SurfacePlasmonResonanceBiosensors Tuncer ÇOBAN
SPR Principle • Linear relationship is found between resonance energy and mass concentration of biochemically relevant molecules.
Surface plasmon resonance (SPR) sensing has been demonstrated in the past decade to bean exceedingly powerful and quantitative probe of the interactions of a variety of biopolymers withvarious ligands, biopolymers, and membranes, including protein:ligand, protein:protein, protein:DNAand protein: membranebinding. • In a typical SPR biosensing experiment, one interactant in the interactant pair (i.e., a ligand orbiomolecule) is immobilized on an SPR-active gold-coated glass slide which forms one wall of a thinflow-cell, and the other interactant in anaqueous buffer solution is induced to flow across this surface,by injecting it through this flow-cell.
Basiccomponents of an instrumentfor SPR biosensing: A glassslide with a thin gold coating is mountedon a prism. Light passes through theprism and slide, reflects off the gold andpasses back through the prism to adetector. • Changes in reflectivity versusangle or wavelength give a signal that isproportional to the volume of biopolymerbound near the surface. A flow cellallows solutions above the gold surfaceto be rapidlychanged.
When light (visible or near infrared) is shined through theglass slide and onto the gold surface at angles and wavelengths near the so-called “surface plasmonresonance” condition, the optical reflectivity of the gold changes very sensitively with the presence ofbiomolecules on the gold surface or in a thin coating on the gold. • The high sensitivity of the opticalresponse is due to the fact that it is a very efficient, collective excitation of conduction electrons nearthegoldsurface.
A typical SPR biosensingexperiment, showingtheopticalresponse versus time: The gold surfacewith immobilized interactant starts in purebuffer at time 0. At 100 s,solutioncontaining the other interactant isintroduced. At 300 s, the flow cell isflushed with pure buffer, and at 420-520s, the starting surface is regenerated with a sequence of reagents. • The extent of binding between the solution-phase interactant and the immobilizedinteractant is easily observed and quantified by monitoring this reflectivity change Anadvantage of SPR is its high sensitivity without any fluorescent or other labeling of the interactants.
Advantage of using gold film • Gold:Non-magnetic, surface plasmon wave is p-polarized, and due to its electromagnetic and surface propagating nature, creates enhanced evanescent wave
TechniquestoInduceSurfacePlasmonResonanceBiosensors • The SPR technique is used in the development andcharacterization of ultra-thin films. SPR sensor system consistsof three parts: the optical system, the sensor system and thedetection system.
Among them, optical system includes thelight source and the optical path which is used to produce theincidence light meeting the performance requirements; then thesensitive information is transformed to the refractive indexchanges of the film by the sensor system, based on theprinciples mentioned above, and which can be transformed tothe changes of the resonance wavelength or the resonancesangle through the optical coupling action; finally by thedetection system the intensity of the reflected light is detectedand the position of the Resonance absorption peak is recordedforthefurtheranalysis.
SPR sensor has the followingadvantages: • High detection sensitivity • Real-time detection • Antiinterferencecapability • Samples without pretreatment • Rapid • High-throughput analysis • Less reagents and samples.
The SPR sensor detection method can be divided into thefollowing four types • Monochromatic incidence light: Changing the incidence angle, then detecting the changes of thenormalized intensity of the reflected light with the change ofthe incidence angle, and finally recording the incidence angle.When the reflected light intensity is minimal, the incidenceangle is also called the resonance angle. • Polychromaticincidence light: The incidence angle is fixed, and the reflectivitycurve is gained with the wavelength being changed, then theresonant wavelength is recorded. • Both the wavelength and the angle of incident light are fixed; The changes of therefractive index are analyzed by measuring the reflected lightintensity. • Both the wavelength and the angle of the incident light are fixed, the phase difference of the incident light andreflectedlight is observed.
The first two types of methods are used commonly amongthese four methods, the third one is less practical, and the lastone has the maximum sensitivity, but with a series of highfrequencycircuits required. • According to the structure of theoptical coupling system, the current SPR sensor system can bedivided into four structural types: optical prism couplers,grating couplers, optical fiber and optical waveguides.
A. Surface plasmon resonance biosensors based onopticalprismcouplers • The most common approach to excitation of surfaceplasmons is by means of a prism coupler and the attenuatedtotal reflection method. There are two configurations of theattenuated total reflection method:Kretschmann geometry andOtto geometry. • To avoid the refractive of the incidence laser inthe surface of the prism, the incidence or reflected laser isgenerally vertical to the prism surface. The prisms have twomain formats: theisosceles triangle prism and the semicylindricalprism.
Fig. 1. Attenuated total reflection method based SPR sensor • The Kretschmann prism is used to measurereactions on a sensor chip attached to a prism. The apparatusconsists of a sensor chip, a light source, a light detector, and aprism also referred to as the Kretschmann Prism (Fig. 1). ForKretschmann structure, a certain thick metal film at the bottomof the prism and the selective sensitive membrane are preparedto be placed above the sample pool, as the attenuated totalreflection occurs in the bottom of prism, the resonance angle orresonance wavelength will be gained by detecting the intensityof reflected light, thus the presence or concentration of this analyte may be determined. Prism coupled is widely used withsimple, sensitive and easy to implement.
B. Surface plasmon resonance biosensors based on opticalwaveguides • Surface plasmons can be also excited by modes of adielectric waveguide, and an example of a waveguidingstructure integrating a dielectric waveguide and a metaldielectricwaveguide is shown in Fig.2. • Its principle is verysimilar to that of the coupling prism of the Kretschmannstructure. A mode of the dielectric waveguide propagates alongthe waveguide and when it enters the region with a metal film,it penetrates through the metal film and couples with a surfaceplasmon at the outer boundary of the metal. If the phase ofSPW accords with the phase of SPW waveguide mode, SPWwill inspire and SPR peak curve can be detected at the outputof waveguide. This structure has a certain value with its meritssuch as easily-controlled, easy miniaturization and goodstability, etc. Fig.2 Dielectric waveguide based SPR sensor
C. Surface plasmon resonance biosensors based onopticalfibers • An optical fiber SPR sensors works by using a largediameter and multimode fiber, Cladding is removed from aportion of the fiber, and a surface plasmon metal layer isdeposited instead (as Fig.3). • When optical fiber SPR sensorsare used, their metal layer part is kept contact with thedetective liquid. In most cases SPR sensor with high sensitivityand high resolutions can be made by improving the singlemodefiber’s structure when it is used. • For it use fiber as itstransmission medium with such advantages as theminiaturization, remote detection and distributed detection,high sensitivity, allowing for chemical and biological sensingin inaccessible locations, and being able to transmit opticalsignals over a long distance makes the use of optical fibers veryattractive. Fig.3 Fibreopticbased SPR sensor
D. Surface plasmon resonance biosensors based ongratingcouplers • Grating coupled SPR sensors is shown in figure 4, if ametal–dielectric interface is periodically distorted, the incidentoptical wave is diffracted forming a series of beams directedaway from the surface at a variety of angles. Fig.4 Grating couple based SPR sensor • Thecomponent of momentum of these diffracted beams along the interface differsfrom that of the incident wave by multiples of the grating wavevector. • If the total component of momentum along the interfaceof a diffracted order is equal to that of the SPW, SPRphenomenon has occurred, thus the intensity of the diffractionluminous will substantially reduce, or even disappear.
Therefore, the Grating coupled SPR sensors can obtain SPRpeak curve by detecting the distribution of the diffractionluminous intensity. • This structure has such advantages that thesenor system can realize micro and batch production by the useof the modern advanced micro-machining technology and itisn’t strict with the thickness of metal films, but mathematicsinvolved in modeling of grating SPR-sensing structures is morecomplex than that for planar prism-based systems, thereforemodeling of the response of grating-based SPR structures andanalysis of sensor data are more difficult. • These disadvantageshave strictly limited the applications of the grating coupled SPR sensors.
SPR Biosensor Systems SPR: A powerful tool for real-time, label-free analysis of biomolecular interactions • The study and characterization of molecular interactions is essential to explore biomolecular structure-function relationships, and it aids our understanding of biological systems in life sciences. • Surface Plasmon Resonance (SPR) biosensors analyze macromolecular interactions in real-time and label-free. • They have proven to be a valuable tool for scientists in many disciplines including immunology, molecular biology, cell biology and biochemistry. • Compared to conventional techniques, SPR biosensors speed up such investigations as drug development, immunoreagent quality control, cell adhesion studies and polymer- biomolecule interactions.
Listed below are some examples of biomolecular interactions which have been successfully studied using SPR: • Peptide/protein - protein • DNA/RNA - protein • protein - cell • receptor - cell • protein - virus/phage • carbohydrate - protein • carbohydrate - cell • liposome - protein • artificial materials - biological matter • drugs - protein • drugs - DNA/RNA. In addition to biomolecular interaction studies, SPR sensors can be used to quantify adsorption and desorption processes in non-biological systems or to follow the course of solid phase-based chemical reactions on the chip surface.
Applications of SPR • Physical applications: measure dielectric properties, adsorption processes, surface degradation or hydration of • Thin organic monolayers or bilayers • Polymer films • Biological applications: as biosensors for specific biological interactions including adsorption and desorption kinetics, antigen-antibody binding and epitope mapping for determination of • Biomolecular structure and interactions of proteins, DNA & Viruses • Lipid Bilayers • Non-specific biomolecular interactions-bio-compatibility • Tissue engineering
SPR: Physical applications Thin organic monolayers or bilayersPolymer film
SPR: Biological applications Epitope mapping