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Claims  |
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What is claimed is:
1. A method for detecting biomolecules, comprising the steps of:
(a) attaching a plurality of biomolecules to a boundary surface, said
boundary surface being between an optically denser and an optically rarer
medium and having a first side adjacent to said optically denser medium
and a second side adjacent to said optically rarer medium, said plurality
of biomolecules being attached by at least one of
(i) adsorbing to said boundary surface;
(ii) depositing on said boundary surface; and
(iii) physically or chemically bonding to said boundary surface, said
boundary surface having a film, the film having a conductive
characteristic being capable of conducting surface plasmon waves,
comprising the steps of:
(b) irradiating p-polarized electromagnetic waves, using light through said
optically denser medium onto said boundary surface, wherein the angle of
incidence (.theta.) of the p-polarized electromagnetic waves impinging
onto said boundary surface is substantially equal to an angle
(.theta..sub.SPR) at which surface plasmon resonance occurs, surface
plasmon waves capable of causing said plurality of biomolecules at said
boundary surface to reflect or generate electromagnetic waves in response
to said surface plasmon wave;
(c) monitoring at least one of the electromagnetic waves reflected and the
electromagnetic waves generated by said plurality of biomolecules at said
boundary surface and determining at least one of:
(i) the presence of said plurality of biomolecules; and
(ii) the concentration of said plurality of biomolecules by analysis of at
least one of said reflected or generated electromagnetic waves, wherein
the intensity of the electromagnetic waves reflected or generated by said
plurality of biomolecules at said boundary surface is detected; and
(d) controlling the angle of incidence (.theta.) of the impinging
p-polarized electromagnetic waves depending on the intensity of reflected
electromagnetic waves at said boundary surface such that said intensity is
substantially maintained at a minimum corresponding to the occurrence of
surface plasmon resonance.
2. The method according to claim 1, wherein said optically denser medium is
a transparent prism.
3. The method according to claim 2, further comprising the step of:
actuating a rotatable support carrying said transparent prism depending on
said intensity.
4. The method according to claim 3, wherein said rotatable support is
rotated in a first direction and then in a second direction opposite to
said first direction in response to an increase in said intensity, or in
case a predetermined time period (T.sub.max) has expired.
5. The method according to claim 1, wherein said electromagnetic waves
reflected at said boundary surface are fed to an array of monitoring
elements comprising at least a central monitoring element and at least a
lateral element at either side of said central monitoring element and a
control signal causing adjustment of said angle of incidence (.theta.) is
generated if the intensity detected by one of said lateral elements is
lower than the intensity detected by said central element.
6. The method according to claim 1, further comprising at least one of:
(i) passing p-polarized electromagnetic waves through a dielectric layer
before passing through said film, and
(ii) passing p-polarized electromagnetic waves through a dielectric layer
after passing through said film.
7. The method according to claim 1, wherein said optically rarer medium is
a solvent in which said plurality of biomolecules are dissolved such that
at least some of said plurality of biomolecules adsorb to, deposit on, or
are physically or chemically bound to said second side of said boundary
surface.
8. The method according to claim 7, wherein at least one of said plurality
of biomolecules and complementary biomolecules which are complementary to
said plurality of biomolecules are labelled with a fluorescent,
phosphorescent, chemiluminescent or electroluminescent substance.
9. The method according to claim 8, wherein said second side of said
boundary surface is coated with capture molecules complementary to said
plurality of biomolecules, said solvent comprising said plurality of
biomolecules and labelled biomolecules complementary to said biomolecules.
10. The method according to claim 9, wherein said second side of said
boundary surface is coated with capture molecules complementary to said
plurality of biomolecules, said solvent comprising said plurality of
biomolecules and labelled biomolecules complementary to said biomolecules.
11. The method according to claim 1, wherein at least one of said plurality
of biomolecules and complementary biomolecules which are complementary to
said plurality of biomolecules are labelled with a fluorescent,
phosphorescent, chemiluminescent or electroluminescent substance.
12. The method according to claim 1, wherein said optically rarer medium is
a solvent in which said plurality of biomolecules are dissolved such that
at least some of said plurality of biomolecules adsorb to, deposit on, or
are physically or chemically bound to said second side of said boundary
surface.
13. The method according to claim 12, wherein at least one of said
plurality of biomolecules and complementary biomolecules which are
complementary to said plurality of biomolecules are labelled with a
fluorescent, phosphorescent, chemiluminescent or electroluminescent
substance.
14. An apparatus for detecting biomolecules at a boundary surface, said
boundary surface being between an optically denser medium and an optically
rarer medium, said biomolecules being attached on or to said boundary
surface, and said boundary surface having a film with a conductive
characteristic coupled thereto, said film being capable of conducting
surface plasmon waves, the apparatus comprising:
a source of electromagnetic waves;
polarizing means for p-polarizing the electromagnetic waves emitted by said
source of electromagnetic waves;
means for directing said p-polarized electromagnetic waves through an
optically denser medium onto said boundary surface, said boundary surface
being between said optically denser medium and an optically rarer medium;
a first monitoring means for monitoring radiation reflected or generated by
said biomolecules at said boundary surface;
a second monitoring means for monitoring electromagnetic waves reflected at
said boundary surface;
an intensity detection means coupled to said first and second monitoring
means for detecting an intensity of said electromagnetic waves reflected
at said boundary surface; and
a control means coupled to said intensity detection means and coupled to
said means for directing said p-polarized electromagnetic waves, said
control means controlling the angle of incidence of the impinging
p-polarized electromagnetic waves depending on the intensity of the
electromagnetic waves reflected at said boundary surface such that said
intensity is substantially maintained at a minimum corresponding to an
occurrence of surface plasmon resonance.
15. The apparatus according to claim 14, wherein said second monitoring
means comprises a detector, the detector being responsive only in the
spectrum of said p-polarized electromagnetic waves.
16. The apparatus according to claim 14, wherein said first monitoring
means together with said second monitoring means comprise a detector
selectively responsive to said p-polarized electromagnetic waves and said
electromagnetic waves reflected at said boundary surface.
17. The apparatus according to claim 14, wherein said second monitoring
means comprises an array of monitoring elements, with at least a central
monitoring element and at least a lateral element at either side of said
central monitoring element, a control signal generating means are
providing a control signal if the intensity detected by one of said
lateral elements is lower than the intensity detected by said central
monitoring element, said control signal being fed to said control means.
18. The apparatus according to claim 14, wherein said means for directing
said p-polarized electromagnetic waves onto the boundary surface comprises
a transparent prism.
19. The apparatus according to claim 18, wherein said transparent prism is
mounted on a support rotatable by operating means, controlled by said
control means, wherein said transparent prism is rotated an angle
(.DELTA..theta.).
20. The apparatus according to claim 19, further comprising:
transmission means for rotating said support around first angle (.theta.)
and for rotating at least one of said first and second monitoring means
twice said first angle (2.DELTA..theta.).
21. The apparatus according to claim 14, wherein said source of
electromagnetic waves is adjustable.
22. A method for detecting biomolecules, comprising the steps of:
(a) attaching a plurality of biomolecules to a boundary surface, said
boundary surface being between an optically denser and an optically rarer
medium and having a first side adjacent to said optically denser medium
and a second side adjacent to said optically rarer medium, said plurality
of biomolecules being attached by at least one of
(i) adsorbing to said boundary surface;
(ii) depositing on said boundary surface; and
(iii) physically or chemically bonding to said boundary surface, said
boundary surface having a film, the film having a conductive
characteristic being capable of conducting surface plasmon waves;
(b) irradiating said plurality of biomolecules so attached to said boundary
surface with impinging electromagnetic waves so that said impinging
electromagnetic waves pass through said optically denser medium onto said
boundary surface, wherein the angle of incidence (.theta.) of the
impinging electromagnetic waves is substantially equal to an angle
(.theta..sub.SPR) at which surface plasmon resonance occurs, surface
plasmon waves capable of causing said plurality of biomolecules at said
boundary surface to at least reflect or generate electromagnetic waves in
response to said surface plasmon wave;
(c) detecting the intensity of at least one of the electromagnetic waves
reflected and the electromagnetic waves generated by said plurality of
biomolecules at said boundary surface;
(d) determining based upon said intensity so detected at least one of:
(i) the presence of said plurality of biomolecules; and
(ii) the concentration of said plurality of biomolecules; and
(e) monitoring shifts in the angle .theta..sub.SPR by detecting the
intensity of the reflected electromagnetic waves;
(f) controlling the angle of incidence (.theta.) of the impinging
p-polarized electromagnetic waves depending on the intensity of reflected
electromagnetic waves so detected to substantially optimize the energy of
the impinging p-polarized electromagnetic waves coupled into said
plurality of biomolecules for making said determination of step (d).
23. The method according to claim 22, wherein said optically denser medium
is a transparent prism.
24. The method according to claim 22, further comprising the step of:
actuating a rotatable support carrying said transparent prism depending on
said intensity.
25. The method according to claim 24, wherein said rotatable support is
rotated in a first direction and then in a second direction opposite to
said first direction in response to an increase in said intensity, or in
case a predetermined time period (T.sub.max) has expired.
26. The method according to claim 22, wherein said electromagnetic waves
reflected at said boundary surface are fed to an array of monitoring
elements comprising at least a central monitoring element and at least a
lateral element at either side of said central monitoring element and a
control signal causing adjustment of said angle of incidence (.theta.) is
generated if the intensity detected by one of said lateral elements is
lower than the intensity detected by said central element.
27. The method according to claim 22, further comprising at least one of:
(i) passing said impinging electromagnetic waves through a dielectric layer
before passing through said film, and
(ii) passing impinging electromagnetic waves through a dielectric layer
after passing through said film.
28. The method according to claim 22, wherein at least one of said
plurality of biomolecules and complementary biomolecules which are
complementary to said plurality of biomolecules are labelled with a
fluorescent, phosphorescent, chemiluminescent or electroluminescent
substance.
29. An apparatus for detecting biomolecules at a boundary surface, said
boundary surface being between an optically denser medium and an optically
rarer medium, said biomolecules being attached on or to said boundary
surface, and said boundary surface having a film with a conductive
characteristic coupled thereto, said film being capable of conducting
surface plasmon waves, the apparatus comprising:
a source of electromagnetic waves;
polarizing means for p-polarizing the electromagnetic waves emitted by said
source of electromagnetic waves;
means for directing said p-polarized electromagnetic waves through an
optically denser medium onto said boundary surface to effect surface
plasmon resonance whereby energy is coupled into said biomolecules, said
boundary surface being between said optically denser medium and an
optically rarer medium and having a first side adjacent to said optically
denser medium and a second side adjacent to said optically rarer medium;
a first monitoring means for monitoring radiation reflected or generated by
said biomolecules at said boundary surface to determine at least one of a
presence and a concentration of said biomolecules;
an intensity detection means coupled to said first monitoring means for
detecting an intensity of said electromagnetic waves reflected at said
boundary surface;
a second monitoring means coupled to said intensity detection means for
monitoring, based on said intensity, a shift in an angle of incidence at
which surface plasmon resonance occurs (.theta..sub.SPR); and
a control means coupled to said second monitoring means and coupled to said
means for directing said p-polarized electromagnetic waves, said control
means controlling the angle of incidence (.theta.) of the impinging
p-polarized electromagnetic waves depending on the intensity of the
electromagnetic waves reflected at said boundary surface to substantially
optimize the energy of the impinging p-polarized electromagnetic waves
coupled into said biomolecules for said determination of said presence or
concentration of said plurality of biomolecules.
30. The apparatus according to claim 29, wherein said second monitoring
means comprises a detector, the detector being responsive only in the
spectrum of said p-polarized electromagnetic waves.
31. The apparatus according to claim 29, wherein said intensity detection
means comprises an array of detection elements, with at least a central
detection element and at least a lateral detection element at either side
of said central detection element, said second monitoring means monitoring
said intensity detected at said detection elements and providing a control
signal if the intensity detected by one of said lateral elements is lower
than the intensity detected by said central monitoring element, said
control signal being fed to said control means.
32. The apparatus according to claim 29, wherein said means for directing
said impinging electromagnetic waves onto said boundary surface comprises
a transparent prism.
33. The apparatus according to claim 32, wherein said transparent prism is
mounted on a support rotatable by operating means, controlled by said
control means, wherein said transparent prism is rotated an angle
(.DELTA..theta.).
34. The apparatus according to claim 33, further comprising:
transmission means for rotating said support around angle (.DELTA..theta.)
and for rotating at least one of said first and second monitoring means
twice said angle (2.DELTA..theta.). |
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Claims |
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Description |
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FIELD OF THE INVENTION
The present invention relates to a method and apparatus for detecting
biomolecules. In particular, the present invention relates to detecting
DNA by irradiating a boundary surface with visible light causing surface
plasmon resonance and detecting excitation energy reflected by the DNA
molecules.
BACKGROUND OF THE INVENTION
Several methods for the detection of biomolecules, in particular of low
concentration in a liquid, are known in the art. One method is based on
so-called evanescent waves. The biomolecules to be detected are in a
liquid solution and are brought into close contact with the surface of a
medium, (e.g. glass), which is optically denser than the liquid used. This
may be performed by adsorption of the biomolecules to complementary
biomolecules immobilized on the glass surface. The biomolecules to be
detected are marked with a fluorescent compound. Such may either be
performed directly (i.e., by means of chemical binding between the
biomolecule and the fluorescent substance), or in that biomolecules
labelled with a fluorescent dye and immobilized or chemically bound to a
part of the detection apparatus compete with the biomolecules to be
detected; i.e., the unknown biomolecule releases a fluorescent
biomolecule, which in turn adsorbs to the complementary biomolecule
immobilized on the glass surface. The latter process is described in
Badley, R. A., et al., "Optical Biosensors For Immunoassays: The
Fluorescence Capillary-fill Device", Phil. Trans. R. Soc. Lond. B 316
(1987), pp. 143-160.
Monochromatic light, from a laser source or a filtered flash lamp, strikes
the boundary surface between the optically denser medium (e.g., glass) and
the optically rarer medium (e.g., an aqueous solution). The light beam is
incident from the optically denser medium, and the angle of incidence is
equal to or larger than the critical angle so that total internal
reflection occurs. When such happens, an evanescent wave is created in the
optically rarer medium (aqueous solution); this evanescent wave penetrates
a fraction of a wavelength into the optically rarer medium. The electric
field amplitude of the evanescent wave is largest at the boundary surface
and decays exponentially with the distance from the interface.
Due to the limited depth of penetration of the evanescent wave, such wave
is suited to monitor the presence of biomolecules at the boundary surface.
It causes the fluorescent appendix of the biomolecules to emit light of a
wavelength longer than the incident wavelength (this is effectively how
fluorescence is defined). The fluorescence signal can be measured directly
by monitoring the scattered light, or by measuring the light coupled back
into the optically denser medium.
It is understood that the above technique is not limited to adsorption to a
glass/aqueous solution interface. Instead, other materials may be used as
well. It is further possible to use other effects than fluorescence which
shifts the wavelength of the incident light to larger wavelengths, (e.g.,
phosphorescence or absorbance). In the latter case, even unlabelled
biomolecules may be detected. The general method of detection is based on
measuring a refractive index change caused by the presence of the
biomolecules monitored by the refracted or reflected light, or, in other
words, by the deviation of the angle of the reflected or refracted light.
It is also known in the art to direct the incident light such that it is
reflected multiple times in a waveguide, so that it strikes the boundary
surface multiple times, see e.g., Sutherland, Ranald M. et al., "Optical
Detection of Antibody-Antigen Reactions at a Glass-Liquid Interface",
Clin. Chem. 30/9 (1984), p. 1533-1538.
Another technique for the detection of biomolecules is based on so-called
surface plasmon resonance (SPR). This method requires a thin metal film,
layer or coating (in more general terms, a conductive or semiconductive
layer) between the glass and the liquid solution. Incident light, if
impinging at a certain angle, causes surface modes (TE and/or TM modes)
associated with collective electron oscillations to propagate along the
interface between the metal film and the optically rarer medium (e.g.,
liquid solution). The incident light is usually coupled into the metal
film by means of a prism or a grating. At a specific wavelength or angle,
resonance occurs resulting in a sharp minimum or dip of reflectivity. The
resonance condition is dependent upon the optical characteristics of the
metal film, its thickness, the refractive indices of the dielectrics on
either side of it (if any) and the angle of the incident light.
The first two of these characteristics remain basically unchanged in a
given apparatus for performing surface plasmon resonance. However, the
refractive index of the optically rarer medium varies with the amount of
biomolecules bound or adsorbed to its surface. This is the property to be
monitored.
In order to detect the presence and the amount of adsorbed biomolecules,
either the variation in reflectivity at a given angle of incident light
may be monitored, or the resonance shift (the reflectivity minimum is
shifted to a different angle of incidence upon the presence of
biomolecules) may be observed.
Surface plasmon resonance may be caused either by a metal grating, or by an
evanescent wave resulting from total internal reflection (see above).
For further details of the surface plasmon resonance technique, reference
is particularly made to Daniels, P. B. at al., "Surface Plasmon Resonance
applied to Immunosensors, Sensors and Actuators", 15 (1988), p 11-18, and
Kooyman, R. P. H. et al., "Surface Plasmon Resonance Immunosensors:
Sensitivity Considerations", Analytica Chimica Acta, 213 (1988), p. 35-45.
Prior art surface plasmon techniques used the change of the refractive
index caused by the biomolecules in order to detect their presence and/or
their concentration. However, it is also known in the art to use the
evanescent wave associated with surface plasmons to excite fluorescence or
phosphorescence in an immunoassay, as described in EP-A-353 937.
A general problem in applying surface plasmon resonance is that the
biomolecules may be "overexcited", i.e. too much energy is transmitted
from the incident electromagnetic wave to the biomolecules. In such case,
the biomolecules may bleach out, i.e., they alter their characteristics
and their physical behavior, such that they may be no longer detectable.
It will be appreciated that this effect impairs the results of the
measurement.
The problem is that there is no control of the amount of energy "pumped"
into the biomolecules. Of course, the energy emitted by the source of
p-polarized electromagnetic waves is known and may be varied. However, the
fraction of the total emitted energy pumped into the biomolecules is
unknown (i.e., although the total emitted energy is known, the portion
thereof passed on to the biomolecules is unknown). The reason is that, in
prior art arrangements for the detection of biomolecules, the angle of
incidence is not the exact angle at which surface plasmon resonance occurs
(this angle will be called .theta..sub.SPR hereinafter) either because
this angle cannot exactly be met, or, even if it were possible to adjust
the apparatus exactly to .theta..sub.SPR, this angle would be subject to
drift caused by temperature effects, binding of unspecific molecules
(i.e., molecules not subject to detection), changes at the metal/liquid
interface, etc. Therefore, even the latter approach would lead to some
deviation of the actual angle of incidence, with respect to the optimum
angle .theta..sub.SPR for surface plasmon resonance.
The consequence of the actual angle of incidence not exactly corresponding
to the optimum angle for surface plasmon resonance is that only a fraction
of the total energy carried by the p-polarized electromagnetic waves is
passed on to the biomolecules. On the other hand, and as outlined above,
this fraction is unknown. Even if it were possible to determine the
deviation of the actual angle versus the ideal angle precisely, such would
not solve the problem, as the fraction of energy coupled into the
biomolecules is not a well-defined function of the deviation.
Therefore, in prior art arrangements, effective control of energy coupled
into the biomolecules has been substantially impossible. It is, of course,
possible to reduce the total amount of energy emitted by the radiation
source such that even a 100% energy coupling would not damage the
biomolecules. However, assume that a deviation between the actual angle of
incidence and the ideal angle .theta..sub.SPR for surface plasmon
resonance causes a reduction of energy transmission of 75%. In such a
case, the remaining energy pumped into the biomolecules would be
insufficient for reliable measurements. On the other hand, if the total
energy output of the radiation source were substantially increased,
operation could be performed reliably even upon a very little energy
coupling ratio, but a 100% coupling would then damage the biomolecules.
SUMMARY OF THE INVENTION
It is an objective of the present invention to provide a method for
detecting the presence and/or the concentration of biomolecules of the
kind described above for the excitation of surface plasmon resonance
waves, wherein the method allows accurate control of the energy coupled
into the biomolecules.
This object is solved in the present invention by monitoring the
electromagnetic waves reflected at the boundary surface and detecting
their intensity, and by controlling the angle of incidence .theta. of the
impinging p-polarized electromagnetic waves depending on the intensity of
the electromagnetic waves reflected at the boundary surface such that the
intensity is substantially kept at a minimum corresponding to the
occurrence of surface plasmon resonance (SPR).
The inventive method uses the fact that, if the actual angle of incidence
corresponds to the ideal angle .theta..sub.SPR for surface plasmon
resonance, then approximately 100% of the incident energy is coupled into
the biomolecules. That is, the invention ensures that these angles are
always maintained substantially equal. Then the total energy output from
the radiation source is transmitted into the biomolecules. By controlling,
the (known) energy transmitted from the radiation source, it is possible
to determine accurately the amount of energy received by the biomolecules.
The energy output of the radiation source may thus be precisely adjusted
to the biomolecules to be detected, in order to ensure optimum detection
without any damage of the biomolecules.
The ideal angle .theta..sub.SPR is kept by monitoring the intensity of the
electromagnetic waves reflected at the boundary surface and controlling
the angle of incidence such that the intensity is basically kept at its
minimum. It should be noted that electromagnetic waves reflected at the
boundary surface have usually another angle than the electromagnetic waves
generated by the biomolecules by fluorescence, phosphorescence and similar
effects. This is because the biomolecules adsorbed to the boundary surface
alter the refractive index. Further, the "induced" radiation generated by
fluorescence, phosphorescence etc. is of generally longer wavelength, or
reduced frequency, as compared to the original electromagnetic wave.
Therefore, two beams have to be monitored. A first beam originates from
the biomolecules, wherein the intensity of this beam indicates their
presence and/or concentration. A second beam is reflected at the boundary
surface (the reflection angle of this second beam being equal to the angle
of incidence). The intensity of this second beam is used to keep the angle
of incidence at .theta..sub.SPR, i.e., to meet the optimum of surface
plasmon wave excitation.
The variation of the angle of incidence, is dependent on the intensity of
the second beam and so that it is kept at a minimum, and may be affected
in any convenient manner. In a preferred embodiment wherein the optically
denser medium is a transparent prism (particularly a glass prism), such
variation may be effected by rotation of a support carrying said
transparent prism. However, there are further possibilities like moving
the radiation source along a circular path around the center of the prism.
As outlined above, the general advantage of this method is that the energy
of the radiation source may be accurately adapted to the biomolecules, in
order to provide optimum measurements whilst avoiding damage of the
biomolecules. However, there are further advantages of the inventive
method. In particular, the apparatus is always kept at its optimum point
of operation, thus saving energy. As approximately 100% of the energy is
coupled into the biomolecules, no secondary effects caused by multiple
reflected waves occur. Further, effects like temperature drift, adsorption
of unspecific molecules, changes of the boundary surface etc. are
eliminated. It is important to note that these advantages may be obtained
even if the energy output of the source of radiation is not variable. Even
in order to obtain the first advantage namely, controlled energy transfer
to the biomolecules, a variable radiation source is not necessarily
required, although such is provided in an advantageous embodiment of the
invention.
In an advantageous embodiment of the invention wherein a transparent prism
is carried by a rotatable support, said support is rotated in a first
direction and then in a second direction opposite to said first direction
in case an increase or a substantial increase in said intensity is
detected, or in case a predetermined time period has expired. An increase
or substantial increase of the reflected light is an indication that the
ideal angle .theta..sub.SPR of surface plasmon resonance has moved to
higher or lower values, so that the regions adjoining the previous angle
should be searched for the new minimum. On the other hand, to ensure
continuous proper operation, it is advantageous to scan the regions
adjoining the present point of operation from time to time, even if no
substantial increases have been noted.
In an alternative embodiment, the electromagnetic waves reflected at the
boundary surface are monitored by several monitoring elements, e.g., an
array sensor comprising elements sensitive to said electromagnetic waves
such as photodiodes or phototransistors. A central sensing or monitoring
element serves as a reference. It is a goal of this embodiment to have the
minimum intensity of the reflected electromagnetic waves focused on this
central element.
Even if the reflected beam is focused on the central element, the incident
and the reflected waves do not merely oscillate strictly in a single
plane, i.e., there are also lateral or stray waves. These are irradiated
onto the boundary surface at an angle slightly different from the optimum
angle .theta..sub.SPR for surface plasmon resonance. Sensing or monitoring
elements arranged laterally with respect to the central element are used
to record such stray waves. If the detection apparatus operates at its
optimum point, i.e., at .theta.=.theta..sub.SPR, the intensity recorded by
the lateral elements is therefore expected to be larger than the intensity
of the central element. If, however, the intensity recorded by the lateral
elements falls below the intensity recorded by the central element, this
is an indication that the apparatus is no longer operating at
.theta.=.theta..sub.SPR, either because of a misadjustment or because the
optimum wavelength for surface plasmon resonance has moved, e.g., caused
by temperature effects or due to the adsorption of unspecific molecules.
Therefore, if this happens, a control signal is generated in order to
readjust the equipment. It is understood that the above arrangement of a
central and two lateral recording elements is for the purpose of
illustration. For more accurate control, more lateral elements may be
used, e.g., an array of 11 photodiodes. It is also possible to arrange the
sensing elements in a 2-dimensional configuration rather than
1-dimensionally as described above.
Advantageously, the p-polarized electromagnetic waves are passed through a
thin dielectric film before and/or after passing through said thin
conductive film. Such dielectric film may, be arranged between a metal
film to which the biomolecules adsorb, and a glass prism. Use of such
dielectric layer sharpens the resonance peak and has other related
advantages. Basically, dielectric layers of this kind have already been
known in the art, (see e.g., EP-A-353 937).
In a further preferred embodiment, the optically rarer medium is a solvent
in which said biomolecules are dissolved such that said biomolecules
adsorb to, deposit on, or are physically or chemically bound to the
optically rarer side of said boundary surface. That is, the biomolecules
are present at the optically rarer side of the boundary surface. This is
the usual (however not the only) way of detecting biomolecules. Several
techniques may be used to establish contact between the biomolecules in
solution and the boundary surface. These techniques, most of them
well-known in the art, use various physical and/or chemical effects. The
most common one is simple adsorption. However, more sophisticated ways
like a competition process between the biomolecules to be detected and
already present, marked biomolecules (see e.g., Badley, R. A. et al,
"Optical Biosensors for Immunoassays: The Fluorescence Capillary-fill
Device", Phil. Trans. R. Soc. Lond. B316, 143-160 (1987)) have also been
used.
Advantageously, the inventive method uses biomolecules marked with a
fluorescent, phosphorescent, chemiluminescent or electroluminescent
substance. Such substances are excited by the surface plasmon wave and
emit a radiation of reduced frequency, as compared to the frequency of the
incident wave. The wave of reduced frequency is easier to detect than
waves of the same frequency (the latter approach, namely to detect only
the refractive index change caused by the biomolecules, i.e., to measure
light of the same frequency as the frequency of the incident wave, has
been used in most prior art publications). However, it has to be noted
that the present invention is not restricted to the advantageous
embodiment incorporating labelled biomolecules. Instead, measurements
based on absorption, Raman spectroscopy and nonlinear effects may be
performed by the method and apparatus according to the present invention
as well.
If labelled biomolecules are used, the biomolecules to be detected (unknown
biomolecules) may be directly labelled by a chemical binding with a
fluorescent substance. However, there are also more sophisticated ways
where the chemical structure of the unknown biomolecules need not be
altered. Usually, such techniques employ the use of further labelled
biomolecules. One such technique, namely a competition process between the
unknown biomolecules and labelled biomolecules, has already been mentioned
above. Instead, it is also possible to use complementary labelled
biomolecules.
An advantageous embodiment based on such complementary labelled
biomolecules will be described now.
According to this embodiment, the optically rarer side of said boundary
surface is coated with capture molecules complementary to the unknown
biomolecules. The solvent contains the unknown biomolecules as well as
labelled biomolecules complementary to the unknown biomolecules in
solution. That is, two kinds of complementary biomolecules are used. The
first is the capture molecules (also referred to as "capture probe")
immobilized on the boundary surface. The second kind are the molecules
labelled, for example, with a fluorescent substance and dissolved in the
solution (also referred to as "label probe"). The unknown biomolecules are
also called "target probe".
During measurement, the target probe adsorbs to the capture probe. On the
other hand, the label probe adsorbs to the target probe, such that its
label is excited by the surface plasmon wave.
The above process is particularly useful if deoxyribonucleic acid (DNA)
sequences are to be detected. In this case, a preferred embodiment of the
inventive method operates as follows. A surface plasmon wave is excited,
which in turn excites fluorescent labels. These fluorescent labels can be
attached to the biomolecules under investigation or the label probe and
will lead to a very sensitive and specific detection system.
To detect, for example, a specific DNA sequence ("target DNA"), it is
proposed first to hybridize the single strand of this target DNA with its
complementary synthetic strand in solution. The synthetic strand (label
probe) has to be longer than 16 bases to guarantee its specificity and is
prelabelled or prepared to be labelled afterwards. This target-label
complex is then immobilized at the metal interface by hybridization to
another complementary synthetic strand (capture probe), which is bound to
the metal interface. By embedding the metal film layer between layers of
different dielectrics one may optimize the strength of the evanescent wave
as well as improve the binding of the capture probe. At a certain angle of
incidence, the reflected beam will show a sharp dip, and this angle is
quasi-identical with the angle of coupling maximum power into the sample
volume with the labelled and immobilized target DNA. This sharp dip is
used as a control value to define optimal conditions for maximum photon
output of the fluorescent label; this, in turn, allows minimum excitation
power, thereby reducing the possibility of photodestruction. To further
improve the signal to noise ratio, a fluorescence label with a long decay
rate and a large Stokes shift can be applied to decrease the influence of
the intrinsic fluorescence and of the excitation light upon the detected
surface plasmon signal. It is possible to couple a great amount of the
fluorescence light which is emitted by the fluorescent molecule back into
the denser medium. The advantage of such device is a complete separation
between sample volume and optic path. In addition, the different
dielectric layers and the metal layer can be deposited onto a thin
coverglass, which itself is matched to the denser medium. Such modified
coverglass may be used as a cheap and disposable device, and the light
source and the detector may be integrated in the denser medium. The denser
medium may advantageously also have the shape of a semicylindrical prism
into which a wedge-shaped light beam with defined minimum and maximum
angle of incidence can be coupled.
A suitable fluorescence label for the above process is, for example, a
metal out of the rare earth elements. The long decay rate of these
elements has the advantage that the emission is still detectable even
after the decay of the intrinsic fluorescence, and the large Stokes shift
will result in optical decoupling between excitation light, intrinsic | | |
