Biosensor achieving AChE oriented immobilization and preparation method and application of biosensor

A biosensor, directional fixation technology, applied in the field of biosensors, can solve the problems of poor reproducibility of biosensors, complex working electrode composition, inability to meet micro-analysis, etc., to achieve good sensitivity and reproducibility, convenient storage and repeated reproduction. Utilize and simplify the effect of preparation

Active Publication Date: 2016-07-27
CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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Problems solved by technology

[0004] The immobilization method used in traditional enzyme biosensors is to randomly combine enzymes with carriers, and its working electrode composition is relatively complicated. First, complex composite nanomaterials (with better conductivity) are synthesized through multiple steps, and then AChE enzymes are immobilized on the composite. On the nanomaterials, the composite material immobilized with the enzyme is finally fixed on the electrode by electrochemical deposition to obtain the working electrode; since multiple steps are required to prepare the working electrode, this may weaken the stability of the enzyme and the reproducibility of the biosensor Poor; in addition, the working electrode is a whole, which is not conducive to preservation, and the reaction volume is usually 10mL, which cannot meet the needs of microanalysis

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  • Biosensor achieving AChE oriented immobilization and preparation method and application of biosensor

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Embodiment 1

[0027] Embodiment 1: a kind of preparation method of the biosensor of AChE enzyme orientation immobilization, it comprises the following steps:

[0028] S1. Preparation of magnetic nanomaterials: FeCl 3 ·6H 2 O and FeCl 2 4H 2 Fe with superparamagnetism obtained by magnetic separation after O reaction 3 o 4 Nanoparticles, adding TEOS reaction to generate silica wrapped Fe 3 o 4 Composite A with a core-shell structure of particles; wherein, the FeCl 3 ·6H 2 O and FeCl 2 4H 2 The weight ratio of O is 1:1; the reaction temperature of adding TEOS is 30°C, the reaction time is 5h, and the pH value of the reaction solution is 9, which is adjusted with ammonia water;

[0029] S2. Modification: first add APTMS to compound A to carry out amino modification reaction, the reaction temperature is 30°C, the reaction time is 6.5h, then add DMF and succinic anhydride magnetic stirring reaction for 2.5h, to obtain a magnetic compound with a carboxyl group modification at the end Na...

Embodiment 2

[0032] Embodiment 2: a kind of preparation method of the biosensor of AChE enzyme orientation immobilization, it comprises the following steps:

[0033] S1. Preparation of magnetic nanomaterials: FeCl 3 ·6H 2 O and FeCl 2 4H 2 Fe with superparamagnetism obtained by magnetic separation after O reaction 3 o 4 Nanoparticles, adding TEOS reaction to generate silica wrapped Fe 3 o 4 Composite A with a core-shell structure of particles; wherein, the FeCl 3 ·6H 2 O and FeCl 2 4H 2 The weight ratio of O is 3.5:1; the reaction temperature of adding TEOS is 40°C, the reaction time is 7h, and the pH value of the reaction solution is 10, which is adjusted with ammonia water;

[0034] S2. Modification: first add APTMS to compound A for amino modification reaction, the reaction temperature is 40°C, the reaction time is 7.5h, then add DMF and succinic anhydride magnetic stirring reaction for 3.5h, and the end is carboxyl modified magnetic Nanomaterial B;

[0035]S3. Preparation o...

Embodiment 3

[0037] Embodiment 3: a kind of preparation method of the biosensor of AChE enzyme orientation immobilization, it comprises the following steps:

[0038] S1. Preparation of magnetic nanomaterials: FeCl 3 ·6H 2 O and FeCl 2 4H 2 Fe with superparamagnetism obtained by magnetic separation after O reaction 3 o 4 Nanoparticles, adding TEOS reaction to generate silica wrapped Fe 3 o 4 Composite A with a core-shell structure of particles; wherein, the FeCl 3 ·6H 2 O and FeCl 2 4H 2 The weight ratio of O is 2.8:1; the reaction temperature of adding TEOS is 33°C, the reaction time is 9h, and the pH value of the reaction solution is 9, which is adjusted with ammonia water;

[0039] S2. Modification: first add APTMS to compound A for amino modification reaction, the reaction temperature is 32 ° C, the reaction time is 7 hours, then add DMF and succinic anhydride magnetic stirring reaction for 3 hours, and obtain the magnetic nanomaterial with carboxyl group modification at the e...

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Abstract

The invention discloses a biosensor achieving AChE oriented immobilization and a preparation method and application of the biosensor. The preparation method of the biosensor comprises the following steps of preparation of a magnetic nano material MNP, modification, MNPs-AChE preparation and sensor construction. The biosensor is used for detecting residual organophosphorus pesticide. According to the biosensor achieving AChE oriented immobilization and the preparation method and application of the biosensor, acetylcholinesterase is covalently immobilized to the surfaces of terminated carboxyl-modified MNP granules, and an obtained composite nano material MNPs-AChE can maintain enzymic activity to the maximum extent; according to the constructed amperometric biosensor, an MNPs-AChE modified SPCE system can be automatically adjusted through an external magnetic field, electrode modification can be achieved only through one step, and the stability and activity of AChE are improved; good sensitivity and repeatability are shown in the process of detecting an organophosphorus reagent, and the reaction volume is small; the method for preparing the biosensor is easy and convenient to operate, preparation is convenient, the cost is low, and the method is suitable for industrial large-scale production.

Description

technical field [0001] The invention belongs to the field of biosensors, and in particular relates to a biosensor for directional immobilization of AChE enzyme, a preparation method and application thereof. Background technique [0002] In recent years, food poisoning incidents caused by pesticide residues have occurred frequently, and food safety has posed a great threat to people's lives and health. Residual pesticides enter the human body along the food chain, can inhibit the activity of cholinesterase in the human body, cause the metabolic disorder of the neurotransmitter-acetylcholine, and cause various acute and chronic poisoning conditions, such as ataxia, coma, paralysis and even death. At present, the most serious pesticide residues in my country's grain, fruit, and vegetable products are organophosphorus and carbamate pesticides. According to data, the proportion of organophosphorus pesticides accounts for more than 70% of all pesticides, seriously endangering peop...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/327
CPCG01N27/327
Inventor 廖循曾琼
Owner CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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