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

Published: September 29, 2023

doi:

Wenqiao An*¹, Zhihao Sun*¹, Pengmei Guo, Xiaobo Wang³, Sanyin Zhang³

¹School of Basic Medical Sciences,Chengdu University of Traditional Chinese Medicine, ²Innovative Institute of Chinese Medicine and Pharmacy,Chengdu University of Traditional Chinese Medicine, ³Research Institute of Integrated Traditional Chinese Medicine and Western Medicine,Chengdu University of Traditional Chinese Medicine

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 method¹^,². The fast response biomolecular detection method³, which monitors the affinity binding between biomolecules, has emerged as a prominent research area.

SPR technology has various applications in the field of drug research and development⁴. One of its uses is in discovering the structural basis of specific drug targets. It can also be employed to identify the active ingredients of Chinese herbs that possess significant pharmacological activities and study their mechanisms for drug screening and verification. Gassner et al. have established a linear dose-response curve for bispecific antibodies through SPR determination, which allows for concentration analysis and quality control⁵. Additionally, SPR can be utilized for conducting clinical immunogenicity tests in pharmacopeia and vaccine development⁶.

One area where it can be utilized is in the detection of pesticide residues, veterinary drug residue, illegal additives, pathogenic bacteria, and heavy metals⁷^,⁸^,⁹^,¹⁰ in agricultural products and food safety testing. By using SPR technology, the accuracy and efficiency of these tests can be improved.

Another area where SPR technology can be applied is in the rapid detection of toxins and antibiotics. This technology allows for the attachment of viral antibodies, small molecule compounds, and aptamers to the SPR biosensor chip. The SPR biosensor chip then detects different concentrations of viral RNA as the analyte¹¹. This method has been used successfully in the rapid detection of viruses such as H5N1, H7N9 avian influenza virus, and novel coronavirus¹²^,¹³^,¹⁴. In addition to these applications, SPR technology is also useful in proteomics, drug screening, real-time detection of related pharmacokinetics, and the study of virus and pathogenic proteins and receptors¹⁵^,¹⁶^,¹⁷^,¹⁸. It is particularly suitable for scientific research and teaching experiments in universities and research institutes and is a valuable tool in various scientific and research settings.

The principle of SPR is the collective oscillatory motion of free electrons at the interface between a metal film and dielectric, caused by incident light waves¹⁹. It is essentially the resonance between the evanescent wave and the plasma wave on the metal surface²⁰. When light transitions from a photodense medium to a photophobic medium, total reflection occurs under certain conditions. From the perspective of wave optics, when the incident light reaches the interface, it does not immediately generate reflected light. Instead, it first passes through the optically phobic medium at a depth of approximately one wavelength. It then flows along the interface for about half a wavelength before returning to the optically dense medium. This wave passing through the optically phobic medium is referred to as an evasive wave, as long as the total energy of the light remains constant. Since metal contains free electron gas, it can be regarded as plasma. The incident light excites the longitudinal vibration of the electron gas, leading to the generation of a charge density wave along the metal-dielectric interface, known as a surface plasma wave. This resonance propagates in the form of exponential attenuation in both media. Consequently, the energy of the reflected light is significantly reduced. The corresponding incidence angle at which the reflected light completely disappears is known as the resonance angle²¹. SPR is highly sensitive to the refractive index of the medium adhering to the metal film surface²⁰. The SPR angle varies with the refractive index of the metal film surface, with the refractive index change being primarily proportional to the molecular mass of the metal film surface²². Any changes in the properties of the surface medium or the amount of adhesion will result in different resonance angles. Thus, the molecular interaction can be analyzed by examining the changes in the resonance angle.

This non-destructive, label-free, real-time optical SPR analysis, based on the above principles, is suitable for research in various fields. Therefore, we demonstrated the angular displacement of the SPR curve and experimental results by multi-cycle analysis, taking the combination of quercetin and calycosin with KCNJ2 recombinant protein as an example.

Protocol

NOTE: The complete experimental sensing curve indicates that the experimental process can be categorized into eight distinct stages. 1. Sample and buffer preparation Prepare sensor chips before the experiment. 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…

Representative Results

To determine whether the protein is fixed on the chip surface, the ordinate (response signal) of the SPR sensor map (Figure 1) is used, while the angular displacement of the SPR curve is obtained. Figure 2 and Figure 3 depict the SPR curve of the interaction between quercetin and calycosin with KCNJ2 recombinant protein on the immobilized surface of KCNJ2 recombinant protein after control reduction at concentrations ranging from 3.9…

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…

Declarações

The authors have nothing to disclose.

Acknowledgements

This work was supported by the Sichuan Provincial Major R&D Project (2022YFS043), the Key Research and Development Program of Ningxia (2023BEG02012), and Xinglin Scholar Research Promotion Project of Chengdu University of TCM (XKTD2022013).

Materials

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

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