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WO2017100442A1 - Amplificateurs de nanoparticules et leurs utilisations - Google Patents

Amplificateurs de nanoparticules et leurs utilisations Download PDF

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Publication number
WO2017100442A1
WO2017100442A1 PCT/US2016/065607 US2016065607W WO2017100442A1 WO 2017100442 A1 WO2017100442 A1 WO 2017100442A1 US 2016065607 W US2016065607 W US 2016065607W WO 2017100442 A1 WO2017100442 A1 WO 2017100442A1
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Prior art keywords
particles
recruiting
nanoparticles
catalyst
binding
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Eric Stern
Alec Nathanson Flyer
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Selux Diagnostics Inc
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Selux Diagnostics Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/682—Signal amplification

Definitions

  • the present invention generally relates to amplification methods useful for high- sensitivity assays.
  • Sequence-specific detection of low concentrations of nucleic acids is typically achieved through the use of enzyme-catalyzed reactions, such as the polymerase chain reaction (PCR).
  • enzyme-catalyzed reactions such as the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • these procedures require precise reaction conditions, detailed sample preparation, and carefully controlled reagent storage.
  • these powerful tools are largely absent from all environments beyond centralized laboratories.
  • Sequence-specific nucleic acid detection is a key underpinning of genomics research in particular and biomedical research overall.
  • This dominant technique, PCR, and all others available including loop-mediated isothermal amplification (LAMP), self- sustained sequence replication (3SR), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), and rolling circle amplification (RCA), require enzymes to amplify the signal.
  • LAMP loop-mediated isothermal amplification
  • NASBA self- sustained sequence replication
  • SDA strand displacement amplification
  • RCA rolling circle amplification
  • the environmental sensitivities of the required enzymes has broadly limited these techniques to laboratory settings and added significant difficulties to their field use.
  • the "template” strand of DNA to be amplified is denatured.
  • oligonucleotides (oligos), bind at the 5' ends of the template strands.
  • a DNA polymerase enzyme elongates the entire template strand in the 5' to 3' direction, beginning where the primers have bound. Each cycle thus doubles the number of DNA strands, resulting in a 2" amplification of the nucleic acid for n cycles.
  • EIAs enzyme-amplified immunoassays
  • these enzymes suffer from similar limitations to those of PCR.
  • the PCR enzymes are used to amplify protein signals. By relying on PCR amplification, these techniques significantly enhance the sensitivities of standard EIAs.
  • their complexities and environmental sensitivities have limited their field use to date.
  • the invention features a method of detecting one or more analytes in a sample comprising:
  • each nanoparticle is functionalized with moieties enabling specific binding of an analyte and a plurality of capture particles is functionalized with moieties capable of binding the same analyte;
  • the invention features a kit for detecting one or more analytes in a sample comprising:
  • each nanoparticle is functionalized with moieties enabling specific binding of an analyte
  • FIG. 1 is a schematic showing the steps in the cascaded-amplification processes used in detecting a specific nucleic acid sequence.
  • FIG. 2 is a primer design for nucleic acids and comparison with PCR primers.
  • FIG. 3 shows the TCO-Bz-Biot heterobifunctional crosslinker structure.
  • FIG. 4 shows FAMP particles comprising the TCO-Bz-Biot crosslinker in a poly(/.-lactic acid) (500 kD) matrix.
  • FIG. 5 shows the release of the TCO-Bz-Biot crosslinker.
  • FIG. 6 shows a comparison of activities of horseradish peroxidase (H RP) enzyme (Sigma Aldrich) and smal l molecule catalyst, metal-tetraamidomacrocyclic ligand complex (MTALC or TAML; GreenOx Catalysts; US 6,100,394).
  • H RP horseradish peroxidase
  • MTALC metal-tetraamidomacrocyclic ligand complex
  • FIG. 7 shows effective containment of MTALC in SAMP particles.
  • FIG. 8 shows specific detection of Listeria Monocytogenes genomic DNA (obtained from ATCC, Inc.) was achieved with the cascaded-amplification process using primers specific for 16S rDNA genes.
  • FIG. 9 shows specific detection of Dengue synthetic RNA (obtained from ATCC, Inc.) was achieved with the cascaded-amplification process using primers given by ATCC.
  • FIG. 10 shows specific detection of Yersinia Pestis genomic DNA (obtained from ATCC, I nc) was achieved.
  • FIG. 11 shows specific detection of Listeria Monocytogenes bacteria was performed in spiked lettuce samples.
  • FIGS. 12A-12C shows a schematic of magnetic fractionation device (1201) (FIG.12A) and fluorescence micrographs of red channel-active magnetic particles 2.85 ⁇ in diameter (1202) (FIG.12B) and green channel-active particles 0.86 ⁇ in diameter (1203) (FIG.12C).
  • FIG.13 shows a multiplexed Y. Pestis and Dengue test was performed at the highest dilution.
  • FIG.14 shows a comparison of human chorionic gonadotropin (hCG) detection with standard ELISA (“HRP”) and with FAMP particle binding and recruiting agent release followed by HRP signal generation (“FAMP”).
  • HRP human chorionic gonadotropin
  • FIGS.15A-15E relate to CAN design and performance.
  • FIG.15A is a schematic
  • FIG.15B is a schematic illustration of (i) CAN binding in a sandwich immunoassay, (ii) subsequent CAN bursting to release the biotin-TCO crosslinker that binds surface Tz groups, and (iii) final binding of streptavidin-HRP for signal development.
  • FIG.15C is a CAN-amplification comparison with standard, HRP-based ELISA for 6-hCG. Error bars represent ⁇ 1 standard deviation.
  • FIG.15D is a CAN-amplification comparison with tyramide amplified "high-sensitivity" ELISA for IL-6. Error bars represent ⁇ 1 standard deviation.
  • FIG.15E is a comparison of tyramide amplified ELISA and CAN amplification for 4 human plasma samples (HS) and 2 IVF embryo culture media samples. These were from a sample that comprised an embryo for 5 days (IVF 1) and a control sample without an embryo (IVF 2). The tyramide amplification data point for sample IVF 2 was below the detection threshold. Error bars represent ⁇ 1 standard deviation. For FIGS.15C-15E, error bars may not be visible due to resolution constraints when the relative error is ⁇ 5%. Experiments were repeated at least 3 times in triplicate with similar results.
  • FIGS.16A-16E relate to SAL design, synthesis and performance.
  • FIG.16A is a
  • FIG.16B is a catalytic comparison of HRP and TAML after 15-minute endpoint measurements. The plot compares results from optimal conditions for each catalyst and the inset shows the TAML chemical structure. The dashed lines are linear best fits for each dataset with R 2 of 0.997 for HRP and 0.987 for TAML. The signal was normalized to "1" at zero
  • FIG.16C shows that during the oil-in-oil emulsion step, the dispersed phase (gray) comprises poly-/.-lactic acid (PLLA) and TAML and is stabilized by a surfactant (blue/black), poly(maleic anhydride-o/f-octadecene).
  • FIG. 16D shows that upon inversion into water, a crosslinked polystyrene shell (blue) comprising styrene and divinyl benzene is polymerized around the nanoparticle cores. These dispersed-phase reactions are stabilized by DSPE-PEG surfactants (green).
  • FIG. 16E is a SAL-alone comparison with H RP- based ELISA for C. difficile Toxin A. The signal was normalized to "1" at zero
  • FIGS. 17A-17D relate to CAN-SAL cascade design and performance.
  • FIG. 17A shows a schematic illustration of CAN-SAL cascade, i-iv depict the reaction scheme, as described in the text.
  • the crosslinker structure is in FIG. 3.
  • B Theoretical amplification abilities of assays based on an ELISA baseline of " ⁇ ,” as described in the text. The amplification of PCR relative to ELISA is based on the 1 pM HRP LoD determined in FIG. 2B, which corresponds to 6xl0 7 molecules.
  • C CAN-SAL detection of L. monocytogenes bacteria using a single CAN oligo sequence. Signals were normalized to "1" at zero DNA concentration and errors were propagated.
  • Error bars represent ⁇ 1 standard deviation.
  • (D) CAN-SAL detection of L. monocytogenes bacteria using four CAN oligo sequences permitting multiple CAN binding events per template strand. Signals were normalized to "1" at zero DNA concentration and errors were propagated. Error bars represent ⁇ 1 standard deviation. Error bars in panels (C) and (D) may not be visible due to resolution constraints when the relative error is ⁇ 5%. Experiments were repeated 5 times in triplicate with similar results. L. monocytogenes isolates were selected on 3 different days.
  • FIGS. 18A-18C relate to CAN-SAL performance with low-cost SiPM detector.
  • FIG. 18A shows CAN-SAL detection of purified L. monocytogenes DNA from buffer and complex media. Arrows show the "Low” (green) and “High” (blue) concentrations used in FIG. 18C. Signals were normalized to "1" at zero DNA concentration and errors were propagated. Error bars represent ⁇ 1 standard deviation.
  • FIG. 18B shows a comparison of fluorescein detection with a PMT (Molecular Devices Spectramax M2) and a SiPM (inset). Signals were normalized to "1" at zero fluorescein concentration.
  • FIG. 18A shows CAN-SAL detection of purified L. monocytogenes DNA from buffer and complex media. Arrows show the "Low” (green) and "High” (blue) concentrations used in FIG. 18C. Signals were normalized to "1" at zero DNA concentration and errors were propagated. Error bars represent ⁇ 1 standard
  • FIG. 18C shows a real-time SiPM fluorescence signal from microfluidic chip assays for the "Low” and "High” L. monocytogenes DNA dilutions from FIG. 18A and a L. monocytogenes DNA-free control sample. Datapoints were collected at a rate of 1 per second and each plotted series represents a single experiment. All experiments were repeated at least four times with similar results.
  • FIGS. 19A-19F relate to multiplex detection of DNA, RNA and proteins.
  • FIG.19A is a schematic showing larger, red particles exhibiting increased magnetic susceptibility and exiting in the upper channel. Smaller, green particles remain in the lower channel.
  • FIGS. 19B and 19C are representative fluorescence optical micrographs of particles schematically illustrated in FIG. 19A. Larger, red fluorescent beads cross to the upper exit port (FIG. 19B), whereas the smaller, green fluorescent beads continue in the original stream (FIG. 19C). Note the red particles bleed into the green fluorescence channel. All experiments were repeated 3 times with similar results.
  • FIG. 19D is a comparison of dengue RNA and Y. pestis DNA detection "alone" (for dengue RNA or Y.
  • FIG. 19E is a comparison of PCR and CAN-SAL assays for 5. aureus and MRSA from human BAL samples. Positive samples are defined as those with normalized signals >1.3 (see FIG. 25B).
  • FIG.19F is a normalized -H Ia results for the same BAL samples. The hashed bar shows the -H la-positive 5. aureus sample. All signals were normalized to "1" for zero-concentration a-H Ia controls and errors were propagated. Error bars represent ⁇ 1 standard deviation. Experiments were repeated 3 times in triplicate with similar results.
  • FIG. 20 shows DLS and NanoSight analyses of SALs. These data show DLS as taken by a Malvern ZetaSizer and a laser optical image taken by a Malvern NanoSight of a representative SAL formulation. The PDI as determined by the ZetaSizer was 0.14.
  • FIG. 21 relates to storage stability of SALs. Comparison of newly synthesized and room temperature-stored USAs for performing a human cardiac troponin (cTnl) sandwich immunoassay. These data demonstrate the stability of the SAL platform. Human cTnl antibodies and a commercial kit were purchased from RayBiotech. An assay similar to that for C. difficile Toxin A described in the Methods section was used.
  • cTnl cardiac troponin
  • FIG. 22 relates to thermal stability of CANs.
  • the CAN-SAL cascade is further designed to give dual-primer/antibody specificity similar to that of PCR or ELISAs.
  • Oligo- functionalized CANs bind to oligo-functionalized magnetic capture beads only if the proper template strand is present, FIG. 15b(i).
  • the oligo sequence comprised a complementary DNA sequence followed by two spacers equivalent to 4-5 basepairs in length, which have been shown to be sufficient spacing from nanoparticles for rapid binding 18 .
  • CANs are designed for stability through 50°C, FIG. 22, and all CAN-template- magbead hybridizations are run at this temperature using standard PCR primers 7 .
  • TCO crosslinker 22 shows a measurement of the realease of TCO crosslinker from CANs after thermal treatments at different temperatures. Measurements were performed by coating a Nunc Maxisorp plate with Tz-BSA and using streptavidin-H RP (ThermoFisher) as the reporter. A standard curve was prepared using known concentrations of biotin-TCO crosslinker. Experiments were repeated 4 times with similar results.
  • FIG. 23 shows a primer design comparison between PCR and CAN-SAL assays.
  • CAN oligos can bind a single DNA/RNA strand, in comparison with PCR primers, where only one may bind each strand. It is important that the sense of CAN "Primer 1" is reverse and complementary that of PCR "Primer ⁇ .”
  • FIGS. 24A-24B show microfluidic chip layouts.
  • FIG.24A is an exploded view and FIG.
  • 24B is a complete view of a microfluidic chip used for L. monocytogenes DNA detection. Up to three assays can be run on a same chip. Recesses in the top layer are for N52- grade niobium magnets and optical windows allow detection of optical signals.
  • FIGS. 25A-25B show CAN-SAL S. aureus and MRSA identification.
  • FIG.25A is a
  • FIG.25B shows raw data from CAN-SAL assay for 5. aureus rRNA and mecA shown in FIG. 19E. DEFINITIONS
  • analyte broadly refers to any substance to be analyzed, detected, measured, or quantified.
  • examples of analytes include, but are not limited to, nucleic acids (e.g., DNA, RNA, PNA, LNA, BNA, and/or combinations thereof), proteins, small molecules, organisms such as microorganisms, viruses, peptides, hormones, haptens, antigens, antibodies, receptors, enzymes, polysaccharides, chemicals, polymers, pathogens, toxins, organic drugs, inorganic drugs, cells, tissues, bacteria, fungi, algae, parasites, allergens, pollutants, and combinations thereof.
  • nucleic acids e.g., DNA, RNA, PNA, LNA, BNA, and/or combinations thereof
  • proteins small molecules
  • organisms such as microorganisms, viruses, peptides, hormones, haptens, antigens, antibodies, receptors, enzymes, polysaccharides, chemicals, polymers, pathogens,
  • a "catalyst” e.g., a transition-metal catalyst
  • a conversion is a substance that increases the rate of a chemical reaction without itself undergoing any permanent chemical change, so as to covert a suitable substrate to a product, wherein the conversion results in a signal change.
  • the conversion leads to presence of increase of a detectable signal, e.g., the product releases a signal while the substrate does not.
  • the conversion leads to the diminishing or reduction of a signal, e.g., the substrate releases a signal while the product does not.
  • transition-metal catalysts described herein are precursors to the catalytically active species in a reaction.
  • SAL nanoparticle synthetic amplifier label
  • POC bedside point-of-care
  • the platform's ability to perform immunoassays as well as DNA and RNA assays - with sensitivities approaching quantitative PCR - by simultaneously identifying Staphylococcus aureus genetically and a specific protein toxin, -hemolysin, by immunoassay is demonstrated.
  • the SAL platform thus represents a novel, ultra-sensitive multiplex detection system that is compatible with current detector technology, potentially permitting rapid integration into high- volume laboratory testing in clinical and research settings.
  • nanoparticle labels are a variety of high-sensitivity assay technologies.
  • Nanolabels have emerged as powerful in vitro assay amplifiers compatible with a myriad of detection platforms - including optical platereaders - offering enhanced sensitivities, dynamic ranges, and multiplexing capabilities (References 7-9,13-17). Nanolabels have been used effectively as bio-barcodes (References 7-9) and with electrochemical (13-15), nuclear magnetic resonance (Reference 16), and time-resolved fluorescence (Reference 17) detection platforms, among other approaches. Many of these approaches have been shown to extend limits of detection (LoDs) beyond the capabilities of natural enzymes. However, nanolabels designed for compatibility with optical detection platforms have, to date, offered little to no improvement over standard ELISA sensitivities (References 18,19).
  • the invention features a method of detecting one or more analytes in a sample comprising:
  • each nanoparticle is functionalized with moieties capable of specific binding of an analyte and a plurality of capture particles is functionalized with moieties capable of binding the same analyte;
  • an analyte to be detected is independently a nucleic acid, a protein, a small molecule, an organism, or a virus.
  • an analyte to be detected is independently a nucleic acid.
  • a nucleic acid is independently a DNA or RNA.
  • moieties on the recruiting agent-comprising nanoparticles and the capture particles bind one or more different regions of the analyte to be detected.
  • particles each falls into the size range of 25 nm to 10 microns (e.g., 25 nm to 1 micron, 25 nm to 500 nm, 25 nm to 250 nm, or 25 nm to 100 nm).
  • a recruiting agent-comprising nanoparticles comprise an enzyme conjugate to facilitate signal generation.
  • an enzyme is H RP, AP, and/or ACE.
  • capture particles are susceptible to an external magnetic, optical, and/or acoustic field and/or a size exclusion gradient.
  • recruiting agents are chemical and/or biochemical crosslinkers, comprising one or more of the same or different functional moieties.
  • a catalyst-comprising species is one or more of particulate
  • a generated signal is optical and/or electronic.
  • particles bound to the analytes are addressable.
  • addressable particles are susceptible to different fields and/or gradients such that multiple analytes are detected in parallel.
  • a method further comprises mixing steps to prevent settling of the particles.
  • a method is performed in a glass, polymeric, or metallic vessel and/or microfluidic platform.
  • moieties on the recruiting agent are bound to a solid support.
  • a solid support is a particle susceptible to an external magnetic, optical, and/or acoustic field and/or a size exclusion gradient.
  • a sample pre-treatment is performed prior to the onset of the assay.
  • one or more of the moieties specific for the analytes to be any one or more of the moieties specific for the analytes to be any one or more of the moieties specific for the analytes to be any one or more of the moieties specific for the analytes to be any one or more of the moieties specific for the analytes to be any one or more of the moieties specific for the analytes to be any one or more of the moieties specific for the analytes to be any combination thereof.
  • detected is not functionalized to the recruiting agent-comprising nanoparticles and/or capture particles at the time of binding but, rather, binds one or more of these particle types after binding the analyte to be detected.
  • a recruiting agent reacts with the catalyst-comprising particles to expose the catalyst.
  • a method comprises adding one or more additional recruiting agent-comprising nanoparticles for multi-tiered amplification.
  • a recruiting agent-comprising nanoparticles or the catalyst- comprising particles do not comprise protein signal precursor molecules and/or carrier protein.
  • the invention features a kit for detecting one or more analytes in a sample comprising:
  • each nanoparticle is functionalized with moieties enabling specific binding of an analyte
  • particles each fall into the size range of 25 nm to 10 microns (e.g., 25 nm to 1 micron, 25 nm to 500 nm, 25 nm to 250 nm, or 25 nm to 100 nm).
  • a recruiting agent-comprising nanoparticles comprise an enzyme conjugate to facilitate signal generation.
  • an enzyme is H RP, AP, and/or ACE.
  • recruiting agents are chemical and/or biochemical crosslinkers, comprising one or more of the same or different functional moieties.
  • a catalyst-comprising species is one or more of particulate
  • kits further comprises a glass, polymeric, or metallic vessel and/or microfluidic platform.
  • kits further comprises a solid support.
  • a recruiting agent-comprising nanoparticles or the catalyst- comprising particles do not comprise protein signal precursor molecules and/or carrier protein.
  • FIG. 1 is a schematic showing the steps in the cascaded-amplification processes used in detecting a specific nucleic acid sequence. Capture particles (101), FAMP particles (102), and SAMP particles (103), are all shown. The recruiting agent payload (104) and MTALC (also referred to as TAML) catalyst (105) are also illustrated. Note in this schematic the solid surface binding partner of the recruiting agent is present on the capture particles themselves.
  • FIG. 2 is a primer design for nucleic acids and comparison with PCR primers.
  • Standard PCR primers are shown as 201 and 202. These are each designed to bind the 3' end of different strands of the template region. Cascaded amplification requires binding to each template strand at least one capture particle and at least one FAM P particle. (Both strands may be used.) Thus primer 203, which binds the capture particle, and 204 and 205, which bind the FAMP particle, all bind a single template strand.
  • FIG. 3 shows the TCO-Bz-Biot heterobifunctional crosslinker structure.
  • the trans- cyclooctene was selected because of its rapid reaction kinetics with tetrazine and the irreversibility of the resulting bond (Patterson et al. ACS Chem. Biol. Vol. 9 (2014), 592).
  • the biotin was selected because of its rapid binding kinetics with avidin (including streptavidin and neutravidin) and the very low dissociation constant of the existing bond (Patterson ef al. ACS Chem. Biol. Vol. 9 (2014), 592).
  • the benzene-comprising linker between the functional endgroups was chosen for its low aqueous solubility.
  • the primers described herein are designed to both bind the same template strand.
  • the "capture” particle is magnetically addressable and the first-amplification "FAMP” particle comprises a "recruiting agent” payload, such as a chemical crosslinker. This crosslinker is sealed within the FAMP particles, which are designed to be stable in neutral aqueous solutions. In order to replicate the specificity of PCR, the particles are designed to permit primer hybridization at 50-55°C.
  • a first-amplification "FAMP" particle is a recruiting agent- comprising nanoparticle as described herein.
  • capture particles are trapped in a
  • template strands tether FAMP particles to the capture particles; thus FAMP particles remain after washing only when template strands are present.
  • FAMP particles with multiple oligomer sequences specific for different regions of each template strand may be used such that multiple FAMP particles bind each template strand.
  • this design may be replicated on both complementary strands. In contrast to enzyme-based amplification methods, such as PCR, this technique permits direct RNA detection, thus there is no need for RNA-to-DNA conversion.
  • the first amplification step occurs when bound FAMP particles are "burst" to release the chemical crosslinker. This may be achieved with one or more of: a change in pH, a non-water solvent, and/or a physical trigger such as light, heat, or sonic energy.
  • the released crosslinker may then bind to a reactive moiety on a solid support, such as tetrazine.
  • a solid support such as tetrazine.
  • This support may be the surface of the chamber or channel or the surface of a particle and/or bead, including the capture particles themselves.
  • the solid support may be functionalized with a chemical moiety capable of reacting with one of the crosslinker's groups.
  • the functionalized solid support may be in the vicinity of the electrode.
  • the crosslinker may be designed for minimal aqueous solubility in the pH range of primer and/or protein binding. Soluble binding partners for the crosslinker may be included in the solution during primer binding to "catch" any crosslinkers released at this stage. In some embodiments, aqueous-soluble crosslinkers may be advantageous. Crosslinker solubility may be tuned by altering the linking groups, which can include benzene, phenyl, or similar aromatics, ethylene oxide groups, or aliphatic groups.
  • Each FAMP particle may comprise 10 2 -10 7 crosslinkers, and more preferably 10 3 -10 6 .
  • the ideal first amplification factor is defined as the number of crosslinkers per FAMP particle times the number of bound FAMP particles.
  • the real amplification factor may be lower than ideal due to incomplete crosslinker binding, crosslinker loss, or other effects.
  • SAMP particles are introduced that bind the free functional moiety of the crosslinker, such as with neutravidin.
  • SAMP particles comprise a signal generating payload, such as a catalyst, as described in US Application Numbers 62/029,270; 62/142,721; 62/053,250; 62/194,038; 62/194,046; 62/194,062; 14/809,116; and PCT/US2015/042133, which are incorporated fully by reference herein.
  • these particles are "burst" to release the catalyst, which catalyzes a signal-generating reaction, such as a reaction that produces an optical and/or electrochemical signal.
  • a second-amplification "SAMP" particle is a particle comprising a signal-generating agent (e.g., a catalyst-comprising particle) as described herein.
  • Each SAMP particle may comprise 10 2 -10 7 signal generating agents, and more
  • the ideal second am plification factor is thus the multiple of the number of catalysts per SAMP particle and the catalytic activity of the catalyst itself.
  • kits for use in performing the methods are provided.
  • kits can comprise one, two, three, four, or five of the following components:
  • each FAMP e.g., a nanoparticle
  • each FAMP is independently functionalized with moieties enabling specific binding of an analyte
  • kits may further comprise other components for performing the method.
  • a kit may further comprise a glass, polymeric, or metallic vessel and/or microfluidic platform.
  • a kit further comprises a solid support.
  • Samples may be derived from any biological, chemical, and/or other source.
  • the detection events may occur, e.g., in one or more of the following platforms: tubes, plates, automated, microfluidic.
  • Capture particles addressable by any external force may be used. Examples include, but are not limited to, particles with magnetic susceptibility; particles separable by size exclusion; particles addressable by optical, acoustic, or electrical trapping and/or gradients; and particles with specific surface functionalities to permit separation.
  • surfaces used for capture may also be macroscale, such as microplate wells. Macroscale surfaces may also be used for binding of a recruiting agent. These may be the same and/or different surfaces from those used for capture.
  • capture particles independently each fall into the size range of about 25 nm to about 10 microns (e.g., about 25 nm to about 1 micron, about 25 nm to about 500 nm, about 25 nm to about 250 nm, or about 25 nm to about 100 nm).
  • capture particles are susceptible to an external magnetic, optical, and/or acoustic field, and/or a size exclusion gradient.
  • capture particles are magnetically addressable (e.g., susceptible to an external magnetic field).
  • two different recruiting agents may be required in order for SAMP particles to bind.
  • two different FAMP particles with different oligo- functionalized surfaces would be required in order for a SAMP particle to bind.
  • capture particles requiring fields of different strength may be used for differential capture.
  • split flow microfluidic cells may be used to multiplex samples.
  • a primer may be of any nucleic acid type known to those skilled in the art
  • Primers comprising, but not limited to, DNA, RNA, PNA, LNA, BNA, and/or combinations.
  • Primers may or may not be fully complementary.
  • Primers may further comprise other molecules with binding partners including, but not limited to, e.g. aptamers, antibodies, receptors, ligands, cofactors, antagonists, glycoproteins, sugars.
  • Template strand(s) may similarly be one or more of any nucleic acid type or multiple types including, but not limited to, DNA, RNA, PNA, LNA, BNA, and/or combinations thereof.
  • the definition of template strands may be further expanded to comprise any molecule and/or species with one or more binding partners including, but not limited to, e.g. aptamers, antibodies, receptors, ligands, cofactors, antagonists, glycoproteins, sugars, viruses, bacteria, fungi, yeast.
  • one or more primers may not be bound to capture and/or FAMP particles but may comprise one or more moieties capable of binding to species functionalized on the surface of capture and/or FAMP particles.
  • moieties may include, but are not limited to, e.g. one or more nucleic acid sequences, antibodies, receptors, other proteins, ligands, cofactors, antagonists, glycoproteins, sugars, reactive groups including thiols, amines, hydrazides, carboxylic acids, aldehydes, ketones, maleimides, tetrazines, alkynes, strained alkynes, azides, trans-cyclooctene.
  • Capture particles may bind each template strand or may bind multiple strands resulting from a restriction digest, as known to those skilled in the art. FAMP particles may bind at multiple sites along a template strand.
  • FAMP particles may range in diameter that is about 25 to a bout 5000 nm. These particles may comprise one or more recruiting agent payloads, one or more matrix- forming agents, and one or more species for specific surface functionalization with primers.
  • a FAMP particle is a recruiting agent-comprising nanoparticle.
  • the matrix forming agent(s) may be designed to maintain integrity at moderate temperatures, 50-60°C, and to release the crosslinker upon introduction of a specific stimulus to provide, e.g., a dissociable FAMP particle (e.g., a dissociable recruiting agent- comprising nanoparticle).
  • a dissociable FAMP particle e.g., a dissociable recruiting agent- comprising nanoparticle.
  • Exemplary dissociable matrices are described in US Application Numbers 62/029,270; 62/142,721; 62/053,250; 62/194,038; 62/194,046; 62/194,062; and 14/809,116; as well as International Application Nos.
  • FAMP particles e.g., recruiting agent-comprising nanoparticles
  • FAMP particles are independently dissociable (e.g., a FAMP particle can be dissociated under, e.g., a physical trigger or a chemical trigger to release, e.g., a crosslinker).
  • a suitable trigger for dissociating a particular FAMP particle e.g., a recruiting agent-comprising nanoparticle
  • a particular FAMP particle e.g., a recruiting agent-comprising nanoparticle
  • Exemplary triggers are described in, e.g., Table 2 of International Application No.
  • PCT/US2016/042589 which is hereby incorporated by reference in its entirety, and include: a change in pH, a non-water solvent, and/or a physical trigger such as light, heat, or sonic energy.
  • a suitable trigger for dissociation of a particular FAMP particle e.g., a recruiting agent-comprising nanoparticle
  • a suitable trigger for dissociation of a particular SAMP particle e.g., a catalyst-comprising species
  • FAMP particles e.g., recruiting agent-comprising nanoparticles
  • FAMP particles independently each fall into the size range of about 25 nm to about 10 microns (e.g., about 25 nm to about 1 micron, about 25 nm to about 500 nm, about 25 nm to about 250 nm, or about 25 nm to about 100 nm).
  • FAMP particles e.g., recruiting agent-comprising nanoparticles
  • FAMP particles comprise an enzyme conjugate to facilitate signal generation.
  • an enzyme is HRP, AP, and/or ACE.
  • a FAMP particle (e.g., a recruiting agent-comprising nanoparticle) independently comprises an oligomer primer or an antibody for binding a target (e.g., a nucleic acid or protein, respectively).
  • a target e.g., a nucleic acid or protein, respectively.
  • said oligomer primer or said antibody binds the same target as a capture particle included in the same kit or to be used in the same method.
  • FAMP particles independently comprise different oligomer sequences specific for different regions of a template strand.
  • a kit comprises two or more (e.g., two or more, three or more, four or more, five or more, six or more, etc.) FAMP particles (e.g., recruiting agent-comprising nanoparticles), each independently comprising a different oligomer sequences specific for a different region of a template strand.
  • a method comprises the use of two or more (e.g., two or more, three or more, four or more, five or more, six or more, etc.) FAMP particles (e.g., recruiting agent-comprising nanoparticles), each independently comprising a different oligomer sequences specific for a different region of a template stra nd.
  • FAMP particles e.g., recruiting agent-comprising nanoparticles
  • a recruiting agent may be a crosslinker, such as a heterobifunctional crosslinker or homobifunctional crosslinker.
  • recruiting agents comprise chemical and/or biochemical
  • crosslinkers comprising one or more of the same or different functional moieties.
  • a recruiting agent comprises a chemical crosslinker.
  • a crosslinker is a heterobifunctional crosslinker.
  • a crosslinker is water-soluble.
  • a crosslinker comprises a functional group that modulates water solubility (e.g., a crosslinker comprises: benzene, phenyl, or similar aromatics; ethylene oxide groups; or aliphatic groups).
  • a crosslinker may also have more than two reactive moieties.
  • comprising two or more of the same reactive moiety may be designed to be sufficiently small to ensure only one moiety can bind its surface-immobilized binding partner.
  • Reactive moieties include, but are not limited to, e.g. one or more of the following: TCO, tetrazine, biotin, avidin, streptavidin, neutravidin, thiobiotin, alkyne, strained alkyne, azide, N HS ester, carboxyl, amine, thiol, maleimide, isocyanate, isothiocyanate.
  • a crosslinker comprises a functional moiety that comprises an amine, carboxylic acid, thiol, azide, alkene (e.g., cycloalkene or terminal alkene), alkyne (e.g., cycloalkyne or terminal alkyne), Ni, histidine, Cu, lysine, maleimide, N HS-ester, biotin, or a biotin-binding protein (e.g., avidin or streptavidin), or a combination thereof.
  • alkene e.g., cycloalkene or terminal alkene
  • alkyne e.g., cycloalkyne or terminal alkyne
  • Ni histidine
  • Cu lysine
  • maleimide N HS-ester
  • biotin e.g., avidin or streptavidin
  • a crosslinker is a biotin-benzyl-trans-cycloolefin (e.g., as described herein).
  • a cross-linker comprises a functional group that can bind to a
  • a recruiting agent-comprising nanoparticle independently
  • crosslinkers e.g., about 10 3 to about 10 s crosslinkers.
  • recruiting agent-comprising nanoparticles do not comprise protein signal precursor molecules and/or carrier protein.
  • Recruiting agents may also release the amplifiers of the SAMP particles (e.g.,
  • catalyst-comprising particles by acting to increase the porosity and/or decrease the integrity of these particles.
  • examples include, but are not limited to, metals that intercalate in the membranes of particles or that adjust the solubility of SAMP particles or constituents by releasing a pH modulating, reducing, oxidizing, or solvent.
  • different FAMP particles may comprise different recruiting agents. At least one functional moiety on each recruiting agent may be different in order to permit such multiplexing. Functional groups for this use may be expanded to include small molecules capable of specific antibody binding, such as steroids, cholesterol, etc.; nucleic acids; polymer nucleic acids; or similar species.
  • Released recruiting agents may bind to the same or different surfaces to which the FAMP particles bound. In embodiments, released recruiting agents may bind to the same template molecules to which the FAMP particles
  • FAMP particles may be prepared to additionally include signaling agents, signaling agent precursors, and/or signal generating agents, as described in US Application Numbers 62/029,270; 62/142,721; 62/053,250; 62/194,038; 62/194,046; 62/194,062; and 14/809,116; as well as
  • a signaling agent in a FAMP particle is a catalyst (e.g., TAML) as described herein.
  • the matrix-forming agents of the FAMP particles may be the recruiting agents
  • particles themselves or may be polymeric, inorganic, waxy, surfactant, etc. These particles may be solid or micellar or water-filled, e.g. liposomal.
  • FAMP species further be comprised of a block-copolymer comprising a primer head- group and a tail-group comprising pendant functional moieties capable of reacting and/or binding to the exposed SAMP functional groups.
  • SAMP particles comprise a signal-generating payload (e.g., a signal-generating payload).
  • a signal-generating payload e.g., a signal-generating payload
  • a SAMP particle is a catalyst-comprising particle.
  • a SAMP particle is a catalyst-comprising nanoparticle.
  • a SAMP particle comprises a catalyst as described in any of US Application Numbers 62/029,270; 62/142,721; 62/053,250; 62/194,038; 62/194,046; 62/194,062; and 14/809,116; as well as International Application Nos.
  • a catalyst is a metalorganic compound, which is a complex
  • a metal core e.g., Fe, Mg, Cu, Mn, Pd, Pt, Ag, Ru, or Ce
  • organic ligands e.g., porphyrin, substituted porphyrins, bipyridyls, bis-diimines, polydentates, ethanediamines, ethylenediamines, pentaaminecarbonatos,
  • tetraaminecarbonatos coumarins.
  • Specific examples include, but are not limited to, iron porphyrins, hemin, ruthenium diimines, ruthenium bipyridyls, iridium-coumarin complexes, bis(l,2-ethanediamine)copper, nickel porphyrin, and/or calcium
  • a catalyst that is a metalorganic compound comprises a metal core that is Fe.
  • a catalyst has a structure such as that described in any of U.S.
  • a catalyst is, comprises, or is formed from Fe(lll)-TAML° (sodium salt) metalorganic compound purchased from GreenOx (see, e.g., US Patent No.
  • a SAMP (e.g., a catalyst-comprising species) is particulate in nature, enzymatic in nature, and/or polymeric in nature.
  • a SAMP e.g., a catalyst-comprising species
  • a SAMP e.g., a catalyst- comprising species
  • a SAMP is polymeric.
  • a SAMP (e.g., a catalyst-comprising species) does not comprise protein signal precursor molecules and/or carrier protein.
  • a SAMP e.g., a catalyst-comprising species
  • a SAMP can be dissociated under, e.g., a physical trigger or a chemical trigger.
  • a suitable trigger for dissociating a particular SAMP e.g., a catalyst-comprising species
  • a SAMP particle that is dissociable may comprise one or more matrix forming agents, which may be designed to maintain integrity at moderate temperatures (e.g., 50-60°C) and to effect dissociation upon introduction of a specific stimulus.
  • matrix forming agents e.g., 50-60°C
  • Exemplary dissociable matrices are described in US Application Numbers 62/029,270; 62/142,721; 62/053,250; 62/194,038; 62/194,046; 62/194,062; and 14/809,116; as well as International
  • PCT/US2016/042589 which is hereby incorporated by reference in its entirety, and include: a change in pH, a non-water solvent, and/or a physical trigger such as light, heat, or sonic energy.
  • a SAMP (e.g., a catalyst-comprising species) comprises about 10 2 to about 10 7 signal generating agents (e.g., about 10 3 to about 10 s ).
  • a second amplification factor is the multiple of the number of
  • a SAMP (e.g., a catalyst-comprising species) comprises a moiety that binds a free (unbound) group on a crosslinker.
  • a crosslinker comprises biotin
  • a catalyst-comprising species comprises a biotin-binding protein such as neutravidin.
  • a crosslinker comprises a biotin-binding protein such as neutravidin
  • a catalyst-comprising species comprises biotin.
  • SAMP particles may be replaced with or used in parallel with one or more enzyme conjugates with reactive binding moieties, such as horseradish peroxidase-streptavidin, alkaline phosphatase-streptavidin, etc. They may also be replaced with a polymer chain with multiple signal generating groups in the backbone or pendant groups of the polymer. The outlined steps of the process need not occur sequentially. The FAMP and SAMP particles may be designed such that multiple steps may be performed in parallel.
  • microparticles and nanoparticles were purchased from Bioclone and Ocean
  • Oligonucleotides terminated with a 3' amino modifier were purchased from Integrated DNA Technologies (IDT). Starting at the 5' termini, these oligos comprised the primer sequence, followed by a "C18" spacer, and finally the 3' amino termination. Oligo-particle conjugation was performed according to the Bioclone instructions: particles at 200 mg/mL were suspended in a 5 g/mL solution of the oligo in the Bioclone suspension buffer. The sample was vortexed to ensure particles were suspended and then reacted overnight at 50°C. After 12 hours, the beads were washed twice with the Bioclone wash buffer using a magnetic separation stand
  • FAMP particles were synthesized with TCO-Bz-Biot (Conju-Probe) as the payload and poly(/.-lactic acid) (PLA; 500 kDa; PolySciTech) as the matrix forming agent.
  • PPA poly(/.-lactic acid)
  • PLA poly(/.-lactic acid)
  • PolySciTech poly(/.-lactic acid)
  • a poly(lactic acid)-poly(ethylene glycol)- maleimide block co-polymer of 30 kD:5 kD size was used (PolySciTech). These materials were dissolved in 2 mL chloroform in a 1:10:2 mass ratio.
  • FIG. 4 shows FAMP particles comprising the TCO-Bz-Biot crosslinker in a poly(/.-lactic acid) (500 kD) matrix were tested for their thermal stability.
  • Dynamic light scattering (DLS) measurements were performed using a Malvern ZetaSizer. These show a monodisperse starting particle population ("start") with a mean diameter of 133 nm and a polydispersity index (PDI) of 0.128. DLS following 50°C treatment for 30 minutes show similar monodispersity (“50°C”), mean diameter (133 nm), and PDI (0.127). DLS following 55°C treatment for 30 minutes again show similar monodispersity ("55°C”), mean diameter (133 nm), and PDI (0.127).
  • start monodisperse starting particle population
  • PDI polydispersity index
  • Bovine serum albumin (BSA) was functionalized with tetrazine using a N HS-reactive tetrazine crosslinker (Conju-Probe) and was bound to a MaxiSorp (Nunc) microplate in pH 9.5 sodium bicarbonate buffer. The filtrate was then introduced to the wells and the reaction was allowed to proceed for 1 hour at room temperature. A control dilution series "standard" was obtained using known concentrations of TCO-Bz- Biot in the same reaction buffer.
  • FIG. 5 shows the release of the TCO-Bz-Biot crosslinker was determined for 30 minute incubations at 50°C and 55°C and compared with storage at room temperature.
  • SAMP particles were formulated according to the methods described in US
  • the final MTALC determination was made by first introducing acetone to the filter and pipetting up-and-down, followed by the addition of 0.1 M sodium bicarbonate buffer (pH ⁇ 10), followed by centrifugation and collection. Standard curves were established with soluble MTALC for quantification and particle loading was determined using a NanoSight to measure particle concentration. The resulting calculations show an MTALC loading of ⁇ 60,000 per nanoparticle.
  • FIG. 6 shows a comparison of activities of horseradish peroxidase (H RP) enzyme (Sigma Aldrich) and smal l molecule catalyst, metal-tetraamidomacrocyclic ligand complex (MTALC or TAML; GreenOx Catalysts; US 6,100,394).
  • H RP horseradish peroxidase
  • MTALC metal-tetraamidomacrocyclic ligand complex
  • the molar activity of H RP is found to be ⁇ 10-fold greater than that of MTALC.
  • the molecular weight of H RP is ⁇ 44,000 g/mol and the molecular weight of MTALC is ⁇ 650 g/mol, thus MTALC has a ⁇ 6.5-fold greater per-mass activity.
  • HRP activity was measured using a commercial 3,3',5,5'-tetramethylbenzidine (TMB) solution comprising hydrogen peroxide at pH ⁇ 5.5 (ThermoFisher). MTALC activity was determined in a 0.1 M sodium bicarbonate buffer (pH ⁇ 10) comprising 30 ⁇ hydrogen peroxide and 600 ⁇ 2,7- dichlorodihydrofluorescein diacetate (DCFH-DA; Santa Cruz Biotechnology; Gomes et al. J. Biochem. Biophys. Methods Vol. 65 (2005), 45). Signals are normalized by dividing by the baseline zero values. All samples were run in triplicate and error bars show standard deviations. [0165] FIG.
  • MTALC-comprising nanoparticles were fabricated using the method disclosed in US Provisional Application entitled "Water-inverted oil-oil nanoparticle formulation for hydrophilic species encapsulation", field on even date. Particles were dialyzed into lx PBS and loaded into a microfuge spin-filter tube with a 20 kD membrane. Samples were spun and the filtrate was collected and tested for MTALC activity according to the procedure above.
  • Each "wash” consists of particle resuspension into an addition of an equal amount of PBST and subsequent centrifugation and filtrate collection. Particles were “burst” using acetone followed by the addition of sodium bicarbonate buffer at pH 10. The baseline fluorescence reading is shown as a dashed line and labeled "baseline.”
  • Genomic material was diluted 5-7 times in a 1:5 dilution series in lx SSC buffer (Sigma-Aldrich) comprising 20 nM salmon sperm DNA (Sigma-Aldrich) and 0.001% Tween 20. Samples were heated for 5 min at 95°C to denature the DNA (note this step was not performed for RNA genomic material) and samples were cooled to 50°C. While at 50°C, magnetic capture
  • microparticles prepared as in Example 1 and TCO-Bz-Biot-comprising NPs (prepared as in Example 2), both functionalized with the appropriate primers, were added.
  • the effective particle-bound primer concentrations were 2 ⁇ . Samples were mixed for 30 minutes during the 50°C hybridization to prevent the microparticles from settling.
  • Example 6 Each sample was sonicated for 10 minutes (Heat Systems - Ultrasonics, Inc) and then treated at 95°C for 15 minutes in order to inactivate the bacteria. The samples were cooled to 50°C and the procedure from Example 6 was then followed for the remainder of the test.
  • the split flow multiplexing chip was designed in-house and the CAD layout is shown in Figure 12.
  • the chip was fabricated by A-Line, Inc.
  • the design consists of a 50 mm channel with two equal channels at both the inlet and outlet, each of which is addressable individually with a hose barb connection.
  • Each inlet was fitted with 1/16" ID silicone tubing fed by a syringe loaded in a syringe pum p (Harvard Apparatus).
  • One syringe was filled with the solution comprising mixed Yersinia Pestis and Dengue samples after the completion of the 50°C hybridization step.
  • the second syringe was filled with lx SSC buffer.
  • the exit ports of the chip were fitted with similar tubing comprising valves, which allowed flows to be set.
  • the tubing and plastic chip were treated with bovine serum albumin to minimize binding.
  • a N52-grade neodymium magnet bar magnet (K&J Magnetics) was positioned ⁇ 2/3 the distance down the channel (from inlet to outlet). This was tuned empirically, together with syringe pump flow rates, such that 2 ⁇ magnetic particles would traverse laterally across the channel, while 200 nm magnetic particles would remain in their original channel region.
  • the Y. Pestis and Dengue samples were returned to 1.5 mL Eppendorf tubes and PBST washing was performed on the magnetic capture stand, followed by the release of the TCO-Bz-Biot crosslinker. The remainder of the procedure in Example 6 was then continued.
  • FAMP particles designed for protein binding were synthesized with TCO-Bz-Biot (Conju-Probe) as the payload and poly(/.-lactic acid) (PLA; 500 kDa; PolySciTech) as the matrix forming agent.
  • PPA poly(/.-lactic acid)
  • PLA poly(/.-lactic acid)
  • PLA poly(/.-lactic acid)
  • PLA poly(/.-lactic acid)
  • DSPE-PEG-biotin 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-/V-[biotinyl( polyethylene glycol)-2000]
  • DSPE-PEG-biotin Laysan Bio
  • the resulting solution was then added to chloroform-saturated deionized water (0.815% chloroform in water) comprising 0.1% sodium dodecyl sulfate.
  • the suspension was homogenized at 7,500 rpm (I KA) forming a milky, stable suspension, and was then processed with a Microfluidics LM lOmicrofluidizer at 3000 psi. This was subsequently added to a >300 mL solution of water, which clarified the solution. After clarification the particle solution was filtered through a 300 kD membrane (Millipore). The particles were concentrated to ⁇ 10 mL and stored in Dl.
  • Example 10 Example 10
  • Tz-BSA tetrazine-functionalized bovine serum albumin
  • the solution was then added to the BSA-Tz-coated microplate and binding was allowed to proceed for 1 hour at room temperature. Following washing, the streptavidin-H RP conjugate provided in the kit was added. Washing, 20 minute TMB development, and stop solution addition then proceeded reading the wells at 450 nm absorbance. For the "H RP control" group samples, following washing after biotinylated antibody binding, the streptavidin-HRP conjugate was then added per the kit instructions, followed by subsequent washing. After 30 minute TM B development and stop solution addition, the wells were read at 450 nm absorbance.
  • FIG. 8 shows specific detection of Listeria Monocytogenes genomic DNA (obtained from ATCC, Inc.) was achieved with the cascaded-amplification process using primers specific for 16S rDNA genes.
  • the specific sequences were 5'-CTA TCC ATT GTA GCA CGT G -modifiers-3' (SEQ I D NO:2) and 5'-modifiers-AGA ATA GTT TTA TGG GAT TAG-3' (SEQ ID N0:1) (Wang et al. Appl. Environ. Microbiol. Vol. 58 (1992), 2827).
  • the number of copies of DNA was determined by quantitative PCR and 1:5 dilutions (v/v) were performed in lx SSC buffer.
  • FIG. 9 shows specific detection of Dengue synthetic RNA (obtained from ATCC, Inc.) was achieved with the cascaded-amplification process using primers given by ATCC.
  • the specific sequences were 5'-TGA GTG CGT GTG TCC AGT CC-modifiers-3' (SEQ ID NO:6) and 5'-modifiers-CAT GTC TCT ACC TTC TCG ACT TGT CT-3' (SEQ I D NO:7) (Gijavanekar ef al. FEBS J. Vol. 278 (2011), 1676).
  • the number of copies of the synthetic RNA genome was determined by quantitative PCR and 1:5 dilutions (v/v) were performed in lx SSC buffer.
  • FIG. 10 shows specific detection of Yersinia Pestis genomic DNA (obtained from
  • FIG. 11 shows specific detection of Listeria Monocytogenes bacteria was performed in spiked lettuce samples.
  • the Kim L. Monocytogenes strain was purchased from ATCC, Inc. and cultured with ATCC Medium 44. Lettuce samples were prepared according to the procedure of Berrada ef al. Int. J. Food Microbiol. Vol. 107 (2006) 202. Briefly, store- bought romaine lettuce was homogenized and the resulting suspension was filtered through a 0.2 ⁇ syringe filter. A serial dilution of bacteria was added to 4 filtered samples. Cascaded amplification was able to distinguish ⁇ 250 colony forming units (CFUs).
  • CFUs colony forming units
  • FIGS. 12A-12C shows a schematic of magnetic fractionation device (1201) (FIG.12A) and fluorescence micrographs of red channel-active magnetic particles 2.85 ⁇ in diameter (1202) (FIG.12B) and green channel-active particles 0.86 ⁇ in diameter (1203) (FIG.12C). (Note the red channel-active particles bleed into the green channel.) The red particles moved across the channels in response to a bar magnet whereas the green particles were not sufficiently influenced to move.
  • This device is a type of field- flow fractionation known as split flow thin-cell fractionation (Giddings ef al. Science Vol. 193 (1976) 1244).
  • Fluorescent microparticles were purchased from Bangs; red fluorescent magnetic particles were 2.85 ⁇ diameter and green fluorescent magnetic particles were 0.86 ⁇ diameter. This design permits magnetic particles of different sizes to be separated into two distinct solutions. This is one method for multiplexing cascaded amplification assays.
  • FIG. 13 shows a multiplexed Y. Pestis and Dengue test was performed at the highest dilution. The multiplexed points are circled (1301 is Dengue and 1302 is Y. Pestis). The data for Y. Pesf/s-alone and Dengue-alone is plotted on the same graph for comparison. This demonstrates the multiplexing capability of the cascaded amplification assay.
  • FIG. 14 shows a comparison of human chorionic gonadotropin (hCG) detection with standard ELISA (“H RP”) and with FAMP particle binding and recruiting agent release followed by H RP signal generation (“FAMP”).
  • H RP human chorionic gonadotropin
  • FAMP FAMP particle binding and recruiting agent release followed by H RP signal generation
  • CAN performance was first evaluated in comparison with a commercial 6-human chorionic gonadotropin (6-hCG) ELISA.
  • Standard and CAN-enhanced ELISAs were run in parallel, after the CAN-plate had been blocked with Tz-conjugated BSA.
  • the CAN enhancement step produced a >100-fold sensitivity gain for the same endpoint absorbance reading. This permitted resolution of 6-hCG levels in in vitro fertilization (IVF) embryo culture media, which requires ultra-sensitivities because of small, ⁇ 20 ⁇ sample volumes.
  • Synthetic amplifier labels (SALs) each comprise >10 4 densely packed small SALs.
  • TAML iron-comprising tetra-amino metalorganic ligand
  • HRP tetra-amino metalorganic ligand
  • FIG. 16B molar activities within 5-fold those of this ELISA-standa rd enzyme
  • TAML is a charged, small molecule and is can pose challenges for
  • C. difficile is now the leading cause of hospital-acquired infections and conventional C. difficile toxin ELISAs have poor LoDs that compromise efficient diagnoses (Reference 37).
  • SALs achieved >50-fold sensitivity enhancements over conventional C. difficile ELISAs (FIG. 16E).
  • these C. difficile SAL results were similar to those achieved using the highest-sensitivity immunoassay platform currently available, digital ELISA (References 14,38).
  • Digital ELISA showed a LoD of 0.028 pg/mL for . difficile toxin A (38), compared with the 0.021 pg/mL LoD obtained for the SAL platform.
  • FIG. 17A(ii) releasing the crosslinkers and conferring biotin functionality to the magnetic beads, FIG. 17A(iii). This permits SAv-functionalized SALs to bind. SAL bursting then releases TAML to produce an optical signal, FIG. 17A(iv).
  • FIG. 17D The data in FIG. 17D were obtained by introducing oligo-functionalized CANs directly into lettuce homogenate samples. These results indicate that CAN-SAL is capable of detecting ⁇ 20 specific DNA sequences directly from complex matrices.
  • the enhanced LoD of FIG. 17D relative to FIG. 17C is due to the use of CANs functionalized with multiple oligo sequences, each of which binds different, specific regions of the template strand (FIG. 23).
  • SiPMs are 1-10 mm 2 components comprised of a 2-D array of silicon avalanche photodiodes operating at a reverse bias of 30-70V in Geiger mode (Reference 45). SiPMs have the further advantage of CMOS-compatibility, which permits easy integration with amplifier and processor chips.
  • a detector prototype fabricated from SiPMs is shown in the inset in FIG. 18B that relies on photon counting, in which the SiPM signal is amplified with a highspeed transimpendance amplifier followed by a signal height discriminator and high speed counter.
  • SiPMs permitted this portable detector to perform equivalent high- sensitivity fluorescence sensing to a state-of-the-art microplate reader incorporating PMTs (FIG. 18B).
  • This detector forms the basis of a microfluidic POC platform shown in FIG. 24 that is designed for CAN-SAL cascade operation.
  • the real-time SiPM-derived signals for two L. monocytogenes DNA samples are shown in FIG. 18C. These samples had the same DNA concentrations as the two points highlighted with arrows in FIG. 18A and showed similar results. The evolution of the signal from the low sample is clearly distinguished from that of the L. monocytogenes DNA-free control.
  • multiplexed biomarker detection is important for maximal device utility (References 37,44).
  • the CAN-SAL cascade is easily adapted for multiplexing using the field flow fractionation (FFF) technique (Reference 46) by using magnetic beads as the solid phase.
  • FFF field flow fractionation
  • each analyte capture species is bound to a magnetic bead of a different size.
  • an external magnetic field draws the larger beads across the channel without affecting the smaller beads (FIG. 19A) achieving size-based separation (FIG. 19B-C).
  • the POC prototype was tested with FFF multiplexing with samples comprising a mixture of dengue RNA and Yersinia pestis DNA, causative agents of severe illnesses of global importance (References 47,48). Reverse transcriptase is unnecessary due to the single DNA-RNA hybridization step necessary for detection.
  • a dengue capture oligo was attached to 2 ⁇ -dianneter magnetic beads and a Y. pestis capture oligo to 200 nm- diameter beads.
  • CAN-SAL assays were performed in parallel.
  • FIG. 19D shows agreement between results obtained individually for dengue RNA or Y. Pestis DNA alone and those obtained simultaneously from the same sample, "multiplex." The slight gain in Y. pestis signal and corresponding loss in dengue signal was likely due to some 2 ⁇ particles remaining in the original stream.
  • Biochemical binding events are commonly measured with amplifier labels.
  • Some ultra-sensitive platforms engineer around the limitations of natural enzymes: two of note are intricate microfluidic and optical infrastructures that capture near-single molecule enzymatic effects (Reference 11) and the coupling of polymerase activity with antibody detection (Reference 12). More commonly, platforms engineer replacements for natural enzyme amplifiers.
  • Nanoparticles have demonstrated particular utility as amplifier labels and have been shown to extend LoDs over 2 orders of magnitude beyond ELISAs for proteins and achieve near-PCR levels of sensitivity for nucleic acids (References 7-9,13-17,20).
  • nanoparticle amplifier LoDs fall to ELISA-like levels when standard platereaders are used (References 22-25).
  • the present work demonstrates that nanolabels can be designed for use with standard optical detection equipment but still achieve similar performance.
  • reactive cargoes for macromolecule detection is that it couples homogeneous optical signal catalysis with a solid-phase binding assay.
  • the solid-phase assay backbone maximizes specificity and selectivity (References 2,3).
  • Maximum sensitivity is then achieved by releasing catalysts from the solid surfaces into solution, where kinetics are vastly improved.
  • This design thus incorporates the best-known sensitivity, specificity, and catalytic paradigms and overcomes diffusion limitations that limit detection sensitivities of bound catalysts (References 20,28).
  • the total amplification factor of the CAN-SAL cascade is the product of the number of crosslinkers per CAN (2xl0 5 ) times the number of TAMLs per SAL (6xl0 4 ) times the signal amplification of each TAML molecule.
  • This theoretical product suggests that the method should give single-molecule sensitivity (FIG. 17B).
  • single- oligo CAN-SAL has LoDs of 100 template strands, three orders of magnitude below the theoretical threshold.
  • this may be due to nanolabels binding simultaneously to multiple adherent species or loss of nanolabels or small molecules during the washing steps. I ncreased understanding of the strength of the nanolabel binding to solid surfaces (54) and, in turn, optimizing washing will be important for achieving the technology's full potential. Additionally, developing improved TAML loading techniques for SALs can offer greater amplification factors.
  • CAN formulation [0210] CAN formulation. CAN particles were synthesized with the Biot-TCO crosslinker (Conju-Probe) as the payload and poly(/.-lactic acid) (PLA; 500 kDa; PolySciTech) as the polymer matrix. A poly(lactic acid)-poly(ethylene glycol)-maleimide block co-polymer of 30 kD:5 kD size (PolySciTech) was used to confer thiol reactivity. These materials were dissolved in 2 mL chloroform (Sigma) in a 1:10:2 mass ratio.
  • the resulting solution was then added to chloroform-saturated deionized water (0.815% chloroform in water) comprising 0.1% sodium dodecyl sulfate (Sigma).
  • the suspension was homogenized at 7,500 rpm forming a milky, stable suspension that was subsequently added to >300 mL of water for clarification. Because of maleimide instability in aqueous solutions, pH of the water was titrated to ⁇ 5.0 with dilute hydrochloric acid.
  • the particle solution was immediately filtered through a 300 kD membrane (Millipore), and CANs were concentrated to ⁇ 10 mL and characterized with DLS and NanoSight techniques.
  • oligonucleotides (Integrated DNA Technologies).
  • the oligo sequence comprised a complementary DNA sequence followed by two "C18" spacers, shown to be sufficient removal from nanoparticles for rapid binding (16), followed by a 3'-dithiol endcap.
  • the thiol-maleimide reaction proceeded at room temperature for 2 h in lx PBS, pH 7.2, with 0.1 M TCEP. Conjugation efficacy was determined by optical density absorbance readings at 280 nm with turbidity controls at 320 nm. Reacted particles were dialyzed into lx SSC buffer with a Slide-A-Lyzer cassette (ThermoFisher) and stored at 4°C.
  • CAN particles designed for protein binding were synthesized as above, and DSPE- PEG-biotin was added to the initial oil-in-water emulsion. The remainder of the CAN fabrication proceeded similarly.
  • particle concentration was determined such that the effective biotin concentration in lx PBS, pH ⁇ 7.2 was 0.1 ⁇ .
  • Neutravidin (ThermoFisher) was added at a concentration of ⁇ 10 ⁇ and binding was allowed to proceed at room temperature for 2 h followed by filtering and concentrating the resulting particles with a 300 kD membrane and storage in lx PBS.
  • 6-hCG assay Assays for 6-hCG were performed using components of a commercially- available microplate ELISA kit and commercial 6-hCG antibodies (Ray Biotech). In order to bind released crosslinkers, a Nunc Maxisorp microplate was coated with tetrazine- functionalized bovine serum albumin (Tz-BSA, 200 ng/mL in sodium bicarbonate buffer, pH 9.6) and the 6-hCG antibody (25 ng/mL).
  • Tz-BSA tetrazine- functionalized bovine serum albumin
  • Tz-BSA was prepared by reacting N HS-Tz (Conju-Probe) with BSA (Sigma) for 2 h at room temperature in PBS, pH 7.2, followed by overnight dialysis with a 3 kD-cutoff membrane (ThermoFisher) into PBS, pH 7.2.
  • IVF embryo culture media samples (CSC media, Irvine Scientific) comprise human serum albumin (Irvine Scientific) per standard clinical practice at Boston IVF. Two de- identified samples were obtained under a Boston IVF IRB: one from a sample that comprised an embryo for 5 days (IVF 1) and a control sample without an embryo (IVF 2).
  • IL-6 assay CAN particles were synthesized as described above. Assays for IL-6 were performed using components of a commercially-available microplate "high-sensitivity" ELISA kit (eBioscience) based on tyramide signal amplification and commercial IL-6 antibodies (R&D Systems). Plate coating was performed as described above for 6-hCG, with the only difference that the I L-6 capture antibody was plated at 15 ng/mL. The CAN assays were performed as described above for 6-hCG and the high-sensitivity IL-6 assay was performed per the manufacturer's instructions. Human plasma samples were purchased commercially de-identified (Discovery Life Sciences) and de-identified IVF embryo culture media samples were provided under an existing I RB of Boston IVF as described above.
  • acetate buffer, pH 5.5 and activity was determined using commercial TMB solution (ThermoFisher). Absorbance was measured after addition of 1 M sulfuric acid at 450 nm.
  • TAML ® GreenOx Catalysts
  • bicarbonate buffer, pH 10 activity was determined using 2,7-dichlorodihydrofluorescein diacetate (Santa Cruz Biotech) and read at 490 nm/545 nm (Ref. 55). All measurements were made using a Molecular Devices SpectraMax M2 microplate reader.
  • C. difficile Toxin A assay A monoclonal antibody pair for C. difficile Toxin A (Meridian Life Sciences; C6555M, C01677M, C01678M) was used for the ELISA and SAL assays.
  • the capture antibody was bound to /V-hydroxysuccinimyl (NHS)-ester activated magnetic beads (ThermoFisher) per the manufacturer's instructions.
  • the detection antibody was biotinylated using sulfo-N HS-LC-biotin (ThermoFisher), and the coupling reaction was performed for 2 h at RT in lx PBS, pH 7.5.
  • Recombinant C. difficile Toxin A R&D Systems
  • streptavidin-H RP (Abeam) or neutravidin-functionalized SALs was added.
  • Streptavidin-H RP labeled assays were developed with TM B solution (Abeam), stopped with 1 M sulfuric acid (Abeam), and absorbance read at 450 nm.
  • SALs were burst with acetone and a solution comprising hydrogen peroxide and 2,7- dihydrodichlorofluorescein diacetate in bicarbonate buffer (pH 10) was then added followed by fluorescence measurement at 490/545 nm.
  • superparamagnetic microparticles and nanoparticles were purchased from Bioclone and Ocean Nanotechnology, respectively. Oligos terminated with a 3'-amino modifier were purchased from I DT. Starting at the 5'-termini, these oligos comprised an amino modifier followed by two "C18" spacers followed by the oligo sequence.
  • a ATCCG G ATA ACG CTTG C (SEQ ID NO:ll).
  • CTTAGTTCTTTAGCGATTGC SEQ I D NO:12
  • Non-sense oligos comprising both an amino and a tetrazine modifier, used for crosslinker binding, were purchased from Bio-Synthesis, I nc.
  • Oligo-particle conjugation was performed according to the Bioclone protocol using magbeads (200 mg/mL) and oligo (5 g/mL) in suspension buffer. In brief, the sample was vortexed, reacted overnight at 50°C, and a 50:1 molar ratio of non-tetrazine- comprising oligo-to-Tz-comprising oligo was used. After 12 h, beads were washed with Bioclone buffer using a magnetic separation stand and then with deionized water at 50°C. Particles were resuspended at 20 mg/mL in lx SSC buffer and stored at 4°C until use. Optical density measurements of the oligo solution before and after reactions at 280 nm were used to confirm conjugation.
  • L. monocytogenes genomic DNA assays Purified genomic L. monocytogenes DNA (ATCC, Inc. 19114D-5) was diluted 5-7-times in a 1:5 dilution series using lx SSC buffer comprising salmon sperm DNA (20 nM; Sigma) and Tween 20 (0.001%). Samples were heated for 5 min at 95°C to denature DNA (note this step was not performed for RNA genomic material) and samples were cooled to 50°C. While at 50°C, magbeads, prepared as above, and CANs, each functionalized with the appropriate primer, were added. The effective particle-bound primer concentration was 2 ⁇ .
  • L. monocytogenes bacteria assays Lettuce samples were prepared according to a previously published procedure (39). Commercially-purchased romaine lettuce was homogenized in a 1:2 (w/w) ratio with deionized water for 5 min at 5,000 rpm. The resulting suspension was sequentially filtered through 0.8 ⁇ and 0.2 ⁇ syringe filters (VWR). Listeria monocytogenes (ATCC 19114) were cultured according to ATCC instructions using agar plates with ATCC ® Medium 44 Brain Heart Infusion Agar/Broth. L. Monocytogenes colonies were transferred from plates to lx SSC buffer and serial dilutions of bacteria in lx SSC buffer were added to four filtered samples. Each sample was sonicated for 10 min (Heat Systems - Ultrasonics, I nc) and then treated at 95°C for 15 min in order to kill bacteria. Samples were cooled to 50°C and the procedure described above was followed.
  • Fractional Flow Filtration The fractional flow filtration chip was designed in-house and fabricated by A-Line, Inc. The design consists of a 5 mm x 50 mm channel with Y junctions at both inlet and outlet (2.5 mm), separated by 200 ⁇ wide and 5 mm long divider to minimize fluid mixing at junctions and a llow straight fluid lines. Each inlet was fitted with 1/16" ID and 1/8" OD silicone tubing fed by a syringe loaded onto a syringe pump (Harvard Apparatus). One syringe was filled with solution comprising a mixture of Y. Pestis and Dengue samples after completing hybridization at 50°C.
  • a second syringe was filled with lx SSC buffer. Exit ports were fitted with similar tubing comprising valves, which allowed flows to be set. Tubing and plastic chip were treated with BSA to minimize nonspecific binding.
  • An N52-grade neodymium magnet bar magnet (K&J Magnetics) was positioned ⁇ 2/3 the distance down the channel (from inlet to outlet). The latter was tuned empirically, together with syringe pump flow rates, such that 2 ⁇ magnetic particles would laterally traverse the channel, while 200 nm magnetic particles would remain in their original channel region.
  • SiPM Fluorimeter An SiPM (Hamamatsu S13360-3050CS, 3 mm x 3 mm chip and 50 x 50 micrometer pixels) was biased at the appropriate reverse voltage above the specified breakdown voltage. The output from the SiPM was run thourgh a singal processing hardware taken from SP5600 unit made by CAEN S. p.A. which provides a variable gain amplifier, comaprator with a variable threshold voltage and a counter. A software was written for a National Instruments Data Acquisition Card to read datastreams and generate all control and synchronization signals for readout electronics, excitation Light Emitting Diode (LED, from LedEngin), pumps and valves.
  • LED Excitation Light Emitting Diode
  • SiPM voltage bias, discriminator threshold and amplifier gain were adjusted to permit the highest sensitivity and dynamic range, and the lowest dark count for a reference fluorescent solution ( ⁇ fluorescein, Sigma, in Dl water).
  • the emission LED was biased using an external circuit providing 250 ns pulses at 1 MHz frequency. Excitation pulse width and frequency were set to provide the highest signal-to-noise ratio for reference fluorescence solution.
  • Excitation (480 nm) and emission filters (530 nm) were purchased from Thorlabs.
  • Optical system made of lenses, optical tubes and holders (Thorlabs) was used to focus light from microfluidic chip onto the SiPM and minimize stray light.
  • the microfluidic assay cartridge was designed in-house and manufactured by A-Line Inc. Fluid control was achieved by off-chip valves and a diaphragm pump (Takasago Fluidics). The pump was placed at the outlet thereby creating negative pressure.
  • the cartridge consisted of 3 parallel channels that allow detection of 3 separate samples. Each channel consisted of 2 zones: one for assay incubation and other for detection.
  • the assay zone was designed to fit a cylindrical (1 ⁇ 4" diameter and %" height) N52 grade neodymium magnet to trap functionalized magnetic particles. Dettection zone of the chip was spotted with BSA-Tz to permit capture of Biotion-TCO from CAN.
  • Biotin-TCO released from CAN in the assay zone was moved to detection zone and incubated for 30 minutes, followed by a wash step. SALs were then introduced to detection zone and incubated for 20 minutes followed by a wash cycle. Release solution was then introduced and signal was measured in real-time using SiPM fluorometer.
  • the detection zone was a ⁇ thick layer made of cyclo olefin copolymer (COC) in order to minimize absorbance and auto-fluorescence.
  • FIGS. 24A-24B show microfluidic chip layouts.
  • FIG.24A is an exploded view and FIG.
  • 24B is a complete view of a microfluidic chip used for L. monocytogenes DNA detection. Up to three assays can be run on a same chip. Recesses in the top layer are for N52- grade niobium magnets and optical windows allow detection of optical signals.
  • Microfluidic assays The sequence of steps followed those for L. monocytogenes detection, with a magnet positioned in a recess on the microfluidic platform used to hold magbeads during wash steps.
  • One exception was that no tetrazine-comprising oligos were used on magbeads. Instead, Tz-BSA was spotted on the microfluidic chip surface. The small channel dimensions of microfluidic channels (300 ⁇ wide and 500 ⁇ high) ensured rapid kinetics. The signals were read by the SiPM fluorimeter.
  • BAL sample preparation Commercial human BAL samples (Discovery Life Sciences) were streaked on LB agar plates (EZ BioResearch), inverted, and grown overnight at 35°C. These samples were chosen based on availability on a single day of clinical BAL samples and were numbered in the order they were removed from the sample bag. Five colonies from each plate were picked and resuspended in PBS.
  • the samples were incuabated with human IgG (1 g/mL; Sigma) for 30 minutes and then the IgG-comprising samples were introduced for the capture step. This procedure was performed to minimize protein A interference. The remainder of the procedure for the a-H Ia toxin assay was similar to that described for C. difficile toxin A.
  • RNA/DNA extraction kit Qiagen QIAmp UCP Pathogen Mini Kit
  • BBI D00314 Jiagen D00314
  • BBAR00374AR mecA gene

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Abstract

L'invention concerne des marqueurs d'amplificateurs synthétiques (« SAL ») de nanoparticules pouvant permettre à des lecteurs de microplaques existants de quantifier des protéines avec une sensibilité accrue par rapport à ELISA. De tels marqueurs peuvent être utilisés dans des dosages immunologiques ainsi que dans des dosages d'ADN et d'ARN, en présentant des sensibilités s'approchant d'une PCR quantitative.
PCT/US2016/065607 2015-12-08 2016-12-08 Amplificateurs de nanoparticules et leurs utilisations Ceased WO2017100442A1 (fr)

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RU2799250C1 (ru) * 2023-04-04 2023-07-04 Федеральное государственное бюджетное учреждение "Центр стратегического планирования и управления медико-биологическими рисками здоровью" Федерального медико-биологического агентства Устройство для автономного обнаружения последовательностей нуклеиновых кислот
CN116640830A (zh) * 2023-05-04 2023-08-25 河北国高生物科技有限公司 一种免疫pcr工作液及其制备方法和应用

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