2 SPR PrincipleLinear relationship is found between resonance energy and mass concentration of biochemically relevant molecules.
3 Surface plasmon resonance (SPR) sensing has been demonstrated in the past decade to be an exceedingly powerful and quantitative probe of the interactions of a variety of biopolymers with various ligands, biopolymers, and membranes, including protein:ligand, protein:protein, protein:DNA and protein: membrane binding.In a typical SPR biosensing experiment, one interactant in the interactant pair (i.e., a ligand or biomolecule) is immobilized on an SPR-active gold-coated glass slide which forms one wall of a thin flow-cell, and the other interactant in an aqueous buffer solution is induced to flow across this surface, by injecting it through this flow-cell.
4 Basic components of an instrument for SPR biosensing: A glass slide with a thin gold coating is mounted on a prism. Light passes through the prism and slide, reflects off the gold and passes back through the prism to a detector.Changes in reflectivity versus angle or wavelength give a signal that is proportional to the volume of biopolymer bound near the surface. A flow cell allows solutions above the gold surface to be rapidly changed.
5 When light (visible or near infrared) is shined through the glass slide and onto the gold surface at angles and wavelengths near the so-called “surface plasmon resonance” condition, the optical reflectivity of the gold changes very sensitively with the presence of biomolecules on the gold surface or in a thin coating on the gold.The high sensitivity of the optical response is due to the fact that it is a very efficient, collective excitation of conduction electrons near the gold surface.
6 The extent of binding between the solution-phase interactant and the immobilized interactant is easily observed and quantified by monitoring this reflectivity change An advantage of SPR is its high sensitivity without any fluorescent or other labeling of the interactants.A typical SPR biosensing experiment, showing the optical response versus time: The gold surface with immobilized interactant starts in pure buffer at time 0. At 100 s,solution containing the other interactant is introduced. At 300 s, the flow cell is flushed with pure buffer, and at s, the starting surface is regenerated with a sequence of reagents.
7 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
8 Techniques to Induce Surface Plasmon Resonance Biosensors The SPR technique is used in the development and characterization of ultra-thin films. SPR sensor system consists of three parts: the optical system, the sensor system and the detection system.
9 Among them, optical system includes the light source and the optical path which is used to produce the incidence light meeting the performance requirements; then the sensitive information is transformed to the refractive index changes of the film by the sensor system, based on the principles mentioned above, and which can be transformed to the changes of the resonance wavelength or the resonances angle through the optical coupling action; finally by the detection system the intensity of the reflected light is detected and the position of the Resonance absorption peak is recorded for the further analysis.
10 SPR sensor has the following advantages: High detection sensitivityReal-time detectionAnti interference capabilitySamples without pretreatmentRapidHigh-throughput analysisLess reagents and samples.
11 The SPR sensor detection method can be divided into the following four types Monochromatic incidence light:Changing the incidence angle, then detecting the changes of the normalized intensity of the reflected light with the change of the incidence angle, and finally recording the incidence angle. When the reflected light intensity is minimal, the incidence angle is also called the resonance angle.Polychromatic incidence light:The incidence angle is fixed, and the reflectivity curve is gained with the wavelength being changed, then the resonant wavelength is recorded.Both the wavelength and the angle of incident light are fixed;The changes of the refractive index are analyzed by measuring the reflected light intensity.Both the wavelength and the angle of the incident light are fixed, the phase difference of the incident light and reflected light is observed.
12 The first two types of methods are used commonly among these four methods, the third one is less practical, and the last one has the maximum sensitivity, but with a series of high frequency circuits required.According to the structure of the optical coupling system, the current SPR sensor system can be divided into four structural types: optical prism couplers, grating couplers, optical fiber and optical waveguides.
13 A. Surface plasmon resonance biosensors based on optical prism couplers The most common approach to excitation of surface plasmons is by means of a prism coupler and the attenuated total reflection method. There are two configurations of the attenuated total reflection method: Kretschmann geometry and Otto geometry.To avoid the refractive of the incidence laser in the surface of the prism, the incidence or reflected laser is generally vertical to the prism surface. The prisms have two main formats: the isosceles triangle prism and the semicylindrical prism.
14 Fig. 1. Attenuated total reflection method based SPR sensor The Kretschmann prism is used to measure reactions on a sensor chip attached to a prism. The apparatus consists of a sensor chip, a light source, a light detector, and a prism also referred to as the Kretschmann Prism (Fig. 1). For Kretschmann structure, a certain thick metal film at the bottom of the prism and the selective sensitive membrane are prepared to be placed above the sample pool, as the attenuated total reflection occurs in the bottom of prism, the resonance angle or resonance wavelength will be gained by detecting the intensity of reflected light, thus the presence or concentration of this analyte may be determined. Prism coupled is widely used with simple, sensitive and easy to implement.Fig. 1. Attenuated total reflection method based SPR sensor
15 B. Surface plasmon resonance biosensors based on optical waveguides Surface plasmons can be also excited by modes of a dielectric waveguide, and an example of a wave guiding structure integrating a dielectric waveguide and a metal dielectric waveguide is shown in Fig.2.Its principle is very similar to that of the coupling prism of the Kretschmann structure. A mode of the dielectric waveguide propagates along the waveguide and when it enters the region with a metal film, it penetrates through the metal film and couples with a surface plasmon at the outer boundary of the metal. If the phase of SPW accords with the phase of SPW waveguide mode, SPW will inspire and SPR peak curve can be detected at the output of waveguide. This structure has a certain value with its merits such as easily-controlled, easy miniaturization and good stability, etc.Fig.2 Dielectric waveguide based SPR sensor
16 C. Surface plasmon resonance biosensors based on optical fibers An optical fiber SPR sensors works by using a large diameter and multimode fiber, Cladding is removed from a portion of the fiber, and a surface plasmon metal layer is deposited instead (as Fig.3).When optical fiber SPR sensors are used, their metal layer part is kept contact with the detective liquid. In most cases SPR sensor with high sensitivity and high resolutions can be made by improving the single mode fiber’s structure when it is used.For it use fiber as its transmission medium with such advantages as the miniaturization, remote detection and distributed detection, high sensitivity, allowing for chemical and biological sensing in inaccessible locations, and being able to transmit optical signals over a long distance makes the use of optical fibers very attractive.Fig.3 Fibre optic based SPR sensor
17 D. Surface plasmon resonance biosensors based on grating couplers Grating coupled SPR sensors is shown in figure 4, if a metal–dielectric interface is periodically distorted, the incident optical wave is diffracted forming a series of beams directed away from the surface at a variety of angles.Fig.4 Grating couple based SPR sensorThe component of momentum of these diffracted beams along the interface differs from that of the incident wave by multiples of the grating wave vector.If the total component of momentum along the interface of a diffracted order is equal to that of the SPW, SPR phenomenon has occurred, thus the intensity of the diffraction luminous will substantially reduce, or even disappear.
18 Therefore, the Grating coupled SPR sensors can obtain SPR peak curve by detecting the distribution of the diffraction luminous intensity.This structure has such advantages that the senor system can realize micro and batch production by the use of the modern advanced micro-machining technology and it isn’t strict with the thickness of metal films, but mathematics involved in modeling of grating SPR-sensing structures is more complex than that for planar prism-based systems, therefore modeling of the response of grating-based SPR structures and analysis of sensor data are more difficult.These disadvantages have strictly limited the applications of the grating coupled SPR sensors.
19 SPR Biosensor SystemsSPR: A powerful tool for real-time, label-free analysis of biomolecular interactionsThe 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.
20 Listed below are some examples of biomolecular interactions which have been successfully studied using SPR:Peptide/protein - proteinDNA/RNA - proteinprotein - cellreceptor - cellprotein - virus/phagecarbohydrate - proteincarbohydrate - cellliposome - proteinartificial materials - biological matterdrugs - proteindrugs - 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.
21 Applications of SPRPhysical applications: measure dielectric properties, adsorption processes, surface degradation or hydration ofThin organic monolayers or bilayersPolymer filmsBiological applications: as biosensors for specific biological interactions including adsorption and desorption kinetics, antigen-antibody binding and epitope mapping for determination ofBiomolecular structure and interactions of proteins, DNA & VirusesLipid BilayersNon-specific biomolecular interactions-bio-compatibilityTissue engineering
22 SPR: Physical applications Thin organic monolayers or bilayers Polymer film