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Applied Surface Science 257 (2011) 7090–7095

Contents lists available at ScienceDirect

Applied Surface Science

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

haracterization of immobilization methods of antiviral antibodies in serum forlectrochemical biosensors

ran Quang Huya,b,∗, Nguyen Thi Hong Hanha, Pham Van Chunga, Dang Duc Anha,han Thi Ngaa, Mai Anh Tuanb

National Institute of Hygiene and Epidemiology (NIHE), N◦1 Yersin St., Hanoi, Viet NamInternational Training Institute for Materials Science (ITIMS), Hanoi University of Science and Technology (HUST), N◦1 Dai Co Viet, Hanoi, Viet Nam

r t i c l e i n f o

rticle history:eceived 21 February 2011eceived in revised form 8 March 2011ccepted 8 March 2011vailable online 16 March 2011

a b s t r a c t

In this paper, we describes different methods to immobilize Japanese encephalitis virus (JEV) antibodiesin human serum onto the interdigitated surface of a microelectrode sensor for optimizing electrochem-ical detection: (1) direct covalent binding to the silanized surface, (2) binding to the silanized surfacevia a cross-linker of glutaraldehyde (GA), (3) binding to glutaraldehyde/silanized surface via goat anti-

eywords:erum antibodies immobilizationiointerfacesrotein Alectrochemical biosensors

human IgG polyclonal antibody and (4) binding to glutaraldehyde/silanized surface via protein A (PrA).Field emission scanning electron microscopy, Fourier transform infrared spectrometry, and fluorescencemicroscopy are used to verify the characteristics of antibodies on the interdigitated surface after the serumantibodies immobilization. The analyzed results indicate that the use of protein A is an effective choicefor immobilization and orientation of antibodies in serum for electrochemical biosensors. This studyprovides an advantageous immobilization method of serum containing antiviral antibodies to develop

rs for

reliminary pathogenic screening electrochemical biosenso

. Introduction

Recent years have seen increasing many emerging infectiousiseases caused by viruses. They are liable to outbreak highlypidemic diseases and endanger public health. Among them,apanese encephalitis virus (JEV) is also a leading cause of child-ood encephalitis in Asia; it has a high mortality and has high risk

or subsequent infections. It is the most significant mosquito-borneiral encephalitis in several Asian countries. Twenty-five countriesre at risk from Japanese encephalitis (JE), and approximately threeillion people, including more than 700 million children underhe age of 15, live in risk areas [1,2]. Therefore, the developmentf rapid diagnostic tests always plays a crucial role for the con-rol and prevention of JE outbreaks as well as other outbreaks.everal conventional diagnostic methods have been developed toetect viral infection; these tests involve virus isolation, detection

f virus antigen, and the presence of antiviral specific antibodiesn serum and other body fluids, enzyme-linked immunosorbentssay (ELISA) and other molecular techniques (PCR) [3,4]. How-ver, these developed diagnosis techniques require a pre-treatment

∗ Corresponding author at: National Institute of Hygiene and EpidemiologyNIHE), N◦1 Yersin St., Hanoi, Viet Nam. Tel.: +84 4 39 71 54 34;ax: +84 4 38 21 08 53.

E-mail addresses: huytq@nihe.org.vn (T.Q. Huy), tuanma-itims@mail.hut.edu.vnM.A. Tuan).

169-4332/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.apsusc.2011.03.051

preliminary screening of viruses in clinical samples from outbreaks.© 2011 Elsevier B.V. All rights reserved.

sample, biological products, and time-consuming analysis to yieldan answer. Nowadays, biosensors (DNA sensor and antibody-basedbiosensor) have been considered as an alternative way to com-pensate for conventional diagnostic methods [5–10]. Nevertheless,rapid and direct detection of viruses using DNA sensor is limitedby the complexity of extraction and denatures of viral DNA orRNA. Consequently, the development of antibody-based biosen-sors has become a topic area of research [11,12]. A major problemin the development of antibody-based biosensors is to overcomethe complexity of binding antibody to the sensor surface. Severalimmobilizing methods of antibody onto sensor surfaces were pro-posed such as physical adsorption, entrapment in a gel, covalentbinding, cross-linking, electrochemical polymerization and con-ducting polymers [13–16]. In spite of these studies, the strategiesof the proper orientation and density of serum antibodies immobi-lized on the sensor surface in antibody-based biosensors were notyet fulfilled. These attempts almost applied for immobilizing puri-fied antibodies in biosensor applications [17,18]. In outbreaks, it isnot easy to have purified specific antibodies against the pathogen.Therefore, human serum containing antiviral specific antibodiescan be an effective choice for the development of antibody-basedbiosensors for preliminary pathogenic screening. Hence, a practical

comparison between different serum antibodies immobilizationstrategies is necessary.

In this paper, we present a comparative study of different meth-ods to immobilize human serum containing antibodies against

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EV onto the interdigitated surface for optimizing electrochemi-al detection. Among the studied methods, the use of protein Aevealed as the best choice for this purpose. The advantage of theethod using protein A makes it ideal for the development of elec-

rochemical biosensors for rapid detection of the viruses in clinicalamples directly from outbreaks.

. Materials and methods

.1. Reagents and electrochemical sensors

Human serum containing antibodies against JEV; inactivatedEV (total antigen of JEV) were provided by the National Institute

f Hygiene and Epidemiology (NIHE) of Vietnam. These biologicalroducts were standardized for MAC-ELISA test kits (certified byinistry of Health, Vietnam) for JEV diagnosis [19].Goat anti-human IgG polyclonal antibody (anti-HIgG); FITC-

onjugated mouse anti-human IgG antibody (FITC-Ab), bovinelbumin serum (BSA), Protein A (PrA), 3-aminopropyl-triethoxy-ilane (APTES), and glutaraldehyde (GA) were purchased fromigma, USA. All other chemicals were of analytical grade.

The electrochemical biosensor based on interdigitated micro-lectrodes was designed and fabricated at the Hanoi Universityf Science and Technology (HUST) [20]. The dual electrode wasabricated using a conventional photolithographic method with anger-width of 10 �m and a gap size of 10 �m. The fingers of inter-igitated electrodes were fabricated by sputtering 10 nm Ti and

00 nm Pt on a layer of silicon dioxide (SiO2) with thickness ofpproximately 100 nm thermally grown on top of a silicon wafer.he configuration of this sensor and a diagram of measuring prin-iple are displayed in Fig. 1.

ig. 1. The configuration of electrochemical sensors (A, B) and the diagram of mea-urement principle (C).

ence 257 (2011) 7090–7095 7091

2.2. Immobilization of human serum on the sensor surface

Sensors were immersed in KOH/MeOH (1:1) solution for 30 minfor surface cleaning and functionalization. These sensors were thenrinsed in de-ionized (DI) water and nitrogen-dried. The silanizationprocess was conducted in 5% APTES/MeOH for 1 h to create aminogroups (NH2), allowing binding between the antibodies and inter-digitated surface [see illustration (1)]. A drop of acetic acid wasadded during the silanization to orientate amino groups outwardfrom the interdigitated surface [21]. Sensors were then washedthree times in DI water, nitrogen-dried, and annealed thermally at120 ◦C for 6–8 min to completely remove excess water moleculeson the surface. The silanized sensors were kept in a dry box at roomtemperature before use [22,23].

(1)

In our present study, four methods were investigated to deter-mine an optimal solution for immobilizing antiviral antibodies inserum onto the surface of interdigitated electrodes.

2.3. APTES-serum (APTES-serum)

Silanized sensor was incubated in 0.1, 0.25, 0.5, 0.75 and 1 mg/mlserum containing antibodies against JEV for 1 h, respectively. Theunsaturated and non-specific binding sites on the surface wereblocked with 2% BSA/PBS for 30 min, washed with PBS (pH 7.0),and air-dried [see illustration (2)].

2.4. APTES-glutaraldehyde-serum (APTES-GA-serum)

The silanized sensor was dipped in 5% glutaraldehyde solutionfor 30 min. The sensor was washed three times in DI water, followedby incubation in 0.1, 0.25, 0.5, 0.75 and 1 mg/ml serum contain-ing antibodies against JEV for 1 h, respectively. The unsaturatedand non-specific binding sites on the surface were blocked with2% BSA/PBS for 30 min, washed with PBS (pH 7.0), and air-dried[see illustration (3)].

2.5. APTES-glutaraldehyde-anti-human IgG-serum(APTES-GA-antiHIgG-serum)

The silanized sensor was dipped in 5% glutaraldehyde for 30 minand washed in DI water three times. Next, 1 mg/ml goat anti-humanIgG polyclonal antibody was added to the surface for 30 min, thenwashed in PBS (pH 7.0) followed by incubation in 0.1, 0.25, 0.5,0.75 and 1 mg/ml serum containing antibodies against JEV for 1 h,respectively. The unsaturated and non-specific binding sites on thesurface were blocked with 2% BSA/PBS for 30 min, washed in PBS(pH 7.0), and air-dried [see illustration (4)].

2.6. APTES-glutaraldehyde-protein A-serum(APTES-GA-PrA-serum)

The silanized sensor was dipped in 5% glutaraldehyde for 30 min

and washed in DI water three times. Next, 5 �l of PrA solution [1 mgPrA/1 ml PBS (pH 7.0)] was added to the surface for 30 min, thenwashed in PBS (pH 7.0) followed by incubation in 0.1, 0.25, 0.5,0.75 and 1 mg/ml serum containing antibodies against JEV for 1 h,respectively. The unsaturated and non-specific binding sites on the

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urface were blocked with 2% BSA/PBS for 30 min, washed with PBSpH 7.0), and air-dried [see illustration (5)].

With different methods of serum antibodies immobilization, theeneral morphology of the interdigitated surfaces was observedy a field emission scanning electron microscope (FESEM, S-4800,itachi, Japan), the effective bindings of antibodies in serum onto

he interdigitated surface were justified by Fourier transformnfrared spectroscopy (FTIR, Nicolet 6700 FTIR machine, Thermo,SA), and the good orientation and immobilization of antibodiesn the interdigitated surface were investigated by fluorescenceicroscopy (Elipse 90i, Nikon, Japan). Finally, the electrochemi-

al detection of these biosensors was tested by a SR 830 Lock-Inmplifier with AC reference signals (10 kHz, 100 mV) at room tem-erature.

. Results and discussion

.1. Characterization of serum antibodies immobilization

A series of serum dilution was used for immobilizing antibod-es on the interdigitated surface using different methods, and thexperimented results revealed that 1 mg/ml serum was an optimaloncentration for comparison.

Fig. 2A shows a FESEM image of the interdigitated electrode sur-ace after silanization with APTES, revealing a smooth and uniformurface. This may lead to a good distribution of free NH2 groupsway from the surface in order to link with other chemicals and/oriomolecules [24]. After serum antibodies immobilization, theorphological changes were recognized for each method. Fig. 2B

hows only a few faint traces of serum can be observed on the sur-ace with the APTES-serum method. The lesser traces of serum fromhis method indicate that most of antibodies might be washed awayuring the surface rising process. In contrast, Fig. 2C shows thatsing APTES-GA-serum method, a layer of serum still remained sig-ificantly on the interdigitated surface after washing steps. In this

ethod, the GA could act as a cross-linker between the silane layer

nd antibodies leading to the tightly binding of antibodies on thenterdigitated surface [16,18,25]. In order to improve the binding ofntiviral antibody from serum on the interdigitated surface, proteinand antiHIgG was used as intermediate linkers. Fig. 2D a complex

ence 257 (2011) 7090–7095

(2)

(3)

(4)

(5)

of dense material layer on the surface using the method of APTES-GA-PrA-serum, this image is similar to the result obtained by usingantiHIgG alternative to PrA. Chih et al. reported that using PrA is agood way of immobilizing antibodies onto the silica-based surface[9], and also another paper reported that 30–70% higher antibodiescould be bound on silica-tagged PrA than on uncoated silica sur-face [26]. Hence, these techniques show promising potential forsensitive improvement of antibody-based biosensors.

FTIR spectroscopy was then used to characterize the presenceof specific chemical groups as well as the effective binding ofantibodies on the silanized interdigitated surface after serum anti-bodies immobilizations and washing steps. In our case, we focusedon the FTIR absorption spectra of protein (serum) in the rangeof 1800–1300 cm−1. The results showed the absorption energypeaks of proteins remaining on the silanized interdigitated surfacefor each method. Previous studies have reported that the proteinrepeat units gave rise to good characteristic infrared absorptionbands, namely, amide A, B, and I–VII. Among these absorptionbonds, the amide I and II bands are the most prominent vibra-tional bands of the protein backbone [27–29]. Fig. 3 shows that thepeaks around 1640 cm−1 are characteristic of amide I band withC O stretching frequency, whereas the peaks around 1550 cm−1

are characteristic of amide II band with C–N stretching and N–Hbending [27]. The peak of the C O vibration mode is also found at1420 cm−1 obtained on the samples after serum antibodies immo-bilization (Fig. 3b–e) [28,29], but it is not found on the interdigitatedsurface treated with APTES only. In the contrary, the vibrationmode of NH2 can be seen at 1590 cm−1 on this surface (Fig. 3a)[30]. In these methods, the highly characteristic peaks of antibod-ies obtained using APTES-GA-PrA-serum that assigned to the mosteffective binding of proteins on the interdigitated surface (Fig. 3e),then APTES-GA-antiHIgG-serum (Fig. 3c) and APTES-GA-serummethods, respectively (Fig. 3d). The weakly characteristic peaks of

antibodies were obtained by using the APTES-serum method thatassigned to a poor binding of proteins on the interdigitated surfaceafter washing steps (Fig. 3b).

In order to identify a good orientation and immobilization ofantibodies on the interdigitated surface, fluorescence microscopy

T.Q. Huy et al. / Applied Surface Science 257 (2011) 7090–7095 7093

Fig. 2. FESEM images of interdigitated surfaces of the electrochemical sensor treated with different methods of serum antibodies immobilization: (A) silanized interdigitatedsurface before serum antibodies immobilization; (B) treated with APTES-serum method; (C) treated with APTES-GA serum method; (D) treated with an intermediate linker(APTES-GA-PrA-serum method).

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ig. 3. FTIR spectrum of interdigitated surfaces before and after serum antibodies ierum; (d) APTES-GA-antiHIgG-serum; (e) APTES-GA-PrA-serum.

as used to compare the results from different methods. Thesexperiments were performed on microscope slides. The serum anti-odies immobilization protocols were similar to the process usedor electrochemical biosensors. In human serum, IgG constitutes5% of the total immunoglobulin in human serum [31], the mainactor for the detection of antigens in immunosensor applications.y using FITC conjugated mouse antihuman IgG antibody to reactith IgG molecules immobilized on the silanized surface, the differ-

nt densities of green fluorescent spots were seen on the surface for

ach method. Fig. 4A shows very few spots on the slide using APTES-erum method, means that not many IgG molecules still remain onhis surface. This is an evidence again for non-specific binding ofntibodies onto the silanized interdigitated surface without usingntermediate linkers. Whereas, Fig. 4B shows the denser fluores-

ilization using different methods: (a) APTES only; (b) APTES-serum; (c) APTES-GA-

cent spots obtained using the APTES-GA-serum method, but someof spots seemed to be agglomerated. In this method, GA plays arole as a cross linker, leading to the tightly binding of many ran-dom antibodies on the surface [29]. In the methods using antiHIgG,fluorescent spots are more separately distributed over the side sur-faces (Fig. 4C). Here, antiHIgG binds selectively with almost IgGmolecules in serum and orients them randomly on the surface[32]. In another attempt, protein A was used instead of antiHIgGto immobilize antibodies in serum on the silanized surface. PrA

was proved to bind specifically to the Fc region of immunoglobulinmolecules without disturbing the binding of antigen [33]. Interest-ingly, a large number of fluorescent spots could be seen evenly overthe surface (Fig. 4D). This is in a good agreement with the previouspublications reported that PrA could select and orient the antigen

7094 T.Q. Huy et al. / Applied Surface Science 257 (2011) 7090–7095

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ig. 4. Fluorescence images of JEV antibodies immobilized on microscope slides userum and (D) APTES-GA-PrA-serum.

inding sites outwards from the surface, leading to the improve-ent of the binding possibility with antigens [26,34,35], and this

hows the potential for a good electrochemical detection of theiosensor.

.2. Detection of JEV antigens

The detective ability of interdigitated biosensors using differ-

nt immobilizing methods of serum containing antibodies againstEV was determined through measurements of total antigen of JEVt room temperature and negative control was performed by PBSolution at pH 7.0 [10,19]. Fig. 5 shows detection signals of JEVntigens using these biosensors with different serum antibodies

ig. 5. Detection signals of electrochemical biosensors using different immobilizingethods of antibodies in human serum for JEV detection: (a) APTES-GA-PrA-serum;

b) APTES-GA-serum; (c) APTES-GA-antiHIgG-serum; (d) APTES-serum; (e) negativeontrol with PBS only.

fferent methods: (A) APTES-serum; (B) APTES-GA-serum; (C) APTES-GA-antiHIgG-

immobilizations. In this study, the APTES-GA-PrA-serum methodwas recognized again as the best effective method in immobilizingserum containing antibodies on the interdigitated sensor (Fig. 5a).The electrochemical detection signal of the biosensor using thismethod was two to three times greater than using other methods.Whereas, the two lower detections obtained by using the meth-ods of APTES-GA-serum (Fig. 5b) and APTES-GA-antiHIgG-serum(Fig. 5c) were almost the same. The detection obtained by directimmobilization of serum on the interdigitated sensor was very poor(Fig. 5d). Noticeably, most of tests exhibited the detection signal ofthese studied biosensors obtained after 5 min and stabilized after20 min when the biosensors were exposed to JEV antigens. Thisshowed a good potential applied for the development of serumbased biosensors for rapid detection of viral antigens. Again, theexperimental detection signals agreed highly with the good anal-ysis of FESEM, FTIR and fluorescence microscopy and previouspublications [35,36].

4. Conclusion

In the present work, a comparative study of different methods toimmobilize serum containing antibodies against JEV for the devel-opment of electrochemical biosensors was presented. The analysisshowed that the immobilization of serum on the interdigitated sur-face using protein A caused a twofold to threefold improvement inthe detection signal of JEV antigens as compared with other immo-bilization methods. The response time obtained quickly after 5 minand stabilized after 20 min when the biosensor was exposed to JEVantigens. This study provided an advantageous and suitable methodof serum antibodies immobilization for the development of electro-chemical biosensors in rapid and direct detection of viruses fromthe clinical sample in outbreaks as preliminary screening.

Acknowledgments

This work was financially supported by Vietnam’s NationalFoundation for Science and Technology Development (NAFOSTED),project code: 106.16.181.09.

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