Patent Application
20220381775
The present disclosure relates generally to the technical field of analyzing and quantifying biological analytes and, more particularly, to systems and methods therefor that provide improved accuracy and sensitivity, greater dynamic range, and simplified work flow.
High-sensitivity analytical measurements are fundamental to modern science and medicine. Protein and other small-particle measurements are also broadly essential to biomedical research and medical diagnostics. However, most biomedical assays lack the sensitivity and precision to accurately measure single analytes, particularly within complex sample matrices. Most protein and small-particle assays are formatted around bulk analyses, and often require signal amplification. While bulk analyses can and do provide useful information regarding the system overall, they generally always have fundamental limitations that prevent their ability to identify and quantify multiple characteristics of analytes or analyte subpopulations. This issue can be particularly problematic with complex samples, such as serum, urine or saliva, where the concentrations of target proteins and other analytes are extremely low, there are many non-target analytes present at concentrations that are several orders of magnitude higher than the target analyte, and yet the precision and accuracy of the data or diagnostic readout is essential.
The most commonly used biomedical assays for quantifying specific proteins include enzyme-linked immunosorbent assays (ELISAs), western blotting, and mass spectrometry. ELISAs function by capturing a target analyte with a specific antibody coated to the surface of a plate or a micro bead, and then labeling the analyte with a specific secondary, enzyme-conjugated antibody that allows for subsequent signal amplification—generally pigmentation, luminescence or fluorescence—in order to detect the antibody-analyte sandwich over a range of concentrations. Typically, ELISAs only provide a limited dynamic range, require extensive sample purification and washing, produce analog data that must be compared to calibration curves, and inevitably produce variable results due to even small changes in incubation times, lot-to-lot variation in reagent quality, loss of the analytes or antibodies due to washing and changes in the binding equilibria at different concentrations, slight differences in experimental conditions like temperature or light, the precision of the concentrations of the many substrate and buffer components that are required to produce the signal, and even differences in the stabilities of the various assay components over time in storage. In most cases, ELISAs have a lower limit of detection (LOD) in the pM range, while it is said that most relevant biomedical analytes are present in the serum in the aM to fM range, and they may be orders of magnitude lower in urine and saliva. Increasing the incubation times for the multiple steps to >10 hrs each, dramatically increasing the capture surface area, and/or using fluorogenic or radioisotope-labeled substrates has been shown to have the potential to lower the LOD down to the zeptomolar (zM) range. However, this is not a practical workflow for diagnostic assays, particularly for point-of-care-type environments or any scenario where there are many patient samples and/or time is of the essence.
Methods like western blotting and mass spectrometry are more qualitative, but quantitation can be attempted by correlating the signal intensities to reference standards. This can be sufficient in some circumstances, particularly where more quantitative methods have not been developed, but it is not a precise approach for diagnostic purposes. These methods are actually pivotal to the development and optimization of ELISAs, though ELISAs are fine-tuned for more precise analytical quantification.
A more recent technological development for single-analyte quantification is the digital ELISA. This method utilizes antibody-coated micro beads in a particular ratio sufficient to capture a single target analyte on a subfraction of the beads, and then, similar to a conventional ELISA, labels the target analytes with enzyme-conjugated detection antibodies. The difference from a conventional ELISA, and what makes the assay digital, is that the individual beads, which optimally capture at most 1 protein, are then strewn into separate femtoliter (fL)-sized wells of a 50K- to 200K-well array (called a single-molecule array), and—after exposure to the enzymatic substrate—are discretely counted by both fluorescence and light microscopy. Since the beads are captured within fL-sized wells, the volume surrounding each individual bead is sufficiently small that it enables the enzymatic signal development from even extremely small concentrations of detection enzyme to increase to the point that positive wells can be differentiated from negative wells. Each bead in a fluorescent well is designated as active, and the percentage of active beads is calculated relative to the overall bead count, as determined by light microscopy.
Patent Cooperation Treaty (“PCT”) published patent application WO 2007/098148 A2 entitled “Methods and Arrays for Target Analyte Detection and Determination of Target Analyte Concentration in Solution” discloses both arrays of single molecules and methods of producing an array of single molecules for defined volumes between 10 attoliters and 50 picoliters. The disclosed method enables detecting and quantitating single molecules for biomolecules such as enzymes for discovering function, detecting binding partners or inhibitors, and/or measuring rate constants. The digital counting, with clear differentiation of 0 vs. 1 or more protein, greatly enhances the sensitivity of protein detection by reducing the noise barrier of a conventional ELISA. Unfortunately, the finite number of micro wells in the digital ELISA limits the dynamic range of the assay. In addition, the enzyme concentration in the assay must be carefully balanced in order to minimize background noise and prevent signal saturation.
One major issue for all ELISA-based methods is that the reagents have to be supplied in excess to the target-analyte concentrations in order to produce consistent labeling. For this reason, a wash process must be used after each labeling step to remove the excess, yet ubiquitous, unbound reagents and, thus, minimize the resulting interference to the final measurements. Washing steps not only consume time and resources, but they also change the equilibrium established between bound and free reagents, which could result in bias and the loss of labeled analytes. In addition, enzymatic amplification may result in variable signal outputs for a variety of reasons, including differences in the enzyme kinetics, nonspecific activity, the precise reagent quality or quantity, lot-to-lot variation, and age-related loss of function.
Therefore, there exists a need in the art for improved analyte quantification, with increased accuracy and sensitivity, greater dynamic range, and a simplified workflow.
The present disclosure provides systems and methods to improve the analysis and quantification of biological analytes having improved accuracy and sensitivity, greater dynamic range, and a simplified work flow.
In the primary embodiment of this disclosure, a system and a method are proposed for detecting and enumerating one type of target analytes in a sample at the single-analyte level. The system consists of two distinguished groups of particles: capture particles and detection particles. The capture particles are conjugated with an analyte-specific reagent that has a specific affinity to a certain binding site or epitope of the target analyte. The detection particles are conjugated with another analyte-specific reagent that has a specific affinity to a secondary binding site or epitope of the same target analyte. When a sample containing the target analyte is mixed with the capture and detection particles, analyte-linked particle complexes may form. If the concentration of the target analyte in the sample mixture is significantly lower than the concentration of the capture and detection particles, then the complexes will be mostly particle doublets, consisting of a single capture particle linked to a single detection particle through a single target analyte. By analyzing the sample mixture in a manner that enables the discrete detection, differentiation and enumeration of the analyte-linked particle doublets versus the non-analyte-bound, henceforth referred to as unbound, singlet particles, the concentration of the target analyte in the sample may be accurately determined.
At higher target-analyte concentrations, higher-order particle complexes containing multiple analytes may form. By analyzing the sample mixture in a manner that enables the discrete detection, differentiation and enumeration of all analyte-linked particle multiplets versus unbound singlet particles, the concentration of the target analyte in the sample may again be accurately determined.
Note that the analyte specificity of the reagents discussed in this disclosure should not be taken literally. The capture particles in this disclosure may be conjugated to a collection of reagents, C, and the detection particles conjugated to another collection of reagents, D, with a portion of C targeting a group of analytes, A, and a portion of D targeting either A or a subgroup of A. It should also be noted here that C and D may be identical, or partially overlapping, or totally different.
In the preceding embodiment, as well as in subsequent embodiments, of this disclosure, it should be apparent to those of ordinary skill in the art that the particular nomenclature for which a particle is referenced, such as capture or detection particle, is used to simplify this disclosure's explanation. In fact, both types of particles can be inter-changed in both form and identity, and the system can instead be considered to comprise two or more groups of particles (e.g., Group 1, Group 2, etc.) that are capable of forming analyte-linked particle complexes that are discretely distinguishable from unbound singlet particles.
In another embodiment of this disclosure, a system and a method are proposed for multiplexed assays to simultaneously detect and enumerate multiple types of target analytes in a sample at the single-analyte level. The system comprises two distinct groups of particles: capture particles and detection particles, with the capture particles further divided into subgroups, wherein each subgroup of capture particles is uniquely labeled with certain physical characteristics. The capture particles are each conjugated with an analyte-specific reagent that has a specific affinity to a certain binding site or epitope of a particular type of target analyte. The detection particles are each conjugated with one of a different set of analyte-specific reagents that has a specific affinity to a secondary binding site or epitope of their respective type of target analyte. When a sample containing target analytes is mixed with the capture and detection particles, multiple groups of analyte-linked particle complexes may form, each associated with one type of analyte and the corresponding detection particles and subgroup of capture particles. By differentiating and grouping according to the capture-particle labels, multiple types of analytes in a sample can be simultaneously analyzed, with each type of analyte analyzed in the same way as proposed in the primary embodiment of this disclosure.
For those of ordinary skill in the art, it should be apparent that the multiplexed-assay embodiment of this disclosure may also be implemented by differentially labeling the detection particles or both the capture and detection particles.
In a related embodiment of this disclosure, the previously proposed multiplexed-assay embodiment may be combined with one or more conventional analog assays. For example, one subgroup of analyte-specific detection particles—corresponding to one subgroup of labeled capture particles—may be replaced by analyte-specific molecular probes, such as the analyte-specific detection reagents directly conjugated with fluorescent molecules. As a result, the concentration of the corresponding analyte will be extracted from the mean fluorescence intensity of the analyte-linked particle-and-molecular-probe sandwiches, instead of from the enumeration of analyte-linked particle complexes. One possible application of such a combined multiplexed assay is to simultaneously measure, in one sample, low-abundance analytes using analyte-linked particle complexes, and high-abundance analytes using analyte-linked particle-and-molecular-probe sandwiches. It should be apparent to those of ordinary skill in the art that this example can be extended to assays that combine multiple types of particle-conjugated and molecular detection reagents.
In yet another embodiment of this disclosure, a system and a method studies analyte-analyte interactions in a sample at the single-analyte level. The system consists of two distinct groups of particles: capture particles and detection particles. The capture particles are conjugated with an analyte-specific reagent that has a specific affinity to a certain binding site or epitope of a primary target analyte. The detection particles are conjugated with an analyte-specific reagent that has a specific affinity to a certain binding site or epitope of a secondary target analyte. When a sample containing the two analytes is mixed with the capture and detection particles, analyte-linked particle complexes may form due to interaction between the two different target analytes. The analyte complex may, therefore, be analyzed in the same way as in the primary embodiment of this disclosure. In some embodiments of this disclosure, another group of detection particles specific to the primary target analyte may also be introduced to simultaneously measure its concentration, and consequently the fractional occupancy. In yet other embodiments of this disclosure, one or more of the target analytes may be directly or indirectly bound or conjugated to the capture and/or detection particles without intermediating capture and/or detection reagents, or the secondary target analyte may actually be a capture and/or detection reagent. It should be apparent to those of ordinary skill in the art that the analyte-analyte-interaction-assay embodiment of this disclosure may also be multiplexed, as discussed in the previous multiplexed-assay embodiments of this disclosure.
While the proposed embodiments of this disclosure focus on measuring the concentration of analytes or analyte complexes through the discrete detection and enumeration of analyte-linked particle complexes, it should be apparent to those of ordinary skill in the art that they can be easily implemented as kinetics and/or dynamics assays to study analyte-reagent or analyte-analyte interactions, including changes due to the introduction of a modulating agent, such as drugs, pharmaceuticals, proteins, kinases, transcription factors, peptides, sugars, oligosaccharides, polysaccharides, nucleic acids, lipids, detergents, hormones, growth factors, cytokines, chemokines, activators, inhibitors, small-molecule activators, small-molecule inhibitors, other modulators, and/or combinations or complexes thereof. Such assays may be used for optimizing pharmaceutical development, determining drug efficacies and selection, elucidating on-target versus off-target responses, identifying effective concentrations in different experimental and physiological conditions, elucidating pharmacodynamics, mapping signaling and metabolic pathways, optimizing antibody manufacturing, and many other research and/or development applications.
The capture and detection particles, as well as the analyte-linked particle complexes discussed in the various embodiments of this disclosure, may be distinctively characterized using any particle-counting techniques that leverage their distinguishable size, mass, chemical, optical, electrical, magnetic, radioisotopic, and/or biological properties. For example, particles with unique optical properties, such as light scattering, absorption and/or fluorescence, may be distinctively counted using multi-parameter particle counters, such as flow cytometers as described by Howard M. Shapiro in Practical Flow Cytometry, 4th ed. (Wiley, 2003), or imaging or laser-scanning microscopes. Particles with unique sizes may be distinctively counted using impedance-based particle counters. Alternatively, any measurement technique may be utilized that enables the extraction, from a sample, of the distinctive counts of the capture and detection particles, as well as the analyte-linked particle complexes described in the various embodiments of this disclosure.
In this disclosure's various embodiments, analyte refers to any test molecule or particle of interest, including, but not limited to: proteins, kinases, transcription factors, antibodies, receptors, peptides, cytokines, chemokines, sugars, oligosaccharides, polysaccharides, DNA, RNA, oligonucleotides, polynucleotides, nucleotide complexes, single-stranded nucleic acid sequences, double-stranded nucleic acid sequences, natural polymers, synthetic polymers, lipids, detergents, hormones, growth factors, micelles, liposomes, lipoproteins, extracellular vesicles, exosomes, oncosomes, viruses, virus-like particles, cells, cell fragments, natural particles, synthetic particles, synthetic compounds, plant-derived compounds, animal-derived compounds, chemicals, drugs, pharmaceuticals, activators, inhibitors, small-molecule activators, small-molecule inhibitors, modulators, and/or combinations or complexes thereof. In some embodiments of this disclosure, the analytes may be targeted to bind to the capture and/or detection particles by cognate capture and/or detection reagents that are conjugated to the particle surface, while in other embodiments the analytes may be directly or indirectly bound or conjugated to the capture and/or detection particles without using intermediating capture and/or detection reagents, such as by use of covalent bonding or affinity tags. The various embodiments of this disclosure are not limited to any particular analyte.
In this disclosure's embodiments, the capture and detection reagents may be anything that binds to a site or epitope of the target analytes, including, but not limited to: antibodies, binding proteins, peptides, polypeptides, protein complexes, sugars, oligosaccharides, polysaccharides, DNA, RNA, oligonucleotides, polynucleotides, nucleotide complexes, single-stranded nucleic acid sequences, double-stranded nucleic acid sequences, aptamers, natural polymers, synthetic polymers, pharmaceuticals, drugs, lipids, detergents, micelles, liposomes, lipoproteins, extracellular vesicles, exosomes, oncosomes, viruses, virus-like particles, cells, cell fragments, chemicals, etc. In some embodiments of this disclosure, one or more of the capture and/or detection reagents may function as target analytes, particularly in analyte-reagent- and/or analyte-analyte-interaction assays. In some embodiments of this disclosure, the binding may be specific, and in other embodiments the binding may be intentionally nonspecific. In some embodiments of this disclosure, the capture and/or detection reagents may be directly conjugated to the capture and/or detection particles, while in other embodiments the capture and/or detection reagents may be indirectly conjugated to the capture and/or detection particles, such as by use of affinity tags. The various embodiments of this disclosure are not intended to be limited to any particular capture or detection reagent.
In this disclosure's embodiments, affinity tags refer to any molecule or element with affinity to, or that can be identified and more generally targeted by, a second molecule or binding partner, and can be used to bring two components together into a complex when differentially conjugated to the pair of components. Such affinity tags include, but are not limited to: biotin, streptavidin, avidin, neutravidin, hemagglutinin, poly-histidine, maltose-binding protein, myc, glutathione-s-transferase, FLAG, protein A, protein G, protein L, DNA, RNA, oligonucleotides, polynucleotides, single-stranded nucleic acid sequences, double-stranded nucleic acid sequences, etc. In other embodiments of this disclosure, if there is a difference in species origin for a particular pair of capture and/or detection reagents, or some general difference that allows for differentiation between the reagents, for example mouse vs. rabbit antibodies, IgG vs. IgM, IgG1 vs. IgG3, etc., then these differences may also be targeted by species-, class- or isotype-specific targeting reagents, such as an anti-mouse-IgG3-specific antibody, conjugated directly or indirectly to the capture and/or detection particles, functioning similar to affinity tags. In this context, and for the embodiments of this disclosure, species-, class- and isotype-specific targeting reagents and/or antibodies constitute affinity tags and/or reagents. In some embodiments of this disclosure, affinity tags may be used to conjugate the capture and/or detection reagents to the capture particles, the detection particles, both particles, or neither particle. In other embodiments of this disclosure, affinity tags may be used to specifically or non-specifically bind the analyte directly to the capture and/or detection particles, in which scenario a particular capture and/or detection reagent may not be necessary. The various embodiments of this disclosure are not limited to any particular affinity tag or reagent.
Finally, in these embodiments of this disclosure, the capture and detection particles may be composed of any inorganic, organic or biological material, or composite of materials, including, but not limited to: polystyrene, silica, glass, metals, magnets, proteins, peptides, polypeptides, protein complexes, sugars, oligosaccharides, polysaccharides, DNA, RNA, oligonucleotides, polynucleotides, nucleotide complexes, single-stranded nucleic acid sequences, double-stranded nucleic acid sequences, aptamers, natural polymers, synthetic polymers, lipids, detergents, micelles, liposomes, lipoproteins, extracellular vesicles, exosomes, oncosomes, viruses, virus-like particles, cells, cell fragments, etc. The capture and detection particles may also be labeled with certain unique physical, chemical and/or biological characteristics. Furthermore, the capture and detection particles may be of any size, shape, or material uniformity, as long as they can be discretely detected and enumerated. In some embodiments of this disclosure, the particles will be 5 nm to 100 μm in diameter. In other embodiments of this disclosure, the particles will be 2 nm to 10 μm in diameter. In yet other embodiments of this disclosure, the particles will be 5 nm to 2 μm in diameter. In the preferred embodiment of this disclosure, the particles will be 10 nm to 1 μm in diameter.
This disclosure presents systems and methods for the discrete detection and quantification of individual analytes in a sample. More specifically, this disclosure employs binding two particles to target analytes, i.e. a primary analyte-specific capture particle and a secondary analyte-specific detection particle thereby forming analyte-linked particle complexes. In some embodiments of this disclosure, one type of analyte may be targeted by one set of capture and detection particles, while in other embodiments multiple types of analytes may be simultaneously targeted using different subgroups of capture and/or detection particles, each uniquely labeled with certain distinguishable physical characteristics.
In one embodiment of this disclosure, individual target analytes are first bound by a capture particle, and then subsequently bound by a detection particle. In other embodiments of this disclosure the capture and detection particles are added to the sample simultaneously, while in yet other embodiments the sample may be added to a solution containing capture and detection particles.
It will be understood by those skilled in the art that no specific sequence or order in mixing a sample with the particles is critical to this disclosure. Analysis of a sample mixture in a manner that enables the discrete detection, differentiation and enumeration of the analyte-linked particle complexes versus unbound singlet particles, together with differentiating any particle subgroups, can then be used to accurately determine the concentration of one or more types of target analytes in a sample.
In the preferred embodiment of this disclosure, the particles and particle complexes will be analyzed by a multi-parameter particle counter, such as a flow cytometer, or an imaging or laser-scanning microscope. Analyte-linked particle doublets represent the discrete detection of a single analyte, which can be directly counted to give the total number of analytes per volume analyzed. Higher-order particle multiplets discretely shift the fluorescence, light-scatter, and/or other optical signature to greater intensities, representing the discrete inclusion of additional analytes. The total number of analytes counted per volume analyzed can, therefore, be derived from the summation of the number of each type of particle complex multiplied by the corresponding number of analytes contained in each complex. In other words, if Na is the total number of analytes counted per volume analyzed, and Na is the number of counted particle complexes containing m analytes, then
Furthermore, with sufficient analyte-binding affinity and an excess of capture and detection particles, Na can be directly converted to the analyte molar concentration. At non-equilibrium conditions, or when using capture and/or detection reagents with lower analyte-binding affinities, Na may be compared with a standard curve to determine the analyte molar concentration.
In the primary embodiment of this disclosure, the system and method pertain to the discrete detection and quantification of one type of analyte within a liquid sample matrix. As shown in
A representative capture or detection reagent for protein analytes would be an analyte-specific antibody, which has the characteristic Y-shape depicted in multiple figures of this disclosure. However, the disclosed system is not limited to any particular type of capture or detection reagent. Fundamentally, the capture or detection reagents can be any molecules or entities that bind to the target analytes, specifically or nonspecifically. The capture and detection reagents would preferably bind to the analytes at two different binding sites, shown as 108 and 109 in
In this disclosure's figures, the capture 102 and detection particles 103 are each depicted with only one or several capture reagents 105 or detection reagents 107 conjugated to their surface in order to simplify an explanation of the particular embodiment of this disclosure. However, in reality, there could be an unlimited number of capture or detection reagents conjugated to each particle without a particular maximum or minimum number of capture or detection reagents. The precise number of capture or detection reagents will depend on
While it may be theoretically possible to conjugate particles with only one copy of a particular reagent, this would actually impose a significant limitation on their functionality in most assays, as it would significantly decrease the probability that an analyte would appropriately contact a capture or detection particle at the location of a capture or detection reagent during any particular collision that occurs between the analytes and the particles in a sample mixture during any interval of time. In contrast, a higher reagent density on the particles means a higher local reagent concentration, which will favor the analyte-reagent bond formation in an equilibrium reaction comparable to a molecular reagent of equivalent concentration that is uniformly distributed in bulk.
While the primary embodiment of this disclosure describes the formation of analyte-linked particle doublets, each comprising a pair of capture and detection particles bonded together by a single analyte,
Another embodiment of this disclosure is a multiplexed assay simultaneously targeting multiple different analytes in a complex sample mixture. As depicted in
It should be apparent to those skilled in the art that
Another embodiment of this disclosure is directed toward studying analyte-analyte interactions.
Further, using a variation of the multiplexed assays proposed in the previous embodiments of this disclosure, detection particles labeled with different physical characteristics may be introduced to target the primary analyte 101-1 in order to simultaneously measure its concentration. Such a multiplexed assay would enable the accurate measurement of the fractional occupancy of the secondary analyte 101-2 bound to the primary analyte 101-1 at the single-analyte level. In some cases, the binding of multiple capture and/or detection particles to an analyte complex may be used to identify the presence of multiple copies of the same analyte within the complex.
It should be apparent to those skilled in the art that the proposed embodiment of this disclosure can be used to study, at the single-analyte level, the binding affinities and/or kinetics of two or more analytes that bind together and form analyte complexes, for example, due to the introduction of particular pharmaceuticals, drugs, or other association-modulating molecules or particles of interest. Such assays may be used to optimize pharmaceutical development, determine drug efficacies and selection, elucidate on-target versus off-target responses, identify effective concentrations in different experimental and physiological conditions, elucidate pharmacodynamics, map signaling and metabolic pathways, optimize antibody manufacturing, and many other research and/or development applications. The preceding examples are merely illustrative and not exhaustive, and this disclosure is not limited to any particular format of analyte-complex detection or analysis, any type of target analyte, or any particular number of different target analytes that are concurrently analyzed.
The unbound particles and analyte-linked particle complexes prepared using the systems and methods of this disclosure can be analyzed in any manner that enables the discrete detection, differentiation and enumeration of particle complexes versus singlet particles. In some embodiments of this disclosure, these methods may include, but are not limited to: imaging or laser-scanning microscopy, resistive-pulse sensing, and flow cytometry. In other embodiments of this disclosure, the combination of signals and/or differences in physical properties produced by the proximity of the capture and detection particles in analyte-linked particle complexes can allow for bulk analyses. For example, the size difference between unbound particles and the various analyte-linked particle complexes may be differentiated using centrifuges or size-selective filters, or even dynamic light scattering or nanoparticle tracking analysis. Energy transfer between the capture and detection particles in analyte-linked particle complexes may be interrogated using spectroscopic methods. The preceding list is certainly not exhaustive. Bulk analyses, however, will not provide the precision and accuracy of particle-by-particle counting and analyses. In the preferred embodiment of this disclosure, the particles and analyte-linked particle complexes will be discretely detected, differentiated and enumerated using a multi-parameter particle counter similar to a flow cytometer, or an imaging or laser-scanning microscope.
Furthermore, forming analyte-linked capture-and-detection-particle complexes causes a discrete shift in the light-scatter intensity, dependent on the number of particles bound.
Note that the illustrations appearing in
If both the capture and detection particles are fluorescently labeled, and are used at similar concentrations, rather than one being used in excess of the other, then the resulting analyte-linked particle complexes may produce a fractal pattern in the two fluorescent dimensions that are used for labeling.
Because all of the populations are discretely resolved, each population can be directly enumerated, which can then be used to calculate the analyte concentration either mathematically, statistically, or by comparison to a standard curve. Furthermore, the natural generation of multiple data points at each concentration by this disclosure's technique, unlike many conventional assays that generate only one data point at a given analyte concentration, enables more accurate measurements.
An empirical example of the gating to enumerate the various populations can be seen in
Similar data analyses may be applied to multiplexed assays performed in accordance with this disclosure.
The system described in the preceding exemplary embodiment is not limited to any particular number or variety of fluorophores or spectral labels. In some embodiments of this disclosure, more than two fluorescence channels may be used for analyte labels. In other embodiments, a variety of fluorescence, scatter and/or other optical or spectral properties may be used as analyte labels. Further, the detection particles may also be fluorescently labeled, for example, using colors different from the color of light produced by the capture-particle labels.
The ability to discretely count analyte-linked particle complexes and unbound particles is a major characteristic that differentiates this disclosure from conventional techniques that ultimately rely on analog signal analyses.
Furthermore, as depicted in
Because data gathered using an assay in accordance with disclosure produce many discrete data points, each with their own characteristic population distributions and aggregation behaviors, these data points provide additional characteristics for determining analyte concentrations, and improve the accuracy and reliability of the assay. For example, one common problem exhibited by immunoassays that generate only one data point at a given analyte concentration is that upon reaching a saturation point for the assay they exhibit a hook or prozone effect, where the addition of further analyte inhibits and actually reduces proper antibody binding or complex formation. This leads to uncertainty as to whether any individual result is located on the increasing or decreasing side of the assay signal vs. concentration curve, and requires either performing an additional step after the assay is complete, where more sample is added and it is then reanalyzed, or examining additional dilution points to determine if the signal increases or decreases for the additional data point(s). As demonstrated in
Unlike conventional assays based on analog signals and molecular reagents, by using the techniques appearing in this disclosure, each analyte-linked capture-and-detection-particle doublet that is enumerated represents a single analyte or analyte complex. Thus, the dynamic range of an assay of this disclosure is directly proportional to the total number of particles counted during an experiment. For example, if 106 particles are analyzed, then the nominal dynamic range would be 6 decades. This would only take minutes to acquire on a basic multi-parameter particle counter, such as a flow cytometer, or an imaging or laser-scanning microscope. In practice, when higher-order particle complexes are taken into consideration, the range could be extended by another decade or more even with the same number of counted particles. Furthermore, since the dynamic range is proportional to the total number of events counted, it could be extended even further by extending the sample acquisition time. For example, acquiring the sample for 10 minutes rather than 1 minute would proportionally increase the dynamic range by another decade. As discussed in the previous embodiments of this disclosure, the practical dynamic range described in this disclosure may be further expanded using a multiplexed assay that combines particle-conjugated and molecular detection reagents in order to respectively measure low- and high-abundance analytes.
Although the preceding disclosure has been made in terms of various embodiments, it is to be understood that such disclosure is purely illustrative and is not to be interpreted as limiting. Although several exemplary embodiments of this disclosure have been described in some detail, in light of the above teaching, it will be apparent to those skilled in the art that many modifications and variations of the described embodiments are possible without departing from the principles and concepts of the disclosures as set forth in the claims. Consequently, without departing from the spirit and scope of the disclosure, various alterations, modifications, and/or alternative applications of the disclosure will, no doubt, be suggested to those skilled in the art after having read the preceding disclosure. Accordingly, it is intended that the following claims be interpreted as encompassing all alterations, modifications, or alternative applications as fall within the true spirit and scope of the disclosure.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/057794 | 10/28/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62926679 | Oct 2019 | US |
