WO1996002823A1

WO1996002823A1 - Surface plasmon resonance sensors and methods of operation

Info

Publication number: WO1996002823A1
Application number: PCT/GB1995/001716
Authority: WO
Inventors: Alan Edward Green; Robert Martin Pettigrew; Colin Dennis Ager; Gordon Malcolm Edge
Original assignee: Scientific Generics Limited
Priority date: 1994-07-20
Filing date: 1995-07-20
Publication date: 1996-02-01
Also published as: AU2987395A; GB9414599D0

Abstract

A biosensor comprises a scanner (3) for applying a scan angle to a light beam and also for applying a high frequency oscillation or dither centred on the scan angle. The scannable oscillating beam is directed to a surface plasmon resonance interface (8) whence light is reflected to a photodetector (10). If the scan angle departs from the resonance angle, the reflected light has a component at the dither frequency. The detector detects this component and derives from it an error signal (5) which is fed back to control the scanner (3) to cause the scan angle to track the resonance angle. A further embodiment applies a high frequency variation in wavelength, the scan angle being fixed.

Description

TITLE: SURFACE PLASMON RESONANCE SENSORS AND

METHODS OF OPERATION

This invention relates to surface plasmon resonance (SPR) sensors and to methods of operation.

Surface plasmon resonance is a collective excitation of electrons at a surface of a metal or semi-conductor, with a characteristic electromagnetic field which penetrates a small distance away from the surface. The dispersion which governs the behaviour of the SPR depends strongly on the dielectric properties of the medium into which these fields penetrate. Thus, if an SPR is excited at a fixed wavelength, changing the dielectric constants of the region close to the surface changes the resonance angle of the SPR.

Biosensors using surface plasmon resonance interfaces are known. Typically, the surface is coated with a specific reagent, for example an antibody. When the target substance which is to be detected by the biosensor is present, it is captured by this reagent and held close to the surface, thus changing the dielectric environment for the SPR. For optical SPR biosensors, this change in the SPR's behaviour is measured by coupling free radiation to the SPR using the attenuated total reflection technique. Light is reflected through a prism at an angle such that it is totally reflected by the internal face of the prism at the SPR interface. The SPR active surface is in close proximity to this internal face, such that the evanescent field of the light extending away from the face outside the prism can couple to the fields of the SPR. If the frequency and angle of incidence of the light are such that this coupling can occur, energy is absorbed from the light by the SPR, reducing the amount of light reflected by the internal face. Generally, the biosensor measures the angle of incidence at which this minimum reflection occurs, this being the angle of resonance.

A conventional method of detecting the angle of resonance is to use a single LED source illuminating the SPR interface over a range of angles. The reflected light is distributed over an array of photodetectors, each measuring the reflected light through a corresponding small range of angle of incidence. The detectors are monitored in parallel and this collective response is compared with a model of the expected SPR absorption line shape to estimate the angle of minimum reflection, ie the angle of resonance.

The problems posed by the need to have an array of detectors are partially solved by GB 2247749 A which discloses a scanner for scanning a beam over an angular range. The scanned beam is directed at the surface plasmon resonance interface and a detector is used to detect the optical density of the reflected light. The detector output is measured as a function of time and, from this, the resonance angle can be determined. Because the scanner is not provided with any information about the resonance angle or any changes therein, the scanning beam must continue to scan across the complete angular range, only momentarily coinciding with the resonance angle. The invention aims to provide a sensor (and a method of operation) in which the scanning angle is actively controlled to permit tracking of the resonance angle. The invention also aims to provide a sensor (and a method of operation) in which wavelength is actively controlled to cause the prevailing wavelength to track the resonance wavelength.

According to one aspect of the invention a sensor for detecting the resonance angle in a surface plasmon resonance interface comprises a light source for producing a light beam, modulating means for applying to the light beam a variable scan angle within an angular range, a surface plasmon resonance interface onto which the scanned beam is directed, detecting means for detecting light reflected from the interface and for deriving an error signal representative of the departure of the scan angle from the resonance angle and feedback means for applying the error signal to the modulating means to cause the scan angle to tend to track the resonance angle. Preferably, the detecting means are a single photodetector used to measure the intensity of light reflected from the interface.

Preferably, the modulating means is a scanner operative to apply to the beam a small amplitude oscillation (or dither) which is superimposed on the scan angle to yield a scannable oscillating beam, in which case the detecting means is operative to detect a received light component at the same frequency as the dither oscillation, the error signal being derived from this component.

Neglecting asymmetrical components in the SPR absorption line shape, the detector signal oscillates at twice the dither frequency if the scanner's set position matches the SPR coupling angle (ie angle of resonance). If the set position does not match the coupling angle, there is an additional component of the detected signal at the same frequency as the dither. The phase of this signal (relative to the dither) indicates in which direction the set position should be moved to bring it closer to the coupling angle. This component of the detected signal thus acts as a correction to the set angle of the scanner.

The scanner may be a galvanometer or, advantageously, an acousto-optic (AO) scanner. In either case, the dither can be applied directly to the scanner's driver signal. Furthermore, an AO scanner has no moving parts, leading to a greater long term stability and enabling high frequency dither to be applied. In another implementation, the dither is applied separately using an additional scanner, for example, a piezo-electric actuated mirror.

Another optional feature of the invention is to program the dither to be non-sinusoidal, allowing compensation for an asymmetric resonance absorption.

Unlike the prior an methods using an array of detectors, all the light reflected by the sample is collected by one detector, giving an improvement in signal-to-noise ratio which allows greater angular resolution and faster measurement. The use of active feedback control results in greater accuracy in the determination of the resonance angle.

The performance limitations of the inventive sensor are determined by the optical power at the detector, the measurement bandwidth and the shape of the SPR resonance absorption. Optimal resolution depends on maximising the intensity of the light, reducing the bandwidth and sharpening the resonance.

Another possibility is to use a reference beam measurement to gain the effect of sharpening the resonance. For example, the SPR absorption is coupled only to the light polarised such that the electric field vector is contained in the plane of incidence (p- polarisation). The phase of the reflected p-polarised component undergoes a shift at the resonance position, while the orthogonal (s-) polarisation is unaffected. An additional measure of the resonance angle can therefore be obtained by illuminating the sample with a mixture of s- and p-polarised light and comparing the phase of the reflected p-polarised component against that of the s-polarised component. Combining the two measurements (intensity and relative phase) may be thought of as effectively sharpening the resonance absorption, thereby increasing the overall measurement resolution.

In a further possibility the resonance tracking is driven by maintaining a constant phase- difference between the s- and p-polarised components. The phase of one polarisation is dithered by transmitting it through a liquid crystal cell and varying the voltage applied across the cell. The relative phase of the two reflected polarisations is then compared to a reference value representing the optimal phase difference. The result of this comparison is an error signal which can be supplied to correct the scanner position.

The error signal may be derived from both the amplitude and phase measurement techniques, and combined to produce a more accurate correction for the scanner position.

According to another aspect of the invention there is provided a method of sensing the resonance angle in a surface plasmon resonance interface, comprising directing onto the interface a light beam at a variable scan angle within an angular range, detecting the light reflected from the interface and thereby deriving an error signal representative of the departure of the scan angle from the resonance angle and feeding back the error signal to control the scan angle in such a manner as to cause the light incident on the interface to track the resonance angle.

Instead of scanning the incident angle, the wavelength may be varied by a high frequency dither. Hence, according to a yet further aspect of the invention there is provided a sensor for detecting the resonant wavelength in a surface plasmon resonance interface, comprising a light source for producing a light beam, the source including or being associated with modulating means for varying the wavelength of the beam, a surface plasmon resonance interface onto which the beam is directed, detecting means for detecting light reflected from the interface and for deriving an error signal representative of the departure of the prevailing wavelength from the resonant wavelength and feedback means for applying the error signal to the modulating means to cause the wavelength to tend to track the resonant wavelength.

The invention also provides a method of detecting the resonant wavelength in a surface plasmon resonance interface, comprising modulating the wavelength of a light beam, directing the modulated light beam onto a surface plasmon resonance interface, detecting the light reflected from the interface and deriving an error signal representative of the departure of the prevailing wavelength from the resonant wavelength, feeding back the error signal, and employing the error signal to modulate the wavelength to cause the prevailing wavelength to track the resonant wavelength.

Two biosensors according to the invention will now be described, by way of example, with reference to the accompanying drawings, in which:

Figure 1 is a diagram of the first biosensor according to the invention,

Figures 2 and 3 are graphs representing signals when the scanning angle corresponds to the resonance angle in the first biosensor,

Figures 4 and 5 correspond to Figures 2 and 3 respectively but show the situation when the scanning angle departs from the resonance angle in the first biosensor,

Figure 6 is a diagram of a error signal in the situation depicted in Figures 4 and 5,

Figure 7 is a diagram of the second biosensor according to the invention, and

Figure 8 is a fragmentary view of an optic fibre interface of the second biosensor.

Referring to Figure 1, the first embodiment of biosensor comprises a light source 1 (a laser or an incoherent source) producing a light beam which is fed to a collimator 2, preferably in the form of a lens or series of lenses. The collimated light beam is directed to a scanner 3 which is capable of altering the direction of the beam through an angular range, as depicted by the arrow 4. The scanner 3 sets the angle of the beam, within the angular range, in accordance with an error signal 5. Hence, as the error signal 5 varies so does the scan angle of the beam leaving the scanner 3.

The scanner 3 applies a further angular variation to the light beam. This further angular variation is a high frequency oscillation at a comparatively low amplitude, superimposed on the scan angle. This high frequency oscillation therefore constitutes a dither to produce a rapid oscillation centred on the prevailing angular position of the beam determined by the error signal 5.

The scannable beam, with its high frequency dither, is focused by a lens 6 and passed through one face of a prism 7 before being incident at a point (or a localised region) on a surface plasmon interface 8. The light reflected from the point is focused by a lens 9 onto a single photodetector 10 which measures the intensity of light reflected from the interface 8. The photodetector 10 thus responds to the light intensity reflected from the interface 8 throughout the angular range of the scan.

If the set angle of the light beam incident on the interface 8 corresponds to the SPR angle of resonance, the light intensity at the photodetector 10 will be a minimum and the intensity variation will oscillate at twice the oscillation frequency of the dither. This situation is illustrated in Figures 2 and 3. Figure 2 shows the detector output signal plotted against scan angle of the beam incident on the interface 8. In Figure 2 the range 12 of the high frequency oscillation or dither is centred on the set angle 13 corresponding to a minimum in the detector output signal. Figure 3 shows the detector output signal varying at twice the frequency of the frequency of the dither signal.

If the resonance angle moves away from the set angle due to a change in the concentration of the target substance at the interface 8, the set angle 13 departs from the intensity minimum (Figure 4). Also the variation with time of the detector output signal becomes distorted, as illustrated in Figure 5. This distortion arises because of the presence at the detector 10 of -an additional signal component at the same frequency as the applied dither signal. The detector 10 has a lock-in amplifier which isolates and removes this additional signal component which is depicted in Figure 6 and which is fed back to the scanner 3.

The additional signal component of Figure 6 is an alternating signal. Its magnitude represents the difference between the set angle and the angle of resonance, and its phase (relative to the phase of the dither signal) represents the direction of the difference. From the additional signal component of Figure 6, the detector 10 derives a corresponding d.c. signal which is the error signal 5.

The magnitude of the error signal 5 represents the difference between the set angle and the angle of resonance. The sign of the error signal 5 represents the direction of this difference. The application of the error signal 5 to the scanner 3 thus provides a feedback action which causes the scanner to track or follow the resonance angle with high accuracy. This angle, which can be obtained from the scanner 3 or from the detector 10, is the useful output of the biosensor because it corresponds to the concentration of the target substance at the interface 8.

The second embodiment of biosensor is shown in Figures 7 and 8. This relies on the property that in certain surface plasmon resonance interface systems the angle of incidence is not variable. The second embodiment relies on varying or dithering the wavelength rather than the scan angle.

Referring to Figure 7, a wavelength tunable source 15 produces a light beam 16 which has a predetermined cental wavelength onto which is superimposed a low amplitude and high frequency modulation which varies the wavelength about the central value. The source 15 may be a broad band source (eg a tungsten halogen lamp) followed by a tunable filter or grating, or a tunable narrow band source such as a laser diode in an external cavity arrangement. A diffraction grating may provide the modulating means which applies the wavelength dither or modulation. The modulated beam 16 from the source 15 passes through a collimator 17 and thence encounters a prism 18 before striking the surface plasmon resonance interface 19. Light reflected from the interface 19 is collected by a lens 20 and directed to a detector 22, eg a single photodetector. The detected signal will correspond to that shown in Figures 2 and 4, except that the horizontal axis will represent wavelength not angle. The detector 22 obtains (from the component comparable with that of Figure 4) a d.c. error signal 23 which is fed back to the source 15. The error signal 23 constitutes the modulating signal which modulates the wavelength, to cause the wavelength of the light beam directed on the interference 19 to track the resonance wavelength. The magnitude of the error signal

23 represents the magnitude of the difference between the prevailing wavelength and the resonance wavelength and the sign of the error signal 23 represents the direction of the difference. Hence, the wavelength of the light beam is controlled by a feedback action comparable with that previously described for the scan angle tracking biosensor of Figures 1 to 6.

The surface plasmon resonance interface 19 is formed between the end of an optical fibre

24 (Figure 8) and the surrounding chemical sample 25 whose biological activity is being sensed by the biosensor. The end of the fibre 24 is stripped of its cladding 26 and provided with coatings 27 on its exposed length and a reflecting mirror 28 on its end. The mirror 28 reflects light back from the extremity of the fibre. The light intensity reaching the detector varies with the wavelength of the incident light, there being a minimum in intensity at the wavelength corresponding to the resonance wavelength.

The advantage of the second embodiment is that there is no requirement for a scanner, and the system can be used in arrangements where the angle of incidence to the SPR surface is fixed by geometry.

Claims

1. A sensor for detecting the resonance angle in a surface plasmon resonance interface, comprising a light source for producing a light beam, modulating means for applying to the light beam a variable scan angle within an angular range, a surface plasmon resonance interface onto which the scanned beam is directed, detecting means for detecting light reflected from the interface and for deriving an error signal representative of the departure of the scan angle from the resonance angle and feedback means for applying the error signal to the modulating means to cause the scan angle to tend to track the resonance angle.

2. A sensor according to claim 1, wherein the modulating means is a scanner operative to apply to the beam a high frequency oscillation or dither which is superimposed on the scan angle.

3. A sensor according to claim 2, wherein the detecting means are operative to detect a component at the frequency of the high frequency oscillation, the error signal being derived from said component.

4. A sensor according to claim 3, wherein the magnitude of said component is representative of the magnitude of the departure of the scan angle from the resonance angle and the phase of said component in relation to the phase of the high frequency oscillation is representative of the direction of departure of the scan angle from the resonance angle.

5. A sensor according to any of the preceding claims, wherein the scanner is an acousto- optic scanner.

6. A sensor according to any of claims 1 to 4, wherein the scanner comprises a galvanometer.

7. A sensor according to any of the preceding claims, wherein the detecting means comprise a photodetector and a lock-in amplifier.

8. A sensor according to claim 1, wherein the source produces an s-polarised component the phase of which is unaffected by reflection at the interface and a p-polarised component the phase of which is shifted at the resonance angle, the detecting means being operative to detect the phase difference between the s-polarised component and the p-polarised component after reflection.

9. A sensor according to claim 1, wherein the source produces an s-polarised component and a p-polarised component and wherein the modulating means applies to one of the components a high frequency phase variation, the detecting means being operative to detect the relative phases of the two components after reflection.

10. A method of sensing the resonance angle in a surface plasmon resonance interface, comprising directing onto the interface a light beam at a variable scan angle within an angular range, detecting the light reflected from the interface and thereby deriving an error signal representative of the departure of the scan angle from the resonance angle and feeding back the error signal to control the scan angle in such a manner as to cause the light incident on the interface to track the resonance angle.

11. A sensor for detecting the resonant wavelength in a surface plasmon resonance interface, comprising a light source for producing a light beam, the source including or being associated with modulating means for varying the wavelength of the beam, a surface plasmon resonance interface onto which the beam is directed, detecting means for detecting light reflected from the interface and for deriving an error signal representative of the departure of the prevailing wavelength from the resonant wavelength and feedback means for applying the error signal to the modulating means to cause the wavelength to tend to track the resonant wavelength.

12. A sensor according to claim 11, wherein the surface plasmon resonance interface comprises an optical fibre having an end stripped of cladding and forming the active interface.

13. A sensor according to claim 11 or 12, wherein the source is a tungsten halogen lamp.

14. A sensor according to any of claims 11 to 13, wherein the modulating means is a tunable filter or grating or a tunable laser diode.

15. A sensor according to any of claims 11 to 14, wherein the modulating means include a diffraction grating to modulate the wavelength of the light beam.

!6. A method of detecting the resonant wavelength in a surface plasmon resonance interface, comprising modulating the wavelength of a light beam, directing the modulated light beam onto a surface plasmon resonance interface, detecting the light reflected from the interface and deriving an error signal representative of the departure of the prevailing wavelength from the resonant wavelength, feeding back the error signal and employing the error signal to modulate the wavelength to cause the prevailing wavelength to track the resonant wavelength.