Electrochemical Biosensor Based On Novel Nanomaterials

Biosensors are a typical cross-product of multisubjects, which is one of the important areas of biotechnology analysis. Over 30 years development, it has become a field of wide-range, multi-subject and cross-intervention which is full of vitality and innovation. Electrochemical biosensor is dominant in biosensors, whose research contents are rich and research fruits are plenty, and applications are wide. With the introduction of nano-technology, the development of biosensors comes to a more advanced stage, showing upgraded performance. Nano effect of the nanomaterials brings about large specific surface area, good chemical stability and biocompatibility, and strong adsorption capacity which can significantly improve the biosensors'performance in aspects of immobilization and signal amplification. In this paper, in order to assemble ordered nano-systems to build new type of electrochemical sensing interface with excellent performance, we use template method and seed-mediated method. The developed electrochemical interfaces can greatly improve the immobilization efficiency and orientation of the biological molecules with good biological activity maintained, and at the same time can provide excellent electron transfer. The developed biosensors have the advantages of simple fabricaiton, excellent performance, good reliability and easy regeneration. The details are summarized as follows:(1) In chapter 2, we report a kind of ordered 3D Au nano-prickle clusters by directly electrodeposited on glassy carbon electrode utilizing the spatial obstruction/direction of the polycarbonate membrane. The proposed 3D nanoclusters are applied to fabricate a sandwich-type electrochemical immunosensor with human IgG as a model analyte. The electrodeposited Au nanoclusters build direct electrical contact and immobilization interface for protein molecules, which does not need post-modification and positioning. Scanning electron microscopy, cyclic voltammetry and alternating current impedance spectroscopy were used to investigate the properties of the modified interface. The deposited Au nanoclusters are stable with good biocompatibility, large specific surface area, and high electron exchange capability. Under the optimized experimental conditions, a wide linear range from 1.0 to 10000.0 ng mL^-1 was reached with a detection limit of 0.5 ng mL-1. The calibration curve fits a second-order polynomial equation very well (R2=0.9914). The developed immunosensor based on Au nano-prickle clusters possesses advantages such as simple fabrication, fast response, low detection limit, wide linear range, easy regeneration, excellent reproducibility and long stability. To our knowledge, the Au nanostructure of special ordered 3D nano-prickle clusters is new for electrochemical immunosensor.(2) In chapter 3, poly(toluidine blue) nanowires (PTBNWs) with the average diameter about 200 nm and length about 5μm were synthesized for the first time using a template-directed electropolymerization strategy with the nanopore polycarbonate membrane template. The morphological characterization was carried out by scanning electron microscopy and transmission electron microscope. By electrochemical polymerization, horseradish peroxidase (HRP) was in situ encapsulated in PTBNWs (denoted as PTBNWs-HRP) for potential biosensor application. In the system obtained, the PTBNWs served as an excellent redox mediator exhibiting high efficiency of electron transfer between the HRP and the GC electrode for the reduction of H2O2. The proposed electrode can be used as an excellent amperometric biosensor for H2O2 at -0.1 V with a linear response range covering from 1μM to 28 mM, a detection limit of 1μM (based on 3 S/N) and a fast response time of less than 8 s.(3) In chapter 4, we report a simple, biomolecular friendly protocol for the fabrication of a hydroxyapatite nanowires array (HANWA) biosensor of spatial positioning and large surface area with abundant adsorbing sites and its application to cyanide sensing. The fabrication of HANWA is performed by template-assisted electrodeposition. The well-aligned hydroxyapatite nanoarray is composed of vertical nanowires with the diameter of about 200 nm and the average length of 1μm. The electrochemical biosensor for the determination of cyanide through its inhibitory effect on horseradish peroxidase (HRP) encapsulated by chitosan (CHIT) on the platform of hydroxyapatite nanowires array is demonstrated. The present organic-inorganic hybrid material provides excellent enzyme-substrate contact with enzyme activity well maintained. The densely distributed HANWA with large surface area and abundant adsorbing sites can provide favorable electrochemical interface for the construction of electrochemical biosensor. A sensitive detection limit of 0.6 ng mL-1 was obtained for cyanide. The proposed HANWA/CHIT-HRP biosensor has the advantages of spatial resolution, high sensitivity, rapid regeneration and fast response associated with individual nanowires. It broadens the possible applications of chemosensors and biosensors and offers an alternative method for toxic substance determination. The new device holds great promise for environmental and food industrial monitoring of toxins. (4) In chapter 5, for the first time, we introduced the seed-mediated method to the growth of cobalt hexacyanoferrate nanoparticles (CoNPs), using 3.5 nm gold nanoparticles as seeds and multiwalled carbon nanotubes (MWCNTs) as growth scaffold which would both show synergistic action toward the reduction of H2O2. Via gold seeds, the one-step fabrication of CoNPs on the glassy carbon electrode is simple without any linking reagents, which will ingeniously exert the electrochemical properties of cobalt hexacyanoferrate. Combined with glucose oxidase, the sensing surface is applied as a biosensor for glucose. The growth of CoNPs is a chemical deposition process around the small Au nanoseed particles. The nanoseeds bridge the CoNPs and CNTs to form a smart nanocomposite. Spherical CoNPs have a relatively moderate dispersion on the three-dimensional network of CNTs with relatively even diameter ca. 100 nm. Whereas, in the control experiment without gold seeds cobalt hexacyanoferrate can only form continuous films, of which the size is far from nano level and the catalytic ability is poor. The synthesis and fabrication/modification is simple and fast without prior preparation of nanoparticles and lengthy process of cross-linking. The amount of the seeds and CNTs, growth time and concentration of growth solution were investigated. Scanning electron microscopy and electrochemical method were used.(5) In chapter 6, a seed-mediated method was reported to grow platinum nanoparticles (PtNPs) onto glass carbon electrode. GC was first covered with CNT solution. Then 3.5 nm gold seed solution (AuNPseed) was dropped onto the CNT modified GC. After drying, the GC was immersed into the growth solution containing 0.05 M H2PtCl6 and 0.05 M ascorbic acid for 4 h to attach the PtNPs. Glucose oxidase was electrodeposited onto the the surface of AuNPseed/PtNP/CNT to fabricate the glucose electrochemical biosensor. The proposed biosensor showed excellent properties, including high sensitivity (4.49μA mM-1), fast response time (2 s), low detection limit (0.5μM) and wide linear range (1μM– 4 mM). This method can be used to fabricate many oxidase-based biosensors.(6) In chapter 7, a reusable electrochemical aptasensing strategy based on Au nano-coral and intercalation of methylene blue (MB) for highly sensitive detection of small biomolecules using adenosine as a model analyte has been proposed. The 1,6-hexanedithiol modified gold electrode was assembled with 12 nm gold nanoparticles and then immersed in the growth solution containing 1.8×10-4 M HAuCl4, 7.4×10-2 M CTAB, and 4×10-4 M NADH in 37°C to get the enlarged Au Nano-coral around the gold seeds. Electrochemical impedance spectroscopy, alternating current voltammetry, scanning electrochemical microscopy, scanning electron microscopy and surface-enhanced Raman scattering were used to investigate the properties of the modified interface. The competitive mechanism between the adenosine and the MB has been discussed. It was demonstrated that the presented method was highly sensitive and specific with a wide detection range of 4 orders of magnitude and a detection limit as low as 1 nM. As there is no special requirement on the aptamer part, it is fairly easy to extend this strategy to detect many targets by using different aptamers.