Note: Descriptions are shown in the official language in which they were submitted.
CA 02440183 2003-09-05
Determination of analytes by fluorescence correlation
spectroscopy
Description
The invention relates to a method for the
determination of an analyte in a sample by fluorescence
correlation spectroscopy, the distance between the
measurement volume in the sample and the optical
excitation/detection direction being >- 1 mm and the
sample liquid being thermally insulated from the
optical instrument. The method is especially suitable
for the measurement of temperature-variable processes,
for example the determination of nucleic acid
hybridization melting curves and/or for carrying out
nucleic acid amplification reactions. A device suitable
for carrying out the method according to the invention
is also disclosed.
The use of fluorescence correlation
spectroscopy (FCS) for the detection of analytes is
known. EP-B-0 679 251 discloses methods and devices for
the detection of analytes by means of fluorescence
spectroscopy, the determination being carried out in a
measurement volume which is part of the sample to be
studied, and the measurement volume being arranged at a
working distance of _< 1000 um from an optical focusing
device. Furthermore, the focusing instrument is either
directly in contact with the sample or separated from
the sample merely by an optically transmissive film. It
has previously been assumed that the short distance and
the direct contact between the sample and the optical
device is a necessary feature of fluorescence
correlation spectroscopy. This arrangement, however,
has the disadvantage that processes involving a
temperature change of the sample can only be studied
with great difficulty.
DE-A-36 42 798 discloses a microscope with a
stand which is displaceably mounted on an object stage,
the drive instrument for vertical displacement of the
CA 02440183 2003-09-05
- 2 -
object stage forming an independent support of the
object stage, separate from the guide device. The
temperature of the microscope can be regulated by the
delivery of cooling air and by fitting thermal
protection filters. There is no indication of use in
fluorescence correlation spectroscopy.
GB-A-2 351 556 describes a method for the
measurement of radiation, a plurality of confocal
optical single-channel systems and photoelectric
detectors being arranged in parallel in order to form a
plurality of read heads, which are arranged next to one
another so as to be able to evaluate radiation from
corresponding regions simultaneously. 10 x microscope
objectives with a numerical aperture of 0.4, a focal
length of about 8 mm and an aperture diameter of 5 mm
may be used as the focusing optics. There is absolutely
no indication that the focusing optics may be thermally
insulated from the sample.
DE-C-42 18 729 discloses capillary
electrophoresis apparatus with an optical position
resolving detection system. The temperature inside the
capillary can be thermostatted. There is no indication
of confocal fluorescence correlation spectroscopy.
DE-A-197 48 211 describes an optical system
with a lens array which can be used for fluorescence
correlation spectroscopy. In order to produce confocal
volume elements in the samples, lenses are used with a
focal length of f - 7.5 and a numerical aperture of
0.6. There is absolutely no indication of thermal
insulation of the focusing optics from the sample.
DE-A-199 19 092 describes an arrangement for
the optical evaluation of an object array, which may be
used for fluorescence analysis. Here again, there is
absolutely no indication of thermal insulation of the
focusing optics and the samples.
It is an object of the present application to
provide methods and devices for carrying out
fluorescence correlation spectroscopy, which at least
partially avoid the disadvantages of the prior art.
CA 02440183 2003-09-05
- 3 -
The invention therefore relates to a method for
the determination of an analyte in a sample by
fluorescence correlation spectroscopy, comprising the
steps of:
(a) preparing a sample liquid in a support,
(b) carrying out a luminescence measurement by optical
stimulation of luminescent molecules in a measurement
volume, which is part of the sample liquid, by using a
light source with focusing optics and a detector for
picking up emission radiation from the measurement
volume,
characterized in that the distance between the
measurement volume in the sample liquid and the
focusing instrument of the light source is >- 1 mm and
in that the sample liquid is thermally insulated from
the light source and, in particular, from the focusing
optics.
An essential advantage of the method according
to the invention is that the temperature of the
support, and therefore of the sample, can be adjusted
and varied independently of the optical
excitation/detection device, and in particular its
focusing optics, for example one or more micro-
objectives.
In other regards, the method may basically be
carried out according to the method described in EP-B-0
679 251. In this case, the measurement of one or a few
analyte molecules is preferably carried out in a
measurement volume, the concentration of the analyte
molecules to be determined preferably being < 10-14
mol/1. Substance-specific parameters are determined,
which are found by luminescence measurement of the
analyte molecules. These parameters may be translation
diffusion coefficients, rotation diffusion coefficients
and/or the excitation wavelength, the emission
wavelength and/or the lifetime of an excited state of a
luminescent molecule or the combination of one or more
of these measurement quantities. For specifics about
CA 02440183 2003-09-05
_ 4 _
equipment details, reference is made to the disclosure
of EP 0 679 251.
An essential feature of the method according to
the invention is that the distance between the
measurement volume in the sample liquid and the
focusing optics of the light source is >- 1 mm,
preferably from 1.5 to 10 mm and particularly
preferably from 2 to 5 mm. It is furthermore preferable
for a gas phase region, which may contain air,
protective gas or vacuum, to be arranged between the
support containing the sample liquid and the optical
focusing instrument.
The support used for carrying out the method
according to the invention is preferably a
microstructure which contains a plurality of preferably
separate containers for holding samples. The volume of
these containers is preferably in the range of -< 10-6 1,
and particularly preferably <- 10-a 1. For instance, the
support may comprise a microwell structure with a
plurality of wells for holding sample liquid, which for
example have a diameter of between 10 and 1000 um.
Suitable microstructures are described, for example, in
DE 100 23 421.6 and DE 100 65 632.3. The support
furthermore preferably comprises at least one
temperature control element, for example a Pettier
element, which allows temperature regulation of the
support and/or individual sample containers in it.
The support used for the method is furthermore
expediently configured in such a way that it allows
optical detection of the sample. A support which is
optically transparent at least in the vicinity of the
sample containers is therefore preferably used. The
support may in this case either be fully optically
transparent or contain an optically transparent base
and an optically opaque cover layer with openings in
the sample containers. Suitable materials for supports
are, for example, composite supports made of metal (for
example silicon for the cover layer) and glass (for the
base). Such supports may, for example, be produced by
CA 02440183 2003-09-05
- 5 -
applying a metal layer with predetermined openings for
the sample containers onto the glass. Plastic supports,
for example made of polystyrene or polymers based on
acrylate or methacrylate may alternatively be used. It
is furthermore preferable for the support to have a
cover for the sample containers, in order to provide a
system which is closed and essentially isolated from
the surroundings during the measurement.
In a particularly preferred embodiment, a
support is used which contains a lens element arranged
in the beam path between the measurement volume and the
light source or the detector of the optical device. For
example, the lens element may be fitted at the bottom
of a microwell structure. Such a lens element may, for
example, be produced by heating and shaping a
photoresist by using a master mold, for example made of
metal such as silicon, and then applied onto the
support. Alternatively - for example when supports made
of a fully plastic structure are being used - the lens
elements may be integrated into the support, for
example produced during production by injection
molding. The numerical aperture of the optical
measuring arrangement may be increased by using a lens
element, preferably a convex lens element. This
numerical aperture is preferably in the range of from
0.5 to 1.2.
The support is furthermore preferably coated
with a transparent antireflection coat, in order to
obtain a higher refractive index. Transparent oxides or
nitrides may, for example, be used as antireflection
coats. Antireflection coatings are preferably also used
on the optics.
Electric fields may furthermore be produced in
the support, especially in the vicinity of the sample
containers, in order to achieve concentration of the
analytes to be determined in the measurement volume.
Examples of electrodes which are suitable for producing
such electric fields are described, for example in DE
101 03 304.4.
CA 02440183 2003-09-05
- 6 -
Owing to the thermal decoupling of the support
and the optics, the method according to the invention
makes it possible to carry out a determination at a
different temperature from the surroundings, and in
particular allows variation of the temperature during
the measurement. The spatial decoupling of the support
and the optics allows simplified scanning of supports,
in particular microwell structures with a plurality of
separate sample containers.
The method according to the invention is in
principle suitable for the detection of any analytes.
One or more substances which bind analytes, and which
carry marking groups that can be detected by
luminescence measurement, especially fluorescent
marking groups, are preferably added to the sample. In
this case, the method according to the invention
preferably comprises determination of the binding of
the marking substance to the analyte to be detected.
This detection may, for example, be carried out by
using a mobility change of the marking group due to the
binding to the analyte or using a change in the
luminescence of the marking group (intensity and/or
decay time) due to the binding to the analyte, or by
the so-called cross-correlation if a plurality of
marking groups are being used. In the cross-correlation
determination, at least two different markings are
used, especially fluorescent markings, whose correlated
signal inside the measurement volume is determined.
This cross-correlation determination is described, for
example, in Schwille et al. (Biophys. J. 72 (1997),
1878-1886) and Rigler et al. (J. Biotechnol. 63 (1998),
97-109) .
The method according to the invention is
especially suitable for the detection of biomolecules,
for example nucleic acids, proteins or other analyte
molecules which occur in living bodies, especially in
mammals such as humans. It is furthermore possible to
detect analytes which have been produced from
biological samples in vitro, for example cDNA molecules
CA 02440183 2003-09-05
- 7 -
which have been produced from mRNA by reverse
transcription, or proteins which have been produced
from mRNA or DNA by in vitro translation. The method is
furthermore suitable for the detection of analytes
which exist as elements of a library and are intended
to show predetermined characteristics, for example
binding to the detection reagent. Examples of such
libraries are phage libraries or ribosomal libraries.
In a particularly preferred embodiment, the
determination comprises a nucleic acid hybridization,
with one or more luminescence-marked probes binding to
a target nucleic acid as the analyte. Such
hybridization methods may, for example, be used for the
analysis of gene expression, for example in order to
determine a gene expression profile, or for the
analysis of mutations, for example single-nucleotide
polymorphisms (SNPs). The method according to the
invention is, however, also suitable for the
determination of enzymatic reactions and/or for the
determination of nucleic acid amplifications,
especially in one or more thermocycling processes.
Preferred methods for the determination of nucleic acid
polymorphisms are described in DE 100 56 226.4 and DE
100 65 631.5. A two-color or multi-color cross-
correlation determination is particularly preferably
carried out in this case.
In a further particular preferred embodiment,
the determination comprises the detection of a protein-
protein or protein-ligand interaction, in which case
low molecular-weight active agents, peptides, nucleic
acids etc . may be used as protein ligands . A two-color
or multi-color correlation method is also preferably
carried out for such determinations.
In an alternative preferred embodiment, so
called "molecular beacon" probes or primers may be
used, which -when they are in the free form - give rise
to a different measurement signal in respect of the
luminescence intensity and/or decay time than in the
bound state.
CA 02440183 2003-09-05
In yet another alternative preferred
embodiment, the determination may comprise the
measurement of an energy transfer, which is caused by
at least one luminescence marker as the energy donor
and at least one luminescence marker as the acceptor.
The donor and acceptor are present in a complex
containing the analyte and one or more detection
reagents, preferably at least 2 detection reagent.
The invention also relates to a device for the
determination of an analyte by means of fluorescence
correlation spectroscopy (FCS), in particular for
carrying out the method, comprising
(a) a support with at least one container for holding a
sample liquid, which contains the analyte to be
determined,
(b) an optical excitation instrument comprising a light
source and focusing optics for the stimulation of
luminescence in a measurement volume, which is part of
the sample liquid, and
(c) an optical detection instrument for the detection
of luminescence from the measurement volume,
characterized in that the distance between the focusing
optics and the measurement volume is ~ 1 mm, and in
that the support is thermally insulated from the
excitation instrument.
The support is preferably a microstructure with
a plurality of, preferably at least 102, containers for
holding a sample liquid, in which case the sample
liquid in the separate containers may come from one or
more sources. The sample liquid may, for example, be
introduced into the containers of the support by means
of a piezoelectric liquid delivery device.
The containers of the support are configured in
such a way that they allow binding of the detection
reagent to the analyte in solution. The containers are
preferably wells in the support surface, in which case
these wells may in principle have any shape, for
example circular, square, rhombic etc. The support may
even comprise 103 or more separate containers.
CA 02440183 2003-09-05
- 9 -
The optical excitation instrument comprises a
strongly focused light source, preferably a laser beam,
which is focused onto the measurement volume in the
sample liquid by means of appropriate optical
instruments. The light source may also contain two or
more laser beams, which are then respectively focused
onto the measurement volume by different optics before
entering the sample liquid. The detection instrument
may, for example, contain a fiber-coupled avalanche
photodiode detector or an electronic detector. It is,
however, also possible to use excitation and/or
detection matrices consisting of a point matrix of
laser points, produced by diffraction optics or a
quantum-well laser, as well as a detector matrix
produced by an avalanche photodiode matrix, or an
electronic detector matrix, for example a CCD camera.
The support may be provided in prefabricated
form, a plurality of separate containers of the support
being filled with luminescence-marked detection
reagents, preferably luminescence-marked hybridization
probes or primers. The support containing the detection
reagents is then advantageously dried.
In a preferred embodiment of the invention, a
prefabricated support is provided which contains a
multiplicity of, for example 100, separate containers
which are respectively filled with different detection
reagents, for example reagents such as primers and/or
probes for the detection of a nucleic acid
hybridization. This support may then be filled with a
sample coming from a body to be studied, for example a
human patient, so that different analytes from a single
sample are determined in the respective containers.
Such supports may, for example, be used to compile a
gene expression profile, for example for the diagnosis
of diseases, or for the determination of nucleic acid
polymorphisms, for example for the detection of a
particular genetic predisposition.
The invention furthermore relates to a method
for the determination of nucleic acid polymorphisms.
CA 02440183 2003-09-05
- 10 -
This method is preferably used in combination with the
aforementioned fluorescence correlation spectroscopy
method, although it may also be used for other types of
single-molecule detection or for conventional detection
methods.
This method comprises the steps of:
(a) preparing a nucleic acid matrix to be studied and
two probes that bind to the matrix under hybridization
conditions, the probes being selected in such a way
that
(i) they bind directly to the matrix next one another,
so that the 3' end of the first probe is directly next
to the 5' end of the second probe,
(ii) positions at which the occurrence of a
polymorphism is to be expected on the matrix are
selected for the 3' end of the first probe and/or the
5' end of the second probe,
(iii) the nucleotides at the 3' end of the first probe
and/or at the 5' end of the second probe are
complementary to the respective positions on the matrix
when it has a first predetermined variant of the
polymorphism, and not complementary to the respective
positions on the matrix when it has another variant of
the polymorphism,
(b) hybridization of the probes onto the matrix,
(c) treatment of the hybridization complex comprising
the matrix and the first and second probes bound to it,
with a ligase under conditions such that ligation of
the first and second probes takes place selectively
only if the nucleotide at the 3' end of the first probe
and the nucleotide at the 5' end of the second probe
are complementary to the respective positions of the
matrix, and
(d) detection of whether a ligation has taken place
between the first and second probes, in order to
determine the variant of the polymorphism present on
the matrix.
In the simplest case, the nucleic acid
polymorphism is a single-nucleotide polymorphism (SNP).
CA 02440183 2003-09-05
- 11 -
The polymorphism may, however, also affect two or more
nucleotides.
DNA of any origin, for example from natural
sources but also recombinantly produced DNA or
synthetic DNA, may be used as nucleic acid matrices.
The DNA preferably comes from a body in which nucleic
acid polymorphisms occur, which are intended to be
determined by the method. The DNA is preferably used in
single-stranded form, for example as single-stranded
cDNA. It is, however, also possible to use double-
stranded DNA, which is separated into single-stranded
DNA by heating and is then used for the hybridization
with the probes.
The hybridization probes preferably consist at
least partially of single-stranded DNA. In any event,
the 3' end of the first probe must be ligatable to the
5' end of the second probe, preferably with the use of
a DNA ligase. The probes may, however, also be formed
by nucleic acid analogs, for example peptide nucleic
acids. Preferably, the first probe contains a free 3'
OH group at the 3' end and the second probe contains a
5' phosphate group at the 5' end.
Preferably, the first and second probes each
carry a different marking group, the joint occurrence
of which can be detected by a cross-correlation
determination. This means that the presence of the two
different marking groups in a single molecule (ligation
product) can be detected separately from presence in
two separate molecules (first and second probe). The
size differences between the ligation product and the
individual probes results in very different
hybridization melting points, and therefore in a high
sensitivity of the method. If, for example, a melting
point curve measurement of the hybrids is carried out,
the cross-correlation for the single probes vanishes
immediately above the lowest melting point of an
individual probe, while the ligation product still
gives a cross-correlated signal up to substantially
CA 02440183 2003-09-05
- 12 -
higher temperatures (up to its melting point, which is
preferably >- 10°C higher).
Fluorescence markings are preferably used as
the marking groups, for instance, fluorescein,
rhodamine, phycoerythrin, CY3, CY5 or derivatives
thereof. The discrimination of the fluorescence marking
groups may be carried out using the emission
wavelength, using the lifetime of the excited states or
using a combination thereof.
As mentioned above, the detection may
preferably be carried out by means of single-molecule
determination, for example with position and/or time-
resolved fluorescence spectroscopy which is capable of
detecting fluorescence signals in a very small volume
element like in a microchannel or a microwell down to
single-photon counts.
For example, the detection may be carried out
by means of confocal single-molecule detection, for
instance by fluorescence correlation spectroscopy.
Alternatively, the detection may also be carried out by
a time-resolved decay measurement, so-called time
gating, in which case the fluorescence molecules inside
a measurement volume are stimulated and a detection
interval on the photodetector is then opened preferably
in a time interval of >- 100 ps. This measurement method
is, for example, described by Rigler et al. in
"Ultrafast Phenomena" D. H. Auston, ed. Springer 1984.
The present invention will also be explained by
the following figures, in which:
Figure 1A shows the schematic representation of
a support (2) which is suitable for carrying out the
method according to the invention, with a multiplicity
of containers (4) for holding sample liquid which are
designed in the form of wells on the support. A support
with an area of 1-2 cmz may, for example, contain up to
104 wells .
For the detection of an analyte in a container
(4), an excitation and detection device preferably
arranged under the support base may be used. This
CA 02440183 2003-09-05
- 13 -
device may contain a light source (6), for example a
laser, with which light can be shone via an optical
focusing instrument (8) into a measurement volume (10)
inside the sample liquid. The luminescence radiation
emitted from the measurement volume is conducted via
the optical focusing instrument (8; 8a) to a detector
(12). The measurement volume (10) is arranged at a
working distance (14) of >- 1 mm, and preferably > 1 mm
from, the focusing instrument (8). The support (2) is
furthermore thermally insulated from the optical
excitation and detection device, for example by
arranging a gas phase region between the optical
focusing instrument (8) and the support (2).
Figure 1B shows the schematic representation of
a particularly preferred embodiment of a support (20)
which is suitable for carrying out the method according
to the invention. The support (20) also has a
multiplicity of separate containers (22) for holding a
sample liquid. A preferably convex lens element (24) is
additionally arranged in the beam path between the
optical excitation and detection device (not shown) and
the measurement volume (not shown) contained in the
sample liquid. This lens element advantageously has an
anti-reflecting coat (26).
Figure 2 shows the schematic representation of
a particularly preferred embodiment of a support (30)
which is suitable for carrying out the method according
to the invention, with a multiplicity of separate
containers (32) for holding sample liquids. The support
furthermore contains a temperature control element
(34), for example a Peltier element. The temperature
control element is preferably arranged at least
partially around the support circumference. In order to
allow a temperature-variable determination, the
containers (32) are provided with a cover (36) from the
surroundings, which essentially insulates the sample
liquid from the surroundings. For example, seals (not
shown) may be used for this purpose.
CA 02440183 2003-09-05
- 14 -
Figure 3 shows a plan view of the support (30)
with the temperature control element (34) represented
in Figure 2. This arrangement may optionally be fitted
on a frame holder (36) with the use of additional
heating or cooling elements (not shown).
Figure 4 shows the introduction of the sample
into a support (40) with separate containers (42a; 42b)
via accesses (44a, 44b). The containers (42a) and (42b)
are separated by a barrier (46). The barrier may be
designed as a permanent barrier or as a valve, for
example a hydrophobic diaphragm valve which can be made
permeable by pressure application. The sample liquid
may be introduced simultaneously into a plurality of
containers, or - after the first container has been
filled - via a valve (46) into the second container.
Figure 5 shows a particularly preferred
embodiment of the introduction of sample liquid into
the support. The sample liquid is conducted from a
reservoir (50), optionally integrated on the support
itself, respectively via feed lines (52) in parallel
into sample containers (54). The sample liquid may
optionally be conducted from the containers (54) via a
valve (56) into a further container (58), that is to
say the parallel filling may be combined with serial
filling.
