DE102006049687A1 - High resolution imaging method for capturing a test substance in a test medium comprises interacting the test substance and a reference substance bounded to a solid-phase and capturing the resulting optical changes by ellipsometer - Google Patents

Abstract

High resolution imaging method for capturing a test substance in a test medium by interacting the test substance and a reference substance bounded to a solid-phase, comprises subjecting the test medium to a solid phase, at whose surface a film is arranged with the reference substance, capturing optical changes emerged by the interaction of the reference- and test substance by an ellipsometer, and optionally comparing the changes with a standard sample, where the ellipsometer is supplied with a brilliant infrared-light source in an average infrared spectral area (400-4000 cm->1>). An independent claim is included for a measuring apparatus for the execution of the above procedure, comprising an ellipsometer, which shows a radiation source, a polarizer, an analyzer, a detector and an evaluator, and a solid phase, at whose surface a film with a reference substance and a test substance adhered to the reference substance are arranged, where the radiation source is a brilliant infra-red light source.

Description

The The invention relates to a method for imaging measurements for quantitative mark-free analysis of thin biofilms, biosensors, Biochips and biolayer systems as well as an arrangement for carrying out the Process. Areas of application of the invention are the life sciences (Life Sciences) and materials science.

Commercial IR ellipsometers can currently only analyze nanometer-sized biological layers with an inadequate spatial resolution for many areas of application since, when using non-brilliant radiation sources, the corresponding sensitivity with high spatial resolution (less than 1 mm ² ) and aperture angles of less than +/- 4 ° in a simple reflection for any substrates currently can not be achieved.

Of the The state of the art is covered by a number of commercial IR ellipsometers SOPRA, SENTECH Instruments, J. A. WOOLLAM.

A general feature of these devices is the use of an interferometer with Globarlichtquelle for the mid-infrared spectral range. For high-resolution ellipsometric measurements, the documents are DE10126152 and US5991037 known; this only applies DE10126152 on ellipsometric measurements on biosensors / biochips, however, the use of a metal layer in the region of the sample to be measured is required. For investigations on biological samples, but without high spatial resolution, is also the document US2005012041 known.

The invention is therefore based on the object to provide a method and a measuring arrangement, the measurement and quantitative examination of a thin layer (1-1000 nm) or a corresponding layer stack of a biosensor, biochip or bioarray in the infrared spectral marker and non-contact with high lateral resolution (less than 1mm ² ) in terms of composition, layer thickness and charge carrier properties without sample specific limitations (metal layer and multiple reflections).

These Task is done according to a The method of claim 1 and a measuring arrangement according to claim 4 solved. The other claims are preferred embodiments the invention.

In the work that led to the invention, it was found quite surprisingly that the use of brilliant infrared light in the mid-infrared spectral range (400-4000 cm ^-1 ) for biosensory measurement of biochips is outstandingly suitable. As a result, metal layers on the sample carriers to be measured are no longer required, as they are used in conventional methods for ellipsometric measurement of biochips.

The invention therefore relates to polarization-dependent spectroscopy, in particular ellipsometry in the mid-infrared spectral range (400 cm ^-1 to 4000 cm ^-1 ) for imaging measurements for quantitative and marker-free analysis of thin biofilms, biosensors, biochips on semiconducting, insulating and metallic substrates and their modification (eg adsorption of molecules). In principle, layer systems of different biological substances, in particular peptides and proteins (eg of a substance covalently coupled to the surface (eg antigen) and of a substance (eg antibody) covalently bound to the surface, can also be detected and measured by the method In addition to the simple identification of one type of molecule, the layer thickness, conductivity and molecular structure of the film are coupled The invention is coupled to the use of a brilliant radiation source (such as synchrotron, laser) The term "brilliant radiation source" in the sense of the invention comprises sources containing more photons radiate into a defined solid angle as a globar source, ie its radiant intensity in Wm ^-2 sr ^{-1 is} greater than that of a globar source, in particular all radiation sources are meant, which have a larger radiant intensity for a vertical and / or horizontal opening angle, which is smaller than 30 It enables a lateral resolution <1mm ² and allows quantitative label-free analysis of films and arrays with layer thicknesses of a few mm to 1 μm.

The core of the method according to the invention is therefore the use of a brilliant infrared light source (11, numbers are given here and below for a better overview) in the middle infrared spectral range (400-4000 cm ^-1 ) for feeding an ellipsometer ( 8th ) for a high resolution process ( 1 ), in particular with a lateral resolution better than 5 × 5 mm ² , and with imaging ( 2 ), which preferably takes place by computer-side representation of the measured data of the high-resolution measured surfaces of a sample which can be displaced in a defined manner in the x and y directions, for the detection of a test substance ( 3 ), which from a test medium ( 4 ) to a solid phase-bound test substance ( 5 ) or interacts with it.

Preference is given as a brilliant infrared light source ( 11 ) selected for feeding the ellipsometer with light in the mid-infrared spectral range (400-4000 cm ^-1 ) a synchrotron or a laser, more preferably a synchrotron radiation source or a molecular gas laser, solid-state or semiconductor laser, quantum cascade laser or differential frequency generation laser. The term "brilliant radiation source" in the sense of the invention comprises sources which emit more photons into a defined solid angle than a globar source, ie whose radiation intensity in Wm ^-2 sr ^{-1 is} greater than that of a globar source. In particular, this means all radiation sources which have a greater beam intensity for a vertical and / or horizontal opening angle which is smaller than 30 mrad.

As used test substance ( 3 ) are in principle all possible, preferably biologically active, particularly preferably at least partially soluble in aqueous medium, substances having at least one adsorption band in the spectral range of 400-4000 cm ^-1 , in particular oligonucleotides, proteins, peptides, lipids, carbohydrates, Pharmaceuticals, biotin, cells, or cell compartments or substances that are a combination thereof, eg, glycoproteins or peptides, membrane-associated proteins or RNA-protein complexes, or fragments thereof, e.g. B. a protein fragment. It is particularly advantageous in particular if, as test substance ( 3 ) DNA, RNA, cDNA, mRNA, cRNA, PNA, protein A, protein G, streptavidin, a receptor, a ligand, an enzyme substrate, an enzyme, an antibody and / or an antigen is selected or if the test substance ( 3 ) comprises or is formed as a derived structure.

As a test medium ( 4 ) are selected in particular gases, liquids, solids or a combination thereof, preferably liquids, particularly preferably aqueous liquids, for. B. a physiological buffer solution or body fluid, in particular a test medium ( 4 ) containing the test substance to be detected ( 3 ), for example as a solution, suspension or emulsion.

• – Beaufschlagung einer Festphase (6), an deren Oberfläche ein Film (7) mit einer Prüfsubstanz (5) angeordnet ist, mit einem Testmedium (4),
• – Vermessung von optischen Veränderungen (9) in dem Film (7), die durch die Wechselwirkung der Prüfsubstanz (5) mit der Testsubstanz (3) verursacht sind, mittels eines Ellipsometers (8), und
• – ggf. Vergleichen der Ergebnisse mit einer Standardprobe (10).

The method according to the invention comprises the steps

• - imposition of a solid phase ( 6 ), on whose surface a film ( 7 ) with a test substance ( 5 ) is arranged with a test medium ( 4 )
• - Measurement of optical changes ( 9 ) in the film ( 7 ) caused by the interaction of the test substance ( 5 ) with the test substance ( 3 ) by means of an ellipsometer ( 8th ), and
• - If necessary, compare the results with a standard sample ( 10 ).

The loading is advantageously carried out by conventional methods for contacting a functionalized surface with a medium to be tested, for example by applying a gas or a liquid, for example by pipetting the test medium ( 4 ) on the solid phase ( 6 ) with the movie ( 7 ) or by wetting or bathing the film ( 7 ) comprehensive solid phase ( 6 ) with the test medium ( 4 ).

The ellipsometric measurement of the sample from solid phase ( 6 ) and movie ( 7 ) is preferably carried out before, during or after the exposure to the test medium ( 4 ), in particular if the sample is washed after the application of washing solution, for example water or buffer, and dried if necessary. The polarized light is thereby applied to the front or the back of the solid phase ( 6 ), especially on the side with the film ( 6 ) and the test substance ( 5 ) with the test medium ( 4 ), and the reflective radiation is measured.

For the process according to the invention, a solid phase ( 6 ), which is formed from a material which has substantially no strong adsorption bands in the spectral range of 400-4000 cm ^-1 , ie in particular has no interfering adsorption bands whose maxima are greater than the maxima of the adsorption bands of the measured sample (film (FIG. 7 ) with the test substance ( 5 ) and / or the movie ( 7 ) attached test substance ( 3 )) in the measured spectral range. In particular for the investigation of ultrathin films, the solid phase ( 6 ) selected a substrate that does not show strong intrinsic absorptions in the spectral region to be examined.

Particular preference is given as solid phase ( 6 ) Silicon, in particular in a conventional embodiment, for example. Amorphous or crystalline silicon, for example a doped monocrystalline silicon wafer or a part thereof. With the method according to the invention, it is now possible in a particularly advantageous manner to use solid phases as sample carriers which have no metal layer in the region of the sample to be measured.

According to the invention, a solid phase ( 6 ) whose surface is at least partially, preferably covalently, with a preferably organic film ( 7 ) containing a test substance ( 5 ), in particular predominantly or completely from the test substance ( 5 ), the test substance ( 5 ) physically, eg via electrostatic and / or hydrophobic interactions, or preferably covalently to the solid phase ( 6 ) or the test substance is physically or preferably covalently linked to a linker, in particular a crosslinker, which is covalently bound to the solid phase. For the process according to the invention, particular preference is given to choosing a film having a thickness / height of 1 nm to 1000 nm.

As immobilized test substance ( 5 ) are in principle all possible, in particular biologically active, preferably organic, substances suitable for use with a test substance to be detected ( 3 ), for example by covalent interaction, enzymatic reaction or in particular association, interaction. Particularly preferred as the test substance ( 5 ) Linker molecules, carbohydrates, lipids, pharmaceuticals, peptides, polypeptides, oligonucleotides, proteins, cell compartments, amino acids, cells, or biotin, or a combination thereof, eg one to a crosslinker, preferably covalently bound to the solid phase ( 6 ), bound oligonucleotide, peptide or protein, or a transmembrane protein incorporated into a lipid film bound to the solid phase, eg via a crosslinker.

It is particularly preferred in particular if several test substances or different amounts of a test substance are used, which are localized on the solid phase ( 6 ) are immobilized, for example as spots, in particular as an array, whereby several interactions, eg with different test substances, within a measurement can also be detected in parallel.

Advantageously, all possible measuring arrangements which are suitable for ellipsometric measurements of a sample are suitable for the method according to the invention. Particularly preferred is an ellipsometer ( 8th ), which is a radiation source ( 12 ), a polarizer ( 13 ), an analyzer ( 14 ), a detector ( 15 ) and an evaluation device ( 16 ), wherein the radiation source is a brilliant infrared light source ( 11 ), which emits light in the mid-infrared spectral range (400-4000 cm ^-1 ). Particularly suitable is the use of a fixed or rotatable polarizer ( 13 ), a fixed or rotatable analyzer ( 4 ) and / or a common detector for the middle infrared. Evaluation device ( 16 ) are all common facilities for evaluating ellipsometric measurements suitable, especially if they include a computer and software.

Particularly advantageous for the inventive method, in particular the use of an ellipsometer ( 6 ), which is a null or a photometric ellipsometer, which operates with fixed or rotatable polarizers or a phase modulator.

As optical changes ( 9 ) in the film ( 7 ) caused by the interaction of the test substance ( 5 ) with the test substance ( 3 In the method according to the invention, those changes in the ellipsometric spectrum that occur due to changed conductivity, structure, bonds and composition are particularly preferably measured.

For different applications of the method according to the invention, it is advantageous if a standard sample ( 10 ) or measured values obtained by measuring a standard sample ( 10 ) obtained with the same measuring method. The measured values of the test medium ( 4 ) fixed phase ( 6 ) are then used with the measured values for the standard sample ( 10 ) and the possibly occurring difference of the measured values as a measure of the interactions between the test substance ( 5 ) and test substance ( 3 ) evaluated. It is advantageous, in particular, if as a standard sample ( 5 ) a solid phase is used, which in its metrological characteristics is substantially identical to the solid phase applied to the detection of the test substance ( 6 ). Particularly favorable is the measurement of the same solid phase ( 6 ), in particular before exposure to the test medium or the use of values previously obtained for comparable measurements, in particular of functionalized and test medium-loaded solid phases, preferably when these values describe known / defined interactions.

It is particularly advantageous for the method according to the invention if the standard sample ( 10 ) on the same solid phase ( 6 ), that is, for example, an area is measured, which is not with the film ( 7 ) and / or the test substance ( 5 ) is occupied.

Another aspect of the invention relates to a measuring arrangement, preferably for carrying out the Method according to one of claims 1-3, with an ellipsometer ( 8th ), which is a radiation source ( 12 ), a polarizer ( 13 ), an analyzer ( 4 ), a detector ( 15 ) and an evaluation device ( 16 ), and a solid phase ( 6 ), on whose surface a film ( 7 ) with a test substance ( 5 ) and one to the test substance ( 5 ), preferably by association or covalent bond-attached test substance ( 3 ), wherein according to the invention the radiation source ( 12 ) a brilliant infrared light source ( 11 ) emitting preferably or predominantly in the mid-infrared spectral range (400-4000 cm ^-1 ) for ellipsometric measurements.

In particular, the arrangement according to the invention is designed such that optical changes ( 9 ) in the film ( 7 ) caused by the interaction of the test substance ( 5 ) with the test substance ( 3 ) are measurable as changes in the ellipsometric spectrum that occur due to altered conductivity, structure, bonds and composition.

In a particularly advantageous embodiment for the most diverse applications, the radiation source ( 12 ) is formed as a synchrotron or a laser, more preferably as a synchrotron radiation source or as a molecular gas laser, solid state or semiconductor laser, quantum cascade laser or differential frequency generation laser.

Particularly preferably, the measuring arrangement includes an ellipsometer ( 8th ), which is a radiation source ( 12 ), a polarizer ( 13 ), an analyzer ( 14 ), a detector ( 15 ) and an evaluation device ( 16 ), wherein the radiation source is a brilliant infrared light source ( 11 ), which emits light in the mid-infrared spectral range (400-4000 cm ^-1 ). It is particularly suitable when the polarizer ( 13 ) and / or the analyzer ( 14 ) is fixed or rotatable and / or the detector ( 15 ) is a common detector for the middle infrared. Evaluation device ( 16 ) are all common facilities for evaluating ellipsometric measurements suitable, especially if they include a computer and software.

Particularly advantageous for a very wide variety of applications is in particular a measuring arrangement according to the invention with an ellipsometer ( 8th ) which is a null or a photometric ellipsometer operating with fixed or rotatable polarizers or a phase modulator.

The radiation source ( 12 ) is advantageously arranged such that that of the ellipsometer ( 8th ) generated light on the front or the back of the functionalized solid phase ( 6 ) is steerable, in particular to the area with the film ( 7 ) with the test substance ( 8th ), and the detector ( 15 ) is advantageously arranged such that it matches that of the film ( 7 ) and / or the test substance ( 5 ) can detect reflected radiation.

The test substance ( 3 ) may advantageously be formed from all possible, preferably biologically active, particularly preferably at least partially soluble, substances in aqueous medium which have at least one adsorption band in the spectral range of 400-4000 cm ^-1 . The test substance ( 3 ) is preferably an oligonucleotide, a protein, a peptide, a lipid, a carbohydrate, a drug, biotin, a cell, a cell compartment or a combination thereof, since these are usually detectable particularly specific. For very different applications of the measuring arrangement, it is particularly advantageous if the test substance ( 3 ), DNA, RNA, cDNA, mRNA, cRNA, PNA, protein A, protein G, streptavidin, a receptor, a ligand, an antibody, or an antigen is or comprises or is a structure derived therefrom.

Particularly advantageous for the arrangement according to the invention have as test substance ( 5 ) organic structures specifically with the test substance ( 3 ), in particular via association or covalent bonds, interact. As immobilized test substance ( 5 ) are particularly suitable linker molecules, preferably crosslinkers, carbohydrates, lipids, pharmaceuticals, peptides, polypeptides, oligonucleotides, proteins, cell compartments, amino acids, cells or biotin or a test substance ( 5 ) which comprises or is a structure derived therefrom, for example one to a crosslinker which is preferably covalently bonded to the solid phase ( 6 ), bound oligonucleotide, peptide or protein, or a transmembrane protein incorporated into a lipid film bound to the solid phase, eg via a crosslinker.

In another particularly advantageous embodiment of the measuring arrangement according to the invention several test substances or different amounts of a test substance are located on the solid phase ( 6 ) are immobilized, for example as spots, in particular as an array, whereby the arrangement also for parallel detection of multiple interactions, eg with different test substances, within a measurement suitable is.

In a particularly preferred embodiment of the arrangement according to the invention, the solid phase ( 6 ) formed from a material having substantially no adsorption bands in the spectral range of 400-4000 cm ^-1 , that is, that the solid phase, in particular in the spectral range suitable for the use of the arrangement has no interfering adsorption bands whose maxima are greater than the maxima of the adsorption bands the measured sample (film ( 7 ) with the test substance ( 5 ) and / or the movie ( 7 ) attached test substance ( 3 )). Especially for the investigation of ultrathin films, the solid phase ( 6 ) is formed from a substrate that does not show strong intrinsic absorptions in the appropriate spectral range.

Particularly preferred is the solid phase ( 6 ) a silicon substrate, in particular in a conventional embodiment, for example. Amorphous or crystalline silicon, for example, a doped monocrystalline silicon wafer or a part thereof. Advantageously, the solid phase ( 6 ) designed such that it has no metal layer in the region of the sample to be measured.

In another particularly suitable embodiment of the arrangement, the surface of the solid phase ( 6 ) with a preferably organic film ( 7 ) containing the test substance ( 5 ), in particular predominantly or completely from the test substance ( 5 ), the test substance ( 5 ) physically, eg via electrostatic and / or hydrophobic interactions, or preferably covalently to the solid phase ( 6 ) or the test substance is physically or preferably covalently linked to a linker, in particular a crosslinker, which is covalently bound to the solid phase. Particularly suitable for the process according to the invention is a film which has a thickness / height of 1 nm to 1000 nm.

For different applications of the arrangement according to the invention, it is also advantageous if the arrangement with a standard sample ( 10 ) or if the evaluation device ( 16 ) Contains measured values obtained by measuring a standard sample ( 10 ) were obtained with the same measuring method. be used. It is particularly advantageous if the standard sample ( 10 ) is a solid phase whose metrological characteristics are substantially the same as the solid phase charged for detecting the test substance ( 6 ), in particular before exposure to the test medium or the evaluation device comprises values that are comparable to the measurements that can be implemented with the device, in particular measured values obtained from functionalized solid phases loaded with test medium, preferably when these values describe known / defined interactions.

In a particularly suitable embodiment of the measuring arrangement, the standard sample ( 10 ) on the same solid phase ( 6 ) is, for example, an area of the sample to be measured, which does not interfere with the film ( 7 ) and / or the test substance ( 5 ) is occupied.

One Another aspect of the invention relates to the method according to the invention in its different embodiments, for the the inventive arrangement is used in their different embodiments.

Of the Advantage of the method according to the invention and the measuring arrangement according to the invention is to destroy and label-free samples, especially biosensors and biochips with high spatial resolution to be quantitatively examined. Another big advantage across from Standard methods (e.g., chemiluminescence imaging) and measuring arrangements their implementation is the high amount of information (layer thickness, Composition, charge carrier properties) the above the pure identification of the material can be achieved can. Synchrotron - or broadband laser sources for the middle infrared are for it just as suitable as single laser lines of molecular gas laser and solid state or semiconductor lasers or narrow frequency ranges of quantum cascade lasers and difference frequency generation lasers. The procedure can be for a wide Range of substrates can be used.

Sufficient for the inventive arrangement and the method according to the invention is basically an infrared ellipsometer with a mapping table when using a brilliant (laser, synchrotron) or high-intensity Radiation source suitable. Compared to other common commercial Methods (e.g., chemiluminescence, fluorescence, radioactive imaging) In this procedure, the sample or test substance to be detected must be not be manipulated (labeled). In addition, with the method according to the invention and the measuring device according to the invention on the pure identification a material-specific information calculating measured spectra in optical models possible.

As a non-exhaustive embodiment, the inventive method and the inventions The arrangement according to the invention is tested with a synchrotron mapping ellipsometer for the examination of a biosensor, but the method and the measuring arrangement can also be implemented with all the technical features given in the present description and the claims in their different combinations

Technical and scientific principles, the mode of operation and the structure, as well as the advantages and improvements over the prior art and the realization by experiments are exemplified by the accompanying manuscript ( Hinrichs et al., Submitted to Analytical Bioanalytical Chemistry (2006) ), which is hereby integrated into the present invention description by the marked passages, in particular concerning the technical features of the ellipsometric method described there and the measuring arrangement used therefor.

Quick Facts

to Time standard methods used to read out microarrays Fluorescence and chemiluminescence techniques. These methods require Labeling of individual components and usually only concentrations certainly. A label-free method, which also provides a quantitative spectral analysis to determine the composition and molecular interactions allows would from great advantage be. This post shows for the first time that the IR Ellipsometry label-free imaging of a biochip before and after Allowed incubation with a peptide. The measurements show that IR ellipsometry is a sensitive tool for lateral resolution Investigations on the identification and determination of the composition is. The required lateral resolution was achieved by Radiation from an infrared synchrotron tube used.

introduction

Some applications of ellipsometry in the life sciences and for investigations of bioarrays are known for the visible spectral range [1, 2]. For biofilms, this method has recently been extended to the infrared spectral range [3-7]. However, in contrast to the visible spectral range (VIS), one can achieve a significantly higher specific contrast in the infrared by absorption of vibrations. It has been shown that the structure, molecular orientation, thicknesses and optical constants can be determined by the evaluation of the IR ellipsometric spectra [7]. From this point of view, we expected IR ellipsometry to be an ideal tool for studying such biosensors and protein adsorption on them. The method seems to be well suited for this purpose, also because of the monolayer sensitivity and the possibility of quantitative evaluation through the use of optical models. We would like to note that IRSE shares some features with other infrared spectral analysis methods, such as Reflection Absorption Infrared Spectroscopy (RAIRS), a brief discussion of this point can be found in Reference 7. For IR ellipsometric mapping studies with lateral resolutions below 1mm ² , more brilliant radiation than standard MIR sources, such as a globar, is needed to achieve monolayer sensitivity in the appropriate measurement times. One source that provides the entire infrared range is infrared synchrotron radiation. For the studies presented, we used the improved IR Mapping Microfocus Ellipsometer [8-9] on the IR beam tube of the synchrotron storage ring at BESSY. [10]

Infrarotellipsometrie construction

IR ellipsometry has recently been established as a method in thin-film analysis [7, 11]. Commercial devices with adequate evaluation software are offered by several manufacturers (eg SOPRA, JA Woollam Co., Inc., SENTECH Instrumente GmbH). The ellipsometer used in this work has been specially developed for the use of infrared radiation on a synchrotron storage ring and can thereby achieve a lateral resolution of less than 300 × 300 μm ² . For a general description of our method we refer to reference 7, and more detailed information about ellipsometry can be found elsewhere [11, 12]. A brief description of the ellipsometric principle is given below. As in 1 As shown, linearly polarized radiation of the incident beam is generally elliptically polarized by reflection at the sample surface. The polarization state can be described by two ellipsometric parameters: Δ (phase shift) and tanψ (amplitude ratio) of mutually perpendicular components of the reflected wave (r _p , r _s ).

1 : Ellipsometric principle (based on Ref. 7) and spot profile of the synchrotron light beam at an angle of incidence of 0 °.

The ellipsometric parameters are defined by the size ρ, which is given by the ratio of the complex reflection coefficients r _s and r _p :

For analysis and determining the optical constants of a thin organic film a substrate can use an optical simulation [7]. It can Structure, thickness, refractive index, absorption index and vibration parameters of organic film to be determined by best-fit simulations, in which the parameters of the simulations are changed as long as until the best match between calculated and measured spectra is achieved [7].

Infrarotellipsometrie results

2 shows ellipsometric spectra along a line for selected characteristic areas on the biochip. In a position on the (a) oxidized silicon substrate, (b) a point on the film of the uncovered linker, (c) a point where the linker is covered with the peptide (Cys-GCN4) (d) and a point with the Peptide (Cys-GCN4) located at the boundary of left-covered to oxidized silicon surface (e) and again a point on the oxidized silicon. Spectra of point A and E were used as reference for linker and peptide-free areas. By comparison to these spectra, specific bands of the linker can be identified at 1288 cm ^-1 and 1667 cm ^-1 (see point B). Specific bands of the "washed" peptide film at 1547 cm ^-1 and 1676 cm ^-1 are easily identified in the spectra of point D, where the amount of linker material is less than for point C. For the following discussion, the band at 1547 cm ^-1 (1288 cm ^-1 ) is used for the unambiguous identification of linker or peptide covered areas.

The band at 1288 cm ^-1 in the linker spectra is assigned to an oscillation of the head group of the linker. For the assignment of other bands, we follow the discussion in reference 21. Bands in the frequency range of 1600-1700 cm ^-1 are assigned to an amide I band, which arises mainly by C = O stretching vibrations and to a lesser extent by CN and CCN deformation vibrations. The band in the range of 1510-1580 cm ^-1 is assigned to the amide II band, which is a mixed mode dominated by the deformation vibration of NH and the CN stretching vibrations, but also consists of a smaller part of C = O and NC deformation vibrations [ 21]. In the range between 1200-1350 cm ^-1 , the amide III band observed for peptides is seen, and is a combination of "in-phase" NH deformation vibration and CC and CN stretching vibrations and a C = O deformation vibration Amide II bands can be masked by vibrations of the side chains of the amino acid, such as vibrations of CH, COOH, -NH2, and -NH3 + groups [13].

2 : A) Ellipsometric tanψ spectra for characteristic areas on the sample. B) Diagram showing the approximate position of the measured points in the different regions of the sample. Characteristic gangs are marked. A resolution of 4 cm ^-1 was used and a 65 ° angle of incidence was set. The slope of the baseline to the smaller wave numbers for points B and C in 2 is probably due to the absorption of free charge carriers [14] in the linker film.

3 Figure 6 shows plots of a 6 mm x 6 mm range of bandshifts of the bands at 1288 cm ^-1 and 1547 cm ^-1 . The amplitudes (tape strokes) have been determined as shown in the inset. Only ellipsometric tanψ spectra have been recorded to reduce the time to measure a single point below 3 minutes. In 3a the change of the band lift of a specific oscillation of the linker is shown while in 3b this is shown for a specific oscillation of the peptide. In both representations, peptide- and left-covered areas can be easily identified. As expected, the peptide is found only on sample areas that have also been spiked with peptide solution, while the non-incubated areas do not show the characteristic peptide vibration at 1547 cm ^-1 . The areas covered with the different materials become easily in the shape plots of 3 identified, with the tape strokes increase to the edge of the elliptical spot. Assuming that molecular orientations in the thin peptide film and in the linker film are isotropically distributed, and structural differences for different layer thicknesses can be neglected, the band-strokes are directly related to the layer thicknesses. The layer thickness which is not homogeneous for the linker region may then result from inhomogeneous illumination conditions and the geometry of the photochemical cell.

3 : 3d representation and contour plots for 6 × 6 mm ² areas of the band amplitude of a handlebar-specific band A) at 1288 cm ^-1 and a peptide band B) at 1547 cm ^-1 . The band sizes are for everyone Point has been determined as shown in the diagram on top. A resolution of 4 cm ^-1 was used and an angle of incidence of 65 ° was set.

The qualitative interpretation of the band forms [6] is consistent with an isotropic one Film structure, but a slight preference for out-of-plane transition dipole moments can not be excluded at this stage of the evaluation. In principle, you can the measured spectra are evaluated more comprehensively by optical simulation become. For such evaluations would the optical constants of each material needed to they as starting parameters for to use the simulation. The knowledge of the constants would be the Calculation of ellipsometric spectra for probable scenarios allow (see example reference 7). The work will be done continued at the moment. In summary, the results presented have shown that IR mapping ellipsometry for spatially resolved composition analysis of Biochips can be used.

Summary

It IR ellipsometry has been shown to be chemically sensitive non-destructive Method for the fab free characterization of a biochip and the connection a polypeptide can be used. Compared to further Common chemiluminescence imaging procedure may be from IR ellipsometric spectra label-free specific information to identify different Materials (linker, peptide) are obtained. Mappingellipsometrie allows the examination of larger sample areas with a local resolution smaller than 300 μm × 300 μm. For the examined Biochip was a heterogeneous layer of the linker and bound Peptide film inferred on the assumption that the film structure isotropic. Further evaluation in optical reference models is in progress. The intention of the future Work is the molecular structure of the organic monomolecular To enlighten films.

references

• [1] Arwin H. (2005) in Handbook of Ellipsometry, HG Tompkins, EA Irene (eds.), William Andrew publishing - Springer, pp. 799-847 ,
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• [7] Hinrichs K., Gensch M., Esser N. (2005) Appl. Spectroscopy 59: 272A-282A ,
• [8th] Gensch M., Korte EH, Esser N., Schade U., Hinrichs K. (2006) Infrared Phys. Technol. in print ,
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• [11] Röseler A. (2005) in Handbook of Ellipsometry, HG Tompkins, EA Irene (eds.), William Andrew publishing - Springer, pp. 763-798 , Tan☐ was determined from the square root of the ratio of p and s polarized reflectances, which were determined by measurements where analyzer and polarizer were set to 0 ° and 90 ° ° respectively.
• [12] Röseler A., Korte EH (2002) Infrared Spectroscopic Ellipsometry, in Handbook of Vibrational Spectroscopy, PR Griffiths, J. Chalmers (eds.), Wiley, UK, Vol. 2 ,
• [13] Handbook of Infrared Spectroscopy of Ultrathin Films (2003) Tolstoy VP, Chernyshova IV, Skryshevsky VA (eds), Wiley & Sons, Inc., pp.618-621 ,
• [14] Handbook of Infrared spectroscopy of Ultrathin films (2003) Tolstoy VP, Chernyshova IV, Skryshevsky VA (eds), Wiley & Sons, Inc., pp.206-209, p.580, p.590 ,

Claims (7)

High-resolution ( 1 ) imaging ( 2 ) Method for detecting a test substance ( 3 ) in a test medium ( 4 ) by the interaction of the test substance ( 3 ) with a solid-phase-bound test substance ( 5 ), in which a solid phase ( 6 ), on whose surface a film ( 7 ) with the test substance ( 5 ), with the test medium ( 4 ) and by means of an ellipsometer ( 8th ) optical changes ( 9 ) in the film ( 7 ) caused by the interaction of the test substance ( 5 ) with the test substance ( 3 ), with or without comparison with a standard sample ( 10 ), characterized in that the ellipsometer ( 8th ) with a brilliant infrared light source in the mid-infrared spectral range (400-4000 cm-1) ( 11 ) is fed. Method according to claim 1, characterized in that - high-resolution ( 1 ) means that the lateral resolution is better than 5 × 5 mm ² , and / or - imaging ( 2 ) is the computer-side representation of the measured data of the high-resolution ( 1 ) measured surfaces of a displaceable in x and y direction defined sample, and / or - as a test substance ( 3 ) an oligonucleotide, a protein, a peptide, a lipid, a carbohydrate, a drug, biotin, a cell, a cell compartment or a combination thereof is used, and / or - as a test medium ( 4 ) a liquid is used, and / or - as a test substance ( 5 ) an organic structure is used, and / or - as a solid phase ( 6 ) a material is used which has essentially no strong adsorption bands in the spectral range of 400-4000 cm ^-1 , and / or - a film ( 7 ) is used, which has a thickness of 1 nm-1000 nm, and / or - an ellipsometer ( 8th ), which is a radiation source ( 12 ), a polarizer ( 13 ), an analyzer ( 14 ), a detector ( 15 ) and an evaluation device ( 16 ), and / or - optical changes ( 9 ) are changes in the ellipsometric spectrum which occur due to changed conductivity, structure, bonds and composition, and / or - as a standard sample ( 10 ) a solid phase ( 6 ) is used, on whose surface a film ( 7 ) with the test substance ( 5 ), and / or - as a brilliant infrared light source ( 11 ) a synchrotron or a laser is used. Method according to one of the preceding claims, characterized in that - as test substance ( 3 ) DNA, RNA, cDNA, mRNA, cRNA, PNA, protein A, protein G, streptavidin, a receptor, a ligand, an enzyme, an enzyme substrate, an antibody, or an antigen or a substance comprising it or a structure derived therefrom becomes; and / or - as test substance ( 5 ) a linker molecule, carbohydrate, lipid, drug, peptide, polypeptide, oligonucleotide, protein, cell compartment, amino acid, cell, or biotin, or a substance comprising the same, or a structure derived therefrom; - as a solid phase ( 6 ) a silicon substrate is used; and / or - a movie ( 7 ), which consists essentially of the test substance ( 5 ) and / or covalently with the solid phase ( 6 ), and / or - a standard sample ( 10 ), which is essentially identical to the solid phase ( 6 ), on whose surface a film ( 7 ) with the test substance ( 5 ), and / or - as a brilliant infrared light source ( 11 ) a synchrotron radiation source or a molecular gas laser, semiconductor laser, quantum cascade laser or differential frequency generation laser is used, and / or - the polarizer ( 13 ) is stationary or rotatable, and / or - the analyzer ( 14 ) is stationary or rotatable, and / or, - the detector ( 15 ) is a common detector in the mid n infrared spectral range; and / or - the ellipsometer ( 8th ) is a zero or photometric ellipsometer which operates with fixed or rotatable polarizers or a phase modulator, and / or - the evaluation device ( 16 ) comprises a computer and software, and / or - the radiation source ( 12 ) an infrared light source in the mid-infrared spectral range (400-4000 cm ^-1 ) ( 11 ). Measuring arrangement for carrying out the method according to one of claims 1-3 with an ellipsometer ( 8th ), which is a radiation source ( 12 ), a polarizer ( 13 ), an analyzer ( 14 ), a detector ( 15 ) and an evaluation device ( 16 ), and a solid phase ( 6 ), on whose surface a film ( 7 ) with a test substance ( 5 ) and one to the test substance ( 5 ) attached test substance ( 3 ), characterized in that the radiation source ( 12 ) a brilliant infrared light source ( 11 ). Measuring arrangement according to claim 4, characterized in that - the test substance ( 3 ) is an oligonucleotide, a protein, a peptide, a lipid, a carbohydrate, a pharmacon, biotin, a cell, a cell compartment or a combination thereof, and / or - the test substance ( 5 ) is an organic structure, and / or - the solid phase ( 6 ) is formed from a material which has substantially no adsorption bands in the spectral range of 400-4000 cm ^-1 , and / or - the radiation source ( 12 ) a brilliant infrared light source ( 11 ) emitting light in the mid-infrared spectral range (400-4000 cm-1), and / or - the film ( 7 ) has a thickness of 1 nm-1000 nm, and / or - the brilliant infrared light source ( 11 ) is a synchrotron or a laser, and / or - the polarizer ( 13 ) is stationary or rotatable, and / or - the analyzer ( 14 ) is stationary or rotatable, and / or The detector ( 15 ) is a common detector for the medium infrared, and / or - the evaluation device ( 16 ) comprises a computer and software; having. Measuring arrangement according to one of claims 4-5, characterized in that - the test substance ( 3 ) DNA, RNA, cDNA, mRNA, cRNA, PNA, protein A, protein G, streptavidin, a receptor, a ligand, an antibody, or an antigen is or comprises or is a structure derived therefrom; and / or - the test substance ( 5 ) is or comprises a linker molecule, carbohydrate, lipid, drug, peptide, polypeptide, oligonucleotide, protein, cell compartment, amino acid, cell, or biotin, or a structure derived therefrom; - the solid phase ( 6 ) is a silicon substrate, and / or - the film ( 7 ) consists essentially of the test substance ( 5 ) and / or covalently with the solid phase ( 6 ), and / or - the ellipsometer ( 8th ) is a zero or photometric ellipsometer which operates with fixed or rotatable polarizers or a phase modulator, and / or - the brilliant infrared light source ( 11 ) a synchrotron radiation source or a molecular gas laser, semiconductor laser, quantum cascade laser or differential frequency generation laser. Method according to one of claims 1-3, characterized that a measuring arrangement according to a the claims 4-6 is used.