WO1998021565A2

WO1998021565A2 - Process and device for optical thin-layer biosensor technology

Info

Publication number: WO1998021565A2
Application number: PCT/DE1997/002646
Authority: WO
Inventors: Alexander W. Koch
Original assignee: Koch Alexander W
Priority date: 1996-11-13
Filing date: 1997-11-12
Publication date: 1998-05-22
Also published as: WO1998021565A3; DE19646770A1

Abstract

The invention concerns a measuring system for detecting chemical and/or biochemical processes along a boundary layer of a transparent substrate which transmits an optical beam. Three media are formed with differing refractive indices. The three media with differing refractive indices (10, 11 and 12) (n0, n1 and n2) have two boundary surfaces (19, 20) with respect to each other. If there is a differential refractive index across the boundary layer, then incident light rays (5) will be partly refracted (8) and partly reflected (6). The overlaying of the rays (8) in the medium (11) with rays (6 or 7) respectively can result in constructive or destructive interference in accord with the difference in the optical paths. If the transmitted or reflected light is focused onto a detector (4) by means of an optical system (3), these intensities can be converted into electrical impulses. While under static conditions, a fixed intensity value is produced, a periodic structure of the detected signal variation (21) in intensity I arises with a variation of the refractive index (11 or 10) and/or the thickness (9)(d). The form of this periodic structure is a sensitive function of the calculation index ratios at the optical boundary surfaces (19 and 20).

Description

Method and device for optical thin-film biosensor technology

description

The present invention relates to a method and a device for determining local properties in biochemical sensors, in particular thin films on substrate surfaces which are coated with biologically and / or chemically active materials.

While a large number of sensors for physical and electrical measurement quantities are on the market as mass articles, the detection of chemical and / or biochemical signals is based on complex measurement principles with correspondingly complex sensors. Against the background of increasing demands on the acquisition of measurement data, in particular in the environmental and medical fields, the need for inexpensive, simple and reproducible chemical and biochemical sensors increases rapidly. Since chemical and / or biochemical signals cannot be directly detected electrically, transducers, so-called transducers, are necessary which transform the respective measured variable into an electrical output signal. Sensors for detecting chemical and / or biochemical signals, so-called chemo and / or biosensors, are based partly on electrical transducers, in which the chemical and / or biochemical signal directly changes an electrical variable, partly on optical transducers, which are based on the optical change in a medium caused by a chemical and / or biochemical signal change, which in turn can be converted into an electrical signal in a simple and efficient manner.

Because of their sensitivity to interference and their high selectivity, optical sensors in bio and chemical sensors (in short: biochemical sensors) are becoming increasingly important. Various optical principles play a role here, such as extrinsic or intrinsic optodes, evanescent field techniques, interferometry and ellipsometry. However, the prior art has various disadvantages when it comes to the practical use of optical measuring devices. For example, DE 43 43 490 describes a fast spectroscopic ellipsometer with high measuring speed, with which in-situ measurements of processes on a substrate surface are possible. These ellipsometric arrangements allow the simultaneous measurement of refractive index and layer thickness on growing and static layers. Significant disadvantages of such interferometers, as are also described in US Pat. No. 5,343, ^J 293, US Pat. No. 5,335,066, WO 94/25823, EP 0527150, DE 4108329, DE 4301889, lie in the complex adjustment of the measuring device and the generally ambiguous evaluation method. Thus, when constructing such egg ellipsometers, the angles of incidence and exit must be kept extremely precisely to at least 0.01 °. In addition, these angles must be kept close to the Brewster angle (approx. 70 ° for transparent glass layers) to increase the sensitivity. Another problem with ellipsometry is that the polarization rotation caused by the measuring principle has to be carried out very quickly and that a high data rate is thus obtained.

Another optical thickness measuring device for layers or for transparent workpieces is described in DE 39 38 113. A working signal is derived from the determination of the focal point condition, which is a direct measure of the thickness of the workpiece. This method is technologically simple to implement and possibly inexpensive, but has the major disadvantage that the refractive index and thickness resolution required for the chemical and biosensor technology cannot be achieved. The required sensor resolution does not allow methods based on the focus point principle and the triangulation principle.

Another interesting layer thickness measurement method is described by Dr. W. Riedel, Fraunhofer Institute for Physical Measurement Techniques (Annual Report 1995: Layer Thickness Measurement with White-Tight Interferoretry, Freiburg). The process is based on the use of white light with its extremely short coherence length. Passing through a focus through a layer system provides interference contributions at precisely the points at which the reference arm and object arm lengths in the interferometer match. During the measuring process, the object or reference path must be modulated in the measuring range using a path encoder. The method works with great precision, but its structure (interferometer, modulation unit, precise geometric arrangement) is too complex for compact biochemical sensors. Conventional thin-film interferometry can always be used excellently when dynamic processes have to be recorded. A dynamic change in layer thickness and / or refractive index occurs, for example, in the case of plasma coating or etching processes. Suitable thin-layer interferometric measurement techniques are described in US Pat. No. 5,371,582, WO 94/28376, US Pat. No. 5,337,144, G 296 07 227.3 and in the publications by Caranto et al. (An optical fiber thin film thickness monitor, Meas. Sei. Technol. 4 (1993) pages 865-869, 1993 IOP Publishing Ltd) and the publication by V. Lange and G. Higelin (Static and dynamic test method for thin mono- and polycrystalline Si-films, Microsystem Technologies 1 (1995) pages 185-190, Springer-Verlag 1995). DE 42 28 535 describes a thin-film interferometer for use in chemical and biochemical measurement and analysis technology with a planar waveguide as an integrated optical sensor element. Here, as is required for chemical and / or biosensor technology, a very simple sensor concept is presented, which combines a cost-effective production of the sensor element with high sensitivity and error-free detection in a robust design suitable for practical use. The main disadvantage of this method is the need to install and assign multiple detectors to detect the interference along a longer or shorter side edge of the sensor element. In this way, the detection is in principle also possible under static conditions, but the signal acquisition and processing is complex.

DE 44 00 689 describes an apparatus and a method for determining local properties of a partially ionized gaseous medium and layer thicknesses which are applied to the sensor head of a probe. The method is also based on thin-film interferometry, i.e. during the vapor deposition there is a change in the optical paths between two adjacent, mutually parallel interfaces. This change in the optical paths is recognizable as an interference structure when vapor deposition or etching away of layers.

Further interferometric arrangements using white light are based on a spectral evaluation of the light reflected back from the thin-film sample. For example, EP 06 31 102 describes a method with which the spectrum via the wavelength dispersion is used for measuring the layer thickness. In addition, the light modulated (EP 0567745) and polarized (EP 0622624). Also interesting is the combination of the previously explained ellipsometric measuring technique with reflectometric measuring technique using a wavelength-resolving detector, as described in the company brochure of SENTECH Instruments GmbH, Rudower Chaussee 6, 12484 Beriin, company catalog FTP 500). For chemical and / or biochemical sensors, these wavelength-selective methods generally have the disadvantage that a wavelength-dispersive element, such as a monochromator with a grating or prism, must be used in the detector. In this way, the sensor becomes bulky and expensive due to the high outlay on the detector side.

Furthermore, the method of thin-layer interferometry can be used to easily detect thermal expansion of thin transparent substrates or thin transparent layers, as described in utility model G 92 18 626.2.

Donnelly and McCaulley (Infrared-laser interferometric thermometry: A nonintrusive technology for measuring semiconductowafer temperatures J. Vac. Sei. Technol. A 8 (1), Jan / Feb 1990, American Vacuum Society, pages 84-92) and Fang et al. (A fiber-optic high-temperature sensor, sensors and actuators A 44 (1994) pages 19-24) use the effect of thin-layer interference to determine temperature changes of a substrate or measurement object via the thermal expansion.

DE 40 17 440 and WO 94/29681 describe a method for measuring the layer thickness and the refractive index of a thin layer on a substrate and a device for carrying out the method, which is based on the use of the thermal radiation of the substrate mentioned. Here, too, only dynamic processes can be measured. Furthermore, a CCD camera with complex data evaluation is used to detect the measurement signal.

All interferometric methods with a simple transmitter beam and a photodiode as a detector for the optical characterization of materials according to the prior art have in common that only dynamic processes can be examined. In many cases, the periodic interference structure also creates the problem of ambiguity. Furthermore, optical methods for chemical and biosensor technology are known which are based on evanescent field technologies, such as, for. B. in the article by Günther Gauglitz (chemical and biosensors with optical transducers, technical measurement, 5/95, page 204-212). The attenuation of the total reflection in a waveguide by changing the refractive index ratios at the boundary layer of the waveguide environment is used as the measurement effect. The field penetrating the biochemical medium in particular is cross-damped or evanescent.

Conventional methods according to the prior art, which are based on the attenuated total reflection, the total internal fluorescence / reflection and the surface plasmon resonance as well as the ellipsometry, generally have the disadvantage of a high outlay on equipment. This high expenditure on equipment is necessary for many applications in the field of bio and chemical sensors

a. Cost efficiency,

b. Miniaturizability,

c. easy handling in harsh industrial and environmental conditions,

d. high time and location resolution

e. easier and faster online data processing

intolerable. Optical measuring methods, which are based on thin-film interferometry, have the advantage of a very compact design in combination with very simple data acquisition and data evaluation. However, conventional thin-film interferometric methods generally have the disadvantage, as explained, that only dynamic processes can be observed, such as, for example, the growth of a transparent layer on a substrate by tracking the interference structure that is being formed. If dynamic processes on the layer to be examined are too slow or too complex, thin-layer interferometric methods for bio and chemical sensors cannot be used. The invention is therefore based on the technical problem

to provide an apparatus and a method for determining the properties of a medium which is applied to a substrate and which does not itself have to be changed to measure its properties. Advantages resulting from the optical-interferometric measuring technique should be used in an advantageous manner for the probe construction and the method according to the invention, i.e. high local and spatial resolution, low sensitivity to interference, electromagnetic compatibility, no influence on the test object to be examined should be achieved.

With the interferometric measuring method according to the invention and the device for carrying out the method, the advantage is achieved in particular that the possible uses of thin-layer interferometry can be extended to non-dynamic or very slow-running chemical and / or biochemical processes.

According to the invention, this technical problem is solved by a measuring method which has the following steps:

the optical path of one or more optical beams is periodically modulated in the substrate; the resulting periodic fluctuation is achieved by superimposing the individual beams which can be brought into interference in the transparent layer; the shape of the resulting periodic intensity fluctuation is a measure of the refractive index ratios of a layer applied to the substrate; an evaluation device connected to the detector head is used to determine the local properties by suitable processing of the optical and electrical signals.

In particular, it is advantageously achieved that the optical parameters of one or more layers applied are detected by suitable dynamic changes in the substrate and that the layer itself remains in a more or less static state can be.

The above technical problem is also solved by a device in which

an optically modulable substrate with an interface on which the chemically and / or biologically sensitive layer is applied acts as a sensor element; a preferably coherent light beam is generated by a transmission unit with one or more beam transmitters; a modulation unit is used to modulate the optically modulatable waveguide; one or more selective receivers are used to record the interference structure; an optical unit is used to detect the radiation transmitted or reflected by the substrate; a data evaluation unit is designed to evaluate the processing and display of the measurement signal and to control the modulation unit.

The measuring device according to the invention is advantageously based on the principle of thin-film interferometry, wherein

a scanning optical beam detects a change in the optical path in a transparent substrate; in addition, modulation of the optical path in the substrate is carried out using suitable methods, i.e. Even in the case of a static measurement object applied to the substrate, a periodic signal shape corresponding to the thin-film interferometry for the intensity of the output signal is advantageous.

In the method according to the invention, the periodic structure of the interference signal is not generated by the optical path change on the measurement object, ie the applied layer, corresponding to the prior art, but by a refractive index variation and / or a variation of the geometric thickness, ie the optical path in the transparent substrate. A periodic variation of the optical path (refractive index x substrate thickness) is generated by the following methods:

a geometric change in substrate thickness, e.g. by applying lateral forces to a deformable substrate, e.g. by means of longitudinal changes in density caused by piezo oscillators, a change in the refractive index, e.g. B. through the use of substrate materials that change their internal structure and thus their macroscopic refractive index when external electrical fields are applied, e.g. is the case with electroreological fluids,

by generating acousto-optical waves in suitable media.

by radiation in an angular range that leads to different propagation paths in the substrate, e.g. by means of convergent radiation.

The modulation of the optical path is advantageously detected with a bundled light beam in order to increase the local resolution. In a simple manner, the light beams on the transmitter side can be designed as laser beams. It is also advantageous to carry out the light irradiation onto the modulating substrate at a flat angle in order to obtain the highest possible sensitivity with regard to the curve shape. The curve shape of the periodic signal depends very sensitively on the covering of the substrate with the biochemical layer and the changes in the layer caused by biochemical processes.

The principle of the curve shape detection is effectively used with the method according to the invention and with the device according to the invention in that, due to the sinusoidal shape of the interference signal, the distances between the rising or falling edges are detected at the points with half the maximum amplitude; that is, a distance ratio is detected. Equal distances mean a pure sine course, while a curve shape change shifts these distances. Other waveform acquisition methods such as

Fourier analysis

Correlation analysis

Wavelet transformation,

Curvature analysis

Inflection point determination by double derivation are used.

With a flat angle of incidence, the previously explained distance ratio is a sensitive function of the refractive index ratios at both interfaces (transition from environment to substrate or transition from substrate to environment). If the ambient conditions on one substrate surface are now kept constant, the conditions according to the invention on the other substrate surface can be measured with high resolution using the method according to the invention. The signal is processed further and can be used, for example, as a monitor signal for a biochemical process.

It is advantageous to carry out averaging over several period durations, so that the optical path is modulated over correspondingly large values. It is also advantageous to modulate the light beam on the primary side at a high frequency in order to filter out undesired constant light interference. Through a freely definable choice of the optical density differences on the primary side (medium 0 - medium 1), a large measuring range with regard to the density differences between medium 1 (substrate) and the measurement object (medium 2) can be achieved.

Further advantageous embodiments of the invention emerge from the attached subclaims. The invention is described in more detail below with reference to its embodiments ^" with reference to the drawings. The drawings show:

Fig. La, lb sketches of an embodiment of the interferometric measuring device of the invention, consisting of a substrate 1, a light source 2 and a detector 4 with pin diode, with the optics 3 and 5b, the modulation control 15, the optical beam paths 5, 6th and 7 are spanned;

2a, 2b, 2c simulations of the signal profiles 21, which are different in FIG

The composition of the test objects according to Table 1 is created with the distance ratio defined by the quotient from distances 22 and 23;

Fig. 3 shows an embodiment of the sensor head consisting of the

Substrate 1 which is modulated with the modulation unit 16 controlled by the modulation controller 15; the biochemical layer 17 applied on one interface 20, while the other interface 19 remains unoccupied; the light source 2, which transmits optical radiation via the primary beam path 5 and the secondary beam path 7 to the detector 4; the refractive indices 10, 11 are set for measurement in accordance with Table 1;

Fig. 4 shows the structure of an embodiment with substrate 1, biochemical

Layer 17 in the block diagram with a modulation controller 15, the modulation unit 16, the light source 2, the detector 4, the oscillator 24 and the data evaluation unit 18, which is equipped with phase-sensitive detection, integrated effective formers and transmittance amplifiers.

In the following, the reference numerals designate the same or similar parts as described with reference to FIG. 1. 1 shows an embodiment of a measuring device according to the invention for generating interference, consisting of a three-phase system. The three media with different refractive indices 10, 11 and 12 (n ₀ , n, and n ^ have the two interfaces 19 and 20 with one another. If there is a different refractive index on both sides of the interfaces, partial refraction 8 and one occurs for an incident light beam 5 Reflection 6. Structural or destructive interference can occur depending on the difference in the optical paths by superimposing the beams 8 in the medium 11 with the beams 6 and 7. If the transmitted or reflected light is imaged onto a detector 4 by means of an optical system 3, These intensities are converted into electrical signals. While there is a fixed intensity value under static conditions, a variation of the refractive index and / or the thickness 9 (d) results in a periodic structure of the detected signal profiles 21 with the intensity Kd ^" ) and the data 2b according to the following relationships:

Φ0 = 88-— dl = 0 nO = 1,362 λ = 670 Δd = 3

180

A0 = sin (ΦO) BO = oos (Φ0) _ / n0-A0 \ ²

A2 -i'-i ^ f __ nl-BO - nO-Al nl-BO -mO Al

180 arccos (Al) = 68,899 180 xp (- ß-d-i) π arccos (A2) = 83,491 ß E (d) = e λ π,. n2 Al - nl A2

• (E (d)) n2 Al nl-A2 iUcn - ^{r01 + r12} ^{"- /} (1 + I01T12-E (Λ) Kd) = (! R (d) |) ²

Fig. 2 shows this periodic waveform 21 for three different refractive index ratios. In this regard, it should be noted that a similar periodic structure can also be produced by varying one of the two media 10 or 12. The conventional thin-layer interferometry for measuring dynamically growing layers of the medium 10 or 12 is based on this principle. In the present case, constant media 10 and 12 are assumed, so that, according to the invention, the periodic structure can be achieved optical thickness of the medium 11 is varied. This has the advantage that static processes at the boundary layers can also be detected via a change in signal shape, defined, for example, by the distance ratio of length 22 to length 23.

In the evaluation method described, which only has to take into account a distance ratio of length 22 to length 23, independence from the signal amplitude is advantageously achieved. This facilitates the adjustment of the overall system. The total amplitude does not have to be known when recording the signal.

Maximum sensitivity is advantageously obtained if, according to another embodiment of the invention, the angle of incidence 5a and thus also the exit angle 14 via the transmitting optics 5b is as large as possible, ie. H. as flat as possible incidence on the surface of the substrate 1 is achieved. The inner angle 13 is determined by the refractive index ratios.

Fig; 2a, 2b, 2c show the simulation results that were achieved with the designated angles and refractive indices. The following information applies to the two exemplary cases:

Table 1:

Fig. Angle of incidence 5a refractive indices: in degrees substrate n, environment n ₀ biochemical layer n ₂

2a 88 1,459 1,362 1,500 2b 88 1,459 1,362 1,370 2c 88 1,459 1,362 1,362

3, a modulation unit 16 is used to modulate the optical path in the substrate 1, controlled with the modulation controller 15. The modulation unit 16 is set in such a way that several periods of the signal are always copied at the same time, so that averaging is possible. The modulation unit 16 is advantageously implemented, for example, by a piezo oscillator, which causes longtudinal density waves in the substrate. Furthermore, modulations are achieved by mechanically changing the path of deformable media. Media that perform refractive index fluctuations due to an external electric field can also be used advantageously. The transparent, electro-rheological liquids are an example of such a medium.

4 finally shows a block diagram of the overall arrangement, the central oscillator 24 being used to implement both the modulation of the light source 2 and the synchronization of the data processing unit 18 and the modulation controller 15. The data processing unit 18 is used for the evaluation, further processing and display of the measurement signal and for controlling the modulation unit 16 via the oscillator 24 and the modulation control (15).

The field of application of the measuring system according to the invention, the device and the method relates to chemical and / or biochemical sensor technology, a key technology in which recording process processes are converted into electrical signals with high sensitivity by means of the optical transducer described. In particular, the measuring system can be used for measuring requirements that are not accessible to conventional biochemical sensors. For example, the measuring system according to the invention can be used for:

optical detection of slow biochemical processes in a thin layer;

Real-time monitoring of pollutants in water

Gas analysis in environmental control;

Patient monitoring in medical technology.

Indoor air monitoring,

Control of water in hydraulic oil,

Composition of dyes,

Flow measurement,

Level indicator. - 14

Claims

Method and device for optical thin-film biosensor technology

1. A method for determining the local properties of one or more layers on a substrate, characterized in that the optical path of one or more optical beams in a substrate (1) is periodically modulated and that one or more split optical beams in a detector (4 ) are united and can be brought to interference.

2. The method according to claim 1, characterized in that an electrically controllable modulation unit specifically changes the optical path between the two boundary layers of the substrate.

3. The method according to claim 1, characterized in that an evaluation unit evaluates the periodic signal curves (21) generated as a result of the optical interference.

4. The method according to claim 1, characterized in that an optical path change in the substrate is caused by a geometric change in the substrate thickness.

5. The method according to claims 1 and 4, characterized in that an optical path change in the substrate is caused by a change in the refractive index of the substrate (1) or the medium (10).

6. The method according to claims 1, 4 and 5, characterized in that an optical path change in the substrate is caused by the generation of acousto-optical waves.

7. The method according to claims 1 and 3, characterized in that the waveform detection methods Fourier analysis, correlation analysis, wavelet transformation, curvature analysis and inflection point to evaluate the waveforms. determination can be used.

8. The method according to claims 1 to 7, characterized in that the modulation unit (16) via the data evaluation unit (18) and the modulation control (15) is controlled such that there is a maximum sensor sensitivity.

9. The method according to claims 1 to 8, characterized in that one or more optical beams to increase the sensitivity of the measuring system at a flat angle (5 a) irradiate on the substrate.

10. The method according to claims 1 to 10, characterized in that a focusing of the optical radiation is provided to increase the local resolution.

11. Device for determining local properties of layers, characterized by the following features:

an optically modulable substrate (1) with an interface (20) on which the chemically and / or biologically sensitive layer (17) is applied; a transmission unit (2) with one or more radiation transmitters; and transmitting optics (5b) a modulation unit (16) for modulating the optically modulable substrate (1); one or more selective receivers (4) for receiving the interference radiation; an optical unit (3) with a receiving unit (4) for detecting the radiation transmitted or reflected by the substrate (1); a data evaluation unit (18) for evaluating, further processing and displaying the measurement signal and for controlling the modulation unit (16) via the oscillator (24) and the modulation control (15).

12. The apparatus according to claim 11, characterized in that the substrate (1) with the layer (17) and the surroundings (10) form the optical interfaces (19) and (20).

13. The device according to claims 1 1 and 12, characterized in that the chemical and / or biological layer (17) to be measured at one of the interfaces (19, 20) causes refractive index differences which are reflected in the signal shape of the detected interference signal.

14. Device according to claims 11 to 13, characterized in that an optical beam (5) is coupled in at the lowest possible angle of incidence (5a) at the interface (19).

15. Device according to claims 11 to 14, characterized in that the modulation control (15) permits modulation of the optical path in the substrate (1) with variable frequencies and / or curve shapes.

16. The device according to claims 11 to 15, characterized in that the evaluation unit (18) carries out a curve shape analysis which enables a determination of the refractive index differences at the interface (20).

17. The method according to claims 1 to 10, characterized in that the transmission beam or beams (5) are amplitude-modulated.

18. The method according to claims 1 to 10, characterized in that a phase-sensitive detection by means of lock-in technology in the data evaluation unit (18) also makes it possible to evaluate signals with a high noise component.

19. The method according to claims 1 to 10, characterized in that on the receiving side an integrated RMS causes the acquisition of a DC signal.

20. Device according to claims 11 to 19, characterized in that a low-noise PIN diode is used as a light detector in the receiving unit 4.

21. The method according to claims 1 to 10, characterized in that the wavelet transformation is used in the data evaluation unit 18 for signal shape recognition.

22. Device according to claims 11 to 21, characterized in that the transmitter optics (5b) comprises a beam splitter array for areal illumination of the measuring range.

23. Device according to claims 11 to 22, characterized in that the coupling and uncoupling of the beams (5) and (7) is carried out by an optical waveguide.

24. Device according to claims 11 to 23, characterized in that a transmittance amplifier integrated in the data evaluation unit (18) effects a gain which is adapted to the size of the input signal.

25. The device according to claims 11 to 24, characterized in that the measuring system structure comprises a variably held substrate carrier which enables a rapid exchange of the substrates (1) provided with different chemically and / or biochemically sensitive layers (17).

26. Device according to claims 11 - 25, characterized in that the substrate 1 is designed as an optical waveguide.