WO2009074933A1

WO2009074933A1 - Biosensor based on frustrated total internal reflection

Info

Publication number: WO2009074933A1
Application number: PCT/IB2008/055116
Authority: WO
Inventors: Coen A. Verschuren
Original assignee: Koninklijke Philips Electronics N. V.
Priority date: 2007-12-13
Filing date: 2008-12-05
Publication date: 2009-06-18

Abstract

The invention provides a Frustrated Total Internal Reflection (FTIR) biosensor device which comprises an optical cavity (10). Light is coupled into the optical cavity from a light source (1), for example a pulsed laser. Inside the optical cavity, the light is directed multiple times along an optical path which includes a light entrance (8), a reflection on the sensor surface (2) of the FTIR biosensor under an angle fulfilling the condition of total internal reflection, and a light exit (9). A predetermined portion of the light beam directed from the sensor surface towards the light exit is redirected back to the sensor surface and subsequently to the light exit by light beam redirecting means (4). The intensity loss due to the total internal reflection on the sensor surface is a measure for the presence of label particles close to the sensor surface. The intensity of the light coupled out from the optical cavity is detected by detecting means (3).

Description

BIOSENSOR BASED ON FRUSTRATED TOTAL INTERNAL REFLECTION

FIELD OF THE INVENTION

The invention relates to an improved optical biosensor device, in particular a biosensor device with optical read-out using Frustrated Total Internal Reflection (FTIR). BACKGROUND OF THE INVENTION

The demand for biosensors is increasingly growing these days. Usually, biosensors allow for the detection of a given specific molecule within an analyte, wherein the amount or concentration of said target molecule is typically small. For example, the amount of drugs or cardiac markers within saliva or blood may be measured. Therefore, label particles, for example paramagnetic label beads, are used which bind to a specific binding site or spot only, if the molecule to be detected is present within the analyte.

In a typical experiment, at least a portion of the bottom of a well is prepared for the detection of the target molecules. The paramagnetic beads are added and the liquid to be investigated, which possibly contains the target molecules, is inserted into the well. To increase the reaction speed, a magnetic field (gradient) below the well can be used to pull the beads towards the bottom of the well. After a predetermined time, the magnetic field is removed, and another magnetic field above the well may be applied to pull the non-bonded beads away from the well bottom. Subsequently, the presence of beads at the binding spots at the bottom of the well may be detected.

The detection of the beads may be done using for example magneto- resistive techniques. A further known technique is to optically detect the magnetic label beads bound to the binding spots using FTIR. Such a FTIR magnetic biosensor may have a geometry as schematically shown in Fig. l(a). The biosensor device has a hemispherical bottom with a radius R. Light emitted from a light source, for example a laser or a LED, is coupled into the sample at an angle of total internal reflection, that is, an angle which is larger than θ_c. If no particles are present close to the sample surface, the light is completely reflected. If, however, label particles are bound to the sample surface, the condition of total internal reflection is violated, and a portion of the light is scattered into the sample and thus the amount of light reflected by the sample surface is decreased. By measuring the intensity of the reflected light with an optical detector, it is possible to estimate the amount of label particles bound to the binding spots on the sample surface.

For certain applications of the above described method using FTIR, like blood testing, near-patient or home testing, a high sensitivity may be required. Using the above described geometry shown in Fig. l(a), the detection signal is directly related to the ratio 1/I₀ of the incident intensity I₀ of the light source 1 and the reflected intensity I as monitored by the photo-detector, where I=I₀(I -α), α being the loss factor due to frustration of the total internal reflection at the sensor surface due to the presence of label particles close to the surface. Since α usually is small, I and Io may be very close, which makes accurate measurements important.

A way to increase the sensitivity of a magneto-optical FTIR biosensor is to allow the light beam emitted by the light source to undergo multiple FTIR reflections on the sample surface, before detecting the intensity in the photo-detector. This may be achieved by providing, for example, a dove-tail configuration as shown in Fig. l(b) with a highly reflective surface on one side of the dove-tail configuration. In this case, the light beam reflected from the sensor surface may again be re-directed to the sensor surface by the reflection on the highly reflecting surface and may then be detected after a further FTIR reflection from the sensor surface. By arranging multiple highly reflective surfaces, multiple-pass reflection on the sensor surface may be possible. In this way, the signal I will be given by the I=Io(I -α)ⁿR^{n l}, where n is the number of FTIR reflections at the sensor surface and R is the reflectivity of the highly reflecting surface. That is, depending on the quality of the reflecting surface, that is, the reflectivity R, the gain in sensitivity can be close to n. However, due to practical limitations, such as beam divergence and mirror lithography accuracy, the maximum number n of reflections, and thus the gain in sensitivity is limited to about 10 for a typical configuration. SUMMARY OF THE INVENTION There is therefore a need for a FTIR biosensor device which allows the detection of a specific target molecule in an analyte with a higher sensitivity.

The FTIR biosensor device according to the invention comprises an optical cavity wherein the optical path through the optical cavity comprises a total internal reflection on a sensor surface of the FTIR biosensor device. Light emitted from a light source coupled into the optical cavity is directed multiple times through the cavity, including the total internal reflection on the sensor surface of the biosensor device. Thus, a substantial increase of the number of reflections on the sensor surface is achieved, resulting in a substantial increase of gain in sensitivity. The light path through the cavity further includes a light entrance and a light exit. A light redirecting means redirects a predetermined portion of the light beam directed from the sensor surface towards the light exit back to the sensor surface and subsequently to the light exit. The light exit may comprise a mirror functioning as light beam redirecting means. The mirror preferably has a high reflectivity to allow a substantial portion of light to be redirected through the optical path to the sensor surface. However, also a small portion of light is coupled out of the optical cavity through the mirror each round-trip inside the cavity. The light coupled out from the cavity is detected by detecting means, for example a fast photo-multiplier or an avalanche photo-diode.

Signal detection methods may be similar to those used in cavity ring- down spectroscopy and related fields. Here, the intensity decay time or ring-down time of the cavity, which is determined by using the change of the intensity of the light detected by the detecting means, is used to characterize the intensity loss at the detection interface. In addition to the intensity loss due to absorption at the sensor surface, the measured intensity decay time depends on the frustration of the total internal reflection on the sensor surface, for example due to the presence of magnetic beads close to the sensor surface.

Typically, the optical cavity comprises two or more high-reflective mirrors acting as light beam redirecting means with appropriate spacing and radii of curvature to form a stable resonator. For example, the optical cavity may have the shape of a dove-tail prism, as shown in Fig. l(b), where high-reflective mirrors are arranged on the inclined surfaces of the prism. The high-reflective mirrors should have a reflectivity of at least 90% so that sufficient light is redirected through the cavity to the sensor surface. Furthermore, the mirrors should have a transmission which may be 5% or smaller in order to allow the light to be coupled in and out of the optical cavity through the mirrors.

When a very short laser pulse from a laser source is coupled into the cavity, one or more cavity modes are excited, which then decay exponentially with the ring-down time. A measurement of differences in the intensity decay time in the presence and absence of label particles on the sensor surface yields the absolute intensity loss, and is therefore a direct measure for the number of particles bound to a binding spot on the sensor surface.

In another embodiment of the invention, the optical cavity may include multiple facets, the optical path through the cavity comprising total internal reflections on some or on all of these facets. Light may be coupled in and out of such a cavity by using a prism-like structure in very close proximity to the entrance and exit facets. One single facet may act as entrance and exit facet, the entrance and exit facets may alternatively also be different facets of the optical cavity. One example of a multi-facet shape of the optical cavity is a regular octagon.

These and other aspects of the invention will be apparent from and elucidated with reference to the embodiments described hereafter.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 schematically shows a FTIR biosensor with (a) a hemispherical bottom and (b) a dove-tail prism shaped bottom for FTIR detection;

Fig. 2 schematically shows the lay-out of a biosensor including an optical cavity according to an embodiment of the invention;

Fig. 3 shows diagrams of the ring-down time versus the light loss; and

Fig. 4 schematically shows an optical cavity in the form of a regular octagon according to another embodiment of the invention. DETAILED DESCRIPTION OF EMBODIMENTS

Fig. 2 schematically illustrates the layout of a FTIR biosensor device comprising an optical cavity 10 according to an embodiment of the present invention. In this case, a structure for sample application similar to the one shown in Fig. l(a) and l(b) may be added on top of the optical cavity 10 shown in Fig. 2.

The general shape of the optical cavity 10 is a dove-tail prism as the one shown in Fig. l(b). The inclined surfaces of the prism are high-reflectivity mirrors 4 as an example of light beam redirecting means, by the reflections on these mirrors 4 and the reflection on the sensor surface 2 the optical cavity 10 is formed.

Light emitted from a laser source 1, which represents in this example the light source 1, is coupled into the optical cavity 10 through one of the high-reflectivity mirrors 4. As indicated in Fig. 2, preferably a short laser pulse is coupled into the optical cavity 10. The intensity of light coupled out of the optical cavity 10 through the other high-reflectivity mirror 4 at the right side of Fig. 2 is measured by detector 3. In case of a very short laser pulse, the intensity detected by detector 3 exponentially decreases with time. In order to measure the decay time of the intensity, the measured intensity may be digitized using a digitizer 6. A trigger signal 5 may be supplied to the digitizer 6 from the laser light source 1. The digitized signal output from the digitizer 6 may be used to extract the decay time, for example in a computer 7. Technically, the extraction of the decay time can be done by fitting the natural logarithm of the digitized data to a straight line, using a weighted least squares fitting algorithm.

When a short pulse from a laser source is coupled into the optical cavity, one or more cavity modes are excited, which then decay exponentially with a ring-down time which is given by τ=t_r / (L₀+Ld_et) where L₀ is the round-trip intrinsic loss, for example due to surface scattering, mirrors 4 and cavity bulk, and La_et is the intensity loss at the detection surface. The round-trip time t_r for light propagating in the optical cavity 10 is given by the round-trip cavity length times the refractive index of the material inside the optical cavity 10 divided by the speed of light in the vacuum. By measuring the difference τ-τo of the intensity decay rates in the presence (τ) and in the absence of label particles (τo) on the sensor surface 2, the absolute intensity loss can be determined. The intensity decay rate is thus a direct measure for the number of label particles on the sample surface.

To estimate the achievable sensitivity, some numbers are given in the following for practical configuration. In current research set-ups for Blu-ray Discs, laser pulse rise times of round 200ps can be achieved and detectors can distinguish timing differences in the order of lOps. Assuming a total mirror 4 and bulk material absorption loss of about Lo= 1%, a refractive index of 1.5, and a cavity length of lcm, the nominal ring-down time τo is 5ns. Light loss at the sensor surface 2 will lead to shorter ring-down times.

To give an indication of the attainable sensitivities, an example is given in Table 1 for the numbers provided above. Fig. 3(a) and (b) show the light loss for the ring-down time and τ-τo, respectively, for the above numbers. As can be seen, the lOps resolution just allows to distinguish the nominal ring-down time from a ring-down time for a light loss which is related to the presence of magnetic particles close to the sensor surface 2, as small as 2xlO^~5, that is L₀xl0ps / τo, which is orders of magnitude better than previous methods which may achieve a sensitivity of 10^~2. For larger bead concentrations close to the sensor surface 2, which accordingly causes a larger light loss, ring-down times go down rapidly, ultimately to values which are too small for the current detectors to capture. However, these small ring-down time fall outside the interesting range.

Table 1. Ringdown times for L=lcm, n=1.5 and Zo=O-Ol.

As it is clear from the above, improvements in the round-trip intrinsic loss L₀ of the optical cavity, either through reduced bulk absorption or improved mirror reflectivity, directly improve the sensitivity. For example, reducing L₀ from 0.01, on which value the numbers in Table 1 are based, to 0.001 already yields a minimum detectable light loss 2xlO^"7, since τ₀ goes up to 50ns. A comparison of L₀ = 0.01 and L₀ = 0.001 is shown in Fig. 3. From these results, it can be seen that using high-quality mirrors 4 is important when a high sensitivity should be achieved. However, when choosing the mirrors 4, the improvements in sensitivity should be balanced with the increase of costs, which is of particular importance for disposable cartridges.

For the highest sensitivity, the photo detectors do not need to be very fast. For the parameters given above, the ring-down time is in the order of 10ns or larger. Therefore, low noise is more important in order to extract the decay time with high accuracy. For further improving the signal quality, time-averaging can be used, that is, an average of multiple decay time measurements may be calculated.

Furthermore, due to the high mirror reflectivity, high-power laser pulses are preferred. Since only a small fraction of the light will be coupled out of the optical cavity 10 and then reach the photodetector, a photodetector with a high sensitivity is required. For example, a fast photo -multiplier tube (PMT) or an avalanche photo-diode arrangement may be used.

For determining decay times which are short or even too short compared to the detector speed, it is preferable to calibrate the laser power. To do so, a time- integration of the detector signal can be used with a fixed or calibrated laser intensity to extract the decay time, even when the decay time itself can no longer be measured directly.

If very short pulses are used, a variation of the laser power intensity is no problem since the decay time is measured and not the intensity itself. However, instead of using pulsed lasers, also continuous lasers can be used. In this case, the light emitted by the continuous laser is rapidly scanned through a small wavelength range. When the wavelength corresponds to one of the resonance frequencies of the optical cavity 10, the detected intensity will show a peak. The total intensity of this peak is also a direct measure of the decay time, and thus a measure for the light loss at the detection surface.

In a further preferred embodiment of the invention, total internal reflection mirrors 4 with high optical grade, ultra-smooth polished surfaces may be used in the optical cavity. With such surfaces, 1-R values of 10^"6 have been achieved, allowing orders of magnitude improvements in sensitivity.

However, for conventional glass with a refractive index n of 1.5, the large value of the critical angle for total internal reflection requires a multi- facet configuration. As an example, a regular octagon configuration is shown in Fig. 4. In order to couple light in and out of the optical cavity 10 formed by the eight facets of the octagon, a prism- like structure 12 in very close proximity to an entrance facet 8 and an exit facet 9 can be used. In the octagon shown in Fig. 4 the entrance facet 8 for coupling in light is at the bottom side of the octagon next to the light source 1, the exit facet 9 for coupling out light is at the right side of the octagon next to the detector 3. At the top of the octagon opposide to the entrance facet 8 the sensor surface 2 is arranged. The five residual facets of the optical cavity 10 formed as an octagon are means for redirecting the light, in this example mirrors 4. The exact distance from the facet determines the efficiency of evanescent coupling from the light source 1 to the optical cavity 10 and from the optical cavity 10 to the detector 3, respectively. This distance can be tuned using piezo-actuators, and also by applying a low-refractive index transparent dielectric with the correct thickness on the mentioned facets may be used. In the latter case, the prisms may simply be pushed in contact with the coated facets. To further elucidate, the course of light inside the cavity 10 is described in the following. Starting from the light entrance facet 8 the light from the light source 1 enters the cavity 10, is reflected by the facets of the octagon in a direction anticlockwise. Small losses of light occur at the sensor surface 2, at the entrance facet 8, and at the exit facet 9. The main part of light is reflected at the facets and keeps going round inside the cavity 10 as indicated by the arrows showing the course of light. At the sensor surface 2 at the top the light looses relatively much intensity, depending on the amount of label beads in the analyte which is correlated to the molecule or substance to be detected by the biosensor. As described the label beads are detected by the detector means or detector 3. As the label beads bind to the molecules or substances, for which detection the biosensor is adopted, the amount of molecules is concluded by the measurement of the label beads. The amount of light coupled out of the cavity 10 at the exit facet 9 is sufficient to execute a sensitive measurement of the amount of label beads at the sensor surface 2, at which a chamber of the biosensor is provided comprising the label beads and the molecules or substances to be measured within the analyte. This chamber comprising the analyte is preferentially arranged outside the cavity 10 at the sensor surface 2. Within the chamber an assay my be provided, to which the label beads and molecules bind in a variety of binding methods known in the art. Generally, an assay is a procedure and a substrate at which a property or concentration of a substance is measured.

While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and non-restrictive; the invention is thus not limited to the disclosed embodiments. Variations to the disclosed embodiments can be understood and effected by those skilled in the art and practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite article "a" or "an" does not exclude a plurality. A single processor or other unit may fulfill the functions of several items recited in the claims. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures can not be used to advantage. Any reference signs in the claims should not be considered as limiting the scope.

Claims

CLAIMS:

1. A FTIR biosensor device comprising

(a) a light source (1) generating a light beam;

(b) an optical cavity (10) comprising an optical path for the light beam between a light entrance, a sensor surface (2) arranged with an angle of the optical path fulfilling the condition of total internal reflection, and a light exit;

(c) light beam redirecting means for redirecting a predetermined portion of the light beam directed from the sensor surface (2) towards the light exit back to the sensor surface (2) and subsequently towards the light exit; and

(d) detecting means (3) for detecting the intensity of light coupled out from the optical cavity (10) through the light exit.

2. The device according to claim 1, wherein the total internal reflection on the sensor surface (2) is frustrated due to magnetic label beads close to the sensor surface (2).

3. The device according to claim 1, wherein the light exit comprises a mirror (4) through which a portion of the light beam is coupled out of the optical cavity (10).

4. The device according to claim 3, wherein the mirror (4) has a reflectivity of at least 90% and a transmission of 5% or less.

5. The device according to claim 1, wherein the optical cavity (10) includes at least two highly reflective mirrors (4).

6. The device according to claim 5, wherein the mirrors (4) have a convex surface.

7. The device according to claim 1, wherein the optical cavity (10) has the shape of a dove-tail prism.

8. The device according to claim 1, wherein the light source (1) is a laser or a LED.

9. The device according to claim 1, wherein the detecting means (3) is a fast photo-multiplier or an avalanche photo-diode.

10. The device according to claim 1, wherein the light source (1) generates a pulsed light beam.

11. The device according to claim 1, wherein the light source (1) is a pulsed laser.

12. The device according to claim 1, wherein the detecting means (3) is adapted to measure the decay time of the intensity of the light coupled out from the optical cavity (10).

13. The device according to claim 1, wherein the optical cavity (10) includes multiple facets, wherein the optical path comprises total internal reflections on the facets.

14. The device according to claim 13, wherein the optical cavity (10) has the shape of a regular octagon.