Nanochannel-based Enzyme Sensor For Detection Of Glucose

Solid-state nanochannels have been broadly studied for their application in the development of innovative detection methods because of their advantages of porous structure with a large surface area,tunable pore size,and easy functionalization.Among the methods base on the nanochannel platform,signals generated from the change of the channel aperture is one of the main approaches.It’s known that enzymes have the advantages of high catalytic activity.However,the research on enzymatic catalysis in nano-channels is rarely reported.Anodic aluminum oxide(AAO)nanochannels were used as platforms,glucose oxidase(GOx)and horse radish peroxidase(HRP)as model enzymes The catalytic properties of enzymes in the nanochannel were determined and then the methods for organic polymer and inorganic nanoparticles catalyzed were compared.Finally several detection methods were developed as the applications of nanochannel sensing for detection of glucose in this dissertation,which are summarized as the follows.1,Study on enzyme catalysis induced polymer growth in nanochannels and enzymatic kineticsA more efficient signal generation method was explored based on enzyme-catalysis inducing current change by pore shrinking.As a model enzyme,HRP was immobilized in the nanochannels through a drying method.In the presence of H2O2,HRP catalyzed o-phenylenediamine(o-PD)to trigger its polymer growth,in turn blocked the ion transport and led to the decrease of the ion current.The system readily achieved the blocking ratio of ion current reaching to 99.6%of the initial.Based on this concept,we developed a new method to evaluate the enzyme catalysis kinetics in nano-confined space.By comparing with those in free state in solution and absorbed on planar surface,HRP confined in the nanochannel presented similar Michaelis constant(Km)values for the substrate H2O2 but much higher Km values for the substrate o-PD.due to the steric hindrance and diffusion suppression.2.Detection of glucose based on double-enzyme catalyzed polymer growth in nanochannelsThis method was developed based on the established method of enzyme-catalyzed polymer-induced growth in nanochannels.GOx and HRP were mixed and immobilized on the inner wall of the nanochannel by a drying method.GOx catalyzed glucose to produce H2O2 in the presence of O2.Then HRP catalyzed the oxidation of o-PD to trigger its polymer growth,which in turn blocked ion transport and reduced the ion current.Based on those strategies,the method was used to detect H2O2 by HRP-catalyzed polymer growth in nanochannels.Meanwhile,the method was used to detect glucose based on GOx/HRP dual-enzyme catalytic polymer growth in nanochannels.The linear detection range of H2O2 was from 2 to 20 mM with a detection limit of 0.15 mM.The linear detection range of glucose was from 8 to 20 mM with a detection limit of 4.36 mM.This method was able to detect glucose with higher concentration as a new alternative method for glucose analysis.3.A colorimetric biosensor based on enzyme-catalysis-induced production of inorganic nanoparticles for sensitive detection of glucose in wineAs a new colorimetric nanoprobe,enzyme-catalysis-induced production of Prussian blue nanoparticles(PBNPs)was employed to develop a colorimetric biosensor which was simple and inexpensive for rapid detection of glucose in white wine.Briefly,glucose as the detection target was added into a solution of GOx,FeC13 and K3Fe(CN)6,turning the solution color from light-yellow to blue within 10 min.Thus,it could be probed by UV-vis spectroscopy.Unlike common colorimetric method based on sole color change mechanism,this method has two paths to generate PBNPs.Because both K3Fe(CN)6 and O2 are involved in the turnover of GOx catalysis,they generate K4Fe(CN)6 and H2O2 that reduces Fe3+,respectively.and both of paths finally produce PBNPs.Between the two paths,the PBNPs was mainly produced in absence of O2.Under optimized conditions,the biosensing method presented a linear detection range of 4 μM-0.5 mM and a limit of detection of 3.29 μM,which is comparable to or better than analogues.The proposed biosensing method is rapid,simple,sensitive and selective,therefore,it is a promising method for daily detection of glucose in wine.4.Detection of glucose based on enzyme-catalyzed-induced inorganic nanoparticles in nanochannelsIn order to improve the efficiency of signal generation in nanochannels,combined with the results of the previous chapter,a method was developed based on enzyme-catalyzed_induced inorganic nanoparticles in nanochannels for glucose detection.It was found that the current was significantly reduced with the addition of glucose,and PBNPs particles were generated on the surface and channels of GOx-AAO membrane using FeCl3/K3Fe(CN)6 as a substrate.The linear range of glucose concentration was from 12 to 30 mM,and the LOD was 11.60 mM.However,the background noise was high.The method provided a new approach for glucose detection.