Methods and apparatus for single biological nanoparticle analysis

Abstract

The present disclosure relates to methods, systems and devices for performing biological nanoparticle analysis. More specifically, the present disclosure relates to methods, systems and devices for performing single biological nanoparticle size determination on a sample as the single biological nanoparticle moves through a microfluidic chip. In other aspects, the present disclosure relates to methods, systems and devices for selectively capturing biological nanoparticles on a coated planar surface, said capture being facilitated by centrifugation.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 657,278, filed April 13, 2018, which is incorporated herein by reference in its entirety for all purposes. Background Art

[0003] Microfluidics has become an essential component of bioanalytical research. Successful microfluidic bioanalytical applications include DNA sequencing and fragment sizing, PCR amplification, protein analysis, immunoassays, and cell sorting and manipulation. Some of these examples take advantage of low sample and reagent volumes and the unique physical properties of micrometer-scale fluid flow to enable new studies. Near-nanoscale microfluidic methods are particularly well suited for studying subcellular organelles, macromolecular complexes, and other nanoscale materials.

[0004] Conventional flow cytometers and sorters have been used for cell sorting in a wide range of biological research and clinical applications, including diagnosis and monitoring of cancer and detection of virus-cell interactions. Micro-flow cytometers have become a low-cost, disposable alternative to traditional flow cytometers and can show higher sensitivity when used to sort objects such as fluorescent beads and E. coli cells. The sensitivity of these micro-cytometers is still not sufficient for analyzing and / or sorting nanoscale systems such as subcellular organelles, and may lack the necessary sorting speed and throughput or information content (e.g., size information).

[0005] Further analysis of subcellular organelles and other bio-nanoparticles is accomplished by concentrating the bio-nanoparticles using, for example, ultracentrifugation. However, such concentration can require a significant amount of time (typically 4-5 hours), typically uses specialized and expensive equipment, involves a centrifugal force of 200,000 times gravity, and typically results in contamination of the concentrate due to lack of specificity in the concentration process. More specific capture and separation of bio-nanoparticles can be achieved using beads, which are coated with capture coatings such as streptavidin or antibodies. However, such methods can still result in sample contamination or poor properties, and any downstream analysis of the captured nanoparticles may require them to be released from the beads, which can result in reduced yields, damage to the bio-nanoparticles, and decreased accuracy of the results. In addition, the use of beads to capture bio-nanoparticles necessarily requires the use of expensive equipment, frequent manual handling, advanced technical skills, and / or long incubation times. SUMMARY OF THE INVENTION

[0007] The present disclosure provides methods, systems and devices for performing biological nanoparticle analysis.

[0008] This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

[0009] In various aspects, the present disclosure provides a method for determining the size of biological nanoparticles in a fluid sample, the method comprising: providing a microfluidic chip, the microfluidic chip comprising at least one microfluidic channel; introducing the fluid sample into the microfluidic chip, the fluid sample comprising a plurality of biological nanoparticles; causing a portion of the plurality of biological nanoparticles to flow through at least one microfluidic channel; irradiating at least one biological nanoparticle from the portion of the plurality of biological nanoparticles on a particle-by-particle basis in the at least one microfluidic channel; detecting the intensity of light emitted from the at least one biological nanoparticle; and assigning a size value to the at least one biological nanoparticle as it moves through the microfluidic chip, wherein the hydrodynamic diameter of the biological nanoparticle is less than 1 μm. In certain aspects, the method comprises irradiating a portion of the plurality of biological nanoparticles. In some aspects, the method comprises irradiating at least 90% of the portion of the plurality of irradiated biological nanoparticles on a particle-by-particle basis in the at least one microfluidic channel.

[0010] In some aspects, the microfluidic chip includes a plurality of parallel microfluidic channels. In some aspects, the method includes irradiating the plurality of parallel microfluidic channels with a single radiation source. In some aspects, the method includes providing a plurality of detectors configured to detect signals from a single microfluidic channel of the plurality of parallel microfluidic channels. In some aspects, the signal includes the intensity of light emitted from the at least one biological nanoparticle. In some aspects, the method includes detecting the intensity of light emitted from the at least one biological nanoparticle with a detector in a detection region.

[0011] In some aspects, the at least one microfluidic channel comprises at least one constriction. In some aspects, the detection region is located within the at least one constriction. In some aspects, the detection region has an area of ​​less than 10 μm 2 In some aspects, the method includes detecting a portion of the plurality of biological nanoparticles. In some aspects, more than 90% of the detected biological nanoparticles in the portion of the plurality of biological nanoparticles are detected particle by particle within the detection region.

[0012] In some aspects, the method includes measuring the intensity of light emitted from the at least one biological nanoparticle. In some aspects, measuring the intensity of light emitted from the at least one biological nanoparticle includes using a time bin. In some aspects, the range of the time bin is less than 2ms and greater than 1μs. In some aspects, the signal-to-noise ratio of the intensity of light emitted from the at least one biological nanoparticle is greater than 10:1.

[0013] In some aspects, irradiating the at least one biological nanoparticle comprises using an irradiation source having a beam width of less than 2 μm. In some aspects, the irradiation source irradiates an area within the at least one microfluidic channel less than 10 μm. 2 area.

[0014] In some aspects, detecting the light intensity includes using a light collection system having a numerical aperture equal to or greater than 1.0 and no greater than 1.5.

[0015] In some aspects, assigning a size value while moving includes assigning a size value while the biological nanoparticle is in a flow state. In some aspects, the flow is uninterrupted.

[0016] In some aspects, the size value is specified according to the light intensity. In some aspects, the light intensity includes fluorescence, scattered light, or any combination thereof. In some aspects, the bio-nanoparticle is combined with a detectable agent. In some aspects, the detectable agent is connected to the surface of the bio-nanoparticle, the detectable agent is in the surface of the bio-nanoparticle, the detectable agent is in the interior of the bio-nanoparticle, or a combination thereof. In some aspects, the detectable agent is a fluorescent detectable agent, the detectable agent is a luminescent detectable agent, or any combination thereof. In some aspects, the bio-nanoparticle is combined with a variety of detectable agents. In some aspects, at least one of the multiple detectable agents is connected to the surface of the bio-nanoparticle, at least one of the multiple detectable agents is in the surface of the bio-nanoparticle, at least one of the multiple detectable agents is in the interior of the bio-nanoparticle, or a combination thereof. In some aspects, the multiple detectable agents have overlapping emission profiles. In some aspects, the multiple detectable agents have the same emission profile. In some aspects, the emission profile has the same peak wavelength. In some aspects, the plurality of detectable agents comprises the same detectable agent. In some aspects, the plurality of detectable agents comprises more than one type of detectable agent.

[0017] In some aspects, the multiple detectable agents have different emission spectra. In some aspects, the emission spectra have different peak wavelengths. In some aspects, the peak wavelengths are separated by more than 10 nanometers, more than 20 nanometers, more than 30 nanometers, more than 40 nanometers, more than 50 nanometers, more than 75 nanometers, more than 100 nanometers, more than 120 nanometers, more than 140 nanometers, more than 160 nanometers, more than 180 nanometers, more than 200 nanometers, more than 300 nanometers, more than 400 nanometers, more than 500 nanometers, more than 600 nanometers, or more than 700 nanometers.

[0018] In some aspects, the multiple detectable agents include fluorescent detectable agents. In some aspects, the light intensity is emitted from the detectable agent. In some aspects, the specified size value includes using the measured light intensity. In some aspects, the specified size value includes using a modulation index. In some aspects, the specified size value includes using the measured light intensity and using a modulation index. In some aspects, the modulation index is an amplitude modulation index. In some aspects, the modulation index is a frequency modulation index. In some aspects, the modulation index is a phase modulation index.

[0019] In some aspects, the size value is a relative size value. In some aspects, the relative size value is determined by a difference in measured light intensity.

[0020] In some aspects, the method includes determining the actual size value of the bio-nanoparticle by calibrating the measured light intensity relative to a standard. In some aspects, the method includes calibrating the measurement of the light intensity with a standard. In some aspects, the calibration occurs before introducing a sample containing at least one bio-nanoparticle. In some aspects, the size value is the actual size value of the bio-nanoparticle. In some aspects, the standard includes gold nanoparticles. In some aspects, the standard includes multiple lipids. In some aspects, the standard includes lipid vesicles. In some aspects, the standard includes silica beads, polystyrene beads, fluorescent beads, polymer beads, polymer nanoparticles or a combination thereof. In some aspects, the fluid sample also includes a second bio-nanoparticle, and wherein the standard includes the second bio-nanoparticle. In some aspects, the size of the standard is measured via dynamic light dispersion.

[0021] In various aspects, the method includes directing the flow of the bio-nanoparticles according to the size value. In some aspects, the directing directs the bio-nanoparticles to a collection, generating a collection of bio-nanoparticles based on the size value. In some aspects, the directing does not exceed 1 ms. In some aspects, the directing of the flow includes flow displacement. In some aspects, the directing of the flow includes electroosmotic flow. In some aspects, the directing of the flow includes the application of pressure.

[0022] In some aspects, the method includes quantifying the number of bio-nanoparticles having the size value. In some aspects, the method includes determining the concentration of the sample. In some aspects, the concentration of the sample is determined by counting the peak frequency of the sample. In some aspects, the method includes comparing the peak frequency of the sample with the peak frequency of a calibration particle standard. In some aspects, the concentration of the sample is determined by counting the peak frequency of the sample and comparing it with the volume of the sample. In some aspects, the concentration of the sample is determined by counting the peak frequency of the sample, comparing it with the peak frequency of the calibration particle standard, and comparing the peak frequency of the sample with the volume of the sample.

[0023] In some aspects, the method includes filtering the sample. In some aspects, the filtering occurs before the specified size value. In some aspects, the filtering removes debris. In some aspects, the filtering prevents clogging.

[0024] In some aspects, the method includes collecting the bio-nanoparticles for analysis. In some aspects, the analysis includes nucleic acid analysis. In some aspects, the nucleic acid analysis includes sequencing, PCR or digital PCR. In some aspects, the analysis includes protein analysis. In some aspects, the protein analysis includes ELISA, digital ELISA or mass spectrometry. In some aspects, the analysis includes lipid analysis. In some aspects, the analysis includes small molecule analysis. In some aspects, the small molecule analysis includes analysis of metabolites, analysis of signal transduction molecules, analysis of drugs or a combination thereof. In some aspects, the analysis includes mass spectrometry. In some aspects, the analysis includes carbohydrate analysis. In some aspects, the carbohydrate analysis includes mass spectrometry, nuclear magnetic resonance, Fourier transform spectroscopy or a combination thereof.

[0025] In some aspects, the microfluidic chip is a planar device and includes at least one microfluidic channel. In some aspects, the at least one microfluidic channel has a maximum height, a maximum width and / or a maximum cross-sectional area. In some aspects, the at least one microfluidic channel includes a constriction. In some aspects, the width of the constriction is less than 25% of the maximum width of the microfluidic channel. In some aspects, the height of the constriction is less than 25% of the maximum height of the microfluidic channel. In some aspects, the cross-sectional area of ​​the constriction is less than 5% of the maximum cross-sectional area of ​​the microfluidic channel. In some aspects, the value of the maximum width is less than 500μm and greater than 10μm. In some aspects, the value of the maximum height is less than 500μm and greater than 10μm. In some aspects, the value of the maximum cross-sectional area is less than 250,000μm 2 And greater than 250μm 2 In some aspects, at least a portion of the at least one microfluidic channel has a width of less than 10 μm, a width of less than 5 μm, or a width of less than 2 μm. In some aspects, at least a portion of the at least one microfluidic channel has a height of less than 10 μm, a height of less than 5 μm, or a height of less than 2 μm. In some aspects, at least a portion of the at least one microfluidic channel has a cross-sectional area of ​​less than 100 μm. 2 , cross-sectional area less than 90μm 2 , cross-sectional area less than 80 μm 2 , cross-sectional area less than 70 μm 2 , cross-sectional area less than 60 μm 2 , cross-sectional area less than 50 μm 2 , cross-sectional area less than 40 μm 2 , cross-sectional area less than 30 μm 2 , cross-sectional area less than 20 μm 2 , cross-sectional area less than 10 μm 2 , cross-sectional area less than 5 μm 2 Or the cross-sectional area is less than 2μm 2 .

[0026] In some aspects, the microfluidic chip includes a plurality of microfluidic channels intersecting at a node. In some aspects, a channel intersects at least 3 different microfluidic channels at a node. In some aspects, the node has no dead volume.

[0027] In some aspects, the light intensity includes scattered light. In some aspects, the scattered light is backscattered light, sidescattered light, or forward scattered light. In some aspects, the light intensity includes scattered light and fluorescence.

[0028] In some aspects, the detection of light intensity from at least one biological nanoparticle is detected while moving through the at least one microfluidic channel. In some aspects, the detection of light intensity has single nanoparticle sensitivity, or the detection of light intensity has single molecule sensitivity. In some aspects, the detection of light intensity detects a single antibody comprising multiple detectable agents.

[0029] In some aspects, the bio-nanoparticle comprises at least one biomarker. In some aspects, the method comprises determining at least one copy number of at least one biomarker.

[0030] In some aspects, the specified size value uses a beam modified by stimulated emission depletion (STED). In some aspects, the size value of the bio-nanoparticle is a hydrodynamic diameter. In some aspects, the hydrodynamic diameter is less than 900 nanometers, less than 800 nanometers, less than 700 nanometers, less than 600 nanometers, less than 500 nanometers, less than 400 nanometers, less than 300 nanometers, less than 200 nanometers, less than 150 nanometers, less than 100 nanometers, less than 90 nanometers, less than 80 nanometers, less than 70 nanometers, less than 60 nanometers, less than 50 nanometers, less than 40 nanometers or less than 30 nanometers. In some aspects, the specified size value uses detection of a duty cycle.

[0031] In some aspects, the bionanoparticles include extracellular vesicles, organelles, microvesicles, cell-derived vesicles, protein aggregates, nucleic acid aggregates, lipid aggregates, viruses, bacteria or exosomes. In some aspects, the bionanoparticles are passed through the microfluidic chip at a rate of more than 1 million particles / hour, more than 2 million particles / hour, more than 3 million particles / hour, more than 4 million particles / hour, more than 5 million particles / hour, more than 6 million particles / hour, more than 7 million particles / hour, more than 8 million particles / hour, more than 9 million particles / hour, more than 10 million particles / hour, more than 15 million particles / hour, more than 20 million particles / hour, more than 25 million particles / hour, more than 30 million particles / hour, more than 35 million particles / hour, more than 40 million particles / hour, more than 45 million particles / hour or more than 50 million particles / hour.

[0032] In some aspects, the method includes sorting the at least one nanoparticle into an enriched population. In some aspects, the sorting includes flow-displacement sorting. In some aspects, the sorting does not include acoustic sorting or the use of a physical barrier. In some aspects, the sorting is determined by the presence of a size value, a biomarker, the detected light intensity, an emission wavelength, a plurality of emission wavelengths, the identification of biological nanoparticles, or a combination thereof. In some aspects, the diameter variance of the enriched population is less than 100%. In some aspects, the enriched population has the following diameter range: within 500% of the diameter value, within 400% of the diameter value, within 300% of the diameter value, within 200% of the diameter value, within 100% of the diameter value, within 50% of the diameter value, or within 25% of the diameter value. In some aspects, the enriched population is greater than 80% of the biological nanoparticles combined with a detectable agent.

[0033] In some aspects, the fluid sample comprises a body fluid. In some aspects, the bio-nanoparticle is an exosome. In some aspects, the body fluid comprises serum, plasma, cerebrospinal fluid, or lymph. In some aspects, the bio-nanoparticle is isolated. In some aspects, the bio-nanoparticle is combined with at least one biomarker.

[0034] In some aspects, the method further comprises: detecting at least one biomarker associated with the bio-nanoparticle; and assigning a biomarker value to the bio-nanoparticle. In some aspects, the biomarker value is assigned by a binary system. In some aspects, the biomarker value is assigned by a plurality of values. In some aspects, the biomarker is a protein biomarker. In some aspects, the protein biomarker is selected from the group consisting of a tetraspanin, a heat shock protein, a membrane transport protein, a cell adhesion protein, a lipid binding protein, a transmembrane protein, an enzyme, or a combination thereof. In some aspects, the biomarker is a nucleic acid biomarker. In some aspects, the nucleic acid biomarker is selected from the group consisting of miR-141, miR-375, miRNA-21, miR-1246, miR-320c, miR-6068, miR-9, miR-107, miRNA-128, miRNA134, miRNA-137, miRNA124, miR-21, miR-141, miR-200a, miR-200b, miR-200c, miR-203, miR-205, miR-214; miR-17, miR-3p, miR-21, miR-20b, miR-223, miR-301, miR-141, miR-375, miR-21, miR-1246; miR-21, let-7f, and other Let-7 miRNAs.

[0035] In some aspects, the method includes directing the flow of the biological nanoparticles according to the size value, the biomarker value, or a combination thereof. In some aspects, the method also includes sorting the biological nanoparticles into enriched populations, wherein the sorting is determined by the size value, the biomarker value, or a combination thereof.

[0036] In some aspects, the biological nanoparticle is bound to at least one tag. In some aspects, the method further comprises: detecting at least one tag bound to the biological nanoparticle; and assigning a tag value to the biological nanoparticle. In some aspects, the tag value is assigned by a binary system. In some aspects, the tag value is assigned by multiple values. In some aspects, the tag is selected from the group consisting of: a signal derived from a protein marker bound to the biological nanoparticle, a signal derived from the size of the biological nanoparticle, a signal derived from a membrane dye bound to the biological nanoparticle, a signal derived from a volume dye bound to the biological nanoparticle, a signal derived from a dye bound to the nucleic acid of the biological nanoparticle, a signal derived from a dye bound to the lipid of the biological nanoparticle, and a signal derived from a dye bound to the biological nanoparticle. In some aspects, the method further comprises directing the flow of the biological nanoparticle according to the size value and / or the tag value. In some aspects, the method further comprises sorting the biological nanoparticle into an enriched population, wherein the sorting is determined by the size value and / or the tag value.

[0037] In various aspects, the present disclosure provides an apparatus for determining the size of biological nanoparticles in a fluid sample, the apparatus comprising: a microfluidic chip; at least one detector configured to detect the presence or absence of biological nanoparticles on a particle-by-particle basis as the biological nanoparticles flow through at least a portion of the microfluidic chip; and a computer containing software for: sorting the biological nanoparticles according to the presence or absence of detectable light intensity emitted by the biological nanoparticles; and measuring the size value of the biological nanoparticles based on the detectable light intensity emitted by the biological nanoparticles.

[0038] In some aspects, the microfluidic chip includes at least one microfluidic channel. In some aspects, the at least one microfluidic channel includes a constriction. In some aspects, the device also includes a source for interrogating. In some aspects, the interrogation source includes an electromagnetic radiation source. In some aspects, the interrogation source is selected from the group consisting of a solid-state laser, a diode-pumped laser, a light-emitting diode (LED), a lamp, an arc discharge, a magnetic pulse, and natural light. In some aspects, the detector detects fluorescence, luminescent light, or any combination thereof. In some aspects, the detector detects a variety of different divergent spectra. In some aspects, the detector can detect scattered light. In some aspects, the detector has single nanoparticle sensitivity, the detector has single molecule sensitivity, or any combination thereof.

[0039] In some aspects, the ranking corresponds to the measured size value. In some aspects, the size value is a relative size value. In some aspects, the size value is measured by a difference in detected light intensity. In some aspects, the size value is measured based on a detected modulation index. In some aspects, the size value is measured based on the detected light intensity and the detected modulation index. In some aspects, the modulation index includes an amplitude modulation index, a frequency modulation index, a phase modulation index, or a combination thereof. In some aspects, the size value is an actual size value.

[0040] In some aspects, the device also includes a mechanism for guiding the flow of biological nanoparticles. In some aspects, the mechanism for guiding the flow of biological nanoparticles can guide the flow in no more than 1ms. In some aspects, the mechanism for guiding the flow uses flow displacement. In some aspects, the mechanism for guiding the flow uses electroosmotic flow, applied pressure or a combination thereof. In some aspects, the device also includes software for quantifying the number of biological nanoparticles with the size value. In some aspects, the device also includes software for determining the concentration of the fluid sample.

[0041] In some aspects, the device further comprises a filter. In some aspects, the microfluidic chip is a planar device and comprises at least one microfluidic channel. In some aspects, at least a portion of the at least one microfluidic channel has a width of less than 10 μm, a width of less than 5 μm, or a width of less than 2 μm. In some aspects, at least a portion of the at least one microfluidic channel has a height of less than 10 μm, a height of less than 5 μm, or a height of less than 2 μm. In some aspects, at least a portion of the at least one microfluidic channel has a cross-sectional area of ​​less than 100 μm 2 , cross-sectional area less than 90μm 2 , cross-sectional area less than 80 μm 2, cross-sectional area less than 70 μm 2 , cross-sectional area less than 60 μm 2 , cross-sectional area less than 50 μm 2 , cross-sectional area less than 40 μm 2 , cross-sectional area less than 30 μm 2 , cross-sectional area less than 20 μm 2 , cross-sectional area less than 10 μm 2 , cross-sectional area less than 5 μm 2 Or the cross-sectional area is less than 2μm 2 .

[0042] In some aspects, the device also includes software for determining at least one copy number of a biomarker. In some aspects, the interrogation source is subjected to stimulated emission depletion (STED) modification. In some aspects, the detection, sorting and measurement occur at the following rate: more than 1 million particles / hour, more than 2 million particles / hour, more than 3 million particles / hour, more than 4 million particles / hour, more than 5 million particles / hour, more than 6 million particles / hour, more than 7 million particles / hour, more than 8 million particles / hour, more than 9 million particles / hour, more than 10 million particles / hour, more than 15 million particles / hour, more than 20 million particles / hour, more than 25 million particles / hour, more than 30 million particles / hour, more than 35 million particles / hour, more than 40 million particles / hour, more than 45 million particles / hour or more than 50 million particles / hour. In some aspects, the rate exceeds 5 million particles / hour.

[0043] In some aspects, the device includes more than one microfluidic channel, and the mechanism for directing flow can sort the bio-nanoparticles into each of two or more microfluidic channels. In some aspects, sorting produces enriched populations. In some aspects, the device also includes software for sorting the bio-nanoparticles. In some aspects, the sorting depends on the size value. In some aspects, the sorting depends on the ranking.

[0044] In various aspects, the present disclosure provides a method for capturing biological nanoparticles on a coated planar surface, the method comprising: providing at least one planar surface having a coating, the coating comprising: a nonspecific adsorption resistant material; and a plurality of capture molecules; contacting a fluid sample comprising a plurality of biological nanoparticles with the coating; centrifuging the fluid sample in contact with the at least one planar surface to facilitate contact of the biological nanoparticles with the coating; and capturing at least some of the plurality of nanoparticles with at least some of the plurality of capture molecules.

[0045] In some aspects, the layer thickness of at least part of the fluid sample is less than 10mm, less than 9mm, less than 5mm, less than 1mm, less than 900μm, less than 800μm, less than 700μm, less than 600μm, less than 500μm, less than 400μm, less than 300μm, less than 200μm or less than 100μm. In some aspects, the at least one planar surface includes glass. In some aspects, the method also includes covering the fluid sample with an evaporation-proof layer. In some aspects, the evaporation-proof layer is a tape. In some aspects, the evaporation-proof layer is mineral oil.

[0046] In some aspects, the method further comprises filtering the fluid sample. In some aspects, the filtering step occurs before the contacting step. In some aspects, the filtering is driven by centrifugal force.

[0047] In some aspects, the method further comprises analyzing the at least one planar surface. In some aspects, the analysis comprises counting the number of bio-nanoparticles bound to the coating. In some aspects, the analysis comprises measuring the intensity of emitted light from the bio-nanoparticles bound to the coating. In some aspects, the analysis comprises imaging the bio-nanoparticles bound to the coating. In some aspects, the non-specific adsorption resistant material comprises polyethylene glycol. In some aspects, the non-specific adsorption resistant material comprises a zwitterionic surface. In some aspects, the zwitterionic surface comprises carboxybetaine. In some aspects, the non-specific adsorption resistant material comprises bovine serum albumin.

[0048] In some aspects, the plurality of capture molecules include antibodies. In some aspects, the antibodies are biotinylated. In some aspects, the plurality of capture molecules include biotin or streptavidin. In some aspects, the plurality of capture molecules include nucleic acids. In some aspects, the plurality of bio-nanoparticles include extracellular vesicles, organelles, microvesicles, cell-derived vesicles, protein aggregates, nucleic acid aggregates, lipid aggregates, viruses, bacteria, exosomes, or combinations thereof. In some aspects, the plurality of bio-nanoparticles include exosomes.

[0049] In some aspects, the diameter of the at least one planar surface is 0.1mm-100cm, 0.1mm-20cm, 1mm-20cm, 5mm-10cm, 1mm-5cm, 10mm-10cm, 5mm-5cm or 1cm-50cm. In some aspects, the diameter of the at least one planar surface is greater than 1cm. In some aspects, the width of the at least one planar surface is 1cm-100cm, 1mm-1cm, 1mm-20cm, 5mm-20cm, 10mm-20cm, 100mm-20cm, 1cm-20cm or 1cm-50cm. In some aspects, the length of the at least one planar surface is 0.1mm-100cm, 1mm-20cm, 1mm-10cm, 5mm-20cm, 10mm-20cm, 100mm-20cm, 1cm-20cm or 1cm-50cm. In some aspects, the at least one planar surface has a surface area, wherein the surface area is 200 cm 2 -100cm 2 , the surface area is 150cm 2 -80cm 2 , the surface area is 150cm 2 -1cm 2 , the surface area is 200cm 2 -1mm 2 , the surface area is 120cm 2 -1mm 2 , the surface area is 80cm 2 -1mm 2 , the surface area is 50cm 2 -1mm 2 , the surface area is 20cm 2 -1mm 2 , the surface area is 1cm 2 -1mm 2 , the surface area is 75mm 2 -1mm 2 , the surface area is 50mm 2 -1mm 2 , the surface area is 25mm 2 -1mm 2 , or the surface area is 10mm 2 -1mm 2 .

[0050] In some aspects, the method further comprises combining a detectable agent with at least one of the plurality of nanoparticles. In some aspects, the detectable agent is selected from the group consisting of a fluorescently labeled antibody, a fluorescently labeled protein, a fluorescently labeled nucleic acid, a fluorescently labeled lipid, a membrane dye, a fluorescent dye, a dye, a polymer dot, and a combination thereof.

[0051] In some aspects, the method further comprises providing a holding device comprising a plurality of compartments, wherein at least one of the plurality of compartments comprises the at least one planar surface and the fluid sample. In some aspects, the holding device is an orifice plate. In some aspects, the plurality of compartments comprises a hole. In some aspects, the holding device is a microfluidic chip. In some aspects, the plurality of compartments comprises a microfluidic channel. In some aspects, the holding device is a block, and the plurality of compartments comprises a plurality of holes.

[0052] In some aspects, the method further comprises connecting the containment device to the at least one planar surface. In some aspects, the containment device is connected to a top device. In some aspects, the top device comprises a filter. In some aspects, the top device comprises a microfluidic device. In some aspects, the top device comprises a multiwell plate.

[0053] In some aspects, the method further comprises removing the at least one planar surface from the containment device. In some aspects, the removal occurs after the capture. In some aspects, the centrifugation occurs for a period of time, and the number of bio-nanoparticles bound to the coating is at least 100% greater than a control experiment omitting the centrifugation step, wherein the control fluid sample is in contact with the coating for the same length of time. In some aspects, the length of time is less than 1 hour, less than 30 minutes, less than 15 minutes, less than 10 minutes, less than 5 minutes, or less than 1 minute.

[0054] In some aspects, at least 10% of the plurality of nanoparticles are captured by the coating.

[0055] In some aspects, the method further comprises enhancing the stability of the coated planar surface. In some aspects, the enhancing stability comprises freeze drying. In some aspects, the enhancing stability occurs prior to capturing at least some of the plurality of nanoparticles with at least some of the plurality of capture molecules bound to the coating.

[0056] In some aspects, the at least one planar surface is compatible with a microscope. In some aspects, the at least one planar surface comprises a cover glass. In some aspects, the centrifugal force is applied to multiple planar surfaces simultaneously.

[0057] In some aspects, the plurality of capture molecules are connected to the planar surface via a connecting portion. In some aspects, the method further comprises cutting the connecting portion. In some aspects, the method further comprises releasing at least some of the plurality of bio-nanoparticles from the coating. In some aspects, the release comprises eluting with biotin. In some aspects, the released bio-nanoparticles are structurally intact. In some aspects, the released bio-nanoparticles are dissolved, and the lysates are collected for analysis. In some aspects, the analysis is selected from the group consisting of nucleic acid analysis, sequencing, polymerase chain reaction, protein analysis, ELISA, mass spectrometry, carbohydrate analysis, small molecule analysis, drug analysis, and lipid analysis.

[0058] In some aspects, the method further comprises washing the planar surface to remove at least some of the fluid sample that is not captured by at least some of the plurality of capture molecules. In some aspects, at least some of the plurality of nanoparticles captured with the coating are imaged. In some aspects, the imaging comprises fluorescence microscopy. In some aspects, the fluorescence microscopy is super-resolution imaging. In some aspects, the fluorescence microscopy uses at least one membrane dye bound to the biological nanoparticles. In some aspects, the at least one membrane dye comprises FM 143, ANEPPS, or a combination thereof. In some aspects, the imaging comprises atomic force microscopy. In some aspects, the imaging comprises transmission electron microscopy. In some aspects, the imaging provides an information set. In some aspects, the information set comprises identifying biomarkers present on the nanoparticles. In some aspects, the information set comprises identifying antibodies present on the nanoparticles. In some aspects, the information set comprises determining the size of the nanoparticles.

[0059] In some aspects, the size of the nanoparticle is a relative size value, and wherein the relative size value is determined by the difference in the measured light intensity. In some aspects, the method also includes calibrating the measured light intensity with a standard to determine the actual size value of the nanoparticle. In some aspects, the calibration occurs before the measured light intensity. In some aspects, the size of the nanoparticle is the actual size value of the bionanoparticle. In some aspects, the standard includes gold nanoparticles. In some aspects, the standard includes multiple lipids. In some aspects, the standard includes lipid vesicles. In some aspects, the standard includes silica beads, polystyrene beads, silicone beads, polymer beads, polymer nanoparticles or a combination thereof.

[0060] In some aspects, the fluid sample further comprises a second bio-nanoparticle, and wherein the standard comprises the second bio-nanoparticle. In some aspects, the size of the standard is measured via dynamic light dispersion. In some aspects, the measured light intensity has single nanoparticle sensitivity. In some aspects, the measured light intensity has single molecule sensitivity. In some aspects, the measured light intensity detects a single antibody comprising a plurality of detectable agents.

[0061] In some aspects, the information group includes identifying the concentration of the sample. In some aspects, the information group includes identifying the copy number of a biomolecule. In some aspects, the bio-nanoparticle comprises at least one biomarker. In some aspects, the bio-nanoparticle is combined with a variety of detectable agents. In some aspects, at least one of the multiple detectable agents is connected to the surface of the bio-nanoparticle, at least one of the multiple detectable agents is in the surface of the bio-nanoparticle, at least one of the multiple detectable agents is in the interior of the bio-nanoparticle, or a combination thereof. In some aspects, a variety of detectable agents include more than one type of detectable agent. In some aspects, the multiple detectable agents have different scatter spectra. In some aspects, the scatter spectra have different peak wavelengths. In some aspects, the peak wavelengths are separated by more than 10 nanometers, more than 20 nanometers, more than 30 nanometers, more than 40 nanometers, more than 50 nanometers, more than 75 nanometers, more than 100 nanometers, more than 120 nanometers, more than 140 nanometers, more than 160 nanometers, more than 180 nanometers, more than 200 nanometers, more than 300 nanometers, more than 400 nanometers, more than 500 nanometers, more than 600 nanometers, or more than 700 nanometers. In some aspects, the multiple detectable agents are co-located. In some aspects, the presence of co-located detectable agents provides an information group. In some aspects, the information group includes the presence of at least one biomarker, the absence of at least one biomarker, or a combination thereof.

[0062] In some aspects, the at least one planar surface is translucent. In some aspects, the at least one planar surface is transparent.

[0063] In various aspects, the present disclosure provides a kit for capturing nanoparticles, the kit comprising: at least one planar surface having a coating, the coating comprising: a nonspecific adsorption resistant material; and a plurality of capture molecules; and a containment device comprising a plurality of compartments, the containment device being compatible with centrifugation.

[0064] In some aspects, the containing device is a well plate. In some aspects, the plurality of compartments include wells. In some aspects, the containing device is a microfluidic chip. In some aspects, the plurality of compartments include microfluidic channels. In some aspects, the containing device is a block and the plurality of compartments include a plurality of holes. In some aspects, the at least one planar surface includes glass. In some aspects, the nonspecific adsorption resistant material includes polyethylene glycol. In some aspects, the nonspecific adsorption resistant material includes a zwitterionic surface. In some aspects, the zwitterionic surface includes carboxybetaine. In some aspects, the nonspecific adsorption resistant material includes bovine serum albumin.

[0065] In some aspects, the plurality of capture molecules comprises antibodies. In some aspects, the antibodies are biotinylated. In some aspects, the plurality of capture molecules comprises biotin or streptavidin. In some aspects, the plurality of capture molecules comprises nucleic acids.

[0066] In some aspects, the kit further comprises a plurality of biological nanoparticles, wherein the plurality of biological nanoparticles comprises extracellular vesicles, organelles, microvesicles, cell-derived vesicles, protein aggregates, nucleic acid aggregates, lipid aggregates, viruses, bacteria, exosomes, or a combination thereof.

[0067] In some aspects, the kit further comprises instructions. In some aspects, the kit further comprises a detectable agent. In some aspects, the detectable agent is a fluorescent detectable agent. In some aspects, the detectable agent is a luminescent detectable agent. In some aspects, the detectable agent is selected from the group consisting of a fluorescently labeled antibody, a fluorescently labeled protein, a fluorescently labeled nucleic acid, a fluorescently labeled lipid, a membrane dye, a fluorescent dye, a dye, a polymer dot, and a combination thereof.

[0068] In some aspects, the kit also includes a variety of detectable agents. In some aspects, the multiple detectable agents have overlapping divergence spectra. In some aspects, the multiple detectable agents have the same divergence spectra. In some aspects, the divergence spectra have the same peak wavelength. In some aspects, the multiple detectable agents include the same detectable agent. In some aspects, the multiple detectable agents include more than one type of detectable agent. In some aspects, the multiple detectable agents have different divergence spectra. In some aspects, the divergence spectra have different peak wavelengths. In some aspects, the peak wavelengths are separated by more than 10 nanometers, more than 20 nanometers, more than 30 nanometers, more than 40 nanometers, more than 50 nanometers, more than 75 nanometers, more than 100 nanometers, more than 120 nanometers, more than 140 nanometers, more than 160 nanometers, more than 180 nanometers, more than 200 nanometers, more than 300 nanometers, more than 400 nanometers, more than 500 nanometers, more than 600 nanometers, or more than 700 nanometers.

[0069] In some aspects, the kit further comprises a standard for calibration. In some aspects, the standard comprises gold nanoparticles. In some aspects, the standard comprises a plurality of lipids. In some aspects, the standard comprises lipid vesicles. In some aspects, the standard comprises silica beads, polystyrene beads, silicone beads, polymer beads, polymer nanoparticles or a combination thereof.

[0070] Incorporation by Reference

[0071] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0072] BRIEF DESCRIPTION OF THE DRAWINGS

[0073] The novel features of the present invention are described in detail in the appended claims. The features and advantages of the present invention may be better understood with reference to the following detailed description, which sets forth illustrative embodiments utilizing the principles of the present invention and the accompanying drawings, in which:

[0074] Figure 1 A device arranged to detect scattered light is shown.

[0075] Figure 2A Such a device is shown, comprising a microfluidic channel with flowing nanoparticles, an inlet, a constriction, and an outlet.

[0076] Figure 2B Such a device is shown, which includes a microfluidic chip with two flow focusing channels to concentrate nanoparticles in the plane of the microfluidic chip.

[0077] Figure 2C Such a device is shown, which includes a microfluidic chip with four flow focusing channels to concentrate nanoparticles in and perpendicular to the plane of the microfluidic chip.

[0078] Figure 3A -E shows the bionanoparticle size determination using surface membrane dyes. Figure 3A A bio-nanoparticle with a dye bound to the surface of the bio-nanoparticle is shown. Figure 3B It was shown that the intensity emitted from a surface dye can be proportional to the surface area. Figure 3C The reference size distribution of the standards is shown. Figure 3D An uncorrected histogram with intensity distribution is depicted. Figure 3E Histograms overlaid with reference size distributions are shown.

[0079] Figure 4A -E shows the bionanoparticle size determination using bulk dye. Figure 4A Bio-nanoparticles with a bulk dye encapsulated by the bio-nanoparticles are shown. Figure 4B It was shown that the intensity of emitted autologous dyes can be proportional to the volume of bionanoparticles. Figure 4C The reference size distribution of the standards is shown. Figure 4D An uncorrected histogram with intensity distribution is depicted. Figure 4E Histograms overlaid with reference size distributions are shown.

[0080] Figure 5A -B shows bionanoparticle size determination using bulk dye and surface membrane dye. Figure 5A Bio-nanoparticles with a bulk dye encapsulated by the bio-nanoparticle and a dye bound to the surface of the bio-nanoparticle are shown. Figure 5B It was shown that the intensity of emitted autologous dyes and surface dyes can be proportional to the surface area and volume of the bionanoparticles.

[0081] Figure 6 Two detection beams are shown, which can be used to determine bionanoparticle size by using duty cycle.

[0082] Figure 7 Shown is the intensity emission output detected when a bio-nanoparticle conjugated with a fluorescent dye passes through the detection beam and is excited.

[0083] Figure 8 The duty cycle that can be used to determine the size of bionanoparticles is shown.

[0084] Fig. 9 It is shown that an equation can be generated to relate the duty cycle to the nanoparticle size.

[0085] Fig.10 Shown is an excitation beam modified by a STED beam to generate a narrow region of efficient excitation.

[0086] Fig.11 Two excitation beams modified by the STED beam are shown to generate two narrow regions of efficient excitation.

[0087] Fig.12 The nanoparticle is shown passing through two excitation beams and zero length.

[0088] Fig.13 Shown is a device for controlling directional flow, which can be used to sort and enrich samples with nanoparticles of interest.

[0089] Fig.14 A device for capturing biological nanoparticles with a coated planar surface is shown.

[0090] Fig.15A -B shows the comparison of bio-nanoparticles captured on the coated flat surface with and without centrifugation. Fig.15A Bionanoparticles displayed on a coated planar surface are shown. Fig. 15B A comparison of the number of bionanoparticles captured on the coated flat surface with and without centrifugation is shown.

[0091] Fig.16 A microfluidic chip that can sort biological nanoparticles is shown.

[0092] Fig.17A -B shows a microfluidic chip and optical device with single dye molecule sensitivity and which utilizes internal calibration (Au nanoparticles) to normalize the detected light signal between different measurements. Fig.17A A microfluidic chip and an optical device are shown that can detect backscattered light from Au nanoparticles for use as an internal calibration and / or for normalizing the detected light signal. Fig. 17B Signal traces are shown, demonstrating that the detected fluorescence has single-dye molecule sensitivity.

[0093] Fig.18 Results are shown for counting the number of biomolecule copies on a single nanoparticle using Au nanoparticles as an internal standard for calibrating the intensity between two measurements.

[0094] Fig.19 A schematic diagram of the optical layout of the instrument used to achieve single-molecule sensitivity detection is shown.

[0095] Fig. 20A -C shows the co-localization determination of different protein biomarkers on single vesicles using different colored antibodies. Fig. 20AA signal time trace showing photon spikes detected from vesicles bound to an antibody against VGlut1 (Protein A) labeled with Alexa561 is shown. Fig. 20B A signal time trace showing photon spikes detected from vesicles bound to an Alexa647-labeled antibody against VATPase (Protein B) is shown. Fig. 20C The results show that the protein A ( Fig. 20A ) and protein B ( Fig. 20B ) were analyzed for co-localization.

[0096] Fig.21A -D shows the co-localization assay of different protein biomarkers on single vesicles using different colored antibodies that specifically bind to different proteins on the vesicles and a membrane dye that reports membrane presence and / or vesicle size. Fig.21A Shown is a signal time trace showing photon spikes detected from vesicles labeled with the membrane dye ANEPPS. Fig. 21B An exemplary signal time trace showing photon spikes detected from vesicles bound to an Alexa647-labeled antibody against CD63 is shown. Fig. 21C Shown is a cross-correlation analysis of colocalization between the membrane dye ANEPPS and anti-CD63 fluorescent antibody on the same vesicles. Fig.21D Shown are the fluorescence intensity distributions of a single free fluorescent antibody and those of antibodies bound to vesicles based on colocalization with a membrane dye.

[0097] Fig.22A -C shows the co-localization of different protein biomarkers on single vesicles using different colored antibodies that specifically bind to different proteins on the vesicles and bulk dyes that report the presence of intact membrane-bound volume and / or vesicle size. Fig.22A A signal time trace showing photon spikes detected from vesicles labeled with the bulk dye Oregon Green Diacetate is shown. Fig. 22B A signal time trace showing photon spikes detected from vesicles bound to an Alexa647-labeled antibody against SV2A is shown. Fig. 22C Shown is a cross-correlation analysis of colocalization between the body dye Oregon Green and anti-SV2A fluorescent antibody on the same vesicle; the percent colocalization was calculated to be 68%.

[0098] Fig.23A -D shows the signal intensity calibration using the backscattered light from gold nanoparticles as an internal standard. Fig.23AA signal time trace showing photon spikes detected from exosomes labeled with the membrane dye ANEPPS is shown. Fig. 23B A signal time trace showing a spike of photons detected from the gold nanoparticles is shown. Fig.23C Exemplary signal intensity histograms from membrane dye-labeled vesicles are shown (n=636 events). Fig.23D Shown are exemplary signal intensity histograms from gold nanoparticles (n=538 events), measured simultaneously with membrane dye-labeled exosomes in the same experiment.

[0099] Fig.24A -C shows a fluorescent image demonstrating size-dependent protein expression on exosomes. Fig.24A The fluorescence intensity of the membrane dye ANEPPS is shown. Fig. 24B Insets showing examples of large vesicles (1), medium vesicles (2), and small vesicles (3) are shown. Fig.24C Co-localization and relative copy number of Alexa647-labeled antibodies, as reflected by the difference in fluorescence intensity measured on exosomes, are shown.

[0100] DETAILED DESCRIPTION OF THE INVENTION

[0101] The present disclosure relates to methods, systems and devices for performing biological nanoparticle analysis. More specifically, in some aspects, the present disclosure relates to methods, systems and devices for single biological nanoparticle size determination of a sample while the biological nanoparticle is moving. In other aspects, the present disclosure relates to methods, systems and devices for selectively capturing biological nanoparticles on a coated planar surface, the capture being facilitated by centrifugation.

[0102] An important application for identifying biological nanoparticles is to sort or enrich the nanoparticles of interest. Body fluids contain multiple biological nanoparticles, which have rich diversity. Many biological nanoparticles can be used in biomedical applications, as biomarkers of disease, or contribute to disease identification. Biological nanoparticles may be rare, and body fluids containing nanoparticles often express them at low concentrations, and the fluid also contains other biological structures or fragments. The separation, purification or enrichment of biological nanoparticles of interest can contribute to biomedical treatment, and can additionally increase the local concentration of biological nanoparticles. Using microfluidic devices can increase efficiency and / or can promote separation, purification or enrichment of biological nanoparticles of interest. Biological nanoparticles can be classified by size, so rapid identification of nanoparticle size has many benefits.

[0103] The capture or separation of rare biological nanoparticles can help research, nanoparticle identification and medical treatment. Since the nanoparticles of interest are often expressed in low concentrations, methods to increase their capture have proven to be beneficial. Beads coated with capture materials have been used to separate particles of interest (see, for example, Lee, Nano Lett, 2014, 14 (1), pages 1-5), but small biological nanoparticles cannot be easily analyzed when on the beads, requiring a cutting step, which destroys the nanoparticles and reduces yield. The use of beads may also lead to problems associated with nonspecific binding, and therefore may lead to insufficient sample purity and / or low recovery efficiency. Low concentrations of biological nanoparticles in body fluids can lead to slow capture or low yield and / or low purity when captured and separated using conventional methods. In addition, the use of beads may tend to cause problems due to nonspecific binding, resulting in "dirty" samples. A method that helps capture or separate biological nanoparticles and reduces time would be beneficial in hospital and laboratory environments.

[0104] In view of the above, there is a need to quickly determine the size of biological nanoparticles as they move through, for example, a microfluidic chip. There is also a need to identify, characterize, separate or enrich the biological nanoparticles of interest. Since most biological nanoparticles are in low concentrations, there is also a need to quickly capture nanoparticles in a manner that provides high recovery efficiency, purity and sensitivity, and which can also be easily analyzed without further processing. The present disclosure provides these needs and more.

[0105] Device and method for determining biological nanoparticle size in transit

[0106] In some embodiments, the present disclosure provides methods, systems, devices and apparatus for determining (also referred to as identifying), manipulating and analyzing biological nanoparticles, which are fast and versatile and can be performed while the nanoparticles are moving. In some embodiments, the methods, systems, devices and apparatus of the present disclosure include a microfluidic chip, which can facilitate the manipulation, detection, analysis, determination and / or identification of biological nanoparticles while moving. Microfluidic chips can be used to process small amounts of fluid samples and have advantages over traditional large devices (e.g., compared to large devices, microfluidic chips only require trace amounts of fluid samples, require fewer reagents, and shorter processing times, increasing efficiency). Microfluidic chips are planar devices and can therefore facilitate the detection and analysis of biological nanoparticles by using an objective lens, lens or light collection system with a high numerical aperture, thereby enhancing light collection and thereby facilitating the detection, analysis, determination and / or identification of biological nanoparticles while moving. Microfluidic chips are planar devices, which enhances their compatibility with microscope settings. In addition, microfluidic chips can allow the design and generation of interconnected fluid networks without dead volumes, which in turn can facilitate the detection and manipulation of biological nanoparticles (e.g., sorting using flow displacements at three or more fluid channel nodes). Dead volume is the portion of the volume within the microfluidic chip outside the flow path (e.g., the volume into which a liquid that may carry sample nanoparticles may diffuse, thereby potentially reducing accuracy). Through microfabrication methods, microfluidic chips can allow the creation of channels with non-spherical or non-square (e.g., rectangular) cross-sections, which can facilitate the detection, analysis, determination, and / or identification of biological nanoparticles while moving. Microfluidic chips can facilitate the creation of channels with different widths or heights along the length of the channel (e.g., contraction or step change of channel width and / or height) to facilitate the manipulation, detection, analysis, determination, and / or identification of biological nanoparticles while moving. Microfluidic chips can be formed by bonding coverslips of desired thickness and with desired material properties (e.g., refractive index) to enhance compatibility with efficient light collection systems (e.g., high numerical aperture objectives requiring appropriate coverslip thickness and refractive index for maximum light collection and / or minimal distortion) to facilitate manipulation, detection, analysis, determination and / or identification of bio-nanoparticles while in motion. Microfluidic chips provide an attractive and versatile platform for manipulation, separation, sorting and / or transport of bio-nanoparticles.

[0107] In certain aspects, nanoparticles traveling through the microfluidic channels of a microfluidic device may be exposed to radiation, including infrared, visible light, and microwave radiation. The effect of exposing the biological nanoparticles to radiation is the resulting light intensity emitted by the nanoparticles or by molecules bound to the nanoparticles. Measuring the light intensity emitted from the nanoparticles provides a corresponding measurement of the nanoparticle size value. The size value may be a relative size value or a true size value. The method of measuring the light intensity emitted when the biological nanoparticles flow through the microfluidic device allows the size value to be assigned to the nanoparticles as the nanoparticles move. In some embodiments, the detector may be configured to detect the presence or absence of the biological nanoparticles particle by particle when the biological nanoparticles flow through at least a portion of the microfluidic chip. In some embodiments, the microfluidic chip includes a microfluidic channel, and the biological nanoparticles flow through the microfluidic channel. In some embodiments, the microfluidic channel includes a constriction, and the biological nanoparticles flow through the constriction. This method provides unexpected benefits for conventional nanoparticle size determination, at least because the use of microfluidic devices provides surprisingly efficient analysis of detection and / or sorting events at high frequency and high throughput. The compact size of the microfluidic chip allows the use of the methods, devices, systems and apparatus of the present disclosure in a variety of settings that do not support traditional laboratory settings, and the accuracy of the data obtained using the microfluidic device is better than expected by simply scaling down traditional large-scale equipment. The determination of nanoparticle size values ​​during flow avoids the need for biological nanoparticle capture and downstream release in existing methods. The methods of the present disclosure can be applied to a variety of devices and systems.

[0108] In some embodiments, the size value is assigned to the bionanoparticle while the bionanoparticle is flowing. The terms "flowing" and "moving" are used interchangeably herein. In some embodiments, the flow is uninterrupted. The terms "uninterrupted flow", "continuous flow" and variants thereof are used interchangeably herein. The term "uninterrupted flow" (and variants thereof) refers to a flow state in which the flow does not stop for more than 1 second. In some aspects, the flow can be directionally assigned.

[0109] As used herein, the term "assignment" refers to the quantitative attribute, qualitative attribute or importance of the biological nanoparticle classification indicated for the biological nanoparticle object being assigned. In one embodiment, a size value can be assigned to a biological nanoparticle. As used herein, the term "size value" refers to a relative size value or an actual size value. The size value provides a true or relative measure of a linear distance. In certain embodiments, the assignment is performed by software and computers representing an assignment algorithm.

[0110] As used herein, the term "ranking" refers to the quantitative characteristics, qualitative characteristics or importance of evaluating nanoparticles by classification. In one embodiment, biological nanoparticles can be ranked as empty (e.g., when the emission intensity of nanoparticles is lower than a detectable threshold) or non-zero (e.g., when nanoparticles are detected). In some embodiments, the ranking is binary. For example, each nanoparticle whose detected light intensity is higher than a threshold limit is assigned a value of 1, and each sample of each measurement whose detected light intensity is not higher than the threshold limit is assigned a value of 0, thereby forming a binary ranking. In other embodiments, nanoparticles can be ranked according to other classifications, for example, about the type of nanoparticles, the presence of detectable features, the presence of distinguishing features, etc. Any number can be assigned to the ranking, which corresponds to one of a plurality of predetermined quantitative or qualitative categories. In other embodiments, the ranking is non-binary, for example, based on the amount of the emission light intensity measured from the biological nanoparticles to assign a value. In certain embodiments, the ranking is performed by software and computers representing a ranking algorithm.

[0111] As used herein, "detectable feature" refers to an observable property associated with a biological nanoparticle, for example, an optical, electrical, biological, or magnetic property associated with or inherent to a biological nanoparticle. In certain embodiments, a "detectable feature" includes the binding of a biological nanoparticle to a detectable agent or biomarker.

[0112] Examples of photoactive properties include, for example, changes in optical intensity (optical reflection, scattering, deflection, transmission, absorbance, or emission) typically caused by biological particle morphology (particle size, internal subcellular structure), fluorescence, luminescence, immunofluorescence, etc. Detection of a photoactive property can, for example, report on the size, mass, surface area, volume, protein content, membrane area, lipid content, enzyme content, metabolite content, carbohydrate content, nucleic acid content, protein species, or nucleic acid species on, in, or associated with the nanoparticle.

[0113] As used herein, the terms "biological nanoparticle", "bionanoparticle" and "bioparticle" are used interchangeably. The general term "nanoparticle" refers to a bionanoparticle. The term "bionanoparticle" refers to a biological unit with a hydrodynamic diameter of less than 1 μm. A non-limiting example of a bionanoparticle is an exosome.

[0114] Microfluidic devices

[0115] In some aspects, the present disclosure provides the use of microfluidic chips. Microfluidic chips can be formed by substrates (e.g., silicon, glass, ceramics, plastics, silicone, quartz or combinations thereof) and can include a network of microfluidic channels through which fluid flows. Microfluidic devices can be used to process small amounts of fluid samples and have advantages over traditional large-scale devices (e.g., requiring significantly less volume of fluid samples, requiring less reagent use, and reducing processing time compared to large-scale devices). Microfluidic chips provide an attractive and versatile platform for manipulating, separating, sorting and / or transporting bio-nano particles. The simplicity of patterning and integration of arrays of microfluidic channels in microfluidic devices makes these microfluidic devices attractive platforms for applications involving bio-nano particles. Microfluidic chips are planar devices, and therefore can facilitate detection and analysis of bio-nano particles by using an objective lens, lens or light collection system with a high numerical aperture, thereby enhancing light collection and thus facilitating detection, analysis, determination and / or identification of bio-nano particles when moving.

[0116] Microfluidic chips are planar devices, and therefore can enhance their compatibility with microscope settings. In addition, microfluidic chips can allow the design and generation of interconnected fluid networks, and there is no dead volume, which in turn can promote the detection and operation of biological nanoparticles (for example, using the flow displacement at three or more fluid channel nodes to sort). Through micro-fabrication methods, microfluidic chips can allow the creation of channels with non-spherical or non-square (for example rectangular) cross-sections, and this can promote the detection, analysis, determination and / or identification of biological nanoparticles when moving. Microfluidic chips can promote the creation of channels with different widths or heights along the length of the channel (for example, contraction or step changes of channel width and / or height), to promote the manipulation, detection, analysis, determination and / or identification of biological nanoparticles when moving. Microfluidic chips can be formed by bonding coverslips of desired thickness and having desired material properties (e.g., refractive index) to enhance compatibility with efficient light collection systems (e.g., high numerical aperture objectives requiring appropriate coverslip thickness and refractive index for maximum light collection and / or minimal distortion) to facilitate manipulation, detection, analysis, determination and / or identification of bio-nanoparticles while moving. Microfluidic chips and devices using microfluidic chips allow large numbers of bio-nanoparticles to be transported, combined, separated, sorted and / or otherwise manipulated in a rapid manner. As used herein, the terms "microfluidic chip," "microfluidic device," and "microfluidic channel device" can be used interchangeably.

[0117] In some embodiments, the microfluidic chip includes an inlet, an outlet, and a microfluidic channel, which can be analyzed using a detection beam to detect nanoparticles (see, e.g., Figure 2AIn certain embodiments, the microfluidic chip includes an inlet, an outlet, and a microfluidic channel that can be analyzed using a detection beam to detect nanoparticles, and two flow focusing channels to focus the nanoparticles in the plane of the microfluidic chip (see, e.g., Figure 2B In some embodiments, the microfluidic chip includes an inlet, an outlet, and a microfluidic channel that can be analyzed using a detection beam to detect nanoparticles, and four flow focusing channels to focus the nanoparticles in the plane of the microfluidic chip and perpendicular to the plane of the microfluidic chip (see, e.g., Figure 2C ).

[0118] In some embodiments, the microfluidic chip includes an input channel, a first output channel, a second output channel, and a directional flow channel. In some embodiments, the microfluidic chip also includes a valve, wherein the valve is separable from the microfluidic chip. In some embodiments, the flow of the first fluid in the directional flow channel is regulated; and the flow of the first fluid in the directional flow channel guides the flow of the second fluid from the input channel to the first output channel, the second output channel, or a combination thereof. In some embodiments, the detector is configured to detect a signal emitted from a biological nanoparticle, and the processor is configured to assign a value to the biological nanoparticle and operate a flow regulation mechanism. In some aspects, the flow regulation mechanism is an electrically actuated mechanism.

[0119] Microfluidic chips can be manufactured to provide efficient active sorting schemes and subsequent purification (e.g., purification chamber) schemes. Microfluidic chips can be composed of two layers on a silicon motherboard and can be manufactured with a one-step molding into a polymer substrate. Microfluidic chips can be refined by bonding to a glass substrate or a polymer substrate.

[0120] In some aspects, the silicon motherboard can be manufactured using two photolithography processes. Features can be designed using standard software (e.g., AutoCAD, Autodesk, San Rafael, CA) and can be written on a chrome mask. In this case, positive resist lithography and deep reactive ion etching (DRIE) can be used to form the first layer. In some aspects, a positive photoresist (e.g., AZ 1512) is obtained by a process that can include a DRIE process. The DRIE process can reach a depth suitable for various features (e.g., 2-5 μm).

[0121] In some aspects, the second layer of microfluidic chip features can be made using a negative photoresist (e.g., SU-8-3050, from MicroChem, Newton, Massachusetts), and the height of the features can be controlled (e.g., 50 μm). The mainboard can be silanized using, for example, tridecafluoro-1,1,2,2-tetrahydrooctyl-1-trichlorosilane (Sigma-Aldrich, St. Louis, Missouri). The silanized mainboard and silicon wafer can be coated with uncured PDMS and baked (e.g., 2 hours at 70°C). In some aspects, the PDMS sheet with the desired micro features can be peeled off from the silicon mainboard, and then the cover glass sheet is bonded using a standard process of plasma oxidation to complete the manufacture of the microfluidic chip.

[0122] In some aspects, the microfluidic chip provided herein can include a flow channel or chamber surrounded by walls, wherein the walls are made of materials including but not limited to: polymer materials (polydimethylsiloxane (PDMS), polyurethane methacrylate (PUMA), polymethyl methacrylate (PMMA), polyethylene, polyester (PET), polytetrafluoroethylene (PTFE), polycarbonate, polyparaxylene, polyvinyl chloride, fluoroethyl propylene, lexan, polystyrene, cyclic olefin polymers, cyclic olefin copolymers, polyurethane, polyester carbonate, polypropylene, polybutylene, polyacrylate, polycaprolactone, polyketone, polyphthalamide, cellulose acetate, polyacrylonitrile, polysulfone, epoxy polymers, thermoplastic materials, fluoropolymers, and polyvinylidene fluoride, polyamide, polyimide), inorganic materials (glass, quartz, silicon, GaAs, silicon nitride), fused silica, ceramics, glass (organic) and / or other materials and combinations thereof.

[0123] In some aspects, the wall material can be made from porous molds, woven or nonwoven fibers of wool (such as cloth or mesh), metal (e.g., stainless steel or monel), glass, paper, or synthetics (e.g., nylon, polypropylene, polycarbonate, polyparaxylene, and various polyesters), sintered stainless steel and other metals, and porous inorganic materials such as alumina, silica, or carbon.

[0124] In some embodiments, a microfluidic chip includes at least one first input channel and at least two outlet channels.

[0125] In certain embodiments, a microfluidic chip is provided and includes a plurality of microfluidic channels. In some embodiments, the microfluidic chip may include a channel for transporting biological nanoparticles based on sorting. In some embodiments, the microfluidic chip may include a channel for transporting biological nanoparticles based on size value. In some of the described, the device may further include electrodes for tracking and manipulating the directional flow of biological particles.

[0126] In certain embodiments, the microfluidic chip provided herein may include a plurality of flow channels, including one or more input flow channels (i.e., channels that bring biological nanoparticles into the detection volume) and one or more output channels (i.e., channels that bring biological nanoparticles away from the detection volume). In some embodiments, the device provided herein may include at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, or more input channels and at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, or more output channels. In certain embodiments, the plurality of flow channels are in a parallel configuration.

[0127] In certain embodiments, a microfluidic chip can include multiple flow channels connected to a main channel to allow the injection of additional fluids to change local velocities.

[0128] The passage of microfluidic chip may intersect at a node. In some aspects, a passage intersects with another passage at a node. In some aspects, a passage intersects with a plurality of different passages at a node. In some aspects, a passage intersects with at least 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95 or 100 different passages at a node. In some aspects, a plurality of passages intersect with another passage at a node. In some aspects, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 different channels intersect with a plurality of channels at a node. In some embodiments, a node does not have any dead volume.

[0129] The passage of microfluidic chip may not intersect at the node.In some aspects, a passage is not the position of node on microfluidic chip and intersects with another passage.In some aspects, a passage is not the position of node on microfluidic chip and intersects with a plurality of other passages.In some aspects, a passage is not the position of node on microfluidic chip and intersects with at least 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95 or 100 different passages.In some aspects, a plurality of passages are not the position of node on microfluidic chip and intersect with another passage. In some aspects, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 different channels intersect with another channel at a location on the microfluidic chip that is not a node.

[0130] In some embodiments, the microfluidic device is a planar device and can be used for high numerical aperture detection or imaging. Planar microfluidic devices prevent image or light collection distortion or aberration (e.g., spherical or chromatic aberration) that often occurs during analysis of non-planar systems (e.g., capillaries, in which the inner and / or outer surfaces of the cylinder can act as cylindrical lenses, thus causing distortion in terms of focusing of light and / or signal collection). The ability to use high numerical aperture objective lenses also maximizes light collection efficiency. In addition, planar microfluidic devices are not subject to the same distance limitations observed when using non-planar systems for detection or imaging. In addition, planar microfluidic devices do not have the material limitations of other systems (e.g., planar microfluidic devices may include coverslips with specific thickness, refractive index, and optical properties), and the effects observed when using non-planar systems for detection or imaging. In some embodiments, detection, imaging, or a combination thereof include epi-illumination, and a light collection system with a numerical aperture greater than 1.0. In some embodiments, inspection or imaging of a microfluidic device uses a light collection system with a numerical aperture of 0.8 or greater, 0.9 or greater, 1.0 or greater, 1.1 or greater, 1.2 or greater, 1.3 or greater, or 1.4 or greater. In some embodiments, inspection or imaging of a microfluidic device uses a light collection system with a numerical aperture of 0.8 or greater and not greater than 1.5, 0.9 or greater and not greater than 1.5, 1.0 or greater and not greater than 1.5, 1.1 or greater and not greater than 1.5, 1.2 or greater and not greater than 1.5, 1.3 or greater and not greater than 1.5, or 1.4 or greater and not greater than 1.5. In a preferred embodiment, a planar microfluidic device can be imaged using a light collection system with a numerical aperture of 1.2 or greater.

[0131] In some embodiments, the microfluidic chip is a planar device and comprises at least one microfluidic channel. In some embodiments, at least a portion of at least one microfluidic channel has a width of less than 500 μm, a width of less than 400 μm, a width of less than 300 μm, a width of less than 200 μm, a width of less than 100 μm, a width of less than 75 μm, a width of less than 50 μm, a width of less than 40 μm, a width of less than 30 μm, a width of less than 25 μm, a width of less than 20 μm, a width of less than 15 μm, a width of less than 10 μm, a width of less than 5 μm, a width of less than 2 μm, a width of less than 1 μm, a width of less than 900 nm, a width of less than 850 nm, a width of less than 800 nm, a width of less than 750 nm, a width of less than 700 nm, a width of less than 650 nm, a width of less than 600 nm, a width of less than 550 nm, or a width of less than 500 nm. In some embodiments, the maximum width of at least one microfluidic channel is less than 500 μm, less than 400 μm, less than 300 μm, less than 200 μm, less than 100 μm, less than 75 μm, less than 50 μm, less than 40 μm, less than 30 μm, less than 25 μm, less than 20 μm, less than 15 μm, less than 10 μm, less than 5 μm, less than 2 μm, less than 1 μm, less than 900 nm, less than 850 nm, less than 800 nm, less than 750 nm, less than 700 nm, less than 650 nm, less than 600 nm, less than 550 nm or less than 500 nm. In a preferred embodiment, at least a portion of at least one microfluidic channel has a width of less than 10 μm, a width of less than 5 μm or a width of less than 2 μm. In a preferred embodiment, at least a portion of at least one microfluidic channel has a width of 1 μm-10 μm.

[0132] In some embodiments, at least a portion of at least one microfluidic channel has a height of less than 500 μm, a height of less than 400 μm, a height of less than 300 μm, a height of less than 200 μm, a height of less than 150 μm, a height of less than 125 μm, a height of less than 100 μm, a height of less than 75 μm, a height of less than 50 μm, a height of less than 40 μm, a height of less than 30 μm, a height of less than 25 μm, a height of less than 20 μm, a height of less than 15 μm, a height of less than 10 μm, a height of less than 5 μm, a height of less than 2 μm or a height of less than 1 μm. In some embodiments, the maximum height of at least one microfluidic channel is less than 500 μm, less than 400 μm, less than 300 μm, less than 200 μm, less than 150 μm, less than 125 μm, less than 100 μm, less than 75 μm, less than 50 μm, less than 40 μm, less than 30 μm, less than 25 μm, less than 20 μm, less than 15 μm, less than 10 μm, less than 5 μm, less than 2 μm or less than 1 μm. In preferred embodiments, at least a portion of at least one microfluidic channel has a height of less than 10 μm, a height of less than 5 μm or a height of less than 2 μm. In preferred embodiments, at least a portion of at least one microfluidic channel has a height of 1 μm-10 μm.

[0133] In some embodiments, the at least one microfluidic channel includes a constriction. In some embodiments, the at least one microfluidic channel includes a plurality of constrictions. In some embodiments, the microfluidic chip includes a plurality of microfluidic channels, which at least partially include a constriction. In some embodiments, the microfluidic chip includes a plurality of microfluidic channels, each of which includes a constriction. The constriction is a portion of a microfluidic channel that is narrower (i.e., constricted) than other portions of the microfluidic channel. The fluid sample can flow through the constriction. In certain embodiments, the constriction includes the narrowest region of the microfluidic channel.

[0134] In some aspects, the width of the constriction is less than the widest part (i.e., the maximum width) of the microfluidic channel. In certain embodiments, the width of the constriction is relative to the widest part of the microfluidic channel. In some embodiments, the width of the constriction is less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2% or less than 1% of the maximum width of the microfluidic channel. As a non-limiting example, a microfluidic channel with a maximum width of 100 μm can have a constriction less than 25% of the maximum width value (i.e., less than 25 μm). In a preferred embodiment, the width of the constriction is less than 10% of the maximum width of the microfluidic channel.

[0135] In certain embodiments, the maximum width of the microfluidic channel has a value of less than 900 μm and greater than 0.1 μm, less than 800 μm and greater than 0.5 μm, less than 700 μm and greater than 1 μm, less than 600 μm and greater than 5 μm, less than 500 μm and greater than 10 μm, less than 1,000 μm and greater than 10 μm, less than 900 μm and greater than 10 μm, less than 800 μm and greater than 10 μm, less than 700 μm and greater than 10 μm, less than 600 μm and greater than 10 μm, less than 500 μm. and greater than 10 μm, less than 400 μm and greater than 10 μm, less than 300 μm and greater than 10 μm, less than 500 μm and greater than 0.1 μm, less than 500 μm and greater than 1 μm, less than 500 μm and greater than 2 μm, less than 500 μm and greater than 5 μm, less than 800 μm and greater than 0.1, less than 700 μm and greater than 0.1, less than 600 μm and greater than 0.1, less than 500 μm and greater than 0.1, less than 400 μm and greater than 0.1 or less than 300 μm and greater than 0.1. In a preferred embodiment, the value of the maximum width of the microfluidic channel is less than 500 μm and greater than 10 μm.

[0136] In some embodiments, the width of the constriction is less than the average width of the microfluidic channel. In certain embodiments, the width of the constriction is relative to the average width of the microfluidic channel. In some embodiments, the width of the constriction is less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2% or less than 1% of the average width of the microfluidic channel.

[0137] In certain embodiments, the average width of the microfluidic channel has a value of less than 900 μm and greater than 0.1 μm, less than 800 μm and greater than 0.5 μm, less than 700 μm and greater than 1 μm, less than 600 μm and greater than 5 μm, less than 500 μm and greater than 10 μm, less than 1,000 μm and greater than 10 μm, less than 900 μm and greater than 10 μm, less than 800 μm and greater than 10 μm, less than 700 μm and greater than 10 μm, less than 600 μm and greater than 10 μm, less than 500 μm. and greater than 10 μm, less than 400 μm and greater than 10 μm, less than 300 μm and greater than 10 μm, less than 500 μm and greater than 0.1 μm, less than 500 μm and greater than 1 μm, less than 500 μm and greater than 2 μm, less than 500 μm and greater than 5 μm, less than 800 μm and greater than 0.1, less than 700 μm and greater than 0.1, less than 600 μm and greater than 0.1, less than 500 μm and greater than 0.1, less than 400 μm and greater than 0.1 or less than 300 μm and greater than 0.1. In a preferred embodiment, the value of the average width of the microfluidic channel is less than 500 μm and greater than 10 μm.

[0138] In some embodiments, the height of the constriction is less than the maximum height value (i.e., maximum height) of the microfluidic channel. In certain embodiments, the height of the constriction is relative to the maximum height of the microfluidic channel. In some embodiments, the height of the constriction is less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2% or less than 1% of the maximum height of the microfluidic channel. As a non-limiting example, a microfluidic channel with a maximum height of 20 μm can have a constriction less than 10% of the maximum height value (i.e., less than 2 μm). In a preferred embodiment, the height of the constriction is less than 25% of the maximum height of the microfluidic channel.

[0139] In certain embodiments, the maximum height of the microfluidic channel has a value of less than 900 μm and greater than 0.1 μm, less than 800 μm and greater than 0.5 μm, less than 700 μm and greater than 1 μm, less than 600 μm and greater than 5 μm, less than 500 μm and greater than 10 μm, less than 1,000 μm and greater than 10 μm, less than 900 μm and greater than 10 μm, less than 800 μm and greater than 10 μm, less than 700 μm and greater than 10 μm, less than 600 μm and greater than 10 μm, less than 500 μm. and greater than 10 μm, less than 400 μm and greater than 10 μm, less than 300 μm and greater than 10 μm, less than 500 μm and greater than 0.1 μm, less than 500 μm and greater than 1 μm, less than 500 μm and greater than 2 μm, less than 500 μm and greater than 5 μm, less than 800 μm and greater than 0.1, less than 700 μm and greater than 0.1, less than 600 μm and greater than 0.1, less than 500 μm and greater than 0.1, less than 400 μm and greater than 0.1 or less than 300 μm and greater than 0.1. In a preferred embodiment, the value of the maximum height of the microfluidic channel is less than 500 μm and greater than 10 μm.

[0140] In some embodiments, the height of the constriction is less than the average height of the microfluidic channel. In certain embodiments, the height of the constriction is relative to the average height of the microfluidic channel. In some embodiments, the height of the constriction is less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2% or less than 1% of the average height of the microfluidic channel.

[0141] In certain embodiments, the average height of the microfluidic channel has a value of less than 900 μm and greater than 0.1 μm, less than 800 μm and greater than 0.5 μm, less than 700 μm and greater than 1 μm, less than 600 μm and greater than 5 μm, less than 500 μm and greater than 10 μm, less than 1,000 μm and greater than 10 μm, less than 900 μm and greater than 10 μm, less than 800 μm and greater than 10 μm, less than 700 μm and greater than 10 μm, less than 600 μm and greater than 10 μm, less than 500 μm. and greater than 10 μm, less than 400 μm and greater than 10 μm, less than 300 μm and greater than 10 μm, less than 500 μm and greater than 0.1 μm, less than 500 μm and greater than 1 μm, less than 500 μm and greater than 2 μm, less than 500 μm and greater than 5 μm, less than 800 μm and greater than 0.1, less than 700 μm and greater than 0.1, less than 600 μm and greater than 0.1, less than 500 μm and greater than 0.1, less than 400 μm and greater than 0.1 or less than 300 μm and greater than 0.1. In a preferred embodiment, the value of the average height of the microfluidic channel is less than 500 μm and greater than 10 μm.

[0142] In some embodiments, the cross-sectional area of ​​the constriction is less than the maximum cross-sectional area (i.e., the maximum cross-sectional area) of the microfluidic channel. In certain embodiments, the cross-sectional area of ​​the constriction is relative to the maximum cross-sectional area of ​​the microfluidic channel. In some embodiments, the cross-sectional area of ​​the constriction is less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 24%, less than 23%, less than 22%, less than 21%, less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.2%, less than 0.1%, less than 0.05%, less than 0.02%, less than 0.01%, less than 0.005%, less than 0.002% or less than 0.001% of the maximum cross-sectional area of ​​the microfluidic channel. As a non-limiting example, the maximum cross-sectional area is 200 μm 2 The microfluidic channel may have a maximum cross-sectional area of ​​less than 10% (i.e., less than 20 μm 2 In a preferred embodiment, the cross-sectional area of ​​the contraction is 10%-0.01% of the maximum cross-sectional area of ​​the microfluidic channel.

[0143] In certain embodiments, the maximum cross-sectional area of ​​a microfluidic channel has a value less than 1,000,000 μm 2 And greater than 10μm 2 , less than 750,000μm2 And greater than 25μm 2 , less than 500,000μm 2 And greater than 100μm 2 , less than 250,000μm 2 And greater than 250μm 2 , less than 900,000μm 2 And greater than 100μm 2 , less than 800,000μm 2 And greater than 100μm 2 , less than 700,000μm 2 And greater than 100μm 2 , less than 600,000μm 2 And greater than 100μm 2 , less than 400,000μm 2 And greater than 100μm 2 , less than 300,000μm 2 And greater than 100μm 2 , less than 200,000μm 2 And greater than 100μm 2 , less than 100,000μm 2 And greater than 100μm 2 , less than 50,000μm 2 And greater than 100μm 2 , less than 25,000μm 2 And greater than 100μm 2 , less than 10,000μm 2 And greater than 100μm 2 , less than 1,000μm 2 And greater than 100μm 2 , less than 2,000,000μm 2 And greater than 250μm 2 , less than 1,000,000μm 2 And greater than 250μm 2 , less than 900,000μm 2 And greater than 250μm 2 , less than 800,000μm 2 And greater than 250μm 2 , less than 700,000μm 2 And greater than 250μm 2 , less than 600,000μm 2 And greater than 250μm 2 , less than 400,000μm 2 And greater than 250μm 2, less than 300,000μm 2 And greater than 250μm 2 , less than 200,000μm 2 And greater than 250μm 2 , less than 100,000μm 2 And greater than 250μm 2 , less than 50,000μm 2 And greater than 250μm 2 , less than 25,000μm 2 And greater than 250μm 2 , less than 10,000μm 2 And greater than 250μm 2 or less than 1,000μm 2 And greater than 250μm 2 In a preferred embodiment, the maximum cross-sectional area of ​​the microfluidic channel has a value less than 250,000 μm 2 And greater than 250μm 2 .

[0144] In some embodiments, the cross-sectional area of ​​the constriction is less than the average cross-sectional area of ​​the microfluidic channel. In certain embodiments, the cross-sectional area of ​​the constriction is relative to the average cross-sectional area of ​​the microfluidic channel. In some embodiments, the cross-sectional area of ​​the constriction is less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 24%, less than 23%, less than 22%, less than 21%, less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.2%, less than 0.1%, less than 0.05%, less than 0.02%, less than 0.01%, less than 0.005%, less than 0.002% or less than 0.001% of the average cross-sectional area of ​​the microfluidic channel.

[0145] In certain embodiments, the average cross-sectional area of ​​the microfluidic channel has a value less than 1,000,000 μm 2 And greater than 10μm 2 , less than 750,000μm 2 And greater than 25μm 2 , less than 500,000μm 2 And greater than 100μm 2 , less than 250,000μm 2 And greater than 250μm 2 , less than 900,000μm2 And greater than 100μm 2 , less than 800,000μm 2 And greater than 100μm 2 , less than 700,000μm 2 And greater than 100μm 2 , less than 600,000μm 2 And greater than 100μm 2 , less than 400,000μm 2 And greater than 100μm 2 , less than 300,000μm 2 And greater than 100μm 2 , less than 200,000μm 2 And greater than 100μm 2 , less than 100,000μm 2 And greater than 100μm 2 , less than 50,000μm 2 And greater than 100μm 2 , less than 25,000μm 2 And greater than 100μm 2 , less than 10,000μm 2 And greater than 100μm 2 , less than 1,000μm 2 And greater than 100μm 2 , less than 2,000,000μm 2 And greater than 250μm 2 , less than 1,000,000μm 2 And greater than 250μm 2 , less than 900,000μm 2 And greater than 250μm 2 , less than 800,000μm 2 And greater than 250μm 2 , less than 700,000μm 2 And greater than 250μm 2 , less than 600,000μm 2 And greater than 250μm 2 , less than 400,000μm 2 And greater than 250μm 2 , less than 300,000μm 2 And greater than 250μm 2 , less than 200,000μm 2 And greater than 250μm 2 , less than 100,000μm 2 And greater than 250μm 2, less than 50,000μm 2 And greater than 250μm 2 , less than 25,000μm 2 And greater than 250μm 2 , less than 10,000μm 2 And greater than 250μm 2 or less than 1,000μm 2 And greater than 250μm 2 In a preferred embodiment, the average cross-sectional area of ​​the microfluidic channel has a value less than 250,000 μm 2 And greater than 250μm 2 .

[0146] In some embodiments, the height of the constriction is less than 10 μm, less than 9 μm, less than 8 μm, less than 7 μm, less than 6 μm, less than 5 μm, less than 4 μm, less than 3 μm, less than 2 μm, or less than 1 μm. In some embodiments, the width of the constriction is less than 10 μm, less than 9 μm, less than 8 μm, less than 7 μm, less than 6 μm, less than 5 μm, less than 4 μm, less than 3 μm, less than 2 μm, or less than 1 μm.

[0147] In some embodiments, at least one microfluidic channel includes one, two, three, four, five, six, seven, eight, nine, ten or more than ten contractions. In some embodiments, the microfluidic chip includes a plurality of microfluidic channels, each of which at least partially includes one, two, three, four, five, six, seven, eight, nine, ten or more than ten contractions. In some embodiments, the microfluidic chip includes a plurality of microfluidic channels, most of which each include one, two, three, four, five, six, seven, eight, nine, ten or more than ten contractions. In some embodiments, the microfluidic chip includes a plurality of microfluidic channels, each of which includes one, two, three, four, five, six, seven, eight, nine, ten or more than ten contractions. In some embodiments, compared to another microfluidic channel with a contraction size, a microfluidic channel including at least one contraction can avoid the establishment of back pressure when a sample flows through the microfluidic channel.

[0148] In some embodiments, at least a portion of at least one microfluidic channel has a cross-sectional area of ​​less than 10,000 μm. 2 , with a cross-sectional area less than 5,000 μm 2 , with a cross-sectional area less than 3,000 μm 2 , with a cross-sectional area less than 1,000 μm 2 , cross-sectional area less than 800μm 2, cross-sectional area less than 600μm 2 , cross-sectional area less than 400 μm 2 , cross-sectional area less than 200 μm 2 Or the cross-sectional area is less than 100 μm 2 In a preferred embodiment, at least part of at least one microfluidic channel has a cross-sectional area of ​​less than 100 μm 2 , cross-sectional area less than 90μm 2 , cross-sectional area less than 80 μm 2 , cross-sectional area less than 70 μm 2 , cross-sectional area less than 60 μm 2 , cross-sectional area less than 50 μm 2 , cross-sectional area less than 40 μm 2 , cross-sectional area less than 30 μm 2 , cross-sectional area less than 20 μm 2 , cross-sectional area less than 10 μm 2 , cross-sectional area less than 5μm 2 , cross-sectional area less than 2μm 2 Or the cross-sectional area is less than 1μm 2 In some embodiments, at least one microfluidic channel has a maximum cross-sectional area of ​​less than 250,000 μm 2 , less than 100,000μm 2 , less than 50,000μm 2 , less than 25,000μm 2 , less than 10,000μm 2 , less than 5,000μm 2 , less than 3,000μm 2 , less than 1,000μm 2 , less than 800μm 2 , less than 600μm 2 , less than 400μm 2 , less than 200μm 2 or less than 100μm 2 In some embodiments, at least one microfluidic channel has a maximum cross-sectional area of ​​less than 100 μm 2 , less than 90μm 2 , less than 80μm 2 , less than 70μm 2 , less than 60μm 2 , less than 50μm 2 , less than 40μm 2 , less than 30μm 2 , less than 20μm 2 , less than 10μm 2 , less than 5μm 2, less than 2μm 2 or less than 1μm 2 In a preferred embodiment, at least part of at least one microfluidic channel has a cross-sectional area of ​​1 μm 2 -100μm 2 In some embodiments, at least one microfluidic channel has a maximum cross-sectional area of ​​100 μm 2 -10,000μm 2 .

[0149] In certain embodiments, at least a portion of at least one microfluidic channel includes a discontinuous change in at least one of its width or height (e.g., implemented using microfabrication techniques). The microfluidic chip used herein may include a microfluidic channel having a step gradient or step change in at least one of its width or height, which contrasts with a microfluidic channel comprising a continuous change in height or width. Channels comprising a continuous change in height or width are common in such devices, which include, for example, glass tubes, which can be implemented by pulling a heating tube. In a specific embodiment, the height and width of at least a portion of at least one microfluidic channel change independently of each other. The independent change of height and width contrasts with, for example, a glass tube, wherein in the glass tube manufacturing process, a reduction in height is accompanied by a corresponding reduction in width (e.g., stretching and thinning a glass tube that has been heated to a temperature close to its melting point).

[0150] In certain embodiments, the devices provided herein may include a flow channel or chamber that has been chemically or biomolecularly pretreated. For example, the channel or chamber may be treated with an anticoagulant compound to prevent or reduce the binding of bio-nanoparticles in a fluid sample, or a compound that prevents or reduces the aggregation or aggregation of bio-nanoparticles in a fluid sample.

[0151] In certain embodiments, the microfluidic chip includes a translucent or transparent feature. For example, a fluid sample containing biological nanoparticles can be provided to a microfluidic chip comprising at least one translucent or transparent surface, and the at least one translucent or transparent surface allows an interrogation source to interact with the biological nanoparticles, thereby causing the biological nanoparticles to emit light intensity that passes through the at least one translucent or transparent surface, thereby being detected.

[0152] equipment

[0153] In one aspect, the present disclosure provides an apparatus for inspecting and measuring biological nanoparticles in a fluid sample. In one embodiment, the apparatus comprises: (a) at least a first input channel; (b) at least two outlet channels; (c) at least one detector capable of detecting one or more biological nanoparticles in the fluid sample; (d) a mechanism for directing the flow of biological nanoparticles; and (e) a sorting device capable of assigning values ​​to biological nanoparticles based on the presence, absence, type, composition, size value, or light emission of the biological nanoparticles, or based on the presence, absence, type, composition, light emission, or amount of a detectable agent bound to the biological nanoparticles, wherein a computer is in communication with the detector and the mechanism for directing the flow of biological nanoparticles. In a preferred embodiment, at least the first input channel and at least two outlet channels are in a microfluidic chip. In a preferred embodiment, the biological nanoparticles do not stop during movement through the microfluidic chip, and detection and / or assignment of values ​​are performed as the biological nanoparticles move through the microfluidic chip.

[0154] In other aspects, the present disclosure provides an apparatus for detecting biological nanoparticles in a fluid sample, the apparatus comprising: (a) one or more detectors for detecting the presence or absence of biological nanoparticles; (b) a computer containing software for sorting the biological nanoparticles based on the intensity of the emitted detectable light; and (c) a microfluidic chip. In some embodiments, the sorting is binary. In some embodiments, the sorting is non-binary. For example, when the apparatus is used to detect biological nanoparticles of multiple types or sizes, non-binary sorting can be used.

[0155] In another aspect, the present disclosure provides an apparatus for determining the size of biological nanoparticles in a fluid sample, the apparatus comprising: (a) a microfluidic chip; (b) at least one detector configured to detect the presence or absence of biological nanoparticles; and (c) a computer comprising software for: (i) sorting the biological nanoparticles according to the presence or absence of emitted detectable light intensity; and (ii) measuring the size value of the biological nanoparticles based on the light intensity emitted by the biological nanoparticles, wherein the detection, sorting and measurement occur as the biological nanoparticles flow through the microfluidic chip.

[0156] In certain embodiments of the devices and apparatus provided herein, the device comprises one or more detectors selected from the group consisting of a camera, an electron multiplier, a charge coupled device (CCD) image sensor, a photomultiplier tube (PMT), a microchannel plate PMT (MCP), a hybrid PMT detector, an avalanche photodiode (APD), a single photon avalanche diode (SPAD), or a complementary metal oxide semiconductor (CMOS) image sensor. In some embodiments, the detector detects fluorescence. In some embodiments, the detector detects luminescent light. In some embodiments, the detector detects a variety of different divergent spectra. In some embodiments, the detector detects scattered light.

[0157] In certain embodiments, the detector has single nanoparticle sensitivity. In preferred embodiments, the detector has single molecule sensitivity.

[0158] In certain embodiments of the devices and apparatus provided herein, the device may further include one or more sources for interrogating one or more biological nanoparticles from a fluid sample. The source for interrogating the biological nanoparticles may be, for example, an electromagnetic radiation source. In a specific embodiment, the one or more sources for interrogating are selected from the group consisting of a laser (solid-state, diode pumped, ion or dye), a light emitting diode (LED), a lamp, an arc discharge, a magnetic pulse or natural light. In other embodiments, when the biological nanoparticles exhibit light emission such as chemiluminescence or bioluminescence, there is no need to interrogate the source of the biological nanoparticles.

[0159] In certain embodiments, the interrogation source is subjected to stimulated emission depletion (STED) modification.

[0160] In some embodiments, the interrogation source causes the bio-nanoparticle to emit light intensity. In a preferred embodiment, the light intensity is detectable and is combined with a size value. In some embodiments, where the bio-nanoparticle inherently exhibits luminescence, or where the bio-nanoparticle is combined with a detectable agent that exhibits luminescence, the device may not require a source for interrogating the bio-nanoparticle. The light intensity signal can have a signal-to-noise ratio, where a signal peak can be observed above the background noise of the light intensity output. A high signal-to-noise ratio can increase accuracy and improve detection and measurement. In some embodiments, the signal-to-noise ratio of light intensity is greater than 3: 1, greater than 4: 1, greater than 5: 1, greater than 6: 1, greater than 7: 1, greater than 8: 1, greater than 9: 1, greater than 10: 1, greater than 11: 1, greater than 12: 1, greater than 13: 1, greater than 14: 1, greater than 15: 1, greater than 16: 1, greater than 17: 1, greater than 18: 1, greater than 19: 1, greater than 20: 1, greater than 30: 1, greater than 40: 1, greater than 50: 1, greater than 60: 1, greater than 70: 1, greater than 80: 1, greater than 90: 1, greater than 100: 1, greater than 125: 1, greater than 150: 1, greater than 175: 1, greater than 200: 1, greater than 250: 1, greater than 500: 1 or greater than 1000: 1. In a preferred embodiment, the signal-to-noise ratio of light intensity is greater than 10: 1.

[0161] In certain embodiments, the bionanoparticles move through the microfluidic chip at a high rate. In a specific embodiment, the detection, sorting and measurement of bionanoparticles occur at a rate of more than 1 million particles / hour, more than 2 million particles / hour, more than 3 million particles / hour, more than 4 million particles / hour, more than 5 million particles / hour, more than 6 million particles / hour, more than 7 million particles / hour, more than 8 million particles / hour, more than 9 million particles / hour, more than 10 million particles / hour, more than 15 million particles / hour, more than 20 million particles / hour, more than 25 million particles / hour, more than 30 million particles / hour, more than 35 million particles / hour, more than 40 million particles / hour, more than 45 million particles / hour or more than 50 million particles / hour. In a preferred embodiment, the rate exceeds 5 million nanoparticles / hour.

[0162] In certain embodiments, the device provided herein may include a flow channel surrounded by a wall and / or microfabricated on a substrate, which has a design feature that minimizes accidental damage to nanoparticles. The flow channel may further include a channel having a fluid dynamics design feature or a filter structure that removes undesirable substances or debris with minimal stress or damage, as described in U.S. Patent Application Nos. 2007 / 0037172 and 2008 / 0248499. Such a channel referred to as having a one-dimensional ("1-D") aperture in the aforementioned patent application reduces the fluid dynamics pressure that the biological nanoparticles experience during the cell exclusion process, and therefore reduces the possibility of cracking. The channel with a 1-D aperture can be strategically arranged in an array according to the "effective filtration" described in U.S. Patent No. 2008 / 0318324, to further redirect, distribute, suppress, or disperse flow, thereby reducing the impact force that the nanoparticles experience when excluding. The walls surrounding the flow channel can be fabricated from a biocompatible substrate material of a medical device grade polymer using a UV curing process according to the methods described in PCT / US2009 / 02426, so the device will comply with regulations governing the manufacture of medical devices.

[0163] In certain embodiments, the mechanism for directing the flow of bio-nanoparticles comprises electrodes, magnetic elements, acoustic elements, piezoelectric elements, electric fields or magnetic fields. In some embodiments, the mechanism for directing the flow of bio-nanoparticles comprises one or more electric valves or pistons, wherein the valves or pistons control the flow of liquid in at least a first directional flow channel, which intersects with a first input channel and two outlet channels at a first node. In one embodiment, the solenoid piston is a subassembly of an electric solenoid valve. In another embodiment, the solenoid piston is embedded in the device by molding. In another embodiment, the embedded solenoid piston can be replaced by a solenoid valve through fluid communication in a tube. In a specific embodiment, the device provided herein may include one or more electrodes for tracking and / or manipulating the trajectory or flow of particles, bio-nanoparticles or fluid samples. In certain embodiments, based on phenomena such as dielectric or electrowetting, the electrodes can enhance the separation of nanoparticles. In embodiments where the hydrodynamic diameter of the bio-nanoparticles is less than 100 nm, the acoustic element and / or sheath flow focusing is insufficient to fully manipulate the trajectory of the bio-nanoparticles of the methods and devices disclosed herein. See, e.g., Optics Express, Vol. 15, No. 10, pp. 6167-6176 (2007), which is incorporated by reference. Thus, in some embodiments, the mechanism for directing the flow of biological nanoparticles excludes sheath flow focusing, acoustic flow focusing, or a combination thereof. In some embodiments, biological nanoparticles are oriented, provided that the orientation does not use acoustic focusing, sheath flow focusing, or a combination thereof.

[0164] In some embodiments, the apparatus provided herein may further include a magnetic element for separating biological nanoparticles having intrinsic magnetism, and / or biological nanoparticles associated with magnetic particles. In certain embodiments, magnetism may enhance the separation of biological nanoparticles based on the magnetic susceptibility of nanoparticles or micromagnetic or nanomagnetic particles combined with biological nanoparticles. In certain embodiments, the apparatus provided herein may include the use of fluid pressure changes, flow rate changes, or electroosmotic flow changes to manipulate the trajectory of selected particles or cells.

[0165] In some embodiments, the mechanism for directing the flow of biological nanoparticles can change trajectory or otherwise direct the flow of biological nanoparticles in less than 10 ms, 5 ms, 1 ms, 500 μs, less than 250 μs, less than 200 μs, less than 150 μs, less than 100 μs, less than 50 μs, less than 40 μs, less than 30 μs, less than 20 μs, less than 10 μs, or less than 5 μs. In a preferred embodiment, the mechanism for directing the flow of biological nanoparticles can direct the flow in no more than 1 ms.

[0166] In certain embodiments, the mechanism for directing flow uses flow displacement. In some embodiments, the mechanism for directing flow uses electroosmotic flow. In some embodiments, the mechanism for directing flow uses application of pressure.

[0167] In some embodiments, the mechanism for directing flow can sort the biological nanoparticles into one of a plurality of microfluidic channels in a microfluidic chip. In certain embodiments, directing flow to one of a plurality of microfluidic channels produces an enriched population of biological nanoparticles.

[0168] In some embodiments, the sorting device can be selected from: a computer, a controller, a chip with an integrated circuit, a circuit board, an electronic component, software, an algorithm or a combination thereof. In some embodiments, the measuring device can be selected from: a computer, a controller, a chip with an integrated circuit, a circuit board, an electronic component, software, an algorithm or a combination thereof. In some embodiments, the sorting device and / or the measuring device integrates a designated device.

[0169] In some embodiments, the ranking corresponds to a measured size value. In certain embodiments, the size value is a relative size value. In other embodiments, the size value is a true size value. In a particular embodiment, the size value is measured by a difference in detected light intensity. In some embodiments, the size value is measured based on a detected modulation index. In certain embodiments, the size value is measured based on a detected light intensity and a detected modulation index. The modulation index may include an amplitude modulation index, a frequency modulation index, a phase modulation index, or a combination thereof.

[0170] In certain embodiments, the apparatus provided herein may further include additional elements that can be used to perform experiments, processes or tests in a coupled manner with the method provided herein. In one embodiment, the apparatus provided herein may further include one or more resistive heating elements to perform cell experiments on a chip, such as polymerase chain reaction (PCR) or real-time polymerase chain reaction (RT-PCR). In certain embodiments, the apparatus provided herein may further include one or more electrodes, for example, to perform chemical experiments on a chip such as electrophoresis or electrochromatography.

[0171] In some embodiments, the device provided herein can further include a filter. In specific embodiments, the filter element can be in the form of a micropost, a microimpactor, a microsieve, a channel with a pore size larger than the bio-nanoparticle, a channel with pores that allow the bio-nanoparticle to freely pass through the filter but larger substances or debris in the fluid sample are blocked by the filter, a microbead, a porous membrane, protrusions on the wall, an adhesive coating, woven or non-woven fibers of wool (such as cloth or mesh), a metal (e.g., stainless steel or monel), glass, paper or a synthetic (e.g., nylon, polypropylene, polycarbonate, polyparaxylene and polyester), sintered stainless steel or other metal, or a porous inorganic material (such as alumina, silica or carbon).

[0172] In some embodiments, the filter is placed so that filtering occurs before the fluid sample is introduced into the microfluidic chip. In other embodiments, the filter is placed so that filtering occurs before detection or sorting. In a preferred embodiment, filtering occurs before a specified size value. In certain embodiments, the method disclosed herein also includes filtering the fluid sample. In a specific embodiment, filtering occurs before a specified size value. In some embodiments, filtering removes debris. In certain embodiments, filtering prevents clogging.

[0173] In another embodiment, the device provided herein can be coupled to a conventional flow cytometer. For example, Fig.16 An outlet channel (1621) is shown which can be fluidically connected to a conventional flow cytometer (with or without a detection region (1622)) for further sequential inspection or sorting of sorted biological nanoparticles (1661) containing a detectable agent (1602) (once per nanoparticle).

[0174] In some aspects of the present disclosure, the system further comprises an imaging device, such as a microscope (e.g., a confocal microscope, a spinning disk microscope, a multiphoton microscope, a planar illumination microscope, a Bessel beam microscope, a differential interference contrast microscope, a phase contrast microscope, an epifluorescence microscope, a transmission electron microscope, or a combination thereof. Optionally, the interrogation source is a component of the imaging device, e.g., providing illumination for imaging. In certain aspects, the imaging device is used to obtain image data of the biological nanoparticles, e.g., while moving through the microfluidic chip. Optionally, the image data is used as a basis for assigning true size values. In some aspects, the process occurs manually, e.g., a user views the image data and enters instructions to assign true size values ​​to biological nanoparticles that may have previously been assigned relative size values. In other aspects, the process occurs automatically, e.g., one or more processors analyze the imaging data, such as by using a computer display or an image analysis algorithm, and assign size values ​​to the biological nanoparticles without requiring user input. In other aspects, the assignment is semi-automatic, e.g., involving some user input and some automatic processing.

[0175] In some aspects, the system described herein includes a computer including one or more processors and a storage device having executable instructions stored thereon. In some aspects, the computer is used to perform the method described herein. In various aspects, a computer can be used to implement any system or method described or shown above. In some aspects, the computer includes a processor that communicates with multiple peripheral subsystems via a bus subsystem. These peripheral subsystems can include a storage subsystem, which includes a memory subsystem and a file storage subsystem, a user interface input device, a user interface output device, and a network interface subsystem.

[0176] In some aspects, the bus subsystem provides a mechanism that enables the various components and subsystems of the computer to communicate with each other as intended. The bus subsystem may include a single bus or multiple buses.

[0177] In some aspects, the network interface subsystem provides an interface with other computers and networks. The network interface subsystem can be used as an interface to receive data from the computer and transmit data to other systems. For example, the network interface subsystem can enable the computer to connect to the Internet and facilitate communications using the Internet.

[0178] In some aspects, a computer includes user interface input devices such as a keyboard, a pointing device such as a mouse, trackball, touch pad or graphics tablet, a scanner, a bar code scanner, a touch screen incorporated into a display, audio input devices such as a voice recognition system, a microphone, and other types of input devices. In general, use of the term "input device" is intended to include all possible types of devices and mechanisms for entering information into a computer.

[0179] In some aspects, the computer includes a storage subsystem that provides a computer-readable storage medium for storing basic programs and data structures. In some aspects, the storage subsystem stores software (programs, code modules, instructions) that provide the functions of the methods and systems described herein when executed by a processor. These software modules or instructions can be executed by one or more processors. The storage subsystem can also provide a repository for storing data used according to the present disclosure. The storage subsystem can include a memory subsystem and a file / disk storage subsystem.

[0180] The software can be used to rank the biological nanoparticles based on the presence or absence of detectable light intensity emitted, and to measure the size value of the biological nanoparticles based on the intensity of light emitted by the biological nanoparticles.

[0181] In some embodiments, the software can be used to quantify the number of biological nanoparticles having a specific size value. In certain embodiments, the software can be used to determine the concentration of a fluid sample. In a specific embodiment, the software can be used to determine the concentration of a specific biological nanoparticle in a fluid sample.

[0182] In some embodiments, the software can be used to determine at least one copy number of a biomarker associated with a biological nanoparticle.

[0183] In certain embodiments, the software can be used to sort biological nanoparticles. In certain embodiments, the sorting depends on size values. In certain embodiments, the sorting depends on ranking.

[0184] In some aspects, the computer includes a memory subsystem, which may include multiple memories including a primary random access memory (RAM) for storing instructions and data during program execution and a read-only memory (ROM) in which fixed instructions are stored. The file storage subsystem provides non-temporary persistent (non-volatile) storage for program and data files, and may include a hard disk drive, a solid-state drive and associated removable media, a compact disk read-only memory (CD-ROM) drive, an optical drive, removable media cartridges, and other similar storage media.

[0185] The computer can be of various types, including a personal computer, a laptop, a tablet, a smartphone, a workstation, a network computer, a mainframe, a kiosk, a server, or any other data processing system, such as a field programmable gate array (FPGA). Due to the ever-changing nature of computers and networks, the descriptions of computers contained herein are intended only as specific examples to illustrate aspects of a computer. Many other configurations with more or fewer components than the system shown herein are possible.

[0186] Detection of biological nanoparticles

[0187] In some aspects, the present disclosure provides a method for detecting the size of biological nanoparticles in a fluid sample, the method steps comprising: (a) providing a microfluidic chip; (b) introducing the fluid sample into the microfluidic chip, the fluid sample comprising at least one biological nanoparticle; (c) measuring the light intensity from the biological nanoparticle; and (d) assigning a size value to the biological nanoparticle as it moves through the microfluidic chip, wherein the hydrodynamic diameter of the biological nanoparticle is less than 1 μm. In some aspects, the microfluidic chip includes a plurality of microfluidic channels. In a specific embodiment, the microfluidic chip includes a mechanism for directing flow, and is used to enrich and separate biological nanoparticles. In some embodiments, the fluid sample includes a plurality of biological nanoparticles. In some embodiments, portions of the plurality of biological nanoparticles are introduced into the microfluidic chip. In some embodiments, portions of the plurality of biological nanoparticles flow through the microfluidic channels.

[0188] In certain aspects, the methods of the present disclosure further provide for controlling flow direction. In some embodiments, directional flow is controlled in response to the ordering of biological nanoparticles. In some embodiments, directional flow is controlled in response to the detection of the light intensity emitted by the biological particles. In a specific embodiment, the flow or collection of nanoparticles is guided based on the size value assigned to the biological nanoparticles. In another specific embodiment, the flow or collection of nanoparticles is guided based on the detection of markers bound to the biological nanoparticles. In another specific embodiment, the flow or collection of nanoparticles is guided based on the size value assigned to the biological nanoparticles and the detection of markers bound to the biological nanoparticles. In certain embodiments, the method may include concentrating biological particles by collecting nanoparticles with similar ordering. In a specific embodiment, the method may include controlling the flow direction using a mechanism for guiding flow.

[0189] In one aspect, the present disclosure provides a method for detecting biological nanoparticles in a fluid sample, the method comprising the steps of: (a) measuring the intensity of light emitted from the biological nanoparticle; and (b) assigning a size value to the biological nanoparticle while moving, the size value being based on the detected intensity of the emitted light. In certain embodiments, the hydrodynamic diameter of the biological nanoparticle is less than 1 μm. In specific embodiments, the measurement is performed while the biological nanoparticle is within a microfluidic chip. Optionally, the assignment is performed while the biological nanoparticle is within a microfluidic chip. Optionally, the measurement and / or assignment is performed while the biological nanoparticle flows through the microfluidic chip.

[0190] In some embodiments, the bionanoparticle is combined with a detectable agent. In certain embodiments, the detectable agent is a luminescent dye, a fluorescent dye, a fluorescently labeled antibody, a fluorescently labeled protein, a fluorescently labeled nucleic acid, a fluorescently labeled lipid, a fluorescently labeled carbohydrate, a fluorescently labeled small molecule, a membrane dye, a fluorescent dye, a dye, a polymer dot, a fluorescent substrate for an enzyme, or a combination thereof. In some embodiments, the detectable agent is a fluorescent detectable agent. In other embodiments, the detectable agent is a luminescent detectable agent. As used herein, "associated" includes interactions via covalent and / or non-covalent interactions. For example, a detectable agent can be covalently linked to a bionanoparticle. Alternatively, a detectable agent can be, for example, embedded in the membrane of a bionanoparticle. In a specific embodiment, a detectable agent can be embedded in the membrane of a bionanoparticle by non-covalent interactions such as van der Waals forces or electrostatic forces.

[0191] In a specific embodiment, the detectable agent is combined with the surface of the bionano particle. In some embodiments, the detectable agent can be covalently and / or non-covalently connected to the surface of the bionano particle. In other embodiments, the detectable agent can be embedded in the surface of the bionano particle. In a specific embodiment, the detectable agent is surrounded by the surface of the bionano particle, for example, a membrane dye embedded in the exosome lipid layer. The relationship between the detectable agent combined with the bionano particle surface provides information about the size of the bionano particle. For example, bionano particles with a large surface area will be combined with a large amount of detectable agents, while bionano particles with a small surface area will be combined with a smaller number of detectable agents. The relationship between the number of detectable agents combined with the bionano particle surface provides the correlation between light intensity and nanoparticle surface area. In this way, the amount of emitted light intensity corresponds to the size of the bionano particle, and specifically corresponds to the surface area of ​​the bionano particle.

[0192] In other embodiments, the detectable agent is combined with the interior of the bionano particle. In some embodiments, the detectable agent is not combined with the surface of the bionano particle, and is embedded in the bionano particle, or is otherwise surrounded by the bionano particle. In a specific embodiment, the detectable agent is surrounded by the bionano particle, but is not combined with the inner surface, for example, a dye that floats freely in an exosome, which is not associated with the interior of its lipid membrane. Internal detectable agents are also referred to as "volume dyes" in this article. The relationship of the volume dyes surrounded by bionano particles provides information about the size of the bionano particles. For example, bionano particles with large volumes will contain a large amount of volume dyes, while bionano particles with small volumes will contain less volume dyes. The relationship between the number of volume dyes in the bionano particles provides the correlation between light intensity and nanoparticle volume. In this way, the amount of emitted light intensity corresponds to the size of the bionano particles, and specifically corresponds to the volume of the bionano particles.

[0193] In some embodiments, the bionanoparticles comprise a detectable agent associated with the surface and a body dye. Nanoparticles comprising body dyes and surface-bound detectable agents can provide information related to the surface area and volume of the bionanoparticles. In some embodiments, the body dye and the surface area detectable agent are the same. In other embodiments, the body dye and the surface area detectable agent are different. In certain embodiments, the body dye can provide information about the type or type of bionanoparticles detected or separated. In some embodiments, the use of body dyes as fluorescent substrates can provide information about the type or type of bionanoparticles detected or separated. In specific embodiments, the use of body dyes as fluorescent substrates for enzymes that are specific to bionanoparticles such as exosomes can further provide information about the type or type of bionanoparticles detected or separated.

[0194] In some embodiments, the bionanoparticles are non-spherical. In some embodiments, information about the shape of the bionanoparticles can be extracted by analyzing the ratio of the signals bound to the bionanoparticles. In a specific embodiment, the bionanoparticles comprising a surface membrane dye and a body dye provide an emissivity. In a specific embodiment, the bionanoparticles comprising a surface membrane dye and a body dye provide a surface area to volume ratio. In some embodiments, the ratio of the surface membrane dye to the body dye provides information about the shape of the bionanoparticles.

[0195] In some embodiments, the detectable agent specifically binds to one or more binding targets associated with the bio-nanoparticle. In certain aspects, the binding target is a polypeptide, such as a protein, and the detectable agent is a fluorescently labeled antibody that specifically binds to the target polypeptide.

[0196] In some embodiments, the bionanoparticle comprises a plurality of detectable agents. In some embodiments, the plurality of detectable agents comprises: luminescent dyes, fluorescent dyes, fluorescently labeled antibodies, fluorescently labeled proteins, fluorescently labeled nucleic acids, fluorescently labeled lipids, fluorescently labeled carbohydrates, fluorescently labeled small molecules, membrane dyes, fluorescent dyes, dyes, polymer dots, fluorescent substrates for enzymes, or combinations thereof. In some embodiments, at least one of the plurality of detectable agents is bound to the surface of the bionanoparticle. In some embodiments, the plurality of detectable agents comprises a body dye. In a specific embodiment, the plurality of detectable agents comprises at least one detectable agent bound to the surface of the bionanoparticle and further comprises a body dye. As used herein, the term "multiple detectable agents" refers to more than one detectable agent bound to a bionanoparticle.

[0197] In some embodiments, a plurality of detectable agents have overlapping divergence spectra. In certain embodiments, a plurality of detectable agents have overlapping divergence and absorption spectra. In certain embodiments, the detectable agents have the same divergence spectra. In certain embodiments, the detectable agents have the same divergence and absorption spectra. In a specific embodiment, the detectable agents have a divergence spectra with the same peak wavelength. In some embodiments, a plurality of detectable agents include the same detectable agents. As a non-limiting example, bionanoparticles can be combined with a plurality of detectable agents, the detectable agents are combined with a surface, wherein all detectable agents are the same (e.g., having the same divergence and absorption spectra and peak wavelength).

[0198] In some embodiments, a plurality of detectable agents include detectable agents of more than one type. In some embodiments, detectable agents have different divergence spectra. In some embodiments, detectable agents have divergence spectra with different peak wavelengths. In a specific embodiment, the peak wavelengths can be separated by more than 10 nanometers, more than 20 nanometers, more than 30 nanometers, more than 40 nanometers, more than 50 nanometers, more than 75 nanometers, more than 100 nanometers, more than 120 nanometers, more than 140 nanometers, more than 160 nanometers, more than 180 nanometers, more than 200 nanometers, more than 300 nanometers, more than 400 nanometers, more than 500 nanometers, more than 600 nanometers, more than 700 nanometers or more than 800 nanometers. In some embodiments, detectable agents have different absorption spectra. In some embodiments, detectable agents have absorption spectra with different peak wavelengths. In a specific embodiment, the peak wavelengths can be separated by more than 10 nanometers, more than 20 nanometers, more than 30 nanometers, more than 40 nanometers, more than 50 nanometers, more than 75 nanometers, more than 100 nanometers, more than 120 nanometers, more than 140 nanometers, more than 160 nanometers, more than 180 nanometers, more than 200 nanometers, more than 300 nanometers, more than 400 nanometers, more than 500 nanometers, more than 600 nanometers, more than 700 nanometers or more than 800 nanometers. In some embodiments, the detectable agents have different divergence and absorption spectra. As a non-limiting example, the bionanoparticle can be combined with a variety of detectable agents, which are combined with a surface, wherein all surface detectable agents are the same (e.g., have the same divergence and absorption spectra and peak wavelength), and the bionanoparticle can further include a variety of body dyes, wherein the multiple body dyes have the same divergence spectrum and peak wavelength, but wherein the peak wavelength of the surface dye is different from the peak wavelength of the body dye. Such biological nanoparticles can use the emission light intensity of the body dye and the emission light intensity of the surface dye to provide information about the biological nanoparticle size, which can improve the accuracy of size determination. As a second non-limiting example, the biological nanoparticle can be combined with a variety of detectable agents, which are combined with the surface, wherein several of the multiple detectable agents are the same (for example, having the same divergence and absorption spectrum and peak wavelength), and at least one of the multiple detectable agents is different (for example, having non-overlapping peak wavelengths). In such an example, at least one of the different multiple detectable agents can be combined with a specific protein on the biological nanoparticle surface, for example. In this second non-limiting example, the combined detectable agent can provide information about the biological nanoparticle size, and nanoparticle species and / or biological nanoparticle surface protein composition.

[0199] In certain embodiments, a plurality of detectable agents can include more than one plurality of detectable agent groupings, wherein each group has a common feature. In some embodiments, a plurality of detectable agents include one detectable agent grouping, two detectable agent groupings, three detectable agent groupings, four detectable agent groupings, five detectable agent groupings, six detectable agent groupings, seven detectable agent groupings, eight detectable agent groupings, nine detectable agent groupings, ten detectable agent groupings or more than ten detectable agent groupings. Each detectable agent grouping can include multiple detectable agents with the same divergence and absorption spectra and peak wavelengths. Each detectable agent grouping can include the same multiple detectable agents. As a non-limiting example, the bionanoparticles can be combined with a first detectable reagent grouping, the first detectable reagent comprising a plurality of detectable reagents bound to a surface, and wherein all surface detectable reagents are identical (e.g., having the same divergence and absorption spectra and peak wavelengths); and the bionanoparticles can further include a second detectable reagent grouping, the second detectable reagent grouping comprising a plurality of body dyes, wherein all body dyes have the same divergence and absorption spectra and peak wavelengths; the peak wavelengths of the first detectable reagent grouping and the second detectable reagent grouping can be different. Such bionanoparticles can provide information about the size of the bionanoparticles using the emission light intensity of the first detectable reagent grouping and the emission light intensity of the second detectable reagent grouping, which can improve the accuracy of size determination. As another non-limiting example, biological nanoparticles can be combined with the following: a first detectable reagent grouping, the first detectable reagent grouping includes a plurality of detectable reagents bound to a surface, a second detectable reagent grouping, the second detectable reagent grouping includes a plurality of detectable reagents bound to a specific protein on the biological nanoparticle, a third detectable reagent grouping, the third detectable reagent grouping includes a plurality of detectable reagents bound to different specific proteins on the biological nanoparticle, and a fourth detectable reagent grouping, the fourth detectable reagent grouping includes a plurality of body dyes, the combination of which can provide accurate size information and type or protein composition of the biological nanoparticles.

[0200] In some embodiments, a detector configured to detect the presence or absence of a biological nanoparticle is used. In certain embodiments, the detector is used to measure the intensity of light emitted by the biological nanoparticle. In specific embodiments, light emitted from the biological nanoparticle is detected by the detector. In certain embodiments, light emitted from a detectable agent bound to the biological nanoparticle is detected by the detector.

[0201] In some aspects, the detector is selected from the group consisting of a camera, an electron multiplier, a charge coupled device (CCD) image sensor, a photomultiplier tube (PMT), a microchannel plate PMT (MCP), a hybrid PMT detector, an avalanche photodiode (APD), a single photon avalanche diode (SPAD), a silicon photomultiplier (SiPM) and a complementary metal oxide semiconductor (CMOS) image sensor.

[0202] In some embodiments, the emitted light comprises luminescent light. In certain embodiments, the emitted light comprises fluorescent light. In some embodiments, the emitted light comprises dispersed light. In specific embodiments, the emitted light comprises luminescent light, fluorescent light, scattered light, or a combination thereof.

[0203] Detection of the emitted light by the detector provides a measure of light intensity. In some aspects, assigning a size value to a biological nanoparticle includes using the measured light intensity. As a non-limiting example, a first biological nanoparticle having a first light intensity that is twice as large as a second light intensity from a second biological nanoparticle will report a relative size difference to an observer, where the first biological nanoparticle is larger than the second biological nanoparticle. In some aspects, the measured light intensity includes fluorescence. In some embodiments, the measured light intensity includes luminescence. In some embodiments, the measured light intensity includes scattered light.

[0204] In some embodiments, the source for interrogating the fluid sample is configured to activate emission of light from the bio-nanoparticles. The source for interrogating the bio-nanoparticles can be an electromagnetic radiation source. In some embodiments, the source for interrogating the fluid sample includes: a laser (solid-state, diode pumped, ion or dye), a light emitting diode (LED), a lamp, an arc discharge, a magnetic pulse or natural light. The source for interrogating the fluid sample can be adjusted to increase the intensity of the light emitted from the bio-nanoparticles. As a non-limiting example, the laser wavelength can be selected to have a wavelength that is consistent with the wavelength of the fluorescent detectable agent. abs The same value, the fluorescent detectable agent is bound to the biological nanoparticle, and the detector can be calibrated to detect the fluorescence λ emitted by the detectable agent. em. This selection of interrogation sources and detection provides advantageous light intensity emission and detection. In some embodiments, the interrogation source activates fluorescence intensity. In certain embodiments, the interrogation source activates scattered light intensity. In specific embodiments, the interrogation source activates backscattered light intensity, side scattered light intensity, forward scattered light intensity, or a combination thereof. In specific embodiments, the interrogation source activates scattered light intensity and fluorescence intensity. As a non-limiting example, a laser can be used to interrogate a fluid sample, and nanoparticles passing through can emit scattered light, and detectable agents bound to the nanoparticles can emit fluorescence. Measurements of scattered light and fluorescence intensity report parameters related to the biological nanoparticles. In embodiments where the emitted light includes luminescent light, an interrogation source may not be required.

[0205] In certain aspects, the interrogation source is a source of electromagnetic radiation (e.g., a light source). In some embodiments, the source of electromagnetic radiation comprises a laser, a lamp (e.g., a mercury lamp, a halogen lamp, a metal halide lamp, or other suitable lamp), an LED, or a combination thereof. In some embodiments, the interrogation source comprises epi-illumination. In some embodiments, the interrogation source comprises line confocal detection (LCD), line illumination (e.g., for use with an array detector), or a combination thereof. In certain embodiments, the illumination of the interrogation source is in the form of a line that intersects the width of the microfluidic channel (e.g., using LCD or line illumination). As a non-limiting example, the interrogation source can use line confocal detection. In some aspects, the peak wavelength emitted from the light source is about 200nm-about 300nm, about 250nm-about 350nm, about 300nm-about 400nm, about 350nm-about 450nm, about 400nm-about 500nm, about 450nm-about 550nm, about 500nm-about 600nm, about 550nm-about 650nm, about 600nm-about 700nm, about 650nm-about 750nm, about 700nm-about 800nm, about 750nm-about 850nm, about 800nm-about 900nm, about 850nm-about 950nm, or about 900nm-about 1000nm. In some aspects, two or more light sources with different peak wavelengths can be used. In some aspects, the light emitted from the light source is spectrally filtered by a light filtering device. In some aspects, the optical filtering device includes a filter, such as a bandpass filter, which only allows wavelengths of light that fall within a certain range to pass through it toward the biological nanoparticles. In some aspects, the optical filtering device includes a polychromatic mirror, which can separate light into different spectral components, thereby allowing only wavelengths of light that fall within a certain range to be directed toward the biological nanoparticles. In some aspects, the longest wavelength passing through the optical filtering device is less than 300nm, less than 400nm, less than 500nm, less than 600nm, less than 700nm, less than 800nm, less than 900nm or less than 1000nm. In some aspects, the shortest wavelength passing through the optical filtering device is greater than 200nm, greater than 300nm, greater than 400nm, greater than 500nm, greater than 600nm, greater than 700nm, greater than 800nm ​​or greater than 900nm.

[0206] In some embodiments, the interrogation source has a beam width of less than 10 μm, less than 9 μm, less than 8 μm, less than 7 μm, less than 6 μm, less than 5.0 μm, less than 4.5 μm, less than 4.0 μm, less than 3.5 μm, less than 3.0 μm, less than 2.5 μm, less than 2.0 μm, less than 1.5 μm, less than 1.0 μm, less than 950 nm, less than 900 nm, less than 850 nm, less than 800 nm, less than 750 nm, less than 700 nm, less than 650 nm, less than 600 nm, less than 550 nm, less than 500 nm, less than 450 nm, less than 400 nm, less than 350 nm, or less than 300 nm. In some embodiments, the interrogation source is a beam of light having a width less than 10 μm, less than 9 μm, less than 8 μm, less than 7 μm, less than 6 μm, less than 5.0 μm, less than 4.5 μm, less than 4.0 μm, less than 3.5 μm, less than 3.0 μm, less than 2.5 μm, less than 2.0 μm, less than 1.5 μm, less than 1.0 μm, less than 950 nm, less than 900 nm, less than 850 nm, less than 800 nm, less than 750 nm, less than 700 nm, less than 650 nm, less than 600 nm, less than 550 nm, less than 500 nm, less than 450 nm, less than 400 nm, less than 350 nm, or less than 300 nm. In certain embodiments, the interrogation source has a beam width greater than or equal to 0.5 μm and less than or equal to 2 μm. In some embodiments, the beam width of the interrogation source is greater than 0.1 μm and less than 20 μm, greater than 0.1 μm and less than 10 μm, greater than 0.1 μm and less than 9 μm, greater than 0.1 μm and less than 8 μm, greater than 0.1 μm and less than 7 μm, greater than 0.1 μm and less than 6 μm, greater than 0.1 μm and less than 5 μm, greater than 0.1 μm and less than 4 μm, greater than 0.1 μm and less than 3 μm, greater than 0.1 μm and less than 2 μm, greater than 0.2 μm and less than 10 μm, greater than 0.2 μm and less than 5 μm, greater than 0.2 μm and less than 4 μm. m, greater than 0.2μm and less than 3μm, greater than 0.2μm and less than 2μm, greater than 0.3μm and less than 5μm, greater than 0.3μm and less than 4μm, greater than 0.3μm and less than 3μm, greater than 0.3μm and less than 2μm, greater than 0.4μm and less than 5μm, greater than 0.4μm and less than 4μm, greater than 0.4μm and less than 3μm, greater than 0.4μm and less than 2μm, greater than 0.5μm and less than 5μm, greater than 0.5μm and less than 4μm, greater than 0.5μm and less than 3μm or greater than 0.5μm and less than 2μm.

[0207] In some embodiments, the source for interrogating at least one biological nanoparticle ("irradiation source" or "illumination source") comprises using an illumination source with a beam width less than 2 μm. In some embodiments, the illumination source has a beam width less than 2 μm at a detection location in a microfluidic channel.

[0208] The measurable light intensity can be the emitted light intensity (e.g., emission peak intensity or emission intensity range), emission wavelength (e.g., emission peak wavelength or emission wavelength range), emission lifetime, excitation wavelength (e.g., excitation peak wavelength or excitation wavelength range), absorption wavelength (e.g., absorption peak wavelength or absorption wavelength range), or spectral intensity. Detecting the emitted light intensity can include measuring the emission intensity (e.g., emission peak intensity or emission intensity range), emission wavelength (e.g., emission peak wavelength or emission wavelength range), emission lifetime, excitation wavelength (e.g., excitation peak wavelength or excitation wavelength range), absorption wavelength (e.g., absorption peak wavelength or absorption wavelength range), spectral intensity, or a combination thereof. In some cases, the spectral intensity can include, for example, a plurality of emission peak intensities, emission peak wavelengths, emission intensity ranges, emission wavelength ranges, emission wavelength spectra, excitation peak wavelengths, excitation wavelength ranges, absorption peak wavelengths, or absorption wavelength ranges. For example, detecting or measuring the spectral intensity of a bio-nanoparticle comprising more than one detectable agent can include detecting or measuring the emission intensity of the detectable agent produced by the probe at two or more wavelengths (e.g., over two or more wavelength ranges), and optionally, calculating the ratio of the intensity at the two or more wavelengths at which the intensity is detected or measured (e.g., the intensity range).

[0209] In some embodiments, the emitted light intensity may include a wavelength or wavelength range that includes at least one wavelength of 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, or a range defined by any two of the values ​​thereof. The detectable emission of light or any aspect thereof (e.g., emission peak intensity, emission intensity range, emission peak wavelength, emission wavelength range, excitation peak wavelength, excitation wavelength range, absorption peak wavelength, absorption wavelength range, emission lifetime, or spectral intensity) may be used to determine the presence or absence of a target bio-nanoparticle. Thus, the methods and systems described herein may be used to facilitate determining, measuring, or indicating the presence or absence of one or more different target bio-nanoparticles in a fluid sample, or may be used to facilitate determining or indicating the presence or absence of one or more detectable agents bound to bio-nanoparticles in a fluid sample.

[0210] Various types of detectable agents are suitable for use in the methods and systems of the present disclosure. In some aspects, the detectable agent includes one or more chromophores (e.g., fluorophores). The chromophores described herein can be used to produce fluorescent emission according to various mechanisms. In some embodiments, converting the detectable agent from a first optical state to a second optical state comprises a method based on Förster resonance energy transfer (FRET). Resonance energy transfer (FRET) quenches one or more chromophores, or removes FRET-based quenching of one or more chromophores. In certain embodiments, the application of light energy triggers compositional and / or structural changes in entities that interact with chromophores. For example, in some aspects, the detectable agent includes at least one photochromic molecule that is converted (e.g., reversibly or irreversibly) into different compositions and / or structures after exposure to light energy. Optionally, the photochromic molecule is a photochromic quencher that controllably quenches the fluorescence of the chromophore according to the conformation of the molecule, thereby producing a change in the optical state of the optical marker. Examples of photochromic molecules include, but are not limited to, azobenzene, stilbene, azostilbene, diarylethene, quinone, nitrone, fulgide, or a derivative or combination thereof. 1,2-Bis(2,4-dimethyl-5-phenyl-3-thienyl)-3,3,4,4,5,5,-hexafluoro-1-cyclopentene (BTE) is an example of a diarylethene suitable for use in accordance with various aspects presented herein. In some aspects, BTE exhibits improved photoswitching kinetics, thermal stability, and fatigue resistance compared to other types of photochromic molecules.

[0211] In some embodiments, when exposed to light energy, the chromophore itself undergoes structural and / or compositional changes, which alter the optical properties of the chromophore. For example, in some aspects, the detectable agent includes at least one photoactivated chromophore that irreversibly changes from a first optical state (e.g., exhibiting relatively low or no fluorescence) to a second optical state (e.g., exhibiting relatively high fluorescence) when light energy is applied. Examples of photoactivated chromophores include, but are not limited to: photoactivated green fluorescent protein (PA-GFP), PA-CFP2, PA-mRFP1, PA-mCherry1, Phamret, caged fluorescent dyes (e.g., 5-carboxymethoxy-2-nitrobenzyl (CMNB) caged fluorescein, CMNB caged carboxyfluorescein) or combinations or derivatives thereof. In some aspects, the photoactivated fluorescent protein can be a genetically encoded and expressed biological nanoparticle.

[0212] In some aspects, the detectable agent includes at least one photoconvertible chromophore that reversibly switches between a first optical state (e.g., showing relatively low fluorescence or no fluorescence) and a second optical state (e.g., showing relatively high fluorescence). In certain aspects, the photoconvertible chromophore switches from the first optical state to the second optical state when exposed to a first light energy (e.g., light of a first wavelength), and switches from the second optical state to the first optical state when exposed to a different second light energy (e.g., light of a second wavelength). Optionally, the photoconvertible chromophore can reversibly switch between the two optical states multiple times with minimal or no photobleaching, for example, at least 3 times, 5 times, 10 times, 20 times, 50 times or more. Examples of photoconvertible chromophores include, but are not limited to, Dronpa, rsFastLime, Padron, bsDronpa, E2GFP, rsCherry, rsCherryRev, or a combination or derivative thereof. In other aspects, as further described herein, the detectable agent includes at least one photoconvertible chromophoric polymer particle.

[0213] In certain embodiments, the chromophore is selected from the group consisting of SYBR green, Evagreen, SYTO-9, SYTO-82, fluorescein, FITC, FAM, rhodamine, HEX, VIC, JOE, TET, TAMRA, ROX, TRITC, Texas Red, GFP, phycoerythrin (PE), Cy3, Cy3.5, Cy5, Cy5.5, PE-Cy5, calcein, BODIPY, Alexa Fluor, DyLight Fluor, ATTO, Quasar, Cal Fluor, TYE, Qdot, Cy, SYSTO, derivatives thereof, chromophoric polymers, semiconductor polymers, semiconductor polymer dots, and combinations thereof.

[0214] Semiconducting polymers of various types and compositions may be suitable for use in accordance with aspects of the present disclosure. Semiconducting polymers may be homopolymers or heteropolymers. For example, many semiconductor polymers are suitable for use in chromophoric polymer particles according to the present invention. Examples of semiconductor polymers include, but are not limited to: polyfluorene-containing polymers, including but not limited to poly(9,9-dihexylfluorenyl-2,7-diyl) (PDHF) and poly(9,9-dioctylfluorenyl-2,7-diyl) (PFO); fluorene-containing copolymers, including but not limited to poly[{9,9-dioctyl-2,7-dienylidene-fluorenyl}-alternating-co-{2-methoxy-5-(2-ethylhexyloxy)-l,4-phenylene}] (PFPV), poly[( 9,9-dioctylfluorenyl-2,7-diyl)-co-(l,4-benzo-{2,1,3}-thiadiazole)] (PFBT), poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(4,7-di-2-thienyl-2,1,3-benzothiadiazole)] (PFTBT) and poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(4,7-di-2-thienyl-2,1,3-benzothiadiazole)] (PF-0.1T BT); phenylene vinylene polymers, including but not limited to poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylene vinylene] (MEH-PPV) and poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-(1-cyano vinylene-1,4-phenylene)] (CN-PPV); phenylene ethynylene polymers, including but not limited to poly(2,5-bis(3',7'-dimethyloctyl)phenylene-1,4-ethynylene) (PPE); or combinations thereof. In certain embodiments, the semiconducting polymer may include but is not limited to a polymer comprising a polyfluorene monomer, a fluorene monomer, a phenylene vinylene monomer, a phenylene ethynylene monomer, any derivatives thereof, or combinations thereof. In some embodiments, the semiconducting polymer comprises a polyfluorene, a polyfluorene derivative, a fluorene, a fluorene derivative, a phenylene vinylene, a phenylene vinylene derivative, a phenylene ethynylene, a phenylene ethynylene derivative, or a combination thereof.

[0215] A variety of chromophoric polymer structures are suitable for use in various aspects according to the present disclosure. In some aspects, the chromophoric polymer is a linear polymer. In some aspects, the chromophoric polymer is a branched polymer. In some aspects, the chromophoric polymer is a dendritic polymer. In some aspects, the chromophoric polymer is a brush polymer. In some aspects, the chromophoric polymer is a star polymer.

[0216] In some aspects, the chromophoric polymer particles described herein comprise copolymers having one or more functionalized monomer units, such as amphiphilic polymers, including but not limited to: polymers based on poly((meth)acrylic acid), such as: poly(acrylic acid-b-acrylamide), poly(acrylic acid-b-methyl methacrylate), poly(acrylic acid-b-N-isopropylacrylamide), poly(n-butyl acrylate-b-acrylic acid), poly(sodium acrylate-b-methyl methacrylate), poly(methacrylic acid-b-neopentyl methacrylate), poly(methyl methacrylate-b-acrylic acid), poly(methyl methacrylate-b-methylacrylic acid), poly(methyl methacrylate-b-N,N-dimethylacrylamide ), poly(methyl methacrylate-b-sodium acrylate), poly(methyl methacrylate-b-sodium methacrylate), poly(neopentyl methacrylate-b-methacrylic acid), poly(tert-butyl methacrylate-b-ethylene oxide), poly(2-acrylamido-2-methylpropanesulfonic acid-b-acrylic acid); copolymers based on polydiene, such as: poly(butadiene (1,2 addition)-b-ethylene oxide), poly(butadiene (1,2 addition)-b-methacrylic acid), poly(butadiene (1,4 addition)-b-acrylic acid), poly(butadiene (1,4 addition)-b-ethylene oxide), poly(butadiene (1,4 addition)-b-sodium acrylate), poly(butadiene (1,4 addition)-bN -methyl 4-vinylpyridinium iodide), poly(isoprene-b-ethylene oxide), poly(isoprene-b-ethylene oxide) and poly(isoprene-bN-methyl 2-vinylpyridinium iodide); copolymers based on poly(ethylene oxide), such as: poly(ethylene oxide-b-acrylic acid), poly(ethylene oxide-b-acrylamide), poly(ethylene oxide-b-butylene oxide), poly(ethylene oxide-bc-caprolactone), poly(ethylene oxide-b-lactide), poly(ethylene oxide-b-lactide), poly(ethylene oxide-b-methacrylic acid), poly(ethylene oxide-b-methyl acrylate), poly(ethylene oxide-bN-isopropylacrylamide), poly(ethylene oxide-b-methacrylic acid acrylate), poly(ethylene oxide-b-nitrobenzyl methacrylate), poly(ethylene oxide-bN,N-dimethylaminoethyl methacrylate), poly(ethylene oxide-b-propylene oxide), poly(ethylene oxide-b-tert-butyl acrylate), poly(ethylene oxide-b-tert-butyl methacrylate), poly(ethylene oxide-b-tetrahydrofurfuryl methacrylate), poly(ethylene oxide-b-2-ethyloxazoline), poly(ethylene oxide-b-2-hydroxyethyl methacrylate), poly(ethylene oxide-b-2-methyloxazoline); copolymers based on polyisobutylene, such as poly(isobutylene-b-acrylic acid), poly(isobutylene-b-ethylene oxide), poly(isobutylene-b-methacrylic acid);Polystyrene-based copolymers such as poly(styrene-b-acrylamide), poly(styrene-b-acrylic acid), poly(cesium styrene-b-acrylate), poly(styrene-b-ethylene oxide), poly(styrene-b-ethylene oxide) acid cleavable at block junctions, poly(styrene-b-methacrylic acid), poly(4-styrenesulfonate-b-ethylene oxide), poly(styrenesulfonate-b-methylbutylene), poly(styrene-bN,N-dimethylacrylamide), poly(styrene-bN-isopropylacrylamide), poly(styrene-bN-methyl 2-vinylpyridinium iodide), poly(styrene-bN-methyl-4-vinylpyridinium iodide), poly(styrene-b-propyl acrylate), poly(sodium styrene-b-acrylate), poly(sodium styrene-b-methacrylate), poly(p-chloromethylstyrene-b-acrylamide), poly(styrene-co-p-chloromethylstyrene-b-acrylamide), poly(styrene-co-p-chloromethylstyrene-b-acrylic acid), poly(styrene- b-methylbutylene-co-isoprene sulfonate); copolymers based on polysiloxane, such as poly(dimethylsiloxane-b-acrylic acid), poly(dimethylsiloxane-b-ethylene oxide), poly(dimethylsiloxane-b-methacrylic acid); copolymers based on poly(ferrocenyldimethylsilane), such as poly(ferrocenyldimethylsilane-b-ethylene oxide); copolymers based on poly(2-vinylnaphthalene), such as poly(2-vinylnaphthalene-b-acrylic acid); copolymers based on poly(vinylpyridine and N-methylvinyl iodide), such as poly(2-vinylpyridine-b-ethylene oxide), poly(2-vinylpyridine-b-methacrylic acid), poly(N-methyl 2-vinylpyridinium iodide-b-ethylene oxide), poly(N-methyl 4-vinylpyridinium iodide-b-methyl methacrylate), poly(4-vinylpyridine-b-ethylene oxide) PEO terminal functional OH; and copolymers based on poly(vinylpyrrolidone), such as poly(vinylpyrrolidone-bD / L-lactide); etc.;

[0217] In certain aspects, the chromophoric polymer comprises a blend of semiconducting polymers. The blend may comprise a combination of homopolymers, copolymers, and oligomers. The polymer blend used to form the chromophoric polymer particles may be selected to adjust the properties of the resulting polymer particles, for example, to achieve a desired excitation or emission spectrum for the polymer particles.

[0218] In addition, the detectable agent can be, for example, a molecule of interest (e.g., a nucleic acid or a biomarker) present in the bionanoparticle to be analyzed. Alternatively, the detectable agent can be a molecule that is combined with a molecule of interest (e.g., a nucleic acid molecule or a biomarker), and the molecule of interest is combined with the bionanoparticle, thereby allowing the detection of nanoparticles. In some embodiments, the detectable agent is a fluorescent detectable agent, and therefore can be detected by fluorescence-based detection methods known in the art. However, other detection methods (e.g., absorbance, chemiluminescence, turbidity and / or scattering) can be used to analyze bionanoparticles. A variety of detectable agents suitable for the present disclosure are well known in the art and can be found, for example, in The Molecular Probes Handbook, 11th edition (2010).

[0219] Using detection methods as described herein, the combination or non-combination of biological nanoparticles with detectable agents can be analyzed. For a given biological nanoparticle sample in a concentration, some biological nanoparticles may include detectable agents, and some may not include. Usually, when compared with biological nanoparticles with a smaller hydrodynamic radius, biological nanoparticles with a larger hydrodynamic radius are combined with a larger amount of detectable agents. Therefore, when compared with biological nanoparticles with a smaller hydrodynamic radius, biological nanoparticles with a larger hydrodynamic radius usually emit higher light intensity.

[0220] In specific embodiments provided herein, detecting the intensity of light from a biological nanoparticle occurs as the nanoparticle moves through a microfluidic chip. In specific embodiments, the biological nanoparticle is detected as it moves through a microfluidic chip, wherein the movement comprises an uninterrupted flow.

[0221] Using the detection methods described herein, biological nanoparticles, detectable agents, labels, etc. can be specifically detected. In some embodiments, the detection, measurement, or detection and measurement of light intensity has single nanoparticle sensitivity. For example, even if the fluid sample includes multiple biological nanoparticles, a single biological nanoparticle can be detected when moving through the microfluidic chip. In some embodiments, a single biological nanoparticle can be detected even in the presence of other similar biological nanoparticles. As used herein, "single nanoparticle sensitivity" refers to the detection of a single biological nanoparticle.

[0222] The irradiation of a single biological nanoparticle may refer to a biological nanoparticle that is irradiated in a fluid sample including multiple biological nanoparticles and in the absence of any of the other biological nanoparticles in the multiple biological nanoparticles. The irradiation of a single biological nanoparticle is different from the irradiation of two or more biological nanoparticles that are randomly co-located in the irradiation area (i.e., two or more biological nanoparticles that just exist in the irradiation area). The irradiation of a single biological nanoparticle is different from the irradiation of the aggregation of biological nanoparticles (i.e., two or more biological nanoparticles). As a non-limiting example, a single biological nanoparticle can pass through a light beam and is therefore irradiated. A single biological nanoparticle can pass through a light beam in the absence of any of the other biological nanoparticles in the multiple biological nanoparticles, so the single biological nanoparticle is irradiated by itself. In some embodiments, a single biological nanoparticle is such a singular nanoparticle that can be interrogated by a light source in the absence of any of the other biological nanoparticles in the fluid sample (e.g., for a given beam width, a single biological nanoparticle is present in the beam, thus allowing it to be irradiated in the absence of any of the other biological nanoparticles in the multiple biological nanoparticles).

[0223] Detecting the light intensity from a single bio-nanoparticle may refer to a bio-nanoparticle that is detected in a fluid sample including multiple bio-nanoparticles and in the absence of any of the other bio-nanoparticles in the multiple bio-nanoparticles. The detection of a single bio-nanoparticle is different from the detection of the aggregation of bio-nanoparticles (i.e., two or more bio-nanoparticles are connected to each other). As a non-limiting example, a single bio-nanoparticle can pass through a light beam, generating a measured light intensity. A single bio-nanoparticle can pass through a light beam in the absence of any of the other bio-nanoparticles in the multiple bio-nanoparticles, so a single bio-nanoparticle is detected by itself. In some embodiments, a single bio-nanoparticle is such a singular nanoparticle that can be interrogated by a light source in the absence of any of the other bio-nanoparticles in the fluid sample (e.g., for a given beam width, a single bio-nanoparticle is present in the beam, thus allowing it to be detected in the absence of any of the other bio-nanoparticles in the multiple bio-nanoparticles).

[0224] In some embodiments, at least a portion of a plurality of biological nanoparticles in a plurality of biological nanoparticles are irradiated (i.e., are irradiated biological nanoparticles). As a non-limiting example, a biological nanoparticle that passes through a beam from an irradiation source can be an irradiated biological nanoparticle. In some embodiments, a majority of biological nanoparticles in a plurality of detected biological nanoparticles are irradiated in the absence of any of the other biological nanoparticles in the plurality of biological nanoparticles. In certain embodiments, a majority of irradiated biological nanoparticles are irradiated individually (i.e., measured as a single biological nanoparticle). In some embodiments, greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99% of the irradiated biological nanoparticles in a plurality of biological nanoparticles are irradiated individually. In a preferred embodiment, greater than 90% of the irradiated biological nanoparticles in a plurality of biological nanoparticles are irradiated individually.

[0225] In some embodiments, at least a portion of a plurality of biological nanoparticles in a plurality of biological nanoparticles is detected (i.e., is a detected biological nanoparticle). As a non-limiting example, a biological nanoparticle that passes through a detection region may be a detected biological nanoparticle. In some embodiments, most of the biological nanoparticles in a plurality of detected biological nanoparticles are detected in the absence of any biological nanoparticle in the other biological nanoparticles of the plurality of biological nanoparticles. In certain embodiments, most of the detected biological nanoparticles are detected individually (i.e., detected as a single biological nanoparticle, also referred to as "individual detection"). In some embodiments, more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98% or more than 99% of the biological nanoparticles detected in a plurality of biological nanoparticles are detected individually. In a preferred embodiment, more than 90% of the biological nanoparticles in a plurality of detected biological nanoparticles are detected individually.

[0226] In some embodiments, at least some of the plurality of biological nanoparticles are detected on a particle-by-particle basis. In some embodiments, at least some of the plurality of biological nanoparticles are irradiated on a particle-by-particle basis. On a particle-by-particle basis describes the observation of a plurality of biological nanoparticles passing through a region (e.g., a light beam having a given width) individually (i.e., one at a time). As a non-limiting example of a particle-by-particle basis, a fluid sample including a plurality of biological nanoparticles can flow through a constriction of a microfluidic channel and through a light beam, such that at least some of the plurality of biological nanoparticles pass through the light beam alone (i.e., in the absence of any of the other biological nanoparticles in the plurality of biological nanoparticles). As another non-limiting example of biological nanoparticles on a particle-by-particle basis, a fluid sample including a plurality of biological nanoparticles can flow through a microchannel and through a light beam, such that no more than one biological nanoparticle passes through the light beam at a time and does not have any overlap with other biological nanoparticles in the plurality of biological nanoparticles. In some specific embodiments, a majority of the biological nanoparticles pass through the light beam, such that no more than one biological nanoparticle passes through the light beam at a time and does not have any overlap with other biological nanoparticles in the plurality of biological nanoparticles. In some embodiments, more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% of the biological nanoparticles in a plurality of irradiated biological nanoparticles are irradiated on a particle-by-particle basis. In a preferred embodiment, more than 90% of the irradiated biological nanoparticles in a plurality of biological nanoparticles are irradiated on a particle-by-particle basis. In some embodiments, more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% of the biological nanoparticles in a plurality of detected biological nanoparticles are detected on a particle-by-particle basis. In a preferred embodiment, greater than 90% of the biological nanoparticles in the plurality of detected biological nanoparticles are detected on a particle-by-particle basis.

[0227] In some embodiments, the detection light intensity has single molecule sensitivity. For example, a single biological nanoparticle can be combined with multiple detectable agents, and a single detectable agent can be detected. As another example, a single biological nanoparticle can be combined with multiple detectable agents of the same type, and individuals in multiple detectable agents can be detected. As used herein, "single molecule sensitivity" refers to the detection of a single molecule, where a single molecule is combined with a biological nanoparticle, or where the biological nanoparticle contains a single molecule. Single molecule detection can include point detection, where the detection is focused on a spatial region (e.g., a point or line), and the detection of light from the spatial region can correspond to the presence of a biological nanoparticle. Point detection can include the use of a point detector, such as an avalanche photodiode (APD), a multi-element photodiode, an electron multiplier, a photomultiplier tube (PMT), a microchannel plate PMT (MCP), a hybrid PMT detector, a single photon avalanche diode (SPAD) or a silicon photomultiplier tube (SiPM). Single molecule detection can include detecting point illumination, or more preferably line illumination, wherein the illumination source is focused on a spatial region (e.g., illuminating a beam of light of a width or a beam illuminating a point), and detecting light from the spatial region can correspond to the presence of a biological nanoparticle. Detection of point illumination or line illumination can include the use of: a camera, an electron multiplier, a charge coupled device (CCD) image sensor, a photomultiplier tube (PMT), a microchannel plate PMT (MCP), a hybrid PMT detector, an avalanche photodiode (APD), a single photon avalanche diode (SPAD), a silicon photomultiplier tube (SiPM), and a complementary metal oxide semiconductor (CMOS) image sensor; as further disclosed above, the source of illumination (i.e., the interrogation source) can include a laser, a lamp (e.g., a mercury lamp, a halogen lamp, a metal halide lamp, or other suitable lamp), an LED, or a combination thereof. In some embodiments, the detector includes a line confocal detector or imager. In certain embodiments, detection of biological nanoparticles uses line confocal detection or imaging.

[0228] In some embodiments, the detection of light from the spatial region includes detecting the intensity of light from the region (also referred to herein as the "detection region") with at least one detector. In some embodiments, the area of ​​the detection region is less than 250 μm 2 , less than 200μm 2 , less than 150μm 2 , less than 100μm 2 , less than 90μm 2 , less than 80μm 2 , less than 70μm 2 , less than 60μm 2 , less than 50μm 2 , less than 45μm 2 , less than 40μm 2, less than 35μm 2 , less than 30μm 2 , less than 25μm 2 , less than 20μm 2 , less than 19μm 2 , less than 18μm 2 , less than 17μm 2 , less than 16μm 2 , less than 15μm 2 , less than 14μm 2 , less than 13μm 2 , less than 12μm 2 , less than 11μm 2 , less than 10μm 2 , less than 9μm 2 , less than 8μm 2 , less than 7μm 2 , less than 6μm 2 , less than 5μm 2 , less than 4μm 2 , less than 3μm 2 , less than 2μm 2 or less than 1μm 2 In a preferred embodiment, the area of ​​the detection region is greater than or equal to 1 μm 2 And less than or equal to 10μm 2 In some embodiments, the area of ​​the detection region is greater than or equal to 0.1 μm 2 And less than or equal to 50μm 2 , greater than or equal to 0.2μm 2 And less than or equal to 45μm 2 , greater than or equal to 0.3μm 2 And less than or equal to 40μm 2 , greater than or equal to 0.4μm 2 And less than or equal to 35μm 2 , greater than or equal to 0.5μm 2 And less than or equal to 30μm 2 , greater than or equal to 0.6μm 2 And less than or equal to 25μm 2 , greater than or equal to 0.7μm 2 And less than or equal to 20μm 2 , greater than or equal to 0.8μm 2 And less than or equal to 15μm 2 , greater than or equal to 0.9μm 2 And less than or equal to 12.5μm 2 or greater than or equal to 1μm 2 And less than or equal to 10μm 2In some embodiments, the detection zones overlap, intersect, and / or are located within at least one microfluidic channel. In some embodiments, the detection zones overlap, intersect, and / or are located within a constriction of at least one microfluidic channel.

[0229] In some embodiments, at least one source for interrogation interrogates a region (also referred to herein as an "irradiation region"). In some embodiments, the source of illumination illuminates the illumination region. In some embodiments, the illumination region has an area of ​​less than 250 μm 2 , less than 200μm 2 , less than 150μm 2 , less than 100μm 2 , less than 90μm 2 , less than 80μm 2 , less than 70μm 2 , less than 60μm 2 , less than 50μm 2 , less than 45μm 2 , less than 40μm 2 , less than 35μm 2 , less than 30μm 2 , less than 25μm 2 , less than 20μm 2 , less than 19μm 2 , less than 18μm 2 , less than 17μm 2 , less than 16μm 2 , less than 15μm 2 , less than 14μm 2 , less than 13μm 2 , less than 12μm 2 , less than 11μm 2 , less than 10μm 2 , less than 9μm 2 , less than 8μm 2 , less than 7μm 2 , less than 6μm 2 , less than 5μm 2 , less than 4μm 2 , less than 3μm 2 , less than 2μm 2 or less than 1μm 2 In a preferred embodiment, the area of ​​the irradiated region is greater than or equal to 1 μm 2 And less than or equal to 100μm 2 In some embodiments, the area of ​​the irradiated region is greater than or equal to 0.1 μm 2 And less than or equal to 500μm 2 , greater than or equal to 0.2μm 2And less than or equal to 450μm 2 , greater than or equal to 0.3μm 2 And less than or equal to 400μm 2 , greater than or equal to 0.4μm 2 And less than or equal to 350μm 2 , greater than or equal to 0.5μm 2 And less than or equal to 300μm 2 , greater than or equal to 0.6μm 2 And less than or equal to 250μm 2 , greater than or equal to 0.7μm 2 And less than or equal to 200μm 2 , greater than or equal to 0.8μm 2 And less than or equal to 150μm 2 , greater than or equal to 0.9μm 2 And less than or equal to 125μm 2 or greater than or equal to 1μm 2 And less than or equal to 100μm 2 .

[0230] In some embodiments, detecting light intensity includes using a time bin. The disclosed apparatus and methods for determining bionanoparticle characteristics can be performed quickly, with a short signal integration time, or a fast bin time. Bin time can be used to evaluate, for example, the start-stop time of interrogation-fluorescence, thereby helping information sorting. A time bin (also referred to herein as a signal integration time) can disclose the time range over which an event occurs or is observed in a histogram. In some embodiments, a time bin is used to detect, measure, and / or interrogate bionanoparticles. In some embodiments, the range of a time bin is less than 10 ms, less than 5 ms, less than 1 ms, less than 0.5 ms, less than 0.1 ms, less than 90 μs, less than 80 μs, less than 70 μs, less than 60 μs, less than 50 μs, less than 40 μs, less than 30 μs, less than 20 μs, less than 10 μs, less than 5 μs, or less than 1 μs. In some embodiments, the value of the time bin is 10ms-0.1μs, 5ms-0.1μs, 1ms-0.1μs, 0.5ms-0.1μs, 0.1ms-0.1μs, 90μs-0.1μs, 80μs-0.1μs, 70μs-0.1μs, 60μs-0.1μs, 50μs-0.1μs, 40μs-0.1μs, 30μs-0.1μs, 20μs-0.1μs, 10μs-0.1μs, 5μs-0.1μs or 1μs-0.1μs. In a preferred embodiment, the time bin range is 1μs-2ms.

[0231] In certain embodiments, detection of light intensity will detect a single antibody bound to a bio-nanoparticle. In specific embodiments, detection of light intensity will detect a single antibody comprising multiple detectable agents, wherein the antibody is bound to a bio-nanoparticle. In some embodiments, detection of light intensity will detect a single antibody bound to at least one detectable agent, wherein the antibody is bound to a bio-nanoparticle.

[0232] Particle-by-particle inspection

[0233] As described herein, a plurality of biological nanoparticles can be detected using a particle-by-particle basis. Detection on a particle-by-particle basis describes the detection of a singular biological nanoparticle from a plurality of biological nanoparticles. In some embodiments, the particle-by-particle basis includes single-file detection, sequentially detecting portions of a plurality of biological nanoparticles as the portions of the plurality of biological nanoparticles pass through a microfluidic channel or a compressed portion of a microfluidic channel. The particle-by-particle basis refers to a physical separation between at least some of the plurality of biological nanoparticles.

[0234] In some embodiments, detecting at least one biological nanoparticle on a particle-by-particle basis includes irradiating a single biological nanoparticle, detecting a single biological nanoparticle, or a combination thereof. As further disclosed herein, the detection and / or irradiation of a single biological nanoparticle may refer to a biological nanoparticle that is irradiated and / or illuminated in a fluid sample that includes a plurality of biological nanoparticles, but in the absence of any of the other biological nanoparticles in the plurality of biological nanoparticles. The detection and / or irradiation of a single biological nanoparticle is different from the detection and / or irradiation of an aggregation of biological nanoparticles (i.e., two or more biological nanoparticles are connected to each other). As a non-limiting example, a single biological nanoparticle may pass through a light beam, generating a light intensity that is detected. A single biological nanoparticle may pass through a light beam in the absence of any of the other biological nanoparticles in the plurality of biological nanoparticles, so that the single biological nanoparticle is detected and / or illuminated by itself.

[0235] In some embodiments, a single biological nanoparticle is surrounded by a solvent or fluid from a sample. In certain embodiments, a single biological nanoparticle is completely surrounded by a solvent or fluid from a sample. The aggregation of two or more biological nanoparticles prevents each biological nanoparticle in the aggregation from being completely surrounded by a solvent or fluid from a sample. Therefore, the aggregation of two or more biological nanoparticles is not a single biological nanoparticle, and a single biological nanoparticle is not an aggregation of two or more biological nanoparticles.

[0236] In some embodiments, a portion of the plurality of biological nanoparticles flows through a microfluidic channel and at least one biological nanoparticle is detected as a single biological nanoparticle. In some embodiments, a portion of the plurality of biological nanoparticles flows through a constriction of a microfluidic channel and at least one biological nanoparticle is detected as a single biological nanoparticle at the constriction. In certain embodiments, at least 1% of the plurality of biological nanoparticles detected in the plurality of biological nanoparticles is detected as a single biological nanoparticle. In some embodiments, at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the plurality of detected biological nanoparticles are detected as single biological nanoparticles (i.e., on a particle-by-particle basis). In a preferred embodiment, at least 90% of the detected biological nanoparticles in the plurality of detected biological nanoparticles are detected as single biological nanoparticles.

[0237] In some embodiments, a portion of the plurality of biological nanoparticles flows through a microfluidic channel and at least one biological nanoparticle is irradiated as a single biological nanoparticle. In some embodiments, a portion of the plurality of biological nanoparticles flows through a constriction of a microfluidic channel and at least one biological nanoparticle is irradiated as a single biological nanoparticle at the constriction. In certain embodiments, at least 1% of the plurality of irradiated biological nanoparticles is irradiated as a single biological nanoparticle. In some embodiments, at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of a portion of a plurality of irradiated biological nanoparticles is irradiated as a single biological nanoparticle (i.e., on a particle-by-particle basis). In a preferred embodiment, at least 90% of the irradiated biological nanoparticles in a portion of a plurality of biological nanoparticles are irradiated as a single biological nanoparticle.

[0238] In some embodiments, a portion of a plurality of biological nanoparticles flows through a microfluidic channel and at least one biological nanoparticle is measured as a single biological nanoparticle. In some embodiments, a portion of a plurality of biological nanoparticles flows through a constriction of a microfluidic channel and at least one biological nanoparticle is measured as a single biological nanoparticle at the constriction. In some embodiments, the detection or measurement occurs at a constriction present in a microfluidic channel. In certain embodiments, at least 1% of the plurality of measured biological nanoparticles is measured as a single biological nanoparticle. In some embodiments, at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the portion of a plurality of measured biological nanoparticles is measured as a single biological nanoparticle (i.e., on a particle-by-particle basis). In a preferred embodiment, at least 90% of the measured biological nanoparticles in the portion of a plurality of biological nanoparticles are measured as a single biological nanoparticle.

[0239] In some embodiments, portions of a plurality of bio-nanoparticles flow through a microfluidic channel and include void volumes between the plurality of bio-nanoparticles. In some embodiments, portions of a plurality of bio-nanoparticles flow through a contraction of a microfluidic channel and include void volumes between the plurality of bio-nanoparticles. The void volume describes the volume between individual bio-nanoparticles in the portion of a plurality of bio-nanoparticles (and in a microfluidic channel having a width, describes the void distance). For example, portions of bio-nanoparticles may be separated from one another (i.e., not aggregated) and have an average void distance between them. The void distance value and the void volume value are directly related. The void volume may refer to the volume of fluid in a microfluidic channel after detecting and / or irradiating one bio-nanoparticle and before detecting and / or irradiating another bio-nanoparticle. In some embodiments, the void volume refers to the volume between bio-nanoparticles that pass sequentially through a microfluidic channel. In some embodiments, the void distance may refer to the distance between individual bio-nanoparticles. In some embodiments, the average interstitial distance between portions of the plurality of bionanoparticles is 0.5 times larger, 1 times larger, 1.1 times larger, 1.2 times larger, 1.3 times larger, 1.4 times larger, 1.5 times larger, 1.6 times larger, 1.7 times larger, 1.8 times larger, 1.9 times larger, 2 times larger, 3 times larger, 4 times larger, 5 times larger, 6 times larger, 7 times larger, 8 times larger, 9 times larger, 10 times larger, 15 times larger, 20 times larger, 25 times larger, 50 times larger, 75 times larger, 100 times larger, 250 times larger, 500 times larger, or 1,000 times larger than the average hydrodynamic diameter of the portions of the plurality of bionanoparticles.

[0240] In some embodiments, the average gap distance is generally sufficient to allow a single individual bio-nanoparticle to pass through the detection region and / or illumination region of the microfluidic channel. In some embodiments, the average gap distance is sufficient to allow at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of a portion of a plurality of bio-nanoparticles to pass through the detection region and / or illumination region on a particle-by-particle basis. In a preferred embodiment, the average interstitial distance is sufficient to allow at least a 90% portion of the plurality of bio-nanoparticles to pass through the detection and / or illumination region particle by particle.

[0241] In some embodiments, the average distance between portions of the plurality of bio-nanoparticles is greater than the width of the detection region, the illumination region, or a combination thereof. The detection region, the illumination region, or a combination thereof can describe an area of ​​at least one microfluidic channel that is illuminated by a radiation source (e.g., an illumination source, thereby forming an illumination region) and / or detected by a detector (thereby forming a detection region). In some embodiments, the microfluidic channel includes a constriction, and the constriction includes the detection region. In some embodiments, the constriction is the detection region. In some embodiments, the average distance between portions of the plurality of bio-nanoparticles is greater than the length of the detection region by 0.5 times, 1 times, 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 25 times, 50 times, 75 times, or 100 times. In some embodiments, the average distance between portions of the plurality of bio-nanoparticles is greater than the length of the irradiation area by 0.5 times, greater than 1 times, greater than 1.1 times, greater than 1.2 times, greater than 1.3 times, greater than 1.4 times, greater than 1.5 times, greater than 1.6 times, greater than 1.7 times, greater than 1.8 times, greater than 1.9 times, greater than 2 times, greater than 3 times, greater than 4 times, greater than 5 times, greater than 6 times, greater than 7 times, greater than 8 times, greater than 9 times, greater than 10 times, greater than 15 times, greater than 20 times, greater than 25 times, greater than 50 times, greater than 75 times, or greater than 100 times. As a non-limiting example, the irradiation area may include a beam having a certain width, and the average distance between portions of the plurality of bio-nanoparticles is greater than the width of the beam.

[0242] In some embodiments, a portion of a plurality of bio-nanoparticles flows through a channel at a rate sufficient to allow detection of a single bio-nanoparticle in the portion of a plurality of bio-nanoparticles. In some embodiments, a portion of a plurality of bio-nanoparticles flows through a channel at a rate of less than 300,000 bio-nanoparticles / second, less than 200,000 bio-nanoparticles / second, less than 100,000 bio-nanoparticles / second, less than 75,000 bio-nanoparticles / second, less than 50,000 bio-nanoparticles / second, or less than 25,000 bio-nanoparticles / second, less than 15,000 bio-nanoparticles / second, less than 10,000 bio-nanoparticles / second, less than 5,000 bio-nanoparticles / second, or less than 1,000 bio-nanoparticles / second. In a preferred embodiment, a portion of a plurality of bio-particles flows through a channel at a rate of less than 50,000 bio-nanoparticles / second.

[0243] In some embodiments, detecting a portion of a plurality of biological nanoparticles particle by particle includes detecting at least two biological nanoparticles sequentially. As a portion of a plurality of biological nanoparticles flowing through a microfluidic channel, an average distance between the plurality of biological nanoparticles (e.g., an interstitial distance, as described above) is sufficient to detect at least two biological nanoparticles individually. In some embodiments, as a portion of a plurality of biological nanoparticles flowing through a constriction of a microfluidic channel, an average distance between the plurality of biological nanoparticles (e.g., an interstitial distance, as described above) is sufficient to detect at least two biological nanoparticles individually. At least two biological nanoparticles can pass through a detection region and / or illumination region of a microfluidic channel one at a time (i.e., not clustered together) and are thus illuminated and / or detected sequentially (i.e., one after another). In some embodiments, at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the biological nanoparticles (i.e., irradiated biological nanoparticles) in a portion of a plurality of irradiated biological nanoparticles are sequentially irradiated, sequentially detected, or a combination thereof. In a preferred embodiment, at least 90% of the biological nanoparticles in a portion of a plurality of irradiated biological nanoparticles are sequentially irradiated, sequentially detected, or a combination thereof.

[0244] In some embodiments, the method includes detecting a signal from at least one of a plurality of bio-nanoparticles, and the signal includes emission from at least one bio-nanoparticle. In some embodiments, the signal includes emission intensity, emission wavelength, or a combination thereof. The signal may include a background noise value. When a plurality of bio-nanoparticles are not detected (e.g., when none of the plurality of bio-nanoparticles pass through a detection region), the background noise may describe the detected signal. In some embodiments, the signal includes the emission light intensity from at least one bio-nanoparticle in a plurality of bio-nanoparticles, and the background noise includes the absence of a signal emitted from at least one bio-nanoparticle. In some embodiments, as the bio-nanoparticles flow through the detection region, the emission light intensity is observed and detected, and as the bio-nanoparticles flow out of the detection region, the observed light intensity decreases to a background noise value. In some embodiments, particle-by-particle detection includes detecting a first light intensity emitted from a first bio-nanoparticle flowing through a microfluidic channel, the detected light intensity decreases to a background noise value, and detecting a second light intensity emitted from a second bio-nanoparticle flowing through a microfluidic channel. In certain embodiments, particle-by-particle detection includes detecting a first light intensity emitted from a first biological nanoparticle flowing through a microfluidic channel, the detected light intensity decreasing to less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of the peak intensity value, and detecting a second light intensity emitted from a second biological nanoparticle flowing through the microfluidic channel. In preferred embodiments, particle-by-particle detection includes detecting a first light intensity emitted from a first biological nanoparticle flowing through a microfluidic channel (or a constriction of a microfluidic channel), the detected light intensity decreasing to less than 10% of the peak intensity value, and detecting a second light intensity emitted from a second biological nanoparticle flowing through the microfluidic channel (or a constriction of a microfluidic channel). In certain embodiments, a first biological nanoparticle is detected, then the light intensity is reduced (eg, to less than 20% of peak light intensity), and then a second biological nanoparticle is detected, which describes a portion of a plurality of biological nanoparticles being detected particle by particle.

[0245] In some embodiments, a particle-by-particle basis includes physical separation between at least some of the plurality of biological nanoparticles. A biological nanoparticle physically separated from other biological nanoparticles is a biological nanoparticle that is not in direct contact with any of the other biological nanoparticles of the plurality of biological nanoparticles. In some embodiments, at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the biological nanoparticles in the portion of the plurality of biological nanoparticles are physically separated from any of the other biological nanoparticles in the portion of the plurality of biological nanoparticles. In a preferred embodiment, at least 90% of the biological nanoparticles in the portion of the plurality of biological nanoparticles are physically separated from any of the other biological nanoparticles in the portion of the plurality of biological nanoparticles.

[0246] In some embodiments, particle-by-particle detection uses a device and / or system that includes a microfluidic chip that includes multiple microfluidic channels, and a radiation source that irradiates the multiple microfluidic channels. In some embodiments, the multiple microfluidic channels are in a parallel arrangement. The system and / or device may include a source of radiation, wherein the source of radiation irradiates multiple microfluidic channels. In some embodiments, the source for radiation irradiates multiple microfluidic channels at an angle perpendicular to the microfluidic channels. In some embodiments, the device and / or system also includes multiple detectors, each detector being configured to detect an area of ​​a single microfluidic channel. As a non-limiting example, the microfluidic chip may include 10 microfluidic channels in parallel, and the source for radiation may irradiate the 10 microfluidic channels; 10 detectors may be arranged to detect signals from each of the 10 microfluidic channels, and each detector is paired with a single microfluidic channel.

[0247] In some embodiments, particle-by-particle detection uses such a device and / or system, which includes a microfluidic chip, the microfluidic chip includes multiple microfluidic channels, and multiple radiation sources, the radiation sources irradiate multiple microfluidic channels. In some embodiments, multiple microfluidic channels are in a parallel arrangement. The system and / or device may include multiple sources of radiation, wherein the source for radiation irradiates multiple microfluidic channels. In some embodiments, the source for radiation irradiates multiple microfluidic channels at an angle perpendicular to the microfluidic channels. In some embodiments, the device and / or system also includes multiple detectors, each detector is configured to detect the area of ​​a single microfluidic channel. As a non-limiting example, the microfluidic chip may include 10 microfluidic channels in parallel, and 4 sources for radiation can irradiate these 10 microfluidic channels at 5 μm intervals; 10 detectors can be arranged to detect signals from each of the 10 microfluidic channels, and each detector is paired with a single microfluidic channel.

[0248] Assigning size values ​​to biological nanoparticles

[0249] As described herein, biological nanoparticles can emit light intensity. In some embodiments, the interrogation source activates the light intensity. The present disclosure provides a method for detecting the light intensity emitted from biological nanoparticles. In a specific embodiment, the present disclosure provides a method for measuring the light intensity emitted from biological nanoparticles and assigning a size value to the biological nanoparticles. In a specific embodiment, the size value corresponds to the light intensity. In some embodiments, the size value is assigned according to the light intensity.

[0250] In a specific embodiment, the present disclosure provides a method for detecting the intensity of light from a biological nanoparticle as the particle moves through a microfluidic chip. In a specific embodiment, the microfluidic chip includes a translucent or transparent feature. For example, a fluid sample containing biological nanoparticles can be provided to a microfluidic chip including at least one translucent or transparent surface, and the at least one translucent or transparent surface allows an interrogation source to interact with the biological nanoparticle, so that the biological nanoparticle emits light intensity that passes through the at least one translucent or transparent surface, thereby being detected. In a specific embodiment, the biological nanoparticle is detected as it moves through the microfluidic chip, wherein the movement includes uninterrupted flow.

[0251] In some embodiments, the specified size value is binary. In a specific embodiment, a positive size value is assigned to biological nanoparticles that emit a light intensity above a threshold, and a negative size value is assigned to the remaining fluid samples. In some embodiments, the threshold is a relative threshold. In certain embodiments, the threshold is greater than 10 detected photons, greater than 100 detected photons, greater than 1,000 detected photons, greater than 10,000 detected photons, greater than 100,000 detected photons, greater than 1,000,000 detected photons, greater than 10,000,000 detected photons, or greater than 100,000,000 detected photons. In a preferred embodiment, the threshold is greater than 10 detected photons. In certain embodiments in which the specified size value is binary, the size value assigned to the biological nanoparticle can be zero or non-zero.

[0252] In some embodiments, a relative size value is assigned to a nanoparticle. In a specific embodiment, the relative size value is related to the relative light intensity emitted by the biological nanoparticle. As a non-limiting example, a first biological nanoparticle that emits a large amount of light intensity can be assigned a high relative size value, while a second biological nanoparticle that emits a small amount of light intensity can be assigned a low relative size value. In some embodiments, a fluid sample includes biological nanoparticles having multiple relative size values. The relative size values ​​can be organized into boxes so that biological nanoparticles that emit light of similar intensity can be grouped together and assigned the same relative size value. The fluid sample may include more than 1 box, more than 2 boxes, more than 3 boxes, more than 4 boxes, more than 5 boxes, more than 6 boxes, more than 7 boxes, more than 8 boxes, more than 9 boxes, more than 10 boxes, more than 15 boxes, more than 20 boxes, more than 25 boxes, more than 30 boxes, more than 40 boxes, more than 50 boxes, more than 60 boxes, more than 70 boxes, more than 80 boxes, more than 90 boxes, more than 100 boxes, more than 200 boxes, more than 300 boxes, more than 500 boxes, more than 1,000 boxes or more than 5,000 boxes. As a non-limiting example, a fluid sample including a plurality of biological nanoparticles may have a light emission scale with a relative minimum of 0 and a relative maximum of 1.0, and 5 bins for relative emission values, wherein the first bin is assigned with biological nanoparticles having an emission of 0.0-0.2, the second bin is assigned with nanoparticles having an emission of 0.21-0.4, the third bin is assigned with nanoparticles having an emission of 0.41-0.6, the fourth bin is assigned with nanoparticles having an emission of 0.61-0.8, and the fifth bin is assigned with nanoparticles having an emission of 0.81-1.0. Analysis of the fluid sample may assign relative size values ​​to the nanoparticles by assigning each detected nanoparticle to its corresponding bin based on its detected light emission. The number of nanoparticles assigned to each bin may be calculated, and a histogram may be generated. In this way, relative size values ​​may be assigned to a plurality of biological nanoparticles, wherein each relative size value is determined by the difference in the measured light intensity.

[0253] In some embodiments, the actual size value of the bio-nanoparticle is determined by calibrating the measured light intensity with a standard. In some embodiments, calibration of the light intensity measurement is performed prior to measuring the light intensity of the bio-nanoparticle, and thereafter, the measured light intensity is correlated with the actual size value of the bio-nanoparticle. In other embodiments, calibration of the light intensity measurement is performed after measuring the light intensity of the bio-nanoparticle, and the measured light intensity is correlated with a relative size value. In certain embodiments, after calibration with a standard, an actual size value can be assigned to the relative size value. In some embodiments, the standard includes gold nanoparticles. In certain embodiments, the standard includes a plurality of lipids. In some embodiments, the standard includes lipid vesicles. In certain embodiments, the standard includes silica beads, polystyrene beads, silicone beads, polymer beads, polymer nanoparticles, or combinations thereof. In some embodiments, where the fluid sample contains a second bio-nanoparticle, the standard contains a second bio-nanoparticle. In some embodiments, the standard is measured via dynamic light scattering.

[0254] In a specific embodiment, where the standard is a second bio-nanoparticle from the fluid sample, the second bio-nanoparticle is the same as the first type of bio-nanoparticle. In other embodiments, the second bio-nanoparticle is different from the first bio-nanoparticle. Methods known in the art can be used to determine the true size value of the second bio-nanoparticle. For example, dynamic light scattering or TEM microscopy, and the light intensity emitted from the second bio-nanoparticle can report the size value of the first bio-nanoparticle. In some embodiments, determination of the size of the second bio-nanoparticle occurs prior to measuring the light intensity from the second bio-nanoparticle. In other embodiments, determination of the size of the second bio-nanoparticle occurs after measuring the light intensity from the second bio-nanoparticle. For example, a first portion of a fluid sample containing a plurality of bio-nanoparticles can be processed through a microfluidic chip using the methods disclosed herein and assigned relative size values to generate a histogram, as further disclosed herein. A second portion of the fluid sample can be analyzed using dynamic light scattering or TEM microscopy and true size values assigned to the bio-nanoparticles. The second portion can then be processed through the microfluidic chip in the same manner as the first portion, and the resulting true size histogram can be used to assign true size values to the bio-nanoparticles in the first portion of the fluid sample. In at least this manner, the standard can include at least one bio-nanoparticle from the fluid sample.

[0255] In assigning size values ​​to biological nanoparticles by relating them to the relative intensity of light emitted by the biological nanoparticles, it is important to ensure that the measured light intensity does not vary from run to run or sample to sample due to measurement variability and that differences in the measured light intensity are indeed caused by differences in the biological nanoparticles. This requirement can be difficult to meet in practice because the measured light intensity can be affected by a number of experimental variabilities that are difficult to control, including the exact z-position of the laser line in the microchannel, or variability that can arise between chips (e.g., the thickness of the cover glass that forms the bottom of the microchannel and contacts the objective), instrument drift (e.g., laser or detector alignment), or small uncontrolled variability in operating conditions (e.g., flow rate). To overcome these problems, internal standards can be used, using the intensity of back-reflected light or scattered light from the gold nanoparticles. Gold nanoparticles are particularly advantageous for this operation because: 1) gold nanoparticles are readily available in different sizes, which allows one to select a size that will best match the desired detected light intensity in the detection channel; 2) the back-reflected light intensity or scattered light intensity of the gold nanoparticles allows them to be used with any laser excitation wavelength and, therefore, any color channel; 3) the size or diameter of the gold nanoparticles can be uniform, which can minimize the distribution of the light intensity detected and, therefore, facilitate internal calibration and improve quantitative accuracy; 4) the surface of the gold nanoparticles can be easily modified, such as with PEG, so that they do not self-aggregate or non-specifically attach to biological nanoparticles or microchannel surfaces; and 5) gold nanoparticles can be robust and will not degrade or aggregate even during long-term (e.g., more than one month) storage.

[0256] In some embodiments, assigning a size value to a biological nanoparticle includes using a modulation index. In a specific embodiment, assigning a size value to a biological nanoparticle includes using an amplitude modulation index. In another embodiment, assigning a size value to a biological nanoparticle includes using a frequency modulation index. In another embodiment, assigning a size value to a biological nanoparticle includes using a phase modulation index. The use of a modulation index indicates how much the modulated emission of light intensity varies around an unmodulated level. As a non-limiting example, a fluid sample may include biological particles that autofluoresce when excited by an interrogation source. The use of a modulation index (e.g., an amplitude modulation index) can account for background light intensity, thereby providing a more accurate size value.

[0257] In certain embodiments, the size value includes a range of values. In some embodiments, the difference between the maximum and minimum values ​​of the range of values ​​is less than 1 micron, 500 nanometers, 200 nanometers, 100 nanometers, less than 80 nanometers, less than 50 nanometers, less than 40 nanometers, less than 30 nanometers, less than 20 nanometers, less than 15 nanometers, less than 10 nanometers or less than 5 nanometers. For example, a range of values ​​including a size value with a hydrodynamic diameter range of 40-100 nanometers is 60 nanometers.

[0258] In some embodiments, the size value is assigned using (a) exciting the biological nanoparticle by an interrogation source, and (b) measuring the light intensity emitted from the biological nanoparticle. As described herein, the light intensity can include scattered light, fluorescence, luminescent light, or a combination thereof. In certain embodiments, the interrogation source uses a light beam modified by stimulated emission depletion (STED). When a size value is assigned to a biological nanoparticle, STED is used to improve accuracy. In certain embodiments, the interrogation source uses multiple light beams, and in certain embodiments, the multiple light beams are modified by STED.

[0259] In certain embodiments, the size value of the bionanoparticle is the hydrodynamic diameter. In specific embodiments, the hydrodynamic diameter is less than 1,000 nanometers, less than 900 nanometers, less than 800 nanometers, less than 700 nanometers, less than 600 nanometers, less than 500 nanometers, less than 400 nanometers, less than 300 nanometers, less than 200 nanometers, less than 150 nanometers, less than 100 nanometers, less than 90 nanometers, less than 80 nanometers, less than 70 nanometers, less than 60 nanometers, less than 50 nanometers, less than 40 nanometers or less than 30 nanometers. In a preferred embodiment, the hydrodynamic diameter is less than 100 nanometers. In certain embodiments, the hydrodynamic diameter is determined by measuring dynamic light scattering (DLS) and refers to the size of a hard sphere that diffuses light in the same manner as the bionanoparticle being measured.

[0260] In some embodiments, the hydrodynamic diameter is between 1,000 nanometers and 10 nanometers, between 900 nanometers and 10 nanometers, between 800 nanometers and 10 nanometers, between 700 nanometers and 10 nanometers, between 600 nanometers and 10 nanometers, between 500 nanometers and 10 nanometers, between 400 nanometers and 10 nanometers, between 300 nanometers and 10 nanometers, between 200 nanometers and 10 nanometers, between 100 nanometers and 10 nanometers, between 90 nanometers and 10 nanometers, between 80 nanometers and 10 nanometers, between 70 nanometers and 10 nanometers, between 60 nanometers and 10 nanometers, between 50 nanometers and 10 nanometers, or between 40 nanometers and 10 nanometers. In certain embodiments, the hydrodynamic diameter is between 1,000 nanometers and 800 nanometers, between 800 nanometers and 600 nanometers, between 600 nanometers and 400 nanometers, between 400 nanometers and 200 nanometers, or between 200 nanometers and 10 nanometers. In preferred embodiments, the hydrodynamic diameter is between 200 nanometers and 20 nanometers. In a more preferred embodiment, the hydrodynamic diameter is between 100 nm and 40 nm. Unless otherwise specified, when used in the context of two values, the term "between" includes outer values ​​(e.g., between 40 nm and 10 nm encompasses all values ​​from 10 nm to 40 nm, including 10 nm and 40 nm).

[0261] In some embodiments, assigning a size value to a biological nanoparticle includes using a duty cycle. A duty cycle can be defined as a detection method with a binary on / off cycle, where an on cycle is associated with detecting a light intensity from a biological nanoparticle above a threshold, and where an off cycle is associated with a lack of detecting a light intensity above a threshold. The duty cycle is the portion of a cycle where detection of a light intensity above a threshold is observed. The duty cycle can be expressed as D=(T 开 / T 总 ), where D is the duty cycle, T 开 is the time during which the observed light intensity is above the threshold, T 总is the total measurement time. For example, a bio-nanoparticle emitting a light intensity above the detection threshold can pass through the interrogation source, and when the light intensity is detected, the duty cycle is in the on state. When the nanoparticle passes through the interrogation source, the light intensity drops below the detection threshold and the duty cycle is in the off state. The size of the interrogation source is its pulse width. In certain embodiments, multiple interrogation sources can be used to calculate the duty cycle, and the multiple interrogation sources are spatially separated. In a specific embodiment, two interrogation sources can be used to calculate the duty cycle. In other embodiments, three interrogation sources can be used to calculate the duty cycle, four interrogation sources can be used to calculate the duty cycle, five interrogation sources can be used to calculate the duty cycle, or more than five interrogation sources can be used to calculate the duty cycle. In some embodiments, multiple interrogation sources include interrogation sources with the same pulse width. In other embodiments, multiple interrogation sources include interrogation sources with different pulse widths. In a preferred embodiment, the spatial interval between multiple interrogation sources includes zero length. As used herein, "zero length" is a region where a bio-nanoparticle travels between interrogation sources, where the light intensity emitted from the bio-nanoparticle is not observed to be above the threshold. The calculated duty cycle can provide a size value for a biological nanoparticle that passes through an interrogation source. In certain embodiments, a small biological nanoparticle will have a small duty cycle (e.g., little time spent in the "on" state compared to the time spent in the "off" state), while a large biological nanoparticle will have a large duty cycle (e.g., much time spent in the "on" state compared to the time spent in the "off" state). The time that the duty cycle is in the off state is T 关 , which is equal to T 总 -T 开 In other embodiments, T 关 , T 开 and T 总 Combinations (such as T 关 / T 总 or T 关 / T 开 ), not D.

[0262] Sorting and analysis of biological nanoparticles

[0263] In one aspect, the present disclosure provides methods and apparatus for measuring, detecting and / or sorting biological nanoparticles in a fluid sample. In one embodiment, the method can be characterized by (i) detecting the presence or absence of biological nanoparticles, (ii) sorting the biological nanoparticles according to size values, and (iii) directing the flow or collection of biological nanoparticles based on a specified sort. In certain embodiments, the detection component can detect single molecules bound to the biological nanoparticles. In some embodiments, the sorting of nanoparticles includes parameters other than size values.

[0264] Non-limiting examples of methods and devices for measuring, detecting and / or sorting biological nanoparticles in a fluid sample can be shown in Fig.16 . A fluid sample containing a plurality of bio-nanoparticles, some of which are bound to a detectable agent (1602), is introduced into a microfluidic chip (1603), which includes an inlet and two outlets connected by microfluidic channels. The bio-nanoparticles flow through an inlet channel (1642) toward a detection area (1604), where the binding (or non-binding) and size values ​​of the detectable agent and the bio-nanoparticles can be determined. The bio-nanoparticles bound to the detectable agent (1641) are assigned positive values ​​and move downstream, where they are sorted (mechanism for directing flow not shown) to a positive microfluidic channel (1621) to be directed to an enrichment container or further processing. The positively sorted bio-nanoparticles (1661) can pass through a second detection area (1622), which can confirm whether the correct sorting has been performed. The bio-nanoparticles that are not bound to the detectable agent (1601) are assigned negative values ​​and move downstream, where they are sorted to a negative microfluidic channel (1611). Negatively sorted bio-nanoparticles (1651) can be transported to a waste container.

[0265] As used herein, "fluid sample" refers to any liquid that may contain the bio-nanoparticles of interest. In a preferred embodiment, the fluid sample comprises a body fluid. In certain embodiments, the fluid sample may be a biological fluid sample, for example, a serum sample, a plasma sample, a saliva sample, a urine sample, a milk sample, a synovial fluid sample, an amniotic sample, a lymph sample, a spinal fluid sample, etc. In a specific embodiment, the body fluid comprises serum, plasma, cerebrospinal fluid, or lymph fluid.

[0266] As used herein, the term "ranking" refers to evaluating the quantitative characteristics, qualitative characteristics or importance of biological nanoparticles by classification. In one embodiment, the biological nanoparticles can be ranked as empty (e.g., when no biological nanoparticles are detected) or non-zero (e.g., when biological nanoparticles are detected). In some embodiments, the ranking can be binary. In other embodiments, the biological nanoparticles can be ranked according to other classifications, for example, about the size value of the biological nanoparticles, the type of nanoparticles, the detectable features of the biological nanoparticles, the detectable agents combined with the biological nanoparticles, etc. In this way, any number of classifications can be specified. Any number (e.g., 0, 1, 2, 3, 4, 5, etc.) corresponding to one of a plurality of predetermined quantitative or qualitative classifications can be specified for these rankings, or a number corresponding to the actual value of the biological nanoparticle (e.g., its size value).

[0267] As used herein, "detectable characteristic" refers to a property associated with a biological nanoparticle of interest, for example, an optically active, electrically active, biologically active, or magnetic property associated with or intrinsic to the biological nanoparticle.

[0268] Examples of photoactive properties include, for example, changes in optical light intensity (optical reflection, scattering, deflection, transmission or absorption) typically caused by biological particle morphology (particle size, granularity or internal subcellular structure), fluorescence, immunofluorescence, etc.

[0269] Examples of biologically active properties include, for example, detectable interactions with enzymes such as esterases, phosphatases, lipases, peroxidases, β-galactosidases, and their fluorescent or fluorogenic substrates, or their chemiluminescent or chemiluminescent substrates.

[0270] In certain embodiments, the moieties that can be used to detect biological nanoparticles include, but are not limited to, antibodies and fragments thereof, fluorescent antibodies, polymer molecules, dye molecules, DNA or RNA molecules (e.g., aptamers), protein molecules, lipid molecules (e.g., fluorescent dyes bound to lipids), and the like.

[0271] As used herein, the term "antibody" refers to a polypeptide or fragment thereof comprising a framework region from an immunoglobulin gene that specifically binds to and recognizes an antigen. Recognized immunoglobulin genes include κ, λ, α, γ, δ, ε, and μ constant region genes, as well as a large number of immunoglobulin variable region genes. Light chains are classified as κ or λ. Heavy chains are classified as γ, μ, α, δ, or ε, which in turn determine the immunoglobulin class, which is IgG, IgM, IgA, IgD, and IgE, respectively. Typically, the antigen binding region of an antibody is the most important in the specificity and affinity of the binding. Antibodies can be polyclonal or monoclonal, derived from serum, hybridoma, or recombinant clones, and can also be chimeric, primatized, or humanized.

[0272] The structural unit of an exemplary immunoglobulin (antibody) comprises a tetramer. Each tetramer comprises two identical pairs of polypeptide chains, each pair comprising one "light" chain (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids that is primarily responsible for antigen recognition. The term light chain variable region (V L ) and the heavy chain variable region (V H ) refer to these light and heavy chains, respectively.

[0273] For example, antibodies exist as intact immunoglobulins or as well-characterized fragments produced by digestion with a variety of peptidases. Thus, for example, pepsin digests an antibody below the disulfide bonds in the hinge region to produce a dimer of Fab, F(ab). 2 Fab itself is connected to V by disulfide bonds H -C H 1 light chain. F(ab') can be reduced under mild conditions 2To break the disulfide bonds in the hinge region, thereby converting F(ab') 2 The dimer is converted into a Fab' monomer. The Fab' monomer is essentially a Fab with a portion of the hinge region attached (see Fundamental Immunology, edited by Paul, 3rd edition, 1993). Although various antibody fragments are defined based on the digestion of intact antibodies, it will be appreciated by those skilled in the art that such fragments can also be synthesized de novo by chemical methods or recombinant DNA methods. Therefore, the term antibody as used herein also includes antibody fragments generated by modifying the entire antibody, or produced by de novo synthesis using a recombinant DNA method (e.g., single-chain Fv), or those antibody fragments identified using a phage display library (see, e.g., McCafferty et al., Nature 348:552-554 (1990)).

[0274] In one embodiment, the antibody is coupled to a label or detectable moiety. In certain embodiments, the antibody is coupled to a bionanoparticle. In a specific embodiment, the bionanoparticle is coupled to an antibody, which comprises a plurality of detectable agents. In certain embodiments, the bionanoparticle is coupled to an antibody, and the antibody is coupled to a plurality of detectable agents. In some embodiments, the bionanoparticle is coupled to an antibody, and the antibody is coupled to at least one detectable agent.

[0275] As used herein, "label" or "detectable moiety" refers to a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical or other physical means. For example, useful labels include, but are not limited to, radionuclides, fluorescent dyes (e.g., fluorescein, fluorescein isothiocyanate (FITC), Oregon Green TM ,Alexa TM , rhodamine, Texas Red, tetrapropylenediamine isothiocyanate (TRITC), Cy3, Cy5, etc.), fluorescent markers (e.g., green fluorescent protein (GFP), phycoerythrin, etc.), auto-quenched fluorescent compounds activated by tumor-associated proteases, enzymes (e.g., luciferase, esterase, lipase, peroxidase, phosphatase, etc.), nanoparticles, biotin, digitoxin, etc.

[0276] In certain embodiments, detection reagents can be perfused to selectively label the bio-nanoparticles of interest. Examples of such reagents include, but are not limited to, fluorescence, immunofluorescence, dye-coupled molecules (such as antibodies, fab fragments, aptamers, polymers, ligands, agonists, antagonists or combinations thereof) magnetic, electroactive, bioactive or photoactive compounds. One example is the use of a stain that reacts with exosomes. Other dye examples include fluorescein isothiocyanate (FITC)-coupled mouse anti-human epithelial antibody (HEA) and phycoerythrin (PE)-coupled anti-CD63. Other examples of dye-coupled antibodies include, but are not limited to, those targeting the following: tetraspanins (e.g., CD9, CD63, and CD81), heat shock proteins (e.g., HSC70 and HSC90), membrane transporters (e.g., GTPases) and lipid-binding proteins, cell-adhesion proteins, lipid-binding proteins, transmembrane proteins, enzymes, pan-cytokeratin antibodies A45B / B3, AE1 / AE3 or CAM5.2 (pan-cytokeratin antibodies that recognize cytokeratin 8 (CK8), cytokeratin 18 (CK18) or cytokeratin 19 (CK19), and are directed against one of the following: breast cancer antigen NY-BR-1 (also known as B726P, ANKRD30A, ankyrin repeat domain 30A); B305D isoform A or C (B305D-A roB305D-C; also known as antigen B305D); Hermes antigen (also known as antigen CD44, PGP1); E-cadherin (also known as uvom adhesion protein (Uvomorulin), cadherin-1, CDH1); carcinoembryonic antigen (CEA; also known as CEACAM5 or carcinoembryonic antigen-related cell adhesion molecule 5); beta-human chorionic gonadotropin (beta-HCG; also known as CGB, chronic gonadotropin, beta polypeptide); cathepsin-D (also known as CTSD); neuropeptide Y receptor Y3 (also known as NPY3R; lipopolysaccharide-associated protein 3, LAP3, fusion protein; chemokine (CXC motif, receptor 4; CXCR4); oncogene ERBB1 (also known as c-erbB-1, epidermal growth factor receptor, EGFR); Her-2 Neu (also known as c-erbB-2 or ERBB2); GABA receptor A, π polypeptide (also known as GABARAP, GABA-A receptor, π polypeptide (GABA A(π), γ-aminobutyric acid type A receptor π subunit) or GABRP); ppGalNac-T(6) (also known as β-1-4-N-acetyl-galactosaminyltransferase 6, GalNAc transferase 6, GalNAcT6, UDP-N-acetyl-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase 6 or GALNT6); CK7 (also known as cytokeratin 7, Sarcolectin, SCL, keratin 7 or KRT7); CK8 (also known as cytokeratin 8, keratin 8 or KRT8); CK18 (also known as cytokeratin 18, keratin 18 or KRT18); CK19 (also known as cytokeratin 19, keratin 19 or KRT19); CK20 (also known as cytokeratin 20, keratin 20 or KRT20); Mage (also known as melanoma antigen family A subtype or MAGE-A subtype); Mage3 (also known as melanoma antigen family A 3 or MAGA3); hepatocyte growth factor receptor (also known as HGFR, renal cell carcinoma papillary type 2, RCCP2, proto-oncogene or MET); mucin-1 (also known as MUC1, cancer antigen 15.3, (CA15.3), cancer antigen 27.29 (CA 27.29); CD227 antigen, Episialin, epithelial membrane antigen (EMA), polymorphic epithelial mucin (PEM), peanut-reactive urinary mucin (PUM), tumor-associated glycoprotein 12 (TAG12); macrocystic disease fluid protein (also known as GCDFP-15, prolactin-induced protein, PIP); urokinase receptor (also known as uPR, CD87 antigen, plasminogen activator receptor urokinase type, PLAUR); PTHrP (parathyroid hormone-related protein; also known as PTHLH); BS106 (also known as B511S, small mammary epithelial mucin or SBEM); prostaglandin-like lipophilin B (LPB, LPHB; also known as antigen BU101, secretory globulin Lactoglobin (MGB); also known as mammaglobin 1, MGB1, mammaglobin A, MGBA, secretoglobin family 2A member 2 or SCGB2A2); mammary gland Serine protease inhibitor (Maspin, also known as serine (or cysteine) protease inhibitor clade B (ovalbumin) member 5, or SERPINB5); prostate epithelial-specific Ets transcription factor (PDEF; also known as ets transcription factor containing a sterile alpha motif tip domain, or SPDEF); tumor-associated calcium signal transducer 1 (also known as colorectal cancer antigen CO17-1A, epithelial glycoprotein 2 (EGP2), epithelial glycoprotein 40 kDa (EGP40), epithelial cell adhesion molecule (EpCAM), epithelial specific antigen (ESA), gastrointestinal tumor-associated antigen 733-2 (GA733-2), KS1 / 4 antigen, MK-1 antigen, MIC18 antigen, TROP-1 antigen, TACSTD1 or membrane component of chromosome 4 surface marker 1 (M4S1); telomerase reverse transcriptase (also known as telomerase catalytic subunit, or TERT); trefoil factor 1 (also known as breast cancer estrogen-induced sequence, BCEI, gastrointestinal trefoil protein, GTF, pS2 protein or TFF1); folate; trefoil factor 3 (also known as intestinal trefoil factor, ITF, p1.B; or TFF3); HSPA8, actin, beta (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), enolase 1, alpha (ENO1), heat shock protein 90, alpha (cytosolic), class A member 1 (HSP90AA1), CD9 antigen (CD9), CD81 antigen (CD81), tyrosine 3-monooxygenase / tryptophan 5-monooxygenase activating protein, zeta polypeptide (YWHAZ), or pyruvate kinase, muscle (PKM2).

[0277] When referring to biological nanoparticles, the phrase "specifically (or selectively) binds" an antibody or "specifically (or selectively) immunoreacts with..." refers to such a binding reaction that determines the presence of the biological nanoparticle of interest, usually in a heterogeneous group of nanoparticles and other biological agents. Therefore, under the specified immunoassay conditions, the binding of a specific antibody to a specific biological nanoparticle is at least twice the background, and more usually 10-100 times the background. Specific binding to an antibody under such conditions requires an antibody selected for its specificity for a specific biological nanoparticle. For example, polyclonal antibodies can be selected to obtain those polyclonal antibodies that specifically immunoreact only with a selected antigen and not with other proteins. This selection can be achieved by subtracting antibodies that cross-react with other molecules.

[0278] In some embodiments of the methods provided herein, multiple parameters are detected in a single measurement. In certain embodiments, multiple parameters include different size values. In some embodiments, multiple parameters include the presence or absence of a detectable agent.

[0279] In some embodiments, the bio-nanoparticle ranking is binary, e.g., if the bio-nanoparticle of interest is not detected, a value of "0" is assigned, and if the bio-nanoparticle is detected, a value of "1" is assigned. In other embodiments, the ranking is non-binary, e.g., a value is assigned based on the size of the bio-nanoparticle or the type of bio-nanoparticle. In certain embodiments, the ranking is performed by software and a computer representing a ranking algorithm. In certain embodiments, the measurement is performed by software and a computer representing a measurement algorithm.

[0280] In some embodiments, the method provided herein further comprises the step of transporting biological nanoparticles based on the sorting of biological nanoparticles. For example, the flow or collection of biological nanoparticles is guided based on the value assigned to the biological nanoparticles. In some embodiments, this is achieved by using an external field or by generating a flow disturbance. In a specific embodiment, this is achieved by using a mechanism for guiding flow. In some embodiments, the guiding of flow and / or control of the mechanism for guiding flow is carried out by a computer and a software representing a guiding algorithm.

[0281] In certain embodiments, the method may include concentrating the biological nanoparticles of interest by collecting and / or pooling biological nanoparticles of similar order. In specific embodiments, the concentration of the biological nanoparticles is a result of directing the flow. For example, biological nanoparticles of a size value of interest may be directed to a specific flow channel and may be collected in a collection device at the end of the flow channel, resulting in an increase in the local concentration of biological nanoparticles of that size value.

[0282] In another aspect, the present disclosure provides a device configured to detect one or more biological nanoparticles in a sample fluid; the device comprising: (a) one or more detectors configured to detect the presence or absence of one or more detectable agents associated with one or more biological nanoparticles, wherein at least one of the one or more biological nanoparticles comprises a plurality of detectable agents; and (b) a computer comprising software for sorting the biological nanoparticles based on the presence or absence of the one or more detectable agents. In some embodiments, the sorting is binary. In other embodiments, wherein the device is used to detect multiple types of biological nanoparticles, and / or wherein the device is used to detect multiple types of detectable agents associated with biological nanoparticles, the sorting is non-binary.

[0283] In another aspect, the present disclosure provides an apparatus configured to detect one or more biological nanoparticles in a sample fluid; the apparatus comprising: (a) one or more detectors for detecting the presence or absence of biological nanoparticles comprising a size value; and (b) a computer comprising software for ranking the biological nanoparticles based on the size value. In some embodiments, the ranking is binary. In other embodiments, where the apparatus is used to detect biological nanoparticles of multiple sizes, the ranking is non-binary.

[0284] In another embodiment, the present disclosure provides a device configured to detect one or more biological nanoparticles in a sample fluid; the device includes: (a) one or more detectors configured to detect the presence or absence of biological nanoparticles comprising a size value and to detect the presence or absence of one or more detectable agents associated with the biological nanoparticles; and (b) a computer containing software for sorting the biological nanoparticles based on the size value and / or the presence or absence of one or more detectable agents associated with the biological nanoparticles. In some embodiments, the sorting is binary. In other embodiments, where the device is used to detect biological nanoparticles of multiple sizes and / or where the device is used to detect multiple types of biological nanoparticles and / or where the device is used to detect multiple types of detectable agents associated with biological nanoparticles, the sorting is non-binary.

[0285] In some embodiments, the microfluidic chip comprises a translucent or transparent material. In some embodiments, at least a portion of the microfluidic chip is transparent or translucent. In some embodiments, at least one side of the microfluidic chip is transparent or translucent. In some embodiments, the microfluidic chip is transparent or translucent. In a specific embodiment, the transparent or translucent properties of the microfluidic chip allow detection of light emitted from biological nanoparticles.

[0286] In certain embodiments, the device may further comprise a channel for delivering said nanoparticles based on said ranking. In certain embodiments, the channel is treated with an anticoagulant or anti-aggregation compound, a compound that preferentially prevents binding of biological nanoparticles, a compound that prevents aggregation of biological nanoparticles, or a combination thereof.

[0287] In some embodiments, the device may further include electrodes for tracking and manipulating the trajectory of the biological nanoparticles. In some embodiments, the device may include a magnetic element for separating biological nanoparticles with attached magnetic particles. In some embodiments, the device may include an acoustic element for tracking and manipulating the trajectory of biological nanoparticles.

[0288] In certain embodiments, the bionanoparticles can be further manipulated or analyzed. For example, the bionanoparticles can be further partitioned or sorted by conventional flow cytometry, or the bionanoparticles of interest can be analyzed to provide further information, such as nucleic acid analysis, protein analysis, lipid analysis, small molecule analysis, carbohydrate analysis, or a combination thereof can independently analyze the DNA, RNA, DNA sequence, metabolites, lipids, carbohydrates, protein content, etc. of the contents of each bionanoparticle. For example, exosome nucleic acids such as microRNAs (miRNA or miR-) can be used for the diagnosis of various tumors. For example, increased levels of exosome miRNAs miR-141 and miR-375 in serum may be associated with the progression of prostate cancer; increased levels of exosome miRNAs-21 and miRNA-1246 may be associated with esophageal cancer. Other exosome miRNAs may be potential diagnostic markers for renal fibrosis or cardiovascular disease. In addition, exosome miRNAs miR-320c and miR-6068 may be upregulated in the urine of patients with diabetic nephropathy. Regarding progressive degenerative diseases, high expression levels of several miRNAs may be associated with Alzheimer's disease, such as miR-9, miR-107, miRNA-128, miRNA134, and miRNA-137miRNA124. Other miRNAs with diagnostic value include, but are not limited to, miR-21, miR-141, miR-200a, miR-200b, miR-200c, miR-203, miR-205, miR-214; miR-17, miR-3p, miR-21, miR-20b, miR-223, miR-301, let-7f; miR-141, miR-375; miR-21, miR-1246; miR-21; and Let-7 family miRNAs.

[0289] In one embodiment, the fluid sample can contain more than one type of bio-nanoparticles, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more types of bio-nanoparticles. Thus, in certain embodiments, the fluid sample is simultaneously contacted with a plurality of distinguishable detectable agents, each of which has a different specificity under conditions sufficient to convert the plurality of detectable agents into a plurality of complexes comprising the detectable agent and a plurality of bio-nanoparticles. In some embodiments, a plurality of complexes are detected simultaneously, such as by using an apparatus comprising more than one interrogation device and / or more than one detection device.

[0290] For example, where two or more distinguishable detectable agents are bound to a bio-nanoparticle and each detectable agent is to be detected simultaneously, each bio-nanoparticle bound to the detectable agent can be detected by one of two or more detection devices. In addition, where the detectable agent includes a fluorescent moiety, two or more interrogation devices can be used (e.g., two lasers radiate at different wavelengths corresponding to the excitation wavelengths of the different fluorescent moieties), and the corresponding fluorescence emission intensities can be detected by two different detection devices. Thus, in one embodiment, the detectable agents can be distinguished by fluorescence of different wavelengths.

[0291] In another embodiment, two or more biological nanoparticles can be detected consecutively. For example, in one embodiment, the method can include detecting a first biological nanoparticle at a first location of the device, and detecting a second biological nanoparticle at a second location of the device. In this way, the biological nanoparticle can be delivered after the first detection step, after the second detection step, or after both detection steps.

[0292] In certain embodiments of the present disclosure, the detection of features from biological nanoparticles can be simultaneous or cumulative over time. For example, features can be detected from biological nanoparticles at once ("simultaneously"). In certain embodiments, wherein the method described is performed in a simultaneous mode, the biological nanoparticles can be carried by a variable speed flow. For example, as the biological nanoparticles traverse the detection volume, they can be carried by a steady flow. Alternatively, the flow can be slowed down or accelerated as the nanoparticles move in the detection volume. The flow can be regulated upstream or downstream of the detection volume using valves, bubbles, electric fields, magnetic fields, light fields, air pressure sources, solid particles, membranes, immiscible droplets, gravity differences, or coatings that change the surface tension of the channel.

[0293] In some embodiments of the methods provided herein, the detection step is performed during the continuous flow of the fluid sample through the flow channel. In a preferred embodiment, the flow is uninterrupted. In certain embodiments, the individual bio-nanoparticles are not physically separated, but are defined by an optical detection step, i.e., the bio-nanoparticles can be determined by a biomarker or detectable agent bound to the bio-nanoparticles present in the detection volume at the moment the detection occurs.

[0294] In certain embodiments, detection events will occur at a regular frequency, depending on the concentration of the bio-nanoparticles and the flow rate of the fluid sample. For example, if the concentration of a particular fluid sample is 1,000 bio-nanoparticles / 100 μL, and the fluid sample flows through the device at a rate of 100 μL / second, then different bio-nanoparticles can be detected at an average of 1 millisecond each, or at an average rate of 1,000 Hz.

[0295] In a preferred embodiment, at least one detection event directly corresponds to measuring the intensity of light emitted from the biological nanoparticle and assigning a size value to the biological nanoparticle. For example, a biological nanoparticle moving through a microfluidic chip may be exposed to two interrogation sources; a first interrogation source causes the biological nanoparticle to emit a light intensity that is measured and a size value can be assigned; a second interrogation source causes a detectable agent bound to the biological nanoparticle to emit fluorescence that is detected and the identity of the bound detectable marker can be determined.

[0296] In certain embodiments, the discrete biological nanoparticles traverse the detection volume at an average rate between 0.1 kHz and 100 MHz, depending on the geometry of the device and the volume of fluid to be processed. In another embodiment, the discrete biological nanoparticles traverse the detection volume at an average rate between about 10 Hz and about 10 MHz. In other embodiments, the discrete biological nanoparticles may traverse the detection volume at an average frequency of about 0.1 kHz to about 100 kHz, or about 1 kHz and about 10 MHz, or about 1 kHz to about 5 MHz, or about 1 kHz and about 1 MHz. In certain embodiments, the average frequency at which the bionanoparticles traverse the detection volume can be at least about 0.1 kHz, or at least about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 200, 250, 300, 400, 500, 600, 700, 800, or 900 kHz, or at least about 1 MHz, or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 MHz.

[0297] In some embodiments of the present disclosure, a fluid sample, such as a biological fluid sample, can be stabilized before detecting the bio-nanoparticles. In certain embodiments, the fluid can be stabilized with a reagent, including, but not limited to, an antiaggregant; an anticoagulant, such as citric acid, heparin, ethylenediaminetetraacetic acid (EDTA), serum albumin, polyethylene glycol (PEG), diethylenetriaminepentaacetic acid (DTPA), 1,2-diaminocyclohexanetetraacetic acid (DCTA) or ethylenebis(oxyethylenetriazine)tetraacetic acid (EGTA); an aldehyde, such as methyl alcohol, a hydroxymethyl derivative of an amine or amide of formaldehyde, diazolinidene urea, imidazolidinyl urea, methenamine, paraformaldehyde, glutaraldehyde or glyoxal, etc.

[0298] In other embodiments of the present disclosure, the methods provided herein can be further coupled to secondary processes that occur after the delivery of the bio-nanoparticles of interest. Examples of processes and / or functions that can be coupled to the methods provided herein include, for example, selective reactions to identify bio-nanoparticle content (e.g., DNA, RNA, microRNA, lipids, metabolites, carbohydrates, or proteins encapsulated in or compounded with bio-nanoparticles). These reactions include polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), isothermal nucleic acid amplification, reactions that determine DNA epigenetic states, single molecule hybridization reactions that determine microRNA and siRNA content, or aptamer (short DNA chains) selective reactions.

[0299] In certain embodiments, after the bio-nanoparticles are sorted or collected, the methods provided herein can be further coupled to experimental protocols. Non-limiting examples of tests that can be coupled to the methods provided herein include nucleic acid-based methods such as RNA extraction (with or without amplification), cDNA synthesis (reverse transcription), gene microarrays, DNA extraction, polymerase chain reaction (PCR) (single, nested, quantitative real-time or linker-linker) or DNA methylation analysis; cell counting methods such as fluorescence in situ hybridization (FISH), flow cytometry, fluorescence activation sorting or comparative genomic hybridization (CGH) studies; chemical test methods such as electrophoresis, Southern blot analysis or enzyme-linked immunosorbent assay (ELISA); tests to determine microRNA and siRNA content; tests to determine DNA / RNA content; tests to determine lipid content; tests to determine carbohydrate content; tests to determine metabolite content; tests to determine protein content; functional tests, etc.

[0300] In some embodiments, after the bio-nanoparticles are sorted or collected, the methods provided herein can be further coupled with analysis. In certain embodiments, the analysis comprises nucleic acid analysis. In specific embodiments, nucleic acid analysis comprises sequencing, PCR or digital PCR. In certain embodiments, the analysis comprises protein analysis. In specific embodiments, protein analysis comprises ELISA, digital ELISA or mass spectrometry. In some embodiments, the analysis comprises lipid analysis. In certain embodiments, the analysis comprises small molecule analysis. In specific embodiments, small molecule analysis comprises analysis of metabolites, analysis of signal transduction molecules, analysis of drugs, mass spectrometry or a combination thereof. In certain embodiments, the analysis comprises mass spectrometry. In some embodiments, the analysis comprises nuclear magnetic resonance spectroscopy. In some embodiments, the analysis comprises carbohydrate analysis. In specific embodiments, carbohydrate analysis comprises mass spectrometry, nuclear magnetic resonance, Fourier transform spectroscopy or a combination thereof.

[0301] In another embodiment, the method that this paper provided can be further coupled with flow cytometry, for example, to further partition or separate the biological nanoparticles present in the fluid sample. In one embodiment, the channel of the device that is used for the method that this paper provides can be communicated with the flow cytometer fluid. In some embodiments, the coupling of device and flow cytometer allows further inspection or continuous sorting of selected biological nanoparticles to further enrich the colony of biological nanoparticles of interest. In some embodiments of the method that this paper provides, this setting allows biological nanoparticles to be carried out upstream rough selection, and only the biological nanoparticles that will comprise the desired size value or the biological nanoparticles that will be combined with a specific detectable agent are guided to downstream processes, such as flow cytometry, thereby reducing time, cost and / or labor.

[0302] Sorting biological nanoparticles

[0303] In one embodiment of the method provided herein, if the biological nanoparticle is combined with a detectable agent, then the first value of the biological nanoparticle detected is specified, or if the biological nanoparticle is not combined with a detectable agent, then the second value of the biological nanoparticle detected is specified. In another embodiment of the method provided herein, if the light intensity emitted by the biological nanoparticle is detected to be higher than a threshold value, then the first value of the biological nanoparticle detected is specified, or if the light intensity emitted by the biological nanoparticle is detected to be not higher than a threshold value, then the second value of the biological nanoparticle detected is specified. In a specific embodiment, the sorting (i.e., the designation of the value) is binary. For example, each biological nanoparticle that emits a light intensity higher than a detectable threshold value is specified as a value of 1, and each biological nanoparticle that does not emit a light intensity higher than a detectable threshold value is specified as a value of 0.

[0304] In another embodiment of the methods provided herein, a value is assigned to a biological nanoparticle based on the detectable agent associated with the biological nanoparticle. For example, a biological nanoparticle comprising 4 detectable agents can be assigned a value of 4. Alternatively, a value for a biological nanoparticle comprising 4 detectable agents can be assigned to correspond to a specific range of detectable agent amounts, for example, 0-5 detectable agents, 1-10 detectable agents, 4-6 detectable agents, etc.

[0305] In another embodiment of the methods provided herein, a value is assigned to a biological nanoparticle based on the intensity of light emitted from the biological nanoparticle. For example, a biological nanoparticle that emits light with an intensity that is 3 times greater than a threshold value can be assigned a value of 3. Alternatively, a value of a biological nanoparticle that emits light with an intensity that is 3 times greater than a threshold value can be assigned a value that corresponds to a specific range of light intensity quantities, for example, 1-5 times greater than a threshold value, 1-10 times greater than a threshold value, 2-3 times greater than a threshold value, etc.

[0306] In yet another embodiment of the methods provided herein, where more than one type of biological nanoparticle is present in a single fluid sample, values ​​are assigned to the biological nanoparticles based on the nanoparticle size value or the type of any detectable agent that binds to the biological nanoparticle. For example, where the fluid sample contains two types of detectable agents A and B, the value of biological nanoparticles that do not bind to either A or B can be 0, the value of biological nanoparticles that bind only to A can be 1, the value of biological nanoparticles that bind only to B can be assigned to 2, and the value of biological nanoparticles that bind to both A and B can be assigned to 3; using the methods disclosed herein, nanoparticles assigned a value of 2 can be selectively separated, thereby obtaining a concentrated sample of biological nanoparticles that specifically bind only to detectable agent B. Therefore, in one embodiment of the methods provided herein, where more than one type of detectable agent is present in a single fluid sample, the ranking (i.e., the assignment of values) is not binary.

[0307] In some embodiments of the method provided herein, wherein there are more than one type of bio-nano particles in the fluid sample, values ​​are assigned to the bio-nano particles according to a plurality of parameters. A plurality of parameters can include bio-nano particle size values, light intensity emitted from the bio-nano particles, detectable agents combined with the bio-nano particles, the types of the bio-nano particles, etc. The sorting or assigned values ​​may have more than one variable. For example, wherein the fluid sample comprises two types of bio-nano particles, and wherein the various types of bio-nano particles exist in a variety of sizes, then a plurality of parameters can be used to assign sorting to the bio-nano particles. Each of the two types of bio-nano particles can be combined with detectable agents A or B; similarly, each of the two types of bio-nano particles can emit such light intensity, and the light intensity is related to the size value that varies between 20-50nm with the hydrodynamic diameter. Thus, sorting can be applied to size (e.g., assigning a value of 1 to a size value of 20-30 nm, a value of 2 to a size value of 31-40 nm, and a value of 3 to a size value of 41-50 nm) and detectable agent binding (e.g., assigning a detectable agent value of "A" when the nanoparticle binds to A; assigning a detectable agent value of "B" when the nanoparticle binds to B, assigning a detectable agent value of "AB" when the nanoparticle binds to both A and B; and assigning a detectable agent value of "Z" when the nanoparticle binds to neither A nor B). Multi-parameter sorting can be applied to biological nanoparticles as they move through a microfluidic chip. Sorting can be applied to a population of desired biological nanoparticles enriched by any one of a plurality of parameters. For example, using the population of biological nanoparticles described above, a counting or sorting event can be performed for all biological nanoparticles having an "A" value, or more specifically, a counting or sorting event can be performed for all biological nanoparticles having a 2-A value (e.g., all biological nanoparticles having a size value corresponding to a hydrodynamic diameter of 31-40 nm and bound to a detectable agent A). In this way, population enrichment, sorting, and / or counting can be performed by multiple parameters. In a specific embodiment, the multiple parameters of the biological nanoparticles include emission light intensity, size value, binding to a detectable agent, type, etc.

[0308] In certain embodiments, the non-zero assigned value may depend on the type of biological nanoparticles or the concentration of biological nanoparticles in the fluid sample.

[0309] In certain embodiments, multiple bio-nanoparticles having the same designated value are pooled or delivered together.

[0310] In one embodiment of the method of the present disclosure, active decision making is required to rank or assign a value to the biological nanoparticles. In certain embodiments, a computer, a controller, a chip with an integrated circuit, a circuit board, an electronic component, software and / or an algorithm is used to rank or assign a value to the biological nanoparticles.

[0311] In some embodiments, at least one of the values ​​assigned to the bio-nanoparticles can be a biomarker value. The biomarker value can be a biomarker type, a count of a biomarker, a count of a specific type of biomarker, a copy number of a biomarker, etc. In a specific embodiment, the bio-nanoparticle contains at least one biomarker. In some embodiments, the bio-nanoparticle is bound to at least one biomarker. The method disclosed herein provides such a step, which detects at least one biomarker bound to the bio-nanoparticle, and assigns a biomarker value to the bio-nanoparticle. In some embodiments, the biomarker value is assigned by a binary system. For example, a plurality of bio-nanoparticles can include a portion of nanoparticles bound to a biomarker A-binding-a binary biomarker value can be applied to the bio-nanoparticles, thereby distinguishing nanoparticles bound to the biomarker from nanoparticles not bound to the biomarker.

[0312] In some embodiments, the present disclosure provides a method comprising determining at least one copy number of at least one biomarker bound to a bio-nanoparticle. The determination of the copy number can provide at least one selection for identifying the bio-nanoparticle and / or the bio-nanoparticle bound to the bio-nanoparticle. The variation of the copy number can also be analyzed, which can involve the increase or loss of the function of the bio-marker and / or the bio-nanoparticle. For example, a change in the copy number of a bio-marker such as a protein bio-marker or a nucleic acid can provide information about the bio-marker and their binding to the bio-nanoparticle. In some embodiments, the present disclosure provides a method comprising determining at least one copy number of at least one bio-marker, wherein the bio-nanoparticle comprises at least one bio-marker. For example, the detected bio-nanoparticle can provide a copy value of the bio-marker, such as the number of trinucleotide repeats present in the bio-marker. In some embodiments, the determination of the copy number uses a restriction enzyme. In certain embodiments, the determination of the copy number uses a standard. In a specific embodiment, the standard for determining the copy number has not been amplified. In some embodiments, the copy number and / or copy number variation of a protein bio-marker is determined. In some embodiments, the copy number and / or copy number variation of a nucleic acid is determined.

[0313] In some embodiments, the biomarker is a protein biomarker. In certain embodiments, the protein biomarker is selected from: epithelial-derived cell-specific proteins, tetraspanins (e.g., CD9, CD63, and CD81), heat shock proteins (e.g., HSC70 and HSC90), membrane transporters (e.g., GTPases) and lipid binding proteins, cell-adhesion proteins, lipid binding proteins, transmembrane proteins, enzymes, pan-cytokeratin antibodies A45B / B3, AE1 / AE3, or CAM5.2 (pan-cytokeratin antibodies that recognize cytokeratin 8 (CK8), cytokeratin 18 (CK18) or cytokeratin 19 (CK19), and are directed against one of the following: breast cancer antigen NY-BR-1 (also known as B726P, ANKRD30A, ankyrin repeat domain 30A); B305D isoform A or C (B305D-A ro B305D-C; also known as antigen B305D); Hermes antigen (also known as antigen CD44, PGP1); E-cadherin (also known as uvom adhesion protein (Uvomorulin), cadherin-1, CDH1); carcinoembryonic antigen (CEA; also known as CEACAM5 or carcinoembryonic antigen-related cell adhesion molecule 5); beta-human chorionic gonadotropin (beta-HCG; also known as CGB, chronic gonadotropin, beta polypeptide); cathepsin-D (also known as CTSD); neuropeptide Y receptor Y3 (also known as NPY3R; lipopolysaccharide-associated protein 3, LAP3, fusion protein); chemokine (CXC motif, receptor 4; CXCR4); oncogene ERBB1 (also known as c-erbB-1, epidermal growth factor receptor, EGFR); Her-2 Neu (also known as c-erbB-2 or ERBB2); GABA receptor A, π polypeptide (also known as GABARAP, GABA-A receptor, π polypeptide (GABA A(π), γ-aminobutyric acid type A receptor π subunit) or GABRP); ppGalNac-T(6) (also known as β-1-4-N-acetyl-galactosaminyltransferase 6, GalNAc transferase 6, GalNAcT6, UDP-N-acetyl-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase 6 or GALNT6); CK7 (also known as cytokeratin 7, Sarcolectin, SCL, keratin 7 or KRT7); CK8 (also known as cytokeratin 8, keratin 8 or KRT8); CK18 (also known as cytokeratin 18, keratin 18 or KRT18); CK19 (also known as cytokeratin 19, keratin 19 or KRT19); CK20 (also known as cytokeratin 20, keratin 20 or KRT20); Mage (also known as melanoma antigen family A subtype or MAGE-A subtype); Mage3 (also known as melanoma antigen family A 3 or MAGA3); hepatocyte growth factor receptor (also known as HGFR, renal cell carcinoma papillary type 2, RCCP2, proto-oncogene or MET); mucin-1 (also known as MUC1, cancer antigen 15.3, (CA15.3), cancer antigen 27.29 (CA 27.29); CD227 antigen, Episialin, epithelial membrane antigen (EMA), polymorphic epithelial mucin (PEM), peanut-reactive urinary mucin (PUM), tumor-associated glycoprotein 12 (TAG12); macrocystic disease fluid protein (also known as GCDFP-15, prolactin-induced protein, PIP); urokinase receptor (also known as uPR, CD87 antigen, plasminogen activator receptor urokinase type, PLAUR); PTHrP (parathyroid hormone-related protein; also known as PTHLH); BS106 (also known as B511S, small mammary epithelial mucin or SBEM); prostaglandin-like lipophilin B (LPB, LPHB; also known as antigen BU101, secretory globulin Lactoglobin (MGB); also known as mammaglobin 1, MGB1, mammaglobin A, MGBA, secretoglobin family 2A member 2 or SCGB2A2); mammary gland Serine protease inhibitor (Maspin, also known as serine (or cysteine) protease inhibitor clade B (ovalbumin) member 5, or SERPINB5); prostate epithelial-specific Ets transcription factor (PDEF; also known as ets transcription factor containing a sterile alpha motif tip domain, or SPDEF); tumor-associated calcium signal transducer 1 (also known as colorectal cancer antigen CO17-1A, epithelial glycoprotein 2 (EGP2), epithelial glycoprotein 40 kDa (EGP40), epithelial cell adhesion molecule (EpCAM), epithelial specific antigen (ESA), gastrointestinal tumor-associated antigen 733-2 (GA733-2), KS1 / 4 antigen, MK-1 antigen, MIC18 antigen, TROP-1 antigen, TACSTD1 or membrane component of chromosome 4 surface marker 1 (M4S1); telomerase reverse transcriptase (also known as telomerase catalytic subunit, or TERT); trefoil factor 1 (also known as breast cancer estrogen-induced sequence, BCEI, gastrointestinal trefoil protein, GTF, pS2 protein or TFF1); folate; trefoil factor 3 (also known as intestinal trefoil factor, ITF, p1.B; or TFF3); HSPA8, actin, beta (ACTB), 3-phosphoglyceraldehyde dehydrogenase (GAPDH), enolase 1, alpha (ENO1), heat shock protein 90, alpha (cytoplasmic), class A member 1 (HSP90AA1), CD9 antigen (CD9), CD81 antigen (CD81), tyrosine 3-monooxygenase / tryptophan 5-monooxygenase activating protein, zeta polypeptide (YWHAZ) or pyruvate kinase, muscle (PKM2), enzymes such as esterases, phosphatases or lipases. In some embodiments, the biomarker is a nucleic acid biomarker. In certain embodiments, the nucleic acid biomarker is selected from microRNA (miRNA or miR-), such as exosomal miRNA, including miR-141, miR-375, miRNA-21, miRNA-1246, miR-320c, miR-6068, miR-9, miR-107, miRNA-128, miRNA134, miRNA-137, miRNA124, miR-21, m iR-141,miR-200a,miR-200b,miR-200c,miR-203,miR-205,miR-214;miR-17,miR-3p,miR-21,mi R-20b,miR-223,miR-301,miR-141,miR-375,miR-21,miR-1246;miR-21,let-7f,or other Let-7miRNA. .

[0314] In some embodiments, at least one of the values ​​assigned to the biological nanoparticle can be a tag value. The tag value can be a tag type, a count of tags, a count of a specific type of tag, etc. In a specific embodiment, the biological nanoparticle contains at least one tag. In some embodiments, the biological nanoparticle is bound to at least one tag. The method disclosed herein provides such a step, which detects at least one tag bound to the biological nanoparticle and assigns a tag value to the biological nanoparticle. In some embodiments, the tag value is assigned by a binary system. For example, a plurality of biological nanoparticles can include a portion of nanoparticles bound to tag A - a binary tag value can be applied to the biological nanoparticles to distinguish nanoparticles bound to the tag from nanoparticles not bound to the tag.

[0315] In a specific embodiment, the present disclosure provides a method comprising determining at least one value of at least one tag associated with a biological nanoparticle. In some embodiments, the present disclosure provides a method comprising determining the type of at least one tag, wherein the biological nanoparticle includes at least one tag. For example, the detected biological nanoparticle can provide the type of the tag.

[0316] In some embodiments, the tag value is specified from a plurality of values. For example, a plurality of biological nanoparticles may include a population bound to any one of six tags, each of which provides a unique tag value that depends on the type of tag. The methods disclosed herein include detection of specific tags and sorting based on tag values. In some embodiments, the tag value is specified by a binary system. In a specific embodiment, the system includes non-zero and zero values. In some embodiments, the system includes positive or negative values ​​(for example, biological nanoparticles bound to a tag of interest can be specified as positive values, while nanoparticles not bound to a tag will be specified as negative values, thereby allowing detection and / or sorting of biological nanoparticles bound to a tag of interest).

[0317] In certain embodiments, the tag is selected from: a signal derived from a protein marker associated with a biological nanoparticle, a signal derived from the size of a biological nanoparticle, a signal derived from a membrane dye associated with a biological nanoparticle, a signal derived from a body dye associated with a biological nanoparticle, a signal derived from a dye associated with a nucleic acid of a biological nanoparticle, a signal derived from a dye associated with a lipid of a biological nanoparticle, or a signal derived from a dye associated with a biological nanoparticle.

[0318] In some embodiments, the biological nanoparticle comprises an extracellular vesicle, an organelle, a microvesicle, a cell-derived vesicle, a protein aggregate, a nucleic acid aggregate, a lipid aggregate, a virus, a bacterium, an exosome, or a combination thereof. In a specific embodiment, the biological nanoparticle comprises an exosome. In a specific embodiment, the biological nanoparticle is an exosome.

[0319] Channeling and Fluid Flow

[0320] In certain embodiments of the present disclosure, the flow or collection of biological nanoparticles is directed based on the values ​​assigned to the biological nanoparticles. For example, biological nanoparticles assigned a null or "0" value can be directed to a first channel (delivered to) or a waste outlet, and biological nanoparticles assigned a positive or "1" value can be directed to a second channel or a collection chamber. For another example, biological nanoparticles having a value specified by any one of a plurality of parameters can be directed to a first channel or a second channel.

[0321] In certain embodiments of the present disclosure, the flow or collection of biological nanoparticles is guided based on the size value. In certain embodiments of the present disclosure, the flow or collection of biological nanoparticles is guided based on the combination of biological nanoparticles with a detectable agent. In certain embodiments of the present disclosure, the flow or collection of biological nanoparticles is guided based on the size value of the biological nanoparticles and the biomarker value of the biological nanoparticles. In some embodiments, the flow or collection of biological nanoparticles is guided to sort the biological nanoparticles into enriched populations. In a preferred embodiment, sorting the biological nanoparticles into enriched populations is determined by the size value and biomarker value of the biological nanoparticles. In certain embodiments of the present disclosure, the flow or collection of biological nanoparticles is guided based on the combination of biological nanoparticles with a label. In certain embodiments of the present disclosure, the flow or collection of biological nanoparticles is guided based on the size value of the biological nanoparticles and the label value of the biological nanoparticles. In certain embodiments, sorting the biological nanoparticles into enriched populations is determined by the size value and label value of the biological nanoparticles. In some embodiments, sorting the biological nanoparticles into enriched populations is determined by the size value of the biological nanoparticles. In certain embodiments, the directing of the fluid directs the biological nanoparticles into a collection, generating a collection of biological nanoparticles based on the size value.

[0322] In certain embodiments, a biomarker value is specified from a plurality of values. For example, a plurality of bio-nanoparticles may include a population bound to any one of four tags, each of which provides a unique bio-marker value that depends on the type of bio-marker. The methods disclosed herein include the detection of specific bio-markers and sorting based on bio-marker values. In some embodiments, the bio-marker value is specified by a binary system. In a specific embodiment, the system includes non-zero and zero values. In some embodiments, the system includes positive or negative values ​​(for example, bio-nanoparticles bound to a bio-marker of interest can be specified as positive values, while nano-particles not bound to a bio-marker will be specified as negative values, thereby allowing detection and / or sorting of bio-nanoparticles bound to a bio-marker of interest).

[0323] In some embodiments, the methods provided herein can further include sorting at least one biological nanoparticle into an enriched population. In a specific embodiment, sorting the biological nanoparticles into an enriched population is determined by: a size value of the biological nanoparticle, the presence of a biomarker associated with the biological nanoparticle, a measured light intensity emitted from the biological nanoparticle, a measured light intensity emitted from a detectable agent associated with the biological nanoparticle material, a wavelength of light emitted by the biological nanoparticle, a wavelength of light emitted by a detectable agent associated with the biological nanoparticle, multiple wavelengths emitted by the biological nanoparticle, multiple wavelengths emitted by a detectable agent associated with the biological nanoparticle, an identification mark of the biological nanoparticle, or a combination thereof. For example, biological nanoparticles having a hydrodynamic diameter of 40-100 nanometers can be selectively enriched by their size value, thereby producing a sample comprising a concentrated population of biological nanoparticles having a hydrodynamic diameter of 40-100 nanometers. In a preferred embodiment, sorting uses a mechanism for directing flow.

[0324] In certain embodiments, the sorting of bionanoparticles into enriched populations is determined by the size values ​​of the bionanoparticles. In some embodiments, the diameter variance of the enriched population is less than 1000%, the diameter variance is less than 900%, the diameter variance is less than 800%, the diameter variance is less than 700%, the diameter variance is less than 600%, the diameter variance is less than 500%, the diameter variance is less than 400%, the diameter variance is less than 300%, the diameter variance is less than 200%, the diameter variance is less than 100%, the diameter variance is less than 75%, the diameter variance is less than 50%, or the diameter variance is less than 25%. In preferred embodiments, the diameter variance of the enriched population is less than 100%. As provided herein, the term "variance" is intended to describe a parameter of a value greater than or less than an indicated value. For example, if the size value of interest is a diameter of 100 nm, an enriched population with a diameter variance of less than 25% will contain values ​​greater than 75 nm to less than 125 nm. One skilled in the art will appreciate that as used herein, variance provides a parameter for measuring an accuracy greater or less than a specified value (eg, a value with a variance less than 20% provides a true value ± less than 20%).

[0325] In some embodiments, the enriched population has the following diameter range: within 500% of the diameter value, within 400% of the diameter value, within 300% of the diameter value, within 200% of the diameter value, within 100% of the diameter value. In a specific embodiment, the diameter range of the enriched population is within 100% of the diameter value. In some embodiments, the enriched population having a diameter variance or diameter range also has a confidence interval for the enriched population, wherein the confidence interval is greater than 99%, greater than 98%, greater than 97%, greater than 96%, greater than 95%, greater than 90%, greater than 80%, greater than 70% or greater than 50%. In a preferred embodiment, the confidence interval is greater than 95%. The confidence interval can be applied to any of the enriched population diameter variance or diameter range. For example, the diameter of the population is within 100% of the diameter value, wherein the diameter value is 75nm, and the confidence interval is 95%, which means that the probability that the diameter of the bionanoparticles of the enriched population is within 100% of the 75nm hydrodynamic diameter is 95%.

[0326] In some embodiments, sorting of biological nanoparticles into enriched populations is determined by binding to at least one detectable agent. In some embodiments, sorting of biological nanoparticles into enriched populations is determined by binding to a plurality of detectable agents. In a specific embodiment of an enriched population, more than 50% of the biological nanoparticles are bound to a detectable agent, more than 60% of the biological nanoparticles are bound to a detectable agent, more than 70% of the biological nanoparticles are bound to a detectable agent, more than 75% of the biological nanoparticles are bound to a detectable agent, more than 80% of the biological nanoparticles are bound to a detectable agent, more than 85% of the biological nanoparticles are bound to a detectable agent, more than 90% of the biological nanoparticles are bound to a detectable agent, more than 93% of the biological nanoparticles are bound to a detectable agent, more than 95% of the biological nanoparticles are bound to a detectable agent, more than 97% of the biological nanoparticles are bound to a detectable agent, more than 98% of the biological nanoparticles are bound to a detectable agent, or more than 99% of the biological nanoparticles are bound to a detectable agent. In a preferred embodiment, more than 80% of the biological nanoparticles in the enriched population are bound to a detectable agent. In preferred embodiments, the detectable agents are of the same type of detectable agent.

[0327] As used herein, the terms "channeling" and "directing" may be used interchangeably. The terms "channeling" and "directing" flow refer to controlling the movement of a fluid in a manner that may force the destination, trajectory, or movement of a bio-nanoparticle.

[0328] In some embodiments of the present disclosure, where more than one type of biological nanoparticle is present in the fluid sample, the biological nanoparticles can be transported based on a specific emission of light intensity from the biological nanoparticles. In one embodiment, if the light intensity emitted by the nanoparticles is below a threshold, the biological nanoparticles can be directed to a first channel or a waste outlet, if the light intensity emitted by the biological nanoparticles is above a threshold corresponding to a first size value, the biological nanoparticles can be directed to a second channel or a first collection chamber, and if the light intensity emitted by the biological nanoparticles is above a threshold corresponding to a second size value, the biological nanoparticles can be directed to a third channel or a second collection chamber.

[0329] In another embodiment of the present disclosure, where more than one type of biological nanoparticle is present in the fluid sample, the biological nanoparticles can be delivered based on the specific detectable agent bound to the biological nanoparticles. In one embodiment, biological nanoparticles that are not bound to a detectable agent can be directed to a first channel or a waste outlet, biological nanoparticles bound to a first type of detectable agent can be directed to a second channel or a first collection chamber, and biological nanoparticles bound to a second type of detectable agent can be directed to a third channel or a second collection chamber.

[0330] In certain embodiments, bio-nanoparticles containing more than one parameter value can be directed to a specific flow channel or collection chamber. Alternatively, the bio-nanoparticles can be directed to a mixing or dilution chamber, and then the mixed or diluted bio-nanoparticles can be further partitioned, so that the bio-nanoparticles are divided into different aliquots. The bio-nanoparticles in the aliquots can then be detected again, so that the bio-nanoparticles can be separated from each other.

[0331] In one embodiment of the methods provided herein, transport (ie, directing the flow or collection of bio-nanoparticles) can be performed by using an external field or by creating a flow disturbance. In specific embodiments, transport uses a mechanism for directing flow.

[0332] In one aspect of the present disclosure, once the bio-nanoparticles are ordered, external fields can be used to change the orientation of the bio-nanoparticles. These fields can include electric, magnetic, electrokinetic, electrophoretic, electroosmotic, dielectrophoretic, hydrodynamic, gravitational, pneumatic, or optical forces. Alternatively, external flow disturbances can be induced by introducing materials that are immiscible with the suspension, such as air, immiscible organic liquids, or microbeads.

[0333] In certain embodiments, flow can be delivered, for example, by methods and devices that introduce fluid dynamic fluid pressure, including but not limited to those that operate based on mechanical principles (e.g., external syringe pumps, pneumatic membrane pumps, vibrating membrane pumps, vacuum devices, centrifugal forces, and capillary action); electrical or magnetic principles (e.g., electroosmotic flow, electric pumps, piezoelectric / ultrasonic pumps, magnetic fluid plugs, electrofluidic pumps, and magnetic fluid pumps); thermodynamic principles (e.g., volume expansion caused by bubble generation / phase change); surface wetting principles (e.g., electrowetting, chemical, thermodynamic, and radioactively induced surface tension gradients); etc.

[0334] In other embodiments, the fluid can be delivered or transported by fluid driving forces provided by: gravity feed, surface tension (such as capillary action), electrostatic forces (electric flow), electroosmotic flow, centrifugal flow (substrate placed on a disc and rotated), acoustic forces, magnetic forces (oscillating ions cause flow), magnetohydrodynamics, and vacuum or pressure differentials.

[0335] In certain embodiments, a fluid flow control device (such as those listed for directing fluid dynamic fluid pressure or fluid driving force methods and devices) can be coupled to an input port or an output port of the present disclosure. In one example, multiple ports are provided in one or both of the inlet or outlet, and one or more ports are coupled to the fluid flow control device.

[0336] In some embodiments, the flow guidance includes flow displacement. As used herein, the terms "flow displacement" and "fluid displacement" are used interchangeably. In some embodiments, flow displacement is a positive displacement, a negative displacement, or a combination thereof. In certain embodiments, flow displacement provides a means for guiding flow with reduced stress on bio-nanoparticles. Flow displacement may cause flow disturbances, and flow disturbances can guide bio-nanoparticles or adjust their trajectories. In some embodiments, a sorting device as described above is used to cause flow displacement. In certain embodiments, sorting includes flow-displacement sorting. In some embodiments, sorting does not use acoustic sorting or physical barriers. In certain embodiments, the premise of sorting is that acoustic sorting is not included or a physical barrier is used.

[0337] In certain embodiments, the directing of flow comprises electroosmotic flow. In some embodiments, the directing of flow comprises application of pressure.

[0338] In some embodiments, the guiding of the biological nanoparticle takes no more than 100 ms, no more than 50 ms, no more than 10 ms, no more than 5 ms, no more than 3 ms, no more than 1 ms, no more than 500 μs, no more than 100 μs, no more than 50 μs, or no more than 10 μs. In a preferred embodiment, the guiding of the biological nanoparticle does not exceed 1 ms. In another preferred embodiment, the guiding of the biological nanoparticle does not exceed 100 μs.

[0339] In certain embodiments, sorting is determined by: a size value of a biological nanoparticle, the presence of a biomarker associated with a biological nanoparticle, measuring the intensity of light emitted from a biological nanoparticle, measuring the intensity of light emitted from a detectable agent associated with a biological nanoparticle material, a wavelength of light emitted by a biological nanoparticle, a wavelength of light emitted by a detectable agent associated with a biological nanoparticle, multiple wavelengths emitted by a biological nanoparticle, multiple wavelengths emitted by a detectable agent associated with a biological nanoparticle, an identification mark of a biological nanoparticle, or a combination thereof.

[0340] In some embodiments of the method provided herein, biological nanoparticles are separated. In specific embodiments, sorting or guiding can be used to separate biological nanoparticles. In some embodiments, biological nanoparticles are separated in a separation chamber. The separation chamber can be, for example, a hole of a multi-well plate, an Eppendorf tube, a vial, etc. In some embodiments, a plurality of biological nanoparticles are separated into a single separation chamber. In specific embodiments, an enriched population of a biological nanoparticle colony is separated in a single separation chamber. For example, a plurality of biological nanoparticles can be sorted, thereby only nanoparticles with a hydrodynamic diameter of 40-100nm are guided into a vial, which can be retained for further processing or identification. In some embodiments, a single biological nanoparticle is separated.

[0341] Counting biological nanoparticles

[0342] In some embodiments of the method provided herein, biological nanoparticles are counted. As used herein, the terms "counting" and "quantitative" are interchangeable. The term "counted" used herein represents that biological nanoparticles have entered the measurement of the amount, such as a counting system (e.g., counter). For example, a fluid sample containing 400 biological nanoparticles of a specific sorting in a microfluidic device provided by the method as described herein can result in each biological nanoparticle with the sorting being counted individually or quantitatively, thereby counting 400 biological nanoparticles. In some embodiments, biological nanoparticles with a size value in a fluid sample can be counted to provide the quantity of biological nanoparticles with the size value in a sample.

[0343] In some embodiments, the method provided by this paper further comprises that the quantity of biological nanoparticles is quantitatively carried out. In a specific embodiment, the method provided by this paper comprises that the quantity of biological nanoparticles with size value is quantitatively carried out. In a preferred embodiment, the method provided by this paper comprises that the quantity of biological nanoparticles with specific size value is quantitatively carried out. In some embodiments, before the fluid sample is introduced into the microfluidic device, the size value is selected.

[0344] In certain embodiments, the method provided herein also includes determining the concentration of the biological nanoparticles of interest in the fluid sample. In some embodiments, the determination of the concentration includes counting the number of biological nanoparticles of interest and dividing the value by the volume of the fluid sample. As a non-limiting example, such a sample fluid can be introduced into a microfluidic chip, the volume of the sample fluid is 1 μL, and multiple biological nanoparticles with a size value of 30-50nm are included; quantifying the number of biological nanoparticles with a size value of 30-50nm can provide a total count of 6,000 detected biological nanoparticles with the same size value; therefore, the concentration of the biological nanoparticles of interest in the sample is measured as 6,000 nanoparticles / μL. In this way, the method provided herein can include determining the concentration of the fluid sample.

[0345] In some embodiments, the method for determining the concentration of a sample includes calculating the peak frequency of a fluid sample. As used herein, the term "peak frequency" refers to the frequency of detecting luminescence from a bio-nanoparticle, wherein the light emission is above a threshold (e.g., when a bio-nanoparticle has a light emission above a threshold when passing through a detection volume, a system including a detector observes and registers a "peak"), or within a desired value range. In certain embodiments, the flow rate is known. In some embodiments, the flow rate is constant. In other embodiments, the flow rate is an average flow rate. The peak frequency corresponds to the number of bio-nanoparticles of interest passing through the detection volume over a period of time. For example, if a fluid sample in a microchannel passes through the detection volume and 10 peaks (e.g., 10 emission signals with a specific value) are detected within a period of 1 second, then the peak frequency is 10 peaks / second. Given the flow rate, the volume of the fluid sample passing through the detection area and / or the total volume of the sample, the concentration can be determined. For example, if the flow rate of the above example is a constant 1 μL / second, and the peak frequency is 10 peaks / second, then the concentration of the bio-nanoparticles of interest is 10 bio-nanoparticles / 1 μL.

[0346] As used herein, a "peak" describes an emission observed within a parameter. A peak can correspond to a size value, light emission intensity, binding to a detectable agent, etc. For example, if the light intensity emitted by the desired bio-nanoparticle exceeds a threshold limit by 4 to 5 times, any bio-nanoparticle observed within these limits will be recorded as a peak. In some embodiments, the number of peaks is counted. In a specific embodiment, the number of peaks is counted over a period of time to determine the peak frequency. The observation of peaks and the calculation of peak frequencies can be performed using the equipment described in the present disclosure.

[0347] In certain embodiments, the peak frequency is associated with a parameter value of the biological nanoparticle. The parameter value can be a size value, the presence of a detectable agent, light emission intensity, etc. For example, by measuring the frequency at which the detectable agent A is observed, the peak frequency of a biological nanoparticle labeled with a detectable agent A can be determined from a plurality of biological nanoparticles.

[0348] In some embodiments, the concentration of the sample is determined by counting the peak frequency of the sample and comparing it to the volume of the sample. In some embodiments, the concentration of the sample is determined by counting the peak frequency of the sample and comparing it to the peak frequency of the calibration particle standard. For example, if the standard has a known concentration of 100 nanoparticles / μL of the biological nanoparticles of interest, and the peak frequency of the standard is 10 peaks / ms, then it can be determined that the concentration of a sample with an unknown concentration of the desired biological nanoparticles analyzed using the same conditions and having a peak frequency of 5 peaks / ms is 50 nanoparticles / μL. In certain embodiments, the concentration of the sample is determined by counting the peak frequency of the sample, comparing it to the calibration particle standard peak frequency, and comparing the peak frequency of the sample to the volume of the sample. As further described in the present disclosure, any of a variety of standard particles can be used. In some embodiments, the calibration particle standard includes gold nanoparticles. In certain embodiments, a gold nanoparticle standard of known concentration (and therefore peak frequency) is mixed with a biological nanoparticle sample of unknown concentration to form a sample solution, whereby the concentration of the biological nanoparticles in the sample solution can be determined by comparing the peak frequency of the gold nanoparticles with the peak frequency of the biological nanoparticles.

[0349] In certain aspects, the bionanoparticles move through the microfluidic chip at a high rate. In a specific embodiment, the bionanoparticles pass through the microfluidic chip at a rate of more than 1 million particles / hour, more than 2 million particles / hour, more than 3 million particles / hour, more than 4 million particles / hour, more than 5 million particles / hour, more than 6 million particles / hour, more than 7 million particles / hour, more than 8 million particles / hour, more than 9 million particles / hour, more than 10 million particles / hour, more than 15 million particles / hour, more than 20 million particles / hour, more than 25 million particles / hour, more than 30 million particles / hour, more than 35 million particles / hour, more than 40 million particles / hour, more than 45 million particles / hour or more than 50 million particles / hour. In a preferred embodiment, the bionanoparticles move through the microfluidic chip at a rate of more than 5 million nanoparticles / hour.

[0350] Device and method for capturing biological nanoparticles

[0351] In some embodiments, the present disclosure provides methods, systems, devices and apparatus for capturing (also referred to as adsorbing), manipulating and analyzing biological nanoparticles on coated planar surfaces. In certain embodiments, capturing biological nanoparticles on a planar surface provides a preferred capture system, at least because analysis can be performed quickly after capture. In contrast to the present disclosure is the use of coated beads, which can be exposed to a sample fluid containing biological nanoparticles and capture biological nanoparticles. Biological nanoparticles captured on beads cannot be easily displayed using, for example, a microscope, so that rapid analysis can be performed. Methods using beads that capture biological nanoparticles often require a step of dissociating or separating the biological nanoparticles from the coated surface, which may reduce the yield of captured biological nanoparticles, or may destroy the biological nanoparticles. The present disclosure provides methods, systems, devices and apparatus for capturing biological nanoparticles in a manner that can be quickly and easily analyzed and / or further processed. In certain embodiments, the present disclosure provides methods for capturing biological nanoparticles of interest from a fluid sample to a planar surface.

[0352] In certain embodiments, the present disclosure provides a method for capturing biological nanoparticles on a planar surface, the method comprising: (a) providing at least one planar surface having a coating; (b) contacting a fluid sample containing a plurality of biological nanoparticles with the coating; (c) providing a force to the fluid sample in contact with the at least one planar surface to promote contact of the biological nanoparticles with the coating; and (d) capturing at least some of the plurality of nanoparticles with the coating.

[0353] In a specific embodiment, the present disclosure provides a method for capturing biological nanoparticles on a coated planar surface, the method comprising (a) providing at least one planar surface having a coating, the coating comprising a nonspecific adsorption resistant material, and a plurality of capture molecules; (b) contacting a fluid sample containing a plurality of biological nanoparticles with the coating; (c) centrifuging the fluid sample in contact with the at least one planar surface to facilitate contact of the biological nanoparticles with the coating; and (d) capturing at least some of the plurality of nanoparticles with at least some of the plurality of capture molecules.

[0354] In other embodiments, the present disclosure provides a method for capturing biological nanoparticles on a coated planar surface, the method comprising (a) providing at least one planar surface having a coating comprising a nonspecific adsorption resistant material, and a plurality of capture molecules; (b) contacting a fluid sample containing a plurality of biological nanoparticles with the coating; (c) centrifuging the fluid sample in contact with the at least one planar surface to facilitate contact of the biological nanoparticles with the coating; (d) capturing at least some of the plurality of nanoparticles with at least some of the plurality of capture molecules; and (e) imaging the plurality of captured biological nanoparticles on the planar surface with a fluorescence microscope.

[0355] The methods, systems, devices and apparatus provided herein can capture biological nanoparticles. Details of biological nanoparticles are further provided herein. In certain embodi...

Claims

1. A method for determining the size of biological nanoparticles in a fluid sample, the method comprising: include: Providing a planar microfluidic chip, the microfluidic chip comprising at least one microfluidic channel; introducing the fluid sample into the microfluidic chip, wherein the fluid sample comprises a plurality of biological nanoparticles; flowing a portion of the plurality of bio-nanoparticles through the at least one microfluidic channel, the at least one microfluidic channel comprising at least one constriction, the detection region being located within the at least one constriction; irradiating at least one biological nanoparticle from the portion of the plurality of biological nanoparticles on a particle-by-particle basis in the at least one microfluidic channel; detecting the intensity of backscattered light or epifluorescence emitted from the at least one biological nanoparticle with a detector in the detection region; and assigning a size value to the at least one illuminated biological nanoparticle based on the detected backscattered light intensity or epifluorescence intensity, wherein biological nanoparticles having a larger hydrodynamic radius generally emit a higher light intensity when compared to biological nanoparticles having a smaller hydrodynamic radius, and The bio-nanoparticles have a hydrodynamic diameter of less than 1 µm. The method of claim 1 , comprising irradiating a portion of the plurality of biological nanoparticles.

3. The method of any one of claims 1-2, comprising irradiating a plurality of irradiated biological nanoparticles on a particle-by-particle basis in the at least one microfluidic channel.

4. The method of any one of claims 1 to 3, wherein the microfluidic chip comprises a plurality of parallel microfluidic channels.

5. The method of claim 4, comprising irradiating the plurality of parallel microfluidic channels with a single radiation source.

6. The method of any one of claims 4-5, further comprising providing a plurality of detectors configured to detect signals from a single microfluidic channel of the plurality of parallel microfluidic channels.

7. The method according to claim 1, in, The detection area is less than 10 µm².

8. The method of claim 1, comprising detecting a portion of the plurality of biological nanoparticles.

9. The method according to any one of claims 1 to 8, in, More than 90% of the detected biological nanoparticles in the portion of the plurality of biological nanoparticles are detected particle by particle within the detection zone.

10. The method according to claim 1, in, The detecting the intensity of backscattered light or epifluorescence emitted from the at least one biological nanoparticle comprises using time binning.

11. The method according to claim 10, in, The time bins range from less than 2 ms to greater than 1 µs.

12. The method according to any one of claims 1 to 11, in, The signal-to-noise ratio of the intensity of light emitted from the at least one biological nanoparticle is greater than 10:

1.

13. The method according to any one of claims 1 to 12, in, Said irradiating said at least one biological nanoparticle comprises using an irradiation source having a beam width less than 2 µm into at least one microfluidic channel and wherein said irradiation source irradiates a detection region having a cross-sectional area less than 10 µm² within said at least one microfluidic channel.

14. The method according to any one of claims 1 to 13, in, Detecting the light intensity includes using a light collection system having a numerical aperture equal to or greater than 1.0 and not greater than 1.

5.

15. The method of claim 1, in, The specifying of the size value comprises specifying the size value when the biological nanoparticle is in a flow state.

16. The method of claim 15, in, The flow is uninterrupted.

17. The method according to any one of claims 1 to 16, in, The bio-nanoparticle is associated with a detectable agent.

18. The method of claim 17, in, The detectable agent is attached to the surface of the biological nanoparticle, the detectable agent is in the surface of the biological nanoparticle, the detectable agent is within the interior of the biological nanoparticle, or a combination thereof.

19. The method according to any one of claims 17 to 18, in, The detectable agent is a fluorescent detectable agent, the detectable agent is a luminescent detectable agent, or any combination thereof.

20. The method according to any one of claims 1 to 19, in, The bio-nanoparticles are conjugated to a variety of detectable agents.

21. The method of claim 20, in, At least one of the plurality of detectable agents is attached to the surface of the biological nanoparticle, at least one of the plurality of detectable agents is in the surface of the biological nanoparticle, at least one of the plurality of detectable agents is within the interior of the biological nanoparticle, or a combination thereof.

22. The method according to any one of claims 20 to 21, in, The plurality of detectable agents have overlapping emission spectra.

23. The method of claim 22, in, The plurality of detectable agents have the same emission profile.

24. The method of claim 22, in, The emission spectra have the same peak wavelength.

25. The method according to any one of claims 20 to 24, in, The plurality of detectable agents comprises the same detectable agent.

26. The method according to any one of claims 20 to 24, in, The plurality of detectable agents includes more than one type of detectable agent.

27. The method of claim 26, in, The plurality of detectable agents have different emission profiles.

28. The method of claim 27, in, The emission spectra have different peak wavelengths.

29. The method of claim 28, in, The peak wavelengths are separated by more than 10 nanometers, more than 20 nanometers, more than 30 nanometers, more than 40 nanometers, more than 50 nanometers, more than 75 nanometers, more than 100 nanometers, more than 120 nanometers, more than 140 nanometers, more than 160 nanometers, more than 180 nanometers, more than 200 nanometers, more than 300 nanometers, more than 400 nanometers, more than 500 nanometers, more than 600 nanometers, or more than 700 nanometers.

30. The method according to any one of claims 20 - 29, wherein, the plurality of detectable reagents includes fluorescent detectable reagents.

31. The method according to any one of claims 17 - 18, wherein, the light intensity is emitted from the detectable reagent.

32. The method according to any one of claims 1 - 30, wherein, the specified size value includes using a modulation index.

33. The method according to claim 32, wherein, the modulation index is an amplitude modulation index.

34. The method according to claim 32, wherein, the modulation index is a frequency modulation index.

35. The method according to claim 32, wherein, the modulation index is a phase modulation index.

36. The method according to any one of claims 1 - 35, wherein, the size value is a relative size value, and the relative size value is determined by the difference in the measured light intensity.

37. The method according to claim 1, wherein the size value is a relative size value, and the method further includes determining the actual size value of the biological nanoparticle by calibrating the measured light intensity relative to a standard with a known size or size distribution.

38. The method according to any one of claims 1 - 34, further including calibrating the light intensity measurement with a standard.

39. The method according to claim 38, wherein, the calibration occurs before introducing the sample containing at least one biological nanoparticle.

40. The method according to any one of claims 38 - 39, wherein, the size value is the actual size value of the biological nanoparticle.

41. The method according to any one of claims 37 - 40, wherein, the standard includes gold nanoparticles.

42. The method according to any one of claims 37 - 41, wherein, the standard includes a variety of lipids.

43. The method according to any one of claims 37 - 42, wherein, the standard includes lipid vesicles.

44. The method according to any one of claims 37 - 43, wherein, the standard includes silica beads, polystyrene beads, fluorescent beads, polymer beads, polymer nanoparticles, or a combination thereof.

45. The method according to any one of claims 37 - 44, wherein, the fluid sample further contains a second biological nanoparticle, and the standard contains the second biological nanoparticle.

46. The method according to any one of claims 37 - 45, wherein, the size of the standard is measured by dynamic light scattering.

47. The method according to claim 1, further including guiding the flow of the biological nanoparticle according to the size value.

48. The method according to claim 47, wherein, The directing directs the biological nanoparticles into a collection, generating a collection of biological nanoparticles based on the size values.

49. The method of any one of claims 47-48, in, The boot time is no more than 1 ms.

50. The method of any one of claims 47 to 49, in, The directing flow includes flow displacement.

51. The method of any one of claims 47 to 50, in, The directed flow includes electroosmotic flow.

52. The method of any one of claims 47 to 51, in, Directing the flow includes applying pressure.

53. The method of any one of claims 1-52, further comprising quantifying the number of biological nanoparticles having said size value.

54. The method of any one of claims 1-53, further comprising determining the concentration of the sample.

55. The method of claim 54, in, The concentration of the sample is determined by counting the peak frequency of the sample.

56. The method of claim 55, further comprising comparing the peak frequency of the sample to a calibration particle standard peak frequency.

57. The method of claim 55, in, The concentration of the sample is determined by counting the peak frequency of the sample and comparing it to the volume of the sample.

58. The method of claim 55, in, The concentration of the sample is determined by counting the peak frequency of the sample, comparing it to the peak frequency of a calibration particle standard, and comparing the peak frequency of the sample to the volume of the sample.

59. The method of any one of claims 1-58, further comprising filtering the sample.

60. The method of claim 59, in, The filtering occurs before the size value is specified.

61. The method of any one of claims 59 to 60, in, The filtering removes debris.

62. The method of any one of claims 59 to 60, in, The filtration prevents clogging.

63. The method of any one of claims 1-62, further comprising collecting the bio-nanoparticles for analysis.

64. The method of claim 63, in, The analysis includes nucleic acid analysis.

65. The method of claim 64, in, The nucleic acid analysis includes sequencing, PCR or digital PCR.

66. The method of any one of claims 63 to 65, in, The analysis includes protein analysis.

67. The method of claim 66, in, The protein analysis includes ELISA, digital ELISA or mass spectrometry.

68. The method of any one of claims 63 to 67, in, The analysis includes lipid analysis.

69. The method of any one of claims 63 to 68, in, The analysis includes small molecule analysis.

70. The method of claim 69, in, The small molecule analysis includes analysis of metabolites, analysis of signal transduction molecules, analysis of drugs or a combination thereof.

71. The method of any one of claims 63 to 70, in, The analysis includes mass spectrometry.

72. The method of any one of claims 63 to 71, in, The analysis includes carbohydrate analysis.

73. The method of claim 72, in, The carbohydrate analysis includes mass spectrometry, nuclear magnetic resonance, Fourier transform spectroscopy or a combination thereof.

74. The method of claim 73, in, The at least one microfluidic channel has a maximum height, a maximum width and / or a maximum cross-sectional area.

75. The method of any one of claims 74, in, The at least one microfluidic channel includes a constriction.

76. The method of claim 75, in, The width of the constriction is less than 25% of the maximum width of the microfluidic channel.

77. The method of any one of claims 75 to 76, in, The height of the constriction is less than 25% of the maximum height of the microfluidic channel.

78. The method of any one of claims 75 to 77, in, The cross-sectional area of ​​the constriction is less than 5% of the maximum cross-sectional area of ​​the microfluidic channel.

79. The method of any one of claims 75 to 78, in, The value of the maximum width is less than 500 µm and greater than 10 µm.

80. The method of any one of claims 75 to 79, in, The value of the maximum height is less than 500 µm and greater than 10 µm.

81. The method of any one of claims 75 to 80, in, The value of the maximum cross-sectional area is less than 250,000 µm² and greater than 250 µm².

82. The method of any one of claims 1 to 81, in, At least a portion of the at least one microfluidic channel has a width less than 10 µm, a width less than 5 µm or a width less than 2 µm.

83. The method of any one of claims 1 to 82, in, At least a portion of the at least one microfluidic channel has a height of less than 10 µm, a height of less than 5 µm or a height of less than 2 µm.

84. The method of any one of claims 1 to 83, in, At least a portion of the at least one microfluidic channel has a cross-sectional area of ​​less than 100 µm², a cross-sectional area of ​​less than 90 µm², a cross-sectional area of ​​less than 80 µm², a cross-sectional area of ​​less than 70 µm², a cross-sectional area of ​​less than 60 µm², a cross-sectional area of ​​less than 50 µm², a cross-sectional area of ​​less than 40 µm², a cross-sectional area of ​​less than 30 µm², a cross-sectional area of ​​less than 20 µm², a cross-sectional area of ​​less than 10 µm², a cross-sectional area of ​​less than 5 µm² or a cross-sectional area of ​​less than 2 µm².

85. The method of any one of claims 1-84, wherein the microfluidic chip comprises a plurality of microfluidic channels intersecting at nodes.

86. The method of claim 85, in, One channel intersects at least three different microfluidic channels at a junction.

87. The method of claim 86, in, The junction has no dead volume.

88. The method according to any one of claims 1-87, wherein the detection of the light intensity from at least one bio-nanoparticle is performed while it is moving through the at least one microfluidic channel.

89. The method according to claim 88, wherein, the detection of the light intensity has single-nanoparticle sensitivity, or the detection of the light intensity has single-molecule sensitivity.

90. The method according to claim 88, wherein, the detection of the light intensity detects a single antibody containing multiple detectable reagents.

91. The method according to any one of claims 1-90, wherein, the bio-nanoparticle contains at least one biomarker.

92. The method according to claim 91, further comprising determining at least one copy number of the at least one biomarker.

93. The method according to any one of claims 1-92, wherein, the specified size value includes using a beam modified by stimulated emission depletion (STED).

94. The method according to claim 1, wherein, the hydrodynamic diameter is less than 900 nm, less than 800 nm, less than 700 nm, less than 600 nm, less than 500 nm, less than 400 nm, less than 300 nm, less than 200 nm, less than 150 nm, less than 100 nm, less than 90 nm, less than 80 nm, less than 70 nm, less than 60 nm, less than 50 nm, less than 40 nm or less than 30 nm.

95. The method according to any one of claims 1-94, wherein, the specified size value includes using a duty cycle detection.

96. The method according to any one of claims 1-95, wherein, the bio-nanoparticle includes extracellular vesicles, organelles, microvesicles, cell-derived vesicles, protein aggregates, nucleic acid aggregates, lipid aggregates, viruses, bacteria or exosomes.

97. The method according to any one of claims 1-96, wherein, the bio-nanoparticles pass through the microfluidic chip at a rate of more than 1 million particles per hour, more than 2 million particles per hour, more than 3 million particles per hour, more than 4 million particles per hour, more than 5 million particles per hour, more than 6 million particles per hour, more than 7 million particles per hour, more than 8 million particles per hour, more than 9 million particles per hour, more than 10 million particles per hour, more than 15 million particles per hour, more than 20 million particles per hour, more than 25 million particles per hour, more than 30 million particles per hour, more than 35 million particles per hour, more than 40 million particles per hour, more than 45 million particles per hour or more than 50 million particles per hour.

98. The method according to any one of claims 1-97, further comprising sorting the at least one nanoparticle into an enriched group.

99. The method according to claim 98, wherein, the sorting includes flow-displacement sorting.

100. The method according to any one of claims 98-99, wherein the sorting does not include acoustic sorting or using a physical barrier.

101. The method of any one of claims 98-100, in, Sorting is determined by size value, presence of a biomarker, detected light intensity, emission wavelength, multiple emission wavelengths, identification of biological nanoparticles, or a combination thereof.

102. The method of any one of claims 98 to 101, in, The diameter variance of the enriched populations is less than 100%.

103. The method of any one of claims 98 to 101, in, The diameter range of the enriched group is: within 500% of the diameter value, within 400% of the diameter value, within 300% of the diameter value, within 200% of the diameter value, within 100% of the diameter value, within 50% of the diameter value or within 25% of the diameter value.

104. The method of any one of claims 98 to 103, in, More than 80% of the bio-nanoparticles in the enriched population were bound to the detectable agent.

105. The method of any one of claims 1 to 104, in, The fluid sample comprises body fluid.

106. The method of claim 105, in, The biological nanoparticles are exosomes.

107. The method of any one of claims 105 to 106, in, The body fluid includes serum, plasma, cerebrospinal fluid or lymphatic fluid.

108. The method of any one of claims 1 to 107, in, The bionanoparticles are isolated.

109. The method of any one of claims 1 to 108, in, The bio-nanoparticle is bound to at least one biomarker.

110. The method of claim 109, further comprising: include: detecting at least one biomarker associated with the biological nanoparticle; and assigning a biomarker value to the biological nanoparticle.

111. The method of claim 110, in, The biomarker values ​​are assigned by a binary system.

112. The method of claim 110, in, The biomarker value is specified by a plurality of values.

113. The method of any one of claims 109 to 112, in, The biomarkers are protein biomarkers.

114. The method of claim 113, in, The protein biomarker is selected from the group consisting of tetraspanins, heat shock proteins, membrane transport proteins, cell adhesion proteins, lipid binding proteins, transmembrane proteins, enzymes, or a combination thereof.

115. The method of any one of claims 109 to 112, in, The biomarkers are nucleic acid biomarkers.

116. The method of claim 115, in, The nucleic acid biomarker is selected from the following group: miR-141, miR-375, miRNA-21, miRNA-1246, miR-320c, miR-6068, miR-9, miR-107, miRNA-128, miRNA134, miRNA-137, miRNA124, miR-21, miR-141, miR-200a, miR-200b, miR-200c, miR-203, miR-205, miR-214; miR-17, miR-3p, miR-21, miR-20b, miR-223, miR-301, miR-141, miR-375, miR-21, miR-1246; miR-21, let-7f, and other Let-7 miRNAs.

117. The method of any one of claims 110-116, further comprising directing the flow of the biological nanoparticles based on the size value, the biomarker value, or a combination thereof.

118. The method of any one of claims 110-117, further comprising sorting the bio-nanoparticles into enriched populations, wherein the sorting is determined by the size value, the biomarker value, or a combination thereof.

119. The method of any one of claims 1 to 118, in, The biological nanoparticle is associated with at least one tag, and the method further comprises: detecting the at least one tag associated with the biological nanoparticle; and assigning a tag value to the biological nanoparticle.

120. The method of claim 119, in, The tag values ​​are specified by a binary system.

121. The method of claim 120, in, The tag value is specified by multiple values.

122. The method of any one of claims 119 to 121, in, The tag is selected from the following group: a signal derived from a protein marker bound to the biological nanoparticle, a signal derived from the size of the biological nanoparticle, a signal derived from a membrane dye bound to the biological nanoparticle, a signal derived from a body dye bound to the biological nanoparticle, a signal derived from a dye bound to the nucleic acid of the biological nanoparticle, a signal derived from a dye bound to the lipid of the biological nanoparticle, and a signal derived from a dye bound to the biological nanoparticle.

123. The method of any one of claims 119-122, further comprising directing the flow of the biological nanoparticles according to the size value and / or the tag value.

124. The method of any one of claims 119-123, further comprising sorting the bio-nanoparticles into enriched populations, wherein the sorting is determined by the size value and / or the tag value.

Images