Study On The Fabrication Of Immunosensor Using Ionic Liquid As Medium

Ionic liquids as a new green solvent have excellent physical and chemical properties, these were widely investigated and used as medium or catalyst for liquid-liquid extraction, organic synthesis and non-aqueous biocatalysis in recent years. However, little report refers to fabrication of immunosensor with ionic liquid and thus study on immunosensors using ionic liquid as medium is of practical significance.The modified procedure using non-solvent and gradually heating was developed for synthesis of ionic liquids with the yield in the range from 88% to 93%, which include 1-butyl-2,3-dimethylimidazole hexafluorophosphate, 1-amyl-2,3-dimethylimidazole hexafluorophosphate, 1-isooct- 2,3-dimethylimidazole hexafluorophosphate. After the crude products were diluted by acetone, they were decolored with activated carbon and almost colorless ionic liquids were obtained. Visible absorption spectrometry of purified samples was measured on the ultra-visible spectrometer, no obvious absorption peak was found in the wavelength range of 400-800nm. Moreover, the results of the IR and 1H-NMR analysis confirmed the structure of the ionic liquids.The density, viscosity, conductivity and solubility in water of ionic liquids were investigated, respectively. The results showed that the density and the conductivity of ionic liquids decreased with increasing the carbon chain of the substitute group. However, a reversible law was observed about the viscosity. As solubility in water of ionic liquids was important to its application, a new spectrophotometry was developed and applied to measure solubility of ionic liquids in water based on the absorbance of imidazole cation at 211nm linearly increasing with the concentration of the ionic liquid in water. It was found the solubility of 1-amyl-2,3-dimethylimidazole hexafluorophosphate is 2.813mg/mL.Silica sol which was hydrolyzed from tetraethoxysilicane in presence of hydrochloric acid, ionic liquid and Aflatoxin B1(AFB₁) antibody were mixed and were dripped onto the surface of glass carbon electrode to fabricate non-labeled immunosensor. In this study, cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were applied to investigate the electrochemistry performance of electrode in which Fe(CN)₆^3-/4-phosphate buffer solution (pH7.4) was empolyed as base solution for test. The results showed that after the immunoreaction the redox peaks decreased while EIS increased. This indicated the immunoreaction hindered the diffusion of Fe(CN)₆^3-/4- occurred on the electrode surface. When monitoring the electrode surface by EIS, the electron transfer resistance of the sensor increased linearly with increasing concentration of AFB₁. Basing on the analysis of EIS plot, we think the reaction on the sensor surface is a electron transfer limited process. In these newly synthesized ionic liquids, 1-amyl-2,3-dimethylimidazole hexafluorophosphate was selected as medium of immunosensor for its good electrochemical response and the smallest electron transfer resistance. Under optimal conditions, the immunosensor was highly sensitive to AFB₁ with a detection limit of 0.1ng/mL(S/N=3) and the linear range was from 1 to 10ng/mL. The immuonsensor had very good stability and could be preserved for 25 days under 4℃. It was successfully applied to determine AFB₁ in several samples.The method similar to those applied to prepare AFB₁ antibody immunosensor was employed to prepare shrimp antibody immunosensor. EIS was employed to investigate changes of electron transfer resistance of immunosensor in different concentration of shrimp allergen. The results showed that the electron transfer resistance of the sensor increased with increasing concentration of shrimp allergen. The linear range was from 1 to 12ng/mL with a detection limit of 0.1ng/mL. The immunosensor could be preserved at least 3 weeks under 4℃. It was successfully applied to determine shrimp allergen in several samples.