dence of

tification

ilized on

ovalently

covered

s mostly

the fact,

50 m/z;

Figure

in enzym

Metallic

removal

(R)GPS

stericall

Since t

nanopar

the enz

decrease

due to t

the part

(SEM)

aggrega

Due to m

active (e

In order

experim

immobi

recycled

with a p

2.4 a) Larg

matic cataly

c wool par

l from the c

SIWDTFTH

ly least hind

the high m

rticles, vibr

zyme-particl

e in Ms of 2

the presence

ticle surfac

(Figure A1

ates once di

mixing effe

exposed) su

r to show,

ment was co

ilized on th

d from 15 L

pump, stirre

ge scale setu

ysis is schem

rtly loaded

cartridge.

HKHPEK(I)

dered).

magnetizatio

rating samp

le conjugat

2 (< 1 % los

e of the bio

ce morpholo

.1). Co/C n

ispersed in

ects, particle

urface area i

that the pr

onducted (F

he magnetic

L suspensio

er and modif

up (20 L gla

matically di

with parti

) is on the o

on saturati

le magneto

tes and the

ss) and very

o-polymer a

ogy was de

nanoparticle

solution as

es bud off a

is more or le

resented ma

Figure 2.4).

c nanopartic

ons (Figure

fied with th

ass tank rea

isplayed; b)

icles (on th

opposite sid

ion is one

ometry (VSM

eir precurso

y little decre

around the p

etected by

es either nak

it can be s

and re-agglo

ess the sam

aterial and

This expe

cles could

2.5). For th

he addition o

actor modifi

Magnetic f

he right) af

de of the enz

e of the k

M) hysteres

ors (Table

ease of 3 (5-

particles. On

means of

ked or imm

seen from b

omerate with

me at any ins

concept ca

riment show

be rapidly

his purpose

of a magnet

ied with a m

filter used in

fter magne

zyme (i.e. a

key feature

sis data wa

A1.2). The

-8 % loss) w

n the other

scanning el

obilized ten

both SEM a

h other clus

stant of time

an be scale

wed that th

and efficie

e a glass tan

ic filter was

magnetic filt

n the separ

etic separat

attack by pr

es of the

as obtained

ere was alm

which was e

hand, no ch

lectron mic

nd to form

and STEM

sters. As a r

e.

ed up, a pil

he β-Glu co

ently separa

nk reactor e

s used.129

41

ter) used

ation; c)

tion and

otease is

metallic

for both

most no

expected

hange in

croscopy

dynamic

pictures.

result the

lot scale

ovalently

ated and

equipped

42

Figure

catalytic

from the

The rea

substrat

magneti

magnet

the part

product

(old wa

was pu

cartridg

magneti

particles

To addr

the cob

spectros

of the in

for 30 m

Finally,

protein

carbodii

(Life tec

measure

and incu

by the s

2.5 β-Glu-c

c generatio

e product m

action starte

te (2 g) so

ic separatio

placed on t

ticles could

t solution. T

s discarded

umped (> 1

ge filled wi

ic field (B)

s could be r

ress the issu

balt leachin

scopy (AAS

nstrument (

min. This is

our cataly

immobili

imide hydro

chnologies™

ed and com

ubation step

supplier. Th

coated nan

on of the ye

mixture (righ

d with the a

olution unde

on experime

the wall of

d be separat

The particle

) and the re

1.5 m3/hour

ith stainles

of 0.5 T (s

removed fro

ue of possib

ng from th

S). The coba

(< 1 mg/L)

well below

st was com

ization on

ochloride (E

™) were pu

mpared to th

ps were per

he enzymati

omagnets in

ellow nitrop

ht) within m

addition of

er continuo

ent the par

the reactor

ted from th

es were then

action took

r) through

s steel wo

ee Appendi

om the react

ble cobalt t

he Co/C--G

alt concentr

when 1 mg

w any toxico

mpared to a

n magnet

EDC) activa

urchased an

he same res

rformed exa

c activity w

n 15 liters

phenol prod

merely 30 se

the catalyst

ous stirring

rticles were

(non-optim

he reaction

n reused by

k place again

a magnetic

ool and fou

ix). Under t

tion mixture

toxicity an

Glu particl

ration was d

g of particle

ologically re

commercia

tic carrier

ation capabl

nd their enzy

sults obtain

actly as des

was measure

of substrat

duct and co

conds.

t (0.6 g β-G

and contin

e separated

mized separa

mixture in

y the additio

n. The secon

c filter uni

ur permane

these optim

e (15 liters)

experiment

les with th

determined

es was shak

elevant level

ally availabl

rs. N-ethy

le Dynabead

ymatic activ

ned with the

scribed in t

ed in an iden

te solution (

ould be ma

Glu loaded n

nued for 18

from the v

ation). Und

18 minutes

on of a new

nd time the

t, which w

ent magnets

mized separa

within only

t was perfor

he help of

to be below

ken in react

l.130, 131

le gold stan

yl-N’-(3-dim

ds®, MyOne

vity and sep

e Co/C--G

he coupling

ntical way a

(left) facilit

agnetically

nanomagnet

8 min. In

volume by

der these con

s to yield a

w substrate

particle sus

was compos

s, which c

ation condit

y 30 second

rmed to inv

atomic ab

w the detect

tion buffer

ndard in the

methylamin

e™ Carboxy

paration spe

Glu. Both ac

g protocol p

as with the

tated the

removed

ts) to the

the first

a small

nditions,

a yellow

solution

spension

sed of a

reated a

ions, the

ds.

vestigate

bsorption

ion limit

(20 mL)

e area of

opropyl)

ylic Acid

eed were

ctivation

provided

Co/C-β-

43

Table 2.2 Quantitative comparison in enzyme loading, activity, cost and magnetization of

Co/C and Dynabeads (DB) as supports for β-Glu immobilization

Sample Enzyme loading (mg protein/g

particles)

Activity per carrier (U/g nanoparticles)

Cost per activity (USD/U activity)

Magnetic saturation (emu/g)

Co/C--Glu 45 585 0.3 133

DB--Glu 30 483 18 25.1

Glu. Comparison between the specific enzymatic activities of both free and immobilized

enzymes is given in Table 2.2. As shown in the table, the β-Glu immobilized on the

Dynabeads showed activity of about 500 U/g, which is 100 U/g less than Co/C-β-Glu.

Magnetic separation speed was compared by placing a small magnet in between suspensions

of both immobilized catalysts (Figure A1.4 in Appendix). As shown on Figure A1.4, when the

magnet was placed at zero distance from the sample vials after one minute of separation the

Co/C suspension was about 85 % clear. At the same timepoint hardly any clearance could be

observed with the Dynabeads. From these experiments it can be concluded that much faster

separation is achieved when the Co/C particles are employed which is especially important in

large volumes. Even with the aid of a bigger magnet (4x4x2 cm) it took 3-4 min to obtain a

sort of clear supernatant in the case of the Dynabeads sample. The slower separation is a

result of the lower magnetic saturation values (Ms) of the Dynabeads when compared to the

magnetic saturation values of the Co/C (Table 2.2, see Appendix). It could be concluded that

the low magnetic saturation (Ms) of the Dynabeads limits their usage mainly to small volumes

whereas the Co/C beads can also be separated from larger (multi liter) volumes. In addition

the Dynabeads are more expensive than the commercially available Co/C particles. Only 20

mg of the Dynabeads ® MyOne™ Carboxylic Acid cost ~150 USD (Life Technologies™).

On the other hand functionalized Co/C nanoparticles are currently available at a price of 410

USD per 2.5 g (e.g. Sigma-Aldrich Product 742406). Calculated per achievable unit of

enzymatic activity the carbon coated carrier is more than 50 times more cost effective.

To show the potential usage as a universal enzyme carrier the magnetic nanoparticles also

served as a support for other enzymes, α-CT and CALB were covalently immobilized

utilizing the same method and their activities were assayed and directly compared to the free

enzyme in solution (Figure 2.1f). The enzymatic assay of the α-CT is based on ester bond

hydrolysis of N-benzoyl-L-tyrosine ethyl ester and N-benzoyl-L-tyrosine detection at 256

nm.132 In the case of CALB the hydrolysis of tributyrin was followed titrimetrically. While

the

44

Table 2.3 Comparison of retained enzymatic activities and Ms of the particle-enzyme

conjugates compared to literature data on magnetic immobilized enzymes; DB-COOH stands

for carboxylic acid functionalized Dynabeads®

Magnetic nanoparticles Enzyme immobilized Ms (emu/g) Retained activity (%) Ref.

Fe3O4/SiO2 lipase 30 18-24 94

Fe3O4/SiO2 α-CT 30 6-18 94

Fe3O4/CS lipase 36 56 112

Fe3O4 YADH a 63 62 108

Fe3O4/SiO2 β-lactamase 76 54 109

Fe3O4/APTES GOD a 85 15-23 110

Fe3O4 ALP a 82 20-43 111

Fe3O4 GOD a - 30 95

-Fe3O4 lipase 61 0.3-0.6 79

Fe3O4 b-DI a 38 22-43 113

Co/C β-Glu 142 55 b

Co/C lipase 142 36 b

Co/C α-CT 142 23 b

DB-COOH β-Glu 26.2 47 b

aGOD: glucose oxidase; ALP: alkaline phosphatase; YADH: yeast alcohol dehydrogenase; b-DI: biotinylated

diaphorase; bthis work

relative activities remained similar to previously reported values on magnetic iron oxide

nanoparticles (Table 2.3), and are in line with the typical loss of enzyme activity when

immobilized,104, 133 the highly increased magnetic properties and reliable magnetic attachment

facilitated a highly increased ease of recyclability.

2.4 Conclusion

Chemical functionalization of carbon surfaces and protein coupling chemistry using activated

carboxylic acids enables the immobilization of enzymes on highly magnetic (metallic)

nanosupports. In terms of activity the covalent immobilization yields good storage stability

45

and recyclability of the conjugates. In terms of applicability the improved magnetic properties

allow the usage of magnetically immobilized enzymes to multi-liter volumes. With this, the

rapidly growing field of chemical biocatalysis can profit from magnetic separation

technology, which is already well established in the fields of analytical immunoprecipitation

and cell separation on the ml scale utilizing metal oxide based particles.

2.5 Contribution of the authors

R. N. Grass initiated and supervised the whole project. V. Zlateski gave ideas, took part in all

of the experimental work (including designing of experiments) and he wrote a scientific

publication with the input and help mainly of R. N. Grass and the other co-authors. W. J.

Stark and F. M. Koehler contributed by giving valuable theoretical input and ideas. R. Fuhrer

assisted with the large-scale experimental set-up. M. Zeltner contributed by giving theoretical

and practical help with the surface chemistry on the magnetic nanoparticles. S. Wharry and T.

S. Moody provided us with the lipase CALB and the enzymatic activity measurement

protocol.

46

3

Immobi

V

ilizing and

V. Zlateski,

d de-imm

Publi

T. C. Keller

RSC Adv.

mobilizing

ished in par

r, J. Pérez-R

2015, 5, 87

enzymes

rts as:

Ramírez and

7706-87712

on mesop

d R. N. Gra

porous sil

ass

lica

47

3.1 Introduction

Mild and environmentally friendly reaction conditions in combination with high

chemo-, regio- and stereoselectivity as well as high turnover rates compared to

synthetic catalysts, contributed to the increased use of enzymes in the last decades, thus

fostering the idea of using sustainable methodologies for chemical reactions.104, 134

Even though enzymes are extremely efficient as biocatalysts for many chemical

reactions, their application is often hampered by the lack of long-term storage stability,

considering the fact that many temperature sensitive enzymes need to be continuously

stored at -20°C. Operational stability, namely inactivation caused by mechanical

treatment or heat denaturation, and difficulties in recovery and recycling are other

every-day problems.105 The effort invested to circumvent these issues led to the

development of enzyme immobilization techniques on solid supports (physical

adsorption or covalent binding), entrapment and cross-linking, which have proven to

enhance enzyme stability and enable re-use.104, 135-137

Physical adsorption is based purely on hydrogen bonds, electrostatic and hydrophobic

interactions between the support surface and the protein of interest. Compared to the

other immobilization methods it is the simplest, with which denaturation/deactivation

of the enzymes can be avoided and good enzymatic activities can be maintained.138

Adsorption of enzymes on pre-fabricated porous inorganic supports, such as

mesoporous silicates (MPS), is currently one of the most attractive enzyme

immobilization methods due to the offered simplicity, support stability and large

surface area.137, 139-142 Despite its high loading, intrinsic problems remain: immobilized

enzymes are less active than free enzymes; activity may be further lost91 due to

enzymes leaching out from the support and due to spatial constraints, the reaction of

immobilized enzymes with large substrates (proteins/DNA/polysaccharaides) is very

limited.

Previous studies have demonstrated attempts to tackle the leaching and stability

problems by fine-tuning the channel to the enzyme size (snug fit).143, 144 However, this

approach is enzyme specific and results in low protein loadings and substrate diffusion

problems.145 Another strategy is based on selective silylation thus reducing the size of

the pore openings of the mesoporous supports. In their work, He and co-workers

managed to slightly reduce the pore opening diameter of the lipase immobilized MPS

by employing 3-(trimethoxysilyl)propyl methacrylate (PMA) and polymerization of

the anchored vinyl groups with free PMA.146 Similar work was done by Ma and co-

48

worker

Howev

the latt

and the

have no

Follow

in anal

literatu

overcom

herein r

(Schem

Scheme

3.2 E

3.2.1 M

Mesoce

modific

(Sigma-

poly(eth

(4 g) w

magneti

Next, m

stirred f

rs, where t

ver, the con

er case) fo

ermal stab

ot solved th

ing on pre

ogy to enc

re knowled

me the dis

report a no

me 3.1).

e 3.1 Silica

Experimen

MCF meso

ellular foam

cations.152 T

-Aldrich), p

hylene glyc

was added to

ic stirring p

mesitylene (3

for 2 h at

hey emplo

nditions use

or most enz

ility of the

he large su

evious unsu

capsulating

dge on enz

sadvantage

ovel appro

entrapment

tal section

oporous sili

(MCF) wa

The mesopor

pluronic P1

ol)) (Sigma

o water (12

plate (Heido

3 g) and 12

room temp

oyed the sa

ed in both

zymes to s

e proteins

bstrate lim

uccessful a

g DNA/RN

zyme imm

es that imm

ach to stab

t and fluorid

n

ica synthesi

s prepared a

rous silica w

23 (poly(et

a-Aldrich) a

0 mL), foll

lph) at 500

N HCl (23.

erature. TE

ame enzym

cases are q

survive and

was not i

mitations.

attempts to

NA (see Ap

mobilization

mobilized

bly entrap

de buffer-tri

is

according to

was synthes

thylene gly

and mesityle

lowed by K

rpm at room

.6 g, Sigma

EOS (8.5 g)

me but a d

quite harsh

d be active

investigate

o directly e

ppendix),14

n on mesop

enzymes h

enzymes a

iggered enzy

o a method

sized from T

ycol)-block-

ene (Sigma

KCl (6.1 g).

m temperatu

-Aldrich) w

) was then

different gr

h (toluene,

e again. Be

d. Both st

encapsulate48-151 we t

porous ma

have durin

and release

zyme release

reported pr

TEOS (tetra

-poly(propy

-Aldrich). F

The mixtu

ure until it b

were added a

added to t

rafting stra

70 °C or 3

esides, mec

trategies h

e proteins i

took advan

aterials. To

ng applicat

e them on

e.

reviously w

aethyl ortho

ylene glycol

First, pluron

ure was stir

became tran

and the mix

the mixture

ategy.147

35 °C in

chanical

owever,

in silica

ntage of

o further

tion, we

demand

ith some

osilicate)

l)-block-

nic P123

red on a

nslucent.

xture was

e and all

49

together stirred vigorously for 30 min at room temperature. The solution was transferred to a

Teflon-lining steel autoclave and was aged for 24 h at 35 °C in an incubator (Binder GmbH).

Later, the solution was subsequently aged for an additional 24 h at 130 °C in a drying oven (T

6030, Heraeus Instruments), filtered and washed with water and ethanol. After all the ethanol

was evaporated, the produced powder was calcined in a furnace (Nabertherm) at a heating rate

of 60 °C/h and held at 500 °C for 6 h.

3.2.2 Mercury intrusion

Hg intrusion in the pressure range of 0.01-400 MPa was carried out in a Micromeritics

Autopore IV 9510 instrument assuming a contact angle = 140 ° with a pressure equilibration

time of 10 s. Pore size distributions were smoothed using a 2nd order Savitzky-Golay filter

over a window of 10 points to eliminate noise from the differentiation.

3.2.3 Nitrogen sorption

Nitrogen sorption at −196 °C was carried out in a Micromeritics TriStar II instrument. The

MCF was evacuated for 3 h at 300 °C, whereas the enzyme-loaded analogues were outgassed

at room temperature. The total surface area (SBET) of the samples was determined by the BET

method, and the t-plot method was used to determine the external surface area (Smeso). Pore

size distributions were determined by applying the BJH model to the adsorption branch of the

isotherm (Table 3.1).

Table 3.1 Structural properties of the mesoporous silica after synthesis (MCF), after β-

glucosidase addition (MCF-β-Glu) and following additional silica growth (MCF-β-Glu-SiO2)

Sample dpore (nm)

Vpore (cm3/g)

Smeso (m²/g)

SBET (m²/g)

MCF 23 1.95 410 434

MCF-β-Glu 21 1.70 323 368

MCF-β-Glu-SiO2 13 0.48 234 341

3.2.4 Small-angle X-ray scattering (SAXS)

The small-angle X-ray scattering (SAXS) curve was recorded on an Empyrean powder

diffractometer (PANalytical B.V., The Netherlands), operating in transmission mode with Cu

Kα radiation (45 kV, 40 mA). The interlayer spacing was calculated by the Bragg`s law (n x λ

= 2 x d x sin(θ), where λ = 0.154 nm.

50

3.2.5 TEM and SEM analysis

For transmission electron microscopy (TEM), the samples were dispersed in ethanol and some

droplets of the suspension were deposited on a lacey carbon foil supported on a Cu grid. TEM

was performed on a Tecnai F30 (FEI, field emission gun (FEG), SuperTwin lens (point

resolution ca. 0.2 nm), operated at 300 kV). For scanning electron microscopy (SEM) the

samples were resuspended in i-PrOH and loaded onto copper/carbon grids. The microscope

(FEI Nova NanoSEM) was operated at 30 kV.

3.2.6 β-glucosidase immobilization, entrapment and release

Immobilization: MCF (20 mg) was suspended in MQH2O (0.5 mL) by 30 s ultrasonicating

and 15 s vortexing. Separately, β-glucosidase from almonds (20 mg, Sigma-Aldrich) was

dissolved in MQH2O (1 mL) and split into two eppendorf tubes (0.5 mL each). In one, the

MCF suspension was added, whereas in the second MQH2O (0.5 mL) was added. Both tubes

were shaken on an orbital shaker (VXR basic, IKA) for 3 h at room temperature followed by

centrifugation at maximum speed for 4 min (CT15E, Hitachi Koki Co., Ltd). The pellet

(mesoporous silica plus enzyme) was washed with MQH2O (1 mL) and finally suspended in

the same volume of MQH2O.

Entrapment: MCF-β-glucosidase (1 mg, corresponding to 50 μL suspension after

immobilization) was suspended in MQH2O (0.45 mL) by 30 s ultrasonicating and 15 s

vortexing, followed by TEOS addition (4 μL). The final mixture was left shaking for 5 days at

500 rpm on an orbital shaker, with subsequent additions of TEOS (4 μL) after 24, 48 and 72

h. Afterwards, the sample was washed twice with MQH2O and stored in the fridge until the

next activity measurement. In parallel, the two control samples (1) β-glucosidase in water and

(2) MCF-β-glucosidase were treated the same way but without any TEOS addition.

Release: The release of the enzyme was triggered by the fluoride buffer, which was

added to the entrapped enzymes in order to dissolve the silica support. The buffer was

prepared in polyethylene, polypropylene or Teflon containers according to the

following protocol: for 10 mL fluoride buffer we dissolved 0.23 g of NH4FHF in 5 ml

of H2O and 0.19 g of NH4F in 5 ml of H2O eventually pooling the two solutions

together (pH∼4; measure pH with pH paper and not with a pH electrode). This solution

is stable at room temperature for at least 2 months. In order to release the enzyme,

enough fluoride buffer was added in order to obtain a clear solution. In the case of the

fluoride buffer at pH 5, the pH was adjusted by carefully adding NaOH (1 M, Merck).

Fluoride comprising waste as collected in a saturated calcium carbonate solution.148

51

3.2.7 Enzymatic activity assays

β-glucosidase enzymatic activity assay: The increase in absorbance (production of p-

nitrophenol) over time at a wavelength of 405 nm was measured on a micro-titer plate reader

(Infinite f200 Tecan) in a transparent flat bottom 96-well plate (TPP) at RT. The assay

mixture of both free and immobilized enzyme contained 4-nitrophenyl β-D-glucopyranoside

(11 mg, 0.037 mmol, Sigma-Aldrich) dissolved in sodium phosphate buffer (1.404 mL, 0.1

M, pH 6.5) to which enzyme solution was added (48 µL). Samples (242 µL) were added to a

stopping sodium carbonate buffer solution (62 µL, 0.5 M, pH 10.8) and transferred to the 96-

well microplate for measurement. Reaction took place over time (final time of 4 min) with

samples being taken after every minute. In the case of the silica-entrapped enzyme we took 10

µL of the reaction mix (as described in the “β-glucosidase entrapment in silica” section) and

diluted up to 100 µL with water (1:10). Then from the dilution we used 48 µL in the assay (as

mentioned above). In the cases where fluoride buffer was utilized for either enzyme release or

fluoride buffer resistance tests, 25 µL of the fluoride buffer were added to the 10 µL of the

enzyme sample, the volume was brought up to 100 µL with water and again 48 µL were taken

for the assay.

α-chymotripsin enzymatic activity assay: The increase in absorbance (production of p-

nitroaniline) over time at a wavelength of 390 nm was measured on a spectrophotometer

(Nanodrop 2000c, Thermo scientific) at RT. The assay mixture contained: tris/HCl buffer pH

7.8 (1.42 mL, 80 mM, Fluka), substrate solution (1.4 mL, 1.18 mM N-benzoyl-L-tyrosine-p-

nitroanilide, Sigma-Aldrich, dissolved in 1:1 water/DMSO mixture), CaCl2 (80 µL, 2 M,

Fluka) and 100 µL of the sample to make up a final volume of 3 mL. Immediately after the

enzyme addition, the mixture was transferred to a plastic disposable cuvette and absorbance

was monitored each minute for up to 30 minutes.

3.2.8 Enzymatic activity calculation

The specific enzymatic activities (U/mg) of both free and immobilized -glucosidase and -

chymotrypsin were calculated with the given formula: Specific Activity = (∆A x Vt x Df)/( x

l x Vs x C) where ∆A = (∆A Test - ∆A Blank)/min at the desired wavelength (390 or 405 nm);

Vt = total volume of the reaction mixture; Df = dilution factor; = extinction coefficient; l =

path length; Vs = volume of the sample; C = protein concentration. The extinction coefficient

in the case of the p-nitrophenol (β-glucosidase assay) was calculated under our assay

conditions = 13394.43 (M-1 x cm-1) and in the case of the p-nitroaniline (α-chymotrypsin

assay) = 12500 (M-1 x cm-1). The path length is l=1 cm.

52

3.2.9 Protein concentration measurement

The amount of protein bound on the mesoporous material was estimated from the C, H, N

percentage mass increase after immobilization obtained by elemental microanalysis

measurement (Vario Micro Cube, Elementar) and from knowing the elemental content of the

enzymes (% N and % C).

3.2.10 Thermal stability test

Two samples, β-glucosidase free in water and β-glucosidase entrapped in the silica material,

were submitted to thermal stress by incubating them for 1 h at 4 different temperatures: room

temperature, 50 °C, 60 °C and 70 °C in a thermomixer (compact, Eppendorf). The samples

were let to cool down to and enzymatic activity was measured where the enzyme entrapped in

silica was first dissolved with a proper amount of fluoride buffer (as described in the “β-

glucosidase assay“enzymatic activity section).

3.2.11 Fluoride buffer influence on enzymatic activity

Both β-glucosidase and α-chymotrypsin free in solution were used to check the influence of

the fluoride buffer on the enzymatic activity. The same concentrations of free enzyme in

solution as used for the immobilization process were used and enzymatic assays were

performed by using 10 μL of the enzyme solutions and 25 μL of the following fluoride buffer

solutions: 1) fluoride buffer pH 4 (preparation steps shown in the “enzyme release” section of

this materials and methods chapter), 2) 1:10 diluted fluoride buffer pH 4 and 3) fluoride

buffer pH 5 (pH adjusted to 5 with NaOH). The enzyme solutions were incubated in the

presence of the respective fluoride buffer for 2-3 minutes and then transferred to a glass vial

for neutralization of the excess F- ions. The enzyme assay solutions were added into the vials

and the desired absorbance was subsequently measured.

3.3 Results and discussion

For entrapment, we combined the well-known advantages of the mesoporous silicas

with the simplicity of the adsorption process in order to obtain high loadings of highly

active enzymes immobilized on the MCF carrier. The ultralarge cage-like mesopores

of this support are ideal for entrapping enzymes of different dimensions in high

loadings.153 Additionally, MCFs possess a three-dimensional, interconnected pore

structur

point, w

by grow

(water)

MCF m

of choi

adsorpti

distribu

IV isoth

and giv

3.1, Tab

with the

attribute

resulted

nitrogen

the App

peaks t

could b

Figure

adsorpt

MCF-β-

re which w

we develop

wing addit

).

material with

ce. The m

ion–desorpt

ution curve.

herm (well d

es an extern

ble 3.1). Th

e microscop

ed to the o

d in a pore w

n sorption w

pendix). A

o any plan

be found (F

3.1 N2 sorp

tion branch

-Glu (blue c

would faci

ped a proce

tional silic

h a mesopor

mesopores o

tion isotherm

(Figure 3.1

defined hys

nal surface

he large uni

py analysis

organic coso

window siz

was proven

Analysis of

ne or space

Figure 3.3).

ption isothe

h by the Ba

circles), and

ilitate subs

edure to fu

ca inside th

re size of 2

of the calci

ms of the d

, black squ

steresis loop

of 410 m2

iform meso

(Figure 3.2

olvent mesi

e of ~ 11 n

to be acces

the scatter

group (ex.

.

erms and po

arret-Joyner

d MCF-β-G

strate diffu

urther entra

he MCF c

3 nm was s

ined MCF

dried sample

uares). N2 so

p) obtained

g-1 with a t

opores (dpore

2, a) and c)

itylene add

nm and 75 %

ssible for H

ring data s

. p6mm) is

ore-size dis

r-Halenda

Glu-SiO2 (red

usion.154, 15

ap enzymes

ells in a p

synthesized

were confi

e and plottin

orption anal

for uniform

total pore v

e = 23 nm),

)) and the X

dition.152, 157

% of the me

g intrusion

shows that

possible a

tribution cu

(BJH) meth

d triangles)

55 Having

s within th

protein-frie

as the imm

firmed by m

ng the corre

lysis eviden

m-size meso

volume of 1

which are

X-ray data (7, 158 Hg in

esopore vol

(Figure A2

no indexin

and no SBA

urves (inset)

hod of: MC

).

that as a

he porous m

endly envir

mobilization

measuring

esponding p

nces the typ

oporous mat

1.95 cm3 g-1

in good ag

(Figure 3.3)

ntrusion exp

lume determ

2.1, Figure

ng of highe

A-15-like s

t) obtained f

CF (black s

53

starting

material,

ronment

n support

nitrogen

pore-size

ical type

terials156 1 (Figure

greement

), can be

periment

mined by

A2.2 in

er order

structure

from the

squares),

54

Figure

The tex

mesopo

β-gluco

entrappe

take pla

Knowin

which i

carried)

changin

Figure

25.6 nm

3.2 TEM a

xture of the

re openings

sidase from

ed in the sil

ace mainly

ng that the

is much low

) values of

ng the pH of

3.3 Small-a

m. No ordere

nd SEM im

e material

s in the orig

m almonds w

lica matrix.

due to the

surface of

wer than th

most prote

f the solutio

angle X-ray

ed structure

mages of the

after silica

ginal materi

was chosen

Typically,

e amino an

the silicas

he isoelectri

eins, we are

on.

y scattering

e is evident.

e MCF (a) a

a depositio

ial were no

n as a mode

proteins en

nd carboxyl

carries a n

ic point (pH

e able to tu

g (SAXS) fro

and c)) and

on drastica

longer visib

el enzyme t

nable bindin

lic acid gro

negative ch

H at which

une the cha

om a MCF

d MCF-β-Gl

lly changed

ble.

to be immo

g interactio

oups presen

arge at pH

h no overal

arge of the

F with an in

lu-SiO2 (b)

d and the

obilized and

ons for adso

nt on their

values abo

ll electric c

protein su

nterlayer sp

and d)).

obvious

d further

rption to

surface.

ove 2,159

charge is

rface by

pacing of

55

However, if the proteins get too much positively charged strong self-repulsion may

occur.139, 160 Taking advantage of improved adsorption in solutions which have a pH

near the pI value of the enzyme, we immobilized β-glucosidase (pI = 7.3) in water at

pH 7. We managed to retain activity up to 95 % after immobilization with an enzyme

loading of ~80 mg/g. The pore network is well-preserved after the loading of the

enzyme, although the size of the mesopores, and consequently their volumes are

reduced (Figure 3.1 (blue circles), Table 3.1).

With the aim to increase the enzyme stability and prevent enzyme leaching, we further

entrapped the enzymes within the matrix. For this we utilized silica sol-gel synthesis, a

well-known and fundamental reaction that brings about the conversion of silicate

precursors (i.e. TEOS) to silica gels.161 In order to minimize any perturbation of the

enzyme integrity, we performed the polycondensation reaction by simply mixing the

immobilized enzyme with highly diluted tetraethyl orthosilicate (TEOS) in water

(opposed to the traditional procedure in alcohol and base catalysis). The entrapment

process was monitored by evaluating the activity of the enzyme after various

timepoints. As shown on Figure 3.4, this entrapment process was relatively slow and

proceeded over several days, which is due to the slow polycondensation reaction of

TEOS in the absence of a suitable catalyst.162 After a silica growth process of 5 days,

the enzyme activity had dropped by > 80% indicating that new silica material had

formed within the MCF cells hindering the substrate diffusion to the enzyme. This

observation was confirmed by nitrogen sorption and electron microscopy, evidencing

that the silica deposition led to a pronounced textural modification (Figure 3.2 & Table

3.1). After the reaction the material evidences both micro- and shallow mesopores. The

mesopore volume is reduced by 75%, and the formerly uniform pore-size is

transformed into a broader distribution centred at around dpore = 13 nm (Figure 3.1, red

triangles), indicating that the cells are gradually filled with amorphous silica.

Furthermore, the broad hysteresis loop points towards the presence of ink-bottle-like

pores, i.e. pores accessible only through a narrow opening, further corroborated by the

increased intensity of the forced closure of the isotherm at p/p0 = 0.45.163, 164

While the above shows that the entrapment of the enzyme within the support was successful,

it also displays that as long as the enzyme is entrapped within the inorganic material, it is not

very active. As both the support structure (MCF) and the additional material grown within the

pores consists entirely of silica, we investigated on a de-encapsulation scheme by dissolving

the silica, again without perturbing the enzyme structure. It is well known that silica dissolves

56

Figure

Glu free

degrada

(mechan

activity.

rapidly

ammon

biochem

implica

in cla

butylam

dangero

from am

product

conside

In orde

with a

evaluat

activity

Figure

that the

The act

3.4 Enzyma

e in solutio

ation during

nical stress

.

in fluori

nium fluor

mistry as

ations with

assical pr

mmonium

ous HF, w

mmonium

ts (fluorid

ered the pro

er to de-en

small volu

te the inta

y of the res

3.5 this pr

e silica sup

tivity of the

atic activitie

on (squares)

g the 5 day

and temper

de compri

ride soluti

they are

h proteins. S

rotection

fluoride) a

e prepared

fluoride an

e gels) co

obable toxi

ncapsulate

ume of buf

ctness of

sulting solu

rocedure re

pport had b

e released

es during th

). In order

y reaction,

rature / but

ising buffe

ion). How

feared du

Still the ap

group ch

and even d

d small volu

nd ammoni

ontain up t

ic dose for

the entrap

ffered oxid

the active

ution was m

esulted in

been dissolv

enzyme we

he sol-gel sy

to rule out

free enzym

t no TEOS a

fers (e.g. b

wever, thes

ue to their

pplication o

hemistry

dentistry.

umes of a

ium hydrog

to 1.23 wt

r humans.14

pped enzym

de etch (50

site of th

measured

a great bo

ved and th

ent up to ~

ynthesis of M

that the los

me was subj

addition) an

buffered o

se reagent

r toxicolog

of fluoride

(e.g. pota

So in orde

4 wt% F-

gen fluorid

t% F- and 48

mes, 20 μg

0 µl), resu

he released

in an appr

oost in the

hat the enzy

~250 % of t

MCF-β-Glu-

ss of activit

bjected to th

nd showed o

oxide etch,

s are very

gical poten

solutions i

assium fl

er to avoid

buffered o

de. For refe

5 mg F-/k

g of the ma

lting in a

d enzyme

opriate buf

catalytic a

yme was re

the entrapp

u-SiO2 (stars

ty is due to

he same co

only a mino

, a pH st

ry rarely u

ntial und

is a comm

luoride o

d the hand

oxide etch s

erence, den

/kg bodyw

aterial was

clear solut

the β-gluc

ffer. As sh

activity, ind

eleased unh

ped one and

s) and β-

o enzyme

onditions

or loss of

abilized

used in

unclear

monplace

or tetra

dling of

solution

ntal care

weight is

s mixed

tion. To

cosidase

hown on

dicating

harmed.

d was

Figure

columns

columns

storage

entrapm

compar

fridge

entrapm

is evide

(Figure

glucosi

widely

compat

measur

buffer a

It is w

display

more t

entrapp

Besides

enzyme

3.5 Highly

s) was treat

s) and β-G

stability w

ment/release

rable to the

(Figure 3

ment/releas

ent when th

e A2.3). A

idase with

used enz

tibility tes

red for both

at pH 4 dilu

worth highl

ying data a

than half

ped enzyme

s the high m

e shows a

active β-G

ted with fluo

lu free in s

was assessed

e scheme for

e activity o

3.5, dense

se process

he immobi

As an addit

fluoride c

zyme, α-ch

t (Figure

h enzymes

uted (1:10)

lighting th

fter 15 day

of its acti

e after the r

mechanica

high resist

Glu (white c

oride-conta

solution (de

d after 15

r enzyme sto

of β-glucos

columns)

itself. It is

lized (not e

ional contr

comprising

hymotrypsi

A2.4). A

s, in the thr

) and fluori

he importa

ys of wet s

ivity, no

release.

al and stora

tance towa

columns) wa

aining buffer

ense diagon

days and fu

orage.

sidase free

), attribut

s worth me

entrapped)

rol to this

buffers (S

in, was su

minor ac

ree cases w

ide buffer

ance of th

storage in

substantial

age stabilit

ards heat tr

as released

r. The activ

nal lines) a

further show

in solutio

ing almos

entioning is

enzyme is

we measu

See Figure

ubmitted to

ctivity dec

where fluor

at pH 5 wa

he second

the fridge.

l activity

ies already

reatment. M

after MCF

ities of the M

are given fo

ws the adva

n after 5 d

st no acti

s that no in

s incubated

ured the co

A2.4). In

o the sam

crease (10-

ride buffer

as used for

compariso

While the

loss was

y discussed

MCF-β-Gl

F-β-Glu-SiO

MCF-β-Glu

or comparis

antage of th

days storag

ivity loss

ncrease in

d in fluorid

ompatibilit

n addition,

me fluoride

-20 %) co

r at pH 4,

incubation

on on Figu

e free enzy

observed

d, the encap

lu-SiO2 an

57

O2 (black

u (sparse

son. The

he silica

ge in the

to the

activity

de buffer

ty of β-

another

e buffer

ould be

fluoride

n.

ure 3.5,

yme lost

for the

psulated

d β-Glu

58

free in

60°C an

measur

activity

% activ

treatme

of the

change

The ent

enzyme

enzyma

faced w

this in c

Since t

nanopo

present

immob

Figure

Glu rel

corresp

solution w

nd 70°C fo

rement. Wh

y at 60 and

vity recove

ent (Figure

silica matr

s inside the

trapment n

es, but one

atic reactio

when worki

connection

there is alr

orous silic

ted here ca

ilized enzy

3.6 Enzyma

leased (whi

onding acti

were subm

ollowed by

hile β-gluc

70°C), β-g

ery at 50 an

e 3.6). This

rix, which

e material.

not only all

e could als

ons with la

ing with so

n with prote

ready exten

a supports

an be adap

ymes.

atic activiti

te columns)

ivities at roo

mitted to 1

y a subsequ

cosidase fre

glucosidase

nd 60°C an

s resistance

prevents t

lows for im

so take the

arge substra

olid suppor

eins, DNA

nsive work

s we anti

ted to man

ies of the β-

) after hea

om tempera

hour of in

uent release

ee in solut

e released

nd a high ~

e to heat ca

the protein

mproved op

e advantag

ates, thus t

rt immobili

and polys

k and know

cipate tha

ny other sy

-Glu free in

at treatment

ature.

ncubation a

e of the enz

tion perfor

from its en

~75 % reta

an be attrib

n undergoin

perational,

ge of the t

tackling so

ization tech

accharides

wledge on

at the fluo

ystems and

n solution (

t; the activ

at room tem

zyme and e

rmed poor

ntrapped sta

ained activ

buted to the

ng extensiv

storage an

triggered r

ome of the

hniques. A

is currentl

the loadin

oride de-en

d will offer

(dense strip

ities given

mperature,

enzymatic

(very low

ate resulted

vity after th

e protectiv

ve conform

nd heat stab

release to p

e biggest pr

Additional w

ly ongoing

ng of enzy

encapsulatio

r new aven

pes columns)

are relativ

, 50 °C,

activity

and no

d in 100

he 70°C

ve effect

mational

bility of

perform

roblems

work on

g.

ymes on

on step

nues for

s) and β-

ve to the

59

3.4 Conclusion

In summary, this work demonstrates the synthesis of a novel enzyme-in-silica material with

improved operational and storage stability, that can undergo enzyme release in solution upon

a chemical trigger. For this purpose, we utilized the well-known advantages of the

mesoporous silicas (high surface area and stability) and the simplicity of enzyme adsorption

and further optimized them according to our needs in order to obtain high loadings of active

β-glucosidase as a model enzyme. Furthermore, we developed a procedure to additionally

silica-entrap the previously immobilized enzymes which led to high mechanical, storage and

heat stability of the biomolecules. Last, we utilized a non-harmful way to dissolve the support

and trigger an immediate release of the enzyme molecules, giving a possibility to select from

both large and small substrates. In this way, one could store enzymes for a long time and

release them upon need. In the future, one could pay special attention to sensitive enzymes,

which are very delicate to handle and require low storage temperatures. The idea of replacing

the freezer with the shelves, eliminating the multiple freeze-thaw cycles and the large number

of sensitive enzymes available on the market, shows a great application potential and is

certainly worth further detailed experimentation.

3.5 Contribution of the authors

R. N. Grass initiated and supervised the whole project. V. Zlateski gave ideas, took part in all

of the experimental work (including designing of experiments) and he wrote a scientific

publication with the input and help mainly of R. N. Grass and the other co-authors. T.C.

Keller and J. Pérez-Ramírez contributed by helping us characterize the mesocellular foam and

interpret the data.

60

4 Sel

mi

lective ssD

icroarray

DNA enri

chemistr

ichment b

ry

by magne

tic up-conncentratioon using g

glass

61

4.1 Introduction

Fostered by the recent breakthroughs in DNA sequencing technologies96,165, sequence-specific

DNA detection (sequencing and especially next generation sequencing (NGS)) has become

increasingly popular especially in medical diagnostics.166,167 Time and cost became limiting in

exploiting the possibilities that the sequencing offers. As a result, molecular biology

procedures have started to target specific genomic regions by enrichment, whereas DNA chips

(solid-based hybridization) have been implemented as part of the recent sequencing

methodologies.168-170 This led to the re-use of the well-known DNA microarrays

technology,97, 98, 171, 172 a concept originally introduced for de novo nucleotide sequencing by

hybridization,173 which is based on specific ssDNA hybridization to a complementary

sequence attached on a flat surface, usually made out of glass, and its subsequent detection.

Several factors contributed to the popularity of glass slides as the substrate of choice for the

microarrays: chemical durability, low intrinsic fluorescence, chemical inertness towards

biomolecules and the established chemistries to functionalize the surface for DNA binding.97,

174-176

Despite the wide use and initial success of the glass DNA microarrays, the expensive

hardware and lack of scalability led to the development of alternative solution-based

hybridization techniques.177 Instead of the flat glass surfaces magnetic particles have been

proposed in order to collect the DNA of interest. One strategy is to fish out pre-hybridized

biotinylated probe-target duplex by the use of streptavidin-functionalized magnetic beads

simply with the aid of a magnet. Gnirke and co-workers used this approach to enrich genetic

material for parallel sequencing.103 In their work they first allowed biotinylated-RNA probes

to bind to the target of interest and then separate it from solution with the help of the

streptavidin-coated beads. Due to the two binding steps involved (binding of DNA to its

complement and binding of biotin to streptavidin) this procedure is long (66h with several

PCR steps required) and the presence of the streptavidin has been reported to result in non-

selective DNA binding.178 In addition to the application in pre-sequencing enrichment, this

hybridization technology serves as a robust platform and proved to be a crucial component in

novel nanotechnology. The use of magnetic particles in DNA analysis has also been utilized

in the work of Mirkin et al., where thiolated target DNA is immobilized on malemide

functionalized particles and further indirectly detected using gold nanoparticles decorated

with DNA bar-codes.179 Also, there are many procedures available for the non-specific

enrichment of DNA using magnetic particles first made popular by Hawkins et al.180, 181

62

In this work, we profit from the surface chemistries developed for DNA microarrays in the

90ies182 and combine these approaches with novel magnetic particles to allow for a magnetic

up-concentration of a target ssDNA without having to modify the DNA strands prior to

capture. Here, the synthesis of novel, highly magnetic and chemically inert iron-carbon (Fe/C)

silica coated nanoparticles (magnetic glass nanobeads) is shown and their usefulness for the

enrichment of DNA is demonstrated. Ultimately, we have managed to develop an easy, fast

and reliable procedure to bind and release a target ssDNA (takes only 40 min) and

subsequently detect and quantify it with a standard laboratory equipment (for ex. Qubit®

fluorimeter in 10 min) in many cases eliminating the need for PCR thanks to the reduction in

volume and increase in concentration achieved.

4.2 Experimental section

4.2.1 Diazonium chemistry (Fe/C-OH)

The Fe/C nanoparticles (1 g, TurboBeads™, Zurich) together with 4-aminobenzyl alcohol

(0.1 g, 0.81 mmol, Sigma-Aldrich) and sodium nitrite (0.1 g, 1.45 mmol, Fluka) were

suspended in dH2O (40 mL) in a 100 mL Schott flask by the use of an ultrasonic bath

(Sonorex RK 106, Bandelin) for 3 min and a high-shear mixer (IKA® T10 basic ULTRA

TURRAX) for 1 min. Concentrated HCl (1.5 ml, Sigma-Aldrich) was added to the particle

suspension followed by another round of high-shear mixing (3 min) and ultrasonication (1 h).

The Fe/C-OH nanoparticles were recovered from the reaction mixture with the aid of a

neodymium based magnet (Q-30-30-15-N, Webcraft GmbH, side length 30 mm) and washed

2x with water, 3x with isopropanol, 7x with ethanol (industrial) and 2x with ethanol (≥ 99.8

%, Fluka). Each washing step consisted of suspending the particles in the solvent,

ultrasonication (1 min), high-shear mixing (1 min) and retracting the particles from the

solvent by the aid of the magnet. After washing the particles were suspended in pure ethanol

(230 mL) and transferred to a 250 mL polyethylene bottle.

4.2.2 Silica coating (Fe/C-SiO2)

This followed directly after the diazonium chemistry step. A hole was made through the cap

of the Fe/C-OH particle suspension bottle so that the dispersing element of the high-shear

mixer could fit through and go deep in the suspension while the bottle is still closed. After the

suspension was simultaneously ultrasonicated and high-shear stirred (3 min), triethoxysilane

63

(TEOS) was added (3 mL, 95%, Sigma-Aldrich) and then another round of simultaneous

ultrasonication and mixing followed (5 min). The suspension was high-shear stirred further

for additional 5 min, ammonium hydroxide solution was added (15 mL, 25% NH3 in H2O,

Sigma-Aldrich) and the final mixture was high-shear stirred for another 3 min. The bottle was

left fixed in the ultrasonication bath (where cooling system was also installed!) and another

stirring device was mounted on top (Heidolph RZR 2102 control drill). The suspension was

continuously ultrasonicated and stirred (1000 rpm) for 24 h. The produced Fe/C-SiO2 particles

were washed 5x with pure ethanol with ultrasonication (1 min) and high-shear mixing (1 min)

in between each washing step. The particles were retracted from the mixture with the aid of a

magnet. Finally, the particles were suspended in 60 mL pure ethanol and transferred to a 100

mL Schott flask. The particles were stored in ethanol and the concentration was determined

by drying out a sample volume of the suspension and recording its mass.

4.2.3 APTES functionalization (Fe/C-SiO2-NH2)

The produced Fe/C-SiO2 particles (100 mg) were suspended in pure ethanol (41.5 mL) in a

Falcon tube by ultrasonication (5 min). (3-Aminopropyl)triethoxysilane or APTES (0.615 mL,

Sigma-Aldrich), TEOS (0.05 mL) and ammonium hydroxide solution (25% NH3, 2.5 mL)

were consecutively added to the suspension and ultrasonicated for 5 min. The suspension was

left to shake on an orbital shaker (VXR basic, IKA) at 1000 rpm for 24 h. The produced Fe/C-

SiO2 particles were washed 5x with pure ethanol. Each washing step consisted of suspending

the particles in the solvent by ultrasonication (1 min), vortexing (15 s, Heidolph REAX top)

and retracting the particles from the solvent by the aid of a magnet. Afterwards, the solvent

was removed and the particles were dried for 3 h in a vacuum furnace at 60 °C.

4.2.4 ssDNA binding (Fe/C-SiO2-ssDNA)

80 μL (100 μM stock) ssDNA (5`-NH2-AAAAAAAAAAATCGGGTTACACTGGCTGAC-

3`, Mw = 9442.5 g/mol, Microsynth) and 320 μL 3x SSC (saline-sodium citrate) were added to

15 mg Fe/C-SiO2-NH2 and suspended by ultrasonication (3 min) and vortexing (10 s) in a 1.5

mL reaction tube. The suspension was put in a thermomixer (Eppendorf, Thermomixer

compact) with the lid open and was incubated at 80 °C for 2.5 h and then at RT for another 24

h both with shaking (1000 rpm). After the reaction the particles were recovererd from the

solvent by the aid of a magnet and the remaining supernatant was discarded. The pellet was

washed with (0.5 mL each): 1x 0.1% SDS, 2x mQH2O, 1x hot (pre-heated to 99 °C) mQH2O

64

(1000 rpm, 3 min, 99 °C), 1x pre-hybridization buffer (5x SSC, 0.1% SDS, 10 mg/mL BSA)

(1000 rpm, 45 min, 42 °C), 2x mQH2O and finally store in 1.5 mL mQH2O at +4 °C.

4.2.5 Second strand hybridization/melting experiments

In the hybridization experiments both the complementary (correct) ssDNA sequence (5`-

GTCAGCCAGTGTAACCCGAT-3`, Mw = 6851.4 g/mol, Microsynth) and the control (false)

ssDNA sequence (5`-TTTTCCCTCTCTCTCCCTTT-3`, Mw = 6637.1 g/mol, Microsynth)

were ordered modified with a fluorescent label (ATTO-488) at the 5`end. All the

hybridization experiments were performed in hybridization buffer (5x SSC, 0.1% SDS) by

first ultrasonicating the suspension (2 min) and vortexing (10 s) followed by incubation with

the second DNA strand for 25 min at 37 °C with shaking (1000 rpm). Afterwards, the

particles were retracted by the aid of a magnet, supernatant was removed and the following

washing steps were performed (0.5 mL each): 1x washing buffer 1 (2x SSC, 0.2% SDS), 1x

washing buffer 2 (2x SSC), 1x washing buffer 3 (0.2x SSC). The particles were suspended in

mQH2O (40 μL). The melting was performed by incubating the particle suspension in a

thermomixer at elevated temperature (1000 rpm, 10 min, 95 °C) followed by a very fast

particle retraction with a magnet and pipetting out of the supernatant (Note: it takes

approximately 5-7 s to remove the tube from the thermomixer, retract the particles and

remove the supernatant). The DNA concentration was determined by measuring the

fluorescence of the supernatants (ex. 485 nm, em. 535 nm). The fluorescence was measured

using 200 μL (1:80 dilution of all samples) pipetted into a black 96-well plate (Infinite F200,

TECAN). The hybridization/melting procedure was conducted as described for all the

samples with certain variations in DNA amount, particle amount or volume depending on the

specific experiment. The respective variations are given below:

In the DNA selective binding experiment 40 μL 500 nM (20 pmol) ssDNA (2 μL ssDNA from

a 10 μM stock and 38 μL hybridization buffer) were mixed with 1 mg of particles.

Experiments with both correct and false DNA sequences were conducted here.

In the upconcentration experiment the amount of DNA (20 pmol) and particles (1 mg) were

kept constant and the following hybridizations were performed in different volumes: 500 nM

(2 μL ssDNA from a 10 μM stock and 38 μL hybridization buffer), 25 nM (2 μL ssDNA from

a 10 μM stock and 798 μL hybridization buffer), 5 nM (2 μL ssDNA from a 10 μM stock and

3998 μL hybridization buffer) and 0.5 nM (2 μL ssDNA from a 10 μM stock and 39998 μL

hybridization buffer).

65

In the particle amount optimization experiment the ssDNA amount (20 pmol) and volume (4

mL) were kept constant (2 μL ssDNA from a 10 μM stock and 3998 μL hybridization buffer)

and added to different amounts of particles: 0.25, 0.5, 1 and 2 mg.

In the ssDNA amount optimization experiment the particle amount (1 mg) and volume (4 mL)

were kept constant and the following DNA amounts were added: 2 pmol (2 μL ssDNA from a

1 μM stock and 3998 μL hybridization buffer), 20 pmol (2 μL ssDNA from a 10 μM stock

and 3998 μL hybridization buffer), 50 pmol (2 μL ssDNA from a 25 μM stock and 3998 μL

hybridization buffer) and 200 pmol (2 μL ssDNA from a 100 μM stock and 3998 μL

hybridization buffer).

In the ssDNA binding kinetics experiment 100 μL 200 nM (20 pmol) ssDNA (2 μL ssDNA

from a 10 μM stock and 98 μL hybridization buffer) were mixed with 1 mg of particles.

During the hybridization reaction 10 μL from the suspension were taken out at the following

time points: 0, 1, 5, 15 and 30 min. The particles were immediately retracted by magnet and

the supernatant was removed and transferred to another tube for analysis.

In the specific DNA extraction from a contaminated tap water sample experiment 2 mL (7 μg/

mL) of salmon sperm DNA (Deoxyribonucleic acid, low molecular weight from salmon

sperm, Sigma-Aldrich) was treated with an ultrasonic processor (horn) (UP50H, Hielscher) at

an amplitude of 100 % for 5 min in order to disrupt the nucleic acid into smaller pieces. The

disrupted DNA solution was mixed with 1.998 mL of hybridization buffer (2x) and 2 μL

ssDNA from a 10 μM stock (5 nM solution) and added to 1 mg of particles. Afterwards the

usual hybridization/melting procedure followed.

4.2.6 FTIR spectroscopy

Samples were prepared in pure KBr (Sigma-Aldrich) (5 wt% particles) and measured by a

Tensor 27 Spectrometer (Bruker Optics, equipped with a diffuse reflectance accessory,

DiffusIR™, Pike technologies).

4.2.7 Nitrogen sorption experiment

Surface areas were determined by applying the Brunauer-Emmett-Teller (BET) model in the

pressure range of p/p0 = 0.05-0.25 to datapoints acquired by Nitrogen sorption at 77 K using a

Quantachrome Quadrasorb-SI analyser on degassed samples (10−1 mbar, 373 K, 3 h). The

particle primary diameter was calculated by assuming a spherical shape and the following

formula was used: DBET = 6000/ (ρ x SBET).

66

4.2.8 STEM analysis

Samples were prepared by dissolving the particles in isopropanol (Sigma-Aldrich) and

pipetting few microliters onto copper/carbon grids. After letting the particles dry on the grid

scanning transmission electron microscopy (STEM) was performed (FEI Nova NanoSEM 450

and FEI Magellan 400 FEG) operated at 30 kV.

4.2.9 X-ray diffraction (XRD)

X-ray diffraction (XRD, X’Pert PRO-MPD, PANalytical) was used with Ni-filtered Cu Kα

radiation (λ = 0.1541 nm) from 10-70 ° in the 2θ scale. The measurement was performed at 40

mA and 40 mV and lasted 8 h per sample. The XRD software X`Pert HighScore Plus was

used to analyze the results and compare the obtained patterns to a reference database. The

reference pattern 01-087-0722 was used for the iron high and the reference pattern 01-089-

2867 shows the iron carbide peaks.

4.2.10 C, H, N elemental analysis

The % change in C, H and N was measured by weighing 2 mg of the dried sample powder in

tin containers. The method used was “2mgchem70s” and the instrument used was a Vario

micro cube, Elementar.

4.3 Results and discussion

In the material design (Scheme 4.1A) the silica coating is expected to combine chemical

robustness and surface chemistry needed for biomolecule attachment and the highly-magnetic

metallic iron core enables particle separation from a large volume within seconds. Following

the established chemistry of microarrays (Polycationic Slide Surfaces)183 probe DNA is first

electrostatically and then covalently attached to the bead surface. To test performance of these

particles, target DNA binding tests have been performed under varying conditions (Scheme

4.1B).

In detail, the metallic core was provided by the iron based carbon-coated magnetic

nanoparticles (Fe/C, Turbobeads™, Zurich) with a magnetic saturation (Ms) of 134 emu/g

measured by vibrating sample magnetometry (VMS) and an average size of 25 nm determined

by BET (Table 4.1). The presence of iron was confirmed by X-ray diffraction (XRD) (Figure

4.1A, black line). In fact, the material showed a combination of two diffraction patterns, one

that cor

another

37.8, 48

Scheme

diazoniu

formatio

ssDNA

immobil

the step

concent

Table 4

measure

particle

nanopar

FeF

Fe/C

rresponds to

correspond

8.70, and 51

e 4.1 An ov

um chemist

on of a sili

to first ads

lized to the

ps involved

tration (and

4.1 C, H, N w

ed by micro

e diameters

rticles (Fe/C

Sample

Fe/C e/C-BnOH Fe/C-SiO2 C-SiO2-NH2

o metallic ir

ding to iron

1.96.

verview of t

try, were in

ica coating

dsorb, which

particle su

d in the bin

d detection)

weight % of

oelemental a

(DBET) and

C) are show

C (%

8.212.29.09.4

ron (Cubic,

n carbide (O

the materia

nvolved in

g. The APTE

h during th

rface. B. A

nding and e

of the targe

f the silica c

analysis. In

d magnetic s

wn

%) H (

21 0.925 0.9

08 143 1.2

, lm-3m) wi

Orthorombi

al synthesis

the silica p

TES function

he baking p

schematic

elution of t

et.

coated nano

addition, th

saturations

(%) N

912 0922 0.1 0

268 0

ith the mos

ic, Pnma) w

is given. A

polycondens

nalization o

process (he

drawing of

target DNA

oparticles (F

he BET surf

(Ms) of the

(%) (

0.07 0.27 0.25 0.62

t prominant

with most p

Alcohol gro

sation reac

of the silica

eating at 80

f the experim

A resulting

(Fe/C-SiO2)

face areas (

e silica coa

SBET m2/g)

DB

38 -

54 -

nt peak at 44

prominent p

oups, introd

ction leadin

a allows th

0 °C) is co

mental set-u

in the des

and the pr

(SBET), BET

ted and unm

BET (nm)

25 -

33 -

67

4.77 and

peaks at:

duced by

ng to the

he probe

ovalently

up shows

ired up-

ecursors

T primary

modified

Ms (emu/g)

134 -

72 -

68

Figure

spectra

images

respecti

Althoug

stability

layers)

reported

a silica

Diazoni

rather i

introduc

silica sy

carbon

accorda

4.1 XRD s

of Fe/C (b

of Fe/C a

ively.

gh the unm

y,184, 185 no

and the nu

d for carbon

a (SiO2) co

ium chemis

inert carbon

ction of alc

ynthesis to

(Table 4.1

ance with lit

spectra of b

(black), Fe/

and Fe/C-S

modified par

on-specific

ucleotide b

n nanotubes

oating in tw

stry is one

n surfaces

ohol group

the particle

1) and the

terature.188

both Fe/C

/C-BnOH (r

iO2 (visible

rticles are

aromatic in

bases result

s.186, 187 Thi

wo steps:

of the few

and its ap

s on the par

e surface a

C-O stretc

(black) and

(red) and F

e silica coa

known for

nteractions

t in non-sp

s unspecific

diazonium

chemistrie

pplication w

article surfac

as described

ches at 10

d Fe/C-SiO

Fe/C-SiO2 (

ating in gr

their high

between t

pecific DNA

c binding w

chemistry

es known fo

with 4-amin

ce which in

d in Schem

20 cm-1 (F

O2 (blue) ar

(blue) are r

ray) are gi

temperatur

the carbon

A binding

was prevente

and silica

or the funct

nobenzyl a

n the second

me 4.1A. Th

Figure 4.1B

re shown. B

represented

iven on C.

re, pH and

surface (g

as also pr

ed by the gr

a polyconde

ctionalizatio

alcohol ena

d step direc

he mass inc

B, red line

B. FTIR

d. STEM

and D.

d solvent

graphene

reviously

rowth of

ensation.

on of the

ables the

cts in the

crease in

) are in

69

As a second step, silica sol-gel synthesis was employed, a well-known and fundamental

polycondensation reaction that brings about the conversion of precursors, in this case

tetraethyl orthosilicate (TEOS) catalysed by ammonia, to silica gels.189,161 The successful

silica coating could be confirmed by microscopy and is seen as a greyish region around the

dark metallic particles in Figure 4.1D. Another evidence for the successful silica

incorporation are the strong IR peaks at 800 and 950 cm-1 which could be ascribed to the

bending and stretching vibration of Si-OH and the prominent peak at 1100 cm-1 could be

assigned to the stretching vibration of the Si–O bond (Figure 4.1B).188

After silica coating, the structural composition of the iron core remained unchanged as

confirmed by the XRD results (Figure 4.1A, blue line). It is worth mentioning that the

materials (both before and after silica coating) contained no measurable amounts of iron

oxides. However, an increase in surface area per particle mass could be observed after the

silica coating (from 38 to 54 m2/g see Table 4.1). This could be mainly attributed to the

differences in density between SiO2 (ca. 2'000 kg/m3) and Fe (ca. 8'000 kg/m3), especially

considering the fact the final material comprises 47 wt% silica determined by the changes in

magnetic saturation (VSM).

The silica-coated magnetic particles did not show any interaction (non-specific binding) with

DNA, as expected from the negative surface charge. In order to be able to bind DNA to the

material, additional surface chemistry is required. Silanization is the most common way to

introduce a variety of functional groups onto glass surfaces by covalently linking organosilane

species with the surface silanol groups, which then bind the capture molecules either

covalently or through electrostatic interactions. Positively charged amino groups for example

have been widely used for DNA attachment to glass surfaces (established microarray

technology).176,190 An amino- functionality was introduced by reacting the silica surface with

ammonia catalysed (3-aminopropyl)triethoxysilane (APTES) in EtOH overnight. The increase

in nitrogen mass recorded by microanalysis clearly confirms the success of functionalization

(Table 4.1). At neutral pH the protonated and therefore positively charged surface amino

group were able to bind (overnight, room temperature) negatively charged short single

stranded probe-DNA sequences of choice. During a subsequent “baking” process (heating up

the sample at 80 °C for 2.5 h), the probe DNA was fixated on the particle surface. Whereas

the details of this covalent linkage are poorly understood, Michael addition to pyrimidine C6

has been proposed183 and also the formation of phosphoramides is conceivable. It has to be

noted that in spite of the unclear nature of the chemistry, this procedure is commercially

applied in the formation of microarray glass slides (e,g, Nexterion® Slide A+, Schott). After

70

multiple

water (a

Figure

(A), co

before

melting

fluoresc

superna

measur

To test

DNA ta

sequenc

volume

resuspe

melt th

(before

shown

the com

recover

e washings

at +4 °C) an

4.2 A. Ag

orrect ssDN

hybridizat

g (recovery

cence of

atants after

rement.

how the n

arget captu

ce (modifi

e of 40 μ

ended in w

he DNA. U

e and after

on Figure

mplementar

ry yield (>

and blockin

nd were read

garose gel

NA after m

tion (D) an

y yields) of

the fluoro

r hybridiza

novel silica

ure we adde

ied with a

μL). The

water (same

Upon remo

r hybridiza

4.2B (spar

ry sequenc

> 90 %). T

ng steps (se

dy to use.

electropho

melting (B)

nd the par

f both corr

ophore-mod

ation and a

a-coated m

ed 1 mg of

fluorophor

hybridized

e volume)

oval of the

ation, after

rse column

ce (correct

This result

ee materials

oresis of: th

B), false ss

rticle stabil

rect and fa

dified ssD

after each w

magnetic be

f particles t

re at the 5

d DNA c

and then

e particles

r washing

ns), the par

ssDNA) u

t was conf

and metho

he correct

sDNA after

lity contro

alse ssDNA

DNA seque

washing st

eads equipp

to 20 pmol

5` end) in

carrying p

heated at

s the fluore

and after

rticles succ

under the g

firmed by

ds) the part

ssDNA bef

r melting

l (E); B. H

A sequence

ences was

tep were al

ped with s

of a comp

a hybridiza

particles w

95 °C for

escence of

melting) w

cessfully bo

given cond

agarose g

ticles were s

efore hybrid

(C), false

Hybridizat

es are show

measured

lso include

ssDNA per

plementary

ation buffe

were wash

a few min

f the supe

was measu

ound and r

ditions with

gel electrop

stored in

dization

ssDNA

ion and

wn. The

d. The

ed in the

rform in

ssDNA

er (final

hed and

nutes to

rnatants

ured. As

released

h a high

phoresis

71

(Figure 4.2A, lanes A and B) where both samples (before hybridization and after

melting) show similar colour intensity and equal DNA lengh. Apart from the excellent

recovery yield the particles showed specificity, meaning they showed no binding to the

non-complementary sequence (false ssDNA, random sequence of equal length) and

nothing could be measured in the supernatant after melting (Figure 4.2B, dense

columns). Once again, the result was confirmed by gel electrophoresis (lanes D and C).

In addition, the particle stability under hybridization conditions was tested (washing

and melting steps included) and no loss of the target DNA could be observed (lane E)

evidencing the successful formation of the covalent linkage between the particles and

the target DNA.

To investigate the speed of the hybridization reaction, further binding experiments

were performed (Figure 4.3A). 20 pmol of the complementary ssDNA were mixed

with 1 mg nanoparticles in a final volume of 100 μl hybridization buffer. Small

volumes were taken at given time points during a 30 min interval. As shown on Figure

4.3A, the hybridization required less than a minute to complete under the given

conditions. Still for further experimentation (especially different volumes and

concentrations) we decided to keep 25 min as a standard hybridization time, giving

more time to circumvent the diffusion limitations in the less concentrated samples.

We then performed a series of optimization reactions by altering either the amount of

DNA or particles mass and keeping the volume constant. For 1 mg of particles the

highest recovery yield was observed when 20 pmol of the complementary sequence

were added (Figure 4.3B, solid squares). From this and the data in Figure 4.3C

(constant amount of DNA, variable particle concentrations) it can be concluded that at

low DNA/particle ratios, mass transport and statistics of DNA/particle binding events

limit optimal performance. At higher DNA/particle ratios the limited capacity of the

particles (ca. 500 ng / mg ~ 1 1011 molecules / cm2)191, 192 prevents optimal binding

yields. Still, over the range of more than one order of magnitude of initial conditions

(10...300 ng DNA / mg particle) recovery yields of > 50% can be obtained.

Keeping the optimized particles to ssDNA ratio constant (1 mg of particles, 20 pmol of

DNA) we performed a series of up-concentration experiments in which the reaction

volumes were gradually increased, and the DNA concentrations were therefore

decreased (Figure 4.3D). Even when starting with DNA concentrations in the sub

nanomolar region (and a volume of 40 ml) we were able to capture > 30 % of the

specific DNA sequence present and release it into a one thousand times smaller volume

72

(40 µl f

instead

yield of

Figure

ssDNA-

differen

(1 mg);

constan

comple

parame

in the g

To sim

extracti

dissolv

ultrason

The tar

for direct d

d of 1 mg (t

f 80 % was

4.3 A. B

-modified p

nt DNA am

; C. Recov

nt (20 pm

ementary se

eter shown

given exper

mulate a rea

ion and d

ed salmon

nication pr

rget strand

detection).

to increase

s obtained.

inding kin

particles;

mounts wer

very yield o

mol) where

equence wh

in red in t

riment.

alistic scen

etection fr

n sperm

rocessor lo

d (20 pmol

When we

e the statist

netics of th

B. Recove

re used wh

of the com

eas the pa

hen the rea

the boxes o

nario in ter

rom a con

DNA in

ong enough

, 0.14 μg)

repeated t

tics of DNA

he complem

ery yield o

hereas the

mplementar

article mas

action volu

on top of ev

rms of spe

ntaminated

tap water

h to break

was added

this experim

A - particl

mentary se

of the com

amount of

ry sequence

ss varied;

ume was inc

very graph

cificity we

d water sam

r (7 μg/mL

it into sho

d to the sh

ment using

e binding e

equence w

mplementar

f particles

e when its

D. Recov

creased up

is the one

e performe

mple. For

L) and tre

orter fragm

heared DN

g 5 mg of p

events) a r

when added

ry sequenc

was kept c

s amount w

very yield

p to 1000-fo

e that was c

ed a specifi

this purp

eated it w

ments (ca 2

NA solution

particles

ecovery

d to the

ce when

constant

was kept

d of the

fold. The

changed

fic DNA

pose we

with an

200 bp).

n one to

one, res

particle

to recov

the spe

and pur

Scheme

terms o

Standar

Qubit®

The de

target s

min to

hybridi

microti

standar

(estima

(Schem

and eve

h). In a

mixture

using th

sulting in a

es was add

ver 70 % o

cificity of

rifying spe

e 4.2 An ov

of time req

rd laborat

ssDNA As

escribed ex

ssDNA seq

o determin

ization, 5 m

iter plate a

rd laborato

ated time o

me 4.2). To

en then ke

addition, PC

e, thus req

he particle

a 100 fold

ed and the

of the comp

the bindin

cific DNA

verview of

quirement.

tory ssDNA

ssay which

xperimenta

quence, sim

ne DNA

min washin

and measur

ory assay

f analysis

o run a qPC

ep in mind

CR is often

quiring a p

based enri

excess of t

e hybridizat

plementary

g and the p

A sequences

f the ssDNA

The enrich

A detection

requires 1

al procedur

mply by m

presence

ng, 10 min

re the fluo

for ssDNA

is 10 min)

CR instead

d the longe

n affected b

pre-treatme

ichment pro

the salmon

tion reactio

y sequence

potential u

s from envi

A enrichme

hment pro

n method

10 min to p

re allows

magnetic u

and conce

n melting a

orescence).

A detectio

, we would

d one need

er time unt

by some co

ent (an add

ocedure).

n sperm DN

on took pla

added to t

sefulness o

ironmental

ent and det

cedure tak

follows up

erform.

for a fast

up-concentr

entration b

and 5 min t

If we com

on, for ex

d need 50 m

ds to posse

til the resu

omponents

ditional ste

NA by mas

ace. Even t

the mix. Th

of the parti

l samples.1

tection met

kes 40 min

pon enrich

and reliab

ration and

by a fluo

to pipette t

mbine our

x. Qubit®

min for the

ess the exp

ult is out (a

found in th

ep we coul

s (14 μg).

then, we m

his result c

icles for co193

thod from

n until com

hment, for

ble detecti

required

orimeter (2

the sample

procedure

ssDNA as

e whole pr

pensive equ

approxima

he original

ld eliminat

73

1 mg of

managed

confirms

ollecting

DNA in

mpletion.

ex. the

on of a

only 45

25 min

es into a

e with a

ssay kit

ocedure

uipment

tely 2-3

l sample

te when

74

4.4 Conclusion

In summary, we demonstrated a synthesis of a novel magnetic nanomaterial that was

successfully used in DNA enrichment experiments. For this purpose, we combined the

well-known advantages of the silica surfaces in biomolecule attachment with the high

magnetic saturation of the carbon-coated iron nanoparticles used as a starting point in

our synthesis. We managed to stably immobilize ssDNAs of interest on the surface of

our newly produced material. Furthermore, we developed a procedure to enrich (up-

concentrate) a target complementary ssDNA of interest from a mixture of DNAs with

high recovery yield and specificity. No binding of a false DNA sequence could be

monitored. Under the tested conditions, we achieved high recovery yields even when

the complementary DNA sample volume was up-concentrated 1000-fold. Coupled

with a standard laboratory ssDNA detection assay kit we demonstrated a possibility to

PCR-free detect low-concentrated ssDNA in significantly less than an hour, thus

eliminating the need for having a PCR machine and the longer times of detection.

For the future we envision that this proof-of-principle of rapid DNA purification and

up-concentration can be extended and proven useful in the areas of pre-sequencing

DNA enrichment, environmental sample analysis, DNA tracing experiments as well as

point of care diagnostics. It may also be useful in combination with other DNA

detection means (e.g. polyvalent DNA gold nanoparticles, lateral flow devices) in

which the concentration and purity of the natural sample may limit reliable DNA

detection.

75

5 Conclusion and outlook

76

To summarise, biomolecule/inorganic particle hybrids were assembled and some of their

applications were presented in the previous chapters. In other words, we managed to bind

proteins and DNAs to nanomaterials and carry them around in solution. We utilized the

advantages magnetic nanoparticles and silicas have to offer in the field of nanobiotechnology

each of them alone but also combined. Firstly, we successfully constructed promising

enzyme/magnetic particles hybrids which showed high enzyme loadings and activity and most

importantly allowed for a multiliter scale re-use of the biocatalyst. With the idea to improve

enzyme shelf storage at RT (having in mind the fragility of most enzymes) we thought of

sealing high amounts of enzymes in the pores of mesocellular foams and releasing them upon

demand with application in biocatalysis or possibly biosensors. By combining these two

materials and their properties we finally produced magnetic NPs/silica composites which were

stably loaded with single stranded DNAs with which we could selectively enrich a target

DNA strand from a DNA mix.

When it comes to magnetic nanoparticles and their application in biocatalysis, they are

becoming increasingly important and receiving more and more attention. The main reason for

their popularity is the ability to separate them from the reaction medium simply with the aid

of a magnet while the other advantages that nanomaterials offer are preserved, like for

example the high surface area to volume ratio. In the last years, iron oxide nanoparticles

(magnetite or maghemite) have been shown to be a possible platform for enzyme

immobilization. As good as it might sound, the iron oxide nanomaterials show only limited

magnetic saturation which prevents them from use on larger scales, for example in industrial

set-ups. Some iron oxide materials have shown questionable stability which is also very

important when it comes to the enzymatic carrier of choice. The carbon-coated cobalt

nanoparticles utilized in this work not only show high pH, temperature and organic solvent

stability but also could be retracted from the reaction medium in a few seconds (mL scale) or

in a few minutes (L scale) thanks to their high magnetic saturation. In addition, the carbon

surface gave us the possibility to link the enzyme covalently to the surface by reliable organic

chemistries in order to prevent leaching. All three enzyme we bound to the nanomaterials

resulted in high activity and loading and enzyme re-use in 20 L reaction tank was

demonstrated. In terms of applicability of the improved magnetic properties, the rapidly

growing field of chemical biocatalysis can profit from magnetic separation technology, which

is already well established in the fields of analytical immunoprecipitation and cell separation

on the milliliter scale utilizing metal oxide based particles.

77

Silica materials have also been extensively utilized in the field of biocatalysis. The channels

of such pre-fabricated porous inorganic materials proved to be very suitable for enzyme

immobilization. Adsorption is especially attractive due to its simplicity but leaching of the

enzyme is inevitable. With the know-how in pore fine-tuning scientists tried tailoring

mesoporous materials with channel sizes big enough to exactly fit the protein of interest.

However, decreased enzyme activity and substrate diffusion problems were evident. Our

strategy was to completely seal the enzyme inside the porous matrix in order to be able to

stably store it at room temperature for a longer period of time without the need of having a

fridge or a freezer. The silica build-up (sol-gel synthesis) by utilizing silicate precursors is a

well-known reaction and we managed to optimize it to be able cover the pore openings of the

mesocellular foam. The novelty we introduced is the mild fluoride buffer solutions (4 % F-)

needed to completely dissolve the nano-support. Such buffers have a limited use in

biochemistry although we saw no effect on the enzyme stability at the concentrations of

buffer used. For comparison, up to 1.23 wt% fluoride-containing dental products (gels) are

nothing uncommon. In the future, one could pay special attention to sensitive enzymes, which

are very delicate to handle and require low storage temperatures. The multiple freeze-thaw

cycles and the need for a freezer could be eliminated by our approach. In addition, the

possibility to perform field studies (as part of biosensors) in regions where temperature is high

shows a great application potential and is worth further experimentation.

By combining the advantages of both so far utilized nanomaterial classes we aimed at

producing magnetic particles-silica nanocomposites for enrichment applications. Despite the

ease of separation provided by the magnetic core, the silica surface proved to be a good

platform for covalent DNA attachment because of its anti-fouling character and the well-

established silane chemistry. Basically, our ssDNA-loaded beads were thrown into larger

volumes, let hybridize with the complementary strand from a mix of DNAs and the strand

was then released into smaller volumes by heating up the samples. Although here we mainly

showed a proof of concept, the very high specificity achieved opens up a window of

opportunities for future applications. One application would be fast DNA detection from

samples with very low concentrations without the need to do PCR. If we combine our

procedure with a standard laboratory assay for ssDNA detection, for ex. Qubit® ssDNA

Assay kit (estimated time of analysis is 10 min), we would need 50 min for the whole

procedure where 3 h is the estimated time one needs to perform a PCR. The expensive PCR

equipment and the fact that PCR is often affected by some components found in the original

sample mixture makes our approach a faster and cheaper alternative. Other applications could

78

involve targeting certain genomic regions prior to sequencing as another alternative of the

solution-based hybridization approach. Such a platform could be also used for enzyme

applications and possibly biocatalysis. ssDNA-modified enzyme could be obtained with a

sequence complementary to the one attached to the particles surface. A boomerang enzyme

system could be created by controlling the release and immobilization of the enzyme by

changing the temperature.

The thesis shows how the fusion of biomolecules and inorganic materials has not only led to

significant progresses in traditional application fields but has additionally opened up new

opportunities. The combination of these two components in the last years has allowed the

design and manufacturing of hybrid materials with new properties to address different

technological problems. In our work we tried to profit from the advantages some novel

nanomaterials have to offer mainly focussing on applications in the fields of biocatalysis and

DNA enrichment and meeting the industrial needs for a re-usable biocatalyst and long-term

RT storage stability of enzymes. Fast and reliable DNA detection without the need for PCR is

promising in many technological areas.

79

Appendix

80

A.1 Su

Scheme

flow. Fi

80 mL)

(unmod

Afterwa

decante

suspend

upporting

e A1.1 Imm

irst, an enzy

of which th

dified or D

ards the pa

ed and the

ded in dH20

g informat

mobilization

yme solution

he enzymati

DSC-activate

articles wer

e residual

(80 mL) an

ion to Cha

(covalent o

n was prepa

c activity w

ed) were a

re separate

enzymatic

nd were used

apter 2

or adsorptio

ared by diss

was measure

added and

ed with the

activity w

d in the imm

on) of enzy

solving the

ed (total act

the immob

e aid of a

was measur

mobilized en

me on a pa

enzyme in w

tivity). Mag

bilization p

magnet, th

red. The n

nzyme activ

article expe

water (fina

gnetic nanop

proceeded f

he supernat

nanoparticl

vity assays.

erimental

l volume

particles

for 5 h.

tant was

es were

81

Table A1.1 (Upper rows): C, H, N weight gain of the Co/C–enzyme conjugates relative to

their precursors determined by elemental microanalysis measurement (Co/C–C: 6.8 %, H:

0.1 %, N: 0 %). (Lower rows): C, H, N weight gain of the DB–β-Glu compared to the -COOH

functionalized DB

Sample C (%) H (%) N (%)

Co/C–Ph-EtOH + 0.8 + 0 + 0

Co/C–DSC activated + 0.4 + 0.1 + 0.1

Co/C–β-Glu

Co/C–α-CT

+ 1.8

+ 2.3

+ 0.3

+ 0.4

+ 0.8

+ 1

Co/C–CALB + 0.8 + 0.2 + 0.2

DB-COOH 36 3.7 3.9

DB-β-Glu 37.5 3.9 4.4

Table A1.2 Vibrating sample magnetometry (VSM) hysteresis data of magnetic particle–

enzyme conjugates compared to their precursors. The enzyme containing particles show

almost as high magnetic saturation as the unmodified raw products

Sample Ms (emu g-1) Hc (Oe) Mr (emu g-1)

Co/C 143 241 31.1

Co/C–Ph-EtOH 142 158 20.8

Co/C–β-Glu

Co/C–α-CT

133

131

178

170

17.7

21.8

Co/C–CALB 136 175 22.4

DB-COOH 26.2 2.3 0.16

DB-β-Glu 25.1 2.5 0.18

82

Table A

detected

Atomic %

Co/C–Ph

Co/C–DS

Figure

microsc

particle

Figure

(red colu

A1.3 XPS an

d elements

%

h-EtOH

SC activated

A1.1 Scann

copy (SEM)

es coated wi

A1.2 LC/M

umns) β-Glu.

nalysis of th

ning transm

) b) of the

ith a polyme

MS/MS analys

. Actual mas

he Co/C func

C

85.8

82.1

mission elec

enzyme co

eric layer (e

sis of tryptic

s versus sign

ctionalized

O

5.

7.

ctron micro

oated magn

enzyme).

c digests of n

nal intensity

nanopartic

O

4

6

oscopy (STE

netic nanop

native (black

is shown.

les-normali

N

0.0

1.1

EM) a) and

particles dis

 

k columns) a

ized atomic

Co

8.8

9.2

d scanning

splaying in

and covalent

% of all

 

electron

ndividual

tly bound

Figure

regions

mass spe

with the

Figure

Co/C) a

placed b

the vials

 

A1.3 Swiss

indicated: g

ectroscopy a

highest sign

A1.4 The

and the bac

between the

s.

-PdbViewer

green (left)–th

analysis of it

al intensity a

relative tra

ckground pl

e vials; Hol

v4.1 image

he region of

ts tryptic dig

after mass sp

ansmission

lotted again

llow square

of the β-Gl

f the native e

gest and red

pectroscopy a

between th

nst time. So

es-Dynabea

lu monomer

enzyme with

(right)-the r

analysis of it

he particle

olid squares

ads when th

simulation w

the highest

region of the

ts tryptic dig

suspension

s–Co/C wh

he magnet w

with two po

signal inten

e immobilized

gest.

ns (Dynabe

hen the mag

was placed

83

olypeptide

nsity after

d enzyme

eads and

gnet was

between

84

A.2 Su

Figure

(MCF).

extrusio

Figure

extrapo

upporting

A2.1 Cum

Solid circ

on.

A2.2 Size o

lated from t

g informat

mulative me

cles indica

of the pore

the mercury

ion to Cha

ercury intru

te the intr

windows of

y intrusion d

apter 3

usion analy

rusion whe

of the MCF

data.

ysis of the

ereas the e

F cells plotte

siliceous

empty coun

ed against t

mesocellul

nterparts st

the pore vo

ar foam

tand for

olume as

Figure

glucosid

inverted

after fl

immobil

Figure

chymotr

fluoride

A2.3 Influe

dase entrap

d triangles)

luoride buff

lized enzym

A2.4 Influe

rypsin after

e buffer pH

ence of the

pped (black

and β-gluc

ffer suppor

me after diss

ence of fluo

r treatment

4 (1:10 dilu

entrapment

k stars) com

cosidase fre

rt dissoluti

olving its su

ride buffers

of the enzy

uted, sparse

t process an

mpared to th

ee in solutio

ion (dashed

upport coul

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