| Electrochemical biosensors combining the specific recognition of biomaterials with the magnification function of electrochemical determination have the advantages of high-sensitivity, nice-selectivity as well as easy miniaturization and automation. They can be applied to a wide range of analytical tasks, such as bioassay, environmental monitoring and industrial analysis. Nanomaterials have special structure, which results in series of interesting physical and chemical properties. In our work, the nanomaterials and composite nanomaterials are used to construct the electrochemical biosensors by means of the combination of biochemistry and electrochemical methods and we aim to develop new types of biosensors based on nanomaterials for the purpose of improving the long-term stability and the higher sensitivity of biosensors. The details are summarized as follows:Chapter One: PrefaceA critical review with regard to the biosensor and nanotechnology is given. In particular, the application of nanotechnology into biosensors is highlighted. After being introduced nanomaterials, the physical and other attributes of biosensors can be improved and analyzing multiple components in vivo is becoming available.Chapter Two(1) Biosensor based on TTF@SiO2 nanoparticles as electron transfer mediatorfor the determination of glucoseIn this section, tetrathiafulvalene (TTF) was doped in SiO2 network and the nanocomposite was used as mediator for the selective detection of glucose. The uniform TTF doped silica (TTF@SiO2) nanoparticles were prepared by a water-in-oil (W/O) micromulsion method, and were characterized by transmission electron microscopy (TEM). The core-shell of structure TTF@SiO2 could prevent TTF from leaching out into the aqueous solutions. Combined with chitosan (CHIT), which served as a scaffold for glucose oxidase and nanocomposite immobilization, the GCE/TTF@SiO2-CHIT-GOx biosensor was developed. Under optimal conditions, the biosensors exhibit a linear range of 1.0×10-5 to 5.0 × 10-3 mol/L with the detection limit down to 5.0 μmol/L (S/N = 3). The excellent selectivity, sensitivity and stabilityof the glucose biosensor show the potential for practical applications.(2) Amperometric Sensor Based on Neutral Red-Doped Silica NanoparticlesCoupled with Microdialysis for the Measurement of Glutamate in the RatStriatumAmperometric sensor based on neutral red-doped silica (NR@SiO2) nanoparticles (NPs) was fabricated and coupled with a microdialysis sampling system for the detection of glutamate (Glu) in the rat striatum. The NR@SiO2 NPs (about 45 ± 3 nm) were prepared with water-in-oil (W/O) microemulsion method, and characterized by transmission electron microscope (TEM) technique. The neutral red (NR) doped in silica network could maintain its high electroactivity and behave as an excellent electron mediator for electrocatalysis of hydrogen dioxide. Furthermore, the silica surface could prevent the leakage of NR, hence, the stability of biosensor was improved. The novel Glu biosensor showed a linear range from 5.0 × 10-7 to 1.5 × 10-4 mol/L, with a detection limit of 5.0 × 10-7 mol/L (S/N = 3).Chapter Three(1) Development of Quantum Dots Modified Acetylcholinesterase Biosensor for the Detection of TrichlorfonPoly (N-vinyl-2-pyrrolidone) (PVP)-capped CdS quantum dots (QCdS-PVP) was synthesized with CdCl2 and Na2S in the presence of PVP. QCdS-PVP has been used for the immobilization and stabilization of the acetylcholinesterase (AChE). The electrocatalytic activity of QCdS-PVP leads to a greatly improved electrochemical detection of the enzymatically generated thiocholine product. In addition, the sensitivity and the stability of the biosensor are also improved. The GCE/QCdS-PVP/AChE biosensor was used for the detection of organophosphate pesticides (OPs), such as trichlorfon. The sensor performance, including pH and inhibition time, was optimized with respect to operating conditions. Under the optimal conditions, the biosensor was used to measure as low as 12 ppb trichlorfon with a 5-min inhibition time.(2) CdS Quantum Dots Derived Reagentless Uric Acid BiosensorA reagentless uric acid (UA) biosensor based on uricase immobilized on PVP-CdS quantum dosts was developed. Direct electrochemistry and thermal stability of immobilized uricase were studied. PVP-CdS quantum dosts show an electrocatalytic activity to the oxidation of uric acid without the presence of an electron mediator. The current response showed a linear dependence on the uric acid concentration ranging from 5.0 μmol/L to 1.0 mmol/L with a detection limit of 2.0 μmol/L at 3a. The attractive features might be attributed to the unique PVP-CdS quantum dosts nanorods matrix, which provided a favorable microenvironment for enzyme loading. Such successful procedures in enzyme immobilization may lead to a novel method in biosensor construction.Chapter Four(1) Biosensor Based on Iron Porphyrin Nanoparticles for Amperometric Detection of GlucoseA new type of iron (III) meso-tetraphenylporphyrin (FeTPP) nanoparticles (NPs) was synthesized and its application in glucose biosensor was described. A glassy carbon (GC) electrode was modified with the FeTPP NPs, and then the glucose oxidase (GOx) was coated on the FeTPP NPs-terminated layer by cross-linking BSA with GOx via glutaraldehyde. The biosensor prepared under optimal conditions, the biosensor showed a fast response time (3s), high sensitivity (3.43 mA mol-1 L cm-2), long-term operational stability, good suppression of interference and low detection limit (5.0 μmol/L). A linear calibration plot was obtained in the wide concentration range from 10 μmol/L to 2.1 mmol/L. A possible mechanism for the operation of the glucose biosensor was also proposed. Furthermore, the level of glucose in rat brain was also detected in order to demonstrate the practical usage of this biosensor.(2) Amperometric biosensor for hypoxanthine based on immobilized xanthine oxidase on iron (III) meso-tetraphenylporphyrin nanoparticles modified glassy carbon electrodeThe preparation and performance of hypoxanhine electrochemical biosensor, which was based on iron (III) weso-tetraphenylporphyrin (FeTPP) nanoparticles (NPs), is reported in this work. FeTPP NPs were prepared by mixing solvent techniques with diameters ca. 25 ～ 45 nm and were used as a mediator. The XOD/FeTPPNP/GC electrode exhibited good amperometric signal for Hx. The biosensor could detect the concentration of Hx up to 0.34 mmol/L with a detection limit of 1.0 μmol/L. The usefulness of this biosensor for the analysis of real samples was also demonstrated by determining Hx in rat brain dialysate coupled with microdialysis. (3) Amperometric biosensor based on immobilization acetylcholinesterase on manganese porphyrin nanoparticles for detection of organosphate pesticides with flow injection systemA highly sensitive flow injection amperometric (FIA) biosensor for the detection of organophosphate pesticides (OPs) is developed. The biosensor was fabricated by immobilized acetylcholinesterase (AChE) on manganese (III) meso-tetraphenylporphyrin (MnTPP) nanoparticles (NPs)-modified glassy carbon (GC) electrode. The MnTPP NPs used in this article were synthesized by mixing solvent techniques. AChE enzyme was immobilized on the MnTPP NPs surface by conjugated with chitosan (CHIT). The electrocatalytic activity of MnTPP NPs led to a greatly improved performance for thiocholine (TCh) product detection. The developed AChE-CHIT/MnTPPNP/GC biosensor integrated with a flow injection system was used to monitor OPs. The sensor displayed a linear response over a concentration range of 1.0 × 10-9mol/L～ 1.0 × 10-3 mol/L for trichlorfon with a detection limit of 0.5 × 10-9 mol/L, over a range 1.0 × 10-7 ～ 9.0 × 10-4 mol/L for phoxim with a detection limit of 5.0 × 10-4mol/L, and a range 2.0 ×10-7 ～1.0 × 10-3 mol/L for dimethoate with a detection limit of 1.0 × 10-7 mol/L.
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