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Acetylcholine detection kit based on protein-inorganic hybrid nanoflower and preparation method thereof

A technology of acetylcholine and protein, which is applied in the field of biosensors, can solve the problems of high concentration of acetylcholine, cumbersome test methods, and low detection sensitivity, and achieve the effects of low cost, easy operation, and simple construction process

Inactive Publication Date: 2019-05-03
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to provide a protein-inorganic based on protein-inorganic Hybrid nanoflowers (BSA-Cu 3 (PO 4 ) 2 ·3H 2 O) Acetylcholine (ACh) Activity Detection Kit

Method used

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  • Acetylcholine detection kit based on protein-inorganic hybrid nanoflower and preparation method thereof
  • Acetylcholine detection kit based on protein-inorganic hybrid nanoflower and preparation method thereof
  • Acetylcholine detection kit based on protein-inorganic hybrid nanoflower and preparation method thereof

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Experimental program
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Effect test

Embodiment 1

[0016] Embodiment 1: protein-inorganic hybrid nanoflower (BSA-Cu 3 (PO 4 ) 2 ·3H 2 O) Synthesis

[0017] Na 2 HPO 4 and NaH 2 PO 4 Dissolved in PBS buffer, prepared into a PBS buffer solution with a pH value of 6.8; the CuSO 4 Soluble in water, prepare 200mmol L -1 CuSO 4aqueous solution; mix 30 μL of the PBS buffer solution prepared above and 20 μL of CuSO 4 The aqueous solution was mixed and shaken for 2 minutes; 100μL, 1mg mL -1 Bovine serum albumin (BSA) aqueous solution was added to the above mixed solution; after standing still for 12 hours, it was centrifuged for 5 minutes, the supernatant was poured off and the lower layer was left, and then 100 μL of deionized water was added to obtain 4 mg mL -1 Protein-inorganic hybrid nanoflowers (BSA-Cu 3 (PO 4 ) 2 ·3H 2 O) solution.

Embodiment 2

[0018] Embodiment 2: the making of kit

[0019] Dissolve 20 mg of agarose in 2.0 mL of boiling water at 100 °C, wait until the temperature of the agarose has dropped to 55 °C, and add 200 μL of protein-inorganic hybrid nanoflowers (BSA-Cu 3 (PO 4 ) 2 ·3H 2 O) solution and 75 μL of 1 mg mL -1 Add the mixed solution of 3,3',5-,5'-tetramethylbenzidine (TMB) into the centrifuge tube containing agarose, stir evenly, take out 200 μL and drop it on the cover of another centrifuge tube, etc. When the temperature drops to room temperature, the agarose solution on the cover of the centrifuge tube is solidified, and 50 μL of 1U mL of -1 of acetylcholinesterase (AChE) and 50 μL of 2U mL -1 Mixed solution of choline oxidase (CHO) to obtain white matter-inorganic hybrid BSA-Cu 3 (PO 4 ) 2 ·3H 2 O's Acetylcholine (ACh) Activity Assay Kit.

Embodiment 3

[0020] Embodiment 3: the application of kit to the detection of acetylcholine (ACh) activity

[0021] The present invention will add concentration in the kit to be 0.1mmol mL -1 100 μL of acetylcholine solution (ACh) for 60 minutes, then close the kit and turn it upside down, the color reaction is triggered, the kit is blue and can keep the color stable for a certain period of time. 100 μL of acetylcholine solutions (ACh) with different concentrations were prepared and added to the prepared protein-inorganic hybrid BSA-Cu 3 (PO 4 ) 2 ·3H 2 In the kit of the O / AChE / CHO system, then close the kit and turn it upside down. With the increase of ACh concentration, the gradient of the color of the kit can be observed from colorless to blue, and by fitting Made a standard curve, and can quantitatively detect acetylcholine (such as figure 1 ). The abscissa is the concentration of ACh, the ordinate is the intensity, different colors correspond to different intensities, and the ord...

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Abstract

The invention relates to an acetylcholine detection kit based on the protein-inorganic hybrid nanoflower and a preparation method thereof and belongs to the technical field of a biosensor. The kit isloaded with the protein-inorganic hybrid nanoflower, acetylcholinesterase, choline oxidase and color developing agent. Through the acetylcholinesterase, the acetylcholine can be catalyzed to produce choline, the choline is further oxidized by the choline oxidase to produce hydrogen peroxide, and the formed hydrogen peroxide is catalytically oxidized by a hydroxyl group to generate a hydroxyl radical to further develop the color developing agent. The kit is advantaged in that the acetylcholine can be rapidly detected by the kit, a direct quantification tool is provided for identification of theacetylcholine, simple and convenient operation, high sensitivity and low cost are realized, the portable on-site test kit is provided for immediate testing of the activity of the acetylcholine, and the needs of frequent screening and diagnostic tracking are met.

Description

technical field [0001] The invention belongs to the technical field of biosensors, in particular to a protein-inorganic hybrid nanoflower (BSA-Cu 3 (PO4) 2 ·3H 2 O) A quantitative and qualitative detection kit for acetylcholine (ACh) activity and a preparation method thereof. Background technique [0002] As a neurotransmitter that transmits signals, acetylcholine has been involved in the neurotransmission of the central and peripheral nervous systems, and plays an important role in biological fields such as learning, memory, movement, and emotion. Abnormal levels of acetylcholine can lead to the termination of cholinergic synaptic neurotransmission and lead to Alzheimer's disease, Parkinson's disease, progressive dementia, schizophrenia, motor dysfunction and other diseases. Driven by the above facts, high-sensitivity assay and on-site screening of ACh have received increasing attention in the field of disease prevention to identify early stages of disease. At the same ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/531
Inventor 卢革宇孔德帅闫旭刘方猛刘晓敏孙鹏高原梁喜双刘凤敏
Owner JILIN UNIV
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