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Sensors and Actuators B 156 (2011) 471–478

Contents lists available at ScienceDirect

Sensors and Actuators B: Chemical

journa l homepage: www.e lsev ier .com/ locate /snb

on-labeled virus detection using inverted triangular Au nano-cavities arrayeds SERS-active substrate

hia-Wei Changa, Jiunn-Der Liaoa,∗, Ai-Li Shiaub, Chih-Kai Yaoa

Department of Materials Science and Engineering, National Cheng Kung University, No. 1, University Road, Tainan 70101, TaiwanDepartment of Microbiology and Immunology, National Cheng Kung University, No. 1, University Road, Tainan 70101, Taiwan

r t i c l e i n f o

rticle history:eceived 23 November 2010eceived in revised form 20 March 2011ccepted 3 April 2011vailable online 12 April 2011

a b s t r a c t

Virus detection is frequently based on antibody-based arrays and polymerase chain reactions. However,these methodologies have time-consuming incubation steps and obtaining clearly isolated target speciesis a complex process. In the present study, inverted triangular Au nano-cavities with various indentationdepths and tip-to-tip displacements are well arrayed as a substrate for qualitative virus detection. Thesubstrate is competent to entrap size-comparable target virus into nano-cavities, which as one, exerts its

eywords:riangular Au nano-cavitiesndentationurface-enhanced Raman scatteringast-screening detection

activity to form surface-enhanced Raman scattering particularly by the edges and cavities of the substrate.Through the induction of the electromagnetic effect by the substrate, the virus can be distinguished fromthe amino acids on its surface. The detectable concentration for encephalomyocarditis virus or adenovirusis 106 PFU/ml and that for influenza virus is 104 PFU/ml. The tailored substrate is enabled for fast-screeningdetection of target virus.

arget virus

. Introduction

The rapid and accurate detection and identification of pathogenst an early stage is of the utmost importance in disease mon-toring and containment systems [1,2]. Several techniques arevailable for monitoring viruses, such as antibody-based arrays thatnclude enzyme-linked immunosorbent assays (ELISA) [3], fluores-ent antibody arrays [4], and serological testing [5]. However, theseethodologies have time-consuming incubation steps [6]. In recent

ears, the development of polymerase chain reaction (PCR) hasrovided alternative approaches, such as single-nucleotide poly-orphism (SNP) analysis via the amplification of the target DNA

equence [7,8]. However, the complexity of isolating target speciesrior to the application of ELISA or the PCR method remains a prob-

em [9,10].A simple process that can detect the target microorganism is

hus desirable. Viral diseases preferably distinguishable at earlytages have been investigated to keep up with the evolution ofirus pathogens [11]. The major difficulties for virus detectionre associated with the purification and detectable quantity [12],pparent size and dimensions [13], and the establishment of a

hemical identification database for a particular virus [14]. If aurification procedure is employed [15,16], the detectable quan-ity for fast-screening detection at the early stage of viral infection

∗ Corresponding author. Tel.: +886 6 2757575x62971; fax: +886 6 2346290.E-mail address: jdliao@mail.ncku.edu.tw (J.-D. Liao).

925-4005/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.snb.2011.04.006

© 2011 Elsevier B.V. All rights reserved.

should be reduced to less than 106 plaque-forming units (PFUs)/ml[17]. The required quantity is difficult to characterize as com-pared to that of target molecular species detected using commonanalytical methods [18]. In addition, since the apparent size anddimensions of a virus are much larger than an identical molecu-lar segment, a partial description of the integrity of a virus maylead to an incomplete characterization. It is thus difficult to estab-lish a chemical identification database for interpreting a completevirus. For example, encephalomyocarditis virus (EMCV) is a non-enveloped, positive sense RNA virus with a diameter of ≈30 nm thatinfects vertebrates [19]. Adenovirus is a non-enveloped double-stranded linear DNA virus that infects mammals and birds; itscapsid consists of three main proteins with a diameter of 80–90 nm[20]. Influenza virus is an enveloped, negative-sense, and seg-mented RNA virus; its envelope consists of lipids with a diameterof 90–120 nm [21]. To date, antigenic differences of the hemag-glutinin (HA) and neuraminidase (NA) envelope glycoproteins areused to classify influenza A viruses into sixteen HA (H1–H16) andnine (N1–N9) subtypes [22]. The influenza A subtypes H1N1 andH3N2 have co-circulated with influenza B viruses since 1977. In2009, new swine-origin influenza A (H1N1) emerged, which raisesconcerns for influenza pandemic [23]. Therefore, rapid detection ofinfluenza A virus is of great importance for the control of influenza.The information about the health impacts and the economic costs

associated with the influences of these three viruses were brieflyintroduced in Supplementary data 1.

Biomolecules or segments can be investigated using resonat-ing mechanical cantilevers [24], evanescent wave biosensors [25],

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72 C.-W. Chang et al. / Sensors an

r scanning using atomic force microscopy [26]. However, thesepproaches are sensitive to the measured quantity and weight.lternatively, labeling method is usually employed to bind withparticular group for detecting microorganisms [27,28]. Never-

heless, it is difficult to prepare samples without contaminations,hich leads to overlapping signals of the labels and target species

29,30]. These technologies are workable in discriminating viruseshrough a slow process that is unsurpassed the requirement of clin-cal detection [1]. It is thus desirable to facilitate the proceduref sample preparation, narrow the spectral bands, and examinehe practical structure of a specific virus [31,32]. Recent develop-

ent on the detection of microorganisms has combined the effectf surface-enhanced Raman scattering (SERS) with the function ofroughened metal surface [33]. The roughened metal surface cane a group of nano-particles [34] or a tailored and nano-structuredrray [35] that is capable of amplifying the effect of SERS and estab-ishing a chemical identification database of viruses. To fabricate

SERS-active substrate for the detection of microorganisms, theffective distance between the tailored surface and the sampleust be addressed [36].Most investigations on the metal-base SERS-active surface

tilize the micro/nano-lithographic technique [37,38] to cre-te a variety of topographical patterns. However, a deepenedicro/nano-scale pattern is still difficult to control in a quick way.

n addition, the lithographic technique to produce SERS-active sub-trate has a potential drawback leading to trace contamination andsually requires the additional cost of wastewater treatment. Onhe other hand, an advanced focused ion beam (FIB) technique cane applied to fabricate a designed nano-scale pattern with highrecision, but the required cost is comparatively high [39]. In thisork, a triangular pyramid-shape tip with a nano-scale diame-

er is indented into a Au layer to form an inverse pyramid cavity.he nano-mechanically made substrate provides a high-densityip-to-tip concave space, which is completely free from residualontamination during fabrication [40,41]. As compared with com-ercially available Klarite substrates [42], present technique is

dvantageous to produce an array with ordered edges and cavitiestructures for facilitating the entrapment of target virus. To exam-ne whether the as-prepared array provides SERS induced effector detecting target microorganism, a molecular probe, rhodamineG (R6G), physically adsorbed upon the tailored surface, was uti-

ized as the index and evaluated using Raman spectroscopy. Theano-indentation depth of the tailored cavities was taken as a geo-etrical factor and purposely fitted with that of target virus for a

pace of one virus. The nano-mechanical method is ideal for theabrication of precise micro/nanostructure surfaces as it allows theontrol of infinitesimal forces and displacements [43,44]. It is antic-pated to discriminate different viruses at very small quantity andstablish an easily and highly reproducible method to make tailoredERS-active substrates.

. Materials and methods

.1. Fabrication of the nAu

Au substrate was prepared by thermal evaporation of ≈200 nmf Au (99.99% purity) onto the polished single crystal silicon (100)afers (Silicon Sense, Germany) primed with a ≈5-nm-thick tita-ium adhesion layer. The as-prepared Au substrate (shortened ashe “apAu”) was polycrystalline with a grain size of 20–50 nm; itsrains predominantly exhibited a (1 1 1) orientation. The grain size

nd the root mean squared roughness (≈0.62 nm) of the apAu wereeasured by scanning probe microscope (SPA300HV, SEIKO, Japan).

he measurement type was the tapping mode with a probe tipUltrasharp silicon cantilevers NSC15, �-masch, Estonia). The cone

uators B 156 (2011) 471–478

angle for the tip was less than 20◦. The typical resonant frequencywas 325 kHz and the force constant was 40 N/m. In order to obtainhigh resolution data in the experiment, low scan rate (i.e., 1 Hz, 512lines) was used.

The inverted triangular Au nano-cavities array (shortened as the“nAu”) was fabricated using one-step loading-unloading mode ofdynamic contact module system (NanoIndenter G200, Agilent Tech-nologies, USA). In the process, a triangular pyramid tip of Berkovichdiamond with a radius of ≈20 nm was used under a controlled rela-tive humidity (≈32%) and room temperature (≈24 ◦C). Before eachnotch, the nano-indentation tip was cleaned by indenting on thesingle crystal aluminum. The loading procedure was controlled tohave a constant drift rate of 0.05 nm/s for stabilizing the fabricationsteps. The applied loading force was in the range of 270–1700 �Nfor indentation depths (Dv) of 50, 70, 90, 120, and 140 nm mea-sured using the vertical displacement. The tip-to-tip displacements(Dt–t) of 500 and 1000 nm, measured using the parallel displace-ment, were used. These two geometrical factors were chosen as themajor parameters for comparison. The approaching velocity andthe harmonic displacement of the nano-indentation tip toward thetarget surface were maintained at 1 nm/s and 1 nm for all the test-ing surfaces. Depending on the unspecified contact points, surfaceapproaching sensitivity was optimized at 10% for the apAu [43].

2.2. Preparation of molecular probes and viruses

R6G solution is commonly used as a tracer dye and exten-sively applied in biotechnology applications such as fluorescencemicroscopy, flow cytometry, and ELISA. In this work, R6G solu-tion was used as the index to examine the nAu induced SERSeffect [45] and furthermore to detect nano-scale virus withinand on the substrate. The R6G (Sigma, Germany) molecules werediluted with phosphate-buffered saline (PBS, Sigma, Germany) toa concentration of 10−4 M. Thereafter the nAu samples with themolecular probes-containing solution were covered with slide andthen immediately measured using Raman spectroscopy.

The target viruses employed for the assessment of the nAusamples were EMCV, adenovirus, and influenza virus. EMCV waspropagated in Vero (African green monkey kidney) cells. An ade-novirus vector derived from wild-type adenovirus type 5, whichwas defective in E1B-55 kD gene, was propagated in 293 (humanembryonic kidney) cells [15]. Influenza A virus/WSN/33 (H1N1)was propagated in MDCK (Madin-Darby canine kidney) cells. Allof the cells were cultured in Dulbecco’s modified Eagles mediumsupplemented with 10% cosmic calf serum (Hyclone, USA). Stan-dard protocols were used to propagate different viruses. Viral titerswere determined by the plaque assay. All of the viruses were storedat −70 ◦C before use. The titers of the original stocks of EMCV,adenovirus, and influenza A virus were 108, 108, and 106 PFU,respectively. They were diluted to different titers in PBS for furtheruse. A quantity of 5 �l from different dilutions of each virus wasplaced within the nAu. Both the apAu surface and the chosen nAusamples were subsequently employed as the substrates for virusdetection.

2.3. Raman spectroscopy and SERS evaluation

The presence of a virus upon the apAu surface or within the cho-sen nAu samples was examined by a confocal microscopic Ramanspectrometer (In Via Raman microscope, RENISHAW, United King-dom) using 633 nm radiations from the excitation of He–Ne laserwith the power of 17 mW. The power of laser on the substrate was

8 mW measured by illumination meter. The scattering light was col-lected by a 50× air objective lens to a CCD detector. A grating of 1800lines/mm was used to disperse the scattered light. All the reportedRaman spectra were the results of a single 10 sec accumulation in

C.-W. Chang et al. / Sensors and Actuators B 156 (2011) 471–478 473

Fig. 1. FESEM micrographs of the typical nAu samples #1 to #10 represent to (a to j). Details of theoretical and experimental calculations of their size and volume were givenin Fig. S-1 and Table S-1 in Supplementary data 2. The corresponding dimension, expressed as Dt–t/Dv , of Raman scattering area was illustrated and marked as e.g., 1000/50for these nAu samples.

4 d Actuators B 156 (2011) 471–478

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Fig. 2. Raman-active peaks of R6G molecules (PR6G) within the nAu samples #1 to

#1 to #10 were estimated to be 5.85 × 107 (Detailed calculationswere described in Supplementary data 3). In the experiment, eachmeasurement was effectively counted. The measured error bars,

74 C.-W. Chang et al. / Sensors an

range of 500–3000 cm−1. Since there was no signal, e.g., organicpecies, found in the measurement range of 2000–3000 cm−1, thepectra in the measurement range of 500–2000 cm−1 were pre-ented. Before each batch, Raman shifts were calibrated using theignal of 520 cm−1 with the same absolute intensity from a standardilicon wafer.

The SERS enhancement factor (EF) was estimated according tohe standard equation [46]:

F =[

ISERS/NSERS

INRS/NNRS

](1)

here ISERS and INRS are SERS and normal Raman scatter-ng intensity, respectively; NSERS and NNRS are the numbers of

olecules contributing to the inelastic scattering intensity, whichre respectively evaluated by SERS and normal Raman scatteringeasurements. Raman intensity was averaged from ten consec-

tive measurements. Note that the apAu and nAu are initiallyaman-inactive and used as the reference substrates for the calcu-

ation of EF when Raman-active R6G molecules are adsorbed withinhe nAu.

.4. Negative staining

The drop-to-drop method [47] was conducted for the fixationf virus and subsequent negative staining. A quantity of 5 �l virus-ontaining liquid was dropped onto the cleaned nAu. At the sameime, a quantity of 10 �l phosphotungstic acid (PTA, 2% PTA inml PBS) was dropped onto a glass slide. On the other hand,

he dried virus-containing nAu surface was tightly clipped, turnedownward, and followed by placing the virus-containing nAu sub-trate upon the PTA-dropped glass slide and staining for 2 min. Thetained nAu sample was rinsed twice using de-ionized water. Afterhe treatment, the entrapped virus within the nAu was examined byeld-emission scanning electron microscope (FE-SEM, JSM-7001,

EOL, Japan).

. Results and discussion

.1. Optimization of the nAu samples as SERS-active substrates

Fig. 1a–j shows FE-SEM micrographs of nAu samples #1 to10, respectively. The geometrical information expressed as cor-

esponding sample numbers and their Dt–t/Dv of the nAu samples1 to #10 were shown in the upper-left side of each figure. Theeometrical information was given in Table S-1 in Supplementaryata 2. In Fig. 2, the nAu samples #1 to #10 containing the opti-ized concentrations of 10−4 M R6G solution were examined by

aman spectroscopy. In this study, He–Ne laser with the wave-ength of 633 nm exhibited sensitive property and enabled thenhancement for the detection of R6G molecules in solution. Theajor peak of the molecule, designated “PR6G”, was clearly found at

360 cm−1 (�(C C), aromatics). Raman intensity of “PR6G”, denoteds Ipeak (R6G), showed signs of the most intense enhancement. The

peak (R6G) for the nAu samples #1 to #10 were recorded for subse-uent comparisons.

According to Eq. (1), SERS EF was estimated by takingSERS = Ipeak (R6G) and NSERS = NR6G. For calculating NR6G, the volumef a cavity (Vc), as described in Fig. S-1 and Table S-1 in Supplemen-ary data 2, for the detectable dimension ≈1 �m3 is multiplied, andhe value NR6G is correlated with the concentration of 10−4 M R6Golution. Note that NNRS of R6G molecules is estimated using ≈5 �l

f 10−2 M R6G solution spreading over 3 mm in diameter, then driedff and measured. Since the spot size of laser beam was focused on1 �m2, the average NNRS was calculated as ≈4.2 × 109 molecules.he INRS was measured as ≈45. Based on the above calculations,

#10 for a laser wavelength of 633 nm. Raman laser wavelengths are compared inFig. S-2 in Supplementary data 4. The PR6G at 1360 cm−1, �(C C) aromatics was takenas the index for the measurement of Ipeak (R6G).

the optimized SERS EF for R6G molecules within the nAu samples

Fig. 3. SERS spectra of EMCV within the chosen nAu samples #6 to #9 were com-pared with that upon the apAu surface. Two EMCV concentrations suspended inPBS: (a) 107 and (b) 106 PFU/ml, were examined. PBS was also examined by the nAusample #8 as the control.

C.-W. Chang et al. / Sensors and Actuators B 156 (2011) 471–478 475

Fig. 4. SERS spectra of adenovirus within the chosen nAu samples #7 to #9 werecpb

a±d

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Fig. 5. SERS spectra of influenza virus within the chosen nAu samples #8 to #10

ompared with that upon the apAu surface. Two adenovirus concentrations sus-ended in PBS: (a) 107 and (b) 106 PFU/ml, were examined. PBS was also examinedy the nAu sample #8 as the control.

s shown in Fig. 2, were controlled in the range of smaller than3%. Accordingly, the nAu samples were presumably reliable inetecting R6G molecules.

Obviously, Raman scattering with the laser wavelength of33 nm for Ipeak (R6G) derived from the nAu samples #6 to #10as of great interest. In addition, these nAu samples with Dt–t of

00 nm that significantly increased Raman intensity were there-fter utilized for virus detection. As a consequence, the nAu samplesith the defined inverted triangular shape and the chosen Dv andt–t were anticipated to adjust the resonance frequency of local-

zed surface Plasmon, to fit with an incident laser wavelength, ando maximize the nano-structures induced electromagnetic (EM)ffect.

.2. Optimization of nAu samples for the detection of virus

Fig. 3a and b shows the results of detecting the presence of EMCVt concentrations of 107 and 106 PFU/ml using the apAu and nAuamples #6 to #9, respectively. No significant signals were foundt both concentrations of EMCV placed upon the apAu. In con-rast, Raman shifts in the range of 1100–1600 cm−1 appeared asMCV was examined by the nAu samples #6 to #9. The peak assign-ents for these SERS spectra for EMCV were given in Table 1. As

hown in Fig. 3a, SERS spectrum for EMCV placed upon the nAuample #8 exhibited the highest intensity, with bands of aminocids at 1558 and 1591 cm−1, a relatively minor band of aminocid at 1165 cm−1, amide III at 1232 cm−1, amide III (i.e., from

were compared with that upon the apAu surface. Two influenza virus concentra-tions suspended in PBS: (a) 105 and (b) 104 PFU/ml, were examined. PBS was alsoexamined by the nAu sample #9 as the control.

protein) at 1302 cm−1, and N–H bending at 1506 cm−1. Ramanpeaks of SERS spectra for EMCV detected using the nAu samples#6 to #9 were all identical. Above all, the nAu sample #8 (i.e.,Dv = 90 nm) combined with EMCV resulted in the highest SERSintensity, which also corresponded well with that coupled with R6Gsolution.

In Fig. 4a and b, the presence of adenovirus at the concentra-tions of 107 and 106 PFU/ml was examined by the apAu and thenAu samples #7 to #9. No significant signals were found at bothconcentrations of adenovirus placed upon the apAu. On the otherhand, Raman shifts in the range of 1200–1650 cm−1 appeared asadenovirus was examined by the nAu samples #7 to #9. The peakassignments for these SERS spectra for adenovirus were referencedin Table 1. SERS spectrum for adenovirus placed upon the nAusample #8 (i.e., Dv = 90 nm) exhibited the highest intensity in therespective band frequencies, making it the most suitable substratefor the detection of adenovirus.

In Fig. 5a and b, the presence of influenza virus at the concen-trations of 105 and 104 PFU/ml was examined by the apAu andthe nAu samples #8 to #10. No significant signals were found atboth concentrations of influenza virus placed upon the apAu. Incontrast, Raman shifts in the range of 1100–1650 cm−1 appeared

when influenza virus was examined using the nAu samples #8 to#10. The peak assignments of these SERS spectra for influenza viruswere referenced in Table 1. The SERS spectrum for influenza virusplaced upon the nAu sample #9 (i.e., Dv = 120 nm) exhibited the

476 C.-W. Chang et al. / Sensors and Actuators B 156 (2011) 471–478

Table 1Raman shifts and their assignments for target viruses (a) EMCV, (b) adenovirus, and (c) influenza virus.

Raman shift (cm−1) Possible assignments Target virus

(a) (b) (c)

1650 Protein amide I adsorption [48]√

1615 Tyrosine [49]√

1591 Phenylalanine [49]√ √

1558 Tryptophan [49]√ √

1506 N–H bending [50]√

1491 NH3+ deformation vibrations from the viral protein coat [51]

√1480 Amide II (due to a coupling of C–N stretching and in-plane bending of the N–H group) [52]

√1360 Tryptophan [49]

√1302 Amide III (from protein) [49]

√ √1260 CH2 in-plane deformation (from lipids) [52]

√1232 Amide III (from coupling of C–N stretching and N–H bonding; it can be mixed with vibrations of side chains) [53]

√√

ht

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etserfait

Fat

1200 Amide III (from C–N stretching and N–H bending) [53]1165 Tyrosine [49]

ighest intensity and corresponded to an adequate substrate forhe detection of influenza virus.

.3. SERS mechanism for the detection of virus within theffective nAu samples

Base on the distinction of SERS spectra, these three viruses weressentially distinguishable using the selected nAu samples. Forhe comparisons, EMCV, adenovirus, or influenza virus-containingolution with an equivalent condition of PBS was respectivelyxamined using the most effective nAu samples #8 and #9. Theesults show that no signals appeared in the testing range. There-

ore, the effect of SERS generated by the nAu samples was mainlyttributed to the presence of each target virus. Moreover, as shownn Fig. 6a–c, EMCV, adenovirus, or influenza virus was possibly cap-ured or entrapped into the nAu sample #8 or #9. Owing to partly

ig. 6. FESEM micrographs of residual stained viruses trapped within the cavity of thedenovirus within the nAu sample #8, (c) influenza virus within the nAu sample #9. Inriangular nano-cavity is illustrated.

√ √

covering among Au nano-cavities over the nAu sample #8 or #9,the apparent width was relatively large (i.e., as described in Fig. S-1 and Table S-1 in Supplementary data 2), however, Dv was shallow.It tends to facilitate the entrapment of a virus within the triangulartip, as illustrated in Fig. 6a, b and d, or the attachment of virus uponthe inverted triangular edge, as shown in Fig. 6c for influenza virus.

For the cases of different virus concentrations, an excess of avirus tends to attach to the pile-up region of the overlapped Aunano-cavities, which contributes very little to SERS effect. With asuitable size and dimensions of the nAu sample #8 or #9, EMCV,adenovirus, or influenza virus was presumably entrapped withinthe nAu and noticeably detected by the SERS effect, as demon-

strated in Figs. 3–5. For each virus at two concentrations, theSERS peak intensities were roughly identical. The tendencies weresimilar among the three viruses, which indicates that SERS peakintensities are independent of the virus-containing concentrations

nAu after negative staining treatment: (a) EMCV within the nAu sample #8, (b)(d), the dimension of virus with respect to the top and side views of the inverted

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C.-W. Chang et al. / Sensors an

f each virus, whereas the effect of SERS is most probably inducedy the nAu with edges and depths of cavity, as the viruses entrapped

nto the effective nAu.In view of the respective size and dimensions of adenovirus and

nfluenza virus, the nAu samples #8 and #9 with a relatively match-ng Dv are found to be effective to induce SERS. The result indicateshat a relatively small Dv cannot include the entire target virus intohe effective SERS-active region; instead, a relatively large Dv withespect to the target virus may contain the unfilled space that influ-nces the intensity of the identified signals. For EMCV with muchmaller size and dimensions than all Dv of Au cavities, the mostffective nAu sample #8 is resulted. Based on the SERS peak assign-ents, the distinction of virus is attributed to the structure of amino

cids, which are mostly from the surface of the target virus. Theesults show that the chosen nAu samples have high potential ascharacterization tool for fast-screening virus detection for very

mall quantity of target virus.

. Conclusion

The chosen nAu samples with the variation of Dv and Dt–t

re optimized by most intense enhancement of Ipeak (R6G). Threeiruses, EMCV, adenovirus, and influenza virus, with different sizesnd dimensions were respectively assessed and compared usinghe nAu samples with the effect of SERS. The SERS peak intensi-ies from each of the viruses was significantly enhanced, which areistinguishable and corresponding to the size and dimensions ofirus with respect to Dv of the nAu. In this work, the detection limitor EMCV or adenovirus can be reduced to 106 PFU/ml, and that fornfluenza virus can be reduced to 104 PFU/ml. As well, the nAu sam-les are appropriate for a qualitative determination of virus and areelatively independent of virus concentration.

cknowledgements

This work was supported by Top 100 University Advancementnd Center for Micro/Nano Science and Technology of Nationalheng Kung University, under grant numbers D98-2740 and D98-700.

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at doi:10.1016/j.snb.2011.04.006.

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Biographies

Chia-Wei Chang is currently pursuing his PhD degree in Materials Science and Engi-neering at National Cheng-Kung University (NCKU), Tainan, Taiwan. He received hisBS and MS degrees in Chemistry and Micro-Electro-Mechanical-System Engineer-ing from NCKU in 2005 and 2006, respectively. His current research interests arefocused on nanofabrication of SERS-active substrates for fast-screening detectionplatform.

Jiunn-Der Liao is currently the Chairman and Professor of Department of Mate-rials Science and Engineering, NCKU, Tainan, Taiwan. He obtained his BS degreeat NCKU (1984), MS degrees at K.U. Leuven (1990 and 1991) in Belgium, andPhD degree at ENS Mines (1994) in France. He also worked as a researcher atUniversity of Heidelberg (1995–1996) in Germany and as an Associate Professorat Chung Yuan Christian University (CYCU) (1996–2002) in Taoyuan, Taiwan. Hiscurrent research interests are focused upon (1) Mechanics of Biomaterials, e.g.,tissue engineering, scaffold materials, cell–surface interactions, nano-indentation,mechano-transduction; (2) Plasma Chemistry and Plasma Processing, e.g., plasmageneration, plasma diagnoses, plasma physics and chemistry, metal vapor vacuumarc, plasma ion immersion; (3) Nano-fabrication and Nano-characterization, e.g.,focused ion beam based nano-fabrication, micro-contact imprinting, synchrotron-based and laboratory-based high resolution analyses.

Ai-Li Shiau is currently the Distinguished Professor at the Department of Microbiol-ogy and Immunology, College of Medicine, NCKU, Taiwan. She is also the professorin the Institute of Clinical Medicine, College of Medicine, NCKU. Dr. Shiau receivedher BS degree (1977) and MS degree (1980) from National Taiwan University,Taiwan, and in 1993 she obtained her PhD in molecular biology at the Universityof Edinburgh, UK. Her research is focused on (1) cancer biology and therapy; (2)development of vaccines and theragnostics for viral diseases; (3) molecular therapyfor autoimmune diseases.

Chih-Kai Yao is currently pursuing his PhD degree in Materials Science and Engi-neering at NCKU, Tainan, Taiwan. He received his BS and MS degrees in MaterialsScience and Engineering from NCKU in 2007 and 2009, respectively. His currentresearch interests are focused on nanofabrication of SERS-active substrates for fast-screening detection platform.