WO2026006161A1

WO2026006161A1 - System and method for characterizing individual particles

Info

Publication number: WO2026006161A1
Application number: PCT/US2025/034749
Authority: WO
Inventors: Ricardo Vazquez; Stephen Jacobson; Sarah COX-VAZQUEZ; Tanner Young; Ethan Douglas CALL; Brigham POPE
Original assignee: Indiana University; Indiana University Bloomington
Current assignee: Indiana University; Indiana University Bloomington
Priority date: 2024-06-24
Filing date: 2025-06-23
Publication date: 2026-01-02
Anticipated expiration: 2026-12-24

Abstract

A system for characterizing individual particles comprises a solution including an individual particle, a reservoir for receiving the solution, and a constriction fluidly coupled to the reservoir. The system further comprises an excitation source configured to excite the individual particle with light energy. A first detector and a second detector of the system concurrently measure resistive pulse sensing data and photoluminescence lifetime data of the individual particle.

Description

SYSTEM AND METHOD FOR CHARACTERIZING INDIVIDUAL PARTICLES

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This patent application claims the benefit of, and priority to, U.S. Provisional Patent Application Ser. No. 63/663,441 , filed June 24, 2024, the disclosure of which is expressly incorporated herein by reference in its entirety.

TECHNICAL FIELD

[0002] The present disclosure relates generally to systems and methods for characterizing individual particles, including, but not limited to, nanoscale and biological particles.

BACKGROUND

[0003] Biological nanoparticles, such as liposomes, lipid nanoparticles (LNPs), viruses, and extracellular vesicles (EVs), are rapidly emerging due to their biomedical relevancy. Currently, most bioanalytical techniques for the characterization of biological nanoparticles at the single entity level, namely nanoflow cytometry (NFC) and nanoparticle tracking analysis (NTA), couple light scattering measurements with intensity-based photoluminescence methods to inform on such properties. However, concerning light scattering measurements, small differences in relative refractive indices result in challenges for accurately characterizing biological nanoparticles with sizes less than 100 nanometers in diameter. Moreover, intensitybased photoluminescence methods relay minimal information about relevant biological environments at the nanoscale level. In addition, the characterization of heterogeneous samples that have been dye labeled is difficult due to fluorescence spectral overlap. As such, bioanalytical methods that interrogate single biological particles and return information, such as particle size, charge, viscosity, membrane composition, surface composition, particle identity, and phenotype efficiency, would be advantageous. SUMMARY

[0004] The present disclosure may comprise one or more of the features recited in the attached claims, and/or one or more of the following features and combinations thereof.

[0005] In a first aspect, a system for characterizing individual particles may comprise a solution including an individual particle; a reservoir for receiving the solution; a constriction fluidically coupled to the reservoir; at least one voltage source configured to apply a potential across the constriction to cause the individual particle to move through the constriction; an excitation source configured to excite the individual particle in the reservoir or in the constriction with light energy; and a first detector and a second detector configured to concurrently measure resistive pulse sensing data and photoluminescence lifetime data of the individual particle, respectively.

[0006] A second aspect includes the features of the first aspect, and wherein the constriction may include one or more pores.

[0007] A third aspect includes the features of the first aspect or the second aspect, and wherein the constriction may comprise a nanopore.

[0008] A fourth aspect includes the features of any of the first aspect through the third aspect, and wherein the reservoir may be a first reservoir upstream of the constriction, and wherein the system may further comprise a second reservoir downstream of the constriction.

[0009] A fifth aspect includes the features of the fourth aspect, and wherein the first and second reservoirs may be microchannels.

[0010] A sixth aspect includes the features of any of the first aspect through the fifth aspect, and wherein the first detector may include a current amplifier configured to measure current within the constriction.

[0011 ] A seventh aspect includes the features of the sixth aspect, and wherein the second detector may include a microscope configured to measure the photoluminescence lifetime data of the individual particle.

[0012] An eighth aspect includes the features of any of the first aspect through the seventh aspect, and wherein the individual particle may have a first size and the constriction may have a second size that is substantially similar to the first size. [0013] A ninth aspect includes the features of any of the first aspect through the eighth aspect, and wherein the constriction may be a first constriction, and wherein the system may further comprise a second constriction in series with the first constriction.

[0014] A tenth aspect includes the features of the ninth aspect, and wherein the first constriction and the second constriction may each include one or more pores.

[0015] An eleventh aspect includes the features of any of the ninth aspect through the tenth aspect, and wherein the first constriction and the second constriction may each include one or more nanopores.

[0016] A twelfth aspect includes the features of the eleventh aspect, and wherein the individual particle may have a first size and the one or more nanopores of the first constriction and the one or more nanopores of the second constriction may each have a second size that is substantially similar to the first size.

[0017] A thirteenth aspect includes the features of any of the ninth aspect through the twelfth aspect, and wherein the system may further comprise a filter arranged upstream of the first constriction.

[0018] A fourteenth aspect includes the features of any of the ninth aspect through the thirteenth aspect, and wherein the first detector may include a current amplifier configured to measure current within the one or more nanopores of the first constriction and the one or more nanopores of the second constriction, and wherein the second detector may include a microscope configured to measure the photoluminescence lifetime data of the individual particle.

[0019] A fifteenth aspect includes the features of any of the ninth aspect through the fourteenth aspect, and wherein the system may further comprise a pump configured to aid in movement of the individual particle through the system.

[0020] A sixteenth aspect includes the features of any of the ninth aspect through the fifteenth aspect, and wherein the reservoir may be a first reservoir upstream of the first and second constrictions, and wherein the system may further comprise a second reservoir downstream of the first and second constrictions.

[0021] A seventeenth aspect includes the features of the sixteenth aspect, and wherein the first and second reservoirs may be microchannels. [0022] An eighteenth aspect includes the features of any of the first aspect through the eighth aspect, and wherein the constriction may be a first constriction, and wherein the system may further comprise a second constriction in parallel with the first constriction.

[0023] A nineteenth aspect includes the features of the eighteenth aspect, and wherein the first constriction and the second constriction may each include one or more pores.

[0024] A twentieth aspect includes the features of any of the eighteenth aspect through the nineteenth aspect, and wherein the first constriction and the second constriction may each include one or more nanopores.

[0025] A twenty first aspect includes the features of the twentieth aspect, and wherein the individual particle may have a first size and the one or more nanopores of the first constriction and the one or more nanopores of the second constriction may each have a second size that is substantially similar to the first size.

[0026] A twenty second aspect includes the features of any of the eighteenth aspect through the twentieth aspect, and wherein the system may further comprise a filter arranged upstream of the first constriction.

[0027] A twenty third aspect includes the features of any of the eighteenth aspect through the twenty second aspect, and wherein the first detector may include a current amplifier configured to measure current within the one or more nanopores of the first constriction and the one or more nanopores of the second constriction, and wherein the second detector may include a microscope configured to measure the photoluminescence lifetime data of the individual particle.

[0028] A twenty fourth aspect includes the features of any of the eighteenth aspect through the twenty third aspect, and wherein the system may further comprise a pump configured to aid in movement of the individual particle through the system.

[0029] A twenty fifth aspect includes the features of any of the eighteenth aspect through the twenty fourth aspect, and wherein the reservoir may be a first reservoir upstream of the first and second constrictions, and wherein the system may further comprise a second reservoir downstream of the first and second constrictions. [0030] A twenty sixth aspect includes the features of the twenty fifth aspect, and wherein the first and second reservoirs may be microchannels.

[0031 ] A twenty seventh aspect includes the features of any of the first aspect through the twenty sixth aspect, and wherein the individual particle may be a biological particle.

[0032] A twenty eighth aspect includes the features of any of the first aspect through the twenty sixth aspect, and wherein the individual particle may be inorganic. [0033] A twenty ninth aspect includes the features of any of the first aspect through the twenty eighth aspect, and wherein the resistive pulse sensing data related to the individual particle may be used to determine physical properties of the individual particle, and wherein the physical properties may include one or more of a particle size, a surface charge, and a particle shape.

[0034] A thirtieth aspect includes the features of the twenty ninth aspect, and wherein the photoluminescence lifetime data related to the individual particle may be used to determine photophysical properties of the individual particle, and wherein the photophysical properties may include one or more of a membrane viscosity, a membrane polarity, and a membrane composition.

[0035] In a thirty first aspect, a method for characterizing individual biological particles may comprise inputting a solution comprising an individual particle into a reservoir; applying a potential across a constriction to cause the individual particle to move through the constriction from the reservoir; exciting the individual particle in the reservoir or in the constriction with light energy; and concurrently measuring resistive pulse sensing data and photoluminescence lifetime data of the individual particle.

[0036] A thirty second aspect includes the features of the thirty first aspect, and wherein the method may further comprise, based on the resistive pulse sensing data, determining one or more of a particle size, a surface charge, and a particle shape of the individual particle.

[0037] A thirty third aspect includes the features of the thirty first aspect or the thirty second aspect, and wherein the method may further comprise, based on the photoluminescence lifetime data, determining one or more of a membrane viscosity, a membrane polarity, and a membrane composition of the individual particle. [0038] A thirty fourth aspect includes the features of any of the thirty first aspect through the thirty third aspect, and wherein the steps of determining may occur concurrently.

BRIEF DESCRIPTION OF THE DRAWINGS

[0039] FIG. 1 A represents intensity data from traditional confocal microscopy;

[0040] FIG. 1 B represents intensity and local microenvironment data collected by what is conventionally named fluorescence lifetime imaging microscopy (FLIM);

[0041] FIG. 1C shows a system for resistive-pulse sensing (RPS) measurements and photoluminescence lifetime measurements;

[0042] FIG. 2A shows another system for resistive-pulse sensing (RPS) measurements and photoluminescence lifetime measurements, the system having two V-shaped microchannels bridged by a nanofilter and a series of nanochannels and nanopores;

[0043] FIG. 2B is a scanning electron microscope (SEM) image of the system shown in FIG. 2A showing the nanofilter placed upstream of the two nanopores in series for RPS measurements and a detection region for acquiring FLIM measurements;

[0044] FIG. 3A is a scatter plot of photoluminescence lifetimes and corresponding photon counts for liposomes (50 nanometers) intercalated with a first dye showing that measurement precision of the photoluminescence lifetime improves with the number of photons collected;

[0045] FIG. 3B is a scatter plot of relative volumes and photon counts for liposomes (50 nanometers) intercalated with the first dye showing that particle size (Ai/i) and number of photons detected by the FLI detector are not correlated;

[0046] FIG. 4A is a current trace from RPS measurements and the correlated photoluminescence intensity from FLIM measurements of three liposomes (100 nanometers) composed of POPC and POPG lipids translocating through the detection region on a two-pore system;

[0047] FIG. 4B is an expanded view of the current trace of FIG. 4A of a single liposome translocating through two pores in series and producing a two-pulse sequence; [0048] FIG. 5A shows distributions of photoluminescence lifetimes of individual liposomes (100 nanometers) intercalated with a first dye with 0%, 15%, and 50% mol cholesterol and measured with RPS-FLIM;

[0049] FIG. 5B shows photoluminescence decay plots of liposomes (100 nanometers) intercalated with a first dye with 0%, 15%, and 50% mol cholesterol;

[0050] FIG. 6A is a scatter plot of photoluminescence lifetimes of liposomes (100 nanometers) intercalated with a first dye with 0%, 15%, 30%, and 50% mol cholesterol, showing that lifetimes increase with increasing concentration of cholesterol;

[0051] FIG. 6B is a scatter plot of photoluminescence lifetimes and relative volumes of liposomes (100 nanometers) intercalated with a first dye with 0%, 15%, and 50% mol cholesterol;

[0052] FIG. 7A shows photoluminescence lifetimes of liposomes (100 nanometers) intercalated with a first dye with 0%, 15%, 30%, and 50% mol cholesterol;

[0053] FIG. 7B shows photoluminescence intensities of the liposomes of FIG. 7A showing that longer photoluminescence lifetimes of a first dye in FIG. 7A coincide with increased photoluminescence intensities in FIG. 7B for increasing cholesterol concentration;

[0054] FIG. 8A shows distributions from RPS experiments of liposomes intercalated with a first dye (50 nanometers) and a second dye (100 nanometers) and two polystyrene standards (70 nanometer and 100 nanometer diameters);

[0055] FIG. 8B shows negative stain transmission electron microscopy (TEM) images of liposomes extruded through membranes with 50 nanometer pores with measured diameters of 75 ± 25 nanometers;

[0056] FIG. 8C shows negative stain TEM images of liposomes extruded through membranes with 100 nanometer pores with measured diameters of 90 ± 39 nanometers;

[0057] FIG. 9A shows bulk photoluminescence lifetime measurements of liposomes intercalated with a first dye or a second dye with 15% mol cholesterol and mixtures of those liposomes at excitation wavelengths of 475 nanometers, 525 nanometers, and 575 nanometers; [0058] FIG. 9B shows the photoluminescence spectra of the liposome samples of FIG. 9A;

[0059] FIG. 10A shows photoluminescence lifetime decay curves of a second dye or a first dye intercalated liposome measured with RPS-FLIM;

[0060] FIG. 10B is a phasor plot of a mixture of liposomes (100 nanometers) intercalated with a second dye or a first dye with lifetimes of 0.9 nanoseconds and 2.6 nanoseconds, respectively;

[0061] FIG. 10C shows distributions of photoluminescence lifetimes of liposomes intercalated with a second dye or a first dye and a mixture of the liposomes and detected in the detection region;

[0062] FIG. 10D shows distributions of the relative pulse amplitude (A///) of liposomes intercalated with a second dye or a first dye and a mixture of the liposomes compared to 70 nanometer and 100 nanometer polystyrene sphere standards;

[0063] FIG. 10E is a scatter plot of the size measurement (A/77) and photoluminescence lifetimes of liposomes intercalated with a second dye or a first dye and a mixture of the liposomes;

[0064] FIG. 11 A shows distributions of dwell times for liposomes intercalated with a first dye or a second dye and a mixture of those liposomes;

[0065] FIG. 11 B shows a scatter plot of dwell times and photoluminescence lifetimes of liposomes intercalated with a first dye or a second dye and a mixture of those liposomes;

[0066] FIG. 12A shows a schematic of another system with two V-shaped microchannels connected by an array of parallel nanochannels;

[0067] FIG. 12B shows a scanning electron microscope (SEM) image of the four nanochannels in parallel;

[0068] FIG. 12C shows a distribution of photoluminescence lifetimes of a mixture of liposomes intercalated with a second dye or a first dye detected within the nanochannel array of FIG. 12A with photoluminescence lifetimes of 0.95 ± 0.8 nanoseconds and 2.6 ± 0.3 nanoseconds, respectively;

[0069] FIG. 12D is an image from FLIM of liposomes intercalated with a second dye or a first dye flowing through the nanochannel array of FIG. 12A; [0070] FIG. 13A shows a schematic of another system with three sample microchannels that are each connected to a respective array of parallel nanochannels;

[0071] FIG. 13B shows F LI M images of four liposomes migrating through one of the arrays of parallel nanochannels of FIG. 13A;

[0072] FIG. 14A shows a two-dimensional plot of fluorescence lifetimes and particle velocities of liposomes with 10%, 30%, and 50% cholesterol and intercalated with Di8 and liposomes with 30% cholesterol and intercalated with COE;

[0073] FIG. 14B shows a two-dimensional plot of fluorescence lifetimes and particle velocities of a mixture of Di8-intercalated liposomes with 30% cholesterol and COE-intercalated liposomes with 30% cholesterol;

[0074] FIG. 15A shows distributions of fluorescence lifetimes for Di8- intercalated liposomes with 30% cholesterol;

[0075] FIG. 15B shows histograms of velocities for Di8-intercalated liposomes with 30% cholesterol at applied potentials of 0.2, 0.5, 0.7, and 1 .0 V;

[0076] FIG. 16A shows distributions of fluorescence lifetimes of COE- and Di8-intercalated liposomes mixed in particle concentration ratios of 4:1 and 7:13 (COE:Di8); and

[0077] FIG 16B shows bulk measurements of the fluorescence lifetimes of COE- and Di8-intercalated liposomes mixed in particle concentration ratios of 4:1 and 7:13 (COE:Di8) and measured with an excitation wavelength of 525 nanometers.

DETAILED DESCRIPTION OF THE DRAWINGS

[0078] For the purposes of promoting an understanding of the principles of the disclosure, reference will now be made to a number of illustrative embodiments illustrated in the drawings and specific language will be used to describe the same.

[0079] The present disclosure combines resistive pulse sensing (RPS) with photoluminescence lifetime imaging microscopy for single-entity particle characterization and analysis. Photoluminescence lifetime imaging microscopy includes fluorescence lifetime imaging microscopy (FLIM). The particle may be an organic particle, an inorganic particle, or a biological particle. The particle may be a microparticle or a nanoparticle. The nanoparticle may have a diameter of about 100 nanometers or less than about 100 nanometers. Although fluorescence lifetime imaging microscopy (FLIM) measurements are described herein, other time-resolved spectroscopic techniques are contemplated. Microfluidics and nanofluidics can temporarily isolate particles smaller than 1 micrometer. FLIM can use the photoluminescence lifetime of a dye to extract information about the local microenvironment of the particle.

[0080] An effective method for characterizing particles, such as biological nanoparticles, at the nanoscale level is RPS. RPS can extract information on a particle size and a surface charge of the biological nanoparticles. RPS is a singleparticle technique that provides precise information about the particle size or volume based on a change in ionic current (Ai) and information about the particle shape based on a pulse duration (td) and/or a shape of a current trace. RPS devices may be designed and fabricated in a plane of a substrate to facilitate the integration of multiple functions and coupling of optical techniques. Having multiple nanopores in series improves the precision of the particle size measurement, and the electrophoretic mobility and surface-charge density (or ^-potential) of particles are calculated from the pulse duration (td), migration time between nanopores, pore-to- pore time (t_PP), or a combination of the same.

[0081] FLIM produces an image based on the differences in the photoluminescence lifetimes of dyes. As a result, photoluminescence lifetimes can be used to extract biological information at the molecular level concerning micro and nanoenvironments, often unattainable for intensity-dependent photoluminescence methods (see FIGS. 1A and 1 B). FIGS. 1 A and 1 B show data visualization comparison between confocal microscopy (FIG. 1A) and FLIM (FIG. 1 B). Traditional confocal microscopy provides information only on intensity, whereas FLIM provides information regarding both intensity and local microenvironments based on variations in photoluminescence lifetimes of associated fluorophores. Note that images similar to FIG. 1 B can be generated with any emissive lifetime and/or photoluminescence lifetime process.

[0082] By implementing a fluorescent dye with a lifetime responsive to local microenvironments such as viscosity, polarity, rigidity, pH, fluidity, and chemical composition, FLIM produces images reporting differences in these micro and nanoenvironments. For instance, subtle changes in the photoluminescence lifetime show a steady increase in cholesterol-dependent membrane fluidity in cancer cells during treatment. Additionally, FLIM experiments have tracked membrane lipid modification upon treatment and monitored cholesterol-dependent membrane viscosity in differentiating stem cells. FLIM measurements using an environmentally sensitive dye, such as Di8 (i.e. , a first dye), may characterize changes in cholesterol concentration due to the impact on the viscosity of the lipid bilayers. In another example, photoluminescence lifetimes of dyes confined in nanochannels shift as the channel dimensions decrease due to lower dielectric constants and higher viscosities in the nanochannels relative to the microchannels.

[0083] RPS-FLIM systems can give information about the size, the shape, the surface charge (or (-potential), and the local microenvironment concurrently. In this way, characterization of individual particles or biological nanoparticles can be significantly improved. RPS-FLIM systems allow for the differentiation of liposomes, among other vesicles or inorganic particles, at the single particle level. RPS measures the particle size or volume, the particle shape, and the particle surfacecharge density. FLIM determines the photoluminescence lifetime of the fluorophore associated with the lipid membrane.

[0084] A system 10 for characterizing at least one individual particle is disclosed herein and shown in FIG. 1C. The system 10 includes a solution 12 comprising at least one individual particle and a reservoir 14 for receiving the solution 12 therein. The system 10 further includes a constriction 20 fluidically coupled to the reservoir 14 to receive the individual particle therein and at least one voltage source 36 configured to apply an electric potential across the constriction 20 to cause the individual particle to move from the reservoir 14 and through the constriction 20. The system 10 further includes an excitation source 42, a first detector 38, and a second detector 40, as shown in FIG. 1C. In some embodiments, the excitation source 42 is included in the second detector 40. The excitation source 42 is configured to excite the individual particle in the reservoir 14 or in the constriction 20 with light energy. The first detector 38 and the second detector 40 concurrently measure resistive pulse sensing data and photoluminescence lifetime data of the individual particle, respectively. The photoluminescence lifetime data may illustratively be or include, but is not limited to, photoluminescence lifetime data and/or phosphorescence lifetime data. The term “concurrently” refers to occurring within the boundaries of the system 10. In some embodiments, the second detector 40 also concurrently measures light scattering data, which may be used to determine particle size. Particle size can be obtained from particles traversing the constriction 20 by collecting scattered light. This is accomplished by overlapping the excitation laser wavelength with the emission detection range, enabling the reflected light from the particles to be captured and analyzed as the particles move through the field of view of the second detector 40. The scattered light may be backscattered reflected light or forward scattered reflected light.

[0085] In some embodiments, the first detector 38 is a current amplifier 38 configured to measure a current pulse as the individual particle passes through the constriction 20. In illustrative embodiments, the resistive pulse sensing data is measured by the first detector 38 while the individual particle is passing through the constriction 20. In some embodiments, the second detector 40 is a microscope 40. The constriction 20 helps to keep the individual particle within the focus of the microscope 40. In illustrative embodiments, the photoluminescence lifetime data is measured by the second detector 40 anywhere in the system 10 downstream of a portion of the reservoir 14. In other words, the photoluminescence lifetime data is measured by the second detector 40 while the individual particle is in the reservoir 14 or in the constriction 20.

[0086] In some embodiments, the system 10 further includes a pump 44 configured to aid in movement of the individual particle through the constriction 20, as shown in FIG. 1C. In some embodiments, the system 10 includes the reservoir 14 as a first reservoir 14 and a second reservoir 16 downstream of the first reservoir 14 and the constriction 20, as shown in FIG. 1C. The reservoirs 14, 16 may be channels, microchannels, vessels, beakers, or any other structure configured to hold the solution 12.

[0087] The individual particle may be an organic particle, a biological particle, or an inorganic particle. In illustrative embodiments, the individual particle has a first size (i.e. , a first diameter), and the constriction 20 has a second size (i.e., a second diameter) that is substantially similar to the first size. In this way, a single particle passes through the constriction 20 at a time. In some embodiments, the system 10 includes a plurality of constrictions, as shown in FIG. 2A and described in more detail below.

[0088] The resistive pulse sensing data measured by the first detector 38 is used to determine the physical properties of the individual particle, such as, but not limited to, a particle size, a surface charge, and a particle shape. The photoluminescence lifetime data measured by the second detector 40 is used to determine the photophysical properties or other identification properties of the individual particle, such as, but not limited to, membrane viscosity, membrane polarity, and membrane composition. The other identification properties may include the presence or absence of a biomolecule and/or properties of inorganic or organic compounds. For example, biomarkers may be identified using antibodies or nucleic acid may be identified using dyes. Any of the physical properties may be determined concurrently with the photophysical properties or the identification properties. In some embodiments, the system 10 includes a processor 24 having a memory 26, as shown in FIG. 1C. The memory 26 has instructions stored therein which, when executed by the processor 24, cause the processor 24 to control the at least one voltage source 36 to apply the electric potential across the constriction 20 and to control the first detector 38 to measure the current passing through the constriction 20. The processor 24 is electrically connected to the first detector 38 and the second detector 40 so as to receive the data therefrom. The processor 24 receives such data and stores the data in the memory 26. In some embodiments, the processor 24 is coupled to a display monitor, a printer, and/or an other output device. The memory 26 has instructions stored therein which, when executed by the processor 24, cause the processor 24 to control one or more of the display monitor, the printer, and/or the other output device to display and/or record analyses of the stored data. The data is processed by the processor 24 to determine any of the particle size, the surface charge, the particle shape, the membrane viscosity, the membrane polarity, the membrane fluidity, the membrane composition, or any combination of the same.

[0089] Another system 110 for characterizing individual particles is disclosed herein. The system 110 includes a solution 112 comprising at least one individual particle, a first microchannel 114 configured to receive the solution 112 therein, and a second microchannel 116 spaced apart from the first microchannel 114, as shown in FIG. 2A. The microchannels 114, 116 may be referred to as reservoirs. The system 110 further includes a plurality of nanochannels 118 arranged between the first microchannel 114 and the second microchannel 116 and a plurality of nanopores 120 arranged between the first microchannel 114 and the second microchannel 116. The nanochannels 118 and the nanopores 120 may be referred to as constrictions.

[0090] In response to an electric potential being applied between the first microchannel 114 and the second microchannel 116 via at least one voltage source 136, the individual particle moves from the first microchannel 114 and through the plurality of nanochannels 118 and the plurality of nanopores 120 toward the second microchannel 116. The rate at which the individual particle passes through the plurality of nanopores 120 may be increased with applied pressure via, for example, a pump or vacuum (such as the pump 44). While the individual particle is moving toward the second microchannel 116, resistive pulse sensing data and photoluminescence lifetime data are concurrently measured so that a current pulse related to the individual particle and a photoluminescence lifetime related to the individual particle are determined. Though shown and described with the individual particle being transported with an electric potential (i.e. , electrokinetically), particles can be transported with pressure-driven flow via the pump, electrokinetically via the at least one voltage source 136, or in combination.

[0091 ] The system 110 includes a first detector 138 for measuring the resistive pulse sensing data and a second detector 140 for measuring the photoluminescence lifetime data. In some embodiments, the second detector 140 also concurrently measures light scattering data, which may be used to determine particle size. Particle size can be obtained from particles traversing the plurality of nanochannels 118 and the plurality of nanopores 120 by collecting scattered light. This is accomplished by overlapping the excitation laser wavelength with the emission detection range, enabling the reflected light from the particles to be captured and analyzed as the particles move through the field of view of the second detector 140. The scattered light may be backscattered reflected light or forward scattered reflected light. The system 110 also includes an excitation source 142 that excites the individual particle in the system 110. The excitation source 142 may be a part of the second detector 140.

[0092] The resistive pulse sensing data is measured in each of the plurality of nanopores 120, and the photoluminescence lifetime data may be measured anywhere in the system 110, such as in the first microchannel 114, in the plurality of nanochannels 118, in the plurality of nanopores 120, and/or in the second microchannel 116.

[0093] In some embodiments, the individual particle is a nanoparticle. In some embodiments, the individual particle is a liposome.

[0094] In some embodiments, the system 110 further comprises a nanofilter 122 arranged between the first microchannel 114 and the second microchannel 116 and upstream of the plurality of nanopores 120 and the plurality of nanochannels 118, as shown in FIGS. 2A and 2B. The nanofilter 122 is configured to prevent debris or aggregates in the solution 112 from entering the plurality of nanochannels 118 from the first microchannel 114.

[0095] In some embodiments, the plurality of nanochannels 118 includes a first nanochannel 118A adjacent to the first microchannel 114, a second nanochannel 118B spaced apart from the first nanochannel 118A, and a third nanochannel 118C spaced apart from the second nanochannel 118B and adjacent the second microchannel 116. Though shown and described with three nanochannels 118, any number of nanochannels 118 is contemplated. In some embodiments, cross-sectional areas of each of the plurality of nanochannels 118 are different to effect different particle velocities within the plurality of nanochannels 118. In some embodiments, a first cross-sectional area of the first nanochannel 118A is less than a third cross-sectional area of the third nanochannel 118C. The third cross- sectional area of the third nanochannel 118C is illustratively larger than the other cross-sectional areas of the first and second nanochannels 118A, 118B to reduce particle velocity and, therefore, increase the number of photons collected by FLIM to improve the precision of the lifetime measurements (see FIG. 3A). FIG. 3A is a scatter plot of the lifetime and photon counts for liposomes (50 nanometers) intercalated with Di8. Measurement precision of the photoluminescence lifetimes improves with the number of photons collected. Though shown and described with nanochannels 118 of fixed or constant dimensions, nanochannels 118 of any dimensions are contemplated.

[0096] FIG. 3B is a scatter plot of the relative volume and photon counts for liposomes (50 nanometers) intercalated with Di8. Particle size (Ai/i) and number of photons detected in the photoluminescence lifetime imaging microscopy detection region 113 are not correlated.

[0097] In some embodiments, the plurality of nanopores 120 includes a first nanopore 120A arranged between the first nanochannel 118A and the second nanochannel 118B and a second nanopore 120B arranged between the second nanochannel 118B and the third nanochannel 118C. The nanopores 120A, 120B are arranged in series. Though shown and described with two nanopores 120, any number of nanopores 120 is contemplated. Though shown and described with nanopores 120 of fixed or constant dimensions, nanopores 120 of any dimensions are contemplated.

[0098] The resistive pulse sensing data (i.e., the current pulse) related to the individual particle is used to determine a particle size, a surface charge, and/or a particle shape of the individual particle. The particle size is related to an amplitude of the current pulse (Ai), as shown in FIG. 4B. The surface charge is related to a time between two current pulses (pore-to-pore time, t_PP), as shown in FIG. 4B. The particle shape and the surface charge are related to a time duration (td) of the current pulse, as shown in FIG. 4B. The particle shape can also be deduced from oscillations in the amplitude, not depicted.

[0099] The photoluminescence lifetime related to the individual particle is used to determine a membrane viscosity, a membrane polarity, and/or a membrane composition of the individual particle. As such, differentiation in a complex mixture comprising a plurality of individual particles can be achieved. The membrane viscosity refers to a viscosity of a membrane or to changes between liquid order or liquid disorder phases, which could determine differences in diffusion through the membrane. Membrane polarity refers to a potential difference across the membrane. Membrane composition refers to proportions of proteins, lipids, carbohydrates, or any combination thereof in the membrane. [00100] The determinations related to the current pulse (e.g., particle size, surface charge, particle shape) occur concurrently with the determinations related to the photoluminescence lifetime (e.g., membrane viscosity, membrane polarity, membrane composition), as defined above.

[00101] In some embodiments, the system 110 for characterizing individual particles includes a resistive pulse sensing device 111 configured to receive the solution 112 therein and a photoluminescence lifetime imaging microscopy detection region 113 integrated with the resistive pulse sensing device 111 , as shown in FIGS. 2A and 2B. In some embodiments, the photoluminescence lifetime imaging microscopy detection region 113 is and/or begins downstream of the first nanopore 120A and/or between the plurality of nanopores 120A, 120B included in the resistive pulse sensing device 111 , as shown in FIG. 2B. In some embodiments, the photoluminescence lifetime imaging microscopy detection region 113 begins in the second nanochannel 11 SB, as shown in FIG. 2B. In some embodiments, the photoluminescence lifetime imaging microscopy detection region 113 begins in the first microchannel 114.

[00102] In some embodiments, the system 110 includes a processor 124 having a memory 126, as shown in FIG. 2A. The memory 126 has instructions stored therein which, when executed by the processor 124, cause the processor 124 to control the at least one voltage source 136 to apply the electric potential between the first microchannel 114 and the second microchannel 116 and to control the first detector 138 to measure the current passing through the plurality of nanopores 120. The processor 124 is electrically connected to the first detector 138 and the second detector 140 so as to receive the current pulse data and the photoluminescence lifetime data therefrom. The processor 124 receives such data and stores the data in the memory 126. In some embodiments, the processor 124 is coupled to a display monitor, a printer, and/or an other output device. The memory 126 has instructions stored therein which, when executed by the processor 124, cause the processor 124 to control one or more of the display monitor, the printer, and/or the other output device to display and/or record analyses of the stored data. The data is processed by the processor 124 to determine any of the particle size, the surface charge, the particle shape, the membrane viscosity, the membrane polarity, the membrane fluidity, the membrane composition, or any combination.

[00103] The RPS-FLIM system 10, 110 for RPS-FLIM measurements is designed and fabricated in the plane of the substrate to conduct resistive-pulse and photoluminescence lifetime measurements of individual dye-intercalated liposomes simultaneously. The solution 112, which contains the at least one individual particle (e.g., liposomes (LPs)), is loaded into reservoirs 128, 130, and an electric potential is applied between the reservoirs 128, 130 and buffer reservoirs 132, 134. FIG. 2B shows a scanning electron microscope (SEM) image of the photoluminescence lifetime imaging microscopy detection region 113 for acquiring FLIM measurements with the nanofilter 122 placed upstream of the plurality of nanopores 120A, 120B in series for RPS measurements.

[00104] A method for characterizing individual particles is also provided herein. The method includes inputting the solution 112 comprising at least one individual particle into the first microchannel 114. The method includes applying the electric potential between the first microchannel 114 and the second microchannel 116 via the at least one voltage source 136. The method includes, in response to the electric potential, moving the individual particle from the first microchannel 114, through the plurality of nanochannels 118 and the plurality of nanopores 120, and into the second microchannel 116. The method includes, in response to the individual particle passing through the plurality of nanopores 120 toward the second microchannel 116, concurrently measuring resistive pulse sensing data of the individual particle and photoluminescence lifetime data of the individual particle.

[00105] The method includes, based on the resistive pulse sensing data, determining one or more of a particle size, a surface charge, and a particle shape of the individual particle. The method includes, based on the photoluminescence lifetime data, determining one or more of a membrane viscosity, a membrane polarity, and a membrane composition of the individual particle. The steps of determining occur concurrently.

[00106] In some embodiments, the step of moving the individual particle includes moving the individual particle from the first microchannel 114, through the first nanochannel 118A adjacent the first microchannel 114, through the first nanopore 120A adjacent the first nanochannel 118A, through the second nanochannel 118B adjacent the first nanopore 120A, through the second nanopore 120B adjacent the second nanochannel 118B, through the third nanochannel 118C adjacent the second nanopore 120B, and into the second microchannel 116, as suggested in FIG. 2B.

[00107] In some embodiments, the resistive pulse sensing device 111 is fabricated in-plane on a glass substrate to facilitate coupling of the resistive pulse sensing device 111 with the photoluminescence lifetime imaging microscopy detection region 113. In some embodiments, the RPS-FLIM system 110 includes a microfluidic device with nanopores coupled to an RPS setup on a Leica Stellaris 8 FLIM on a 63X objective.

[00108] In some embodiments, biological particles, such as liposomes, containing various cholesterol concentrations with membrane-intercalated Di8 (i.e., a first dye) may be inserted into the system 10, 110. In other words, the solution 12, 112 may comprise biological particles and a plurality of dyes. With the system 10, 110, increasing cholesterol concentrations in the liposomes from 0% to 50% increases the photoluminescence lifetimes from 2.1 ± 0.4 nanoseconds to 3.4 ± 0.5 nanoseconds, respectively. Moreover, the RPS-FLIM system 10, 110 may discern liposome populations with the same cholesterol concentration labeled with dyes with photoluminescence spectral overlap but that have different photoluminescence lifetimes (Di8 and COE-S6, a first dye and a second dye, respectively). Thus, the RPS-FLIM system 10, 110 may parse two particle populations with statistically identical volumes, cholesterol concentration, and lipid composition, but different fluorophores.

[00109] As an example, liposomes composed of a 3:1 ratio of 1 -palmitoyl-2- oleoyl-glycero-3-phosphocholine (POPC) to 1-palmitoyl-2-oleoylsn-glycero-3- phospho-(T-rac-glycerol) (POPG), different cholesterol concentrations, and 1% membrane-intercalated dye (Di8 and COE-S6) were characterized by the RPS-FLIM system 10, 110. The RPS-FLIM system 10, 110 distinguished liposomes based on different cholesterol concentrations due to the photoluminescence lifetime sensitivity of Di8 to membrane environments. RPS-FLIM measurements also differentiated photoluminescence lifetimes of liposomes labeled with Di8 or COE and mixtures of those liposomes. The inability of bulk measurements to accurately characterize complex mixtures highlights the advantage of the RPS-FLIM system 10, 110 for single-entity biological characterization, which concurrently reports changes in particle size and membrane composition.

[00110] To correlate the FLIM signal detected by the second detector 40, 140 with the resistive-pulse signal detected by the first detector 38, 138, a TTL pulse may be sent from software of the second detector 40, 140 to a digitizer of the first detector 38, 138 for a current amplifier signal during the first frame, and subsequent pulses may be sent every 320 frames (or about every 12 seconds). After the first frame is recorded with the second detector 40, 140 and the TTL pulse is recorded with the current amplifier software of the first detector 38, 138, the lifetime and current signals are aligned. FIG. 4A shows the alignment of a series of three current pulses, and photoluminescence bursts from about 1 second of data of three liposomes (100 nanometers) composed of POPC and POPG lipids translocating through the detection region in a two-nanopore system 10, 110. The current pulses from the RPS data and the photoluminescence bursts from the photoluminescence lifetime data are correlated. FIG. 4B shows an expanded view of the current trace from the dashed box in FIG. 4A of a single liposome translocating through two nanopores in series and producing a two-pulse sequence. Labeled in FIG. 4B are the current pulse amplitude or size (Ai), pore-to-pore time (t_PP), and dwell time (td). For particle size distributions, the pulse amplitude (Ai) is divided by the baseline current (i; not labeled) to normalize data across systems 10, 110. To be considered a correlated event, a minimum threshold of 100 photons per frame for the photoluminescence signal was set. Across all experiments, the degree of correlation of resistive-pulse and FLIM signals ranged from 45% to 85% of recorded events. At lower applied potentials, e.g., 0.5 V, the degree of correlation is closer to 85% because the particles traveled more slowly through the photoluminescence lifetime imaging microscopy detection region 113, which allowed more photons to be collected from individual particles. Also, the scan region of the second detector 40 was set to about 50 pm² compared to about 10 pm² for the photoluminescence lifetime imaging microscopy detection region 113 on the nanofluidic device (FIG. 2B), which led to a low duty cycle of 20% over the detection region and loss of collected photons.

[00111] One advantage of sensing photoluminescence lifetimes versus photoluminescence intensity (see FIGS. 1 A and 1 B) is the ability to detect subtle differences in local molecular environments, regardless of dye concentration. One biomolecule of interest is cholesterol, which plays a role in modulating physico- electrochemical properties in membranes and is associated with cellular communication, signaling, dysfunction, and trafficking. There is a positive correlation between the photoluminescence lifetime of Di8 and cholesterol concentration in membranes. With a similar experimental approach, liposomes comprised of a 3:1 ratio of POPC:POPG with 0% to 50% mol cholesterol and with 1 % mol dye intercalated into the lipid bilayer were extruded through membranes with pore diameters of 50 nanometers and 100 nanometers and characterized with the RPS- FLIM system 10, 110.

[00112] Results revealed distributions with a positive correlation between cholesterol concentration and the photoluminescence lifetime of the Di8-intercalated liposomes. For example, when cholesterol concentrations were 0%, 15%, and 50%, the lifetimes shifted to averages of 2.1 ± 0.4, 2.5 ± 0.4, and 3.4 ± 0.5 nanoseconds, respectively (FIGS. 5A and 6A).

[00113] FIG. 5A shows distributions of photoluminescence lifetimes of individual liposomes (100 nanometers) intercalated with Di8 with 0%, 15%, and 50% mol cholesterol and measured with the RPS-FLIM system 10, 110. Average photoluminescence lifetimes of Di8 were 2.1 ± 0.4, 2.5 ± 0.4, and 3.4 ± 0.5 nanoseconds for 0%, 15%, and 50% mol cholesterol, respectively. FIG. 5B shows photoluminescence decay plots of liposomes (100 nanometers) intercalated with Di8 with 0%, 15%, and 50% mol cholesterol. The lifetimes for the liposomes were 2.22 nanoseconds, 2.54 nanoseconds, and 3.30 nanoseconds, respectively.

[00114] FIG. 6A is a scatter plot of photoluminescence lifetimes of liposomes (100 nanometers) intercalated with Di8 with 0%, 15%, 30%, and 50% mol cholesterol. Lifetimes increase with increasing concentration of cholesterol. FIG. 6B is a scatter plot of photoluminescence lifetimes and relative volumes of liposomes (100 nanometers) intercalated with Di8 with 0%, 15%, and 50% mol cholesterol. The average lifetimes and counts for 0%, 15%, 30%, and 50% mol cholesterol were 2.1 ± 0.4 nanoseconds, 2.5 ± 0.4 nanoseconds, 3.1 ± 0.6 nanoseconds, and 3.4 ± 0.5 nanoseconds and 1239, 2011 , 573, and 859, respectively.

[00115] The increase in photoluminescence lifetimes upon increasing cholesterol concentrations is visualized with images of individual liposomes for each condition and the corresponding photoluminescence decay curve (see FIG. 5B). The trend of increasing photoluminescence lifetimes of Di8 with increased cholesterol levels can be explained by a reduction in membrane fluidity. Additionally, a hypsochromic shift reduces the full-width at half maximum (FWHM) in the photoluminescence spectra of Di8 (see FIG. 7B). In all cases, RPS-FLIM measured particle sizes from about 70 nanometers to about 140 nanometers in diameter for liposomes extruded with the 100 nanometers membrane and about 50 nanometers to about 100 nanometers in diameter for the liposomes extruded with the 50 nanometers membrane (see FIG. 8A). These values were further validated with TEM, which revealed a size of 90 ± 39 nanometers for the 100 nanometers extruded liposomes and 75 ± 25 nanometers for the 50 nanometers extruded liposomes (FIGS. 8B and 8C). Measurements from the TEM and RPS had comparably broad distributions for the particle sizes.

[00116] FIG. 7A shows bulk photoluminescence lifetimes of liposomes (100 nanometers) intercalated with Di8 with 0%, 15%, 30%, and 50% mol cholesterol and FIG. 7B shows the liposomes’ respective photoluminescence intensities. Longer photoluminescence lifetimes of Di8 in FIG. 7A coincides with increased photoluminescence intensities in FIG. 7B for increasing cholesterol concentration. In addition, a blue shift (hypsochromic) is concomitant with a reduction in the full-width at half-maximum (FWHM) obtained when cholesterol concentration is increased. IRF in FIG. 7A is the instrument response function.

[00117] Distributions from RPS experiments of liposomes intercalated with Di8 (50 nanometers) and COE (100 nanometers) and two polystyrene standards (70 and 100 nanometers diameters) are shown in FIG. 8A. Negative stain TEM images of liposomes extruded through membranes with 50 nanometers pores (FIG. 8B) and 100 nanometers pores (FIG. 8C) measured diameters of 75 ± 25 nanometers and 90 ± 39 nanometers, respectively. [00118] Photoluminescence lifetimes of Di8-intercalated liposomes obtained with RPS-FLIM were validated with time-correlated single photon counting (TCSPC), which can resolve changes in photoluminescence lifetime on the nanosecond timescale. Bulk measurements also corroborate the positive correlation between cholesterol concentrations in the liposomes and the photoluminescence lifetime of Di8 (FIG. 7A). For example, when the cholesterol concentrations were systematically increased from 0% to 50%, the photoluminescence lifetime of Di8 increased from 2.22 ± 0.03 nanoseconds to 3.62 nanoseconds ± 0.03, respectively. These trends aligned well with those obtained with single particle measurements by RPS-FLIM (see FIG. 5A and Table 1).

[00119] Table 1. Photoluminescence Lifetimes of Di8 from Bulk and Single-Particle Measurements.

[00120] Liposome characterization of a solution 12, 112 containing a single fluorophore with varying cholesterol concentrations is quantifiable for both singleparticle and bulk measurements. However, data interpretation from bulk measurements becomes more challenging when the solution 12, 112 becomes more complex (e.g., a mixture of liposomes intercalated with two different fluorophores). For example, bulk measurements of a mixture of liposomes intercalated with COE or Di8 return an average lifetime of the two fluorophores instead of their individual lifetimes (see FIG. 9A). Additionally, for TCSPC, one wavelength is selected for detection at a time, resulting in a shift in the lifetime that will more closely resemble the lifetime of the more excited fluorophore (see FIG. 9A). Additionally, for bulk TCSPC characterization, one wavelength is selected for detection at a time, which may make it impossible to avoid photoluminescence spectra overlap, resulting in a shift in the lifetime that will more closely resemble the lifetime of the more excited fluorophore (see FIG. 9A).

[00121] FIG. 9A shows bulk photoluminescence lifetime measurements of liposomes intercalated with Di8 or COE with 15% mol cholesterol and mixtures of those liposomes at excitation wavelengths of 475 nanometers, 525 nanometers, and 575 nanometers. Liposomes with COE had lifetimes of 1.0 nanoseconds, 1.0 nanoseconds, and 1 .2 nanoseconds at wavelengths of 475 nanometers, 525 nanometers, and 575 nanometers, respectively. Liposomes with Di8 had a lifetime of 2.6 nanoseconds at 575 nanometers. The mixture of liposomes with COE or Di8 had lifetimes of 1 .0 nanoseconds, 2.4 nanoseconds, and 2.8 nanoseconds at 475 nanometers, 525 nanometers, and 575 nanometers, respectively. The difference in lifetimes was caused by the mixture of liposomes having overlapping photoluminescence spectra (i.e. , photoluminescence spectra overlap), resulting in an incorrect assessment of the lifetimes. FIG. 9B shows the photoluminescence spectra overlap of the dye-intercalated liposome samples of FIG. 9A.

[00122] RPS-FLIM can resolve photoluminescence lifetime information while creating an image with that photoluminescence lifetime at the single particle levels isolated from more complex samples. For example, FIG. 10A shows FLIM images isolated from a mixture of liposomes (100 nanometers) intercalated with COE or Di8 with 30% mol cholesterol. The color scale bar represents lifetimes from 0.8 nanoseconds to 4.0 nanoseconds, and the image scale bar is 1 .0 pm. With RPS- FLIM, two individual particles in close proximity exhibiting unique photoluminescence lifetimes were resolved with COE (top trace of FIG. 10A; 0.91 nanoseconds) having a shorter lifetime than Di8 (bottom trace of FIG. 10A; 2.74 nanoseconds). From the phasor plot, two well-resolved populations from the same mixture of liposomes had estimated lifetimes of 2.7 nanoseconds and 0.9 nanoseconds corresponding to Di8 and COE, respectively, which were obtained over 5 minutes of data acquisition with the RPS-FLIM system 10, 110 (see FIG. 10B). [00123] Moreover, as shown in FIG. 10C, well-defined and fully resolved photoluminescence lifetime distributions of the mixture of liposomes were in line with those obtained when the liposomes were measured individually. FIG. 10C shows distributions of photoluminescence lifetimes of liposomes intercalated with COE or Di8 and a mixture of those liposomes and detected in the photoluminescence lifetime imaging microscopy detection region 113. For example, when measured together with RPS-FLIM, the photoluminescence lifetimes of liposomes intercalated with Di8 or COE were 2.8 ± 0.4 nanoseconds and 1.1 ± 0.2 nanoseconds, respectively. When measured separately, the photoluminescence lifetimes were 3.1 ± 0.6 nanoseconds for Di8 and 0.9 ± 0.12 nanoseconds for COE. Differences in data tabulation between the phasor plots (see FIG. 10B) and RPS-FLIM lifetime distributions (see FIG. 10C) may explain the small discrepancy in the observed average lifetimes. For the phasor plots, every pixel with greater than or equal to 2 photons from FLIM frames is represented as a data point and counted for the photoluminescence lifetime analysis (see FIG. 6B). Alternatively, for the RPS-FLIM analysis, a minimum threshold of 100 photons in a single frame is set for photoluminescence lifetime analysis. However, in all cases, the photoluminescence lifetime trends extracted by RPS-FLIM are consistent with those obtained with TCSPC when samples are measured separately (see FIG. 9A), but bulk measurements with TCSPC could not resolve the lifetimes of Di8 and COE in a mixture.

[00124] Because the sizes of the liposomes (see FIG. 10D) are measured concurrently with their photoluminescence lifetimes (see FIG. 10C), the data can be compiled in a scatter plot of relative volume and photoluminescence lifetime for each measured particle, as shown in FIG. 10E. FIG. 10E is a scatter plot of the size measurement (Ai/i) and photoluminescence lifetimes of liposomes intercalated with COE or Di8 and a mixture of those liposomes. Particle counts were 728, 573, and 1208, respectively.

[00125] The liposome size is not impacted by the intercalated dye and does not change when in a mixture. All three samples exhibited broad distributions with diameters ranging from 70 nanometers up to 200 nanometers when calibrated with polystyrene standards with diameters of 70 nanometers and 100 nanometers. FIG. 10D shows distributions of the relative pulse amplitude (Ai/i) of liposomes intercalated with COE or Di8 and a mixture of those liposomes. Particle size distributions detected inside the RPS region show no statistical differences with intercalated dye. Moreover, the photoluminescence lifetimes for each dye were constant with particle size, and the lifetimes of COE and Di8 were fully resolved when measured separately (top panel of FIG. 10E) or as a mixture (bottom panel of FIG. 10E), which highlights the advantage of single-particle characterization.

[00126] FIGS. 11A and 11 B show dwell times (td) and lifetimes for liposomes intercalated with Di8 or COE. Particle velocity through the nanofluidic region of the resistive pulse sensing device 111 depends on the electric field strength within the nanofluidic region and surface-charge density on the particle (or ^-potential). Similar to particle size and particle shape, particle charge is a physical property of the particle and can be measured with resistive-pulse sensing. The dwell time (td) is the time that the particle resides in the nanopore 120, and the pore-to-pore time (t_PP) is the time that the particle takes to travel between two adjacent nanopores 120A, 120B. Both the dwell time and the pore-to-pore time can be used to calculate the velocity, the electrokinetic mobility, and the zeta potential of the particle. For example, extracellular vesicles from bovine milk and human cancer cell line MDA- MB-468 have unique zeta potentials. When analyzing liposomes (100 nanometers) intercalated with COE or Di8, COE-labeled liposomes had an average dwell time of 0.32 ± 0.04 milliseconds, whereas Di8-labeled liposomes had an average dwell time of 0.43 ± 0.06 milliseconds (FIG. 11 A). The shift in dwell times between the two liposome samples can be attributed to the different charges on the COE and Di8 dyes. When the Di8 is intercalated into the lipid bilayer, one negatively charged sulfonate headgroup is oriented toward the exterior of the liposome. When COE-S6 is intercalated into a lipid bilayer, three positively charged quaternary amines on the ends of the molecule are oriented towards the exterior of the liposome. This bridged orientation results in a greater number of positive charges on the outer surface of the liposome in comparison to Di8. When the dwell time and photoluminescence lifetime measurements of liposomes intercalated with COE or Di8 are compared, the lifetimes are fully resolved (FIG. 11 B) with values of 1 .0 ± 0.2 nanoseconds and 3.1 ± 0.6 nanoseconds, respectively. When these samples are mixed, the dwell times are partially resolved, and a slight shift is observed (FIG. 11 B); however, the lifetimes remain fully resolved with values of 1 .1 ± 0.2 nanoseconds and 2.8 ± 0.3 nanoseconds, respectively.

[00127] FIGS. 12A-12D shows another system 210 having a nanochannel array for a high throughput characterization of particles (e.g., liposomes) with FLIM. A method to increase the number of particles characterized by FLIM is to pass the particles in parallel through an array of nanochannels 220 while in the photoluminescence lifetime imaging microscopy detection region 213. A schematic of the system 210 is shown in FIG. 12A, where two V-shaped microchannels 214, 216 are connected by four nanochannels 220 placed in parallel. An SEM image of the nanochannel 220 array is shown in FIG. 12B. By placing nanochannels 220 in parallel, solution throughput is increased, and therefore, the number of particles characterized is increased. An electric potential may be applied across the nanochannels 220 via at least one voltage source 236.

[00128] As an example, over a 1 -minute experiment, 229 liposomes intercalated with COE or Di8 and 30% cholesterol were analyzed, and photoluminescence lifetimes of 0.95 ± 0.08 nanoseconds and 2.6 ± 0.3 nanoseconds, respectively, were determined (FIG. 12C). The system 210 can be operated with an applied potential, a pressure-driven flow, or in combination. In some embodiments, the system 210 can be operated in FLIM mode during which only imaging data is collected via the detector 240. In some embodiments, the second detector 240 also concurrently measures light scattering data, which may be used to determine particle size. Particle size can be obtained from particles traversing the nanochannels 220 by collecting scattered light. This is accomplished by overlapping the excitation laser wavelength with the emission detection range, enabling the reflected light from the particles to be captured and analyzed as the particles move through the field of view of the second detector 240. The scattered light may be backscattered reflected light or forward scattered reflected light.

[00129] An image of four particles (three particles labeled with COE and one particle labeled with Di8) passing through a four-nanochannel 220 array is shown in FIG. 12D. To extract data at the single particle level, a region of interest spanning the full width and half the length of each nanochannel 220 isolated particles translocating individual nanochannels 220 while minimizing double particle events from being counted. COE-intercalated liposomes appear bluer in color, whereas Di8- intercalated liposomes appear greener in color, which are respective to color-coded lifetimes. These photoluminescence lifetimes agree well with lifetimes determined by RPS-FLIM. These experiments highlight the ability to increase the number of particles analyzed by FLIM at a single particle level.

[00130] With ten nanochannels 220 in parallel, counts of more than 600 per minute may be possible, effectively increasing the sample throughput by an order of magnitude compared to a single nanochannel. The surface charge density of the particle can be extracted from the streak length of the particle in the image or distance traveled by the particle over two or more frames. Also, resistive-pulse sensing can be integrated into the nanochannel 220 array. To distinguish from which nanochannel 220 the resistive-pulse signal is being generated, each nanochannel 220 can have a different number of pores in series, a different spacing among pores, or a combination thereof. Because each nanochannel 220 generates a unique signal, a single detector (i.e., current amplifier) 238 or multiple current amplifiers 238 can be used to measure the current.

[00131] The nanochannel 220 arrays may be milled with a focused ion beam instrument (Auriga 60 CrossBeam, Carl Zeiss) with a beam current of 200 pA, spot size of 40 nanometers, and dose of 2.0 nC/pm². The channels 220 may be 470 nanometers deep, 525 nanometers wide, 8 millimeters long, and 1 millimeter center- to-center. FLIM measurements may be taken with a zoom of 13x, field of view of 14.10 micrometers x 14.10 micrometers, pixel array of 64 x 64, pixel size of about 220 nanometers, and frame rate of 17 frames per second. Each nanochannel 220 may be isolated as a region of interest (ROI) that spans half the length of the nanochannel 220 to minimize multiple particles from being analyzed in one ROI.

[00132] FIG. 13A shows another system 310 having three nanochannel arrays 315A, 315B, 315C for a high throughput characterization of particles (e.g., liposomes) with FLIM. The system 310 includes three nanochannel arrays 315A, 315B, 315C, and each nanochannel array 315A, 315B, 315C includes a plurality of nanochannels 318. Though shown and described with three nanochannel arrays 315A, 315B, 315C, any number of nanochannel arrays 315A, 315B, 315C is contemplated. For example, each of the nanochannel arrays 315A, 315B, 315C may include six nanochannels 318 arranged in parallel. Though shown and described with six nanochannels 318 in each nanochannel array 315A, 315B, 315C, any number of nanochannels 318 is contemplated. An advantage of the nanochannel arrays 315A, 315B, 315C is being able to take multiple images of the individual particle as it passes through the constriction (i.e. , the nanochannel 318). Multiple images improves the precision of luminescence and velocity measurements.

[00133] Each of the nanochannel arrays 315A, 315B, 315C is coupled to a respective microchannel 314A, 314B, 314C upstream of the corresponding nanochannel array 315A, 315B, 3150, as shown in FIG. 13A. The system 310 also includes a solution 312 comprising at least one individual particle. The solution 312 is received in one or more of the microchannels 314A, 314B, 314C. In some embodiments, the same solution 312 is received in the one or more of the microchannels 314A, 314B, 314C. In some embodiments, a different solution 312 is received in the one or more of the microchannels 314A, 314B, 314C. In the illustrative embodiment, the microchannels 314A, 314B, 314C are separate from one another and not in fluid communication with one another.

[00134] Each microchannel 314A, 314B, 314C includes two reservoirs 328, 330, as shown in FIG. 13A. The solution 312, which contains the at least one individual particle, is loaded into the reservoirs 328, 330 of the one or more microchannels 314A, 314B, 314C, and an electric potential is applied between the reservoirs 328, 330 and buffer reservoirs 332, 334.

[00135] The system 310 further includes a microchannel 316 spaced apart from each of the microchannels 314A, 314B, 314C, as shown in FIG. 13A. The microchannels 314A, 314B, 314C, 316 may be referred to as reservoirs. The microchannel 316 is coupled downstream to each of the nanochannel arrays 315A, 315B, 315C. The nanochannel arrays 315A, 315B, 315C (and the nanochannels 318 of each nanochannel array 315A, 315B, 315C) are arranged between the respective microchannel 314A, 314B, 314C and the microchannel 316. The nanochannels 318 may be referred to as constrictions.

[00136] In response to an electric potential being applied between the one or more microchannels 314A, 314B, 314C and the microchannel 316 via at least one voltage source 336, the individual particles move from the one or more microchannels 314A, 314B, 314C and through the corresponding plurality of nanochannels 318 toward the microchannel 316. The electric potential may range from about 0.2 V to about 1 V. The rate at which the individual particles pass through the plurality of nanochannels 318 may be increased with applied pressure via, for example, a pump or vacuum (such as the pump 44). In some embodiments, the same voltage source 336 is coupled to each of the microchannels 314A, 314B, 314C. In some embodiments, a different voltage source 336 is coupled to each of the microchannels 314A, 314B, 314C. In some embodiments, an electric potential is applied to each of the microchannels 314A, 314B, 314C simultaneously. In some embodiments, an electric potential is applied to each of the microchannels 314A, 314B, 314C sequentially.

[00137] While the individual particles are moving toward the microchannel 316, resistive pulse sensing data and photoluminescence lifetime data are concurrently measured so that a current pulse related to the individual particles and a photoluminescence lifetime related to the individual particles are determined. Though shown and described with the individual particle being transported with an electric potential (i.e., electrokinetically), particles can be transported with pressure-driven flow via the pump, electrokinetically via the at least one voltage source 336, or in combination.

[00138] The system 310 includes a first detector 338 for measuring the resistive pulse sensing data and a second detector 340 for measuring the photoluminescence lifetime data. In some embodiments, the second detector 340 also concurrently measures light scattering data, which may be used to determine particle size. Particle size can be obtained from particles traversing the nanochannels 318 by collecting scattered light. This is accomplished by overlapping the excitation laser wavelength with the emission detection range, enabling the reflected light from the particles to be captured and analyzed as the particles move through the field of view of the second detector 340. The scattered light may be backscattered reflected light or forward scattered reflected light. In some embodiments, the same first detector 338 is coupled to each of the nanochannels 318. In some embodiments, a different first detector 338 is coupled to each of the nanochannels 318. To distinguish from which nanochannel 318 the resistive-pulse signal is being generated, each nanochannel 318 can have a different number of pores in series, a different spacing among pores, or a combination thereof. Because each nanochannel 318 generates a unique signal, a single first detector (i.e., current amplifier) 338 or multiple first detectors 338 can be used to measure the current. [00139] The system 310 also includes an excitation source 342 that excites the individual particles in the system 310. The excitation source 342 may be a part of the second detector 340 (i.e., microscope). As the particles traverse the nanoarray 315A, 315B, 315C, the fluorescence and fluorescence lifetimes of the intercalated dyes (Di8 or COE) may be measured with the second detector 340 (for example, a Stellaris 8 confocal microscope (Leica Microsystems) equipped with a 63x waterimmersion objective). The region of interest (ROI ) on the second detector 340 may be set to be 10.5 pm x 10.5 pm, which allows for all six nanochannels 318 in the nanoarray 315A, 315B, 315C to be imaged simultaneously. In this way, each nanoarray 315A, 315B, 315C may be imaged sequentially, while each nanochannel 318 within one nanoarray 315A, 315B, 315C may be imaged simultaneously. For the FLIM measurements, the excitation wavelength may be about 440 nanometers, and the emission wavelengths detected may be between about 450 and 720 nanometers.

[00140] The resistive pulse sensing data is measured in each of the plurality of nanochannels 318, and the photoluminescence lifetime data may be measured anywhere in the system 310, such as in the microchannel 314A, 314B, 314C, in the plurality of nanochannels 318, and/or in the microchannel 316.

[00141] In some embodiments, the system 310 includes a processor 324 having a memory 326, as shown in FIG. 13A. The memory 326 has instructions stored therein which, when executed by the processor 324, cause the processor 324 to control the at least one voltage source 336 to apply the electric potential between the microchannels 314A, 314B, 314C and the microchannel 316 and to control the first detector 338 to measure the current passing through the plurality of nanochannels 318. The processor 324 is electrically connected to the first detector 338 and the second detector 340 so as to receive the current pulse data and the photoluminescence lifetime data therefrom. The processor 324 receives such data and stores the data in the memory 326. [00142] By placing nanochannels 318 in parallel and by including more than one nanochannel array 315A, 315B, 315C, solution 312 throughput is increased, and therefore, the number of particles characterized is increased.

[00143] In some embodiments, each nanochannel 318 is about 500 nanometers wide, about 350 nanometers deep, and about 15 nanometers long. The nanochannels 318 are designed to transport particles through the observation region of the second detector 340. The width and depth of the nanochannels 318 keep the particles in focus as they traverse the field of view, and these dimensions can be increased or decreased to accommodate larger or smaller particles, respectively. Having particles flow through the detection region in a straight line simplifies the data analysis, especially tracking a single particle, averaging multiple measurements of the fluorescence lifetime, and determining the velocity of the particle from a series of images. Higher throughput for particle analysis is achieved by having particles flow through the detection region and multiple nanochannels 318 in parallel.

[00144] FIG. 13B shows a fluorescence lifetime image of four 100 nanometer liposomes migrating through one of the nanochannel arrays 315A, 315B, 315C. Three of the particles were liposomes intercalated with COE (nanochannel numbers 2, 3, and 6), and one was a liposome intercalated with Di8 (nanochannel number 5). As the liposomes migrate through the nanochannels 318, multiple images of the liposomes may be collected and analyzed. The analysis code may track individual particles as the individual particles traverse the nanochannels 318. By keeping track of individual particles across multiple frames, the fluorescence lifetimes from a single particle may be averaged across those frames to improve the precision of the measurement. Similarly, the position of the particle traversing the nanochannels 318 and a timestamp from each image may be used to calculate the velocity of each particle. From the velocity, the electrophoretic mobility and, subsequently, the zeta potential can be calculated.

[00145] FIG. 14A shows a two-dimensional plot of the fluorescence lifetimes and velocities of liposomes with 10%, 30%, and 50% cholesterol and intercalated with Di8 and liposomes with 30% cholesterol and intercalated with COE. These liposome samples were run individually. The fluorescence lifetimes of the Di8- labeled liposomes with 10%, 30%, and 50% cholesterol and COE-labeled liposomes with 30% cholesterol were 2.26 ± 0.05, 2.58 ± 0.08, 2.88 ± 0.07, and 0.98 ± 0.03 nanoseconds, respectively. Particle velocities for the Di8-labeled liposomes with 10%, 30%, and 50% cholesterol and COE-labeled liposomes with 30% cholesterol were 56 ± 8, 54 ± 6, 62 ± 8, and 109 ± 9 pm/s, respectively. The fluorescence lifetimes and particle velocities are projected onto their respective axes as histograms. As seen in FIG. 14A, the lifetime distributions for the Di8-intercalated liposomes with 10%, 30%, and 50% cholesterol were well resolved. The lifetime and velocity distributions of the Di8- and COE-intercalated liposomes are well-resolved from each other. Particle counts were 2532, 1349, 2447, and 2061 for Di8-labeled liposomes with 10%, 30%, and 50% cholesterol and COE-labeled liposomes with 30% cholesterol, respectively.

[00146] FIG. 14B shows a two-dimensional plot of fluorescence lifetimes and particle velocities of a mixture of Di8-intercalated liposomes with 30% cholesterol and COE-intercalated liposomes with 30% cholesterol. The fluorescence lifetimes of the Di8- and COE-intercalated liposomes with 30% cholesterol were 2.66 ± 0.09 and 0.99 ± 0.02 nanoseconds, respectively. Particle velocities for the Di8- and COE- labeled liposomes in the mixture were 46 ± 7 and 103 ± 10 pm/s, respectively. The total particle count for the mixture was 3038. Both the fluorescence lifetime and particle velocity distributions are fully resolved.

[00147] COE intercalated into liposomes exhibited fluorescence lifetimes of 1.0 nanoseconds, which did not vary with cholesterol concentration. However, fluorescence lifetimes of Di8 are sensitive to cholesterol concentration within a liposome and range from 2.0 to 3.4 nanoseconds for 0% to 50% cholesterol, respectively. Consequently, the fluorescence lifetime of COE can be used as a control, and the fluorescence lifetime of Di8 can monitor the cholesterol concentration.

[00148] Because multiple frames for each particle were collected, the particle velocity was calculated for each sample (FIG. 14B). Particle velocities did not change with cholesterol concentration and were 56 ± 8 pm/s, 54 ± 6 pm/s, 62 ± 8 pm/s for Di8-intercalated liposomes with 10%, 30%, and 50% cholesterol, respectively. However, the particle velocity was impacted with the intercalated dye. Liposomes with 30% cholesterol and intercalated with Di8 or COE had average velocities of 54 ± 6 pm/s and 109 ± 9 pm/s, respectively. COE is positively charged, whereas Di8 is negatively charged. Consequently, when intercalated into liposomes, COE makes the surface of the liposome less negatively charged.

[00149] With the nanochannel arrays 315A, 315B, 315C, multiple particles can be imaged simultaneously in the same nanochannel 318 or in adjacent nanochannels 318. With the six-channel array 315A, 315B, 315C, particle frequencies of greater than 1 ,000 min-1 were achievable. Most data was collected at particle frequencies of about 400 to about 800 min-1. The framerate of the system 310, velocity of particles in the nanochannel arrays 315A, 315B, 315C, and desired precision of the measurements impact the particle throughput. Measurement precision is improved through signal averaging. For the measurements described here, an applied potential of 0.5 V permitted a balance between particle throughput and measurement precision.

[00150] FIG. 15A compares average fluorescence lifetimes for Di8-intercalated liposomes with 30% cholesterol and standard deviations for one measurement, an average of two measurements, and an average of five measurements, which were 2.58 ± 0.13, 2.58 ± 0.10, and 2.58 ± 0.08 nanoseconds, respectively. The average remained constant, but the standard deviation decreased with an increasing number of measurements. Particle counts were 1385, 1349, and 1382 for one, two, and five measurements, respectively.

[00151] FIG. 15B shows particle velocity distributions for Di8-intercalated liposomes with 30% cholesterol when 0.2, 0.5, 0.7, and 1 .0 V were applied across the nanochannel arrays 315A, 315B, 315C. The particle velocities were 22 ± 4, 53 ± 7, 74 ± 10, and 105 ± 14 pm/s, respectively. With 0.2, 0.5, 0.7, and 1.0 V applied, the relative standard deviations were 18%, 13%, 14%, and 13%. Particle counts were 933, 1985, 2892, and 4102, respectively.

[00152] FIGS. 16A and 16B demonstrate the ability to determine the relative abundances of two dye-intercalated liposomes with FLIM with the system 310. FIG. 16A shows fluorescence lifetimes of mixtures of COE- or Di8-intercalated liposomes in ratios of 4:1 and 7:13 (COE:Di8). As the fraction of Di8-intercalated liposomes increased, the number of counts of Di8-intercalated liposomes increases relative to the COE-intercalated liposomes. The lifetimes for COE and Di8 were 1 .02 ± 0.02 and 2.5 ± 0.1 nanoseconds for the 4:1 mixture and 1.01 ± 0.03 and 2.4 ± 0.1 nanoseconds for the 7:13 mixture, respectively. As the concentration of Di8-labeled liposomes in the ratio increased, their fraction of counts increased. Particle counts were 2091 and 2890 for the ratios 4:1 and 7:13 (COE:Di8), respectively.

[00153] To compare single-particle and bulk measurements, photoluminescence measurements were collected. FIG. 16B highlights the inability for bulk measurements to accurately quantify the fluorescence lifetimes of a mixture of liposomes intercalated with either Di8 or COE. The emission wavelength for the data was set at 525 nanometers, at which both Di8 and COE emit. The fluorescence decay curve qualitatively showed an increased abundance of a longer lifetime species with increased concentration of Di8-labeled liposomes relative to COE- labeled liposomes. However, when fitting the data, the lifetimes were not representative of either COE or Di8, and no information regarding relative abundance was obtained.

[00154] Nanoarray FLIM can distinguish multiple populations based on both fluorescence lifetime and velocity in liposomes intercalated with either COE or Di8 as the liposomes translocate through parallel nanochannels 318. In addition to resolving these populations, this approach enables accurate quantification of the relative abundances of each species. This technique is highly applicable to biologically derived nanoparticles with heterogeneous populations, such as extracellular vesicles, viruses, and lipid nanoparticles. Biophysical and biochemical characteristics of these particles, such as membrane fluidity, nucleic acids, and protein biomarkers, can be probed using fluorophores with intrinsically distinct lifetimes. Moreover, the inability of traditional photoluminescence spectroscopy to extract meaningful information from mixtures of two species underscores the need for a single-particle FLIM approach to nanoparticle analysis.

[00155] Coupling resistive-pulse sensing with photoluminescence lifetime imaging microscopy (RPS-FLIM) provides concurrent and correlated information about the size and membrane composition of individual dye-intercalated biological particles, such as liposomes. With RPS-FLIM, liposomes with different lipid-bilayer compositions can be differentiated by subtle changes in the photoluminescence lifetimes of the dye. In addition, in samples in which multiple dye-intercalated liposomes are studied, photoluminescence lifetimes may be resolved and appropriately assigned at the single particle level. RPS-FLIM overcomes limitations with spectral overlap often seen with bulk measurements. Moreover, RPS-FLIM can differentiate liposomes with the same membrane composition but different sizes. RPS-FLIM may be used for the biophysical interrogation of liposomes and other biological nanoparticles at the single entity level, including determining membrane composition and dynamics, analyzing FRET of cargo-loaded biomolecules, and extracting mechanical properties of individual particles, all of which might go unnoticed in bulk measurements.

[00156] Examples.

[00157] Example 1. Liposome Preparation. All lipids were purchased from Avanti Polar Lipids. Di-8-ANEPPS (Di8) was purchased from Biotium. COE-S6 (COE) was synthesized according to previously published protocols. 1-palmitoyl-2- oleoyl-glycero-3-phosphocholine (POPC), 1 -palmitoyl-2-oleoylsn-glycero-3-phospho- (T-rac-glycerol) sodium (POPG), and cholesterol (plant-derived) were dissolved in chloroform. Di-8 and COE were dissolved in ethanol. These solutions were mixed in a glass vial in molar ratios listed in Table 1 . The overall POPC and POPG concentration decreased as cholesterol concentration increased, but the POPC: POPG ratio was maintained at 3:1 so as not to drastically change the overall charge of the liposomes. The lipid and dye mixtures were dried under a gentle stream of argon in a fume hood and were further dried in a vacuum oven at 100°C for 30 minutes to obtain a thin lipid film. To prepare liposomes, the dried film was rehydrated by adding 1x PBS (phosphate-buffered saline, GIBCO) at 60°C to a final lipid concentration of 3.125 mg/mL and vortexed for 1 minute. With an Avanti Mini Extruder, the liposomes were extruded through a series of membranes with decreasing pore diameters. The 100 nanometer liposomes were extruded sequentially through membranes with pore diameters of 200 nanometers and 100 nanometers. The 50 nanometer liposomes were extruded sequentially through membranes with pore diameters of 200 nanometers, 100 nanometers, and 50 nanometers. All liposomes were extruded through each membrane twenty-one times at room temperature and were stored at 4°C until further use (all data shown were collected within 7 days of extrusion). [00158] Example 2. Fabrication of Nanofluidic Devices. The nanofludic devices (see FIGS. 2A, 2B, 12A, 13A) were made through a combination of micro- and nanofabrication techniques. Glass substrates (D263, Precision Glass & Optics) were coated with a chromium film (40 nanometers thick) by thermal evaporation (Auto 306, BOC Edwards). Photoresist (1.3 micrometers thick; Microposit S1813 G2, Kayaku Advanced Materials) was spin-coated on top of the chromium film. The microchannel pattern was transferred into the photoresist through a photomask (HTA Photomask) with a UV exposure system (Model 200, Optical Associates, Inc.) and developed (Microposit MF-319, Kayaku Advanced Materials). The chromium film was etched (Chromium Etchant CE-8002-A, Transene Co., Inc.) followed by etching of the microchannels (Buffered HF Improved, Transene Co., Inc.). Microchannels were etched to a depth of about 7 micrometers and measured with a stylus-based profiler (KLA Tencor T-7).

[00159] The nanochannels, nanopores, and nanofilters were designed in CAD software (Nanopatterning and Visualization Engine, Fibics, Inc.) and milled with a focused ion beam (FIB) instrument (Auriga 60, Carl Zeiss, GmbH). A 30-kV ion beam with a 50-pA beam current was used for milling the nanofluidic detection region (see FIG. 2B). The nanochannels were milled with a dose of 1 .58 nC/pm² to 750 nanometers wide, 315 nanometers deep, and 1500 nanometers long. The pore-to- pore regions between the nanochannels and nanopores were milled with a dose of 1.33 nC/pm² to 550 nanometers wide, 260 nanometers deep, and 600 nanometers long. The nanopores and nanofilters were milled with a dose of 0.9 or 1 .0 nC/pm² to 180 or 200 nanometers wide, 200 or 215 nanometers deep, respectively, and 450 nanometers long. After milling, the remaining chromium film was removed (Chromium Etchant 1020, Transene Co., Inc.). The dimensions of the nanopores, nanochannels, and nanofilters were measured with an atomic force microscope (AFM; MFP-3D, Asylum Research, Inc.).

[00160] Each sample microchannel 314A, 314B, 314C was connected to the buffer microchannel 316 through an array of six nanochannels 318 (FIG. 13A) milled with a focused ion beam instrument (Auriga 60, Carl Zeiss, GmbH) and a beam current of 200 pA. The nanochannels 318 in the array were milled with a dose of 1.6 nC/pm2 to a width of 500 nanometers, depth of 350 nanometers, and length of about 15 micrometers. Nanochannels 318 were spaced 1.5 micrometers center-to-center. Nanochannel 318 depths were measured with an atomic force microscope (AFM; MFP-3D, Asylum Research, Inc.).

[00161] Access holes were sandblasted (Air Eraser, Paasche Airbrush Co.) through the substrates at the ends of the microchannels prior to bonding the substrate and cover plate. The substrates were plasma cleaned on medium power for 90 seconds (Harrick PDC-32G) and soaked in 0.1 M NaOH (VWR, Inc.) at room temperature for 10 minutes. No. 1 .5 coverslips (VWR, Inc.) were cleaned in 1 M NaOH at 85°C for 10 minutes. Substrates and cover plates were sonicated in ultrapure water for 10 minutes, rinsed with ultrapure water, and brought into contact with each other while wet. Devices were dried at 90°C for at least 4 hours and annealed at 545°C for 12 hours. Glass reservoirs were epoxied over the access holes (353NDT, Epoxy Technology).

[00162] Example 3. Resistive-Pulse Sensing. The microchannels and nanochannels were filled with a solution by applying a vacuum to the liposome reservoirs and buffer reservoirs (see FIG. 2A). Prior to the first use, each device was sequentially rinsed with ultrapure water, 0.1 M NaOH, ultrapure water, and buffer solution (1x PBS) for 10 minutes each. All solutions were passed through a 200 nanometer syringe filter. Finally, samples were placed into the liposomes reservoir and drawn into the microchannels and nanochannels with vacuum. Each nanofluidic device was calibrated with polystyrene nanospheres with diameters of 70 nanometers and 100 nanometers (3070A and 3100A, Thermo Fisher Scientific).

[00163] Resistive-pulse measurements were conducted inside a copper mesh Faraday cage constructed on an aluminum stage insert for a Leica Stellaris 8 confocal microscope. An Axopatch 200B current amplifier (Molecular Devices, Inc.) was used to apply a potential between the liposomes reservoirs and buffer reservoirs through Ag/AgCI electrodes and to measure the resulting current. For all experiments, a 1 .0 V potential was applied. The settings on the current amplifier were a sampling frequency of 100 kHz, gain of a = 1 , head stage amplification of = 0.1 , and 10 kHz low-pass Bessel filter. The current signal was digitized (Digidata 1550B, Molecular Devices, Inc.) and collected with pCLAMP software (Molecular Devices, Inc.). [00164] Example 4. Photoluminescence Lifetime Measurements.

Fluorescence lifetime imaging microscopy (FLIM) images were recorded on a Leica Stellaris 8 confocal microscope at a scan speed of 1 kHz, zoom of 13.68 x, and 256 x 64 lines for an average frame rate of 27 frames per second. The software (Leica Application Suite X version 4.6.1 .27508 with Live Data Mode) outputs a 3.3 V TTL pulse at the start of FLIM acquisition and every about 12 seconds thereafter for alignment with the resistive-pulse data. The timing pulse was sent from the DMI8 Stellaris 8 to a second input on the Digidata 1550B separate from the current signal from the Axopatch 200B. For the excitation of Di8 and COE, a white-light laser set at 440 nanometers, 95% power, and 100% intensity in the software was sufficient for a consistent signal from the 50 nanometer and 100 nanometer liposomes. A photoluminescence spectrometer (FLS-1000, Edinburgh Instruments) was used for bulk photoluminescence lifetime measurements. All measurements were carried out at room temperature. In all cases, the lifetimes were measured at the Amax of the photoluminescence spectra with an excitation wavelength of 456 nanometers, and the repetition rate of the diodes for collecting the data was 5 kHz.

[00165] Example 5. Analysis of RPS-FLIM Data. Raw data from the resistive- pulse measurements and FLIM measurements were imported separately into MatLab R2020b (Mathworks, Inc.). The current pulse amplitude (Ai), pulse width (w), and baseline current (i) were extracted from the resistive-pulse data with a modified version of Open Nanopore 1.4.36. Frames from the FLIM data with intensities between 100 counts and 10,000 counts were kept for further analysis. These thresholds were set to remove low signal-to-noise frames and high-intensity artifacts. Because the photoluminescence lifetimes were generated from fitting the measured intensity versus time, frames with %² values greater than 1 .4 were also removed. The resistive-pulse and FLIM measurements were aligned in time by setting the initial time of the resistive-pulse data to the trailing edge of the first TTL pulse. FLIM frames within ± 0.1 millisecond of an RPS event were identified as a match to the resistive-pulse data and were recorded with the associated resistive-pulse measurement. The pulse amplitude (Ai), pulse width (w), baseline current (i), photoluminescence intensity, and photoluminescence lifetime were exported to

Excel, where two-pulse events indicating a single particle event were identified and averaged. Likewise, pore-to-pore times (t_pp) were identified and averaged. Uncorrelated events, e.g., series of current pulses produced by more than one particle in the detection region, were not analyzed further and excluded from the pulse amplitude and pore-to-pore time distributions. The baseline current was an average of 200 data points prior to the first of two pulses in a two-pulse sequence. The averaged pulse amplitude from each pair of pulses was divided by the average baseline current and multiplied by 100 to report the relative pulse amplitude (Ai/i) as a percentage.

[00166] Example 6. Transmission Electron Microscopy (TEM). Liposomes were prepared as described and extruded sequentially through polycarbonate membranes with pore diameters of 200 nanometers, 100 nanometers, and/or 50 nanometers and imaged the same day. The TEM grids (Carbon Square Mesh, Cu, 300 Mesh, UL; Electron Microscopy Sciences) were glow discharged with a PELCO easiGLOW (Ted Pella, Inc.). The liposomes were added to the TEM grid for 5 minutes, then wicked away. Uranyl acetate (2% v/v) in water (4 pL) was added to the grid for 30 seconds and then removed. Grids were imaged immediately on a JEOL JEM 1010 plus transmission electron microscope (JEOL, Inc.) at 80 kV with a 1 k x 1 k Gatan CCD camera (MegaScan model 794). At least 350 intact particle diameters were measured with ImageJ for each sample (NIH).

[00167] Example 7. Data Analysis.

[00168] Code to analyze the FLIM data measured by the system 310 was written in MATLAB. Input to the analysis program includes two data videos, one containing the lifetime data and one containing the intensity data, along with parameters from the measurement (e.g., framerate and pixel size). After defining a background intensity from the intensity video, any number of analysis regions-of- interest (ROI) are defined; for example, each of the six nanochannels 318 in the array is defined as a separate ROI. An image compiled from the data video is generated to aid in defining the x- and y-boundaries of the nanochannel 318. Each ROI, which is a 2-D array of pixels, is averaged along the direction orthogonal to particle flow to compact the data into two 1-D datasets along the length of the nanochannel 318, one representing the intensity data and one representing the RGB data. [00169] For each frame of the video, peaks in the 1-D intensity data are identified as regions with intensity 10 times the background intensity. The location, integrated intensity, and average RGB values for each peak are recorded. A series of logic tests are applied to link identified peaks from adjacent frames as the same particle. First, because particles are flowing through defined nanochannels 318 in a uniform direction, the peak in a given frame is further down the channel than the peak in the previous frame to be assigned as the same particle. Second, fluorescence lifetimes (RGB values) are assumed not to vary by more than 20% from frame to frame. Lastly, it is assumed that overtake events do not occur where one particle both enters the ROI after and exits before another particle. If multiple peaks in the frame fit the previous criteria, the peak that maintains the particle order is preferred. If a peak passes all logical checks, the resulting peak data are correlated to the same particle.

[00170] When a particle exits the ROI, the position data from every correlated peak is fitted to a line to calculate the particle velocity. Framerate and pixel-size parameters are used to convert the velocity to micrometers per second, while nanochannel dimensions and applied potential are used to convert the velocity to electrokinetic mobility and, subsequently, to zeta potential. The compiled intensity and RGB data from the correlated peaks are averaged, and the RGB data are converted to a fluorescence lifetime via comparison to the lifetime-to-color RGB scale output from the measurement software on the Stellaris microscope.

[00171] Lastly, the program provides a user interface wherein the user can set filters on the output data. Data can be filtered to exclude particles of insufficient brightness, particles that were detected in the ROI for an insufficient number of frames, or particles whose statistical uncertainty in mobility and lifetime measurements are too high. The user can also define the division between lifetime populations. With filters applied, the program provides figures and statistics comparing the defined particle populations and outputs spreadsheets containing the values for each particle.

[00172] While the disclosure has been illustrated and described in detail in the foregoing drawings and description, the same is to be considered as exemplary and not restrictive in character, it being understood that only illustrative embodiments thereof have been shown and described and that all changes and modifications that come within the spirit of the disclosure are desired to be protected. For example, it will be understood that any of the constructions in any of the illustrated embodiments may include one or more pores, as described above with respect to the embodiment illustrated in FIGS. 12A-12B, and/or that any of the constructions may be or include at least one nanopore.

[00173] It will be understood that any of the above described systems 10, 110, 210, 310 may be used to measure, determine, and/or detect resistive pulse sensing data and photoluminescence lifetime data of the individual particle concurrently. It will be understood that any of the above described systems 10, 110, 210, 310 may be used to measure, determine, and/or detect resistive pulse sensing data only without measuring, determining, and/or detecting photoluminescence lifetime data. It will be understood that any of the above described systems 10, 110, 210, 310 may be used to measure, determine, and/or detect photoluminescence lifetime data only without measuring, determining, and/or detecting resistive pulse sensing data.

Claims

What is claimed is:

1 . A system for characterizing individual particles, the system comprising: a solution including an individual particle, a reservoir for receiving the solution, a constriction fluidically coupled to the reservoir, at least one voltage source configured to apply a potential across the constriction to cause the individual particle to move through the constriction, an excitation source configured to excite the individual particle in the reservoir or in the constriction with light energy, and a first detector and a second detector configured to concurrently measure resistive pulse sensing data and photoluminescence lifetime data of the individual particle, respectively.

2. The system of claim 1 , wherein the constriction includes one or more pores.

3. The system of claims 1 or 2, wherein the constriction comprises a nanopore.

4. The system of any of claims 1-3, wherein the reservoir is a first reservoir upstream of the constriction, and wherein the system further comprises a second reservoir downstream of the constriction.

5. The system of claim 4, wherein the first and second reservoirs are microchannels.

6. The system of any of claims 1 -5, wherein the first detector includes a current amplifier configured to measure current within the constriction.

7. The system of claim 6, wherein the second detector includes a microscope configured to measure the photoluminescence lifetime data of the individual particle.

8. The system of any of claims 1-7, wherein the individual particle has a first size and the constriction has a second size that is substantially similar to the first size.

9. The system of any of claims 1-8, wherein the constriction is a first constriction, and wherein the system further comprises a second constriction in series with the first constriction.

10. The system of claim 9, wherein the first constriction and the second constriction each include one or more pores.

11 . The system of any of claims 9-10, wherein the first constriction and the second constriction each include one or more nanopores.

12. The system of claim 11 , wherein the individual particle has a first size and the one or more nanopores of the first constriction and the one or more nanopores of the second constriction each have a second size that is substantially similar to the first size.

13. The system of any of claims 9-12, further comprising a filter arranged upstream of the first constriction.

14. The system of any of claims 9-13, wherein the first detector includes a current amplifier configured to measure current within the one or more nanopores of the first constriction and the one or more nanopores of the second constriction, and wherein the second detector includes a microscope configured to measure the photoluminescence lifetime data of the individual particle.

15. The system of any of claims 9-14, further comprising a pump configured to aid in movement of the individual particle through the system.

16. The system of any of claims 9-15, wherein the reservoir is a first reservoir upstream of the first and second constrictions, and wherein the system further comprises a second reservoir downstream of the first and second constrictions.

17. The system of claim 16, wherein the first and second reservoirs are microchannels.

18. The system of any of claims 1 -8, wherein the constriction is a first constriction, and wherein the system further comprises a second constriction in parallel with the first constriction.

19. The system of claim 18, wherein the first constriction and the second constriction each include one or more pores.

20. The system of any of claims 18-19, wherein the first constriction and the second constriction each include one or more nanopores.

21 . The system of claim 20, wherein the individual particle has a first size and the one or more nanopores of the first constriction and the one or more nanopores of the second constriction each have a second size that is substantially similar to the first size.

22. The system of any of claims 18-21 , further comprising a filter arranged upstream of the first constriction.

23. The system of any of claims 18-22, wherein the first detector includes a current amplifier configured to measure current within the one or more nanopores of the first constriction and the one or more nanopores of the second constriction, and wherein the second detector includes a microscope configured to measure the photoluminescence lifetime data of the individual particle.

24. The system of any of claims 18-23, further comprising a pump configured to aid in movement of the individual particle through the system.

25. The system of any of claims 18-24, wherein the reservoir is a first reservoir upstream of the first and second constrictions, and wherein the system further comprises a second reservoir downstream of the first and second constrictions.

26. The system of claim 25, wherein the first and second reservoirs are microchannels.

27. The system of any of claims 1-26, wherein the individual particle is a biological particle.

28. The system of any of claims 1-26, wherein the individual particle is inorganic.

29. The system of any of claims 1-28, wherein the resistive pulse sensing data related to the individual particle is used to determine physical properties of the individual particle, and wherein the physical properties include one or more of a particle size, a surface charge, and a particle shape.

30. The system of claim 29, wherein the photoluminescence lifetime data related to the individual particle is used to determine photophysical properties of the individual particle, and wherein the photophysical properties include one or more of a membrane viscosity, a membrane polarity, and a membrane composition.

31 . A method for characterizing individual biological particles, the method comprising: inputting a solution comprising an individual particle into a reservoir, applying a potential across a constriction to cause the individual particle to move through the constriction from the reservoir, exciting the individual particle in the reservoir or in the constriction with light energy, and concurrently measuring resistive pulse sensing data and photoluminescence lifetime data of the individual particle.

32. The method of claim 31 , further comprising, based on the resistive pulse sensing data, determining one or more of a particle size, a surface charge, and a particle shape of the individual particle.

33. The method of claim 31 or 32, further comprising, based on the photoluminescence lifetime data, determining one or more of a membrane viscosity, a membrane polarity, and a membrane composition of the individual particle.

34. The method of any of claims 31-33, wherein the steps of determining occur concurrently.