||||||IIIHHHHIII USOO5313264A

United States Patent 19 (11) Patent Number: 5,313,264

Ivarsson et al. 45) Date of Patent: May 17, 1994
(54) OPTICAL BIOSENSORSYSTEM Measurement of Water in Process' K. Matsubara, et all
(75) Inventors; Bengt Ivarsson, Balinge; Jönsson Ulf, vol. 2, No. 8, 1988. . ... ". .
Upsala; Stefan Sjölander, Upsala; "Optical Chemical Sensor Based on Surface Plasmon
Ralph Ståhlberg, Upsala; Hakan Measurement' K. Matsubara, et al. vol. 27, No. 6, Mar.
Sjödin, Uppsala, all of Sweden 15, 1988.
(73) Assignee: Pharmacia Biosensor AB, Upsala, "Internal Reflection Spectroscopy: Review and Supple
Sweden ment' F. Mirabella, Jr., et al. 1985.
"Reflectometry as a Technique To Study the Adsorp
21 Appl. No.: 681,533 tion of Human Fibrinogen at the Silica/Solution Inter
(22) PCT Filed: Nov. 9, 1989 face' P. Schaaf, et al, Langmuir 1987.
86 PCT No.: PCT/SE89/00641 "Polymer Concentration Profile Near a Liquid-Solid
Interface: Evanescent Wave Ellipsometry Study" M.
S371 Date: May 10, 1991 W. Kim, et al, Macromolecules 1989.
S 102(e) Date: May 10, 1991 (List continued on next page.)
87 PCT Pub. No.: WO90/05295
PCT Pub. Date: May 17, 1990
(30) Foreign Application Priority Data Primary Examiner-F. L. Evans
Nov. 10, 1988 CH Switzerland...................... 8804075-3 (57) ABSTRACT
51) Int. Cl....................... G01N 21/17; G01N 33/53 An optical biosensor system using internal reflection
(52) U.S. C. ................................... 356/73; 250/458.1; versus angle of incidence determination for the detec
356/318; 356/446; 356/246; 356/445 tion of biomolecules, the system comprising a sensor
58) Field of Search ................. 356/73,317, 318,338, unit (10) with at least two sensing surfaces (39A-D), a
356/343, 417, 446, 246, 445; 250/458.1, 459.1, source of light (1), and lens means (2) for forming a
461.1, 461.2 convergent beam of light which is focused in wedge
(56) References Cited
shape fashion to form a streak of light (5) extending
transversely over all the sensing surfaces; a photodetec
U.S. PATENT DOCUMENTS tor device (7) in the form of a two-dimensional matrix of
4,304,257 12/1981 Webster .
individual photodetector; optical imaging instrumenta
tion in the form of an anamorphic lens system (6) for the
FOREIGN PATENT DOCUMENTS purpose of imaging rays of reflected light from the
0067921 6/1981 European Pat. Off. . sensing surfaces on each its own column of photodetec
020202 1/1986 European Pat. Off. . tors, so that for each sensing surface there is a corre
0226470 12/1986 European Pat. Off. . sponding set of columns of photodetectors; and an eval
0254430 6/1987 European Pat. Off. . uation unit (8) for determining the minimum reflectance
027.8577 2/1988 European Pat. Off. . or the resonance angle at each of the sensing surfaces.
0257955 3/1988 European Pat. Off. . The invention also relates to a method for calibrating
0286.195 10/1988 European Pat. Off. . the biosensor system, a method for correcting for base
0305109 3/1989 European Pat. Off. . line drift as well as a method for temperature regulation
05417 6/1989 PCT Int'l Appl. .
2197065 5/1988 United Kingdom . of thermostat means in the biosensor system.
2197068 5/1988 United Kingdom.
OTHER PUBLICATIONS
"A Compact Surface Plasmon Resonance Senso for 53 Claims, 12 Drawing Sheets

iA2.2227

T.
ATA councATION
 5,313,264
Page 2

OTHER PUBLICATIONS "Surface Plasmon Resonance For Gas Detection and

"A New Immunoassay Based on Fluorescence Excita Biosensing' B. Liedberg, et al Sensors and Actuators
tion by Internal Reflection Spectroscopy" M. N. Kro (1983).
nick, et al, Journal of Immunological Methods, (1975). "The ATR Method With Focused Ligth-Application
“Total Internal Reflection Fluorescence: A Technique to Guided Waves on a Grating' E. Kretschmann Optics
for Examining Interactions of Macromolecules with Communications vol. 26, No. 1 (1978).
Solid Surfaces' B. K. Lok, et al, Journal of Colloid and "Angular Emission Profiles of Dye Molecules Excited
Interface Sciences, vol. 91, No. 1, (1983). by Surface Plasmon Waves at a Metal Surface' R. Ben
"Evanescent Detection of Adsorbed Protein Concen ner, et al Optics Communication vol. 30, No. 2, (1979).
tration-Distance Profiles: Fit of Simple Models to "Plasmon Surface Polariton Dispersion by Direct Opti
Variable-Angle Total Internal Reflection Fluorescence cal Observation' J. D. Swalen, et al Am. J. Phys. (1980)
Data' W. M. Reichert, et al Applied Spectroscopy vol. vol. 48, No. 8.
41, No. 3, (1987). "Flow Injection Analysis From Test Tube to Inte
"Physics of Thin Films' H. Raether, et al Academic grated Microconduits' Ruzicka Analytical Chemistry,
Press 1977. vol. 55, No. 11 1983.
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and a phase-modulated ellipsometer, has been used for
OPTICAL BIOSENSORSYSTEM studying the polymer (polystyrene) concentration pro
file near a prism/liquid interface; see Kim, M. W., Mac
The present invention relates to an optical multi romolecules, 22, (1989) 2682-2685. Furthermore, total
analyte biosensor system employing the principle of 5 internal reflection ellipsometry in the form of stationary
internal reflection of polarized light for use in biologi optical means at a single angle of incidence has been
cal, biochemical and chemical analysis and in particular suggested for quantification of immunological reac
for detecting a specific molecule, for example antigens. tions; see EP-A1-0 067921 (1981), and EP-A1-0278.577
The detection method used in the biosensor system may (1988).
be based on the evanescent wave phenomenon at total 10 By a detection employing evanescent wave excitation
internal reflection, such as surface plasmon resonance fluorescence through an angle of incidence dependent
(SPR), critical angle refractometry, total internal reflec TIRF, the intensity and wavelength of the radiation
tion fluorescence (TIRF), total internal reflection phos emitted from the either natively fluorescent or fluores
phorescence, total internal reflection light scattering, cence labeled sample molecules within the sensing layer
and evanescent wave ellipsometry. Furthermore, the 15 is measured. Total internal reflection fluorescence
detection method may be based on Brewster angle re (TIRF) was suggested for a immunoassay in 1974, in
flectometry. that a separate antigen-coated quartz slide was brought
The main advantage of the internal reflection based into optical contact with a quartz prism via a drop of
techniques is that the sensitivity region for the specific index-matching solution and by using a Teflon O-ring
substance is restricted to the extension length of an 20 sealed plexiglas cell, Kronick, M. M. et al., J. Immunol.
evanescent wave, i.e. the depth of an electromagnetic Meth., 8, (1975) 235-240. Now total internal reflection
wave penetrating into the liquid medium from the sens fluorescence is an established technique to examine
ing surface side. Consequently, there will be a minimum interactions of macromolecules with solid surfaces, the
of influence on the response connected to specifically surface being generally one side of the coupling prism;
bound analyte molecules from non-bound sample mole 25 see Lok, B. K. et al., J. Colloid Interf. Sci., 91, (1983)
cules. Moreover, the penetration depth of the evanes 87-103. Furthermore, variable-angle total internal re
cent wave for a totally reflected ray of light depends on flection fluorescence has been used to study adsorbed
the angle of incidence for the ray. For a comprehensive protein concentration profiles at a prism surface; see
treatment of the concept of internal reflection one is Reichert, W. M. et al., Applied Spectroscopy, 3, (1987)
referred to Mirabella and Harrick, Internal Reflection 30 503-508.
Spectroscopy, Harrick Scientific Corporation, N.Y. In a detection employing evanescent wave excitation
1985. scattered light, through an angle of incidence depen
The optical response induced by either the primary dent evanescent wave penetration depth, the intensity
evanescent wave, or by a secondary evanescent wave of the radiation scattered within the sensing layer due to
excited in turn by said primary evanescent wave, may 35 its interaction with the specific substance is measured.
be measured as changes in the reflectance or in the state Scattered total internal reflectance (STIR) has been
of polarization of the incident light wave upon reflec suggested for utilization in immunoassays, and in a pre
tion, or as the fluorescence or phosphorescence or light ferred embodiment, colloidal gold is used as a label for
scatter of radiation, as a result of a specific substance the solution phase immunologically active component;
interaction with a sensing layer at the sensing surface. 40 see EP-A2-0 254430 (1987).
The optical response related to the specific substance With regard to the use of surface plasmon resonance
may be measured as the reflected intensity as a function (SPR), in somewhat simplified terms SPR may be said
of angle of incidence of p-polarized light, being applica to be a technique according to which changes in the
ble for surface plasmon resonance, Brewster angle re refractive index of a layer close to a thin free-electron
flectometry, and critical-angle refractometry, in that 45 metal film are detected by way of consequential
the angle of minimum reflectance is determined and changes in the intensity of a p-polarized light beam
related to a refractive index and surface concentration reflected from the metal film (see for example Raether
of the bound substance at the sensing surface. H, Physics of Thin Films, Academic Press, N.Y., 9
With regard a detection using surface plasmon reso (1977) 145.
nance, this will be treated in more detail hereinafter. 50 In the first publication indicating the possibilities of
Internal multiple-angle Brewster angle reflectometry, SPR technology in biochemical analysis, Liedberg Bet
based on rotating optical means and a rotating prism for al., Sensors and Actuators, 4 (1983) 299 have at first
the variation of incident angle, has been shown to pro adsorbed a monolayer of IgG to a silver surface and
vide information to characterize the adsorption of pro then adsorbed to said monolayer a layer of anti-IgG, in
teins on a silica prism/solution interface; see Schaaf, P. 55 order to then study the effect of the resultant change in
et al. Langmuir, 3, 1131-1135 (1987). the resonance angle. EP 202 021 describes a biosensor
Critical-angle refractometry has so far been used employing movable optical instrumentation for deter
mainly to measure the concentration or density of solu mining the angle-henceforth called the resonance an
tions in process streams. gle-at which surface plasmon resonance occurs. Such
In a detection employing evanescent wave ellipsome 60 movable optical units are not suitable for commercial
try, the optical response related to the specific sub type instruments because (i) when readings of the reso
stance is measured as changes in the state of polarization nance angle are to be taken this will require manual
of elliptically polarized light upon reflection, in that this operations, and (ii) technical manufacturing tolerances
state of polarization is related to a refractive index, in the suspension mechanism of the movable optical
thickness, and surface concentration of a bound sample 65 system contribute to errors occurring in the measure
at the sensing surface. Multiple-angle evanescent wave ments of the resonance angle. EP 257955 describes
ellipsometry, in the form of using rotating optical means another optical system which is scanned mechanically
and a rotating prism for the variation of incident angle, for determining the resonance angle. GB 2 197 068
 5,313,264
3 4.
describes an optical sensor employing a divergent beam sensor surface over a range of angles) and of collection
of rays for irradiating the sensitized surface, this latter of the reflected beams (over a range of angles) by an
being a metal film with receptors or ligands which inter array of angularly spaced detectors.
act selectively with one or more biomolecules. The Furthermore, the transparent block described in EP
optical system is stationary, so the above-mentioned A1-0305 109 may take the form of a hemicylinder creat
drawbacks of movable optical systems are avoided. ing a wedge-shaped beam, giving a line of a small illumi
A source of light is employed for irradiating a sensi nated area on the sensing surface. The hemicylindrical
tized surface which is subject to the action of a sample lens has the advantage that it can be used to perform
solution while another source of light is employed for several tests simultaneously on a single sample. To this
irradiating another sensitized surface which is subject to O end, the sensing surface takes the form of a series of
the action of a reference solution. The light sources and sensitive areas, each comprising a different antibody,
sensitized surfaces are arranged in such a way that the with each separate area being monitored by its own
reflected divergent beams will strike a photodetector detector in a detector array. The cylindrical focusing
matrix. By means of alternate activations of each one of principle used to produce an identical angular range of
the two light sources, the resonance angle obtained S light beams along a focused line for SPR of separate
from each of the two sensitized surfaces can be mea surface areas has been published by Benner, R. E. et al.
sured with precision, and the difference between the Optics Communications 30 (1979) 145-149, and Swalen,
two resonance angles at each of the two sensitized sur J D et al. Am J. Phys. 48 (1980) 669-672.
faces will be a measure of the amount of the specific Further, a focusing lens in EP-A1-0305 109 creates a
biomolecule bound on the sensitive layer. The disad 20 substantially parallel-sided beam incident upon the de
vantage of this apparatus resides in the use of two indi tector, or a beam of at least of reduced divergence con
vidual sources of light-one for the reference solution, pared to the fan-shaped spread of light reflected from
one for the sample solution-as this will tend to make the sensing surface with the object to reduce stray light
the measuring result uncertain in view of the fact that reflections in the detector array. The disadvantages of
the resonance angle is highly dependent on the spectral 25 this apparatus, however, are as follows. The approach,
character of the light source. Another drawback of this to use a small illuminated area in relation to the sensitive
known optical sensor resides in the positioning thereof layer for sensing, in order to reduce effects due to inevi
directly on the prism of the optical system, and in hav table variations in a commercially produced metal film
ing light directed to the sensitized surface via an immer and coating of antibody. In fact, the surface concentra
sion oil that has a suitable refractive index. Such a use of 30 tion of bound sample molecules will in general also be
light-coupling oils will involve much practical inconve non-uniform across the sensitive layer and strongly
nience when the sensor unit, comprising a sensitized dependent on mass transport conditions. Thus, the small
metal layer coated on a transparent plate, is to be re sensing area will be very sensitive to local variations of
placed by a new sensor unit with a sensing surface hav the sensing surface and its sample surface concentration
ing an affinity for a different specific biomolecule. The 35 resulting in low accuracy of the SPR response. Due to
replacement operations will inevitably give rise to oil stray light arising from the coupling optics and a reflec
smears, and the prism has to be cleaned before a new tion in the sensor surface, it is possible to use the de
sensing surface can be analyzed. Manipulation of the scribed optical system for monitoring beams from sepa
instrument will thus be a somewhat messy business. As rate sensitive areas simultaneously by its own detector
to the actual structure of the analytical instrument, this in an array only under the condition that the array can
is not disclosed in the aforesaid GB patent specification. be conveniently placed close to the exit surface of the
EP 226470 describes an apparatus for microchemical hemicylinder or attached to or deposited on that sur
analyses comprising two glass plates with a gel placed face. This leads to limitations in the resolution of indi
between them. The apparatus is of the disposable type, vidual sensitive areas on the detector array, expensive
to be used only once. One of the two glass plates serves 45 optoelectronic constructions, complicated production
as a platform on which the sample liquid is applied. process (detector alignment, optimization of collected
Capillary force will then draw the sample liquid into the angle span etc.). Optical oil or grease may be used to
capillary cell that has been formed between the plates. ensure good optical coupling between the hemisphere
A device of this type, the dimensions of which are about and the sensor substrate (glass support plate or slide).
3x1.5 cm, requires the use of tweezers or the like for 50 The an approach of a disposable hemicylinder as
handling. It is difficult to determine the volume of the optional technical solution for the change of sensor
liquid sample, and this device is therefore unsuitable for surface is unpractical due to the high costs of an ade
quantitative analyses. quate optical quality of this component and the critical
EP-A1-0 305 109 (published after the priority date optical alignment relative to the light source.
claimed in the present application) describes a SPR 55 With regard to the use of internal reflection proce
sensor system employing a focused (fan-shaped) light dures, most of the present publications laboratory
beam to illuminate the sensitive surface through a equipment for a singular determination of a specific
curved transparent block and via an index matching substance at a time. Such equipments, however, are not
fluid. The beam enters the transparent block in a direc suitable as practical commercial instruments; they are
tion orthogonal to the tangent of the surface of the too complex and cumbersome in their construction for
transparent block. sample and liquid handling, detection, and evaluation,
As was published by Kretschmann, E., Optics Com and the analysis procedures take quite a long time and
munications, 26, (1978) 41-44, the problem of slow moreover require highly skilled operators in order to
speed of operation relative to changes in reflectance and obtain accurate results.
the insufficient precision in the resonance angle deter 65 By the present invention a new analytical system is
mination related with SPR procedures based on move provided for at least one specifically sensing surface
able mechanics, is solved by the use of a fan-shaped comprising the simultaneous detection of a plurality of
beam (equivalent to several beams incident upon the specific interactions, in that it is adaptable for detection
 5,313,264
5 6
techniques based on cylindrically focused total internal functional/structural information of macromolecules,
reflection and internal Brewster angle reflectometry. kinetic information about molecular interactions,
Thus, in one embodiment of the present invention, specific functionalization of at least one sensing surface
means for employing internal multiple-angle Brewster on the sensor unit,
angle reflectometry on an exchangeable sensing surface regeneration or changing of the specific functionaliza
for a new analytical system by use of a stationary optical tion of at least one sensing surface on the sensor unit.
system is provided. In accordance with the above, the optical biosensor
By another embodiment of the present invention, system of this invention comprises at least one, but
means for employing multiple-angle evanescent ellip preferably a number of sensing surfaces or areas ar
sometry on an exchangeable sensing surface is provided 10 ranged in side-by-side relationship for being exposed to
for a new analytical system by use of a stationary optical sample liquid passing over them. These sensing surfaces
system. are analyzed by the above-mentioned optical techniques
In another embodiment of the present invention, a based on a single source of light so that due to the use of
new analytical system is provided enabling variable-. one identical set of wavelengths for all the sensing sur
angle total internal reflection fluorescence on an ex 15 faces it is now possible to obtain a calibration, reference
changeable sensing surface by use of a stationary optical and evaluation process of such high analytical perfor
system. mance that this biosensor system becomes commer
In a still further embodiment of the present invention cially useful. The system is also such that the sample
a new analytical system is provided enabling variable liquid can be made to sweep over the sensing surfaces
angle scattered total internal reflectance (STIR) on an all at the same time or in a sequence. Measurements are
exchangeable sensing surface by use of a stationary made under controlled temperature conditions. At the
optical system. time when the measurement is being performed the
According to a preferred embodiment the present temperature at all the sensing surfaces is to be the same
biosensor system uses a new mean-value procedure, and is to be kept constant during the measuring opera
utilizing an anamorphic lens system, for the detected 25 tion.
reflectrometric, emitted or scattered light due to a spe The sensing surface or surfaces of the sensor unit will
cific interaction at a sensing surface that is optimized lend themselves readily and simply to functionalization
relative to the flow cell geometry, permitting inherently individually, for selective interaction with the desired
accurate and highly sensitive results to be obtained. biomolecules; that is, it will be easy to bestow different
Furthermore, in a preferred embodiment a the pres 30 affinity properties on these sensing surfaces.
ent invention provides a biosensor analytical system The provision, in accordance with the above men
that eliminates the drawbacks related with more or less tioned preferred embodiment, of a block unit for liquid
stationary sensing surfaces, generally being coated on handling using automated microprocessor-controlled
expensive prisms or wave guides, by introducing a sepa sample insertion and sample guiding valves will permit
rate sensor unit that is semi-automatically exchangeable 35 precise and reproducible dispersion of the sample zone
in the instrument. Such a sensor unit having at least two and accurately determined amounts and flow rates of
sensing surfaces, as well as a method for its functionali sample solutions, reagent solutions or reference solu
zation, is the object of our copending PCT-application tions.
entitled "Sensor unit and its use in biosensor systems', The advantages of an integrated conduit liquid han
now PCT Publication No. WO 90/05305 (based upon dling system situated in a permanent rigid and planar
Swedish patent application No. 8804074-6), the disclo structure has been described by Ruzicka (Ruzicka, J.,
sure of which is incorporated by reference herein. Analytical Chemistry, 55, 1041A (1983). Hence, the
Moreover, the present biosensor system provides a in rigidity of such a structure and the possibility to inte
a preferred embodiment thereof, an automated inte grate and control sample injection ensures repeatability
grated liquid handling block, micro-processor con 45 of dispersion of the sample zone. Further, the small
trolled valves therein, preferably membrane valves, dimensions of the microconduits reduces sample rea
enabling complex liquid handling tasks comprising spe gent consumption to the microliter level. Furthermore,
cific sequences of addition of various ligands, macro the versatile combination of engraved grooves as con
molecules, and reactants for interaction at the sensing, duits within laminated parallel layers, interconnected
ult. 50 by perpendicular channels, provides the means for inte
The biosensor system of the present invention enables grating a number of highly controlled solution handling
new applications related to the field of characterizing tasks. The technical limit on possible miniaturisation of
biomolecules to be performed, e.g. as disclosed in our such flow injection analysis systems has, however, been
copending PCT-application entitled "A method of the availability of detectors suitable for sub-microliter
characterizing macromolecules' now PCT Publication 55 detector volumes. The present invention comprises in
No. WO 90/05306 (based upon Swedish patent applica one particular embodiment thereof a multi-analytical
tion No. 8902043-2), the disclosure of which is incorpo system enabling a detector volume of and below 60
rated by reference herein; as well as in our aforesaid nanoliter.
copending PCT-application entitled "Sensor unit and Thus the liquid handling block unit of the present
its use in biosensor systems'. Such new applications biosensor system contains conduits or channels having
made possible by the present invention comprise: one or more portions which, when the measuring opera
qualitative as well as quantitative specific determination tion is to be performed, are pressed against the sensor
of at least one biomolecule in a sample either simulta unit to thus form one or more flow cells, the arrange
neously or sequentially, ment being such that these flow cells can either be cou
qualitative as well as quantitative structural information 65 pled in series or be made to each receive its own sample
of macromolecules through the detection of interac solution separately. A flow cell may contain a single
tions between surface-exposed structural elements on sensing surface or alternatively a plurality of sensing
the macromolecule and various ligands, surfaces; in this latter case, the sensing surfaces lie in a
 7
5,313,264
8
row in the longitudinal flow direction of the flow cell. of an electronic mean value of the mass of biomolecules
The block unit for liquid handling is to be employed bound to the sensing surface, as seen in the longitudinal
both when the sensing surfaces on the sensor unit are direction of the beam of light,
being functionalized and when analysis is carried out. FIG. 10 is a schematic perspective view of the flow
Furthermore, the optical biosensor system according cell, illustrating the flow conditions therein and the
to this invention provides for a stationary optical instru creation of an integrated optical mean value of the mass
mentation, without any movable parts. Thereby the of bound biomolecules,
optical system may already in the course of its manufac FIG. 11 shows a meridian section of the light source,
ture in the factory be given a fixed, "once for all" stan a coupling prism and the anamorphic imaging system,
dard setting so that this setting does not need to be O and the first and second lens means; and a sagittal view
altered during the subsequent use of the system. In view of the same items,
of this fixed setting it is possible to have the optical FIG. 12 is a perspective view of a carrier and sensor
system mounted inside a dust- and light-tight housing. unit in relation to a block unit for liquid handling,
Moreover no smeary oils need to be employed for cou FIG. 13 shows a plan view of the underside of the
pling the light to the replaceable sensor unit. Instead a 15 base plate in the FIG. 1 block unit for liquid handling,
replaceable opto-interface may be used for coupling FIGS. 14A and 14B taken together show a plan view
light from the light source to the sensor unit. Such an of the base plate according to another embodiment of a
opto-interface is disclosed in our copending PCT block unit for liquid handling,
application entitled "Optical interface means', the dis FIG. 15 schematically shows the functions of the
closure of which is incorporated by reference herein. 20 block unit for liquid handling according to FIGS. 14A
As mentioned above the measurement accuracy may and 14B, and
be still further increased by establishing an optical and, FIG. 16 shows another embodiment of a flow cell in
respectively, electronic mean value procedure as will be longitudinal or parallel mode in relation to light streak
described in detail below; this is so because the estab 5.
lishment of such mean values will permit an accurate 25 FIG. 1 shows the main components of the optical
determination of the quantitative or relative amount of biosensor system according to this invention, in the
specific biomolecules that have adhered to the individ form of an exploded view. The system comprises a
ual sensing surfaces. source of light 1, first lens means 2 for directing a trans
With the features mentioned above, an optical biosen versely extending convergent beam 3 toward a prism 4
sor-based analytical system is obtained that permits fast 30 whereby the beam is focused in the bottom surface of
automated high-precision measurements of extremely the prism to thus form a streak 5 of light. Rays of light
low concentrations, nanomolar and even down to reflected from the sensitized surfaces are imaged via an
picomolar, and with a high response dynamics. In, for anamorphic lens system 6 on a two-dimensional photo
example, the SPR biosensor system the resonance angle detector device 7. The electric signals created by the
may be measured over a wide range of angles. 35 photodetectors are processed in an evaluation device 8
The invention will now be described in greater detail in the form of a computer. By means of the prism and an
below, with reference to the attached drawings in opto-interface 9 light from streak 5 is directed to a sen
which sor unit 10 which is to lie in contact with a number of
FIG. 1 is a schematic exploded view of an optical parallel, upwardly open portions 11A-D of flow chan
biosensor system according to the present invention, nels 14A to 14D respectively; only one of these, 14A, is
FIG. 1A is a partial cross-sectional view of FIG. 1 shown. The flow channels form part of a block unit 15
showing a valve in its open position, and for liquid handling, this unit being provided with sche
FIG. 1B is a corresponding view showing the valve matically indicated inlet connection means 16 and 96
in its closed condition, and outlet connection means 101 and 92.
FIG. 1C is an illustration of an electromagnetic valve 45 FIG. 2 is a perspective view of the analytical appara
in its closed condition. tus, generally designated 18, which is the heart of the
FIG. 2 is a perspective view of the central part of the optical sensor system according to the invention. The
biosensor system, apparatus comprises a support 19 on which the block
FIG. 3 is a cross-sectional view of a sensor unit, unit 15 for liquid handling is mounted in a fixed position,
FIG. 4 is a top view of an irradiated sensor unit, 50 and furthermore comprises a housing 20 in which the
FIG. 5 is a perspective view of a carrier plate for the light source 1, the first lens means 2, the prism 4, the
sensor unit, imaging optical unit 6 and the photodetector device 7
FIG. 6 is a plan view of an opto-interface, are accommodated in fixed positions. An aperture 21 in
FIG. 7 is a sectional view of the opto-interface me the bottom portion of the housing is to be covered by
chanically coupled to the sensor unit, 55 the opto-interface 9 which is retained in its desired
FIG. 8 is a schematic perspective view of the refrac position by means of two guide pins 22, 23. The housing
tive characteristics of the anamorphic lens system, 20 is hinged to the support by means of a system of
showing the creation of an optical mean value of the articulated arms 24, 25 pivotally attached to the housing
mass of biomolecules bound to the sensing surface, as and to the support respectively. The block unit 15 for
seen along the width of the streak of light, this being liquid handling carries a number of holder means 26 for
achieved in that light rays in the plane P reflected at a the sensor unit in its carrier plate. As shown in detail in
certain specific angle irrespective of the point of reflec FIG. 12 the holder means are L-shaped angle pieces on
tion on the sensitized surface are imaged on a row of top of the upper face of the block unit for liquid han
detector elements, and how in the plane S light rays dling, thus forming guide means enabling the sensor unit
from an individual sensing surface are imaged on a 65 to be slid over said block unit. A thermostat means, not
column of detector elements, shown in FIG. 2, surrounds prism 4. This thermostat
FIG. 9 is a schematic plan view in the S plane of the means consists of channels for liquid in a heat exchanger
incident collimated beam of light, showing the creation block which maintains the metal frame around aperture
 5,313,264
10
21 at a constant temperature. A similar thermostat above description regarding the activation of dextran
means 27 placed between the block unit for liquid han film 38, sensing surfaces 39A-D are formed each corre
dling and the support is to maintain the liquids in the sponding to the length of the open portions 11A-D
channeling system of said block unit at the same con (provided the sensor unit is attached to the liquid han
stant temperature as that stated above. The photodetec dling block unit with the same orientation both in the
tor device is accommodated on a printed circuit card 28 analysis phase and in the sensitation phase). Light streak
mounted in a fixed position at one of the end faces of the 5 extends transversely over the sensing surfaces 39A-D
housing 20. Guide pins 22, 23 are received in openings and has a width at least equal to the length of portions
29, 30 provided in the block unit for liquid handling. A 11A-D.
transverse beam member 31 extending between the 10. In FIG. 4, the metal surface 37 is presented on a
arms 24, 25 carries a projecting peg 32 for rotating the magnified scale. In actual practice the metal surface will
housing 20 back and forth between two positions, viz., be a square of say an order of magnitude of about
an analysis position and a loading position. These will 10x10 mm, the dimensions of the transparent plate then
be described later in greater detail. An electric motor 33 being somewhat larger, e.g. 11 X11 mm. Such a con
which is attached to the support has an output shaft struct is difficult to handle without tweezers or other
carrying a disk 34 excentrically, and the peg 32 is in seizing means. But such procedures involving the use of
contact with the periphery of that disk 34. By activation seizing tools are undesirable because they tend to
of the electric motor so that the excentrically mounted thwart high-precision measurement. What is proposed
disk 34 rotates through a predetermined angle the hous instead, according to the present invention, is that each
ing is automatically swung into and out of these analysis 20 sensor unit is mounted on a carrier plate which can
and loading positions without any manual operation. A easily be seized between forefinger and thumb. A plan
flat cable 35, shown schematically, goes from the view of the carrier plate is shown in FIG. 5; the plate
printed circuit card 28 to the evaluation device 8. comprises an elongated sheet of plastic 42 with a grip
FIG. 3 shows the sensor unit 10 in cross section. In area 43. An opening 44 through the carrier plate has a
our copending Swedish patent application entitled 25 flange 45 on which the sensor unit will be resting.
"Sensor unit and its use in biosensor systems' this sen Clasps 46 projecting laterally from the flange surface
sor unit is described in greater detail. The sensor unit serve to retain the sensor unit between the flange and
comprises a transparent plate 36 of glass, plastics or clasps. The sheet 42 has two guide holes 47, 48 through
other transparent material. On one of its faces the plate which the guide pins 22, 23 will pass in the analysis
is provided with a metal film 37 that has been applied by 30 position of the apparatus. When the apparatus is in its
e.g. sputtering. A dielectric film 38 is attached to the loading position the guide pins are retracted both from
metal film. In the preferred embodiment of the inven said guide holes 47, 48 and from the openings 29, 30, the
tion, the dielectric film is a layer of dextranbound to the carrier plate being now free to be slid into its position on
metal film. By means of a coupling technique such as is the block unit for liquid handling or, respectively, to be
known in biotechnology, ligands are bound to the dex 35 drawn off therefrom.
tran film which serve to interact with specific bi Sheet 42 also has control springs 49 for (i) facilitating
omolecules present in the sample solution. For attach introduction of the sensor unit into the liquid handling
ing the ligands the sensor unit with its metal film and the block unit and (ii) protecting the sensor unit from dam
dextran layer thereon is contacted sealingly with the age or e.g. scratches in case the carrier plate is uninten
upwardly open portions 11A-D of all the flow channels 40 tionally set down on a table or the like. It is preferable
14A-D. By pumping a solution containing a specific to accommodate the carrier plate and its sensor unit in
ligand L1 into one of the channels, 14A, another spe a housing which comprises top and botton walls, two
cific ligand L2 into the second channel, 14B, a third side walls and one end wall. The interior faces of the top
specific ligand L3 into the third channel, 14C etc., a and bottom walls are provided with pairs of opposite
corresponding number of sensing surfaces may be pro 45 ledges extending longitudinally of the housing. They
duced on a single metal film with each its own individ serve to maintain a clearance space between the carrier
ual affinity for a (respective) specific biomolecule. The plate and the top and bottom walls.
ligands may for example be bifunctional or polyfunc FIG. 6 shows a plan view of an opto-interface plate
tional molecules having one active portion consisting of according to the present invention. The plate is fastened
antidextran and another portion consisting of an antigen 50 on a metal frame which has two projecting tongues 55,
to the antibody that is to be detected. As a result of this 56, said tongues being provided with one hole each,
functionalization, the optical biosensor system of the these holes 53, 54 extending all through the thickness of
invention may be tailored individually by the user so as the tongues and serving to receive the guide pins 22, 23.
to be rendered useful for detection of just those bi The frame has two flanges 51, 52 against which a trans
omolecules in which he is interested. This functionaliza 55 parent plate 57 of glass or plastic has been applied. As
tion will permit inter alia qualitative analyses. The func shown in FIG. 7, on one face plate 57 is provided a
tionalized sensor unit is introduced into the analytical number of longitudinally extending parallel ridges 58 in
apparatus or functionalized in situ, whereupon the anal side-by-side relationship. On its opposite face the plate
ysis is carried out. When the analysis operation has been has a corresponding number of parallel longitudinal
finished the sensor unit is regenerated in that the sensing ridges 59 lying opposite the ridges of the first-named
surfaces are "washed" with a solution that will disrupt plate face. The ridges are made of a transparent elastic
the coupling between the ligands and the dextran film material and spaced apart a distance corresponding to
38. that between the upwardly open portions 11A-D of the
FIG. 4 is a top view of the sensor unit 10 of FIG. 3. flow channels. As seen more clearly from FIG. 7 the
The sensor unit has been placed on top of the upwardly 65 ridges 58, 59 have longitudinally extending stepped
open portions 11A-D, and the streak 5 of light arriving portions 60 at each side to thus form a structure having
from lens system 2 of light source 1 irradiates the re in cross section the configuration of a flight of stairs, the
verse side of metal film 37. As will be seen from the uppermost step or top platform of each of these stepped
 5,313,264
11 12
structures being capable of being pressed resiliently adjusting the degree of magnification of the resonance
against the transparent plate 36 of the sensor unit and, angle range along photodetector columns.
respectively, the prism 4. This stepped configuration Light rays having a different plane of incidence paral
prevents air pockets from being formed between the lel to the plane of incidence P will in a similar way be
interfaces contiguous to the prism and, respectively, imaged on individual photodetectors belonging to other
plate 36 of the sensor unit. FIG.7 shows the prism 4, the columns of the two-dimensional photodetector device.
opto-interface plate and the sensor unit in the analysis Every photodetector of a row thus corresponds to one
position of the analytical apparatus 18. Ridges 58, 59 are specific angle of incidence. As against this, conditions
spaced apart in such a manner that the distances be are different in the case of the reflected light obtained
tween them correspond to the distances between the 10 from light that is reflected in an S plane, i.e. in planes
upwardly open portions 11A-D of the flow channels differing from the plane of incidence P of the conver
14A-D. gent beam. Such light will image a point on the sensi
This arrangement of the ridges 58, 59 on the opto tized surface as a real image element on detector pixel
interface plate, the holes 53, 54 for the guide pins 22, 23, 61D. Thus to each detector column corresponds a re
the holes 47, 48 on the carrier plate 42 of the sensor unit 15 spective part of the sensing surface as seen in the trans
and the stationary mounting of the upwardly open por verse direction of the conduit portion. Depending on
tions 11A-D of the block unit for liquid handling ensure the width of the channel, the surface dimensions of the
that the lower ridges 59 will serve as sources of light individual photodetectors, and the spaces between
which lie directly above each of the corresponding them, a particular number of photodetector columns
channel portions 11A-D. No scattered light from 20 may be required for imaging the total width of the chan
neighboring ridges 59 will interfere with the resonance nel portion in question. In a preferred embodiment of
angle determination for the individual sensing surfaces. the invention a reduced scale image of a large portion of
In this manner it is possible to have a great number of the flow cell width is created on one single column of
these channel portions packed next to one another. As detectors.
an example, it may be mentioned that up to 20 of such 25 Upon this introductory description of the anamor
upwardly open channel portions can be packed to phic lens system will now follow a description of the
gether within a breadth of about 10 mm without any mass uptake on a sensing surface, e.g. sensing surface
scattered light interfering with the measuring operation. 39D (see FIG. 9). The rate of flow is zero at the side
FIGS. 8-11 illustrate the optical system in the ana walls of the channel, and then increases toward the
lytic apparatus according to the invention. The basic 30 center of the channel. The flow profile is shown sche
principle of the anamorphic lens system 6 is shown in matically at 70 where arrow lengths correspond to the
FIG.8. The broad beam of light which is wedge-shaped flow rates occurring. The channel width is assumed to
or convergent strikes the illustrated sensitized surface, be about 300 m. In order to avoid measuring results
e.g. at sensing surface 39, at angles of incidence of from becoming distorted by the outer channel regions where
62 to 78 degrees. Rays with all the intermediate (be 35 no sample liquid is flowing the anamorphic lens system
tween 62 and 78) angles of incidence are present in the is arranged so as to produce an image of only a narrow
beam. Consider only one incident plane P. All the rays portion, say of an order of magnitude of 50% or more of
incident with for example an angle of 62 are indicated the width of the sensing surface, on a single column of
by white arrows, are reflected on the sensitized surface photodetectors; e.g. a 150 in central portion of a sensi
and will be imaged on only one single photodetector tized surface whose total width is 300 um is imaged on
61A by the anamorphic lens system. Similarly, all the a column pixel 61D having a width of 90 m. This will
rays incident with an angle of 78 and indicated by black electronically produce a mean value of the mass uptake
arrows will be imaged by the anamorphic lens system within this central part of the sensing surface. Without
on one single photodetector 61G of the photodetector such a creation of a mean value of the amount of bound
device 7. Light rays having incident angle values inter 45 biomolecule as seen in the transverse direction of the
mediate between 62 and 78 degrees will similarly strike channel it would not be possible to obtain such a high
those photodetectors which are situated between detec degree of reproducibility of the response curve of any
tors 61A and 61G in the same photodetector column; in given sample concentration of the biomolecule as is
FIG. 8 this is illustrated as being a vertical column. required in a commercial-type analytical instrument
Light source 1, e.g. a light emitting diode, emits a 50 except perhaps by putting unrealistically high demands
type of light that is substantially monochromatic in on high reproducibility and control of (i) flow profile in
character (E30 nm), and furthermore is incoherent and the sample liquid (thickness variation of diffusion layer
has a center wavelength of an order of magnitude of transversely of the channel), (ii) analysis position of the
about 650 to about 850 nm. Alternatively, the light instrument for optical units 2, 6 relatively to the position
source 1 is a laser, e.g. a semiconductor laser, a dye laser 55 of the flow channel, (iii) homogeneousness in respect of
or a gas laser, emitting substantially monochromatic ligand density and in respect of ligand accessibility
and coherent light. The light passes through the first within the dielectric film on the sensing surfaces.
lens means 2, which in the case that light source 1 is a FIG. 10 illustrates the flow conditions prevailing in
light emitting diode includes an interference filter 303 the channel portion below the sensing surface. The flow
for forming a wedge-shaped convergent elongated rate profile of the sample liquid is illustrated by means
bean which passes through a plane polarizer 63 indi of arrows of different lengths. In the 90' bending region
cated schematically in FIG. 11 and then on toward the of the flow channel a large amount of biomolecules may
bottom face of prism 4. In that bottom face and on the bind to the sensing surface. Reference numeral 75 desig
underside of metal film 37 a streak of light is formed nates the distribution of the amount of bound biomole
having a width which is adjustable by means of a cylin 65 cule. The amount thus bound decreases downstream of
drical lens 64. The optical imaging system comprises the the inlet. It will be appreciated that if the resonance
anamorphic lens system 6 the function of which has angle were to be determined on the basis of only one
been described above. It contains inter alia a lens 66 for section, indicated by arrows 76, this would result in one
 5,313,264 14
13
particular value being obtained, while if the resonance It will now be understood that when in the analysis
angle were determined on the basis of the section indi position of the analytical apparatus the sensor unit 10 is
cated by arrows 77 this would result in another value pressed against layer 80 by the opto-interface 9 the
therefor being obtained. In order to make sure that a upwardly open portions 11A-D in layer 80 will be
reliable mean value of the resonance angle is obtained sealed in liquid-tight relationship against the sensor unit
the width of light streak 5 is made to agree, according to 10 and form four flow cells. For the sake of simplicity,
the invention, with the length of the upwardly open these flow cells too are designated 11A-D.
channel portion. In this manner all the contributions There now follows a description of the principle
from the amount of bound biomolecule will be included according to which a liquid sample is made to pass
when the mean value of the resonance angle is being O through flow cell 11A: By means of a pump (not shown)
determined. An optically created mean value is thus sample liquid is pumped to inlet tube 16, passes through
obtained, as seen in the direction transversely of the an inlet channel 87 past an open valve 88 and then goes
path of the light beam. on through a primary channel 89 having a fixed and
Without creation of a mean value of the amount of well-defined volume, until it reaches a closed valve 90
bound biomolecule as seen in the longitudinal direction 15 whence it is directed into a waste channel 91 communi
of the channel it would not be possible to obtain repro cating via a connecting tube 92 with waste disposal
ducibility of the response curve of any given sample means 93.
concentration of the biomolecule, within a concentra Next, a valve (not shown) at the upstream end of
tion range of interest for analysis purposes, as is re 20 waste channel 91 is closed. At the same time valve 88 is
quired in a commercial-type analytical instrument-ex ready toSample
closed.
be
liquid in the primary volume is now
pumped into the flow cell 11A. This is done
cept perhaps by putting unrealistically high demands on with the aid of an eluent solution 94 which is pumped by
high reproducibility and control of (i) flow profile in the a pump 95 through an inlet tube 96 to an eluent conduit
sample liquid in the longitudinal direction of the chan 97 ending in a valve (not shown in FIG. 1, for the sake
nel (thickness variation of diffusion layer in the direc of clarity) which is now opened, together with valve 90.
tion of flow), (ii) sample dispersion in the direction of 25 Continued pumping of the eluent solution will result in
flow, (iii) homogeneousness with respect to ligand den that the advancing eluent solution will press forward
sity and with respect to ligand accessibility within the against the primary volume of the sample liquid and
dielectric film on the sensing surface, and (iv) the posi force it to advance upwardly through a riser duct 98 in
tion of the focused light streak along the channel. With 30 the plateau 81, thence into the flow cell 11A, and then
the creation of these mean values, it is possible to elimi down through a riser duct 99 in the plateau and out
nate such deterioration of reproducibility as will other through an exhaust duct 100 and an outlet tube 101; then
wise occur due to variations in the position of the streak from this outlet tube at first the sample solution and then
of light caused by variation in the thickness of the sen the eluent solution will pass on to a second waste dis
sor plate. Maximum analytical sensitivity in the curve of 35 posal means 102 for sample and eluent solutions. When
response vs. concentration, and a minimum sample con the sample solution which has a predetermined volume
centration for the detection, are obtained for a light is passing along flow cell 11A the chemical action ex
streak width and thus mean value creation covering the erted by the sample solution on the sensor unit is ana
sensing surface at the riser duct that opens out into the lyzed optically.
flow cell, but with a reduced detectable maximum sam 40 Valves 88, 90 and the other valves which have not
ple concentration. been shown in the drawing are identical in construction;
The width of light streak 5 is adjustable by means of therefore, only valve 88 will be described. The valve
a cylindrical lens 64 which is shown in FIG. 11. Alter comprises a diaphragm 103 opposite a channel or
natively, light source 1 and lens means 2 are moved through opening 104 in the support plate 85. The valve
mutually fixed along the optical axis. FIG. 11 illustrates 45 diaphragm is an integral part of layer 84. A valve seat
the anamorphic optical system in the form of both a 105 formed in layer 83 is an integral part of that layer
meridian section, uppermost part of the Figure, and a and has the shape of a projection 105. The portion of
sagittal section, lowermost part of the Figure. channel 104 leading to the valve is filled with a short
The block unit for liquid handling will now be de liquid column 106 of e.g. glycerol. Via a compressed air
scribed. 50 supply line 107 and an electromagnetically operated
FIG. 1 shows a flow channel 14A belonging to the compressed-air valve 108 (shown only schematically)
upwardly open portion 11A. The flow channels 14B-D the channel 104 communicates with a source 109 of
belonging to the respective upwardly open portions compressed air. The compressed-air valve 108 acts in
11B-D have not been shown here, for the sake of clar response to electric signals from the evaluation device 8
ity. A first layer 80 of sealing elastomer material, e.g. 55 which is here embodied in the form of a computer. FIG.
silicone rubber, has a number of cuts or slits extending 1A shows the valve 88 in its open position while FIG.
therethrough, corresponding to the upwardly open 1B shows the same valve in its closed position. Were it
portions 11A-D. The layer has been cast onto a plateau not for the liquid column 106 the compressed air would
81 which is integral with a base plate 82. This plate is penetrate through 84 and pass into the channel system
preferably a solid member made of e.g. plastic, metal, in the form of undesirable air bubbles.
ceramics, silicon. A second layer 83 (FIG. 1A) of elasto FIG. 13 shows an embodiment of the channel system
mer material, for example silicone rubber, has been in a block unit for liquid handling. Dotted lines 11A-D
applied by e.g. casting to the underside of base plate 82. symbolize the four flow cells each having their riser
This layer 83 is provided with a system of flow channels tube connection. The small circular rings indicate
formed by casting. A third layer 84, preferably of the 65 valves each of which has a construction as explained
same material as layer 83, has been cast onto a support above. Eluent conduit 97 contains valves 110, 111, 112
plate 85 of solid material, preferably the same as that of in the places where shown. Waste duct 91 has a valve
base plate 82. 113. Immediately upstream of riser tubes 98A-D there
 5,313,264
15 16
are valves 114A-D, simply designated by the common sample solution plus eluent solution as shown to the
numeral 114 in FIG. 13, for the sake of clarity. Immedi right in the Figure, 101.
ately downstream of riser tubes 99A-D there are valves Valves having the same numeral in FIG. 15 are all
115A-D; here again the valves are symbolized by only controlled by one common compressed-air valve. Thus
one common numeral, 115. A second waste duct, desig for instance, when valves 208 are pressurized, all the
nated 116, has valves 117A-D in the places where valves 208A, 208B, 208C and 208D are pressurized
shown; their one common numeral is 117. Moreover the simultaneously.
outlet tube 100 has a group of valves 118A-D corre If sample solution is to be pumped into the lowermost
sponding to valves 117. Two different eluant solutions channel system of FIG. 15 then valves 209B, 214, 215,
may be introduced into the system through conduits 10 209D and 213 are maintained in closed positions so that
119, 120 leading to connecting tubes 121 for a mixing the sample liquid can pass through the open valves
chamber (not shown) the outlet of which communicates 208D to the primary volume 122 and thence to the
with eluent conduit 97. After the primary volume 89 has secondary volume, designated by 123. If this secondary
been filled in, the sample liquid can be directed to any volume, too, is to be filled valve 213 is kept closed.
one of the flow cells 11A-D with the aid of the eluent 15 Valves 212A and B are kept open, 209C closed, 208C
liquid, the valves 117 and 118 being maintained open open, the sample liquid being discharged via the waste
and closed in appropriate combinations. For instance, if disposal duct 124 of the lowermost channel system.
the sample solution is to be passed on to flow cell 11C, When sample liquid in the primary and secondary vol
then valves 114A, B and D are kept closed, 110 closed, umes is to be analyzed, valves 208 are closed, valves 209
117A and B open, 117C closed, 111,118A and B closed, and 212 are opened, 213 is closed; and eluent solution is
and 118C and D open. 115A, B and D should also be now pumped through the lower eluent channel 125 to
closed. force sample solution in the primary and secondary
An especially advantageous feature of the liquid han volumes through a desired flow cell 11A-D. Valves
dling block unit according to FIG. 13 is that flow cells 201-207 serve as switch mechanisms for directing sam
11A-D can be coupled in series with each other. In that 25 ple liquid to the desired flow cell. Sample and eluent
case one and the same sample liquid will pass through solutions will then be discharged through an exhaust
the flow cells one after the other. The number of such duct 126 which is common to both channel systems.
serially arranged flow cells may be 2, 3 or 4 depending Correspondingly the uppermost channel system has
on how the valves are set. For example, if all the four an eluent channel 127 and a waste conduit 128, a pri
flow cells are to be coupled in series the following 30 mary volume 132 and a secondary volume 133.
valves are maintained in the closed position when the Valves 208B and D, valves 209B and D, and valves
eluent solution is forcing the sample solution out of the 214, 215 serve as means for selecting the desired channel
primary volume 89: Valves 110, 111, 113, 117A, 117C, system, i.e. causing sample liquid to be pumped either
118B, 118D, 88; while the following are maintained in into the lowermost channel system 135 or into the up
the open position: Valves 114A-D, 115A-D, 112, 90, 35 permost channel system 136. The sample liquid is
118A, 118C, 117B, 117D. The sample liquid and eluent pumped in through a common inlet channel 129.
solution run off to the waste disposal means via duct In order to avoid sample carry-over, the T-section of
16. the channel 129 can be washed. When the channel sys
Another embodiment of the block unit for liquid tem 136 is washed, a small volume of the washing liquid
handling is shown in FIGS. 14A, B and 15. This em is released through the waste disposal channel 130 by
bodiment differs from that of FIG. 13 in that a second opening the valve 215 for a short time. When the chan
ary volume for liquid samples has been added and the nel system 135 is washed, a small volume of the washing
liquid channel system has been duplicated. The second liquid is released through the waste disposal channel
ary volume, which has an exactly defined volume, pref. 131 by opening the valve 214 for a short time.
erably different from that of the primary volume, can be 45 Although not shown in FIGS. 14A, B and 15 the
coupled in series with the primary volume if and as block unit for liquid handling may be provided with
desired. In this manner it thus becomes possible to ana valves corresponding to valves 115, 18 in FIG. 13 to
lyze two sample liquid volumes which differ inter se thus enable the flow cells to be coupled in series with
and which both are well-defined. The duplicate channel one another.
system is a time-saving feature when analyses are car 50 By means of separating the valve functions so that
ried out-for while the sample liquid of one channel every valve, i.e. also each of the valves 208A, 208B,
system is being pumped into the flow cells for analysis 208C and 208D, is actuated by a separate compressed
the other channel system may at the same time be air valve of its own, possibilities are provided to pump
cleaned and filled with fresh sample liquid; so that when either the primary volume or the secondary volume, or
analysis of the sample liquid of the first channel system 55 the primary plus secondary volume, through a desired
has been finalized the sample liquid of the second chan flow cell.
nel system is immediately ready and available for analy As may be seen from FIGS. 14A and 14B, the inlet
SS. channel 129 for sample liquid and the inlet channels 125,
The small rings in FIGS. 14 and 15 designate valves 127 for eluent solution all have a considerable length.
201, 202, 203, 204, 205, 206, 207, 208A-B, 209A-C, This is so because the temperature is to be kept constant
20A-B, 211, 212A-B, 214, 215. Furthermore, riser and equal in both of these solutions with the aid of the
ducts are provided which are to be connected-via e.g. thermostat means 27 (shown in FIG. 2) which is situ
a block (not shown) having flexible tube connections ated below these channel portions and serves as a kind
and planar sealings around the riser duct ends on its of a heat exchanger. The thermostat means 27 and the
underside-to the pump 96, to the pump (not shown) 65 thermostat means in housing 2 are both of the liquid
for sample liquid, to the sample solution waste disposal operated type, their liquid flows emanating preferably
means 93, to the waste disposal means from the two from one common liquid bath which is maintained
channel systems and to the waste disposal means for under strict temperature control.
 5,313,264
17 18
In the above-described embodiments of the invention Solenoid-activated valves may be employed instead
a plurality of flow cells-up to 20-are arranged side by of the pneumatically activated valves. As illustrated in
side and are irradiated simultaneously by a streak of FIG. 1C, a solenoid-activated valve may consist, ac
light extending transversely over the flow cells and cording to the invention, of a wire 302 of superelastic
their sensing surfaces. 5 alloy attached to the solenoid armature. The wire ex
According to the invention it is also possible to em tends through holes in the support plate into contact
ploy just one flow cell the longitudinal direction of with the layer 84. When the solenoid 301 is activated
which is parallel to the streak of light, with a number of the end of the wire 302 will push up the diaphragm 84
mutually spaced sensing surfaces which are arranged of the valve against the valve seat 105 in a similar way
within the flow cell, are irradiated simultaneously by 10 as shown in FIG. 1B. Typically the valve openings in
the light streak and are each functionalized in the man the support plate 85 have a diameter of 0.5 mm; conse
ner described above. After functionalization in an appa quently the superelastic alloy wires 302 should have a
ratus with flow channels similar to that shown in FIG. diameter of an order of magnitude of 0.4 mm. Superelas
1 the sensing surfaces 40A-D are oriented as shown in 5 ticity in this case means that the wires 302 may be
FIG. 16. As will be seen from this Figure, the block unit formed with sharp bends but nevertheless remain stiff.
FIG. 12 shows the region around plateau 81 of the
for liquid handling 140 which is then used in the analysis
phase may have just one single flow cell 141 instead of block unit for liquid handling. As will be seen, there are
the four cells shown earlier. The chief advantage of a number of L-shaped angle pieces 101-105 on each side
such an arrangement resides in obtaining a short and of the plateau for retaining carrier plate 42 in its position
well-defined interface between the sample liquid and 0 over the sheet 80.
eluent solution, as compared to the interface formed It will be appreciated that when the analytical appa
when the flow cells are coupled in series and the sample ratus 18 is in its loading position, the housing being
liquid travels along a winding path through the serial raised and the guide pins being retracted from openings
cells; in this case the interface tends to become elon 29, 30, a carrier plate 42 can be slid in between the
gated each bend of 180. 25 L-shaped angle pieces 100-105. Then when the electric
From the above description of the block unit for motor rotates and lowers its head the prism 4 in aper
liquid handling it will be evident that the number of ture 21 of housing 20 will be pressed against the opto
primary volumes may be increased and that such addi interface plate and thus also sensor unit 10 against layer
tional volumes may each be given any size desired. 80 in a liquid-tight manner due to the weight of the
According to still another embodiment of the channel
30 housing. When the metal film 37 of the sensor unit 10
system in a liquid handling block unit of the type having and the dielectric film 38 on metal film 37 are sealingly
a plurality of flow cells in side-by-side relationship, all pressed against layer 80, flow cells will be created as
formed by the aforesaid upwardly open portions
the flow cells are simultaneously fed, each with one 11A-D.
liquid of its own. In this manner the liquids can be ana- is For initial calibration of the analytical apparatus, salt
lyzed simultaneously in the analytical apparatus. The solutions each having a known refractive index are
liquids may be sample solutions of different types or of pumped one after the other past each of the sensing
the same type. They may also be a number of sample surfaces. The electric signals from the detectors of the
solutions plus one or a plurality of reference solutions. photodetector columns corresponding to each of the
This manner of parallelly analyzing a plurality of liquids 40 sensing surfaces are stored in a memory, separately for
is conducive to shortening the time required for analy each one of the salt solutions. By curve fitting of the
sis. Still another variant form of the invention resides in response values from each of the photodetectors all the
(i) providing a plurality of sensing surfaces in each of photodetector members of the analytical apparatus are
the flow cells lying next to each other side by side, and calibrated so that a linear relationship is established
(ii) feeding all the flow cells simultaneously each with 45 between the salt concentrations and the measured re
one liquid of its own. In this case the time required for fractive indices. Next, the sample solution is pumped
analysis is shortened to a still greater extent, and a possi
through a flow channel 14 which serves as a reference
bility is provided of qualitatively analyzing a greater channel; its sensing surface has no affinity for bi
number of substances simultaneously. omolecules at this particular time when the calibration
According to an alternative embodiment the layer 80 50 is carried out. The electric signals obtained from the
may be a plate of silicon into which slits extending photodetector rows corresponding to the resonance
therethrough and corresponding to the upwardly open angle of the sample solution are stored in another loca
channel portions 11A-D have been etched by means of tion of the memory. The purpose of this calibration is to
a technique such as is known within the field of mi determine the refractive index of the sample solution.
cromechanics. 55 The stored signal corresponding to that index is then
Knowing the cross sectional area of the flow channel corrected from the signals obtained when the sample
and also the volume capacity of the pump (e.g. in ul per solution is pumped through a flow channel with an
unit time) one is able to carry out a time-controlled activated sensing surface in order to determine a differ
pumping operation whereby a liquid volume chosen as ence in refractive index due to the presence of the target
desired is pumped along the sensing surfaces. Now 60 biomolecule in the sample solution. The same reference
looking at FIG. 15, suppose primary volume 122 has a channel may also be used for temperature monitoring of
capacity of 5ul and secondary volume 123 has a capac the analytical procedure; in case unnatural temperature
ity of 45 ul: By means of opening valve 213 and closing variations are detected the pumps are stopped and anal
valves 212A-B, alternatively opening valve 211 and ysis is discontinued.
close valves 210A-B, after a predetermined period of 65 By coating at least one of the sealing areas of layer 80
time corresponding to the desired liquid volume to be between the flow channels with a dielectricum of a
analyzed it is thus possible to feed any desired volume suitable refractive index in order to gain SPR response,
of between 5 and 50 ul along a sensing surface. the temperature at layer 80 can be monitored. The re
 5,313,264
19 20
fractive index of most organic materials is sensitive to wave stimulated fluorescence or phosphorescence,
temperature, and the dielectricum should have a known and light originating from scattering at said sensing
temperature dependence of its refractive index. Light surface, is imaged on a photodetector device; and
beams reflected at the metal film 37 at areas thereof an evaluation unit for determining at least one of an
opposite to the film areas pressed in close optical 5 angle of minimum reflectance, a state of polariza
contact with the liquid sealing areas of the dielectricum tion, and a collected light intensity;
will be imaged by the anamorphic lens system 6 onto wherein at least one sensing surface is arranged on the
corresponding columns of detector elements, e.g. situ sensor unit;
ated between columns of detector elements correspond wherein said at least one sensing surface, in order to
ing to flow channels. The electrical signal representing O form an elongated flow cell, is pressed over an
the resonance angle as measured at the sealing areas upwardly open portion of at least one channel for
between the flow channels will represent an actual tem the sample solution which is to be analyzed and
perature value at the dielectric layer. By guidance from affects at least one of the angle of minimum reflec
the measured difference between the corrected signal tance, the polarization state for minimum reflec
and the actual value, the thermostat system can be con 15 tance, and the collected light intensity, at which
trolled towards the temperature set point selected. The the evanescent wave interacts with the sample;
measured electric signal may also, as an alternative, or wherein said first lens means forms a convergent
complement, to the above described method, be used to beam of light focused in a wedge-shape fashion to
correct for the baseline drift caused by temperature form a streak of light extending transversely over
disturbances due to the thermostat means not being 20
all of said at least one sensing surface;
positioned in the flow cell. wherein the photodetector device, at least in the case
The optical biosensor system instrument schemati of more than one sensing surface, is a two-dimen
cally described for a surface plasmon resonance detec sional matrix of individual photodetectors;
tion in FIGS. 1 and 8, may also be adapted for detection wherein said second lens means is a lens system for
using critical angle refractometry, multiple angle eva 25 imaging rays of one of said reflected light from said
nescent wave ellipsometry, and Brewster angle reflec at least one sensing surface, said light emitted from
tometry, in that the first lens means 62 and the second the sample, and said light originating from scatter
lens means 65 are generally similar for these various ing at the sensing surface onto a corresponding
detection principles. It should be noted, however, that column of photodetectors, so that said at least one
the addition of specific optical components are required 30
for the employment of ellipsometry in the optical bio sensing surface has at least one corresponding col
sensor system. umn of photodetectors;
Additionally, the optical biosensor system instrument wherein said evaluation unit determines at least one
described in FIGS. 1 and 8, may be arranged for em of the angle of minimum reflectance, the state of
ploying variable angle total internal reflection fluores 35 polarization, and the collected light intensity, for
cence, variable angle total internal reflection phospho said at least one sensing surface.
rescence, variable angle total internal reflection light 2. The optical biosensor system of claim 1, further
scattering. More specifically, the second lens means 65 comprising:
may be positioned for detection of in-line fluorescence a support in which the open portion of the at least one
or phosphorescence, or as indicated in FIG. 1, by the 40 channel is arranged in a fixed position;
second lens means 165 and photodetectors 161 for de a housing in which said light source, said optical
tection of right-angle fluorescence orphosphorescence, imaging instrumentation, the photodetector device,
or of right angle light scattering. and a coupling prism are arranged in fixed posi
The above-described embodiments of the invention tions;
may be modified and varied in many ways within the 45 an aperture provided in said housing below said cou
scope of the inventive concept. pling prism and covered by an opto-interface plate
We claim: for coupling incident and reflected light and/or
1. An optical biosensor system using an internal re scattered or emitted light from the sample, respec
flection versus angle of incidence determination for tively, to said sensor unit and said optical imaging
detecting a specific biomolecule, comprising: 50 instrumentation; and
a sensor unit including a transparent plate, at least a holder means by which said sensor unit is detachably
portion of one of the faces of said plate having a attached below said aperture,
dielectric film adhered thereto, or being primarily said housing being placed on top of said sensor unit
coated with a metal film to which has been adhered for pressing the opto-interface plate against an
a dielectric film, said dielectric film having an affin 55 opposite face of said sensor unit.
ity for the specific biomolecule at the time the 3. The optical biosensor system of claim 2, wherein
measurement is carried out; said housing is hinged to said support by means of a
a light source, including first lens means, for directing system of articulated arms extending from each lateral
a beam of light at an interface of the transparent face of said housing and said support, respectively.
plate and said metal film or said dielectric film 4. The optical biosensor system of claim 2, further
directly adhered thereto to produce an evanescent comprising thermostat means provided in said housing
wave at a sensing surface of said dielectric film and at the at least one channel, for thermostatically
when a sample solution contacts said dielectric film controlling said at least one sensing surface and the at
on the transparent plate; least one channel, respectively.
optical imaging instrumentation including second 65 5. The optical biosensor system of claim 1, wherein
lens means by which one of light reflected from said at least one sensing surface includes a plurality of
said sensing surface of the transparent plate, light sensing surfaces, arranged in a side-by-side relationship
originating from the sample through evanescent on said sensor unit.
 21
5,313,264
22
6. The optical biosensor system of claim 1, wherein second plurality of channels are filled while said sample
the transparent plate of said sensor unit is of glass. liquid of said plurality of channels is analyzed.
7. The optical biosensor system of claim 1, wherein 16. The optical biosensor system of 13, wherein pneu
the dielectric film comprises a dextran layer. matic valves are formed in through openings in said
8. The optical biosensor system of claim 1 wherein planar support plate, by the second elastomer layer
the at least one channel includes a plurality of channels above these through openings, and by valve seats in the
arranged in a parallel side-by-side relationship, wherein form of projections from the third elastomer layer of
each of the plurality of channels has a corresponding said base plate.
upwardly open portion, wherein the corresponding 17. The optical biosensor system of claim 13, wherein
upwardly open portions are all covered by said sensor 10 each of said plurality of individually controllable valve
unit when a measurement is performed, and wherein in means comprise solenoids actuating respective wires,
said measuring stage, wherein each of said at least one each wire being of superelastic material and extending
sensing surfaces is situated above its corresponding through the through opening in said support plate to act
upwardly open portion. upon the second elastomer layer.
9. The optical biosensor system of claim 8, wherein 15
18. The optical biosensor system of claim 8, wherein
each of the corresponding upwardly open portions are the corresponding upwardly open portions have a
slits extending through a first layer of elastomer mate length of about 800 um, a width of about 300 um, and
rial or silicon. a height of about 30 a.m.
10. The optical biosensor system of claim 9, wherein 20 19. The optical biosensor system of claim 2, further
the first layer is made of a rubber material. comprising:
11. The optical biosensor system of claim 10, wherein a carrier plate for said sensor unit, wherein an open
said plurality of channels are formed in a liquid handling ing in said carrier plate accommodates said sensor
block unit of a solid material selected from the group unit and wherein said sensor unit rests on flange
consisting of plastics, metals and ceramics. 25 means at a lower end of said opening.
12. The optical biosensor system of claim 8 wherein 20. The optical biosensor system of claim 1, wherein
means are provided which permit said plurality of chan said second lens means is an anamorphic second lens
nels to be coupled in series with each other. system for -
13. The optical biosensor system of claim 9, wherein imaging reflected rays of light from surface elements
said plurality of channels are formed in a liquid handling 30 in a longitudinal direction of the streak of light, in
block unit of a solid material selected from the group the form of real image elements corresponding to
consisting of plastic, metal, and ceramics, and wherein rows and columns of photodetectors where each
said liquid handling block unit comprises: column corresponds to part of said at least one
a planar support plate having a second elastomer sensing surface in a direction of the streak of light,
layer on its upper face; 35 and
a base plate positioned on top of the second elastomer imaging reflected rays of light from surface elements
layer including, transverse to the longitudinal direction of the
a third elastomer layer positioned on an underside of streak of light, onto pixels situated along the col
said base plate, . . umns of photodetectors, such that mutually paral
a first number of riser ducts which correspond to a 40 lel incident rays lying in a plane of incidence and
number of the corresponding upwardly open chan aligned transversely to the longitudinal direction of
nel portions, extend through said base plate, and the streak of light are focused toward a single pho
connect with one end of each of the corresponding todetector in a column, so that each detector in a
upwardly open channel portions, and column corresponds to a one particular angle of
a second number of riser ducts which correspond to 45 incidence.
the number of corresponding upwardly open chan 21. The optical biosensor system of claim 20, wherein
nel portions and connect with an opposite end of said internal reflection versus angle of incidence deter
each of the corresponding upwardly open channel mination is based upon surface plasmon resonance.
portions; 22. The optical biosensor system of claim 21, wherein
a pattern of channels formed in the third elastomer 50 a convergent beam of light strikes a reverse side of the
layer including, metal film with angles of incidence of between 62 and
at least one primary volume for a sample liquid, 78 degrees.
said primary volume having a first exactly deter 23. The optical biosensor system of claim 1, wherein
mined volume, said second lens means is arranged for collecting one of
at least one inlet channel for said sample liquid, 55 rays of light emitted from a sample and rays of light
at least one inlet channel for eluent liquid, and scattered from surface elements in a longitudinal direc
an outlet channel for said sample and eluent liquids; tion of the streak of light, onto columns of photodetec
and - tors where each column corresponds to part of said at
a plurality of individually controllable valve means in least one sensing surface in the direction of the streak of
said planar support plate for directing the liquid 60 light, and for
flow in said plurality of channels. collecting rays of light from surface elements trans
14. The optical biosensor system of claim 13, further verse to the longitudinal direction of the streak of
comprising a secondary volume for said sample liquid, light, onto pixels situated along the columns of
said secondary volume having a second exactly deter photodetectors, such that mutually parallel inci
mined volume which differs from said first exactly de 65 dent rays lying in a plane of incidence and aligned
termined volume. - transverse to the longitudinal direction of the
15. The optical biosensor system of claim 14, further streak of light are focused toward a single photode
comprising a second plurality of channels so that said tector in a column, so that each photodetector in a
 5,313,264
23 24
column corresponds to one particular angle of 34. The optical biosensor system of claim 23, wherein
incidence. said evaluation unit is arranged such that when the mass
24. The optical biosensor system of claim 1, wherein of bound specific biomolecules is calculated, the calcu
said at least one sensing surface includes a plurality of lation includes light emitted from samples or scattered
surfaces, a width of the streak of light irradiating said 5 from said at least one sensing surface from only a partial
plurality of sensing surface is substantially equal to an band of the width of said at least one sensing surface and
extent of said plurality of sensing surfaces transverse to positioned centrally therein, this positioning being ef.
the longitudinal direction of the streak of light to pro fectuated by evaluation of electric signals obtained from
duce an integrated mean value of a mass of bi the photodetectors in a column which corresponds to a
omolecules binding to the dielectric film transverse to 10 corresponding partial band on each of said at least one
the longitudinal direction of the streak of light, thereby sensing surface.
permitting a reproducible quantitative analysis or alter 35. The optical biosensor system of claim 25, wherein
natively a reproducible analysis of the relative amount said evaluation unit is arranged such that when the mass
of the particular biomolecule. of bound specific biomolecules is calculated, the calcu
25. The optical biosensor system of claim 24, wherein 15 lation includes reflected light from only a narrow band.
an extent of said plurality of sensing surfaces in the 36. The optical biosensor system of claim 35, wherein
longitudinal direction of the streak of light are imaged said evaluation unit is arranged for calculating parame
in reduced size on a single photodetector, to produce an ters for a surface plasmon resonance curve for a deter
integrated mean value of the mass of biomolecules bind mination including a reflectance minimum and a reso
ing to the dielectric film in the longitudinal direction of 20 nance angle and a displacement thereof for each flow
the streak of light. cell, by curve fitting response values obtained from
26. The optical biosensor system of claim 20, wherein column detector members corresponding to the reso
said second lens means reduces an image element in S nance angle.
planes with a linear degree of magnification m=0.56 37. The optical biosensor system of claim 2, wherein
when the extent is of the order of magnitude of about 25
said coupling prism is a plane-sided prism placed over
300 m and the photodetector has dimensions of about the aperture in a bottom wall of said housing, wherein
90x90 um. refractive properties of said plane-sided prism deter
27. The optical biosensor system of claim 1, wherein mine a layout of said first lens means in order to form a
said first lens means of said light source includes, a lens convergent beam of light focused in a wedge-shaped
member for varying the width of the streak of light 30
fashion into the streak of light extending transversely
irradiating each of said at least one sensing surfaces. over all sensitized areas.
28. The optical biosensor system of claim 2, wherein
said coupling prism is a truncated straight hemicylinder 38. The optical biosensor system of claim 37, wherein
placed over said aperture in a bottom wall of the hous the upwardly open channel portion of at least one chan
ing. 35 nel is a wall-jet cell, from which a jet flow is directed
29. The optical biosensor system of claim 22, wherein against a centre of a circular sensing surface.
said light source in combination with a filter emits sub 39. A method of calibrating the optical biosensor
stantially monochromatic and incoherent light. system of claim 1, comprising the steps of:
30. The optical biosensor system of claim 22, wherein directing a beam of light from said light source to
said light source emits substantially monochromatic and form a streak of light extending transversely over
coherent light. all of said at least one sensing surface;
31. The optical biosensor system of claim 2, further pumping a plurality of calibration solutions without
comprising positioning means for determining relative affinity to said at least one sensing surface, each
positions of said housing, said sensor unit and said sup with a known refractive index one after the other,
port. 45 through a flow cell,
32. The optical biosensor system of claim 2 wherein storing electric signals from the detectors of the pho
said opto-interface plate includes, todetector rows corresponding to said at least one
a second transparent plate of glass including longitu sensing surface in a memory, for each of the plural
dinally extending parallel ridges on one face ity of calibration solutions, so that a known rela
aligned with longitudinally extending parallel. 50 tionship is obtained between a resonance angle and
ridges on an opposite face of said second transpar a refractive index of the dielectric film, and
ent plate, said ridges being made of an elastic trans calibrating the response from each of the photodetec
parent material, spaced apart with distances be tor OWS.
tween corresponding to distances between each of 40. The method of claim 39, further comprising the
said at least one sensing surfaces with a length at 55 steps of:
least sufficient to couple an entire cross section of passing the sample solution over a separate sensing
the convergent beam of light to said sensor unit and surface that has no affinity for any biomolecule and
pressed against said transparent plate of said sensor is situated on said metal film;
unit directly opposite each of said at least one sens storing the electric signals obtained from the photo
ing surfaces. dector rows corresponding to the separate, non
33. The optical biosensor system of claim 32, wherein affinity sensing surface in another location of the
each of said longitudinally extending parallel ridges has memory;
a number of longitudinally extending stepped portions and correcting the stored signals obtained when the
on each side to form a structure having, in cross section, sample solution is passed along said at least one
a configuration of a flight of stairs, the uppermost steps 65 sensing surface, to determine a difference between
thereof being pressed resiliently against said sensor unit the refractive indices due to the presence of the
and said coupling prism without formation of enclosed particular biomolecule for said at least one sensing
air pockets. surface.
 5,313,264
25 26
41. A method of correcting for baseline drift in sur 45. The optical biosensor system of claim 29, wherein
face plasmon resonance (SPR) analysis procedures said light source includes a light emitting diode and a
using the optical biosensor system of claim 1, compris interference filter.
ing the steps of: 46. The optical biosensor system of claim 30, wherein
establishing a predetermined temperature set point said light source includes one of a semiconductor diode
for the sample solution; laser, a dye laser, and a gas laser.
creating an electric signal representing an actual tem 47. The optical biosensor system of claim 36, wherein
perature value of the sample solution, by utilizing the response values are curve fitted using a reflectance
electric signals which represent the resonance minimum of the resonance angle.
10 48. The optical biosensor system of claim 1, wherein
angle as measured in a flow cell, a sensing surface said at least one sensing surface includes a plurality of
of which has no affinity for the specific molecule surfaces, a width of the streak of light irradiating said
and through which a liquid is fed which is con plurality of sensing surfaces is substantially equal to an
veyed through a liquid flow handling unit; extent of said plurality of sensing surfaces transverse to
correcting the signals from the sample solution with 15 the longitudinal direction of the streak of light to pro
respect to a temperature disturbance caused by not duce an integrated mean value of a mass of bi
positioning a thermostat in the flow cell; and omolecules binding to the dielectric film transverse to
controlling said thermostat by measuring difference the longitudinal direction of the streak of light, thereby
between the corrected signal and the actual value 20 permitting a reproducible quantitative analysis or alter
in order to adjust the temperature set point. natively a reproducible analysis of the relative amount
42. A method of measuring the actual feedback value of the particular biomolecule.
of the temperature at the flow channels for regulation of 49. The optical biosensor system of claim 43, wherein
a thermostat system by surface plasmon resonance an extent of each of said at least one sensing surfaces in
(SPR) analysis procedures using the optical biosensor 25 the longitudinal direction of the streak of light are in
system of claim 1 comprising the steps of: aged in reduced size on a single photodetector, to pro
creating an electrical signal representing an actual duce an integrated mean value of the mass of bi
temperature value of the sample solution, by utiliz omolecules binding to the dielectric film in the longitu
ing electric signals which represent the resonance dinal direction of the streak of light.
50. The optical biosensor system of claim 1, wherein
angle as measured at a flow cell, a sensing surface 30 said second lens means is an anamorphic lens system for
of which is in contact with at least one sealing area imaging rays of reflected light from said at least one
adjacent or between the flow channels, wherein sensing surface, and said evanescent wave is produced
said sealing area is coated with a dielectricum of by surface plasmon resonance.
suitable material and refractive index properties in 51. The optical biosensor system of claim 1, wherein
order to obtain a surface plasmon resonance (SPR) 35 said second lens means is a lens system for imaging one
response from said sealing area, said dielectricum of rays of light emitted from the sample and rays scat
having a known temperature dependence of its tered at the sensing surface, said emission and said scat
refractive index, light beams reflected at areas of tering of said light rays being generated by said evanes
the metal film opposite to areas of the film pressed 40 cent Wave.
in close optical contact with the liquid sealing areas 52. The optical biosensor system of claim 35, wherein
of said dielectricum being imaged by the second said narrow band is preferably about 50% of the width
lens means, being anamorphic onto corresponding of each of said plurality of sensing surfaces, said narrow
columns of detector elements; and band extending substantially along an entire length of
controlling said thermostat system by measuring a 45 each of said plurality of sensing surfaces and positioned
centrally therein, this positioning being effectuated by
difference between the corrected signal and the evaluation of electric signals obtained from the photo
actual value in order to adjust the temperature detectors in a column which corresponds to a corre
toward the temperature set point. sponding narrow band on each of said plurality of sens
43. The optical biosensor system of claim 1, wherein ing surfaces.
said optical imaging instrumentation further including 50 53. The method of claim 39, wherein said plurality of
optical filter means. calibration solutions are salt solutions and/or suitable
44. The optical biosensor system of claim 10, wherein organic solutions. r ax x k k
the rubber material is silicon rubber.
55

65
 UNITED STATES PATENT AND TRADEMARK OFFICE
CERTIFICATE OF CORRECTION
PATENT NO. : 5,313, 264
DATED : May 17, 1994
INVENTOR(S) : Ivarsson et al.
it is certified that error appears in the above-indentified patent and that said Letters Patent is hereby
Corrected as shown below:
On the title page: Item 75)
Change second Inventor's name from "Jönsson Ulf" to --Ulf
J8nsson--
Change residences
"Upsala' of second, third, and fourth Inventors from
to-Uppsala--
Change the address
--Uppsala, of the Assignee from 'Upsala, Sweden' to
Sweden.--

Signed and Sealed this

Sixteenth Day of August, 1994

(a teen BRUCE LEHMAN

Attesting Officer Commissioner of Patents and Trademarks
 UNITED STATES PATENT AND TRADEMARK OFFICE
CERTIFICATE OF CORRECTION

PATENT NO. : 5,313.264 Page 1 of 1

APPLICATIONNO. : 07/681533
DATED : May 17, 1994
INVENTOR(S) : Bengt Ivarsson et al.
It is certified that error appears in the above-identified patent and that said Letters Patent is
hereby corrected as shown below:

On the Title Page

Item (30)
Foreign Application Priorty Data “Nov. 10, 1988 CHSwitzerland....... 8804075-3
should read as -- Nov. 10, 1988 SE. Sweden....... 8804075-3-

Signed and Sealed this

First Day of May, 2007

WDJ
JON. W. DUDAS
Director of the United States Patent and Trademark Office