Design Of Malate Dehydrogenase Biosensor Based On Screen-printed Electrode

L-malate is an organic acid that has a set of pivotal biological functions and has been widely found in animals, plants and microorganisms. It has great uses in industries of food, pharmaceuticals and chemistry. For quantification of L-malate, many methods, include chromatography, spectroscopy and enzymatic analysis, etc., have been successfully established to date. However, these methods are often too complicated and non-cost-saving, despite they can make the measurements in precise and accurate ways.Malate dehydrogenase(MDH) is responsible for converting malic acid under physiological conditions when its coenzyme, NAD(P)+ is presented. By making use of electrochemical sensors that are NAD(P)H-sensitive, both qualifications and quantifications of L-malate can be realized. According to this principle, amperometric enzyme electrode can be constructed via immobilizing malate dehydrogenase onto the surface of the NADH-sensitive electrode and thus method for detecting L-malate can be established.In this thesis, enzyme biosensor was constructed by first preparing the NADH-sensitive electrochemical substrate with the modification of carbon nanotube, onto which malate dehydrogenase was then immobilized through integrating after processed with chitosan and glutaraldehyde:(1) The experiment first prepared carbon nanotube modified screen-printed electrode Pr/CNT/SPE through modifying the screen-printed electrode(SPE) with a biocomposite acquired from processing carbon nanotube(CNT) along with protamine(Pr). It is found by microscopic characterization results that the use of protamine led successfully to make CNTs forming a stable and compact structure onto the base electrode. This gave rise to the improvement in the NADH sensitivity of the modified electrode. The modified electrode presented a linear response when NADH concentration was among 2-1500 μmol/L, and it has the regressive equation of i(μA) =-1.8764c(mmol/L)- 0.0516, R2 = 0.984. Its response time was 6 s, sensitivity was 1.8764μA·L/mmol with the limit of detection of 0.05μmol/L(S/N=3). Additionally, the experiment also improved the formula of the Pr/CNT preparation. Results shown the optimal sensing effect of Pr/CNT/SPE can be reached when the mass fraction of Pr-CNT was 0.5.(2) L-malate sensitive electrode was then fabricated based on the Pr/CNT/SPE. After processing chitosan(CHIT) with glutaric aldehyde(GDI), enzyme immobilizer with free aldehyde groups was thereby obtained. Immobilized MDH with this immobilizer in appropriate recipe to obtain the immobilized enzyme CHIT-GDI-MDH. Then, modified the base electrode Pr/CNT/SPE with the immobilized enzyme and finally protect it by application of nucleapore membrane, so the integrated enzyme electrode, CHIT-GDI-MDH/Pr/CNT/SPE was thus gotten. The enzyme electrode shown linear responses when L-malate in the range of 5-300 μmol/L and has a regressive equation of i(μA) =-0.69c(mmol/L)- 0.0024(R2 = 0.995); the response time was 6-8s, sensitivity was 0.69μA·L/mmol and limit of detection was 0.03 μmol/L(S/N=3).(3) Optimizations of the preparation method and detecting conditions of the enzyme electrode. Select CHIT-GDI:MDH ratio, p H, working potential and testing temperature as the 4 factors to be the variables for next experiments. After performing single factor experiment through which their effects on the enzyme electrode were identified separately, they were integrated to make an orthogonal test so as to obtain the refined parameters for the enzyme electrode. Results reveal that the optimal preparation method and detecting conditions for the enzyme electrode are: mix 30 U MDH with 100μL CHIT-GDI to prepare CHIT-GDI-MDH; select 7.5 as p H condition of the buffering system; 45℃ as the testing temperature and 0.36 V as the working potential.This enzyme electrode has good stability and reliability, and is adaptive to be applied to make analysis of L-malate in real samples. It is suitable for industrialized productions for easy to be prepared and used.