Label free electrochemical immunosensor based on cerium oxide nanowires for vibrio cholerae o1 detection

Ờ Å ỊÙ× Ư Ờ Label-free electrochemical immunosensor based on cerium oxide nanowires for Vibrio cholerae O1 detection Phuong Dinh Tam, Cao Xuan Thang PII: DOI: Reference: S0928-4931(15)30353-2 doi: 10.1016/j.msec.2015.09.027 MSC 5739 To appear in: Materials Science & Engineering C Received date: Revised date: Accepted date: February 2015 27 July 2015 September 2015 Please cite this article as: Phuong Dinh Tam, Cao Xuan Thang, Label-free electrochemical immunosensor based on cerium oxide nanowires for Vibrio cholerae O1 detection, Materials Science & Engineering C (2015), doi: 10.1016/j.msec.2015.09.027 This is a PDF file of an unedited manuscript that has been accepted for publication As a service to our customers we are providing this early version of the manuscript The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain ACCEPTED MANUSCRIPT Label-free electrochemical immunosensor based on cerium oxide nanowires RI P T for Vibrio cholerae O1 detection Phuong Dinh Tam*, Cao Xuan Thang MA NU SC Advanced Institute for Science and Technology (AIST), Hanoi University of Science and Technology, Vietnam ED *Corresponding author Phuong Dinh Tam, Ph.D PT Advanced Institute for Science and Technology (AIST), AC CE Hanoi University of Science and Technology (HUST) Phone: 84 36231524 Fax: 84 36230293 E-mail: tam.phuongdinh@hust.edu.vn or phuongdinhtam@gmail.com Postal address: No.1 Dai Co Viet, Hanoi, Vietnam ACCEPTED MANUSCRIPT Abstract This paper developed a label-free immunosensor based on cerium oxide nanowire for Vibrio cholerae O1 detection application The CeO2 nanowires were synthesized by T hydrothermal reaction The immobilization of Anti-V cholerae O1 onto CeO2 nanowire- RI P deposited sensor was performed via an amino ester, which was created by using 1-ethyl-3-(3dimethylaminopropyl) carbodiimide, and sulfo-N-hydroxysuccinimide The electrochemical SC responses of the immunosensor were studied by electrochemical impedance spectroscopy with [Fe (CN) 6] 3−/4− as redox probe A linear response in electron transfer resistance for cell NU of V cholerae O1 concentration was found in the range of 1.0 × 102 CFU/mL to 1.0 × 104 CFU/mL The detection limit of the immunosensor was 1.0 × 102 CFU/mL The MA immunosensor sensitivity was 56.82 /CFU.mL-1 Furthermore, the parameters affecting ED immunosensor response were also investigated, as follows: pH value, immunoreaction time, incubation temperature, and anti-V cholerae O1 concentration AC CE PT Keywords: antibody, antigen, immunosensor, nanowire, CeO2 ACCEPTED MANUSCRIPT INTRODUCTION Vibrio cholerae O1, a member of the family Vibrionaceae, is a Gram-negative with about 1.4- T 2.6 m long It is well determined by biochemical tests and DNA homology invetigattions RI P [1] To date, a number of approaches for detection of Vibrio cholerae O1 have been developed such as polymerase chain reaction (PCR) [2], Immunofluorescent-Aggregation Assay [3], SC DNA sensor [4] and immunosensor [5] Immunosensors are most commonly used as analytical tools for clinical diagnosis [6–8], food security [9-12], and environmental pollutants NU [13–15] because they are simple, highly sensitive, and easy to use Several materials have MA been studied to fabricate immunosensors, such as conducting polymers [16–19], carbon nanotubes [20, 21], graphene [22, 23], and metal nanoparticles [21, 24–26] Recent studies ED show that nanostructured semiconductor metal oxides, such as zinc oxide, titanium oxide, tin oxide, tungsten oxide, and cerium oxide, have been studied for immunosensor fabrication PT [27–33] Kyu et al [27] studied a titanium dioxide nanotube array-based immunosensor They used protein A capture to immobilize antibodies on the inner pore walls of the nanotube by AC CE electrostatic adsorption The fundamental response of material to liquid infiltration was determined The aqueous stabilities of porous TiO2 and SiO2 were compared in the pH range of to 12 The response signals of immunosensor were observed by reflectivity spectra measurement Ronghui Wang et al [28] studied a TiO2 nanowire bundle-based immunosensor for rapid and sensitive detection of Listeria monocytogenes concentration TiO2 nanowire bundle was prepared by a hydrothermal reaction of alkali with TiO2 powder Monoclonal antibodies were immobilized on the surface of TiO2 nanowire bundle to specifically capture L monocytogenes The TiO2 nanowire bundle-based immunosensor could detect L monocytogenes at a concentration as low as 4.7 × 102 CFU/mL and at a response time of 50 Chi-Chang Lin et al [29] constructed an immunosensor of antibodies/conducting polymer/TiO2 nanowire composite film TiO2 nanowires were synthesized by hydrothermal ACCEPTED MANUSCRIPT method and spin-coated on a gold microelectrode Conducting polymer and antibodies were electrochemically polymerized on patterned nanowire The immunosensor responses were T characterized by measuring changes in current-voltage As a result, immunosensors could RI P detect anti-rabbit IgG within a linear range of 11.2 g/mL to 112 g/mL, the immunosensor sensitivity was -0.64 A/(g/mL) Pavel and co-worker [30] reported a zinc oxide thin film SC transistor-based immunosensor Primary monoclonal antibodies were attached to the ZnO channel surface Detection of antibody and antigen interactions was performed by channel NU carrier modulation via pseudo double gating field effect, which was caused by the MA biochemical reaction The immunosensor sensitivity was 10 fM Anees et al [31] developed a nanostructured nano zinc oxide film immunosensor for mycotoxin detection The antibodies ED and bovine serum albumin were co-immobilized on zinc oxide film Fourier transform infrared spectroscopy, scanning electron microscopy, and electrochemical impedance PT spectroscopy were used to analyze the immobilization characterizations The immunosensor response was characterized by electrochemical method with a detection limit of 0.006 AC CE nM/dm3, response time of 25 s, and sensitivity of 189 /nM Michael et al [32] developed an immunosensor based on iridium oxide thin film matrices Antibodies were attached to iridium oxide by physical entrapment in the 3D matrix The immunosensor displayed a linear range for IgG concentrations (10 and 200 ng/mL) and a low detection limit of ng/mL An immunosensor with cerium oxide as medium material for food-borne mycotoxin detection was studied by Ajeet et al [33] Here, the co-immobilization of r-IgG and bovine serum albumin (BSA) onto nano cerium oxide film was prepared Electrochemical studies confirmed that the immunosensor exhibited a detection limit of 0.25 ng/dl and a response time of 25 s Pratima et al [34] developed a cerium oxide film based label – free capacitive immunosensor for detection of human chorionic gonadotropin hormone (hCG) The nano CeO2 film was fabricated onto indium tin oxide (ITO), which was used for the immobilization of anti-hCG ACCEPTED MANUSCRIPT antibody (Abs) Acorrding to Pratima group, the nano CeO2 film has roled in higher loading antibodies led to improve the immunosensing response The sensitivity immunosensor T obtained of 0.838 pF/mIU/mL in the detection range of 0–500 mIU/ mL The storage stability RI P of immunosensor exhibits 95% response after about week with relative standard deviation (RSD=3.4%) An electrochemical immunoassay for the prostate specific antigen (PSA) using SC ceria mesoporous nanospheres was investigated by Juan Peng and coworker [35] A glassy carbon electrode was coated by multiwalled carbon nanotube, poly(dimethyldiallylammonium NU chloride), CeO2 and PSA using layer by layer method for immunosensor application A linear MA relationship between the decrease in current and concentration of PSA was found in the range from 0.01 pg/mL to 1.000 pg/mL The detection limit was fg/mL Thus, many researchers have studied semiconductor metal oxide-based immunosensors However, published ED information is lacking on immunosensors that use CeO2 nanowires for label-free detection of PT Vibrio cholerae O1 bacteria AC CE In this paper, we reported a CeO2 nanowire-based immunosensor for label-free detection of V cholerae O1 bacteria The CeO2 nanowires are fabricated by hydrothermal method using Teflon autoclave The covalent method was performed to immobilize anti-V cholerae O1 on CeO2 nanowire-modified sensor surface Electrochemical impedance spectroscopy was used to detect V cholerae O1 cell concentration with [Fe (CN) 6] 3−/4− as redox probe Electron transfer resistance (Ret) increased linearly in the range of 1.0 × 102 CFU/mL to 1.0 × 104 CFU/mL after interaction with V cholerae O1 cells The CeO2 nanowire-based immunosensor exhibited low detection limit, highly sensitivity and easy to use ACCEPTED MANUSCRIPT EXPERIMENTAL 1) Chemical reagents saline (PBS 1×, pH 7.4), 1-ethyl-3-(3-dimethylaminopropyl) RI P Phosphate-buffered T CeO(NO3)3.6H2O, H2O2, toluene, anti-V cholerae O1 were provide by Invitrogen Co carbodiimide hydrochloride (EDC), sulfo-N-hydroxysuccinimide (NHS), bovine serum SC albumin BSA, H2SO4 98%, KCr2O7, , and 3-aminopropyl triethoxy-silane (APTES) were purchased from Sigma-Aldrich Potassium ferrocyanide and potassium derricyanide were NU purchased from Beijing Chemical Reagent (China) All solutions were prepared with de- 2) CeO2 nanowires synthesis MA ionized (DI) water We transferred 10 mL of mol/L of CeO(NO3)3.6H2O, H2O2, and toluene into a 50 mL ED Teflon lined stainless steel autoclave that was placed into a furnace The temperature was PT controlled to react at 160 °C for 72 h The obtained product was directly precipitated on the silicon substrate, which was placed in the autoclave Subsequently, the nanomaterial could be AC CE dispersed in ethanol after the silicon substrate was removed The products were dried in an oven for 12 h at 80 °C before antibody immobilization 3) Immunosensor fabrication In this work, the microelectrode was utilized as a sensor for the electrochemical impedance spectroscopy measurement The sensor was fabricated by sputtering 10 nm Cr and 200 nm Pt on a ~100 nm thick silicon dioxide (SiO2) layer thermally grown on top of a silicon wafer Then, the surface of the sensor was initially cleaned with KCr2O7 in 98% H2SO4, followed by cyclic voltammograms (swept potential from -1 V to +2 V; scan rate of 50 mV/s) in 0.5 M H2SO4 to activate the sensor surface Subsequently, 10 µL of silanized-CeO2 nanowires were drop-coated on the sensor surface using APTES and were dried in a desiccator The sensor was then immersed in the mixture of 100 mM EDC and 50 mM NHS in H2O for 60 at ACCEPTED MANUSCRIPT room temperature to shift the terminal carboxylic group to activate NHS ester The modified sensor was rinsed with DI water to remove EDC and NHS molecules, which were not T covalently bound to the surface of the sensor and then dried in nitrogen flow Subsequently, RI P g/mL of anti-V cholerae O1 was passed on the sensor surface The NHS moiety reacted spontaneously with a primary amine group in anti-V cholerae O1 Covalent bonding between SC anti-V cholerae O1 and matrix was formed Afterward, the immunosensor surface was rinsed with double-distilled water and dried in nitrogen flow Finally, mg/mL BSA was added to NU the modified immunosensor’s surface to block nonspecific sites The immunosensor was kept at °C in the refrigerator ED 4) Bacterial binding measurement MA rinsed with DI water and dried in nitrogen flow When not in use, the immunosensors were IM6-impedance analyzer with IM6-THALES software was used to detect PT concentration of cell of V cholerae O1 In this work, anti-V cholerae O1 modified sensor was immersed in a measuring cells and was filled with mL of mM PBS solution (pH 7.3) AC CE containing defined concentration of cells of V cholerae O1 for 90 at room temperature to form an antibody-antigen complex The immunosensor was rinsed thrice with buffer solution to remove the non-specifically adsorbed cells The immunosensor responses were monitored by dipping the modified sensor in mL of mM PBS solution containing 20 mM [Fe(CN)6]3/4- as an indicator probe The detected immunosensor was connected to the test and sense probe, and Pt electrode was connected to the counter electrode on the IM6 impedance analyzer Ag/AgCl electrode was used as a reference electrode All tests were conducted in an open circuit The tested frequency range was from Hz to 100 kHz with an amplitude of 5mV The Nyquist was recorded The differences in electron transfer resistance (Ret) were considered the signal produced by the interaction reaction between antibodies and cells ACCEPTED MANUSCRIPT RESULTS AND DISCUSSION Electrochemical impedance spectroscopy characterizations of the immunosensor T The schematic diagram of the immunosensor fabrication and cells of V cholerae O1 binding RI P is displayed in Figure As mentioned in [36], the impedance spectra included a semicircle portion that corresponds to the electron transfer process and a linear portion that corresponds SC to the diffusion process The semicircle diameter is the electron transfer resistance This resistance restrains the electron transfer kinetics of redox-probe at the interface of the sensor NU In this work, impedance spectra used to test interaction between antibodies and cells of V with 20 mM [Fe (CN) 6] 3−/4− MA cholerae O1 The anti-V cholerae O1 modified sensor was dipped in mM PBS solution as an indicating probe The impedance measurements were carried out at the open circuit voltage The tested frequency range was from Hz to 100 kHz ED with an amplitude of ±5 mV The Nyquist frequency was recorded The difference between PT the electron transfer resistance (Ret) before and after immunoreaction was considered as the signal produced A Nyquist plots for bare sensor, CeO2 nanowire modified sensor, anti-V AC CE cholerae O1/CeO2 nanowire modified sensor, control sample, and detection of V cholerae O1 cells are presented in Figure From the Nyquist plots, we can observe that when the bare sensor was immersed in an electrolyte solution containing the redox probe, the reduction process of the redox probe occurred, 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 sensor’s surface Ret was determined to be 437  On the surface of CeO2 nanowire-modified sensor, a thin film was formed, and this film could hinder electron transfer of hexacyanoferrates into the conductive sensor surface A small semicircle domain was formed, which corresponded to Ret = 479  On anti-V cholerae O1/CeO2 nanowiremodified sensor surface, a thinner film was formed, and the electron transfer of hexacyanoferrates was continuously inhibited Ret was determined to be 632  Thus, the ACCEPTED MANUSCRIPT impedance change showed that antibodies were attached to CeO2 nanowire-modified sensor surface The antibody immobilization was continuously confirmed by interaction tests T between antibodies and cells, as indicated in Figure The diameter of the semicircle RI P increased continuously Ret value was 764 k when antibodies/cells reaction occurred because of the creation of a thick barrier layer, which blocked the access of the redox probe to immunosensor exposed to the control sample SC the sensor surface By contrast, no significant signal change was observed for the NU Based on these results, an equivalent circuit of the system based on the models of Randles MA [37] was simulated and presented in the inset of Figure This equivalent circuit includes the following: ohmic resistance of the electrolyte solution (Rs), depending on the ionic ED concentration, type of ions, temperature, and the geometry of the area in which current is carried; and the Warburg impedance (Zw), which is the impedance caused by the diffusion of PT the redox probe to the interface from the electrolyte bulk Two elements were unaffected by the reaction on the sensor surface The double layer capacitance (Cdl) represented the AC CE electrical double layer at the electrode/solution interface that was formed as ions from the solution attached to the electrode surface The value of the double layer capacitance depends on many variables, including electrode potential, temperature, ionic concentrations, type of ionic, and electrode roughness The Ret is the electron transfer resistance that shows electron transfer kinetics of the redox probe at the electrode diffusion layer The Cdl and Ret represent the interface properties of the sensor By fitting the electrochemical impedance spectra to the equivalent circuit, the value of each electrical element in the equivalent circuit was obtained, as shown in Table As mentioned above, the double layer capacitance and electron transfer resistance described the interface properties of the sensor/electrolyte and changed because of the modified sensor surface As shown in Table 1, the change of double layer capacitance was not as high as the ACCEPTED MANUSCRIPT observed With these results, we can conclude that the immunosensor has acceptable stability (Figure 5b) T Specificity of the immunosensor is a crucial factor in the development of microorganism RI P detection tools To investigate the specificity of the immunosensor, we used herpes simplex virus, Salmonella, and E coli O157: H7 bacterium as control sample In Figure 5c, the SC response signal of the immunosensor was not detected for herpes simplex, Salmonella, and E coli O157: H7 The shift in Ret value was ~1200 ohm for the detection of V cholerae O1, NU thereby indicating that the specificity of immunosensor was quite good MA Regeneration is a significant factor in the development of immunosensor for in-field/on-site detection To study the regeneration of the immunosensor, anti-V cholerae O1-immobilized sensor was immersed in a buffer solution containing V cholerae O1 for 90 Washing was ED performed by using a PBS buffer solution and DI water, and drying was performed by using PT nitrogen gas The V cholerae O1 cell concentration was determined by the change in measurement of Ret, as presented in Figure 5d After detection of cells of V cholerae O1, the AC CE immunosensor was dipped into the glycine-HCl buffer (pH 2.8) for about 10 to remove cells Subsequently, the sensor was washed with PBS buffer solution and DI water, and drying was conducted by using nitrogen gas The immunosensor was again measured with cells of V cholerae O1 under the same conditions The obtained results in Figure d indicated that the response signal decreased by approximately 5% on average because biomolecules (antibodies) could be denatured or destroyed by using glycine-HCl buffer CONCLUSION In summary, a CeO2 nanowire-based electrochemical immunosensor for label-free detection of V cholerae O1 cell was developed Electrochemical impedance spectroscopy was used to detect V cholerae O1 cell concentration with [Fe (CN) 6] 3−/4− as redox probe A linear relationship between electron transfer resistance and V cholerae O1 cell concentration was 13 ACCEPTED MANUSCRIPT found in the range of 1.0 × 102 CFU/mL to 1.0 × 104 CFU/mL The detection limit of immunosensor was 1.0 × 102 CFU/mL The immunosensor sensitivity was 58.82 /CFU.mL This immunosensor benefits from the use of a label-free detection method, which is easy to T RI P use and simple The immunosensor could be useful for saving time and reducing costs in clinical testing and could be applied to portable devices used for home tests, as well as for in- improving sensitivity of the immunosensor NU ACKNOWLEDGMENTS SC field/outside detection Thus, future research would focus on the reduction of the size and MA The work was supported by the Ministry of Education and Training under research Project code: B2014.01.78 ED REFERENCES Baumann, P, Furniss, A L and Lee, J V., Genus I Vibrio Pacini 1854 In Bergey’s PT Manual of Systematic Bacteriology, (1984) 518–538 Edited by N R Krieg and J AC CE G Holt Baltimore: Williams and Wilkins Walter H.koch, William L.payne, barry A.wentz, Thomas A.cebula, Rapid Polymerase Chain Reaction Method for Detection of Vibrio cholerae in Foods, Applied and environmental microbiology, 59 (1993) 556-560 Duochun Wang, Xuebin Xu, Xiaoling Deng, Changyi Chen, Baisheng Li, Hailing Tan, Haibo Wang, Song Tang, Haiyan Qiu, Jingdiao Chen, Bixia Ke, Changwen Ke, Biao Kan, Detection of Vibrio cholerae O1 and O139 in Environmental Water Samples by an Immunofluorescent-Aggregation Assay, Applied and environmental microbiology, 76 (2010) 5520-5525 Khemthongcharoen, Wonglumsom, Suppat, Jaruwongrungsee, Tuantranont, Promptmas , Piezoresistive microcantilever-based DNA sensor for sensitive detection 14 ACCEPTED MANUSCRIPT of pathogenic Vibrio cholerae O1 in food sample, Biosensors and Bioelectronics, 63 (2015) 347-353 T Jy-Young Jyoung, SaHyun Hong, Woochang Lee, Jeong-Woo Choi, Immunosensor RI P for the detection of Vibrio cholerae O1 using surface plasmon resonance, Biosensors and Bioelectronics, 21 (2006) 2315-2319 SC Shaohuang Weng, Min Chen, Chengfei Zhao, Ailin Liu, Liqing Lin, Qicai Liu, Jianhua Lin, Xinhua Lin, Label-free electrochemical immunosensor based on NU K3[Fe(CN)6] as signal for facile and sensitive determination of tumor necrosis factor- MA alpha, Sensors and Actuators B 184 (2013) 1–7 Chao Song, Guoming Xie, Li Wang, Lingzhi Liu, Guang Tian, Hua Xiang, DNAbased hybridization chain reaction for an ultrasensitive cancer marker EBNA-1 ED electrochemical immunosensor, Biosensors and Bioelectronics 58 (2014) 68–74 PT Xiaobo Wang, Jingjing Miao, Qing Xia, Kai Yang, Xiaohua Huang, Wenbo Zhao, Jian Shen, A high-sensitivity immunosensor for detection of tumor marker based on AC CE functionalized mesoporous silica nanoparticles, Electrochimica Acta 112 (2013) 473– 479 ia A es, Fi i a B Pimen e , Bego a Gon enri P.A ou s, a ue B ar ues, ar a e -Garc a, M Beatriz P.P Oliveira, Cristina Delerue-Matos, Detection of Ara h (a major peanut allergen) in food using an electrochemical gold nanoparticle-coated screen-printed immunosensor, Biosensors and Bioelectronics 64 (2015) 19–24 10 Gongjun Yang, Wenjie Jin, Liping Wu, Qianqian Wang, Hongxia Shao, Aijian Qin, Bing Yu, Dongming Li, Baoliang Cai, Development of an impedimetric immunosensor for the determination of 3-amino-2-oxazolidone residue in food samples, Analytica Chimica Acta 706 (2011) 120–127 15 ACCEPTED MANUSCRIPT 11 Wenjie Jin, Gongjun Yang, Hongxia Shao, Aijian Qin, A label-free impedimetric immunosensor for detection of 1-aminohydantoin residue in food samples based on T sol–gel embedding antibody, Food Control 39 (2014) 185–191 RI P 12 Qin Wei, Yanfang Zhao, Bin Du, Dan Wua, He Li, Minghui Yang, Ultrasensitive detection of kanamycin in animal derived foods by label-free electrochemical SC immunosensor, Food Chemistry 134 (2012) 1601–1606 13 F Long, M He, H.C Shi, A.N Zhu, Development of evanescent wave all-fiber (2008) 952–958 iumasu, Pe mer, Cristina M Weber, Gunther Kolb, David MA 14 Ioan NU immunosensor for environmental water analysis, Biosensors and Bioelectronics 23 Tiemann, Stefan Windisch, Ines Frese, Antonius A Kettrup, A new, versatile field ED immunosensor for environmental pollutants Development and proof of principle with PT TNT, diuron, and atrazine, Biosensors and Bioelectronics 21 (2005) 354–364 15 E Mauriz, A Calle, A Montoy, L.M Lechug, Determination of environmental AC CE organic pollutants with a portable optical immunosensor, Talanta 69 (2006) 359–364 16 A Ramanavicius, Y.Oztkin, A Ramanaviciene, Electrochemical formation of polypyrrole-based layer for immunosensor design, Sensors and Actuators B 197 (2014) 237–243 17 Dong-Min Kim, Md Aminur Rahman, Minh Hien Do, Changill Ban, Yoon-Bo Shim, An amperometric chloramphenicol immunosensor based on cadmium sulfide nanoparticles modified-dendrimer bonded conducting polymer, Biosensors and Bioelectronics 25 (2010) 1781–1788 18 Farzana Darain, Doeg Su Park, Jang-Su Park, Seung-Cheol Chang, Yoon-Bo Shim, A separation-free amperometric immunosensor for vitellogenin based on screen-printed 16 ACCEPTED MANUSCRIPT carbon arrays modified with a conductive polymer, Biosensors and Bioelectronics 20 (2005) 1780–1787 T 19 Kanika Singh, Md Aminur Rahman, Jung Ik Son, Kyung Chun Kim, Yoon-Bo Shim, RI P An amperometric immunosensor for osteoproteogerin based on gold nanoparticles deposited conducting polymer, Biosensors and Bioelectronics 23 (2008) 1595–1601 SC 20 B Garcinuno, I.Ojeda, M.Moreno-Guzman, A.Gonzales-cortes, P.Yanez-Sedeno, J.M.Pingarron, Amperometric immunosensor for the determination of ceruloplasmin NU in human serum and urine based on covalent binding to carbon nanotubes-modified MA screen-printed electrodes, Talanta 118 (2014) 61–67 21 Siming Liu, Ruo Yuan, Yaqin Chai, Huilan Su, A label-free amperometric ED immunosensor based on horseradish peroxidase functionalized carbon nanotubes and bilayer gold nanoparticles, Sensors and Actuators B 156 (2011) 388–394 PT 22 Hee Dong Jang, Sun Kyung Kim, Hankwon Chang, Jeong-Woo Choi, 3D label-free AC CE prostate specific antigen (PSA) immunosensor based on graphene–gold composites, Biosensors and Bioelectronics 63 (2015) 546–551 23 Lifang Zhao, Qin Wei, Hua Wu, Jinke Dou, He Li, Ionic liquid functionalized graphene based immunosensor for sensitive detection of carbohydrate antigen 15-3 integrated with Cd2 þ -functionalized nanoporous TiO2 as labels, Biosensors and Bioelectronics 59 (2014) 75–80 24 Lei Liu, Yingjun Chao, Wei Cao, Yulan Wang, Chuannan Luo, Xuehui Pang, Dawei Fan, Qin Wei, A label-free amperometric immunosensor for detection of zearalenone based on trimetallic Au-core/AgPt-shell nanorattles and mesoporous carbon, Analytica Chimica Acta 847 (2014) 29–36 25 Na Li, Yulan Wang, Yueyun Li, Wei Cao, Hongmin Ma, Dan Wua, Bin Dua, Qin Wei, A label-free electrochemical immunosensor based on Au@Pd/Ag yolk-bimetallic 17 ACCEPTED MANUSCRIPT shell nanoparticles and amination graphene for detection of nuclear matrix protein 22, Sensors and Actuators B 202 (2014) 67–73 T 26 Minghui Yang, Cunchang Wang, Label-free immunosensor based on gold RI P nanoparticle silver enhancement, Analytical Biochemistry 385 (2009) 128–131 27 Kyu-Shik Mun, Sara D Alvarez, Won-Youl Choi, Michael J Sailor, A Stable, Label- SC free Optical Interferometric Biosensor Based on TiO2 Nanotube Arrays, ACS Nano (2010) 2070–2076 NU 28 Ronghui Wang, Chuanmin Ruan, Damira Kanayeva, Kentu Lassiter, Yanbin Li, TiO2 MA Nanowire Bundle Microelectrode Based Impedance Immunosensor for Rapid and Sensitive Detection of Listeria monocytogenes, Nano Letters (2008) 2625–2631 29 Chi-Chang Lin, Yung-Ming Chu, Hsien-Chang Chang, In situ encapsulation of ED antibody on TiO2 nanowire immunosensor via electro-polymerization of polypyrrole PT propylic acid, Sensors and Actuators B 187 (2013) 533–539 30 Pavel Ivanoff Reyes, Chieh-Jen Ku, Ziqing Duan, Yicheng Lu, Aniruddh Solanki, Ki- AC CE Bum Lee, ZnO thin film transistor immunosensor with high sensitivity and selectivity, Applied Physics Letters 98 (2011) 173702 31 Anees A Ansari, Ajeet Kaushik, Pratima R Solanki, B.D Malhotra, Nanostructured zinc oxide platform for mycotoxin detection, Bioelectrochemistry 77 (2010) 75–81 32 Michael S Wilson, R David Rauh, Novel amperometric immunosensors based on iridium oxide matrices, Biosensors and Bioelectronics 19 (2004) 693–699 33 Ajeet Kaushik, Pratima R Solanki, M K Pandey, Sharif Ahmad, Bansi D M alhotra, Cerium oxide-chitosan based nanobiocomposite for food borne mycotoxin detection, Applied Physics letters 95 (2009) 173703 18 ACCEPTED MANUSCRIPT 34 Pratima R Solanki, Md AzaharAli, Ajeet Kaushik, B D Malhotra, Label-Free Capacitive Immunosensor Based on Nanostructured Cerium Oxide, Advanced T Electrochemistry 1(2014), 1–6 RI P 35 Juan Peng, Ying-Di Zhu, Xing-Hua Li, Li-Ping Jiang, E S Abdel-Halim, Jun-Jie Zhu, Electrochemical immunoassay for the prostate specific antigen using ceria mesoporous SC nanospheres, Microchim Acta 181 (2014) 1505–1512 36 Joseph Wang, analytical electrochemistry, John Wiley & Sons, 2006 MA Faraday Soc 1(1947) 11 – 19 NU 37 J E B Randles, Kinetics of rapid electrode reactions, J E B Randles Discuss 38 Usa Sungkanak, Assawapong Sappat, Anurat Wisitsoraat, Chamras Promptmas, ED Adisorn Tuantranont, Ultrasensitive detection of Vibrio cholerae O1 using microcantilever-based biosensor with dynamic force microscopy, Biosensors and PT Bioelectronics 26 (2010) 784–789 AC CE 39 Vepa K Rao, Mukesh K Sharma, Ajay K Goel, Lokendra Singh, and Krishnamurthi Sekhar, Amperometric Immunosensor for the Detection of Vibrio cholerae O1 Using Disposable Screen-printed Electrodes, Analytical sciences, 22 (2006), 1207-1211 40 M K Sharma, A K Goel, L Singh, V K Rao, Immunological Biosensor for Detection of Vibrio cholerae O1 in Environmental Water Samples, World Journal of Microbiology and Biotechnology, (22) 2006, 1155-1159 41 Zhu Qiang, Ruo Yuan, Yaqin Chai, Na Wang, Ying Zhuo, Ying Zhang, Xuelian Li, A ne o en iome ric immunosensor for de ermina ion of α-fetoprotein based on improved gelatin–silver complex film, Electrochimica Acta 51 (2006) 3763–3768 42 Guangying Zhao, Xuejia Zhan, Wenchao Dou, A disposable immunosensor for Shigella flexneri based on multiwalled carbon nanotube/sodium alginate composite electrode, Analytical Biochemistry 408 (2011) 53–58 19 ACCEPTED MANUSCRIPT 43 Farzana Darain, Sang-Un Park, Yoon-Bo Shim, Disposable amperometric immunosensor system for rabbit IgG using a conducting polymer modified screen- T printed electrode, Biosensors and Bioelectronics 18 (2003) 773-780 RI P 44 Lu Qiao, Xiangyou Wang, Xia Sun, A Novel Label-Free Amperometric Immunosensor Based on Graphene Sheets-Methylene Blue Nanocomposite/Gold SC Nanoparticles, Int J Electrochem Sci., (2014) 1399 – 1414 45 Ning Gan, Linghua Meng, Tianhua Li, Yuting Cao, Lei Zheng, A Magnet-Controlled NU and Renewable Amperometric Immunosensor for Carcinoembryonic Antigen Based MA on Magnetic Fe3O4(core)/Au(shell) Nanoparticles Modified Screen-Printed Carbon Electrode, Asian Journal of Chemistry 23 (2011) 3261-3267 46 Xia Sun, Lu Qiao, Xiangyou Wang, A Novel Immunosensor Based on Au Nanopar- ED ticles and Polyaniline/Multiwall Carbon Nano- tubes/Chitosan Nanocomposite Film AC CE PT Function- alized Interface, Nano- Micro Lett (2013) 191-201 20 ACCEPTED MANUSCRIPT 47