The Constructions And Applications Of Inositol Hexakisphosphoric Micelles Enhanced Biosensors

Recently, with the increasing development of nano-technology, the exploration of nano-biosensors has been attracting more and more attention. Enzyme sensor as a kind of biosensors, because of its unique specificity toward the detected substances and high detection sensitivity toward the biological materials, such as hydrogen peroxide, glucose, cholesterol, ascorbic acid, nitrite, which make it into the widespread application. Typically, the performance of enzymatic biosensor depends on the enzymes biological activity. The method of fixing and the search of fixing carriers thus become a key problemon how to maintain the activity of enzyme. Therefore, a simple, fast, stable and controllable method for biosensor preparation method is becoming a hot research topic.Accurate, rapid, and reagentless determination of hydrogen peroxide (H₂O₂) is very important in fields of food, industry, environmental protection, clinical control and so on. Based on its simplicity, high selectivity and intrinsic sensitivity, the electrochemical tracking of biological targets by way of enzyme based H₂O₂ detection is of special attention.Inositol hexakisphosphoric (IP₆), also called phytic acid, a kind of naturally occurring specie as the principal storage form of phosphorus in many plant tissues, especially in brans and seeds, presents unique benefits such as biocompatibility, The nontoxicity, and"green"to the environment. These properties constitute favorable conditions for enzyme or protein immobilization. IP₆ has six phosphates to react easily with the enzyme. Hence, IP₆ should be used to capture enzyme as amolecular binder instead of employing a polymeric binder. Quite recently, IP₆ molecules have been found to self-assemble into a micelle sphere structure under boiling condition. This unique structure is very similar to the structure of biological membranes, in which the constituent lipids are arranged in the tail-to-tail configuration with the hydrophilic head groups toward outside and proteins are adsorbed onto the surface of or imbedded into the layers. Therefore, botanical IP₆ micelle is expected to offer a promising template for enzyme-based biosensor fabrication because of its satisfying biocompatibility and well-distribution in water for easy spread onto substrate. In the present study, by using the nanotechnology and self-assembly technology, the electrochemical sensors constructed with the use of IP₆ and its nano-materials were prepared in order to achieve the determination of H₂O₂. The specific preparation of the three kinds of sensors has been highlighted as follows:(1) Horseradish peroxidase sensor constructed on a glassy carbon electrode substrate by using IP₆ micelle membrane: Two areas are worth of noticing. First, the IP₆ micelles as the basement membrane, absorb large amount horseradish peroxidase. Second, IP₆ micelles constructed by the enzyme sensor successfully achieved the direct electron transfer, and the stable signal as well as the high sensitivity detection of H₂O₂. Nafion/HRP/IP₆ micelles/GCE modified electrode constructed with IP₆ micelles, not only adsorbed HRP on the micelles, but also entrapped HRP inside the spherical micelles. This greatly improved the loading of HRP enzyme on the electrode surface, and the enzyme provides a good biological compatibility environment, to further maintain its biological activity, which thus leading to achieve the accurate determination of H₂O₂. The as-developed preparation method is very facile, environmentally friendly, economy, and directly prepared from plants and can be used for constructing IP₆ micelles biosensor. The horseradish peroxidase achieved the direct electron transfer; showed short response time to hydrogen peroxide, low detection limit, high sensitivity and the smaller Michaelis constant. The current response of H₂O₂ shows two linear relationships within the concentration range from 0.1 to 0.5μmol L^-1 with a correlation coefficient of 0.998 (n = 5) and from 0.6 to 1.6μmol L^-1 with a correlation coefficient of 0.997 (n = 11). The detection limit of 0.1μmol L^-1 is estimated at signal-to-noise ration of 3 (S/N = 3), Michaelis constant of 0.0016 mmol L^-1.(2) A hybrid and bio-compatible sensor constructed with IP₆ micelles phosphate bonded horseradish peroxidase and gold nanoparticles: The nano-composite meterials can not only keep the biological activity of horseradish peroxidase, but also achieve the high electron density, dielectric properties and catalytic performance of gold nanoparticles to facilitate the direct electron transfer. Whereas, Nafion membrane can improve the selectivity and stability. The characterizations of UV - visible absorption spectroscopy and transmission electron microscopy showed that the high conductivity and high catalytic properties of nanoparticles together with the excellent biocompatibility and high enzyme loading IP₆ micelles made the adsorption of HRP maintain its activity. Nafion/HRP-IP₆ micelles-GNPs/GCE biosensor can electrocatalytically reduce H₂O₂. The current response and the concentration of H₂O₂ has a linear relationship within the concentration range of 5×10^-7 1.15×10^-5 mol L^-1 (linear correlation coefficient R = 0.993, n = 9), the detection limit was 0.1μmol L^-1 (signal to noise ratio S / N = 3), Michaelis constant of 0.0024 mmol L^-1.(3) A biosensor integrated with poly (diallyldimethylammonium chloride) (PDDA) and PA-MWCNTs composite film together with the embedding horseradish peroxidase: The sensor was firstly fabricated by the electrostatic self-assembly technology, the positively charged PDDA and negatively charged PA-MWCNTs composite film, and followed by the assembly onto the electrode surface. Subsequently, HRP was adsorbed on the PA-MWCNTs membrane by the electrostatic attraction. The study indicated that HRP composite membrane showed its good catalytic activity toward hydrogen peroxide reduction properties. The current response and the concentration of H₂O₂ has a linear relationship within the concentration range of 8.9×10^-7 to 1.1×10^-5 mol L^-1, the detection limit was 0.1μmol L^-1 (signal to noise ratio S/N =3), is 0.01 mmol L^-1. The constructed sensor shows good stability and reproducibility, and is a sensitive hydrogen peroxide biosensor without the redox medium.