Enzymes Immobilized On Superparamagnetic Fe₃O₄ Microspheres And The Application Of Biological Sensors

Biosensor is a new subjects developed by multi-discipline mutual penetration, which demonstrates faster, more accurate, real-time than other detection methods in the detection. It becomes a hot research now. The construction of interface on the sensor substrate surface is a crucial step for development and application of biosensors. Magnetic microspheres which were formed from coating various of function groups on the superparamagnetic Fe₃O₄ surface are used to connect to biologically active substances and to maintain their activity. The magnetic microspheres have been a wide range of applications in biosensors. Because of their application improves the detection sensitivity of sensor greatly, reduces the time of biochemistry reactions, and increasing the detection throughout. We aimed to study new methods of enzyme immobilizing on the superparamagnetic nanoparticles and develop a new biosensor on the above basis.Preparation of Fe₃O₄ nanoparticles. The Fe₃O₄ nanoparticles were obtained by the co-precipitation method. Fourier transform infrared spectroscopy, X-ray diffraction and transmission electron microscope analysis confirm the product obtained is Fe₃O₄ magnetic nanoparticles. The result is that the magnetic nanoparticles which are the face centered cubic structure are the particles we want to get. They are spherical in shape, and the particles size are less than 20 nm. The nanoparticles are fabricated with desired size and good dispersion. The magnetic nanoparticles are superparamagnetic that they have a high magnetic susceptibility in magnetic fields and no residual magnetism after removing external magnetic fields.Immobilized enzyme by embedding. The enzyme was immobilized on magnetic microspheres by using chitosan embedding method. The Fe₃O₄ dispersed in water well mixed with chitosan solution, lipase powder then added to above the mixture solution when it was evaporatted to sticky paste and mixed well. Finally, Fe₃O₄ magnetic nanoparticles embedded enzyme was dried in a freezer dryer. Then, the transesterification reaction of (R, S)-1-phenylethanol was employed as a model to investigate the effect various factors on the reaction, and further to observe the characteristics of the physical methods. The result showed that immobilized enzyme and pure enzyme had almost the same activity. The immobilized enzyme can separate from the reaction substrate in an external magnetic field and turn to next reaction. The characteristics of this method are simplicity in used and relatively big loading capacity of the enzyme, but the enzyme is easy to fall off in this way. This method can be used in non-water catalytic reaction.Immobilized enzyme by chemical cross-linking. We coated silica on superparamagnetic nanoparticles by hydrolysis and the polycondensation of tetraethyl orthosilicate in alcohol-alkali solution. Characterized the magnetic microspheres by fourier transform infrared spectroscopy, X-ray diffraction and vibration sample magnetometer to see their structure, appearance and magnetic properties. Then the peroxidase fixed on Fe₃O₄/SiO₂ composite magnetic beads with the crosslinker, glutaraldehyde. The immobilization peroxidase was able to activate H2O₂ and oxidize N,N-diethyl-p-phenylenediamine sulfate to a colored product. The result of the characterization about magnetic microspheres showed that Fe₃O₄ nanoparticles were coated by a layer of SiO2, having the same structure and lower saturation magnetization, but still showed superparamagnetic and had good magnetic responsiveness in magnetic field. Result from the factor about amount of enzyme, catalyst, color reagent, pH and temperature was that the microspheres maintained high activity, enhanced the stability of enzyme and made enzyme easy to separate. This method is more firmly fixed enzyme.Production of biosensors. Compared the above two ways for enzyme immobilization, chemically was the right one. We used gold nanoparticles instead of the poor conductivity of silicon dioxide as the modified materials. The Au-Fe₃O₄ microspheres gained by citric acid reduction, combined with cysteine to get amino, immobilized peroxidase by glutaraldehyde which was a cross-linking agent and then modified gold electrodes through cysteine. EIS was used to study electrode surface properties and the oxidation peak in current of cyclic voltammetry was used to quantitate. The sensor had good response to hydrogen peroxide. The detection limit was 2.8×10-6mol/L and the linear range was 10²00μmol/L. This sensor had high sensitivity, simply in use and good stability.