Preparation Of Disposable Saliva α-Amylase Biosensor

ObjectiveFollowing rapid social progress and serious competition, mental illnesses are as common as influenza. Mental stress that is not treated can lead to serious illnesses. Several literatures revealed obvious increases in the salivaryα-amylase activity following psychosocial stress could identify people's mental stress condition.1. A disposable screen-printed electrode has been developed and Glucose oxidase,alpha-glucosidase were immobilized on it to examineα-amylase activity. 2. A disposableα-amylase biosensor was developed based on ferrocene as an electron transfer mediator and Glucose oxidase,alpha-glucosidase were immobilized by the indirect cross-linking method. The biosensor could detect the concentration ofα-amylase quickly and accurately. This will build the foundation for a new method justifying people's stressful condition.Methods(1)The PVC plate was coated with the sliver ink as the conductive track. Aside from the two ends of the PVC plate, the PVC ink was deposited onto the conductive track as the insulating layer. The one end of the plate uncoated with PVC ink was an electrical contact site, while the other end was printed with the carbon ink as the working electrode. The electrode performance experiment were carried out in conventional three-electrode cell containing KCL and [Fe(CN)6]3- /[Fe(CN)6]4- solution with an SCE as the reference, a platinum wire as the counter electrode, and screen-print electrode as the working electrode.(2)A disposableα-amylase biosensor based on ferrocene as an electron transfer mediator has been developed by the screenprinting technique. Glucose oxidase and alpha-glucosidase were immobilized on the screen-printed electrode modified with the ferrocene and Nafion. The gelatin solution was dipped onto the electrode and dried in air. The electrode was soaked into gelatin solution at last. Thus, enzymes were immobilized on the electrode by the indirect cross-linking method. All experiments were performed in a thermostat, electrochemical cell containing 10 ml of 2% (m/v) starch solution diluted with 0.1 M phosphate buffer solution (pH 7.0). The current intensity was recorded when it reacted for 5 min. The proposed biosensor detects the generated current to obtain the activity ofα-amylase.(3)The operational stability and accuracy of theα-amylase biosensor were investigated by recovery test. Results(1)The proposal screen-printing electrode display cheap,good reproducibility,collection and easy to batch production.(2)A disposableα-amylase biosensor was developed based on ferrocene as an electron transfer mediator and Glucose oxidase,alpha-glucosidase were immobilized by the indirect cross-linking method. The proposed biosensor presented good repeatability, stability, and accuracy allowing alpha-amylase quantification at levels down to 17.0 U L-1. Moreover, the biosensor showed a wide linear response range (from 60 to 840 U L-1 for alpha-amylase). The biosensor response time was very short, reaching 95% of the steady-state current in less than 30 seconds. The proposed biosensor presented good repeatability, stability, and accuracy.ConclusionIn this study, a foundation for a new method to examine the salivaryα-amylase activity was established to identify people's mental stress conditions conveniently and accurately. A disposableα-amylase biosensor was developed based on ferrocene as an electron transfer mediator and Glucose oxidase,alpha-glucosidase were immobilized by the indirect cross-linking method. The operational stability of the biosensor was investigated by recording 10 successive assays of 300 IU L-1α-amylase with the result of the 1.3% relative standard deviation. The storage stability at 4oC was examined by periodical checking. The storage stability at 4 oC was examined by checking periodically its relative activity. The activity was maintained at 100.0% approximately for 15 days and at 86.8% for a month.