ISyPeM

[email protected] EDMI Research Day 2012

ISyPeM IIGiulia CappiÉcole polytechnique fédérale de Lausanne

Annual Plenary Meeting of the Nano-Tera programMay 4th, 2015

[email protected] EDMI Research Day 2012ISyPeM II - G. Cappi

Therapeutic drug monitoring (TDM)

time after dose (hrs)

Imati

nib

expo

sure

(ng/

ml)

Courtesy of N. Widmer

Modern anticancer agents

Immunosuppressants

Antiretrovirals

Antibiotics

2

Candidates for therapeutic drug monitoring

[email protected] EDMI Research Day 2012ISyPeM II - G. Cappi 3

Therapeutic Drug Monitoring Today

Abbott "Architect ci8200"

Blood sample taken at the doctor’s office or

at the hospital

Transfer to and measurement at

central clinical labsInterpretation of the

result by a specialized doctor

Today this procedure severely restricts the applicability of TDM

Treatment duration (days)

Drug

Con

cent

ratio

n

Patient

Target

?

http://e-sante.futura-sciences.com


Drug quantification in body fluids

LC-MS/MS

Agilent

Used in Clinical facilities

Abbott "Architect ci8200”

FPIA (fluorescence polarization immunoassay) CMIA (chemiluminescent microparticle immunoassay) MIA (magnetic immunoassay)

Point-of-need

Syva RapidTest®

EMIT (Enzyme multiplied immunoassay technique)


Point-of-need therapeutic drug monitoring

iSTAT, Abbott

ClonitStMicroelectronicsCobas product

lines Roche Diagnostics

Accu-Chek product lines. Roche Diagnostics

ISyPeM approach toTDM

Top bench

Hand-held


ISyPeM. A solution to enable drug-based therapeutic drug monitoring

• Low-sample volumes

• Miniaturized reader

• Cartridge that can be stored with stable reagents

• Monitoring and prediction interface for clinicians and practitioners

• Local or remote

• Extension of eHealth medical standards

• Data integrity


ISyPeM: A solution to enable drug-based therapeutic drug monitoring

• Cartridge that can be stored with stable reagents

In vitro selection of capture molecules for drug targets

HOMOGENEOUS AND HETEROGENEOUS ASSAYS


In vitro selection of monoclonal antibodies for Tacrolimus

Heinis LabAntibody phage display

Sangram Kale

NO

OH

O

NO

O

H2NO COOH

NaOAc, rtdry MeOH, 16 h

HN NH

S

O

HN

OO

OO

HN

HN NH

S

O

HNOO

OOH2N

HATU, HOBt, DMF, DIEA, 5 h

Target:tacrolimus

Phage-antibodies were successfully selected for Tacrolimus and expressed


In vitro selection of DNA-aptamers for Tobramycin

Spiga F. M. et al. ACS Comb. Sci. 2015, featured on the cover of May Issue

Guiducci Lab

F. M. SpigaNow with Creoptix Sensors

No drug derivatization High affinitySpecificFunctional in serum

DNA beacon aptamers Capture Selex


Low-volume sample preparation on chip

FPIA of Tobramycin performed in glass microcapillaries

Squared glass capillary

Plasma extraction from whole blood

Yield outperforms any passive on-chip method reported to date http://dbs-system.ch

300 µm

0 2 4 6 8 10 120.140

0.160

0.180

0.200

0.220

0.240

0.260

Concentration [µg/ml]

Aniso

trop

y

Diana BurgheleaJ-M Segura

reference systemcapillary

David Forchelet

Renaud Lab


Measurement system: miniaturization of an established assay technique

25 cm

20 cm

Fluorescence Polarization ImmunoassayCompetitive assayHighly-sensitiveCompatible with small volumes

Requires derivatization of drug molecules

Martial Geiser team

*


Label-free quantification of drug from serum samples

200 nm

Layers of functionalized gold nanoislands

Heterogeneous assay based on binding kineticsNo drug derivatizationNo additional reagents

Giulia Cappi

Transmission Surface Plasmon ResonanceChange of optical properties of the interface

Cappi G. et al. Sensors and Actuators 2013

Guiducci Lab


Hue readout of surface plasmonics

Hue readout of surface plasmonics

CMOS image sensorGuiducci Lab

Compact read-out system

Cappi G. et al. Analyt. Chem. 2015


Measurement of binding kinetics of DNA aptamer-Tobramycin

KD = 200 nM, same as extracted from commercial SPR

Transmission SPR signal


Guiducci Lab


Tobramycin quantification from serum

Jang, H. R., et al. Anal. Chem. 86, 814–819 (2013)

Chang, A. L. et al., Anal. Chem. 2014, 86, 3273−3278.

✔ Small molecules✔ Complex Matrix

✖ Small molecules✔ Complex Matrix

✔ Small molecules✖ Complex Matrix



Supporting clinical interpretation

Population-based percentiles

Individual concentration vs time profile

Dosing schedule suggestion

Div. Clin. Pharmac.

Aline Fuchs

Yann Thoma

EzeCHiel interface


Data management

MOLIS, CHUVEzeCHielRSDB

“translate” from one representation to another

Data Exchange between EzeCHiel and DB of the

Medical Institution

Health data

POC

Medical DB

Dubovitskaya, A. Privacy Preserving Interoperability for Personalized Medicine, in: Swiss Medical Informatics, Swiss Society for Medical Informatics, Switzerland, 2014

Alevtina DubovitskayaData securityDynamic Health Data Aggregation


Population pharmacokinetics

Div. Clin. Pharmac.

Br J Clin Pharmacol. 2014 Nov;78(5):1090-101

Meta-analysis. Implementation and validation

Pharmacokinetic models

Aziz ChaouchAline Fuchs


Acknowledgements

Guiducci Lab


BACK UP


Filtration and FPIA on paper

FPIA for tacrolimus with antibody selected by EPFL (tacrolimus fluorescent derivate needed)

Aptamer for imatinib

EzeCHiel start up and beta testers

Interoperability with MOLIS database

Population pharmacokinetic studies on tacrolimus and tobramycin


Personalized Medicine and TDM Therapeutic drug monitoring (TDM) is foundational to the concept of

personalized medicine. The concept has later evolved to include pharmacogenomic and

other biomarker-driven strategies for patient segmentation

In the clinics: TDM transformed drug therapy by affording the ability to characterize sources of variability in drug disposition and response to individualize drug dosing. Initially, TDM formed the key conceptual basis for personalized medicine

Interest from Pharma:Personalized medicine takes into account the fact that 30% of drugs investigated in clinical trials fail because of lack of efficacy*, and its premise is that stratifying patients and diseases into molecular subtypes and treating with subtype-specific drugs will improve drug efficacy.

JD Momper and JA Wagner, Clinical pharmacology & therapeutics, VOLUME 95 NUMBER 2 2014 Li and Jones Genome Medicine 2012, 4:27 T. Buclin, Who is in charge of assessing therapeutic drug monitoring? The case of imatinib. Lancet Oncology 2012Kola I, Landis J: Can the pharmaceutical industry reduce attrition rates? Nat Rev Drug Discov 2004, 3:711-715


Research level drug quantification approaches

MEDICUCSB

Griss R, Schena A, Reymond L, Patiny L, Werner D, Tinberg CE, Baker D, Johnsson K. | . Nature Chemical Biology

The sensor molecule works by binding the drug circulating in the patient’s bloodstream and changing color accordingly. The molecule itself is made up of four components. One component is a receptor protein, which can bind the molecules of the target drug. The second component is a small molecule similar to the target drug, which can bind the drug receptor. The third component is a light-producing enzyme called luciferase, and the fourth is a fluorophore molecule that can modify the color of the luciferase’s light when it comes close to it.

LUCIDEPFL

We conjugated its 3 end to a methylene blue (MB) reporter and its 5 ′ ′end to an alkane thiol for attachment to gold working electrodes within the MEDIC chip micro- channel . Target binding induces a conformational change in the aptamer that modulates electron transfer between MB and the electrode . This modulation is expected to produce a readily measurable change in current at the MB reduction peak when the sensor is interrogated using square-wave voltammetry (SWV).

Real-Time, Aptamer-Based Tracking of Circulating Therapeutic Agents in Living Animals, Brian Scott Ferguson et al.Sci Transl Med 5, 213ra165 (2013)


Where we stand. Why we are unique.

Well-defined application objective (in the short-medium term) candidate diseases clearly identified point-of-care use for therapeutic drug monitoring in clinical or private

practice settings support to MDs. Development of comprehensive, flexible, but user

friendly code

Unique analytical approach, combining development of novel in vitro selection protocols of drug capture

molecules (small drugs, even with no receptor or antibody available) assay development (new molecules employed in traditional or

alternative detection techniques)


Where we stand. Why we are unique. (cont.)

Committed to the feasibility of our solutions Data management: compliancy and interoperability Assay development: integration, sample size, sensor characteristics

responding to realistic requirements for development

Leveraging nearby companies’ potential interest Pharma: Novartis, Roche, DebiopharmAssays/Systems: STMicroelectronics, Abionics, Mycartis, DBS systems, …


PROJECT STRUCTURE AND TIMELINE


Research areas – Internal structureNovel capture probes and quantification in complex matricesC. Guiducci, CHUV, STMicroelectronics and C. Heinis

Miniaturization of sample preparation and FPIAJ.M Segura, C. Heinis, Ph. Renaud, CHUV

Interpretation and dose adjustmentDatabase exchange and interoperabilityY. Thoma, CHUV, M. Schumacher


Internal organization in teams and team leaders

System integrationM. Pfeifer

Assays for drug quantification/sem

i-quantificationJ-M Segura

Data interpretation and data

managementY. Thoma


Milestones expected by May 2015WORKPACKAGE 1 - Sample preparationLMSI- EPFL, LDI-HES-SO Valais (Prof. Marc Pfeifer), CHUVM1.1: demonstration of volumetric sampling of blood plasma, concept validation of FP measurements in paper. Concept of the whole system architecture.

WORKPACKAGE 2 - Highly selective capturing molecules for the target drugsLPTT-EPFL, CLSE-EPFL and CHUVM2.1: Phage selection and characterization of antibody specific for imatinib (will be provided to Segura for FP assay development); expression of tacrolimus-specific Ab and characterization (will be provided to Segura).M2.4: report on SELEX-driven convergence of the DNA library and choice of the 10 (aptamer) candidates

WORKPACKAGE 3 - Drug detection by miniaturized systemsCLSE-EPFL, LDI-HES-SO Valais, STMicroelectronics, CHUVM3.1: fluorescent and biotin derivatesM3.2: validated FPIA protocols for tacrolimus and tobramycinM3.5: choice of an opto-electronic device (including light source and detection) for the FP method and functioning table-top system.


Milestones expected by May 2015 (cont.d)

WORKPACKAGE 4 - Data Analysis, Interoperability and Intelligent DatabasesHEIG-VD, AISLab HES-SO Valais, CHUV

M4.1: Local connected version of the database integrated with EzeCHiel

WORKPACKAGE 5 - DemonstratorsLDI-HES-SO Valais, STMicroelectronics, CHUV

WORKPACKAGE 6 - Consolidation of pharmacokinetic/pharmacodynamic reference data, clinical exploitation of results and validation of TDM at point of careCHUV, ASILab HES-SO Valais, HEIG-VDM6.1: theoretical elaboration of a PKPD meta-analysis concept

M6.4: validation report about the interpretation algorithms as implemented in the EzeCHieL tool


FORTHCOMING MILESTONES


Milestones expected by Oct 2015WORKPACKAGE 1 - Sample preparationLMSI- EPFL, LDI-HES-SO Valais (Prof. Marc Pfeifer), CHUVM1.2: concept validation of fluid transfer between two paper layers by contact and sample concentration by evaporation

WORKPACKAGE 2 - Highly selective capturing molecules for the target drugsLPTT-EPFL, CLSE-EPFL and CHUVM2.5: report on the chosen aptamer binding characteristics towards the target drug M2.2: Affinity and/or stability maturation of imatinib-specific antibody according to the needs of Segura; CDR grafting of tacrolimus-specific Ab into stable IgG framework (will be provided to Segura).

WORKPACKAGE 3 - Drug detection by miniaturized systemsCLSE-EPFL, LDI-HES-SO Valais, STMicroelectronics, CHUVM3.3: comparison of assays in Microsystems M3.7: full description of the requirement specification for the demonstrators to be deployed.


Milestones expected by Oct 2015 (cont.d)

WORKPACKAGE 4 - Data Analysis, Interoperability and Intelligent DatabasesHEIG-VD, AISLab HES-SO Valais, CHUV M4.2: Interoperable server prototype with eHealth standards

WORKPACKAGE 5 - DemonstratorsLDI-HES-SO Valais, STMicroelectronics, CHUV M5.1:Advanced Breadboard (MAY 2016)

WORKPACKAGE 6 - Consolidation of pharmacokinetic/pharmacodynamic reference data, clinical exploitation of results and validation of TDM at point of careCHUV, ASILab HES-SO Valais, HEIG-VDM6.2: publication of a computer too implementing PKPD meta-analysis M6.5: validation report about the measurement results produced by the analytical tool


Timeline for demonstrators1ST YEAR

Nov 2013 – Oct 20142ND YEAR

Nov 2014 – Oct 2015

FUNCTIONAL MODULES PRE-PROTOTYPES

FIRST ASSEMBLED PROTOTYPE FIRST

TESTING PHASE

SYSTEM PROTOTYPE

SECONDTESTING PHASE

MODELING SOFTWARE

INTELLIGENT DATABASES

3RD YEARNov 2015 – Oct 2016

4th YEARNov 2016 – Oct 2017

ADVANCED PROTOTYPE


PROJECT CONTEXT AND AIMS


DEADLINES AND DATES


Deadlines

28th of March send out my presentation for the REVIEW MEETING (6th of May in Bern)

13th of April updated scientific report which will cover (Nov. 1st, 2013 – March 2015). Please, send me your updated contribution, according to the usual template:https://www.dropbox.com/sh/5asho7dmnqmcowv/AADjc7ULkxvbZV9Ds2wBYyDva?dl=0

https://www.dropbox.com/sh/5asho7dmnqmcowv/AADjc7ULkxvbZV9Ds2wBYyDva?dl=0




Mark your calendars

REVIEW MEETING 6th of May (afternoon) in Bern. Co-PIs

ANNUAL N-T MEETING 4-5th of May

in Bern. All ISyPeM participants. We have to choose a PhD student to present existing achievements of our work!


CLSE and ISyPeM

DNA capture molecules

Analysis of matrix

effects

Label-free measurement techniques

Nano-patterned surfaces

CHUV• Identification of drug candidates• Comparison with standard measurement systems• Samples

J-M/HES-SO• Use of DNA aptamers

for FPIA assays

Philippe Renaud• Integration of sample

preparationSTMicroelectronics

• Optical readout


aptamers


Challenges in small molecules assays

Lack of suitable capture molecules ANTIBODIES

150 kDaAPTAMERS10 kDa - 30 kDa

Sensing in matrices:• Non specific

adsorption • Interference


Selection strategy for DNA aptamer

Ligand-binding oligos amplified by PCRLonger randomized region

Tobramycin.Selected best binders (CLSE) better or comparable to existing aptamers

Fabio M. Spiga, Paolo Maietta and Carlotta Guiducci, “More DNA−aptamers for small drugs: a capture−SELEX coupled with Surface Plasmon Resonance and High Throughput Sequencing”, ACS Combinatorial Science, accepted for publication


Minimizing the number of cycles

SPR monitoring Specificity arises for sublibrary at cycle 8

High throughput sequencingThe most enriched sequences are already visible after only two capture-SELEX cycles

Tobramycin

Fabio M. Spiga, Paolo Maietta and Carlotta Guiducci, “More DNA−aptamers for small drugs: a capture−SELEX coupled with Surface Plasmon Resonance and High Throughput Sequencing”, ACS Combinatorial Science, accepted for publication


Selectivity towards Kanamycin


Surface preparation45

Controlaptam

er

Passivation

Analysis

50μm

500μm

~2m

m

Specific

aptamer

Detection is label-free Does not require drug derivatization


46Binding kinetics of Tobramycin on SPR in serum Tobramycin concentration range of initial serum samples: [5µM

- 100µM] Derived sample characteristics:

Serum 10%, Tween 20 0.01%, Tobr [0.5µM - 10µM]

Detection of sample concentration is preceded by calibration


R2=0.993

Determination of Tobramycin concentration in serum

47

Tobramycin concentration range of initial serum samples: [5µM - 100µM]

Derived sample characteristics:Serum 10%, Tween 20 0.01%, Tobr [0.5µM -

10µM]• LoD 0.15 µM


Conclusive remarks48

Tobramycin detection [0.5µM - 100µM] in serum samples Linearity up to 10 µM Label-free, direct detection Volume of serum needed: 20µl

Sample preparation time: 45 min per sample (from serum) Analysis time: half an hour per sample

Tobramycin-specific DNA aptamers successfully selected with KD <1 µM

No drug derivatization Relatively simple and fast aptamer selection protocol


Direct detection of drug molecules in patient sera

6% error24% error(Tolerance in this range: 20%)

Standard addition methodDirect detection by SPR based on immobilized DNA aptamers specific for Tobramycin


Classic Capture

• Evolution-like iterative selection system• Long(er) sequences, immobilised oligo• More specificity - more possibilities for binding detection• And they are selected on a surface

New SELEX concept50


Design of the assay

Sensitivity in the physiological range Specificity in presence of concomitant

drugs Simple and fast sample preparation Transferability of the analytical

approach to other drugs Avoid drug derivatization Straightforward selection of new probes

Sample size efficiency

51

Specifications for a companion diagnostic device in TDM


Serum sample preparation for drug concentration analysis

Precipitation upon protein denaturation

Filtering with low cut-off (eg: < 3 kDa)

Anti-fouling solutions to prevent non specific adsorption on the surface

52


Determination of Tobramycin concentration53

Tobramycin concentration range of initial serum samples: [5µM - 100µM]

Derived sample characteristics:Serum 10%, Tween 20 0.01%, Tobr [0.5µM -

10µM]Some numbers: Volume of patient serum needed: 20µl

Sensing region covered by aptamers: 1.05 mm2

Moles of aptamers required: 150pmol Manipulation time: ~10min per sample Run time: ~12min per cycle (2 cycles per

sample)


Variability in treatment response

54

T. Buclin, et al.“Who is in charge of assessing therapeutic drug monitoring? The case of imatinib”. Lancet Oncol. 2011;12(1):9-11. 0 1 2 3 4 5 6 7 8 9 10 11 12

Treatment duration (days)

Drug

Con

cent

ratio

n (μ

g/m

L)

0.0

0.4

0.8

1.2

1.6

2.0

2.4

Patient

Target

Risk of adverse effects

Risk of inefficacy

Imatinib 400 mg qd

800 mg qd


Therapeutic drug monitoring at the point-of-care Compatibility/

Suitability Acceptable and suitable

setting for in-field drug measurement, considering disease, frequency of monitoring, treatment toxicity, costs

Need for stable and specific molecular assay for drug measurement, compatible with on field biosensors

55

Information and Interpretation

Missing statistical population dataNeed for formal and accessible models of interpretationData exchange

DA RISITEMARE


tSPR


Transmission SPR. Direct detection in undiluted serum.

0 5 10 15 200

2

4

6

8

10

12

14

16

x 10-4

Tobramycin concentration (µM)

hue

Aptamer (average)Langmuir interpolationControl

KD= 0.26 µM

G. Cappi, E. Accastelli, V. Cantale, M. A. Rampi, L. Benini, C. Guiducci ., Sensors and Actuators B: Chemical, 2013G. Cappi, Spiga, F.M., Moncada, Y., Ferretti, A., Beyeler, M., Bianchessi, M., T. Buclin, L. Decosterd, Guiducci, C. Analytical Chemistry, accepted for publications

Undiluted serum


Portable system for array measurements

Power supply

Plasmonic

sensor

Computer

White LED

CMOS image sensor

Diffuser

Lens

58

• Power supply via USB• Real-time display of images

registered

Regions of interest


Do Hue and Peak have the same plasmonic information?

• Evaluation of the response of the NIs to different RI• Test with glucose/sucrose solutions alternated with water• Elaboration from the same RGB raw data


Validation of the hue for plasmonic evaluation

Bulk refractive index change

Surface binding events Small molecules in saline

buffer Small molecules in serum

matrix

60

• Tobramycin 10 μM in TE 1X buffer

• DNA aptamer spot

10 20 30 40 50 60 7000.198

0.1985

0.199

0.1995

0.2

0.2005

0.201

0.2015

0.202

Hue

580

580.5

581

581.5

582

582.5

583

583.5

584

Pea

k lo

catio

n (n

m)

Time (min)


Hue-peak correlation to surface events

580 581 582 583 5840.2

0.201

0.202

0.203

0.204

0.205

Peak location (nm)

Hue

h33 correlation hue peak in TE

Surface RI change in TE buffer

580 580.1 580.2 580.3 580.40.2001

0.2002

0.2003

0.2004

Peak location (nm)

Hue

0.0715 nm

1E-4

35


Small molecules detection in TE buffer

37

0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 60.195

0.196

0.197

0.198

0.199

0.2

Time (hours)

Hue

AptamerControl

TE 1X

Serum (control)

NaCl 1M

TE 1X

TE + Tobramycin

0.5 µM

1 µM

2 µM

5 µM

10 µM


DNA aptamer and control spots Array of aptamers/control HCR/blank NIs 2-channels microfluidics

Ref FTO

NIs

SH-aptamControl HCRBlank NIs

NaCl

NaCl

NaCl

TE TE TE TE TETobr

20µM

Tobr200µM

500 1000 1500 2000 2500 3000 3500 4000 4500 50000.184

0.186

0.188

0.19

0.192

0.194

0.196

0.198

Numero Immagine

Valo

re P

ixel

Tinta in FTO & NIs

HFTO

HCONTR

HDNA

HDNA

HNIS


THERAPEUTIC DRUG MONITORING FOR PERSONALIZED MEDICINE

ISyPeM


MULTI-SCALE APPROACH FOR POINT-OF-CARE TDM

POINT-OF-CARE TESTING Low volume

blood testing Quantitative Specificity

towards metabolites

Miniaturized

INTERPRETATION AND DOSE ADJUSTMENT Is the result

expected? Is the drug still

suitable? Prediction and dose

adjustment

DATA EXCHANGE AND INTEROPERABILITY Upload patient’s data Extension of eHealth

medical standards Data integrity


INNOVATE IN VITRO ASSAYS

DNA aptamers

Monoclonal antibodies

In-Check - STMicroelectronics

Aptamer-based analysis of tobramycin in serum samples


SUPPORT INTERPRETATION OF DRUG CONCENTRATION DATA

ezeCHiel


EXPLOITATION PARTNERS

Division of Clinical Pharmacology Collect patient samples

Validation in field conditions

Elaborate a protocol for large-scale randomized clinical trials

Readers and prototyping

Contribution in the development of a demonstrator


Candidate treatments for therapeutic drug monitoring

69

Modern anticancer agents

Antiretrovirals

Immunosuppressant

Antibiotics

IMATINIB 1000 ng/ml493.60 Da

TACROLIMUS 10 ng/ml804.02 Da

EFAVIRENZ 2000 ng/ml315.70 Da

TOBRAMYCIN 1000 ng/ml467.5 Da


From surface confined to volume confined systems


Clinical Pharmacology, UNIL CHUV (CH)Thierry Buclin, DirectorLaurent Decosterd

European Institute of Oncology, Milan (I)Marco Giorgio

Ludwig Institute for Cancer Research (CH)Immanuel Luescher

CEA LETI (F)Thomas Ernst

EPFL

Microelectronic Systems Laboratory, Yusuf Leblebici

CMi

LMIS4, Philippe Renaud

Nanophotonics and Metrology Laboratory Olivier Martin


• Dielectric properties of single cell : Discrimination- Main leukocyte sub-populations (monocytes, lymphocytes, neutrophils) in human blood(Label-Free Differential Leukocyte Counts Using a Microfabricated, Single-Cell Impedance Spectrometer, D Holmes et al.)- Red blood Cells ghost and RBCs fixed in glutaraldehyde (Impedance spectroscopy flow cytometry: On-chip label-free cell differentiation, K. Cheung et al.) - Monocytes and dendritic cells(On-chip non-invasive and label-free cell discrimination by impedance spectroscopy, G.Schade-Kampmann et al.) viability - MFC-7 cell death (Label-free single cell analysis with a chip-based impedance flow cytometer, A.Pierzchalski et al)- Living and dead yeast cells (Multiple-frequency impedance measurements in continuous flow for automated evaluation of yeast cell lysis , G. Mernier et al) Infection - Babesia bovis infected erythrocytes (Label-free detection of Babesia bovis infected red blood cells using impedance spectroscopy on a microfabricated flow cytometer, C. Küttel )

• Dielectric properties of labels decorating the cell Polysterene particles (Single Cell Impedance Cytometry for Identification and Counting of CD4 T-Cells in

human Blood Using Impedance Labels, D. Holmes et al.)

• Change in ionic force of medium cell lysis detection- CD4+ T (Cell detection and counting through cell lysate impedance spectroscopy in microfluidic devices , X. Cheng)


Back up


Quantification of small molecules in serum

76

EMIT(Enzyme Multiplied Immunoassay Technique )

requires derivatization of the drug molecules Drug of abuse

FPIA: (Fluorescence Polarization Immunoassay)

hard to integrate, based on antibodies requires derivatization of the drug molecules Therapeutic drug monitoring: employed only for

specific diseases (difficult to interpret and measure)

Syva RapidTest®

FPIA

Carlotta Guiducci

I do nto have to convince you that aptamers are It is veru clear that the challenges are : probes adapted tosmall moelcules, smaller than antobodies. detection techniques avoiding drug derivatization


New approaches to small molecules quantification. DNA aptamer based

cANTIBODIES

150 kDaAPTAMERS

10 kDa - 30 kDa YFerguson, B. S. et al. (2013). "Real-Time, Aptamer-Based Tracking of Circulating Therapeutic Agents in Living Animals." Science Translational Medicine 5(213): 213ra165.

Chang, A. L. et al., Anal. Chem. 2014, 86, 3273−3278.


Do our aptamerswork in complex matrices?

78

Test on SPR label-free


Point of care biosensor

79

Localised Surface Plasmon Resonance

Cappi, G., Accastelli, E., Spiga, F.M., Cantale, V., Rampi, M.A., Benini, L., Guiducci, C. (2013) Mat Res Soc Symp Proc


DNA Aptamer Selection against tobramycin

Tobramycin

Aminoglycoside antibiotic Adverse effects on kidney

and ears

Tested in buffer:• 0.23 M KD

• No affinity toward carbenicillin

• 5 time higher KD toward kanamycin

80


Tobramycin on T-LSPR in undiluted serum

0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5

-0.4

0

0.4

0.8

1.2

1.6

Time (hours)

Nor

mal

ized

sig

nal

Aptamer

10 µM 20 µM 40 µM 60 µM 80 µM0 µM 0 µM 0 µM 0 µM


Point of care biosensor:can monitor small molecules?

82

Tobramycin on T-LSPR

• T-LSPR setup: Kd= 0.26 µM

• Biacore SPR: Kd= 0.23 µM

0 5 10 15 200

2

4

6

8

10

12

14

16

x 10-4

Tobramycin concentration (µM)

hue

Aptamer (average)Langmuir interpolationControl

Cappi, G., Spiga, F.M., Moncada, Y., Ferretti, A., Beyeler, M., Bianchessi, M., Guiducci, C. (2014) ACS Nano (in prep.)


Point of care biosensor:small molecules in complex matrices?

83

Tobramycin on T-LSPR in undiluted serum

Linear trend Theoretical minimum

resolvable concentration of tobramycin 3.4 µM.

Good linearity in almost all the analytical range (1-80µM)Cappi, G., Spiga, F.M., Moncada, Y., Ferretti, A., Beyeler, M., Bianchessi, M., Guiducci, C. (2014) ACS Nano (in prep.)


Making aptamers - SELEX

84

Classic Capture

In-vitro evolution-like process

No need to immobilize the target: good for small molecules!

Spiga, F.M., Maietta, P., Guiducci, C. (2014) JACS (in submission)


Capture-SELEX with control

85

In stream control of library affinity via Surface Plasmon Resonance

Spiga, F.M., Maietta, P., Guiducci, C. (2014) JACS (in submission)


Tobramycin Carbenicillin

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