阅读说明：本技术 一种基于葡萄糖氧化酶和类***脱氧核酶构成的荧光型葡萄糖传感器 (Fluorescent glucose sensor based on glucose oxidase and pistol-like deoxyribozyme ) 是由 姜大志 许家翠 于文倩 刘程程 郭艳杰 于 2020-06-28 设计创作，主要内容包括：本发明公开一种基于葡萄糖氧化酶和类手枪脱氧核酶构成的荧光型葡萄糖传感器,属于生物化学检测领域。在类手枪脱氧核酶5’端化学修饰荧光淬灭分子,在其DNA底物5’和3’端分别化学修饰荧光淬灭分子和荧光分子。当检测液中含有葡萄糖时,葡萄糖氧化酶催化葡萄糖生成过氧化氢和葡萄糖酸,过氧化氢与铜离子作用促使类手枪脱氧核酶催化底物断裂,同时产生荧光信号。该传感器检测葡萄糖下限为50纳摩尔/升,葡萄糖浓度和荧光信号之间曲线拟合符合Extreme方程,对葡萄糖检测显示出专一性。使用本发明可以实现对唾液中葡萄糖含量检测。(The invention discloses a fluorescent glucose sensor based on glucose oxidase and pistol-like deoxyribozyme, and belongs to the field of biochemical detection. The 5 ' end of the pistol-like deoxyribozyme is chemically modified with a fluorescence quenching molecule, and the 5 ' end and the 3 ' end of the DNA substrate are respectively chemically modified with a fluorescence quenching molecule and a fluorescence molecule. When the detection liquid contains glucose, the glucose oxidase catalyzes the glucose to generate hydrogen peroxide and gluconic acid, and the hydrogen peroxide and copper ions act to promote the catalytic substrate of the pistol-like deoxyribozyme to break, and simultaneously generate a fluorescence signal. The lower limit of the sensor for detecting the glucose is 50 nanomole/liter, curve fitting between the glucose concentration and the fluorescence signal accords with an Extreme equation, and specificity is shown for detecting the glucose. The invention can realize the detection of the glucose content in the saliva.)

1. A fluorescent glucose sensor based on glucose oxidase and pistol-like deoxyribozyme is characterized in that: the sensor consists of glucose oxidase, pistol-like deoxyribozyme DNA substrate, copper ions, sodium chloride and a pH buffer system.

2. The fluorogenic glucose sensor of claim 1, wherein said fluorogenic glucose sensor comprises a glucose oxidase and a pistol-like deoxyribozyme, and wherein: the pistol-like deoxyribozyme contains a DNA sequence of 5 '-ctgggcc-3'.

3. The fluorogenic glucose sensor of claim 1, wherein said fluorogenic glucose sensor comprises a glucose oxidase and a pistol-like deoxyribozyme, and wherein: the quenching molecule chemically modified at the 5' end of the pistol-like deoxyribozyme is a black hole quenching molecule 1(BlackHole Quencher-1).

4. The fluorogenic glucose sensor of claim 1, wherein said fluorogenic glucose sensor comprises a glucose oxidase and a pistol-like deoxyribozyme, and wherein: the pistol-like deoxyribozyme DNA substrate has the sequence of 5 '-aagacag-3'.

5. The fluorogenic glucose sensor of claim 1, wherein said fluorogenic glucose sensor comprises a glucose oxidase and a pistol-like deoxyribozyme, and wherein: the quenching molecule chemically modified at the 5 'end of the DNA substrate is Black hole quenching molecule 1(Black HoleQuencer-1), and the fluorescent molecule chemically modified at the 3' end is Carboxyfluorescein (Carboxyfluorescein).

6. The fluorogenic glucose sensor of claim 1, wherein said fluorogenic glucose sensor comprises a glucose oxidase and a pistol-like deoxyribozyme, and wherein: the glucose oxidase concentration was 0.01 unit/microliter.

7. The fluorogenic glucose sensor of claim 1, wherein said fluorogenic glucose sensor comprises a glucose oxidase and a pistol-like deoxyribozyme, and wherein: the copper ions were used at a concentration of 1. mu. mol/l.

8. The fluorogenic glucose sensor of claim 1, wherein said fluorogenic glucose sensor comprises a glucose oxidase and a pistol-like deoxyribozyme, and wherein: the pistol-like deoxyribozyme was used at the same concentration as the DNA substrate of 50 nmol/liter.

9. The fluorogenic glucose sensor of claim 1, wherein said fluorogenic glucose sensor comprises a glucose oxidase and a pistol-like deoxyribozyme, and wherein: the sodium chloride concentration was 800 mmol/l.

10. The fluorogenic glucose sensor of claim 1, wherein said fluorogenic glucose sensor comprises a glucose oxidase and a pistol-like deoxyribozyme, and wherein: the buffer system was 50 mmol/l MES buffer (pH 6.0).

Technical Field

The invention belongs to the field of biochemical detection, relates to a glucose sensor, and particularly relates to a fluorescent sensor for detecting the content of glucose in saliva based on glucose oxidase and pistol-like deoxyribozyme.

Background

Worldwide, diabetes has become an important chronic disease threatening human health. The incidence of diseases is rising continuously in China, and the number of patients is the first in the world. In order to treat or prevent diabetes, diabetics and susceptible people need to constantly monitor their glucose levels in their bodies in daily life. The detection mode is blood detection, and the process needs to take blood by acupuncture, which is easy to cause pain and discomfort of patients. To solve this problem, diabetes detection is rapidly progressing towards non-invasive detection, and the detection target gradually changes from blood to body metabolites such as saliva, tears, sweat, and the like. The saliva/tear/sweat detection belongs to non-invasive detection technology (non-invasive technology) which is newly developed in recent years, can obtain required detection data under the condition of not causing body injury or pain, and has particularly important practical significance for diabetic patients needing frequent detection.

Disclosure of Invention

The invention aims to provide a fluorescent sensor for detecting the content of glucose in saliva based on glucose oxidase and pistol-like deoxyribozyme.

To achieve the above object, the present invention is achieved by the following techniques:

detection principle of the fluorescent glucose sensor:

the fluorescent glucose sensor mainly comprises glucose oxidase, pistol-like deoxyribozyme and DNA substrate thereof.

The 3 ' end of the pistol-like deoxyribozyme is marked with a quenching molecule, and the 5 ' end and the 3 ' end of the substrate are respectively marked with a quenching molecule and a fluorescent molecule.

When the deoxyribozyme is combined with the substrate recognition, the fluorescent molecule and the quenching molecule are close to each other, the fluorescence quenching phenomenon occurs, and no fluorescence signal is emitted.

In the detection process, glucose is used as a primary signal molecule, glucose oxidase catalyzes glucose to generate gluconic acid and hydrogen peroxide, and hydrogen peroxide is used as a secondary signal molecule and is recognized by the pistol-like deoxyribozyme to perform catalytic substrate cleavage reaction.

The substrate is separated from the pistol-like deoxyribozyme, meanwhile, the fluorescent molecule marked by the substrate is separated from the enzyme-marked quenching molecule, the separated fluorescent molecule emits a fluorescent signal, the fluorescent intensity of the fluorescent signal is positively correlated with the concentration of hydrogen peroxide, and further, the quantitative relation exists between the fluorescent intensity and the concentration of glucose (see figure 1).

Detection conditions of the fluorescent glucose sensor:

the glucose oxidase concentration was 0.01 unit/microliter, the pistol-like deoxyribozyme concentration and its DNA substrate were 50 nmol/liter, the sodium chloride concentration was 800 mmol/liter, 50 mmol/liter MES buffer (pH 6.0), the total volume of the detection solution was 100 microliter, wherein the saliva-containing sample was 50 microliter, and the detection temperature was 23 ℃.

Detection range of the fluorescent glucose sensor:

the lower limit of the detected copper ion concentration of the sensor is 50 nanomoles/liter, and a curve fitting the glucose concentration and the sensor fluorescence signal intensity in the range of 50 nanomoles/liter to 1 millimole/liter accords with an Extreme equation (see figure 2).

Detection specificity of the fluorescent glucose sensor:

maltose, galactose, mannose, trehalose, gulose, fructose, sucrose, lactose and glucose were selected for the experiments. The results showed that there was signal output for maltose, galactose, mannose, sucrose, but the interference signal for these 4 sugars was much weaker than the signal for glucose at the same concentration (see FIG. 3).

Because the saliva secreted by the oral cavity does not contain maltose, galactose, mannose and sucrose, the detection of the glucose content in the saliva by the glucose fluorescence type sensor can not be interfered.

Detecting the saliva sample by the fluorescent glucose sensor:

10 saliva samples were selected and samples were collected about 2 hours after breakfast. And (4) detecting the glucose content of the saliva after meals by using a glucose sensor. The glucose concentration of the sample is 149.4 +/-12.9 micromoles/liter at the highest, and 26.9 +/-8.5 micromoles/liter at the lowest.

Drawings

FIG. 1 is a schematic diagram of a fluorescent glucose sensor. Letters indicate key DNA sequences. F represents a fluorescent molecule, Q represents a fluorescence quenching molecule, PLQ represents a pistol-like deoxyribozyme, and SQF represents a DNA substrate.

FIG. 2 is a graph showing the relationship between the detection range and the fluorescence signal of the fluorescent glucose sensor.

FIG. 3 is a diagram showing the detection specificity of a fluorescent glucose sensor.

Detailed Description

The instrument used in the experiment of the invention is a Tecan Infinite F200 multifunctional microplate reader.

Glucose oxidase used was purchased from Sigma, and pistol-like deoxyribozyme and its DNA substrate were purchased from bio-engineering (shanghai) gmbh.

The fluorescent glucose sensor of the present invention will be further described with reference to specific embodiments.

Detecting the saliva sample by the fluorescent glucose sensor:

the detection system comprises a saliva sample of 50 microliter, glucose oxidase of 0.01 unit/microliter, pistol-like deoxyribozyme of 50 nanomole/liter, pistol-like deoxyribozyme substrate of 50 nanomole/liter, sodium chloride of 800 millimole/liter, MES buffer (pH 6.0) of 50 millimole/liter, and the total volume is 100 microliter.

After reacting for 15 minutes in a water bath at 23 ℃, transferring the detection liquid into a 96-hole fluorescence detection plate by using a micropipette, detecting a fluorescence signal by using a Tecan Infinite F200 multifunctional microplate reader, and then substituting the fluorescence value into a formula to calculate the glucose concentration.

Sequence listing

<110> Jilin university

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