Zinc Oxide Tetrapods Based Biohybrid Interface for Voltammetric

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Functional Nanostructured Materials (including low-D carbon)

Zinc Oxide Tetrapods Based Biohybrid Interface for Voltammetric Sensing of Helicobacter pylori Nidhi Chauhan, Shaivya Gupta, Devesh Kumar Avasthi, Rainer Adelung, Yogendra Kumar Mishra, and Utkarsh Jain ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b08901 • Publication Date (Web): 21 Aug 2018 Downloaded from http://pubs.acs.org on August 22, 2018

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ACS Applied Materials & Interfaces

Zinc Oxide Tetrapods Based Biohybrid Interface for Voltammetric Sensing of Helicobacter pylori Nidhi Chauhan,† Shaivya Gupta,† Devesh K. Avasthi,† Rainer Adelung,‡ Yogendra Kumar † Mishra,‡* Utkarsh Jain, * †

Amity Institute of Nanotechnology (AINT), Amity University, Noida - 201303, Uttar Pradesh, India

‡

Functional Nanomaterials, Institute for Materials Science, Kiel University, Kaiserstr. 2, D-24143 Kiel, Germany

Keywords: Zinc oxide tetrapods, ion beam modification, H. pylori, CagA antigen, immunosensor, screen printed electrode

*Corresponding authors: UJ ([email protected]), YKM ([email protected])

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ABSTRACT Helicobacter pylori is a gram negative, spiral shaped, microaerophilic bacteria colonizes human gastric mucosa which causes various gastric diseases. In this work, the utilization of ion irradiated zinc oxide tetrapods (ZnO-T) based biohybrid interface accentuates the development of an electrochemical immunosensor for the fast and sensitive detection of H. pylori. After coating of (ZnO-T) over the surface of screen printed electrode (SP-AuE) through electrodeposition, the ZnO-T/SP-AuE was irradiated with N2+ ion of energy 100 keV. The ion irradiation significantly enhances the conductivity of ZnO-T coated SP-AuE. The revamped SP-AuE is further used for establishing an immunosensor interface based upon immobilization of the CagA antigen on ZnO-T electrodeposited over the surface of SP-AuE. The sensing interface demonstrated good linearity (0.2 ng/ml to 50 ng/ml) and limit of detection (0.2 ng/ml). The ion beam irradiated ZnO-T based immunosensor showed significantly high conductivity and enhanced the analytical properties of the working electrode in terms of the sensitivity, detection limit and response time. A study on the comparison of irradiated and pristine electrode is performed for amperometric sensing of H. pylori. In addition, the significance of work conducted on ion irradiated ZnO-T based interfaces provides a basis of further development of electrochemical immunosensors.

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1. INTRODUCTION Helicobacter pylori (H. pylori) infects the inner lining of stomach of more than 80% of the world population where diagnosis of the presence of bacterium is indispensable.1 It is reported that H. pylori is considered to be as the significant cause for duodenal ulcers, gastritis and gastric adenocarcinoma.2-5 Gastric tumor is the second driving reason for cancer related deaths around the world.6-8 Being a standout amongst the most widely recognized diseases is gastric cancer with H. pylori infection, however it develops over the years after continuous colonization.9 The promising rationale for the difference in the outcomes can be variations in the virulence of H. pylori strains in addition to host, environmental and dietary factors.10 Biomarker for the cancer is an indication for the risk to check the existence of a particular disease or to check the efficiency of a drug therapy.11 Moreover, biomarkers denote the protein concentrations which are present in the blood, serum or tissue for a clinical diagnosis. A considerable biomarker of H. pylori’s virulence factor cytotoxin associated gene A (CagA) protein12 is used herein for the preparation of bio hybrid interface in order to develop H. pylori immunosensor. Although a few conventional methods for the detection of H. pylori are available, however, these methods suffer with drawbacks like long time consumptions, high costs, and very short shelf-life. In order to overcome with these issues, a label free immunosensor has been contemplated for its high sensitivity, cost effectiveness, minimal limit of detection, quick and simple handling.

In context of sensing gas, chemical or biomolecule, the material choice is the first and foremost requirement which is quite well fulfilled by nanoscale structures, such as nanorods, nanowires, nanotubes etc. from various materials because of their extraordinary large surface to volume ratios13. Since the role of parent material directly decides the overall features of its any nanostructure along with additional nanoscale properties, metal oxides have been the most trusted one among the entire family of materials. Among various oxides semiconductors, zinc oxide nanostructures showed superior properties.14-16 Ultra violet (UV) light sensitivity, wide band gap (~3.37 eV), elevated exciton binding energy (~ 60 meV) are the intresting attributes of the ZnO nanostructures which provide enhancements of conductive properties.17 ZnO structural configuration can be moulded into numerous shapes and these configured structures utilizes in various functions and applications. The selection of the ZnOT lies in the fact that emergence of tetrapods, nanorods and nanowires have recently attracted 3



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extensive attention exhibiting novel properties and potential applications in revolutionary nanotechnology.18 The tetrapodal shape illustrates three- dimesnional configuration that has connective four arms from the core center at an average angle of ~ 110° nanoscale features from their arms with respect to each other.19-22 It can be used in different functional way including development of vast, permeable, mechanically adaptable and high temperature stable network. Such interconnected systems make it imperative materials for applications in various interfaces for development of new sorts of multifunctional 3D networks.23-28 Because of the complex 3D structure, the ZnO-T are better than 1D nanostructures considering no agglomeration, unique composites, high conductivity, easy fabrication of electrodes and sensing devices.

In order to make ZnO tetrapods more functional, additional treatements (physical, chemcial, biological, etc.) can be done or even their surface can be hybridzed with other foreign nanostructures (inorganic, carbon, organic, etc.) which lead to a better response29. Here, we have demonstarted for the first time that low energy ion beam (which offers a unique physical treatment to nanostructures) irradiated zinc oxide tetrapods (ZnO-T) coated on SP-AuE substantiate a highly sensitive immunosensor for the detection of test organism Helicobacter pylori.30 Irradiated ZnO-T has uniqueness for the sensing of H. pylori because of enhanced electrochemical properties due to synergistic effect of ion beam irradiation and ZnO-T which in addition pave the way for diverse nanotechnological functions. ZnO-T has several benefits over other ZnO materials due to the three-dimensional distribution of tetrapodal legs. One of the main advantages of tetrapod over other nanostructured ZnO materials is that it gets automatically oriented on the substrate with one of its arms directed normal to the substrate surface29. The ion beam treatment improved electrochemical properties of the electrodes made of the ZnO tetrapod to creation of edge defects or decreased the ZnO-T size and thus sufficient accessibility to electrons between electrolyte and electrodes is facilitated. The smaller the particle size, the higher the depth of space charge region. Greater space charge regions provides better electronic conductance and thereby capacitance. The high surface area of ZnO-T nanomaterials is employed to immobilize the antibodies due to its strong adsorption capability. These three-dimensional tetrapods is more appropriate for the 4


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immobilization of antibody used further in immunological reaction. In the present work, the electroconductivity of SP-AuE was firstly enhanced through depositing ZnO-T on the Au surface. ZnO-T offer large surface (with good electrical conductivity) accessible to antigen or antibodies interaction. The low energy ion beam ion irradiation was performed before antigen loading over the modified electrode. Electrical conductivity is the most important parameters towards sensing performance of any materials based device. The increased conductivity of the sensing material play a crucial role in enhanced sensitivity of the device, which can either be achieved by modification like doping or ion irradiations.32 Additionally the irradiation further improves electrochemical property of ZnO-T@ SP-AuE. The present work analyses the outcome (improved analytical properties) of low energy ion treatment on ZnO-T modified SP-AuE. The results obtained after various steps of surface modifications, the biological interface was prepared for immunosensing having high sensitivity, wide linearity, low limit of detection (LOD) with good precision and analytical recovery. The analysis prevails the importance of ZnO-T@ SP-AuE sensing interface suitable for biological samples analysis.

2. MATERIALS AND METHODS 2.1 Materials. CagA antibody and antigen (200 μg/ml) were purchased from Santa Cruz Biotechnology, Santa Cruz, California, USA. ZnO-T were synthesized by flame transport synthesis (FTS) approach developed, Kiel University, Germany. Bovine serum albumin (BSA) was supplied by Sigma Aldrich Co. (St. Louis, MO, USA). K3Fe(CN)6, K4Fe(CN)6, Na3PO4 (dibasic and monobasic) was obtained from Sisco Research Laboratory (SRL), Mumbai, India. SP-AuE was procured from Dropsens. Double distilled was used throughout the experiments. All electrochemical behaviours including cyclic voltammetry (CV), impedance spectroscopy measurements and electrodeposition of conducting materials were carried out using potentiostat (Bio-Logic – Science Instruments SP-200 with EC-Lab software with an impedance analysis module) (Figure S1). Scanning electron microscopy (SEM; ZEISS EVOHD with EDX detector) characterization of the modiﬁed electrodes were carried out at Indian Institute of Technology, New Delhi and the low energy ion beam facility (LEIBF) was performed at Inter University Accelerator Centre, New Delhi. 5



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2.2 Synthesis of ZnO-T. For synthesis of ZnO-T, zinc microparticles and PVB (polyvinyl butyral) were mixed together in a ratio of 2:1. The mixture was filled by ¾ volume into the crucible and was then heated in muffle furnace for 30-90 minutes at 900 °C. After preheating the furnace, at 450 °C temperature was followed by the insertion of crucible. However, it was observed that tetrapods started instigating at 600 °C. As the temperature of the furnace was very high, PVB started burning and in result providing Zn microparticles in the form of flames which later on converted into Zn nano and microstructures. Through controlled Flame transport synthesis (FTS) approach, formation of ZnO tetrapods with diverse arm morphologies and their interlinked networks were attained.32-34

2.3 Treatment of ZnO-T by UV irradiation. To increase the conductivity of ZnO-T, it was illuminated with long wave UV light of 365 nm for 30-40 mins. By treating with UV light, oxygen vacancies can be created on the ZnO surface, which lead to significant change in its intrinsic properties e.g., increase in conductivity.35 The UV treated ZnO-T were proceeded further electrochemical studies. ZnO-T were dissolved in ethanol in 1:10 ratio36 for subsequent electrochemical study.

2.4 Electrodeposition of ZnO-T on the surface of SP-AuE. The eletrodeposition was performed with a Bio-Logic (Science Instruments SP-200) electrochemical workstation. A standard three electrode configuration was used. The SP-AuE was used as the working electrode, a Pt wire as counter electrode, and an Ag/AgCl (KCl saturated) as the reference electrode. The electrochemical cell contains 5mg/ml ZnO-T suspension, 0.5 mM sodium phosphate buffer (pH 7.5) and 5 mM potassium ferricyanide and potassium ferrocyanide. The modification of SP-AuE was done with ZnO-T through electrodeposition technique (cyclic voltammetry) with potential range of -1.0 V to -1.2 V for 10 cycles at 50 mVs−1. After deposition, the samples were rinsed with DI water and dried under a stream of compressed air.

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2.5 Ion beam treatment of the ZnO-T@ SP-AuE. Irradiation of ZnO-T@SP-AuE films was performed by 100 keV N+ ions at two fluences viz. 1 x 1015 and 1 x 1016 ions cm-2 at a current of 1 µA cm-2 using the low energy ion beam facility (LEIBF) at IUAC, New Delhi. Ion beam was incident on the scanned area of 1 x 1 cm2 at an angle of 90° (normal to sample surface). 2.6 Immobilization of CagA antigen over the ZnO-T@ SP-AuE. After ion beam treatment, the modified electrode’s surface was covered with 10.0 µl CagA antigen and was kept for 12 h incubation at 4 ºC. By slow dipping the electrode into the PBS buffer (pH 7.5), the unbound antigens were removed. Subsequently, with 1% BSA-PBS the modified electrode was treated for 1 h to obstruct the non-specific and unreacted sites. Finally, the electrode was rinsed and dried for the further electrochemical characterization.

2.7. Electrochemical measurements. All the electrochemical studies were performed at room temperature in a three-electrode system (Figure S1). In SP-AuE, Au coated portion was used as a working electrode. Other 2 semi circles were served as reference and counter electrode respectively. Cyclic Voltammetry (CV) and Electrochemical Impedance Spectroscopy (EIS) were carried out in potassium ferricyanide and potassium ferrocyanide electrolyte supplemented with 0.5 mM sodium phosphate buffer (pH 7.5). The setup for electrochemical workstation with SP-AuE was showed in Supporting Information (Figure S1).

3. Results and Discussion 3.1 Characterization evidences for interface preparation. The morphology and size characterization of modified electrodes were studied. Figure 1 a – 1c depicts bare SP-AuE, treated ZnO-T and CagA antigen@ZnO-T/SP-AuE. Figure 1d exhibits the EDX image of the ZnO-T modified electrode. The bare SP-AuE has a uniform and smooth surface (image a). Figure 1b reveals ZnO-T structure after electrodeposition onto SP-AuE. It is observed that there is no shape deformation of ZnO-T. In the inset of Figure 1b which is the zoomed image of ZnO-T powder proves that there is no shape deformation after the UV treatment. It persists the tetrapodal structure even after the UV light treatment. In Figure 1c, CagA antigen has 7



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covered whole interfacial surface by its thick globular structure. EDX spectrum in Figure 1d of ZnO-T/SP-AuE (irradiated with ɸ = 1015; N+ ions) represents the pointers of carbon, nitrogen, oxygen, zinc and gold with percentage weight of 14.16, 9.77, 10.16, 4.15 and 61.76 % respectively. The availability of Zn pointer with other elements proved the existence of ZnO-T onto the SP-AuE (Figure 1, image d). Figure 1 e depicts ZnO-T/SP-AuE after ion beam irradiation with ɸ = 1015; N+ ions. It shows that after treatment of ion beam the tetrapodal structure has been broken and changed into cuboidal structure.

3.2 Electrochemical signal amplification strategy. The electrochemical response of ion beam treated working electrode at different 3 fluences are shown in Figure S2 and the working electrode irradiated with ɸ = 1015 ion/cm2 and ɸ = 1016 ion/cm2, gave the best quasi reversible (oxidation and reduction) performance therefore these fluences were selected for further electrochemical study. Figure 2A gives the CV studies of electrodes before and after N2+ ions treatment (ion beam irradiation with fluences 1×1015 and 1×1016 ions cm-2). Herein, cyclic voltammetry graphs represented the ZnO-T/SP-AuE (unirradiated), ZnO-T/SP-AuE (ɸ = 1×1015), ZnO-T/SP-AuE (ɸ = 1×1016), CagA antigen@ZnO-T/SP-AuE (ɸ = 1015) and CagA antigen@ZnO-T/SP-AuE (ɸ = 1016) in electrolytic solution that contains 0.5 mM sodium phosphate buffer (pH 7.5) and 5 mM potassium ferricyanide and potassium ferrocyanide at 50 mVs−1 . The cyclic voltammetric behavior of unirradiated and irradiated ZnO-T modified SP-AuE are compared. The redox response of the irradiated ZnO-T/SP-AuE with fluence 1×1015 and 1×1016 shows an increase in magnitude of oxidation and reduction at the same potential + 0.08 V and -0.12 V respectively. However, the maximum current response is observed at fluence 1×1015. After immobilization of the antigen onto the irradiated ZnO-T/SP-AuE electrode with fluence 1×1015 and 1×1016 the redox peak are decreased due to the hindrance of electron transfer process in the presence of biological materials (antigen).37 The electrolyte solution and the experimental conditions remains same in all the CV graphs. Oxidation peak current (Ipa) values of CagA antigen@ZnO-T/SP-AuE (ɸ = 1015) and CagA antigen@ZnOT/SP-AuE (ɸ = 1016) electrodes are 0.15 mA and 0.09 mA correspondingly as shown in Figure 2. A significant increase in peak current after irradiation was observed however the highest peak current was observed in the ZnO-T/SP-AuE (irradiated at fluence of ɸ = 1015 8


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ion/cm2). Significant changes in the properties of material occurs after ion irradiation treatment. The irradiation served as an effective tool to control modify the electrode to improve the electrochemical response. The ion irradiation is known to produce defect in controlled manner and modify the properties of material.38-39 Due to nuclear energy loss (Sn), ion energies in keV causes damage while damage governed by electronic energy loss (Se) is in MeV range. The energy exchange of Sn is sufficient to cause nuclear relocations and thereby changes the material properties. The benefit of using low energy ion is economically feasible in contrast to high energy ion beam. At lower energies, the ion irradiation results in defects causing modification of materials by elastic collisions.40

3.3 Electrochemical impedance spectroscopy (EIS) characterization of immunosensor. EIS is an important technique which provides information about the resistance within the electrode material and also the resistance in between the electrolyte and the electrode.41 Figure 2B. represents the impedance curves of bare SP-AuE, ZnO-T/SP-AuE, ZnO-T/SPAuE (ɸ = 1015), ZnO-T/SP-AuE (ɸ = 1016), CagA antigen@ZnO-T/SP-AuE (ɸ = 1015) and CagA antigen@ZnO-T/SP-AuE (ɸ = 1016) obtained in electrolytic solution that contains 0.5 mM sodium phosphate buffer (pH 7.5) and 5 mM potassium ferricyanide and potassium ferrocyanide. Bare SP-AuE possess the highest Ret and once the modification with ZnO-T (unirradiated) occurred, a differentiable drop in the Ret value (1800 Ω) was observed. Subsequently, after ion beam irradiation with different fluences of N+ ions (1×1015 and 1×1016) Ret gradually decreases having the lowest Ret of 160 Ω, recorded for ZnO-T/SP-AuE (ɸ = 1015). The modified electrode irradiation with ɸ = 1015 (low fluence) results in improved charge transfer owing to breakage of H -bonds resulting in creation of active sites enhancing diffusion of Ferricyanide and Ferrocyanide ions towards the surface of modified electrode.42-43 After immobilization of antigens over the irradiated electrodes [CagA antigen@ZnO-T/SP-AuE (ɸ = 1015) and CagA antigen@ZnO-T/SP-AuE (ɸ = 1016)] having large diameter showed greater hindrance to electron transfer as compared to the small semicircle of ZnO-T modified SPelectrodes (irradiated). Both CV and EIS are consistence in showing the modification of the electrode by ion irradiation. As a result, CagA antigen@ZnO-T/SP-AuE (ɸ = 1015) is used for the subsequent studies due to its high conductivity.

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3.4 Analytical performance of the immunosensor. Figure 3A illustrates the CV image of the effect of different concentrations of CagA antibody over the CagA antigen@ZnO-T/SPAuE (ɸ = 1015) at 50 mV/s scan rate with -0.4V to +0.4V potential range obtained in electrolytic solution that contains 0.5 mM sodium phosphate buffer (pH 7.5). The graph provided the information that with the increase in the concentration of CagA antibody current response decreases from 0.2 ng/ml to 50 ng/ml. It was observed that the detection limit of the present immunosensor is improved as compared to the other biosensors based on H. Pylori.4445

.

In Figure 3B calibration curve was plotted and correlation coefficient was calculated, which exhibiting an excellent linear correlation between Ab concentration range (ng/ml) and obtained current (mA). The change in the current of developed immunosensor is linearly relative to antibody concentration in the range of 0.2 to 50 ng/ml. The linear equation (Equation 1) is given below: y = -0.001x + 0.058; R² = 0.982

------------------------------------------------------ (1)

From the formula y = mx + c, where m is the slope of the line and b is the intercept. Correlation Coefficient is 0.982 and slope of the line is -0.001. The experiment was repeated 3 times to check the reproducibility and limit of detection was found to be 0.2 ng/ml. Figure 3C corresponds to 3D image plot of developed immunosensor with different concentration of CagA antibody. By increasing concentration of CagA antibody from 0.2 ng/ml to 50 ng/ml, the peak current gradually decreased. Subsequent decrease in the peak current is attributed to the immunocomplex formation between CagA antibody and CagA antigen over ZnO-T/SP-AuE (ɸ = 1015). This was observed since protein covers the surface of the immunoelectrode which hinders the flow of electron or redox reaction.46-48 Figure 4 shows the square wave voltammetry (SWV) study for different concentrations of CagA antibody over the ion beam treated electrode (CagA antigen@ZnO-T/SP-AuE with ɸ = 1015). This study was adopted because the peaks were sharper and better defined at lower concentrations of the antibody than those obtained by cyclic voltammetry. According to obtained result shown in the figure, it indicates that because of rise in the concentration of 10


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antibody, the peak current reduces linearly.49-51The optimum conditions are kept similar to cyclic voltammetry studies. 3.5 Biosensing response studies with different scan rates. The typical CV curves in the Figure 5A is the biosensing response studies of the CagA antigen@ZnO-T/SP-AuE (irradiated) in 5mM potassium ferricyanide and potassium ferrocyanide electrolyte and 0.5 mM PBS (pH 7.5) at different scan rates ranging from 10 to 120 mV/s and the potential ranging from -0.4V to +0.4V. It was observed that a pair of almost symmetrical oxidation and reduction peaks is formed that had increased at approximately same potential, which shows the high stability of the working electrode. In addition, there is increase in the peak to peak separation with increase in the scan rate upto 120 mV/s. Figure 5B demonstrates that the square root of scan rates is directly proportional to the redox peak current from 10 mV/s to 120 mV/s. The oxidation and reduction peak currents are linearly relative to the square root of the relevant scan rate. The equations 2 and 3 between current and square root of scan rate has been expressed as: y = 0.003x + 0.037; R² = 0.991 (Ipa) --------------------------------------- (2) y = -0.002x – 0.025; R² = 0.937 (Ipc) --------------------------------------- (3)

3.6 Effect of pH and temperature. The different parameters of the immunosensor were optimized with pH and temperature studies. Figure 6A depicts the electrochemical behaviour of the immunosensor in the presence of 0.2 ng/ml CagA antibody with 1 ml solution of the buffer (pH ranging from 6 to 8.5) containing electrolyte solution in electrochemical cell. The amperometric response of immunosensor decreases with increasing the pH rate. However, 7.5 pH was selected as the optimum pH value for H. pylori detection as it gave the lowest oxidation and reduction peaks among all the pH values. This is due to the formation of maximum immunocomplex on interface at lower acidic or alkaline surroundings.52 Figure 6B shows the effect of temperature on immunosensor, which is carried out in sodium phosphate buffer and electrolytic solution containing 0.2 ng/ml CagA antibody. The electrolytic solution is kept at various temperatures ranging from 10 °C to 50 °C at an interval

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of 5 °C. It is found that the peak current response gradually decreases when the temperature increases from 10 °C to 50 °C. However, the minimum current response is observed at 30 °C.

3.7 Selectivity, Reproducibility and stability. Selectivity is the important indicators of specificity and anti-interference ability of immunosensor. Selectivity studies were performed by using Cag A, Bab A, Vac A, BSA, and alpha-fetoprotein (AFP) solutions (0.2 ng/ml) as interfering substances. The results were shown in Figure 7. When results compared with the current response caused by CagA Ag, the variation in current generated by other interferents was less than 20%, even in the condition that the concentration of the interfering agents was 5 times higher than that of CagA. This study demonstrated that the present immunosensor was able to detect the CagA effectively with high specificity and anti-interference ability. The shelf-life of the CagA antigen@ZnO-T/SP-AuE (ɸ = 1015) was examined by calculating the current response through cyclic voltammetry in the presence of 0.2 ng/ml standard CagA antibody solution containing phosphate buffer at 7 days interval. It is observed that developed electrode keeps holding the activity upto 90% for the period of 8-9 weeks when stored at 4 o

C, which is higher than earlier reported electrochemical44 and voltammetric45 sensors for H.

pylori. The observation concludes that the loss in activity of immunoelectrode is due to denaturing of the CagA protein and not due to the instability of ZnO-T/SP-AuE (ɸ = 1015). 3.8 Serum sample analysis. From Bio-diagnostics Lab, Rohini, New Delhi, five H. pylori positive human serum samples were collected for the clinical estimation of H. pylori using CagA antigen@ZnO-T/SP-AuE (ɸ = 1015). The relative standard deviation (RSD) method was chosen for the calculation of the precision, having CagA antibodies were analysed 5 times in human serum samples. With the repetition of the same experiments for five times, the average recovery was calculated. The RSD calculated found to be 2.2% and the recovery is 98.6%.

4. CONCLUSION For the first time, the ion irradiated zinc oxide tetrapods based biohybrid interface work with novel CagA antigen@ZnO-T/SP-AuE is shown to the effectively integrated in a simple way to build an immunosensor for Helicobacter pylori detection. It was observed that once 12


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unirradiated and irradiated ZnO-T/SP-AuE compared with respect to electroconductivity, the irradiated electrode shows excellent electroconductivity by significantly enhancing the peak currents and found to be more specific and sensitive. Results from the studies reveal that CagA antigen@ZnO-T/SP-AuE can detect CagA antibody in a wide concentration range of 0.2 – 50 ng/ml with low limit of detection 0.2 ng/ml. Considering the consistency and performance of proposed immunosensor, it can be further explored in clinical diagnosis of Helicobacter pylori through CagA antigen@ZnO-T/SP-AuE.

 ACKNOWLEDGEMENT We would like to express our sincere thanks to IUAC, New Delhi for doing ion beam irradiation and Amity Institute of Advanced Research and Studies (AIARS) for performing SEM. We are grateful for their cooperation during the period of our experiment. Kiel authors acknowledge the financial support from Deutsche Forschungsgemeinschaft (DFG) under schemes SBF 677 (C14) and GRK 2154 (Project P3).

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(13) Mishra, Y. K.; Adelung, R. ZnO Tetrapod Materials for Functional Applications. Mater. Today 2018, DOI: 10.1016/j.mattod.2017.11.003. (14) Jones, N.; Ray, B.; Ranjit, K. T.; Manna, A. C. Antibacterial Activity of ZnO Nanoparticle Suspensions on A Broad Spectrum of Microorganisms. FEMS Microbiol. Lett. 2008, 279 (1), 71-76. (15) Saad, L.; Riad, M. Characterization of Various Zinc Oxide Catalysts and Their Activity in The Dehydration-Dehydrogenation of Isobutanol. J. Serb. Chem. Soc. 2008, 73 (10), 9971009. (16) Janotti, A.; Van de Walle, C. G. Fundamentals of Zinc Oxide as A Semiconductor. Rep. Prog. Phys. 2009, 72 (12), 126501. (17) Wang, Z. L. ZnO Nanowire and Nanobelt Platform for Nanotechnology. Mater. Sci. Eng. : R: Reports 2009, 64 (3-4), 33-71. (18) Bhakat, C.; Singh, P. P. Zinc oxide Nanorods: Synthesis and Its Applications in Solar Cell. Int J Mod Eng Res 2012, 2, 2452-2454. (19) Lao, J. Y.; Wen, J. G.; Ren, Z. F. Hierarchical ZnO Nanostructures. Nano Letters 2002, 2 (11), 1287-1291. (20) Tian, Z. R.; Voigt, J. A.; Liu, J.; Mckenzie, B.; Mcdermott, M. J.; Rodriguez, M. A.; Konishi, H.; Xu, H. Complex and Oriented ZnO Nanostructures. Nat. Mater. 2003, 2 (12), 821. (21) Tsivion, D.; Schvartzman, M.; Popovitz-Biro, R.; Joselevich, E. Guided Growth of Horizontal ZnO Nanowires with Controlled Orientations on Flat and Faceted Sapphire Surfaces. ACS nano 2012, 6 (7), 6433-6445. (22) Nasajpour, A.; Ansari, S.; Rinoldi, C.; Rad, A. S.; Aghaloo, T.; Shin, S. R.; Mishra, Y. K.; Adelung, R.; Swieszkowski, W.; Annabi, N. A Multifunctional Polymeric Periodontal Membrane with Osteogenic and Antibacterial Characteristics. Adv. Funct. Mater. 2018, 28 (3), 1703437. (23) Tiwari, S.; Vinchurkar, M.; Rao, V. R.; Garnier, G. Zinc Oxide Nanorods Functionalized Paper for Protein Preconcentration in Biodiagnostics. Sci. Rep. 2017, 7, 43905. (24) Paulowicz, I.; Postica, V.; Lupan, O.; Wolff, N.; Shree, S.; Cojocaru, A.; Deng, M.; Mishra, Y. K.; Tiginyanu, I.; Kienle, L. Zinc Oxide Nanotetrapods with Four Different Arm Morphologies for Versatile Nanosensors. Sens. Actuators, B 2018, 262, 425-435.

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(25) Wang, J.; Wang, Z.; Huang, B.; Ma, Y.; Liu, Y.; Qin, X.; Zhang, X.; Dai, Y. Oxygen Vacancy Induced Band-Gap Narrowing and Enhanced Visible Light Photocatalytic Activity of ZnO. ACS Appl. Mater. Interfaces 2012, 4 (8), 4024-4030. (26) Mishra, Y. K.; Kaps, S.; Schuchardt, A.; Paulowicz, I.; Jin, X.; Gedamu, D.; Freitag, S.; Claus, M.; Wille, S.; Kovalev, A. Fabrication of Macroscopically Flexible and Highly Porous 3D Semiconductor Networks From Interpenetrating Nanostructures By A Simple Flame Transport Approach. Part. Part. Syst. Charact. 2013, 30 (9), 775-783. (27) Mishra, Y. K.; Modi, G.; Cretu, V.; Postica, V.; Lupan, O.; Reimer, T.; Paulowicz, I.; Hrkac, V.; Benecke, W.; Kienle, L. Direct Growth of Freestanding ZnO Tetrapod Networks for Multifunctional Applications in Photocatalysis, UV Photodetection, and Gas Sensing. ACS Appl. Mater. Interfaces 2015, 7 (26), 14303-14316. (28) Costa, S. V.; Gonçalves, A. S.; Zaguete, M. A.; Mazon, T.; Nogueira, A. F. ZnO Nanostructures Directly Grown on Paper And Bacterial Cellulose Substrates Without Any Surface Modification Layer. Chem. Commun. 2013, 49 (73), 8096-8098. (29) Smazna, D.; Rodrigues, J.; Shree, S.; Postica, V.; Neubüser, G.; Martins, A.; Sedrine, N. B.; Jena, N. K.; Siebert, L.; Schütt, F. Buckminsterfullerene Hybridized Zinc Oxide Tetrapods: Defects and Charge Transfer Induced Optical and Electrical Response. Nanoscale 2018, 10, 10050-10062. (30) Chauhan, N.; Jain, U. Influence of Zinc Oxide Nanorods on the Sensitivity of A Glycated Hemoglobin Biosensor. Adv. Mater. 2016, 7 (8), 666-672. (31) Modi, G. Zinc Oxide Tetrapod: A Morphology with Multifunctional Applications. Adv. Nat. Sci.: Nanosci. Nanotechnol. 2015, 6 (3), 033002. (32) Qiu, Y.; Yan, K.; Deng, H.; Yang, S. Secondary Branching and Nitrogen Doping of ZnO Nanotetrapods: Building A Highly Active Network for Photoelectrochemical Water Splitting. Nano Lett. 2011, 12 (1), 407-413. (33) Mishra, Y. K.; Kaps, S.; Schuchardt, A.; Paulowicz, I.; Jin, X.; Gedamu, D.; Wille, S.; Lupan, O.; Adelung, R. Versatile Fabrication of Complex Shaped Metal Oxide NanoMicrostructures and Their Interconnected Networks for Multifunctional Applications. KONA Powder Part. J. 2014, 31, 92-110. (34) Antoine, T. E.; Hadigal, S. R.; Yakoub, A. M.; Mishra, Y. K.; Bhattacharya, P.; Haddad, C.; Valyi-Nagy, T.; Adelung, R.; Prabhakar, B. S.; Shukla, D. Intravaginal Zinc Oxide Tetrapod Nanoparticles as Novel Immunoprotective Agents Against Genital Herpes. J. Immunol. 2016, 196 (11), 4566-4575. 16


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(35) Vai, A. T.; Kuznetsov, V. L.; Dilworth, J. R.; Edwards, P. P. UV-induced improvement in ZnO thin film conductivity: a new in situ approach. J. Mater. Chem. 2014, 2 (45), 96439652. (36) Lupan, O.; Chow, L.; Chai, G. A Single ZnO Tetrapod-Based Sensor. Sens. Actuators, B 2009, 141 (2), 511-517. (37) Jain, U.; Chauhan, N. Glycated Hemoglobin Detection with Electrochemical Sensing Amplified by Gold Nanoparticles Embedded N-doped Graphene Nanosheet. Biosens. Bioelectron. 2017, 89, 578-584. (38) Naundorf, V. Diffusion in Metals and Alloys Under Irradiation. Int. J. Mod. Phys. B 1992, 6 (18), 2925-2986. (39) Verma, A.; Srivastav, A.; Sharma, D.; Banerjee, A.; Sharma, S.; Satsangi, V. R.; Shrivastav, R.; Avasthi, D. K.; Dass, S. A Study on The Effect of Low Energy Ion Beam Irradiation on Au/TiO2 System for Its Application In Photoelectrochemical Splitting of Water. Nucl. Instrum. Methods Phys. Res. B 2016, 379, 255-261. (40) Prakash, J.; Tripathi, A.; Rigato, V.; Pivin, J.; Tripathi, J.; Chae, K. H.; Gautam, S.; Kumar, P.; Asokan, K.; Avasthi, D. Synthesis of Au Nanoparticles at the Surface and Embedded in Carbonaceous Matrix by 150 keV Ar ion irradiation. J. Phys. D: Appl. Phys. 2011, 44 (12), 125302. (41) Palanisamy, S.; Vilian, A. E.; Chen, S.-M. Direct Electrochemistry of Glucose Oxidase at Reduced Graphene Oxide/Zinc Oxide Composite Modified Electrode for Glucose Sensor. Int. J. Electrochem. Sci 2012, 7 (3), 2153-2163. (42) Joshi, N.; Sharma, A.; Asokan, K.; Rawat, K.; Kanjilal, D. Effect of Hydrogen Ion Implantation on Cholesterol Sensing Using Enzyme-Free LAPONITE®-Montmorillonite Electrodes. RSC Adv. 2016, 6 (27), 22664-22672. (43) Si, X.; Wei, Y.; Wang, C.; Li, L.; Ding, Y. Sensitive Electrochemical Sensor For Ofloxacin Based on Graphene/Zinc Oxide Composite Film. Anal. Methods 2018, 10, 19611967. (44) Del Pozo, M.; Alonso, C.; Pariente, F.; Lorenzo, E. DNA Biosensor for Detection of Helicobacter Pylori Using Phen-Dione As the Electrochemically Active Ligand in Osmium Complexes. Anal. Chem. 2005, 77 (8), 2550-2557. (45) Ly, S. Y.; Yoo, H.-S.; Choa, S. H. Diagnosis of Helicobacter Pylori Bacterial Infections Using A Voltammetric Biosensor. J. Microbiol. Methods 2011, 87 (1), 44-48.

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(46) Choudhary, M.; Kumar, V.; Singh, A.; Singh, M.; Kaur, S.; Reddy, G.; Pasricha, R.; Singh, S.; Arora, K. Graphene Oxide Based Label Free Ultrasensitive Immunosensor for Lung Cancer Biomarker, hTERT. Biosens. Bioelectron. 2013, 4 (4), 1-9. (47) Jain, U.; Gupta, S.; Chauhan, N. Detection of Glycated Hemoglobin with Voltammetric Sensing Amplified by 3D-Structured Nanocomposites. Int. J. Biol. Macromol. 2017, 101, 896-903. (48) Jain, U.; Gupta, S.; Chauhan, N. Construction of An Amperometric Glycated Hemoglobin Biosensor Based on Au–Pt Bimetallic Nanoparticles and Poly (indole-5carboxylic acid) Modified Au Electrode. Int. J. Biol. Macromol. 2017, 105, 549-555. (49) Ciltas, U.; Yilmaz, B.; Kaban, S.; Akcay, B. K.; Nazik, G. Square Wave Voltammetric Determination of Diclofenac in Pharmaceutical Preparations and Human Serum. Iran. J. Pharm. Res. 2015, 14 (3), 715. (50) Wang, J. Electroanalytical Techniques In Clinical Chemistry And Laboratory Medicine, John Wiley & Sons: 1988. (51) Kissinger, P.; Heineman, W. R. Laboratory Techniques In Electroanalytical Chemistry, Revised And Expanded, CRC press: 1996. (52) Li, C.; Chen, X.; Wang, N.; Zhang, B. An Ultrasensitive and Label-Free Electrochemical DNA Biosensor for Detection of DNase I Activity. RSC Adv. 2017, 7 (35), 21666-21670.

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Graphic for manuscript

19



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Captions to Figures Scheme 1.

Schematic illustration of the steps involved in the preparation of the CagA antigen @ZnO-T/SP-AuE immunosensor.

Figure 1

SEM micrograph image of (a) bare SP-AuE, (b) UV treated ZnO-T electrodeposited onto SP-AuE; Inset : Satellite Image of UV treated ZnO-T powder, (c) CagA antigen@ZnO-T/SP-AuE and (d) EDX spectrum ZnO-T/SPAuE.

Figure 2A

CV curves of ZnO-T/SP-AuE (unirradiated), ZnO-T/SP-AuE (ɸ = 1×1015), ZnOT/SP-AuE (ɸ = 1×1016), CagA antigen@ZnO-T/SP-AuE (ɸ = 1015) and CagA antigen@ZnO-T/SP-AuE (ɸ = 1016) obtained with 5mM [Fe(CN)6]3−/4− electrolyte containing 0.5 mM sodium phosphate buffer (pH 7.5) at 50 mVs−1.

Figure 2B

Electrochemical impedance plots (Nyquist plots) for stepwise assembly of bare SP-AuE, ZnO-T/SP-AuE,ZnO-T/SP-AuE (ɸ = 1015), ZnO-T/SP-AuE (ɸ = 1016), CagA antigen@ZnO-T/SP-AuE (ɸ = 1015) and CagA antigen@ZnO-T/SP-AuE (ɸ = 1016) in [Fe(CN)6]3−/4− electrolyte containing 0.5 mM sodium phosphate buffer (pH 7.5). The frequency range is between 0.01 - 1,00,000 Hz with amplitude of 5 mV.

Figure 3A

Biosensing response studies (CV) of CagA antigen@ZnO-T/SP-AuE (ɸ=1015) with different CagA antibody concentrations at 50 mVs−1 between -0.4 and +0.4 V in 5mM [Fe(CN)6]3−/4−electrolyte containing 0.5 mM sodium phosphate buffer (pH 7.5).

Figure 3B

Standard calibaration curve of CagA atigen@ZnO-T/SP-AuE (ɸ=1015) with different CagA antibody concentrations.

Figure 3C

3D representation of the cyclic voltammogram data for CagA antigen@ZnO-T/SPAuE (ɸ=1015) with different CagA antibody concentrations.

Figure 4

Biosensing response studies through Square wave voltammetry (SWV) of CagA 20


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antigen@ZnO-T/SP-AuE (ɸ=1015) with different Anti CagA concentrations in 5mM [Fe(CN)6]3−/4−electrolyte containing 0.5 mM sodium phosphate buffer (pH 7.5).

Figure 5A

The CV response of CagA antigen@ZnO-T/SP-AuE (ɸ=1015) scanned in 5 mM [Fe(CN)6]3−/4− electrolyte containing 0.5 mM sodium phosphate buffer (pH7.5) with 0.2 ng/ml Anti CagA at different scan rates from 10 to 120 m V s-1.

Figure 5B

The dependency of peak currents on variation of peak oxidation (Ipa) and reduction (Ipc) as a function of square root of scan rate.

Figure 6A

Biosensing response studies (CV) to check the pH effect (pH 6 – pH 8.5) on the current response of CagA antigen@ZnO-T/SP-AuE (ɸ=1015) with 0.2ng/ml CagAantibody at 50 mVs−1 between -0.4 and +0.4 V in 5mM [Fe(CN)6]3−/4− electrolyte containing 0.5 mM sodium phosphate buffer (pH 7.5).

Figure 6B

Biosensing response studies (CV) to check temperature effect (10– 50°C) on the current response of CagA antigen@ZnO-T/SP-AuE (ɸ=1015) with 0.2ng/ml Anti CagAat 50 mVs−1 between -0.4 and +0.4 V in 5mM [Fe(CN)6]3−/4− electrolyte containing 0.5 mM sodium phosphate buffer (pH 7.5).

Figure 7

Plot of interferents studies of CagA antigen@ZnO-T/SP-AuE (ɸ=1015) in 0.5 mM sodium phosphate buffer (pH 7.5).

21



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Scheme 1

22


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2

Figure 1

1 µm 23



0.3 0.2

Current (mA)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 49

0.1 0.0 -0.1 ZnO-T/SPE CagA Antigen@ZnO-T/SP-AuE (  1015; N2+ ions)

-0.2

CagA Antigen@ZnO-T/SP-AuE ( 1016; N2+ ions) ZnO-T/SP-AuE (  1015; N2+ ions)

-0.3 -0.4

ZnO-T/SP-AuE (  1016; N2+ ions)

-0.2

0.0

0.2

0.4

Potential (V)

Figure 2A

24


Page 25 of 49

ZnO-T/SP-AuE CagA antigen@ZnO-T/SP-AuE (  1016;N2+ ions)

6000

CagA antigen@ZnO-T/SP-AuE ( 1015;N2+ ions)

5000

Bare SP-AuE ZnO-T/SP-AuE (  1015;N2+ ions) ZnO-T/SP-AuE (  1016;N2+ ions)

4000

-Im (Z)/Ohm

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


3000 2000 1000 0 0

1000

2000

3000

4000

Re (Z)/Ohm

Figure 2B

25



0.06

0.04

0.02

Current (mA)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 49

0.2 ng/ml 50 ng/ml 40 ng/ml 30 ng/ml 20 ng/ml 10 ng/ml 1 ng/ml 0.5 ng/ml 0.2 ng/ml

50 ng/ml

0.00

-0.02

-0.04

-0.06 -0.4

-0.2

0.0

0.2

0.4

Potential (V)

Figure 3A

26


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Figure 3B

27



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Figure 3C

28


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120

100

Current (µA)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.2 ng/ml 0.5 ng/ml 1 ng/ml 10 ng/ml 20 ng/ml 30 ng/ml 40 ng/ml 50 ng/ml

0.2 ng/ml

80

50 ng/ml 60

40

20 -0.4

-0.2

0.0

0.2

0.4

Potential (V)

Figure 4

29



0.08 0.06 0.04

Current (mA)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 49

0.02

10 mV/s 20 mV/s 40 mV/s 60 mV/s 80 mV/s 100 mV/s 120 mV/s

120 mV/s

10 mV/s

0.00 -0.02 -0.04 -0.06 -0.4

-0.2

0.0

0.2

0.4

Potential (V)

Figure 5A

30


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Figure 5B

31



0.3

0.2

Current (mA)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 49

0.1

pH 6.0 pH 6.5 pH 7.0 pH 7.5 pH 8.0 pH 8.5

0.0

-0.1

-0.2 -0.4

-0.2

0.0

0.2

0.4

Potential (V)

Figure 6A

32


Page 33 of 49

0.4 0.3 0.2

Current (mA)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.1

10 20 25 30 35 40 45 50

0.0 -0.1 -0.2 -0.3 -0.4

-0.2

0.0

0.2

0.4

Potential (V)

Figure 6B

33



Page 34 of 49

Figure 7

34


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Captions to Figures Scheme 1.

Schematic illustration of the steps involved in the preparation of the CagA antigen @ZnO-T/SP-AuE immunosensor.

Figure 1

SEM micrograph image of (a) bare SP-AuE, (b) UV treated ZnO-T electrodeposited onto SP-AuE; Inset : Satellite Image of UV treated ZnO-T powder, (c) CagA antigen@ZnO-T/SP-AuE and (d) EDX spectrum ZnO-T/SPAuE.

Figure 2A

CV curves of ZnO-T/SP-AuE (unirradiated), ZnO-T/SP-AuE (ɸ = 1×1015), ZnOT/SP-AuE (ɸ = 1×1016), CagA antigen@ZnO-T/SP-AuE (ɸ = 1015) and CagA antigen@ZnO-T/SP-AuE (ɸ = 1016) obtained with 5mM [Fe(CN)6]3−/4− electrolyte containing 0.5 mM sodium phosphate buffer (pH 7.5) at 50 mVs−1.

Figure 2B

Electrochemical impedance plots (Nyquist plots) for stepwise assembly of bare SP-AuE, ZnO-T/SP-AuE,ZnO-T/SP-AuE (ɸ = 1015), ZnO-T/SP-AuE (ɸ = 1016), CagA antigen@ZnO-T/SP-AuE (ɸ = 1015) and CagA antigen@ZnO-T/SP-AuE (ɸ = 1016) in [Fe(CN)6]3−/4− electrolyte containing 0.5 mM sodium phosphate buffer (pH 7.5). The frequency range is between 0.01 - 1,00,000 Hz with amplitude of 5 mV.

Figure 3A

Biosensing response studies (CV) of CagA antigen@ZnO-T/SP-AuE (ɸ=1015) with different CagA antibody concentrations at 50 mVs−1 between -0.4 and +0.4 V in 5mM [Fe(CN)6]3−/4−electrolyte containing 0.5 mM sodium phosphate buffer (pH 7.5).

Figure 3B

Standard calibaration curve of CagA atigen@ZnO-T/SP-AuE (ɸ=1015) with different CagA antibody concentrations.

Figure 3C

3D representation of the cyclic voltammogram data for CagA antigen@ZnO-T/SPAuE (ɸ=1015) with different CagA antibody concentrations.

Figure 4

Biosensing response studies through Square wave voltammetry (SWV) of CagA antigen@ZnO-T/SP-AuE (ɸ=1015) with different Anti CagA concentrations in



Page 36 of 49

5mM [Fe(CN)6]3−/4−electrolyte containing 0.5 mM sodium phosphate buffer (pH 7.5).

Figure 5A

The CV response of CagA antigen@ZnO-T/SP-AuE (ɸ=1015) scanned in 5 mM [Fe(CN)6]3−/4− electrolyte containing 0.5 mM sodium phosphate buffer (pH7.5) with 0.2 ng/ml Anti CagA at different scan rates from 10 to 120 m V s-1.

Figure 5B

The dependency of peak currents on variation of peak oxidation (Ipa) and reduction (Ipc) as a function of square root of scan rate.

Figure 6A

Biosensing response studies (CV) to check the pH effect (pH 6 – pH 8.5) on the current response of CagA antigen@ZnO-T/SP-AuE (ɸ=1015) with 0.2ng/ml CagAantibody at 50 mVs−1 between -0.4 and +0.4 V in 5mM [Fe(CN)6]3−/4− electrolyte containing 0.5 mM sodium phosphate buffer (pH 7.5).

Figure 6B

Biosensing response studies (CV) to check temperature effect (10– 50°C) on the current response of CagA antigen@ZnO-T/SP-AuE (ɸ=1015) with 0.2ng/ml Anti CagAat 50 mVs−1 between -0.4 and +0.4 V in 5mM [Fe(CN)6]3−/4− electrolyte containing 0.5 mM sodium phosphate buffer (pH 7.5).

Figure 7

Plot of interferents studies of CagA antigen@ZnO-T/SP-AuE (ɸ=1015) in 0.5 mM sodium phosphate buffer (pH 7.5).


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Scheme 1



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2

Figure 1

1 µm


Page 39 of 49

0.3 0.2

Current (mA)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.1 0.0 -0.1 ZnO-T/SPE CagA Antigen@ZnO-T/SP-AuE (  1015; N2+ ions)

-0.2

CagA Antigen@ZnO-T/SP-AuE ( 1016; N2+ ions) ZnO-T/SP-AuE (  1015; N2+ ions)

-0.3 -0.4

ZnO-T/SP-AuE (  1016; N2+ ions)

-0.2

0.0

0.2

Potential (V)

Figure 2A


0.4


ZnO-T/SP-AuE CagA antigen@ZnO-T/SP-AuE (  1016;N2+ ions)

6000

CagA antigen@ZnO-T/SP-AuE ( 1015;N2+ ions)

5000

Bare SP-AuE ZnO-T/SP-AuE (  1015;N2+ ions) ZnO-T/SP-AuE (  1016;N2+ ions)

4000

-Im (Z)/Ohm

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 49

3000 2000 1000 0 0

1000

2000

3000

Re (Z)/Ohm

Figure 2B


4000

Page 41 of 49

0.06

0.04

0.02

Current (mA)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.2 ng/ml 50 ng/ml 40 ng/ml 30 ng/ml 20 ng/ml 10 ng/ml 1 ng/ml 0.5 ng/ml 0.2 ng/ml

50 ng/ml

0.00

-0.02

-0.04

-0.06 -0.4

-0.2

0.0

0.2

Potential (V)

Figure 3A


0.4


Figure 3B


Page 42 of 49

Page 43 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3C



120

100

Current (µA)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

0.2 ng/ml 0.5 ng/ml 1 ng/ml 10 ng/ml 20 ng/ml 30 ng/ml 40 ng/ml 50 ng/ml

Page 44 of 49

0.2 ng/ml

80

50 ng/ml 60

40

20 -0.4

-0.2

0.0

0.2

Potential (V)

Figure 4


0.4

Page 45 of 49

0.08 0.06 0.04

Current (mA)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.02

10 mV/s 20 mV/s 40 mV/s 60 mV/s 80 mV/s 100 mV/s 120 mV/s

120 mV/s

10 mV/s

0.00 -0.02 -0.04 -0.06 -0.4

-0.2

0.0

0.2

Potential (V)

Figure 5A


0.4


Figure 5B


Page 46 of 49

Page 47 of 49

0.3

0.2

Current (mA)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.1

pH 6.0 pH 6.5 pH 7.0 pH 7.5 pH 8.0 pH 8.5

0.0

-0.1

-0.2 -0.4

-0.2

0.0

0.2

Potential (V)

Figure 6A


0.4


0.4 0.3 0.2

Current (mA)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 48 of 49

0.1

10 20 25 30 35 40 45 50

0.0 -0.1 -0.2 -0.3 -0.4

-0.2

0.0

0.2

Potential (V)

Figure 6B


0.4

Page 49 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 7


Zinc Oxide Tetrapods Based Biohybrid Interface for Voltammetric

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