6.6 Surface plasmon resonance

Surface plasmon resonance is a powerful optical technique for studying biomolecular interactions at the nanoscale. It uses the collective oscillation of electrons at metal-dielectric interfaces to detect changes in refractive index caused by molecular binding events.

SPR sensors rely on principles like resonance conditions and evanescent waves to achieve label-free, real-time detection. Various configurations like prism-coupled and grating-coupled SPR enable applications in drug discovery, biomarker detection, and fundamental interaction analysis.

Surface plasmon resonance fundamentals

• Surface plasmon resonance (SPR) is an optical phenomenon that occurs at the interface between a metal and a dielectric medium, making it a powerful tool for studying biomolecular interactions at the nanoscale
• SPR is highly sensitive to changes in the refractive index near the metal surface, enabling label-free detection of biomolecules and their interactions
• The fundamental principles of SPR involve the collective oscillation of electrons, resonance conditions, evanescent waves, and penetration depth, which together determine the sensitivity and specificity of SPR-based sensors

Collective oscillation of electrons

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• In SPR, the free electrons in a metal (typically gold or silver) oscillate collectively in response to an incident electromagnetic wave
• These oscillations, known as surface plasmons, propagate along the metal-dielectric interface and are highly sensitive to changes in the dielectric environment
• The collective oscillation of electrons is responsible for the enhanced electromagnetic fields near the metal surface, which enables the detection of biomolecular interactions

Resonance condition

• SPR occurs when the wavevector of the incident light matches the wavevector of the surface plasmons, leading to a resonance condition
• The resonance condition depends on factors such as the wavelength and angle of the incident light, the refractive indices of the metal and dielectric medium, and the thickness of the metal film
• By monitoring changes in the resonance condition (e.g., shifts in the resonance angle or wavelength), SPR sensors can detect biomolecular interactions in real-time

Evanescent wave

• When the resonance condition is met, an evanescent wave is generated that propagates into the dielectric medium adjacent to the metal surface
• The evanescent wave has a limited penetration depth (typically 100-200 nm) and decays exponentially with distance from the metal surface
• Biomolecules that bind to the metal surface or to immobilized ligands within the evanescent field cause a change in the local refractive index, which is detected by the SPR sensor

Penetration depth

• The penetration depth of the evanescent wave determines the sensing volume of an SPR sensor
• A shorter penetration depth provides higher surface sensitivity but may limit the detection of larger biomolecules or complexes
• The penetration depth can be tuned by adjusting the wavelength of the incident light or the properties of the metal film (e.g., thickness, composition)
• Optimizing the penetration depth is crucial for achieving high sensitivity and specificity in SPR-based biosensing applications

SPR sensing principles

• SPR sensing relies on the sensitivity of surface plasmons to changes in the refractive index near the metal surface, which can be induced by the adsorption of biomolecules or changes in the bulk solution
• The key sensing principles in SPR include refractive index sensitivity, resonance angle shift, resonance wavelength shift, and the adsorption of molecules on the metal surface
• Understanding these principles is essential for designing and optimizing SPR sensors for various nanobiotechnology applications, such as biomolecular interaction analysis, biomarker detection, and drug discovery

Refractive index sensitivity

• SPR sensors are highly sensitive to changes in the refractive index near the metal surface, which can be caused by the adsorption of biomolecules or changes in the bulk solution composition
• The refractive index sensitivity of an SPR sensor depends on factors such as the properties of the metal film (e.g., composition, thickness), the wavelength of the incident light, and the refractive index of the dielectric medium
• Higher refractive index sensitivity enables the detection of smaller changes in the local environment, leading to improved detection limits and resolution

Resonance angle shift

• In angular-interrogation SPR sensors, the resonance condition is monitored by measuring the shift in the resonance angle (the angle at which minimum reflectivity occurs) as biomolecules adsorb onto the metal surface
• The magnitude of the resonance angle shift is proportional to the change in the refractive index near the metal surface, which is related to the mass and optical properties of the adsorbed biomolecules
• By tracking the resonance angle shift over time, SPR sensors can provide real-time, label-free monitoring of biomolecular interactions and kinetics

Resonance wavelength shift

• In wavelength-interrogation SPR sensors, the resonance condition is monitored by measuring the shift in the resonance wavelength (the wavelength at which minimum reflectivity occurs) as biomolecules adsorb onto the metal surface
• The resonance wavelength shift is also proportional to the change in the refractive index near the metal surface, providing an alternative method for detecting biomolecular interactions
• Wavelength-interrogation SPR sensors offer the advantage of using a fixed angle of incidence, which simplifies the instrumentation and enables miniaturization

Adsorption of molecules on metal surface

• The adsorption of biomolecules onto the metal surface is a key process in SPR sensing, as it leads to a change in the local refractive index that is detected by the sensor
• Biomolecules can adsorb onto the metal surface either directly (e.g., through electrostatic or hydrophobic interactions) or indirectly (e.g., by binding to immobilized ligands or receptors)
• The kinetics and affinity of biomolecular adsorption depend on factors such as the surface chemistry, the concentration and mass transport of the biomolecules, and the experimental conditions (e.g., temperature, pH, ionic strength)
• Optimizing the surface chemistry and experimental conditions is crucial for achieving specific and sensitive detection of target biomolecules while minimizing non-specific adsorption and background signals

SPR sensor configurations

• SPR sensors can be implemented in various configurations, each with its own advantages and limitations for nanobiotechnology applications
• The main SPR sensor configurations include prism-coupled SPR, grating-coupled SPR, waveguide-coupled SPR, and fiber optic-based SPR
• The choice of sensor configuration depends on factors such as the required sensitivity, sample volume, ease of integration with other techniques, and the specific application requirements

Prism-coupled SPR

• Prism-coupled SPR is the most common configuration, in which a thin metal film is deposited on a glass prism and the incident light is coupled to the surface plasmons through the prism
• The prism provides a high refractive index medium that enables the excitation of surface plasmons at a specific angle of incidence (Kretschmann or Otto configuration)
• Prism-coupled SPR offers high sensitivity and is well-suited for lab-based applications, but the bulky prism and precise angle control requirements limit its potential for miniaturization and point-of-care use

Grating-coupled SPR

• Grating-coupled SPR uses a periodic grating structure on the metal surface to couple the incident light to the surface plasmons
• The grating provides a wavevector matching condition that allows the excitation of surface plasmons at a specific wavelength and angle of incidence
• Grating-coupled SPR offers a more compact and integrated solution compared to prism-coupled SPR, as it eliminates the need for a separate coupling prism
• However, the fabrication of high-quality grating structures can be challenging, and the sensitivity may be lower than prism-coupled SPR

Waveguide-coupled SPR

• Waveguide-coupled SPR uses a planar waveguide structure to guide the incident light and couple it to the surface plasmons on a thin metal film deposited on the waveguide surface
• The waveguide provides a confined optical path that enables the excitation of surface plasmons over a longer interaction length, leading to enhanced sensitivity
• Waveguide-coupled SPR offers the potential for miniaturization and integration with other waveguide-based optical components, making it attractive for lab-on-a-chip and point-of-care applications
• However, the fabrication of high-quality waveguide structures and the efficient coupling of light into the waveguide can be challenging

Fiber optic-based SPR

• Fiber optic-based SPR uses an optical fiber with a thin metal coating on the fiber core or tip to excite surface plasmons and detect biomolecular interactions
• The optical fiber provides a compact and flexible platform for SPR sensing, enabling remote sensing and integration with other fiber optic components
• Fiber optic-based SPR offers the potential for miniaturization, multiplexing, and in-situ sensing applications, such as in-vivo monitoring and environmental analysis
• However, the sensitivity of fiber optic-based SPR may be lower than other configurations due to the limited interaction length and the challenges in achieving a uniform and stable metal coating on the fiber surface

SPR instrumentation

• SPR instrumentation involves the integration of various components, such as light sources, detectors, fluidic systems, and temperature control, to enable reliable and sensitive detection of biomolecular interactions
• The choice of instrumentation components depends on the specific SPR sensor configuration, the required sensitivity and resolution, and the application requirements
• Advances in SPR instrumentation have led to the development of compact, automated, and high-throughput systems that are well-suited for various nanobiotechnology applications, such as drug discovery, biomarker detection, and real-time monitoring of biological processes

Light sources for SPR

• The light source is a critical component in SPR instrumentation, as it provides the incident light that excites the surface plasmons and probes the biomolecular interactions
• Common light sources for SPR include monochromatic lasers (e.g., He-Ne, diode lasers) and broadband sources (e.g., halogen lamps, LEDs) coupled with a monochromator or a narrow bandpass filter
• The choice of light source depends on the SPR sensor configuration (angular or wavelength interrogation), the required sensitivity and resolution, and the spectral properties of the metal film and the biomolecules
• Factors such as the wavelength, power, stability, and beam quality of the light source can significantly influence the performance of the SPR sensor

Detectors for SPR

• The detector is responsible for measuring the changes in the reflected light intensity or spectrum that occur due to the biomolecular interactions at the metal surface
• Common detectors for SPR include photodiodes, CCD or CMOS cameras, and spectrometers, depending on the SPR sensor configuration and the required sensitivity and resolution
• Photodiodes are often used in angular-interrogation SPR sensors to measure the intensity of the reflected light at a fixed wavelength as a function of the angle of incidence
• CCD or CMOS cameras are used in SPR imaging (SPRi) to provide spatial resolution and enable the simultaneous monitoring of multiple biomolecular interactions on a single sensor surface
• Spectrometers are used in wavelength-interrogation SPR sensors to measure the reflectivity spectrum and detect shifts in the resonance wavelength

Fluidic systems for sample delivery

• Fluidic systems are essential for delivering the sample solution containing the biomolecules to the SPR sensor surface and controlling the flow rate, volume, and timing of the sample injection
• Common fluidic systems for SPR include microfluidic channels, flow cells, and automated liquid handling systems, depending on the sample volume, flow rate, and the level of automation required
• Microfluidic channels and flow cells enable the precise control of sample delivery and minimize sample consumption, which is particularly important for precious or limited-volume samples
• Automated liquid handling systems, such as robotic pipetting or microfluidic valves, enable high-throughput and reproducible sample delivery, which is essential for applications such as drug discovery and screening

Temperature control in SPR

• Temperature control is crucial in SPR experiments, as temperature fluctuations can affect the refractive index of the sample solution, the stability of the biomolecular interactions, and the overall sensor performance
• SPR instrumentation often incorporates temperature control elements, such as Peltier devices or temperature-controlled flow cells, to maintain a stable temperature during the experiment
• Precise temperature control is particularly important for measuring the thermodynamics and kinetics of biomolecular interactions, as well as for studying temperature-dependent conformational changes or phase transitions
• Advances in temperature control systems have enabled the development of high-resolution SPR thermodynamic and kinetic analysis methods, such as SPR-based thermodynamic profiling and temperature-jump SPR

Applications of SPR in nanobiotechnology

• SPR has emerged as a powerful tool for various nanobiotechnology applications, owing to its label-free, real-time, and sensitive detection capabilities
• The main applications of SPR in nanobiotechnology include biomolecular interaction analysis, affinity and kinetics determination, biomarker detection, and drug discovery and screening
• SPR-based methods have greatly advanced our understanding of the fundamental principles governing biomolecular interactions and have accelerated the development of new diagnostic and therapeutic technologies

Biomolecular interaction analysis

• SPR is widely used for studying the interactions between biomolecules, such as proteins, nucleic acids, lipids, and carbohydrates, at the molecular level
• By immobilizing one of the interacting partners (ligand) on the sensor surface and flowing the other partner (analyte) over the surface, SPR can monitor the association and dissociation of the biomolecular complex in real-time
• SPR-based biomolecular interaction analysis has provided valuable insights into the mechanisms of protein-protein interactions, enzyme-substrate recognition, antibody-antigen binding, and DNA hybridization
• SPR has also been used to study the interactions between biomolecules and nanostructured materials, such as nanoparticles, nanowires, and graphene, which is essential for developing novel nanobiosensors and nanomedicine applications

Affinity and kinetics determination

• SPR is a powerful tool for determining the affinity and kinetics of biomolecular interactions, which are critical parameters for understanding the specificity, stability, and functionality of biomolecular complexes
• By measuring the association and dissociation rates of the biomolecular complex at different analyte concentrations, SPR can provide quantitative information on the binding affinity (equilibrium dissociation constant, KD) and the kinetic rate constants (association rate constant, ka, and dissociation rate constant, kd)
• SPR-based affinity and kinetics determination has been applied to a wide range of biomolecular systems, including antibody-antigen interactions, protein-small molecule binding, and aptamer-target recognition
• The high sensitivity and throughput of SPR have made it a valuable tool for screening and optimizing antibodies, aptamers, and other affinity reagents for diagnostic and therapeutic applications

Biomarker detection

• SPR has been extensively used for the detection of various biomarkers, such as proteins, nucleic acids, and small molecules, which are indicative of disease states or biological processes
• By functionalizing the SPR sensor surface with specific recognition elements (e.g., antibodies, aptamers, or molecularly imprinted polymers), SPR can selectively detect the target biomarkers in complex biological samples, such as blood, serum, or urine
• SPR-based biomarker detection has shown great potential for early diagnosis, monitoring, and prognosis of diseases, such as cancer, cardiovascular disorders, and infectious diseases
• The label-free and real-time detection capabilities of SPR have enabled the development of rapid, sensitive, and multiplexed biomarker assays that can be integrated with point-of-care and personalized medicine applications

Drug discovery and screening

• SPR has emerged as a valuable tool for drug discovery and screening, owing to its ability to measure the interactions between small molecules and their protein targets in a label-free and high-throughput manner
• By immobilizing the protein targets on the SPR sensor surface and screening libraries of small molecules, SPR can identify hit compounds that bind to the target with high affinity and specificity
• SPR-based drug screening has been applied to various therapeutic targets, such as enzymes, receptors, and ion channels, and has led to the discovery of novel lead compounds and drug candidates
• SPR has also been used to characterize the binding kinetics, thermodynamics, and structure-activity relationships of drug-target interactions, which is essential for optimizing the potency, selectivity, and pharmacokinetic properties of drug candidates
• The integration of SPR with other techniques, such as mass spectrometry and X-ray crystallography, has provided a powerful platform for structure-based drug design and fragment-based drug discovery

Advances in SPR technology

• Recent advances in SPR technology have led to the development of new sensing configurations, imaging capabilities, and multiplexing strategies that have greatly expanded the applications of SPR in nanobiotechnology
• These advances include localized surface plasmon resonance (LSPR), SPR imaging (SPRi), multiplex SPR sensing, and the integration of SPR with other analytical techniques
• These new SPR technologies have enabled the detection of smaller analytes, the spatial resolution of biomolecular interactions, the simultaneous monitoring of multiple targets, and the correlation of SPR data with complementary information, such as mass, structure, or functionality

Localized surface plasmon resonance (LSPR)

• LSPR is a variant of SPR that utilizes the localized surface plasmon oscillations in metal nanostructures, such as nanoparticles, nanorods, or nanoholes, to detect biomolecular interactions
• LSPR offers several advantages over conventional SPR, including higher sensitivity to small analytes, lower sample volumes, and the potential for multiplexing and imaging
• LSPR-based sensors have been developed using various nanostructure geometries and materials, such as gold nanorods, silver nanotriangles, and aluminum nanoholes, which can be tuned to specific wavelengths and refractive index sensitivities
• LSPR has been applied to the detection