Real-Time Detection of Dynamic Affinity between Biomolecules Using Surface Plasmon Resonance (SPR) Technology

Summary

The present study aims to elucidate the principle and methodology of surface plasmon resonance (SPR) technology, which finds versatile applications across multiple domains. This article describes SPR technology, its operational simplicity, and its remarkable efficacy, with the goal of fostering broader awareness and adoption of this technology among readers.

Abstract

Surface plasmon resonance (SPR) technology is a sensitive precise method for detecting viruses, pathogenic molecular proteins, and receptors, determining blood types, and detecting food adulteration, among other biomolecular detections. This technology allows for the rapid identification of potential binding between biomolecules, facilitating fast and user-friendly, non-invasive screening of various indicators without the need for labeling. Additionally, SPR technology facilitates real-time detection for high-throughput drug screening. In this program, the application field and basic principles of SPR technology are briefly introduced. The operation process is outlined in detail, starting with instrument calibration and basic system operation, followed by ligand capture and multi-cycle analysis of the analyte. The real-time curve and experimental results of binding quercetin and calycosin to KCNJ2 protein were elaborated upon. Overall, SPR technology provides a highly specific, simple, sensitive, and rapid method for drug screening, real-time detection of related pharmacokinetics, virus detection, and environmental and food safety identification.

Introduction

Surface plasmon resonance (SPR) technology is an optical detection technique that eliminates the need for labeling the analyte. It enables real-time and dynamic monitoring of quantitative binding affinity, kinetics, and thermodynamics. This high-throughput capacity is highly sensitive and reproducible, allowing for the measurement of various open rates, off rates, and affinity. Additionally, the small sample quantity required further enhances the utility of this method^1,2. The fast response biomolecular detection method³, which monitors the affinity binding between....

Protocol

NOTE: The complete experimental sensing curve indicates that the experimental process can be categorized into eight distinct stages.

1. Sample and buffer preparation

1. Prepare sensor chips before the experiment.
   1. Treat the chips with the piranha solution (30% H₂O₂: H₂SO₄=1:3; v/v) for 2 min. Subsequently, clean the chip thoroughly with a large amount of deionized water and then soak it in anhydrous ethanol, allow.......

Discussion

The SPR analysis cycle is divided into four stages. The first stage, the baseline, involves the injection of the buffer. Following that is the second stage, ligand capturing. The sensor chip COOH is activated with EDC/NHS (1:1) at a flow rate of 20 µL/min. The chip is then deactivated using 1 M ethanolamine hydrochloride-NaOH at a flow rate of 20 µL/min. Moving on to the third stage, the multi-cycle analyte method. The analyte is injected into the channel at a flow rate of 20 µL/min for an association phas.......

Materials

Name	Company	Catalog Number	Comments
1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)	Nan Jing Reagent,Nanjing,China	C08296594
Anhydrous ethanol	Merck Chemical Technologies Ltd., Shanghai, China	459836
BIAnormalizing solution	Merck Chemical Technologies Ltd., Shanghai, China	49781
Blocking solution	Bosheng Biotechnology Co.,Ltd., Shanghai, China	110050
Bromoacetic acid	Merck Chemical Technologies Ltd., Shanghai, China	17000
Calycosin	Push Bio-technology Co., Ltd., Chengdu, China	PU0124-0025
Dextran	Canspec Scientific Instruments Co., Ltd.,Shanghai, China	PM10036
Epichlorohydrin	Merck Chemical Technologies Ltd., Shanghai, China	492515
Ethanolamine hydrochloride	Yuanye Biotech Co., Ltd., Shanghai, China	S44235
Glycine-HCl	Merck Chemical Technologies Ltd., Shanghai, China	G2879
H2O2	Merck Chemical Technologies Ltd., Shanghai, China	3587191
H2SO4	Nantong high-tech Industrial Development Zone,China	2020001150C
HEPES	Xiya Reagent Co., Ltd., Shandong, China	S3872
KCNJ2 (Human) Recombinant Protein	Abnova,West Meijie Technology Co., Ltd., Beijing, China	H00003759-Q01
MUOH	Jizhi Biochemical Technology Co., Ltd., Shanghai, China	M40590
NaOH	Merck Chemical Technologies Ltd., Shanghai, China	SX0603
N-Hydroxysuccinimide(NHS)	Yuanye Biotech Co., Ltd., Shanghai, China	S13005
OpenSPRTM	Nicoya
Quercetin	Push Bio-technology Co., Ltd., Chengdu, China	PU0041-0025
Sensor Chip COOH	Nicoya
Sodium Acetate	Merck Chemical Technologies Ltd., Shanghai, China	229873