EP1448798A1

EP1448798A1 - Nanostructure, in particular for analysing individual molecules

Info

Publication number: EP1448798A1
Application number: EP02792817A
Authority: EP
Inventors: Thomas Ericson
Original assignee: Gnothis Holding SA
Current assignee: Gnothis Holding SA
Priority date: 2001-11-27
Filing date: 2002-11-27
Publication date: 2004-08-25
Also published as: WO2003046216A1; US20050164181A1; AU2002358552A1

Abstract

The invention relates to a nanostructure and to a method for analysing or synthesising a small quantity of molecules or individual molecules, in particular for the sequencing of individual molecules of nucleic acids.

Description

Nanostructure, especially for the analysis of single molecules

description

The invention relates to a nanostructure and its use for the synthesis and analysis of molecules, in particular single molecules, for. B. for single molecule sequencing of nucleic acids.

The sequencing of the human genome consisting of approx. 3 x 10 ⁹ bases or the genome of other organisms and the determination and comparison of individual sequence variants requires the provision of sequencing methods which are fast on the one hand and can be used routinely and at low cost on the other hand. In recent years great efforts have been made to use common sequencing methods, for example the enzymatic chain termination method according to Sanger et al. (Proc. Natl. Acad. Sci. USA 74 (1 977) 5463), especially through automation (Adams et al., Automated DNA Sequencing and Analysis (1 994), New York, Academic Press). A maximum of 500,000 bases per day can currently be determined with a sequencer (Mega Bace from Applied Biosystems). However, the conventional sequencing methods are less suitable for some applications.

Over the past few years, new approaches to overcome the

Limitations of conventional sequencing methods have been developed, including sequencing by scanning tunneling microscopy (Lindsay and Phillip,

Gene. Anal. Tech. Appl. 8 (1 991), 8-1 3), by highly parallelized capillary electrophoresis (Huang et al., Anal. Chem. 64 (1 992), 2149-21 54; Kambara and Takahashi, Nature 361 (1 993), 565-566 ), by oligonucleotide hybridization (Drmanac et al., Genomics 4 (1 989), 1 14-1 28; Khrapko et al., FEBS Let. 256 (1 989), 1 1 8-1 22; Maskos and Southern, Nucleic Acids Res. 20 (1 992), 1 675-1 678 and 1 679-1 684) and by matrix-assisted laser desorption / ionization mass spectroscopy (Hillenkamp et al., Anal. Chem. 63 (1 991 ), 1 1 93A- 1 203A).

Another approach is single molecule sequencing (Dörre et al., Bioimaging 5 (1 997), 139-1 52), in which the sequence of nucleic acids is achieved by the progressive enzymatic degradation of fluorescence-labeled single-stranded DNA molecules and detection of the sequentially released monomer molecules in a microstructure channel takes place in which the monomer molecules are guided electroosmotically by pumps. The advantage of this method is that only a single molecule of the target nucleic acid is sufficient to carry out a sequence determination.

A disadvantage of the Dörre et al. However, the method described is that the sequentially released monomer molecules can interact with the walls of the microstructures, which can lead to considerable problems in the evaluation.

WO 99/36766 describes methods and devices for regulating laminar fluid flows in a flow cell. Discrete sensor areas are generated that can be used to detect analytes in a sample liquid.

PCT / EP01 / 07460 proposes methods and devices for single-molecule sequencing. For this purpose, a system of microchannels in fluid communication is used, in which liquids are guided through the microchannels by means of a hydrodynamic flow. The object underlying the present invention was to provide a device and a method for the synthesis and analysis of molecules, in particular for single-molecule sequencing of nucleic acids, which represent a further improvement over the prior art and in particular enable a simplified analysis.

This object is achieved by providing an apparatus comprising:

(a) comprising a system of fluidic communication, at least partially optically transparent microchannels

(i) a first microchannel for introducing a first fluid stream, comprising a carrier particle with at least one molecule immobilized thereon,

(ii) at least one second microchannel for introducing at least one second fluid stream, comprising one

Reactants for the immobilized molecule,

(iii) optionally at least one third microchannel for introducing at least one third fluid stream,

(iv) at least one fourth microchannel, the microchannels (i), (ii) and (iii) opening into the fourth microchannel and wherein at least in a section of the fourth microchannel at least the first and second fluid flows in the

Flow essentially without mixing,

(b) means for holding the carrier particle at a predetermined position in the region of the section of the fourth microchannel in which the fluid streams flow essentially without mixing,

(c) means for contacting the retained carrier particle with the second fluid stream containing the reactants and

(d) optionally means for detecting a reaction of the immobilized molecule with the reactant. The device according to the invention is based on the principle that a carrier is provided with a plurality of microchannels in fluidic communication, at least two and preferably three or four or more microchannels being provided for introducing different fluid flows into the carrier. These microchannels open into another microchannel in which the immobilized molecule reacts with one or more reactants. A carrier particle loaded with one or more immobilized molecules is introduced into the device through a first microchannel in a first fluid stream. Second fluid streams, each containing reactants for the immobilized molecule, were introduced into the device in one or more second microchannels. One or more third microchannels are preferably provided, which are used to introduce one or more third fluid streams. These third fluid streams can contain buffer solutions, for example. B. serve to separate the first fluid stream from one or more second fluid streams. The geometry of the device is designed in such a way that the fluid streams flowing into the fourth microchannel are essentially not mixed at least in a section of the fourth microchannel, in particular in the region of the mouth zone, the individual fluid streams preferably flowing in a laminar flow.

After introduction into the device, the loaded particle is by suitable means at a first predetermined position in the fourth microchannel, for. B. in the area of the mouth zone. At this position, the held particle can be brought into contact by suitable measures with one or more second fluid streams, which contain free reactants for the immobilized molecule, so that a reaction between the immobilized molecule and one or more free reactants can take place. The presence of separate, preferably laminar, fluid flow zones in the fourth microchannel allows the contacting between the molecule and the reactants to be controlled in a targeted manner become. The reaction can preferably be detected downstream in the fourth microchannel.

In a first embodiment, the held carrier particle is subjected to a transport within the fourth microchannel from the first predetermined position to a second predetermined position. This transport takes place from the zone of the first fluid stream into the zone of a second fluid stream and preferably across the zone of a third fluid stream. It is further preferred that the transport takes place essentially transversely to the direction of flow.

In a further embodiment, the carrier particle held does not have to be transported, but the carrier particle can be brought into contact with the second fluid stream by changing the flow conditions in the fourth microchannel, e.g. can be achieved by changing the flow velocities in one or more microchannels, so that the captured carrier particle, which is initially in a zone corresponding to the first fluid flow, reaches a zone by changing the flow conditions in which it comes into contact with the second fluid flow. Of course, the two aforementioned embodiments can also be combined with one another.

In addition to the first and the one or more second microchannels which transport the carrier particle with the immobilized molecule and the reactant (s) into the device, one or more third microchannels are preferably also provided which, for example, inert fluid flows, such as buffer solutions, into the device transport. The third microchannel (s) can be arranged in such a way that one or more third fluid streams are created in the fourth microchannel, which are arranged between the first and second fluid streams. The device according to the invention can be used for the analysis of molecules. When using carrier particles on which only a single molecule is immobilized, the device is suitable for carrying out single-molecule analyzes, for example for characterizing biomolecules. A particularly preferred area is the sequencing of nucleic acids. In addition, the device can also be used for the synthesis of molecules, a starting product being immobilized on the particle carrier which is brought into contact with one or more reaction partners simultaneously or sequentially in the device according to the invention. In this way, for example, organic compounds, e.g. B. complex organic compounds or biopolymers such as peptides, polypeptides, nucleic acids and nucleic acid analogs can be synthesized. The device is particularly suitable for the synthesis of individual molecules.

If the device for analytical applications, e.g. B. is used for nucleic acid sequencing, it advantageously contains a means for detecting a reaction of the immobilized molecule with the reactant, for example a suitable detector. If the device is used for synthetic purposes, it advantageously contains means for isolating the molecule synthesized on the carrier particle, if necessary after cleavage from the carrier particle.

Furthermore, the device can contain means for introducing fluid flows into the microchannels, means for discharging fluid flows from the microchannels and reservoirs for first, second and possibly third fluid flows. In addition, it preferably contains automatic manipulation devices, heating or cooling devices such as Peltier elements, means for sorting carrier particles and / or synthesis products, reagents and electronic control and evaluation devices. Another object of the invention is a method for performing a reaction between an immobilized molecule and a free reactant, e.g. B. for single molecule analysis and in particular for single molecule sequencing, comprising: (a) providing a carrier particle with a molecule immobilized thereon,

(b) introducing the carrier particle into a system of fluidic communication, at least partially optically transparent microchannels, the device comprising: (i) a first microchannel for introducing a first

Fluid stream comprising the carrier particle, (ii) at least one second microchannel for introducing at least one second fluid stream comprising a reactant for the molecule to be immobilized, (iii) optionally at least a third microchannel for

Introducing a third fluid flow,

(iv) at least a fourth microchannel, the microchannels

(i), (ii) and (iii) open into the fourth microchannel and wherein at least in a section of the fourth microchannel at least the first and second fluid flows in the

Flow essentially without mixing,

(c) holding the carrier particle at a predetermined position in the region of the section of the fourth microchannel in which the fluid streams flow essentially without mixing, (d) bringing the retained carrier particle into contact with at least a second fluid stream containing the reactants and (e) optionally detecting the reaction between the immobilized molecule and the reactant.

The method according to the invention is preferably a sequencing method in which a single nucleic acid molecule immobilized on a carrier is examined. However, the method is suitable for other analytical and synthetic reactions with a small number of z. B. can be carried out up to 1,000 molecules or even only with single molecules. It is particularly preferred to carry out analytical reactions which can be detected within the device by optical methods.

The carrier particle used for the method has a size that enables movement in microchannels and retention in a desired position within a sequencer. The particle size is preferably in the range from 0.5 to 10 μm and particularly preferably from 1 to 3 μm. Examples of suitable materials for carrier particles are plastics such as polystyrene, polymethyl methacrylate, polypropylene, polycarbonate or copolymers thereof, glass, quartz, metals or semimetals such as silicon, metal oxides such as silicon dioxide or composite materials which contain several of the aforementioned components. Optically transparent carrier particles, for example of plastics or glass, particles of silicon or particles with a plastic core and a silicon or silicon dioxide shell are particularly preferably used.

Nucleic acid molecules are preferably immobilized on the carrier particle via their δ 'ends. The nucleic acid molecules can be bound to the support by covalent or non-covalent interactions. For example, the binding of the polynucleotides to the carrier can be mediated by high-affinity interactions between the partners of a specific binding pair, for example biotin / streptavidin or avidin, hapten / anti-hapten antibodies, sugar / lectin etc. In this way, biotinylated nucleic acid molecules can be coupled to streptavidin-coated supports. Alternatively, the nucleic acid molecules can also be bound to the support by adsorption. For example, nucleic acid molecules modified by incorporation of alkanethiol groups can be bound to metallic supports, for example gold supports. Yet another alternative is covalent immobilization, where the binding of the Polynucleotides can be mediated via reactive silane groups on a silica surface ^•.

Carrier particles to which only a single molecule, e.g. a nucleic acid molecule is bound. Such carrier particles can be generated by the molecules to be analyzed, e.g. the nucleic acid molecules intended for sequencing, in a molar ratio of preferably 1: 5 to 1:20, e.g. 1:10, are brought into contact with the carrier particles under conditions in which the molecules are immobilized on the carrier. The resulting carrier particles are then e.g. sorted on the basis of fluorescent marking groups contained on the molecules and separated from particles to which no molecule is bound. This sorting and separation can, for example, according to the in Holm et al. (Analytical Methods and Instrumentation, Special Issue μTAS 96, 85-87), Eigen and Rigler (Proc. Natl. Acad. Sei. USA 91 (1 994), 5740-5747) or Rigler (J. Biotech. 41 (1 995 ), 1 77-1 86) described methods that involve detection with a confocal microscope.

The nucleic acid molecules bound to a carrier, for example DNA molecules or RNA molecules, can be in single-stranded or double-stranded form. In the case of double-stranded molecules, it must be ensured that labeled nucleotide building blocks can only be split off from a single strand. In the nucleic acid strands to be sequenced, essentially all, for example at least 90%, preferably at least 95% of all nucleotide building blocks of at least one base type carry a fluorescent labeling group. Preferably, essentially all nucleotide building blocks of at least two base types, for example two, three or four base types, carry a fluorescent label, each base type advantageously carrying a different fluorescent label group. Nucleic acids marked in this way can be obtained by enzymatic primer extension on a nucleic acid template using a suitable polymerase, for example a DNA polymerase such as a DNA polymerase from Thermococcus gorgonarius or other thermostable organisms (Hopfner et al., PNAS USA 96 (1 999), 3600-3605) or a mutated Taq polymerase (Patel and Loeb, PNAS USA 97 (2000), 5095-510) using fluorescence-labeled nucleotide building blocks. The labeled nucleic acid strands can also be produced by amplification reactions, for example PCR. In the case of an asymmetrical PCR, this results in amplification products in which only a single strand contains fluorescent labels. Such asymmetrical amplification products can be sequenced in double-stranded form. Nucleic acid fragments in which both strands are fluorescence-labeled are produced by symmetrical PCR. These two fluorescence-labeled strands can be separated and immobilized separately in single-stranded form on carrier particles, so that the sequence of one or both complementary strands can be determined separately. Alternatively, one of the two strands at the 3 'end can be modified in this way, for example by installing a PNA clamp, so that it is no longer possible to split off monomer units. In this case, double-strand sequencing is possible.

If necessary, a "sequence identifier", i.e. a labeled nucleic acid of known sequence, e.g. by enzymatic reaction with ligase or / and terminal transferase, so that at the beginning of the sequencing a known fluorescence pattern is obtained first and only then the fluorescence pattern corresponding to the unknown sequence to be examined is obtained.

The nucleic acid template, the sequence of which is to be determined, can be selected, for example, from DNA templates such as genomic DNA fragments, cDNA molecules, plasmids etc., but also from RNA templates such as mRNA molecules. The fluorescent labeling groups can be used for labeling biopolyers, e.g. B. The use of fluorescent labeling groups such as fluorescein, rhodamine, phycoerythrin, Cy3, Cy5 or derivatives thereof etc. can be selected.

Step (b) of the method comprises introducing a loaded carrier particle into a device according to the invention, e.g. a sequencer.

The introduction of the carrier particle through the first microchannel and the introduction of further fluid flows through the second and third microchannels can take place by hydrodynamic and / or electroosmotic flow. A hydrodynamic flow is preferably used. The control of the flow in the feed channels can be done together or separately by using suitable means, e.g. Pumps, valves and / or gravity-controlled supply take place. The diameter of the first, second and third microchannels can be the same or different and is e.g. in the range from 5 to 500 μm, particularly preferably in the range from 10 to 100 μm and most preferably 25-75 μm.

With the help of a capture laser, e.g. an IR laser, the carrier particle can be held in the mouth area of the fourth microchannel according to method step (c). Such methods are described, for example, by Ashkin et al. (Nature 330 (1987), 24-31) and Chu (Science 253 (1991), 861-866).

The carrier particle is preferably held in place by an automated process. For this purpose, the carrier particles are passed through the first microchannel, passing through a detection element that activates the capture laser. Then, after capture and displacement in the fluid stream containing a reactant, the reaction is carried out on the immobilized carrier particle. A washing away from remaining carrier particles are not necessary in the method according to the invention, since the captured carrier particle is shifted from the first fluid stream into a second fluid stream by active transport and / or by changing the flow conditions. It is therefore also possible to trap a further carrier particle as soon as the first carrier particle held has been removed from the first fluid stream. This leads to a considerable increase in the process speed.

The sequencing reaction of the method according to the invention comprises the progressive cleavage of individual nucleotide building blocks from the immobilized nucleic acid molecules. An enzymatic cleavage is preferably carried out using an exonuclease, single-strand or double-strand exonucleases which degrade in the 5 '→ 3' direction or in the 3 '→ 5' direction - depending on the type of immobilization of the nucleic acid strands on the support - can be used. T7 DNA polymerase, E. coli exonuclease I or E. coli exonuclease III are particularly preferably used as exonucleases.

The reaction products, e.g. B. the nucleotide building blocks released by the cleavage reaction are then e.g. by means of a hydrodynamic and / or electroosmotic flow through the fourth

Microchannel directed and preferably determined during the flow through the fourth microchannel. Preferably a hydrodynamic one

Flow that allows an increase in the flow rate, which in turn increases the detection probability of a

Reaction product leads. Furthermore, one can by the hydrodynamic

River which e.g. generated by suction or application of pressure, reduce the occurrence of wall effects compared to electroosmotic pumps. The hydrodynamic flow preferably has a parabolic flow profile, i.e. the flow rate is maximum in

Center of the fluid flow and increases in a parabolic function

Edge up to a minimum speed. The flow rate the maximum is preferably in the range from 1 to 50 mm / s, particularly preferably in the range from 5 to 10 mm / s. The diameter of the fourth microchannel in the region of the section in which the fluid streams flow essentially without mixing is preferably in the range from 5 to 1000 μm, particularly preferably from 20 to 500 μm.

In some cases it may be preferred that the fourth microchannel has a larger diameter than the first, second and third microchannels. However, this is not absolutely necessary. For example, the flow velocity can be controlled in a targeted manner by local constrictions or / and extensions. For example, the flow rate can be increased by narrowing the channel and the flow rate can be reduced in a targeted manner by widening.

The identification of reaction products, e.g. B. of fluorescence-labeled nucleotide building blocks, according to step (e) of the method according to the invention can be by means of any measurement method, e.g. with a spatially and / or time-resolved fluorescence spectroscopy, which is able to detect fluorescence signals down to single photon counting in a very small volume element such as is present in a microchannel.

For example, the detection can be carried out by means of confocal single-molecule detection, such as by fluorescence correlation spectroscopy, with a very small, preferably confocal volume element, for example 0.1, 1 × 10 ^{~ 15} to 20 × 10 ^"12 I, through the microchannel flowing sample liquid is exposed to an excitation light of a laser which excites the receptors located in this measurement volume to emit fluorescent light, the emitted fluorescent light from the measurement volume being measured by means of a photodetector, and a correlation between the temporal change in the measured emission and the relative flow velocity of the involved molecules is created, so that with correspondingly strong dilution individual molecules in the measurement volume can be identified. Reference is made to the disclosure of European Patent 0 679 251 for details of the implementation of the method and apparatus details for the devices used for the detection. Confocal single molecule determination is further described by Rigler and Mets (Soc.Photo-Opt.lnstrum.Eng. 1 921 (1 993), 239 ff.) And Mets and Rigler (J.Fluoresc. 4 (1 994) 259-264) ,

Alternatively or additionally, the detection can also be carried out by a time-resolved decay measurement, a so-called time gating, as described, for example, by Rigler et al., "Picosecond Single Photon Fluorescence Spetroscopy of Nucleic Acids", in: "Ultrafast Phenomenes", D.H. Auston, Ed., Springer 1984. The fluorescence molecules are excited within a measurement volume and then - preferably at a time interval of> 100 ps - the detection interval is opened at the photodetector. In this way, background signals generated by Raman effects can be kept sufficiently low to enable essentially interference-free detection.

In a particularly preferred embodiment, the detection is carried out using a laser device which has a diffraction element or a phase-modulating element in the beam path of the laser, which optionally in combination with one or more optical

Imaging elements is set up from the laser beam

To produce diffraction patterns in the form of a linear or two-dimensional array of focal areas in the microchannel, the optical

Arrangement is set up to confocal each focal area for the

Imaging fluorescence detection by the photodetector arrangement. In a further preferred embodiment, the detection device is integrated in two mutually opposite sides of the microchannel walls, one wall having an array of laser elements emitting into the microchannel as the fluorescent excitation light source and the other an array of the laser elements in each case has opposite assigned photodetector elements as fluorescent light detectors. These two embodiments are disclosed in detail in patent application DE 100 23 423.2.

An increase in the detection probability of nucleotide building blocks and thus an improvement in sensitivity can be achieved by a hydrodynamic flow profile in the fluid streams of the first, second and third microchannels and in the fluid sub-streams of the fourth microchannel of the sequencing device. The hydrodynamic flow can be controlled by suitable control devices, e.g. B. can be set and controlled by controllable pumps and / or by special geometric design. In addition to the hydrodynamic flow, electrophoretic and electroosmotic methods for the transport of reagents can also be used in the sequencing device. The method according to the invention also allows the parallel sequencing of several nucleic acid molecules bound to carrier particles in different, preferably parallel, microchannel systems.

In a particularly preferred embodiment, the nucleic acid coupled to a carrier particle is essentially in the middle of a

Partial fluid stream recorded, the cleaved fluorescence-labeled

Nucleotides are conducted downstream in the laminar flow to a detection volume element, which is essentially via the fluid

Partial flow and in particular over its middle, where the highest flow rate prevails, is positioned. The flow rate is preferably so great that regardless of the thermal broadening of the

Flow trajectories by Brownian diffusion the nucleotide arrives in the detector field and is registered. The detector field is kept as small as possible so that the reaction products, e.g. B. the nucleotide bases are completely detected, while only the smallest possible fraction of the background contamination (ratio of the detector cross section to

Channel or fluid partial flow cross section) occurs in the detector. The invention is further illustrated by the following figures. Show it:

Figure 1 is a schematic representation of a preferred embodiment of the device according to the invention. In a first microchannel (2), a carrier particle (4) with a molecule immobilized thereon, e.g. B. a nucleic acid molecule in a first fluid stream introduced into the device. A second microchannel (6) is used to introduce a second fluid stream which contains a reactant, e.g. B. contains a reactant provided for the degradation of the nucleic acid molecule. Furthermore, a third microchannel (8) is provided which is used to introduce a third fluid flow, e.g. B. serves a buffer solution. The third fluid stream advantageously causes a separation of the first and the second fluid stream. The first, second and third microchannels (2, 6, 8) open into a fourth microchannel (10). At least in the region of the junction with the fourth microchannel (10) there are separate fluid zones (2a, 6a, 8a) for the fluid streams originating from the microchannels (2, 6, 8).

The carrier particle (4) introduced through the first microchannel (2) is held in the fourth microchannel (10) at a first predetermined position (1 2) in the zone of the first fluid flow (2a). The microparticle held in place at the first predetermined position (1 2) can then be transported, for example using the capture laser, to a second predetermined position (14), which is in the region of the second fluid flow (6a) emerging from the second microchannel (6 ) comes from. At this second predetermined position (14) the reaction, e.g. B. the digestion of the immobilized on the carrier particle nucleic acid molecule with the degradation enzyme present in the second fluid stream, preferably an exonuclease. The reaction products, e.g. B. the nucleotide building blocks which are separated sequentially by the enzymatic digestion are preferably from the fluid flow in the fourth microchannel (10) to a detection element (1 6) a confocal detection element, transported and detected there.

The flow rate in the system, in particular in the fourth microchannel, is set so that the broadening of the migration path of the split-off nucleotide building blocks caused by Brownian molecular movement is so low that they can be detected with sufficient statistical probability in the detection volume (16).

The diameter of the first, second and third microchannels (2, 6, 8) is preferably in the range of approximately 50 μm. The fourth microchannel (10) preferably has a width of approximately 150 μm and a depth of approximately 50 μm. However, the shapes and dimensions of the microchannels can be varied considerably provided that a mixture-free flow of several separate fluid streams is ensured at least in a section of the fourth microchannel.

The sequencing device according to the invention can the arrangement shown in Figure 1 several times, e.g. arranged in parallel or / and sequentially, so that a parallel or / and sequential determination of several molecules e.g. Nucleotide sequences per device is possible.

Advantages of the sequencing device according to the invention are, in particular, that simple control and separation of several different fluid flows is possible. In addition to the specified application in nucleic acid sequencing, the device can also be used for other analyzes, e.g. for single molecule detection, but also for synthetic processes.

In deviation from the embodiment shown in FIG. 1, devices can also be used in which the third microchannel (8) is missing, or in which a plurality of second and / or third microchannels are present. An example for such an embodiment with two third ^{microchannels'} is shown in FIG. 2 The embodiment of the device according to the invention shown in FIG. 2 allows the molecule immobilized on the carrier particle to be detected without the carrier particle having to be transported within the sequencing device after being captured.

For this purpose, the carrier particle is first introduced into the device through the first microchannel (20) in the setting shown in FIG. 2A and is held at a predetermined position (30) in the fourth microchannel (28) by a trapping laser. The predetermined position (30) is initially in the region of the fluid flow (20a) originating from the first microchannel (20). The device also contains a second microchannel (22) for introducing the reactant and two third microchannels (24, 26) for introducing buffer solution.

After capturing the microparticle at position (30), the flow conditions in the device are changed, e.g. by reducing or switching off the first fluid flow from the first microchannel and by switching on or increasing the fluid flows from the second microchannel (22) and a third microchannel (26). In this way, the fluid flow zones associated with the respective microchannels change in the fourth microchannel (28). In the setting shown in FIG. 2B, the microparticle held at position (30) thus reaches the region of the second fluid stream (22a) that comes from the second microchannel (22) and contains the degrading enzyme.

Of course, the embodiments shown in FIG. 1 and FIG. 2 can also be combined with one another. This means that both a transport of the captured microparticle from a first predetermined position to a second predetermined position and a change in the fluid flow conditions in the region of the Mouth zone in the fourth microchannel from a "capture" setting to ^• an "analysis" setting.

FIGS. 3a, 3b and 3c finally show 3 specific embodiments of the device according to the invention which have already been used in practice.

Claims

claims

Device comprising:

(i) a first microchannel for introducing a first fluid stream comprising a carrier particle with at least one molecule immobilized thereon, (ii) at least one second microchannel for introducing at least one second fluid stream comprising a reactant for the immobilized molecule, (iii) optionally at least a third microchannel for introducing at least a third

Fluid flow, (iv) at least a fourth microchannel, the

Microchannels (i), (ii) and (iii) open into the fourth microchannel, and at least in a section of the fourth microchannel at least the first and second fluid streams essentially without

Flow mixing,

(b) means for holding the carrier particle at a predetermined position in the region of the section of the fourth

Microchannel in which the fluid streams flow essentially without mixing,

(d) optionally means for detecting a reaction of the immobilized molecule with the reactant.

2. Device according to claim 1, characterized in that the first, second and third fluid flows have an essentially laminar flow at least in a section of the fourth microchannel.

3. The apparatus of claim 1 or 2, further comprising means for transporting the retained carrier particle from a first predetermined position in the fourth microchannel to a second predetermined position in the region of a second fluid flow.

4. Device according to one of claims 1 to 3, further comprising means for changing the flow conditions in the fourth microchannel, so that the retained carrier particle reaches the area of a second fluid flow.

5. Device according to one of claims 1 to 4 further comprising:

Means for introducing fluid flows into the microchannels,

- Means for deriving fluid flows from the microchannels, - Reservoirs for first, second and optionally third fluid flows.

6. Use of a device according to one of claims 1 to 5 for the analysis of molecules.

7. Use according to claim 6 for single molecule analysis.

8. Use according to claim 6 or claim 7 for sequencing nucleic acids.

9. Use of a device according to one of claims 1 to 5 for the synthesis of molecules.

0. Use according to claim 9 for the synthesis of single molecules.

1 . Use according to claim 10 for the synthesis of organic compounds or biopolymers.

2. A method for performing a reaction between an immobilized molecule and a free reactant, comprising: (a) providing a carrier particle with at least one molecule immobilized thereon, (b) introducing the carrier particle into a fluidic system

Communication, at least partially optically transparent microchannels, the device comprising: (i) a first microchannel for introducing a first fluid stream, comprising the carrier particle, (ii) at least one second microchannel for introducing at least a second fluid stream, comprising a reactant for the immobilized molecule, (iii) optionally at least one third microchannel for introducing at least one third fluid stream,

(iv) at least a fourth microchannel, the

Flow mixing, (c) holding the carrier particle at a predetermined position in the region of the section of the fourth microchannel in which the fluid streams flow essentially without mixing, (d) contacting the retained carrier particle with at least one second fluid stream containing the reactants and

(e) optionally, detecting the reaction between the immobilized molecule and the reactant.

1 3. The method according to claim 1 2, characterized in that one uses a carrier particle made of plastic, glass, quartz, metals, semimetals, metal oxides or from a composite material.

14. The method according to claim 1 2 or 13, characterized in that one uses a carrier particle made of plastic, glass or silicon or a composite material thereof.

1 5. The method according to any one of claims 1 2 to 14, characterized in that the carrier particle has a diameter of 0.5 to 1 0 microns.

1 6. The method according to any one of claims 12 to 1 5, characterized in that one uses a carrier particle with a single molecule immobilized thereon

1 7. The method according to any one of claims 1 2 to 1 6, characterized in that one through the at least one third microchannel

Conducts liquid flow to separate the first and second liquid flows.

1 8. The method according to any one of claims 1 2 to 1 7 for the analysis of molecules.

1 9. The method according to any one of claims 1 2 to 1 8 for sequencing nucleic acids, comprising: providing a carrier particle with a nucleic acid molecule immobilized thereon, wherein essentially all nucleotide building blocks of at least one base type carry a fluorescent label in at least one strand of the nucleic acid molecule, progressively Cleaving off individual nucleotide building blocks from the immobilized nucleic acid molecule, passing the cleaved nucleotide building blocks through the fourth microchannel and determining the base sequence of the nucleic acid molecule based on the sequence of the cleaved nucleotide building blocks.

20. The method according to claim 1 9, characterized in that the nucleic acid molecule is immobilized on the carrier particle via its 5'-terminus by means of bioaffine interactions.

21. Method according to one of claims 1 9 to 20, characterized in that essentially all nucleotide building blocks of at least two base types carry a fluorescent label.

22. The method according to any one of claims 1 9 to 21, characterized in that the cleavage of individual nucleotide components is carried out by an exonuclease.

23. The method according to any one of claims 1 2 to 1 7 for the synthesis of molecules.

24. The method according to claim 23 for the synthesis of organic ^' molecules or biopolymers

25. The method according to any one of claims 12 to 24, characterized in that the carrier particle is held in place with a capturing laser.

26. The method according to any one of claims 1 2 to 25, characterized in that the detection of the reaction is carried out by a confocal fluorescence measurement in a detection volume element.

27. The method according to claim 26, characterized in that the detection is carried out by confocal single molecule detection such as fluorescence correlation spectroscopy.

28. The method according to any one of claims 1 2 to 27, characterized in that the detection is carried out by a time-resolved decay measurement or time gating in a detection volume element.

29. The method according to any one of claims 12 to 28, characterized in that a parallel and / or sequential reaction is carried out on molecules in one or more microchannels.

30. The method according to any one of claims 1 2 to 29, characterized in that the carrier particles are held substantially in the middle of a partial fluid flow and the detection by conducting Reaction products takes place in a laminar flow to a • detection volume element, which extends over the partial fluid stream in which the carrier particle is held.

31 A method according to claim 30, characterized in that the detection volume element is kept as small as possible in order to just detect all reaction products.

32. The method according to any one of claims 1 2 to 31, comprising transporting the retained carrier particle in the fourth microchannel from a first predetermined position to a second predetermined position which is in the region of the second fluid flow.

33. The method according to any one of claims 1 2 to 32, comprising changing the flow conditions in the fourth microchannel, so that the retained carrier particle reaches the region of the second fluid flow.