| Objective:Alcohol is the most common poisonous substance related to clinical medicine and ingesting overdose alcohol can lead to the damage of central nerve. At the same time, the incidence of traffic accidents caused by drinking more is increasing. Measurement of blood alcohol concentration (BAC) is seen as the most convincing evidence to judge drunk driving. So, the accurate measurement of BAC quickly and quantitatively is very important for forensic medicine and communication lawsuit.1. Three sliver strips were printed on polyvinyl chloride (PVC) basal lamina. Then carbonic working electrode, carbonic counter electrode and Ag/AgCl reference electrode were completed in order. The prepared screen-printed electrodes (SPE) were stable and had satisfactory performance.2. GNPs-MWCNT-Nafion compounds were prepared and the electrochemistry behaviors of Nafion/SPE, MWCNT-Nafion/SPE and GNPs-MWCNT-Nafion/SPE were discussed. 3. An alcohol biosensor was prepared and the factors which influenced the detection performance of the biosensor were optimized. The detectivity of the biosensor for alcohol was discussed and the technology evaluation was done.Methods:1. To prepare screen-printed electrodes which contain carbonic working electrode, carbonic counter electrode and Ag/AgCl reference electrode. The electrochemistry performances of the prepared screen-printed electrodes were done in [Fe(CN)6]3- /[Fe(CN)6]4- containing NaCl used the method of cyclic voltammetry.2. Gold colloid solution was prepared and Nafion-117 solution was added into the obtained GNPs (volume ratio 1:4) with ultrasonic agitation for 10 min, and then 1 mg MWCNT was dispersed in the prepared GNPs-Nafion suspension by ultrasonic 40 min. A homogeneous, well distributed 1 mg/mL solution of GNPs-MWCNT-Nafion hybrid composites was obtained and the SPE was modified by the compounds. Meanwhile, the Nafion, MWCNT-Nafion and GNPs-MWCNT-Nafion modified SPE were prepared. All modified electrodes were dip into MB solution to absorb MB at cyclic voltammetry scan and the electrochemistry performances of the different modified electrodes were discussed in 0.1 mol/L PBS with the method of cyclic voltammetry.3. The ADH enzyme solution was modified on the working electrode. Hydrophilic membrane with an eyehole in center was placed on the outermost of the working area to make a reaction tank of 5μL. An integrity alcohol biosensor was fabricated eventually. Optimum conditions of the biosensor were experimentally determined by varying several important parameters: solution pH value, environmental temperature, working potential. Quantitation and microamount of standard blood alcohol solution was taken by siphonage and measured by the alcohol biosensor. The relationship of electric current and alcohol concentration was analyzed used the method of chronoamperometry and the standard curve was done to determine the linear response range and the detection limit of the biosensor. The samples were measured used the alcohol biosensor and the stability, the performance of resisting interfering substance were discussed.Results:1. The prepared SPE was length of 29 mm,width of 10 mm,thickness of 0.5 mm and had good electrochemistry performances. The anodic peak currents (ipa) and cathodic peak currents (ipc) were direct ratio with the square root of scan rates when the electrode was experimented in [Fe(CN)6]3-/[Fe(CN)6]4- solution used the method of cyclic voltammetry. So this indicated the prepared SPE correspond to the demand of the experimental study.2. The GNPs-MWCNT-Nafion was homogeneous and the cyclic voltammograms of all modified SPE were obtained at different scan rates in PBS solution. The anodic peak currents and cathodic peak currents were direct ratio with the square root of scan rates which indicated that the electrode reaction was a surface diffusion controlled process. Also, the GNPs-MWCNT-Nafion compounds modified SPE has the highest current response in all prepared.3. The prepared biosensor had steady detection performances for alcohol at optimum experiment conditions and the detection linear response range was 2.0×10-4 25×10-3 mol/L and the detection limit of the biosensor was 5.0×10-5 mol/L (S/N=3). The prepared biosensor need small samples and had favourable reproducibility, stability and anti-interference ability.Conclusion:1. The GNPs-MWCNT-Nafion compounds possess stable performance and can adsorb MB firmly. Also, the compounds can enhance the detection performance because it has the effect of enlarging the current response.2. Sticking the hydrophilic membrane at the outermost of the working area to make a reaction tank achieves the purpose of microassay detection for the determination of BAC. The prepared blood alcohol biosensor can measure alcohol quickly and has a favourable detection performance as alcohol dehydrogenase can catalyze alcohol specific.
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