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Preparation method and application of array sensor for instantaneously identifying drug-induced HK-2 cell damage

An array sensor and cell damage technology, applied in the field of biosensing, can solve the problems of cumbersome time-consuming, low sensitivity, huge equipment, etc., and achieve the effects of low cost, wide application range and simple preparation method

Active Publication Date: 2020-11-20
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the shortcomings of the prior art, such as cumbersome time-consuming, low sensitivity, bulky equipment, and high cost, the present invention provides a preparation method and application of a multi-channel sensor for instantaneously identifying HK-2 cell damage induced by different drugs. The sensor is easy to prepare, the reaction conditions are mild, and the cost is low, and it is easy to prepare in batches. The constructed sensor can be added to the cell to obtain the response result instantaneously.

Method used

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  • Preparation method and application of array sensor for instantaneously identifying drug-induced HK-2 cell damage
  • Preparation method and application of array sensor for instantaneously identifying drug-induced HK-2 cell damage
  • Preparation method and application of array sensor for instantaneously identifying drug-induced HK-2 cell damage

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preparation example Construction

[0036] Such as figure 1 The preparation method of an array sensor that transiently recognizes drug-induced HK-2 cell damage, the specific steps are as follows:

[0037] Step (1.1), preparation of polydopamine-polyethyleneimine (PDA-PEI) copolymer carrier: Add dopamine hydrochloride and polyethyleneimine to Tris-HCl buffer, and stir at room temperature 10-30°C in the dark After 2-8 hours, filter and place in a dialysis bag for dialysis for 24-36 hours; remove unreacted dopamine hydrochloride and polyethyleneimine to obtain a PDA-PEI copolymer carrier;

[0038] Specifically, 1. Dilute 100 μL Tris-HCl (1M, pH=7.4) buffer solution with ultrapure water to 10 mL for later use;

[0039] 2. Weigh 10mg of dopamine hydrochloride (DA·HCl) and 10mg of polyethyleneimine (PEI, M.W.600Da) respectively, add the two into 10mL Tris-HCl buffer solution, and place on a magnetic stirrer at room temperature 10-30°C to avoid Light stirring for 2-8h;

[0040] 3. Filter the above solution with a 0....

Embodiment 1

[0058] Construction of PDA-PEI / QDs sensor and verification of successful synthesis

[0059] 1. Preparation of PDA-PEI carrier

[0060] (1) Dilute 100 μL of Tris-HCl (1M, pH=7.4) buffer solution with ultrapure water to 10 mL for later use;

[0061] (2) Weigh 10mg of dopamine hydrochloride (DA·HCl) and 10mg of polyethyleneimine (PEI, M.W.600Da) respectively, add the two to 10mL Tris-HCl buffer solution, and place on a magnetic stirrer at room temperature 10-30°C Stir in the dark for 2-8 hours;

[0062] (3), filter the above solution with a 0.22 μm cellulose ester membrane, then place it in a dialysis bag (molecular weight cut-off 1000Da) and dialyze for 24-36h to remove unreacted DA and PEI to obtain polydopamine-polyethyleneimine (PDA-PEI ) copolymer carrier;

[0063] During the preparation of PDA-PEI, the successful preparation of the PDA-PEI carrier was verified by the color changes before and after the reaction, transmission electron microscope images, ultraviolet spectra...

Embodiment 2

[0078] Administration (taking isoniazid as an example), the IC is obtained by the classic MTT method 50 value:

[0079] 1. The preparation process of the sensor is consistent with Example 1;

[0080] 2. To study the cytotoxicity of isoniazid on HK-2 cells by MTT method. Include the following steps:

[0081] (1), cell plate: HK-2 cells in 4 × 10 per well 3 Density inoculation into 96-well plates, in which 200 μL of NEAA-containing MEM basal medium with a volume fraction of 12.5% ​​FBS was added to each well (edge ​​wells were filled with sterile PBS at pH = 7.4), at 37°C, 5% CO 2 Cultivate in the incubator for 24h;

[0082] (2), administration: After aspirating the culture solution, add 200 μ L of isoniazid solutions (without containing FBS (dissolved in MEM basal medium) was incubated at 37°C for 24 hours, and the cell morphology was observed under an inverted microscope;

[0083] (3) MTT administration: after the incubation is completed and the culture medium is sucked ...

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Abstract

The invention discloses a preparation method and application of an array sensor for instantaneously recognizing drug-induced HK-2 cell damage, and the preparation method comprises the following steps:adding dopamine hydrochloride and polyethyleneimine into a buffer solution, uniformly mixing and stirring, filtering and dialyzing to obtain a polydopamine-polyethyleneimine copolymer carrier with excellent quenching effect; wherein three kinds of QDs with different emission wavelengths can be adsorbed through electrostatic interaction, fluorescence of the QDs is quenched, and the multi-channel sensor is formed. Different renal toxicity drugs act on cells, substances on the surfaces of cell membranes are changed, the sensor QDs is induced to dissociate, a characteristic fluorescence fingerprint spectrum is formed, and a drug damage mechanism is rapidly identified by means of a multivariate statistical method. Based on the time-effect relationship of drug damage, fluorescence signals at different times are detected, a dynamic fluorescence fingerprint is drawn, and accurate typing of drug-induced renal cell damage is further realized. The sensor provides a new tool and a new method forrapidly exploring a renal toxicity mechanism of a drug.

Description

technical field [0001] The invention belongs to the field of biosensing, and relates to a preparation method and application of an array sensor for instantly identifying drug-induced HK-2 cell damage, in particular to a multi-channel sensor for instantaneously identifying different drug-induced HK-2 cell damage based on fluorescent fingerprints The preparation method of the sensor and its application. Background technique [0002] Drug-induced renal injury refers to the abnormal structure or function of the kidney caused by drugs, mainly manifested as acute tubular necrosis, acute interstitial nephritis and osmotic nephropathy. Many drugs are metabolized or excreted by the kidneys, and tend to accumulate in the kidneys and damage glomeruli, renal tubules or renal basement membranes. In addition, some drugs can cause a sharp decline in the filtering function of bilateral kidneys in a short period of time, causing the accumulation of toxins in the body, which may seriously le...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C08L79/02C08L79/04C08K3/30C08G73/06G01N21/64
CPCC08L79/02C08L79/04C08G73/0672G01N21/6428G01N2021/6432C08L2205/02C08L2205/03C08K2003/3036C08K3/30
Inventor 余伯阳田蒋为白雪斐喻谢安
Owner CHINA PHARM UNIV
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