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

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 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 metals^7,8,9,10 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 coronavirus^12,13,14. 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 receptors^15,16,17,18. 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

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, allowing it to dry naturally.
   2. Next, soak the chip overnight in a solution of 11-mercapto-l-undecanol (MUOH) with a concentration of 50 M. Afterward, place the chip in an epichlorohydrin solution and allow it to react at 25 °C for 4 h.
   3. Remove the chip from the solution and wash it thoroughly with distilled water and anhydrous ethanol. Finally, drop the dextran basic solution onto the chip's gold film surface and allow it to react at 25°C for 20 h. Clean the chip with deionized water and then immerse it in a bromoacetic acid solution to achieve the dextran hydroxyl carboxymethylation modification. Use quercetin and calycosin with purity ≥98.5% and ≥99%, respectively.
2. Prepare all buffers containing running buffer, activator, immobilization buffer (10 mM sodium acetate), and regeneration solution 10 mM Glycine-HCl, pH 1.5. The activator contains 115 mg N-Hydroxysuccinimide (NHS), 750 mg 1-Ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC), and 10.5 mL of 1 M ethanolamine hydrochloride-NaOH at pH 8.5. Dissolve the EDC and NHS by adding 10 mL of filtered deionized water to each vial (see Table of Materials).
   ​NOTE: Store EDC and NHS aliquots at -18 °C or below, use aliquots within 2 months. All solutions should be degassed before use.

2. Instrument calibration

1. Draw a standard curve of alcohol concentration and peak value injecting alcohol at different concentrations (2%, 1%, 0.5%, 0.2%, and 0.1% volume ratio) on bare gold and comparing the maximum peak value of alcohol.
   NOTE: Injecting at a flow rate of 50 µL/min, by origin data analysis software obtained a standard curve.
2. According to the technical manual, calculate the correction factor of the instrument, represented by K, using the formula:
   K=Δθ/Δ[(A-B)/(A+B)]
   In this formula, A and B represent the signal values of the respective detectors. The theoretical slope value, as indicated in the manual, is 0.06. Therefore, the correction factor, K, can be calculated as K=0.06/R, (where R is the slope of the linear regression equation obtained from the standard curve).
3. Multiply the measured Δ[(A-B)/(A+B)] value by the correction factor to get the actual Δθ value.

3. Basic system operation

1. To start the system, turn on the power switch and wait until the detection unit reaches the preset temperature (usually 25 °C).
2. Open the Control Software by clicking Start > Program > OpenSPRTM Control Software, and the software program will automatically connect to the host system after running.
3. Follow the standard operating procedure of the SPR instrument to install the COOH chip (glucan-based chip). Firstly, put a drop of cedar oil on the back of the SPR biochip. Then, attach the COOH chip to the prism surface of the sensor, ensuring there are no air bubbles between the chip and the prism. Finally, install the flow pool and tighten it to the light spot.
   NOTE: Position the flow pool into the designated slot and securely fasten it using the fixing screws. The channel dimensions are 10 mm x 1 mm x 1 mm.
4. Chip placement
   1. To open the chip hatch, select Insert Chip option from the Tools menu. If there is already a chip in the chip bay, click Eject Chip option in the Tools menu.
   2. Select the corresponding Chip Type from the chip type drop-down menu if using a new chip and fill in the chip ID with the experimental information related to the chip. The chip lot number can be filled in (optional). If it is a used chip, select Reuse Chip and find the corresponding chip information in the chip ID drop-down menu.
   3. Hold the chip with the word side facing up, gently push the chip into the card slot following the direction of the arrow on the chip, and finally close the door of the chip compartment.
   4. Click Dock Chip button. The system will automatically switch to the standby state after the chip is placed.
   5. To flush the entire internal flow system at a high flow rate, select Tools > Prime command > Start. The entire process takes 6-7 min. Click Close to automatically switch the system to the standby state.
5. Press and hold the Black Button on the right side of the tube holder and open the metal cover to the left. After placing the appropriate test tube, gently close the metal lid. If you hear a click sound, the metal cover is locked.
6. Gently push the test tube holder along the card slot into the sample compartment and hear a click sound, indicating that the test tube holder is in the correct position and locked.
7. Click OK in the Eject Rack Tray dialog box to automatically feed the rack into the sample compartment and close the door.
   NOTE: There is a time limit when the sample cabin door is opened. The door will automatically close after 60 s of opening. In the last 15 s, the countdown in the dialog box appears in red font and flashes. Wait for the door to close before re-opening it to avoid pinching your hand.
8. Normalization of response signals
   1. Select Tool > More Tools, select Normalize in the Maintenance Tools directory, and click Start > Next.
   2. Add 120 µL of 70% BIAnormalizing solution to a 7 mm test tube. Place the test tube in the specified position shown in the program (R2F6). After confirming that the tube holder lid is properly closed, place the tube holder in the sample compartment and close the cabin door, and click Start to run the program.

4. Ligand capturing

1. pH measurement
   1. Before fixing ligands on the chip, screen a suitable ligand buffer pH to enrich ligands near the surface of the chip through electrostatic adsorption to achieve a better coupling effect. The general ligand concentration is 10-100 µg/mL, and for the first experiment, try using 20 µg/mL.
   2. Screen the ligand from 10 mM sodium acetate with pH values of 5.5, 5.0, 4.5, and 4. To set the process parameters, select Run-Wizard, Surface Preparation-Immobilization pH Scouting, and click New.
   3. Select the chip channel, which can be chosen from channels 1, 2, 3, or 4. The buffer has four preset conditions: 10 mM sodium acetate, pH 5.5, 5.0, 4.5, and 4. Next, enter the ligand name, contact time (180 s), and flow rate (5 µL/min).
   4. After the surface is regenerated with 50 mM NaOH, set the chip surface temperature and the temperature of the sample chamber. Next, select sample and reagent rack1 on the left, and place the solution according to the corresponding position in the sample rack. Save the experimental method and start the experiment by clicking Next.
2. Ligand immobilization
   1. Select Run-Wizard, Surface Preparation-Immobilization and click New. Set the mobilization capability to four channels: FC1, FC2, FC3, and FC4. Generally, channel 1 is used as the reference when pairing channels 1 and 2, and channel 3 is the reference when mobilizing all four channels.
   2. Use the reference channels for blank contrast or activation-closed ligand-free fixation, using channels 1 and 2 to couple ligands on channel 2.
      ​NOTE: There are two coupling modes available: aim for immobilized level and specify contact time. In the aim for immobilized level mode, enter the target coupling level, and the software will automatically achieve the corresponding level. The contact time mode requires the input of injection time and flow rate and is generally used based on pre-experiments.

5. Multi-cycle analyte method

1. Run Prime and fill the syringe and chamber with a fixed buffer. Select Run-Manual run, select Channels 1-2 in series on the flow path, and click Start.
2. Begin running at the maximum flow rate (150 µL/min) and detect the buffer which is HEPES (pH 7.4). When a stable baseline is reached, flush the sample ring with buffer and empty the sample ring.
   NOTE: If the baseline (a line of equilibrium formed on the sensing curve when the buffer is run) is not reached within 10 min, try injecting 10 mM of sodium hydroxide for 30 s.
3. Activate the chip with EDC/NHS (1:1, 50 µL each) solution.
4. Run 200 µL of ligand diluted with the activated buffer on the sample for 4 min. Once the binding is stabilized, wash the sample ring with buffer.
5. Sample 200 µL of blocking solution, rinse the sample ring with buffer, and empty it with air. Observe the baseline for 5 min to ensure stability.
6. Dilute the analytes with buffer solution, then take 3.9 µM, 7.8 µM, 15.625 µM, and 31.25 µM quercetin, and 15.625 µM, 31.25 µM, 62.5 µM, 125 µM, and 250 µM calycosin, as samples at 20 µL/min.
7. The binding time of protein (10 µg/mL-50 µg/mL) and ligand is 240 s, with a natural dissociation time of 360 s. Increase flow rate to 150 µL/min and inject appropriate regeneration buffer to remove analyte.

6. Regeneration

1. For regeneration, use 10 mM Glycine-HCl, high salt, or acid-base solutions of varying pH levels (e.g., 2.5, 4.5, 5.0, 5.5, etc.).
2. To screen the regeneration conditions, use run-wizard-assay development-regeneration scouting and click New. Select 1-2 channels in series on the flow path, select the chip, and click Next.
3. To warm up the chip, click on Startup, which should be done more than 3x. Enter the name of the analyte and set the flow rate of the regenerated solution to 150 µL/min.

7. Data analysis

1. Analyze the experimental results using an SPR data analysis software, using the One to One analysis model.
   ​NOTE: The whole process is conducted in the running buffer. The other buffers used in the SPR assay process are the same as the injection buffer.

8. System maintenance

1. After completing the experiment, remove the detection chip and replace it with the maintenance chip. Replace the running buffer with enough fresh pure water and flush the system with prime. Once primed, the system will automatically switch to standby mode to clean the waste liquid bottle and change the needle water.

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 µM to 250 µM. To minimize inaccuracies resulting from mass transport effects, the KCNJ2 recombinant protein molecules were not only fixed at low concentrations but also subjected to a higher flow rate of 20 µL/min during the kinetic test. Consequently, the dose-response sensory maps of quercetin and calycosin were obtained. Table 1 and Table 2 display the calculation results of kinetic rate constants (association rate constant, Ka; dissociation rate constant, Kd) and dissociation equilibrium constant (KD). When comparing both, it was observed that the Ka constant for calycosin was higher than that of quercetin, indicating that less time was required to form the complex. Conversely, the Kd constant for quercetin was lower than that of calycosin, demonstrating that more time was needed for quercetin to degrade the protein when a greater amount of KCNJ2 recombinant protein was fixed to the surface. The low KD content confirmed the high molecular affinity between the analyte and KCNJ2 recombinant protein, which is likely closely related to the channel in which this protein is found.

Figure 1: SPR sensing image of KCNJ2 recombinant protein immobilized on COOH sensor mount with glucan group modification. Activation: EDC/NHS 1:1 mixture activation (injection time: 1 min, flow rate: 20 µL/min). Fixation: 0.06 mg/mL KCNJ2 recombinant protein fixation (injection time: 5 min, flow rate: 20 µL/min). Inactivation: Remaining active NHS ester inactivated with ethanolamine (flow rate: 20 µL/min). Please click here to view a larger version of this figure.

Figure 2: Sensory map of interaction between quercetin and immobilized KCNJ2 recombinant protein. Four concentrations were tested: 3.9 µM, 7.8 µM, 15.625 µM and 31.25 µM. The affinity constant between quercetin and KCNJ2 recombinant protein was 20.5 µM. Please click here to view a larger version of this figure.

Figure 3: Sensory map of interaction between calycosin and immobilized KCNJ2 recombinant protein. Five concentrations were tested: 15.625 µM, 31.25 µM, 62.5 µM, 125 µM and 250 µM.The affinity constant between calycosin and KCNJ2 recombinant protein was 204 µM. Please click here to view a larger version of this figure.

Parameter	Paraphrase	Result
ka (1/(M*s))	Association rate constant	1.84 x 102
kd (1/s)	Dissociation rate constant	3.78 x 10-3
KD (M)	Dissociation equilibrium constant Affinity constant	2.05 x 10-5

Table 1: Kinetic measurements of quercetin interaction with KCNJ2 recombinant protein.

Parameter	Paraphrase	Result
ka (1/(M*s))	Association rate constant	1.21 x 102
kd (1/s)	Dissociation rate constant	2.46 x 10-2
KD (M)	Dissociation equilibrium constant Affinity constant	2.04 x 10-4

Table 2: Kinetic measurements of calycosin interaction with KCNJ2 recombinant protein.

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 phase of 240 s, followed by a dissociation period of 360 s. Both the association and dissociation processes are conducted in the running buffer. The cycles of the analyte are repeated according to analyte concentrations in ascending order. After each cycle of interaction analysis, the sensor chip surface needs to be completely regenerated using 10 mM glycine-HCl as the injection buffer at a flow rate of 150 µL/min to remove the analyte. Then, the next concentration cycle of the analyte is repeated by conducting analyte injection and regeneration steps. Lastly, in the fourth step, the chip is regenerated by injecting 10 mM glycine-HCl at a flow rate of 150 µL/min in the running buffer. This cycle enables the rapid and non-invasive screening of various indicators, facilitating high-throughput drug screening²³.

Additionally, it can detect early-stage antibody reactions and predict possible outcomes²⁴. Compared to other technologies, SPR has simple operation steps that do not require complex procedures such as electrophoresis and membrane transfer. It offers several advantages including a short detection cycle of 15 min²⁵, real-time detection without the need for labeling^26,27, minimizing human error, and high repeatability. Moreover, its ability to acquire multi-data such as dynamic parameters like Ka, Kd, and KD allows for accurate determination of molecular interactions, particularly in complex systems with dense molecules^28,29.

However, there are limitations to consider. Firstly, SPR chips are relatively expensive, as they are typically composed of a gold film mixed with other metal elements, with strict requirements for the thickness of the gold film. Secondly, kinetic detection may be influenced by the mass transfer rate. Thirdly, the SPR sensor chip may experience certain non-specific adsorption. Lastly, SPR faces challenges in accurately detecting substances with small molecular weight³⁰. To address this, methods such as mass labeling and indirect competition can enhance the sensitivity of small molecule detection, offering wide applications and promising prospects.

SPR finds extensive applications in environmental research, food safety³¹, and early diagnosis and treatment of heart disease, dementia, and cancer. Moreover, researchers have started combining SPR with various technologies such as electrochemistry³², fluorescence spectroscopy³³, and scanning electrochemical microscopy to broaden its analytical detection capabilities.

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