JP2003523185A - Arrays based on ferrofluids - Google Patents

Abstract

  (57) [Summary] Disclosed are magnetically derived microarrays based on ferrofluids, which are useful in high-throughput screening assays for identification, characterization and analysis of target substances in test specimens.

Description

Detailed Description of the Invention

CROSS REFERENCE TO RELATED PATENT APPLICATION This application describes 35U. S. C. 119 (e), claiming priority to US Provisional Application No. 60/175828 filed January 13, 2000,
The disclosure of which is incorporated herein by reference.

FIELD OF THE INVENTION The present invention relates to the field of molecular biology and analytical methods. More specifically, the present invention provides magnetically aligned arrays of ferrofluids on a solid support having at least one member of a specific binding pair that has an affinity for a target biomaterial. Such arrays provide protein / protein, drug / protein, drug / oligonucleotide, complementary oligonucleotide hybridization, complementary (cDNA) for screening, detection and analysis of target substances in test samples.
, RNA) nucleic acids as well as protein / oligonucleotide binding reactions, providing excellent support for assessing binding reactions.

BACKGROUND OF THE INVENTION In order to more fully describe the state of the art in the fields to which the present invention pertains, several publications and patents are shown within parentheses herein. The disclosures of each of these publications and patents are incorporated herein by reference.

Many laboratory and clinical procedures use specific affinity-based interactions to isolate and analyze target substances or species, which are herein referred to as Defined as a substance or analyte in a test sample of physiological, biological or chemical origin. Usually in diagnostic tests and drug screening, or in a wide range of soluble and particulate target substances or analytes, especially biological substances such as antigens, antibodies, proteins, nucleic acid sequences, oligonucleotides, cells, bacteria, viruses etc. Such affinity interactions have been used for the separation of

Various methods are available for analyzing or separating the target substance from a test sample based on the complex formation between the target substance and a specific binding substance having an affinity for the target. Selective separation of such complexes from unbound material can be accomplished by gravity, eg, by resting, or alternatively, by centrifuging small particles or beads with material having binding affinity for the bound target species. It can be done by If desired, and preferably, such particles or beads may be magnetized to facilitate separation of the bound fraction on the beads and the free fraction in the supernatant liquid. Limited applications of non-magnetic particles (and latex particles) in microarray applications are described in "DNA Microassays, M. Schena, ed.
., pages 7-18, 1999 ”.

[0006] Many different methods can be used to deposit biological material on the microarray solid support. These methods include capillary attachment, and US Pat.
Includes photolithography / capillary attachment as described in US Pat. Alternatively,
Multiple inkjets can also be used to deposit reactive species onto conventional microarrays. Using the jet method, a small volume of concentrated and therefore viscous binding reagent is deposited in defined areas on a non-reactive or chemically reactive planar surface. Such a method is also described in US Pat. No. 6,110,426.
This approach provides a very small droplet volume, minimizes reagents used and therefore minimizes associated costs. Moreover, the printing process can be accelerated to provide thousands of drops per second, which makes it possible to process many reaction vessels. Reactive species may be sequentially applied to the array to obtain the desired probe sequence or analyte (on-chip synthesis). Alternatively, the complete pre-synthesized sequence or analyte may be applied to the chip in a precisely controlled local area (
Off-chip synthesis). Such localized arrays come from several companies (Erie
Scientific Company, Portsmouth, NH is an example). Inkjet deposition methods are further classified by the way the reagents are delivered. Such methods include, for example, piezoelectric capillaries, piezoelectric cavities, thermal, acoustic, and continuous flow.

Application of the binding reagent to the chemically reactive surface causes the reagent to covalently attach to the microarray surface, typically irreversible attachment. In contrast, non-reactive attachment of binding reagents often results in the attachment of conformationally altered or modified coatings, reducing analyte recognition and stability. Such non-reactive binding reagents also tend to be washed out during incubation, which limits the stringency of incubation conditions during hybridization. Problems of denaturation using single-point, directed, or site-specific conjugation, as is commonly done in reactive coating with monofunctionalized oligonucleotide probes, or using, for example, biotinavidin-conjugated pairs. Can be minimized. However, functionalization of members of the desired binding pair is not always available. Moreover, when the oligonucleotides are attached directly to a solid surface, electrostatic interactions with the solid support tend to interfere with effective hybridization between the immobilized oligonucleotides and the target diffusion sequence. There is. Clearly, an improved method that minimizes the conformational changes in the members of the binding pair during attachment on the microarray surface is highly desirable.

Magnetic particles are well known in the art as they have been used in immunity and other specific affinity interactions. For example, US Pat. No. 4,554.
088 and Immunoassays for Clinical Chemistry, pp. 147-162, Hunter e.
See t al., eds., Churchill Livingston, Edinburgh (1983). In general, any material that facilitates magnetic or gravitational separation can be used for this purpose. However, it is clear that a magnetic separation means is the method of choice.

Magnetic particles are large (1.5 μm to about 50 μm) and small (0 μm) based on size.
． 7 μm to about 1.5 μm), or colloidal (<200 nm), and are also referred to herein as magnetic nanoparticles, ferrofluids, and colloidal or superparamagnetic particles. These nanoparticles are composed of aggregates of small magnetite or magnetite crystals, the latter being the oxidized form of magnetite.

Magnetic nanoparticles of the type described herein are conventionally coated with members of a specific binding pair to provide high surface area and rapid reaction kinetics so that they have specific affinity interactions. It is very useful in action-based analysis. Magnetic particles in the range of 0.7 μm to 1.5 μm have been described in the patent literature, eg, US Pat. Nos. 3,970,518, 4018886, 4230685, 4267.
234, 4452773, 4554088, and 4659678. Certain of these particles are disclosed to be useful solid supports for immunological reagents.

The small magnetic particles described herein generally fall into two broad categories. The first category includes permanently magnetizable particles or ferromagnetic particles. The second category includes particles that exhibit bulk magnetic behavior only when in a magnetic field. The latter, called magnetically responsive or superparamagnetic particles, are composed of magnetite or hemagnetite crystals with a diameter of 30 nm or less and can form the large agglomerates used in the present invention.

Similar to the small magnetic particles described above, large magnetic particles (> 1.5 μm to about 50 μm) in which magnetite particles are dispersed or encapsulated in an organic polymer may also exhibit superparamagnetic behavior. A typical example of such a material is Ugelstad, US Pat. No. 4,654,267.
And manufactured by Dynal (Oslo, Norway). Ugels
The method for producing tad is characterized in that polymer particles are synthesized by microemulsion polymerization to obtain polymer beads, which are swollen and absorb magnetite particles. It can also be achieved by synthesizing polymer particles in the presence of dispersed magnetite crystals.
． Magnetic particles in the same size range of 5 to 20 μm can be produced. Such synthesis causes trapping of magnetite crystals into the polymer matrix, thus magnetizing the resulting beads. In both cases, the polymer magnetic particles exhibit superparamagnetic behavior, which is evidenced by the fact that they can be easily dispersed when the magnetic field is removed. Unlike the magnetic colloidal nanoparticles described above and further detailed below, these large magnetic beads as well as large magnetite particles are simple low-gradient magnetic separation devices due to the relatively high magnet mass per particle. Is easily separated using. Therefore, in the case of test tubes or microtiter wells, the separation of these large particles in a conventional magnetic rack separator using a gradient in the range of hundreds of Gauss / cm to about 1.5 kilogauss / cm. You can On the other hand, colloidal magnetic particles less than about 200 nm in size require a substantially high magnetic gradient. Because of their dispersion energy, small magnet mass per particle, and Stokes drag, all of which must be overcome by the use of high-gradient magnetic separation devices that exert effective magnetic collecting power. This is because it must be done.

Another standard for detecting and measuring target analytes in a sample is PCT / US
98/05911. These methods magnetically label the target,
The target-magnetic particle complex is then characterized by attracting it to a collection surface having target binding capacity. This analytical method is based on two specific binding interactions for the target, the first interaction is by the particle and the second interaction is by the collecting surface. However, such a method is applicable only if the characteristic determinants of the target are known to allow specific binding interactions. Furthermore, a method for specifically binding a target to a position defined by the magnetic sequence used is described, but how to create a position that can be specifically aimed (ie aim To make a defined array).

Although in the prior art, magnetic particle assays and applications in single test or low volume formats have been described and illustrated, it is highly desirable to develop methods for performing such magnetic assays in high throughput screening models. Such a method should facilitate the identification, characterization and analysis of biological or pharmacological target substances in test samples.

SUMMARY OF THE INVENTION According to the present invention, methods and devices for immobilizing and isolating target substances on a solid support for use in high throughput screening assays are disclosed. A typical method is to use a magnetically aligned array in bound compartments to prepare a solid support to which the first member of a specific binding pair is attached and to which magnetic particles are added. And immobilizing and identifying the target substance in the test sample by adding to the attached magnetic particles a plurality of second members of a specific binding pair having a binding affinity for the first member. Characterize. Magnetic particles are then concentrated in the array by magnetic means inside the compartment, such that between the members of the first binding pair on the solid support and the second binding members on the magnetic particles. Interact with each other to immobilize the magnetically responsive particles essentially irreversibly. The particles immobilized in the compartment on the solid support are then contacted with a first nucleic acid sequence operably linked to a first member of the specific binding pair, whereby A first nucleic acid sequence is immobilized on the immobilized particles. The immobilized particles bound by the first nucleic acid sequence are then contacted with a test sample that may contain a detectably labeled target substance having an affinity for the first nucleic acid sequence. By measuring the detectable signal of the bound labeled target substance, the binding between the nucleic acid sequence of 1 and the target substance present in the test sample is detected and optionally quantified.

A suitable solid support is selected from the group consisting of plastic surfaces, glass surfaces, silica surfaces, metal surfaces, membranes, microtiter plates, microtiter strips, and microtiter wells. The solid support may include a bound compartment overlying the pin location. In one embodiment, such a compartment is formed by drawing a hydrophobic line on the solid surface. Alternatively, the solid support may comprise particles selected from the group consisting of polymer particles, latex particles and inorganic particles in the size range of about 0.5 μm to about 20 μm. Magnetic particles suitable for use in the present invention are selected from the group consisting of colloidal ferrofluids, magnetic nanoparticles, magnetic microparticles and ferrofluid-coated microparticles.

In another embodiment of the invention, the first nucleic acid sequence is operably linked to a biotin species and placed in a separate compartment on the array. The array is then contacted with a test sample containing a mixed population of detectably labeled nucleic acids. A subset of nucleic acids is suspected of being complementary to the first immobilized nucleic acid. The immobilized first nucleic acid sequence and the test sample are subjected to conditions that allow hybridization between complementary nucleic acids, which allows the detection of multiple complementary target nucleic acids in the test sample. Become. In another embodiment, multiple nucleic acid sequences may be attached to the array.

In a preferred embodiment of the invention, the first member of the specific binding pair is biotin, or an analogue thereof having a considerable or slightly lower affinity for avidin or an avidin species, eg desthiobiotin. And the second member of the specific binding pair is streptavidin, or an avidin species with considerable or slightly lower affinity for biotin or biotin analogues, such as avidin or nitro-avidin.

A suitable device for measuring the immobilized detectable label is selected from the group consisting of a fluorometer, a spectrophotometer, a scintillation counter, a gravimetric biosensor, and a surface plasmon resonance biosensor. .

In a further embodiment of the invention, an apparatus is provided for detecting binding events between members of a specific binding pair. A typical device of the invention is a means for defining a magnetically aligned responsive array on a non-magnetizable solid support surface, a plurality of magnetizable magnets in direct contact with the lower surface of the solid support. Includes an array template with array pins. Further, the solid support is divided into bonded compartments overlying the array pins, and an array template connected to a rare earth magnet or electromagnet, which results in a high magnetic gradient near the tip of the array. The gradient enables the collection of magnetic particles in the magnetically responsive array.
In a preferred embodiment, the array is from about 2 to about 1000 per square inch.
Pin density in the range of 00 pins, more preferably about 4 chairs per square inch and about 200
It is a microarray with a pin density in the range of 0.

The utility of the novel magnetic microarrays of the present invention is further facilitated by the use of an external magnetic gradient that allows for simultaneous detection or screening of many target substances in a sample specimen. Alternatively, multiple interactions of a single substance with multiple different specific binding substances, each of which may exhibit a biological function, may be identified and quantified, as exemplified by screening drug candidates.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides ordered micro-sized ferrofluid particles on a suitable planar or particulate solid support for use as microarrays containing so-called biochip geometries. Although it relates to an array fabrication method and multiple applications, many other variations are feasible. In one embodiment, the microarray is formed by means of static magnetizing pins that form a ferrofluidic spot at a location on the microarray defined by the magnetizing pin and the coupling compartment corresponding to the pin position. Such arrays can be used to advantage in many applications, including gene expression analysis, hybridization assays for mutation detection, polymorphism analysis, genomic mapping for drug mechanism studies, lead compound identification and Includes, but is not limited to, optimization, combinatorial synthesis, array immunoassays, toxicity studies in pharmacodynamics and pharmacogenomics. Preferably, all of the above reactions are performed using the High Throughput Screening (HTS) method.

One particular promising method for analyzing a nucleic acid-containing biological sample is D in a sample.
It uses a DNA-based microarray that produces a hybridization pattern that is characteristic of NA. Briefly, conventional DNA microarrays contained single-stranded DNA fragments bound to a solid support,
The methods and arrays of the present invention use magnetic particles and magnetic fields to facilitate the accuracy, reproducibility and detailed analysis of the binding reactions to be evaluated. Each element in the array contains tens to millions of copies of the same single-stranded nucleic acid sequence. The same or different fragments of nucleic acid may be provided to each of the different elements of the array. In other words, position (1,1) may comprise a single-stranded nucleic acid fragment different from position (1,2), position (1,2) also being different from position (1,3). Good (same below). Chemiluminescence, fluorescence, or electrical phenomena may be used to assess interactions between nucleic acid molecules.

Various devices are known for detecting such interactions. The most basic of these are purely chemical / enzymatic assays in which the presence or amount of analyte is assayed by measuring or quantifying detectable reaction products such as gold immunoparticles. The interaction of specific binding pairs can also be detected and quantified by radiolabeling assays. In contrast, in biosensor diagnostic devices, the assay substrate and detector surface are integrated in a single device. One common type of biosensor uses, for example, an electrode surface combined with a current or impedance measuring element to detect changes in current or impedance in response to the presence of a ligand-receptor binding event. This type of biosensor is disclosed in US Pat. No. 5,567,301.

Mass measurement biosensors use piezoelectric crystals to generate surface acoustic waves, the frequency, wavelength and / or resonance state of which is sensitive to the surface mass relative to the crystal surface. Therefore, changes in acoustic properties are indicative of changes in surface mass due to, for example, interaction events of specific binding pairs. U.S. Pat. Nos. 5,478,756 and 4,789,804 describe mass measuring biosensors of this type.

Biosensors based on the surface plasmon resonance (SPR) effect have also been proposed, for example in US Pat. Nos. 5,485,277 and 5,492,840. These devices use changes in the SPR surface reflection angle that occur with perturbations, such as binding events at the SPR interface. Finally, various biosensors are known that use changes in the optical properties of the biosensor surface, such as US Pat. No. 5,268,305. Biosensors have many potential advantages over binding assay systems with individual reaction substrates and reading devices. One important advantage is that microchip manufacturing methods such as those described in US Pat. Nos. 5,200,521 and 5,212,050 can be used to manufacture biosensor units that are small but highly reproducible. . Another advantage is the potentially large number of different analyte detection areas that can be integrated into a single biosensor unit, allowing sensitive detection of some analytes with very small amounts of biological liquid samples. it can. Both of these advantages can lead to substantial savings in cost per test.

Unlike other DNA array systems in which complementary nucleic acid sequences are synthesized in situ on an array biochip, the present invention allows direct attachment of specific complementary oligonucleotides onto a solid support. To do. In this embodiment, the oligonucleotides are operably linked to magnetic particles, which are then magnetically focused onto the array into a single spot. As such, a simple unknown sample can be analyzed in bulk fashion as a simple microarray composed of multiple magnetic particle spots, each spot containing a different complementary oligonucleotide sequence of interest. Similarly, multiple unknown samples may be analyzed by specifically dispensing such samples on individual spots.

In a typical method of the invention in which fluorescence imaging is used, first the target DNA sample to be analyzed is fluorescently labeled. Each DNA sample is then denatured and hybridized to the microarray, after which the formation of complementary duplexes is measured by fluorescence. Each sequence target controls the stringency of hybridization so that it only hybridizes to its complementary nucleic acid sequence already attached to the microarray. Nucleic acid sequences that are not complementary to any nucleic acid present on the microarray do not hybridize to any part of the microarray and are washed away during the subsequent washing step.

Thus, only those positions of the microarray containing probe sequences that hybridize to complementary sequences in the target DNA sample will receive the fluorescent molecules. Typically, a fluorescent source is then applied to the microarray to obtain a fluorescence image to determine which elements of the microarray bind, eg, to a patient DNA sample, or which elements do not. The image is then analyzed to determine which specific DNA fragment was contained in the original sample, which results in the presence of a particular disease, mutation or other condition in the patient sample. Decide whether or not

For example, a specific element of a microarray may be a DNA that represents a specific type of cancer.
Expose to array. When the element of the array fluoresces under fluorescent illumination, the sample DNA contains a DNA sequence indicative of the particular type of cancer. It can therefore be drawn that the patient who provided the sample already has, or is likely to be predisposed to, that type of cancer. It will be appreciated that by providing various known DNA fragments on a microarray, the resulting fluorescence image can be analyzed to identify a wide range of conditions.

In general, there are two types of DNA microarrays: passive hybridization microarrays and active hybridization microarrays. In passive hybridization, solid phase-probe and solution phase-
As a target, the oligonucleotides that characterize the DNA sample are simply applied to the DNA microarray, and the target passively forms complementary duplexes with the array probe. In active hybridization, for example, using a magnetic method,
The DNA array is arranged to externally facilitate the interaction between the fragments of the DNA sample and the fragments attached to the microarray. It should be understood that currently either passive hybridization steps or active hybridization steps, but not both, are typically used for any individual microarray. Referring first to passive hybridization, a DNA microarray chip with oligonucleotides of interest attached or operably linked to the microarray by chemical or magnetic forces is prefabricated. The labeled sample target sequence is hybridized to the microarray. In one embodiment, labeled sample target sequences that match any of the probe oligonucleotides attached to the microarray hybridize while their fluorescent labels are retained, thus forming a duplex with the sample target. Only locations in the microarray fluoresce. It should be noted that high stringency hybridization conditions ensure that the duplex is formed and represents the correct sequence hybrid match. When fluorescent light is applied,
An exact match fluoresces most efficiently, and an inexact match fluoresces fairly weakly or not at all.

A DNA microarray loaded with a sample is accompanied by chemicals to facilitate the hybridization reaction, ie, chemicals to facilitate hybridization of the sample sequence with the corresponding complementary probe in the microarray. Place it in the fluidics station. The microarray is then exposed to the fluorescence, perhaps that produced using an ion-argon laser, and the resulting fluorescence pattern is digitized and recorded. Alternatively, the fluorescent pattern may be photographed, developed and then scanned into a computer to obtain a digital representation of the fluorescent pattern. In either case, the digitized pattern is processed using a dedicated software program that operates to focus on the digital pattern, quantify the pattern, and obtain fluorescence intensity values for each array in the microarray pattern. Calculate the average intensity value at each array position and perform the required normalization,
The resulting array pattern is processed with an additional software program that provides color correction and scanning. After this step, a digitized fluorescence pattern is obtained to identify the position in the microarray where the particular gene sequence from the DNA sample formed a double-stranded hybrid.

As already mentioned, the invention is not limited to the evaluation of interactions between complementary nucleic acid molecules. The methods disclosed herein may be used to assess binding interactions between members of any specific binding pair.

As used herein, the term “target substance” refers to various soluble and particulate analytes or substances of biological or pharmacological interest. Examples of soluble target substances include hormones, proteins, peptides, lectins, drugs, chemicals, oligonucleotides, nucleic acids (eg RNA and / or DNA) and particles including biological particles such as cells, viruses, bacteria and the like. Target substances.

The term “determinant”, when referring to any of the above target substances, refers to a “binding substance” having a specific binding affinity for the determinant or a portion of the target bound by any molecule or Refers to the group. Such binding agents may or may not include one member of a specific binding pair. A determinant may consist of an antigenic epitope recognized by an "antibody binding agent", a nucleic acid sequence recognized by a complementary nucleic acid "binding agent", or any molecule recognized by a particular binding partner. In basic terms, a determinant is defined as the region of molecular contact that is recognized in the interaction of a specific binding pair. Preferred determinants are unique to the target.

The expressions “specific binding pair” and “specific binding partner” are used interchangeably,
Two substances that selectively recognize and interact with each other are meant to substantially exclude other substances present in the sample medium. Specific binding partners comprising members of the first and second binding pairs include antigens and antibodies, receptors and hormones, receptors and ligands, agonists and receptors, antagonists and receptors,
Lectins and carbohydrates, complementary nucleic acids (RNA or DNA) and peptides-
Nucleic acid (PNA) sequences, Fc receptors and mouse IgG-Protein A, biotin- or biotin analogues and avidin, biotin or biotin analogues and streptavidin, metal chelators and metal ions (ie EDT
A or DTPA and Fe ³⁺ , Cr ³⁺ , or Ni ²⁺ ), enzymes and substrates, and viruses and virus binding proteins. Various other combinations of specific binding pairs are contemplated for practicing the methods of the present invention and will be readily apparent to those skilled in the art. Also contemplated for use in the present invention are peptides, agents, nucleic acids, or analogs thereof that specifically recognize determinants with similar specificity to traditional antibodies.

The term “oligonucleotide” as used herein refers to the primers and probes of the invention and is defined as a nucleic acid molecule containing two or more, preferably more than three ribo- or deoxyribonucleotides. The exact size of the oligonucleotide will depend on various factors and on the particular application and use of the oligonucleotide.

The term “probe” as used herein may be a naturally-occurring substance that is a purified digestion product of a restriction enzyme or a synthetically-produced product, and is not defined as Oligonucleotides, polynucleotides or nucleic acids (RNA that can anneal or specifically hybridize to nucleic acids having complementary sequences)
Or DNA). The probe may be single-stranded or double-stranded. The exact lengths of the probes will depend on many factors, including temperature, source of probe and method of use. For example, for diagnostic applications, the oligonucleotide will typically contain 15-25 or more nucleotides, although fewer oligonucleotides may be included, depending on the complexity of the target sequence. The probes of the invention are selected to be "substantially" complementary to different strands of an individual target nucleic acid sequence.
This means that under a set of predetermined conditions the probes must be sufficiently complementary to "specifically hybridize" or anneal to their respective target strands. Means Therefore, the probe sequence need not reflect the exact complementary sequence of the target. For example,
A non-complementary nucleotide fragment is attached to the 5'or 3'end of the probe,
The remaining portion of the probe sequence may be complementary to the target strand. Alternatively, non-complementary bases or longer sequences may be inserted in the probe, provided that the probe sequence has sufficient complementarity to the target nucleic acid sequence and specifically anneals to it. Is. The probe may include a detectable label.

The term “specifically hybridize” refers to the association between two single-stranded nucleic acid molecules that have sufficiently complementary sequences and is a pre-determined condition commonly used in the art. The term below refers to those that allow such hybridization (sometimes referred to as "substantially complementary"). In particular, the term refers to the single chain D of the invention.
Hybridization of an oligonucleotide with a substantially complementary sequence contained in an NA or RNA molecule, which excludes hybridization of a single-stranded nucleic acid with a non-complementary sequence with the oligonucleotide.

As used herein, the term “primer” refers to RNA or DNA, single-stranded or double-stranded, derived from a biological system, obtained by restriction enzyme digestion, or obtained synthetically, and is a suitable oligonucleotide. When placed in the environment, it acts functionally as an initiator of template-dependent nucleic acid synthesis. Primers by the addition of nucleotides by the action of a polymerase or similar activity when provided with a suitable nucleic acid template, a suitable nucleoside triphosphate precursor polymerase enzyme for nucleic acids, a suitable cofactor and conditions such as temperature and pH. May be extended at its 3'end to give a primer extension product. The length of the primer may vary depending on the particular conditions and requirements of the application. For example, for diagnostic applications, otide primers are typically 15-25 or more nucleotides in length. The primer must be sufficiently complementary to the desired template to prime the synthesis of the desired extension product, ie, the 3 of the primer in a juxtaposed position suitable for use in initiating synthesis by a polymerase or similar enzyme. 'It must be able to anneal to the desired template strand in a manner that provides a hydroxyl moiety. The primer sequence need not be an exact complement to the desired template.
For example, a non-complementary nucleotide sequence may be attached to the 5'end of a complementary primer. Alternatively, non-complementary bases may be dispersed in the oligonucleotide primer sequence, but the primer sequence is sufficiently complementary to the desired template sequence that the template-primer complex for extension product synthesis is obtained. The condition is that it produces the body. The primer may include a detectable label.

The phrase “inkjet method” refers to a method suitable for depositing a desired reagent on a solid support. The advantage of this deposition method is that it allows very small amounts of reagent (<1 μl) to be deposited at precisely controlled locations as shown in FIG. Depending on the chemical reaction conditions used, such attachment may be reversible or irreversible.

The terms “operably linked” and “attached thereto” are used interchangeably and refer herein to the covalent or non-covalent association of two molecules with no loss of function, or of a molecule and a solid support. .

The expression “detectable label” as used herein refers to a substance whose direct or indirect detection or measurement by physical or magnetic means is indicative of the presence of the target substance in the test sample. Typical examples of useful detectable labels are optically detectable molecules or ions with light absorption, fluorescence, refractive index, light scattering, phosphorescence, or luminescence properties; molecules or ions detectable by radioactivity; nuclei. Includes, but is not limited to, molecules or ions detectable by magnetic resonance, remanence or paramagnetism. Quenched fluorescent label (quen that does not fluoresce unless hybridized to the target sequence)
ched fluorescent labels) are a group of molecules that can be indirectly detected based on light absorption or fluorescence. A suitable reagent for this purpose is the "Molecular Beacon" sold by RESEARCH GENETICS.

Molecular beacons, as used in the preferred embodiment, are useful probes for the detection of specific nucleic acids in homogeneous solution. A probe based on the molecular beacon theory is a single-stranded nucleic acid molecule having a stem-and-loop structure. The loop portion of the molecule is a probe sequence that is complementary to a predetermined sequence in the target nucleic acid. The stem is formed by annealing two complementary arm sequences on either side of the probe sequence. The arm sequence is independent of the target sequence. The fluorescent moiety is attached to the end of one arm and the non-fluorescent quencher is attached to the end of the other arm. The stem keeps these two parts in close proximity to each other and causes fluorescence resonance energy transfer to quench the fluorescence of the fluorophore. As a result, the fluorophore is unable to generate fluorescence. When the probe encounters the target molecule, it forms a stable hybrid for a longer time than the hybrid formed by the arm sequences. Thus, the probe undergoes a spontaneous conformational change that separates the arm sequences, separating the fluorophore and quencher sequence from each other.
This change in proximity can cause the fluorophore to fluoresce when illuminated with light of the appropriate wavelength. These "molecular beacons" can only emit an increase in background fluorescence signal when hybridized to their target molecule. In a preferred embodiment, the molecular beacon is an analyte that is specific for, competes with, or binds to the complementary oligonucleotide sequence as the target substance, thereby determining the amount of the target substance in the test solution. Provides an inversely proportional signal. Therefore, regarding a detectable signal, "quenched" refers to an undetectable signal, and "not quenched" refers to a detectable signal. Molecular Beacons: Probes that Fluoresce upon Hybridization, Sanjay Tyag
See i and Fred R. Kramer, Nature Biotechnology, volume 14 March, 1996. Also included are non-detectable enzyme substrates that are converted by enzymatic action from undetectable to detectable light absorbing or fluorescent molecules.

The phrase “substantially excludes” refers to the specificity of a binding reaction between a specific binding substance in a specific binding pair and its corresponding binding partner that includes a specific target substance. Specific binding substances have a substantially selective binding affinity for their target substance, but at detectable levels of non-specific binding to other non-target substances or components in the test sample. Also shows.

As used herein, the term “species” refers collectively to a family of related substances consisting of isomers, analogs, and derivatives of the specified substance.

The term “microarray” or “biochip” refers to a position on an array or x
-Refers to an array generated by a magnetic force on a planar surface that constitutes a plurality of separate reactions or incubation compartments identifiable by the y-coordinate. Such arrays are suitable for use in assays to assess specific binding properties between members of a specific binding pair.

The phrase "high throughput screen" of the present invention is defined as an analytical system capable of processing a large number of test specimens for a large number of target substances contained within a short period of time. Alternatively, a high throughput screen may be used to characterize the biological properties of a substance by assessing its binding to multiple potential binding partners. For example, Affymax Corporation uses photolithography to create a number of unique D
Manufactures DNA chips or microarrays containing NA probes. An immunoassay array panel carrying immobilized binding partners is disclosed in US Pat.
70 and 4829010.

The term “conformational change” as used in connection with attachment of binding species on a microarray includes minimal to global denaturation and also refers to undesired changes in the tertiary structure of the binding reagent. Such changes are particularly problematic in protein binding molecules, which can lose up to 90% of their binding capacity by coating reactive or non-reactive surfaces by multipoint binding to the surface.

In the present invention, “magnetization pin” or “pin” means a protrusion arranged on the surface of one of a three-dimensional flat object capable of magnetizing or non-magnetizing or a protrusion processed by a machine.

Preferred magnetized particles used in the practice of the present invention are in the range of 0.05 to 5 μm,
More preferred are particles with a size in the colloidal range of 0.05 to 0.5 μm, most preferably 0.09 to 0.15 μm (90 to 150 nm). Colloidal particles are characterized by stability when left for a long time and / or resistance to gravitational separation. In addition to many other advantages, particles in this size range rarely interfere with most detection methods. For example, such particles do not interfere with the microscopic analysis of target cells. Within the range of 90 to 150 nm, preferably 5%
Particles having a magnet mass in the range of to 99%, more preferably between 10% and 95%, most preferably between 20% and 90% are contemplated for use in the present invention. Suitable magnetic particles include a crystalline core of magnetite or maghematite, which is coated or bonded to the magnetic core, eg,
It is physically adsorbed or covalently bound and surrounded by molecules that impart stable colloidal properties. Preferably, the coating material should be applied in an amount or thickness effective to sufficiently shield the magnetic core from non-specific interactions with biological macromolecules in the test sample. Such biological macromolecules may include one member of a specific binding pair, immunoglobulins, albumins, non-target groups sialic acid residues on the cell surface, lectins, glycoproteins, and other cell membrane components.

Furthermore, the material should contain as much magnet mass as possible per particle.
The size of the magnetic crystal containing the particulate core is small enough not to contain the complete magnetic domain. Nanoparticles are also small enough that their Brownian kinetic energy exceeds their magnetic moment. As a result, the N / S pole arrangement and the resulting mutual attraction / repulsion of these colloidal magnetic particles occurs minimally even in moderately strong magnetic fields, thereby contributing to their resistance to sedimentation. To do. Eventually, magnetic particles should be separated in a high field gradient external magnetic field separation device characterized by generating a strong magnetic field in close proximity, but from the outside of the test sample. Magnetic particles having the above properties can be produced by modifying the base materials described in US Pat. Nos. 4,795,698, 5597531, and 5698271.

There are many ways to create an array, which will be described in more detail in the examples. However, each method relies on magnetically collecting particles, which can be bound to the collecting surface as precisely positioned spots. The magnetic configuration used will determine the physical layout of the array. The collection surface is coated with one member of the specific binding pair and the magnetic particles with a second member. As the particles are drawn into the collecting structure, they become bound to the surface.

In one embodiment of the present invention, compartments were formed on a flat flat surface containing up to 500 μL from the containment gasket as described in Examples 3 and 4. Smaller surface compartments suitable for volumes in the range of 1 to 100 μL can be easily constructed by conventional mechanical or lithographic line forming means to prevent intermixing of the contents of adjacent array compartments with hydrophobic lines or " A fence is used.

Such a patterned support with compartments is available from Erie Scientific
Commercially available from companies such as Company, Portsmouth, NH in the form of microarray glass plates, 96, 384, 1536 and other substrates custom printed with more patterned hydrophobic inks. There is. A robust and durable hydrophobic pattern for making the microwell compartments can be deposited, which makes the method very compatible and applicable to the disclosed magnetic microarray concept.

When using such arrays, ferrofluidic particles, reagents and test solutions can be dispensed by conventional nanoliter or microliter delivery systems. Such systems include the single or multi-channel precision inkjet dispensers described above manufactured by DIE-MARK, San Diego, CA. The reagents may be deposited directly in the planar surface compartment or in microtiter wells. As shown in FIGS. 5 and 6, high precision inkjet dispensers can deliver reagents to the entire sample or to specific locations on the array. Pre-binding members of a specific binding pair with characteristic determinants that are complementary to a region of interest or sequence on a target species to ferrofluid particles prior to attachment or array formation by magnetic pinning means You can Figure 5 shows a device suitable for assaying multiple samples for a determinant of 1. An important feature of the apparatus and method that distinguishes FIG. 6 from FIG. 5 is the deposition and formation of ferrofluid spots containing ferromagnetic particles of different properties so that multiple target determinants can be detected from one unknown sample. It is possible.

In a further embodiment of the invention, polymerase chain reaction (PCR), ligation chain reaction (LCR), in vitro RNA polymerase / promoter amplification (a
Hybridization signals can be further amplified by conventional nucleic acid amplification methods such as RNA, TMA, 3SR and the like), and rolling circle amplification method (RCAT). Furthermore, standard signal amplification methods can amplify the signal of selected compartments.
Possible hybridization buffers include any and all suitable reaction mixtures, including volume excluders, hybridization rate enhancers and sensitivity enhancers (ie dextran sulfate, polyethylene glycol, phenol, etc.). Including reagents.

Unique benefits are provided when ferrofluids are used as substrates for the fabrication of indexed microarrays as disclosed in the present invention. Such advantages include: 1) Using conventional pumps or inkjet delivery systems, colloidal ferrofluids are dispensed like regular liquids. This is because the particle concentration and the resulting viscosity are low. 2) Dispensed ferrofluid particles can be concentrated from solution in many typical forms, such as very strong dots, groups of dots, or lines, significantly dilute solutions and low reagent concentrations. Can be used, and in addition, very precise control of the exact shape and size is possible. 3) A thin layer of coupled ferrofluid (reversible as a three-dimensional pattern using a non-bonding support held by magnetic force, or a two-dimensional pattern that remains firmly bonded even after the removal of a magnetic field The ferrofluid can be irreversibly deposited using a binding support that provides a monolayer of particles. 4) on a flat, non-porous support, or 96, 384, 1
Ferrofluid can be dispensed into preformed wells of a microtiter plate containing 536 and a larger number of discrete spots, eg, about 2000 spots per square inch as shown in FIG. . 5) Ferrofluids allow the use of magnetic field detectors for nondestructive measurement of the mass and hence the number of attached particles, thus providing an internal standard which allows normalization of the detectable signal. Created. 6) Reversibly opening and re-capturing the three-dimensional magnetic microarray spots by magnetic means allows the ferromagnetic particles to mix locally in suspension in the absence of a magnetic field. Increases the kinetics of the affinity reaction; applying a magnetic field before substantial dispersion of the magnetic particles occurs causes the ferrofluid particles to return to their original spot configuration.

One method for making the microarrays of the present invention involves magnetic focusing using magnetic pins that produce "points" of ferrofluid particles in a planar pattern in a suitable magnetic field. It is used. This method allows each position to be uniquely defined and probed, thus allowing the binding interactions with individual target substances to be traced.

As shown in FIG. 5, the “magnetization pin” forms a protrusion mounted on the surface of one of the three-dimensional planar object which can be magnetized or cannot be magnetized, or a protrusion which is machined. Such non-magnetic pins (54) become magnetized when exposed to the magnetic field from the magnet applied to the pin ends. The magnet (55) may be an electromagnet,
Alternatively, it may be a removable rare earth type strong permanent magnet,
53) Inducing a strong magnetic gradient near the tip of the pin that collects the magnetized ferrofluid particles (52) near the pin, thereby forming an array pattern. Once collected, the streptavidin-loaded ferrofluidic particles in the array are reversibly immobilized (three-dimensional thin layer) depending on the absence or presence of members of the specific binding pair covalently bound to the surface. (As a layer) or irreversibly immobilized (as a two-dimensional monolayer after washing the unbound ferrofluid). A typical inkjet dispenser (51) is also shown.

FIG. 6 illustrates the synthesis or formation of a single ferrofluid spot. Ferromagnetic particles (
62) covalently bound ligand with affinity for complementary moieties on (
A derivatized thin solid support (64) containing 63) is placed on top of the magnetizing pin.
Dispenser tip (61) placed very close above the magnetising pin is shown,
The arrow indicates a very rapid movement of the ferrofluid particle, which is ejected from the tip towards the magnetic pin. The concentration of ferromagnetic particles in this manner allows the formation of very small spots whose diameter is limited only by the diameter of the pin used. In this way, ferromagnetic spots in the submicron to micron size diameter range can be easily formed. A feature of FIG. 6 that distinguishes it from FIG. 5 is the ability to deposit and form ferrofluid spots containing ferrofluid particles of different specificities so that multiple targets can be detected from one unknown sample.

This will allow the determination of the relative concentration of the target molecule, as well as its presence or absence. This can be done because the signal of the spot will be known and can be compared to the relative intensity of the detectable label corresponding to the amount of target molecule present. The limit of detection of the target substance in the fluorometric immunoassay can be further extended, for example by the ability to concentrate the signal in a very small area. In contrast, conventional fluorometric methods focus the light beam only on a portion of the incubation mixture and may not be able to detect all the fluorophore present.

An alternative method is a detectably labeled small non-magnetic carrying one or more encapsulated fluorophores and further carrying a surface covalently bound to the first member of the specific binding pair. Microspheres, for example, non-magnetic microspheres having a size of 0.5 to 10 μm are used. The magnetic particle carries the second member of the specific binding pair,
It can form bound pairs and immobilize particles. The resulting binding layer on the magnetic particles renders the microspheres magnetically responsive and allows the binding of additional binding partners or labels. Such magnetic microspheres can also be handled magnetically,
It can be concentrated in a point or linear array. After binding of the members of the specific binding pair, one of them contains a detectable label, and a device such as a scanning laser spectrophotometer in a high throughput screening mode can be used to analyze the microparticles on the microarray. it can. As an internal standard for magnetic collection efficiency, magnetic particle detectors, such as the MAR described by Quantum Design
II (WO99 / 27369) may be used to determine the mass or number of magnetic particles at a given array site.

A typical assay involves the sequence of the following steps. The binding partner for the target is bound to the magnetic particle surface. This complex is mixed with the sample to facilitate the capture of the target on the magnetic particles. The magnetic particle-label complex is then concentrated and collected on the solid support collection surface for signal acquisition and analysis.

Ferrofluids provide an excellent substrate for molecular interactions. This is because the reaction kinetics of dispersed ferrofluids with target or labeled species can be substantially increased in reversible array mode compared to irreversible array or solid phase modes in conventional microarray assays. is there. This reversible array mode is possible if the arrayed particles do not bind to the surface and thus can be processed by magnetic field removal and recovery. When the coercive force is removed, the ferrofluid particles diffuse to the surface, allowing more efficient interaction of the reactive species. However, reapplying the magnetic force before the substantial diffusion has taken place reforms the spot in its original position.

In addition to optical detection or quantification of target species on the array by conventional means, non-destructive measurements of attached magnet mass, which allows calculation of the number of magnetic particles, can also be made. Miniaturized and sensitive residual or residual magnetic field detection device (eg Quan
Magnetic Assay Reader, M, manufactured by tum Design, San Diego
With recent advances in AR II), such dense ferrofluid arrays are easily localized and quantifiable down to the level of 100 to 10,000 ferrofluid particles per spot. This non-destructive method for analyzing spots makes it possible to correlate relative fluorescence measurements with magnetic field measurements as an internal standard, normalizing the measured signal with respect to variations in the amount of ferrofluid particles collected. Or, correction allows for more accurate target species determination. The following examples are provided to illustrate various embodiments of the present invention. They do not limit the invention in any way.

Example 1 Use of 5.5 μm Diameter Magnetic Biotinylated Polystyrene Beads as a Solid Support to Perform Hybridization This example illustrates the use of a ferrofluid reagent for use as a substrate in DNA hybridization. The preparation will be described. Biotinylated oligonucleotides were added to streptavidin ferrofluid in small portions, followed by biotinylated polystyrene microsphere beads. The magnetically collected microspheres were visualized and counted by brightfield microscopy. By increasing the amount of 26-mer 5'-biotinylated oligomer (molecular weight = 8684D) (OPERON TECHNOLOGIES INC., Alameda, CA) encoding the protein β-actin, a constant amount (50 μg of iron) of streptavidin was detected. Magnetic fluid (Cat. # F3107; I
MMUNICON CORP., Huntingdon Valley, PA) and mixed and incubated. Next, 5.5 μm biotinylated microspheres (Cat. # CP10N; BANGS LABORATORIES I
NC., Fishers, IN) 1 × 10 ⁶ were added to each mixture and incubated at room temperature for 48 hours with mixing. Next, Microwell Magnetic Protein Separator
(Cat. # GS4108; IMMUNICON CORP., Huntingdon Valley, PA) with the help of
All the above mixtures were magnetically separated for 10 minutes, washed in a magnetic field and finally finally resuspended in TE buffer (TRIS / EDTA / MgCl _2, pH 7.0). afterwards,
Each magnetic bead mixture was counted with a hemacytometer to quantify the number of magnetic beads collected. The results show that the maximum binding capacity is about 13 units of biotin oligomer per 50 μg of streptavidin ferrofluid.

The results of this experiment are shown in FIG. The data demonstrate that magnetized streptavidin beads can be successfully synthesized, which, after conjugation to biotinylated oligonucleotides, retain residual biotin binding sites for further immobilization on biotinylated substrates or solid phases. Indicates that The method can be extended to cover multiple oligonucleotides of different complementarity or specificity to analyze multiple target analytes.

Example 2 Quantification of Hybridization Reactions on Beads Hybridization reactions were performed using several magnetized biotin-oligomer-ferrofluid bead mixtures prepared in Example 1. This is because these bead mixtures have streptavidin available for attachment of additional biotinylated species.

An oligomer complementary to the β-actin sequence containing an incompletely saturated magnetic biotin-bead-streptavidin complex (about 50% saturated with biotin-oligo-26mer) containing a quenched chromophore. (Does not fluoresce when not hybridized (Molecular Beacon from RESEARCH GENETICS, Huntsville, AL)
) Was added at a 26-mer level of about 10% (Fig. 2C). After incubation for a sufficient period of time, each mixture, including the appropriate controls, was subjected to FACSCalibur Flow for fluorogenic events (such detectable events indicate successful hybridization).
Analyzed by Cytometer (BECTON DICKINSON, San Jose, CA). Matched controls without microbeads (Figure 2A) and without biotin-oligomers (Figure 2).
B) was also analyzed. FIG. 2 shows those three bar graphs. The hybridized sample (FIG. 2C) showed a significant amount of fluorescent events (> 99%), indicating that the ferromagnetic immobilized biotin-oligomer undergoes an efficient hybridization reaction.

The magnetic microbeads described above have been successfully used to mount a focusing pin (focusing pin) mounted in a non-magnetic planar support below and in contact with a planar surface.
Arrays can be formed by immobilizing single or multiple populations of magnetized microbeads on a planar surface in an ordered manner by means of s). This can be done by magnetically attracting and concentrating the magnetic particles to the array points using a moveable magnet or electromagnet located below the magnetizable concentrating pin. Precise pipetting of relatively viscous ferrofluid suspensions allows consistent loading of spots. Ferrofluidic beads can be reversibly or irreversibly attached to a solid support depending on the absence or presence of biotin or other complementary binding partners on the support surface.

Example 3 Effect of Standard "Hybridization Conditions" on the Integrity of Ferrofluidic Particles The robustness and functionality of ferrofluidic complexes by exposure to the relatively rough hybridization conditions shown in Table 1. Was tested. A stock solution of a 26-mer 5'-biotinylated oligomer / streptavidin-ferrofluid complex was made using 200 picomoles of biotin-oligomer as described in Example 1. A portion of this complex was then resuspended and incubated in various hybridization buffers for 1 hour. Each mixture was then magnetically washed using a Microwell Magnetic Protein Separator and resuspended in TE buffer. Then, each washed mixture was reacted with 0.5 × 10 ⁶ 5.5 μm biotinylated microspheres for 12 hours at room temperature. Then 100 μl of the resulting biotin-oligomer / streptavidin-ferrofluid complex and biotin-microspheres was added to 100 μl of 0.1 μM M.
Mixed with olecular Beacon ^™ , incubated for 60 minutes at room temperature and finally analyzed by flow cytometer for fluorescence events. The results of this experiment are shown in Table 1.
Shown in. It can be readily seen that ferrofluid particles retain a fluorescence intensity greater than 98% and are therefore stable under these rough hybridization conditions.

[0073]

[Table 1] ¹ ExpressHyB (Clontech, Palo Alto, CA) -As formulated ² SSC-a 6x solution contains 0.9M sodium chloride and 0.09M sodium citrate

Example 4 Preparation and Use of Ferrofluidic Array-Based Glass Substrates This procedure describes the preparation of irreversibly bound streptavidin-ferrofluidic spots on biotinylated glass substrates, but with polystyrene. Any of a number of types of organic or inorganic solid phases such as, polycarbonate, mylar, silicon wafer, etc. can be used as well. When excess unbound ferrofluid is washed away, the resulting streptavidin-ferrofluid layer irreversibly binds to the surface,
Does not require magnetic separation. Spot arrays in planar or multiwell formats are stable and can be prefabricated for use in high throughput screening assays of target species applied as ultrathin layers to reduce diffusion times.

In this example, first, a # 1 thick glass coverslip (42 mm x
40 mm) (Cat. # C7931; SIGMA CHEMICAL COMPANY, St. Louis, MO) is acid-etched with concentrated hydrochloric acid for 20 minutes, washed with deionized water, then treated with concentrated nitric acid for 20 minutes and re-treated with deionized water. Wash, briefly soak in methanol, then 10 ° C at 90 ° C.
Allow to dry for minutes. Next, the etched coverslip was treated with 10% N-2-aminoethyl) -3-aminopropyltrimethoxysilane (Cat. # 80379; PIERCE, Roc
kford, IL) treated with ethanolic solution for 16 hours at room temperature. The coverslips were then heated at 90 ° C for 1 hour, allowed to cool, washed with deionized water, ethanol, then deionized water, and finally dried at 90 ° C for 1 hour. These aminosilanized coverslips were treated with 10 mg / ml biotin-succinimide ester (Cat.
# S-1582; MOLECULAR PROBES INC., Eugene, OR) was biotinylated for 4 hours at room temperature. After similarly washing with a buffer of deionized water, 20 mM HEPES, then 0.15 M sodium chloride, 0.1% sodium azide, pH 7.5, the coverslips are 5% bovine serum albumin, phosphate buffered saline. , 0.1% sodium azide solution at room temperature for 4 hours. After further washing with HEPES buffer, the coated coverslips were left to dry at room temperature.

Next, the plastic gasket (which forms the shallow incubation chamber
25 mm x 20 mm x 2.5 mm) on one of the coverslips and place the assembly on a 4-point array of four 13 mm long claws fixed to a table that cannot be magnetized, and cover the tip of the point claws with a slip. Underneath. The assembly was then placed on top of a 13mm x 13mm x 50mm neodymium / iron / boron permanent magnet with the claw head in contact with the magnet. The tip of the dotted nail is about 0
． Enclose a 3 mm circle.

400 μl of 10 μ in the incubation chamber surrounded by the gasket
It was filled with g / ml streptavidin ferrofluid solution and left for 10 minutes to form a ferrofluid spot array on the coverslip surface. The coverslips were then removed from the magnetized nail bed and washed repeatedly with deionized water followed by HEPES buffer to remove loosely bound ferrofluid and then dried at room temperature for several hours before use.

To one of these coverslips fitted with a plastic gasket was added 400 μl of a 10 μM 26-mer biotin oligomer solution in TE buffer and left to incubate for 2 hours at room temperature. The coverslips were washed with deionized water, then HEPES buffer, then dried at room temperature and 400 μl of 1 μM molecular beacon solution was added. 20 minutes later, FOTODY NE-UV equipped with a camera
A coverslip was placed in the box to monitor and record the fluorescence emission from the hybridization reaction. Negative controls, where biotin-oligomer incubation was omitted, were also processed on separate coverslips containing ferrofluid "spots".

FIG. 3B shows the results of this experiment and it is readily apparent that effective hybridization occurred in the four ferrofluid “spots” immobilized with biotin-oligomers, producing bright white “spots”. is there. Little fluorescence was seen on the negative control coverslip and in the region adjacent to the ferrofluid spot (
FIG. 3A).

Example 5 Construction and Use of a Microarray Pin Template In FIG. 4A, a biotin-coated glass surface defined by pin positions that irreversibly concentrate streptavidin-ferrofluid and have minimal binding adjacent nonmagnetic regions. Immobilized on top. The reaction components are shown diagrammatically in FIG. 4B.

A microarray pin mold was constructed from a 25 mm x 25 mm x 25 mm cube block of magnetizable stainless steel that was machined so that one surface had an array of about 45 x 45 pins. Each pin has a diameter of 0.050 mm and a spacing of 0.50 mm.
The pin pattern had about 2000 spots per square inch. As in Example 3, the pin mold was sandwiched between a biotinylated coverslip (placed on the pin) and a rare earth magnet (13 mm x 13 mm x 50 mm) (contacting the lower cube surface away from the pin pattern).

Streptavidin-ferrofluid was placed on a biotinylated coverslip sealed with a gasket and incubated for 10 minutes to concentrate the ferrofluid in separate spots. The coverslips were removed and excess loosely bound ferrofluid was flushed out as in Example 3. After drying, the sections of the microarray were magnified about 100 times under a brightfield microscope and photographed (Figure 4A). The microarray pattern was clearly shown, with minimal binding to unconcentrated adjacent regions.

Alternatively, a preformed biotinylated complex of streptavidin can be used that carries a biotinylated binding partner or a detectable biotinylated label that can be simultaneously aggregated on the same particle. This is because the particles have a relatively high binding ability as shown in Example 1.

The gasket enclosure used in these examples holds about 500 μl of solution. Smaller volumes in the range of 1-100 μl are placed in a rectangular compartment delimited by hydrophobic boundaries or “fences” that have been mechanically or lithographically processed to prevent intermixing of substances in adjacent array sites. You can put it in. Similarly, larger magnetic particles prepared by coating conventional biotinylated microspheres, eg, 0.5-10 μm, with streptavidin-ferrofluid, are used to deposit the magnetic layer on the microspheres. It is thus possible to subject them to a magnetic treatment.

Example 6 Immobilization and Identification of a Single Target Agent in a Microarray Containing Multiple Binders with Potential Binding Affinity On a non-binding surface or 96, 384, which can be magnetically treated. 1
Similar but reversibly bound patterns can be obtained in 536 well microtiter wells or larger arrays. Using conventional inkjet or other means of implementation, different binders or magnetic binder complexes are deposited in separate wells to allow multiple target species with affinity for different binders, as in gene profiling. Effective means for high throughput screening of test samples for may be obtained. Alternatively, and conversely, a single target substance may be tested with multiple different binding agents, such as drug profiling and toxicity studies. Hybridization studies with RNA and DNA substrates, templates or amplicons can also be performed by the methods of the invention. Many more modifications to the theories, methodologies and processes disclosed in the present invention are possible.

Exemplary embodiments were first described with reference to diagrams that illustrate important features of the embodiments. Each method step may be a hardware or software component for performing the corresponding step. Not all of the components or methods to fully implement an actual system are described in detail. Rather, only those components or method steps necessary for an understanding of the present invention have been illustrated and described in detail. More steps or components may be used in the actual implementation, or fewer steps or components may be involved. Thus, while the preferred embodiments of the invention have been described and illustrated individually, the invention is not limited to such embodiments. Various modifications may be made to the invention without departing from the scope and spirit of the invention as claimed.

[Brief description of drawings]

FIG. 1 is a graph showing the inhibition of binding of 5.5 μm biotin beads to streptavidin-ferrofluid. The amount of biotin oligomer was increased to determine the optimal loading of biotin beads on the maximum loading capacity of streptavidin-ferrofluid particles.

2A-2C are a series of flow cytometric bar graphs,
5 shows the increase in fluorescence resulting from the formation of a specific binding complex between members of a specific binding pair by the hybridization reaction.

3A and 3B are fluorescent images showing the effect of non-specific hybridization (FIG. 3A) and magnetically focused specific oligonucleotide hybridization (FIG. 3B) on a 4-point microarray of the invention. is there.

FIG. 4A shows a magnified view (about 100 ×) of a streptavidin-ferrofluidic microarray irreversibly bound to a biotinylated support placed on top of a magnetizable microarray pin template. The pin mold is made of steel,
It has 0.05 mm diameter circular pins spaced 50 mm apart and they are located on top of the rare earth magnets. This microarray has about 2000 pins per square inch. FIG. 4B is a diagrammatic representation of the reaction components shown in the microarray shown in FIG. 4A.

FIG. 5 is an apparatus for making a homogeneous array using a dispenser that delivers particles, which are attracted to a collection surface. Such methods and devices may be used to make arrays suitable for assaying multiple samples for the presence of a single determinant.

FIG. 6 is a device for creating a specifically targeted array in which particles are delivered directly to localized spots on a collection structure. Such methods and devices may be used to create arrays for assaying a sample for the presence of multiple determinants.

─────────────────────────────────────────────────── ─── Continued front page    (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE, TR), OA (BF , BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, G M, KE, LS, MW, MZ, SD, SL, SZ, TZ , UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, B Z, CA, CH, CN, CR, CU, CZ, DE, DK , DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, J P, KE, KG, KP, KR, KZ, LC, LK, LR , LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, R O, RU, SD, SE, SG, SI, SK, SL, TJ , TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Sean Mark O'Hara             United States 19002 A Pennsylvania             Humbler, Isaacs Court No. 1519 (72) Inventor Gala Chandra Rao             United States 08540 New Jersey             Princeton, Heritage Boulevard             No. 6 (72) Inventor Anthony Barnes             31411 Savannah, Georgia, United States             Wilford Drive No. 1 (72) Inventor Leon W. M. M. Turs             Tappen             United States 19006 Ha, Pennsylvania             Ntingdon Valley, Old Four             De Road 1354 (72) Inventor Herman Ratner             United States 19040 Ha, Pennsylvania             Tutboro, Apartment 609, South             Pen Street No. 50 F-term (reference) 4B024 AA11 BA80 CA01 HA12                 4B029 AA07 AA21 AA23 BB20 CC03                       FA01 FA12                 4B063 QA01 QA18 QQ42 QR32 QR50                       QR55 QS34 QX01

Claims (20)

[Claims] 1. A method for immobilizing and identifying a target substance in a test sample by using a magnetically aligned array in a bound compartment, comprising the steps of: a) specific binding. Providing a solid support to which the first member of the pair is attached; b) immobilizing a plurality of second members of the specific binding pair having binding affinity for the first member of the specific binding pair. C) focusing the magnetic particles into an array by magnetic means inside the compartment, the members of the first binding pair on a solid support and the second on the magnetic particles. Immobilize the magnetically responsive particles by irreversible interactions with members of a binding pair of d); d) immobilize the immobilized particles in the compartment on the solid support. Contacting with a first nucleic acid sequence operably linked to a first member of the specific binding pair; e) bringing the immobilized particle and the first nucleic acid sequence into contact with the first nucleic acid sequence. Contacting with a test sample which may contain a target substance having affinity; then f) binding between said first nucleic acid sequence and said target substance present in the test sample by binding of said bound labeled target substance. A method comprising detecting by measuring a detectable signal. 2. The solid support is a support selected from the group consisting of plastic surfaces, glass surfaces, silica surfaces, metal surfaces, membranes, microtiter plates, microtiter strips, and microtiter wells. The method described. 3. The solid support carries bound compartments that overlap pin locations, the compartments being formed by depositing hydrophobic lines on the surface of the solid support. The method of claim 1. 4. The method of claim 1, wherein the solid support is selected from polymer particles, latex particles and inorganic particles in the size range of about 0.5 μm to about 20 μm. 5. The method of claim 4, wherein the particles are in the size range of about 0.5 μm to about 5 μm. 6. The method of claim 1, wherein the magnetic particles are selected from the group consisting of colloidal ferrofluids, magnetic nanoparticles, magnetic microparticles and ferrofluid-coated microparticles. 7. The first member of the specific binding bear is a biotin species,
The method of claim 1, wherein the second member of the specific binding bear is a streptavidin species. 8. The method of claim 1, further comprising quantifying the amount of target biological material present in the test sample. 9. The first nucleic acid sequence is operably linked to a biotin species and is located in a separate compartment on the array, the test sample comprising a detectably labeled mixed population of nucleic acids. , The subset may be complementary to the first nucleic acid and the first nucleic acid sequence and the test sample are subjected to conditions that allow hybridization between complementary nucleic acids, whereby The method according to claim 1, which enables detection of a plurality of target nucleic acids in the test sample. 10. The method of claim 1, wherein the detectable label is selected from the group consisting of fluorescent labels, radioactive labels, chemiluminescent labels and magnetic labels. 11. The detectable label is detected using a device selected from the group consisting of a fluorimeter, a spectrophotometer, a scintillation counter, a mass-measuring biosensor, and a surface plasmon resonance biosensor. the method of. 12. An apparatus for detecting binding events between specific binding pairs comprising means for defining a magnetically aligned magnetic response array on the surface of a non-magnetizable solid support, The array template carries a plurality of magnetizable array pins in direct contact with the underside of the SEQ ID NO: solid support; the solid support is divided into bound compartments overlapping the array pins; the array template further comprises a rare earth magnet Alternatively, an apparatus that is connected to an electromagnet, which provides a high field gradient close to the array pin tip, which gradient collects the magnetic particles in the magnetically responsive array. 13. A device according to claim 12, wherein the compartments are joined by hydrophobic lines made by mechanical or lithographic means. 14. The device of claim 12 wherein the array is a microarray having a pin density in the range of about 2 to about 100,000 pins per inch. 15. The device of claim 14, wherein the pin density range is from about 4 to about 2000 pins per inch. 16. The apparatus of claim 12, wherein the array mold comprises magnetizable pins mounted on the mold that cannot be magnetized. 17. The pin is mounted on the surface of one of the magnetizable array molds by means selected from the group consisting of mechanical means and lithographic means.
The device according to 2. 18. The apparatus of claim 12, wherein the magnetically responsive array is processed by changing a magnetic field proximate the pin. 19. The apparatus of claim 12, wherein the collected magnetic particles are analyzed by optical means. 20. The device of claim 12, wherein the collected magnetic particles are analyzed by measuring magnetic signals.