DE60023862T2 - MICROMASSETTE DEVICE FOR CHEMICAL ANALYSIS - Google Patents

Abstract

The invention relates to an apparatus for performing chemical assays in an aqueous medium. The apparatus contains a reaction chamber(s) and a liquid in-flow channel connected to each chamber. The flow of liquid through the fluid in-flow channel to the reaction chamber is controlled by the presence of a hydrophobic inner surface on the walls of the in-flow channel. Under normal conditions fluid will not flow through the channel. However, application of an external force pushes the liquid through said channel into the reaction chamber. The invention is applicable to the monitoring of many different molecular interactions, in particular molecular recognition between an immobilised affinity partner and a species in solution, such as immunoglobulin/antigen interaction, DNA hybridisation, haptamer-protein interaction, drug and virus detection, high throughput screening of synthetic molecules and for determining the concentration and reaction kinetics of target species.

Description

These The invention relates to a device for detecting presence a target species in an aqueous Sample and also a device for determining the concentration and reaction kinetics of target species. The invention is to the monitoring (monitoring) of many different molecular interactions applicable, in particular to molecular recognition between a immobilized affinity partner and a species in solution, like Immunoglobulin / antigen interaction, DNA hybridization, haptamer-protein interaction, Drug and virus detection and high throughput synthetic screening Molecules.

There many affinity complexations between two reactants are diffusion controlled, depends time required to reach the reaction equilibrium directly from the mass transport of the molecule. The diffusion time of a molecule in one solution is proportional to the square of the path length; usually needs one small molecule less than one second to diffuse through 10 μm, while it takes two hours to travel one millimeter. To reduce the equilibrium time of the reaction, the chemical partners therefore as close as possible applied to each other become; the equilibrium time can be dramatically reduced by the reactor size on microdimensions is reduced, a partner immobilized on the surface of the reactor and the reactor is filled with the second partner.

The use of microreactors not only increases the speed of the affinity assays, but also facilitates obtaining information related to reaction kinetics, which is important for understanding the thermodynamic stability of complexes. The affinity constant K _d is the ratio between the forward and backward reaction rate constants k ₊ and k _- , which stand for the association and dissociation constants, respectively. Strong complex formation is characterized by a very rapid association and very slow dissociation, which in the specific case of sorbent affinity assays are adsorption and desorption from the surface of the microreactor. Understanding these thermodynamic properties can be used to study cross-reactivity between multiple antigens or nonspecific adsorption of a matrix element during an affinity assay. Complex formation of higher affinity partners is favored by modulating incubation time of the solution and nonspecific adsorption is then minimized. This fact may be an important factor because of the minimization of the background signal in decreasing the detection limit in immunosorbent assays. This method can also be used to monitor the adsorption of antigens of different molecular weight.

Since the diffusion coefficient of a molecule is proportional to its mass, the diffusion time of the molecule through the reaction chamber differs for small and large molecules. In the case of molecules having different molecular weights and the same epitope (z. B. fibrin degradation products), the K _d for all molecules to be the same, while the diffusion coefficient is different for each of them. When a kinetic experiment is performed, the smaller molecules quantitatively reach the antibodies before the larger ones. Monitoring the affinity response resulting signal as a function of time may thus provide useful information about the kinetics, which may aid in understanding the degradation process. These kinetic events can be tracked by modulating the residence time of molecules in contact with their reactants, which is most easily achieved by immobilizing an antibody on the walls of a series of microreactors and incubating different solutions of the analyte of interest for different periods of time.

In the past, analytical methods of the type described above (such as enzyme-linked immunosorbent assays - ELISAs) have been performed with microtiter plates and have been relatively slow. Great efforts have been made in recent years to reduce the size of micrometer scale analyzers to reduce response times: these miniaturized systems have been termed "full-scale microanalysis systems" (μ-TAS) ¹ , and they are already useful Means for manipulating and analyzing small sample quantities known ^2-8 . Most μ-TAS devices have heretofore been made by photolithography, wet-chemical etching, or thin-film deposition on substrates such as glass, quartz, and silicon ^9,10 . In order to reduce production costs, plastic substrates have also been machine-micromachined using either casting of silicone rubber ^11-14 , injection molding ¹⁵ , stamping ^{16, 17,} or laser photoablation ¹⁸ . These structures are planar devices with channels of micrometer size, often sealed by thermal or anodic bonding to a glass cover. Interconnected channels are easy to fabricate, allowing for rapid separation and reactions in volumes of a few picoliters. Other advantages of μ-TAS are the reduction in sample and reagent consumption, and the higher accuracy and reproducibility compared to laboratory-scale devices ^21,22 .

Competitive immunoassays have also been performed on microchips ^23-25 , but the microchannels have been used only to electrophoretically separate free and bound forms of antigen or antibody. In these assays, antibody and labeled antigen are added in specific amounts to the sample to be analyzed. The sample is then incubated with a mixture of the labeled and native antigens that compete for a limited number of antibody binding sites. The microchannel is then used to separate the free labeled antigen from the complex by capillary electrophoresis, and the quantification is performed by luminescence (fluorescence or chemiluminescence) at the end of the separation channel. The measured amount of free labeled antigen is then related to the analyte concentration in the sample using a previously determined calibration curve. It is essential in this type of assay to avoid adsorption of a reactant on the microchannel walls.

For the simultaneous analysis of several samples another type of immunoassay device has been developed ²⁶ . In this case, biotin-labeled antibodies were patterned on an avidin-coated waveguide to form a grouping of six vertically oriented strips of fixed antibodies immobilized on the waveguide surface by avitin-biotin bridges. The samples were then analyzed by a sandwich immunoassay format by overlaying another array of six horizontally oriented lines containing the corresponding fluorescently labeled antigen at various concentrations. Fluorescence complexes on the surface of the waveguide were then excited by a diode laser, and the fluorescence intensities of 36 square dots were detected by a CCD camera. This immunosensor enables the parallel analysis of multiple samples and the simultaneous detection of more than one analyte per sample.

Numerous analytical methods use luminescence to detect an analyte of interest. Luminescence is the generic term that refers to the emission of electromagnetic radiation (UV, visible light, or IR) by an excited molecule that relaxes to its ground state, and can be induced by photoexcitation (photoluminescence) or chemical reaction (chemiluminescence and electrochemiluminescence) become. Chemifluorescence (CF) is another class of luminescent reactions that combines the reaction mechanism of both PL and CL. In this case, a fluorogenic substrate A is converted by chemical reaction into a fluorescent product C, and the luminescence is generated by exciting this product: A + B → C + D, hv 1 + C → C * and C * → C + hv 2

For analytical purposes, one of the reactants of the assay system that can produce luminescence can be bound to a molecule to specifically "tag" it. The presence or absence of an observable label bound to one or more of the binding materials is then used as an indicator of the existence of an analyte of interest. There has been developed a wide range of experiments for detecting and quantifying trace amounts of pharmaceuticals, microorganisms, hormones, viruses, antibodies, nucleic acids and other proteins according to such methods. For example, in clinical diagnostics, competitive and sandwich immunoassays utilizing luminescence detection are now being used routinely ^27,28 .

In enzyme-mediated immunoassays, a molecule is labeled with an enzyme that catalyzes the luminescence reaction. Typical examples are the detection of immunoreagents labeled with horseradish peroxidase (HRP) or alkaline phosphatase (ALP), which in the presence of hydrogen peroxide and hydroxide ions facilitate the oxidation of luminol and dioxetanes or the hydrolysis of phosphate-containing reagents. ALP has been similarly used in CF assays to cleave a phosphate group from a fluorogenic substrate to obtain a product with high fluorescence ²⁹ .

Luminescent assay methods are widely used for the analysis of peptides, proteins and nucleic acids. It is shown that CL is a highly sensitive detection method in both flow injection analysis (FIA) and high performance liquid chromatography ^30-32 , and it is also ^useful in capillary electrophoresis (CE) ^33,34 for the detection of amino acids, neurotransmitters ³⁵ , rare earth metal ions ³⁶ or labeled proteins ³⁷ have been used. However, in immunoassays luminescence is the most commonly used detection ^{method 27, 34, 38-43} .

Under the method of the prior art for measuring the enzymatic Reaction rate, US-A-4,621,059 discloses a method in which the light emitted by a luminescent substance being passed through a capillary column flows and reacts with an immobilized enzyme by multiple optical Fibers are collected around the longitudinal direction of the column are arranged to the enzyme activity or the amount of interest To determine analytes from the distribution of luminescence intensity.

US-A-5,624,850 describes a method for performing immunoassays in capillaries, wherein Fluorescence for detecting an analyte of interest in translucent Capillaries with an inner diameter of ~ 0.1 microns to 1.0 mm is used. Homogeneous chemiluminescent immunoassays can be performed in a similar manner as, for example in US-A-5,017,473, wherein a light absorbent material and a luminescently labeled tracer the analyte / anti-analyte complex are incubated so that the entire emitted light except that which belongs to the bound tracer, by the light absorbing Material is absorbed.

In US-A-5,585,069 discloses a method in which two or more Samples are processed in parallel in one system, the more one Has troughs connected by one or more channels are to make a sample with mechanical or electrokinetic pumps to move from one hollow to another. In this device, the channels simply used as connections between two hollows not used as reaction or detection chambers.

A the main difficulties associated with the use of μ-TAS devices is the right one, because of the low Reynolds numbers of the currents Mixing the reagents. Another problem lies in the exact Timing of fluid entry, indicating kinetic studies is essential. The applicants have, as they are aware of these problems have found that the fluid enters a reaction chamber (For example, a microchannel) by means of a hydrophobic barrier can be controlled exactly.

The present application therefore provides in one aspect a device comprising: at least one reaction chamber, at least one fluid inlet channel in conjunction with the or each reaction chamber, and barrier means, for preventing the passage of aqueous fluid through the fluid inlet channel (s) Fluid inflow channels in the reaction chamber (s) are adapted to a fluid inlet force acting on the fluid, wherein the barrier means at least one Part of the or each fluid inlet channel having a hydrophobic inner surface comprises.

The Device preferably has a plurality of reaction chambers, which are the shape assume of microchannels, each one having an associated one Fluid inlet channel has. Alternatively, multiple microchannels can pass through a single inflow channel operated in a common Feed pipeline that communicates with the microchannels. In some preferred embodiments the fluid entry force is delivered by aspiration means, which are connected to a common pipeline connected to their away from the inflow channel to each microchannel stands. In other embodiments the device comprises a rotatable support element, the fluid entry force is provided by centrifugal pressure when the substrate for Rotating is brought. The carrier element can appropriate that Form substrate of the microchannel device, wherein the microchannels in general are arranged radially. The rotatable support member may alternatively as a carrier for one or multiple devices with parallel microchannels are used.

Of the Advantage of a common source of fluid inlet force for all micro-channels is in that simultaneous filling guaranteed can be, with the fluid samples through the hydrophobic barrier means on entering the microchannels be prevented until the fluid inlet force is applied. The degree of fluid entry force can also be easily controlled be to fast filling the microchannels and adequate To ensure mixing. The microchannels can also by exercise a reinforced Force on the fluid in the channels, for example, by increasing the degree of aspiration or by increasing the rotational speed the rotation support element, be emptied in an efficient and rapid way. Therefore, it is possible reach an exact endpoint of an assay. It is beneficial in many cases the sample before monitoring to eject bound target species.

If desired, can be a liquid Reagent or a washing fluid provided in a sealed cavity which forms a reservoir, preferably one such Reservoir per micro channel gives. The reservoirs can be arranged so that she over normally closed valves communicate with their respective microchannels stand, and can be ejected through such valves to eject their contents, when respective pistons act on them. Alternatively it can be give single reservoir that over a normally closed valve with a common pipe which feeds into all microchannels.

Detection of target species with the microchannels can be achieved by conventional means. For example, preferred embodiments of the device are configured such that at least a portion of the surface of the microchannel is formed of an electrically conductive material to facilitate electrochemical detection. This may be, for example, a conductive polymer material or an electrode. In some embodiments, at least a portion of the microchannel walls may be formed from a semiconductor material, such as indium oxide. The semiconductor material is preferably transparent. The detection can alternatively be performed by luminescence or fluorescent means, in which case an electromagnetic radiation detector such as a photodiode or a secondary electron multiplier is provided.

One particular advantage of the invention is that chemical reagents on the insides of the microchannels can be immobilized thus the possibility of ELISA-type assays in a μ-TAS type system. On the microchannel walls can a Number of different types of reagents, for example oligonucleotides, Polypeptides, proteins (such as enzymes) or other natural or synthetic molecules. These can appropriate on the surface the microchannel walls be adsorbed or covalently bound thereto (for example by amide bond formation with succinimide) or electrostatically be bound (for example with a crosslinker such as polylysine). The inside of the microchannel and / or the fluid inflow channel can also be provided with chemical-functional groups by chemical or physical treatment have been formed.

The The invention also extends to a method of manufacture a device as defined above, comprising the following steps, which can be performed in any order or simultaneously: Form at least one reaction chamber and forming at least one fluid inflow channel in connection with the reaction chamber or the reaction chambers, wherein at least a portion of the or each fluid inflow channel a hydrophobic inner surface which is adapted to act as a barrier means, the passage of fluid through the fluid inlet channel into the reaction chamber (s) as long as to prevent a fluid entry force on this fluid acts.

The Device is for ease of manufacture, preferably in two main parts formed: a substrate in which the microchannels (and possibly also the inflow channels) as Wells are formed (for example by injection molding, hot stamping, photoablation, to water or polymerization on a mold) and a topcoat overlying the substrate and over the wells is applied to the microchannels (and optionally also the inflow channels). In embodiments, in which the inflow channels not prepared in the substrate, they can be produced, for example be pierced by a laminated cover layer with a laser is or above the inlet of the reaction chamber a Joint is applied, which consists of a hydrophobic material as polydimethylsiloxane (PDMS) is made.

The Device may be formed of any suitable material For example, ceramics, glass, semiconductors, polymers or Combinations of it. In a particularly preferred embodiment Both the substrate and the lamination layer are made Polymer material formed, not only easy formation of the microchannels (for example by photoablation), but also the merging of the two components by means of a thermal lamination technology allows. It is for this purpose preferred that at least one of the polymers is of a material, which has a relatively low melting point, for example polyethylene with a melting point below 200 ° C. The lamination layer may advantageously be made of an elastomeric material such as polydimethylsiloxane (PDMS). It is in devices that to be used together with optical detection means, preferred that the lamination layer of a substantially transparent material is formed and the substrate is made of a essentially opaque material (such as a ceramic material or carbon-filled Polymer) is formed.

In In one aspect, the invention extends to a method for Operating a device as defined, comprising the steps: Applying at least one sample of an aqueous to be tested solution at the end of at least one fluid inlet channel away from at least a reaction chamber, induce the entry of the sample into the reaction chamber (s) via the Fluid inflow channel or the fluid flow channels by exerting a Fluid entry force and monitoring the sample in the reaction chamber or the reaction chambers for presence or concentration of a target substance.

In embodiments the device with Aspirationsmitteln is the fluid inlet force Preferably, by activating the Aspirationsmittels exercised for a Period in the range of 0.1 to 100 s reduced pressure on the microchannels exercise. The aspirants can then be activated to evacuate the microchannels to the microchannels yet lower pressure available too optionally in combination with the supply of washing fluid from a reservoir.

In embodiments the device constructed with rotatable substrates or supports, For example, the fluid entry force is preferably achieved by rotating the substrate or the carrier at an angular velocity in the range of 1 to 1000 revolutions per minute for provided a period in the range of 1 to 100 s. The microchannels can then by rotating the substrate or carrier at an increased angular velocity in the range of 10 to 100,000 revolutions per minute for one Period can be evacuated in the range of 1 to 100 seconds.

The Invention will follow in more detail with reference to the attached drawings as examples in which:

1 Figure 3 is a schematic cross section of one embodiment of a device according to the invention, which is shown a) after settling an aqueous sample drop on the hydrophobic barrier and b) after loading the sample.

2 Figure 10 is a schematic plan view of an embodiment of a device according to the invention, showing the steps of parallel sampling, loading, and washing achieved by aspiration.

3 is a schematic plan view of an alternative embodiment of a device according to the invention, in which parallel filling and washing stages are achieved by centrifugal force.

4 is a schematic plan view of another embodiment of a device according to the invention, in which the solution is charged by slight aspiration and washed by strong aspiration.

5a is a partial plan view of an embodiment of a device according to the invention, which includes a fluid reservoir adjacent to the fluid inlet channel.

5b consists of two vertical cross sections of the device of 5a together with an associated piston, which illustrates the action of the piston, which penetrates into the sealed reservoir and ejects its contents via a valve into the reaction chamber.

6 FIG. 11 is a top plan view of one embodiment of a device made by UV laser photoablation of a polycarbonate CD, which embodiment is essentially similar to the device of FIG 3 is constructed.

7 Fig. 13 is a calibration curve obtained from a fluorescence imaging apparatus using ALP-DDi solution in the microchannel of one embodiment of a device according to the invention.

8th Figure 4 is a graph illustrating fluorescence results showing the level of binding between DDi-ALP in a test involving the use of two microchannels of the device of the invention, one with BSA only and the other with BSA and anti-DDi (shown in the Figure BSA + Ab indicated) was incubated.

9 is a fluorescence image generated using a device of the type described in U.S. Pat 6 was obtained, wherein different microchannels were incubated with different concentration of DDi-ALP.

10 Figure 4 is a graph illustrating the fluorescence intensity resulting from varying concentrations of ALP-DDi in a device of the present invention 6 of the type shown with antibody-coated microchannels.

11 Figure 4 is a graph illustrating the variation in fluorescence intensity over time from incubation of ALP-DDi in one embodiment of the device of the invention with at least one antibody-coated microchannel, and

12 Figure 3 is a graph illustrating fluorescence intensity (showing bound ALP-DDi) in a competitive immunoassay with D-dimer using one embodiment of the device of the invention having at least one antibody-coated microchannel.

The microchannel devices of 1 to 6 are produced by LTV laser photoablation of commercially available polymers such as PET or polycarbonate. The photoablation process is carried out in a known manner, for example as previously described by the applicants of the present invention. Briefly, a polymeric film was rinsed with distilled water and ethanol and then mounted on an X, Y work platform (Microcontrol, France). Thereafter, UV laser pulses (193 nm) (Lambda Physik LPX 205 i, Germany) were bombarded by a photomask and a 10: 1 lens at a frequency of 50 Hz at 200 mJ / pulse onto the polymer substrate target, giving a fluorescence of 1 J / cm ² on the surface. During the photoablation process, the polymer substrate was moved horizontally with a X, Y stepping motor (Microcontrol, France) at a speed of 0.2 mm / s, resulting in 22 mm long linear channels. These microchannels are typically between 1 and 1000 microns wide, and in this example they were about 100 microns wide. The depth of the channels was fixed at 40 μm by controlling the number of laser pulses used (each pulse was approximately 150 nm by photoablation). The channels were then sealed by thermal lamination of a polyethylene layer over the base polymer film, the channels then showed a trapezoidal shape with three walls of the substrate polymer (PET or polycarbonate) and the lamination (polyethylene) blanket. Fluid inflow channels (or "barriers") (1) are opened either by bombardment with sufficient laser pulses, or are mechanically drilled through the hydrophobic lamination layer. The barriers, which may have a diameter between 10 μm and 10 mm, have hydrophobic insides due to the nature of the polymer and therefore inhibit the passage of aqueous fluids.

The exact arrangement of the microchannels is not critical to the operation of the invention, although two general geometries have been developed and tested by the Applicants and have proven to be advantageous. In the first several microchannels are arranged parallel to each other, expediently on a generally rectangular substrate. The inflow channel "barriers" of the various microchannels are aligned to allow rapid and efficient loading of test solutions from a linear multi-pipetting device (see US Pat 2 ). In the second configuration, the microchannels are radially disposed on a generally circular substrate, with the inflow channel barriers either toward the center of the circle and the opposite (outflow) ends of the microchannels in the direction of the circumference (FIG. 3 and 6 ), or the other way round ( 4 ).

Many different means of providing the fluid entry force may be used, the preferred means being aspiration and centrifugal force. In the device of 2 becomes a common pipeline ( 3 ) at the outflow of the microchannels ( 2 ) to which a reduced pressure is applied during operation of the device to draw fluid through the fluid flow barriers into the microchannels. As in the last presentation in 2 As can be seen, the aspirating agent can also be used to exert a stronger aspiration force to eject the contents of the microchannels into a drain, optionally together with the supply of a washing fluid. In the 4 The device shown operates in a similar manner, with the aspiration at the common outflow ( 6 ) is created. In the in the 3 and 6 As shown, fluid is caused to pass through the fluid inlet barriers and into the microchannels by rotating the substrate to produce centrifugal force. In the illustrated arrangement, each microchannel has its own drain ( 7 ).

In an aspiration-driven device (as in Figs 2 and 4 ) is usually a 2 ul sample by pipette on each barrier ( 1 ) applied. The solution is then removed by a short aspiration from the common pipeline ( 3 ; 6 ) loaded into the microchannel. The technique ensures homogeneity of the solution across the entire microchannel. The microchannel is aspirated after incubation and rinsed three times with 2 μl. It is worth noting that the wash solution volume is much larger than the volume of the microchannel (about 100 nl) to ensure efficient washing. The filling and washing processes can be accomplished using centrifugally-driven devices by placing over each barrier ( 1 ) 2 μl of solution are applied. Slow rotation results in loading the sample into the microchannel or microchannels, and faster spinning is then used to eject the sample from the microchannel (s).

5 illustrates an optional modification of the device according to the invention, wherein each microchannel has an associated fluid reservoir (FIG. 10 ) formed by a sealed cavity adjacent to the fluid inlet barrier ( 1 ) is located. The reservoir is connected by means of a normally closed valve ( 12 ), the valve element ( 13 ) under pressure in well ( 14 ) can be deformed with microchannels ( 2 ) in connection. Reservoir ( 10 ) is sealed by seal ( 15 ), which can be broken by downward pressure applied by pistons ( 11 ) in close fitting in reservoir ( 10 ) is shaped. The downward movement of pistons ( 11 ) in reservoir ( 10 ) increases the fluid pressure in the reservoir, whereby valve ( 12 ) is opened and fluid from the reservoir can enter the microchannel. Depending on the requirements of any particular assay, the reservoir may be filled with either a reagent or washing fluid.

Various tests have been carried out, by way of example, to determine the usefulness of the device according to the invention when carrying out an immunoassay for D-dimer. D-dimer is used as a diagnostic indicator in thromboembolic events: deep vein thrombosis and pulmonary embolism can be diagnosed by monitoring blood D-dimer levels. In the past, the most reliable assay on D-dimer was performed by ELISA techniques, for example the "Asserachrom D-Di" from Diagnostica Stago. However, the standard ELISA techniques are unsuitable for emergency situations, and alternative membrane-based techniques have been developed using color-based detection systems ⁴⁸ . However, these suffer from the disadvantage that the detection mechanism is too subjective.

In the present tests, the detection of the enzyme was effected by a chemiluminescent substrate solution (VCR, Amersham). This system is based on fluorescence detection of the AttoPhos substrate, which is hydrolyzed by ALP. The microchannels were then exposed to a fluorescent imaging screen (MP840, Molecular Dynamics) and each channel was read 1 minute. The image was then quantified using Image Quant software (Molecular Dynamics). Calibration of the enzyme in the microchannel was achieved by mixing the substrate solution at different enzyme concentrations and incubating for 5 minutes. The microchannels were then filled with the mixtures and analyzed with the fluorescence imaging device. In the actual tests, the enzyme was immobilized on the surface of the microchannels and the VCR solution was added to the channels, measuring the fluorescence 5 minutes later.

immobilization the proteins became by one hour Physisorption at room temperature achieved. The IgG antibody of Mouse (Serbio, France) was immobilized by either 10 or 100 μg / ml applied in the microchannel and one hour in a moist Chamber were incubated. The surface was then washed with PBS and 20% Tween (Tween / water: 0.2 ml / L, Fischer, Germany) and an hour with a solution of 50 μg / ml wärmegeschocktem BSA (Sigma, USA) blocked in the wash buffer solution. After a further washing step, the channels were individually filled with the antigen solution. To five minutes (except at the kinetic experiment, with other periods below were specified), the microchannels were rinsed, and a solution of 10 μg / ml Alkaline phosphatase labeled antigen (ALD-DDi) was introduced and after five Rinsed again for a few minutes.

The fluorescence dependence of the enzyme concentration after 5 minutes of incubation is graphically in 7 shown. The detection limit is reached in the range of 1 ng · ml ^-1 . The non-linear detection range results from the fact that the hydrolysis product is not very soluble and may precipitate at higher surface concentrations. This system can still be used to quantify the enzyme concentration in the microchannel.

To study the activity of the adsorbed antibodies in the microchannel, two different incubation procedures were used. First, some channels were only incubated with BSA. Second, several more channels were incubated with Ab and then with BSA. Thereafter, each channel was filled with the DDi-ALP, incubated for one hour and washed by aspiration according to the procedure already described. The fluorescence intensity of each channel was then measured and the results are in 8th shown. The BSA-only incubated channels showed low fluorescence, which did not differ significantly from that of the polymer substrate itself. In contrast, the channels incubated with the antibodies showed much more fluorescence, indicating that DDi-ALP was adsorbed on the antibodies. This experiment shows that some of the adsorbed antibodies are still active on the surface, and that BSA is an effective blocking agent for nonspecific adsorption of the DDi-ALP complex.

9 shows the fluorescence of the substrate in the channels after adsorption of various concentrations of ALP-DDi on the adsorbed from 10 ug · ml ^-1 antibodies. The fluorescence intensity of the microchannel lines clearly shows the concentration gradient in the various microchannels. The relative intensity of each microchannel is in 10 shown graphically. The saturation of the channels is achieved at about 30 μg / ml.

11 shows the fluorescence intensity of microchannels that have been incubated for different periods of time. For short incubation times (<5 minutes), the intensity increases linearly, indicating that the antigens are captured by the antibodies very rapidly. It is believed that not all antigens have reached the surface by diffusion. This first tilt roughly follows the diffusion of the molecules to the walls. The molecules then react quickly, and the reaction is quasi-diffusion-controlled. After 5 minutes of incubation, the reaction is controlled by slower kinetics driven by two different phenomena. First, large molecules diffuse more slowly and therefore reach the surface after a long time. In this case, the molecules may be partially degraded fibrin products whose molecular weight may be greater than 1000 kD. Second, there is a tendency for nonspecific adsorption, such reactions are much slower than immunological recognition and are driven by electrostatic or hydrophobic interactions requiring reorganization at the molecular level. This type of nonspecific adsorption can therefore be excluded by short incubation times.

12 shows the fluorescence dependence of the D-dimer concentration after a competitive immunoassay. In the lower concentration range, most of the immobilized antibody sites are not occupied by the DDi; whereby the DDi-ALP can be present in a large amount and therefore hydrolyze more fluorescent substrates. At concentrations above 1000 ng.ml ^-1 , D-dimer molecules are present at most of the antibody sites, and therefore only a few sites are available for DDi-ALP. The middle part of the concentration range (100 to 1000 ng.ml ^-1 ) shows the strong concentration dependence of the system, with the two orders of magnitude of detection range in the range of interest for diagnostic applications ⁴⁹ .

These Experiments show the feasibility of ELISA techniques in microchannels. The required to complete an assay (including calibration) Time can be reduced to less than 10 minutes compared with a typical time of 3 hours for an ELISA in a microtiter plate, because rapid equilibrium times and rapid filling and rinsing procedures can be used. It can if desired Hundreds of microchannels be provided on a substrate, and the ability to simultaneous filling to ensure, offers the possibility for highly efficient parallel assays.

References:

• (1) A. Manz; N. Graber; H.M. Widmer, Sens. Actuators B, 1990, 244.
• (2) A. Manz; D.J. Harrison; E.M. J. Verpoorte; J.C. Fettinger; A. Paulus; H. Lüdi; H.M. Widmer, J. Chromatogr., 1992, 593, 253.
• (3) D.J. Harrison; K. Fluri; K. Seiler; Z. Fan; C. S. Effenhauser; A. Manz, Science, 1993, 261, 895-897.
• (4) S.C. Jacobson; J.M. Ramsey, Electrophoresis, 1995, 16, 481st
• (5) D.E. Raymond; A. Manz; H.M. Widmer, Anal. Chem., 1994, 66, 2858.
• (6) A.T. Woolley; R.A. Mathies, Proc. Natl. Acad. Sci. USA, 1994, 91, 11348-11352.
• (7) C.S. Effenhauser; A. Paulus; A. Manz; H.M. Widmer, Anal. Chem., 1994, 66, 2949.
• (8) A.W. Moore; S.C. Jacobson; J.M. Ramsey, Anal. Chem., 1995, 67, 4184-4189.
• (9) S.D. Collins, J. Electrochem. Soc, 1997, 144, 2242-2262.
• (10) A. Manz; H. Becker; Microsystem Technology in Chemistry and life science; Springer: Berlin, 1998.
• (11) C.S. Effenhauser; G.J.M. Bruin; A. Paulus; M. Ehrat, Proceedings μ-TAS '96, Anal. Methods Instrum. Special Issue, 1996, 124-125.
• (12) D.S. Soane; Z.M. Soane, 1998 US-A-5,750,015.
• (13) B. Eckström; G. Jacobson; O. Ohman; H. Sjodin, 1991, PCT application WO-A-91/16966.
• (14) C.S. Effenhauser; G.J.M. Bruin; A. Paulus; M. Ehrat, Anal. Chem., 1997, 69, 3451-3457.
• (15) R.M. McCormick; R.J. Nelson; M.G. Alonso-Amigo; D.J. Benvegnu; H.H. Hooper, Anal. Chem., 1997, 69, 2626-2630.
• (16) H. Becker; W. Dietz; P. Dannberg In D.J. Harrison and A. van den Berg, publisher, "Proceedings of the μ-TAS '98 workshop hero in Banff, Canada, 13-16 October 1998 "; Kluwer Academic Publishing, Dordrecht, 1998; 253-256.
• (17) L. Martynova; L.E. Locascio; M. Gaitan; G.W. Kramer; R.G. Christensen; W.A. MacCrehan, Anal. Chem., 1997, 69, 4783-4789.
• (18) M.A. Roberts; J.S. Rossier; P. Bercier; H. Girault, Anal. Chem., 1997, 69, 2035-2042.
• (19) N.C. Chiem; C. Colyer; J.D. Harrison In Transducers' 1997, 1997; Part 1, Pages 183-186.
• (20) U.D. Larsen; J. Branebjerg; G. Blankenstein In Special Issue on Micro-TAS; H.M. Widmer, publisher; Ciba Geigy, Basel, 1996, pages 228-230.
• (21) K. Seiler; D.J. Harrison; A. Manz, Anal. Chem., 1993, 65, 1481.
• (22) S.C. Jacobson; R. Hergenroeder; L. B. Koutny; R.J. Warmack; J.M. Ramsey, Anal. Chem., 1994, 66, 1107-1113.
• (23) L. B. Koutny; D. Schmalzing; T.A. Taylor; M. Fox, Anal. Chem., 1996, 68, 18-22.
• (24) N. Chiem; D.J. Harrison, Anal. Chem., 1997, 69, 373-378.
• (25) S.D. Mangru; D.J. Harrison, Electrophoresis, 1998, 19, 2301-2307.
• (26) C.A. Rowe; S. B. Scruggs; M.J. Feldstein; J.P. Golden; F. S. Ligler, Anal. Chem., 1999, 71, 433-439.
• (27) L.J. Kricka In Textbook of Immunological Assays; E.P. Diamandis, T.K. Christopoulos, Editor; Academic Press, San Diego, 1996, pages 337-353.
• (28) J.W. Hasting; L.J. Kricka; P. E. Stanley, Editor, "Bioluminescence and Chemiluminescence: Molecular Reporting with Photons "Wiley, Chichester, 1997.
• (29) J.L. Cherry; H. Young; L.J. Di Sera; F.M. Ferguson; A.W. Kimball; D.M. Dunn; R.F. Gesteland; R. B. Weiss, Genomics, 1994, 20, 68-74.
• (30) P.J.M. Kwakman; U. A. T. Brikman, Anal. Chim. Acta, 1992, 266, 175.
• (31) K. Robards; P.J. Worsfold, Anal. Chim. Acta, 1992, 266, 147th
• (32) J. Cepas; M. Silva; D. Perez-Bendito, J. Chromatogr. A. 1996 749, 73-80.
• (33) A.M.G. Campana; W.R.G. Baeyens; Y. Zhao, Anal. Chem., 1997, 69, 83A-89A.
• (34) S.A. Soper; I.M. Warner; L.B. Mcgown, Anal. Chem., 1998, 70, R477-R494.
• (35) Y. Zhang; Huang; J.K. Cheng, Anal. Chim. Acta, 1998, 363, 157-163.
• (36) Y. Zhang; J. Cheng, J. Chromatogr. A, 1998, 813, 361-368.
• (37) K. Tsukagoshi; A. Tanaka; R. Nakajima; T. Hara, Anal. Sci. 1996, 12, 525-528.
• (38) H.G. Thorpe; L.J. Kricka; S. B. Moseley; T. P. Whitehead, Clin. Chem., 1985, 31, 1335-1341.
• (39) L.J. Kricka, J. Clin. Immunoassay, 1993, 16, 267-271.
• (40) T.A. Nieman In Encyclopedia of Analytical Science; Academic Press, 1995; Volume 1, pages 608-621.
• (41) D. Perez-Bendito; A. Gomez-Hens; M. Silva, J. Pharm. Biomed. Anal., 1996, 14, 917-930.
• (42) M.A. Cousino; W.R. Heineman; H. B. Halsall: Annali Di Chimica 1997, 87, 93-101.
• (43) A.R. Bowie; M.G. Sanders; P.J. Worsfold, J. Biolumin. Chemilumine, 1996, 11, 61-90.
• (44) M.A. Roberts; J.S. Rossier; P. Bercier; H.H. Girault, Anal. Chem., 1997, 69, 2035-2042.
• (45) JS Rossier; P. Bercier; A. Black; S. Loridant; HH Girault, "Topography, Crystallinity and Wettability of Photoablated PET Surfaces".
• (46) A. Black; J.S. Rossier; M.A. Roberts; H.H. Girault and E. Roulet; H. Mermod, Langmuir, 1998, 14, 5526-5531.
• (47) J.S. Rossier; M.A. Roberts; R. Ferrigno; H. H. Girault, "Electrochemical Detection in Polymer Microfluidic Devices ", submitted.
• (48) A.-M. Vissac; M. Grimaux; S. Chartier; F.A. Chan; B. Chambrette; J. Amiral, Thrombosis Research, 1995, 78 (4), 341-352.
• (49) G. Reber; P. Demoerloose; C. Coquoz; H. Bounameaux, BLOOD COAGULATION & FIBRINOLYSIS, 1998, 9, 387-388.

Claims (59)

Apparatus for carrying out chemical assays which aqueous Include fluids, the device comprising at least one Reaction chamber, at least one fluid inlet channel in conjunction with the or each reaction chamber, and barrier means used for Preventing the flow of aqueous fluid through the fluid inlet channel (s) Fluid inflow channels in the reaction chamber (s) are adapted to a fluid inlet force acting on the fluid, wherein the reaction chamber is a hydrophilic palm and the barrier means at least part of or each Fluid inflow channel having a hydrophobic inner surface comprises. Apparatus according to claim 1, wherein the reaction chamber a microchannel with at least one dimension in the range of 1 up to 1000 μm includes. Apparatus according to claim 1 or claim 2, wherein the fluid inlet channel is formed in a substrate, of which at least a part consists of a hydrophobic material. Apparatus according to claim 1 or claim 2, wherein the fluid inlet channel is formed in a substrate, of which at least a part has been physically or chemically treated to it to make hydrophobic. Device according to one of the preceding claims, wherein the fluid inlet channel has a cross-sectional area in the range of 10 microns ² to 1000 mm ² . Device according to one of the preceding claims, wherein the fluid inflow channel is shaped as a complementary form to a standard pipette (for example of the Eppendorf ^(RTM) type). Device according to one of the preceding claims, wherein the fluid entry force is provided by piston pressure. Device according to one of the preceding claims, comprising several separate reaction chambers, each with an inflow channel and related Barrier means in connection. Apparatus according to claim 8, wherein each reaction chamber equipped with a separate inflow channel. Apparatus according to claim 8, wherein an inflow channel present, which provides a common pipeline for all reaction chambers. Device according to claims 8, 9 or 10, as far as dependent on Claim 2, wherein each microchannel is at its from the inflow channel distant end with a common pipe in communication stands, with the common piping to an aspirating agent connected, which is adapted to selectively reduced pressure to put on the pipeline and so in operation fluid through the microchannel to draw. Apparatus according to claim 10 or claim 11, wherein the common conduit has a cross-sectional area in the range of 0.01 mm ² to 25 cm ² . Device according to one of the preceding claims, wherein the microchannels are arranged generally parallel to each other. Apparatus according to claim 13, as far as dependent on one of the claims 10 to 12, the microchannels are arranged generally perpendicular to the common pipe. Device comprising multiple devices according to claim 14 mounted together on a tape. Apparatus according to claim 11, which is a substantially circular Substrate, wherein the microchannels arranged substantially radially are, with their respective inflow channel towards the periphery of the circle points and its opposite end with a central one Chamber communicates connected to the aspiration agent is. Apparatus according to claim 8 or claim 9 which a substantially circular Substrate, wherein the microchannels arranged substantially radially are, the inflow channel or the inflow channels in the direction of the center of Circle are arranged and each microchannel a waste chamber on having opposite end in the direction of the periphery of the circle. Apparatus according to claim 16 or claim 17, wherein the thickness of the substantially circular substrate in the area from 50 to 5000 microns lies. The device of claim 16, wherein the circular substrate is rotatable and the fluid inlet force provided by centrifugal pressure becomes when the substrate is rotated. Device comprising multiple devices according to claim 13 or claim 14 comprises arranged on a rotatable support member, and wherein the fluid entry force is provided by centrifugal pressure when the carrier element is made to rotate. Device according to one of the preceding claims, wherein then seal the or each reaction chamber with a sealed one Cavity is equipped, which is filled with an aqueous fluid reservoir forms. The apparatus of claim 21, wherein the reservoir via a normally closed valve communicating with the reaction chamber stands by exercising of heightened Pressure on the watery Fluid in the cavity for opening can be brought. The apparatus of claim 22, further comprising a piston member with an external profile which is shaped so that it fits into the cavity, wherein the Cavity through a rupturable Seal closed, which ruptured during operation by the piston is, whereby the movement of the piston into the cavity the necessary increased Pressure provides to the watery Fluid from the cavity over to push the valve into the reaction chamber. Device according to one of the preceding claims, wherein at least part of the surface of the Reaction chamber formed of an electrically conductive material and the device further comprises electrical detection circuits which are connected to the conductive part to the detection of a Target species in the reaction chamber by electrochemical means to enable. The device of claim 24, wherein the conductive Part is formed of a conductive polymer material. The device of claim 24, wherein the conductive Part is formed by an electrode. The device of claim 26, wherein the electrode made of a semiconductor material. The device of claim 27, wherein the semiconductor material is essentially transparent. The device of claim 28, wherein the semiconductor material Indium oxide is. Apparatus according to any one of claims 1 to 23, further comprising electromagnetic Radiation detection means adapted to from a target species detect radiation emitted in the reaction chamber. The device of claim 30, wherein the detecting means at least one photodiode or at least one secondary electron multiplier group that covers over at least a part of the reaction chamber is arranged. Device according to one of the preceding claims, wherein a chemical reagent on at least a portion of the inner surface of the Reaction chamber is immobilized, with the reagent for interaction is adapted to a target species, their presence or concentration should be determined. The device of claim 32, wherein the reagent an oligonucleotide, a polypeptide, a protein or other natural or synthetic molecule includes. Apparatus according to claim 32 or claim 33, wherein the reagent on the inner surface the reaction chamber is adsorbed. Apparatus according to claim 32 or claim 33, wherein the reagent is covalently bound to the inner surface of the reaction chamber is. The device of claim 35, wherein the covalent Binding over a succinimide-binding agent is reached. Apparatus according to claim 32 or claim 33, wherein electrostatically transfer the reagent a crosslinker to the inner surface the reaction chamber is bound. The device of claim 37, wherein the crosslinker Polylysine is. Device according to one of the preceding claims, wherein at least a part of the inner surface the reaction chamber and / or the fluid inlet channel with chemical functional groups equipped by chemical or physical treatment of the surface are formed. Device according to one of the preceding claims, which comprises a substrate in which the reaction chamber and / or the fluid supply channels as well (s) are formed, wherein the reaction chamber and / or the fluid inlet channel are sealed by a cover layer applied over the substrate is. The device of claim 40, wherein the substrate and the cover layer are formed of polymeric materials, wherein the melting point of at least one of the materials is sufficient is low to enable that the substrate and the cover layer by thermal lamination be sealed together. Apparatus according to claim 41, wherein said at least a material is polyethylene. Apparatus according to claim 40 or claim 41, wherein the cover layer is formed from an elastomeric material. The device of claim 43, wherein the elastomeric Material polydimethylsiloxane (PDMS) is. Apparatus according to claim 40, as far as dependent on Claim 30 or 31, wherein at least a part of the substrate a substantially opaque material is formed and the cover layer is formed of a substantially transparent material. The device of claim 45, wherein the substantially opaque material is a carbon filled polymer or ceramic material includes. Method for producing a device according to one of the claims 1 to 46, comprising the following stages, in any order or performed simultaneously can be: Forming at least one reaction chamber having a hydrophilic inner surface and Forming at least one fluid inlet channel in conjunction with the Reaction chamber or the reaction chambers, wherein at least one Part of the or each fluid inlet channel has a hydrophobic inner surface, which is adapted so that it acts as a barrier means to the Passage of fluid through the fluid inlet channel into the reaction chamber as long as to prevent a fluid entry force on this fluid acts. The method of claim 47, wherein the device is formed of polymeric material. The method of claim 48, wherein the device by injection molding, Hot stamping, photoablation, to water or polymerization is formed on a mold. The method of claim 48 or claim 49, comprising the steps: forming a substrate with at least one recess and applying a topcoat over the substrate around or To seal each well, so at least one fluid inlet channel and / or at least one reaction chamber. The method of claim 50, wherein the cover layer is sealed to the substrate by thermal lamination. The method of claim 47, wherein at least one Part of the device made of a ceramic material, glass, a conductor or a semiconductor material is formed. Method for operating a device according to the claims 1 to 46, comprising the steps of applying at least one Sample of an aqueous to be tested solution at the end of at least one fluid inlet channel away from at least a reaction chamber, wherein at least a portion of the or each Fluid inlet channel has a hydrophobic inner surface; Induce the Entering the sample into the reaction chamber (s) via the fluid inlet channel or through the fluid flow channels exercise a fluid inlet force and monitoring the sample in the reaction chamber or the reaction chambers for presence or concentration of a target substance. The method of claim 53, wherein the sample (s) for Leaving the reaction chamber (s) is brought or before the reaction chamber (s) or ejected sample for presence or concentration of a target substance is monitored. A method according to claims 53 or 54, wherein the or each sample by means of a pipette, a syringe or a syringe electrically operated injection device is applied. Method according to one of claims 53 to 55 for operating An apparatus according to claim 11 or any claim dependent thereon, wherein the fluid entry force is provided by aspiration means, wherein the aspirating agent for applying reduced pressure to the or each reaction chamber for a period in the range of 0.1 to 100 s are activated. Method according to one of claims 53 to 55 for operating a device according to claim 19 or claim 20 or any depend on it Claim, wherein the fluid inlet force by rotating the substrate or the carrier element at an angular velocity in the range of 1 to 1000 revolutions per minute for a period in the range of 1 to 100 s is provided. The method of claim 57, wherein the sample comprises the reaction chamber is ejected by the substrate with a higher Angular velocity in the range of 10 to 100,000 revolutions per minute for a period in the range of 1 to 100 s is rotated. Method according to one of claims 53 to 55 for operating a device according to claim 7 or any claim dependent thereon, wherein the fluid entry force is provided by piston pressure.