Detection of vibrio cholerae o1 by using cerium oxide nanowires based immunosensor with different antibody immobilization methods

Journal of the Korean Physical Society, Vol 68, No 10, May 2016, pp 1235∼1245 Detection of Vibrio cholerae O1 by Using Cerium Oxide Nanowires - Based Immunosensor with Different Antibody Immobilization Methods Phuong Dinh Tam,∗ Nguyen Luong Hoang, Hoang Lan and Pham Hung Vuong Advanced Institute for Science and Technology (AIST), Hanoi University of Science and Technology (HUST), No Dai Co Viet St., Hanoi, Viet Nam Ta Thi Nhat Anh Vinhphuc Technology Economic Colleges, No 10 Hung Vuong St., Vinhphuc, Viet Nam Tran Quang Huy and Nguyen Thanh Thuy National Institute of Hygiene and Epidemiology (NIHE), No.1 Yersin St., Hanoi, Viet Nam (Received 18 March 2016, in final form 18 April 2016) In this work, we evaluated the effects of different antibody immobilization strategies on the response of a CeO2 -nanowires (NWs)-based immunosensor for V ibrio cholerae O1 detection Accordingly, the changes in the electron-transfer resistance (Ret ) from before to after cells bind to an antibody-modified electrode prepared by using three different methods of antibody immobilization were determined The values were 16.2%, 8.3%, and 6.65% for the method that utilized protein A, antibodies activated by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)/Nhydroxysuccinimide (NHS), and absorption, respectively Cyclic voltammetry confirmed that the change in the current was highest for the immunosensors prepared using protein A (11%), followed by those prepared with EDC/NHS-activated antibodies (9%), and finally, those prepared through absorption (7.5%) The order of the antibody immobilization strategies in terms of resulting immunosensor detection limit and sensitivity was as follows order: absorption (3.2 × 103 CFU/mL; 45.1 Ω/CFU·mL−1 ) < EDC/NHS-activated antibody (1.0 × 103 CFU/mL; 50.6 Ω/CFU·mL−1 ) < protein A (1.0 × 102 CFU/mL; 65.8 Ω/CFU·mL−1 ) Thus, we confirmed that the protein A mediated method showed significantly high cell binding efficiencies compared to the random immobilization method PACS numbers: 81.05.Cy, 82.45.Tv, 81.16.-c Keywords: Antibody, Protein A, Immunosensor, Nanowire, CeO2 DOI: 10.3938/jkps.68.1235 I INTRODUCTION Immunosensors are currently attracting much attention because of their promising applications, particularly, in clinical diagnostics [1–5], the food industry [6–8], and environmental monitoring [9–12] Many kinds of immunosensors, such as electrochemical, optical, and mechanical immunosensors, are used for various purposes However, lectures report that immunosensor performance is significantly affected by antibody immobilization approaches Therefore, the selectivity of the immobilization methods used to improve immunosensor performance is very important To date, several techniques for antibody immobilization have been reported, including physicochemical absorption [13], covalent attachment ∗ E-mail: tam.phuongdinh@hust.edu.vn, phuongdinhtam@gmail com; Fax: +84-4-3623-0293 [14, 15] or Langmuir Blodgett method [16], and other methods The physicochemical adsorption strategy relies on weak binding, such as van der Waals, hydrophobic, or electrostatic interactions, to attach antibody molecules For example, Buijs and coworkers [13] studied the effect of adsorption on antigen binding by IgG and its F(ab )2 fragments The group performed antibody absorption on hydrophilic silica and hydrophobic methylated silica surfaces The electrostatic interactions were studied by varying pH values and ionic strength Experimental results showed that the orientation of the adsorbed antibodies could be strongly influenced by electrostatic interactions Similar results were attained by Chen et al [17] An ultrasensitive microcantilever immunosensor based on antibody chemical adsorption was developed by Sungkanak et al [18] In the study, linkers, including 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and sulfo-N-hydroxysuccinimide -1235- -1236- Journal of the Korean Physical Society, Vol 68, No 10, May 2016 (sulfo-NHS), were used to activate carboxylic groups to form peptide bonds with the primary amines of the antibodies Monoclonal antibodies in the PBS solution were spread onto the sensor surface, and were achieved a detection limit of 1.0 × 103 CFU/mL and a mass sensitivity of 146.5 pg/Hz As demonstrated, the adsorption approach was used to attach antibodies onto the transducer surface This method is simple, easy to perform, and cost effective However, fabricating high-performance immunosensors is difficult because of the random antibody orientations achieved from this method, leading to a low density of antigen-binding sites [19] To address this issue, sensor performance was improved by using the covalent attachment methods developed by various research groups [14, 15, 20] These techniques rely on linker materials to fasten antibodies onto electrode surfaces Wang et al [20] reported an orientation-controlled immobilization method based on protein A for immunosensor design Their study showed the good binding ability of protein A with gold nanoparticles The inclusion of amineterminated plasma polymerization also led to enhanced antibody binding capabilities, which improved sensor performance In another study, Franco and coworkers [21] implemented an oriented immobilization of antibodies on gold surfaces by using protein A to fabricate a surface plasmon resonance immunosensor The protein A-gold binding domain consist of a gold-binding peptide coupled with the immunoglobulin-binding domains of staphylococcal protein A This coupling facilitated the oriented immobilization of antibodies The fabricated immunosensor achieved a detection limit of 90 ng/mL, with an interchip variability of lower than 7% Many other studies used different linkers, such as (3-glycidyloxypropyl)trimethoxysilane (GPS) [14], graphene paper [15], carboxylmethyl dextran [22], and semiconductor metal oxide [23–30], to immobilize antibodies on electrode surfaces To date, studies comparing antibody immobilization techniques have been conducted Babacan et al [31] evaluated different antibody immobilization methods for piezoelectric immunosensor application In the study, two linkers (protein A and polyethylenimine/glutaraldehyde, PEI/GA) were assessed In the PEI/GA method, the antibodies were immobilized in random orientations through the surface aldehyde group of GA on a PEI-coated quartz crystal Otherwise, antibodies were bound to protein A, which was directly immobilized on the quartz crystal’s surface by van der Waals interactions The group showed that antibody immobilization onto piezoelectric quartz crystals through protein A showed better results than immobilization through PEI/GA Meanwhile, Danczyk et al [32] investigated three different antibody immobilization methods: adsorption, aminosilance, and Ngamma-maleimidobutyryloxy-succinimide ester (GMBS) linkers They demonstrated that the presence of protein A improved the antigen capture ability of the ad- sorbed antibodies and the GMBS surface However, protein A did not increase the antigenic capture of the aminophase surfaces Additionally, the aminosilance surface exhibited the highest level of nonspecific binding Vashist et al [33] investigated antibody immobilization by using EDC, EDC/N-hydroxysuccinimide (NHS), and EDC/sulfo-NHS for immunosensor applications At pH 7.4, EDC crosslinks antibodies to the 3-aminopropyl triethoxy-silane (APTS)-modified surface of SPR more efficiently than EDC/NHS and EDC/sulfo-NHS Recently, Lee and co-workers [34] compared two antibodyoriented immobilization methods that adopted thiolconjugated secondary antibodies and thiolated-protein A/G linkers The secondary antibody-mediated attachment method provided better antigen-binding efficiency compared with the other strategy As discussed, studies of various antibody immobilization approaches have been extensively reported in the literature However, no consensus has been achieved on recommendations for antibody attachment approaches Furthermore, the use of antibody immobilization to improve immunosensor performance continues to challenge the immunosensor fabrication process Therefore, a suitable immobilization method for each immunosensor application needs to be found In this paper, we report three different approaches for immobilizing antibodies on the surface of electrode modified by using CeO2 nanowires to detect V ibrio cholerae O1 These methods include adsorption, the use of EDC/NHS-activated antibodies, and the use of protein A Our main aim was to evaluate the effects of the different antibody immobilization strategies on the response of the CeO2 NWsbased immunosensor The results of this study would provide insight into the best immobilization method for the design of CeO2 NWs-based immunosensors, as well as immunosensors in general II EXPERIMENTS CeO(NO3 )3 ·6H2 O, H2 O2 , toluene, and antibodies against V cholerae O1 (anti-V cholerae O1) were provided by Invitrogen Co Phosphate buffered saline PBS (0.01 M, pH 7.4), EDC, NHS, bovine serum albumin (BSA), 98% H2 SO4 , KCr2 O7 , protein A, and were purchased from Sigma-Aldrich Potassium ferrocyanide and potassium derricyanide were acquired from Beijing Chemical Reagent (China) All solutions were prepared with de-ionized (DI) water (18.2 MΩ·cm) In this work, a microelectrode was utilized as a sensor for electrochemical measurements Briefly, the sensor was fabricated by sputtering 10 nm Cr and 200 nm Pt onto a ∼150 nm thick silicon-dioxide (SiO2 ) layer thermally grown on top of a silicon wafer The sensor surface was initially cleaned with KCr2 O7 in 98% H2 SO4 , followed by cyclic voltammograms sweep from −1 V to +2.1 V, at a scan rate of 25 mV/s in 0.5 M H2 SO4 Detection of Vibrio cholerae O1 by Using Cerium Oxide Nanowires · · · – Phuong Dinh Tam et al Fig (Color online) (a) schematic of an electrode structure with a photograph of the electrodes, (b) fingers of the electrode image with higher magnification, (c) the FE-SEM image of CeO2 nanowires deposited on the electrode, and (d) the FE-SEM image of CeO2 nanowires with higher magnification to activate the sensor’s surface CeO2 NWs were prepared by a reaction with 1% (v/v) APTS solution in ethanol for 36 h at room temperature Afterward, a mixture of APTS/CeO2 NWs was centrifuged for 60 at 3500 rpm and washed thrice with ethanol Subsequently, APTS/CeO2 NWs were sprayed onto the activated electrode’s surface (Fig 1) The APTS/CeO2 -modified electrode was incubated in PBS solution (PBS 0.01 M, pH 7.4) containing 12 μg/mL of anti-V cholerae O1 and then stored at ◦ C overnight Continuously, the electrode’s surface was rinsed with double-distilled water and dried under nitrogen flow To block nonspecific sites, we immersed the immunosensor in PBS solution containing 1% BSA for 30 min, washed it with DI water, and dried under nitrogen flow Figure 2(a) shows a schematic illustration of antibody immobilization on the surface of the CeO2 NWs-deposited electrode by absorption For the EDC/NHS - activated antibody method, first 12 μg/mL of anti-V cholerae O1 was incubated in 50 μL of solution containing mg/mL of EDC and 10 mg/mL of NHS in 0.01 M PBS buffer for 20 at pH 7.4 and room temperature The EDC binds carboxyl groups to primary amines, forming an O-acylisourea intermediate This product is unstable in aqueous solutions Thus, NHS is required for stabilization by converting the intermediate to an amine-reactive NHS ester [33,35] Second, the electrode modified with APTS/CeO2 NWs was immersed in a solution of EDC/NHS-activated antibodies for 24 h at room temperature In this step, the activated antibodies crosslinked with the free amino groups on the surface of the electrode modified with APTS/CeO2 NWs Afterward, the immunosensor was rinsed with doubledistilled water and dried under nitrogen flow Finally, -1237- 1% BSA was added to the modified nanowire’s surface to block nonspecific sites as previously described The immunosensor was rinsed with DI water and dried under nitrogen flow When not in use, the immunosensors were stored at ◦ C in a refrigerator Figure 2(b) displays a schematic illustration of the antibody immobilization on the surface of the APTS/CeO2 NWs-deposited electrode using EDC/NHS-activated antibodies Similarly, protein A was activated with EDC/NHS as mentioned in Ref [22] Then, an electrode modified with APTS/CeO2 NWs was dipped in a solution of EDC/NHS-activated protein A for 24 h at room temperature Subsequently, 50 μL of anti-V cholerae O1 (12 μg/mL) was dropped on the electrode modified with protein A/APTS/CeO2 NWs Finally, the electrode was treated with 1% BSA for 30 to prevent nonspecific binding, as previously described In each step, the electrode was rinsed with double-distilled water and dried under nitrogen flow Figure 2(c) shows a schematic of the antibody immobilization on the surface of APTS/CeO2 NWs-deposited electrode using protein A For the electrochemical impedance spectroscopy (EIS) measurements, an IM6-impedance analyzer with IM6THALES software was used to detect the cell concentrations of V cholerae O1 In this work, the electrode modified with anti-V cholerae O1 was immersed in a measuring cell that was filled with mL of 0.01 M PBS solution (pH 7.4) containing a defined cell concentration of V cholerae O1 for 90 at room temperature to form an antibody-antigen complexes The immunosensor was rinsed with buffer solution to remove the nonspecifically adsorbed cells The immunosensor responses were monitored by dipping the modified sensor in mL of 0.01 M PBS solution containing 20 mM [Fe(CN)6 ]3−/4− as an indicator probe The detected immunosensor was connected to the test and sense probes, and the Pt electrode was connected to the counter electrode on the IM6impedance analyzer An Ag/AgCl electrode was used as a reference electrode All tests were conducted in an open circuit The tested frequency range was Hz to 100 kHz, with an amplitude of ±5 mV The Nyquist plots were recorded The differences in the electron-transfer resistance (Ret ) were considered as the signal produced by the interaction between the antibodies and the cells For cyclic voltammetry (C-V) measurements, an IM6impedance analyzer with IM6-THALES software was used under the C-V program The electrode modified with anti-V cholerae O1 was immersed in a measuring cell that was filled with mL of 0.01 M PBS solution (pH 7.4) containing a defined concentration of V cholerae O1 cells for 90 at room temperature to form antibodyantigen complexes The immunosensor was rinsed with buffer solution to remove nonspecifically adsorbed cells Immunosensor responses were monitored by dipping the modified sensor in mL of 0.01 M PBS solution containing 20 mM [Fe(CN)6 ]3−/4− as an indicator probe The detected immunosensor was connected to the test and sense probes, and the Pt electrode was connected to -1238- Journal of the Korean Physical Society, Vol 68, No 10, May 2016 Fig (Color online) Schematic illustration of antibody immobilization on the surface of a CeO2 - nanowire - modified electrode: (a) antibody absorption immobilization, (b) EDC/NHS-activated antibody method and (c) antibody immobilization via protein A the counter electrode on the IM6-impedance analyzer An Ag/AgCl electrode was used as a reference electrode The potential was scanned from −0.2 V to 0.67 V at a scan rate of 100 mV·s−1 The differences in the current peak were regarded as the signal produced by the interaction between the antibodies and the cells III RESULTS In this work, Fourier transforms infrared (FTIR) spectroscopy was used to verify the existence of CeO2 nanowires and anti-V cholerae O1 on the sensor’s surface Figure shows the FTIR spectra of (a) the Detection of Vibrio cholerae O1 by Using Cerium Oxide Nanowires · · · – Phuong Dinh Tam et al -1239- Fig (Color online) Fluorescence images of antibody immobilized electrode: (a) ATPS/CeO2 NWSs modified electrode, (b) absorption, (c) EDC/NHS - activated antibody, and (d) protein A-mediated immobilization methods Fig (Color online) FTIR spectra of antibody immobilized electrode’s surface: (a) ATPS/CeO2 NWSs modified electrode, (b) absorption, (c) EDC/NHS - activated antibody, and (d) protein A-mediated immobilization method ATPS/CeO2 nanowires, (b) immobilization via absorption antibody, (c) immobilization via EDC/NHS activated antibody and (d) protein A-mediated immobilization The FTIR spectral features of the CeO2 nanowires sample displays in Fig 3(a) The intense band observed round 825 cm−1 is due to the Ce−O−C stretching vibration The peak at 3410 cm−1 is related to hydrogen bond of − OH groups of water molecules or surface − OH groups The peak around 542 cm−1 is assigned to the Ce−O stretching band The presence of APTS was confirmed by the aliphatic C-N character around 1230 cm−1 , and the peak at 1589 cm−1 confirmed primary N-H bending When the antibody is absorbed on the electrode’s surface, peaks corresponding to C=O stretching and NH bending of amin I form at 1671 cm−1 , 1695 cm−1 , respectively The band at 1548 − 1586 cm−1 is assigned to the N-H bending of amin II vibration Meanwhile, the peak around 467 cm−1 is assigned to the Ce−O stretching band, and the peak at 1430 cm−1 is assigned to the bending vibration of C-H stretching of CeO2 (Fig 3(b)) Figure 3(c) shows the FTIR spectrum for antiboby immobilization via EDC/NHS-activated antibody It can be seen that the peak at 1673 cm−1 corresponding to the C=O stretching of amin I The peak around 610 cm−1 is assigned to the Ce−O stretching band, the intense band round 825 cm−1 is due to the Ce−O−C stretching vibration In the case of protein A-mediated immobilization (Fig 3(d)), the presence of a 825 cm−1 peak, which is clearly seen in the spectrum corresponds to Ce−O−C stretching vibration, the peak at 695 cm−1 is assigned to the Ce−O stretching band, and peak at 556 cm−1 is assigned to the Ce−O stretching band The peak at 1675 cm−1 corresponds to the C=O stretching of amin I, confirming the immobilization of antibodies Additionally, the peak at 3435 cm−1 is related to the O-H stretching vibration of H2 O in sample The density of antibodies on an APTS-CeO2 NWs modified electrode was studied by using fluorescence microscopy, and the results are shown in Fig APTS/CeO2 NWs deposited electrode’s surface was clearly black (Fig 4(a)) while the surface of the electrode with antibodies immobilized by using absorption (Fig 4(b)), EDC/NHS activated antibody (Fig 4(c)), and protein A-mediated immobilization (Fig 4(d)) clearly shown green fluorescence spots This could confirm that antibodies were immobilized on the electrode’s surface In this work, we used EIS to determine the immunosensor response defined by the interaction between the antibodies and the V cholerae O1 cells The responses of the immunosensors prepared by using the three-immobilization methods were compared and evaluated to determine the best immobilization method for the CeO2 NWs-based immunosensor As mentioned in a previous study [36], EIS is an effective technique for developing biosensors that detect bacteria The principle underlying the function of the EIS-based immunosensor depends on measurements of electrochemical Faradaic impedance with [Fe(CN)6 ]3−/4− as the indicator probe The electrode was immersed in PBS solution containing [Fe(CN)6 ]3−/4− , and an alternating current potential of V was applied to the electrode Consequently, oxidation and reduction of the [Fe(CN)6 ]3−/4− occurred The electrons were transferred between the two fingers of the electrode array When linkers modified the elec- -1240- Journal of the Korean Physical Society, Vol 68, No 10, May 2016 Fig (Color online) Nyquist diagrams for impedance measurements of immunosensors in the presence of 20 mM [Fe(CN)6 ]3−/4− in a mM PBS solution: (A) Antibody absorption method: (a) bare electrode, (b) APTS-CeO2 nanowiremodified electrode, (c) antibodies/APTS-CeO2 NWs-modified electrode, (d) BSA/antibodies/APTS-CeO2 NWs-modified electrode, and (e) cells/BSA/antibodies/APTS-CeO2 NWs-modified electrode (B) Immobilization method via EDC/NHS activated antibody: (a) bare electrode, (b) APTS-CeO2 NWs-modified electrode, (c) antibodies-EDC-NHS/APTS-CeO2 NWsmodified electrode, (d) BSA/antibodies-EDC-NHS/APTS-CeO2 NWs-modified electrode, and (e) cells/ BSA/antibodies-EDCNHS/APTS-CeO2 NWs-modified electrode (C) Immobilization method via protein A: (a) bare electrode, (b) APTS-CeO2 NWs-modified electrode, (c) protein A/APTS-CeO2 NWs-modified electrode, (d) antibodies/protein A/APTS-CeO2 NWsmodified electrode, (e) BSA/antibodies/protein A/APTS-CeO2 NWs-modified electrode, and (f) cells/BSA/antibodies/Protein A/APTS-CeO2 NWs-modified electrode (D) Comparison of the immunosensor responses for the three different antibody immobilization methods trode’s surface, a thin film layer was formed, leading to the inhibition of electron transfer between the fingers Thereby, an increase in electron-transfer resistance was detected Figure presents the Nyquist plots for bacterial cell detection by immunosensors prepared by using the three different immobilization methods: (A) absorption, (B) EDC/NHS-activated antibody, and (C) protein A-mediated methods Meanwhile, Fig 5(D) compares the immunosensor output signals for the three different immobilization methods As observed in Fig 5(A), when the bare electrode was immersed in PBS solution containing the redox probe, the reduction process of the redox probe was initiated, and electrons were transferred between the two electrodes through the redox probe [Fe(CN)6 ]3−/4− The electron transfer was not blocked by any monolayer on the electrode’s surface Thus, the electron-transfer resistance (Ret) was determined to be 745 Ω, as shown in Fig 5A(a), thereby indi- cating high electron-transfer kinetics of the redox probe at the electrode interface After the APTS/CeO2 NWs modified electrode surface, a thin film layer that could have hindered electron transfer from [Fe(CN)6 ]3−/4− to the conductive electrode’s surface was formed However, the positively-charged APTS/CeO2 nanowire promoted transfer of the negative redox probe to the electrode’s surface by electrostatic attraction Thus, the value of Ret decreased to 695 Ω, as shown in Fig 5A(b) When the antibodies were absorbed directly onto the electrode’s surface modified with APTS/CeO2 nanowires, a layer of antibodies was formed on the electrode surface This layer inhibited the electron transfer between the fingers of the electrode An increase in the electron-transfer resistance of 812 Ω was noted, as shown in Fig 5A(c) To block the unreacted and nonspecific sites, the electrode surface modified with antibodies/APTS/CeO2 nanowires was treated with 1% Detection of Vibrio cholerae O1 by Using Cerium Oxide Nanowires · · · – Phuong Dinh Tam et al -1241- Table Comparison of the analytical parameters of the developed immunosensors for V cholera O1 detection by using the three different immobilization methods Methods Absorption EDC/NHS Protein A Detection limit CFU/mL 3.2 × 103 1.0 × 103 1.0 × 102 Sensitivity Ω/CFUmL−1 45.1 50.6 65.8 BSA for 30 A Ret of 858 Ω was obtained, as shown in Fig 5A(d) When the cells are bound to the modified electrode’s surface, a reaction between the antibodies and the cells potentially occurs Consequently, immunocomplexes would then attach to the electrode’s surface, forming an additional barrier that would further increase the electron-transfer resistance The Ret was 915 Ω, as shown in Fig 5A(e) The changes in electron-transfer resistance before and after cell detection were calculated by the following equation: %ΔRet = 100(Ret cell/antibody −Ret antibody )/Ret antibody (1) Where Ret antibody and Ret cells/antibody are the Ret values before and after the cells bind to the antibodymodified electrode’s surface, respectively A 6.65% increase in the electron-transfer resistance was observed after cell binding Antibodies were confirmed to be immobilized successfully on the electrode’s surface by using the adsorption method Figure 5B illustrates the Nyquist plots for the immunosensors prepared by using the EDC/NHS-activated antibody approach As previously mentioned, the value of Ret for the bare electrode and the electrode surface modified with APTS/CeO2 NWs were 745 and 695 Ω, respectively The Ret increased to 847 Ω when EDC/NHS-activated antibodies were immobilized on the surface of the electrode modified with APTS/CeO2 NWs, as shown in Fig 2B(c) When the modified electrode’s surface was blocked with 1% BSA for 30 min, the value of Ret increased to 892 Ω, as shown in Fig 5B(d) The value of Ret increased significantly (965 Ω) when cells were bound to the antibodymodified surface of the electrode, as shown in Fig 5B(e) By using Eq (1), we calculated the change in Ret to be about 8.3% Figure 5C shows the response of the immunosensor prepared by antibody immobilization through protein A In this work, protein A was immobilized on the electrode modified with APTS/CeO2 nanowires, as described in a previous study [22] The terminal carboxyl groups of protein A were activated by incubation in EDC/NHS solution for 20 to produce an active intermediate of the NHS ester (Fig 2(c)) Afterward, the electrode modified with APTS/CeO2 NWs was immersed in a solution Linearity range CFU/mL 3.2 × 103 − 1.0 × 107 1.0 × 103 − 1.0 × 107 1.0 × 102 − 1.0 × 107 of EDC/NHS-activated protein A for 24 h at room temperature This step led to a crosslinking of the activated protein A with the free amino groups on the electrode’s surface modified with APTS/CeO2 nanowires A Ret of 870 Ω was obtained for protein A immobilization on the modified electrode (Fig 5C(c)) When anti-V cholerae O1 was dropped on the protein A/APTS/CeO2 -NWmodified electrode, the value of Ret increased to 910 Ω, as shown in Fig 5C(d) To avoid the binding of other proteins to the unreacted NHS ester, the electrode was blocked with 1% BSA for 30 min, which resulted in an increase in the value of Ret to 955 Ω, as shown in Fig 5C(e) When the cells interacted with the antibodies immobilized on the electrode surface, a Ret of 1110 Ω was noted By using Eq (1), we calculated the change in Ret to be 16.2% A comparison among the responses of the immunosensors prepared by using the three different antibody immobilization methods is shown in Fig 5D The highest and the lowest responses were achieved by the immunosensors prepared by antibody immobilization with protein A (16.2% according to Eq (1)) and with adsorption (6.65%) The response of the immunosensor prepared using EDC/NHSactivated antibodies was 8.3% The analytical parameters of the developed immunosensors for V cholera O1 detection were also compared (Table 1) As shown in Table 1, the order of the antibody immobilization strategies in terms of resultant immunosensor detection limit and sensitivity is as follows: adsorption immobilization < immobilization using EDC/NHS-activated antibodies < protein A-mediated immobilization Meanwhile, the linearity range changed insignificantly C-V studies on the immunosensor detection of bacterial cells were performed in PBS solution containing 20 mM [Fe(CN)6 ]3−/4− at a scan rate of 100 mV·s−1 versus a Ag/AgCl reference electrode Figure presents the C-V characterizations of the immunosensors prepared by antibody immobilization through (A) absorption, (B) the use of EDC/NHS-activated antibodies, and (C) the use of protein A Meanwhile, Fig 6D displays a comparison of the responses of the immunosensors prepared by using the three different methods As presented in Fig 6A(a, b), the C-V response of the APTS/CeO2 NWs-modified electrode is shifted relative to that of the bare electrode The peak current obviously increased up to 84 μA because the APTS/CeO2 NWs film could -1242- Journal of the Korean Physical Society, Vol 68, No 10, May 2016 Fig (Color online) Cyclic voltammetry of the sensor in the presence of 20 mM [Fe(CN)6 ]3−/4− in a 1mM PBS solution, at a scan rate 100 mV/s (A) Antibody absorption method: (a) bare electrode, (b) APTS-CeO2 NWs-modified electrode, (c) antibodies/APTS-CeO2 NWs-modified electrode, (d) BSA/antibodies/APTS-CeO2 NWs-modified electrode, and (e) cells/BSA/antibodies/APTS-CeO2 NWs-modified electrode (B) Immobilization method via EDC/NHS - activated antibody: (a) bare electrode, (b) APTS-CeO2 NWs-modified electrode, (c) antibodies-EDC-NHS/APTS-CeO2 NWs-modified electrode, (d) BSA/antibodies-EDC-NHS/APTS-CeO2 NWs-modified electrode, and (e) cells/BSA/antibodies-EDC-NHS/APTS-CeO2 NWs-modified electrode (C) Immobilization method via protein A: (a) bare electrode, (b) APTS-CeO2 NWs-modified electrode, (c) protein A/APTS-CeO2 NWs-modified electrode, (d) antibodies/protein A/APTS-CeO2 NWs-modified electrode, (e) BSA/antibodies/protein A/APTS-CeO2 NWs-modified electrode, and (f) cells/BSA/antibodies/Protein A/APTS-CeO2 NWsmodified electrode (D) Comparison of the immunosensor responses for the three different antibody immobilization methods promote electron transfer of [Fe(CN)6 ]3−/4− A slight decrease in the peak current (77 μA) and a separation of the peak potential (0.38 V) were observed for antibody absorption on the APTS/CeO2 NWs-modified electrode surface, as shown in Fig 6A(c) When the antibody-modified electrode’s surface was blocked with 1% BSA for 30 min, the current value decreased continuously by 75 ΩA An insignificant separation of the peak potential (0.36 V) was observed, as shown in Fig 6A(d) When the cells interacted with the modified electrode’s surface, a decrease in the peak current of 70 μA was observed, and this decrease corresponded to a signal variation of about 7.5% compared with that of the modified electrode, as shown in Fig 6A(e) In the case of EDC/NHS activated antibody immobilization method, a decrease in the peak current of 76.5 μA was observed when EDC/NHS-activated antibodies were bound to the APTS/CeO2 -NW-modified electrode’s surface, as shown in Fig 6B(c) A change of 69 μA was observed continuously after blocking with 1% BSA for 30 (Fig 6B(d)) When cells were bound on the electrode’s surface, a further decrease in the peak current was found at 63 μA, as shown in Fig 6B(e) Therefore, the current value varied by about 9% compared with the signal for the antibody-modified electrode For the immunosensors prepared using protein A, a slight decrease in the peak current (70 μA) was observed under protein A immobilization on the APTS/CeO2 -NWs-modified electrode surface, as shown in Fig 6C(c) When antibodies attached onto the protein A/APTS/CeO2 -NWs-modified electrode, a further decrease of 68 μA in peak current was noted, as shown Detection of Vibrio cholerae O1 by Using Cerium Oxide Nanowires · · · – Phuong Dinh Tam et al in Fig 6C(d) A drop in the peak current of 66.7 μA was obtained continuously when the sensor’s surface was blocked with 1% BSA for 30 min, as shown in Fig 6C(e) When cells bound to the electrode surface, a decrease in the peak current (11%) was clearly observed (Fig 6C(f)) Thus, the voltammetric behavior of the redox probe was demonstrated to be affected by electrode’s surface modification Changes in the current peak were observed because of electrode’s surface modification, which created another barrier layer that blocked access of the redox probe to the electrode’s surface within the applied potential The change in current was highest for the immunosensors with antibodies immobilized using protein A (11%), followed by those prepared using EDC/NHSactivated antibodies (9%), and finally, by those prepared by direct adsorption (7.5%), as shown in Fig 6D The stability of the immunosensors prepared by using different immobilization methods was studied and evaluated The nine immunosensors that utilized different antibody immobilization methods were stored in 0.1 M PBS (pH 7.4) at ◦ C for 135 days and subsequently analyzed at different times (45 day/times), as shown in Fig 7(a) Repeatable signals were observed for up to 45 days for all the immunosensors At day 90, the signal response of the stored immunosensors was changed by approximately 11.4% for those prepared by using direct antibody absorption, 6.1% for those prepared by using EDC/NHS-activated antibodies, and 5.6% for protein A-mediated immobilization No response signal was detected for all of the immunosensors after they had been stored for 135 days, indicating a loss of biological activity of the anti-V cholerae O1, which could have been denatured by that time; thus, no binding with the bacterial cells was exhibited By these results, we can conclude that the immunosensors exhibited acceptable stability and that the immunosensor using protein A showed the greatest stability The specificity of the immunosensors was also studied by using Salmonella and Escherichia coli O157:H7 bacteria as control samples No response signals of the immunosensors were detected for either bacterial species Shifts in the value of Ret of 900, 975, and 1010 Ω corresponding to 5.9%, 14.7%, and 18.8%, respectively, after V cholerae O1 detection were noted after antibody immobilization by adsorption, EDC/NHS activation, and protein A, respectively (Fig 7(b)) These results show that the specificities of the immunosensors prepared under the three approaches were all high Regeneration is a significant aspect in the development of immunosensors for in-field/on-site detection To study the regeneration of the immunosensors, antibodyimmobilized electrodes were immersed in a buffer solution containing V cholerae O1 for 90 Then, the immunosensors were washed with PBS buffer solution and DI water and dried with nitrogen gas The V cholerae O1 cell concentration was determined from the change in the measure value of Ret After detection of cells of V cholerae O1, the immunosensor was dipped into a -1243- Fig (Color online) (a) Stability of the immunosensor at different times for 135 day, (b) specificity of the immunosensor toward E coli O157: H7, salmonella, and V cholerae O1 bacterium, and (c) the regeneration performance of the immunosensor glycine-HCl buffer (pH 2.8) for about 10 to remove cells Subsequently, the immunosensor was washed with PBS buffer solution, DI water and dried with nitrogen gas The immunosensor was again measured with cells of V cholerae O1 under the same conditions The obtained results indicate that the signal responses had decreased by approximately 25%, 18%, and 11% for the immunosensors prepared by using adsorption, EDC/NHS- -1244- Journal of the Korean Physical Society, Vol 68, No 10, May 2016 activated antibodies, and protein A-mediated immobilization, respectively (Fig 7(c)) among those of all methods tested Thus, CeO2 -NWsbased immunosensors should be prepared by using the protein A-mediated immobilization method to enhance immunosensor performance IV DISCUSSION The three-immobilization methods were compared to determine the best immobilization method to prepare CeO2 -NWs-based immunosensors for V cholerae O1 detection As mentioned above, the changes in the immunosensor’s response after the use of the three different immobilization methods were approximately 6.65%, 8.3%, and 16.2% for the antibody adsorption, the EDC/NHS-activated antibody, and the protein Amediated immobilization methods, respectively The variation in the immunosensor’s signal was also determined by using cyclic voltammetry The peak current changed by about 7.5% for antibody adsorption, 9% for EDC/NHS-activated antibody immobilization, and 11% for antibody immobilization via protein A Antibody adsorption exerted no significant effect on the immunosensor’s response In contrast, the immunosensor’s response increased significantly when antibodies were immobilized by using EDC/NHS activation and protein A As previously discussed, the change in the immunosensor’s response could be attributed to several factors including the following: 1) the random orientation of antibodies and steric hindrance caused by improper antibody orientation with respect to the electrode’s surface, 2) denaturation of immobilized antibodies by the environmental pH and temperature, and 3) biocompatibility Several researchers [31, 37–39] have reported that protein A could specifically bind with the Fc fragment of antibodies and control antibody orientation This attribute does not block the antibody’s active sites for antigen binding, resulting in high cell-binding capacities Additionally, protein A’s biocompatibility conferred the immunosensor with enhanced detection performance In the immunosensors prepared with EDC/NHS-activated antibodies, the antibodies could be crosslinked to the amino groups on the surface of the APTS/CeO2 -NWsmodified electrode This occurrence would remove some biological activity and prevent some of the random orientations of the antibodies Several binding sites of antibodies could be blocked because of steric hindrance of the antigen-binding domains For this reason, the immunosensor’s response was lower than that of the immunosensors prepared via immobilization using protein A For direct antibody absorption, antibodies were absorbed on the sensor’s surface by weak binding, such as van der Waals or electrostatic interactions However, these interactions can be denatured by thermal and acidbase environmental or analytical chemistry conditions Furthermore, antibody orientation was random, leading to low cell-binding capacity and low cell detection Therefore, the immunosensor response was the lowest V CONCLUSION Our study mainly aims to evaluate the effects of different antibody immobilization strategies on the response of CeO2 -NWs-based immunosensors We determined the best immobilization method to apply for the design of CeO2 -NWs-based immunosensors and immunosensors in general In this study, we compared the performances of immunosensors prepared by using three antibody immobilization methods: adsorption, the use of EDC/NHSactivated antibodies, and the use of protein A-mediated antibodies Both the adsorption and the EDC/NHSactivated antibody methods are simple, cost effective, and appropriate for large-scale production However, improving sensor performance using these approaches was difficult because of low cell-binding capacity from random antibody orientations and denaturation under environmental conditions In contrast, protein A-mediated immobilization yielded the best orientation and antigenbinding efficiency among the tested methods This approach was then confirmed as the most suitable strategy for preparing CeO2 -NWs-based immunosensors for V cholerae O1 detection ACKNOWLEDGMENTS This work was supported by the Ministry of Education and Training under the research project code B2014.01.78 REFERENCES [1] S Weng, M Chen, C Zhao, A Liu, L Lin, Q Liu, J Lin and X Lin, Sensors and Actuators B 184, (2013) [2] C Song, G Xie, L Wang, L Liu, G Tian and H Xiang, Biosensors and Bioelectronics 58, 68 (2014) [3] X Wang, J Miao, Q Xia, K 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Vibrio cholerae O1 by Using Cerium Oxide Nanowires · · · – Phuong Dinh Tam et al -1 24 1- Table Comparison of the analytical parameters of the developed immunosensors for V cholera O1 detection by using. .. effects of different antibody immobilization strategies on the response of CeO2 -NWs -based immunosensors We determined the best immobilization method to apply for the design of CeO2 -NWs -based immunosensors