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High-throughput screening method for targeted drug

A screening method and high-throughput technology, applied in the field of high-throughput screening of targeted drugs, can solve problems such as the application of Raman signal detection technology, and achieve the effects of simple operation, strong signal, and small sample volume

Pending Publication Date: 2021-07-30
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, so far, no Raman signal detection technology has been applied to the drug screening of small molecule inhibitors

Method used

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  • High-throughput screening method for targeted drug
  • High-throughput screening method for targeted drug
  • High-throughput screening method for targeted drug

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1 prepares gold (silver) nanoparticles

[0045] 1. Preparation of Gold Nanoparticles

[0046] Gold nanoparticles (AuNPs): take a 250mL three-neck flask, wash it with aqua regia (1 part of concentrated nitric acid and 3 parts of concentrated hydrochloric acid), take it out the next day, rinse it, and then wash it with water for injection three times to ensure that the wall is pure and free of impurities . Place in oven to dry and cool to room temperature. Add 2 mL of 1% HAuCl to the flask 4Add water to 200mL, connect a condenser, heat to 150°C with an electric heating mantle, stir and heat to reflux until slightly boiling. Add 2mL of 1% sodium citrate, control the temperature, observe the color change, from light yellow to black (110°C-120°C), and then to red (90°C), lower the temperature to 90°C, and keep the temperature for 40min to obtain gold nanoparticles decentralized system.

[0047] 2. Preparation of Silver Nanoparticles

[0048] Silver nanoparti...

Embodiment 2

[0049] The preparation of embodiment 2 magnetic nanoparticles

[0050] The commercial carboxyl magnetic beads produced by Biomag are prepared at a concentration of 0.5 mg / mL, washed with magnetic bead preservation solution and magnetic separation repeated three times, resuspended in the preservation solution to prevent magnetic beads from aggregating, and stored at 4°C for later use.

Embodiment 3

[0051] Example 3 Synthesis of two Raman probes related to the screening of PDEδ protein inhibitors (when Raman signal molecules are coupled to magnetic beads)

[0052] Specific process such as figure 1 as shown in:

[0053] (1) Preparation of AgNPs@Fc fusion KRAS silver probe

[0054] Take 1 mL of 0.29nM silver nanoparticles (AgNPs), add 10 μL Tween 20, react for 30 min, add 5.0 μL 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) ( 2.5mM) and 5.0μL N-hydroxysuccinimide (NHS) (2.5mM), react for 1h, centrifuge to remove excess activator, and resuspend. Add 4 μL of 0.05 mg / mL Protein A (Domain B) of Protein A (a protein on the surface of Staphylococcus aureus), react for 2 hours, centrifuge to discard the supernatant and resuspend. Add 5 μL ethanolamine (15.0 mM) to block, react for 30 min, centrifuge to discard the supernatant and resuspend. Add 5 μL of 0.5 mg / mL KRAS protein fused to express the Fc segment, react for 2 h, centrifuge to discard the supernata...

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Abstract

The invention discloses a high-throughput screening technology of a targeted drug, which comprises the following steps: respectively coupling a metal nanoparticle probe or a magnetic bead probe to a Raman signal molecule, a target protein or a target protein binding receptor protein, simultaneously adding the substances into a compound to be screened, incubating, finally detecting a Raman signal, and judging whether the compound to be screened is a targeted inhibitor or not, the targeted inhibitor having the characteristic of inhibiting target protein related biological activity. Raman signal detection is applied to the technical field of targeted new drug screening for the first time, and metal nanoparticles and magnetic beads are combined to form a simple and sensitive new drug screening method. Meanwhile, the method is small in measurement interference, small in required sample amount, strong in signal, high in sensitivity and easy and convenient to operate, cleaning is not needed in the whole detection process, and a new strategy is provided for a targeted drug screening approach.

Description

technical field [0001] The invention belongs to the technical field of drug screening, in particular to a high-throughput screening method for targeted drugs. Background technique [0002] High-throughput drug screening is based on experimental methods at the molecular and cellular levels, using microplates and other forms as experimental tool carriers, using automated operating systems to execute experimental processes, and using sensitive and fast detection instruments to collect experimental results and data. It is a technical system that analyzes and processes experimental data, detects a large number of samples at the same time, and supports the operation of the entire system with a corresponding database. With the emergence of more and more new targets, the development of synthetic chemistry and the application of new detection technologies, many high-throughput drug screening methods have emerged, such as differential scanning fluorimetry, surface plasmon resonance ( ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/65
CPCG01N21/658
Inventor 梁重阳徐抒平戴璐
Owner JILIN UNIV
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