Surface plasmon resonance (SPR) is an optical phenomenon. When the substance or the amount of substance on the metal surface changes, the refractive index changes accordingly. The resonance angle of the SPR spectrum to the refractive index of the medium in contact with the metal.

SPR affinity biosensors are devices that consist of three main subsystems: sensor hardware (optical reader), biorecognition element, and sample preparation and delivery system. In the optical reader of SPR sensor, the light wave excites the special electromagnetic field mode of surface plasmon (SP). The surface plasma propagates along the thin metal film, and its electric field detects the medium near the metal surface. Any change in the refractive index near the metal surface will lead to a change in the propagation velocity of the surface plasma. This change in propagation can be determined by the optical wave characteristics coupled to the surface plasmon. Biorecognition elements specific to the analyte molecules are immobilized on the metal surface. When the liquid sample is in contact with the sensor surface, the analyte molecules will be captured by the biorecognition molecules. This combination leads to a change in the refractive index near the surface of the sensor, which can be measured by an optical reader. The liquid sample is introduced into the sensor surface through a sample preparation and delivery system. In SPR affinity biosensors, the biomolecular recognition element is immobilized on the solid surface of the SPR sensor reader and captures analyte molecules.

Figure 1. Principle of operation of SPR affinity biosensors. (Reference source: Surface Plasmon Resonance Biosensing.)

After incubation, unbound proteins and non-specifically bound proteins need to be removed by washing steps. Washing buffers usually contain a certain concentration of salts (such as NaCl) and detergents (such as Triton X-100) to reduce non-specific binding. The washing step is the key to the Pull-down experiment, which determines the specificity and signal-to-noise ratio of the experiment.

The bound protein complex needs to be released from the solid support by elution buffer. The elution method depends on the characteristics of the label and the solid support:

Ligands are directly or indirectly immobilized on the chip surface. 

The ligand is covalently coupled to the chip surface, commonly used amino coupling method.

The capture molecule is covalently coupled to the chip surface, and the capture molecule is coupled to the ligand by affinity during each cycle. Activate the chip, dilute the ligand in the fixed buffer, and inject the sample channel. The chip was inactivated for some time. 

Biofilm interferometry (BLI) is a technique to detect the surface reaction of the sensor by detecting the displacement change of the interference spectrum. When a beam of visible light is emitted from the spectrometer, two reflection spectra are formed at the two interfaces of the optical film at the end of the sensor, and an interference spectrum is formed. Any change in film thickness and density due to molecular binding or dissociation can be reflected by the displacement value of the interference spectrum, and a real-time response monitoring map can be made through this displacement value.

• 1 BLI sensor chips (e.g. Ni-NTA, Protein A / G, Streptavidin, etc., selected according to experimental requirements) 
• 2 Ligand : molecules immobilized on the sensor 
• 3 Analyte : An interacting molecule to be detected
• 4 Binding buffers (such as PBS, HBS-EP, etc.) 
• 5 Elution buffer (remove unconjugated)
• 6 Regeneration buffer (for sensor regeneration)

The sensor was pre-wetted in an appropriate buffer solution to reduce non-specific binding and improve signal stability. 

The sensor is immersed in the analyte solution to be tested to monitor its binding to the ligand. Record the change of optical interference signal (combined phase). 

The sensor was moved into the binding buffer to dissociate the analyte from the ligand and monitor the dissociation rate. 

The regenerated buffer was used to remove the ligand-analyte conjugates, making the sensor reusable. 

The binding rate (ka), dissociation rate (kd) and affinity (KD) were calculated by the sensor signal curve (binding phase, dissociation phase).

Figure 2. A Schematic of the equilibration, association, and regeneration steps in each assay cycle. (Reference source: Detection of deoxynivalenol using biolayer interferometry.)

Alpha Lifetech provides SPR affinity assay and BLI affinity assay. Customers can choose the appropriate affinity assay according to their own needs. At the same time, Alpha Lifetech also provides other affinity measurement services, such as competitive ELISA affinity detection services, to help customers better research.