APPARATUS FOR THE SPECTROSCOPIC DETERMINATION OF THE BINDING KINETICS OF AN ANALYTE

Abstract

The invention relates to a device for the label-free quantitative spectroscopic determination of the binding kinetics of an analyte. Essential components of the device, namely a light source (2), optical elements (5; 6; 7; 8; 9; 13; 13′) for beam guidance and for optically influencing the light of the light source (2) and light modes emitted by a microsensor (functionalized spherical microparticle) retained in a microstructure (3) as a result of the exposure to the light of the light source (2), a spectrometer, which consists of an optical receiver (10) for the emitted light modes and an evaluation unit, actuators (14; 15) for positioning a carrier (4) with the microstructure (3) arranged thereon, and at least one control unit, are jointly arranged in an apparatus (1) having an apparatus housing (11). The light, namely the light of the light source (2) and the light modes emitted by a microparticle in question as a result of the exposure to said light, is guided in three different planes within the apparatus housing (11) by means of the optical elements (5; 6; 7; 8; 9; 13; 13), in particular by means of a first optical deflecting element (6) and by means of a second optical deflecting element (7).

Claims

1. An apparatus for the label-free quantitative spectroscopic determination of the binding kinetics of an analyte, comprising a light source for the emission of light used for the spectroscopic analysis, a fluidics module, composed of a movable carrier and of a microstructure which is arranged on this carrier and through which a fluid can flow, having at least one spherical microparticle that is held in this microstructure and functions as an optically active microsensor that is designed for the adsorption of an analyte that is carried in a fluid to the microstructure or for the release of an analyte that binds to its surface into a fluid carried to the microstructure, and for the emission of light modes as a result of exposure to the light of the light source, optical elements for beam guidance and for the optical influencing of the light that is emitted from the light source as well as light modes that are emitted by the microsensor held in the microstructure and impinge on an objective lens of the optical elements, an optical receiver for the reception of light modes that are emitted by a microsensor held in the microstructure and guided via the objective lens, actuators for the positioning of the carrier of the fluidics module, means for the movement and carrying of a fluid to the fluidics module, an analysis unit that, together with the optical receiver, forms a spectrometer for the determination of the binding kinetics of the particular analyte observed in this respect by analysis of the light modes received through the optical receiver, at least one control unit for control of the light source, for control of actuators for the positioning of the carrier with the microstructure, and for control of the means for the movement and carrying of fluid, wherein the analysis unit and the at least one control unit can constitute a common unit, is hereby characterized in that at least the light source, the optical elements, the optical receiver, and the actuators for the positioning of the carrier are arranged together in an instrument with an instrument housing, and in that the light is guided within the instrument housing by means of the optical elements in three different planes by deflecting the light that is emitted by the light source and initially guided in a first plane by means of a first optical deflection element to a second plane for the exposure of a microsensor held by the microstructure, and by deflecting the light modes, which are guided initially in the opposite direction likewise in this second plane and are emitted by the microsensor held in the microstructure as a result of light exposure and which are taken for the determination of the binding kinetics of the analyte, by means of a second optical deflection element to a third plane that is different from the first plane and the second plane and by guiding these light modes via further optical elements to the optical receiver.

2. The apparatus according to claim 1, further characterized in that the fluidics module is arranged outside of the instrument housing at a housing wall of the instrument, wherein, via a connecting means provided for this purpose by way of at least one cutout in the housing wall, the carrier is brought into an operative connection with complementary connecting means, which can be moved by means of the actuators serving for the positioning of the carrier with the microstructure arranged on it.

3. The apparatus according to claim 1, further characterized in that the light emitted by the light source is guided initially within the instrument housing along a first coordinate axis of the chamber and is then deflected by means of a first beam splitter forming the first optical deflection element in a wavelength-selective manner and is guided along a second coordinate axis, which is orthogonal to the first coordinate axis, within the space of the microstructure, and in that the light modes emitted by a microsensor held in the microstructure as a result of the light exposure are guided initially along the aforementioned second coordinate axis in the opposite direction to the light guided onto the microstructure for light exposure of the microsensor and then deflected by means of a second beam splitter that forms the second optical deflection element in a wavelength-selective manner, and guided along a third coordinate axis in the space, which is orthogonal to both to the first and second coordinate axis, via an optical slit aperture to an optical grating, and finally the light that is reflected by the grating and fanned out according to wavelength is guided to the optical receiver.

4. The apparatus according to claim 3, further characterized in that, arranged also in the common instrument, is a camera, which can be connected to an imaging system via signal connection terminals placed on the instrument housing, and light components passing the second beam splitter without deflection are guided to this camera.

5. The apparatus according to claim 4, further characterized in that a lighting means is arranged on the instrument housing, for an additional illumination of the fluidics module for the purpose of its graphic detection by means of the camera.

6. The apparatus according to claim 5, further characterized in that at least one diffuse light source is involved in the additional lighting means.

7. The apparatus according to claim 1, further characterized in that the objective lens that captures the light modes emitted by the microsensor held in the microstructure involves a long-distance dry objective lens.

8. The apparatus according to claim 7, further characterized in that the objective lens is an objective lens with a 10× to 40× magnification, preferably with a 20× magnification, and with a numerical aperture NA of between 0.6 and 1.2, preferably ≥0.75.

9. The apparatus according to claim 1, further characterized in that the light source for the emission of the light used for the spectroscopic analysis is a pulse-width-modulated laser with a power of between 0.1 mW and 10 mW, preferably of between 0.5 mW and 1.5 mW.

10. The apparatus according to claim 9, further characterized in that the laser that constitutes the light source emits light with a wavelength in the range of between 350 nm and 600 nm, preferably of between 400 nm and 500 nm.

11. The apparatus according to claim 9, further characterized in that the light exposure of a microsensor held by the microstructure takes place with the light of the light source only for the duration of a measurement operation relating to the microsensor in question.

12. The apparatus according to claim 1, further characterized in that the optical receiver for reception of the light modes taken for the determination of the binding kinetics of the analyte involves a CCD or CMOS line scan camera.

13. The apparatus according to claim 1, further characterized in that the optical receiver is surrounded by a double-wall housing, and in that it is accommodated separately within the instrument housing by a further housing.

14. The apparatus according to claim 1, further characterized in that, in the instrument housing, is also arranged the analysis unit for the determination of the binding kinetics of the analyte and/or the at least one control unit for control of the light source, for control of the actuators for the positioning of the carrier with the microstructure, and for control of the means for the movement and carrying of fluid.

Description

[0037] In regard to the apparatus according to the invention, or, to be more exact, in regard to the instrument accommodating the key components that essentially represent this apparatus, such as the optical elements, an exemplary embodiment will be presented and discussed below on the basis of drawings.

[0038] Shown in the appended drawings are:

[0039] FIG. 1: an isometric illustration of the instrument with a cutout in the instrument housing,

[0040] FIG. 2: the instrument with a fluidics module fixed in place thereon in plan view with a cutout on the top side of the housing,

[0041] FIG. 3: an example for the influencing of the spectrum detected by the optical receiver occurring during adsorption operation,

[0042] FIG. 4: an example for the time-dependent shift of the mode position during an adsorption operation.

[0043] FIG. 1 shows an isometric illustration of an exemplary embodiment for the key component part of the apparatus according to the invention, namely, for the instrument 1, which essentially constitutes and characterizes this apparatus and is designed in accordance with the invention, by way of which key components, in particular the optical elements 5, 6, 7, 8, 9, 13, 13′ of the claimed apparatus are accommodated in a common instrument housing 11. In the illustration, the instrument 1 is shown as viewed at an angle from obliquely in front with a cutout made in the instrument housing 11. On account of the cutout made in the illustration, the key components of the apparatus according to the invention that are combined in the common instrument housing 11 are readily seen in the illustration.

[0044] Key components of the apparatus that are accommodated by the instrument housing 11 are accordingly a light source 2 for the emission of the light serving for the spectroscopic analysis, or, to be more exact, for the excitation of the dye contained in the microparticles (also not shown in FIG. 1 because of their tiny size) or in the microsensors of the microstructure 3, respectively, and optical elements 5, 6, 7, 8, 9, 13, 13′ for beam guidance and for the optical influencing of the light emitted from this light source 2 as well as of the light modes emitted by the microsensor in the microstructure 3 irradiated currently by the light source 2. What is involved here are a first optical deflection element 6 (in the following, the first beam splitter 6) and a second optical deflection element 7 (in the following, the second beam splitter 7), an objective lens 5, an optical slit aperture 8, an optical grating 9, and two lenses 13, 13′, which serve for focusing the beam. The optical receiver 10, which is likewise a key optical element, that is, a key optical component, is not visible in this drawing, because it is concealed by an intervening wall. However, the optical receiver 10 can be readily seen in FIG. 2, which is yet to be explained below.

[0045] A further key component of the apparatus according to the invention, illustrated in FIG. 1, which, however, in the exemplary embodiment shown, is not accommodated by the instrument housing 11, is the fluidics module 3, 4. As can be seen from the figure, it is arranged outside of the instrument housing 11 on the top side of the instrument 1 in immediate proximity to the upper housing wall 12. The fluidics module 3, 4, which is comprised of a carrier 4 that can move in the three spatial dimensions x, y and z, and the microstructure 3 arranged on it, is joined to the instrument 1 by way of suitable means of connection, which are not illustrated in detail in the drawing and which have complementary means of connection, which are likewise not shown, via at least one opening (not shown) in the upper housing wall 12 and, via these complementary connecting elements, are brought into an operative connection with actuators 14, 15 serving for movement of the carrier 4.

[0046] The actuators 14, 15 are linear motors and mechanical elements transmitting their movement onto connecting elements coupled to the fluidics module 3, 4. By means of the actuators 14, 15 (xyz stage), the carrier 4 of the fluidics module 3, 4 and, with it, the microstructure 3 arranged on it can be positioned in a controlled manner by a control unit, which is not shown, with respect to the three spatial dimensions in a highly precise manner with an accuracy in the submicrometer range. For the carrying of fluid, by means of which functionalized microparticles suspended therein and/or the respective analyte to be investigated and to be bound to the microparticles introduced beforehand in the microchannels of the microstructure 3, corresponding ports, which are not shown here, are provided at the fluidics module 3, 4. The respective fluid is delivered by means of a pump to the fluidics module 3, 4 via ports formed on the fluidics module for this purpose and are not shown here and via connecting lines attached to these ports, Also not shown in the drawing are the aforementioned connecting lines, the sealing elements required for their connection to the ports of the fluidics module 3, 4, and the mentioned pump.

[0047] The emitting light source 2 used for the spectroscopic investigation is, in accordance with the example shown here, a pulse-width-modulated laser, which emits a laser beam in the violet region of the spectrum, namely, with a wavelength of 405 nm. This laser beam, which is guided initially along the coordinate axis x, is deflected by the first beam splitter 6, which is designed in a wavelength-selective manner, namely, is adjusted in this regard to a wavelength of 405 nm, to a second plane and is guided in this second plane along the coordinate axis y onto the microstructure 3 of the fluidics module 3, 4 or, to be more exact, onto a microparticle held and correspondingly positioned therein. Here, in a prototype of the instrument 1 realized in accordance with the exemplary embodiment shown, the light beam of the laser impinges with the formation of a light spot with a diameter of approximately 10 μm and with a power of approximately 1 mW. When the laser beam impinges, the dye contained in the microparticle in question is excited to glow. In the process, a plurality of resonant light modes of different wavelengths in the region of the fluorescence band of the dye used are formed, such as, for example, with wavelengths in the range between 470 and 520 nm, whereby, depending on the resonance wavelengths, two modes that belong to each other, namely, a TE mode and a TM mode, are formed, which, in regard to their electric field components and their magnetic field components, are polarized orthogonally to each other. Portions of this light finally exit the microparticle and, in this respect, are emitted from it.

[0048] A portion of the light having the light modes emitted by a microparticle currently exposed to the light of the light source 2 is captured by a recess in the housing wall 12 by the objective lens 5 and, via the latter, is guided, opposite to the direction of the light of the light source 2 that impinges on the microparticle, likewise along the coordinate axis y in the instrument 1. Here, these modes penetrate, that is, pass, initially the first beam splitter 6, which does not reflect the wavelengths of these modes, and finally impinge on the second beam splitter 7, which is designed to be selective in regard to these wavelengths. By way of the second beam splitter 7, light with wavelengths of less than 550 nm is deflected, in turn, to another plane and guided there along the coordinate axis z, initially via the optical slit aperture 8 serving for beam formation and then onto the optical grating 9. Light with wavelengths of greater than 550 nm, in contrast, also passes the second beam splitter 7 and is guided below the beam splitter 7 through a deflecting mirror 18 to a camera 16 utilized for imaging.

[0049] The arrows inscribed in the figure are intended to highlight the above-described light pathways, whereby a corresponding double arrow is intended to make visible the fact that the segment between the first beam splitter 6 and the microstructure 3 is passed by light in a changing direction, namely, on the one hand, by the light of the light source 2 that has been deflected by the first beam splitter 6 along the coordinate axis y in the direction of the microstructure and, on the other hand, by the light modes that are emitted by the respective microparticle of the microstructure 3 exposed to this light and, after entering the instrument housing 11, pass the first beam splitter 6 via the objective lens 5—symbolized by the arrow extension between the first beam splitter 6 and the second beam splitter 7.

[0050] The light modes that are guided onto the optical grating 9 and are utilized for the spectroscopic investigation are reflected by the grating and the reflected light is thereby fanned out in terms of its wavelength spectrum and finally guided to the optical receiver 10 (see FIG. 2), which cannot be seen here. A converging lens 13, 13′, which serves for focusing the beam, is arranged between the optical slit aperture 8 and the optical grating 9, on the one hand, as well as between the optical grating 9 and the optical receiver 10, on the other hand. In the case of the pair of converging lenses 13, 13′, two lenses are involved that are identical in regard to their optical properties. The light captured by the optical receiver 10 is analyzed, in turn, by means of a processing unit, which is not shown here. In the process, in regard to modes that belong to each other (TM modes and TE modes, which are polarized orthogonally to each other), at least the modes of maximum light intensity are taken for analysis in each instance.

[0051] In addition, in terms of the shift of the respective peak wavelength that occurs for these modes over time on account of the binding of an analyte to the microsensor that emits these modes (analysis of binding kinetics relative to an adsorption rate) or on account of the release of an analyte that is bound to the microsensor already at the start of the analysis operation (analysis of binding kinetics relative to a desorption rate), a plurality of modes of different resonance wavelengths are evaluated. This makes it possible to determine the exact particle size (the particle diameter) of the microsensor being observed and to take this into account in the determination of the adsorption rate or desorption rate, as a result of which, finally, a higher resolution of the measurement result is obtained.

[0052] Thus, for example, the adsorption of a polymer layer with a thickness of 2 nm to 3 nm on the sensor surface produces a shift in the mode wavelengths of approximately 200 pm to 300 pm depending on the refractive index of the polymer. In order to be able to detect the adsorption of a few molecules on the surface in an effective manner, therefore, it is necessary to detect shifts of at least 20 pm or better. This necessitates an extremely high-resolution spectroscopic arrangement, which is realized with the apparatus according to the invention. In tests, by use of an apparatus in accordance with the exemplary embodiment explained here, it was possible to achieve resolutions of less than 10 pm.

[0053] As already discussed in the general illustration of the invention, the last-mentioned analysis unit and the control unit or the control units for control of the light source 2, for control of the actuators 14, 15 for the positioning of the fluidics module 3, 4, and for control of the means for the movement and carrying of the fluid containing the microparticles and/or of the fluid containing the analyte can be realized jointly by a microcontroller system.

[0054] The operations of exposure of a microsensor (microparticle) held in the microstructure 3 to the light of the light source 2 and of guiding the light modes emitted as a result of this exposure of this microsensor onto the optical receiver 10, operations which have become manifest from the preceding discussions, are repeated several times during an analysis process. A corresponding microparticle that is functionalized for the analyte to be investigated is initially irradiated here, in the absence of the analyte, with the light of the pulse-width-modulated laser (light source 2), and an analysis of the light modes that are emitted from the microparticle as a result thereof is carried out. Afterwards, the fluidics module 3, 4, that is, the microstructure 3 thereof, is flushed by the fluid containing the analyte to be investigated. During this process, the measurement at a respectively observed microparticle of the microstructure 3 is repeated, namely, as desired, in accordance with the expected rate of the binding-kinetics processes that are to be detected, at a scanning rate of up to 25 Hz. This means that the microparticle in question is exposed repeatedly to the light of the laser and, in each instance, the light modes incident on the optical receiver 10 are analyzed. During this operation, on account of ongoing binding of the analyte to the microparticle, leading to saturation, the peak wavelength of the light modes emitted by the microparticle shifts. From this shift, as highlighted by way of example in the spectrum shown in FIG. 3, the time course of the binding of the analyte to the microparticle, that is, the adsorption rate of the analyte, is automatically calculated. An exemplary result of this calculation is highlighted by FIG. 4.

[0055] The light emitted in each case by a microparticle of the microstructure 3, as already discussed, is captured by means of the objective lens 5. In accordance with the exemplary embodiment, the objective lens 5 is a special objective lens with a 20× magnification and a numerical aperture of at least 0.75.

[0056] The special beam guidance of the light within the instrument housing 11 makes possible a very compact construction for the instrument 1, while ensuring that it is possible to determine adsorption rates very precisely by means of the apparatus according to the invention with the instrument 1 as its main component part. In the case of the already mentioned prototype of the instrument 1, given an edge length of the instrument housing 11 of approximately 223 mm in width, approximately 193 mm in height, and approximately 568 mm in depth (length), a beam path length of approximately 400 mm of the light modes guided to the optical receiver 10 is realized for the light emitted by the irradiated microparticle for the determination of the adsorption rate. The instrument or the apparatus, respectively, thereby makes it possible under the constraints already mentioned above (in particular: light source 2=pulse-modulated laser with a wavelength of 405 nm, exposure of a microparticle of the microstructure with a light power of 1 mW for a light spot that is approximately 10 μm in diameter, emission of light modes by the respectively irradiated microparticle held in the microstructure 3 in the range of 470 nm to 520 nm, and use of a 20× magnification objective lens with NA≥0.75) for the determination of the respective peak wavelengths of the light modes incident at the optical receiver, an optical resolution of <10 pm, which, by way of interpolation, namely, by way of a modeling of the curves to a Lorentz distribution, is increased to a resolution of approximately 5 pm.

[0057] The already mentioned camera 16, which, in the illustration, is arranged at the bottom right within the instrument housing 11, serves, in combination with a display (not shown here) connected to it, for the optical monitoring of the position of the fluidics module 3, 4 and for a service technician or an operator to monitor the course of the adsorption operation. For support of the imaging in the design in accordance with the exemplary embodiment shown, a diffuse light source (for example, an LED-based light source) is arranged above the fluidics module 3, 4, likewise outside of the instrument housing 11, as an additional lighting means 17.

[0058] Besides its compact construction, the instrument 1 is additionally characterized by a high robustness and a low service requirement. On account of the arrangement of the fluidics module 3, 4 outside of the instrument housing 11, its simple exchange is possible without intrusion in the instrument 1, which, at the same time, brings with it a high ease of use. For operation of the instrument 1 in carrying out an analysis operation for the determination of an adsorption rate, there are, in the instrument 1 shown in the example, on its top side, operating and connecting elements 19, namely, an on-off switch and a USB port for data exchange. In regard to the complexity and relatively high sensitivity of the optics arranged in the interior of the instrument 1, there is a further advantage in this context in that only very few adjustment possibilities for adjusting the components and elements of these optics are provided, which limit the calibration adjustments that are unavoidable in the individual case. Thus, for example, there exists in regard to the latter, preferably for a service technician, the possibility of calibrating the actuators 14, 15 for the positioning of the carrier 4 with the microstructure 3 of the fluidics module 3, 4 arranged on it. The service technician is thereby assisted by the graphic reproduction of partial regions of the microstructure by means of the camera 16.

[0059] FIG. 2 shows in plan view the instrument 1 illustrated in FIG. 1 and explained previously once again, whereby the housing wall 12 on the instrument top side has been partially cut out for the illustration. In this illustration, besides the optical slit aperture 8, the optical grating 9, and the two identical converging lenses 13, 13′, in particular the spectrometer with the optical receiver 10 can also be seen. In order to avoid residual light influence due to surrounding light, which might impair the accuracy of the measurement results, the spectrometer is accommodated within the instrument housing 11 by yet a further housing. The fluidics module 3, 4 can also likewise be readily seen once again in FIG. 2.

[0060] FIG. 3 shows, by way of example, the shift of the peak wavelengths, such as they can be observed for light modes emitted by a microparticle with a diameter of 7 pm during an adsorption operation. The solid line shows the spectrum of the light modes that are emitted by the microparticle prior to the start of adsorption for an exposure to the light of the light source 2 and are detected by the optical receiver 10. The dashed line shows the corresponding spectrum at the end of the adsorption of streptavidin in PBS buffer (PBS=phosphate buffered saline) to the biotinylated particle surface.

[0061] FIG. 4 illustrates, by way of example, the time-dependent shift of the mode position or of the peak wavelengths, respectively, of the modes that are emitted by a 10-μm microparticle during the adsorption of a solution of streptavidin in PBS buffer to the biotinylated particle surface and are detected by means of the optical receiver 10.

LIST OF REFERENCE SYMBOLS

[0062] 1 instrument

[0063] 2 light source

[0064] 3, 4 fluidics module comprising the microstructure 3 and the carrier 4

[0065] 5 objective lens

[0066] 6 first optical deflection element (first beam splitter)

[0067] 7 second optical deflection element (second beam splitter)

[0068] 8 optical slit aperture

[0069] 9 optical grating

[0070] 10 optical receiver

[0071] 11 instrument housing

[0072] 12 housing wall

[0073] 13, 13′ converging lens

[0074] 14, 15 actuators for movement in the x, y, z direction

[0075] 16 camera

[0076] 17 (additional) lighting means

[0077] 18 deflecting mirror

[0078] 19 operating and connecting elements