U.S. patent application number 11/015179 was filed with the patent office on 2005-11-10 for method and device for the selective withdrawal of components from complex mixtures.
This patent application is currently assigned to Evotec BioSystems GmbH. Invention is credited to Eigen, Manfred, Henco, Karsten, Rigler, Rudolf.
Application Number | 20050250157 11/015179 |
Document ID | / |
Family ID | 25937730 |
Filed Date | 2005-11-10 |
United States Patent
Application |
20050250157 |
Kind Code |
A1 |
Eigen, Manfred ; et
al. |
November 10, 2005 |
Method and device for the selective withdrawal of components from
complex mixtures
Abstract
The method according to the invention permits a selected
withdrawal of one or a few molecularly disperse or cellular
components of a system, such as molecules, molecular complexes,
vesicles, micelles, cells, optionally together with an associated
volume element V having a size of 10.sup.-9
l.gtoreq.V.gtoreq.10.sup.-18 l from a larger sample volume. The
selected transfer of the sought component to another environment is
effected by defining the space and time of withdrawal by means of a
signal correlating with the small component to be withdrawn. The
method is particularly useful for the withdrawal of non-abundant
components the existence of which can be detected in a preceding
step by a scanning process. The method is also useful for the
withdrawal of per se unidentified components.
Inventors: |
Eigen, Manfred; (Gottingen,
DE) ; Rigler, Rudolf; (Dandoryd, SE) ; Henco,
Karsten; (Erkrath, DE) |
Correspondence
Address: |
JACOBSON HOLMAN PLLC
400 SEVENTH STREET N.W.
SUITE 600
WASHINGTON
DC
20004
US
|
Assignee: |
Evotec BioSystems GmbH
|
Family ID: |
25937730 |
Appl. No.: |
11/015179 |
Filed: |
December 20, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11015179 |
Dec 20, 2004 |
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08750715 |
Mar 19, 1997 |
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6849461 |
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08750715 |
Mar 19, 1997 |
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PCT/EP95/02344 |
Jun 16, 1995 |
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Current U.S.
Class: |
435/7.1 |
Current CPC
Class: |
C12M 33/07 20130101;
Y10T 436/2575 20150115; G01N 15/10 20130101; B01L 3/0268
20130101 |
Class at
Publication: |
435/007.1 |
International
Class: |
G01N 033/53 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 17, 1994 |
DE |
P 44 22 313.7 |
Jun 25, 1994 |
DE |
P 44 22 290.4 |
Claims
1-32. (canceled)
33. A method for identifying pharmacological target molecules
comprising the steps of: contacting a target molecule with a
pharmacologically active substance in a sample compartment;
irradiating that sample compartment to generate a signal
functionally related to the interaction of said substance with said
target molecule using a confocal optical system; and withdrawing a
withdrawal volume element comprising said interacting substance and
said target molecule to a receptor compartment wherein the
withdrawal is triggered by said signal.
34. The method of claim 33, wherein said confocal optical system
comprises a multitude of confocal pinhole apertures in the image
plane.
35. The method of claim 33, wherein said confocal optical system
comprises optical waveguides in the image plane.
36. The method of claim 33, wherein said confocal optical system
comprises multiarray detectors in the image plane.
37. The method of claim 33, wherein said signal is produced by a
correlated analytical system.
38. The method of claim 37, wherein said correlated analytical
system is a fluorescence correlation spectroscopy system.
39. The method of claim 33, wherein said contacting is in the
presence of a fluorescently labeled ligand.
40. The method of claim 33, wherein withdrawing said withdrawal
volume element is by receptor means selected from the group
consisting of a capillary tube or a membrane.
41. The method of claim 33, wherein said capillary tube has a tip
connecting said sample compartment to said receptor
compartment.
42. The method of claim 41, wherein said tip has an aperture with
size D according to the formula 100 .mu.m.gtoreq.D.gtoreq.0.1
.mu.m.
43. The method of claim 40, wherein said membrane has a pore
connecting said sample compartment to said receptor
compartment.
44. The method of claim 43, wherein said pore has an aperture with
size D according to the formula 100 .mu.m.gtoreq.D.gtoreq.0.1
.mu.m.
45. The method of claim 33, wherein said signal generating and
withdrawing steps are repeated in series, whereby separately
withdrawn volume elements are gathered in said receptor
compartment.
46. The method of claim 33, wherein said withdrawing step is
performed by a procedure selected from the group consisting of
inducing an electrical field between a sample fluid in said sample
compartment and a receptor fluid in said receptor compartment,
inducing in said sample compartment a pressure greater than in said
receptor compartment, inducing a light pressure impulse; and
combinations thereof.
47. The method of claim 46, wherein said withdrawing step is
performed by briefly applying an electrical field between first and
second electrodes, wherein said first electrode contacts said
sample fluid in said sample compartment and said second electrode
contacts said receptor fluid in said receptor compartment.
48. The method of claim 46, wherein said withdrawing step is
performed by inducing a pressure differential by increasing
pressure inside said sample compartment and/or by reducing pressure
inside said receptor compartment.
49. The method of claim 48, wherein said pressure differential is
caused (a) by reducing pressure using a piezo-controlled dispenser
module having a filling volume inside said receptor compartment or
(b) increasing pressure or reducing pressure caused by change of
piston position of a coupled piston pump device.
50. The method of claim 49, wherein piston pump device is
controlled by a stepping motor and the pressure increase amount is
controlled by the number of droplets dispensed by steps of the
stepping motor.
51. The method of claim 33, wherein said optical system detects
said signal, analyzes specific molecular properties of ingredients
of said sample, and time-controls the withdrawing on-line under
control of computer software.
52. A device for performing the method according to claim 33
comprising sample compartment and a receptor compartment connected
by receptor means; confocal optical system including signal
generating means cooperating with withdrawing means, connected to
said receptor means, said withdrawing means is controlled
mechanically, optically or electrically.
Description
[0001] The present invention pertains to a method for the
withdrawal of one or a few components of a system, a device for
performing said method, and uses thereof.
[0002] The functional characterization of single molecules or
molecular complexes, viruses or individual cells is possible by
means of the methods described in Rigler et al. (PCT/EP 94/00117).
By means of the method described, information can be obtained about
whether, in a complex mixture of a solution or suspension or in a
two-dimensional layer, there are contained within very small volume
elements (<10.sup.-12 l) individual molecules or molecular
complexes which are subject to particular interactions with defined
target molecules.
[0003] For many analytical and synthetic problems, it is already a
great advantage to know that a sought molecule does exist in a
mixture being analyzed. Frequently, however, the problem to be
solved by the present invention arises, that is to selectively
remove the molecule once recognized as being the desired one in
terms of a preparative enrichment, e.g., in order to avoid or to
facilitate cloning steps. Another problem in particularly diluted
solutions having a concentration of 10.sup.-12 M is to quickly find
a volume element as a part of the sample volume in which a
representant of the sought substance is present. It is by no means
necessary that the sought substance be known. When unknown
pathogens or active substances are sought, it may be that only
interactions with known substances are known, or interactions with
optionally present detector molecules can be postulated.
[0004] Methods and devices have been described for the withdrawal
of whole cells. The corresponding devices are known as cell
sorters. For example, certain cell types of a blood picture can be
identified from particular parameters of light scattering or
fluorescence, and the cells of a defined specification counted. The
cells can be stained with fluorescence-labeled antibodies and
differentiated by their surface antigens or classsified by
hybridization methods (in situ) due to their nucleic acid contents.
After their isolation in droplets, the cells are analyzed in flow
and may be selectively separated or fractioned by selective
electrostatic deflection of single droplets. Appropriate devices
are commercially available and are of great importance in clinical
diagnostics and research. PCT/EP 93/03077 discloses a method for
separating individual, linearly separated volume elements from a
capillary flow.
[0005] The object of the present invention is to provide a method
which permits to withdraw a small volume element of a solution or
suspension from a larger volume element of a sample volume of said
solution or suspension at defined space coordinates wherein said
small volume element contains a target substance. Such a small
volume element can be defined using analytical methods as described
in the Rigler et al. application (PCT/EP 94/00117). This involves
analyzing extremely small measuring volumes as a part of a larger,
surrounding sample volume by confocal illumination of a volume
element, excitation by the light used for said illumination, and/or
registration of specific fluorescence signals, and concluding
therefrom the presence of particular components. Alternatively,
said illumination may also be achieved, for example, by the method
of near field spectroscopy using apertures which are smaller than
the wavelength of the irradiated electromagnetic radiation. The
object, to selectively withdraw a sought, detected substance, a
particular molecule, a molecular complex or a cell due to their
spectral properties, is also to be achieved.
[0006] The method according to the invention may be employed to
particular advantage for detecting as yet non-identified, unknown
pathogens or immunogens. (Unknown) pathogens or immunogens can be
characterized and optionally obtained in a preparative manner. A
particularly advantageous procedure according to the invention
makes use of the fact that pathogens will first proliferate after
having infected a host organism without being prevented therefrom
by a ready immune defense. Only after a certain period of time has
elapsed, the immune defense will establish a humoral immune
response, e.g., by the synthesis of various immune globulins
(principally IgM, followed by IgG), and later a cellular immune
response. Patients suspected to have undergone an infection or
contact with an as yet unknown pathogen or immunogen without
detectability of known antigen properties with respect to a
particular reaction or detectability with known
antiserums/antibodies are the starting material for pathogen
isolation. Later, in the phase of chronic disease, it is considered
that antibodies against the suspected pathogen, e.g., a virus, will
have formed in the meantime with the virus level, however, having
largely decreased. Serums from this phase of the disease serve to
prepare the immuneglobulins.
[0007] The fraction of immuneglobulins normally contains only a
minor percentage of antibodies directed against the unknown
pathogen. The major part of the antibodies is directed against a
variety of antigen structures which are not related with the sought
specific pathogen/immunogen. Therefore, it is more difficult to
characterize the pathogen through an immune complex using serums
from one single patient.
[0008] However, the sought immune complex can be isolated by
recurring to some expected characteristics:
[0009] The immune complex contains the mass-determining major
portion of a pathogen the mobility of which is between that of a
small RNA virus and that of a bacterium.
[0010] The pathogen has several antigenic binding sites which are
occupied by more than one dye-labeled antibody.
[0011] In a preferred procedure (FIG. 5), the antibodies from two
patients in the condition of the hypothetically chronic phase can
be prepared separately and labeled with different dyes or with dyes
to be differentiated by cross correlations. Said at least two
patients are selected such that there is a high probability that
both patients have been infected by the same pathogen. Since
pathogens usually carry a large number of antigenic determinants on
the surface/viral envelope, it is likely that the immune complexes
formed after reaction with a mixture of differently labeled
antiserums will simultaneously comprise the different labeling
dyes.
[0012] The experiments are performed as illustrated in FIG. 5.
[0013] Detection of Individual Bacteria by the Binding Specifities
of Surface-Expressing Bacteria
[0014] For a large number of important applications in modern
biotechnological research, it would be extremely advantageous and
efficient if the detection of a functional biomolecule in a given
sample volume in a method could be replaced by the detection of an
individual bacterium or virus having functional surface proteins.
The decisive advantage is the interesting coupling of a
phenotypical expression product, e.g., a natural or recombinant
surface protein, to its genetic construction plan. The method, in
particular, is to be seen in the context of a preparative use
according to the invention by means of which cells or molecular
complexes determined to be the sought ones can be separated from an
environment.
[0015] Determination and Preparative Recovery of the Immunogenic
Epitopes of Microorganisms
[0016] The genome of microorganisms comprises about 10.sup.7
nucleotides.
[0017] Subgenic fragments having an average length of 100 amino
acids can be expressed according to the method described by
shot-gun expression. Taking into account the variation of reading
frames (factor 3) and an assumed non-coding counter-strand,
10.sup.8 recombinant bacterial clones contain 100 copies of each
segment. 10.sup.8 recombinant bacterial clones are contained in 1
ml of a suspension of 1 OD which can be examined individually
within about 24 hours, by the method according to the invention,
e.g., in terms of their binding properties with IgE or IgE bearing
cells from an allergic patient. According to the invention, the
correspondingly characterized bacteria are separated, or at least
highly enriched, biologically expanded, or the corresponding genome
segment is amplified and characterized by enzymatic amplification
methods.
[0018] If a corresponding bacterium is detected in the measuring
volume element, it can be immediately withdrawn from the mixture,
according to the invention, by sucking the volume element
surrounding the bacterium/molecular complex through a capillary or
separating the molecular complex by electrophoresis, electroosmosis
or dipole induction (FIGS. 1 and 2).
[0019] An idea on which the invention is based is to employ an
electrically, optically or mechanically controlled sucking device
the aperture of which is large as compared to the measuring volume,
but small as compared to the dimensions of the sample volume, in
order to achieve the object of the invention. According to the
invention, the electrical, optical or mechanical pumping system is
controlled by an FCS-controlled pulse-generator so that a small
fraction of the sample volume is separated from the total volume in
such a way that a back diffusion can be essentially excluded. This
is achieved by electrophoresis, induced dipole moments,
electroosmosis, mechanically induced pressure jump, or the pressure
of light.
[0020] The method permits the withdrawal of one or a few components
of a system, such as molecules, molecular complexes, vesicles,
micelles, cells, optionally embedded in an associated volume
element (withdrawal volume), V
(10.sup.-9l.gtoreq.V.gtoreq.10.sup.-18 l). This volume element is
part of a larger volume of an environment which contains the small
components to be withdrawn (sample volume) The withdrawal is
effected by transferring the component or components to another
environment wherein space and time of the withdrawal are determined
by an analytical signal which is correlated with the small
component to be withdrawn. The analytical methods which may be used
are those by which the molecular contents of smallest volume
elements (10.sup.31 14 l) can be analyzed as described in the
International Application of Rigler et al., PCT/EP 94/00117. The
sample volume is connected with a receptor means through a pore of
a capillary or a pore of a membrane wall whose smallest aperture D
is given by 100 .mu.m.ltoreq.D.ltoreq.0.1 .mu.m.
[0021] In a preferred embodiment, a capillary is employed as
described in Neher et al., Methods in Enzymology, vol. 207, 3-14.
This capillary is connected, e.g., with a conveying means, such as
a pump, which is capable of drawing off the solution volume
surrounding the molecular complex. It is preferred according to the
invention to use a mechanically, light pressure or electrically
controlled sucking system or a (piezo/solenoid) pump controlled
dispenser system.
[0022] Several withdrawals to the same receptor means can be
performed in one or more steps wherein the individual withdrawal
processes may be performed independently in terms of a gathering
process.
[0023] In many cases, the electrically controlled withdrawal
through a directed transport of the volume element by means of at
least one electrical voltage or field strength impulse is
preferred. Other embodiments operate mechanically by means of at
least one pressure difference impulse and/or by means of at least
one light pressure impulse in the direction of the pore aperture
using methods as published, e.g., by Weber and Greulich, Int.
Rev.
[0024] Cytol., 1992, 133, pages 1-41. The receptor means may be a
capillary the lumen of which is preferably larger than the diameter
of the pore or capillary tip the aperture of which is in direct
contact with the sample volume. In constantly thin capillaries,
electroosmosis can be performed as is common in capillay
elektrophoresis since otherwise, in mechanical volume transport,
the flow resistance could become disadvantageously large.
[0025] In the case of an electrically mediated withdrawal, the
sought component in the measuring volume is selectively transferred
to the receptor means preferably by means of an electrical field
strength impulse by shortly applying an electrical field at least
once for electrophoresis of electrically charged components and/or
for electroosmosis with coupled transport of electrically neutral
molecules. One electrode may be placed in electrically conducting
contact with the solution on the side of the sample volume while
the other electrode is placed in electrically conducting contact
with the solution on the side of the receptor means and the
conducting contact between the two compartments is established
through the pore. When the withdrawal is effected by means of a
well-aimed pressure pulse, the desired transport is caused by at
least one short increase of the pressure in the volume outside the
receptor compartment as compared to the pressure inside the
receptor compartment, and/or by a short reduction of the pressure
inside the receptor compartment. Per se known dispensers
(Microdrop) or pumping/sucking devices (solenoid pumps, stepping
motor controlled pumps) have been found to be useful. Volume
elements as small as .ltoreq.1 nl can thereby be transferred from a
larger volume of a solution or suspension to a receptor compartment
by transferring the small volume element through a pore having a
diameter of .ltoreq.100 .mu.m wherein the time and the space
coordinate of the withdrawal process is controlled by a correlated
analytical system, such as fluorescence correlation spectroscopy
(FCS).
[0026] Especially a reduced pressure pulse from a piezo-controlled
dispenser module the filling volume of which is inside the receptor
compartment, and/or a pressure pulse and/or reduced pressure pulse
causes that the volume of the receptor means is enlarged, or the
sample volume is diminished in favor of the receptor means.
According to the invention, transport to the receptor volume
ensues. The size of the received volume is controlled by the number
of the dispensed droplets or the steps of the stepping motor or the
length and intensity of the light pressure pulse.
[0027] The hardware/software-coupled on-line operating analyzing
system triggers the withdrawal time through the received
correlating signal. The withdrawal is performed in the moment when
the particle or particles to be withdrawn is/are present within the
withdrawal volume with high probability. This needs not necessarily
occur in a virtually simultaneous manner with the reception of the
positive analytical result. When a molecule, molecular complex,
virus or cell has been identified by FCS as a component to be
withdrawn, it may be withdrawn immediately or later. This is based
on the assumption that the respective component
[0028] either is present, due to forced transport as described or
electrophoretical migration, in a particular place at a defined
time shortly after the measurement and can be separated from there
electrophoretically or mechanically according to FIGS. 1 and 2;
or,
[0029] when there is no forced translation, is still present near
the detection volume from which the component can be separated
electrophoretically, optically by light pressure, or
mechanically.
[0030] Commercial cell sorters are equipped with a system for the
isolation of cells following prior analysis which may be coupled,
according to the invention, with the analytical method of
fluorescence correlation spectroscopy. Usually, cells are guided in
a liquid-coated capillary flow through a cuvette which is coupled
to a conventional fluorescence measuring device and/or a
light-scattering measuring device. In a defined space and time
interval behind the measuring device, a standardized continuous
division of the thin liquid jet into individual exiting droplets is
provided at the end of the capillary by applying a continuous sound
frequency. When a cell of a desired cell type passes the cuvette,
this cell will be present in one of these droplets after a defined
period of time which droplet can be selectively deflected from its
trajectory after the isolation by means of a coupled signal, e.g.,
by means of a pulse in an electrostatic field, and thus can be
separated in a separate receptor means. The drawback of this method
resides in the fact that in a flowing stream, only an integral
fluorescence signal in terms of a pure intensity measurement can be
obtained. In contrast, the coupling with the method of fluorescence
correlation spectroscopy in small volume elements according to the
invention enables a differentiation of the fluorescence signals
obtained by their assignment to different molecular sizes and
mobilities. This is required, e.g., for distinguishing
receptor-bound ligands from free ligands present. According to the
invention, the method of fluorescence correlation spectroscopical
detection of fluorescing particles, such as molecules, vesicles,
cells or molecular complexes, in a mechanically induced capillary
flow is employed with a selective sorter device for withdrawal
through a pore.
[0031] The actual separating process may take place after the
passage of individual volume elements through the pore by
transferring the measuring volumes yielding a positive registration
with an associated volume element to separate receptor means
following a defined space and time correlation.
[0032] In another preferred embodiment, the FCS measuring process
may also take place prior to the exit from the pore of a capillary
tip which is coupled with a microdispenser means wherein small
measuring volumes with an associated volume element of the
environment are collected as droplets in different receptor means
by various mechanical or field-induced deflections.
[0033] In particular, the time and/or space specific correlation
between the analysis of a subvolume of the sample volume (measuring
volume) within volume element V and the withdrawal of a desired
component by removing the volume element V is effected with the aid
of hardware/software it is effected in such a way that at least one
component which has been positively identified in volume element V
will be present in the withdrawn volume element V during the
withdrawal process. The pore of the receptor means is mechanically
approached to the volume element, and/or the volume element V or
parts thereof are transported to the pore of the receptor
compartment with a predetermined time correlation by transport in
the flow or by electrostatic or magnetic field gradients. The
analysis may also be performed immediately in front of the pore of
the receptor compartment. Preferably, the subvolume (measuring
volume) is smaller than the volume element V. This increases the
probability that the positively identified component is withdrawn
before it has moved too far out of the measuring volume.
[0034] The analytical method must meet certain requirements in
order that it may be employed in the present method. For example,
the signal correlating with the withdrawal process is detected by
an optical analytical system which is capable of analyzing specific
molecular properties in volume elements as small as <10.sup.-14
l. This is preferably done by analysis systems based on confocal
laser correlation spectroscopy or fluorescence correlation
analytics based on near field spectroscopy the signal of which
time-controls a selective withdrawal process on-line and in a
software-controlled fashion. A number of coupled analytical and
withdrawal processes can be sequentially employed in a cascade-like
manner according to the invention wherein the withdrawn sample
volumes are subsequently again subjected to an analysis with or
without an intermediate dilution step, and are again withdrawn
after completion of the analysis in enriched form by a second
and/or further withdrawal unit.
[0035] Such components can be withdrawn which form a complex with
at least one reaction partner capable of being spectroscopically
detected (indirectly), or which have themselves sufficient
fluorescence properties (directly).
[0036] Of particular interest is the method in combination with the
procedure for withdrawing as yet unidentified (unknown) pathogens
according to the invention. The object is to proliferate an
isolated pathogen in vivo or to proliferate the genetic material of
the pathogen or parts thereof in vitro by amplification of the
nucleic acid contained therein in terms of a shot-gun method, and
its characterization by sequencing.
[0037] Also important is the selected withdrawal of components
which have been unknown to date with respect to their molecular
nature, such as molecules, cells, vesicles, molecular complexes,
which can be functionally identified, e.g., through an enzymatic
activity or complex formation.
[0038] As outlined in FIG. 5, unknown particles, such as pathogens
or immunogens, are withdrawn according to the invention by
collecting serums from at least one organism which is infected with
the unidentified pathogen. At least one serum (serum 1) is obtained
from the phase of an acute infection by the as yet unknown pathogen
or immunogen, and at least one other serum (serum 2) is obtained
from the same or at least one other organism with the same or a
homologous infection in the phase of chronical disease.
[0039] The unknown pathogen or immunogen from serum 1 is complexed
with indirectly or immediately fluorescence-dye labeled antibodies
from serum 2, and the complex formed is measured. Important in this
connection is cross-correlation, such as described in PCT/EP
94/00117, by which, e.g., the simultaneous binding of different
fluorescing ligands, such as antibodies from different organisms,
can be measured. The labeling of antibodies may be done immediately
by at least one reaction with dyes capable of coupling, or
indirectly by reaction with dye-labeled antibody binding domains,
in particular protein A derivatives or protein G derivatives. The
unidentified pathogenic components may prove to be per se known
microorganisms. The detected characteristics are specific
interactions with surface-expressed or cytosolic-expressed
structural elements of natural or recombinant proteins or peptides
or enzymatic activities with fluorescence-labeled target
molecules.
[0040] The method according to the invention may cause the
detection of molecules in very low concentrations. For example,
scanning of fetal cells in the maternal blood may be performed.
With the method according to the invention, very low concentrations
(<10.sup.-12 M) of fluorescing molecules can be determined.
However, the method may become impracticable if, for establishing
one or more measuring volumes, too long waiting with unchanged
space coordinates is necessary until a sought molecule passes the
space element of the measuring volume. This problem also arises
with higher concentrations (>10.sup.-12 M) if the diffusion
times are very short as is the case, e.g., with cells and
cell-bound molecules. In such cases, the method according to the
invention may be modified so that the actual measuring process is
preceded by a scanning process in which the space coordinates are
variied in a measuring technique continuous or discontinuous in
time until a signal of the desired quality is detected. This may
be, for instance, the common occurrence of a correlated
fluorescence with two different emissions when the
cross-correlation method is used. When a signal is detected, the
FCS measuring process is started. The duration of a scanning
process may be less than one millisecond per measuring process.
This is sufficient to establish that the scanned measuring volume
or the measuring volumes measured in parallel do not contain the
sought component. In this approach, it has to be considered that
the absolute values of the average characteristic diffusion times
are influenced in a calculatable manner. For example, it may be
that fixed molecules (e.g. on fixed bacterial cells) directly and
exclusively reflect the variation in time of the relative change of
the space coordinates of the measuring volume with respect to the
coordinates of the sample volume, or about half of the average
dwelling time with mobile small molecules and abrupt changes of the
space coordinates since the molecules are already within the
measuring volume at the beginning of the measuring process.
[0041] Scanning processes preceding the actual measurement become
important, e.g., when cell populations are to be analyzed wherein
only a fraction of the cells, molecules or molecular complexes bear
the properties which determine the withdrawal. This is the case,
e.g., in the analysis of evolutively prepared mutant populations of
recombinant cells, but also in the analysis of maternal blood for
the presence of fetal nucleated erythrocytes which are to be
analyzed for particular genes or chromosomal anomalies.
[0042] The method is suitable for a method which will be referred
to as functional gene extraction in the following. This means the
preparation of genetic probes for the
identification/detection/cloning of specific functions which are
encoded in a whole genome or in a cDNA library. Its application may
be exemplified by the functional genome analysis by using phage or
bacterial display systems, as well as corresponding applications in
evolutive biotechnology. Both examples involve the detection and
selection of cells or phages having specific binding properties to
particular ligands before a background of non-reacting phages or
bacteria.
[0043] Thus, the number of screened volume elements significantly
increases. In combination with cross-correlation, many volume
elements can thus be screened in the As to nanosecond range in
single and multiarray operation. The dislocation is only
interrupted, e.g., when differently colored, correlating signals
can be detected in the measured volume element. When this is the
case, material parameters of the components, e.g., translational
diffusion, can be determined. This time is statistically shorter by
a time element to be calculated (about 50%) as compared to the case
that a particle must penetrate into the volume element by itself or
by forced diffusion. Once a particle is detected, it can be
detected a second time by scanning the immediate environment.
[0044] According to the invention, the measuring volume may be
composed of measuring subvolumes illuminated in parallel by
simultaneously illuminating a multitude of measuring volumes by at
least one electromagnetic radiation source using at least one
holographic grid for generating a multitude of volume elements.
[0045] The illumination of a multitude of volume elements in
parallel with confocal optics is described in DE 40 35 799. A
parallel illumination of measuring volumes the relative distances
of which are in the submicron range is not or only unsatisfactorily
accomplished by the devices described. The illuminations to be
provided in the method according to the invention having dimensions
in the lower .mu.m range and below are accomplished by using
holographic grids.
[0046] Extended arrays of small volume elements can be illuminated
by using holographic grids. According to the invention, the
measuring volumes are measured confocally for fluorescence
properties of molecules contained therein by using a multitude of
pinhole apertures in the object plane, by positioning multiarray
detector elements in the object plane, or by using optical fiber
bundles to which the light is coupled in the object plane and
transferred to photon detectors.
[0047] In the highly parallelized illumination of small volume
elements, there is the problem of registration of the emitted
fluorescence signals from the individual volume elements. In the
patent application PCT/EP 94/00117, it is reported that it is
possible to illuminate small space elements in parallel and to
focus the respective fluorescence signals individually on
multiarray detectors by using confocal pinhole aperture systems in
the object plane, or to couple the signals into optical waveguides
at the position and in lieu of the pinhole apertures and to guide
them onto detector elements, or to position the multiarray
detectors themselves in lieu of and at the position of the pinhole
apertures.
[0048] There is also described the possibility to illuminate a
larger volume element and to combine it with the above described
confocal, parallel focussing of small subvolume elements.
[0049] However, in high parallelizations, the requirements on the
number of detector arrays and the computation effort associated
with the data received in the parallel processing become
considerable. According to the invention, these problems are solved
by collecting the signals integrated over a number of space
elements with a registrating device in another mode of coupling
small space elements illuminated in parallel. This approach is
especially useful in such application in which a large number of
volume elements is to be screened;
[0050] the computation effort is to be minimized in favor of the
computing capacity employed and the computing time;
[0051] the number of the volume elements measured in one
measurement and thus the total volume measured is to be
maximized;
[0052] signals from molecules, molecular complexes or cells are to
be analyzed in high dilution;
[0053] the precision of the space-resolved detection is of minor
importance;
[0054] the number of the emitted light quantums during diffusion
through a single space element is sufficient for a correlation.
[0055] According to the invention, at least two measuring volumes
in common or assembled in groups are focussed confocally onto at
least one detector element of a photon-registrating measuring
element in the object plane in the signal registration.
[0056] For the detection of fluorescing molecules in very low
concentrations, the sample volume is subjected to a scanning
process according to the invention prior to the actual measurement
and withdrawal of at least one component wherein by a continuous or
discontinuous variation in time of the space coordinates of the
measuring volume relatively to the space coordinates of the sample
volume, the time for detecting a sought particle is shortened. The
time interval .delta.t for the measurement of one or more volume
elements having defined space coordinates prior to the detection of
a sought molecule by its fluorescence measuring signal is shorter
than the average dwelling time of the sought molecule within a
measuring volume.
[0057] A device for performing the method is characterized in that
a pore of a porous receptor means is approached closely to the
optical measuring volume and said receptor means is connected with
a mechanically, optically or electrically controllable withdrawal
device. It comprises the arrangement of a closed or open container
for receiving a sample volume, coupled with a measuring device for
the illumination and/or measurement of a small volume element
(measuring volume) by electromagnetic radiation, and at least one
connection to at least one second volume element in a receptor
means which is in direct contact with the sample volume through an
aperture and a liquid phase wherein said aperture is preferably
immediately adjacent to said measuring volume.
[0058] The method according to the invention may be used, in
particular, for the preparative recovery of unknown pathogens,
immunogens or organisms which functionally express parts of a
genome, and for the analysis and preparative recovery of nucleated
fetal cells from maternal blood.
[0059] Such problems are connected with methods for the evolutive
optimization of peptides and/or proteins by using mutagenesis
methods and selection methods, as suggested, for instance, in the
International Patent Application PCT/EP 94/00117. It is possible to
screen about 10.sup.9 bacteria for their binding properties to
specific dye-labeled substances within 24 hours, e.g., for the
presence of a bacterium which expresses a surface protein/peptide
capable of interacting with the target molecule of a predetermined
concentration. The corresponding bacterium can be cloned from such
a reaction mixture according to conventional methods or directly
isolated therefrom with the method according to the invention.
[0060] Another important field of applications results from the
so-called genome project for the functional mapping of gene
segments from genomic libraries, cDNA libraries or libraries of
subgenic structural elements (Shape Space). In this way, genomic
and/or subgenomic segments can be determined among extended
collectives by their functions, e.g. their functional binding
behavior to target molecules.
[0061] The use of the described method of functional assignment of
genetically coded peptide segments may become of great importance
particularly in allergological research. The assignment of
immunodominant epitopes on allergens (e.g. Aspergillus, milk
protein, .alpha.-amylase) is of extraordinarily great importance
and to date has been a problem difficult to solve. Typical problems
in practice are:
[0062] Determination of the IgE-binding molecules among a mixture
of substances which is undefined in most cases. For example, it is
important to answer the question as to which components of soybean
lecithin are immunogenic: the pure substance alone, the pure
substance in its interaction with contaminations of the
preparation, or the interaction with structures of the receptor
organism. According to the invention, the different immunogenic
substances can be differentiated in the mixture by means of labeled
IgE from patients.
[0063] By the expression of subgenic gene segments,
[0064] the immunodominant epitopes can be narrowed down,
characterized and preparatively recovered with the method mentioned
above. With these results,
[0065] and with the methods described in DE 41 12 440 C2,
evolutively analogous functional molecules can be generated which
lack the corresponding immunodominant regions, e.g., attenuated
immunogenic .alpha.-amylase or washing agent proteases;
[0066] the specific epitopes can be readily prepared according to
standard methods of genetic engineering, and employed as pure
detecting reagents or used for desensibilization.
[0067] By means of the method according to the invention, certain
problems can be solved which could not be solved to date or which
could only be solved with an unreasonably large effort.
[0068] Screening of pharmacologically active substances by the
binding of known fluorescence-labeled ligands to per se known
receptors which may be present on cells or natural or artificial
vesicle structures.
[0069] There are natural and chemically synthesized drugs with
pharmacological activity whose target molecules are unknown. These
target molecules can be extracellular molecules (e.g. protease
inhibitors), surface membrane receptors (e.g. insulin), soluble
receptors as mediators (steroid hormone binding receptors), or
cellular soluble structural proteins or enzymes.
[0070] Thus, according to the invention, the extremely important
problem can be solved, to find, characterize and optionally to
isolate the pharmacologically important target molecule of a known
drug:
[0071] search for orphan receptors;
[0072] elucidation of mechanisms of pharmacological action;
[0073] search for analogous active substances;
[0074] search and differentiation of different receptor molecules,
preferably in differentiatable biological targets (different cell
differentiation, tumor/non-tumor, disease-associated,
non-disease-associated, etc.).
LEGEND OF THE FIGURES
[0075] FIG. 1
[0076] (Molecule Collector)
[0077] The schematical drawing describes the principle of the
device in which the components of a small volume element can be
transferred from compartment A to a compartment B through a pore
which is the open connection between compartments A and B by means
of a pressure or reduced pressure pulse or an electrical pulse. One
part of this volume element is the measuring volume of
<10.sup.-14 l represented in dark immediately in front of the
pore in which the FCS analysis takes place. A cell or a
macromolecular complex may also be accessible by an appropriately
directed light pressure by means of a laser pulse which is
generated perpendicular to the pore diameter and can be directed to
transport a complex recognized as a desired one to the receptor
means of compartment B.
[0078] FIG. 2
[0079] (FCS Selection of Individual Microorganisms with Selected
Fractioning)
[0080] The figure shows the FCS selection of individual
microorganisms according to the invention with selected fractioning
from a continuously or discontinuosuly moved sample volume. The FCS
measuring volume is located immediately in fromt of the aperture of
a capillary pore and is represented by a hatched column within the
focussing conus of the FCS illumination or near field illumination.
In consecutive steps, measuring volumes identified as positive can
each be transported from the sample container to the receptor means
together with the surrounding volume in a computer-controlled
process. This can be accomplished, e.g., by connecting a microdrop
dispenser device or, in a simple embodiment, e.g., by coupling a
stepping motor controlled syringe (nl reception/step), or an
electrical pulse. Thus, several volume elements can be accumulated
in the receptor means during a measurement.
[0081] FIG. 3
[0082] (Cascade Enrichment)
[0083] By serially connecting separation devices as described in
FIG. 2, the separation performance can be increased. This is
important, for example, when single particles are to be separated
free of background, if possible, from a highly complex mixture
(10.sup.12 particles) of, e.g., recombinant bacteria or phages in
high concentrations.
[0084] FIG. 4
[0085] (FCS Selection of Individual Microorganisms)
[0086] The figure describes a device for separating bacteria
expressing particular properties which can be measured by FCS from
mixtures. A mixture of at first non-induced bacteria is fed to a
capillary flow system. In a mixing chamber, IPTG, for example, is
supplied as an expression inducing reagent. After a sufficiently
long flow time, the expression product is supplied with an assay
system comprising marker molecules which can subsequently be
measured at a defined position by their interaction with a possible
expression product. In addition, the figure is to indicate that a
positively identified measuring volume may also be withdrawn at a
position distant with respect to space and time provided that the
space/time coordinates are in a known and defined relationship to
one another.
[0087] FIG. 5
[0088] (Detection and Preparation of New Pathogens)
[0089] The figure demonstrates the procedure according to the
invention when unknown pathogens are selected which can be detected
via cross-correlation by means of FCS wherein the differently
labeled antibodies directed against a particular pathogen may
preferably be derived from different patients.
* * * * *