WO1995029398A1

WO1995029398A1 - Analytical device

Info

Publication number: WO1995029398A1
Application number: PCT/GB1995/000907
Authority: WO
Inventors: James Oscar Molloy
Original assignee: Fisons Plc
Priority date: 1994-04-23
Filing date: 1995-04-21
Publication date: 1995-11-02
Also published as: GB9408107D0

Abstract

Apparatus for the testing of a sample containing chemical or biochemical species includes an optical sensor (1-4) such as a biosensor based on the principles of frustrated total reflection. A source (13) of monochromatic light is arranged to generate an incident light beam which orbits about the sensor (1-4), preferably in a plane orthogonal to the active surface. A stationary detector (20) is provided to monitor light reflected by the sensor (1-4) as a function of the angle of incidence. The apparatus can be used for the qualitative or quantitative determination of chemical or biochemical species in the sample, or to monitor the interaction of such species with other molecules.

Description

Title : Analytical Device

This invention relates to sensors, especially those termed biosensors, ie devices for the analysis of biologically active species such as antigens and antibodies in samples of biological origin. In particular, the invention relates to biosensors based on resonant optical phenomena, eg surface plasmon resonance or resonant attenuated or frustrated total internal reflection.

Many devices for the automatic determination of biochemical analytes in solution have been proposed in recent years. Typically, such devices (biosensors) include a sensitised coating layer which is located in the evanescent region of a resonant field. Detection of the analyte typically utilizes optical techniques such as, for example, surface plasmon resonance (SPR), and is based on changes in the thickness and/or refractive index of the coating layer resulting from interaction of that layer with the analyte. This causes a change, eg in the angular position of the resonance.

Other optical biosensors include a waveguide in which a beam of light is propagated. The^"optical characteristics of the device are influenced by changes occurring at the surface of the waveguide. One form of optical biosensor is based on frustrated total reflection. The principles of frustrated total reflection (FTR) are well-known; the technique is described, for example, by Bosacchi and Oehrle [Applied Optics (1982), 2_1, 2167-2173]. An FTR device for use in immunoassay is disclosed in European Patent Application No 0205236A and comprises a cavity layer bounded on one side by the sample under investigation and on the other side by a spacer layer which in turn is mounted on a substrate. The substrate-spacer layer interface is irradiated with monochromatic radiation such that total reflection occurs, the associated evanescent field penetrating through the spacer layer. If the thickness of the spacer layer is correct and the incident parallel wave vector matches one of the resonant mode propagation constants, the total reflection is frustrated and radiation is coupled into the cavity layer. The cavity layer must be composed of material which has a higher refractive index than the spacer layer and which is transparent at the wavelength of the incident radiation.

In devices of this kind, the position of resonance is monitored by varying the angle at which light is incident on the sensor. The scanning of angle may be performed either sequentially or simultaneously ie by varying the angle of incidence of a parallel beam of light or by simultaneously irradiating over a range of angles using a fan-shaped beam of light as described (in connection with SPR) in European Patent Application No 0305109A. In the former case, prior proposals have involved a single- channel detector which is mechanically scanned over a range of angles; this necessitates synchronisation of the movement of the light source and the detector. In the latter case, in which a range of angles is irradiated simultaneously, it is generally necessary to use a multi-channel detector having angular resolution. This leads to relatively high manufacturing costs.

International Patent Application WO 93/14391 discloses an arrangement in which a laser light source is mounted on a pivotally-mounted arm which is reciprocated through a range of angles by a rotating cam. This arrangement suffers from the disadvantage that the rate of change of the angle of incidence, and hence the resolution of the determination of that angle, varies sinusoidally with time. Also, the absolute position of the angle of incidence at which resonance occurs may vary quite substantially from one measurement to another. The angular range over which the pivoting arm moves must be great enough to cover all possible positions of the resonance.

There has now been devised an apparatus involving the use of a resonant optical sensor for the testing of a sample containing chemical or biochemical species, which overcomes or substantially mitigates some or all of the disadvantages of the prior art arrangements described above. According to the invention, there is provided apparatus for the testing of a sample containing chemical or biochemical species, comprising an optical sensor having an active surface, a source of monochromatic light arranged to generate an incident light beam which orbits the sensor, a stationary detector adapted to measure some characteristic of light reflected by the sensor, and means for monitoring the instantaneous angle of incidence.

The apparatus according to the invention is advantageous primarily in that, provided the light beam orbits about the sensor at constant angular velocity, the rate of change of the angle of incidence with time is constant. The resolution of the determination of the instantaneous angle is thus also constant. Also -.he f.;paratus is of relatively simple construction and uses only „- single-channel detector. The means for monitoring the instantaneous angle of incidence provides an accurate correlation of the output characteristics of the light beam with that angle.

The source of monochromatic light itself is preferably mounted for orbital motion about the sensor. Most preferably, the source is mounted on a wheel which preferably acts as a flywheel. The wheel may be driven at cc- £:tant speed by any suitably means, most preferably by an electric motor. The wheel is preferably rotated at a frequency of at least lOHz (600rpm), more preferably about 100Hz (6000rpm).

In other embodiments, the orbiting incident light beam may be generated by suitable optical elements, eg mirrors or other reflective elements, the source being fixed or else performing non-orbital motion.

For most applications, the diameter of the source's orbit may be in the range 5-20cm, eg about 10cm.

Preferably, the incident light beam orbits the sensor in a plane which is orthogonal, or substantially orthogonal, to the active surface. However, in other arrangements, the orbital plane may be tilted or inclined relative to the active surface, eg in order to facilitate access to the active surface.

The detector may have any suitable form, eg a linear or two- dimensional array of charge coupled devices (CCDs).

A single detector may be provided, measurements being made once in every complete revolution of the source. Alternatively, two detectors may be provided, symmetrically disposed about the optical sensor. In this case, two measurements are made per revolution, the radiation travelling in opposite directions along substantially the same path.

Any convenient source of monochromatic light may be used. The choice of source will depend inter alia on the particular form of sensor used. In this context, 'light' may include not only visible light but also wavelengths above and below this range, eg in the ultra-violet and infra-red. The source is most preferably a laser light source.

Where the source, or an optical element used to cause orbital motion of the incident light beam, is mounted on a wheel or the like, the means for monitoring the instantaneous angle of incidence of the light on the sensor may comprise means for determining the instantaneous position of the wheel. Most preferably, an opto-coupler device is provided to identify the passage of a fixed point on the wheel past a fixed point adjacent or radially outward of the wheel. Since the wheel rotates at constant angular velocity, this is sufficient to provide a complete record of the position of the motion of the wheel (and hence the angle of incidence) in time. However, more than one opto-coupler device may be provided if desired.

The characteristic of the light which is monitored may be any characteristic which changes at resonance, eg the phase of reflected radiation or, in some cases, the intensity. The sensor is preferably an FTR sensor. Such a sensor will generally include an optical structure comprising a) a cavity layer of transparent dielectric material of refractive index n₃, b) a dielectric substrate of refractive index n_x, and c) interposed between the cavity layer and the substrate, a dielectric spacer layer of refractive index n₂.

In use, the interface between the substrate and the spacer layer is irradiated with light such that internal reflection occurs. Resonant propagation of a guided mode in the cavity layer will occur, for a given wavelength, at a particular angle of incidence of the exciting radiation.

The angular position of the resonant effect depends on various parameters of the sensor device, such as the refractive indices and thicknesses of the various layers. In general, it is a pre¬ requisite that the refractive index n₃ of the cavity layer and the refractive index n_x of the substrate should both exceed the refractive index n₂ of the spacer layer. Also, since at least one mode must exist in the cavity to achieve resonance, the cavity layer must exceed a certain minimum thickness.

The cavity layer is preferably a thin-film of dielectric material. Suitable materials for the cavity layer include hafnium dioxide, zirconium dioxide, titanium dioxide, aluminium oxide and tantalum oxide.

The cavity layer may be prepared by known techniques, eg vacuum evaporation, sputtering, chemical vapour deposition or in- diffusion.

The dielectric spacer layer ι»ys have a lower refractive index than both the cavity layer and the substrate. The layer may, for example, comprise an evaporated or sputtered layer of magnesium fluoride. In this case an infra-red light injection laser may be used as light source. The light from such a source typically has a wavelength around 800nm. Other suitable materials include lithium fluoride and silicon dioxide. Apart from the evaporation and sputtering techniques mentioned above, the spacer layer may be deposited on the substrate by a sol-gel process, or be formed by chemical reaction with the substrate.

The sol-gel process is particularly preferred where the spacer layer is of silicon dioxide.

The refractive index of the substrate ( n_λ ) must be greater than that (n₂) of the spacer layer but the thickness of the substrate is generally not critical.

By contrast, the thickness of the cavity layer must be so chosen that resonance occurs within an appropriate range of coupling angles. The spacer layer will typically have a thickness of the order of several hundred nanometres, say from 200nm to 2000nm, more preferably 500 to 1500nm, eg lOOOnm. The cavity layer typically has a thickness of a few tens of nanometres, say 10 to 200nm, more preferably 30 to 150nm, eg lOOnm.

It is particularly preferred that the cavity layer has a thickness of 30 to 150nm and comprises a material selected from hafnium dioxide, zirconium dioxide, titanium dioxide, tantalum oxide and aluminium oxide, and the spacer layer has a thickness of 500 to 1500nm and comprises a material selected from magnesium fluoride, lithium fluoride and silicon dioxide, the choice of materials being such that the refractive index of the spacer layer is less than that of the cavity layer.

Preferred materials for the cavity layer and the spacer layer are titanium dioxide and silicon dioxide respectively.

At resonance, the incident light is coupled into the cavity layer by FTR, propagates a certain distance along the cavity layer, and couples back out (also by FTR) . The propagation distance depends on the various device parameters but is typically of the order of 1 or 2mm.

At resonance the reflected light will undergo a phase change, and it is this which may be detected. Alternatively, as described in our co-pending International Patent Application No PCT/GB91/01161 the cavity layer and/or spacer layer may absorb at resonance, resulting in a reduction in the intensity of the reflected light.

The sensor may be used for the qualitative or quantitative determination of chemical or biochemical species, or to monitor the interaction of such species with other molecules. In such cases, the surface of the sensor, ie the surface of the cavity layer in the case of an FTR sensor, will generally be sensitised by having biomolecules, eg specific binding partners for analyte(s) under test, immobilised upon it. The immobilised biochemicals may be covalently bound to the sensor surface (either directly or through intermediate molecules, or embedded in a molecular matrix deposited on the sensor surface) by methods which are well known to those skilled in the art.

The invention will now be described in more detail, by way of illustration only, with reference to the accompanying drawings in which

Figure 1 is a schematic side view (not to scale) of an apparatus according to the invention.

Figure 2 is a schematic view along the arrow II in Figure 1, and

Figure 3 depicts the dependence of the intensity of the detected light on the angle of incidence.

Referring first to Figure 1, a biosensor comprises a glass prism 1 coated over an area of its base with a first coating 2 of silicon dioxide and a second coating 3 of titanium dioxide. The prism 1 and first and second coatings 2,3 together constitute a resonant optical structure, the first coating 2 acting as a spacer layer and the second coating 3 as a cavity layer. The first coating 2 has a thickness of approximately lOOOnm and the second coating 3 a thickness of approximately lOOnm.

Immobilised on the surface of the second coating 3 is a layer 4 of immobilised biochemicals, which act as specific binding partner for the analyte under test.

The interface between the base of the prism 1 and the first coating 2 lies in a plane which is intersected by the axis of rotation A of an annular flywheel 10. Mounted on the flywheel 10 is a light-source unit 12 which includes a laser 13, collimating optics 14 and a polariser 15. The polariser 15 is arranged to produce linearly polarised light with two components: transverse electric (TE) and transverse magnetic (TM) . The polariser is set at 45° to the TE and TM transmission axes and thus provides equal components of TE and TM light.

Monochromatic light from the source 12 is incident on the interface between the base of the prism 1 and the first coating 2 and is reflected to a detector unit 20 mounted radially outward of the flywheel 10. The detector unit 20 comprises several components through which the reflected light passes sequentially: a compensator 21, a polarisation analyser 22, a cylindrical condenser lens arrangement 23 and a CCD-array detector 24. The compensator 21, which may be of any conventional form, is manually adjusted to remove any phase difference which is introduced into the TE and TM components on reflection and by birefringence in the optical path.

The analyser 22 is arranged at 90° to the polariser 15. The TE and TM components are interfered at the analyser to allow the phase change which occurs at resonance to be detected. Off resonance both components undergo a similar phase shift on reflection and the relative phase between the components is adjusted by the compensator 21 to give zero transmission through the analyser 22. This will apply for all angles except near resonance. Near resonance of either component, the phase shift between the TE and TM components will vary rapidly with angle, resulting in maximum throughput of light through the analyser 22 at resonance.

Light passing through the analyser 22 is focused by the cylindrical condenser lens arrangement 23 onto the detector 24. The condenser lens arrangement 23 is located so as to collect light from all incident angles of interest onto the detector 24. This minimises the effects of positioning errors.

In use, the flywheel 10 is rotated in the direction shown by the curved arrows in Figure 1 at a constant 6000rpm by an electric motor (not shown) . An opto-coupler device 30 is positioned adjacent the rim of the flywheel 10 to monitor the instantaneous position of the flywheel 10 and hence of the light-source unit 12. Over most of the rotation of the flywheel 10, light from the light-source unit 12 is prevented from reaching the biosensor by a cylindrical shield 40 (omitted from Figure 2 for clarity). However, the shield 40 is provided with a pair of cut-outs 41,42 which permit the light to reach the biosensor and be reflected to the detector unit 20 over a range of angles of incidence of about 20° about the position shown in Figure 1. This range includes all angles at which resonance may occur.

During each rotation of the flywheel 10, the incident light beam is therefore scanned through a range of incident angles including the resonant angle. Off-resonance no light intensity is detected at the detector 24; as resonance is approached, the detected light intensity increases and then falls. The increase in intensity is correlated with the angle of incidence, enabling the angular position of the resonance to be determined. The instantaneous angle of incidence is determined from the elapsed time since the opto-coupler device 30 has identified the passage of a fixed point on the flywheel 10. When the layer of immobilised biochemicals 4 is contacted with a sample containing the analyte under test, specific binding occurs between the biochemicals and the analyte molecules, resulting in a change in the refractive index in the vicinity of the surface of the device. This in turn results in a shift in the position of the resonance. Figure 3 shows a plot of the measured signal intensity against angle of incidence before and (dotted line) after complexation of the immobilised biochemicals with the analyte. Shifts in the position of the resonance can be used to monitor the presence and/or concentration of the analyte, or to provide an indication of the nature or strength of the interaction between the analyte and the immobilised biochemicals.

As can be seen in Figure 2, a second detector unit 20a identical in form to the detector unit 20, is positioned in radial alignment with the position of the light source unit 12 in Figure 1. This permits a second measurement to be made during each revolution of the flywheel 10, when the light-source unit 12 is in the position 12a shown by broken lines.

In alternative embodiments, the light source does not necessarily rotate in the plane which includes the sensor. Instead, the plane of rotation may be offset from that plane, light from the source being directed into the required plane by suitable optical components such as prisms or mirrors. In such a case, the initial path of the emitted light may not lie in the plane of rotation, but may for example be perpendicular to that plane.

Claims

1. Apparatus for the testing of a sample containing chemical or biochemical species, comprising an optical sensor having an active surface, a source of monochromatic light arranged to generate an incident light beam which orbits the sensor, a stationary detector adapted to measure some characteristic of light reflected by the sensor, and means for monitoring the instantaneous angle of incidence.

2. Apparatus as claimed in Claim 1, wherein the source of monochromatic light is mounted for orbital motion about the sensor.

3. Apparatus as claimed in Claim 2, wherein the source is mounted on a flywheel.

4. Apparatus as claimed in Claim 2 or Claim 3, wherein the diameter of the source's orbit is in the range 5-20cm.

5. Apparatus as claimed in any preceding claim, wherein the incident light beam orbits the sensor in a plane which is orthogonal to the active surface of the sensor.

6. Apparatus as claimed in any preceding claim, wherein the detector comprises a linear or two-dimensional array of charge coupled devices.

7. Apparatus as claimed in any preceding claim, wherein two detectors are provided, symmetrically disposed about the optical sensor.

8. Apparatus as claimed in Claim 3, wherein the means for monitoring the instantaneous angle of incidence comprises an opto-coupler device arranged to identify the passage of a fixed point on the flywheel past a fixed point adjacent or radially outward of the flywheel.

9. Apparatus as claimed in any preceding claim, wherein the sensor is an FTR sensor comprising

a) a cavity layer of transparent dielectric material of refractive index n₃, b) a dielectric substrate of refractive index n_{l f} and c) interposed between the cavity layer and the substrate, a dielectric spacer layer of refractive index n₂,

wherein both j_. and n₃ exceed n₂.

10. Apparatus as claimed in Claim 9, wherein the cavity layer has a thickness of 30 to 150nm and comprises a material selected from hafnium dioxide, zirconium dioxide, titanium dioxide, tantalum oxide and aluminium oxide, and the spacer layer has a thickness of 500 to 1500nm and comprises a material selected from magnesium fluoride, lithium fluoride and silicon dioxide, the choice of materials being such that the refractive index of the spacer layer is less than that of the cavity layer.