WO2019106056A1

WO2019106056A1 - Compact device and method for poct diagnostics by means of sers

Info

Publication number: WO2019106056A1
Application number: PCT/EP2018/082911
Authority: WO
Inventors: Bernd WALKENFORT; Sebastian Schlücker
Original assignee: Universität Duisburg-Essen
Priority date: 2017-11-30
Filing date: 2018-11-28
Publication date: 2019-06-06
Also published as: DE102017128443A1

Abstract

The invention relates to a device (1) for diagnostics, particularly for quantitative diagnostics, of a sample (2) which comprises at least one type of SERS-active particles, each type having a specific Raman band (16, 20, 21, 22), by means of surface-enhanced Raman scattering (SERS), wherein the device (1) has a light source (3) for substantially monochromatic light, an optical probe (4) for illuminating the sample (2) to be examined with light from the light source (3) and for collecting scattered light from the sample (2), and a Raman photo detector (5, 5a, 5b, 5c, 5d) as well as a reference photo detector (11) for detecting the light collected by the probe (4) and scattered by the sample (2), wherein an optical filter element (8) for separating elastic scattered light from inelastic scattered light within the specific Raman band (16, 20, 21, 22) is arranged in the beam path of the light scattered by the sample (2), wherein the inelastic scattered light is conducted and the elastic scattered light is blocked by the optical filter element (8), and an additional filter and reflection element (9, 9a, 9b, 9c, 9d, 9e) for separating a reference band (10) specific to a substrate of the sample from the specific Raman band (16, 20, 21, 22) of the SERS-active particles is arranged in the beam path behind the filter element (8) for separating elastic scattered light from inelastic scattered light, wherein the reference band is reflected and directed to the Raman photo detector (5, 5a, 5b, 5c, 5d) and the reference band is allowed through and directed to the reference photo detector (11). The invention also relates to a corresponding method.

Description

Compact device and procedure for POCT diagnostics

by means of SERS

The present invention relates to a readout device and a method for quantitative pati gene near diagnostic, for example in the context of so-called POC (Point Of Care Test) by means of surfaces enhanced Raman scattering (SERS, surface-enhanced Raman scattering) of Raman reporters molecularly functionalized SERS labeling particles (SERS labels / nanotags), preferably noble metal nanoparticles. More specifically, it relates to a device for diagnostics, in particular for quantitative diagnosis, of a sample comprising at least one kind / type of SERS-active particles, each with a specific Raman band, by means of surface-enhanced Raman scattering, the device being a light source for substantially monochromatic light, an optical probe for illuminating the sample to be examined with light from the light source and for collecting light scattered from the sample, and a photodetector for detecting the light collected by the probe and scattered by the sample. It also relates to a method for diagnostics, in particular for quantitative diagnostics, of a sample comprising at least one species / type of SERS-active particles, each with a specific Raman band, by means of surface-enhanced Raman scattering, wherein substantially monochromatic light is transformed into an optical one Probe is irradiated, which illuminates the sample to be examined with light from the light source and collects the light scattered by the sample, wherein scattered light from the sample is passed to a photodetector for detecting the light collected by the probe and scattered by the sample.

For the biochemical detection of substances with antibodies, test strips, also referred to as LEA (lateral flow assay), are generally known. With such test strips, target proteins can be detected by means of nanoparticle-labeled antibodies with the naked eye (red test or control line).

In Raman spectroscopy, samples can be analyzed by studying the inelastic scattering of light on molecules of a sample (Raman scattering). The state of the art is inter alia the so-called Raman microscopy, in optical microscopy, For example, using a light microscope, combined with Raman spectroscopy, for example, using a Raman spectrometer. In Raman microscopy, a sample to be examined is irradiated with monochromatic light, for example in the form of a laser beam. For example, a laser can be focused with a microscope on the sample to be analyzed. Upon irradiation, there is an interaction between the light and molecules of the sample, also known as the Raman effect, whereby light is scattered inelastically on molecules or atoms. There is an energy transfer between the exciting light (a stimulating photon) and the thus irradiated (excited) matter of the sample, wherein in principle energy transfer in both directions is possible borrowed, ie from the light to the sample or vice versa. The energy difference between incident light and inelastically scattered light from the sample is characteristic of the scattering matter, so that conclusions can be drawn from an analysis of the radiation emitted by the sample on the irradiated matter and in particular their molecules can be identified. The sample area recorded in a single examination (point measurement) mainly depends on the magnification of the microscope objective. Typical focus sizes are a few hundred nanometers to a few microns.

In order to achieve a spatially resolved detection, the entire area of the sample to be examined must be snapped with a displaceable microscope stage. This can be done according to the state of the art by means of so-called spatially resolved Raman microscopy, but is disadvantageously quite time-consuming, since the total measurement time scales with the product of the time per point measurement and the number of spatial points to be scanned in two dimensions. There are also known methods and apparatus for Raman spectroscopy based on the use of surface enhanced Raman scattering. It is possible, using nanometallic particles (in the following, SERS nanoparticles), in particular with gold or silver nanoparticles, to label antibodies in a plurality of different timings in parallel. to detect different target proteins qualitatively and quantitatively in very low concentrations. It is exploited that certain SERS nanoparticles each have certain specific spectral signatures, that is, Raman bands or Raman spectra due to inelastic scattering, which are known. For reading and analyzing a suitable reader (reader) is used.

A device and a method for surface-enhanced Raman spectroscopy (SERS) are known, for example, from US Pat. No. 7,688,440. In this case, an excitation and a collection of Raman scattered light with a fiber-optic probe. A spectrally resolving detection of the Raman scattered light is carried out by means of a grating spectrometer (also referred to as a CCD system). The probe is placed appropriately over the sample to be examined. The size of the examined section depends on the focus of the lens used and a spatially resolved detection is possible only by screening the entire area of the sample to be examined with a positionable microscope stage and a plurality of individual examinations. Due to the use of the grating spectrometer, the entire frequency range of the scattered light of the sample is examined. The disadvantage here is that the use of a lattice Raman spectrometer with multi-channel detection (CCD) is more expensive and expensive.

All of the above-described devices and methods have in common that exploitation of known Raman signatures of certain known SERS gold nanoparticles does not take place.

In view of the above-described prior art, the object of the invention is based on the object to reduce or avoid the disadvantages mentioned, in particular to provide a device and a method in which samples in particular with LFA test strips quantitatively and also more sensitive, faster and cost-effective than can be studied and analyzed in the prior art. This object is solved by the subject matters of the independent claims. Before ferred developments of the invention are described in the subclaims.

According to the invention, therefore, a device for diagnostics, in particular for quantitative diagnosis, of a sample is provided which comprises at least one type of SERS-active particle, each with a specific Raman band, by means of surface-enhanced Raman scattering (SESR), the device being a light source for substantially monochromatic light, an optical probe for illuminating the sample to be examined with light from the light source and for collecting scattered light from the sample and a reference photodetector and a Raman photodetector for detecting the collected by the probe and the Sample scattered light has, wherein in the beam path of the light scattered by the sample, an optical Lilterelement for separating elastic scattered light of inelastic scattered light of the speci fied Raman band is arranged, wherein passed from the optical Lilterelement the unelasti cal scattered light and blocked the elastic scattered light is, and in the beam path hinte the Lilterelement for separating elastic scattered light from inelastic scattering light, another Lilter- and reflection element for separating a sample specific for a substrate reference band of the specific Raman band of the SERS active particles is arranged, the reference band reflected and the reference Photodetektor gelei tet and the Raman band is passed and directed to the Raman photodetector.

The above-mentioned optical filter element for separating stray elastic light from inelastic stray light of the specific Raman band is also referred to as Rayleigh filter element for the sake of simplicity and preferably has a high optical density in the wavelength range of the used monochromatic light. The light source for monochromatic light is preferably a laser. Under a sample according to the invention is to be analyzed material. This is preferably applied to a substrate or sample holder, for example an LLA test strip. The term of the scattered light scattered by the sample therefore also includes such scattered light, the not directly from the material to be analyzed, but from the substrate / sample carrier was scattered. In addition to the incident frequency (Rayleigh scattering or elastic scattering), there are other frequencies or bands in the spectrum of the light scattered on the sample and on the substrate. The differences in frequency with respect to the incident light correspond to the energies of rotational, vibrational, phonon or spin-flip processes characteristic of the molecules of the sample. From the spectrum of the scattered light emitted by the sample conclusions can be drawn on the examined substance.

By using the Rayleigh filter element in the context of the present invention, the analysis and evaluation of the light emitted by the sample can be limited in a particularly advantageous manner to the proportion of inelastic scattered light. Since the elastic cal fraction of the scattered light is separated from the inelastic portion, the units used for the evaluation of the device can be designed simpler than in the prior art. In particular, no use of a lattice Raman spectrometer with expensive and time-consuming multi-channel detection (CCD) is required. The use of the Rayleigh filter element makes it possible, in particular, to evaluate the inelastic scattered light originating from the sample by means of inexpensive single-channel detectors. It can also be said that the evaluation can be carried out limited to the known specific Raman bands of the SERS nanoparticles used and present in the sample, ie to inelastic scattered radiation. Thus, the evaluation of the light scattered by the sample is facilitated, since not as in the initially described prior art, the entire frequency spectrum of the sample stam menden scattered light is evaluated.

The Rayleigh filter element for separating elastic and inelastic scattered light is designed to filter out stray elastic light and to transmit inelastic scattering light of the specific Raman band. It is preferably designed as a long-pass filter element such that the Rayleigh scattering, that is, elastically scattered light, is blocked. Preferably, it is in the beam path of the light emitted by the sample between the Probe and the photodetector arranged. Alternatively, it may be net before or in the probe.

According to the invention in the beam path behind the filter element for separating elastic scattered light of inelastic scattered light another filter and reflection element for Tren nen a specific reference to a substrate of the sample reference band of the specific Raman band of SERS active particles arranged. This filter and reflection element is designed and arranged in such a way that the reference band is reflected and directed onto the reference photodetector and the Raman band is passed through and directed onto the Raman photodetector. In reflection, the filter and reflection element thus acts practically as a bandpass for the reference band, while the filter and reflection element in Transmis sion acts as a notch filter for the other frequencies.

In one embodiment of the invention, several filter and reflection elements of the type described above are arranged in the beam path of the scattered light of the sample behind the filter element for separating elastic scattered light from inelastic scattered light of the specific Raman band and in front of the photodetector. At least the first filter and reflection element in the beam path, several or all filter and reflection elements is or are designed and arranged for separating a reference band specific for a substrate of the sample from a Raman specific for one type of SERS-active particle - gang. With this embodiment of the invention it is advantageously possible to detect several different target molecules in a single measurement. Each target molecule is an appropriate antibody with a particular type of SERS nanoparticle having a spectral signature typical of this species, i. with a respective specific Raman band assigned. Each of the plurality of filter and reflection elements is therefore intended for the constitution of one of the specific specific signatures.

For example, antibodies matching a first type of target molecule are associated with a signature Fl-type SERS nanoparticle and antibodies suitable for a second type of target molecule a signature F2 SERS nanoparticle. The two filter and reflection elements required for simultaneous detection are such that the one on the Raman band of the signature Fl and the other on the Raman band of the signature F2 is true abge. Since the respective Raman bands Fl and F2 are known, it can be concluded from a detection of signals in the corresponding Raman bands on the presence of corresponding target molecules. Under a target molecule is in the context of the invention, in principle, a target atom to understand.

According to the invention, the device has at least one reference photodetector, to which the reference band specific for the substrate of the sample is directed in order to provide a reference signal for evaluating the detected inelastic scattered light. The signal of this reference photodetector can be used, for example, by means of evaluation electronics for correction of other signals, in particular for correction of the detected Raman bands, in order to obtain e.g. Fluctuations in fiber performance, fluorescence of the substrate or deviations of the focusing of the fiber beam on the sample compensate.

An embodiment of the invention is characterized in that the device comprises a positioning device for in particular automatic one-, two- or multi-dimensional positioning of the sample relative to the optical probe, in particular orthogonal to its beam axis. In particular, the positioning device can receive and hold a sample carrier or a substrate in such a positionable manner. The sample can thus be moved relative to the probe so that it is particularly easy to perform a surface or area scan of sections of the sample or the sample carrier or even the entire surface. Preferably, the Po sitioniereinrichtung is coupled to a control and evaluation of the device, so that an automatic vote of sample position and detected Raman bands made and can be taken into account in the evaluation. The optical probe according to the invention may comprise a filter element which is angeord net and formed so that the sample with a divergent beam which strikes with an oblique angle of incidence (not equal to 0 ° and 90 °) to the sample is illuminated. In addition, the optical probe can have a collection array for scattered light emitted by the sample, which is aligned and arranged in a coordinated manner to the angle of incidence. Collection array and Filterele element are preferably positioned and aligned so that there is a maximum overlap between lighting and detection area. The former filter element may in particular be a bandpass filter. A further embodiment of the invention is characterized in that the device has a linear fiber bundle for coupling light from the light source into the optical probe. In addition, it may have a linear fiber bundle for coupling stray light from the sample into the probe. The optical probe may comprise an optical element, in particular a lens, which produces an image of the fiber bundle at infinity. This image illuminates the sample. In addition, the optical probe may have a dichroic ele ment passed over the sample passed to the light as well as scattered light from the sample.

Finally, the optical probe may comprise an optical system or element, for example a cylindrical lens, for producing a substantially line-shaped illumination region on the sample.

According to the invention, the abovementioned object is also achieved by a method for diagnostics, in particular for quantitative diagnostics, of a sample comprising at least one type of SERS-active particle, each with a specific Raman band, by means of surface-enhanced Raman scattering (SESR). in particular by means of a device as described above, wherein essentially monochromatic light is emitted to an optical probe, which illuminates the sample to be examined with light from the light source and the scattered light. collecting light from the sample, scattered light scattered from the sample being passed to a reference photodetector or to a Raman photodetector for detecting the light collected by the probe and scattered by the sample, wherein light scattered from the sample is reflected by an incident light source Beam path in front of the photodetector and the Raman photodetector arranged Lilterelement optical element is arranged, the elastic scattered light of inelastic scattering light of the specific Raman band, whereby the inelastic scattered light is passed by the optical Lilterelement and the elastic scattered light is blocked, and the light below is passed in the beam path behind the Lilterelement to another Lilter- and re flexionselement for separating a reference band specific for a substrate of the sample of the specific Raman band of SERS active particles, wherein the Refe rence band directed to the reference photodetector and the Raman Band on the Raman photodetector et will. Preferred developments of the method according to the invention result analogously to the preferred developments of the device according to the invention described above.

In summary, it can be said that within the scope of the present invention, the complete sample surface to be examined is tailor-made and the un elastic scattered radiation emitted thereby can be evaluated in a particularly simple and effective manner. In addition, the sample-derived Raman scattered light can be analyzed by a series of selectively selected long-pass filters or bandpass filters tuned to the known Raman signatures of the SERS gold nanoparticles used in the sample and conveniently imaged on inexpensive single-channel detectors. The inven tion thus provides a possibility for rapid quantitative and highly sensitive pa tientennahe diagnostics using surface-enhanced Raman scattering and can be particularly advantageous in a so-called POCT (point of care testing) can be used. An advantage of section-wise planar or even complete illumination of the sample to be analyzed is that measurements, in particular spatially resolved measurements, can be carried out particularly quickly and sensitively. The inventive use of long-pass filters / Bandpassfiltem and Einkanaldetek factors favors a lower cost compared to the prior art measurement. The invention is particularly suitable for use by hospitals, medical practices, laboratory physicians, veterinarians and pharmaceutical companies. In laboratory medicine, using SERS technology, for example, a fast measuring procedure can be applied directly on-site to the patient or animal, which even allows for absolute quantification through the use of internal standards. The invention is also particularly suitable for use by public authorities of internal and external security (Bundeswehr, police, etc.), for example, for a safety-relevant hazardous substance analysis for the identification of chemical and biological weapons. Another aspect of the invention may be closing investigations in water and food analysis.

Further features and advantages of the present invention will become apparent from the following exemplary and non-limiting description of the invention with reference to figures. These are merely schematic in nature and are only for understanding the invention. As shown in:

Fig. 1 is a schematic representation of a device according to the invention in a first

embodiment,

2 shows a position-voltage diagram for the sample shown in FIG. 1,

3 shows a frequency-intensity diagram as an example of a Raman spectrum acquired in relation to the sample shown in FIG. 1,

Fig. 4 is a schematic representation of a device according to the invention in a second

embodiment, 5 shows a position-voltage diagram for the sample shown in FIG. 4,

6 shows a frequency-intensity diagram as an example of three Raman spectra recorded for the sample shown in FIG. 3, FIG.

Fig. 7 is a schematic representation of a device according to the invention in a third

8, a position-voltage diagram for the sample shown in FIG. 7, FIG.

9 is a frequency-intensity diagram as an example of that shown in FIG

10 shows a schematic representation of a first embodiment of an optical probe for a device according to the invention, FIG.

Fig. 11 is a schematic representation of a second embodiment of an optical

Probe for a device according to the invention,

Fig. 12 is a schematic representation of a third embodiment of an optical

Probe for a device according to the invention and Fig. 13 is a further schematic representation of an apparatus according to the invention.

FIG. 1 shows a schematic representation of a device 1 according to the invention for the quantitative diagnosis of a sample 2 with a single sort of SERS nanoparticles in several Measuring lines I, II, III and IV. The sample 2 is applied to a known LFA test strip 13 as a sample substrate (lateral flow essay), whose operation is well known and therefore will not be described here. The test strip 13 has four test lines I, II, III and IV, at which each - if any - target proteins to be detected on SERS nanogold particles labeled antibodies accumulate. The immobilization of Fängeranti body on the LFA membrane (often nitrocellulose) is carried out with established methods. Instead of the normal gold nanoparticle detection antibody conjugates, in a SERS-based LFA, the corresponding SERS nanoparticle detection antibody conjugates occur. The test strip 13 is mounted on a positioning device (not shown in detail in the figures) 14, which permits linear positioning in the direction of the arrow 15 shown in FIG. 1 (and subsequently back again) during a measurement of the sample. The sample 2 to be measured is placed with the test strip 13 in the plane of the focus of the measuring probe 4 and moved in the direction 15 perpendicular to the axis of the focus. Depending on the sample position, the signal of a Raman photodetector 5 or of a reference photodetector 11 is detected and processed by evaluation electronics 12.

Figure 13 shows an embodiment in which the positioning device 14 is shown in more detail. It comprises a stepping motor 33, a linear guide 34 and a sample holder 35, which holds a sample applied to a test strip 13 as a substrate. By means of the stepping motor 33 and the linear guide 34 is the sample holder together with the therein received

Probe 2 in the direction of arrow 15 relative to the probe 4 positionable. In this embodiment, a reference photodetector 11 and a total of four Raman photodetectors 5a, 5b, 5c, 5d, four filter and reflection elements 9a, 9b, 9c, 9d, which as described above act as a dielectric bandpass or notch filter, and a deflection mirror 36 is used.

The device 1 has a light source 3, which he essentially testifies monochromatic light, in the present case, a laser 3. It further comprises an optical probe 4 to the sample to be examined 2 on the substrate 13 with light of the laser 3 light and to collect stray light from the sample 2 and the substrate 13. In addition, the device 1 has a photodetector 5 for detecting inelastic scattered light from the sample 2, that is, from an emitted Raman band of the scattered light. A laser beam generated in the laser 3 is conducted to the probe 4 via a first optical waveguide 6. Light collected by the probe 4 is coupled into a second optical waveguide 7.

In the spectrum of light scattered on the sample 2 and on the substrate 13, in addition to the radiated frequency (Rayleigh scattering or elastic scattering), there are also other frequencies or bands. The differences in frequency with respect to the incident light correspond to the energies of rotational, vibrational, phonon or spin-lip processes characteristic of the molecules of the sample. From the spectrum of the scattered light emitted by the sample 2 conclusions can be drawn on the examined substance.

The light emitted from the second optical waveguide 7 at the end opposite the probe 4 is collimated by an optical system and directed to a filter element 8 in the form of a longpass filter 8. The long-pass filter 8 has a high optical density in the range of the laser wavelength and is designed and arranged such that the largest possible proportion of elastic scattered light, preferably substantially the entire proportion of the elas tical scattered light is blocked. It can therefore be said that the long-pass filter 8 separates elastic scattered light from inelastic scattered light. In the present embodiment, the long-pass filter 8 passes inelastic scattered light and reflects and blocks elas table stray light. In the beam path after the long-pass filter 8 is therefore only unelasti cal scattered light before. The long-pass filter 8 is also referred to as Rayleigh filter element 8 in the context of the invention.

In the further beam path, a Lilter- and reflection elements 9 is arranged after the long-pass filter 8. This reflects from the inelastic scattered light guided through the Rayleigh filter element 8 the respective Raman signals onto the associated detector elements 5 The spectral components around a substrate-specific reference band 10 are transmitted through the reflection elements 9 and then pass directly or via the mirror 36 to the reference detector 11. The signal of the reference photodetector 11 is represented by an evaluation electronics 12 of the device, shown schematically in FIGS 1 used for a correction of the other signals, for example, to compensate for fluctuations in the power of the laser 3, a fluorescence of the substrate or deviations of the focusing of the laser beam on the sample.

The evaluation and control unit 12 amplifies and processes signals originating from the photodetectors 5 and 11 (shown in FIG. 2 in a position-voltage diagram). For putting in, it is control technology connected to the positioning device 14 for the test strip 13. The interaction with a user via the evaluation and Steuerein unit 12.

FIG. 3 shows the Raman spectrum recorded with the device 1 of FIG. 1 for the test strip 13 shown there. Shown is the intensity peak of the reference band 10 and the SERS signature 16 of the nanoparticle type used in the sample 2 on the lines I, II, III and IV.

FIG. 4 shows a schematic representation of a device 1 according to the invention for quantitatively diagnosing a sample 2 with three different types of SERS nanoparticles on a plurality of measurement lines I, II, III and IV. In contrast to the exemplary embodiment of FIGS Each of the measuring lines I, II and III each have a different type of SERS nanoparticles, for example on the measuring line I SERSl nanoparticles, on the measuring line II SERS2 nanoparticles and on the measuring line III SERS3 nanoparticles. On line IV are all three different SERS nanoparticles. The device 1 corresponds wesentli chen the embodiment shown in Figure 1, so that in the following only the differences are listed. In the beam path between the long-pass filter 8 and the Pho todetektor 5 are a total of three other optical filter and reflection elements 9a, 9b, 9c arranged serially one behind the other. The filter and reflection elements 9a, 9b, 9c are now each true abge on the specific Raman band of one of the three SERS nanoparticles used. The filter and reflection element 9a is matched, for example, to SERSl nanoparticles, the filter and reflection element 9b to SERS2 nanoparticles and the filter and reflection element 9a to SERS3 nanoparticles. The filter and reflection element 9a is associated with a detector 5a, the filter and reflection element 9b, a detector 5b, and the filter and reflection element 9c with a detector 5c. For the filter and reflection elements 9a, 9b and 9c it is true that this passes through the reference band 10 and leads to the reference photodetector 11 and the respective Raman bands of the fi lings I, II, III and IV to the respective Raman photodetectors 5a 5b and 5c reflected.

FIG. 5 shows a position-voltage diagram for the sample shown in FIG. Since the signal of the detector 5a by means of the graph 17, the signal of the detector 5b by means of the graph 18 and the signal of the detector 5c by means of the graph 19 is shown.

FIG. 6 shows the Raman spectrum recorded with the device 1 of FIG. 4 for the test strip 13 shown there. Shown are the intensity peaks of the reference band 10 as well as the SERS 1 signature 20, the SERS2 signature 21 and the SERS3 signature 22 of the three different nanoparticle types used in the sample 2 on the fi les I, II, III and IV.

FIG. 7 shows a schematic representation of a device 1 according to the invention for the quantitative diagnosis of a sample 2 with three different types of SERS nanoparticles on a single measurement line I. In contrast to the exemplary embodiments of FIGS. 1 to 3 and 4 to 6, each is here Measurement line I and IV three different types of SERS nanoparticles, namely on the measurement line I as well as on the measurement line IV SERSl nanoparticles, SERS2 nanoparticles and SERS3 nanoparticles. FIG. 8 shows a position-voltage diagram for the sample shown in FIG. Since the signal of the detector 5a by means of the graph 17, the signal of the detector 5b by means of the graph 18 and the signal of the detector 5c by means of the graph 19 is shown. FIG. 9 shows the Raman spectrum taken with the device 1 of FIG. 7 for the test strip 13 shown there. Shown are the intensity peaks of the reference band 10 as well as the SERS 1 signature 20, the SERS 2 signature 21 and the SERS 3 signature 22 of the three different nanoparticle types used in the sample 2 on the lines I and IV. FIGS. 10, 11 and 12 show Embodiments of the probe 4. Figure 10 shows an embodiment of the probe 4, in which the laser is coupled into a linear fiber bundle 6, at the end of which a bandpass filter 23 is located, which passes only a narrow spectral range around the laser line. The fiber bundle 6 and the notch filter 23 are arranged and aligned such that the outgoing beam is divergent and hits the sample at an oblique angle of incidence. The light scattered on the sample 2 as well as on the substrate 13 is collected via a linear array 24 which is attached to the sample surface so that there is a maximum overlap between illumination and detection range. In front of the Fa sereingang a long-pass filter 25 is mounted, which blocks elastically scattered light. This represents an alternative embodiment to the fact that such a filter is provided as a Rayleigh filter element 8 after the probe, as shown for example in Fig. 1.

FIG. 11 shows a further embodiment of the probe 4. Here, too, the laser is coupled into a linear fiber bundle 6, at the end of which there is a notch filter 23. Through a lens 26, an image of the fiber bundle 6 is generated at infinity. Via a mirror 27 and a dichroic 28 which only reflects light of the laser wavelength, an image of the light emerging from the fiber bundle is generated via an objective lens 29. The scattered light is collected by the objective lens 29 or detil detil the infinite. The elastic scattering components are filtered out by a longpass filter 30. By a lens 31, the image of the fiber input bundle is mapped congruent to the Faserausgangsbün del 7

FIG. 12 shows a further embodiment of the probe 4. Compared with the two previous examples of FIGS. 10 and 11, a linear fiber bundle is not used here for illumination, but instead a cylindrical lens 32 or a more complex optical system for producing a linear illumination. and detection range on the sample 2. The coupling is done by simple optical waveguides 6, 7.

LIST OF REFERENCE NUMBERS

1 device

2 samples

3 light source, laser

4 probe

5, 5a-e Raman photodetectors

6 first optical waveguide

7 second optical waveguide

8 filter element, Rayleigh filter element

9, 9a-e filter and reflection element

10 reference band of the substrate 13

11 reference photodetector

12 evaluation and control unit

13 substrate

14 positioning device

15 Positioning device

16 SERS signature

17 Graph SERS1

18 Graph SERS2

19 Graph SERS3

20 SERS 1 signature

21 SERS2 signature

22 SERS3 signature

23 notch filters

24 linear array

25 long-pass filters

26 lens 27 mirrors

28 dichroics

29 objective lens

30 long-pass filter 31 lens

32 cylindrical lens

33 stepper motor

34 linear guide

35 sample holder 36 deflection mirror

Claims

claims

1. Device (1) for diagnosis, in particular for quantitative diagnosis, of a sample (2) comprising at least one type of SERS-active particle, each with a specific Raman band (16, 20, 21, 22), by means of surface-enhanced Raman Scattering (SERS), where the

Apparatus (1) a light source (3) for substantially monochromatic light, an optical probe (4) for illuminating the sample (2) to be examined with light from the light source (3) and for collecting scattered light from the sample (2) and a Raman photodetector (5, 5a, 5b, 5c, 5d) and a reference photodetector (11) for detecting the collected light from the probe (4) and scattered by the sample (2), wherein

in the beam path of the light scattered from the sample (2), an optical filter element (8) for separating stray elastic light from inelastic stray light of the specific Raman bands (16, 20, 21, 22) is arranged, wherein the optical filter element (8) the inelastic scattered light is separated from the elastic scattered light, and

in the beam path behind the Lilterelement (8) for separating elastic scattered light from inelastic scattered light another Lilter- and reflection element (9, 9a, 9b, 9c, 9d, 9e) for separating a specific reference to the substrate of the sample reference band (10) of the specific Raman bands (16, 20, 21, 22) of the SERS-active particles is arranged, the Raman band being reflected and directed onto the Raman photodetectors (5, 5a, 5b, 5c, 5d) and the reference Passed band and directed to the reference photodetector (11).

2. Device (1) according to claim 1, characterized in that the Lilterelement (8) for separating elastic scattered light from inelastic scattered light of the specific Raman band (16, 20, 21, 22) is a long-pass filter (8), the one has high optical density in the range of the wavelength of the monochromatic light.

3. Device (1) according to any one of the preceding claims, characterized in that in the beam path of the sample (2) emitted scattered light behind the Lilterelement (8) for separating elastic scattered light from inelastic scattered light more optical Lilter- and Reflection elements (9, 9a, 9b, 9c, 9d, 9e) are arranged, each filter and Reflexi onselement (9, 9a, 9b, 9c, 9d, 9e) each for separating a specific for a substrate of the sample (2) Reference band (10) is formed by a Raman band (16, 20, 21, 22) specific for each type of SERS-active particle and a respective filter and reflection element (9, 9a, 9b, 9c, 9d, 9e) a Raman photodetector associated with the filter and reflection element (9, 9a, 9b, 9c, 9d, 9e) and the filter and reflection elements (9, 9a, 9b, 9c, 9d, 9e) together comprise the reference photodetector (11) is assigned to the jewei time reference band or the respective Raman band is passed.

4. Device (1) according to any one of the preceding claims, characterized in that the ses a positioning device (14) for one-, two- or multi-dimensional positioning of the sample (2) relative to the optical probe (4), in particular orthogonal to the beam axis includes.

5. Device (1) according to one of the preceding claims, characterized in that the optical probe (4) has a filter element (23) which is arranged and formed such that the sample (2) with a divergent beam, with a oblique angle of incidence on the sample (2) is illuminated, and that the optical probe (4) has a collection array (24) for scattered light from the sample (2), which is aligned and arranged in accordance with the angle of incidence.

6. Device (1) according to any one of the preceding claims, characterized in that the SES a linear fiber bundle (6) for coupling Ficht the spruce source (3) in the optical probe (4), wherein the optical probe (4) a in particular a fin (26) which produces an image of the fiber bundle (6) at infinity, illuminating the sample (2), and wherein the optical probe (4) comprises a dichroic element (28 ), over which the sample (2) directed spruce as well as scattered light from the sample (2) is passed.

7. Device (1) according to one of claims 1 to 5, characterized in that the opti cal probe (4) an optical system or element (31), for example a cylindrical lens (31), for generating a substantially linear illumination area the sample (2).

8. Method for diagnostics, in particular for quantitative diagnostics, of a sample (2) comprising at least one type of SERS-active particles each having a specific Raman band (16, 20, 21, 22) by means of surface-enhanced Raman scattering ( SESR), in particular by means of a device (1) according to one of the preceding claims, wherein substantially monochromatic light is conducted to an optical probe (4) which illuminates the sample (2) to be examined with light from the light source (3). and the scattered light from the sample (2) collects, scattered light from the sample (2) to a Raman photodetector (5, 5a, 5b, 5c, 5d) and to a reference photodetector (11) for detecting of the light collected by the probe (4) and scattered by the sample (2), wherein

light scattered from the sample (2) is passed through an optical filter element (8) arranged in the beam path in front of the Raman photodetector (5, 5a, 5b, 5c, 5d) and the reference photodetector (11), the stray elastic light of inelastic scattered light of the specific Raman band (16, 20, 21, 22) separates, whereby the inelastic scattered light is transmitted by the optical filter element (8) and the elastic stray light is blocked, and

the light following in the beam path behind the Lilterelement (8) on another Lilter- and reflection element (9, 9a, 9b, 9c, 9d, 9e) for separating a specific reference to a substrate of the sample reference band (10) of the specific Raman band (16, 20, 21, 22) of the SERS-active particles, the reference band being directed to the Raman photodetector (5, 5a, 5b, 5c, 5d) and the Raman band being directed to the reference photodetector (11). is directed.