Patent Application
20160231324
This invention generally relates to bioanalysis, and detection and screening methodologies. In particular, in alternative embodiments, the invention provides high throughput, multiplexed systems or methods for detecting a biological, a physiological or a pathological marker, or a single molecule or a single cell using a droplet microfluidics system, or an emulsifier, integrated with use of a sensor or a sensing system, an aptamer, or a DNAzyme. In alternative embodiments, the sensor or sensing system comprises a nucleic acid based, an antibody based, an enzyme based or a chemical based sensor or sensing system. In alternative embodiments, the invention provides methods for detecting a biological, a physiological or a pathological marker, or a single molecule or a single cell using a droplet or emulsion system integrated with rapid and sensitive fluorescence detection systems including, in particular, a 3D Particle Detector. In alternative embodiments, the invention presents methods for high throughput screening of small molecules and biomolecules, including aptamers, such as oligonucleic acid and peptide aptamers, and related, e.g., aptamer-based, sensors and therapeutics.
Recent advances in genomics, proteomics, cellomics and metabolomics have provided us with large libraries of biological and chemical compounds that modulate various biological processes. Such developments have necessitated the need for high throughput analysis/screening where millions of biochemical, genetic or pharmacological assays are performed and analyzed in a parallel fashion to find active compounds against biological targets. In addition, the analysis, detection, identification and quantification of these markers provide powerful new means to study biology and pathology and to develop new diagnostics and therapeutics.
Many biological and disease markers, such as e.g., molecules and cells such as cancer cells, exist at low concentrations in biological samples, yet play important roles in biological and pathological processes. The ability to rapidly and selectively detect low abundance is critically important to elucidate new biology, to monitor, detect a disease or disorder, and to monitor therapeutic responses and to develop new therapeutics.
Early identification, screening and monitoring of cancer, Alzheimer's Disease (AD) and other diseases and conditions, e.g., before a person has any symptoms, has proven to be a powerful and often necessary step to effectively prevent, treat and eradicate the disease. Traditional imaging tools (e.g., computed tomography (CT) scans and magnetic resonance imaging (MRI)) and biopsy analysis are unfortunately too complicated, expensive and/or invasive for routine disease screening; most importantly, they typically do not possess the sensitivity and specificity to identify the diseases at the early stage. Therefore, recent effort has been focused on developing assays targeting specific molecular biomarkers (e.g., nucleic acids and proteins) and cellular markers (e.g., cancer cells) existed in biological samples (e.g., blood, urine, saliva, tear, and cerebrospinal fluid (CSF)) that distinguish disease from normal samples.
Unfortunately, discovering disease biomarkers and translating them into clinical assays has proven to be an enormous challenge. First, despite the advances in genomic and proteomic technologies (e.g., sequencing, mass spectrometry (MS), and bioinformatics) which are sophisticated and costly, very few reliable disease biomarkers have been discovered. These technologies are limited by their intrinsic, high false discovery rate and the fact that modest differences between normal and diseased samples and large heterogeneity of biomarkers in the diseased samples exist. It has widely been accepted that a single biomarker typically lacks the sensitivity and specificity that is necessary for useful diagnosis. Additionally, even once biomarkers are identified, the implementation and clinical assay development in the next phase is also time-consuming, expensive and sometimes infeasible. For instance, if one wants to develop an ELISA assay to detect prostate-specific antigen (PSA) as a biomarker for prostate cancer, the antibodies for PSA have to already exist with sufficient specificity and selectivity. This is particularly problematic when multiple biomarker assays are required.
Another important area that requires sensitive, rapid and high throughput biomarker identification and detection is infections by pathogens (e.g., bacteria such as tuberculosis (TB), viruses (e.g., HIV), and parasites such as malaria). For instance, bacterial infection is a major health problem and a major cause of sepsis, which annually affects over 18 million people worldwide and 700,000 in the U.S., with a mortality rate of 30-40%. Sepsis and other aggressive bacterial infections are managed within intensive care units with associated high costs, which impose significant healthcare, economic and social burdens. For instance, each septic patient in the US incurs costs of approximately US $25,000 during hospitalization, corresponding to $17 billion annually. In particular, antimicrobial resistance is a growing health problem in the United States and worldwide. According to the Centers for Disease Control and Prevention (CDC), more than two million people are infected annually with antibiotic-resistant infections, with greater than 23,000 deaths1. Aggressive bacterial infections associated with antimicrobial resistance are often managed within intensive care units (ICUs) with high associated costs, which impose significant healthcare, economic and social burdens. The Alliance for the Prudent Use of Antibiotics (APUA) estimates the antibiotic-resistant infections cost the US healthcare system over $20 billion each year.
The high mortality of blood infections is associated with the ineffectiveness and time-consuming process of bacteria diagnosis and treatment. It is widely recognized that effective detection and routine monitoring of infectious bacteria in patients to diagnose diseases at an early-stage have a profound effect on survival rates. Unfortunately, blood culture, the gold standard for identification of bacteria in blood, takes days to obtain results. New molecular diagnosis methods, such as polymerase chain reaction (PCR), can reduce the assay time to hours but are often not sensitive enough to detect bacteria that occur at low concentrations in blood (1-100 colony-forming unit (CFU)/mL). Importantly, PCR-based methods require sample processing, such as lysis and isolation of nucleic acids, for the amplification reaction. Moreover, all these techniques are sophisticated and expensive, and therefore not suited for routine monitoring of bacteria in patients. Therefore, simple methods are urgently needed for rapid and sensitive identification of bacteria in blood, which will significantly reduce the mortality rate and the cost of medical care associated with blood infections.
Microfluidic systems have recently emerged as a promising platform for performing a diverse range of experiments for biological and chemical applications. Microfluidic-based methods have several advantages compared to conventional high throughput screening methods. These include negligible evaporation of reagents, minimal consumption of expensive biological reagents, low fabrication costs, reduced analysis time and the ability to integrate various functional components on a single chip.
In particular, the developments of droplet based microfluidic systems present a promising opportunity for high-throughput biological analysis. In these systems, microdroplets containing nano- to picolitre volumes can be generated at kilohertz frequencies and each droplet serves as a ‘test tube’ for reactions. Because of the small volume of each droplet, reactions between bio-molecules such as protein-protein interaction or DNA hybridization and cell-drug or cell-cell interactions can be performed using 109 times smaller amounts than conventional biological methods such as 96 microwell plate based Enzyme-linked immunosorbent assay (ELISA). In addition, droplet confinement of targets e.g., cells and its immediate environment into a small volume allows us to analyze secreted markers and use them as “markers” for single cell detection and sorting. By contrast, existing techniques, e.g., ELISA, typically measure secreted proteins in bulk and therefore miss key dynamic information at a single cell level. Fluorescence activated cell sorting (FACS) typically rely on cell surface and intracellular markers, rather than secreted markers, for cell sorting. Furthermore, droplet based microfluidic systems have additional advantages compared to continuous microfluidic systems such as reducing the reagent interaction with channel walls and inhibiting dispersion of samples by compartmentalization. In addition, it allows independent control of each droplet including droplet generation, coalescence, sorting, incubation and analysis in a short period of time.
In alternative embodiments, the invention provides high throughput, multiplexed systems or devices, or methods, for detecting, identifying and/or quantifying a target; a target molecule; a virus; a biological, a physiological or a pathological marker; a single molecule; or a single cell or cell-derived particle, e.g., a single pathogen, parasite, bacterial cell, virus or fungus, using a droplet or emulsion-based microfluidics system, a 3D particle detector and/or a 3D particle counting system, integrated with use of an assay, a sensor or a sensing system comprising use of: a small molecule, a biomolecule, an aptamer, a DNAzyme, a nucleic acid, a protein, a peptide, an enzyme, an antibody, or a chemical or small molecule, comprising:
(a) providing an assay, a sensor, a detecting or a sensing system capable of specifically binding to or detecting directly or indirectly a target, a target molecule, a nucleic acid, a protein, a peptide, a virus (e.g., a lentivirus such as HIV, or Ebola virus disease (EVD)), a cell-derived particle or a cell, wherein optionally the cell is a bacterial cell (optionally a slowly-growing organism such as Mycobacterium tuberculosis), a parasite cell or a fungal cell, or optionally the cell is a mammalian cell or a human cell;
wherein optionally the assay, sensor, detecting or sensing system comprises or comprises use of: an aptamer, a DNAzyme (also called a deoxyribozyme, a DNA enzyme or a catalytic DNA), a nucleic acid, a protein, a peptide, an enzyme, an antibody, a chemical or small molecule, a single nucleic acid molecule amplification optionally comprising an EXPonential Amplification Reaction (EXPAR), a Rolling Circle Amplification (RCA), an aptamer Inhibitor-DNA-Enzyme (IDE), or an aptamer-IDE system,
and optionally the target comprises an amplified target, which optionally is a nucleic acid target amplified using Rolling Circle Amplification (RCA) or EXPAR,
wherein the specific binding to, or the direct or indirect detecting of, the target molecule, virus, cell-derived particle or cell, by the assay, sensor, detecting or sensing system results in, or generates, a detectable signal, which optionally comprises a fluorophore signal or a fluorescence,
wherein optionally the nucleic acid, aptamer, aptamer-IDE system, or DNAzyme comprises a RNA-cleaving DNA motif that can cleave a DNA-RNA chimeric substrate at a single ribonucleotide junction, and the ribonucleotide cleavage site is flanked by a fluorophore and a quencher, and optionally binding of the nucleic acid, aptamer, or DNAzyme to its target molecule, virus, cell-derived particle or cell causes cleavage of a ribonucleotide cleavage site to release the quencher from the fluorophore or a fluorescence activator, wherein the fluorescence activator optionally comprises an enzyme capable of when in active form generating a detectable signal such as a fluorophore signal,
and optionally the sensor or sensing system, aptamer, a DNAzyme, an aptamer inhibitor-DNA-enzyme (IDE) molecular complex (also called an aptamer-IDE system), which optionally comprises a structure as set forth in
and optionally the assay, sensor, detecting or sensing system comprises a nucleic acid based, an antibody based, a protein based, a peptide based, an enzyme based or a chemical or small molecule-based assay, sensor, detecting or sensing system, or any combination thereof,
wherein optionally the specific binding of the assay, sensor, detecting or sensing system, to the target triggers an amplification-based or non-amplification-based fluorescence signal,
and optionally the target molecule (optionally a purified or complex target) can be screened, selected and/or isolated from a nucleic acid, peptide or chemical library,
and optionally the target molecule comprises a nucleic acid or a polypeptide, optionally the polypeptide is a diagnostic for a disease or condition, or is a cell surface marker, or is an enzyme, wherein optionally the enzyme is a marker for the detection of a particular disease or is a marker, optionally the enzyme is a beta-lactamase, such as a carbapenemase, optionally for the detection of extended spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae and carbapenem-resistant Enterobacteriaceae (CRE), TB and other antimicrobial resistant pathogens,
and optionally the target molecule, virus, cell-derived particle or cell or bacteria, parasite or fungus, comprises one or a plurality of biological, physiological or pathological markers, or comprises a single or a plurality of molecules or a single cell or a plurality of cells, or a single or a plurality of virus or a cell-derived particles or molecules;
(b) optionally providing a plurality of droplets or microdroplets, or emulsions,
wherein optionally the droplets or microdroplets, or emulsions, are generated by a droplet microfluidics system or a microdroplet-manipulating assay or device, or an emulsifier, or an equivalent device or system,
and optionally droplet size can range from between about 5 to 50 μm in diameter, between about 1 μm to 300 μm, or between about 10 μm to 100 μm,
and optionally providing labels or stains, wherein optionally the target or the amplified target are stained or labeled, optionally with a dye, a nanoparticle, a bead, or an equivalent or combination thereof,
and optionally providing a plurality of particles or nanoparticles, wherein the target consists of, comprises or is contained in the particles or nanoparticles;
(c) providing a sample, wherein optionally the sample comprises or is derived from a biological or an environmental sample,
and optionally the sample comprises the target, or is suspected of comprising the target to be detected,
and optionally the target is or comprises a target molecule, a nucleic acid, a protein, a peptide, a virus, a cell-derived particle or a cell, wherein optionally the cell is a bacterial cell, a parasite cell or a fungal cell, or optionally the cell is a mammalian cell or a human cell;
(d) optionally encapsulating or microencapsulating the sample (comprising or consisting of the target), optionally together with the assay, sensor, detecting or sensing system,
and optionally associating, encasing, or binding the target or the sample with or within the plurality of particles or nanoparticles,
wherein optionally the encapsulating or microencapsulating comprises encapsulating or microencapsulating into a plurality of droplets or microdroplets, or emulsions,
and optionally the target-detecting or sensing system comprises an aptamer-IDE system, and optionally when the aptamer-IDE system comprises use of an enzyme, or a combination of enzymes, that can generate a detectable signal, such as a fluorescent signal, by interacting or processing the detectable signal, the encapsulating or microencapsulating further comprises encapsulating or microencapsulating a substrate or a detectable signal activated by the enzyme,
and optionally processing or making the encapsulated or microencapsulated sample or target, or processing or making the droplets or microdroplets, or emulsions, comprising the encapsulated or microencapsulated sample, comprises use of a droplet microfluidics system or microdroplet-manipulating device, or a high-throughput droplet generator, optionally a 256 channel cartridge system, or an emulsifier,
and optionally labeling or staining the target or the amplified target, optionally with a dye, a nanoparticle, a bead, or an equivalent or combination thereof, and
(e) detecting the presence of a detectable signal, which optionally comprises a fluorophore signal or a fluorescence, or a dye, a nanoparticle, a bead, or an equivalent or combination thereof,
wherein optionally the detecting, identifying and/or quantifying of the presence of a detectable signal is in each encapsulated or microencapsulated sample, or in each droplet or microdroplet, or emulsion, or is in each particle or nanoparticle,
and optionally detecting the presence of a detectable signal detects, identifies and/or quantifies the target molecule, virus, cell-derived particle or cell, wherein optionally the cell is a mammalian cell, a human cell, a bacterial cell, a parasite cell, a fungal cell,
wherein the detection of a fluorophore signal or fluorescence, which optionally is in an encapsulated or microencapsulated sample, or a droplet or microdroplet, or an emulsion, or is in each particle or nanoparticle, indicates the presence of the target molecule, virus, cell-derived particle, cell, parasite, fungus or mammalian or human cell in the sample,
and optionally the detecting and/or quantifying the target molecule, a virus or a cell-derived particle or a cell comprises use of a 3D particle detector or a 3D particle counting system.
In alternative embodiments, the target detected is encapsulated (or microencapsulated) within a droplet or microdroplet or an emulsion, or is associated with or within a particle or a nanoparticle, or alternatively, the target (which can be, for example, in addition to a droplet or microdroplet, a bead, a nanoparticle, an amplified nucleic acid, an inhibitor-DNA-enzyme (IDE) molecular complex, and equivalents) is/are directly detected and/or counted by the 3D particle detector, 3D particle counting system, or equivalent system; e.g., as illustrated in
In alternative embodiments, the cell is a mammalian cell, a human cell, a circulating tumor cell, a circulating melanoma cell, or a bacterial cell.
In alternative embodiments, the droplet microfluidics system, or emulsifier, can generate: (a) picoliter droplets or droplets of between about 1 μm to 300 μm, or between about 10 μm to 100 μm, in diameter; and/or (b) monodisperse, picoliter-sized liquid droplets in an immiscible carrier oil fluid.
In alternative embodiments, the biological sample comprises a biopsy, a blood, serum, saliva, tear, urine or a CSF sample from a patient, or a sample obtained from a food, water, soil, or an air source.
In alternative embodiments, the target molecule detected is or comprises a nucleic acid, a nucleic acid point mutation, or a single-nucleotide polymorphism (SNP), or a microRNA (miRNA) or a small inhibitory RNA (siRNA); or, the target molecule is a protein, a lipid, a carbohydrate, a polysaccharide, a small molecule or a metal complex.
In alternative embodiments, the target molecule is or comprises a polypeptide or a nucleic acid, a polypeptide or a nucleic acid point mutation, or a single-nucleotide polymorphism (SNP), a cell marker (a marker specific or identifying for a particular cell type, genotype or phenotype); or a nucleic acid disease (e.g., diabetes, Alzheimer's disease, and the like) or cancer marker, optionally a breast cancer biomarker,
and optionally detection of the target molecule is diagnostic for the disease (e.g., diabetes, Alzheimer's disease, and the like) or cancer (e.g., prostate, melanoma, breast cancer, optionally the target is prostate-specific antigen (PSA)), or is used for routine disease or cancer screening, early stage disease or cancer diagnosis and/or prognosis, for monitoring disease or cancer progression and/or recurrence, and/or for monitoring drug effectiveness and safety.
In alternative embodiments, the fluorophore comprises a fluorescein-dT and the quencher is a DABCYL-dT™ (Dabcyl-dT); and/or a fluorescence resonance energy transfer (FRET) dye pair; and/or a target-binding dye.
In alternative embodiments, the fluorescence is detected by an APD (photon avalanche diode), a PMT (photomultiplier tubes), a EMCCD (Electron Multiplying Charge Coupled Device), or a MCP (Microchannel plate) or other equivalent detector, optionally in a high throughput manner.
In alternative embodiments, the aptamer is an oligonucleotide, a nucleic acid or a peptide aptamer; or, the aptamer: specifically modulates stem cell differentiation into a particular lineage, or is directly coupled to a downstream signaling pathway.
In alternative embodiments, the aptamer binds to a target as agonist or antagonist or turns on a fluorescence signal as a sensor.
In alternative embodiments, the sensor comprises a DNA strand displacement strategy, or equivalents, as described e.g., in Li et al. (2013) J. Am. Chem. Soc. 2013, 135, 2443-2446; or a proximity ligation assay, or a binding induced DNA assembly assay, as described e.g., in Li et al. (2012) Angew. Chem., Int. Ed. 51, 9317; or Zhang (2012) Anal. Chem. 84:877.
In alternative embodiments, the sensor comprises a fluorogenic substrate or probe, or equivalents that binds to a target to produce fluorescence.
In alternative embodiments, the high throughput, multiplexed system or device, or method, of the invention further comprise detecting and/or quantifying the target, e.g., one or a plurality of biological, physiological or pathological markers, or a single molecule (as the target), or a single cell integration, comprising use of a 3D particle detector, a 3D particle counting system, or equivalent systems. In alternative embodiments, the target detected is encapsulated (or microencapsulated) within a droplet or microdroplet or an emulsion, or is associated with or within a particle or a nanoparticle, or alternatively, the target is directly detected and/or counted by the 3D particle detector, 3D particle counting system, or equivalent system. In alternative embodiments, the high throughput, multiplexed system or device, or method, of the invention comprise use of a DNA-bead or a DNA-bead droplet library or FACS based screening for molecules that bind to a target of interest, for example, a disease or a cancer cell, or a disease or a cell marker, e.g., a nucleic acid or a polypeptide, e.g., a membrane, marker.
In alternative embodiments, the high throughput, multiplexed system is engineered to comprise one or any of: desirable portability (for example, packaged as backpacks), automating fluid handing (i.e., droplet generation and auto sampling), and integrating electronics including a diode laser (light source), APD (detector), Operating (vinci, ISS Inc.) and/or data analyzing software (SimFCS), display, with a 3D particle counting system, e.g., as illustrated in
In alternative embodiments, the high throughput, multiplexed system or device, or method, of the invention further comprise disposable microfluidic “cartridges,” permitting multiplex and rapid detection of multiple types of targets simultaneously, and optionally the high throughput, multiplexed system or device is fully automated, or is fabricated as an all-in-one system or with modular components, or is linked to an electronic device, e.g., a portable device, e.g., a smart phone and/or a Bluetooth, for point-of-care applications, as illustrated in
In alternative embodiments of the high throughput, multiplexed system or device, or method, of the invention, the assay, sensor or sensor system comprises: a nucleic acid based assay; an antibody based assay; an enzyme based assay; a chemical based assay; a nucleic acid based assay; a hybridization; a molecular beacon; an aptamer; a DNAzyme; a real-time fluorescent sensor; an antibody-based assay; an ELISA; a sandwich based assay; an immunostaining assay; an antibody capture assay; a secondary antibody amplification assay; a proximity ligation based assay; an enzyme based assay comprising use of a PCR, RT-PCR, RCA, loop-mediated isothermal amplification (LAMP), nicking, strand displacement and/or an exponential isothermal amplification; or any combination thereof,
wherein optionally the high throughput, multiplexed system or device, or method detects low concentration targets without using droplets,
and optionally nucleic acid targets are detected using signal amplification processes, optionally Rolling Circle Amplification (RCA),which are then stained by dye probes or nanoparticles and measured, optionally by a 3D particle counter.
In alternative embodiments of the high throughput, multiplexed system or device, or method, of the invention, the encapsulated or microencapsulated emulsions or droplets are made by using an emulsifier or by droplet based microfluidics; or the emulsions or droplets comprise water-in-oil formulations, or the droplets comprise water-in-oil-in-water (W/O/W) double emulsion formulations, or the emulsions or droplets comprise liquid droplets, optionally comprising an agarose or a PEG, or optionally the droplets can be gelled or solidified to form droplet particles;
and optionally droplets comprise sizes ranging from between about 10 nm to 100 microns, optionally droplets are monodispersed or polydispersed, and optionally droplets are heated or cooled (e.g., for PCR), merged, split, sorted and/or prepared for long-term storage,
and optionally the emulsions or droplets, optionally fluorescent emulsions or droplets, that contain a target are sorted in a 3D particle counting system, optionally using an optical tweezer, an optical trap, an optical lattice, gradient centrifugation or any combination or an equivalent thereof This enables the sorted target(s) to be further processed and analyzed,
and optionally droplets are analyzed by conventional 1D on-chip or 2D analysis, or by a 3D particle counter.
In alternative embodiments of the high throughput, multiplexed system or device, or method, of the invention, the cell-derived particle comprises an exosome, a microvesicle, an apoptotic body, or any combination thereof; or the target molecule comprises a nucleic acid, a protein, a peptide, a carbohydrate, a lipid, a small molecules, or a metal ion.
In alternative embodiments the invention provides methods of identifying and isolating an enzyme-based target detection system for high through-put detection of specific target, comprising:
(a) providing a library of enzyme-based target detection system molecules designed to bind to and detect one specific target or a plurality of specific targets, the target to which the enzyme-based target detection system designed to detect, and a substrate comprising a detectable moiety,
wherein when the enzyme-based target detection system is not bound to its target, the enzyme is inactive, and when the enzyme-based target detection system binds to its specific target, the enzyme is activated to act on the substrate to generate a detectable signal,
wherein optionally the generated detectable signal comprises a fluorescent signal, and optionally the enzyme-based target detection system is an aptamer inhibitor-DNA-enzyme (IDE) system molecule, optionally as illustrated in
and optionally the enzyme-based target detection system is a nucleic acid initiator triggered signal amplification cascade, optionally as illustrated in
(b) encapsulating the sample, an enzyme-based target detection system and substrate in an immiscible carrier oil fluid such that the encapsulation generates a plurality of droplets, wherein droplet each comprises a plurality of sample, an enzyme-based target detection system and substrate,
wherein optionally the encapsulating comprises pumping the sample, an enzyme-based target detection system and substrate through an oil stream, and optionally the plurality of droplets are picoliter sized droplets;
(c) passing the plurality of droplets generated in (b) through a sorter, which directs the droplets having a detectable signal into a separate channel where the sorted droplets are lysed or broken, diluted, and re-encapsulated with additionally added target and substrate at a concentration of about 1 enzyme-based target detection system molecule per drop with in each droplet one or more of substrate and target,
wherein optionally the sorted droplets are lysed or broken optionally using an optical tweezer, an optical trap, an optical lattice, gradient centrifugation or any combination or an equivalent thereof,
wherein optionally the generated detectable signal comprises a fluorescent signal and the sorter is a FACS,
and optionally the generated detectable signal comprises a fluorescent signal and the sorter is a microfluidic device; and
(d) further sorting out droplets having a detectable signal into a separate channel,
thereby identifying and isolating an enzyme-based target detection system or molecule for high through-put detection of the specific target,
wherein optionally the enzyme-based target detection system or molecule comprises a aptamer inhibitor-DNA-enzyme (IDE) system molecule and the isolated IDE molecule is sequenced.
In alternative embodiments, the invention provides drug or aptamer screening and in vitro selection platforms based on one type of molecule/one bead or one type of molecule/one droplet strategy, wherein DNA, RNA, polypeptides and/or peptides are synthesized in a droplet library, comprising:
providing a high throughput, multiplexed system or device, or method, of the invention, and DNA on microbeads for generating a target or a binder to a target,
wherein the DNA on microbeads, or DNA-bread library, is used for screening drug or aptamer that possesses a function, e.g., binding to target molecule or modulate a molecular or cellular function, and optionally wherein the DNA on microbeads is encapsulated in the droplets or microdroplets, optionally picoliter droplets, optionally about 20 μm in diameter,
amplifying the on-bead DNA by PCR to generate a droplet DNA library,
transcribing and/or translating within the droplets the amplified DNA to form RNA and/or polypeptide or peptide libraries,
optionally the identity/sequence of transcribed RNA, and/or the translated polypeptides or peptides, are barcoded in the same droplet using the nucleic acid sequences, for subsequent screening and biomarker discovery,
and optionally the RNA and/or polypeptides or peptides are detected and/or quantified as the target using the high throughput, multiplexed system or device, or method, of the invention.
In alternative embodiments the invention provides an Integrated Comprehensive Droplet Digital Detection (IC 3D) System comprising a system as set forth in
In alternative embodiments the invention provides a multiplexed system comprising a microencapsulation droplet system integrated with a 3D particle detector as illustrated in
In alternative embodiments the invention provides multiplexed portable systems comprising: an integrated micro-encapsulator and a 3D particle counting system for detecting. identifying or quantifying a target by using a method of the invention, and optionally comprising a multiplexed portable system as illustrated in
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
All publications, patents, patent applications cited herein are hereby expressly incorporated by reference for all purposes.
Like reference symbols in the various drawings indicate like elements, unless otherwise stated.
In
For
The invention provides powerful, high throughput analytical platforms which can monitor a liquid sample (e.g., whole human blood, serum, saline or water, or any environmental sample) to detect biological, physiological and pathological markers with extremely high sensitivity (e. g., a single molecule or a single cell), and methods for making and using them. In alternative embodiments, system integrates novel sensor, e.g., biosensor, technology and a high throughput particle or droplet microfluidics platform. In alternative embodiments, the biosensors are short oligonucleotides, antibodies, peptides or other sensing elements that are engineered to specifically react with the targets, leading to a rapid fluorescence signal. In alternative embodiments, signals can be amplified using standard, conventional assays including PCR, rolling circular amplification, proximity ligation assays and EXPonential Amplification Reaction (EXPAR).
In alternative embodiments, exemplary platforms or systems of the invention enable rapid and sensitive detecting of a small molecule, or a biological, a physiological or a pathological marker, or a single molecule or a single cell using a microencapsulation droplet system integrated with a 3D particle detector (termed “Integrated Comprehensive Droplet Digital Detection (IC 3D)”), where the core concept of the integrated droplet encapsulation and 3D particle detector for the detection and bioanalysis of: low concentration biological markers, or for the detection and diagnosis of complex diseases including infectious diseases, cancer, diabetes, Alzheimer's disease, and the like, is schematically illustrated in
In alternative embodiments, the invention provides high throughput, multiplexed systems or methods for detecting a small molecule, or a biological, a physiological or a pathological marker, or a single molecule or a single cell using a particle or a droplet-based microfluidics system integrated with use of a sensor, e.g., a nucleic acid such as a DNAzyme. In alternative embodiments, the sensors, e.g., the DNAzymes, used to practice this invention are capable of specifically binding to a target molecule or a specific cell. In alternative embodiments, the target molecule or cell comprises a biological, physiological or pathological marker, or a single molecule or a single cell.
We demonstrated the effectiveness of an exemplary system of the invention comprising droplet microfluidics system integrated with a sensor, e.g., a DNAzyme. DNAzymes, also called deoxyribozymes or DNA enzymes or catalytic DNA, are DNA molecules that have the ability to perform a chemical reaction or catalyze a reaction. In practicing these exemplary systems and methods of the invention, the sensor, or the DNAzyme sensor (
In alternative embodiments, systems of the invention can be used to detect rare circulating tumor cells in blood. In alternative embodiments, systems of the invention can specifically assess gene expression, point mutations, miRNAs and SNPs using droplet-PCR, droplet RT-PCR or droplet-EXPonential Amplification Reaction (EXPAR) (see, e.g.,
In alternative embodiments, the invention provides systems comprising Integrated Comprehensive Droplet Digital Detection (IC 3D) (e.g., as illustrated in
In alternative embodiments, exemplary platforms or systems of the invention comprise use of multiple rounds of enrichment using, e.g., disease and/or normal samples as positive and negative selection targets, respectively (see, e.g.,
We have completed exemplary DNAzyme screening processes of the invention for lung cancer, and obtained several DNAzyme sensors (as illustrated, e.g., in
In alternative embodiments, exemplary platforms or systems of the invention can exploit powerful in vitro selection to generate reliable, nucleic acid binders, agonist or antagonist or DNA sensors and diagnostics for complex diseases including cancer, diabetes Alzheimer's disease, and the like (as illustrated e.g., in
In alternative embodiments, the invention's droplet microfluidic systems are significantly more effective, more sensitive, easier to make, and more tunable compared to existing ones to monitor biomarkers for diagnostics and prognostics. In alternative embodiments, the droplet libraries generated by methods and systems of the invention can significantly increase the chance to find drugs candidates and new biomarkers with small sample amount and also can reduce the screening time.
In alternative embodiments, exemplary platforms or systems of the invention includes a method called ENcapsulated ScreeNing of Aptamers by Reporter Amplification (ENSNARA) for identification of aptamers by employing allosteric control over a reporter enzyme or an enzyme system in the droplet; e.g., as illustrated in
For this exemplary ENSNARA protocol, in the first stage there are 106 IDE per drop. A sorter (for example, a FACS, as illustrated in the figure) directs any fluorescent droplets into a separate channel, where they are lysed, diluted, and re-encapsulated at a concentration of 1 aptamer inhibitor-DNA-enzyme (IDE) per drop, and supplemented with substrate and target molecule (substrate and target molecule are added to, or incorporated within, the re-encapsulated 1 IDE per drop microdroplets). The aptamer-containing droplets that produce a fluorescent signal are then collected, and optionally the aptamer can be sequenced.
In alternative embodiments, the systems and methods of the invention can use any form or variation of microfluidic systems for making, using and/or transporting microdroplets to practice this invention.
For example, a microfluidic transport system for transporting microdroplets in three spatial dimensions can be used as described e.g., in U.S. patent app. pub. No. 2013/0213812. In alternative embodiments, the systems and methods of the invention can use microdroplet-manipulating devices coupled to a movement and placement device as described e.g., in U.S. patent app. pub. No. 20130149710, which also described PCR reactions in the microdroplets. U.S. patent app. pub. No. 20130139477 describes use of microdroplets as “microreactors” for controlled processing of the contents, wherein very small amounts of material are encapsulated in a microdroplet in a quantized amount. U.S. patent app. pub. No. 20130130919 describes a microdroplet-based method for sequencing large polynucleotide templates. Microdroplets can be made e.g., by an apparatus as described in U.S. patent app. pub. No. 20130129581.
In alternative embodiments, the systems and methods of the invention can use microdroplet-manipulating devices as described e.g., in: U.S. Pat. No. 8,529,026, describing devices for passively periodically perturbing a flow field within a microfluidic device to cause regular droplet formation at high speed; or U.S. Pat. No. 8,528,589, describing methods for assessing one or more predetermined characteristics or properties of a microfluidic droplet within a microfluidic channel, and regulating one or more fluid flow rates within that channel to selectively alter the predetermined microdroplet characteristic or property using a feedback control; or U.S. Pat. No. 8,492,168, describing droplet-based affinity assays, e.g., detecting a target analyte in a sample by combining affinity-based assay reagents on a droplet microactuator with a sample/target analyte to generate a signal indicative of the presence, absence and/or quantity of analyte; or U.S. Pat. No. 8,470,606, describing methods of circulating magnetically responsive beads within a droplet in a droplet actuator, and methods for splitting droplets; or, U.S. Pat. No. 8,524,457, describing methods for screening specific affinity molecules to target molecules using a homogeneous non-competitive assays using e.g., microdroplets created e.g., using micro- or nanofluidics.
In alternative embodiments, in practicing methods and systems of the invention, microencapsulated emulsions or droplets can be made using traditional methods, or by using an emulsifier (see for example: Griffiths, A. D. & Tawfik, D. S. Miniaturising the laboratory in emulsion droplets. Trends Biotechnol. 24, 395-402 (2006)). In alternative embodiments, methods and systems of the invention comprise use of droplet based microfluidics including high throughput droplet generators or multi-channel devices such as the TELOS SYSTEM™ from Dolomite Microfluidics (Royston, Herts, UK). In alternative embodiments, liquid droplets containing, for example, agarose or PEG, can be gelled or solidified to form droplet particles (see for example: Anal Chem. 2012 Jan. 3; 84(1):350-355). In alternative embodiments, in practicing methods and systems of the invention, highly parallel single-molecule amplification approach based on agarose droplet polymerase chain reaction can also be used for efficient and cost-effective aptamer selection, see, e.g.,).
In alternative embodiments, the invention provides a drug screening and in vitro selection platform based on one type of molecule/one droplet strategy (see, e.g.,
In alternative embodiments, DNAzymes, also called “DNA enzymes” or “deoxyribozymes”, are used to practice the methods and systems of the invention. They are synthetic single-stranded (ss) DNA oligonucleotides with catalytic activities.11,12 In alternative embodiments, catalytic DNA molecules used to practice the invention can be generated in vitro from a vast random library using a combinatorial approach called in vitro evolution or selection13,14 where the properties of the molecules to be selected can be tailored and pre-defined.
In alternative embodiments, DNAzymes used to practice the methods and systems of the invention have diverse catalytic activities, including DNA/RNA cleavage, phosphorylation, and RNA ligation.12 DNAzymes used to practice this invention can be made using any known technique, e.g., as described in U.S. Pat. Nos. 8,329,394; 8,450,103.
In alternative embodiments, DNAzymes used to practice the methods and systems of the invention is a RNA-cleaving DNA motif that can cleave a DNA-RNA chimeric substrate at a single ribonucleotide junction (see e.g.,: Fluorogenic DNAzyme probes as bacterial indicators. Ali M M, Aguirre S D, Lazim H, Li Y. Angew Chem Int Ed Engl. 2011, Apr. 11; 50(16):3751-4.).10,15 In alternative embodiments, this unique property allows use of DNAzymes as a platform for real-time fluorescent sensors (see e.g., Catalytic nucleic acid probes as microbial indicators CA 2829275 A1, PCT/CA2012/000205).
Microencapsulation Droplet Systems Integrated with a 3D Particle Detector
In alternative embodiments, 3D particle detectors or counters are used to practice the methods and systems of the invention, see e.g., and as described in, e.g., Gratton, et al. U.S. Pat. No. 7,973,294 (2011); U.S. Pat. No. 7,528,834 (2009); J. P. Skinner, et al., Rev Sci Instrum 2013, 84; I. Altamore, et al., Meas Sci Technol 2013, 24. In alternative embodiments, the invention provides microencapsulation droplet systems integrated with a 3D particle detector as illustrated, e.g., in
A 3D particle counter used to practice this invention can detect fluorescent particles from mL volumes at single-particle sensitivity within minutes. Briefly, as shown in
This system can robustly detect few particles/mL using fluorescent microbeads or Sytox orange-stained E. coli (see, e.g., Skinner, et al., Rev Sci Instrum 2013, 84; I. Altamore, et al., Meas Sci Technol 2013, 24)
In alternative embodiments, the invention provides methods and systems comprising a microencapsulation droplet system integrated with a 3D particle detector, e.g., as illustrated in
In alternative embodiments, the methods and systems of the invention comprise the following unique features, including some that cannot be easily achieved by traditional detection assays:
In alternative embodiments, methods and systems of the invention can analyze a biological sample which can comprise a biopsy sample, or a blood, serum, saliva, tear, stool, urine or CSF sample from an individual or a patient. In alternative embodiments, methods and systems of the invention can analyze any samples obtained from a food, water, soil, or an air source.
In alternative embodiments, in practicing methods and systems of the invention, the samples can be directly assayed with no or minimal (e.g., dilution) processing. Standard, established biological sample preparation processes including dilution, purification, enrichment, extraction, centrifugation, magnetic bead assays, and washing steps, although not required, can be integrated into assays, methods and systems of the invention.
In alternative embodiments, assays, methods and systems of the invention, can detect and analyze any target, including e.g., but not limited to: cells (e.g., cancer cell, stem/progenitor cell, immune cell), pathogens (e.g., bacteria, multi-drug resistant organisms (MDRO), tuberculosis (TB)), parasites, fungi, viruses (e.g., HIV), cell-derived vesicles (e.g., exosome, microvesicles, apoptotic bodies), nucleic acids (e.g., SNPs, mutations, expression), proteins (e.g., PSA), enzymes (e.g., MMPs), peptides, lipids, carbohydrates, polysaccharides, small molecules or metal ions.
In alternative embodiments, forms of target species detected by assays, methods and systems of the invention include e.g., cell surface (e.g., EpCAM, N-cadherin, CD44, CD24), intracellular, and secreted markers (cell secretome), cell free circulating markers (e.g., miRNA, DNA, protein markers), metabolic markers, mechanical markers (e.g. cell deformability, stiffness, cytoskeleton, etc).
In alternative embodiments, methods and systems of the invention can be used to detect or monitor a biological event, e.g. DNA hybridization, protein receptor-ligand interaction, enzyme-substrate interaction, and cell surface receptor dimerization (including both homo and hetero-clustering), co-localization, or interaction with soluble ligands and drugs and another cells.
In alternative embodiments, methods and systems of the invention comprise use of a variety of detection assays for analyzing or measuring a target in a droplet. For example, methods and systems of the invention comprise use of a wide variety of established fluorescence bioassays, to e.g., selectively detect the targets within droplets for, e.g., the exemplary 3D particle counter analysis embodiments. Such assays include, both not limited to: nucleic acid based assays, antibody based assays, enzyme based assays, or chemical based assays or assays used in combination; or, nucleic acid based assays, including e.g., hybridization, molecular beacons, aptamer, DNAzyme, or other real-time fluorescent sensors; or, antibody-based assays, including, e.g., ELISA, sandwich based, immunostaining, antibody capture, secondary antibody amplification, or proximity ligation based; including e.g., enzyme based assays, including, e.g., PCR, RT-PCR, RCA, loop-mediated isothermal amplification (LAMP), nicking (e.g., EXPonential Amplification Reaction (EXPAR)), strand displacement, and exponential isothermal amplification (e.g., see Lab Chip, 2012, 12, 2469-2486) (a few examples are illustrated in
In alternative embodiments, in practicing methods and systems of the invention, microencapsulated emulsions or droplets can be made using traditional methods, or by using an emulsifier or by droplet based microfluidics. In alternative embodiments, methods and systems of the invention comprise use of droplet based microfluidics including high throughput droplet generators or multi-channel devices (see
In alternative embodiments droplets are made in different sizes ranging from 10 nm to 100 s microns. Droplets can be manipulated in numerous ways including heating/cooling (for PCR), merging, splitting, sorting and long-term storage. Droplets can be analyzed by conventional 1D on-chip or 2D analysis, or by, in this invention, a 3D particle counter.
In alternative embodiments, in practicing methods and systems of the invention, any 3D particle counter can be used, e.g., comprising an instrument system as shown e.g., in
In alternative embodiments, the invention provides integrated systems, e.g., systems engineered to comprise one or any of: desirable portability (for example, packaged as backpacks), automating fluid handing (i.e., droplet generation and auto sampling), and integrating electronics including a diode laser (light source), APD (detector), Operating (vinci, ISS Inc.) and/or data analyzing software (SimFCS), display, computer interface, smart phone, with a 3D particle counting system; e.g., as illustrated in
In alternative embodiments, this exemplary device is integrated with multiple disposable microfluidic “cartridges,” permitting multiplex and rapid detection of multiple types of targets simultaneously. The device can be fully automated, and can be fabricated as an all-in-one system or with modular components. It can also be linked to smart phone and bluetooth etc for point-of-care applications, as illustrated in
In alternative embodiments, to enable multiplex and parallel detection of multiple targets, our device can be comprised of multiple laser sources and detectors capable of reading at different wavelengths. The multiplex system permits simultaneous reading of multiple sensors (labeled in different colors) that are coded for different targets. In alternative embodiments, a carousel can be integrated in our apparatus to accommodate multiple sample vials for carrying out parallel tests.
Applications of Microencapsulation Droplet Systems Integrated with 3D Particle Detectors, or Integrated Comprehensive Droplet Digital Detection (IC 3D) Systems of the Invention
In alternative embodiments, the exemplary systems of the invention comprising an integrating droplet system and a 3D particle counter system, including the so-called “Integrated Comprehensive Droplet Digital Detection (IC 3D) system of the invention” (see e.g.,
Infectious disease pathogens (e.g., bacteria, viruses, fungi, etc), including skin infection, wound infections, diabetic ulcer infections, HIV, bacteria, TB, MDROs (e.g. MRSA);
Cancer;
Diabetes;
Alzheimer disease (e.g., Amyloid beta, Tau proteins);
Brain injury and disorders (e.g., S100B, a glial-specific protein, where elevated S100B levels accurately reflect the presence of neuropathological conditions including traumatic head injury or neurodegenerative diseases)
Inflammatory and autoimmune diseases (e.g., CD4 T cell, immune cell count);
Stem cell and regenerative medicine (e.g., mesenchymal stromal cells, endothelial progenitor cells, hematopoietic stem cell, cells can include both endogenous and exogenously transplanted cells);
Cardiovascular diseases (e.g., C-Reactive Protein (CRP), B-type natriuretic peptide (BNP), troponin, Cystatin C, IL-6);
Drug and abuse (e.g. Tetrahydrocannabinol, THC);
Newborn screening (e.g., phenylalanine).
In alternative embodiments, the exemplary systems of the invention are used to study new biology, cell-drug interactions and drug susceptibility, to develop new drugs and therapeutics and monitor disease progress and treatment efficacy or used as companion diagnostics and to be used in sequencing, personalized diagnostics and medicine. In addition to medical applications, exemplary systems of the invention can also be used for other areas including food industry, agriculture, water systems, air systems, and defense applications.
The invention provides systems and methods for the rapid and sensitive identification of bacteria in blood, which will significantly reduce the mortality rate and the cost of medical care associated with blood infections.
In alternative embodiments, the invention provides rapid and sensitive methods to detect blood stream infections in order to expedite blood infection diagnosis and treatment.
In alternative embodiments, the invention provides rapid and sensitive methods to detect antimicrobial resistances including extended spectrum beta-lactamase (ESBL) and carbapenem-resistant Enterobacteriaceae (CREs).
In alternative embodiments, the invention provides rapid and sensitive methods to detect cancer cells, e.g., to detect a metastasis, or a dissemination of cancer cells from the primary tumor to other organ sites, e.g., to detect the formation and growth of a primary tumor, e.g., to detect cancer cells that are shed from the primary tumor into the circulation known as circulating tumor cells (CTCs). In alternative embodiments, the invention provides methods for the analysis and quantification of CTCs for early-stage diagnosis, prognosis and monitoring disease course. In alternative embodiments, the invention provides methods for detecting cancer markers such as proteins (e.g., Prostate-Specific Antigen (PSA)), cell-free nucleic acids (e.g., DNA, mRNA, miRNA), cell derived particles (e.g., exosomes, microvesicles, apoptotic bodies). In alternative embodiments, the invention provides methods for detecting very rare markers, for example, where one CTC is present per 107 leukocytes. Methods of the invention can be used with or in place of heterogeneous, traditional flow cytometry, DNA and RNA sequencing, and immunological approaches (e.g., a CELLSEARCH™ platform) to, e.g., reliably detect cancer markers such as CTC or PSA in clinical settings.
In alternative embodiments, the invention provides single-cell detection methods that can offer a way to dissect the heterogeneity of cancer cells. In alternative embodiments, the invention provides the ability to detect and analyze rare cells at a single cell level, including detection of nucleic acids, proteins, and metabolites for personalized diagnostics and treatment.
In alternative embodiments, the invention provides methods for detecting established biomarkers for neurological and central nervous system (CNS) diseases and brain tumors, trauma and injury. In alternative embodiments, the invention provides methods for detecting the accumulation of amyloid-β (Aβ) peptides (i.e., Aβ42 and tau proteins, which are two key neuropathological features characterizing the Alzheimer's disease (AD) brain and may be important biomarkers that are detected in CSF characterizing AD pathogenesis. In alternative embodiments, the invention provides methods to detect and quantitate these biomarkers, which can be invaluable to studies that aim to use Aβ and tau proteins as biomarkers to 1) screen and monitor AD, 2) better understand the molecular biology and pathology of the disease, and 3) evaluate therapeutic interventions. In alternative embodiments, the methods of the invention can be used in place of or with existing assays including ELISA to e.g., detect Aβ and tau protein. In alternative embodiments, the invention provides screening and detection of such markers in blood and urine, including any marker such as S100B (S100 calcium binding protein B) that is at a very low concentration, and often cannot be detected by existing assays because of the blood brain barrier (BBB).
In alternative embodiments, the invention provides methods for detecting and characterizing retroviruses, e.g., human immunodeficiency virus (HIV), HIV/antibody complexes and rare reservoir cells containing HIV. Recently, there were a few incidents where HIV patients seemed to be cured by new treatments including bone marrow transplantation. However, HIV returned after several months. A major challenge is that during therapy the viral particle concentration can often go below the detection limit of existing technologies, which appears to be “cured” but actually not. Therefore, methods of this invention can detect extremely low numbers of viral particles to aid in this therapy and prognoses.
In alternative embodiments, the invention provides methods and systems comprising use of droplet emulsion encapsulation (e.g., water-in-oil), which is an established method to compartmentalize samples and agents in small volumes for a variety of purposes including bioassays, drugs and food industry. In alternative embodiments, the invention provides methods comprising use of multiphase flows in microfluidic systems as a platform for ultra-sensitive and high-throughput screening and experimentation.
In alternative embodiments, methods of the invention use “droplet microfluidics” to enable the generation and manipulation of monodisperse, microdroplet, e.g., picoliter-sized, liquid droplets in an immiscible carrier oil fluid (e.g., water-in-oil emulsion) (see e.g., “Droplet microfluidics for single-molecule and single-cell analysis for cancer research, diagnosis and therapy”, Dong-Ku Kang et al. Trends in Analytical Chemistry, 2014). In alternative embodiments, methods of the invention utilize compartmentalization in picoliter droplets (e.g., 1 to 300 μm in diameter) to increase assay sensitivity and decrease assay time by increasing the effective concentration of target species.
In alternative embodiments, droplet microfluidics is used for high-throughput and multiplex detection and analysis of low concentrations of targets such as single cells; and detection of gene expression, cell viability and proliferation, cell-cell and cell-drug interactions at a single-cell level. In alternative embodiments, droplets are manipulated in numerous ways, including heating/cooling (for PCR), merging, splitting, sorting and long-term storage.
In alternative embodiments, methods of the invention comprise multiple (for example, up to 256) droplet generating channels which is able to convert 1 mL sample into droplets within several min.
In alternative embodiments, methods of the invention comprise encapsulation of gelable materials, such as agarose, which can be easily fabricated to form hydrogel droplets for different purposes including repetitive washing and reaction steps and flow cytometry analysis; droplets can be detected on-chip and efficiently sorted with high-throughput, for example, at greater than 1000 droplets/second (s).
In alternative embodiments, methods of the invention comprise use of a 3D particle detector, also called a Rare Event Detector, a 3D particle scanner or a fluorescence correlation spectroscopy (FCS), e.g., as described in U.S. Pat. No. 7,528,384; U.S. Patent application publication no 20090230324; U.S. Pat. No. 7,528,384. In alternative embodiments, such 3D Particle Detectors are able to achieve a throughput that is clinically relevant. In alternative embodiments, methods of the invention comprise use of 3D particle counting techniques that can detect particles (e.g., fluorescent beads or dye-stained cells) from milliliter (mL) volume at single-particle sensitivity within minutes.
In alternative embodiments, methods of the invention comprise use of 3D particle counting techniques that can rapidly scan of one mL of fluid by moving a tube containing the fluid in a spiral motion in front of an objective of the confocal microscope. The optics of the microscope can be designed to measure a relatively large volume (100 pL) in about 0.01 ms. The rotation of the tube in a spiral motion for about 100 seconds effectively explores about 1 ml of the tube. The rapid passage of the fluorescent particle in the volume of excitation produces a very strong signal with signal-to-noise ratio (S/N) greater than 100. Since only the fast signals are detected, the slow modulation of the fluorescence signal due to the imperfections in the mechanical construction of the rotating tube has no effect on the S/N, this system can robustly detect few particles/mL using fluorescent microbeads or Sytox orange-stained E. coli, e.g., as described in Skinner, Rev Sci Instrum., 84(7), 074301; Altamore (2013) FCS. Meas Sci Technol., 24(6), 65702.
The invention will be further described with reference to the following examples; however, it is to be understood that the invention is not limited to such examples.
Real-time fluorescent DNAzyme sensors: In one embodiment, a DNA library containing approximately 1014 random sequences (e.g., chemically synthesized) are used 5 for selecting and/or isolating DNAzyme sensors. The library can consist of a variable sequence, e.g., of about 40 nucleotides that is ligated to a fluorogenic, DNA-RNA chimeric substrate (see
In vitro selection can be by incubating a starting library with a target bacterial lysate for about 10 min in HEPES buffer. The cleaved molecules can be gel isolated, amplified by primer-specific PCR, ligated to the substrate and then used in the next round of selection. Bacterial lysates from non-target bacteria can be included as a negative selection to remove nonspecific DNAzymes and ensure assay specificity. In our experience, 8 to 15 rounds of selection (approximately 1-3 months) are needed for the completion of a selection.7 The final round of the DNA pool can be sequenced. Using this selection approach, real-time DNAzyme sensors that rapidly detect a variety of bacteria including E. coli, Listeria, Salmonella and Clostridium difficile have been isolated. Such high selectivity demonstrates that by including appropriate negative selection targets in the selection process, it is feasible to generate DNAzyme sensors that specifically detect a particular bacterium, MDRO or other pathogens. In alternative embodiments, methods and systems of the invention incorporate any known method using fluorogenic DNAzyme probes as a cell, e.g., a bacterial, indicator, e.g., as described in Ali et al, Angew Chem Int Ed Engl. 2011 Apr. 11; 50(16):3751-4; or, Li et al., WO/2012/119231.
We used these rapid, fluorogenic DNAzyme sensors as an example in our system. As shown in
Since our goal is to develop a “mix-and-read” assay that uses whole blood with no or minimal sample processing, we further tested the sensor performance in whole blood and found that our Fluorescein/Dabcyl modified DNAzyme sensors produced a sufficiently high fluorescence signal in response to E. coli spiked in blood that was diluted by sensor solution to various volume ratios (
Given that blood stream infections (BSIs), sepsis and antimicrobial resistance can be caused by several different types of pathogens, the sensor set can be expanded through in vitro DNAzyme sensor selection described above to detect the other pathogen species. In particular, the nonbiased screening using bacteria as a complex target without prior knowledge of any specific target molecules bypasses the tedious process of purifying and identifying target molecules from extremely complex mixtures and permits the rapid development of sensors for new bacterial strains in an unanticipated outbreak. This addresses a major challenge faced by existing techniques including PCR that rely on the detection of pre-identified target genes or other biomarkers given the rapid and complex evolving mechanisms associated with bacteria. Although the identification of specific bacteria biomarkers that bind to DNAzymes to trigger substrate cleavage is not necessary for our assay to operate, they can be identified in using affinity purification coupled with mass spectrometry.
In alternative embodiments the invention uses a panel of real-time, fluorogenic DNAzyme sensors, which can be make via in vitro selection using e.g., major blood-infection bacteria or drug resistant organisms as targets including e.g., Staphylococcus aureus (S. aureus), Enterococcus faecalis (E. faecalis), coagulase-negative Staphylococci, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter species and extra-intestinal pathogenic Escherichia coli, ESBLs, CREs, methicillin-resistant Staphylococcus aureus (MRSA), and fungal pathogens.
Droplet microfluidics: In alternative embodiments, the systems and methods of the invention manipulate multiphase flows in microfluidic systems as a platform for ultra-sensitive and high-throughput screening and diagnostics. These systems, called “droplet microfluidics”, enable the generation and manipulation of monodisperse, picoliter-sized liquid droplets in an immiscible carrier oil fluid (i.e., water-in-oil emulsion).11-14 The ability to controllably generate droplets with variable analyte composition, and at high rates, makes droplet microfluidics a powerful tool to address a range of chemical and biological applications including enzymatic assays, protein crystallization, nanomaterial synthesis, and cell-based assays.11-14 The compartmentalization in picoliter droplets (which is alternative embodiment can be between about 1 to 300 μm, or 10 to 100 μm, in diameter) increases assay sensitivity and decreases assay time by increasing the effective concentration of target species.11 Therefore, in alternative embodiments droplet microfluidics is particularly suited for high-throughput and multiplex detection and analysis of low concentrations of targets such as single cells. Indeed, gene expression, cell viability and proliferation, cell-cell and cell-drug interactions at a single-cell level have been demonstrated using droplet microfluidics.12 In alternative embodiments, droplets are manipulated in numerous ways including heating/cooling (for PCR), merging, splitting, sorting and long-term storage. In alternative embodiments, droplets can be detected on-chip and efficiently sorted with high-throughput (>1000 droplets/s).11
In alternative embodiments, the systems and methods of the invention can detect bacteria in patient blood at single-cell sensitivity within minutes, as illustrated in
In alternative embodiments, blood sample and DNAzyme sensors are mixed and then encapsulated in hundreds of millions to billions of micron-sized droplets. DNAzyme sensors produce an instantaneous signal in the droplets that contain bacterium, which will be counted and analyzed. In alternative embodiments, patient blood is mixed with DNAzyme sensor solution, including bacteria lysis buffer, within the microfluidic channel, which can be encapsulated in millions of individual picoliter droplets (
In alternative embodiments, the in vitro selection technique can generate multiple DNAzyme sensors for various major pathogenic bacteria, making multiplex bacterial detection possible. The compartmentalization of a single bacterium in a droplet significantly increases the concentration of target molecules, permitting rapid detection and single-cell sensitivity. The significantly shortened assay time (i.e., minutes instead of hours to days in the conventional techniques) allows blood infections to be treated timely and effectively.
In alternative embodiments, an exemplary platform of the invention can also be easily integrated with drug susceptibility screening assays to identify the best antibiotics regimen for patient-specific treatment. Such rapid detection and early intervention can significantly improve the chances of treating blood infections and reduce mortality. Thus, the invention can significantly increase the survival of patients with blood infections and decrease the financial costs associated with patient care.
In alternative embodiments the rapid and single-cell detection methods and systems of the invention can serve as a platform for the detection and screening for slowly-growing species (e.g., Mycobacterium tuberculosis) and other rare cells in blood such as circulating tumor cells.
Device fabrication: Droplet microfluidics can be fabricated and operated following any known and established procedures, e.g., as discussed above.26 For example, in one embodiment, a poly(dimethylsiloxane) (PDMS) chip with 20 μm-depth and 15 μm-width channels is fabricated using standard soft lithography, and mounted on a glass microscope slide. As illustrated in
In alternative embodiments, the droplets in our system can be made via high throughput droplet generator with multiple droplet generation challenges or structures. In alternative embodiments, the high throughput droplet generator permits conversion of a milliliter sample into droplets within several minutes. As illustrated in
In alternative embodiments, the use of larger droplet and smaller blood dilution factor can further significantly reduce the droplet generation time.
In alternative embodiments, droplets can be gelable materials, such as agarose, which can be easily fabricated to form hydrogel particles for different purposes including repetitive washing and reaction steps and flow cytometry analysis.
Droplet detection and quantification: Fluorescence measurement of droplets can be carried out by using a custom-built confocal microscope (Observer Z1™, Zeiss). This confocal setup consists of 488 and 561 nm diode lasers as excitation sources, and an electron multiplying charge coupled device (QuantEM:512SC, Photometics) for fluorescence detection. In order to maximize the scanning speed, a CSU-XI spinning disk (CSU-X1, Yokogawa, Japan) is integrated into the confocal microscope. Typically, droplets will be measured at a rate of 100 s to 1000 s droplets per second and the data can be analyzed using ImageJ. In addition to confocal microscopy, standard flow cytometry can also be used to analyze, quantify and sort fluorescent droplets in a high throughput manner.35
High Throughput Droplet Detection:
To achieve high throughput detection, In alternative embodiments, an optical system which incorporates a highly sensitive APD detector with a dual-band emission filter (z488/635, Chroma Technology Corporation, USA) and dichroic mirror (630dcxr, Chroma Technology Corporation, USA) is used; this can count droplets at a throughput of ˜3000 droplets/s (see
Detection of bacteria in droplets using DNAzyme sensors: Droplet microfluidic systems integrated with DNAzyme sensors can be optimized to detect bacteria in reaction buffer, using e.g., 50 mM HEPES, pH 7.5, 150 mM NaCl, 15 mM MgCl2, and 0.01% Tween 20. Two important properties can be targeted: sensitivity and detection time. As bacteria exist at low numbers in patient's blood (typically 1-100 CFU/ml), when encapsulated in picoliter droplets, each droplet will contain one or no bacterium. Therefore, significant that the systems of this invention can detect bacteria at a single-cell sensitivity. In alternative embodiments, target bacteria such as E. coli, are encapsulated together with their respective DNAzyme sensors (e.g., at 100 nM, modified with Fluorescein and Dabcyl) into droplets. Control experiments including mutant DNAzyme/target bacteria and DNAzyme sensor/non-target bacteria can be included to assess the specificity of a droplet assay. Lysozyme (1 mg/mL), a bacteria lysis agent, can be pre-mixed in the DNAzyme sensor solution. The use of lysis agents allows the target molecules to be rapidly released from bacteria, which will further decrease the assay time. Bacteria lysis conditions can be systemically optimized using various agents including e.g., Triton X-100, IGEPAL, SDS and lysozyme alone or in combination, and identified that lysozyme most efficiently lyses bacteria without interfering with droplet formation or DNAzyme sensor function.
Bacteria can be statistically diluted and compartmentalized in droplets at a range of concentrations. For example, for a 50 μm droplet, the initial cell concentration will be 3, 30, and 300×106/mL bacteria in order to form 1, 10, and 100 bacteria per droplet. When the initial bacteria solution is extremely diluted (<3×106/mL), the formed droplets will contain single or no bacteria per droplet. Bacteria can be stained with Syto9 (green) or Syto17 (red), which allows better visualization of them within droplets to quantify the numbers of cells per droplet using confocal microscopy. Staining bacteria with a different color allows co-localization with the DNAzyme sensor signal in the same droplet in the detection assay.
Bacterium-containing droplets can be easily detected due to the intense fluorescence signal produced by DNAzyme sensors. We have shown that an exemplary E. coli sensor of the invention can detect bacteria in droplets, with the signal directly correlated to the number of cells per droplet (
Optimization: Optimal detection time and signal/background ratio of the droplet assay for a particular assay or target can be achieved by optimizing two parameters: droplet volume (or size) and DNAzyme sensor concentration. As smaller droplet sizes lead to higher target concentrations from single cells, increasing the signal/background ratio and decreasing the detection time, the performance of three different droplet sizes of e.g., 10, 20 and 50 μm can be specifically compared. For the droplet assay, a DNAzyme sensor concentration of 100 nM can be a starting point, which has been shown to be optimal in a bulk assay. DNAzyme sensor concentrations, e.g., at 10, 50, 100, 200 and 500 nM, can be optimized to reach the best balance of detection time and signal/background ratio.
Examine and optimize bacteria detection in spiked blood: In alternative embodiments, exemplary systems of the invention are used to detect bacteria in unprocessed (or diluted) blood. DNAzyme sensors can be modified with dye-quencher pairs that are compatible with blood detection. For titrating and optimization, bacteria can be spiked in undiluted whole blood in various concentrations, which will be encapsulated along with DNAzyme sensor solution into droplets as described above. To prevent clotting and precipitation of blood sample during injection, a 2 mm magnetic bar can be placed inside the syringe with a portable magnetic stirrer placed on the top.
Whole blood containing bacteria can be directly encapsulated into droplets, as illustrated in
In alternative embodiments, the invention provides compositions and methods comprising use of droplet microfluidics with a DNAzyme sensor system to selectively detect single bacterium, e.g., in buffer and/or spiked blood. Using fluorescent microscopy (
In case a single emulsion droplet (water-in-oil) is not compatible with a flow cytometry system, a water-in-oil-in-water double emulsion droplets can be used (fabricated) for that set of flow cytometry measurements. Water-in-oil-in-water double emulsion droplets can be easily fabricated using two flow-focus junction devices, and have been widely used for flow cytometry analysis and sorting. We did not observe blood clogging in the channel before encapsulation in the droplets. If necessary, that part of channel can be coated with non-fouling polyethylene glycol (PEG) or heparin to further minimize undesirable clogging of blood components.36,37
Detecting bacteria from clinical specimens: In alternative embodiments, the invention provides compositions and methods having clinical applicability.
Using patient samples: In alternative embodiments, the invention provides compositions and methods, including devices, able to determine the presence of bacteria with very high sensitivity and specificity. By determining the type and/or presence of bacteria, appropriate antibiotic treatment can be determined—and monitored during the course of therapy. An aliquot from a blood culture is transferred to a sterile 15 ml conical tube; patient blood (e.g., about 1 mL) that may contain or contains a particular type of bacteria can be encapsulated into droplets with its respective DNAzyme sensors, e.g., following the optimized protocols, as discussed above. The fluorescent droplets can be counted by the high throughput APD detector. We can analyze a total of e.g., 10 patient samples for each bacteria target. A set of experiments can be performed to allow determination of whether any particular system can reliably detect bacteria in patient blood samples, e.g., the false positive and negative rate.
Thus, in alternative embodiments, methods, systems and devices of the invention can reliably detect bacteria from patient samples with high sensitivity and selectivity (<10% false positive and false negative rates).
Portable system: In alternative embodiments device are portable and provide automating fluid handing (i.e., droplet generation), and integrating electronics including a light source (thin film LED), diode detector, and detector display (
In alternative embodiments, methods, systems and devices of the invention can detect multidrug-resistant organism (MDRO) or antimicrobial resistant infections, which are a major global health problem and pose a particular challenge to the care of combat- and trauma-wounded personnel.1-2 In alternative embodiments, methods, systems and devices of the invention provide early identification of MDROs, which is crucial for improving patient care by preventing the spread of disease and identifying appropriate antibiotic treatment.3 In alternative embodiments, methods, systems and devices of the invention can be used in place of, or to supplement, bacterial cultures (which require days to get a result) and/or amplification-based molecular diagnosis methods such as polymerase chain reaction (PCR; which can reduce the assay time to hours but are still not sensitive enough to detect bacteria that often occur at low concentrations, e.g., 1-100 colony-forming unit (CFU)/mL in infected blood.4,5 In alternative embodiments, methods, systems and devices of the invention can be used routine screening of MDROs, or under resource-limited environments, such as in third world countries, emergencies, disaster situations or battlefields.
The following describes an exemplary method of the invention for detecting a blood stream infection (BSI), and rapidly detect, identify and thus treat bacteria in the early stages of infection.
We have demonstrated that the integrated droplet system and 3D particle counter system of this invention allows selective detection of bacteria in unprocessed or minimally processed buffer and blood samples at single-cell sensitivity within minutes to several hours. In this example, our system integrates DNAzyme sensor technology, droplet microfluidics and a high throughput 3D particle counting system (i.e., Integrated Comprehensive Droplet Digital Detection (IC 3D)) (
In alternative embodiments, patient whole blood or other biological samples such as urine are mixed with a DNAzyme sensor solution, including bacteria lysis buffer, within the microfluidic channel, which will be encapsulated in hundreds of millions to billions of individual picoliter droplets, as illustrated e.g., in
DNAzyme sensors are short catalytic oligonucleotides that are identified by in vitro selection to specificity react with the lysates of target bacteria, leading to a rapid, real-time fluorescence signal. In alternative embodiments, E. coli-specific DNAzyme sensors are used in this example to selectively detect E. coli (
In alternative embodiments, the compartmentalization of a single bacterium in a droplet significantly increases the concentration of target molecules, permitting rapid detection and single-cell sensitivity without signal amplification processes such as PCR. In alternative embodiments, such compartmentalized, target-specific reaction is a critically necessary step to “light up” the droplets that contain target bacteria so that they can be detected by the 3D particle counting system. In alternative embodiments, the exceptional reliability and accuracy of exemplary 3D particle counting systems of the invention for single droplet analysis in mL volume within minutes bypass many challenges faced by current particle counting techniques, especially flow cytometry that suffers from limited sensitivity and high false positive rates.
In alternative embodiments, fluorescent droplets that contain a target can be sorted in the 3D particle counting system using e.g., optical tweezer, optical trap and optical lattice. This enables the sorted target(s) to be further processed and analyzed.
The existing 1D on-chip droplet counting system (which is also used in the droplet digital PCR system) and other particle counting systems including flow cytometry suffer from low throughput: they typically operate at 1000 s particles s−1 and are only able to analyze a total of 100,000 s to 1 million droplets (or a total sample volume of ˜tens of microliter).31, 34 Therefore, the existing droplet detection systems inevitably require sample preparation to purify and enrich targets and reduce sample volume before droplet encapsulation. In our system, however, we want to rapidly analyze unprocessed biological samples (e.g., blood) with a clinical sample volume of typically milliliters that translates up to billions of droplets. To effectively analyze these many droplets in a short period of time and detect single fluorescent, bacteria-containing droplets among millions of empty ones, in our invented Integrated Comprehensive Droplet Digital Detection (IC 3D) system, we integrated a 3D particle counter21 as we described earlier that can detect fluorescent particles from milliliter volumes at single-particle sensitivity within minutes.
In alternative embodiments, the apparatus comprises a small microscope that has a horizontal geometry and a mechanical sleeve that holds a cylindrical cuvette of diameter 1 cm. Two motors provide rotational and vertical motion of the cuvette. The software allows the rotational speed to be varied in the 10-1100 rpm range and the vertical speed in the 1-15 mm s−1 range. The vertical and rotational motions are produced respectively by the Linear Actuator and a VEXTA stepping motor model PK233PB. These motors are connected to a stage holding the transparent cuvette containing the sample. The excitation light generated by lasers is focused at the volume of observation (see photo). The excitation focus is positioned inside the cuvette and relatively close to the wall of the cuvette, at a distance of about 1 mm from the wall. This distance can be adjusted so that detection of particles and analysis could be done even in highly scattering media. The excitation sources are two diode lasers emitting at 469 nm or at 532 nm. Thus, a particle fluoresces when in the volume of observation. The use of a confocal microscope in combination with simple mechanical motions of the sample container in front of the objective provides the means to move and analyze a sample containing particles through an observation region without requiring a complex optical system comprised of moveable optical components, such as translating optical sources, mirrors or photodetectors. The excitation light from the two lasers are combined in one path through a set of dichroic filters ZT532nbdc and Z470rdc and directed through a 20×0.4 NA air objective to the same volume of excitation. Fluorescence emitted from the sample is collected by the same objective, transmitted through the set of dichroic filters, focused by a lens into a large pinhole (diameter =2 mm), and then collimated by a second lens to the detectors. A dichroic beam splitter T5501pxr-25mmNR separates the emission beam into two light paths prior to its detection by two photomultiplier tubes (PMT). Two emission filters (FF01-HQ 500/24-25 and LP5600) are located in front of each PMT. The signal from the PMT is sent to the analog to digital converter (ADC) and to the acquisition card. The sampling frequency is set to 100,000 Hz, corresponding to a time resolution of 10 μs.
In alternative embodiments, the optics of the microscope is designed to measure a relatively large volume (100 pL) in about 0.01 ms. The rotation of the tube in a spiral motion for about 100 seconds allows us to effectively explore about 1 ml of the tube. When using this exemplary optical setup, the device is penetrating only 150 μm into the sample. Therefore, strongly scattering samples such as whole blood (even before dilution) that have a transmittance at 500 nm of about 10% for a 250 μm path length can be easily handled.
In alternative embodiments, the invention provides alternative designs of the exemplary IC 3D system of the invention (
We encapsulated bacteria spiked blood and DNAzyme sensors into droplets as we described previously (see Example 1). Compartmentalization of target-specific reactions is a critical step to “light up” the droplet “reactors” that contain target bacteria so that they can be detected by the 3D particle counting system. Droplets were collected in a cuvette (
In alternative embodiments, the invention includes a pattern recognition algorithm (
To determine the minimal DNAzyme reaction time that is required in our IC 3D system to detect bacteria in unprocessed blood, we monitored the signal from a 2 ml droplet solution over time using our 3D particle counter (
We next demonstrate that our system can provide absolute quantification of target bacteria at a broad range of extremely low concentration from 1 to 10,000 bacteria ml−1 with single-cell sensitivity and an exceptional limit of detection (LOD) in the single digit regime (
To demonstrate the potential clinical applicability, we tested our system using clinical bacterial isolates obtained from positive blood cultures. We found that our IC 3D system can selectively and robustly detect clinical E. coli isolates with a performance similar to what we observed for positive control E. coli K12 (
In alternative embodiments, exemplary methods and systems of the invention comprising single-cell detection serve as a platform for the detection and screening for slowly-growing organisms (e.g., Mycobacterium tuberculosis).
In alternative embodiments, exemplary methods and systems of the invention serve as a platform technology where other types of sensors can be employed to selectively and sensitively detect almost any type of rare species in the blood including cells (e.g., bacteria, circulating tumor cells and stem cells), viruses, and other low abundant molecular targets.
In alternative embodiments, in addition to DNAzyme sensors, other sensing systems (e.g., digital PCR) for known target genes or molecules can also be integrated with our droplet microfluidics and 3D particle counting system for rapid single bacteria detection.
In alternative embodiments, target bacteria can be further cultured and proliferated in the droplets to amplify the signal before measurement (
In alternative embodiments, a one or more parameters, including droplet size, reaction time, sensor concentration, fluorophore/quencher pair, blood dilution factor scanning time (1-10 min), RPM (200-1000) and PMT (photomultiplier tube) (200-800), can be optimized to achieve optimal performance (i.e., signal/background ratio, sensitivity, LOD and assay time), as illustrated e.g., in
In alternative embodiments, this invention provides a fully integrated IC 3D system that is a bench-top, single-step, sample-to-result diagnostic consisting three major components (
As a platform technology, the IC 3D system can integrate other sensing methods (e.g., enzymatic assays, PCR and isothermal signal amplifications) with droplet microfluidics and a 3D particle counter can serve as a platform for rapid detection and analysis of almost any type of low abundant markers in biological samples including cells (e.g., bacteria, circulating tumor cells and stem cells), extracellular vesicles (e.g., exosomes), viruses (e.g., HIV), and molecular markers (e.g., nucleic acids and proteins) (
In alternative embodiments, the invention provides IC 3D tests for antimicrobial resistance using fluorogenic substrates36 for beta-lactamases and carbapenemases, see e.g.
In alternative embodiments, the invention provides IC 3D test for routine detection and monitoring of cancer circulating tumor cells (CTCs), others markers and cancers e.g., nucleic acids, proteins, peptides, carbohydrates, lipids, small molecules, metal ions (
For many carcinomas, e.g., breast cancer, over 90% of deaths are due to metastasis to distant organs. As metastasis is a multistep process in which disseminating cancer cells must survive transport through the systemic circulatory system, attention has recently been directed towards analysis and quantification of CTCs for early-stage diagnosis, prognosis and monitoring disease course. As CTCs are very rare (one CTC per 107 leukocytes) and heterogeneous, traditional flow cytometry and immunological approaches (e.g., CELLSEARCH™ platform) are complex, expensive and time-consuming, and importantly, lack the sensitivity and specificity to reliably detect CTC in clinical settings. In alternative embodiments the invention provides a platform technology that selectively detects CTCs in non- or minimally processed patients' blood samples at single-cell sensitivity within minutes to hours. In alternative embodiments the invention provides a system that integrates novel fluorescent sensor technology, droplet microencapsulation and a 3D particle counter (i.e., the IC 3D). These sensors including e.g., DNA sensors are engineered to specifically react with the lysates of or intact target CTCs, leading to a rapid, real-time fluorescence signal. Patient samples (e.g., blood) can be mixed with sensor solution including cell lysis buffer within a microfluidic channel, which can be encapsulated in millions of individual picoliter droplets. While the invention is not limited by any mechanism of action, the confinement of CTCs in droplets significantly increases the concentration of target molecules (e.g., Her2 and EpCAM) that can be detected by the sensors in a rapid, real-time fashion. Therefore, the methods and systems of this invention represent a new paradigm in CTC detection which will potentially become a powerful tool for cancer diagnosis and prognosis, and monitoring disease progress and drug efficacy during therapy.
In alternative embodiments the invention provides microencapsulated sensor systems to detect rare cancer CTCs in clinical settings. In alternative embodiments, droplet microfluidics is integrated with sensors for rapid cancer CTC detection at single-cell sensitivity. In alternative embodiments, fluorogenic DNA sensors identified to specifically detect cancer biomarkers (e.g., Her2, EpCAM, CK19, and MUC1) are integrated with the droplet microfluidics system; where confinement of single CTC in droplets can significantly increase the sensitivity and shorten the detection time. Single-cell detection of CTCs from both buffer and spiked whole blood can be optimized.
To validate the ability of an exemplary device to detect CTCs from clinical specimens: patient blood specimens are used in correlation with patient diagnosis to determine the assay selectivity and specificity. Head-to-head comparisons are made using flow cytometry and CELLSEARCH™ platforms with respect to CTC detection selectivity, specificity and assay time.
The invention provides a platform technology that is suited for rapid and robust CTC detection and cancer e.g., breast cancer screening on a routine basis. In alternative embodiments, compositions, systems and methods of the invention are used for sequencing, personalized diagnostics and medicine, e.g., for detecting CTCs.
In alternative embodiments, compositions, systems and methods of the invention are used in genetic analysis, e.g., to detect a single cell gene or residue mutation, or to detect mRNA expression. In alternative embodiments, compositions, systems and methods of the invention are used to study and detect single cell heterogeneity based, e.g., on a gene or residue mutation or an mRNA expression level.
In alternative embodiments, cells are kept intact without lysis, which make it feasible for also using other tests or assays, e.g., such as immune staining or protein profiling. When used as “intact” cells while reagents (e.g., sensors, enzymes) can be delivered to the cell via viral or non-viral routes (e.g., transfection reagent, nanoparticles). In alternative embodiments, this invention includes a method to perform high throughput cell engineering at a single-cell level within the droplet. For instance, we have demonstrated that MCF7 cells can be encapsulated with transfection reagent containing GFP expression vector and engineered to express GFP (
In alternative embodiments multiple enzyme reactions are used, which can give strong and high specific signal. In alternative embodiments, isothermal reactions including e.g. rolling circle amplification (RCA) reaction can be done in serum, facilitating direct CTCs detection in blood (see
In alternative embodiments, cancer cells, e.g., CTCs, can be characterized or detected by their cell surface, intracellular and secreted markers (see e.g.,
In alternative embodiments, cancer cells, e.g., CTCs, can be characterized or detected by detecting cancer markers, e.g., a cancer protein (e.g., Prostate-Specific Antigen (PSA), Her2, EpCAM, CK19, and MUC1), a cell-free nucleic acids (e.g., DNA, mRNA, miRNA and SNPs), a cell derived particles (e.g., exosomes, microvesicles, apoptotic bodies), lipids, carbohydrates, peptides, enzymes, small molecules and ions (
In alternative embodiments, methods of the invention further comprise use (can be used in combination with) detection of cancer cells and markers by known assays, including nucleic acid based, antibody based, enzyme based, or chemical based, and the like. Biological samples can be first processed to reduce the volume and improve the purity by, for example, gradient centrifugation, washing, enrichment, cell lysis, magnetic bead capture and separation, and extraction, prior to the droplet encapsulation and subsequent analysis.
In alternative embodiments, methods of the invention includes detection, track, monitor single transplanted cells including e.g., stem cells and cancer stem cells. In alternative embodiments, the to-be-transplanted cells can be engineered with probes (e.g., enzymes, proteins) that can be secreted to blood or urine where they can be detected by the IC 3D. In alternative embodiments, the to-be-transplanted cells can be engineered with probes to be at downstream of a biological signaling event so the probes can only be activated and produced when a biological signaling event is turned on.
In alternative embodiments, methods of the invention further comprise detection of nucleic acids markers (both intracellular and cell-free circulating forms) including mRNA, DNA, miRNA, SNPs, and the like, which can be detected by PCR, RT-PCR, RCA, loop-mediated isothermal amplification (LAMP), nicking (e.g., EXPonential Amplification Reaction (EXPAR)), strand displacement, exponential isothermal amplification and hybridization, molecular beacons, aptamer, DNAzyme, or other real-time fluorescent sensors. In alternative embodiments, RCA combined with molecular beacon and nicking enzyme reactions can be used to detect nucleic acids markers and their mutations, see e.g.,
In alternative embodiments, methods of the invention further comprise an exemplary method of the invention comprising testing and optimization of AMPLIGASE™ (EPICENTRE, Madison, Wis.) ligation followed by nicking enzyme reaction for DNA mutation detection.
In alternative embodiments, methods of the invention further comprise an exemplary method of the invention comprising testing and optimization of T4 ligase ligation followed by nicking enzyme reaction.
In alternative embodiments, methods of the invention further comprise an exemplary method of the invention comprising testing and optimization of E.coli ligase ligation followed by nicking enzyme reaction.
In alternative embodiments, methods of the invention further comprise mRNA BRAF V600E mutation detection as an example. In such assays, following RCA reaction, signal can be amplified and produced by a variety of methods including DNAzyme based, strand displacement, or nicking enzymes.
Nucleic acid markers and mutations can also be detected by PCR and RT-PCR. For example, we have demonstrated the reactions of using PCR to detect BRAF V600E mutation and BRAF G464V, see, e.g.,
We have demonstrated PCR can be performed with plasma and blood samples, see, e.g.,
We have also demonstrated that Let-7a miRNA using exponential amplification reaction (EXPAR) by a combination of polymerase strand extension and single-strand nicking reactions, see e.g.,
In alternative embodiments, methods of the invention are used to detect protein markers (on cell surface or secreted), e.g., they can be detected by antibody-based ELISA, sandwich based, immunostaining, antibody capture, secondary antibody amplification, proximity ligation based, aptamer, DNAzyme, or other real-time fluorescent sensors.
In alternative embodiments, methods of the invention are used to detect cell surface or free protein markers, for example, detecting EpCAM and Her2, e.g., by standard proximity ligation based assays that can be followed by signal amplification. In alternative embodiments, PSA can be detected by a real-time DNA sensor, or using fluorogenic substrates.
In alternative embodiments, the invention provides rapid and sensitive systems or methods for detecting a biological, a physiological or a pathological maker, or a single molecule or a single cell using a target detection process with and without signal amplification integrated directly with a 3D particle detector (
Features:
Our systems possess the following unique features that cannot be easily achieved by traditional detection assays:
1) Low abundance markers (e.g., 1-1 million/mL)
2) Able to interrogate large sample volume (μLs to mLs) and high throughput
3) Rapid (minutes to hours)
4) Broad detection range
5) Multiplexable
6) No or minimal sample preparation is required.
Samples:
1) wherein the biological sample comprises a blood, serum, saliva, tear, stool, urine or CSF sample from a patient
2) wherein the samples are obtained from food, water and air.
Sample Preparation
The samples can be directly assayed with no or minimal (e.g., dilution) processing.
Standard, established biological sample preparation processes including dilution, purification, enrichment, extraction, centrifugation, cell lysis, magnetic bead assays, and washing steps, although not required, can be integrated into the invented assays.
Targets:
The target species that can be detected and analyzed by the invented systems include, but not limited to (
Cells (e.g., cancer cell, stem/progenitor cell, immune cell), pathogens (e.g., bacteria, multi-drug resistant organisms (MDRO), tuberculosis (TB)), viruses (e.g., HIV), cell-derived vesicles (e.g., exosome, microvesicles, apoptotic bodies), nucleic acids (e.g., SNPs, mutations, expression), proteins (e.g., PSA), enzymes (e.g., MMPs), peptides, lipids, carbohydrates, polysaccharides, small molecules or metal ions.
The forms of target species include cell surface (e.g., EpCAM, N-cadherin, CD44, CD24), intracellular, and secreted markers (cell secretome), cell free circulating markers (e.g., miRNA, DNA, protein markers), metabolic markers, mechanical markers (e.g. cell deformability, stiffness, cytoskeleton, etc).
In addition of the expression, the invented systems can also be used to detect or monitor a biological event, e.g. DNA hybridization, protein receptor-ligand interaction, enzyme-substrate interaction, and cell surface receptor dimerization (including both homo and hetero-clustering), co-localization, or interaction with soluble ligands and drugs and another cells.
Target Detection Assays
There are a wide variety of established fluorescence bioassays that can be utilized in our system to selectively detect the targets for 3D particle counter analysis. Such assays include, both not limited to, (
Some more specific examples are given below:
Nucleic acid based assays including hybridization, molecular beacons, aptamer, DNAzyme, or other real-time fluorescent sensors.
Antibody-based assay include ELISA, sandwich based, immunostaining, antibody capture, secondary antibody amplification, or proximity ligation based.
In RCA-based detection, the target recognition binder is a biological or chemical moiety including aptamer or antibody. RCA can be a linear or branched (i.e., exponential amplification). RCA products can be loaded, stained and analyzed by dyes, nanoparticle or quantum dots.
3D Particle Counter
3D particle counter can be an instrument system as shown in
Integrated Exemplary Systems of the Invention
Our systems can be engineered with desirable portability, automating fluid handling, and integrating electronics including a diode laser (light source), APD (detector), Operating (vinci, ISS Inc.) & data analyzing software (SimFCS), display, with a 3D particle counting system. This envisioned device can also be integrated with multiple disposable microfluidic “cartridges,” permitting multiplex and rapid detection of multiple types of targets simultaneously. The device can be fully automated, and can be fabricated as an all-in-one system or with modular components. It can also be linked to smart phone and bluetooth etc for point-of-care applications (
Applications
The invention's novel approach of target detection process (with or without signal amplification) and the 3D particle counter system is innovative and powerful: it permits selective detection of target species in biological samples in mL volume within minutes that is currently not possible. Therefore, we believe our technology has the potential to revolutionize how we detect and analyze low concentration biological particles and markers and can be utilized in a large variety of detection bioanalysis and diagnosis applications including, but not limited to:
Infectious diseases Pathogens (bacteria, viruses, fungi, etc). Skin infection, wound, diabetic ulcer, HIV, bacteria, TB, MDROs (e.g. MRSA)
Cancer
Diabetes
Alzheimer disease (e.g., Amyloid beta, Tau proteins)
Inflammatory and autoimmune diseases (e.g., CD4 T cell, immune cell count)
Stem cell and regenerative medicine (e.g., mesenchymal stromal cells, endothelial progenitor cells, hematopoietic stem cells, or the cells can be endogenous or exogenously transplanted cells)
Cardiovascular diseases (e.g., C-Reactive Protein (CRP), B-type natriuretic peptide (BNP), troponin, Cystatin C, IL-6)
Drug and abuse (e.g. Tetrahydrocannabinol, THC)
Newborn screening
The system can also be used to study new biology, cell-drug interactions and drug susceptibility, to develop new drugs and therapeutics and monitor disease progress and treatment efficacy or used as companion diagnostics, and to be used in sequencing, personalized diagnostics and medicine.
In addition to medical applications, our system can also be used for other areas including food industry, agriculture, water systems, air systems, and defense applications.
Rolling Circle Amplification Coupled Detection with 3D Particle Counter:
In alternative embodiments, this invention includes a novel detection system that integrates rolling circle amplification (RCA) and a 3D particle counter (
Cancer Cell Detection Using 3D Particle Counter:
Cells, for example cancer cells, in biological samples can be stained, processed and directed detected by 3D particle counter (
The following describes an exemplary method of the invention comprising in vitro evolution to generate cancer-specific DNAzyme sensors.
The invention provides a technology that exploits powerful in vitro evolution to generate reliable, DNAzyme sensor-based cancer diagnostics, as illustrated in
In alternative embodiments, the methods and systems of the invention can be used in the clinic to detect almost any kind of cancer (
In alternative embodiments, the methods and systems of the invention for e.g. cancer sensor screening have many innovative features compared to current technologies (e.g., proteomic biomarker technology). The combination of powerful in vitro selection techniques and targeting the complex cancerous sera as a whole allows us to develop generic and reliable diagnostics without the need for identification of any specific disease biomarkers. The activator of a given DNAzyme can be a protein, a nucleic acid, a small molecule, or metal ions, etc. This is particularly advantageous as it allows us to bypass the tedious process of purifying the target molecules from extremely complex mixtures for developing detection methods: i.e., once isolated, the DNAzyme sensors can be immediately used for cancer detection. The multiple rounds of enrichment and amplification necessary for identification of DNAzyme sensors not only minimizes the high rates of false positive and negative results inherent in traditional methods of biomarker discovery (e.g. 2D gel electrophoresis coupled with MS)1-3 but also allow us to identify the modest differences existing between some cancers and normal tissue. We can also mix multiple patients' serum samples together as the target in order to bypass the non-specific heterogeneity between patients, and therefore truly identify the molecular differences that uniquely discriminate cancer and normal samples. Additionally, our system has the potential to generate multiple DNAzyme sensors simultaneously in the same enriched library pool that respond to a panel of molecular signatures that collectively detect cancer with significantly higher sensitivity and specificity than other single biomarker based assays. Finally, our resultant assay has many appealing features, one of which is its inherently rapid, real-time, mix-and-read nature, which is ideal for rapid screening and monitoring of cancers on a routine basis.
In alternative embodiments, DNAzyme sensors can be optimized towards optimal performance, e.g., signal/background ratio and stability, for e.g., working in whole blood. In alternative embodiments, the invention provides blood-based diagnostics to distinguish established cancer cases from healthy controls with respect to sensitivity and specificity. Retrospective and longitudinal studies can be performed to further validate and test an assay performance in correlation to standard clinical diagnosis and blood tests (for example, ELISA for potential protein biomarkers found in the literature). DNAzyme sensor sensitivity and specificity can be optimized by an iterative, re-selection process.
In vitro Evolution.
Library design. A DNA library containing approximately 1014 random sequences is used for isolating DNAzyme sensors. As illustrated in
Positive and negative targets. Non-small cell lung cancer (NSCLC) was used as a model system because of its high mortality and urgent demand for early-stage diagnostics.1-3 Age- and gender-matched, nonsmoking healthy donor samples will be obtained. We choose to mix multiple patients' samples together in order to minimize non-specific variation between patients and preanalytical variability, and therefore only select the DNAzyme sensors that are universal (for same stage/type of cancer) and specific (between cancer patients and healthy donors). To avoid blood type antigen incompatibility, serum samples are used in the selection process. Mixing serum samples is commonly used in biomarker discovery and does not produce adverse effects (i.e., no immunogenic response is observed).38 Specifically, 10 NSCLC patient serum samples (0.5 ml each) (or healthy control serum samples) are mixed thoroughly, aliquoted, stored at −80° C., and used throughout in the entire selection process.
Selection. As illustrated in
Using this approach, we obtained 19 classes of DNAzyme sensors that exhibited consistently higher activity in NSCLC samples than in healthy donor sera (see
Characterize and engineer DNAzyme sequences towards optimal performance in blood. The identified DNAzyme sequences can be validated individually to make sure they are indeed capable of cleaving the substrate in the presence of target cancer but not normal sera. Additionally, while sera are used as the target during selection, clinical assays can also be performed using whole blood without any processing (i.e., mix-and-read). Therefore, we can characterize and modify identified DNAzyme sensors towards optimal performance with respect to signal/background ratio and stability in whole blood before we validate them clinically as cancer diagnostics.
Sequence performance analysis. In our experience, in vitro selection typically leads to 5-20 different classes (clones) of sequences.10 We can synthesize a representative sequence from each class from IDT. Each sequence can be tested for cleavage performance in the mixed cancerous patient and healthy sera separately. Two parameters, specificity (fluorescence signal ratio between cancer and normal sera) and kinetics (% of cleavage over time) will be studied. Specifically, the cleavage reactions can be conducted in a 96-well plate in 100 μL serum sample mixed in the selection buffer containing 100 nM DNAzyme sensors, and the cleavage activity can be monitored by plate reader based on the fluorescence signal enhancement in real-time. To further prove whether the signal is indeed due the cleavage at the cleavage site, the reaction mixtures can be analyzed by polyacrylamide gel electrophoresis. Because we hypothesize that in vitro selection may identify multiple DNAzyme sequences that define a unique panel of cancer biomarkers, we will carry forward all the sequences that meet the following criteria: 1) fluorescence signal ratio between cancer and normal sera >3, and 2) >50% molecules are cleaved in 1 h. The molecules that meet the above criteria will be combined and carried forward as a homogenous sensing solution in the following tasks.
Signal/background ratio of DNAzyme sensors in blood. The nature of our DNAzyme sensor (i.e., fluorophore and quencher are placed in close proximity and separated before and after adding target) warrants an extremely low background in the absence of target, but high signal in the presence of target.10 We typically obtain DNAzyme sensors that possess a signal/background ratio of >6-10 in buffer.10 When used in blood however, the autofluorescence of blood and interference of dyes (e.g., quenching) from the complex environment in the blood may compromise the signal/background ratio. Fluorescein and Dabcyl are initially chosen as fluorophore and quencher respectively in the selection process because of their simplicity, low-cost and the fact that the cleavage event is monitored by gel during selection. However, fluorescein/Dabcyl may not be ideal for using in the blood due to above-mentioned reasons. In this set of experiments, the fluorophore-quencher pairs including Cy3/BHQ2, Alexa 647/QSY21, TAMRA/BHQ2, Texas red/BHQ2 and Alexa 546/QSY9 (Glen research) are optimized to identify the one that is compatible with fluorescence detection in blood (i.e., not interfered with by blood autofluorescence) and reproducibly produces the highest signal/background ratio (i.e., >5).
Stability of DNAzyme sensors in blood. Since the DNAzymes are evolved directly in serum, we expect that they will be nuclease-resistant and stable in blood for at least the amount of time (i.e., 10 min) we use for selection. We can chemically modify the termini or backbone of DNAzymes (i.e., inverted T and phosphorothioates) which are established to increase the half-life of nucleic acids to up to hours or days in blood without compromising their functions.15 Alternatively, to protect the degradation of RNA linkage in the DNAzyme sensor, we can also include RNase inhibitor (ribolock, Fermentas) in the assay buffer.
Validate DNAzyme sensor specificity and selectivity across all stages of NSCLC. The isolated and optimized DNAzyme sensors can be tested for whether they are able to distinguish between people with NSCLC and healthy controls. Again, blood samples from established NSCLC patients at different stages are obtained and each sample is analyzed in triplicate with a numerical value of fluorescence for each sample before and after addition of the DNAzyme determined with a fluorescence plate reader. Samples can be normalized to background and analyzed to determine 1) specificity, 2) selectivity, and 3) response across different stages of NSCLC. DNAzymes can detect early (Stage 1) NSCLC for early detection of NSCLC. For all samples, head-to-head comparison can be made with ELISAs for carcinoembryonic antigen (CEA) and cytokeratin 19 fragment
(CYFRA 21-1), two biomarkers previously established as relatively sensitive and specific for NSCLC, although not fully clinically validated.1,2 Significance of experimental results can be determined with T-test.
In alternative embodiments, in practicing the invention, a re-selection component in the DNAzyme sensor development is integrated in order to optimize the properties of the DNAzyme sensors (i.e., 90% for both sensitivity and specificity). Re-selection is a process whereby the identified DNA sequence is partially randomized to provide the starting library for a new selection process where more stringent selection criteria will be enforced.15 Re-selection operates more efficiently with fewer rounds required than the first selection to generate desirable molecules. Indeed, re-selection has been used to improve the sensitivity and specificity of DNAzymes.15
If the sensitivity and specificity of our DNAzyme assays do not meet the 90% criteria in clinical tests, a re-selection process can be performed whereby DNAzyme sequences identified are partially randomized (30% mutation at each base position; for example if the original base is A, it will be kept 70% A, 10% each of C, T, and G), and chemically synthesized by IDT. The in vitro selection procedure is repeated as described above, except that more stringent and selective positive and negative selection targets are used. For instance, the group of patent samples that failed to be detected by initial DNAzyme sensors are segregated and used as the target for selection. In order to more effectively discriminate between cancer patients at different stages, one of them is used as the negative selection target for the other instead of using the healthy donor. The optimization using re-selection can allow selection of DNAzyme sensors that are universal (for same stage/type of cancer) and specific (between cancer, healthy donors or other disorders that share similar symptoms (e.g., lung inflammation), and between cancer at different stages).
Thus, the invention provides methods for making optimized DNAzyme sensors for sensitivity and selectivity (both >90%). DNAzyme sensors of the invention can be used as screening tools to identify patients at high risk of cancers at earlier stages than existing technologies. To definitively confirm and stage cancer, other traditional diagnosis tools, especially imaging techniques including CT and MRI can be used following our screening assays.
In alternative embodiments, we developed a drug screening and in vitro selection platform based on one type of molecule one droplet strategy, e.g.,
In alternative embodiments, this invention presents an exemplary method termed “ENcapsulated ScreeNing of Aptamers by Reporter Amplification (ENSNARA)” for aptamer screening. As shown in
In alternative embodiment, an exemplary allosteric enzyme sensing system comprises a covalently linked inhibitor-DNA-enzyme (IDE) complex, which can be similar to a previously described constructs, for example, as described by Saghatelian, et al. “DNA detection and signal amplification via an engineered allosteric enzyme”, J. Am. Chem. Soc. 125, 344-5 (2003); Gianneschi, et al. Design of molecular logic devices based on a programmable DNA-regulated semisynthetic enzyme, Angew. Chem. Int. Ed. Engl. 46, 3955-8 (2007), and the like).
As shown in
In alternative embodiments of the aptamer IDE system of the invention, the DNA can be a synthetic DNA or other nucleic acid, e.g., a synthetic, non-naturally occurring nucleotide or a nucleic acid analogue, such as a peptide nucleic acid (PNA) containing non-ionic backbones, oligonucleotides having phosphorothioate linkages, or oligonucleotides having synthetic DNA backbone analogues such as phosphoro-dithioate, methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate, 3′-thioacetal, methylene(methylimino), 3′-N-carbamate, and morpholino carbamate nucleic acids.
In alternative embodiments of the aptamer IDE system of the invention, the complex can be designed to maximize “switching” (or on/off) ability; and libraries are designed to screen for aptamers of desired properties, for example, a structure where the γ-segment duplex dissociating is controlled by the aptamer binding affinity and the formed aptamer/target tertiary structure. Therefore, by incorporating γ-segment with different lengths in screening, aptamers with distinct affinity and switching efficiency are obtained. The dissociation of inhibitor from the catalytic site of the enzyme may or may not involve breaking the duplex DNA domain.
For example, in making exemplary IDE constructs for practicing this invention, the activity of each exemplary IDE construct can be measured in real-time by fluorescence detection in the presence of target ATP or thrombin (1 pM-100 μM). Addition of a 25-mer DNA complementary to the α-loop can be included as a positive control. Likewise, scrambled sequences in α-loop and GTP (for ATP) or albumin (for thrombin) can be used as negative controls. Outcome parameters used to quantify the performance of each aptamer IDE can include signal-to-background ratio, response time, sensitivity (or affinity, Kd), specificity and dynamic range. Kinetic parameters (Kcat and Km) can be further determined by measuring the reaction kinetics between the IDE construct and the fluorogenic substrate at different concentration ranging from 1 nM to 500 μM to construct velocity-substrate curves.
In one embodiment, a poly(dimethylsiloxane) (PDMS) chip containing channels with depth of 15-50 μm and width of 30 μm is fabricated using standard soft lithography, and mounted on a glass microscope slide. The PDMS device can have one oil inlet and two aqueous inlets (one for IDE library solution with the other one for target and substrate). Standard pressure infuse/withdraw syringe pumps can be used to deliver reagents and oil at flow rates ranging from 0.5 to 2 μL/min. Uniform picoliter-sized droplets can be generated at a rate of approximately 1,000 Hz by flow focusing of the resulting stream with HFE-7500 fluorinated oil containing 2% (w/w) EA surfactant. Droplets can be generated with different sizes (5, 10, 20 and 30 μm in diameter), which can be easily achieved by tuning the microfluidic channel size and flow rate. For FACS sorting, the formed water-in-oil (W/O) single-emulsion droplets can be introduced into 2nd microfluidic device with hydrophilic channels for the formation of water-in-oil-in-water (W/O/W) double-emulsion droplets. In order to minimize the effect of droplet generation time on the enzyme assay, a multi-layer microfluidic device that contains multiple, parallel droplet generating structures which is able to generate about 107 droplets within several minutes can be used. Fluorescent droplets can be imaged and detected using a confocal microscope which consists of 488/561/633 nm argon lasers and PMT detectors. Droplets can be sorted by FACS using a BD FACSAria II™ cell sorter which typically operate at a throughput of >107 droplets/hour.
For identifying a specific IDE for use in a particular assay or protocol, in one embodiment, an IDE library is encapsulated into droplets (which can be size optimized) using droplet microfluidics; for example, an initial library of about 1012 molecules can be co-encapsulated with target molecules (ATP or glutamate) and fluorogenic enzyme substrate (DABCYL-βAla-Ala-Gly-Leu-Ala-βAla-EDANS in about 107 drops (i.e., 105 IDE/droplet). After incubation, the fluorescent droplets that contain aptamer(s) can be sorted by FACS. The correlation between droplet fluorescence and aptamer affinity and switching properties enables identification and sorting of aptamers with defined properties simply by adjusting FACS gating parameters. Sorted droplets can then be collected in an Eppendorf tube held on ice and subsequently broken by adding an equal volume of 1H,1H,2H,2H-Perfluoro-1-octanol (Aldrich). Fresh substrate-containing buffer can be added to dilute the solution and also to increase the separation efficiency from the oil phase. The aqueous phase can be collected and re-encapsulated. After this partitioning procedure, it can be expected that only a single molecule IDE is contained within any given droplet. Once the aptamer-containing droplets are identified by the fluorescent signal, they can be separated individually by FACS, e.g., to a 384 well plate. Finally, after the droplet is lysed in the well, single aptamer molecules can be PCR amplified from IDE directly, and can be sequenced. A negative selection component where the IDE library is first incubated with control molecules (a mixture of GTP, TTP and CTP for ATP; a mixture of glutamine and asparagine for glutamate) can be used to eliminate IDE molecules that are not completely inhibited in the initial stage or DNA sequences that can turn on fluorescence signal via cross-reactivity or nonspecific binding. This negative screening step can enable generation of aptamers that are highly specific to the targets.
The identified aptamer sequences can be characterized, e.g., the identified aptamer sequences can be validated individually to 1) ensure that they specifically bind to and are capable of switching in the presence of targets but not controls, and 2) identify the sequences that generate optimal properties (i.e., affinity, specificity, response time and switching efficiency). The fluorescence signals of each sensor can be monitored in the presence of target (e.g., ATP or glutamate) or their respective controls in a range of concentration (e.g., 1 pM to 100 μM) in real-time using a plate reader. This identifies key properties of identified aptamers/sensors including affinity (Kd), sensitivity, selectivity, signal/background ratio, response time, and dynamic range. Surface plasmon resonance (SPR) (BIAcore 3000™) can be used to further evaluate the binding kinetics (Kon and Koff) and reversibility of the identified aptamers. For example, this set of tests can identify a sensor construct for neurotransmitter imaging, e.g., identifying rapid ligand association and dissociation sensors that permit analysis of the transient (on the order of ms) pulses of neurotransmitters for synaptic transmission.
As illustrated in
In alternative embodiments, ENSNARA can utilize IDE with different structures, architectures and compositions. In alternative embodiments, ENSNARA can employ other signaling amplification processes including e.g., EXPonential Amplification Reaction (EXPAR). In alternative embodiments, ENSNARA can be optimized by numerous parameters including droplet size, reaction time and molecular concentrations in the droplet. In alternative, droplet size can range from between about 5 to 50 μm in diameter.
While the invention is not limited by any particular mechanism of action, in alternative ENSNARA embodiments:
(i) the aptamer conjugated to the IDE can dissociate the inhibitor from enzyme catalytic site to produce a fluorescent signal in response to the binding of target molecules. This is supported by:
ii) that the fluorescence signal triggered by a single aptamer switch can be detected in droplet due to the enzyme reporter signal amplification. This is supported by extensive previous studies including digital PCR and data presented in this invention that the compartmentalization of target enzymes in picoliter droplets permit single molecule detection by increasing the effective target concentration and signal-to-background ratio.
In alternative embodiments, exemplary ENSNARA systems and methods of the invention offer unparalleled sensitivity and throughput for rapid screening of aptamers with defined properties. In particular, the ability to detect single molecule in picoliter (pL)-sized droplet, and this invention's droplet “Break-Dilute-ReEncapsulate” partitioning procedure, allows direct screening of a library with a diversity of as high as approximately 1012 in a single round. In alternative embodiments, exemplary ENSNARA circumvents the lengthy amplification steps necessitated by traditional SELEX (Systematic Evolution of Ligands by EXponential enrichment).
In alternative embodiments, once the aptamers are identified, they can be directly used as structure switching sensors without the need for additional modification and optimization66,68. In addition, the IDE system itself is not only a powerful aptamer screening platform but can also serve as a standalone, ultrasensitive and reversible sensor.
In alternative embodiments, the ENSNARA system or protocol of the invention is automated, e.g., in a microfluidic device; for example, by automating this system in a microfluidic device multiple targets can be selected for simultaneously.
In alternative embodiments, the ENSNARA systems or protocols of the invention comprise a single-round screening approach, which can circumvent a need for PCR amplification; and can also allow for the initial library to be composed of modified nucleotides, which can further increase the diversity and screening efficiency for high-quality aptamers.
In alternative embodiments, the ENSNARA systems or protocols of the invention comprise a new aptamer screening technology that can create a toolbox of real-time sensors for studying molecule and cellular signaling in vitro and in vivo, thus elucidating the biology and developing new therapeutics. In alternative embodiments, the ENSNARA systems or protocols of the invention comprise a rapid and reversible aptamer sensor system that permits continuous and real-time monitoring of neurotransmitters with high spatiotemporal resolution. In alternative embodiments, the ENSNARA systems or protocols of the invention comprise a platform for the design of many aptamers that can be used as probes to study complex biology, or as diagnostics and therapeutics.
A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US14/57282 | 9/24/2014 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61881827 | Sep 2013 | US | |
| 61987078 | May 2014 | US |
