Fabrication Of Novel Electrochemical Biosensors And Their Application In Bio/Chemical Analysis

A biosensor is a device for the detection of an analyte that combines a biological component with a physicochemical detector component, which transforms the signal resulting from the interaction of the analyte with the biological element into easily measured and quantified signal. As a hot topic in analytical chemistry, biosensor is an interdisciplinary approach including chemistry, biology, medical science, physics and electronics. Due to their high sensitivity, rapidity, simplicity, low-cost and potential ability for real-time and on-site analysis, biosensors have attracted wide attentions and applied widely in various areas including environmental monitoring, clinical diagnosis, food industry and so on. Focusing on the key issue of biosensors fabrication, how to immobilize biological component onto transducer surface with high stability and high activity, this dissertation concentrated on the use of various materials and modification methods to prepare novel biosensors and electrochemically nonenzymatic sensor. Then, the fabricated electrochemical sensors were applied to bio/chemical analysis. Their structure, characteristics and performances have been investigated by electrochemical methods such as cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and current-time technique (i-t), ultraviolet-visible spectrophotometry (UV-vis), scanning electron microscopy (SEM), transmission electron microscopy (TEM) and energy dispersive X-ray spectroscopy (EDS), etc. The main points of this dissertation are summarized as follows:(1) Semi-interpenetrating polymer network (semi-IPN) hydrogel based on polyacrylamide (PAM) and chitosan was prepared to immobilize redox protein hemoglobin (Hb). The Hb-PAM-chitosan hydrogel film obtained has been investigated by scanning electron microscopy (SEM), UV-vis spectroscopy and cyclic voltammetry. UV-vis spectroscopy showed that Hb kept its secondary structure similar to its native state in the Hb-PAM-chitosan hydrogel film. Cyclic voltammogram of Hb-PAM-chitosan film modified glass carbon (GC) electrode showed that direct electron transfer between Hb and GC electrode occurred. The electron-transfer rate constant was about 5.51±0.30 s-1 in pH 7.0 buffers. Additionally, Hb in the semi-IPN hydrogel film retained its bioactivity and showed excellent electrocatalytic activity toward H2O2. The electrocatalytic current values were linear with increasing concentration of H2O2 in a wide range of 5-420μM. Compared with PAM hydrogel, Hb entrapped in PAM-chitosan semi-IPN hydrogel could exhibit better stability.(2) SWCNTs were functionalized by DNA through theπ-πinteractions between the nanotube sidewalls and the nucleic acid bases. Then the resulted DNA-SWCNTS hybrids were used to immobilize horseradish peroxidase (HRP) on glassy carbon (GC) electrode. Cyclic voltammetry showed that the direct electrochemistry of HRP immobilized on DNA-SWCNTs hybrids was achieved. The DNA interlayer between the SWCNTs and HRP could be used to keep the activity of HRP. Compared with HRP-SWCNTs/GC and HRP-DNA/GC electrodes, the prepared HRP-DNA-SWCNTs/GC electrode exhibited more excellent electrochemical properties. Thus, the prepared HRP-DNA-SWCNTs/GC electrode was proposed as a third-generation H2O2 biosensor. The effect of pH and applied potential on the performance of the biosensor was discussed in detail. Under the optimal conditions, a wide linear range of the propose biosensor for the detection of H2O2 was observed from 6.0×10-7 to 1.8×10-3 M. The detection limit was found to be 3.0×10-7 M. Furthermore, the proposed biosensor displayed very good reproducibility, high stability, and can be used to detect H2O2 in real samples.(3) Layer-by-layer assembly of Hb with DNA functionalized singlewall carbon nanotubes (DNA-SWCNTs) was achieved on glassy carbon electrode surface based on the electrostatic attraction between positively charged Hb and negatively charged DNA-SWCNTs hybrids. Cyclic voltammogram of (Hb/DNA-SWCNTs)n films modified electrodes indicated that direct electrochemistry of Hb was achieved. The dependence of the formal potential on solution pH indicated that one-proton transfer was coupled to each electron transfer in the direct electron transfer reaction. Additionally, Hb in the multilayer films retained its bioactivity and showed excellent electrocatalytic activity toward H2O2, suggesting that such multilayer films could be used as reagentless biosensors. The layer-by-layer assembly of enzymes with nano-hybrids provided a general and useful way to construct sensitive biosensors without using mediators. On the other hand, this would be used as an easily prepared experimental model to fabricate functional nanostructured biointerfaces.(4) First, nano-gold electrode was constructed by electrochemically depositing gold nanoparticles onto a flat gold electrode surface. Then the nano-gold electrode was immersed in the bath containing p-benzoquinone (BQ), chitosan (CS), glucose oxidase (GOD) and ionic liquid (IL) for fabrication of a sensitive glucose biosensor through electrodeposition. The proton consumption during electroreduction of BQ increased the local solution pH near the electrode surface and led to the deposition of CS hydrogel on the electrode surface. Co-deposition of GOD and IL with the CS hydrogel was achieved. The proposed biosensor exhibited a fast amperometric response (<5 s) to glucose. Under the optimal conditions, the proposed biosensor exhibited a high current sensitivity (14.33μA mM-1 cm-2), which was 2.8 times of the biosensor prepared by electrodepositing CS-IL-GOD biocomposite on flat gold electrode. The detection limit for glucose was 1.5μM, which was 20-fold lower compared to the biosensor prepared on flat gold electrode. Moreover, the proposed biosensor exhibited a wide linear range, high reproducibility, long-time storage stability and satisfactory anti-interference ability. The proposed biosensor can applied to detect glucose concentration in serum samples.(5) DNA-Cu2+ complex were immobilized on the surface of GC electrode through electrodeposition under controlled dc potential. The electrodeposited DNA-Cu2+ complex exhibited excellent electrocatalytic behavior towards H2O2. Thus, a nonenzymatic H2O2 sensor was fabricated. The effects of Cu2+ concentration, electrodeposition time and determination conditions such as pH value, applied potential on the current response of the DNA-Cu2+/GC electrode toward H2O2 were optimized to obtain the maximal sensitivity. Under the optimal conditions, the linear range for the detection of the H2O2 is 8.0×10-7 M to 4.5×10-3 M with a high sensitivity of 40.25μA mM-1, a low detection limit of 2.5×10-7 M and a fast response time of within 4 s. Compared with the traditional enzymic sensor, the nonenzymatic H2O2 sensor exhibited better stability and reproducibility.(6) A novel hybrid, based on the combination of CNTs, Nafion and copper nanoparticles (Cunano) was synthesized and used to determination of nitrite. Nafion was used to disperse CNTs and displayed interaction with Cu2+. Then, Cunano were deposited onto CNTs by electrochemically reduction of Cu2+. SEM, TEM and EDS were used to characterize the resulted Cunano/CNTs-Nf hybrid. The results demonstrated that Cunano uniformly coated on CNTs with an average size of 30 nm. The electrochemical experiments indicated that the Cunano/CNTs-Nf hybrid modified electrode showed high electrocatalytic activity for the reduction of nitrite and could used to detect nitrite under a low applied potential of-0.05 V. Under the optimal conditions, The linear range of the determination of nitrite at the Cunano/CNTs-Nf hybrid modified electrode was from 1.0×106 M to 6.0×10-4 M, and the detection limit was found to be 8.0×10-8 M. The low detection potential gived the Cunano/CNTs-Nf hybrid modified electrode good stability and anti-interferent ability.