Optical biosensor system - Patent Review 5313264

We claim:

1. An optical biosensor system using an internal reflection versus angle of incidence determination for detecting a specific biomolecule, comprising:

a sensor unit including a transparent plate, at least a portion of one of the faces of said plate having a dielectric film adhered thereto, or being primarily coated with a metal film to which has been adhered a dielectric film, said dielectric film having an affinity for the specific biomolecule at the time the measurement is carried out;

a light source, including first lens means, for directing a beam of light at an interface of the transparent plate and said metal film or said dielectric film directly adhered thereto to produce an evanescent wave at a sensing surface of said dielectric film when a sample solution contacts said dielectric film on the transparent plate;

optical imaging instrumentation including second lens means by which one of light reflected from said sensing surface of the transparent plate, light originating from the sample through evanescent wave stimulated fluorescence or phosphorescence, and light originating from scattering at said sensing surface, is imaged on a photodetector device; and

an evaluation unit for determining at least one of an angle of minimum reflectance, a state of polarization, and a collected light intensity;

wherein at least one sensing surface is arranged on the sensor unit;

wherein said at least one sensing surface, in order to form an elongated flow cell, is pressed over an upwardly open portion of at least one channel for the sample solution which is to be analyzed and affects at least one of the angle of minimum reflectance, the polarization state for minimum reflectance, and the collected light intensity, at which the evanescent wave interacts with the sample;

wherein said first lens means forms a convergent beam of light focused in a wedge-shape fashion to form a streak of light extending transversely over all of said at least one sensing surface;

wherein the photodetector device, at least in the case of more than one sensing surface, is a two-dimensional matrix of individual photodetectors;

wherein said second lens means is a lens system for imaging rays of one of said reflected light from said at least one sensing surface, said light emitted from the sample, and said light originating from scattering at the sensing surface onto a corresponding column of photodetectors, so that said at least one sensing surface has at least one corresponding column of photodetectors;

wherein said evaluation unit determines at least one of the angle of minimum reflectance, the state of polarization, and the collected light intensity, for said at least one sensing surface.

2. The optical biosensor system of claim 1, further comprising:

a support in which the open portion of the at least one channel is arranged in a fixed position;

a housing in which said light source, said optical imaging instrumentation, the photodetector device, and a coupling prism are arranged in fixed positions;

an aperture provided in said housing below said coupling prism and covered by an opto-interface plate for coupling incident and reflected light and/or scattered or emitted light from the sample, respectively, to said sensor unit and said optical imaging instrumentation; and

holder means by which said sensor unit is detachably attached below said aperture,

said housing being placed on top of said sensor unit for pressing the opto-interface plate against an opposite face of said sensor unit.

3. The optical biosensor system of claim 2, wherein said housing is hinged to said support by means of a system of articulated arms extending from each lateral face of said housing and said support, respectively.

4. The optical biosensor system of claim 2, further comprising thermostat means provided in said housing and at the at least one channel, for thermostatically controlling said at least one sensing surface and the at least one channel, respectively.

5. The optical biosensor system of claim 1, wherein said at least one sensing surface includes a plurality of sensing surfaces, arranged in a side-by-side relationship on said sensor unit.

6. The optical biosensor system of claim 1, wherein the transparent plate of said sensor unit is of glass.

7. The optical biosensor system of claim 1, wherein the dielectric film comprises a dextran layer.

8. The optical biosensor system of claim 1 wherein the at least one channel includes a plurality of channels arranged in a parallel side-by-side relationship, wherein each of the plurality of channels has a corresponding upwardly open portion, wherein the corresponding upwardly open portions are all covered by said sensor unit when a measurement is performed, and wherein in said measuring stage, wherein each of said at least one sensing surfaces is situated above its corresponding upwardly open portion.

9. The optical biosensor system of claim 8, wherein each of the corresponding upwardly open portions are slits extending through a first layer of elastomer material or silicon.

10. The optical biosensor system of claim 9, wherein the first layer is made of a rubber material.

11. The optical biosensor system of claim 10, wherein said plurality of channels are formed in a liquid handling block unit of a solid material selected from the group consisting of plastics, metals and ceramics.

12. The optical biosensor system of claim 8 wherein means are provided which permit said plurality of channels to be coupled in series with each other.

13. The optical biosensor system of claim 9, wherein said plurality of channels are formed in a liquid handling block unit of a solid material selected from the group consisting of plastic, metal, and ceramics, and wherein said liquid handling block unit comprises:

a planar support plate having a second elastomer layer on its upper face;

a base plate positioned on top of the second elastomer layer including,

a third elastomer layer positioned on an underside of said base plate,

a first number of riser ducts which correspond to a number of the corresponding upwardly open channel portions, extend through said base plate, and connect with one end of each of the corresponding upwardly open channel portions, and

a second number of riser ducts which correspond to the number of corresponding upwardly open channel portions and connect with an opposite end of each of the corresponding upwardly open channel portions;

a pattern of channels formed in the third elastomer layer including,

at least one primary volume for a sample liquid, said primary volume having a first exactly determined volume,

at least one inlet channel for said sample liquid,

at least one inlet channel for eluent liquid, and

an outlet channel for said sample and eluent liquids; and

a plurality of individually controllable valve means in said planar support plate for directing the liquid flow in said plurality of channels.

14. The optical biosensor system of claim 13, further comprising a secondary volume for said sample liquid, said secondary volume having a second exactly determined volume which differs from said first exactly determined volume.

15. The optical biosensor system of claim 14, further comprising a second plurality of channels so that said second plurality of channels are filled while said sample liquid of said plurality of channels is analyzed.

16. The optical biosensor system of 13, wherein pneumatic valves are formed in through openings in said planar support plate, by the second elastomer layer above these through openings, and by valve seats in the form of projections from the third elastomer layer of said base plate.

17. The optical biosensor system of claim 13, wherein each of said plurality of individually controllable valve means comprise solenoids actuating respective wires, each wire being of superelastic material and extending through the through opening in said support plate to act upon the second elastomer layer.

18. The optical biosensor system of claim 8, wherein the corresponding upwardly open portions have a length of about 800 .mu.m, a width of about 300 .mu.m, and a height of about 30 .mu.m.

19. The optical biosensor system of claim 2, further comprising:

a carrier plate for said sensor unit, wherein an opening in said carrier plate accommodates said sensor unit and wherein said sensor unit rests on flange means at a lower end of said opening.

20. The optical biosensor system of claim 1, wherein said second lens means is an anamorphic second lens system for

imaging reflected rays of light from surface elements in a longitudinal direction of the streak of light, in the form of real image elements corresponding to rows and columns of photodetectors where each column corresponds to part of said at least one sensing surface in a direction of the streak of light, and

imaging reflected rays of light from surface elements transverse to the longitudinal direction of the streak of light, onto pixels situated along the columns of photodetectors, such that mutually parallel incident rays lying in a plane of incidence and aligned transversely to the longitudinal direction of the streak of light are focused toward a single photodetector in a column, so that each detector in a column corresponds to a one particular angle of incidence.

21. The optical biosensor system of claim 20, wherein said internal reflection versus angle of incidence determination is based upon surface plasmon resonance.

22. The optical biosensor system of claim 21, wherein a convergent beam of light strikes a reverse side of the metal film with angles of incidence of between 62 and 78 degrees.

23. The optical biosensor system of claim 1, wherein said second lens means is arranged for collecting one of rays of light emitted from a sample and rays of light scattered from surface elements in a longitudinal direction of the streak of light, onto columns of photodetectors where each column corresponds to part of said at least one sensing surface in the direction of the streak of light, and for

collecting rays of light from surface elements transverse to the longitudinal direction of the streak of light, onto pixels situated along the columns of photodetectors, such that mutually parallel incident rays lying in a plane of incidence and aligned transverse to the longitudinal direction of the streak of light are focused toward a single photodetector in a column, so that each photodetector in a column corresponds to one particular angle of incidence.

24. The optical biosensor system of claim 1, wherein said at least one sensing surface includes a plurality of surfaces, a width of the streak of light irradiating said plurality of sensing surface is substantially equal to an extent of said plurality of sensing surfaces transverse to the longitudinal direction of the streak of light to produce an integrated mean value of a mass of biomolecules binding to the dielectric film transverse to the longitudinal direction of the streak of light, thereby permitting a reproducible quantitative analysis or alternatively a reproducible analysis of the relative amount of the particular biomolecule.

25. The optical biosensor system of claim 24, wherein an extent of said plurality of sensing surfaces in the longitudinal direction of the streak of light are imaged in reduced size on a single photodetector, to produce an integrated mean value of the mass of biomolecules binding to the dielectric film in the longitudinal direction of the streak of light.

26. The optical biosensor system of claim 20, wherein said second lens means reduces an image element in S planes with a linear degree of magnification m=0.56 when the extent is of the order of magnitude of about 300 .mu.m and the photodetector has dimensions of about 90.times.90 .mu.m.

27. The optical biosensor system of claim 1, wherein said first lens means of said light source includes a lens member for varying the width of the streak of light irradiating each of said at least one sensing surfaces.

28. The optical biosensor system of claim 2, wherein said coupling prism is a truncated straight hemicylinder placed over said aperture in a bottom wall of the housing.

29. The optical biosensor system of claim 22, wherein said light source in combination with a filter emits substantially monochromatic and incoherent light.

30. The optical biosensor system of claim 22, wherein said light source emits substantially monochromatic and coherent light.

31. The optical biosensor system of claim 2, further comprising positioning means for determining relative positions of said housing, said sensor unit and said support.

32. The optical biosensor system of claim 2 wherein said opto-interface plate includes,

a second transparent plate of glass including longitudinally extending parallel ridges on one face aligned with longitudinally extending parallel ridges on an opposite face of said second transparent plate, said ridges being made of an elastic transparent material, spaced apart with distances between corresponding to distances between each of said at least one sensing surfaces with a length at least sufficient to couple an entire cross section of the convergent beam of light to said sensor unit and pressed against said transparent plate of said sensor unit directly opposite each of said at least one sensing surfaces.

33. The optical biosensor system of claim 32, wherein each of said longitudinally extending parallel ridges has a number of longitudinally extending stepped portions on each side to form a structure having, in cross section, a configuration of a flight of stairs, the uppermost steps thereof being pressed resiliently against said sensor unit and said coupling prism without formation of enclosed air pockets.

34. The optical biosensor system of claim 23, wherein said evaluation unit is arranged such that when the mass of bound specific biomolecules is calculated, the calculation includes light emitted from samples or scattered from said at least one sensing surface from only a partial band of the width of said at least one sensing surface and positioned centrally therein, this positioning being effectuated by evaluation of electric signals obtained from the photodetectors in a column which corresponds to a corresponding partial band on each of said at least one sensing surface.

35. The optical biosensor system of claim 25, wherein said evaluation unit is arranged such that when the mass of bound specific biomolecules is calculated, the calculation includes reflected light from only a narrow band.

36. The optical biosensor system of claim 35, wherein said evaluation unit is arranged for calculating parameters for a surface plasmon resonance curve for a determination including a reflectance minimum and a resonance angle and a displacement thereof for each flow cell, by curve fitting response values obtained from column detector members corresponding to the resonance angle.

37. The optical biosensor system of claim 2, wherein said coupling prism is a plane-sided prism placed over the aperture in a bottom wall of said housing, wherein refractive properties of said plane-sided prism determine a layout of said first lens means in order to form a convergent beam of light focused in a wedge-shaped fashion into the streak of light extending transversely over all sensitized areas.

38. The optical biosensor system of claim 37, wherein the upwardly open channel portion of at least one channel is a wall-jet cell, from which a jet flow is directed against a centre of a circular sensing surface.

39. A method of calibrating the optical biosensor system of claim 1, comprising the steps of:

directing a beam of light from said light source to form a streak of light extending transversely over all of said at least one sensing surface;

pumping a plurality of calibration solutions without affinity to said at least one sensing surface, each with a known refractive index one after the other, through a flow cell,

storing electric signals from the detectors of the photodetector rows corresponding to said at least one sensing surface in a memory, for each of the plurality of calibration solutions, so that a known relationship is obtained between a resonance angle and a refractive index of the dielectric film, and

calibrating the response from each of the photodetector rows.

40. The method of claim 39, further comprising the steps of:

passing the sample solution over a separate sensing surface that has no affinity for any biomolecule and is situated on said metal film;

storing the electric signals obtained from the photodector rows corresponding to the separate, non-affinity sensing surface in another location of the memory;

and correcting the stored signals obtained when the sample solution is passed along said at least one sensing surface, to determine a difference between the refractive indices due to the presence of the particular biomolecule for said at least one sensing surface.

41. A method of correcting for baseline drift in surface plasmon resonance (SPR) analysis procedures using the optical biosensor system of claim 1, comprising the steps of:

establishing a predetermined temperature set point for the sample solution;

creating an electric signal representing an actual temperature value of the sample solution, by utilizing electric signals which represent the resonance angle as measured in a flow cell, a sensing surface of which has no affinity for the specific molecule and through which a liquid is fed which is conveyed through a liquid flow handling unit;

correcting the signals from the sample solution with respect to a temperature disturbance caused by not positioning a thermostat in the flow cell; and

controlling said thermostat by measuring difference between the corrected signal and the actual value in order to adjust the temperature set point.

42. A method of measuring the actual feedback value of the temperature at the flow channels for regulation of a thermostat system by surface plasmon resonance (SPR) analysis procedures using the optical biosensor system of claim 1 comprising the steps of:

creating an electrical signal representing an actual temperature value of the sample solution, by utilizing electric signals which represent the resonance angle as measured at a flow cell, a sensing surface of which is in contact with at least one sealing area adjacent or between the flow channels, wherein said sealing area is coated with a dielectricum of suitable material and refractive index properties in order to obtain a surface plasmon resonance (SPR) response from said sealing area, said dielectricum having a known temperature dependence of its refractive index, light beams reflected at areas of the metal film opposite to areas of the film pressed in close optical contact with the liquid sealing areas of said dielectricum being imaged by the second lens means, being anamorphic onto corresponding columns of detector elements; and

controlling said thermostat system by measuring a difference between the corrected signal and the actual value in order to adjust the temperature toward the temperature set point.

43. The optical biosensor system of claim 1, wherein said optical imaging instrumentation further including optical filter means.

44. The optical biosensor system of claim 10, wherein the rubber material is silicon rubber.

45. The optical biosensor system of claim 29, wherein said light source includes a light emitting diode and a interference filter.

46. The optical biosensor system of claim 30, wherein said light source includes one of a semiconductor diode laser, a dye laser, and a gas laser.

47. The optical biosensor system of claim 36, wherein the response values are curve fitted using a reflectance minimum of the resonance angle.

48. The optical biosensor system of claim 1, wherein said at least one sensing surface includes a plurality of surfaces, a width of the streak of light irradiating said plurality of sensing surfaces is substantially equal to an extent of said plurality of sensing surfaces transverse to the longitudinal direction of the streak of light to produce an integrated mean value of a mass of biomolecules binding to the dielectric film transverse to the longitudinal direction of the streak of light, thereby permitting a reproducible quantitative analysis or alternatively a reproducible analysis of the relative amount of the particular biomolecule.

49. The optical biosensor system of claim 43, wherein an extent of each of said at least one sensing surfaces in the longitudinal direction of the streak of light are imaged in reduced size on a single photodetector, to produce an integrated mean value of the mass of biomolecules binding to the dielectric film in the longitudinal direction of the streak of light.

50. The optical biosensor system of claim 1, wherein said second lens means is an anamorphic lens system for imaging rays of reflected light from said at least one sensing surface, and said evanescent wave is produced by surface plasmon resonance.

51. The optical biosensor system of claim 1, wherein said second lens means is a lens system for imaging one of rays of light emitted from the sample and rays scattered at the sensing surface, said emission and said scattering of said light rays being generated by said evanescent wave.

52. The optical biosensor system of claim 35, wherein said narrow band is preferably about 50% of the width of each of said plurality of sensing surfaces, said narrow band extending substantially along an entire length of each of said plurality of sensing surfaces and positioned centrally therein, this positioning being effectuated by evaluation of electric signals obtained from the photodetectors in a column which corresponds to a corresponding narrow band on each of said plurality of sensing surfaces.

53. The method of claim 39, wherein said plurality of calibration solutions are salt solutions and/or suitable organic solutions.

Description Submit all comments and votes

The present invention relates to an optical multi-analyte biosensor system employing the principle of internal reflection of polarized light for use in biological, biochemical and chemical analysis and in particular for detecting a specific molecule, for example antigens. The detection method used in the biosensor system may be based on the evanescent wave phenomenon at total internal reflection, such as surface plasmon resonance (SPR), critical angle refractometry, total internal reflection fluorescence (TIRF), total internal reflection phosphorescence, total internal reflection light scattering, and evanescent wave ellipsometry. Furthermore, the detection method may be based on Brewster angle reflectometry.

The main advantage of the internal reflection based techniques is that the sensitivity region for the specific substance is restricted to the extension length of an evanescent wave, i.e. the depth of an electromagnetic wave penetrating into the liquid medium from the sensing surface side. Consequently, there will be a minimum of influence on the response connected to specifically bound analyte molecules from non-bound sample molecules. Moreover, the penetration depth of the evanescent wave for a totally reflected ray of light depends on the angle of incidence for the ray. For a comprehensive treatment of the concept of internal reflection one is referred to Mirabella and Harrick, Internal Reflection Spectroscopy, Harrick Scientific Corporation, N.Y. 1985.

The optical response induced by either the primary evanescent wave, or by a secondary evanescent wave excited in turn by said primary evanescent wave, may be measured as changes in the reflectance or in the state of polarization of the incident light wave upon reflection, or as the fluorescence or phosphorescence or light scatter of radiation, as a result of a specific substance interaction with a sensing layer at the sensing surface.

The optical response related to the specific substance may be measured as the reflected intensity as a function of angle of incidence of p-polarized light, being applicable for surface plasmon resonance, Brewster angle reflectometry, and critical-angle refractometry, in that the angle of minimum reflectance is determined and related to a refractive index and surface concentration of the bound substance at the sensing surface.

With regard a detection using surface plasmon resonance, this will be treated in more detail hereinafter.

Internal multiple-angle Brewster angle reflectometry, based on rotating optical means and a rotating prism for the variation of incident angle, has been shown to provide information to characterize the adsorption of proteins on a silica prism/solution interface; see Schaaf, P. et al. Langmuir, 3, 1131-1135 (1987).

Critical-angle refractometry has so far been used mainly to measure the concentration or density of solutions in process streams.

In a detection employing evanescent wave ellipsometry, the optical response related to the specific substance is measured as changes in the state of polarization of elliptically polarized light upon reflection, in that this state of polarization is related to a refractive index, thickness, and surface concentration of a bound sample at the sensing surface. Multiple-angle evanescent wave ellipsometry, in the form of using rotating optical means and a rotating prism for the variation of incident angle, and a phase-modulated ellipsometer, has been used for studying the polymer (polystyrene) concentration profile near a prism/liquid interface; see Kim, M. W., Macromolecules, 22, (1989) 2682-2685. Furthermore, total internal reflection ellipsometry in the form of stationary optical means at a single angle of incidence has been suggested for quantification of immunological reactions; see EP-A1-0 067 921 (1981), and EP-A1-0 278 577 (1988).

By a detection employing evanescent wave excitation fluorescence through an angle of incidence dependent TIRF, the intensity and wavelength of the radiation emitted from the either natively fluorescent or fluorescence labeled sample molecules within the sensing layer is measured. Total internal reflection fluorescence (TIRF) was suggested for a immunoassay in 1974, in that a separate antigen-coated quartz slide was brought into optical contact with a quartz prism via a drop of index-matching solution and by using a Teflon O-ring sealed plexiglas cell, Kronick, M. M. et al., J. Immunol. Meth., 8, (1975) 235-240. Now total internal reflection fluorescence is an established technique to examine interactions of macromolecules with solid surfaces, the surface being generally one side of the coupling prism; see Lok, B. K. et al., J. Colloid Interf. Sci., 91, (1983) 87-103. Furthermore, variable-angle total internal reflection fluorescence has been used to study adsorbed protein concentration profiles at a prism surface; see Reichert, W. M. et al., Applied Spectroscopy, 3, (1987) 503-508.

In a detection employing evanescent wave excitation scattered light, through an angle of incidence dependent evanescent wave penetration depth, the intensity of the radiation scattered within the sensing layer due to its interaction with the specific substance is measured. Scattered total internal reflectance (STIR) has been suggested for utilization in immunoassays, and in a preferred embodiment, colloidal gold is used as a label for the solution phase immunologically active component; see EP-A2-0 254 430 (1987).

With regard to the use of surface plasmon resonance (SPR), in somewhat simplified terms SPR may be said to be a technique according to which changes in the refractive index of a layer close to a thin free-electron metal film are detected by way of consequential changes in the intensity of a p-polarized light beam reflected from the metal film (see for example Raether H, Physics of Thin Films, Academic Press, N.Y., 9 (1977) 145.

In the first publication indicating the possibilities of SPR technology in biochemical analysis, Liedberg B et al., Sensors and Actuators, 4 (1983) 299 have at first adsorbed a monolayer of IgG to a silver surface and then adsorbed to said monolayer a layer of anti-IgG, in order to then study the effect of the resultant change in the resonance angle. EP 202 021 describes a biosensor employing movable optical instrumentation for determining the angle--henceforth called the resonance angle--at which surface plasmon resonance occurs. Such movable optical units are not suitable for commercial-type instruments because (i) when readings of the resonance angle are to be taken this will require manual operations, and (ii) technical manufacturing tolerances in the suspension mechanism of the movable optical system contribute to errors occurring in the measurements of the resonance angle. EP 257 955 describes another optical system which is scanned mechanically for determining the resonance angle. GB 2 197 068 describes an optical sensor employing a divergent beam of rays for irradiating the sensitized surface, this latter being a metal film with receptors or ligands which interact selectively with one or more biomolecules. The optical system is stationary, so the above-mentioned drawbacks of movable optical systems are avoided.

A source of light is employed for irradiating a sensitized surface which is subject to the action of a sample solution while another source of light is employed for irradiating another sensitized surface which is subject to the action of a reference solution. The light sources and sensitized surfaces are arranged in such a way that the reflected divergent beams will strike a photodetector matrix. By means of alternate activations of each one of the two light sources, the resonance angle obtained from each of the two sensitized surfaces can be measured with precision, and the difference between the two resonance angles at each of the two sensitized surfaces will be a measure of the amount of the specific biomolecule bound on the sensitive layer. The disadvantage of this apparatus resides in the use of two individual sources of light--one for the reference solution, one for the sample solution--as this will tend to make the measuring result uncertain in view of the fact that the resonance angle is highly dependent on the spectral character of the light source. Another drawback of this known optical sensor resides in the positioning thereof directly on the prism of the optical system, and in having light directed to the sensitized surface via an immersion oil that has a suitable refractive index. Such a use of light-coupling oils will involve much practical inconvenience when the sensor unit, comprising a sensitized metal layer coated on a transparent plate, is to be replaced by a new sensor unit with a sensing surface having an affinity for a different specific biomolecule. The replacement operations will inevitably give rise to oil smears, and the prism has to be cleaned before a new sensing surface can be analyzed. Manipulation of the instrument will thus be a somewhat messy business. As to the actual structure of the analytical instrument, this is not disclosed in the aforesaid GB patent specification.

EP 226 470 describes an apparatus for microchemical analyses comprising two glass plates with a gel placed between them. The apparatus is of the disposable type, to be used only once. One of the two glass plates serves as a platform on which the sample liquid is applied. Capillary force will then draw the sample liquid into the capillary cell that has been formed between the plates. A device of this type, the dimensions of which are about 3.times.1.5 cm, requires the use of tweezers or the like for handling. It is difficult to determine the volume of the liquid sample, and this device is therefore unsuitable for quantitative analyses.

EP-A1-0 305 109 (published after the priority date claimed in the present application) describes a SPR sensor system employing a focused (fan-shaped) light beam to illuminate the sensitive surface through a curved transparent block and via an index matching fluid. The beam enters the transparent block in a direction orthogonal to the tangent of the surface of the transparent block.

As was published by Kretschmann, E., Optics Communications, 26, (1978) 41-44, the problem of slow speed of operation relative to changes in reflectance and the insufficient precision in the resonance angle determination related with SPR procedures based on moveable mechanics, is solved by the use of a fan-shaped beam (equivalent to several beams incident upon the sensor surface over a range of angles) and of collection of the reflected beams (over a range of angles) by an array of angularly spaced detectors.

Furthermore, the transparent block described in EP-A1-0 305 109 may take the form of a hemicylinder creating a wedge-shaped beam, giving a line of a small illuminated area on the sensing surface. The hemicylindrical lens has the advantage that it can be used to perform several tests simultaneously on a single sample. To this end, the sensing surface takes the form of a series of sensitive areas, each comprising a different antibody, with each separate area being monitored by its own detector in a detector array. The cylindrical focusing principle used to produce an identical angular range of light beams along a focused line for SPR of separate surface areas has been published by Benner, R. E. et al. Optics Communications 30 (1979) 145-149, and Swalen, J D et al. Am J. Phys. 48 (1980) 669-672.

Further, a focusing lens in EP-A1-0 305 109 creates a substantially parallel-sided beam incident upon the detector, or a beam of at least of reduced divergence compared to the fan-shaped spread of light reflected from the sensing surface with the object to reduce stray light reflections in the detector array. The disadvantages of this apparatus, however, are as follows. The approach, to use a small illuminated area in relation to the sensitive layer for sensing, in order to reduce effects due to inevitable variations in a commercially produced metal film and coating of antibody. In fact, the surface concentration of bound sample molecules will in general also be non-uniform across the sensitive layer and strongly dependent on mass transport conditions. Thus, the small sensing area will be very sensitive to local variations of the sensing surface and its sample surface concentration resulting in low accuracy of the SPR response. Due to stray light arising from the coupling optics and a reflection in the sensor surface, it is possible to use the described optical system for monitoring beams from separate sensitive areas simultaneously by its own detector in an array only under the condition that the array can be conveniently placed close to the exit surface of the hemicylinder or attached to or deposited on that surface. This leads to limitations in the resolution of individual sensitive areas on the detector array, expensive optoelectronic constructions, complicated production process (detector alignment, optimization of collected angle span etc.). Optical oil or grease may be used to ensure good optical coupling between the hemisphere and the sensor substrate (glass support plate or slide).

The an approach of a disposable hemicylinder as optional technical solution for the change of sensor surface is unpractical due to the high costs of an adequate optical quality of this component and the critical optical alignment relative to the light source.

With regard to the use of internal reflection procedures, most of the present publications laboratory equipment for a singular determination of a specific substance at a time. Such equipments, however, are not suitable as practical commercial instruments; they are too complex and cumbersome in their construction for sample and liquid handling, detection, and evaluation, and the analysis procedures take quite a long time and moreover require highly skilled operators in order to obtain accurate results.

By the present invention a new analytical system is provided for at least one specifically sensing surface comprising the simultaneous detection of a plurality of specific interactions, in that it is adaptable for detection techniques based on cylindrically focused total internal reflection and internal Brewster angle reflectometry.

Thus, in one embodiment of the present invention, means for employing internal multiple-angle Brewster angle reflectometry on an exchangeable sensing surface for a new analytical system by use of a stationary optical system is provided.

By another embodiment of the present invention, means for employing multiple-angle evanescent ellipsometry on an exchangeable sensing surface is provided for a new analytical system by use of a stationary optical system.

In another embodiment of the present invention, a new analytical system is provided enabling variable-angle total internal reflection fluorescence on an exchangeable sensing surface by use of a stationary optical system.

In a still further embodiment of the present invention a new analytical system is provided enabling variable-angle scattered total internal reflectance (STIR) on an exchangeable sensing surface by use of a stationary optical system.

According to a preferred embodiment the present biosensor system uses a new mean-value procedure, utilizing an anamorphic lens system, for the detected reflectrometric, emitted or scattered light due to a specific interaction at a sensing surface that is optimized relative to the flow cell geometry, permitting inherently accurate and highly sensitive results to be obtained.

Furthermore, in a preferred embodiment a the present invention provides a biosensor analytical system that eliminates the drawbacks related with more or less stationary sensing surfaces, generally being coated on expensive prisms or wave guides, by introducing a separate sensor unit that is semi-automatically exchangeable in the instrument. Such a sensor unit having at least two sensing surfaces, as well as a method for its functionalization, is the object of our copending PCT-application entitled "Sensor unit and its use in biosensor systems", now PCT Publication No. WO 90/05305 (based upon Swedish patent application No. 8804074-6), the disclosure of which is incorporated by reference herein.

Moreover, the present biosensor system provides a in a preferred embodiment thereof, an automated integrated liquid handling block, micro-processor controlled valves therein, preferably membrane valves, enabling complex liquid handling tasks comprising specific sequences of addition of various ligands, macromolecules, and reactants for interaction at the sensing unit.

The biosensor system of the present invention enables new applications related to the field of characterizing biomolecules to be performed, e.g. as disclosed in our copending PCT-application entitled "A method of characterizing macromolecules" now PCT Publication No. WO 90/05306 (based upon Swedish patent application No. 8902043-2), the disclosure of which is incorporated by reference herein; as well as in our aforesaid copending PCT-application entitled "Sensor unit and its use in biosensor systems". Such new applications made possible by the present invention comprise:

qualitative as well as quantitative specific determination of at least one biomolecule in a sample either simultaneously or sequentially,

qualitative as well as quantitative structural information of macromolecules through the detection of interactions between surface-exposed structural elements on the macromolecule and various ligands,

functional/structural information of macromolecules,

kinetic information about molecular interactions,

specific functionalization of at least one sensing surface on the sensor unit,

regeneration or changing of the specific functionalization of at least one sensing surface on the sensor unit.

In accordance with the above, the optical biosensor system of this invention comprises at least one, but preferably a number of sensing surfaces or areas arranged in side-by-side relationship for being exposed to sample liquid passing over them. These sensing surfaces are analyzed by the above-mentioned optical techniques based on a single source of light so that due to the use of one identical set of wavelengths for all the sensing surfaces it is now possible to obtain a calibration, reference and evaluation process of such high analytical performance that this biosensor system becomes commercially useful. The system is also such that the sample liquid can be made to sweep over the sensing surfaces all at the same time or in a sequence. Measurements are made under controlled temperature conditions. At the time when the measurement is being performed the temperature at all the sensing surfaces is to be the same and is to be kept constant during the measuring operation.

The sensing surface or surfaces of the sensor unit will lend themselves readily and simply to functionalization individually, for selective interaction with the desired biomolecules; that is, it will be easy to bestow different affinity properties on these sensing surfaces.

The provision, in accordance with the above mentioned preferred embodiment, of a block unit for liquid handling using automated microprocessor-controlled sample insertion and sample guiding valves will permit precise and reproducible dispersion of the sample zone and accurately determined amounts and flow rates of sample solutions, reagent solutions or reference solutions.

The advantages of an integrated conduit liquid handling system situated in a permanent rigid and planar structure has been described by Ruzicka (Ruzicka, J., Analytical Chemistry, 55, 1041A (1983)). Hence, the rigidity of such a structure and the possibility to integrate and control sample injection ensures repeatability of dispersion of the sample zone. Further, the small dimensions of the microconduits reduces sample reagent consumption to the microliter level. Furthermore, the versatile combination of engraved grooves as conduits within laminated parallel layers, interconnected by perpendicular channels, provides the means for integrating a number of highly controlled solution handling tasks. The technical limit on possible miniaturisation of such flow injection analysis systems has, however, been the availability of detectors suitable for sub-microliter detector volumes. The present invention comprises in one particular embodiment thereof a multi-analytical system enabling a detector volume of and below 60 nanoliter.

Thus the liquid handling block unit of the present biosensor system contains conduits or channels having one or more portions which, when the measuring operation is to be performed, are pressed against the sensor unit to thus form one or more flow cells, the arrangement being such that these flow cells can either be coupled in series or be made to each receive its own sample solution separately. A flow cell may contain a single sensing surface or alternatively a plurality of sensing surfaces; in this latter case, the sensing surfaces lie in a row in the longitudinal flow direction of the flow cell. The block unit for liquid handling is to be employed both when the sensing surfaces on the sensor unit are being functionalized and when analysis is carried out.

Furthermore, the optical biosensor system according to this invention provides for a stationary optical instrumentation, without any movable parts. Thereby the optical system may already in the course of its manufacture in the factory be given a fixed, "once for all" standard setting so that this setting does not need to be altered during the subsequent use of the system. In view of this fixed setting it is possible to have the optical system mounted inside a dust- and light-tight housing. Moreover no smeary oils need to be employed for coupling the light to the replaceable sensor unit. Instead a replaceable opto-interface may be used for coupling light from the light source to the sensor unit. Such an opto-interface is disclosed in our copending PCT-application entitled "Optical interface means", the disclosure of which is incorporated by reference herein.

As mentioned above the measurement accuracy may be still further increased by establishing an optical and, respectively, el