osensor based ondielectrophoretically-deposited carbon nanotubesfor detection of influenza virus H1N1†
Renu Singh,a Abhinav Sharma,a Seongkyeol Honga and Jaesung Jang*abc
The influenza virus has received extensive attention due to the recent H1N1 pandemics originating from
swine. This study reports a label-free, highly sensitive, and selective electrical immunosensor for the
detection of influenza virus H1N1 based on dielectrophoretically deposited single-walled carbon
nanotubes (SWCNTs). COOH-functionalized SWCNTs were deposited on a self-assembled monolayer of
polyelectrolyte polydiallyldimethyl-ammonium chloride (PDDA) between two gold electrodes by
dielectrophoretic and electrostatic forces, which resulted in reproducible, uniform, aligned, and
aggregation-free SWCNT channels (2–10 mm in length). Avidin was immobilized onto the PDDA–SWCNT
channels, and viral antibodies were immobilized using biotin–avidin coupling. The resistance of the
channels increased with the binding of the influenza viruses to the antibodies. These immunosensors
showed linear behavior as the virus concentration was varied from 1 to 104 PFU ml�1 along with a
detection time of 30 min. The immunosensors with a 2 mm channel length detected 1 PFU ml�1 of the
influenza virus accurately (R2 ¼ 0.99) and selectively from MS2 bacteriophages. These immunosensors
have the potential to become an important component of a point-of-care test kit that will enable a rapid
clinical diagnosis.
1. Introduction
Continuous outbreaks of highly pathogenic inuenza virusH1N1 and many cases of human infection have caused signi-cant international concern. Inuenza viruses spread easily byair transmission, and infection through the respiratory systemis quickly acquired, resulting in an urgent need to detect theinuenza virus rapidly and reliably.1 Conventional virus detec-tion methods such as diagnostic test kits, enzyme-linkedimmunosorbent assay, and virus isolation and polymerasechain reaction are either poor in specicity, low in sensitivity,time-consuming, expensive, or require a laboratory and atrained technician.2–6 Hence, it is highly desirable to develop asimple, sensitive, and inexpensive sensor to detect the virusrapidly and accurately.7
Immunosensors are analytical devices that yield measurablesignals in response to specic antigen–antibody interactions,thereby showing a quantity of the antigens in a sample. For
virus detection, many immunosensors have been developedusing electrochemical,8–15 frequency change,16–18 optical,19,20 andelectrical21–23 properties. The electrical detection technique hasseveral advantages such as simple and convenient measure-ments, which enablesminiaturized and inexpensive biosensors.Therefore, it has the potential to revolutionize traditionallaboratory techniques for virus detection.
Carbon nanotubes (CNTs) have proven to be a promisingplatform for ultrasensitive and miniaturized immunosensorsfor disease diagnosis because of superior mechanical andconductive properties such as high actuating stresses, lowdriving voltages, and high energy densities.24 However, thedevelopment of effective functionalization methods that canintroduce homogeneous surface functional groups and alsocause less or no structural damage to CNTs remains a majorchallenge.25 Polydiallyldimethylammonium chloride (PDDA)–CNTs have been used in biosensing applications due to theirgood lm-forming ability and susceptibility to chemical modi-cations.26,27 The strong adsorption of the positively chargedPDDA on CNTs may result from the p–p interaction betweenPDDA and the basal plane of graphite of CNTs. This non-cova-lent polyelectrolyte functionalization leads to homogeneoussurface functional groups on CNTs and also preserves theintrinsic properties of CNTs without damaging their perfectsurface structure.25 The uniformly distributed CNTs realizedonto PDDA can offer much higher surface areas and electro-catalytic activity for virus detection.
Dielectrophoresis (DEP) has been considered one of thereliable, inexpensive, and efficient CNT deposition techniques,and it involves depositing solution-dispersed CNTs betweenelectrodes and aligning them by applying AC electric elds.Although chemical vapor deposition (CVD) is a commonmethod for the direct growth of CNTs or a network of CNTs, andCVD-grown CNTs have shown the best performance, the DEPtechnique is generally much simpler and more cost-effective,and does not require specialized materials and high tempera-ture for the growth. Moreover, the alignment and density of thedeposited CNTs can be controlled by the AC frequency and theconcentration of CNTs.28–30
Here, we present a label-free and highly sensitive electricalimmunosensor to detect inuenza virus H1N1 using single-walled carbon nanotubes (SWCNTs) deposited on a PDDA self-assembled monolayer (SAM) by DEP. Avidin was immobilizedon the SWCNTs, and viral antibodies were then immobilizedusing biotin–avidin coupling. The resistance shi of theSWCNT channels was measured as the concentration of thevirus was varied from 1 to 104 PFU ml�1, and the selectivity ofthe immunosensor was also tested against high-concentrationMS2 bacteriophages.
Previous DEP-deposited CNT sensors constructed for virusdetection were not optimally designed with respect to uniformand aligned SWCNTs. Garcıa-Aljaro et al.22 developed CNT-basedimmunosensors for the detection of bacteriophage T7 yieldingapproximately 10 MU resistance without a SAM, and it wasnecessary to anneal the immunosensors in order to have goodcontact. Lee et al.30 created CNT-based inuenza virus immuno-sensors using the layer-by-layer assembly method, but thisculminated in a random, broken, and dense network of SWCNTswhere the tube–tube junction may limit the charge transport. Inthe current study, the DEP technique was extended in conjunc-tion with a PDDA SAM and the piranha treatment, which intro-duced abundant surface hydroxyl groups (–OH) via thehydroxylation process. That is, the SWCNTs were deposited byboth dielectrophoretic and electrostatic forces that were exertedbetween the positively charged –NH2 groups from PDDA and thenegatively charged (–COOH– functionalized) SWCNTs, resultingin reproducible, uniform, and aligned SWCNT deposition.
2. Materials and methods2.1. Biomolecules and chemical reagents
CNTs (90% SWCNTs, diameter: 1–2 nm, length: 5–30 mm,COOH content: approximately 2.75 wt%) were purchased fromM K Impex Corp. (Canada). PDDA (20 wt%,Mw ¼ 200–350 kDa),avidin (A9275), bovine serum albumin (BSA) (A2153), N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide hydrochloride(EDC) (03449), N-hydroxysuccinimide (NHS) (130672), glutaral-dehyde (G765), osmium tetroxide (75632), and isoamylacetate(112674) were obtained from Sigma-Aldrich (USA). Dime-thylformamide (DMF) (98%, D1021) and phosphate bufferedsaline (PBS) (1�, pH 7.4) containing 0.1% Tween 20 (P2006)were purchased from Biosesang Inc. (South Korea). PBS (10�,pH 7.4, 70011-044) was purchased from Invitrogen Life Tech-nologies (USA). Biotin-conjugated mouse anti-inuenza A
Analyst
monoclonal antibody (bs-1261M) was purchased from GentaurMolecular Products (USA). Inuenza virus H1N1 (KBPV-VR-33)was procured from the Bank of Pathogenic Viruses (SouthKorea). Bacteriophage MS2 (ATCC® 15597-B1™, 1 � 109 PFUml�1) was procured from Koram Biogen Corp. (South Korea).Deionized water (dH2O) (resistance: approximately 18.2 MU)from the Millipore Milli-Q water purication system wasutilized for preparation of the desired aqueous solutions(molecular biology grade). All the solutions and glassware wereautoclaved prior to being used.
2.2. Microelectrode fabrication
A standard 6-inch silicon (Si) wafer with thermally grown silicondioxide (SiO2, thickness: 500 nm) was cleaned with a piranhasolution (H2SO4 : H2O2 ¼ 2 : 1) at room temperature for 15 min,rinsed thoroughly with a copious amount of dH2O, and driedwith a nitrogen gas stream. Two electrodes for the source andthe drain were patterned using photolithography, and thenchromium (thickness: 20 nm) and gold (thickness: 200 nm)were sequentially deposited with an electron beam evaporatoronto the Si/SiO2 substrate. The source and the drain electrodeswere tapered as shown in Fig. 1 to maximize the electric eld atthe edges and to increase the amount of uniformly distributedSWCNT connections during the DEP assembly. The gaps, whichare referred to as channel lengths, between these two electrodeswere 2 mm, 5 mm, and 10 mm, and the width of the facing elec-trodes was 100 mm. The silicon wafer was then diced into chips(10 mm � 10 mm). The chips were cleaned in piranha solutionfollowed by rinsing with dH2O. These chips were then driedwith nitrogen gas and either used immediately or stored undervacuum in a desiccator.
2.3. DEP assembly of SWCNT channels
Firstly, a SAM of PDDA was made as a precursor layer bypipetting onto the channel area for the charge enhancementand incubating for 15 min at 25 �C prior to the DEP depositionstep. A SWCNT suspension was prepared by sonicating theSWCNTs in DMF (10 mg ml�1) in a water bath for 90 min fol-lowed by centrifugation at 5000 rpm for 1 h, and the superna-tant liquid was discarded. The remaining SWCNT suspension isviable for at least 1 month, and can be used when needed aersonication for 10 min. The DEP assembly of the SWCNTs wasperformed by dropping 20 ml of the SWCNT suspension onto thechannel area and applying 10 V (peak-to-peak value) at 200 kHzfor a few second. Finally, a thin lm of uniformly distributed,parallelly aligned, and aggregation-free SWCNTs was formed asa channel between the source and the drain electrodes. Thechips were rinsed with DMF and then dH2O, followed by mildN2 drying.
2.4. Immunofunctionalization and virus attachment
Firstly, 90 ml of avidin (1 mg ml�1) was mixed with 5 ml of EDC(15 mM) and 5 ml of NHS (30 mM), and the mixture was incu-bated for 2 h at 25 �C. Avidin was then immobilized onto theSWCNTs by depositing 10 ml of the mixture and incubating for30 min. The SWCNT channels were washed with dH2O and
Fig. 1 Schematic illustration of the single walled carbon nanotube-based immunosensor for H1N1 virus detection. The inset shows an opticalimage of dielectrophoretically deposited SWCNTs onto a PDDA SAM.
Paper Analyst
Publ
ishe
d on
02
Sept
embe
r 20
14. D
ownl
oade
d by
Uls
an N
atio
nal I
nstit
ute
of S
cien
ce &
Tec
hnol
ogy
(UN
IST
) on
18/
09/2
014
03:2
0:31
. View Article Online
subjected to 10 ml of biotinylated monoclonal antibodies (10 mgml�1) specic to inuenza virus H1N1 in an incubator at 37 �Cfor 2 h. Aer washing with PBS (1�, pH 7.4), the SWCNTchannels were incubated for 30 min in BSA (1 mg ml�1) toprevent nonspecic binding followed by washing with PBS (1�,pH 7.4) containing 0.1% Tween 20 to remove loosely attachedPDDA and SWCNTs. The immunosensors were then incubatedin 10 ml of various concentrations of the viruses in PBS (1�, pH7.4) for 30 min followed by rinsing, air-drying, and resistancemeasurement. The inuenza viruses were inactivated usingUltraviolet Crosslinker (CL-1000 UV crosslinker, UVP, Upland,CA, USA) prior to being used. A schematic of the developedSWCNT immunosensor and its test setup are shown in Fig. 1.The selectivity of the immunosensor was assessed by incubatingthe immunosensor with highly concentrated MS2 bacterio-phages andmeasuring the resistance change due to the bindingto the SWCNT channels.
2.5. Electrical measurements
The electrical measurement of the immunosensors was con-ducted by collecting the current–voltage (I–V) data from theimmunosensors. The immunosensor response due to the virusattachment was measured as the normalized increase in resis-tance (NIR), [DR ¼ (RVirus � R)]/R, where RVirus is the resistanceof the immunosensor aer the exposure to the viruses and R isthe immunosensor resistance aer antibody immobilization.The resistance of the immunosensors was measured aer everyfunctionalization step. The applied voltage was varied from�1.0 to +1.0 V, and current was recorded using a Source Meter®(2400, Keithley, Cleveland, OH).
2.6. Morphological and structural characterizations
Atomic force microscopy (AFM) and scanning electron micro-copy (SEM) were used for morphological characterizations ofthe immunosensors. AFM images were obtained using a
Dimension AFM 3100 (Veeco, USA). The chemical xation wasperformed to image the inuenza viruses captured on theSWCNT channel. First, the immunosensors were immersed in2.5% glutaraldehyde for 2 h and washed in 1� PBS buffer for10 min. They were then immersed in 1% osmium tetroxide indistilled water for 2 h and washed again in 1� PBS buffer twicefor 10 min each. The immunosensors were then placed in 25,50, 70, 90, 100, and 100% ethanol sequentially for 10 min eachfor dehydration. They were sequentially treated with a mixtureof isoamylacetate and ethanol at ratios of 1 : 3, 1 : 1, 1 : 0, and1 : 0 for 10 min each for inltration. The immunosensors werethen placed in a critical point dryer (SPI Supplies) at 35 �C at1200 psi and coated with a thin layer of platinum for imagingwith a scanning electronmicroscope (s-4800, Hitachi). A Fouriertransform infrared (FTIR) Varian 4100 (Agilent, USA) spec-trometer was used for structural characterization without anyfurther treatment of the samples (ESI Fig. 1†).
3. Results and discussion3.1. Morphological characterization – SEM
Fig. 2a and b show SEM images of the uniformly distributed,aligned, and aggregation-free SWCNTs aer the PDDA SAM andDEP assembly, demonstrating that the majority of the indi-vidual SWCNTs were reasonably aligned in parallel to the elec-trodes along with a few misaligned and tilted nanotubes. Thesealigned SWCNTs contrast with a non-aligned and densenetwork of SWCNTs made by sedimentation (Fig. 2c). There wasreproducibility in the deposition due to the PDDA SAM. Thisreproducible formation of uniform and aligned SWCNTs can beattributed to the combined effects of the DEP assembly and theSAM treatment. From these images, the average linear densityof the SWCNT arrays were estimated to be approximately 8SWCNT per mm, which may strongly inuence the performanceof the fabricated SWCNT immunosensor.31 Aer the avidin andbiotinylated antibody immobilization, uniform morphology
Fig. 2 SEM images of (a) PDDA–SWCNTs, (b) PDDA–SWCNTs at higher magnification, (c) SWCNTs deposited by sedimentation, (d) H1N1antibody immobilized on PDDA–SWCNTs, and (e) H1N1 antibody immobilized on PDDA–SWCNTs after capturing a single influenza virus.
Analyst Paper
Publ
ishe
d on
02
Sept
embe
r 20
14. D
ownl
oade
d by
Uls
an N
atio
nal I
nstit
ute
of S
cien
ce &
Tec
hnol
ogy
(UN
IST
) on
18/
09/2
014
03:2
0:31
. View Article Online
appeared due to the interaction of avidin with the carboxylfunctionalized SWCNT surfaces and biotinylated antibodies,revealing the successful immobilization of avidin and bio-tinylated antibodies onto the SWCNT surfaces (Fig. 2d). Fig. 2eshows a single inuenza virus captured by the antibodies thatwere distributed on the SWCNTs, with several virus aggregatesobserved as well. The diameter of a single inuenza virus hasbeen reported to be 80–120 nm,32 and previous studies haveshown that SWCNTs functionalized with specic antibodieswere able to capture viruses.23
3.2. Morphological characterization – AFM
Fig. 3 illustrates the schematic of the SWCNT deposition aerthe PDDA SAM formation along with the AFM micrographs of(a) PDDA, (b) PDDA–SWCNTs, and (c) PDDA–SWCNT–avidin-
Fig. 3 Schematic of PDDA and SWCNT interaction along with represent(c) H1N1 antibody immobilized on PDDA–SWCNTs.
Analyst
biotinylated antibody. An initial monolayer of adsorbed PDDAwas relatively smooth with surface roughness of 1.27 nm(Fig. 3a). Fig. 3b exhibits a layer of the DEP-deposited SWCNTswith an increased surface roughness of 80.2 nm. Fig. 3c showsan AFM image aer the antibody was covalently linked onto theSWCNTs using avidin–biotin coupling. The spiky nanotubefeatures disappeared, and a globular surface generally remi-niscent of thin antibody coatings was seen.33 It is clearly visiblefrom Fig. 3c that a remarkable decrease in surface roughness(15.4 nm) was the result of antibody attachment.
3.3. Resistance measurements and incubation timedependency
Fig. 4a shows the resistance measurement of the immuno-sensors aer PDDA–SWCNT deposition for various channel
ative AFM (tapping mode) images of (a) PDDA, (b) PDDA–SWCNTs, and
lengths of 2, 5, and 10 mm. The resistance was found to linearlyincrease with the channel length, demonstrating a relationshipof R ¼ 9.97 � L + 10.8, where R and L are expressed in kU andmm, respectively. The measurements also showed good repeat-ability, where the relative variations of the measurements wereapproximately 3%. Garcıa-Aljaro et al.22 obtained approximately10 MU resistance for DEP-deposited SWCNTs, and the chipswere annealed in order to have good contact. We used PDDA asa precursor for the charge enhancement and achieved 1–100 kUfor a thin lm of uniformly distributed, aligned, and aggrega-tion-free SWCNTs at 2–10 mm channel lengths. I–V character-istics for the SWCNT immunosensors are shown in the ESIFig. 2.†
The next experiment was focused on determining therequired incubation time for maximum resistance changebecause the resistance can increase with the attachment ofanalytes. To this end, the SWCNT immunosensors were incu-bated in 104 PFU ml�1 of inuenza virus at room temperature,and NIR was measured with incubation time. Fig. 4b shows thatthe NIR increased with incubation time up to 30 min, attaininga plateau value of 1.5 with respect to the initial resistance.Accordingly, 30 min incubation time was used in the subse-quent experiments.
3.4. Resistance measurements aer each functionalization
The resistance measurements of the SWCNT immunosensorswere performed aer each step of the functionalization: PDDA–SWCNT, avidin, antibodies, and H1N1 virus (102 PFU ml�1)(Fig. 5a). The antibodies immobilized on the avidin did notchange the resistance as much as avidin did on the PDDA–SWCNTs. According to the measurements, there must be anelectrostatic and/or structural change to induce an observableresistance shi when avidin is immobilized on the SWCNTsurfaces.34 This suggests that the resistance changes observedaer the immobilization of inuenza virus were probably due totheir charge carrier donating/accepting property and/or thestructural change of the SWCNTs due to the huge structure ofthe viruses. That is, the attachment of the viruses signicantly
Fig. 4 (a) Resistance of the PDDA–SWCNTs with various channel lengdependent response of the PDDA–SWCNT immunosensors after exposu100 mm. The error bars indicate standard deviations from 4 sets of meas
inuenced the electrical properties of the SWCNTs so thatresistivity increased.
3.5. Selectivity and sensitivity
To investigate the selectivity of the SWCNT immunosensors,MS2 bacteriophages with high concentration (109 PFU ml�1)were used with 2, 5, and 10 mm channel length chips (Fig. 5b).The averages and standard deviations were determined from 4sets of immunoassays. The NIRs for MS2 bacteriophages were0.093, 0.148, and 0.188 for 2, 5 and 10 mm channel lengths,respectively, while the NIRs for inuenza viruses (102 PFUml�1)were 0.899, 1.79, and 4.95 for 2, 5, and 10 mm channel lengths,respectively. The NIRs due to the MS2 bacteriophage attach-ment were 10–4% of those due to the inuenza virus attach-ment even with the much higher concentration of MS2bacteriophages. This test showed that the SWCNT immuno-sensors were highly specic to inuenza viruses against MS2bacteriophages.
Fig. 6 shows the calibration plots of the immunosensors asthe inuenza virus concentration was varied from 1 to 104 PFUml�1. The higher the inuenza virus concentration, the largerthe resistance shis due to more inuenza viruses adsorbedon the channel surface. The immunosensor response, NIR,was a linear function of the logarithm of viral concentrationsbetween 1 and 104 PFU ml�1 (R2 ¼ 0.99, 0.94, and 0.89 for 2, 5,and 10 mm channel lengths, respectively). The immunosensorlinearity or accuracy decreased as the channel lengthincreased. It was reported that longer channel chips showedlarger measurement variations due to the attachment of othermolecules in a virus solution on open binding sites.30
However, the longer SWCNT channel chips showed a largerincrease in the NIR with the increasing virus concentration,and they have larger sensing areas, both of which are favorablefor a biosensing platform.
According to the measurements, the shortest channelimmunosensor was the most precise and accurate. The NIR forhighly concentrated MS2 bacteriophages was 0.093 while theNIR was 0.17 for 1 PFU ml�1 of the inuenza viruses.
ths of 2, 5, and 10 mm and constant width of 100 mm, and (b) time-re to H1N1 virus (104 PFUml�1) with a channel length of 2 mm and widthurements.
Fig. 5 (a) Surface-binding studies of PDDA–SWCNT, avidin, biotinylated antibody, and H1N1 virus (102 PFUml�1) for various channel lengths of 2,5, and 10 mmwith a constant width of 100 mm. (b) Selectivity tests of the SWCNT immunosensor against MS2 bacteriophages (109 PFUml�1). Theerror bars indicate standard deviations from 4 sets of measurements.
Fig. 6 Calibration plots of the SWCNT immunosensors showing thatNIRs increased with the logarithm of the virus concentrations, wherethe channel length was 2, 5, and 10 mm. The error bars indicatestandard deviations of 4 sets of measurements. X and Y represent thevirus concentration and the NIR, respectively. The NIR was 0.05 forPBS buffer (1�, pH 7.4).
Analyst Paper
Publ
ishe
d on
02
Sept
embe
r 20
14. D
ownl
oade
d by
Uls
an N
atio
nal I
nstit
ute
of S
cien
ce &
Tec
hnol
ogy
(UN
IST
) on
18/
09/2
014
03:2
0:31
. View Article Online
Moreover, the NIR for PBS buffer (1�, pH 7.4) was 0.05,demonstrating that this immunosensor can detect 1 PFU ml�1
of the inuenza virus selectively from MS2 bacteriophages.This is highly sensitive considering that the range of inuenzaviral particles found in infected swine nasal samples is 103 to105 TCID50 per ml (50% tissue culture infective dose), and alimit of detection of approximately 102 TCID50 per ml wasrecently reported.30 Garcıa-Aljaro et al.22 demonstrated adetection limit of 103 PFU ml�1 for bacteriophage T7 usingSWCNT-based immunosensors. Enhanced limit of detectionin the present study may be attributed to uniformly distrib-uted, aligned, and aggregation-free SWCNTs. The reproduc-ible generation of uniform and aligned SWCNTs on a PDDASAM by dielectrophoretic and electrostatic forces has impor-tant implications for the large-scale fabrication of SWCNT-based biosensors.
Analyst
4. Conclusions
In this study, we demonstrated a label-free, highly sensitive, andselective electrical immunosensor to detect whole inuenzaviruses using dielectrophoretically deposited SWCNTs. Thereproducible formation of a uniform, aligned, and aggregation-free SWCNT thin lm between the source and the drain elec-trodes was observed on a PDDA SAM by applying both dielec-trophoretic and electrostatic forces, which showed anadvantage over applying either as in previous studies. Thisimmunosensor showed linear behavior from 1 to 104 PFU ml�1,with a detection time of 30 min. The shortest channel (2 mm inlength) immunosensor detected 1 PFU ml�1 of the inuenzavirus selectively from MS2 bacteriophages. This SWCNT-basedelectrical immunosensor has potential applications in a point-of-care test kit for rapid and simple clinical diagnosis or acomponent of a portable lab-on-a-chip system.
Acknowledgements
This research was supported by the Basic Science ResearchProgram through the National Research Foundation of Korea(NRF) funded by the Ministry of Education, Science and Tech-nology (2012R1A2A2A01012528).
References
1 L. T. Kao, L. Shankar, T. G. Kang, G. Zhang, G. K. I. Taym,S. R. M. Rafei and C. W. H. Lee, Biosens. Bioelectron., 2011,26, 2006–2011.
2 B. Leuwerke, P. Kitikoon, R. Evans and E. Thacker, J. Vet.Diagn. Invest., 2008, 20, 426–432.
3 Q. G. He, S. Velumani, Q. Y. Du, C. W. Lim, F. K. Ng, R. Donisand J. Kwang, Clin. Vaccine Immunol., 2007, 14, 617–623.
4 M. B. Townsend, E. D. Dawson, M. Mehlmann, J. A. Smagala,D. M. Dankbar, C. L. Moore, C. B. Smith, N. J. Cox andR. D. Kuchta, J. Clin. Microbiol., 2006, 44, 2863–2871.
5 Y. Amano and Q. Cheng, Anal. Bioanal. Chem., 2005, 381,156–164.
