Method and apparatus for detecting the presence and/or concentration of biomolecules

Abstract: An apparatus for detecting the presence and/or concentration of biomolecules (41a-41c), in particular DNA, comprises a boundary surface (39) between an optically denser medium (here a glass prism 35) and an optically rarer medium (here a solvent 40). Biomolecules (41a-41c) adsorb to said boundary surface (39) and alter the characteristics of impinging light. The apparatus uses surface plasmon resonance (SPR), e.g. to excite the biomolecules (41a-41c). In order to operate the apparatus at ϑ = ϑ SPR (ϑ being the angle of incidence) and thus control the energy transferred to the biomolecules (41a-41c), the excitation light reflected at the boundary surface (39) is monitored (detector 46). The angle of incidence (ϑ) is controlled (loop 49) - e.g. by rotation of a desk - such that the intensity of the reflected light is always kept at a minimum, thus ensuring that ϑ = ϑ SPR . The disclosure also relates to a method of controlling the angle of incidence ϑ.

Description: The present invention relates to a method for detecting the presence and/or the concentration of biomolecules, in particular desoxyribonucleic acid molecules, wherein said biomolecules directly or indirectly adsorb to, deposit on, or are physically or chemically bound to a boundary surface between an optically denser and an optically rarer medium, said boundary surface including a thin conductive or semiconductive film, preferably a metal film, comprising the steps of irradiating p-polarized electromagnetic waves, preferably in the spectrum of visible light, through said optically denser medium onto said boundary surface, wherein the angle of incidence of the electromagnetic waves impinging onto said boundary surface is basically equal to the angle at which surface plasmon resonance occurs, the surface plasmon wave causing said biomolecules at said boundary surface to reflect or generate radiation in response to said surface plasmon wave, monitoring the radiation reflected or generated by said biomolecules at said boundary surface and determining their presence and/or their concentration by analysis of said reflected or generated radiation. The invention also relates to an apparatus to perform this method. Several methods for the detection of biomolecules, in particular of low concentration in a liquid, are known in the art. One method is based on so-called evanescent waves. The biomolecules to be detected are in a liquid solution and are brought into close contact with the surface of a medium, e.g. glass, which is optically denser than the liquid used. This may e.g. be performed by adsorption of the biomolecules to complementary biomolecules immobilized on the glass surface. The biomolecules to be detected are marked with a fluorescent compound. Such may either be performed directly - i.e. by means of chemical binding between the biomolecule and the fluorescent substance -, or in that biomolecules labelled with a fluorescent dye and immobilized or chemically bound to a part of the detection apparatus compete with the biomolecules to be detected; i.e., the unknown biomolecule releases a fluorescent biomolecule, which in turn adsorbs to the complementary biomolecule immobilized on the glass surface. The latter process is e.g. described in Badley, R.A., et al., Optical biosensors for immunoassays: The fluorescence capillary-fill device, Phil. Trans. R. Soc. Lond. B 316 (1987), p. 143-160. Monochromatic light, e.g. from a laser source or a filtered flash lamp, strikes the boundary surface between the optically denser medium (e.g. glass) and the optically rarer medium (e.g. an aqueous solution). The light beam is incident from the optically denser medium, and the angle of incidence is equal to or larger than the critical angle so that total internal reflection occurs. When such happens, an evanescent wave is created in the optically rarer medium (aqueous solution); this evanescent wave penetrates a fraction of a wavelength into the optically rarer medium. The electric field amplitude of the evanescent wave is largest at the boundary surface and decays exponentially with the distance from the interface. Due to the limited depth of penetration of the evanescent wave, such wave is suited to monitor the presence of biomolecules at the boundary surface. It causes the fluorescent appendix of the biomolecules to emit light of a wavelength longer than the incident wavelength (this is effectively how fluorescence is defined). The fluorescence signal can be measured directly by monitoring the scattered light, or by measuring the light coupled back into the optically denser medium. It is understood that the above technique is not limited to adsorption to a glass/aqueous solution interface. Instead, other materials may be used as well. It is further possible to use other effects than fluorescence (which shifts the wavelength of the incident light to larger wavelengths), e.g. phosphorescence or absorbance. In the latter case, even unlabelled biomolecules may be detected. The general method of detection is based on measuring a refractive index change caused by the presence of the biomolecules monitored by the refracted or reflected light, or, in other words, by the deviation of the angle of the reflected or refracted light. It is also known in the art to direct the incident light such that it is reflected multiple times in a waveguide, so that it strikes the boundary surface multiple times, see e.g. Sutherland, Ranald M. et al., Optical Detection of Antibody-Antigen Reactions at a Glass-Liquid Interface, Clin. Chem. 30/9 (1984), p. 1533-1538. Another technique for the detection of biomolecules is based on so-called surface plasmon resonance (SPR). This method requires a thin metal film, layer or coating (in more general terms, a conductive or semiconductive layer) between the glass and the liquid solution. Incident light, if impinging at a certain angle, causes surface modes (TE and/or TM modes) associated with collective electron oscillations to propagate along the interface between the metal film and the optically rarer medium (e.g. liquid solution). The incident light is usually coupled into the metal film by means of a prism or a grating. At a specific wavelength or angle, resonance occurs resulting in a sharp minimum or dip of reflectivity. The resonance condition is dependent upon the optical characteristics of the metal film, its thickness, the refractive indices of the dielectrics on either side of it (if any) and the angle of the incident light. The first two of these characteristics remain basically unchanged in a given apparatus for performing surface plasmon resonance. However, the refractive index of the optically rarer medium varies with the amount of biomolecules bound or adsorbed to its surface. This is the property to be monitored. In order to detect the presence and the amount of adsorbed biomolecules, either the variation in reflectivity at a given angle of incident light may be monitored, or the resonance shift (the reflectivity minimum is shifted to a different angle of incidence upon the presence of biomolecules) may be observed. Surface plasmon resonance may be caused either by a metal grating, or by an evanescent wave resulting from total internal reflection (see above). For further details of the surface plasmon resonance technique, reference is particularly made to Daniels, P.B. at al., Surface Plasmon Resonance applied to Immunosensors, Sensors and Actuators, 15 (1988), p. 11-18, and Kooyman, R.P.H. et al., Surface Plasmon Resonance Immunosensors: Sensitivity Considerations, Analytica Chimica Acta, 213 (1988), p. 35-45. Prior art surface plasmon techniques used the change of the refractive index caused by the biomolecules in order to detect their presence and/or their concentration. However, it is also known in the art to use surface plasmon resonance (specifically: The evanescent wave associated with surface plasmons) to excite fluorescence or phosphorescence in an immunoassay, as e.g. described in EP-A-353 937. A general problem in applying surface plasmon resonance is that the biomolecules may be "overexcited", i.e. too much energy is transmitted from the incident electromagnetic wave to the biomolecules. In such case, the biomolecules may bleach out, i.e. they alter their characteristics and their physical behaviour, such that they may be no longer detectable. It will be appreciated that this effect impairs the results of the measurement. The problem is that there is no control of the amount of energy "pumped" into the biomolecules. Of course, the energy emitted by the source of p-polarized electromagnetic waves is known and may be varied. However, the fraction of the total emitted energy pumped into the biomolecules is unknown - i.e., although the total emitted energy is known, the portion thereof passed on to the biomolecules is unknown. The reason is that, in prior art arrangements for the detection of biomolecules, the angle of incidence is not exactly the angle at which surface plasmon resonance occurs (this angle will be called ϑ SPR hereinafter) - either because this angle cannot exactly be met; or, even if it were possible to adjust the apparatus exactly to ϑ SPR , this angle would be subject to drift e.g. caused by temperature effects, binding of unspecific molecules (=molecules not subject to detection), changes at the metal/liquid interface, etc. Therefore, even the latter approach would lead to some deviation of the actual angle of incidence, with respect to the optimum angle ϑ SPR for surface plasmon resonance. The consequence of the actual angle of incidence not exactly corresponding to the optimum angle for surface plasmon resonance is that only a fraction of the total energy carried by the p-polarized electromagnetic waves is passed on to the biomolecules. On the other hand - and as outlined above -, this fraction is unknown. Even if it were possible to determine the deviation of the actual angle versus the ideal angle precisely, such would not solve the problem, as the fraction of energy coupled into the biomolecules is not a well-defined function of the deviation. Therefore, in prior art arrangements, effective control of energy coupled into the biomolecules has been impossible. It is, of course, possible to reduce the total amount of energy emitted by the radiation source such that even a 100% energy coupling would not damage the biomolecules. However, assume that a deviation between the actual angle of incidence and the ideal angle ϑ SPR for surface plasmon resonance causes a reduction of energy transmission of 75%: In such case, the remaining energy pumped into the biomolecules would be insufficient for reliable measurements. On the other hand, if the total energy output of the radiation source were substantially increased, operation could be performed reliably even upon a very little energy coupling ratio, but a 100% coupling would then damage the biomolecules. It is a major objective of the present invention to provide a method for detecting the presence and/or the concentration of biomolecules of the kind described above for the excitation of surface plasmon resonance waves, wherein said method allows accurate control of the energy coupled into the biomolecules. According to the invention, this object is solved by the following steps: monitoring the electromagnetic waves reflected at said boundary surface and detecting their intensity, controlling the angle of incidence of the impinging p-polarized electromagnetic waves in dependence of the intensity of the electromagnetic waves reflected at said boundary surface such that said intensity is substantially kept at a minimum corresponding to the occurence of surface plasmon resonance. The inventive method uses the fact that, if the actual angle of incidence corresponds to the ideal angle ϑ SPR for surface plasmon resonance, then approximately 100% of the incident energy is coupled into the biomolecules. That is, the invention ensures that these angles are always equal, in which case the total energy output of the radiation source is transmitted into the biomolecules; by control of the (known) energy of the radiation source, it is therefore possible to determine exactly the amount of energy received by the biomolecules. The energy output of the radiation source may thus be exactly adjusted to the biomolecules to be detected, in order to ensure optimum detection without any damage of the biomolecules. The ideal angle ϑ SPR is kept by monitoring the intensity of the electromagnetic waves reflected at the boundary surface and controlling the angle of incidence such that said intensity is basically kept at its minimum. It should be noted that electromagnetic waves reflected at the boundary surface have usually another angle than the electromagnetic waves generated by the biomolecules by fluorescence, phosphorescence and similar effects. This is because the biomolecules adsorbed to the boundary surface alter the refractive index. Further, the "induced" radiation generated by fluorescence, phosphorescence etc. is of generally longer wavelength, or reduced frequency, as compared to the original electromagnetic wave. Therefore, two beams have to be monitored: A first beam originating from the biomolecules, wherein the intensity of this beam indicates their presence and/or concentration, and a second beam reflected at the boundary surface (the reflection angle of this second beam being equal to the angle of incidence); the intensity of this second beam is used to keep the angle of incidence at ϑ SPR , i.e. to meet the optimum of surface plasmon wave excitation. The variation of the angle of incidence, in dependence of the intensity of the second beam and such that this intensity is kept at a minimum, may be effected in any convenient manner. In a preferred embodiment wherein the optically denser medium is a transparent prism (particularly a glass prism), such variation may be effected by rotation of a support carrying said transparent prism. However, there are further possibilities like moving the radiation source along a circular path around the center of the prism, etc. As outlined above, the general advantage of this method is that the energy of the radiation source may be accurately adapted to the biomolecules, in order to provide optimum measurements whilst avoiding damage of the biomolecules. However, there are further advantages of the inventive method. In particular, the apparatus is always kept at its optimum point of operation, thus saving energy. As approximately 100% of the energy is coupled into the biomolecules, no secondary effects caused by multiple reflected waves etc. occur. Further, effects like temperature drift, adsorption of unspecific molecules, changes of the boundary surface etc. are eliminated. It is important to note that these advantages may be obtained even if the energy output of the source of radiation is not variable. Even in order to obtain the first advantage - namely controlled energy transfer to the biomolecules -, a variable radiation source is not necessarily required (although such is provided in an advantageous embodiment of the invention). In an advantageous embodiment of the invention wherein a transparent prism is carried by a rotatable support, said support is rotated in a first direction and then in a second direction opposite to said first direction in case an increase or a substantial increase in said intensity is detected, or in case a predetermined time period has expired. An increase or substantial increase of the reflected light is an indication that the ideal angle ϑ SPR of surface plasmon resonance has moved to higher or lower values, so that the regions adjoining the previous angle should be searched for the new minimum. On the other hand, to ensure continuous proper operation, it is advantageous to scan the regions adjoining the present point of operation from time to time, even if no substantial increases have been noted. In an alternative embodiment, the electromagnetic waves reflected at the boundary surface are monitored by several monitoring elements, e.g. by means of an array sensor comprising elements sensitive to said electromagnetic waves such as photodiodes or phototransistors. A central sensing or monitoring element serves as reference; it is the goal of this embodiment to have the minimum intensity of the reflected electromagnetic waves focused on this central element. Even if the reflected beam is focused on the central element, the incident and the reflected waves do not merely oscillate strictly in a single plane, i.e. there are also lateral or stray waves. These are irradiated onto the boundary surface at an angle slightly different from the optimum angle ϑ SPR for surface plasmon resonance. Sensing or monitoring elements arranged laterally with respect to the central element are used to record such stray waves. If the detection apparatus operates at its optimum point, i.e. at ϑ = ϑ SPR , the intensity recorded by the lateral elements is therefore expected to be larger than the intensity of the central element. If, however, the intensity recorded by the lateral elements falls below the intensity recorded by the central element, this is an indication that the apparatus does no longer operate at ϑ = ϑ SPR , either caused by misadjustment or because the optimum wavelength for surface plasmon resonance has moved, e.g. caused by temperature effects or due to the adsorption of unspecific molecules. Therefore, if such happens, a control signal is generated in order to readjust the equipment. It is understood that the above arrangement of a central and two lateral recording elements is for the purpose of illustration; for more accurate control, more lateral elements may be used, e.g. an array of 11 photodiodes or the like. It is also possible to arrange the sensing elements in a 2-dimensional order, rather than in the 1-dimensional order described above. Advantageously, the p-polarized electromagnetic waves are passed through a thin dielectric film before and/or after passing through said thin conductive film. Such dielectric film may e.g. be arranged between a metal film to which the biomolecules adsorb, and a glass prism. Use of such dielectric layer sharpens the resonance peak and has other related advantages. Basically, dielectric layers of this kind have already been known in the art, see e.g. EP-A-353 937. In a further preferred embodiment, the optically rarer medium is a solvent in which said biomolecules are dissolved such that said biomolecules adsorb to, deposit on, or are physically or chemically bound to the optically rarer side of said boundary surface. That is, the biomolecules are present at the optically rarer side of the boundary surface. This is the usual (however not the only) way of detecting biomolecules. Several techniques may be used to establish contact between the biomolecules in solution and the boundary surface. These techniques, most of them well-known in the art, use various physical and/or chemical effects. The most common one is simple adsorption. However, more sophisticated ways like a competition process between the biomolecules to be detected and already present, marked biomolecules (see e.g. Badley, R.A. et al, Optical Biosensors for Immunoassays: The Fluorescence Capillary-fill Device, Phil. Trans. R. Soc. Lond. B316, 143-160 (1987)) have also been used. Advantageously, the inventive method uses biomolecules marked with a fluorescent, phosphorescent, chemiluminescent or electroluminescent substance. Such substances are excited by the surface plasmon wave and emit a radiation of reduced frequency, as compared to the frequency of the incident wave. The wave of reduced frequency is easier to detect than waves of the same frequency (the latter approach, namely to detect only the refractive index change caused by the biomolecules, i.e. to measure light of the same frequency as the frequency of the incident wave, has been used in most prior art publications). However, it has to be noted that the present invention is not restricted to the advantageous embodiment incorporating marked biomolecules. Instead, measurements based on absorption, Raman spectroscopy and nonlinear effects may be performed by the method and apparatus according to the present invention as well. If marked biomolecules are used, the biomolecules to be detected (unknown biomolecules) may be directly marked, e.g. by a chemical binding with a fluorescent substance. However, there are also more sophisticated ways where the chemical structure of the unknown biomolecules need not be altered. Usually, such techniques employ the use of further (marked) biomolecules. One such technique, namely a competition process between the unknown biomolecules and marked (or labeled) biomolecules, has already been mentioned above. Instead, is is also possible to use complementary labeled biomolecules. An advantageous embodiment based on such complementary labeled biomolecules will be described now. According to this embodiment, the optically rarer side of said boundary surface is coated with capture molecules complementary to the unknown biomolecules. The solvent contains the unknown biomolecules as well as labeled biomolecules complementary to the unknown biomolecules in solution. That is, two kinds of complementary biomolecules are used: The capture molecules (sometimes also referred to as "capture probe") immobilized on the boundary surface, and the molecules marked e.g. with a fluorescent substance and dissolved in the solution (sometimes also referred to as "label probe"). The unknown biomolecules are also called "target probe" hereinafter. During measurement, the target probe adsorbs to the capture probe. On the other hand, the label probe adsorbs to the target probe, such that its label is excited by the surface plasmon wave. The above process is particularly useful if desoxyribonucleic acid (DNA) sequences are to be detected. In this case, a preferred embodiment of the inventive method operates as follows: A surface plasmon wave is excited, which in turn excites fluorescent labels. These fluorescent labels can be attached to the biomolecules under investigation or the label probe and will lead to a very sensitive and specific detection system. To detect, for example, a specific DNA sequence ("target DNA"), it is proposed first to hybridize the single strand of this target DNA with its complementary synthetic strand in solution. The synthetic strand (label probe) has to be longer than 16 bases to guarantee its specificity and is prelabelled or prepared to be labelled afterwards. This target-label complex is then immobilized at the metal interface by hybridization to another complementary synthetic strand (capture probe), which is bound to the metal interface. By embedding the metal film layer between layers of different dielectrics one may optimize the strength of the evanescent wave as well as improve the binding of the capture probe. At a certain angle of incidence, the reflected beam will show a sharp dip, and this angle is quasi-identical with the angle of coupling maximum power into the sample volume with the labelled and immobilized target DNA. This sharp dip is used as a control value to define optimal conditions for maximum photon output of the fluorescent label; this, in turn, allows minimum excitation power, thereby reducing the possibility of photo-destruction. To further improve the signal to noise ratio, a fluorescence label with a long decay rate and a large Stokes shift can be applied to decrease the influence of the intrinsic fluorescence and of the excitation light upon the detected surface plasmon signal. It is possible to couple a great amount of the fluorescence light (which is emitted by the fluorescent molecule) back into the denser medium. The advantage of such device is a complete separation between sample volume and optic path. In addition, the different dielectric layers and the metal layer can be deposited onto a thin coverglass, which itself is matched to the denser medium. Such modified coverglass may be used as a cheap and disposable device, and the light source and the detector may be integrated in the denser medium. The denser medium may advantageously also have the shape of a semicylindrical prism into which a wedge-shaped light beam with defined minimum and maximum angle of incidence can be coupled. A suitable fluorescence label for the above process is e.g. a metal out of the rare earth elements. The long decay rate of these elements has the advantage that the emission is still detectable even after the decay of the intrinsic fluorescence, and the large Stokes shift will result in optical decoupling between excitation light, intrinsic fluorescence and emitted light. In order to apply such fluorescence labels, proteins like biotin, digoxigenine or other suitable substances are inserted into the DNA strand. Such proteins bind specifically to molecules like avidin or antibodies which comprise in turn fluorescent, phosporescent etc. molecules. A typical capture DNA, as well as a label DNA, is a synthetic oligonucleotide, complementary to the target DNA and comprising approximately 20 bases. The present invention also relates to an apparatus suited to perform the above method. According to one aspect of the invention, such apparatus for the detection of the presence and/or concentration of biomolecules comprises a source of electromagnetic waves, preferably in the spectrum of visible light, polarizing means for p-polarizing the electromagnetic waves emitted by said source of electromagnetic waves, means for directing said p-polarized electromagnetic waves through an optically denser medium onto the boundary surface between an said optically denser and an optically rarer medium, a thin conductive or semiconductive film, preferably a metal film, on said boundary surface, first monitoring means for monitoring radiation reflected or generated by said biomolecules at said boundary surface, and is characterized by second monitoring means for monitoring electromagnetic waves reflected at said boundary surface, intensity detection means for detecting the intensity of said electromagnetic waves reflected at said boundary surface, control means controlling the angle of incidence of the impinging p-polarized electromagnetic waves in dependence of the intensity of the electromagnetic waves reflected at said boundary surface such that said intensity is substantially kept at a minimum which corresponds to the occurence of surface plasmon resonance. According to the inventive apparatus, the second monitoring means record the electromagnetic waves reflected at the boundary surface. The intensity of the reflected electromagnetic waves is then used to control the angle of incidence. This is performed by the control or feedback means. It has to be noted that the second monitoring means and the intensity detection means mentioned above may be integrated in one component, or to have both tasks performed by a common component. Likewise, the first and second monitoring means may be a common component. Such is particularly the case if absorbance is used to detect biomolecules; the angles of the electromagnetic waves reflected by the boundary surface, and the angles of the electromagnetic waves reflected by the biomolecules, differ then only slightly. However, in case fluorescence or phosphorescence is used, where there is a notable difference between the above angles, different monitoring means may be used. In general, the first monitoring means, the second monitoring means and the intensity detection means may be combined in any suitable manner. For example, if the above angles differ only slightly, the same detector could be used to record the electromagnetic waves reflected from the boundary surface and the electromagnetic waves (e.g. light) emitted by the biomolecules. A diode array could be a suitable detector for such environment. Filters could be used as well. There are various alternatives for controlling (or correcting) the angle of incidence. In a preferred embodiment, wherein the optically denser medium is a transparent (e.g. glass) prism, such glass prism may be mounted on a rotatable support, wherein a motor (such as a stepper motor) rotates the support in dependence of the control signal generated by the control means. If the angle of incidence is rotated for Δϑ, then the second monitoring means is rotated for 2*Δϑ. Therefore, it is advantageous to have transmission means, e.g. a gear which rotates the second monitoring means for 2*Δϑ when the glass prism is rotated around Δϑ. Tables or desks performing this function are well-known in the art and commercially available. However, it should be noted that there are further possibilities of changing the angle of incidence in dependence of the control signal, e.g. by moving the radiation source along a circular path around the center of the prism or using an array of radiation sources, wherein only one of these sources is active (=emitting) at a certain point in time, in dependence of the control signal. There are also several advantageous ways of generating a control signal. One of these solutions (which may be realized in software) has already been described above, namely to search the regions adjoining the present point of operation for an intensity minimum. One way to perform such search will be discussed in the detailed description. According to a further suitable solution, the second monitoring means comprises an array of monitoring elements, preferably photodiodes or phototransistors, with at least a central monitoring element and at least a lateral element at either side of said central monitoring element, wherein control signal generating means are provided generating at least a control signal if the intensity recorded by one of said lateral elements is lower than the intensity recorded by said central element, said control signal being fed to said control means. As already outlined at the beginning, it is a major goal of the present invention to control exactly the amount of energy coupled into the biomolecules. As the invention ensures that the transmission rate is always approximately 100%, it is further advantageous to have an adjustable radiation source, in order to adapt its energy output to the biomolecules under investigation. However, this is not a mandatory feature - suppose that an apparatus is destined for examination of always the same biomolecules; in such case, the radiation source needs not be adjustable. Further features and advantages of the present invention will become apparent from the dependent claims and the detailed description. The above method and apparatus have proven especially useful and suited for the detection of desoxyribonucleic acid molecules. However, it will be appreciated that other kinds of biomolecules may be reliably detected by such method and apparatus as well. An embodiment of the invention will now be described, by way of a non-limiting example, with reference to the accompanying drawings, in which: Fig. 1 depicts a prior art arrangement for exciting evanescent waves and for measuring the concentration of biomolecules, Fig. 2 shows a prior art arrangement for exciting surface plasmon resonance (SPR) and for measuring the concentration of biomolecules, Fig. 3 is a diagram showing the reflectivity of p-polarized light, Fig. 4 depicts schematically the adsorbance mechanism of DNA strands, Fig. 5 is a first environment of an apparatus according to the present invention, Fig. 6 depicts a second environment, Fig. 7 is a flowchart for searching the intensity minimum of reflected light, and Fig. 8 depicts an alternative control mechanism based on a photodiode array. In the prior art arrangement for exciting evanescent waves according to Fig. 1, reference number 1 indicates a source of electromagnetic radiation, here of p-polarized light in the visible spectrum. The light is fed (light ray 2) through a lens 1a into a semicircular glass prism 3. A solvent 4 contains dissolved DNA molecules (three of which are labelled 5a, 5b and 5c in Fig. 1) to be detected. Glass prism 3 acts, with reference to solvent 4, as the optically denser medium; the solvent is the optically rarer medium. The angle of incidence ϑ is selected such that total internal reflection occurs, i.e. ϑ ≧ ϑ Cr (Critical angle) When total internal reflection occurs, an evanescent wave is created, which penetrates a fraction of a wavelength into the optically rarer medium (solvent 4). This evanescent wave is diagrammatically indicated by reference number 6. Although there is no net flow of energy into solvent 4, the electric field amplitude of the evanescent wave 6 is largest at the boundary surface 7 between glass prism 3 and solvent 4 and decays exponentially with the distance from the boundary surface. In the environment shown in Fig. 1, DNA molecules 5a to 5c are labelled with a fluorescent substance. Evanescent wave 6 excites the fluorescent substance, which in turn emits light of lower frequency (due to the losses), i.e. ν emitted ≦ ν impinging wherein ν emitted indicates the frequency of the light emitted by the fluorescent substance, and ν impinging indicates the frequency of the impinging light (reference number 2). One could also write ν emitted = ν impinging - Δν wherein Δν represents the losses in the fluorescent substance. The light emitted by the fluorescent substance is then coupled back into glass prism 3, as indicated by light ray 8. Due to the different frequency, the angle of the emitted light  is not exactly identical to the angle of incidence ϑ (it has to be noted that this is not "reflected" light in the common meaning of this term !). Light ray 8 then reaches an optical filter 9 which permits only light of frequency ν emitted to pass, i.e. light not originating from fluorescence is blocked. The filtered light is fed (light ray 10) to a detector 11. The intensity of the light impinging on detector 11 is now directly proportional to the amount of DNA molecules marked with a fluorescent substance and adsorbed to boundary surface 7. Fig. 2 depicts a prior art arrangement for the excitation of surface plasmon waves. P-polarized light generated by radiation source 12 is fed to filter 13 and then to glass prism 14 (light ray 15). A major difference between the environments of Figs. 1 and 2 is that a thin metal layer 16 is provided between glass prism 14 and solvent 17. Further, a dielectric layer 18 separates metal layer 16 from solvent 17 (although not shown in Fig. 2, a similar dielectric layer may be provided between metal layer 16 and glass prism 14). If the incident light impinges on boundary surface 19 at an angle ϑ ≈ ϑ SPR , surface plasmon resonance (SPR) occurs, i.e. surface modes associated with collective electron oscillations in the metal film are exited. The surface plasmon wave is a "bound" wave (i.e. no energy is radiated) propagating along boundary surface 20 (the boundary surface to solvent 17). The surface plasmon wave propagates in metal film 16 as well as along the surface of solvent 17. Dielectric film 18 reduces the losses due to propagation in metal film 16. In general and as will be shown below, ϑ SPR > ϑ Cr DNA molecules 21a to 21c adsorb to boundary surface 20, thereby altering the refractive index at the boundary surface. This change in the refractive index can be monitored by detector 22. The light ray to the detector is denoted as 23 in Fig. 2; this light ray passes a further filter 24. The angle  of the light fed to the detector is different from the angle of incidence ϑ, due to the refractive index change caused by DNA molecules 21a to 21c, i.e.  ≠ ϑ There have also been attempts to use labelled biomolecules (no DNA molecules) excited by surface plasmon resonance; this works equally well. Fig. 3 depicts the reflectivity R of p-polarized light in dependence of the angle of incidence, ϑ. At the critical angle ϑ Cr (critical angle which is greater than the Brewster angle ϑ Br ), total internal reflection occurs; at this angle, the reflectivity of p-polarized light is nearly one. A sharp dip in reflectivity is observed at ϑ = ϑ SPR . This is the angle of incidence where surface plasmon resonance occurs. It will be noted that the minimum at ϑ = ϑ SPR is quite small. That is, in case the angle of incidence is further increased, i.e. ϑ > ϑ SPR , no surface plasmon resonance will occur. The same is true for ϑ < ϑ SPR . A basic approach to have DNA molecules adsorb to the boundary surface in the present invention is shown in Fig. 4. The metal film is labelled as 25 in this figure; 26 is the dielectric layer. The "target DNA", i.e. the DNA to be detected, is shown as 27. Dielectric layer 26 carries a "coating" of capture DNA 28. Capture DNA 28 is immobilized on dielectric layer 26 and of complementary structure with respect to target DNA 27. During measurement, target DNA 27 as well as a further complementary DNA, namely label DNA 29, are dissolved in a solvent like water. Part of target DNA 27 hybridizes with label DNA 29 in solution; thereafter, another part of target DNA 27 hybridizes with capture DNA 28, such that the whole complex is immobilized on the boundary surface. It is understood that the first hybridization (target DNA with label DNA) runs faster, namely in three dimensions in the solvent; the 2-dimensional hybridization target DNA/capture DNA is slower. Label DNA 29 is chemically bound to a fluorescent substance 30, in the way described above. The amount of fluorescent substance is thus a measure of the amount of target DNA in the solution. In the embodiment of the invention shown in Fig. 5, the source of light 31 is adjustable, as indicated by control means 32. Control means 32 controls the energy output of light source 31, i.e. the intensity of the emitted beam. This beam is fed through a polarizer 33, in order to generate p-polarized light. Incident light beam 34 of p-polarized light then reaches glass prism 35. In the environment of Fig. 5, three layers adjoin glass prism 35, namely a first dielectric layer 36, a metal layer 37 and a second dielectric layer 38. It has to be noted that the thickness of these layers is - as well as in the preceding figures - overdrawn for graphical purposes. In a real environment, their thickness would only be several nanometers. Therefore, they form a boundary surface 39 to a solvent 40 containing target DNA and label DNA in solution, as described above. These DNA's adsorb to the boundary surface, also in the manner described above. Some adsorbed DNA molecules have been drawn for the purpose of illustration, see reference numbers 41a, 41b and 41c. Light beam 42 impinges on the boundary surface at an angle of incidence ϑ (this is the angle between the incident light and the normal on the boundary surface). ϑ is selected such that surface plasmon resonance occurs, i.e. ϑ = ϑ SPR However, it will be noted that there are two light rays 43,44 emerging from the point where the incident light beam hits the boundary surface. Light ray 43 corresponds to excitation light reflected at the boundary surface. I.e., angle δ under which light ray 43 is reflected equals the angle of incidence ϑ, δ = ϑ Assuming the label DNA is marked with a fluorescent label, then the surface plasmon wave excites the fluorescent labels and causes them to emit light of increased wavelength and decreased frequency, i.e. λ emitted > λ impinging and ν emitted < ν impinging (λ: Wavelength, ν: Frequency). This is due to energy losses in the fluorescent molecules. The light emitted by the fluorescent labels is coupled back into glass prism 35 by means of further surface plasmon wave (i.e. the emitted light generates a surface plasmon wave at the boundary surface, which in turn generates a light wave propagating through the glass prism). This is the light denoted as light beam 44 in Fig. 5. Due to the reduced frequency, its angle of reflecitivity  is different from the angle of incidence. Usually, the difference of angles δ and  is only about 1⁰ to 2⁰, i.e.  - δ ≈ 1⁰ to 2⁰ Light rays 43 and 44 are recorded by different detectors 45 and 46 (other embodiments will be discussed below). The intensity of the light ray received by detector 45 is used to determine the concentration of target DNA; this process will not be explained in detail here. The signal of detector 46 is fed to an intensity detector 47. Goal of the invention is to keep this intensity as low as possible, in order to operate the apparatus at optimum condition for surface plasmon resonance, i.e. ϑ = ϑ SPR This step is performed by a control unit 48, which in turn controls the angle of incidence ϑ (feedback 49). The variation and control of the angle of incidence may be achieved in different ways; details will be discussed below. Fig. 5 outlines the basic structure of an apparatus according to the present invention. A more detailed explanation will now be given with reference to Fig. 6. According to Fig. 6, light of a laser 50, e.g. a Helium-Neon laser, is fed to a polarizer 51 and further to an iris 52 (the iris is used to reduce stray light). A first mirror 53 and a second mirror 54 feed the impinging light further to a second iris 55 and to glass prism 56. Mirror 54 is a semipermeable mirror. Some of the photons impinging on this mirror will not be reflected in the direction to iris 55, but pass mirror 54 instead. These photons hit a detector 57, which in turn is connected with a computer 58. The signal recorded by detector 57 is used as a reference signal, e.g. in order to control the energy of laser 50 or to normalize the measured DNA concentration. The details of glass prism 56, and the way how target and label DNA adsorb to the boundary surface, are identical to the environment of Fig. 5 and will not be discussed here. The boundary surface with adsorbed DNA molecules is schematically indicated by reference number 59 in Fig. 6. Glass prism 56 is mounted on a rotatable desk or table 60. Purpose of the rotation is to keep the angle of incident light ϑ at the optimum angle for surface plasmon resonance, i.e. ϑ = ϑ SPR . The shaft of rotatable desk 60 is connected with suitable means for rotation, e.g. a stepper motor (not shown). Further, a transmission (e.g. with gear wheels) is provided which rotates a second detector 61 at twice the rate or angle at which rotatable desk 60 is rotated. I.e., if rotatable desk 60 is rotated for an increment Δϑ, then detector 61 will be rotated for an increment 2*Δϑ. Rotatable desks performing this function are known in the art; their details are therefore not explicitely shown here. Detector 61 contains means for recording the light reflected at the boundary surface, as well as the light emitted by the fluorescent labels. It will be noted that no separate detectors (like in the environment of Fig. 5) are provided. Due to the small angular difference between these light beams, a single detector may be used. In fact, light ray 62 represents both light beams (the light reflected at the boundary surface, as well as the light emitted by the fluorescent labels). The above mentioned light beams are, however, easy to distinguish. One possibility is, for example, to use two detector elements in detector 61 slightly offset in their angular position. Another possibility is to use appropriate filters (one filter permitting the excitation light to pass, and the other adapted to the fluorescent wavelength). Advantageously, the filters may be operated in a time-multiplexed mode, such that a single detector receives the excitation light at a certain point in time, and the fluorescent wavelength at another point in time. Other arrangements will be available to the skilled man. The signals recorded by detector 61 are fed to computer 58, which uses the intensity of the reflected excitation light to control the drive means (e.g. stepper motor) of rotatable desk 60 to keep the angle of incidence ϑ at its optimum position for surface plasmon resonance, as indicated by feedback loop 63. The intensity of the fluorescent light is, in turn, used to calculate the concentration of DNA molecules at the boundary surface of glass prism 56. Computer 58 is further connected with a plotter 64, in order to record the results on paper. The flowchart of Fig. 7 depicts how the computer may keep the angle of incidence at its optimum position for surface plasmon resonance. The routine shown in Fig. 7 is processed in discrete time intervals and e.g. initiated by interrupts. It is entered at label "START" (reference number 65). The routine first checks whether the intensity of the reflected excitation signal, I n , is greater than the intensity I n-1 measured the last time the flowchart was processed (reference number 66). If the answer is yes, this means that the apparatus is probably not operating at the optimum angle of incidence, and operation proceeds to box 67. It has to be noted that a possible increase of the intensity, i.e. I n > I n-1 , does not necessarily mean that the angle of incidence is not at its optimum - the increase of intensity may have been caused by temperature effects, binding of unspecific molecules to the boundary surface, or the like, without shifting the optimum angle of incidence. However, there is a considerable probability that one of the above effects has caused the intensity minimum to move to another angle; likewise, the apparatus may simply be misadjusted. If no increase of the intensity has been noted, it might still happen that the point of operation is not optimal. Assume, for example, that the level of minimum intensity has decreased, and that, at the same time, the minimum has been slightly shifted. In such case, the intensity at a certain point of operation may depict no increase or even a slight decrease, although this point of operation is no longer the point at which ϑ = ϑ SPR . The proposed routine therefore checks from time to time whether the present point of operation is still optimal, regardless whether there is any indication that the intensity has increased. This is shown by box 68. If the time since the last check, T, exceeds a preset value T max , the next check is required. T max may e.g. be selected as several minutes or even hours. If T max has not been exceeded, nor the intensity has increased, no readjustment is necessary. Operation proceeds to box 69 (increase of timer T) and then to the exit (line 70, "RETURN" label 71). Otherwise, timer T is set to zero (box 72). In case a readjustment may be necessary, the routine scans the regions adjoining the present point of operation for new minima. This part of the routine starting with box 67 where a temporary counter m is set up (for the purpose of this part of the routine only). In box 73, counter m is increased, and the angle of incidence ϑ is decreased by a preset value Δϑ (e.g. corresponding to a single step of a stepper motor). With reference to Fig. 6, this may be a rotation of rotation desk 60 in clockwise direction. The intensity at the new point of operation, I m , is then compared with its previous value (reference number 74). If there is a decrease (line 75), this means that the present angle of incidence is nearer to the absolute minimum than its previous value, and rotation continues in the same direction. If, however, the intensity at the new angle exceeds the intensity at the previous angle, the rotatable desk has been rotated too far; the last step is therefore corrected (box 76) by rotating the desk in the opposite direction. In similar manner, the angle of incidence is now increased (counterclockwise rotation of rotatable desk 60), step 77, in order to check whether there is any minimum in the other direction. Steps 78 and 79 correspond to steps 74 and 76. It should be noted that, at the end of the routine, the angle of incidence may well correspond to the previous angle. This is e.g. the case when the search was initiated by expiration of timer T, but without any movement of the intensity minimum. The flowchart in Fig. 7 depicts a quite simple routine to control the angle of incidence. More sophisticated programs may be developed by the skilled man, e.g. considering the derivative of the intensity, dI/dt, searching for a global minimum even in the presence of local minima, etc. Another possibility of controlling the angle of incidence is depicted in Fig. 8. A photodiode array 80 with a central photodiode 80a and two lateral photodiodes 80b and 80c receives the reflected excitation light. The photodiodes are slightly spaced with respect to each other. The stray light received by photodiodes 80b and 80c represents a slightly different angle of incidence. As long as the central photodiode 80a receives the lowest intensity of all photodiodes, the apparatus operates at ϑ = ϑ SPR , i.e. at its optimum condition. In such case, comparators 81 and 82 will generate a negative or zero signal, which will not cause the stepper motor to rotate desk 60; i.e., nothing happens. (Comparator 81 receives the intensity signal of photodiode 80c at its inverting input - line 83 - and the intensity signal of photodiode 80a at its non-inverting input - line 84; likewise, comparator 82 receives the intensity signal of photodiode 80b at its inverting input - line 85 - and the intensity signal of photodiode 80a at its non-inverting input - line 86). Assume now, for example, that the intensity at photodiode 80c is lower than the intensity indicated by central photodiode 80a. This means that the apparatus is misadjusted. Comparator 81 will now generate a positive control signal on line 87 in order to rotate the desk (if photodiode array 80 is part of detector 61 in Fig. 6, this means that the desk has to be rotated to the right or clockwise in order to decrease the angle of incidence; the intensity minimum will then be shifted to the center of photodiode array 80, i.e. in the direction of central photodiode 80a). Rotation continues until central photodiode 80a records the minimum of intensity. In similar manner, the desk is rotated counterclockwise (via control line 88) if photodiode 80b records an intensity minimum. The environment of Fig. 8 is again a simplified, exemplary embodiment. It is understood that e.g. the number of lateral photodiodes may be increased, that light-sensitive elements other than photodiodes may be used, etc.

Claims: Method for detecting the presence and/or the concentration of biomolecules (41a-41c), in particular desoxyribonucleic acid molecules, wherein said biomolecules directly or indirectly adsorb to, deposit on, or are physically or chemically bound to a boundary surface (39) between an optically denser and an optically rarer medium, said boundary surface (39) including a thin conductive or semiconductive film, preferably a metal film (37), comprising the steps of (1.1) irradiating p-polarized electromagnetic waves, preferably in the spectrum of visible light, through said optically denser medium onto said boundary surface, wherein (1.2) the angle of incidence of the electromagnetic waves impinging onto said boundary surface (39) is basically equal to the angle (ϑ SPR ) at which surface plasmon resonance occurs, the surface plasmon wave causing said biomolecules (41a-41c) at said boundary surface (39) to reflect or generate radiation in response to said surface plasmon wave, (1.3) monitoring the radiation reflected or generated by said biomolecules (41a-41c) at said boundary surface (39) and determining their presence and/or their concentration by analysis of said reflected or generated radiation, characterized by the steps of (1.4) monitoring the electromagnetic waves reflected at said boundary surface (39) and detecting their intensity, (1.4) controlling the angle of incidence (ϑ) of the impinging p-polarized electromagnetic waves in dependence of the intensity of the electromagnetic waves reflected at said boundary surface (39) such that said intensity is substantially kept at a minimum corresponding to the occurence of surface plasmon resonance. Method according to claim 1, characterized in that said optically denser medium is a transparent prism, preferably a glass prism (35,56). Method according to claim 1 and 2, characterized by acutating a rotatable support (60) carrying said transparent prism (56) in dependence of said intensity. Method according to claim 3, characterized in that said support (60) is rotated in a first direction and then in a second direction opposite to said first direction in case an increase or a substantial increase in said intensity is detected, or in case a predetermined time period (T max ) has expired. Method according to any of claims 1 to 3, characterized in that said electromagnetic waves reflected at said boundary surface (39) are fed to an array (80) of monitoring elements comprising at least a central monitoring element (80a) and at least a lateral element (80b,80c) at either side of said central monitoring element (80a), and that a control signal causing adjustment of said angle of incidence is generated if the intensity recorded by one of said lateral elements (80b,80c) is lower than the intensity recorded by said central element (80a). Method according to claim any of the preceding claims, characterized in that said p-polarized electromagnetic waves are passed through a thin dielectric film (36,38) before and/or after passing through said thin conductive film. Method according to any of the preceding claims, characterized in that said optically rarer medium is a solvent (40) in which said biomolecules (41a-41c) are dissolved such that said biomolecules (41a-41c) adsorb to, deposit on, or are physically or chemically bound to the optically rarer side of said boundary surface (39). Method according to any of the preceding claims, characterized in that said biomolecules (27) or labeled biomolecules complementary to said biomolecules (27) are marked with a fluorescent, phophorescent, chemiluminescent or electroluminescent substance (30). Method according to claims 7 and 8, characterized in that (9.1) the optically rarer side of said boundary surface (39) is coated with capture molecules (28) complementary to said biomolecules (27), (9.2) said solvent contains said biomolecules (27) and (9.3) labeled biomolecules (29) complemetary to said biomolecules (27). Apparatus for detecting the presence and/or concentration of biomolecules (41a-41c), in particular desoxyribonucleic acid molecules, comprising (10.1) a source (31,50) of electromagnetic waves, preferably in the spectrum of visible light, (10.2) polarizing means (33,51) for p-polarizing the electromagnetic waves emitted by said source (31,50) of electromagnetic waves, (10.3) means for directing said p-polarized electromagnetic waves through an optically denser medium onto the boundary surface (39) between an said optically denser and an optically rarer medium, (10.4) a thin conductive or semiconductive film, preferably a metal film (37), on said boundary surface (39), (10.5) first monitoring means (45;61) for monitoring radiation reflected or generated by said biomolecules (41a-41c) at said boundary surface (39), characterized by (10.6) second monitoring means (46;61) for monitoring electromagnetic waves reflected at said boundary surface (39), (10.7) intensity detection means (47) for detecting the intensity of said electromagnetic waves reflected at said boundary surface (39), (10.8) control means (48) controlling the angle of incidence of the impinging p-polarized electromagnetic waves in dependence of the intensity of the electromagnetic waves reflected at said boundary surface (39) such that said intensity is substantially kept at a minimum which corresponds to the occurence of surface plasmon resonance. Apparatus according to claim 10, characterized in that said second monitoring means (46;61) comprises a detector, preferably a detector responsive in the spectrum of said p-polarized electromagnetic waves only. Apparatus according to claim 10, characterized in that said first monitoring means (45;61) and said second monitoring means (46;61) are a detector selectively responsive to said p-polarized electromagnetic waves and/or said electromagnetic waves reflected at said boundary surface (39). Apparatus according to any of claims 10 to 12, characterized in that said second monitoring means comprises an array (80) of monitoring elements, preferably photodiodes or phototransistors, with at least a central monitoring element (80a) and at least a lateral element (80b,80c) at either side of said central monitoring element (80a), and that control signal generating means (81,82) are provided generating at least a control signal (87,88) if the intensity recorded by one of said lateral elements (80b,80c) is lower than the intensity recorded by said central monitoring element (80a), said control signal (87,88) being fed to said control means. Apparatus according to any of claims 10 to 13, characterized in that said means for directing said p-polarized waves onto the boundary surface (39) comprises a transparent prism, preferably a glass prism (35,56). Apparatus according to claim 14, characterized in that said transparent prism (56) is mounted on a support (60) rotatable by operating means, preferably a motor, which are controlled by said control means. Apparatus according to claim 15, characterized by transmission means which rotates said support (60) around a first angle (ϑ) and said first and/or said second monitoring means (61) around twice said first angle (2ϑ). Apparatus according to any of claims 10 to 16, characterized by a thin dielectric film (36,38) at the optically denser and/or the optically rarer side of said boundary surface (39). Apparatus according to any of claims 10 to 17, characterized in that said source (31) of electromagnetic waves is adjustable.

Inventors: SEHER JENS-PETER, SEHER, JENS-PETER
Applicants: HEWLETT PACKARD GMBH, HEWLETT-PACKARD GMBH
Application year: 1991
Main classification: G01N21
IPC codes: G01N21, G01N33