FULL ARTICLE Plasmonic nanoparticles-decorated diatomite biosilica: extending...

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FULL ARTICLE

Plasmonic nanoparticles-decorated diatomite biosilica:extending the horizon of on-chip chromatography andlabel-free biosensingXianming Kong, Erwen Li, Kenny Squire, Ye Liu, Bo Wu, Li-Jing Cheng, and AlanX. Wang*

School of Electrical Engineering and Computer Science, Oregon State University, Corvallis, OR, 97331, USA

Received 21 February 2017, revised 10 April 2017, accepted 18 April 2017

Keywords: diatomite biosilica, lab-on-a-chip, on-chip chromatography, surface-enhanced raman scattering, photoniccrystals, plasmonics

Diatomite consists of fossilized remains of ancient dia-toms and is a type of naturally abundant photonic crys-tal biosilica with multiple unique physical and chemicalfunctionalities. In this paper, we explored the fluidicproperties of diatomite as the matrix for on-chip chro-matography and, simultaneously, the photonic crystal ef-fects to enhance the plasmonic resonances of metallicnanoparticles for surface-enhanced Raman scattering(SERS) biosensing. The plasmonic nanoparticle-deco-rated diatomite biosilica provides a lab-on-a-chip capa-bility to separate and detect small molecules from mix-ture samples with ultra-high detection sensitivity downto 1 ppm. We demonstrate the significant potential forbiomedical applications by screening toxins in real bio-fluid, achieving simultaneous label-free biosensing of

phenethylamine and miR21cDNA in human plasmawith unprecedented sensitivity and specificity. To thebest of our knowledge, this is the first time demon-stration to detect target molecules from real biofluids byon-chip chromatography-SERS techniques.

1. Introduction

Diatoms are unicellular, photosynthetic bio-mineralization marine organisms that possess a bio-silica shell, which is called the frustule, with two di-mensional (2-D) periodic pores [1,2]. The uniqueoptical, physical, and chemical properties of diatomfrustules have attracted long-lasting interests formore than two centuries [3–5]. For instance, the hi-erarchical nanoscale photonic crystal features canreflect vivid colors and enhance the local optical

fields at the surface of diatom frustules [6,7]. Theabundant hydroxyl groups at the frustule surfacemake diatom biosilica very hydrophilic [8]. Thenano-corrugated surface, which possess a large sur-face-to-volume ratio of 200 m2/g, induces micro- andnano-fluidic properties that can achieve unique flu-idic control [9,10].

In recent years, there has been an escalating trendto detect biological targets with ultra-high sensitivityusing diatom biosilica [11]. Zhen et.al. developed PL-based diatom biosensors that have been successfully

* Corresponding author: e-mail: [email protected]

Supporting information for this article is available on the WWW under https://doi.org/10.1002/jbio.201700045

J. Biophotonics 1–12 (2017) / DOI 10.1002/jbio.201700045

© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

https://doi.org/10.1002/jbio.201700045

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applied for TNT sensing [12]. De Stefano et al. modi-fied the diatom frustules (Coscinodiscus concinnus)chemically and then attached antibodies as a highly-se-lective bioprobe for immunocomplex biosensing [13].Our previous studies have proved that metallic nano-particles (NPs) located near or inside the periodicnanopores of diatoms can form hybrid photonic-plas-monic modes via theoretical analysis and experimentalresults [14–16]. These photonic-plasmonic modes fur-ther increase the local optical field near the plasmonicNPs and additional SERS enhancement was achieved.Furthermore, our group has utilized diatom biosilicaas a platform for a sandwich-type immunoassay [17],which can detect large biomolecules such as antigensfrom complex biological samples. In the developed im-munoassay, the diatom-Ag substrate was first function-alized with an antibody. With the presence of targetantigens, the Au NPs labeled with Raman probe mole-cules and the antibody could crosslink onto the dia-tom-Ag substrate through specific recognition be-tween the antibody and the antigen. That studyshowed that diatom frustules improved the detectionlimit of the antigen to 10 pg/mL, which is nearly100 times better than conventional colloidal SERS im-munoassay on a flat glass substrate.

However, immunoassay is not very effective at de-tecting small molecules due to the weak change of thesignals and the time-consuming labeling processes. Inprinciple, surface enhanced Raman spectroscopy(SERS) [18] can provide unique identification to a tar-get analyte, especially small molecules such as illicitdrugs [19,20]. In practice, however, this is not alwaystrue for real-world samples due to various forms of in-terference [21]. For instance, it is extremely difficult todirectly detect small molecules using SERS from bio-fluid or physiological environments containing highconcentration of salts, because the saline has a stronginfluence on the stability of both the metallic colloidsand the biomolecules. Furthermore, the macro-molecules such as proteins and DNAs in biofluidwould block the interaction between the plasmonicsubstrate and small molecules. Therefore, it is highlydesirable to develop a lab-on-chip device that can si-multaneously perform on-chip chromatography to sep-arate the small molecules and detect them in a label-free manner such as SERS.

In this study, we explore plasmonic NPs-deco-rated diatomite biosilica film as a lab-on-a-chip de-vice for on-chip chromatography and label-free bio-sensing of small molecules from complex biologicalsamples. Diatomite consists of fossilized remains ofancient diatoms and is a type of naturally abundantphotonic crystal biosilica which has been widelyused in industry as water filters, adsorbents, andmedicine [22–24]. As geological deposits with bil-lions of tons of reserve on earth, diatomite has sim-

ilar properties to diatoms such as highly porousstructure, excellent adsorption capacity, and pho-tonic crystal effects [25,26]. Most importantly, dia-tomite can be easily spin-coated on a substrate toform a dense film with extremely low cost. Differentthan our previous research focusing on the photoniccrystal effect and microfluidic properties of in-dividual diatom frustule, this paper utilizes the flu-idic property of diatomite film decorated with goldnanoparticles (Au NPs), which can perform on-chipchromatography and simultaneously, serve as an ul-tra-sensitive SERS substrate to detect the targetmolecules. In this research, Phenethylamine (PEA)is chosen as the target analyte in our study. PEA andsubstituted PEAs are a broad class of compoundsthat affect the central nervous system. The phene-thylamine derivatives are infrequently encounteredby forensic toxicology laboratories. There have beenincreasing reports of the availability, distribution,and use of these compounds, which have caused at-tention by the U.S. Department of Justice [27,28].Herein, we used the hybrid plasmonic-diatomite bi-osilica as the matrix for on-chip chromatographyand SERS substrate to separate and detect PEAfrom real biofluid. The diatomite on the chromatog-raphy plate not only functions as the stationaryphase, but also provides additional Raman enhance-ment to improve the SERS sensitivity, resulting inmore than 10 3 better LOD than commercial chro-matography plates.

2. Experimental section

2.1 Materials and reagents

Tetrachloroauric acid (HAuCl4) was purchased fromAlfa Aesar. Trisodium citrate (Na3C6H5O7), anhy-drous ethanol, ammonium hydroxide (NH4OH,28 %), hexane and ethyl acetate were purchasedfrom Macron. Diatomite (Celite209), pyrene, 4-mer-captobenzoic acid (MBA), nile blue (NB), plasmaand phenethylamine (PEA) were obtained from Sig-ma-Aldrich. Rhodamine6G (R6G) was purchasedfrom TCI. The chemical reagents used were of ana-lytical grade. Water used in all experiments was de-ionized and further purified by a Millipore SynergyUV Unit to a resistivity of ~18.2 MW cm.

2.2 Preparation and Characterization ofAu NPs

The glassware used in the nanoparticle synthesisprocess was cleaned with aqua regia (HNO3/HCl,1:3, v/v) followed by rinsing thoroughly with Milli-Q

2 A. X. Wang et al.: Diatomite on-chip chromatography biosensor

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water. Au NPs with an average diameter of 60 nmwere prepared using sodium citrate as the reducingand stabilizing agent according to the literature withlittle modification [29]. Briefly, a total of 100 mL of1 mM chloroauric acid aqueous solution was heatedto boil under vigorous stirring. After adding 4.2 mLof 1 % trisodium citrate, the pale yellow solutionturned fuchsia within several minutes. The colloidswere kept under refluxing for another 20 min to en-sure complete reduction of Au ions followed bycooling to room temperature.

2.3 Fabrication of diatomite biosilica chip

The diatomite chip for chromatography sensingwere fabricated by spin coating diatomite on glassslides. The diatomite was dried at 150 8C for 6 h inan oven before spreading on the glass slides. Aftercooling to room temperature, 6 g of diatomite wasfirst dispersed in 10 mL of 0.5 % aqueous solution ofcarboxymethyl cellulose and then transferred ontothe glass slide for spin coating at 1200 rpm for20 seconds. The chips were placed in the shade todry and then activated at 110 8C for 3 h to improvethe adhesion of diatomite on glass slides.

2.4 On–chip chromatography method

The on-chip chromatography SERS method was de-signed for detection of analytes from mixtures or bi-ofluid as shown in Scheme 1. First, 1 mL liquid sam-ple was spotted at 12 mm from the edge of thechromatography plate. After drying in air, the chro-matography plate was kept in the glass chromatog-raphy development chamber with different types ofmobile phase eluent. After separation of the analy-tes, the chromatography plates were then dried inthe oven under 60 8C for 1 min. The separated ana-lyte spots were marked under ultraviolet illumina-tion at 380 nm wavelength and visualized by iodinecolorimetry. The retention factor (Rf: equal to the

ratio of the distance migrated by the target analyteand the solvent on the chromatography plate) of theanalytes on chromatography plates were calculatedand marked on the chromatography plates so thatthe analytes spots could be traced even when theyare invisible at low concentrations. Then 2 mL con-centrated Au NPs were added directly three times.A Horiba Jobin Yvon Lab Ram HR800 Raman mi-croscope equipped with a CCD detector was used toacquire the SERS spectra, and a 50 3 objective lenswas used to focus the laser onto the SERS sub-strates. The excitation wavelength was 785 nm, andthe laser spot size was 2 mm in diameter. The con-focal pinhole was set to a diameter of 200 mm. SERSmapping images were recorded with a 10 3 10-pointmapping array. They were collected using the Du-oScan module with a 2.0 mm step size, 0.5 s accumu-lation time, and collected in the Raman spectralrange from 800 cm�1 to 1800 cm�1. The acquireddata were processed with Horiba LabSpec 5 soft-ware.

2.5 Other Instruments

UV-vis absorption spectra were recorded on Nano-Drop 2000 UV-Vis spectrophotometer (Thermo Sci-entific) using a quartz cell of 1 cm optical path. At-tenuated total reflectance (ATR) infrared spectrawere recorded on a Nicolet 6700 Fourier transforminfrared (FT-IR) spectrometer (Thermo Scientific)and Smart iTR diamond ATR accessory with liquidnitrogen-cooled MCT detector. Scanning electronmicroscopy (SEM) images were acquired on FEIQuanta 600 FEG SEM with 15–30 kV acceleratingvoltage. The microscopy images were acquired onthe Horiba Jobin Yvon Lab Ram HR800 Ramanmicroscope with 50 3 objective lens for thin layer onchromatography plate and 100 3 objective lens fornear field image of single diatomite, halogen lampswere used as light source.

3. Results and discussion

3.1 Characterization and evaluation ofSERS-active gold colloidal substrates

Scanning electron microscopy (SEM) and UV-visabsorption spectroscopy were employed to charac-terize the morphology and properties of the pre-pared gold colloidal nanoparticles. The SEM images(Figure S1) indicate that the Au NPs have a spher-ical shape with uniform size distribution and theirdiameters are estimated to be 60 nm. The SERS per-formance of prepared gold colloids was verified

Scheme 1 Schematic representation of the on-chip chro-matography-SERS detection of target molecules frommixtures based on plasmonic NPs-decorated diatomite bi-osilica.

J. Biophotonics (2017) 3

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through the contrast of SERS spectrum of MBA sol-ution, a typical Raman probe molecule (Figure S2).From the UV-vis spectrum (Figure S3), the LSPRband of the prepared Au colloids located at 545 nmwith a narrow bandwidth. These values correspondto relatively uniform, mono-dispersed Au colloidswith diameters of approximately 50–60 nm. The con-centration of Au nanoparticles was calculated to beapproximately 1 3 10�10 M, which is estimated usingthe basis of the Lambert’s law based on UV-visspectroscopy (Figure S3) with a molar extinction co-efficient of 3.4 3 1010 M�1 cm�1 [30].

3.2 Microstructures of the diatomitechromatography chip

SEM was employed to characterize the morphologyof the diatomite used in our experiment as shown inFigure 1. The main component of the commercial di-atomite is disk-shaped with periodic pore structures(Figure 1. a). The size distribution of diatomite rang-es from 10–30 mm. The diatomite is spin coated andworks as the stationary phase on the chromatog-raphy plate. The SEM image of the diatomite layeron the glass substrate is shown in Figure S4. In orderto verify the photonic crystal effect of diatomite, wetook the optical image of a single diatom frustule

from the diatomite, which is shown in Figure 1 (b).The regular light pattern comes from the high orderdiffraction of the photonic crystal, which agrees withthe results from another group [31]. Therefore wecan conclude that diatomite does have photoniccrystal effects, although not perfect. The thickness ofthe diatomite on glass is monitored by optical micro-scopy as shown in Figure 1c. The thickness of the di-atomite is very uniform and measured to be 20 mm,which is much thinner than the commercial silica gelchromatography plate of 60–100 mm (Figure S5). Af-ter deposition of Au NPs onto chromatographyplate, the NPs were distributed on the surface of dia-tomite as shown in Figure 1d.

3.3 SERS analysis of the chromatographyplate

When collecting the SERS spectra of the analyte onchromatography plates, the background SERS sig-nals originating from the blank stationary phaseshould be determined first. Therefore, it is necessaryto investigate the SERS signals from different chro-matography plate matrices. The measured results(Figure S6) indicate that both the diatomite and thesilica gel chromatography plates exhibit a weak,broad spectral background, with no obvious Ramanpeaks. Thus it is conceivable to use these chroma-tography plates for SERS detection.

3.4 Effect of the colloidal gold concentration

The concentration of metallic colloids is of pivotalsignificance to the enhancement of Raman signals.SERS spectra of 100 ppm MBA on diatomite chro-matography plates from 2 mL casted gold colloidswith six different concentrations were collected. Asshown in Figure S7 of the supporting information,the SERS spectrum intensity of MBA clearly in-creases as the colloidal gold concentration increasesfrom 1 to 100 times. If the concentration increasesmore than 200 times, the measured SERS intensitywill decrease. The explanation is that higher concen-tration of metallic nanoparticle colloids results in ac-cretion of denser monolayer coverage, which in-creases the SERS signals. However, if theconcentration exceeds the requirement of mono-layer formation, multilayer nanoparticle accumu-lation leads to the reduction of SERS signals [21].Therefore, 100 fold concentrated (1 3 10�8 M) goldcolloids were chosen for subsequent measurements.

Figure 1 SEM image of the diatomite with honeycombstructure (a), optical image of a single diatom under theoptical microscope, showing the diffraction pattern fromthe photonic crystal structure (b), the microscopy image ofthe cross section of the diatomite biosilica film (c) consist-ing of diatomite by spin coating, and the SEM image ofthe diatomite after Au NPs deposition (d).



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3.5 SERS of PAHs from mixtures

PAHs are a class of organic compounds consisting oftwo or more aromatic or heterocyclic rings. The de-tection of various PAHs has significant engineeringpotential as PAHs could pose a potential hazard tohealth in the environment. Unfortunately, the lowbinding affinity between PAHs and surfaces of met-allic substrates prevents efficient SERS detection ofPAHs from mixtures as the spectra from co-existingcomponents interfere with the spectrum from thePAHs. In this work, pyrene was mixed with threeRaman probe molecules (MBA, R6G and NB) re-spectively to form different mixtures. Figure 2ashows the SERS spectra of the pure substances. Spe-cifically: pyrene, the peak at 590 cm�1 is assigned tothe skeletal stretching vibration and 1230 cm�1 is as-sociated with the C–C stretching/C–H in-planebending of pyrene [32]; MBA, the peaks located at1074 and 1587 cm�1 are associated with C�C ring-

breathing modes of MBA [33]; R6G, the peak at607 cm�1 is associated with C�C-C ring in plane vi-brations, while the peaks at 1360 and 1508 cm�1 areassociated with aromatic C�C stretching vibrationsof R6G [34]; NB, the peak at 594 cm�1 is assigned tothe in-plane deformation vibration of the NB. Thepeaks labeled with asterisks are used to representthe substances respectively. The SERS spectra ofthese three mixtures are shown in Figure 2b. ForMixture 1 (Pyrene and MBA 1/1), the metallic sur-face coverage was dominated by MBA because thecovalent bonds can be formed easily between the AuNPs and mercapto group of MBA. Thus only a veryweak Raman peak from pyrene was observed fromthe SERS spectra of mixture 1. For Mixture 2 (Pyr-ene and R6G 1/1), R6G is a typical Raman probemolecule because of its affinity with metallic sur-faces and intense Raman signals. The in plane vi-brations of R6G are located at 607 cm�1 are nearmain characteristic peak of pyrene and it is hardto distinguish the Raman peak of pyrene from themixture due to the SERS signals from R6G. ForMixture 3 (Pyrene and R6G 1/1), NB is anotherkind of Raman probe molecule that is often usedfor evaluating the SERS performance of the sub-strates. The intense band located at 594 cm�1 isusually used as the characteristic peak of NB indetection, which overlaps with the main character-istic peak of pyrene. Therefore, only molecule in-formation of NB can be observed from the SERSspectra of mixture 3.

The chromatography performance of the diatom-ite and commercial silica gel chromatography plateswas evaluated using the aforementioned three mix-tures. Hexane and ethyl acetate (v/v =3:1) were usedas the eluent for the separation of pyrene from themixtures. After separation, a UV lamp and iodinecolorimetry was used to detect different analytespots corresponding to various substances as illus-trated in Figure 3. Pyrene traveled at faster speedsand located further from the original droppingpoints because of the low molecular polarity. Threedifferent types of mixtures have been successfullyseparated as shown in Figure 3. The Rf values of themixing points were obtained by the UV light scan-ner and iodine colorimetric, which are 0/0.8 on silicagel plate and 0/0.9 on diatomite plate. The separa-tion efficiency of silica gel and diatomite chroma-tography is similar to each other in our experi-ment from the measured Rf. The diatomite platesshow a slightly better separation effect than com-mercial chromatography plates. The SERS spectraat different spots were measured on the diatomiteplate after the chromatography separation asshown in Figure 4. The characteristic peaks of pyr-ene at 590 cm�1 and 1230 cm�1 are clearly ob-

Figure 2 SERS spectra of pure substance of Nile blue,MBA, pyrene and R6G (a) and the corresponding mixture(b).



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served, which means that the diatomite plate cansuccessfully be used as the matrix for on-chipChromatography-SERS method. In this study, therepeatability of the device was also studied bymeasuring 5 random spots on 8 different chips us-ing 200 ppm of pyrene solution as shown in Fig-ure S8. The relative standard deviation (RSD) ofthe intensity of the Raman peak at 590 cm�1 is cal-culated to be 5 % from spot-to-spot on each chip.The RSD from chip-to-chip is only 3.5 %. There-fore we conclude that the repeatability of the on-chip chromatography-SERS sensors is comparablewith other SERS techniques [35].

We compared the SERS spectra obtained fromthe diatomite plate (Figure 5a, b) with commercialsilica-gel chromatography plate (Figure 5c, d). InFigure 5, all the characteristic bands of MBA andpyrene exhibited monotonous decreasement in in-tensity as the mixture concentration decreases. Thelowest detection limit from pyrene/MBA mixture is

between 20 and 100 ppm on the commercial chro-matography plate, and below 2 ppm on the diatom-ite chromatography plate. The experimental resultsdemonstrate more than 10 times improvement ofsensitivity using the diatomite-based chromatog-raphy plate compared to commercial silica-gel chro-matography plates. We attribute this improvementto two contributions from the diatomite plate. First,in our on-chip chromatography-SERS method, themetallic nanoparticles are casted onto the analytespots after chromatography separation. The SERSspectra collected from each spot will only comefrom the target molecules at the surface of the chro-matography plate. This means that the overall sensi-tivity will be compromised because a significant por-tion of the analyte inside the chromatography platescannot be detected. The thickness of the diatomitechromatography plates fabricated by spin coating isaround 20 mm, which is only one third of the thick-ness of the commercial silica-gel chromatographyplate. The thinner diatomite layer will achieve high-er analyte concentration at the surface of the chro-matography plate. Second, diatomite consists of fos-silized remains of diatoms. The two dimensional(2-D) periodic pores on diatomite disk possess hier-archical nanoscale photonic crystal features [7,36].The hybrid photonic-plasmonic modes are formedwhen Au NPs are deposited onto the surface of dia-tomite, which will further increase the local opticalfield of Au NPs. Additional SERS enhancement canbe obtained, similar to our previous work on dia-toms biosilica through theoretical calculation andexperimental characterization [14,15]. The ppm lev-el sensitivity that we achieved is comparable withother very advanced platforms developed by Lu andZhao‘s groups [37, 38], which require complicate andexpensive fabrication processes such as oblique an-gle deposition and molecularly imprinted technol-ogy.

Figure 3 Photographic images of the mixtures (1: pyreneand MBA, 2: pyrene and R6G, 3: pyrene and Nile blue)separated by diatomite biosilica and silica chromatog-raphy plates. The spots after separation are visualized withUV light (a) and iodine colorimetry (b).

Figure 4 SERS spectra of the substances in the mixtures after separation through diatomite biosilica film.



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3.6 Optical simulation of plasmonic NPs ondiatomite

To investigate the photonic property of diatomiteand its contribution to the SERS signals, we usedFullWAVE module from Rsoft photonic componentdesign suite, which is based on three-dimensional(3D) finite-difference time-domain (FDTD) algo-rithm. According to the SEM image, we modeledthe diatomite substrate as a 2-D photonic crystal sili-ca slab with a hexagonal lattice of air holes. As na-ture created diatomite consists of mixed diatom spe-cies with various photonic crystal periods, we onlyconsider the periodic photonic structure that canprovide the largest SERS signal enhancement in oursimulation, which means that the guided mode reso-nance (GMR) peak wavelength of the photoniccrystal structure matches the wavelength of the ex-citation light [14]. Figure 6 (a) plots the schematic ofthe diatomite substrate with labeled structure pa-

rameters. The hybrid plasmonic-photonic crystalnanostructure was achieved by integrating randomlydistributed Au nanoparticles (NPs) on the diatomitesubstrate as shown in the inset of Figure 6 (a). Thediameter of the Au NPs is 70 nm and the minimumgap width between the Au NPs is 5 nm, which matchthe experiment condition. The inset of Figure 6 (a)also indicates the simulation domain of our FDTDcalculation. We use periodic boundary condition inthe horizontal direction (X�Y plane) in order to ex-cite the GMRs. A mesh size of 2.5 nm is used, whichis small enough to qualitatively characterize theelectrical field “hot spots” generated by localizedsurface plasmonic resonances (LSPR) between theAu NPs’ gap. To compare the contribution of dia-tomite, the same distribution of Au NPs is placed ona flat silica substrate as reference. The excitationwas a 785 nm plane wave. Both TE and TM polar-ized incident light was simulated, and the simulationresults were based on equal average of both polar-

Figure 5 SERS spectra of mixture 1 at different concentrations separated by diatomite (a, b) and silica gel (c, d) chroma-tography plates.



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izations. We know that the SERS enhancement fac-tor (EF) is proportional to the four power of the lo-cal electrical field[39]. However, in reality, the meas-ured SERS EF varies from the analyteconcentration and even the analyte molecule dis-tribution in the case of low concentration. To sim-plify the analysis, we only focus on the optical prop-erties of the diatomic earth chromatography plate.So we use the normalized electric field intensity asthe main parameter in the following discussion,which is defined as jETE/E0 j 2/2+ jETM/E0 j 2/2, whereETE and ETM corresponds to the amplitude of the lo-cal electrical field under TE and TM polarized in-cident light and E0 is the amplitude of the incidentlight. Figure 6 (b) and (c) the electric field intensity

distribution of the Au NPs layer. The value wasaveraged over the vertical direction. Since photoniccrystal structure supports GMRs, which increasesthe local electrical field intensity, the electrical field“hot spots” generated LSPR from Au NPs are cou-pled with the GMRs when Au NPs are placed insidethe air holes. As a result, we can see that the E4-EFof many “hot spots” inside the air holes are en-hanced. To quantitatively describe the enhancementeffect, we count the “hot spots” statistical dis-tribution over the electric field intensity of both thediatomite substrate and the reference substrate. Fig-ure 6 (d) plots the enhancement ratio between thetwo distributions on different substrates, which is de-fined as (#on diatomite/# on silica), where #on dia-

Figure 6 (a) Schematic of the simulation model for diatomite substrate. Inset: randomly distributed Au NPs on diatomite.The white background represents the place where the Au NPs are placed in the air holes on the bottom silica substrate,and the blue background means where the Au NPs are placed on the top surface of the silica photonic crystal slab. For thereference silica substrate, all the Au NPs are on the substrate surface. Electric field intensity distribution of the Au NPslayer on a diatomite substrate (b) and on a silica substrate (c). (d) enhancement ratio of the “hot spots” distribution overelectric field intensity, which is defined as the ratio of numbers of “hot spots” at each intensity level.



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tomite/silica means the number of “hot spots” ateach intensity level on the diatomite/silica substrate.We can group the “hot spots” into different levelsaccording to their electric field intensity, as is shownin Figure 6 (d). Clearly, both the intensity and thedensity of “hot spots” are enhanced by the couplingbetween LSPRs and GMRs. The enhancement ratioincreases as the “hot spots” intensity increases. Forexample, enhancement raito of strong “hot spots” ofwhich the electric field intensity is larger 102.5 morethan 14 times. And these strong “hot spots” are cru-cial to detect low analyte molecules. To conclude, di-atomite substrate with Au NPs can increase theSERS sensitivity and detection limit due to the cou-pling between LSPRs of Au NPs and GMR effect ofthe periodic diatomite structure.

Mapping images of the Raman signals visualizedthe distribution of analytes on the chromatographyplate. The SERS mapping image of 10 ppm pureMBA on the diatomite chromatography plate as

well as the silica gel chromatography plate areshown in Figure 7a and b. MBA provides an intenseRaman peak at 1074 cm�1, which is assigned to thering-breathing modes. The SERS mapping imagewas recorded using the integrated peak intensity at1060–1090 cm�1. Diatomite chromatography plateshowed a much stronger and more uniform SERSsignals of MBA than the silica gel chromatographyplate. The intensity distribution in the mapping areais shown in Figure 7 (c) and (d). Low variability isobserved on diatomite plate, in which the coefficientof variation (CV) is 0.39, and is almost 1.0 on silicagel plate. The highly porous structure and more uni-form pore size of the diatomite has lower flow resist-ance [40], which enables more homogenous liquidflows into the pore on diatomite. Therefore, the elu-ent migrates smoothly and uniformly on the surfaceof the stationary phase during the chromatographydevelopment. For the commercial silica gel chroma-tography plate we used, the liquid flow mainly pass-

Figure 7 Raman mapping images of MBA(10 ppm) on (a) diatomite (CV~0.39) and (b) silica gel (CV~1) chromatog-raphy plates and the relative intensity distributions of representative chromatography plates (c) and (d).



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es through the big particle gaps of the silica gel, andthe analyte molecules located mainly in the inter-particle regions. Thus the distribution of the analyteson the diatomite plate is more uniform comparedwith that on the silica gel plate.

3.7 SERS of PEA from biofluid

There have been very few reports to detect analytesin biofluid using SERS because biofluid is complexand the high saline concentration makes metalliccolloids unstable. Here we used this on-chip Chro-matography-SERS method for on-site detection ofbiogenic amines from plasma. Ammonium hydrox-ide and ethanol (v/v =1:1) were used as the eluentfor the separation of PEA from plasma. The bio-molecules such as albumin in plasma cannot diffuseon the chromatography plate due to the high molec-ular weight. We compared the SERS spectra ob-tained from diatomite plate Figure 8 and commer-cial silica-gel chromatography plate (Figure S9). TheRaman peak at 1002 cm�1 was assigned to the phen-yl ring breathing vibration of PEA. The lowest de-tection limit for PEA/plasma was 10 ppm on the dia-tomite chromatography plate. As a comparison,there were no detectable SERS signals of PEA onthe silica gel chromatography plate even when theconcentration of PEA is 100 ppm. The experimentalresults demonstrate more than 10 times improve-ment of LOD using the diatomite-based chromatog-raphy plate over the commercial silica-gel chroma-tography plate. It is difficult to obtain SERS signalsof proteins that have no conjugated chromophore[41]. FT-IR was used to verify the protein in plasmaduring the chromatography process (Figure S10).The peaks at 1645 cm�1 and 1540 cm�1 are assignedto amide I and amide II bands of protein in plasma,and the peak at 1585 was assigned to C=C stretchingvibration of PEA. To further demonstrate the multi-plex sensing capabilities of on-chip Chromatog-raphy-SERS, miR21cDNA was added into the plas-ma (5 3 10�6 M). SERS spectra of DNA wasobserved at the initial spot after the chromatog-raphy separation as shown in Figure 8b, and thepeak at 750 and 790 cm�1 were assigned to the ringbreathing modes of thymine [42] and cytosine [43],respectively. This also verifies that DNA has a verysmall retention factor and almost does not migrateon the chromatography plate.

4. Conclusions

In this study, we demonstrated plasmonic NPs-deco-rated diatomite biosilica as a lab-on-a-chip device

for on-chip chromatography and label-free SERSsensing to detect small molecules from complex bio-logical samples. The experimental results achieved adetection sensitivity down to 1 ppm, an improve-ment of more than 10 times when compared to thecommercial silica-gel chromatography plate. To thebest of our knowledge, this is the first time demon-strating separation and detection of target moleculesfrom biofluids by on-chip chromatography-SERStechniques. To demonstrate the significant engineer-ing potentials for biomedical sensing, we have ach-ieved multiplex sensing of PEA and DNA in humanplasma with LOD of 10 ppm and 5 mM respectivelysimultaneously. This facile lab-on-chip device usinghybrid plasmonic-diatomite biosilica proves itself tobe cost-effective and ultra-sensitive with multiplexsensing capabilities, which will play pivotal roles for

Figure 8 SERS spectra of human plasma with differentconcentrations of PEA separated by diatomite chromatog-raphy plates (a) and with DNA added (b).



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the monitoring of pollutants and toxins in complexenvironments and screening illicit drugs in biofluid.

Supporting Information

Additional supporting information may be found inthe online version of this article at the publisher’swebsite.

Figure S1: SEM image of the prepared Au col-loids.

Figure S2 Raman spectra of 4-mercaptobenzoicacid (MBA, 10 ppm) with and without Au colloids.

Figure S3. The UV-Vis absorption spectrum ofthe prepared Au colloids.

Figure S4. SEM image of the surface of the dia-tomite biosilica film.

Figure S5. Microscopic image of the cross sectionof commercial silica gel chromatography plate.

Figure S6. Raman spectrum of the diatomitebased chromatography plate.

Figure S7. SERS spectrums of MBA (100 ppm)on diatomite-based chromatography plate with dif-ferent concentrations of Au colloids (a) and the de-pendence of the intensity of the Raman peak at1074 cm�1 acquired from the SERS spectra (b).

Figure S8. The peak intensity at 590 cm�1 of200 ppm pyrene versus the different analyzed spotsfrom different diatomite chips.

Figure S9. SERS spectra of plasma with 100 ppmPEA separated by commercial silica-gel chromatog-raphy plate.

Figure S10. FTIR spectra from different spots ondiatomite chromatography plate after the separationof human plasma sample.

Acknowledgement The authors would like to acknowl-edge the support from the National Institutes of Healthunder Grant No. 1R03EB018893 and the Oregon StateUniversity Venture Development Fund.

Author biographies Please see Supporting Informationonline.

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FULL ARTICLE

As nature-created photoniccrystals, diatomite biosilicapossesses unique optical andfluidic properties. A lab-on-a-chipbiosensor combining the functionof on-chip chromatography andsurface-enhanced Raman scatter-ing (SERS) is experimentallydemonstrated using diatomitebiosilica decorated with plasmonicnanoparticles. Owing to its excep-tional capabilities to separate anddetect small molecules frommixture samples, we successfullydetected toxins in real humanplasma with ultra-high sensitivity.

X. Kong, E. Li, K. Squire, Y. Liu,B. Wu, L.-J. Cheng, A. X. Wang*

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Plasmonic nanoparticles-decorated diatomite biosilica:extending the horizon of on-chipchromatography and label-free bi-osensing


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