Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
  • G01N21/55—Specular reflectivity
  • G01N21/552—Attenuated total reflection
  • G01N21/553—Attenuated total reflection and using surface plasmons
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
  • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
  • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
  • G01N21/35—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
  • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
  • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
  • G01N21/35—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light
  • G01N21/3577—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light for analysing liquids, e.g. polluted water

Definitions

• the present invention relates to a system for detecting a physical, chemical or biochemical reactions, and in particular to a system in which surface electromagnetic waves (SEWs) interact with a specimen involved in the reaction.
• SEWs surface electromagnetic waves
• Biosensors incorporating surface electromagnetic wave technology are increasingly gaining popularity in pharmaceutical, medical and environmental applications as well as in biochemical research. These type of sensors require no labelling and offer the possibility of real-time monitoring of binding events. They are based on the sensitivity of surface electromagnetic waves (SEW) to the refractive index of the thin layer adjacent to the surface where the SEW propagates.
• SEW surface electromagnetic waves
• one binding partner is immobilized on the surface (often called a target) and the other partner is flowed across it. As binding occurs, the accumulation or redistribution of mass on the surface changes the local refractive index that can be monitored in real time by the sensor.
• Prior art interferometric devices such as a Mach Zehnder device have been configured to measure variations in the refractive index at the sensor surface via phase shifts. This is disclosed in WO01/20295.
• the configuration requires four independent components and is sensitive to sub- wavelength relative displacements of these components and hence very small mechanical and environmental perturbations.
• a mechanically more robust monolithic interferometric design is outlined in WO03014715.
• the theoretical limit for the sensitivity can be as good as 10 "8 refractive index units
• the sensitivity of real systems is limited to 10 "6 due to fluctuations in the temperature and chemical composition of the buffer surrounding the sample.
• a temperature stability better than 10 "3 °C would be required. This is due to the fact that the influence of changes in the refractive index of the surrounding buffer cannot be isolated from the influence of changes in thickness and refractive index of the analyte absorbed on sensor surface using the methods and systems of the prior art.
• An object of the present invention is to provide a surface electromagnetic wave (SEW) sensor system that can compensate for changes in the bulk refractive index of a buffer or allows the contribution of the bulk refractive index to an interference pattern to be separated from the contribution of an analyte absorbed on the sensor surface.
• SEW surface electromagnetic wave
• a system for detecting a physical, chemical or biochemical reaction comprises: a coherent radiation source for producing an incident wave; a carrier surface for supporting a specimen to be analysed, the carrier surface mounted on a substrate and capable of supporting surface electromagnetic waves (SEW); means for splitting the incident wave into an SEW and a first scattered wave, wherein the SEW propagates along the carrier surface and interacts with the specimen; means for generating a second scattered wave from the SEW; and, a detector for monitoring the interference between the first scattered wave and the second scattered wave.
• SEW surface electromagnetic waves
• a carrier chip for a specimen to be monitored comprises: a dielectric substrate; and a conductive film formed on the surface of the substrate suitable for carrying the specimen; wherein the conductive film comprises first means for splitting an incident wave into a first scattered wave and a surface electromagnetic wave (SEW), the SEW propagating along the carrier surface and interacting with the specimen, and a second means for generating a second scattered wave from the SEW.
• SEW surface electromagnetic wave
• a method of monitoring a specimen undergoing a physical, chemical or biochemical reaction occurring on a surface supporting surface electromagnetic waves comprises the steps of: splitting an incident wave into a first scattered wave and SEW such that the SEW propagates along the surface and interacts with the specimen; splitting the SEW which has interacted with the specimen to generate a second scattered wave; and, monitoring the interference pattern between the first and second scattered waves.
• Figure 1 is a schematic illustration of an apparatus according to the present invention for detecting a physical, chemical or biochemical reaction
• Figure 2 illustrates a first embodiment of a system according to the present invention in which changes in bulk refractive index are compensated for a particular angle so that the system is only sensitive to the changes in thickness or refractive index of an analyte absorbed on the sensor surface;
• Figure 3 illustrates a second embodiment of a system according to the present invention
• FIG. 4 shows detail of a carrier chip according to the present invention
• Figure 5 shows another embodiment of a carrier chip according to the present invention
• Figure 6 shows the calculated variation of the interference fringe position at the optimal angle in the embodiment of Figure 2 versus bulk refractive index of a surrounding buffer
• Figure 7 illustrates measurements made using the embodiment of Figure2 where the buffer (water) surrounding the chip was cooled down from 46 °C to 22 °C.
• the peak position (“phase") is insensitive to the bulk refractive index changes associated with heating while peaks separation (“frequency”) is.
• Figure 8 illustrates a further carrier chip according to the present invention
• Figure 9 illustrates an embodiment of a multi-point array detection system system according to the present invention in which the whole sensor surface is simultaneously illuminated using a line source and a 2-D CCD-array is used for detection;
• Figure 10 shows an example of an image observed on the CCD-array shown in Figure 9;
• Figure 11 shows multi-track analysis of streptavidin binding to a carboxymethylated surface along the sensor line. Tracks on the graph correspond to points along the sensor line separated by approximately 40 ⁇ m; and,
• Figure 12 shows an embodiment of a 2-D sensor array.
• FIG. 1 shows schematically a system for monitoring a physical, chemical or biochemical interaction in accordance with a first aspect of the present invention.
• a coherent optical beam generated by a monochromatic laser is focused using a lens, onto the edge of a metallic film able to support surface electromagnetic waves (SEWs).
• SEWs surface electromagnetic waves
• the optical beam passes through the glass prism on which the metallic film is mounted.
• a near-infrared laser 11 is used as the illumination source.
• Using a near-infrared source has the advantage of long propagation length for surface plasmons in gold and silver while conventional optics can be still used for imaging and illumination.
• other monochromatic sources are suitable and may be used.
• the laser provides a p-polarised beam.
• the p-polarised laser beam passes through the focusing lens 12 and then through the glass prism 13 on which a substrate 14 with a microfabricated metal film is attached, using an index matching liquid or gel in a fluidic cell.
• the index matching gel reduces light scattering and creates a continuous optical path.
• the glass prism may be a triangular prism as shown or a hemi-cylindrical prism.
• the laser beam is focused on an edge of the structure 13.
• the laser beam falls on the glass/liquid interface at an incidence angle larger than the angle of total internal reflection, so that the laser beam is totally reflected except at a small area around the edge of the metal structure.
• the evanescent light wave formed on reflection is partly scattered into light 15 propagating through the fluidic cell and partly scattered into a plasmon wave 16 propagating along the metal structure.
• the plasmon wave is further scattered by the structure to produce lightwave 17. Waves 15 and 17 propagate through the liquid cell and produce an interference fringe pattern on the measurement device 18.
• the metal structure can be formed from gold or silver, or any other metal capable of supporting surface plasmons or a combination of them, or alternatively a dielectric multilayer supporting a SEW. It is preferred to use either gold or silver/gold multilayer to increase surface plasmon propagation length.
• the metal structure can be deposited on the prism using a lithographic process. The metal structure is described below in more detail with reference to Figures 4 and 5 below.
• the method of the measurement is further illustrated by Figure 2.
• the phase is conserved during scattering processes the volume radiation beams 25 and 27 can be brought to interference.
• the phase difference between the beams depends on a surface plasmon (or SEW) wave vector k sp , the distance between the two scattering points a and the refractive index of the solution n.
• SEW surface plasmon
• the shift of the -interferogram will be-detected by a sensor 28 that can be either 2-section photodiode, 1-D photodetector or CCD array, or 2-D photodetector or CCD array.
• phase difference between beams 25 and 27 can be written as 2- ⁇ -a-(n sp -n-cos( ⁇ ))/ ⁇ , where ⁇ - is the wavelength of the light and A? SP is related to n via following equation:
• the bulk refractive index n of the buffer fluid and the local refractive index n t next to the metal surface are distinct and can differ due to layers physically or chemically absorbed on the metal surface (i.e. bound analyte). It can be assumed that:
• the direction ⁇ of this particular point depends both on n and ⁇ n, but expanding the above equation into a series and differentiating it can be found that changes in bulk refractive index in this geometry are partly compensated and the system is more sensitive to the refractive index changes on the surface by a factor of:
• n and ⁇ n separated by varying the shape of the fluidic cell can be equalized as shown in Figure 2.
• This equation can be used to find an angle ⁇ where the variation in n is not reflected in ⁇ ' by solving: d_
• Figure 7 shows the experimentally observed variation of a peak position at the optimal angle while cooling the water surrounding the silver microstructure from 46°C down to 22°C (refractive index variation -2*10 "3 ).
• the lower line shows that the peak position (phase) is insensitive to bulk refractive index changes, whilst the upper line shows that the peak separation (frequency) varies considerably.
• the bulk refractive index can be measured simultaneously and subtracted during data analysis.
• the interference fringes on the detector 38 are equidistant (this simplifies harmonic analysis of the pattern) and the distance between interference fringes ("frequency") depends only on the refractive index of the buffer while fringe position depends both on n and ⁇ n.
• FIG 4 shows a close up of the profile of a film for use in the apparatus of Figure 1 , Figure 2 or Figure 3 in accordance with a second aspect of the present -invention.
• a carrier film is mounted on the surface of a supporting transparent dielectric material.
• the carrier film includes a first section 41 of a first thickness and a second section 42 of greater thickness.
• Coherent radiation 43 incident on the edge of the first section in an attenuated total reflection geometry (ATR), i.e. incident under angle larger than the angle of total internal reflection, will partly scatter into volume radiation 45 and partly generate a SEW 44.
• This SEW 44 will propagate along section 41 of the carrier film until it reaches the boundary between sections 41 and 42.
• ATR attenuated total reflection geometry
• Figure 8 shows another possible embodiment of a microfabricated sensor with a built-in reference area.
• the incident beam is scattered not only on the metal film 81 (as in Fig. 4) but also on the film 87 (which can be both metallic or non metallic) generating a third volume wave 88.
• the relative phase of the wave 88 will depend on the bulk refractive index only. If the gap between 81 and 87 is different from the length of 81 , the contribution of the wave 88 to the fringe pattern can be separated by harmonic analysis.
• FIG. 9 A possible embodiment of such system is shown on Figure 9.
• a laser beam 92 generated by a laser 91 goes through a beam expander and conditioner 93 and is focused into a line by the cylindrical lens 94.
• Scanning mechanism 95 can switch the laser line between a number of microfabricated structures located on a substrate 96.
• the interference pattern generated by the microstructures is imaged and projected on a CCD-camera 98 by an optical system 97.
• the image on the CCD-camera is composed of a set of interference patterns generated by every point along the structure. This is shown in FigurelO.
• each particular location can be traced during a biochemical experiment.
• the system can be used to monitor binding of different analytes to target areas in DNA or protein arrays.
• the substrate can be spotted with different targets, illustrated schematically in Figure 12, where target material 121 is spotted on a plurality of microstructures 122 fabricated, for example, according to embodiments of Figures 4, 5. These microstructures can be interrogated either sequentially or simultaneously.
• a complimentary DNA strand can be produced using a polymerase and a parent DNA strand.
• a DNA strand is built from four base blocks, and binding of each of these four blocks to a DNA strand will lead to a characteristic charge distribution in a polymerase on the surface of the film. This in turn will lead to a characteristic change in the phase velocity of the SEW and hence a characteristic change in the interference pattern.
• the use of surface plasmon resonance in the detection of nucleotide incorporation during DNA synthesis is disclosed in WO-A-99/05315, the content of which is hereby incorporated by reference.

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• Physics & Mathematics (AREA)
• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Analytical Chemistry (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• General Physics & Mathematics (AREA)
• Immunology (AREA)
• Pathology (AREA)
• Spectroscopy & Molecular Physics (AREA)
• Investigating Or Analysing Materials By Optical Means (AREA)

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• 2003
  • 2003-09-02 WO PCT/GB2003/003803 patent/WO2004020985A1/en not_active Application Discontinuation
  • 2003-09-02 CA CA002497289A patent/CA2497289A1/en not_active Abandoned
  • 2003-09-02 EP EP03791065A patent/EP1535050A1/de not_active Withdrawn
  • 2003-09-02 AU AU2003263300A patent/AU2003263300A1/en not_active Abandoned

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