Method used for detecting carbohydrates based on microorganism surface controllable co-display sequential enzyme electrochemical biosensor

A sensor and co-display technology, applied in the field of electrochemical biosensing, can solve the problems of complex fusion protein construction, low sensor sensitivity, and low enzyme expression efficiency, and achieve the effects of fast and sensitive response, sensitive detection, and good reproducibility

Active Publication Date: 2018-02-23
QINGDAO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are obvious deficiencies in the random co-display of enzymes, such as complex construction of fusion proteins, low enzyme expression efficiency, and chemical cross-linking agents are still used when sequential enzyme fusion proteins are immobilized on electrodes. high

Method used

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  • Method used for detecting carbohydrates based on microorganism surface controllable co-display sequential enzyme electrochemical biosensor
  • Method used for detecting carbohydrates based on microorganism surface controllable co-display sequential enzyme electrochemical biosensor
  • Method used for detecting carbohydrates based on microorganism surface controllable co-display sequential enzyme electrochemical biosensor

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Experimental program
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Effect test

Embodiment 1

[0045] The sequential enzyme sensor of the present invention is to co-display glucoamylase (GA) and glucose oxidase (GOx) (yeast–GA&GOx (n:1, n=1,2,3) on the yeast surface in proportion and modify them on the electrode (see figure 1 ). The glucoamylase (GA) is derived from the glucoamylase (GA) encoding gene of Saccharomyces fumigatus and the glucose oxidase (GOx) is derived from the glucose oxidase (GOx) encoding gene of Aspergillus niger and the dockerin structure with different specificities Domains DocC and DocT encoding genes were fused.

[0046] Specifically:

[0047] Step 1, constructing a yeast surface display system for scaffold proteins: display cohesin domains CohC and CohT with different specificities on the surface of yeast cells by using a-lectin anchoring protein. Coh-Doc proteins from C.cellulolyticumH10 and C.thermocellum ATCC27405 were cloned, and the gene fragments were cohc / docc and coht / doct, respectively. The gene encoding Coh was fused to the C-termin...

Embodiment 2

[0058] In 5mL of 0.1M citrate buffer solution (pH 5.0), under the working potential of -0.4V, use the i-t method to measure the current response of the sequential enzyme sensor prepared by the above-mentioned optimal experimental conditions to the soluble starch standard solution ( see image 3 A) and working curve (see image 3 B).

[0059] Depend on image 3 It can be seen that the response time and sensitivity of the whole-cell modified electrode with proportional co-display are better than those of the non-co-display system. After adding the same concentration of starch, the 2:1 co-display GA&GOx modified electrode on the yeast surface had the largest current step value, followed by the 3:1 co-display GA&GOx modified electrode on the yeast surface, and the 1:1 co-display yeast-GA&GOx modification on the yeast surface Electrodes, indicating that the optimal ratio of co-display GA&GOx on the surface is 2:1. Compared with the non-co-display system, the signal response adva...

Embodiment 3

[0061] In 5mL of 0.1M citrate buffer (pH 5.0), apply a potential of –0.4V vs SCE, and use the i–t method to determine the above optimal experimental conditions to prepare yeast–GA&GOx(2:1) / RGO / GCE pair Current response to glucose standard concentration (see Figure 4 A) and working curve (see Figure 4 B).

[0062] It can be seen from the figure that with the addition of glucose, the oxidation current signal increases rapidly and reaches 95% of the stable value within 5s ( Figure 4 A). The linear range of detecting glucose is 0.0002-0.01%.

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Abstract

The invention belongs to the field of electrochemical biosensor, and more specifically relates to a method used for detecting carbohydrates through construction of a sequential enzyme electrochemicalbiosensor based on microorganism surface controllable co-display sequential enzymes. According to the method, glucose oxidase (GOx) single-display on yeast strain surface is carried out, or glucoamylase (GA) and GOx co-display at a certain ratio on yeast strain surface is carried out; the yeast strain treated via single-display or co-display are modified onto a treated electrode so as to obtain anelectrochemical glucose biosensor or an electrochemical starch biosensor, and realize quantitative detection on glucose or starch in sample solutions. The obtained biosensors are capable of providingan effective approach to solve a problem that component analysis using a single enzyme sensor is difficult to perform, and the method can be widely used in the fields such as optimization and controlof biomass raw material pretreatment, biological energy source, food fermentation, medicine, and chemical industry, and clinical detection.

Description

technical field [0001] The invention belongs to the field of electrochemical biosensing, and specifically refers to a method for constructing a sequence enzyme electrochemical sensor to detect sugar based on the controllable co-display sequence enzyme on the surface of microorganisms. Background technique [0002] Since the advent of enzyme electrodes in the 1960s, electrochemical biosensors have achieved tremendous development in their technical research and applications. The immobilized enzyme electrochemical biosensor has the advantages of strong specificity, good stability, high accuracy, fast response speed, low measurement cost, and accurate results, and can realize specific biochemical components in complex samples (turbidity, color, etc.) rapid determination. Sequential enzyme biosensor (sequential enzyme biosensor) refers to a device that completes a series of catalytic reactions in sequence and coordination through a variety of related enzymes, and combines with a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/26G01N27/327
CPCG01N27/26G01N27/3272G01N27/3275
Inventor 刘爱骅
Owner QINGDAO UNIV
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