JP2002527751A - Microarray system and method for performing biochemical reaction - Google Patents

Abstract

(57) Summary A particulate or dynamic microarray system is created by immobilizing reactants on particles arranged in at least three distinct groups depending on the number of reactants used. Preferably, the groups have no immobilized reactant molecules or are separated by an intervening group having a particular signal molecule. Further, following the reaction or reaction between the analyte and the immobilized reagent, the particles are suspended in a solidifiable medium. The system of the present invention provides better kinetic properties than currently used spatially static microarray systems, where the reactant molecules are immobilized, for example, on a gel or chip.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a new concept for performing biochemical reactions and to systems and products for this purpose. The present invention further discloses a method of making the product. In particular, the present invention relates to a dynamic microarray system comprising a discrete group of particulate carrier matrices.

BACKGROUND OF THE INVENTION If a sample is available in large quantities, it may be divided into several identical sample aliquots. These aliquots may then be subjected to multiple tests, for example, by dispensing the aliquots into microtiter plates, where each well has the function of one reaction vessel and represents one reaction. Other samples, which are only available in limited quantities, should be subjected to multiple analyzes, one at a time and in the same reaction vessel at the same time. This is referred to as "multiplex analysis" in the following description.

The rapidly evolving technology, known as microarray technology, has recently become a promising tool for laboratories involved in biochemical and genetic engineering research and development. Microarrays are used for multiplex analysis of single samples. Typical applications are high throughput screening of pharmaceutical compounds, medical diagnosis based on genetic engineering methods, gene discovery, gene expression monitoring, gene mapping and sequencing of deoxyribonucleic acid (DNA) molecules. Traditional and currently used formats for multiplex analysis of a single sample include various types of techniques called dot blot analysis and dipstick-analysis, fragment analysis, and the like.

Recently, new methods of making such microarrays have been developed. In the field of genetic engineering, microarrays use DNA or peptide nucleic acids (PEA)
By the so-called in situ (on-chip) synthesis of oligomers of Another approach is to spot and immobilize oligonucleotide DNA or PEA molecules on a solid glass or nylon substrate using the piezo-electric printing method used in inkjet printers. The sample nucleic acid hybridizes to the complementary sequence on the array, either directly or after amplification, for example, by the polymerase chain reaction (PCR). The hybridization is usually detected by laser scanning using a fluorescent label incorporated into the hybridizing molecule.
A state-of-the-art review of DNA-related microarray technology was published by Ramsay [Nature
Biotechnology, 16, 1998, 40-44].

[0005] The essence of applying microarray technology to the above applications consists in specifying a set of individual locations with respect to their unique coordinates in a two-dimensional matrix. At each location, some events of special interest will occur and be recorded. Since the unique coordinates of each location are known, these events can be observed individually and compared to the nature of the events at other locations. In this context, an event is typically a chemical reaction of particular interest, for example, the process by which molecules chemically bond to each other, or the formation of various chemical complexes. This may be, for example, a hybridization, polymerization or transformation process spontaneously or induced by other processes. Any of the relevant processes may be of photochemical or electrochemical nature.

[0006] Despite being chemistry, current microarray technology uses discrete locations at specific solid surface areas to provide the individual identification required to separate different observations. These separated locations are called "dots" and the solid surface area where these dots are located is called a "matrix". One single two-dimensional xy coordinate system covers the matrix, thus providing the necessary reference to give each dot its unique x and y value. An "array" is defined as a complete set of dots in such a two-dimensional matrix, according to conventional microarray technology.

A special advantage is keeping the distance between dots in the matrix as short as possible.
is there. One advantage is that if the dot-to-dot distance is as short as possible,
Is more rapidly equilibrated. Another advantage is that
If the density of loose dots, that is, the number of dots per area unit is high,
The ability to reduce the overall size of equipment based on array technology. Present
In currently used systems, approximately 20-30.000 probes / cm ² Density, which is the theoretical limit of 10⁶Will be discussed. However, the dot
There is a lower limit on the distance between which is to obtain a separate observation on the matrix
Is defined by the resolution of the means used.

[0008] As a result of both efforts to keep the interdot distance short and to obtain high resolution, it is important that the solid surface on which the matrix is placed is rigid. This may be because the location of the obscured dots with respect to the reference matrix can cause confusion in identifying the particular observation event. Thus, conventionally used microarrays are fabricated on such rigid solid surfaces. Thus, these microarrays can be described as spatially static microarrays, and are referred to herein as "static" microarrays.

[0009] However, there are problems with static microarrays. This is due to a slow chemical reaction. The kinetic properties of the chemical reactions that take place in the liquid phase are such that the reactant molecules are properly positioned relative to each other so that the molecular interactions between the reactants required for a particular chemical reaction occur. Respond to time. It is true that the faster the relevant reactant molecule moves through the chemical environment, on average, the faster the chemical reaction. This is called fast kinetics versus slow kinetics. If one of the reactant molecules is fixed at a particular location, the kinetics will be slower than if all the reactant molecules were able to move.

[0010] Unfortunately, slow kinetics is a significant drawback in most microarray applications. Slow kinetics is associated with low resolution of individual observations as well as low sensitivity of assays based on chemical binding events. When used for sequencing by hybridization, low resolution will lead to imprecise or incorrect sequencing. When used for various mRNA-display analyses, slow kinetics may lead to incorrect conclusions or false clinical diagnosis. When used for high-throughput screening of drug leads, important drug target molecules will be ignored due to their slow kinetic properties.

In microarray-based approaches, it is important to be able to separate a large number, for example hundreds or thousands, of different chemical reactions from each other.
Unfortunately, it is not easy to observe different chemical reactions and distinguish them from other chemical reactions that occur in the same solution. The first chemical reaction may be, for example, a hybridization between a set of identical nucleic acid molecules and another nucleic acid molecule that is complementary to the set. The second chemical reaction will be a hybridization between a second set of nucleic acid molecules and their complementary parts.

[0012]

[Prior art]

U.S. Pat. No. 4,721,681 to Lentrichia et al. Discloses a binary system for so-called competition or inhibition assays, comprising first and second particles with complementary binding pair members. In an embodiment of the disclosed invention, the first heavy particle adsorbs the appropriate antigen and the second light particle adsorbs the corresponding antibody.
After some or complete immunochemical reaction, centrifugation causes the heavy particles and any heavy / light combinations to move outward in a centrifugal field. By waiting for such particles to move outward beyond the center of the light path, only a certain concentration of light particles should remain suspended in the reaction medium. By measuring absorption or light scattering, the quantity for the concentration of light particles is obtained. This value is then related to the analyte concentration with the aid of a pre-made standard curve.

The concept of multiple bead groups is described in WO97 / 35201 by Markman and Paleyov.
Is disclosed. In the description and examples, each bead group is defined as having at least one commonly recognized physical property. However, only the bead shape, size and color are described. Further, the bead size is 0.
It is specified by an upper limit of 5 to 10 μm. In addition, WO97 / 35201
The concept disclosed in U.S. Pat.
Entails a sorter adapted to classify. The sorter is further defined as a fluorescence activated cell sorter, which sorts the beads according to the fluorescence emission from them.

In view of the above, there remains a need for a recognition system for individual chemical reactions that is easily automated and has better kinetic properties than currently known microarray methods. Is clear. Also, spatial limitations and the limited accessibility of the reactants, which currently prevent the use of two-dimensional arrays,
We need to address the issue of cted accessibility of the reactant).

[0015]

Summary of the Invention

The present invention solves the problems and disadvantages outlined above of currently used microarray systems by introducing a novel concept for performing the claimed microarray-type assays. The microarray systems, methods and kits according to the concepts of the present invention not only provide better kinetic properties, but also can simplify handling of reactants and samples, provide faster and easier detection of results, and Integrates measurements in traditional laboratory practices, such as incubation and centrifugation.

The present invention is described in more detail in the following description, examples and figures.

[0017]

DESCRIPTION OF THE INVENTION

A suspension of particles in a solution, such as an aqueous solution, exhibits a similarity to a true solution in which the particles move more or less freely in the solution. In addition, the particle suspension is in contact with the aqueous solution exposing a large total surface area. The present invention relates to a method wherein the individual particles in a particle suspension bind simultaneously to one or more reactants and said particles are clearly recognized,
That is, it is based on the idea that it is possible to develop a microarray based on such a system if it is separated from other particles by any means.

The present invention comprises a microarray system comprising a reactant and a carrier matrix, wherein the carrier matrix comprises at least three multiple groups of particulate matter,
Each group has distinct properties, and the reactants are immobilized on the particles,
Each reactant is characterized in that it corresponds to a specific group with its specific characteristics. The particular property is selected from particle size or diameter, particle density, sedimentation properties, magnetic properties, radioactivity, color, chemical affinity, or a combination thereof. According to a preferred embodiment, the selected property is particle density.

Further, the present invention includes the above-mentioned system, and is characterized in that a group of densities having immobilized reactants is separated by an intermediate group having no immobilized reactants. Preferably, the density groups carrying the immobilized reactants are separated by a specific density of signal molecules, such as colorimetric reagents, fluorophores or chromophores.

One embodiment of the present invention is a kit for performing a multiplex assay, wherein the kit comprises a number of groups of particles having distinct densities, each group comprising a reagent that binds to the particles of the group. Is carried. The various particle populations can be separately transported to separate containers and an environment facilitating storage. This will facilitate "tailoring" a particular test by combining the particles with the immobilized reactants according to the desired measurements. Observing that single criterion, the resulting test must contain a separate group for density or selected property. This will provide users with a great deal of flexibility compared to using static microarrays.

It is also conceivable that a set of particles is distributed together with the corresponding reagents in a “ready-to-use” kit. The kit according to the invention further comprises a reaction medium, one or more suitable reaction vessels, instructions for performing the analysis and instructions for interpreting the results.

According to a preferred embodiment of the present invention, the reaction medium has the following characteristics: it is liquid during the performance of the analysis and does not impose any restrictions on the immobilization of the particulate carrier matrix; and after the necessary reactions have taken place. And, after performing the required separation of separate groups, such as separation by centrifugation, the medium can be forcibly solidified, thus preserving the order of the separated groups.

Optionally, the medium is a buffered solution having a composition that preserves, protects, or maintains the functional moiety that is subjected to or used in the measurement. If it is desired to include a pre-processing step, this can be incorporated into the analysis by adapting the medium. According to this embodiment, the medium comprises, for example, a lysis buffer. In a process according to this particular embodiment, a cell sample is mixed with a medium containing labeled particles, swirled, and centrifuged. Cell debris is separated during centrifugation and the reactants are allowed to bind to the particles. During or after the centrifugation step, the results of the analysis are interpreted by determining which population of particles containing a particular reagent has reacted with the compound present in the sample.

According to the present invention, one possibility is the use of an aqueous solution that freezes by lowering the temperature after completion of the centrifugation. Other suitable media are those containing polymeric or polymerizable components. Crosslinking or polymerization can then be initiated without interfering with the order of the separated groups, for example by irradiation. Other media used according to the invention are viscous or quite viscous at room temperature and are dilute or mobile liquids at elevated temperatures. Inevitably, elevated temperatures, preferably in the range of about 50 ° C. to about 100 ° C., more preferably about 60 ° C.
Incubate and centrifuge at a temperature between 0 ° C and about 90 ° C.

A well documented and highly developed particle technology is the so-called nanobead technology. Technologies based on microparticles, silica beads, polystyrene beads, latex beads, glass milk, paramagnetic particles and combinations thereof, among others, belong to this category of technology. However, observing the individual particles and separating them separately from each other is very difficult and time consuming. For example, WO97 / 3
See FACS apparatus and method described in 5201. Also, specific equipment such as a microscope or typical signal amplification means would have to be used. However,
Observation of groups of particles, each group consisting of hundreds or thousands of similar particles, is made much easier.

There are at least two distinct properties of such particles and can be used for identification. One is particle size or diameter. Another is particle density. In addition, sedimentation velocity and other behavioral characteristics may be used. The particles may be stained with color markers, fluorescent dyes, or associated with other markers, such as magnetic or radioactive markers. All of these properties can be used to separate each other into several categories.

However, according to a preferred embodiment of the present invention, density is selected as a property to characterize the large number of categories required for microarray technology. Thus, the present invention provides a microarray based on a density gradient of suspended particulates in which the reactants are immobilized and capable of reacting with other reactants in solution. Preferably, the density gradient is adapted to the sample to be investigated. According to a preferred embodiment, an adapted gradient is used with a medium having some function in addition to a mere suspension of particles, such as a medium involved in sample preparation, such as a lysis medium. Thus, pre-processing steps can be easily incorporated into this analysis.

Particles having a specific density are separated from other particles having other densities. Reactant molecules specific to this density group are immobilized on the particle surface of the particle group of each density. The entire population of particles is then pooled together into the same container containing the solution and the reactor molecules that are the counterparts of the immobilized reactor. After a sufficient time has passed for the reaction, the different density groups are separated from one another by centrifugation. This collects the particles with the highest density at the bottom of the container. The next particle category is the second highest density and so on.

The particles can also be concentrated by gravity standing, diafiltration or evaporation. An important property of the obtained gradient is its separate distribution for the selected particle properties. The individual groups divided for particle characteristics are clearly and clearly identified for the detection method chosen.

In one embodiment of the invention in which the various groups are separated by centrifugation, an important step is the establishment or stabilization of the particle layer achieved by said centrifugation. this is,
For example, it can be done by suspending the particulate carrier matrix in an aqueous medium. After the sample has been mixed and the reaction has taken place, the sample-matrix-suspension is then treated in a manner that leads to a clear separation of the particles. In embodiments where the particles have distinct and different densities, centrifugation is the preferred way to achieve this.

One problem associated with centrifugation of finely divided materials, such as cell fragments, or particles that show only a small density in current applications, is the vibrations that occur during rotor deceleration. . If the centrifugation is performed and the rotor speed is slowly reduced, when the speed passes a certain critical level,
Rotors often vibrate. This is related to the natural resonance frequency of the rotor. These vibrations overturn or at least disrupt the fine layers achieved by the centrifugation. To overcome this problem, we disclose the use and method of media in which the layer structure is fixed at the completion of centrifugation, but not before the rotor speed is reduced. This is achieved by suspending the particles in a solidifiable medium. Examples include aqueous media that can be frozen, media that can initiate the polymerization process or crosslinking by light or radiation, for example, by photopolymerization. Preferably, solidification of the medium can be initiated in a non-invasive manner, which achieves the solidification without adversely affecting the analyte and / or reactant and without distorting the analytical results. means. In this aspect, freezing the aqueous medium is suitable for most applications. Photopolymerization of a polymerizable material containing a suitable initiator is also suitable.

Examples of particles suitable for use in accordance with the present invention include latex beads, polystyrene beads, mineral particles, silica particles, and polymers and macromolecules of various origins. A currently available type of particle is polystyrene latex beads obtained from Sigma Inc. Silicon dioxide particles ranging from 0.5 to 10 microns are available from Sigma Inc. In addition, methacrylate particles having a density of about 1.2 and resorcinol / formaldehyde particles having a density of about 1.3-1.4 are available from Dyno Specialty Polymers AS, Norway.

Generally, microparticles are available in many different sizes and densities. Commercially available silica particles range from 0.01 to 20 μm. Latex particles are available in at least the following sizes: 0.05-0.
. 10 to 0.15 μm, 0.15 to 0.2 μm, 0.20 to 0.25 μm,
25-0.30 μm, 0.3-0.4 μm, 0.4-0.5 μm, 0.5-0.
6 μm, 0.6-0.7 μm, 0.7-0.8 μm, 0.8-0.9 μm, 0.
9-1.0 μm, 2-2.5 μm, 3-5 μm, and 5-10 μm (standard Dow latex).

[0034] The conventional fluorescent or luminescent reporter systems of the type used in current microarray technology can then be used to identify different particle density groups corresponding to different chemical reactions. In addition, higher resolution is obtained by combining the use of different densities of microbeads / microparticles with signal molecules, eg, signal molecules also known as fluorophores. Currently the number of available fluorophores is on the order of 5 to 10. Only 5
A vertical array containing three fluorophores of three distinct densities or sizes (a ve
Only 15 discriminative tests can be obtained by simply combining rtical arrays. The combination of the discrete physical properties of the microparticles, such as density or diameter, and the signal system, such as a fluorophore, allows for the production of multiplexed analysis kits with high resolution. In particular, the present invention allows for more efficient use of available fluorophores.

The upper limit of the resolution that can be obtained with the vertical or particle microarrays of the invention depends, of course, on the number of distinct fractions that can be obtained and the number of markers that are available. In addition to fluorophores, other markers, such as radioactive markers, may be used. Possible resolution limits for the system of the invention would be positions / samples of more than 1000 orders of magnitude.

[0036]

【Example】

Production Example 1: A suspension of silica particles with different densities ("raw" gradient) is centrifuged in a medium to separate the particles for different densities. Thus, a more or less continuous density gradient is formed. The centrifugal medium, which is in the liquid phase during centrifugation, is solidified, for example, by lowering the temperature or as a result of some chemical reaction, such as polymerization, preferably photoinitiated polymerization. The solidification should be a reversible process. The centrifuge vessel will preferably be a rectangular, closed plastic tube at its end. When the centrifugation medium is solid, the centrifuge vessel can be cut into individual segments and they can be referred to as segment 1, segment 2, etc., where segment 1 is closest to the end of the centrifuge vessel and segment 2 Is the second closest to the end, and so on.
Thereafter, the segments 1, 3 and 5 are put into one single centrifuge tube. The centrifuged medium in the segment is then reliquefied and another centrifugation cycle, solidification, sedimentation and segment classification is performed. By repeating the process a number of times, a discrete particle density gradient is formed. The density groups are called density group 1, density group 2, etc., and the names correspond to the segment names. See FIG. 2 for a schematic diagram.

On the particle surface of the density group 1, a reactant molecule specific to the density group is immobilized. However, if the second set of reactant molecules are immobilized on particles belonging to adjacent density groups, ie density group 2, it is difficult to separate these groups from each other. Alternatively, if the second set of reactant molecules are immobilized on particles belonging to density group 3, there are gaps between the density groups that make them easier to distinguish. Even higher densities are achieved when density groups 4, 5 or more are used for immobilization of the second set of reactant molecules. If further resolution is required, specific signal molecules can be immobilized in the density groups or groups used as spacers between groups carrying reactant molecules. In this way, the third and further sets of reactant molecules are immobilized on successively lower density groups of particles separated from each other by separating regions according to the resolution required to make the analysis feasible. can do. The preferred method of analysis is laser scanning, similar to conventional bar code reading systems. The identity of each density group is determined from its position relative to an adjacent density group that has a signal and is referred to as a space, or has no signal and will also be referred to as a space.

Production Example 2: A homogeneous mother liquor of particulate matter in solution is divided into multiple aliquots. Preferably,
Use polymeric material. To the first aliquot is added a particle size reducing agent, for example, an acid or alkali that digests the particulate material. Either add the same reagent to the next aliquot, but at a lower dose, or add the same dose but supply a shorter duration of action. Further lower doses are added to the next aliquot until an aliquot to which no size reducing agent is added is reached. Thereafter, a size-increasing agent, such as a polymer, substrate, coating, etc., is added to the next aliquot in increasing volumes. See FIG. 3 for a schematic diagram. Alternatively, the various aliquots are subjected to heat treatment, ultrasonic or chemical / enzymatic treatment which affects the particle size. After obtaining different densities in different aliquots, they are reacted with appropriate reagents and signal molecules. Then, by combining the particles with the desired properties,
A vertical microarray system can be assembled.

Production Example 3: A suspension of particulate matter containing large variations in density is centrifuged and the different layers are collected in separate vessels. To achieve sufficient resolution, each second fraction is collected for use in the same kit or for the same purpose. See FIG. 4 for a schematic diagram.

Example 4: The analysis according to the invention is carried out as follows: a small sample is taken from a patient, for example a small body fluid to be subjected to a diagnostic analysis. If desired, the sample is pre-treated, eg, filtered to remove solid particles. Select an appropriate test kit in which the required reagents are immobilized on separate particles. If desired, the test kit may be, for example, by adding a medium or by thawing a freezing medium and / or by, for example, vortexing a container containing the particle suspension to suspend the particles. , Should be in a prepared state. The sample is then added to the reaction vessel, the contents mixed, and after sufficient time for the reaction to occur, the reaction vessel is centrifuged. Once the particles have separated into distinct layers, solidification of the medium is initiated without interrupting centrifugation. In one preferred embodiment, solidification is achieved by using an aqueous medium and reducing the temperature below the freezing or solidification temperature of the suspension. Once the medium has solidified, the centrifugation is interrupted by slowing down the rotation speed. Finally, the reaction results, for example,
The layer is scanned and read by a suitable instrument, for example a suitably modified bar code reader or similar.

Although the present invention has been described in terms of its preferred embodiments, it constitutes the best mode known to the present inventor and departs from the scope of the invention as set forth in the appended claims. It should be understood that various changes and modifications can be made without obvious to those skilled in the art.

[Brief description of the drawings]

FIG. 1 schematically shows how different locations represent distinct groups of particles carrying different reagents.

FIG. 2 illustrates the principle described in Production Example 1.

FIG. 3 illustrates the principle described in Production Example 2;

FIG. 4 illustrates the principle described in Production Example 3;

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Claims (11)

[Claims] 1. A microarray system comprising a reactant, a carrier matrix and a medium, wherein the carrier matrix comprises particulate matter in at least three multiple groups, each group having distinct physical properties; Is immobilized on the particles, wherein one reactant corresponds to one particular group. 2. The method of claim 1, wherein said distinct property is selected from the following: particle size, particle density, sedimentation properties, magnetic properties, radioactivity properties, color, chemical affinity, or a combination thereof. A microarray system as described. 3. The microarray system according to claim 1, wherein said distinct characteristic is particle density. 4. The microarray system according to claim 1, wherein said medium is a solidifiable medium. 5. The microarray system according to claim 1, wherein the separate groups having immobilized reactants are separated by an intermediate group in which the reactants do not have immobilized reactants. . 6. The method according to claim 1, wherein the separate groups having the immobilized reactants are separated by an intermediate group having a specific signal molecule, such as a colorimetric fluorescent chromophore or a chromophore. 5. The microarray system according to any one of items 4 to 4. 7. A method for performing multiplex analysis on a large number of reagents and a particulate carrier matrix, comprising the steps of: adding a sample to a suspension of carrier particles comprising distinct groups with respect to their physical properties. Each reagent is immobilized in a separate group, and the sample is placed in fluid contact with the particles for a time sufficient for a reaction between the sample and the reagent to occur, and the suspension is suspended until the individual group separates. Centrifuging the liquid, and then measuring the reaction result for each reagent or group of particles. 8. A method for performing multiplex analysis on a large number of reagents and a particulate carrier matrix, comprising the steps of: adding a sample to a suspension of carrier particles comprising distinct groups with respect to their physical properties. Each reagent is immobilized in a separate group, and the sample is placed in fluid contact with the particles for a time sufficient for the reaction between the sample and the reagent to occur, and the suspension is suspended until the individual group separates. Centrifuging the liquid, solidifying the suspension, and then measuring the reaction result for each reagent or group of particles. 9. A kit for performing a multiplex analysis, wherein the kit comprises a number of particles having distinct characteristics, each particle carrying a reagent bound to the particles of the group; suspending the particles. Reaction medium, said reaction medium after centrifugation following the reaction between analyte and reagent,
Said kit selected to be capable of solidifying; and comprising instructions for performing said analysis. 10. The kit according to claim 9, wherein the medium is a medium that solidifies when the temperature is lowered for the purpose of fixing the particles after the analysis is performed. 11. The kit according to claim 9, wherein the medium is a medium capable of initiating its polymerization non-invasively by irradiation.