METHODS FOR DETERMINING THE CONCENTRATION OF AN ANALYTE IN SOLUTION
Disclosed is a method for measuring the concentration of an analyte or analytes in a solution. Although the methods can be conducted using a number of different assay formats, in one embodiment, the assays are conducted in reaction vessels defined, at least in part, by the distal ends of fiber optic strands.
Latest Trustees of Tufts College Patents:
The United States government may have certain rights in this invention pursuant to Contract No. N00014-01-1 awarded by the Department of Defense, Defense Advanced Research Projects Agency (DARPA) Office of Naval Research.
BACKGROUNDMethods that implement high-sensitivity and low-level analyte detection in conjunction with rapid and reproducible experimental protocols are the cornerstone of modern analytical measurements. Currently, most known techniques for quantifying low levels of analyte in a sample matrix use amplification procedures to increase the number of reporter molecules and thereby provide a measurable signal. These known processes include enzyme-linked immunosorbent assays (ELISA) for amplifying the signal in antibody-based assays, as well as the polymerase chain reaction (PCR) for amplifying target DNA strands in DNA-based assays. A more sensitive but indirect protein target amplification technique, called immuno-PCR (see Sano, T.; Smith, C. L.; Cantor, C. R. Science 1992, 258, 120-122), makes use of oligonucleotide markers, which can subsequently be amplified using PCR and detected using a DNA assay (see Nam, J. M.; Thaxton, C. S.; Mirkin, C. A. Science 2003, 301, 1884-1886; Niemeyer, C. M.; Adler, M.; Pignataro, B.; Lenhert, S.; Gao, S.; Chi, L. F.; Fuchs, H.; Blohm, D. Nucleic Acids Research 1999, 27, 4553-4561; and Zhou, H.; Fisher, R. J.; Papas, T. S. Nucleic Acids Research 1993, 21, 6038-6039). While the immuno-PCR method permits ultra low-level protein detection, it is a complex assay procedure, and can be prone to false-positive signal generation (see Niemeyer, C. M.; Adler, M.; Wacker, R. Trends in Biotechnology 2005, 23, 208-216).
One disadvantage of known methods for accurately quantifying the concentration of a particular analyte in solution is that they are all based on ensemble responses in which many analyte molecules give rise to the measured signal. Most detection schemes require that a large number of molecules are present in the ensemble for the aggregate signal to be above the detection threshold. This disadvantage limits the sensitivity of most detection techniques and the dynamic range (i.e., the range of concentrations that can be detected).
Therefore, there is a need in the art for an improved method and system of analyte detection. Specifically methods that detect and measure individual molecules rather than an ensemble of molecules would improve the sensitivity, dynamic range and accuracy of analyte detection. The invention described here divides the sample being analyzed into small samples in which only one or zero analyte molecules are present statistically. These analyte molecules are then detected and counted in a digital fashion to determine the concentration of analyte.
BRIEF SUMMARY OF THE INVENTIONThe invention relates to methods, systems and devices for measuring the concentration of an analyte or analytes in a fluid sample. The methods can be performed using a variety of assay platforms, reagents, detectable labels, reaction conditions, and detection systems which are described in detail herein.
In one aspect, the invention provides a method of determining the concentration of an analyte in a fluid sample. The method comprises the steps of: (a) partitioning at least a portion of the analyte molecules in the fluid sample across a plurality of reaction vessels so that a statistically significant fraction of the reaction vessels contain analyte and a statistically significant fraction of the reaction vessels contain no analyte; (b) determining the presence or absence of the analyte in each reaction vessel to identify the number of reaction vessels that contain analyte and/or to identify the number of reaction vessels that contain no analyte; and (c) determining the concentration of the analyte in the fluid sample from the number of reaction vessels that contain the analyte.
In this method, at least 95%, optionally 90%, optionally 80%, optionally 40%, optionally 5%, of the reaction vessels contain no analyte. Depending upon the reaction conditions chosen and the number of wells that contain one or more molecules of analyte, the concentration of the analyte in the fluid sample can be determined by either Poisson distribution analysis or Gaussian distribution analysis of the number of reaction vessels that contain the analyte.
The reaction vessels can be adapted to capture the analyte of interest. For example, depending upon the assay format chosen, the reaction vessel can comprise a microwell and an optional sealing component that connects and seals fluid within the microwell. Accordingly, a capture component for capturing the analyte can be immobilized on a sample contacting surface of the microwell or a sample contacting surface of the sealing component. Alternatively, the capture component can be immobilized on the surface of a particle disposed within the reaction vessel.
The methods preferably are performed using at least 1000 reaction vessels. More specifically, under certain circumstances the methods are performed simultaneously using from about 10,000 to about 200,000 different reaction vessels, more preferably from about 50,000 to about 100,000 different reaction vessels. In addition, in certain embodiments, at least a portion of the reaction vessel is defined by a distal end of an optical fiber. As a result, light from a light source, for example, a laser or lamp, can be transmitted along the optical fiber to the reaction vessel located at the distal end of the optical fiber. Thereupon, depending up the label chosen for a given assay, photoexcitation of the label produces a signal, which then can be detected by a suitable detector.
While multiple embodiments are disclosed, still other embodiments of the present invention will become apparent to those skilled in the art from the following detailed description, which shows and describes illustrative embodiments of the invention. As will be realized, the invention is capable of modifications in various obvious aspects, all without departing from the spirit and scope of the present invention. Accordingly, the drawings and detailed description are to be regarded as illustrative in nature and not restrictive.
The present invention relates to methods, systems, and devices for the detection and quantification of an analyte or analytes in a sample. It is contemplated that the methods described herein can be performed in a number of different formats using a variety of different detectable labels, reagents, reaction conditions, and detection systems.
In one aspect, the invention provides a method of determining the concentration of an analyte in a fluid sample to be tested. The method comprises the steps of (a) partitioning at least a portion of the analyte molecules in the fluid sample across a plurality of reaction vessels so that a statistically significant fraction of the reaction vessels contain analyte and a statistically significant fraction of the reaction vessels contain no analyte; (b) determining the presence or absence of the analyte in each reaction vessel to identify the number of reaction vessels that contain analyte and/or to identify the number of reaction vessels that contain no analyte; and (c) determining the concentration of the analyte in the fluid sample from the number of reaction vessels that contain the analyte.
In another aspect, the invention provides a method of determining the concentration of an analyte in a fluid sample to be tested. The method comprises the steps of: (a) exposing the fluid sample to a plurality of reaction vessels under conditions so that analyte is captured in a statistically significant fraction of the reaction vessels, wherein each reaction vessel comprises a microwell and an optional sealing component and each reaction vessel defines a binding surface that has a capture component immobilized thereon; (b) determining the presence or absence of the analyte in each reaction vessel so as to identify the number of reaction vessels that contain captured analyte and/or the number of reaction vessels that do not contain captured analyte; and (c) determining the concentration of the analyte in the fluid sample to be tested from the number of reaction vessels that contain and/or do not contain the analyte.
In another aspect, the invention provides a method of determining the concentration of an analyte in a fluid sample to be tested. The method comprises the steps of: (a) partitioning at least a portion of the analyte molecules in the fluid sample into a plurality of reaction vessels, so that, for substantially all of the reaction vessels, each reaction vessel contains either no analyte or a single molecule of the analyte; (b) determining the presence or absence of the analyte in each reaction vessel to provide a number of the reaction vessels that contain the analyte; and (c) determining the concentration of the analyte in the fluid sample from the number of reaction vessels that contain the analyte.
In another aspect, the invention provides a method of determining the concentration of an analyte in a fluid sample to be tested. The method comprises the steps of: (a) partitioning the sample to be tested into a plurality of second, smaller fluid samples of equal volume so that a statistically significant fraction of the second, smaller fluid samples contain either a single molecule of the analyte or no analyte; (b) determining the presence or absence of the analyte in each of the second samples so as to identify the number of second samples that contain the analyte; and (c) determining the concentration of the analyte in the sample to be tested from the number of second samples that contain the analyte.
It is understood that the foregoing methods can be performed using a number of different assay formats, different reaction conditions, detectable labels and detection systems. In one embodiment, for example, the invention permits the detection and/or quantification of analytes using arrays of micron- to nanoscale-sized reaction vessels. In one approach, the reaction vessels contain capture components that capture the analyte within the reaction vessel. In such an approach, an array of reaction vessels containing an immobilized capture component is contacted with a sample containing at least one analyte. Following capture of the analyte, depending upon what label is used, the presence of analyte can be detected directly or indirectly.
The direct approach can include, for example, the situation where the analyte is an enzyme. In this case, a chromogenic, fluorogenic, or chemoluminescent enzymatic substrate is included in the reaction mixture and the enzymatic activity of the analyte produces a detectable product. The indirect approach can include, for example, the situation where the analyte does not have intrinsic enzymatic activity and a label is permitted to bind, for example, via a secondary binding ligand, to the analyte. For example, a detectable label conjugated to a secondary binding ligand (for example, a capture component different from the one used to capture the analyte) is added to the captured analyte under conditions to permit the labeled secondary binding ligand to bind to the immobilized analyte. Thereafter, the presence of the label can then be determined to provide an indication of whether the analyte is present in the reaction vessel. It is understood that a variety of different labels, as will be discussed in more detail below, may be used in the practice of the invention. To the extent that an enzymatic label is employed, an enzymatic substrate can be added to the reaction vessel whereupon the enzyme converts the substrate into a chromogenic, fluorogenic, or chemoluminescent detectable product to permit the detection of the analyte. The number or percentage of reaction vessels with captured analytes can be used to calculate the amount of analyte in the sample using a binary readout method.
Similarly, it is understood that in certain assay formats, some or all of the reaction vessels can be defined on a planar support rather than within the recesses of an array of microwells. For example, the sample containing analyte is divided into a plurality of second, smaller fluid samples of equal volume. The second fluid samples then are applied as droplets to the surface of a planar binding surface to create an array of droplets. Each droplet can be applied to a region of the planar binding surface having a capture component immobilized thereon. The samples then are incubated to permit the analyte to bind to the capture component immobilized on the planar binding surface. Afterwards, depending upon the label being employed, the presence of the analyte can be determined directly or indirectly so as to permit the identification of the number of fluid samples that contain the analyte and the number of fluid samples that do not contain the analyte. In certain embodiments, for example, when the label is an enzymatic label, an enzymatic substrate can be added to the surface of the planar binding surface, and the various regions containing the capture component (in the presence or absence of analyte). The planar binding substrate then is sealed against an array of microwells to form an array of reaction vessels to permit the development of a detectable product in each reaction vessel. From the number of reaction vessels containing the analyte in the array of reaction vessels it is possible to determine the concentration of analyte in the test solution.
In one embodiment, the present invention uses an array of micron- to nanoscale-sized reaction vessels specifically functionalized with capture components and capable of capturing analyte molecules. The ability to capture the analyte allows the use of washing steps and indirect assays, as outlined below. In one embodiment, the label used to detect the captured analyte is an enzyme. Once captured in individual reaction vessels, the enzyme catalyzes the production of a sufficient number of chromogenic, fluorogenic, or chemoluminescent product molecules to generate a detectable signal. In accordance with one embodiment relating to samples having low analyte concentrations, only a portion of the reaction vessels capture an analyte molecule, thereby enabling a binary readout of analyte concentration from the array of reaction vessels. The direct enzymatic amplification in the method and system of the present invention allows for direct amplification of a detectable signal.
The present invention allows for detection of low concentrations of analytes, such as proteins. At low concentrations, a statistically significant number of reaction vessels contain zero, one, or several molecules of the target analyte. By observing the presence or absence of a detectable label present in each reaction vessel, a binary readout method can be used to identify the number of reaction vessels that contain one or more molecules of the analyte. As a result, the percentage of reaction vessels occupied by analyte can be used to calculate the original bulk concentration of the analyte in the sample of interest. The following sections discuss exemplary arrays of reaction vessels, capture components, analytes that can be detected by the invention, detectable labels and signal detection protocols, assay methods, and exemplary uses of the invention.
I. Arrays of Reaction VesselsThe present invention utilizes an array of reaction vessels to carry out steps in an assay utilized to determine the concentration of an analyte of interest. The purpose of the array of reaction vessels is to allow a reaction volume to be partitioned into a plurality of discrete reaction volumes during one or more steps in an assay. By “array” herein is meant a plurality of similar reaction vessels.
The reaction vessels may all have the same volume or may be of differing, but known volumes. The volume of each individual reaction vessel can range from attoliters to nanoliters depending upon the analyte of interest and the expected concentration of that analyte in a solution. In one embodiment, the size of the reaction vessel may be selected such that at the concentration of interest between zero and ten molecules of the analyte of interest would be expected to be found in each reaction vessel. In accordance with one embodiment of the present invention, the reaction vessels have a volume ranging from about 10 attoliters to about 50 picoliters. Alternatively, the reaction vessels range in size from about 1 picoliter to about 50 picoliters. In a further alternative, the reaction vessels range in size from about 1 femtoliter to about 1 picoliter. In a further alternative, the reaction vessels range from about 30 femtoliters to about 60 femtoliters.
The number of reaction vessels in the array will depend on the composition and end use of the array. Arrays containing from about 2 to many billions of reaction vessels can be made by utilizing a variety of techniques and materials. Increasing the number of reaction vessels in the array can be used to increase the dynamic range of an assay or to allow multiple samples or multiple analytes to be assayed in parallel. Generally, the array will comprise between one thousand and one million reaction vessels per sample to be analyzed. Arrays used to simultaneously analyze multiple samples will generally contain between 10 thousand and 10 billion reaction vessels.
The array of reaction vessels may be arranged on a planar structure or in some 3 dimensional arrangement. They may be in a regular design or randomly distributed. A preferred embodiment utilizes a regular pattern of sites on a planar structure such that the sites may be addressed in the X-Y coordinate plane.
The reaction vessels can be formed in a solid material. As will be appreciated by those in the art, the number of possible materials are very large, and include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, Teflon™, etc.), polysaccharides, nylon or nitrocellulose, composite materials, ceramics, and plastic resins, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses, plastics, optical fiber bundles, and a variety of other polymers. In general, the substrates allow optical detection and do not appreciably fluoresce.
The reaction vessels can also be formed in a liquid provided that the confining liquid does not mix with the liquid defining the reaction vessel, and that the reaction vessel is stable. Examples of suitable liquids for confining aqueous reactions include, but are not limited to, water-in-oil emulsions, extruded lipid aggregates, stable suspensions of lipids, liquid-crystal aggregates, micelles in water, inverted micelles in oil, and suspensions of cells, bacteria and viruses.
Individual reaction vessels may contain a binding surface. The binding surface may be all or a portion of the interior surface of the reaction vessel or may be on the surface of something that is confined within the reaction vessel such as a bead, or a particle (for example, a micro-particle or a nanoparticle).
In one embodiment, the array of reaction vessels is formed by mating an array of microwells with a sealing component. Microwells are small depressions in a first surface of a supporting material. The microwells may be formed as is generally known in the art using a variety of techniques, including, but not limited to, photolithography, stamping techniques, molding techniques and microetching techniques. As will be appreciated by those in the art, the technique used will depend on the composition and shape of the supporting material. The sealing component has a second surface with the same topology as the first surface that when brought into contact with the first surface creates an array of sealed reaction vessels. Either the first surface or the second surface may be fabricated from a compliant material to aid in sealing. Either or both surfaces may be hydrophobic or contain hydrophobic regions to minimize leakage from the microreactors.
In another embodiment, the array of reaction vessels is formed utilizing a microfluidic device that contains channels and valves that can be closed to isolate discrete reaction volumes.
In one preferred embodiment, an array of reaction vessels is formed creating microwells on the distal end of a fiber optic bundle and utilizing a planar compliant surface as a sealing component. Such an array of reaction vessels can be made as follows. First, an array of microwells are etched into the distal end of a polished fiber optic bundle. The diameter of the optical fibers and the depth of the etch can be varied to create microwells of the desired volume. The etching process creates the microwells in the core material of the individual glass fibers in the bundle such that each well is aligned with a single fiber and isolated from adjacent wells by the slower etching cladding material. One advantage of the fiber optic array format is that it can produce thousands to millions of reaction vessels without the need for complicated microfabrication procedures and provides the ability to observe many reaction vessels simultaneously.
Each microwell is aligned with an optical fiber in the bundle so that the fiber optic bundle can carry both excitation and emission light to and from the wells, enabling remote interrogation of the well contents. Further, an array of optical fibers provides the capability for simultaneous excitation of molecules in adjacent vessels, without signal “cross-talk” between fibers. That is, excitation light transmitted in one fiber does not escape to a neighboring fiber. In one aspect of the present invention, the physical alterations can be made as taught in U.S. Pat. Nos. 6,023,540, 6,327,410, and 6,858,394, which are each incorporated by reference herein in their entirety. Either the surface of the glass microwells, the surface of the sealing component, or particles can be functionalized to create a binding surface.
Alternatively, the equivalent structures can be fabricated using other methods that do not comprise the ends of an optical fiber bundle. For example, the array may be a spotted, printed or photolithographic substrate known in the art; see for example WO 95/25116; WO 95/35505; PCT US98/09163; U.S. Pat. Nos. 5,700,637; 5,807,522 and 5,445,934; and U.S. Ser. Nos. 08/851,203 and 09/187,289, all of which are expressly incorporated by reference.
II. Capture ComponentsDuring practice of the invention, the binding surfaces within the reaction vessels (including microwells defined in fiber optical bundles) incorporate at least one capture component. A capture component (also commonly referred to as a “capture binding ligand,” “binding ligand,” “capture binding species,” or “capture probe”) is any molecule, compound, or solid support modification that can be used to probe for, attach, bind or otherwise capture an analyte disposed upon a solid support, such that the analyte is immobilized during the assay. Generally, the capture component allows the attachment of an analyte to a solid support (that is, the surface of the microwells, the sealing component or nanoparticle) for the purposes of detection, quantification, or other analysis.
As will be appreciated by those in the art, the composition of the capture component will depend on the composition of the analyte. Capture components for a wide variety of analytes are known or can be readily found using known techniques. For example, when the analyte is a protein, the capture components include proteins (particularly including antibodies or fragments thereof (FAbs, etc.)) or small molecules. Preferred capture component proteins include peptides. For example, when the analyte is an enzyme, suitable capture components include substrates and inhibitors. Antigen-antibody pairs, receptor-ligands, and carbohydrates and their binding partners are also suitable analyte-capture component pairs. In addition, when the analyte is a single-stranded nucleic acid, the capture component may be a complementary nucleic acid. Similarly, the analyte may be a nucleic acid binding protein and the capture component is either single-stranded or double-stranded nucleic acid; alternatively, the capture component may be a nucleic acid-binding protein when the analyte is a single or double-stranded nucleic acid. Alternatively, as is generally described in U.S. Pat. Nos. 5,270,163, 5,475,096, 5,567,588, 5,595,877, 5,637,459, 5,683,867, 5,705,337, and related patents, hereby incorporated by reference, nucleic acid “aptamers” can be developed for capturing virtually any analyte. As will be appreciated by those in the art, any two molecules that will associate may be used, either as an analyte or as the capture component. Similarly, when the analyte is a carbohydrate, suitable capture components include antibodies and lectins. It is understood that there is a wide body of literature relating to the development of capture components based on combinatorial chemistry methods.
Suitable analyte/capture component pairs include, but are not limited to, antibodies/antigens, receptors/ligands, proteins/nucleic acid, enzymes/substrates and/or inhibitors, carbohydrates (including glycoproteins and glycolipids)/lectins, proteins/proteins, proteins/small molecules; and carbohydrates and their binding partners are also suitable analyte-capture component pairs. These may be wild-type or derivative sequences. According to one embodiment, the capture components are portions (particularly the extracellular portions) of cell surface receptors that are known to multimerize, such as the growth hormone receptor, glucose transporters (particularly GLUT 4 receptor), and T-cell receptors.
In a preferred embodiment, the capture component is attached to a binding surface (for example, the surface of a microwell) via an “attachment component” (also referred to herein as an “attachment linker”). An “attachment component,” as used herein, is defined as any component, functionalization, or modification of the binding surface that results in the attachment of the capture component, and can include bonds and/or linkers. Alternatively, the capture component may utilize a capture extender component. In this embodiment, the capture component comprises a first portion that will bind the analyte and a second portion that can be used for attachment to the binding surface.
This attachment component can also provide a protective layer that screens the binding surface, such as the glass of a fiber optic bundle, from the assay solution. As a result, the attachment component minimizes non-specific attachment of non-target molecules to the binding surface during the assay and detection that could lead to undesired fluorescent wells and false positive signals.
The method of attachment of the capture component to the attachment component will generally be done as is known in the art, and will depend on the composition of the attachment component and the capture component. In general, the capture components are attached to the attachment component through the use of functional groups on each that can then be used for attachment. According to one embodiment, the functional group is a chemical functionality. That is, the binding surface is derivatized such that a chemical functionality is presented at the binding surface. Preferred functional groups for attachment are amino groups, carboxy groups, epoxide groups, maleimide groups, oxo groups and thiol groups. These functional groups can be attached, either directly or through the use of a linker, the combination of which is sometimes referred to herein as a “cross-linker.” Linkers are known in the art; for example, homo-or hetero-bifunctional linkers as are well known (see 1994 Pierce Chemical. Company catalog, technical section on cross-linkers, pages 155-200, incorporated herein by reference). Preferred linkers include, but are not limited to, alkyl groups (including substituted alkyl groups and alkyl groups containing heteroatom moieties), with short alkyl groups, esters, amide, amine, epoxy groups and ethylene glycol and derivatives being preferred. Linkers may also be a sulfone group, forming sulfonamide.
According to one embodiment, the functional group is a light-activated functional group. That is, the functional group can be activated by light to attach to the capture component or to the attachment component. One example is the PhotoLink™ technology available from SurModics, Inc. in Eden Prairie, Minn.
Alternatively, the attachment component is added without covalently modifying the binding surface. That is, the attachment component can be added to the binding surface by using a molecule that has both a functional group and a group that has binding affinity for the binding surface. Alternatively, the attachment component is any protein capable of binding or sticking to the binding surface. In a further alternative, the attachment component is any molecule capable of binding or sticking to the vessel surface. In one example, the attachment component is serum albumin with free amine groups on its surface. A crosslinker can then be added to attach the amine groups of the albumin to the capture component.
According to one embodiment in which the capture component is a chemical crosslinker, the analyte is captured on the binding surface using chemical crosslinking in the following manner. First, the binding surface is derivatized with a functional group, such as, an amine group. Next, a crosslinker and the analyte are placed in contact with the binding surface such that one end of the crosslinker attaches to the amine group and the analyte attaches to the other end of the crosslinker. In an alternative embodiment described in further detail below in which the analyte is not an enzyme, a label having an enzymatic component can also be attached to the analyte.
In this way, capture components comprising proteins, lectins, nucleic acids, small organic molecules, carbohydrates can be added.
One embodiment utilizes proteinaceous capture components. As is known in the art, any number of techniques may be used to attach a proteinaceous capture component. “Protein” in this context includes proteins, polypeptides, peptides, including, for example, enzymes and antibodies. A wide variety of techniques are known to add moieties to proteins. One preferred method is outlined in U.S. Pat. No. 5,620,850, hereby incorporated by reference in its entirety. The attachment of proteins to surfaces is known; see also Heller, Acc. Chem. Res. 23:128 (1990), and related work.
An alternative embodiment utilizes nucleic acids as the capture component, for example for when the analyte is a nucleic acid or a nucleic acid binding protein, or when the nucleic acid serves as an aptamer for binding a protein, as is well known in the art.
According to one embodiment, each binding surface presents a plurality of capture component molecules. The plurality of capture component molecules, in one aspect of the invention, are distributed on the binding surface like a “lawn.” Alternatively, the capture components are distributed in any known fashion.
The binding between the capture component and the analyte, in accordance with one embodiment, is specific and the capture component is part of a binding pair. That is, the capture component is a target-specific capture component that specifically binds with or has specificity for the analyte. More specifically, the capture component binds specifically and directly to the analyte. By “specifically bind” or “binding specificity” herein is meant that the capture component binds the analyte with specificity sufficient to differentiate between the analyte and other components or contaminants of the test sample. For example, the capture component according to one embodiment is an antibody that binds specifically to some portion of the analyte. The antibody, according to one embodiment, can be any antibody capable of binding specifically to an analyte. For example, appropriate antibodies include, but are not limited to, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to as “antibody mimetics”), chimeric antibodies, humanized antibodies, antibody fusions (sometimes referred to as “antibody conjugates”), and fragments of each, respectively.
However, as will be appreciated by those in the art, it is possible to detect analytes using binding that is not highly specific. For example, the systems may use different capture components such as, for example, a panel of different binding ligands, and detection of any particular analyte is via its “signature” of binding to this panel of binding ligands, similar to the manner in which “electronic noses” work. This finds particular utility in the detection of chemical analytes. The binding affinity between analyte and capture component should be sufficient to remain bound under the conditions of the assay, including wash steps to remove non-specific binding. In some embodiments, for example in the detection of certain biomolecules, the binding constants of the analyte to the capture component will be at least about 104-106 M−1, with at least about 105 to 109 M−1 being preferred and at 107-109 M−1 being particularly preferred.
According to one embodiment in which the analyte is a cell, including, for example, bacterial cells, the capture component can be an adhesin receptor molecule. In use, the adhesin receptor molecule binds with a surface protein called an adhesin on the extracellular surface of the target cell, thereby immobilizing or capturing the cell. Alternatively, in embodiments in which the analyte is another type of cell (a non-bacterial cell), the capture component is an appropriate cell surface receptor that binds the analyte cell. In a further embodiment in which the analyte is a cell, the capture component is fibronectin. For example, fibronectin can be used when the analyte is a nerve cell.
Alternatively, the capture component is a non-specific capture component. That is, the capture component does not bind specifically to an analyte, but rather binds to a corresponding binding partner associated with or attached to the analyte. For example, the non-specific capture component according to one embodiment is a chemical cross-linker as described above. According to one embodiment, every peptide molecule in a sample of analyte can attach to the chemical cross-linker. In one example of a non-specific capture component according to one embodiment, the capture component is streptavidin, which binds with high affinity to biotin, and thus captures any molecule to which biotin has been attached. Alternatively, the capture component is biotin, and streptavidin is attached to or associated with the analyte such that the analyte can be captured by the biotin.
According to one embodiment, the capture component is added to the binding surface in the following manner. First, an array of microwells on the distal end of a fiber optic bundle are prepared for attachment of the capture component(s). That is, the binding surface of the microwells are modified or an attachment component is added to the binding surface of the microwells such that the capture component(s) attach to the binding surface of the microwells. In one embodiment, the binding surface of the microwells are derivatized with a chemical functionality as described above. Next, the capture component is added.
One example of capture component attachment is depicted in
After the attachment component 28 has been added to the binding surface of the microwells, the capture component can be attached. The capture component can be biotin. For example, with respect to
Examples of capture components within the microwell 10 are depicted in
Another example of a capture component attachment is depicted in
As discussed herein, the array of reaction vessels in the present invention provides for detection, quantification, and further analysis of analytes. By “analyte” or grammatical equivalents herein is meant any atom, molecule, ion, molecular ion, compound, particle, cell, or virus to be either detected or evaluated.
According to one embodiment, the analyte is an enzyme. For example, the enzyme can be an enzyme from any of the following enzyme classifications: oxidoreductases, transferases, kinases, hydrolases, lyases, isomerases, and ligases. Thus, appropriate enzymes include, but are not limited to, polymerases, cathepsins, calpains, amino-transferases such as, for example, AST and ALT, proteases such as, for example, caspases, nucleotide cyclases, transferases, lipases, enzymes associated with heart attacks, and the like. When the system of the present invention is used to detect viral or bacterial targets, appropriate enzymes include viral or bacterial polymerases and other such enzymes, including viral or bacterial proteases.
Alternatively, the analyte has an enzymatic component. For example, the analyte can be a cell having an enzyme or enzymatic component present on its extracellular surface. Alternatively, the analyte is a cell having no enzymatic component on its surface. Such a cell is typically identified using an indirect assaying method described below such as a “sandwich” assay.
In accordance with another embodiment, the analyte is not an enzyme. As will be appreciated by those in the art, a large number of analytes may be used in the present invention; basically, any analyte can be used which binds a capture component and/or a secondary binding ligand. As will be explained in further detail below, these analytes are typically identified using an indirect assay such as a “sandwich” assay. As mentioned above, one suitable analyte is a cell. In addition, suitable analytes include organic and inorganic molecules, including biomolecules. In a preferred embodiment, the analyte is a protein. As will be appreciated by those in the art, there are a large number of possible proteinaceous analytes that may be detected or evaluated for binding partners using the present invention. In addition to enzymes as discussed above, suitable protein analytes include, but are not limited to, (1) immunoglobulins; (2) hormones and cytokines (many of which serve as ligands for cellular receptors); (3) cancer markers; and (4) other proteins.
According to one embodiment in which the analyte is not an enzyme and a sandwich assay is performed as described in further detail below, the enzymatic label as described in further detail below can be beta-galactosidase. Alternatively, the enzyme label can be, but is not limited to, alkaline phosphatase or horseradish peroxidase.
Further suitable analytes include, but are not limited to, an environmental pollutant (including pesticides, insecticides, toxins, etc.); a chemical (including solvents, polymers, organic materials, etc.); therapeutic molecules (including therapeutic and abused drugs, antibiotics, etc.); biomolecules (including hormones, cytokines, proteins, nucleic acids, lipids, carbohydrates, cellular membrane antigens and receptors (neural, hormonal, nutrient, and cell surface receptors) or their ligands, etc); whole cells (including procaryotic (such as pathogenic bacteria) and eukaryotic cells, including mammalian tumor cells); viruses (including retroviruses, herpesviruses, adenoviruses, lentiviruses, etc.); and spores; etc.
In certain embodiments, the analyte may be a post-translationally modified protein (e.g., phosphorylation, methylation, glycosylation) and the capture component may be an antibody specific to a post-translational modification. Modified proteins may be captured with a multiplicity of specific antibodies and then visualized with use of a specific-secondary antibody to a post-translational modification. Alternatively, modified proteins may be captured with an antibody specific for a post-translational modification and then visualized with specific antibodies to each modified protein.
In another embodiment, the analyte is a nucleic acid. A nucleic acid may be captured with a capture nucleic acid (e.g., an oligonucleotide) and then visualized with a different specifically labeled oligonucleotide.
IV. Detectable Labels and Signal DetectionIt is understood that a variety of detectable labels can be used in the practice of the methods described herein. For example, it is understood that a variety of colored labels (for example, metallic nanoparticles (for example, gold nanoparticles), semiconductor nanoparticles, semiconductor nanocrystals (for example, quantum dots), spectroscopic labels (for example, fluorescent labels), radiolabels, and enzymatic labels may be used in the practice of the invention.
Depending upon the particular assay format, the detectable labels are indirectly attached to the analyte after it has been captured by the capture component, for example, via a secondary binding ligand that is conjugated to a detectable label and binds to the captured analyte. Once the label has become attached to the analyte, the presence of the label can be detected using suitable detection systems, for example, optical detectors (for example, intensified CCD cameras), fluorescence detectors known in the art.
In one embodiment, the labels are enzymatic labels. In this case, a chromogenic, fluorogenic, or chemiluminescent enzyme substrate is contacted with the enzyme to produce a detectable product. It is understood in the art that chromogenic, fluorogenic, or chemiluminescent enzyme substrates are known or can be made for many different enzymes. Thus, any known chromogenic, fluorogenic, or chemiluminescent enzyme substrate capable of producing a detectable product in a reaction with a particular enzyme can be used in the present invention, including any of the chromogenic, fluorogenic, or chemiluminescent enzyme substrates disclosed in The Handbook—A Guide to Fluorescent Probes and Labeling Technologies, Tenth Ed., Chapter 10, http://probes.invitrogen.com/handbook/sections/1000.html, which is incorporated herein by reference in its entirety.
According to one embodiment in which the analyte is detected using a sandwich assay as described further herein in which the enzyme label is β-galactosidase, the enzyme substrate added to the array is a β-galactosidase substrate such as resorufin-β-D-galactopyranoside.
V. Assay MethodsThe arrays of reaction vessels of the present invention can be used for several different assay methods. More specifically, the present invention provides for both (a) analyte detection and (b) quantification of analyte concentration in a sample.
Generally, the array of microwells of the present invention is exposed to an analyte of interest (or contacted with a sample containing an analyte of interest) and the analyte is captured by a capture component on the binding surface within each microwell under conditions suitable for capture of the analyte by at least one of the capture components, i.e. generally physiological conditions. For purposes of the present application, the term “immobilized” means captured, attached, bound, or affixed to a capture component on the binding surface. For example, in the context of using microwells, the interaction between any analyte molecule and the capture component on the binding surface of a microwell results in immobilization of the analyte molecule within that microwell.
According to one aspect of the invention, the sample of interest is placed in contact with the array of microwells of the present invention (or the array of the microwells is incubated in the sample) for a period of from about 30 minutes to about 12 hours depending upon the concentration of the analyte and the sample volume. In one embodiment, the array of microwells and sample are contacted for a period of from about 50 minutes to about 70 minutes. In a further embodiment, the incubation period is about 1 hour.
According to one embodiment, a wash step is performed after contacting the array of microwells with the sample. The wash step is intended to wash away any analytes or non-analyte molecules that are not bound to a capture component. Alternatively, no wash step is needed.
In one aspect of the invention, a secondary binding ligand then is added to the array of microwells. Generally, the secondary binding ligand is added if the method of detecting the analyte is an indirect assay such as a “sandwich assay” (when the analyte is not an enzyme), as described in further detail herein. The secondary binding ligand, as discussed above, will associate with or bind to the captured analyte and comprises detectable label, for example, an enzymatic component. The secondary binding ligand is added in an amount sufficient to ensure that a secondary binding ligand comes into contact with every bound analyte on the binding surface of the array of microwells. Alternatively, no secondary binding ligand is added, such as, for example, when the analyte is going to be detected directly.
When colored labels and spectroscopic labels are used, the labels can be detected using an appropriate detection system. In contrast, when the label is an enzymatic label, the enzymatic label is contacted with chromogenic fluorogenic, or chemiluminescent enzyme substrate to produce a detectable product.
In the case of enzymatic labels, a chromogenic, fluorogenic, or chemiluminescent enzymatic substrate as described above then is introduced or added to the array of microwells. The chromogenic, fluorogenic, or chemiluminescent enzymatic substrate is provided in an amount sufficient to contact any captured analyte that is either an enzyme itself or is coupled to an enzyme, for example, via an enzyme-labeled antibody. The chosen enzyme substrate reacts with or is modified by the enzymatic component such that the reaction produces a chromogenic, fluorogenic, or chemiluminescent product and thus an optical signal. The presence of the chromogenic, fluorogenic, or chemiluminescent product in the array of reaction vessels (as described below) can provide information about the identity and/or concentration of an analyte based on the interaction of the analyte with the capture component and the enzymatic substrate (and the secondary binding ligand, in some cases).
In one embodiment of the present invention, the array of microwells are sealed after the enzymatic substrate is added to form an array of reaction vessels. That is, a sealing component is placed in contact with the face of the array of microwells, thereby fluidly isolating each microwell and sealing its contents therein. A “sealing component,” as used herein, is defined as any material or device large enough to cover the entire array of microwells and capable of contacting the exterior surface of the array of microwells (for example, the cladding of a fiber optic bundle) such that each reaction vessel thus formed is sealed or isolated such that the contents of each reaction vessel cannot escape the reaction vessel. According to one embodiment, the sealing component is a silicone elastomer gasket that is placed against the array of microwells with a uniform pressure across the entire substrate. By sealing the contents in each microwell, the enzymatic reaction can proceed within the reaction vessel thus formed, thereby producing a detectable amount of the chromogenic, fluorogenic, or chemiluminescent product that is retained in the reaction vessel for detection purposes. That is, the enzyme converts the substrate into a chromogenic, fluorogenic, or chemiluminescent product that builds up to a locally high concentration in each sealed reaction vessel, generating a detectable chromogenic, fluorogenic, or chemiluminescent signal.
According to one embodiment, the present invention provides for a microscope system equipped with a mechanical platform that applies the sealing component. The platform is positioned beneath the microscope stage on the microscopy system. After the enzymatic substrate have been added to the array of microwells, the sealing component is sandwiched between a flat surface (such as, for example, a microscope slide) and the array of microwells using uniform pressure applied by the mechanical platform.
The assays for detecting analytes may be run under a variety of experimental conditions, as will be appreciated by those in the art. A variety of other reagents may be included in the screening assays. These include reagents like salts, neutral proteins, e.g. albumin, detergents, etc, which may be used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Also, reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., may be used. The mixture of components may be added in any order that provides for the requisite binding. Various blocking and washing steps may be utilized as is known in the art.
The reaction vessels exhibiting enzymatic activity or changes in their optical signature may be identified by a conventional optical train and optical detection system. Depending on the particular enzymatic substrates used and the operative wavelengths of their products, optical filters designed for a particular wavelengths may be employed for optical interrogation of the reaction vessels. In a preferred embodiment, the array of reaction vessels of the present invention are formed directly as part of a fiber optic bundle.
According to one embodiment, the array of reaction vessels of the present invention can be used in conjunction with an optical detection system such as the system described in U.S. application Ser. No. 09/816,651, which is incorporated herein by reference in its entirety. For example, according to one embodiment, the array of reaction vessels of the present invention is the distal end of a fiber optic assembly comprising a fiber optic bundle constructed of clad fibers so that light does not mix between fibers. As depicted in the Ser. No. 09/816,651 Application, the proximal end of the bundle is received by a z-translation stage and x-y micropositioner.
The optical detection system of U.S. application Ser. No. 09/816,651 operates as follows. Light returning from the distal end of the fiber optic bundle is passed by the attachment to a magnification changer which enables adjustment of the image size of the fiber's proximal or distal end. Light passing through the magnification changer is then shuttered and filtered by a second wheel. The light then is imaged on a charge coupled device (CCD) camera. A computer executes imaging processing software to process the information from the CCD camera and also possibly control the first and second shutter and filter wheels.
The array of reaction vessels of the present invention may be integral or attached to the distal end of the fiber optic bundle using a variety of compatible processes. Microwells are formed at the center of each individual fiber of the fiber optic bundle and reaction vessels are formed by sealing this array of microwells with a sealing component. Thus, each optical fiber of the fiber optic bundle conveys light from the single microwell formed at the center of the fiber's distal end. This feature is necessary to enable the interrogation of the optical signature of individual reaction vessels to identify reactions in each microwell. Consequently, by imaging the end of the bundle onto the CCD array, the optical signatures of the reaction vessels are individually interrogatable.
A. Detection
As discussed previously, the detection system will depend upon the label being detected. For example, when the label is an optical label, spectroscopic label or radiolabel, the presence of the label can be determined using commercially available detectors known in the art.
When the label is an enzymatic label, the label can be detected directly (for example, when the analyte has inherent enzyme activity) or indirectly (for example, when the analyte does not exhibit enzyme activity) via techniques known in the art. In other words, if the analyte is an enzyme, the analyte can be identified by a direct method of detection. Alternatively, if the target analyte is not an enzyme and thus cannot produce a chromogenic, fluorogenic, or chemiluminescent product in the presence of an enzymatic substrate, the analyte is identified by an indirect method of detection.
The direct method of detection, which involves a analyte that is an enzyme, proceeds as follows. First, the sample of interest and the array of microwells are placed in contact as described in further detail above under suitable conditions. The enzymatic substrate is added either simultaneously with the sample or after addition of the sample.
After sealing the microwells, the presence or absence of the analyte in any given reaction vessel then is detected by optical interrogation. That is, any change in the optical signal caused by production of a chromogenic, fluorogenic, or chemiluminescent product is detected. In any reaction vessel containing the analyte, the analyte modifies or acts upon the enzymatic substrate in some way, thereby resulting in the release of a chromogenic, fluorogenic, or chemiluminescent product, resulting in a change in the optical signal from the reaction vessel. The chromogenic, fluorogenic, or chemiluminescent product is then optically detected.
In one embodiment of the present invention, the array of microwells are sealed with the sealing component to form reaction vessels after the enzymatic substrate is added, as described above.
The indirect methods of detection involve an analyte that does not have enzymatic properties. Two indirect methods that can be used with the present invention are the “sandwich” assay and the “competitive” assay. It is contemplated that similar assay formats can be employed when the labels are optical labels, spectroscopic labels and radiolabels.
A sandwich assay can be performed as depicted in
Next, a “secondary binding ligand” 18 is added to the array of microwells 10, as shown in
A secondary binding ligand 18 has two components, a binding component 22 and an enzymatic label or a label that can capture an enzyme 24. The binding component 22 is the portion of the secondary binding ligand 18 that binds to the analyte 12. Typically, the secondary binding ligand 18 binds to a different portion of the analyte 12 than the capture component 16, because if both the capture component 16 and secondary binding ligand 18 were to bind to the same portion, the secondary binding ligand 18 would not be capable of binding to a captured analyte 12. Thus, the chosen secondary binding ligand 18 can bind to the analyte 12 while the analyte 12 is bound to the binding surface 14 via a capture component 16.
The label 24 is the portion of the secondary binding ligand 18 that exhibits enzymatic activity or can be used to subsequently to capture an enzyme. According to one embodiment, the label 24 is an enzyme attached to the secondary binding ligand 18.
Subsequently, the enzymatic substrate is added. In one embodiment of the present invention, the array of microwells are sealed after the enzymatic substrate is added to form reaction vessels, as described above. The presence or absence of the analyte in any given reaction vessel then is detected by optical interrogation. That is, any change in the optical signal caused by production of a chromogenic, fluorogenic, or chemiluminescent product is detected. In any reaction vessel containing the analyte and the secondary binding ligand, the enzyme associated with the secondary binding ligand modifies or acts upon the enzymatic substrate in some way, thereby producing a chromogenic, fluorogenic, or chemiluminescent product, resulting in a change in the optical signal from the reaction vessel. The product then is optically detected.
The competitive assay operates as follows. First, a labelled molecule is added to the array of microwells of the present invention, wherein the label is a enzyme or enzymatic component. In this embodiment, the chosen labelled molecule binds with the capture component such that the addition of the labelled molecule to the array results in labelled molecules being bound to capture components on the binding surface of the microwells.
Next, the sample of interest and the array of microwells are placed in contact as described in further detail above. The presence of the analyte in the array of microwells causes the displacement of the labelled molecule and binding of the analyte to the capture components. The displacement occurs for the following reason: in this embodiment, the chosen capture component is capable of binding to either of the labeled molecule or the analyte, thus resulting in a competitive binding situation. As a result, if a labelled molecule is bound to a capture component on the binding surface of a microwell and an analyte is added, the analyte will displace the labeled molecule under suitable conditions.
According to one embodiment, a wash step is then performed to remove any non-bound labeled molecules from the binding surface of the array of microwells.
Subsequently, the enzymatic substrate is added. As discussed above, according to one aspect of the invention, the array of microwells are sealed after the enzymatic substrate is added to form reaction vessels. Alternatively, the array of microwells are not sealed. The presence or absence of the analyte in any given reaction vessel then is detected by optical interrogation. But unlike the optical interrogations that are described above, in this interrogation it is the lack of a chromogenic, fluorogenic, or chemiluminescent product that indicates the presence of the analyte in the reaction vessel. In any reaction vessel containing the analyte, no enzymatic action occurs and no change occurs in the optical signal from the reaction vessel. In contrast, in any reaction vessel in which the labelled molecule is still present, an optical signal is detected.
In an alternative version of the competitive assay embodiment, both the labelled molecule and sample of interest are added to the array of microwells at the same time in fixed volumes. In this version, the analyte and labelled molecule compete directly for the binding sites on the capture components.
In addition, it is understood that the methods and compositions described herein can be used in a variety of multiplexed assays to detect the presence of one or more analytes in a sample. Exemplary multiplex assays are described in the following sections.
1. Multiple Different Capture Components to Same Target Analyte
The reaction vessel array of the present invention according to one embodiment utilizes a plurality of capture components that are directed to a single target analyte but are not identical. In other words, the binding components bind to different binding sites on an analyte. This embodiment thus provides for more than one different capture component on each binding surface or different capture components on different binding surfaces. In one example, a single analyte is interrogated by two or more capture components, each of which is capable of binding to different sites on the analyte. This adds a level of confidence to the assay as non-specific binding interactions can be statistically minimized. In this embodiment, when proteinaceous analytes are evaluated, preferred embodiments utilize capture components that bind to different parts of the target. For example, when two or more antibodies (or antibody fragments) to different portions of the same analyte protein are used as capture components, preferred embodiments utilize antibodies to different epitopes. Similarly, when nucleic acid analytes are to be evaluated, the redundant nucleic acid probes may be overlapping, adjacent, or spatially separated. However, it is preferred that two probes do not compete for a single binding site, so adjacent or separated probes are preferred.
In this embodiment, a plurality of different capture components may be used, with from about 2 to about 20 being preferred, and from about 2 to about 10 being more preferred, and from 2 to about 5 being particularly preferred, including 2, 3, 4, or 5. However, as above, more may also be used, depending on the application.
2. Multiple Different Capture Components to Multiple Target Analytes
In another embodiment, the reaction vessel array of the present invention uses a plurality of different capture components that are directed to a plurality of target analytes, which can be the same or different. This embodiment includes more than one different capture component on each binding surface or different capture components on different binding surfaces. In one example, two or more analytes may be provided to which two or more capture components on the same binding surfaces or on different binding surfaces are capable of binding.
In this embodiment, more than one target analyte can be identified. For example, two or more target analytes can be identified so long as, in the case of direct assays, each different analyte is a different enzyme or, in the case of indirect assays, each target is coupled to binding moieties conjugated with different labels. In one embodiment, the analytes are identified using multiple enzymatic substrates wherein each substrate produces a different color upon interaction with the appropriate enzyme. Thus, each target analyte can be distinguished based on the color produced by reaction with the enzymatic substrate. When labels other than enzymes are used, the different labels should have different characteristics so that each different label can be identified with the appropriate detector.
In an alternative approach, referred to as a sequential approach, the analytes are identified using the same or different labels. In this approach, the one or more analytes of interest are captured on one or more binding surfaces. Then a first labeled binding moiety that binds the analyte is permitted to bind to a first immobilized analyte. The presence of the label can be detected with an appropriate detection system. Thereafter, the first labeled binding moiety is removed, for example, by washing. Then a second labeled binding moiety that binds to a second, different analyte is permitted to bind to the second analyte. The presence of the label can be detected with an appropriate detection system. Thereafter, the second labeled binding moiety can be removed by washing and the process repeated as desired. It is understood that, in this approach, the same or different labels can be present in the first and second labeled binding moieties. It is understood that all the labels described herein can be used in this approach. When the label is a enzyme, it is understood that the sequential reactions can be performed using substrates, each of which produce the same or different colors.
In this embodiment, a plurality of different capture components can be used, with from about 2 to about 100 being preferred, and from about 2 to about 20 being especially preferred, and from 2 to about 10 being particularly preferred, including 2, 3, 4 or 5, 6, 7, 8, 9. However, as above, more may also be used, depending on the application.
In an another approach, referred to as a spatial approach, it is possible to use either (i) two different capture components disposed within separate regions of a binding surface, (ii) two different labeled binding moieties that are applied to an separate regions of a binding surface, or (iii) a combination or (i) and (ii).
In the first example of the spatial approach, different capture components are applied to different regions of the binding surface. For example, this can be achieved by placing micro-droplets on different regions of a fiber microwell array if it is used as a binding surface or to the sealing component if it is used as the binding surface. Each micro-droplet would contain a different capture component that would functionalize the surface it contacts with the different capture component. In this way, depending on the size of the droplets, it is possible to make a array of different capture components on the binding surface that are spatially discrete. The number of multiplexed analytes that could be detected in this manner would depend on the size of the different capture component regions and the size of the array of reaction vessels.
In the second example of the spatial approach, it is understood that the analytes of interest can be captured uniformly across a binding surface or within discrete regions of the binding surface. In this approach, however a first labeled binding moiety is applied to a first region of the binding surface and the presence of a first analyte is detected. Simultaneously or sequentially, a second labeled binding moiety is applied to a second region of the binding surface and the presence of a second analyte is detected.
It is understood that that each of the different assay configurations above, including the capture components directed to different analytes and the plurality of capture components directed to the same analyte, can also be utilized for quantification as described below.
In addition, under certain circumstances it is understood that the array of microwells may be reused during a subsequent assay. In this approach, a log of the wells that contained analyte in a first assay is recorded and then in a subsequent assay the wells that previously contained analyte are mathematically subtracted from the array of microwells used in the subsequent assay.
B. Quantification
According to one embodiment of the present invention, the methods, systems, and devices of the invention can be used to both detect the presence and concentration of an analyte in the sample. That is, there is a correlation between the percentage of reaction vessels containing one or more analyte molecules and the concentration of the analyte in the sample. Thus, the quantification method of the present invention allows for calculation of the amount of a analyte in a sample based on the percentage of reaction vessels that contain an analyte molecule.
The present invention is distinguished by its ability to quantify low numbers of individual molecules and is, therefore, well suited to measuring very low concentrations of target analyte. This ability is achieved by spatially isolating single analyte molecules, for example, in an array of reaction vessels, and then optionally generating and confining a chromogenic product, for example, a fluorescent label, in the reaction vessels. The presence of an analyte molecule can easily be counted in a binary fashion (zero when a molecule is absent; one when a molecule is present) due to the high concentration of chromogenic product in any reaction vessel that contains at least one analyte molecule.
Without being limited by theory, the quantification method is driven in part by the fact that the number and volume of reaction vessels employed govern the dynamic range of concentrations that can be determined by this technique. That is, based on the number and volume of the reaction vessels in an array of the present invention, an estimate can be made of the range of concentrations of target analyte in solution that allow for the concentration to be determined using the method of the present invention.
For example, for an array as disclosed in Example 2 with reaction vessels each having a volume of 46 fL, a solution having a concentration of 3.6×10−11 M β-galactosidase will yield, on average, one enzyme molecule per vessel. However, it is important to note that distributing a solution having a target analyte concentration within the appropriate range into an array of reaction vessels will not result in the distribution of exactly one enzyme molecule per vessel; statistically, some vessels will have multiple molecules while others will have zero. In the case where the number of vessels containing one or more enzyme molecules to the number of vessels containing no enzyme molecule is high, the data can be fit to a Gaussian distribution. As the ratio of reaction vessels containing an enzyme molecule to the number of vessels containing no enzyme molecule approaches zero, the Poisson distribution applies. This limiting distribution is used to calculate the probability of rare events occurring in a large number of trials. For example, based on Poisson statistics, for a concentration of 3.6×10−11 M, a distribution between zero and five enzyme molecules per container is predicted, with the most probable values being zero and one.
Equation 1 can be used to determine the probability of observing v events based on the expected average number of events per trial, μ
Pμ(v)=e−μ(μv/v!) Equation 1:
If the concentrations used are much less than 3.6×10−11 M, the expected average becomes exceptionally low, the distribution is narrowed, and the probability of observing anything other than 0 or 1 events per trial is improbable in all experimental cases. At these low concentrations, the relationship between the percentage of active reaction vessels and the bulk enzyme concentration is approximately linear. Thus, based on this knowledge, the array of the present invention can be used to determine the concentration of a target analyte in a sample by a simple digital readout system, i.e., “counting” of active wells, as described herein.
According to one embodiment, the quantification method of the present invention can be performed as follows. The method is a digital readout system (also referred to as a “binary readout system”) that includes first detecting the analytes in the array of reaction vessels by any detection method described above. The number of reaction vessels is then counted and a percentage of the total number of reaction vessels is calculated. That is, utilization of a yes or no response, in conjunction with the high-density array of reaction vessels, permits the digital readout of bulk concentrations of analyte molecules labeled with a label of interest, for example, the enzyme, β-galactosidase. This readout is accomplished by counting the vessels containing an active enzyme molecule across the array, with the resulting “active well” percentage correlating to the concentration of analyte assuming one enzyme label per target molecule. Given the large number of vessels simultaneously interrogated in the array of the present invention, the ratio of enzyme molecules to reaction vessels could be 1:1000 or less, as the large number of wells provides a statistically significant signal even at this low ratio.
Without being limited by theory, it is believed that the quantification method of the present invention is only limited by the number of individual reaction vessels that can be fabricated and interrogated. Thus, expanding the number of reaction vessels will increase both the dynamic range and the sensitivity of the assay.
The limitations of this technique are realized above and below the thresholds of the dynamic range. As the concentration goes below the lower limit of the dynamic range, the number of enzyme molecules is too low to observe sufficient occupied wells and, therefore, the number of wells must be increased in order to make sure that a statistically significant number of them are occupied by enzyme molecules. Results for extremely dilute concentrations have large relative errors associated with them, due to the very small number of reaction vessels that are expected to show activity. Slight deviation from the expected Poisson value, in this case, will result in a large error. The ultimate upper limit to this technique occurs when 100% of the reaction vessels contain at least one enzyme molecule. At this limit, discrimination between two solutions of high enzyme concentrations is not feasible.
In the range where the fraction of reaction vessels containing at least one molecule is less than about 20%, the probability that any well contains two or more analyte molecules is very small and the number of analyte molecules closely matches the number of occupied reaction vessels. Between 20% occupied and 100% occupied, an increasing number of wells will contain more than one molecule, however Gaussian statistics can still be used to correlate occupancy fraction with concentration until the occupancy fraction reaches 100%.
In practice, non-specific signal creates a lower “noise floor” and faulty reaction vessels create an upper “noise ceiling.” Furthermore, with reference to the number of wells occupied or not occupied by analyte, statistically significant occupancy can be determined using standard statistical methods known in the art. For example, a statistically significant measurement can be one that is three times the standard deviation from the mean.
If desired, the practical dynamic range of the method can be increased in several ways. In one approach, the sample is diluted by a factor of 10 or more. Both the sample and the diluted sample can be assayed concurrently using the method of this invention. The dynamic ranges of the two assays will overlap, but be offset by the dilution factor, hence extending the dynamic range.
In a second approach, the amount of signal in each reaction vessel is measured at multiple time points prior to the time when all of the reaction vessels are positive for analyte. The fraction of wells that are positive at an early timepoint can be calibrated against a known reference standard to determine the concentration of the sample being analyzed. In the embodiment of the invention that utilizes a fiber optic bundle and a CCD camera, it is straightforward to image all of the wells in near real-time.
In a third approach, multiple arrays of reaction vessels can be used with differing volumes, differing areas of binding surface, or differing density of concentration of capture components on the binding surface. These arrays can be constructed as either distinct arrays or as one large array with sub-arrays with varying characteristics. Since the probability of an analyte molecule being detected in a given reaction vessel is related to volume, binding surface, and capture component density, the sub-arrays may be designed to cover different sensititivy ranges. If those ranges overlap, the effective range of the combined array can be extended.
In another aspect, the invention provides a method of determining the concentration of an analyte in a fluid sample to be tested. The method comprises the steps of (a) dividing the fluid sample into an array of wells, so that, for substantially all of the wells, each well contains either no analyte or a single molecule of the analyte; (b) determining the presence or absence of the analyte in each well to provide a number of wells in the array that contain the analyte; and (c) determining the concentration of the analyte in the fluid sample to be tested from the number of wells in the array that contain the analyte.
In one embodiment, less than about 20% of the total number of wells contain at least one molecule. Under these circumstances, the number of wells containing at least one molecule falls within the linear range of a Poisson distribution. In another embodiment, less than about 20% of the total number of wells contain about one molecule. In another embodiment, more than about 20% but less than about 60% of the total number of wells contain at least one molecule. Under these circumstances, the number of wells containing at least one molecule falls within the non-linear range of a Poisson distribution. In another embodiment, more than about 20% but less than about 60% of the total number of wells contain about one molecule. In another embodiment, more than about 60% but less than about 95% of the total number of wells contain at least one molecule. Under these circumstances, the number of wells containing at least one molecule falls within the highly non-linear range of a Poisson distribution. In another embodiment, more than about 60% but less than about 95% of the total number of wells contain about one molecule.
In another aspect, the invention provides a method of determining the concentration of an analyte in a fluid sample to be tested. The method comprises the steps of: (a) dividing the sample to be tested into a plurality of second, smaller fluid samples of equal volume so that a statistically significant fraction of the second, smaller fluid samples contain either no analyte or a single molecule of the analyte; (b) determining the presence or absence of the analyte in each of the second, smaller fluid samples so as to identify the number of second, smaller fluid samples that contain the analyte; and (c) determining the concentration of the analyte in the sample to be tested from the number of second, smaller samples that contain the analyte.
In this approach, the statistically significant fraction of the second, smaller fluid samples that contain either no analyte or a single molecule of the analyte is defined as 50%, optionally 75%, optionally 95% of the total number of second fluid samples that contain either no analyte or a single molecule of analyte.
In one aspect of the present invention, the array can also be used to analyze enzyme kinetics. “Enzyme kinetics” as used herein refers to the study of the rates of enzyme-controlled reactions. It is understood in the art of enzyme kinetics that the rate of an enzymatic reaction at low substrate concentrations is proportional to the substrate concentration (is “substrate dependent”). This is referred to as first order. It is further understood that the rate of the reaction at high substrate concentrations reaches a maximum rate and is independent of substrate concentration because the reaction becomes saturated. Thus, if reaction velocity is plotted as a function of substrate concentration, the line initially increases linearly with an increase in substrate and then begins to level off as substrate concentration approaches saturation.
Thus, according to one embodiment, the kinetics of any particular enzyme can be studied using the present system and array. Reaction velocity varies across enzymes for various reasons, including, for example, reaction inhibition caused by allosteric inhibition. The array of the present invention allows for study of these varied kinetic characteristics.
According to one embodiment, kinetics are examined in the following fashion. The target analyte is allowed to bind to the capture component, the substrate is added, and the reaction vessel is sealed. Given that a finite amount of substrate is present in the reaction vessel and that no further substrate can be added due to the sealing of the vessel, the reaction velocity can be determined based on the amount of chromogenic product detected over time.
VI. Exemplary Uses of the Present InventionThe system and array of the present invention has many uses. For example, the array has application to fundamental enzymology studies, as well as digital concentration measurements. Further, the array permits studies with multiple different enzymes and extends the limits of ultra-low detection for protein and DNA targets. With the ability to simultaneously monitor a large array of reaction vessels, single molecule enzymology can be used to resolve individual enzyme molecule behavior from bulk kinetic signal.
Another use, for example, is environmental monitoring of bacteria or viruses or both. An environmental sample potentially containing certain bacteria can be placed in contact with an array of the present invention. To detect the bacteria, the bacteria cells are lysed and a bacterial enzyme (or more than one enzyme) is targeted for detection. According to one embodiment, the cells are lysed prior to being added to the array. Alternatively, the cells are captured and a lysing step occurs on the array prior to detection. In a further alternative, no lysis may be necessary if a cell surface marker is targeted. For example, the bacteria or virus of interest can be captured with an antibody that is specific to a surface marker on the target, and then the capture can be detected with a sandwich-type assay by adding an enzyme-labelled antibody that binds to the target in another location.
Another use, for example, involves measuring gene expression. In one embodiment, the target analytes are cells and these are introduced into the reaction vessels of the array under conditions such that some reaction vessels contain single cells. This is the first step in measuring gene expression, which can be done if desired at the whole cell level by detecting surface proteins and secreted proteins. Alternatively, the cells can be lysed and transcripts (or other components) from the cells can be interrogated. In one embodiment, each site contains one or no transcripts. In one embodiment, normal and cancer cells are compared. The cell to cell variation in gene expression can be addressed by examining gene expression at the single cell level.
Although the present invention has been described herein with reference to preferred embodiments, persons skilled in the art will recognize that changes may be made in form and detail without departing from the spirit and scope of the invention.
EXAMPLES Example 1In this example, a proof-of-concept binding assay is performed using enzymatic signal amplification in an array of femtoliter sized reaction vessels. More specifically, various assays are performed to detect varying amounts of streptavidin-β-galactosidase (SβG) in solution using a biotinylated array of the present invention and then the correlation between the number of wells with captured SβG molecules and the concentration of the SβG in the sample is examined.
In this example, an etched fiber optic array is used to create a collection of femtoliter sized reaction vessels, each specifically functionalized and capable of capturing enzyme-labeled target molecules. Single enzyme molecules are confined to individual reaction vessels and catalyze the production of a sufficient number of fluorescent product molecules to generate a positive signal. At low target molecule concentrations, only a percentage of the capture sites bind a target molecule, enabling a binary readout of target concentration from the high-density array.
MaterialsThe reactor vessel arrays in this example are generated using an acid etch of the distal face of a polished 1 mm fiber optic array, consisting of 24,000 individual 4.5 μm optical fibers. The core fiber material is silica, and the cladding around each fiber is germania-doped silica, which etches at a slower rate. The 4.5 μm fibers are etched to a depth of 2.9 μm, creating an array of reactor vessels, each with a 46 fL volume (see
The fibers were first modified with an aminopropyl silane bound to both the core and cladding surfaces (see
First, the effectiveness of the capture component was tested. To test the effectiveness of the biotinylation of the substrate, streptavidin Alexa Fluor 568® was attached directly to the biotin groups on the surfaces of both a polished and an unpolished fiber, followed by image acquisition of the modified surface (see
Subsequent to array modification, the biotinylated fiber arrays were incubated for 1 hour at room temperature in 150 μL PBS buffer containing varying amounts of SβG. The concentration of the SβG was chosen so that during the incubation time, statistically either one molecule or no molecules would bind to each well. The arrays were then washed repeatedly in PBS buffer, to ensure that unbound target was removed.
For a binary readout of SβG binding, the fiber array was loaded and secured on an upright microscope system equipped with a mechanical platform. A solution of β-galactosidase substrate, resorufin-β-D-galactopyranoside (RDG), was introduced to the distal end of the fiber containing the reaction vessels, and subsequently sealed. The substrate was sealed using a 0.01-inch thick silicone elastomer gasket sandwiched between a microscope slide and the fiber array by means of a mechanical platform located beneath the microscope stage. This platform applied a uniform pressure to the gasket material, across the entire bundle, sealing off each reaction chamber and enabling well to well interrogation of enzyme activity. β-galactosidase hydrolyzes RDG to form resorufin, which builds up to a locally high concentration in each sealed reaction vessel, generating a detectable fluorescent signal (
Analysis of over 5000 reaction vessels for each experiment allowed for a correlation between the percentage of reaction vessels that captured an enzyme molecule and the amount of enzyme present in the interrogated sample. The variation seen in the intensity differences from active well to active well is most likely a result of molecule-to-molecule variation in catalytic activity, in combination with surface effects, which may modulate the relative activities of enzyme molecules based on their orientation to the reaction chamber surface.
Two control experiments were also conducted to ensure that the binding of enzyme to the surface of the reactors was based exclusively on the biotin-streptavidin interaction, and not on non-specific binding to the glass surface. One control experiment consisted of an etched, unmodified fiber incubated with the most concentrated SβG target solution (128 amol in 150 μL). The second control experiment was performed using the modified fiber incubated in a solution of β-galactosidase lacking streptavidin (128 amol in 150 μL). Both control experiments generated a negligible active well percentage (less than 0.06%, versus 0.2% for the 2.6 amol experiment discussed below).
Results
It is also interesting to note that the lowest limit of detection (LOD) for binding streptavidin-β-galactosidase (SβG) to a biotinylated femtoliter array in this example was 2.6 amoles (150 μL of 17 fM solution) using a target incubation time of 1 hour.
Example 2In this example, single molecules of β-galactosidase were monitored using a 1 mm diameter fiber optic bundle with over 2.0×105 individually sealed, femtoliter microwell reactors. By observing the buildup of fluorescent products from single enzyme molecule catalysis over the array of reaction vessels and by applying a Poisson statistical analysis, a digital concentration readout was obtained.
Materials1 mm bundled 4.5 μm optical fibers were purchased from Illumina (San Diego, Calif.). β-galactosidase and Ru(bpy)3Cl2 was obtained from Sigma-Aldrich (St. Louis, Mo.). Resorufin-D-β-galactopyranoside was purchased from Molecular Probes (Eugene, Oreg.). 0.01-inch non-reinforced gloss silicone sheeting material was purchased from Specialty Manufacturing Inc. (Saginaw, Mich.). All other chemicals used were of reagent grade and obtained from Sigma-Aldrich (St. Louis, Mo.).
A custom-built, upright epifluorescence imaging system acquired all fluorescence images using a mercury light source, excitation and emission filter wheels, microscope objectives, and a CCD camera (QE, Sensicam). Filter wheels and shutters were computer controlled and analysis was performed with IPlab software (Scanalytics, Fairfax, Va.). The system was equipped with a fastening device to fix the fiber optic array onto the system through the entire experiment. A mechanical platform beneath the stage was used to house the silicone-sealing layer, which was subsequently brought into contact with the distal end of the fiber array, sealing off each reaction vessel. All measurements were performed with femtowell arrays at the distal end of the optical fiber bundle.
Optical fiber bundles containing approximately 2.4×105 individual 4.5 μm diameter optical fibers were used as the substrate for fabricating femtoliter reaction vessel arrays. The well volume can be precisely controlled, as etch depth varies with etch time and etchant concentration. The optical fibers used in these experiments were etched to a depth of approximately 2.9 μm, yielding a 46 fL well volume.
Assay. For the β-galactosidase assay, the substrate used was resorufin-β-D-galactopyranoside. After the individual wells in the array were sealed in the presence of enzyme and substrate, the fluorescence intensity was monitored across the array of vessels for the enzymatic product, resorufin (ex 558 nm/em 573 nm). A 100 μM solution of resorufin-D-β-galactopyranoside (RDG) was prepared in 100 mM Tris buffer pH 8.0 containing 2.0 mM KCl and 0.1 mM MgCl2. All enzyme solutions were prepared from previously aliquoted and frozen stock samples in the same reaction buffer. Just prior to experimentation, the two samples were centrifuged for 2 min at 7000 RPM to remove any particulate material that could interfere with the mechanics of the silicone seal. Approximately 1 cm2 of silicone and a microscope slide were cleaned with absolute ethanol. The silicone sheeting was placed on the surface of the glass, to which it adhered. Subsequently, 75 μL volumes of enzyme and RDG solutions were mixed on the silicone gasket using a pipette. The gasket was mechanically raised towards the distal end of the fiber bundle until it experienced resistance, suggesting that a seal was formed. An initial fluorescence image was acquired, followed by periodic image acquisition for approximately 2 hr.
Sealing component. To seal the femtoliter array, a 0.01-inch thick silicone elastomer gasket was sandwiched between a microscope slide and the fiber array using a mechanical platform. This platform applied uniform pressure to the gasket material, across the entire bundle, sealing off each microwell to create the reaction vessels.
The silicone/glass seal used to create and isolate the femtoliter containers was inspected for its sealing ability by performing a photobleaching experiment (see
Photobleaching Experiment. A solution of 1 mM Ru(bpy)3Cl2 in DI water was used for the photobleaching experiments. A piece of silicone, approximately 1 cm2, and a microscope slide were cleaned with absolute ethanol using lint-free swabs. The silicone sheeting was placed on the surface of the glass, to which it adhered. 50 μL of the Ru(bpy)3Cl2 solution was placed on the silicone, and subsequently brought into contact with the fiber bundle, to enclose the solution in the individual vessels. Using a field stop on the imaging system, UV light was used to illuminate a small portion of the array for 10 minutes, photobleaching the Ru(bpy)3Cl2. The field stop was then opened, and an image was acquired, displaying the difference in fluorescence. The array was then allowed to rest with the seal maintained. A final image was taken after 60 minutes, confirming the integrity of the seal.
As discussed above, the number and volume of reaction vessels employed govern the dynamic range of concentrations that can be determined by this technique. The reaction vessel volumes employed in this example were 46 fL (vide infra); therefore, it was calculated that a solution of 3.6×10−11 M β-galactosidase will yield, on average, one enzyme molecule per vessel. As also discussed above, if the concentrations used are much less than 3.6×10−11 M, the expected average becomes exceptionally low, the distribution is narrowed, and the probability of observing anything other than 0 or 1 events per trial is improbable in all experimental cases. At these low concentrations, the relationship between the percentage of active reaction vessels and the bulk enzyme concentration is approximately linear. After waiting for sufficient time to allow enzyme catalysis to occur, individual vessels were interrogated for an on/off response, correlating to each vessel either possessing or lacking enzymatic activity.
The substrate resorufin-D-β-galactopyranoside (RDG) was used as the substrate for experiments, which was sealed into all the vessels, along with the trapped enzyme molecules, using a silicone gasket material and mechanical arm. The expected percentages of active wells were calculated for each concentration used by applying the Poisson distribution statistics.
ResultsAs shown in
Table 1 is a comparison of each experimental result with the percentage of occupied vessels calculated from the Poisson distribution. As shown by the data in the table, the array measurements successfully correlated with the number of single enzyme β-galactosidase molecules over the entire range of interrogated concentrations. There is minor disparity in the observed signals as a result of molecule-to-molecule variation in catalytic activity. This result is most likely due to the inherent stochastic nature of enzymes, in addition to surface effects, resulting in modulation of enzyme activity.
The variation between the calculated and experimental results can be attributed to the intrinsic variability associated with the probability distribution, as well as experimental error in the preparation of enzyme solutions.
Example 3In this example, a proof-of-concept sandwich assay is performed to detect a non-enzymatic target protein using an array of femtoliter sized reaction vessels (see,
The reactor vessel arrays in this example were generated using an acid etch of the distal face of a polished 1 mm fiber optic array, consisting of 24,000 individual 4.5 μm optical fibers. The core fiber material was silica, and the cladding around each fiber was germania-doped silica, which etches at a slower rate. The 4.5 μm fibers were etched to a depth of 2.9 μm, creating an array of reactor vessels, each with a 46 fL volume (see
The fibers were first modified with an aminopropyl silane (see
Subsequent to array modification, the fiber arrays having anti-IP-10 antibody disposed thereon were incubated for 1 hour at room temperature in 150 μL PBS buffer containing varying amounts of IP-10. The captured IP-10 arrays then were incubated with 2 μg/mL of a secondary binding ligand, i.e., the detection anti-IP-10 antibody labeled with biotin. These arrays then were incubated with 300 μM of SβG to label the bound IP-10 molecules with enzyme. The arrays then were washed repeatedly after each incubation in PBS buffer, to ensure that unbound target, antibody and SβG was removed.
For a binary readout of SβG bound to IP-10, the fiber array was loaded and secured on an upright microscope system equipped with a mechanical platform. A solution of β-galactosidase substrate, resorufin-β-D-galactopyranoside (RDG), was introduced to the distal end of the fiber containing the reaction vessels, and subsequently sealed. The substrate was sealed using a 0.01-inch thick silicone elastomer gasket sandwiched between a microscope slide and the fiber array by means of a mechanical platform located beneath the microscope stage. This platform applied a uniform pressure to the gasket material, across the entire bundle, sealing off each reaction chamber and enabling well to well interrogation of enzyme activity. β-galactosidase hydrolyzes RDG to form resorufin, which builds up to a locally high concentration in each sealed reaction vessel, generating a detectable fluorescent signal.
Analysis of over 5000 reaction vessels for each experiment allowed for a correlation between the percentage of reaction vessels that captured an enzyme molecule and the amount of IP-10 present in the interrogated sample.
ResultsEach of the following references is incorporated by reference in their entirety, for all purposes.
-
- Sano, T.; Smith, C. L.; Cantor, C. R. Science 1992, 258, 120-122.
- Nam, J. M.; Thaxton, C. S.; Mirkin, C. A. Science 2003, 301, 1884-1886.
- Niemeyer, C. M.; Adler, M.; Pignataro, B.; Lenhert, S.; Gao, S.; Chi, L. F.; Fuchs, H.; Blohm, D. Nucleic Acids Research 1999, 27, 4553-4561.
- Zhou, H.; Fisher, R. J.; Papas, T. S. Nucleic Acids Research 1993, 21, 6038-6039.
- Niemeyer, C. M.; Adler, M.; Wacker, R. Trends in Biotechnology 2005, 23, 208-216.
- Whitesides, G. M. Nature Biotechnology 2003, 21, 1161-1165.
- Rondelez, Y.; Tresset, G.; Tabata, K. V.; Arata, H.; Fujita, H.; Takeuchi, S.; Noji, H. Nature Biotechnology 2005, 23, 361-365.
- Nakano, M.; Komatsu, J.; Matsuura, S.; Takashima, K.; Katsura, S.; Mizuno, A. Journal of Biotechnology 2003, 102, 117-124.
- Nagai, H.; Murakami, Y.; Yokoyama, K.; Tamiya, E. Biosensors and Bioelectronics 2001, 16, 1015-1019.
- Lipman, A. E.; Shuler, B.; Bakajin, O.; Eaton, W. A. Science 2003, 301, 1233-1235.
- Chiu, D. T.; Wilson, C. F.; Ryttsen, F.; Stromberg, A.; Farre, C.; Karlsson, A.; Nordholm, S.; Gaggar, A.; Modi, B. P.; Moscho, A.; Garza-Lopez, R. A.; Orwar, O.; Zare, R. N. Science 1999, 283, 1892-1895.
- Rissin, D. M.; Walt, D. R. Journal of the American Chemical Society 2007, 128(19) 6286-6287.
- Pantano, P.; Walt, D. R. Chemistry of Materials 1996, 8, 2832-2835.
- Monk, D. J.; Ueberfeld, J.; Walt, D. R. Journal of Materials Chemistry 2005, 15, 4361-4366.
- Song, L. N.; Ahn, S.; Walt, D. R. Emerging Infectious Diseases 2005, 11, 1629-1632.
- Lee, J. Y.; Li, H. W.; Yeung, E. S. Journal of Chromatography A 2004, 1053, 173-179.
- Xue, Q. F.; Yeung, E. S. Nature 1995, 373, 681-683.
- Foquet, M.; Korlach, J.; Zipfel, W. R.; Webb, W. W.; Craighead, H. G. Analytical Chemistry 2004, 76, 1618-1626.
- Gratzl, M.; Lu, H.; Matsimoto, T.; Yi, C.; Bright, G. R. Analytical Chemistry 1999, 71, 2751-2756.
- Stamou, D.; Duschl, C.; Delamarche, E.; Vogel, H. Angewandte Chemie-International Edition 2003, 42, 5580-5583.
- Gosalia, D. N.; Diamond, S. L. Proceedings of the National Academy of Sciences USA 2003, 100, 8721-8726.
- Lu, H. P.; Xun, L. Y.; Xie, X. S. Science 1998, 282, 1877-1882.
- Taylor, J. R. An Introduction to Error Analysis; Second Addition ed.; University Science Books: Sausalito, Calif., 1997.
- Wheeler, A. R.; Throndset, W. R.; Whelan, R. J.; Leach, A. M.; Zare, R. N.; Liao, Y. H.; Farrell, K.; Manger, I. D.; Daridon, A. Analytical Chemistry 2003, 75, 3581-3586.
Claims
1. A method of determining the concentration of an analyte in a fluid sample to be tested, the method comprising the steps of:
- (a) partitioning at least a portion of the analyte molecules in the fluid sample across a plurality of reaction vessels so that a statistically significant fraction of the reaction vessels contain analyte and a statistically significant fraction of the reaction vessels contain no analyte;
- (b) determining the presence or absence of the analyte in each reaction vessel to identify the number of reaction vessels that contain analyte and/or to identify the number of reaction vessels that contain no analyte; and
- (c) determining the concentration of the analyte in the fluid sample from the number of reaction vessels that contain the analyte.
2-11. (canceled)
12. A method of determining the concentration of an analyte in a fluid sample to be tested, the method comprising the steps of:
- (a) exposing the fluid sample to a plurality of reaction vessels under conditions so that analyte is captured in a statistically significant fraction of the reaction vessels, wherein each reaction vessel comprises a microwell and each reaction vessel defines a binding surface that has a capture component immobilized thereon;
- (b) determining the presence or absence of the analyte in each reaction vessel so as to identify the number of reaction vessels that contain captured analyte and/or the number of reaction vessels that do not contain captured analyte; and
- (c) determining the concentration of the analyte in the fluid sample to be tested from the number of reaction vessels that contain and/or do not contain the analyte.
13-16. (canceled)
17. A method of determining the concentration of an analyte in a fluid sample to be tested, the method comprising the steps of:
- (a) partitioning at least a portion of the analyte molecules in the fluid sample into a plurality of reaction vessels, so that, for substantially all of the reaction vessels, each reaction vessel contains either no analyte or a single molecule of the analyte;
- (b) determining the presence or absence of the analyte in each reaction vessel to provide a number of the reaction vessels that contain the analyte; and
- (c) determining the concentration of the analyte in the fluid sample from the number of reaction vessels that contain the analyte.
18-20. (canceled)
21. A method of determining the concentration of an analyte in a fluid sample to be tested, the method comprising the steps of:
- (a) partitioning the sample to be tested into a plurality of second, smaller fluid samples of equal volume so that a statistically significant fraction of the second, smaller fluid samples contain either a single molecule of the analyte or no analyte;
- (b) determining the presence or absence of the analyte in each of the second samples so as to identify the number of second samples that contain the analyte; and
- (c) determining the concentration of the analyte in the sample to be tested from the number of second samples that contain the analyte.
22-35. (canceled)
36. A method of detecting analyte molecules or particles in a fluid sample, comprising:
- partitioning a fluid sample containing or suspected of containing analyte molecules or particles across an array comprising a plurality of reaction vessels, so that at least some of the reaction vessels contain no analyte molecules or particles and at least some of the reaction vessels contain at least one analyte molecule or particle;
- immobilizing within each reaction vessel containing at least one analyte molecule or particle, a first binding ligand comprising a label;
- applying an enzyme to the array and capturing the enzyme with the label;
- contacting the enzyme with an enzymatic substrate, wherein the enzymatic substrate produces a detectable product upon contact with the enzyme;
- detecting the detectable product; and
- determining from detecting the presence of the detectable product the number of reaction vessels which contain an analyte molecule or particle.
37. A method of detecting analyte molecules or particles in a fluid, comprising:
- partitioning a fluid containing or suspected of containing analyte molecules or particles across an array comprising a plurality of reaction vessels, so that at least some of the reaction vessels contain no analyte molecules or particles and at least some of the reaction vessels contain at least one analyte molecule or particle;
- exposing the reaction vessels to an enzymatic substrate, wherein the enzymatic substrate produces a detectable product in reaction vessels containing at least one analyte molecule or particle;
- detecting the detectable product; and
- determining from detecting the presence of the detectable product the number of reaction vessels which contain at least one analyte molecule or particle.
38. A method of detecting analyte molecules or particles in a fluid, comprising:
- immobilizing with respect to each analyte molecules or particles, an enzyme molecule;
- partitioning the enzyme molecules across an array comprising a plurality of reaction vessels, so that at least some of the reaction vessels contain no enzyme molecules and at least some of the reaction vessels contain at least one enzyme molecule;
- exposing the reaction vessels to an enzymatic substrate, wherein the enzymatic substrate produces a detectable product in reaction vessels containing at least one enzyme molecule; and
- determining from detecting the presence of the detectable product the number of reaction vessels which contain at least one enzyme molecule.
39. The method of claim 1, wherein the analyte molecules are exposed to a plurality of secondary binding ligands after the analyte molecules have been partitioned across the plurality of reaction vessels, wherein at least one secondary binding ligand becomes associated with each analyte molecule.
40. The method of claim 39, wherein the presence or absence of the analyte in each reaction vessel is determined by determining the presence or absence of a secondary binding ligand in each reaction vessel.
41. The method of claim 40, wherein the number of reaction vessels that contain or do not contain the analyte is determined by determining the number of reaction vessels that contain or do not contain the secondary binding ligand.
42. The method of claim 1, wherein at least one wash step is performed.
43. The method of claim 1, wherein the reaction vessels are sealed during at least step (b).
44. The method of claim 1, wherein each reaction vessel is exposed to a plurality of reaction components, wherein at least one of a chromogenic, fluorogenic, or chemiluminescent product is produced upon exposure of plurality of reaction components to the secondary binding ligand.
45. The method of claim 44, wherein the presence or absence of the analyte in each reaction vessel is determined by determining the presence or absence of at least one chromogenic, fluorogenic, or chemiluminescent product in each reaction vessel.
46. The method of claim 45, wherein the concentration of the analyte in the fluid sample is determined from determining the number of reaction vessels that contain or do not contain at least one chromogenic, fluorogenic, or chemiluminescent product.
47. The method of claim 1, wherein the partitioning of at least a portion of the analyte molecules in the fluid sample across a plurality of reaction vessels comprises partitioning the fluid sample across a plurality of wells of an array comprising the plurality of wells.
48. The method of claim 1, wherein at least one analyte of a first type and at least one analyte of a second type are partitioned across the plurality of reaction vessels.
49. The method of claim 48, wherein the reaction vessels comprises at least one capture component per reaction vessel, wherein the capture component is able to immobilize at least one analyte molecule.
50. The method of claim 49, wherein the first analyte or the second analyte is able to be immobilized with respect to a first capture component.
51. The method of claim 50, wherein the reaction vessels comprise at least one capture component of a first type per reaction vessel and at least one capture component of a second type per reaction vessel.
Type: Application
Filed: Aug 30, 2007
Publication Date: Aug 11, 2011
Applicant: Trustees of Tufts College (Boston, MA)
Inventors: David R. Walt (Boston, MA), David M. Rissin (Somerville, MA)
Application Number: 12/675,686
International Classification: C40B 30/04 (20060101);
