FIELD OF THE INVENTION

The invention relates to an integrated optical or fibre optical interferometer based sensor device The invention also relates to uses of said device and methods for adjusting the output signal of said device

BACKGROUND ART

Both in basic and applied biological science investigating protein interactions, with special respect to medical diagnostics, there is an urgent need for high-throughput, sensitive measuring devices proper to analyse microscopic amounts (typically femtoliters) of samples Common to most of the methods applied today is the requirement for an optical label (e g the ELISA technique), increasing the complexity and thus the duration and costs for analysis It is, therefore, highly desirable to develop label-free detection techniques The already existing label-free technology (e g , "Biacore", based on the surface plasmon resonance phenomenon), on the other hand, requires rather expensive instrumentation In order to overcome these problems, we chose a rapid and sensitive label-free technique to be applied for monitoring protein intei actions

Although, realizations of Mach-Zehnder (MZ) based biosensors have been reported [1 ,2] in the art, to our knowledge, there is no commercially available biosensor based on this technique The main difficulty in applying MZ interferometers in practice is their, apparently inherent, instability due to the drift of the "working point" of the interferometer (a consequence of high sensitivity) The ideal working point of the interferometer is where the value transmission function is the highest For example, in GB 2437 543 A (Inventor Yaping Zhang) MZ and Young mterfeiometer based sensor devices are used in a multi-channelled waveguide system used for chemical and biochemical sensing The system is claimed to be useful in detecting biochemical association- dissociation processes However, no solution for a flexible and dynamic working point adjustment is provided In EP 0862 075 (Castoldi A and Bosso S) a LiNbO3 MZ electrooptical interfeiometi ic modulator device is disclosed which works based on electrooptical principle

In US 6137576 (Detlef P et al ) an optical transducer for measuring a contaminant in a gas is provided which is also based on a MZ interferometer The inventors used an electrooptical effect to detect a change in the sample via liquid crystal No adjuster means integi ated on the MZ interferometei is disclosed To overcome this problem, we developed a unique technique for optical adjustment of the "working point" of the device, that is expected to allow the use of MZ interferometers in piactice

The idea behind the method is the proper preadjustment of the phase shift of the light propagating in the reference branch of the MZ interferometer This can be realized by changing the refi active index of an ad-layer deposited on top of the reference blanch of the interferometer via an external control signal According to the physical nature of the control signal, one can distinguish a photo-optical and an electi o-optical solution

BRIEF DESCRIPTION OF THE INVENTION

The invention provides for the following embodiments \n integrated optical or fibre optical interferometer based sensor device, said device comprising

- an optical waveguide comprising a light path, said light path comprising a lead-in portion, a first junction with an input and at least two outputs, said input being connected to said lead-in portion, 5 a lead-out portion, a second junction with at least two inputs and an output, said output being connected to said lead-out portion at least a first arm and a second arm, each arm extending between one output of said first junction and one input of said second junction, 0 said first junction being capable of splitting a light beam, that is coupled into said lead-in portion, into at least first and second light beams propagating in said first and second arms, respectively, and said second junction being capable of recombining said first and second light beams and thereby creating an interference pat- ternthereof resulting in an output signal,

- at least one container for receiving a sample, said at least one container being directly or indirectly at- 5 tached to a first portion of at least one of the arms in such a way that any change taking place within said sample present in said at least one container affects a wave propagation property of said light beam propagating (travelling) in the at least one arm of said optical waveguide to which said at least one container is attached,

- at least one adjuster membei , said adjuster member having an ad-layer of non-linear optical property, an optical property of said ad-layer which being susceptible of a change upon exposition to an external effect, 0 said ad-layer being in contact with a second portion ot at least one of said first and second arms, wherein said first and said second portions of any of the arms are interchangeable and are spatially separated from each other

The term wave propagation property is meant to include any property of the propagating light which can be detected or described by a mathematical equation, preferably it includes at least the wavelength, frequency, 5 intensity, phase and any combination thereof of the propagating light

The term light is understood herein as an electromagnetic radiation in the infrared, visible or ultraviolet range, i e an infrared, visible or ultraviolet light

An integrated optical intei ferometer based sensor device, said device comprising at least the following paits 0 - an optical waveguide arrangement comprising a lead-in portion, a first Y junction, a first and a second arm, a second Y junction and a lead-out poi tion, wherein it a propagating light beam is inti oduced into the lead- in portion, it can be split at the first Y junction into a first light beam and a second light beam propagating in the first arm and in the second ai m, respectively, and i ecombine at the second junction resulting in an interference pattern and in turn an output signal, i b - at least one photooptical oi eleαrooptical adjuster member said adjuster member comprising an ad- layer comprising a non-linear optical matenal whei ein an optical property of the ad-layei can be changed by an external effect, preferably said ad-layer comprising a photooptical material capable of changing its refractive index upon illumination by a control light (beam), or an electrooptical material capable of changing its refractive index upon an electronic effect, said ad-layer in said adjuster member being in contact with a second poi tion of at least one of the first and the second arms, wherein the phase of the propagating light in said arm can be adjusted if the optical property, preferably refractive index, of the coating is changed,

- at least one container for receiving a sample, said at least one container being dii ectly or indirectly attached to a first portion of either the first or the second arm in an arrangement whei em an alteration of the sample can effect the wave propagation property, e g phase and/or wavelength of the propagating light beam in said first or second arm

The sensor device accoi ding to claim 1 wherein said sensor device comprises

- a first container for receiving a first sample, said first container being directly or indirectly attached to the first portion of the first ai m, and

- a second container for receiving a second sample, said second container being directly or indirectly at- tached to the first portion of the second arm, wherein preferably

The sensor device accoi ding to the invention which comprises a) a single adjuster member having an ad-layer being in contact with the second portion of the first arm or the second portion of the second arm, whereas said portions are interchangeable, b) - a first adjuster member having an ad-layer being in contact with the second portion of the first arm, and

- a second adjuster member having an ad-layer being in contact with the second portion of the second arm, whereas the first and second portions are interchangeable

The sensor device according to the invention wherein said adjuster member comprises an ad-layer an optical property of which being susceptible of a change upon exposition to an electromagnetic radiation as an ex- ternal effect, preferably to light, preferably to a lasei light

The sensor device according to the invention wherein said ad-layer comprises a material of non-linear optical property selected from the following group photochromic proteins, polymers of non-linear optical property The sensor device according to the invention wherein the photochromic protein is a protein comprising cis retinal, preferably a rhodopson protein, more prefei ably bactei iorhodopsin

If the photochromic protein is bacterid hodopsin, the conti ol light is a light of a wavelength which is suitable to change the moleculai state of bactei ioi hodopsm The skilled pei son will be able to select an appropriate wavelength The sensor device according to the invention wherein said adjuster member comprises an ad-layer an optical property of which being susceptible of a change upon exposition to an electric field This embodiment is less advantageous, however Thus, in a preferred embodiment the optical property of the ad-layer is susceptible of a change upon exposition to an electromagnetic radiation Preferably, it is not susceptible of a change upon exposition to an electric field

The use of a sensor device of the invention for detecting a change in a sample comprised in said first container, wherein prior to and/or after the change in the sample the at least one adjuster member of said sensor device is exposed to a controlled external effect thereby an optical property of said ad-layei in said adjuster member is changed and in turn a wave propagation property of said light beam propagating in said at least one arm is affected, and thereby the output signal is effected, wherein the controlled external effect is set in such a way that the output signal has a predetermined value

The use of a sensor device of the invention for detecting a change in a biological sample A method for adjusting an output signal of a sensor device, preferably an integrated optical interferometer based sensor device, comprising the steps of i) providing a sensor device according to the invention, ii) exposing said at least one adjuster member of said sensor device to a controlled external effect thereby changing an optical property of said ad-layer in said adjuster member and in turn affecting a wave propagation property of said light beam propagating in said at least one arm, in) detecting a change in the interference created by the recombined fu st and second light beams and thereby iv) affecting the output signal, whei ein the conti oiled external effect is set in such a way that the output signal has a predetermined value The method of the invention wherein the controlled external effect is set in such a way that the output signal is essentially constant

The method of the invention wherein the controlled external effect is set in such a way that the predetermined value of the output signal is between the maximal value and the minimal value of said output signal

The method of the invention wherein a transmission function of the output signal is measured or defined as a function of the controlled external signal and the output signal is set to a value wherein the value of the slope (the first derivative of) of the transmission function is at least 70%, at least 80%, at least 90 %, at least 95% of the maximum value of said slope or prefei ably is essentially the of the maximum value of said slope

A method for detecting a change in a sample by a sensor device according to the invention, comprising the steps of - adjusting the output signal of a sensor device according to the invention,

- placing a sample in said at least one container,

- effecting a change in the sample or allowing a change in the sample to occur,

- detecting said change as a change in the output signal

The method of the invention wherein said sensoi device comprises - a first container for l eceiving a first sample, said first container being directly or indirectly attached to the first poition of the first ai m, and - a second container for receiving a second sample, said second container being directly or indirectly attached to the first portion of the second arm, and the change in said sample is effected or allowed to occur in the first sample whereas the second sample is unchanged and is considered as a reference sample. The method of the invention wherein the first container is a measuring cuvette and the second container is reference cuvette and the at least one adjuster member is on the first portion of the said second arm of the ligh path in said sensor device.

The method or use of the invention wherein said ad-layer comprises a material of non-linear optical property selected from the following group: photochromic proteins, polymers of non-linear optical property.

The method of the invention wherein the photochromic protein is a protein comprising cis retinal, preferably a rhodopson protein, more preferably bacteriorhodopsin.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 a Scheme of an integrated optical Mach-Zehnder interferometer

Figure Ib A schematic representation of an example for a sensor device according to an embodiment of the invention comprising a measuring cuvette and a reference cuvette and a single adjuster member the ad- layer of which comprises a bacteriorhodopsin film. Figure 2. Sinusoidal modulation of the transmitted red light as a function of exciting light intensity.

Figure 3. Probe signals measured at the minimum (a), the maximum (b), and the highest slope (c) of the transmission function. Figure 4. Photograph of a sensor chip. Red light of a diode laser was coupled in and out from and to single mode optical fibers. The length of the MZ structure itself was 5mm. Figure 5. Light intensity change of the biosensor due to binding of biotinated mouse antibodies added at the time point of 65s.

DETAILED DESCRIPTION OF THE INVENTION Methodology The basic element of the method is an integrated of fibre optical device, which comprises an interferometer, e.g. a Mach-Zehnder interferometer (Fig. Ia). Preferably the interferometer which is utilized in the present sensor is an integrated optical device. Because of the high sensitivity of Mach-Zehnder (MZ) interferometers. The present inventors surprisingly found that even a biochemical reaction accompanying with association processes, i.e. mild changes, (e.g., enzyme-substrate interactions, ligand binding to macromolecules, supramolecular assemblies or whole cells) can be monitored by this optical technique. In an embodiment, the sensor device according to the invention is an integrated optical interferometer based sensor device shown on Figure I b, said device comprising an optical waveguide arrangement comprising a light path, said light path comprising a lead-in portion 1 , a first Y junction 3 with an input and at least two outputs, said input being connected to said lead-in portion

1, a lead-out poition 2, a second Y junction 4 with at least two inputs and an output, said output being connected to said lead-out portion 2 a first arm 5 and a second arm 6, each arm extending between one output of said first Y junction 3 and one input of said second Y junction 4, said first Y junction being capable of splitting a light beam, that is coupled into said lead-in portion I , into at least first and second light beams propagating in said first arm 5 and second arm 6, respectively, and said second Y junction 4 being capable of recombining said fu st and second light beams and thereby creating an lnter- ference pattern thereof i esulting in an output signal 12,

- an adjuster member 9, said adjuster member having an ad-layer 10 of non-linear optical property, said optical property, preferably refractive index, being susceptible of a change upon exposition to an electromagnetic radiation, preferably to light, and said ad-layer 10 being in contact with a second poi tion of at least one of said first and second arms (52 or 62), - a first container 7 and a second container 8, each one for receiving a sample, said first container 7 being directly or indirectly attached to a first portion of the first arm 5 1 and said second container 8 being directly or indirectly attached to the first portion of the second arm 61 in such a way that any change taking place within said sample present in any of the containers affects a wave propagation property of said light beam propagating in the respective arm of said optical waveguide to which container is attached, wherein said first and said second portions of any of the arms are spatially separated from each other and are interchangeable

The basis of operation is that whenever a physical, chemical or biochemical, e g an adsorption process takes place on only one branch oi arm of the interfei ometei (in other words, an ad-layer develops there or the refractive index of an ad-layer is changed), the effective l efractive index of that pai t of the waveguide changes giving use to a phase difference between the two branches The same occurs when processes of different type or level take place on different branches or ai ms The resulting phase difference of the two branches results in a change in the interference of the recombined light beam The subsequent intensity change can then be sensitively detected MZ interferometers, thei efore, can be pre-treated by a chosen reactant such that the surface of only one branch is coated with a given ligand, and this way a high specificity of the sensor is also realized It is also possible to integrate several MZ intei ferometers on a single substrate, in ordei to allow multiple simultaneous measurements Combination of such structures with microfluidics straighttoi ward is also possible By using a conti ol electromagnetic radiation e g light the output signal 12 of the interferometer based sensor device can be adjusted an thus any change in the interference created by the recombined first and second light beams can be detected Thereby, the controlled electromagnetic radiation can be set in such a way that the output signal 12 has a predetermined value It can be either essentially constant or can be is between a prede- termined maximal value and a minimal value or can follow a pi edetermined function Thereby the working point of the interferometer can be dynamically set For example, the output signal can be set to a value wherein the value of the slope, i e the first derivative of the transmission function is at least 70%, at least 80%, at least 90 % at least 95 % at least 98 % or at least 99% of the maximum value of said slope or preferably is essentially at the maximum value ot said slope If a change in a sample in one of the containers occui s, either a change in the output signal can be detected or the output signal can be kept at an essentially constant level by applying a control light of controlled and changing intensity or wavelength (frequency) and the control light intensity or wavelength (frequency) function is recorded

In a preferred embodiment said sensor device comprises - a first container for l eceiving a first sample, said first container being directly or indii ectly attached to the first portion of the first ai m, and

- a second container for receiving a second sample, said second container being directly or indirectly attached to the first portion of the second ai m, and the change in said sample is effected or allowed to occur in the first sample whereas the second sample is unchanged and is considered as a reference sample

In practice the adjustment of the working point can be perfoi med in various ways

For example, it is possible to take a calibration curve in advance and calculate the extent of the external effect, e g an illuminating controlling light or controlling electric field to achieve a desn ed change in the output signal, e g the output light intensity, i e the value of the transmission function Preferably, a drift in the output signal is compensated thereby

It is also possible to adjust the output signal, e g the working point of the sensor according to a predetermined function to compensate the dnft of the output signal, eg the working point

Automation of the woi king point adjustment can be achieved by e g using a control automatic system, which at first changes the intensity of the controlled external effect, e g control signal, e g the control light (e g laser) in a wide range and measures the output signal, e g output light intensity or the derivative (slope) thereot Then the automatic system sets the output signal value between the two extremes (where the derivative is 0, i e the maximum and the minimum) by setting the control led external effect, e g conti ol signal, e g the control light as necessaiy

If the drift is too fast, i e its i ate is comparable to the reaction rate of the change in the sample it may present a problem A possible solution would be to correct the woi king point based on known and predetei mined chai acteristics of working point drift undei the same or similar conditions The success of such correction can be checked after the measurement and any ei ror can be corrected by mathematical calculation However, the drift is in most cases l elatively slow to allow a measurement of even a few minutes This can be improved by setting the temperature, pressui e etc

Preferably, the device is small for this reason and for sake of portability It can be connected to other integrated optical devices or integrated into microfluidical devices Expenmental examples

Example 1

The photo-optical solution would allow the realization of an all-optical device The basis of operation is that upon light excitation of the ad-layer (which should consist of a non-linear optical material), its refractive index changes that gives rise to a phase shift of the guided light In the following, we report on experiments when the ad-layer was a made of a photochromic protein, bacteπorhodopsin, having unique non-linear optical properties [3]

A MZ interferometer was pi epared by a photopolymeπzation technique we formerly adapted for the prepai ation of micromachines and integrated optical structures [4, 5]

A thin (ca, 10 urn) film of bacteriorhodopsin was deposited on the top of the reference waveguide branch by layering [5] The light of a diode laser (633 nm, 10 mW) was coupled into a single-mode optical waveguide whose other end was matched to the input of the MZ interferometei by a mici opositionei , and its optimal position was fixed by a transparent glue Another light beam was directed to the bacteriorhodopsin film, so as to control light-induced l etractive index change in the ad-layer

The principle of tuning the working point is shown in Figure 2 The effect is based on a reversible change of the refractive index of the bacterid hodopsin film covei ing one of the arms of the interferometer upon illumination By changing the intensity of the exciting light, the intei ference of the measuring light at the output can be changed from constructive to destructive or reverse [5]

The measurement of the transfer characteristics of the interferometer was done under a microscope Without exposure, the optical environment and the effective refractive index was the same in each arm When illuminating, only one of the covered arms was exposed, therefore only thei e was a variation in the index of refraction leading to a phase unbalance in the two arms, and interference at the output of the interferometer The output optical fiber guided the transmitted i ed light to a photomultiplier, and the signal was recorded by a storage oscilloscope

By a probe illumination, we can test the sensitivity of the device Fig 3 shows that, depending on where we tune the baseline level, the sensitivity is very different It is supposed to follow the first derivative of the sinusoidal ti ansmissioπ function If we ai e at any ot the two exti eme, the signal is much smaller than in the middle, where the value of the derivative is the biggest This gives a chance to fine-tune the sensitivity of the device prior to a measui ement This is especially useful because of the inhei ent dnft of the MZ intei ferometers, due to their sensitivity to changes of the environmental parameters (humidity, temperature, etc ) Instead ot bacterioi hodopsin, other non-l inear optical matenals (dyes, photochi omic ciystals or other photochromic protein films) could also be used The pi incipal requirement is ti ansparency at the wavelength of the measuring light, and a reversible light-induced refractive index change ot 10 ' or greater The speed of the recovery of the refractive index change should ideally be less than a few seconds The eligibility of a given material can be tested according to the same measurement protocol described above

Example 2 - Electro-optically induced refractive index change A similar effect can, in principle, be achieved when an electro-optically induced refractive index change is used to control the phase shift in the reference branch, instead of the photo-optical one Electro-optical mate- nals (e g liquid crystals) can be placed on the top ot the reference branch ot the waveguide, and the electric field is supposed to be switched via two (micro)electrodes The eligibility cπtei ia are the same as given above, also for the electro-optical materials However, in this case the arrangement shall comprise electrodes which presents an additional complexity Moreover, setting of the working point is subject to chemical reactions which are most probably less precisely controllable Electromagnetic radiation as a control means of the adjuster member, preferably control light, is preferred

Example 3 - Detection of antigen-antibody interaction In order to test our concept in practice, we created a device that can serve as a potential alternative of

ELISA assay To a MZ interferometer desci ibed above, a removable PDMS cuvette was attached to the interferometer with two identical compartments above its arms, separated from the barter iorhodops in film (Fig 3)

The chamber above one of the ai ms was soaked by a buffer solution of mouse monoclonal antibodies, while the other, reference arm with BSA Prior to the measurement, the woi king point of the MZ interferometer has been adjusted, as desci ibed above Having then removed the excess solutions with a washing buffer, both arms were subjected to a treatment with a solution of antimouse-biotin complex that formed an ad-layer due to binding on the antibody-covered arm of the MZ interferometer, while no binding occui red on the BSA pre- -treated arm The subsequent asymmetric effective refractive index changes on the two arms gave rise to intensity changes at the output of the interferometer (Fig 4) The experiments proved that our device is sensitive enough to detect the development of a monomolecu- lar layer due to a specific antigen-antibody reaction We surprisingly found that by using a photochromic protein as a non-linear optical material highly sensitive device could be obtained, wherein an intensity of the control electromagnetic radiation as low as 1 mW/cni2 was sufficient

A furthei major advantage of our biosensor is that, the oppoi tunity of a pi oper adjustment of the working point of the MZ interferometer makes the i esults highly reproducible At the same time it can be set in a flexible manner

Industi ial applicabililty utilization of the i esults

We expect our method to become a cost-efficient alternative of the commonly used measuring tools in protein research As a specific, label-free technique, it should also be utilized in medical diagnostics, e g , for detecting disease mai kei proteins oi infected cells Reference numbei s

1 lead-in portion

2 lead-out portion 3 first Y junction

4 second Y junction

5 first arm

6 second arm

51 first portion of the fu st arm 52 second portion of the first arm

61 first portion ot the second arm

62 second portion of the second ai m

7 first container

8 second container 9 adjuster member

10 optical waveguide

1 1 input l ight

12 output signal

13 control light

References

1 Shew, B Y , Kuo, C H Tsai, Y H Ultra-sensitive biosensoi based on SU-8 planar interferometer IEEE Transducer 2005, Seoul, Korea, June 5-9

2 Shew, B Y , Kuo, C H , Huang, Y C , Tsai, Y H UV-LIGA interfei ometer biosensor based on the SU-8 optical waveguide Sensors and Actuators A 120 (2005) 383-389

3 Ormos, P , Fabian, L , Oi oszi, L , Wolff, E K , Ramsden, J J , Der, A Protein-based integrated optical switching and modulation Appl Phys Lett 80 (2002) 4060-4062 4 Galajda, P And Ormos P Complex micromachines produced and driven by light Appl Phys Lett 78 (2001 ) 249-253

5 Der, A , Fabian, L , Valkai, S , Ramsden, J J , Ormos, P and Wolff, E K Integrated optical switching based on the piotein bacteriorhodopsin Photochem Photobio 83 (2007) 393-396