Application of Hydrothermally Grown ZnO Nanorods for Electrochemical Biosensors

ZnO nanorods have been used on Au work ­ ing electrodes of biosensors for enhancing bi ­ osensor characteristics. ZnO nanorods grown on working electrodes have been employed for fabricating not only glucose sensors but also electrochemical immunosensors for detecting Legionella pneumophilia. The sensitivity of the ­ se biosensors was enhanced substantially compared to typical electrochemical biosen ­ sors based on Au working electrodes.


Introduction
It is often important to measure quantity of an analyte precisely and electrochemical biosensors can be very useful in determining quantity of the an alyte because the output of an electrochemical sen sor is usually proportional to the amount of the ana lyte. Electrochemical sensing method has several advantages including its simplicity in detection, fast response, high selectivity, and relatively low cost [1]. Therefore, an electrochemical sensors have been under development for 50 years, ever since Clark reported upon the first enzyme electrode on which glucose ozidase (GOx) was immobilized [2].
In enzymatic electrochemical sensors, a good enzyme immobilization on the sensor electrode is of vital importance for the realization of high perfor mance sensors. This is because this type of sensors measures the redox current of the products, such as hydrogen peroxide, which are produced by enzyme substrate reactions. Recently, nanostructured metal oxides, such as zinc oxide (ZnO), nickel oxide (NiO), and copper oxide (CuO), have been employed as a part of the working electrode of enzyme biosensors because enzymes with a low isoelectric point (IEP, ~4.2) can be effectively immobilized on the nanostructured metal oxides with high lEPs ( > 9) by electrostatic interactions [3,4] ZnO nanorods have been considered for the biosensors due to large surface area, nontoxicity, biocompatibility, high electrochemical activity, and fast electron transfer [5,6]. The ZnO nanorodsbased matrix film with large surface area has at tracted considerable interest for applications of the biosensors because the sensitivity of an electro chemical biosensor is closely related to the working electrode surface area Chen et. al. and Gupta et.
al. have tried to fabricate a enzyme biosensor such as a glucose biosensor by using ZnO composite matrix film in order to improve the sensitivity [7,8]. In this paper, the effect of the matrix-based ZnO nanorods without composite material is described using both an electrochemical immunosensor for Legionella pneumophila detection and an electro chemical glucose sensor.

Glucose Biosensor Using Hydrothermally Grown ZnO Nanorods
A 500 pm thick 4" 7740 glass wafer was used as a substrate for the fabrication of the electro chemical glucose biosensor based on the ZnO na norod arrays and the network-shaped ZnO nano rods. The 0.02 cm2 area Au/Ti electrodes were pat terned on a glass wafer, and then a polyimide pas sivation layer was patterned onto the Au/Tipatterned substrate. The photoresist was patterned on the substrate for the lift-off process and the lift off was used for patterning the ZnO nanorods The ZnO nanorod seed solution [9] seed solution was coated onto the substrate by a spray method. The hydrothermal growth of the ZnO nanorods was car ried out by keeping the seed solution-coated sub strate in a growth solution at 90°C for 4 hours [9,10]. The substrate was then rinsed with deion ized water and dried with N2 gas. The ZnO nano rods were piled on the substrate. Finally, the pat terned ZnO nanorod electrode was obtained, as shown in Fig. 1 In order to immobilize the GOx on the patterned ZnO nanorod electrode, a 1.5 pL GOx solution was dropped onto the patterned ZnO nanorod electrode, and then the electrode was kept at 4°C overnight It is believed that the GOx immobilization on the pat terned ZnO nanorods was enhanced by an electro static interaction. After the immobilization process, the electrode was washed to remove the unimmobi lized GOx The as-prepared GOx/ZnO/Au/Ti work-ing electrode, a platinum counter electrode, and an Ag/AgCI reference electrode made up the three electrodes of the fabricated glucose biosensor. They were used for the measurement of the output redox currents by using a potentiostat.
The crystallographic information and the chem ical composition of the as-grown network-shaped ZnO nanorods were examined by XRD patterning and EDX spectroscopy, respectively. In Fig. 3(a), all of the diffraction peaks of the ZnO nanorod ar rays and network-shaped ZnO nanorods can be ndexed to the hexagonal wurtzite phase of ZnO, which matches well to the standard XRD data file. The dominant peaks for the ZnO nanorod arrays and the network-shaped ZnO nanorods are (100) and (101) peaks, in contrast to the vertically grown ZnO, where the dominant peak is (002) [11]. Figure  3(b) shows that the chemical composition of the grown ZnO nanorods is the sum of the Zn and O without any other impurities, which confirms the ~igh purity of the grown ZnO nanorods (Zn:O=1:1.18).

0 (Degree) = 5 2. The results of (a) the XRD scan and (b) the = DX spectrometry of the as-grown network-shaped ZnO nanorods
schematic drawing of the working principle of the g ucose biosensor based on the ZnO nanorod elec tee is shown in Fig. 3. The electrochemical reac-:~.s for the detection of the glucose in presence of glucose oxidase are as follows [12]: When a positive potential is applied to the working electrode with respect to the reference electrode, the hydrogen peroxide is oxidized on the working electrode surface, according to Eqn (2). The electrochemical experiments were carried out by using the as-prepared GOx/ZnO/Au/Ti elec trode at various concentrations of glucose and GOx. At various concentrations of glucose level ranging from 0 to 50 mM, the cyclic voltammograms (CVs) of GOx/ZnO/Au/Ti electrode are shown in Fig. 4(a). It is observed from the CVs that the redox current increases with glucose concen tration because the oxidation current of H2O2 in creases with glucose concentration. Fig. 4(b) shows a calibration curve for the redox current measured at + 0.75 V versus the reference elec trode at various glucose concentrations. It can be seen that the response current increases with the glucose concentration, but the response time satu rates at a certain high glucose concentration due to the active sites of the enzymes saturating [13]. The fabricated glucose biosensor showed a good linearity from 10 pM to 7 mM glucose concentration levels with the correlation coefficient of 0.99842 and a high sensitivity of 41.76 pAcm'2mM'1, which is re markably higher than previously reported data. Limit-of-detection (LOD) of the fabricated biosensor was found to be about 1 pM, which is one of the lowest values compared to the previously reported glucose biosensors. It is believed that the high sensitivity and the low LOD of the fabricated bio sensor are attributed to the large surface area of the network-shaped ZnO nanorods and to the di rect electron transfer from the vertically grown ZnO nanorod arrays on the substrate.

Electrochemical immunosensor based on ZnO nanorods for detecting Legionella pneumophila
This part describes the fabrication of an elec trochemical immunosensor based on ZnO nano rods matrix for L. pneumophila detection as a rapid and sensitive method for on-site diagnosis. In this study, a primary antibody was immobilized on the ZnO nanorods matrix and a second antibody con jugated to an enzyme label such as horse radish peroxide (HRP) was used as the detection anti body. The fabricated immunosensor was evaluated in acetate buffer containing 3,3',5,5'-tetramethylbenzidine (TMB), which is a good electron transfer mediator, with H2O2 by using an electrochemical measurement system. A 800 pm thick 3" 7740 glass wafer was used as the substrate for realizing the electrochemical immunosensor. Au/Ti was deposited on a glass wafer and the Au/Ti electrode was patterned by the photolithography. Then, polyimide as the pas sivation layer was patterned on the Au/Ti-patterned substrate. The photoresist was patterned on the substrate for realizing the ZnO nanorods-based electrode. ZnO nanorod seed solution was coated on the substrate by a spray method. Hydrothermal ZnO nanorods growth was carried out by suspend ing the seed solution-coated substrate in a growth solution at 90°C for 4 h in an oven. The substrate was then rinsed with deionized water and dried with N2 gas. Finally, the patterned ZnO nanorods electrode was obtained after removing the photore sist with acetone and dried with N2gas.
A primary antibody as polyclonal, antigen (pep tidoglycan-associated lipoprotein, PAL), and poly clonal antibody-HRP of Legionella were obtained from Prof. Min Ja Kim at Korea University. For im mobilizing the primary antibody on the ZnO nanorods matrix-based electrode, various primary anti body solutions (1 ~ 4 pg/mL) were prepared by di lution. A primary diluted antibody solution was dropped on the ZnO nanorods matrix-based elec trode for immobilizing the antibody on the elec trode, and then the electrode was kept at 37 °C for 1 hour. After washing step, the primary antibody of various concentrations was immobilized on the ma trix-based ZnO. Then, a BSA/PBS solution was dropped on the electrode for blocking the blank space of the electrode, on which the primary anti body was immobilized, and then the electrode was kept at 37 °C for 1 hour for immobilizing the BSA on the ZnO nanorods. Afterward, various concen tration antigen solutions as PAL proteins were pre pared by the dilution. An antigen solution was dropped on the electrode in order to bind the anti gen to the antibody. The electrode was kept at 37°C for 1 hour and washed in the PBS. A polyclo nal antibody-HRP was dropped on the electrode in order to bind the antibody conjugated HRP to the antigen. The electrode was kept at 37 °C for 1 hour and washed in the PBS. Figure 5 shows schematic pictures of the fabricated electrochemical im munosensor. The electrochemical measurements were car ried out in 0.1 M acetate buffer solution using a po tentiostat. According to the following Eqn. (1), elec trochemical reaction for the detection of the antigen as Legionella was proposed to be following [14]: TMB as an electroactive molecule can be oxi dized by HRP or reduced by electrochemical reac tion. The oxidized TMB (TMB+) is generated by the HRP catalytic reaction, and TMB is reduced by the electrochemical reaction. When reducing to TMB, the reduction current value would be related to the antigen concentration. A ZnO nanorod working electrode of the immonosensor Pt wire counter electrode, and Ag/AgCI reference electrode were used in cyclic voltammetry.
The hydrothermally grown ZnO nanorods were successfully patterned on the Au electrode. The diameter of ZnO nanorods was about 700 ~ 900 nm, and the length of ZnO nanorods is about 5 ~ 7 pm The crystal structures of ZnO nanorods were analyzed by X-ray diffraction technique (XRD). All the diffraction peaks of the matrix-based ZnO na norods can be indexed to the wurtzite ZnO with h gh crytallinity. The main peaks for the matrixcased ZnO nanorods are (100) and (101) peaks, in contrast to vertically grown ZnO, where the main peak is (002) [15].
The electrochemical experiments were carried out by using the fabricated immunosensors, on which the primary and second antibodies of 2 pg/mL and various antigen concentrations When various concentrations of antigen (PAL protein) were applied to the working electrode, in case of the bare elec trode, no electrochemical reaction was observed in absence of the oxidized TMB It can be seen that the reduction current increases as the antigen con centration increased, and the reduction peak cur rents of the oxidized TMB were observed at + 0.25 V The reduction currents of the oxidized TMB in creased with antigen concentration.
It is important to immobilize the antibody on the electrode for antigen detection using the fabricated mmunosensors. The electrochemical characteristics of the fabricated immunosensors were evaluated at various antibody concentrations by fixing antigen concentration at 5 ng/mL. The output currents of the *abricated immunosensors increased with the anti body concentration up to 2 pg/mL. However, at -igher concentration of antibody, at 4 pg/mL, the output current was lower than the current at 2 pg/mL antibody as shown in Fig. 6(a). It is believed that this result was attributed to the increase of the re sistance and double layer capacitance of the ZnO nanorods-based electrode because of the decrease of ratio of sensing sites in the ZnO nanorods-based electrode [16,17].
The fabricated immunosensors with the various concentrations of the antibody and the antigen showed the response currents in Fig. 6(b). The fab ricated immunosensors based at 1 pg/mL and 2 pg/mL antibodies showed the response currents in creasing with the antigen concentration. The re sponse currents of the fabricated immunosensors based at 2 pg/mL antibody was higher than those based at 1 pg/mL antibody with increasing the anti gen concentration A limit of detection of the fabri cated immunosensor can be detected under 1 pg/mL antigen. In case of the fabricated im munosensors based at a 4 pg/mL antibody, the re sponse current increased with the antibody concen tration up to 100 pg/mL, and it gradually decreased at higher antigen concentrations because of increas ing the resistance and double layer capacitance of the ZnO nanorods-based electrode [16,17].

Conclusion
An electrochemical immunosensor fabricated on matrix-based ZnO nanorods for detecting glu cose or a Legionella pneumophila was introduced The matrix-based ZnO nanorods were successfully grown on the Au electrode hydrothermally at low temperature and were patterned by lift-off as shown in FESEM images XRD scan result exhibit ed that the ZnO nanorods were grown to the Zn and O without impurity. The antibody and GOx can be effectively immobilized on the ZnO nanorods with by electrostatic interaction. The fabricated electrochemical sensors showed higher sensitivity than previously reported electrochemical sensors.