Dependence of seed layer thickness on sensitivity of nano-ZnO cholesterol biosensor

The anemone-like ZnO nanostructures have been synthesized by hydrothermal method and were further adsorbed immobilized cholesterol oxidase (ChOx) as a nano-biosensor. In this study, the sensitivity of biosensor were improved by varying the thickness of the ZnO seed layer. The SEM analysis showed changes in thickness of seed layer will not affect the morphologies of anemone-like ZnO nanostructures. The X-ray Diffraction patterns showed that the (002) plane of anemone-like ZnO grown on various thickness of the seed layer was more prouded than other crystal plane. Abioelectrode (ChOx/ZnO/ITO/glass) grown on the 30nm of ZnO seed layer with high sensitivity of 57.533μAmM-1cm-2 (1.488 μA (mg/dl) -1cm-2), a wide sensitive range from 25 to 500 mg/dl. It is concluded that the thinner sputtered ZnO seed layer for growing anemone-like ZnO nanostructure can effectively improve the sensitivity of the ZnO biosensor.


Introduction
According to the World Health Organization [1], heart and cerebrovascular diseases accounted for 20% of the world's cause of death. Many clinical diseases have a strong correlation with cholesterol concentration in the blood, such as heart disease, coronary artery disease, atherosclerosis, hypertension, thrombosis, etc. [2][3].Excess cholesterol is a major cause of heart disease and cerebrovascular disease, and therefore it is important to monitor cholesterol levels in the blood. Development of fast and accurate cholesterol biosensor is extremely important to find those diseases. Due to their exceptional optical and electrical properties, the materials with nanostructures are receiving great attention for enzyme immobilization to improve stability and sensitivity of biosensors [4][5].
Enzymes directly immobilized on the electrode surface is easy to bedeteriorated [6].In order to improve this situation, using appropriate intermediate to modify the electrode,such as polymer, metal oxide or organic-inorganic composite film is necessary. These modifications can be used to secure the enzyme, keep the essence of the enzyme and increase the electron transfer.Therefore selection a suitable intermediate is very important for biosensor. Zinc oxide (ZnO) is an excellent intermediate in the enzyme electrode due toits unique characters [7][8][9][10][11]：(1) It is a semiconductor material with a wide bandgap of 3.37eV. (2) ZnO has a high excitation binding energy of 60mV. (3) The Zn elementis easy to be obtained and cheaper. (4) Zinc oxides can be grown using various deposition methodsat a low temperatures. (6)It has excellent biological safety and biocompatibility. (7) Zinc oxide will be positively charged in a neutral solution due to its high isoelectric point (IEP ~ 9.5).It can let protein or enzyme adsorbed by electrostatic attraction force.
There have been a number of reports on the fabrication of cholesterol biosensor using ZnO thin films and nanostructures [12][13][14], but the sensitivity is too low or the detection range is too narrow. In this study, thesensitivity of ZnO biosensor was improved by varying the thickness of the ZnOseed layer.The anemone-like ZnO nanostructures had been grown on the seed layer by hydrothermal method. The cholesterol oxidase (ChOx) was immobilized on the anemone-like ZnO nanostructures as a cholesterol nano-biosensor.

Experimental
ZnO seeds layer was pre-sputtered on the surface of ITO (4.4Ω/□) glasses to grow a high orientation anemone-like ZnO nanostructures. Different thickness of seed layers with 30nm,50nm and 100nm were prepared by changing sputtering time. In hydrothermal growth, all deionized water was preheated to a temperature of 90 o C before the hydrothermal reaction executed. The 0.05 M zinc nitrate solution made in 45 ml deionized water was mixed with 0.05M hexamethylenetetramine (HMTA) solution made in 45 ml deionized water in serum bottles. Then the electrodes were dipped in solution at 90 o C for 9 hrs. After synthesis was completed, the samples were rinsed with deionized water and dried. After drying the samples, 10μl of ChOx solution (1.0 mg/mL) prepared in phosphate buffer saline(PBS)(50mM,pH ∼7.4,0.9% NaCl) was spread over the surfaces of anemone-like ZnO structures and dried at room temperature for about an hour. The sample was then kept overnight for enzyme immobilization at a temperature of 4 o C by physical adsorption technique and subsequently washed with buffer solution to remove the unbound enzyme molecules. After hydrothermal growth, all the sample were stored at 4 o C.Electrochemical analysis is carried out on an Jiehan 5000 potentiostat/galvanostat using a three-electrode configuration with Ag/AgCl electrode as a reference electrode in PBS solution(50 mM, pH ~7.4,0.9% NaCl) containing 5 mM [FeCN 6 ] 3−/4−. The ZnO sample with nanostructures was used as the working electrode and platinum foil as a counter electrode. The cholesterol solutions have been prepared from PBS solution(50 mM, pH ~7.4,0.9% NaCl) containing 1% of triton-X100 and stored at 4 o C. Fig.1 shows the results of grown ZnOnanostructures by a modified hydrothermal method in this study. The SEM images show the morphologies of these ZnO nanostructures which look like anemones appearances. Fig.1(a) shows that each anemone-like structure has three tofive elongated tentacles.There are lots of spacesamong the tentacles which provide plenty of surfaces for enzyme adsorption. These anemone-like structures basically can be divided into two parts,the stubbybasement structures and elongated tentacles structures, such as shown in Fig.1(b)(d)(f). TheseZnO nanostructures were densely and well arranged on the surfaces of ITO substrates.Thepre-sputtered ZnO seeds layerson ITO top surfaces help to overcome the activation energy for heterogeneous nucleation of ZnO nanostructures on the ITO surfaces.

Materials analysis
Different thicknesses of ZnO seed layer were prepared by changing the sputtering time.TheZnO seed layers of 30nm,50nm and 100nmwere prepared for following hydrothermal growth. The thickness of the seed layer did not have obvious effect on the morphologies of ZnO nanostructures. The morphologies are all similar as shown in Fig.1 except with different lengths.All the appearanceslook like anemones. The variations of ZnOaspect ratio(length/diameter) of anemone tentacles depend on seed layer thickness. The aspect ratio decreases with decreasing the thickness of seed layer as seen in Fig.1(b),(d),(f).A lower aspect ratio leads to lower sensitivity when the ZnO nanostructures were assembled as cholesterol biosensors according to the literature [15].   2 was the X-ray diffraction patterns of anemone-like ZnO nanostructures grown on 100nm ZnO seed layer,50nmZnO seed layer and 30nm ZnO seed layer. The results show that thecrystallinities of anemone-likeZnO were good because the diffraction peaks are sharp and strong. A prouded(002) peakswere observed in the XRD patterns imply the ZnO nanostructures have a strong growth directions along the c axis direction. The SEM and XRD results were consistent as presented in Fig.1 and Fig.2. In Fig.2, there are some other small peaks(denoted by triangles)observed in the Figures which are the diffraction peaks of ITO substrates.

Figure1
.Shows X-ray diffraction patterns of anemone-like ZnO nanostructuresgrown on 100nm ZnO seed layer,50nmZnO seed layer and 30nm ZnO seed layer respectively

Electochemical analysis
The anemone-like ZnO nanostructures were assembled as cholesterol biosensor. The sensitivity and linear sensing range of the biosensor were used to calculate the value of the Michaelis-Menten constant (Km). The electrochemical measurement was employed in a PBS solution(50 mM, pH ~7.4,0.9% NaCl) containing 5 mM [FeCN 6 ] 3−/4− ,the scan range of cyclic voltammetry from -0.2V to 0.6V and with sweep rate of 50 mV/s. Fig.3 show thesethree biosensors had good current responses. Thepotential difference between reduction and oxidation peaksinversely proportional to the number of electrons transported between anode and cathodein the solution [16].The thinner the thickness of seed layer, the less electrical resistance it is which means more electronic transportation in the solution. The oxidation current increases with decreasing the thickness of the seed layer due to less electrical resistance with thinner seed layer as seen in Fig.3 from approximate 1×10-4 to around 3×10-4amper. The anemone-like ZnO nanostructures grown on 30nmZnO seed layer has the maximum amount of oxidation current will lead to a higher sensitivity as shown in Fig.4. The sensitivity is defined as the peak oxidation current change per unit cholesterol concentration in the linear range as shown in the following equation: Sensitivity=ΔI pa /(ΔC ×A) (1) Where ΔIpais the variation of oxidation current peak.△C is theanalyte concentration change.A is the working area of electrode. The sensitivity has increased significantly by reducing the thickness of theZnO seed layer from 100nm to 30nm. The best sensitivity obtained in this study is 57.5 µA/mM/cm2 as the ZnO nanostructure prepared on the 30nm seed layer.

Conclusion
In this study,the thinner sputtered ZnO seed layer for growing anemone-like ZnO nanostructure can effectively improve the sensitivity of the ZnO biosensor. The best sensitivity can be obtained in this study is 57.5 µA/mM/cm2 as the ZnO nanostructure grown on the 30nm seed layer. Thelinear sensitive range is from 25 to 500 mg/dl.