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Label-free optical nano sensor for beta-glucosidase activity detection and application

A glucosidase and nanosensor technology, which is applied in the field of label-free optical nanosensors for β-glucosidase activity detection, can solve the problems of poor effectiveness and complicated operation, and achieves simple preparation, strong catalytic activity and low cost. Effect

Pending Publication Date: 2021-11-30
NORTHEAST NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In view of this, the present invention provides a label-free optical nanosensor for the detection of β-glucosidase activity and its application to solve the problems of complex operation and poor effectiveness in the detection of β-glucosidase activity in the past.

Method used

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  • Label-free optical nano sensor for beta-glucosidase activity detection and application
  • Label-free optical nano sensor for beta-glucosidase activity detection and application
  • Label-free optical nano sensor for beta-glucosidase activity detection and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: BSA-Cu 3 (PO 4 ) 2 ·3H 2 Preparation of O NFs

[0028] BSA-Cu 3 (PO 4 ) 2 ·3H 2 The specific synthetic route of O NFs is as follows [see figure 1 (A)]: First, mix 200mmol·L -1 CuSO 4 The solution was injected into 1690μL containing 0.1mg·mL -1 50mmol·L of BSA -1 , pH 6.8 phosphate buffered saline (PBS), and then left at room temperature for 12 hours. The prepared BSA-Cu 3 (PO 4 ) 2 ·3H 2 O NFs were centrifuged at 10,000 rpm for 10 min, and then washed 3 times with ultrapure water for purification. Finally, the collected blue-green precipitate was re-dispersed in 200 μL of PBS, and stored at 4° C. for the next experiment.

Embodiment 2

[0029] Example 2: BSA-Cu 3 (PO 4 ) 2 ·3H 2 Study on catalytic performance of O NFs

[0030] Use H 2 o 2 The BSA-Cu 3 (PO 4 ) 2 ·3H 2 Mimic peroxidase catalytic performance of O NFs. Such as figure 2 Shown, in the control experiment, Amplex Red solution (curve a), Amplex Red and NFs mixed solution (curve b) and Amplex Red and H 2 o 2 The mixed solution (curve c) has no obvious fluorescence emission peak between 565-800nm. When BSA-Cu 3 (PO 4 ) 2 ·3H 2 O NFs, Amplex Red and H 2 o 2 When the three are mixed, the mixed solution shows a significant fluorescence enhancement at 584nm (curve d), which comes from the oxidation product Resorufin of Amplex Red, and the background fluorescence is extremely low and can be ignored. The above results indicated that BSA-Cu 3 (PO 4 ) 2 ·3H 2 O NFs have excellent peroxidase-mimicking catalytic activity. When the excitation wavelength is 550nm, the maximum fluorescence emission wavelength of the reaction system is about ...

Embodiment 3

[0033] Example 3: Construction of Optical Nanosensor and Its Response to β-Glu

[0034] First, prepare 20000U·L -1 β-Glu stock solution. 150 μL of NFs solution and 50 μL of amygdalin solution (20 mmol L -1 ) were added a series of 50 μL of β-Glu solutions of different concentrations, mixed thoroughly, and incubated at room temperature for 1 h to carry out the enzymatic reaction. Then, 20 μmol·L -1 Amplex Red solution and 200mmol L -1 H 2 o 2 solution, use phosphate buffered saline (10mmol·L -1 , pH=6.8) to 1.5mL. After mixing well, incubate at room temperature for 90 minutes. Under the condition of 550nm excitation light, measure the fluorescence emission spectrum of above-mentioned mixed solution in the range of 565-800nm, the result is as follows Image 6 shown. The excitation and emission slits are both 5 nm. from Image 6 It can be observed in 0-1500.0U·L -1range, with the increase of β-Glu content, BSA-Cu 3 (PO 4 ) 2 ·3H 2 O NFs-H 2 o 2 -The fluorescen...

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Abstract

The invention discloses a label-free optical nano sensor for beta-glucosidase activity detection and an application. In the sensor, a protein-inorganic hybrid nanoflower can catalytically convert a fluorescent red dye into a dark purple red resorufin with strong fluorescence through electron transfer. When beta-Glu is added into the system, due to the existence of cyanogenic glycoside, beta-Glu can specifically hydrolyze cyanogenic glycoside and generate CN <->. The CN <-> can effectively inhibit the catalytic activity of the protein-inorganic hybrid nanoflower so that the fluorescence intensity of the system is weakened, and meanwhile, the color of a solution becomes light. The optical double-signal detection of beta-Glu can be realized by monitoring the color change of the system and the ''Turn on-off '' of fluorescence. The constructed fluorescent nano sensor can also be used for quantitative detection of beta-Glu in soil. In addition, the sensor has certain application potential in the aspect of screening beta-Glu inhibitors.

Description

technical field [0001] The invention discloses the technical field of beta-glucosidase activity detection, in particular to a label-free optical nanosensor for beta-glucosidase activity detection and its application. Background technique [0002] β-glucosidase (β-Glu), a class of glycosyl hydrolases that can specifically catalyze the hydrolysis of β-glycosidic bonds, is of great significance for plants, animals and microorganisms to produce functional secondary metabolites, glycosides. In the past two decades, β-Glu has been widely used in medicine, bioenergy, environment, food and other fields. For example, β-Glu activity levels have been associated with metabolic diseases such as diabetes, bacterial or viral infections, and cancer [Lillelund, V.H.; Jensen, H.H.; Liang, X.; Bols, M. Recent developments of transition-state analogue glycosidase inhibitors of non- natural product origin. Chem Rev, 2002, 102, 515-553]. In terms of biotechnology, β-Glu activity plays an extrem...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
CPCG01N21/6428G01N2021/6432
Inventor 刘自平刘莎莎田野周帅
Owner NORTHEAST NORMAL UNIVERSITY
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