WO2025024762A1 - Composition, methods, and kits for direct calibration of fluorescence sensitivity in a flow cytometer - Google Patents

Abstract

The present disclosure provides systems, methods, and kits to directly calibrate fluorescence sensitivity in a flow cytometer using lipid enveloped droplets comprising first, second, and third known concentrations of analytes encapsulated in lipid enveloped droplets, and wherein the analytes have a fluorescent dye. In embodiments, the analytes encapsulated in lipid enveloped droplets comprise a fluorescent dye, a fluorescent dye conjugated to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), a hydrophilic partner that can prevent the analyte from crossing the lipid envelop of the droplet, protein, antibody, antibody fragment, nanobody, or a combination thereof.

Description

COMPOSITION, METHODS, AND KITS FOR DIRECT CALIBRATION OF FLUORESCENCE SENSITIVITY IN A FLOW CYTOMETER

CROSS REFERENCE TO RELATED APPLICATIONS

[01] This application is being filed as a PCT International patent application and claims the benefit of and priority to U.S. Provisional Patent Application Serial Number 63/515,788 filed July 26, 2023, the subject matter of which is hereby incorporated by reference in its entirety.

INTRODUCTION

[02] In flow cytometry, single cells in solution flow through a beam of laser light resulting in light scattered in the forward and the side directions. The scattered light is then collected by photodetectors, analyzed, and the cells are counted according to the analyzed characteristics. In addition to measuring scattered light, flow cytometers can also measure fluorescence, e.g., fluorescence labeled antibodies or markers. Flow cytometers have become more sensitive and capable of measuring more parameters as the technology has matured.

[03] The fluorescent intensity of a signal (e.g., a fluorescence labeled antibody) is one of the important parameters measured and analyzed in flow cytometry. It represents the measure of an analyzed marker expression on a cell or other biological particle and is reported as mean or median fluorescent intensity (MFI). MFI values, however, are strongly instrument-dependent because they are influenced by many instrumentspecific factors, e.g., laser power, detector sensitivity, optical configuration, and optical alignment. Because of a need to introduce a standardized, instrument-independent fluorescent scale for flow cytometry and a need to characterize fluorescence sensitivity of different instruments using a common instrument agnostic calibrator, the concept of Molecules of Equivalent Fluorochrome (MEF) (also know n as Molecules of Equivalent Soluble Fluorochrome (MESF) or Equivalent of Reference Fluorochrome (ERF)) was introduced and adopted in flow cytometry.

[04] The current problem w ith measuring MESF values is that all the current solutions are based on indirect calibration of a flow cytometer, i.e., where stained beads are calibrated externally using a reference solution of fluorophores. The most common indirect calibration method is via the generation of a standard curve using a fluorometer and solutions of fluorochrome in several different concentration. Then during an experiment, the intensity of a labeled particle, e.g., antibody or cell, is measured by the flow cytometer during experimentation and compared to the standard curve to arrive at an equivalent concentration of fluorochrome in the experimental sample. This value is then divided by the known number of particles to generate a MESF value.

[05] This indirect calibration of a flow cytometer introduces error into the experimental measurement of MESF values in several ways. First, it is common practice to use polystyrene (PS) beads that are hard stained with multiple fluorophores to generate a multicolor bead, often referred to as Rainbow Calibration Particles (RCPs), which are available from Spherotech, 27845 Irma Lee Circle, Unit 101, Lake Forest, IL 60045. Notably, however, the dyes used in the PS beads are different spectrally from the calibrator, which introduces an additional source of error when extrapolating MESF values to a blank bead for determining instrument sensitivity. Second, in the case of single dyes conjugated to a bead surface, the quantum yield of fluorophores attached to a bead can be different from the quantum yield of the same fluorophore in solution. Third, spectrofluorometers used to assign MESF values are not sensitive enough to calibrate PS beads with lower amounts of dye, i.e.. lower dye loads. Finally, PS beads often have high autofluorescence, which makes it challenging to assign low MESF values that will be within or below autofluorescence range. The ability to assign low MESF values is especially important when calibrating flow cytometers for fluorescence analysis of nanoparticles.

[06] The present disclosure addresses the pitfalls of the current indirect methods for calibrating a flow cytometer by providing a direct method for calibrating a flow cytometer using lipid enveloped droplets to encapsulate a known concentration of dye, or reagent with dye, e.g., antibody-fluorescent dye conjugates. Knowing the internal volume of a microdrop (e.g., droplet), the concentration of encapsulated antibody, which can be precisely controlled, and the number of fluorophores per antibody (F/P), allow s an accurate and direct calculation of the number of fluorescent molecules per droplet. Accordingly, this provides a direct method for fluorescent calibration of a flow cytometer without relying on assumptions and the need for additional indirect calibration. The antibody per cell (ABC) can also be directly and precisely calculated based on the know n number of antibody molecules in the droplet.

[07] Embodiments of the present invention include systems for direct calibration of fluorescence in a flow cytometer, methods for direct calibration of fluorescence in a flow cytometer, and kits for direct calibration of fluorescence in a flow cytometer. [08] An embodiment of the present disclosure is a system for calibrating a flow cytometer using lipid enveloped droplets comprising a first reagent, wherein the first reagent comprises a first known concentration of analytes encapsulated in lipid enveloped droplets, wherein the analytes have a fluorescent dye. In embodiments, the analytes encapsulated in lipid enveloped droplets comprise fluorescent dye, a fluorescent dye conjugated to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), protein, antibody, antibody fragment, nanobody, a hydrophilic partner that can prevent the analyte from crossing the lipid envelop of the droplet, or a combination thereof. [09] An embodiment of the present disclosure is a method for calibrating a flow cytometer using lipid enveloped droplets comprising (a) loading a first reagent, wherein the first reagent comprises a first known concentration of analytes encapsulated in lipid enveloped droplets, wherein the analytes have a fluorescent dye, into a flow cytometer; (b) evaluating fluorescence sensitivity (MFI) of at least one fluorescent channel of at least one laser in the flow cytometer; loading at least one additional reagent, wherein the at least one additional reagent comprise a different known concentration of the same analytes as in (a) encapsulated in lipid enveloped droplets and repeating (b); (c) making a calibration curve, allowing a conversion of MFI values to MESF values; and optionally the calculation of ABC values. In an embodiment, step (b) is repeated such that (c) uses at least three different reagents of lipid enveloped droplets, each containing different known concentrations of the same analyte. In an embodiment, the at least three reagents are loaded into the flow- cytometer in serial. In another embodiment, the at least three reagents are loaded into the flow⁷ cytometer together at the same time. [010] An embodiment of the present disclosure is a kit for performing a method for calibrating a flow cytometer using lipid enveloped droplets comprising (a) loading a first reagent, w herein the first reagent comprises a first known concentration of analytes encapsulated in lipid enveloped droplets, wherein the analytes have a fluorescent dye, into a flow cytometer; (b) evaluating fluorescence sensitivity (MFI) of at least one fluorescent channel of at least one laser in the flow cytometer; loading at least one additional reagent, w herein the at least one additional reagent comprises a different known concentration of the same analytes as in (a) encapsulated in lipid enveloped droplets and repeating (b); (c) making a calibration curve allowing a conversion of MFI values to MESF values; and optionally the calculation of ABC values. In an embodiment, step (b) is repeated such that (c) uses at least three different reagents of lipid enveloped droplets, each containing different known concentrations of the same analyte. In an embodiment, the at least three reagents are loaded into the flow cytometer in serial. In another embodiment, the at least three reagents are loaded into the flow cytometer together at the same time. In an embodiment, the kit comprises a system for calibrating a flow cytometer using lipid enveloped droplets comprising a at least a first reagent, wherein the first reagent comprises a first known concentration of analytes encapsulated in lipid enveloped droplets, wherein the analytes have a fluorescent dye; at least one vial to hold the first reagent; and instructions for using the kit.

BRIEF SUMMARY OF THE INVENTION

[OH] The systems, methods, and kits of the present disclosure offer significant advantages over the current indirect methods for calibrating a flow cytometer byproviding a direct method for calibrating a flow cytometer using lipid enveloped droplets to encapsulate a know n concentration of reagent with dye. e.g., antibody- fluorescent dye conjugates. The systems, methods, and kits of the present disclosure provide a direct method for fluorescent calibration of a flow cytometer without relying on assumptions and the need for additional indirect calibration. Accordingly, for example, the antibody per cell (ABC) is directly and precisely calculated based on the known number of antibody molecules in a droplet.

Systems for calibrating a flow cytometer.

[012] An embodiment of the present disclosure is a system for calibrating a flow cytometer using lipid enveloped droplets comprising a first reagent, wherein the first reagent comprises a first known concentration of analytes encapsulated in lipid enveloped droplets, wherein the analytes have a fluorescent dye. In embodiments, the analytes encapsulated in lipid enveloped droplets comprise fluorescent dye, a fluorescent dye conjugated to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), protein, antibody, antibody fragment, nanobody, a hydrophilic partner that can prevent the analyte from crossing the lipid envelop of the droplet, or a combination thereof. In embodiments, the analytes encapsulated in lipid enveloped droplets comprise fluorescent dye, protein-fluorescent dye conjugates, antibody-fluorescent dye conjugates, antibody fragment-fluorescent dye conjugates, nanobody-fluorescent dye conjugates, DNA-fluorescent dye conjugates, RNA-fluorescent dye conjugates, other hydrophilic partner-fluorescent dye conjugates, or a combination thereof. In embodiments, a single lipid enveloped droplet may contain a single analyte or multiple analytes.

[013] Embodiments may further comprise at least a second reagent, wherein the second reagent comprises a second known concentration of the analytes encapsulated in lipid enveloped droplets, and the second know n concentration of the analytes is different than the first known concentration of the analytes. Embodiments may further comprise at least a third reagent, wherein the third reagent comprises a third known concentration of the analytes encapsulated in lipid enveloped droplets, and the third know n concentration of the analytes is different than the first known concentration and second known concentration of the analytes. Embodiments may further comprise at least a fourth reagent, wherein the fourth reagent comprises a fourth known concentration of the analytes encapsulated in lipid enveloped droplets, and the fourth known concentration of the analytes is different than the first known concentration, second known concentration, and the third known concentration of the analytes. Embodiments may further comprise at least a fifth reagent, wherein the fifth reagent comprises a fifth known concentration of the analytes encapsulated in lipid enveloped droplets, and the fifth known concentration of the analytes is different than the first known concentration, second known concentration, the third known, and the fourth known concentration of the analytes.

[014] In embodiments, the fluorescent dye comprises a small organic dye, a phycobiliprotein, quantum dots, a polymer dye, a fluorescent protein, a tandem dye, or a combination thereof. In embodiments, the fluorescent dye is capable of excitation by a laser channel between 325 to 808 nm. In embodiments, the fluorescent dyes are capable of being excited by lasers having the following wavelengths: 355 nm (UV), 405 nm (Violet), 488 nm (Blue), 561 nm (Y ellow-Green), 638 nm (Red), and 808 nm (Infrared).

[015] In embodiments, the lipid enveloped droplets are single-core double emulsions, single droplet double emulsion (DE), a double emulsion, a liposome, or a combination thereof. In embodiments, the lipid enveloped droplets are water-oil-water single droplet double emulsion. In embodiments, the lipid enveloped droplets are polymer-based vesicles.

[016] In embodiments, the lipid enveloped droplet reagents are produced in bulk using a microfluidic device. In embodiments, a microfluidic chip design controls the size of the lipid enveloped drops. In embodiments, the concentration of the analyte per drop is defined and is controlled by the microfluidic device.

[017] In embodiments, the lipid enveloped droplets have a diameter of between about 1 pm and about 1000 pm, of between about 3 pm and about 500 pm, of between about 5 pm and about 100pm, of between about 7 pm and about 50 pm, between about 10 pm and about 30 pm, or between about 15 pm and about 22 pm. In certain embodiments, the lipid enveloped droplets have a diameter between 0.5 pm and 2 pm. In certain embodiments, the lipid enveloped droplets have a diameter of greater than about 2 pm, greater than about 5 pm, greater than about 10 pm, greater than about 15 pm, greater than about 20 pm, greater than about 30 pm, greater than about 50 pm, or greater than about 100 pm. In certain embodiments, the lipid enveloped droplets have a diameter of less than about 100 pm, less than about 50 pm, less than about 30 pm, less than about 20 pm, less than about 15 pm, less than about 10 pm, or less than about 5 pm.

Methods for calibrating a flow cytometer.

[018] An embodiment of the present disclosure is a method for calibrating a flow cytometer using lipid enveloped droplets comprising (a) loading at least a first reagent, a second reagent, and a third reagent, wherein each reagent comprises a different known concentration of analytes encapsulated in lipid enveloped droplets, wherein the analytes have a fluorescent dye, into a flow cytometer; (b) making a calibration curve allowing a conversion of MFI values to MESF values; and optionally the calculation of ABC values. In some embodiments, the first reagent, the second reagent, and the third reagent are in the same vial or container and are added simultaneously.

[019] In certain embodiments, the evaluation in step (b) or (e) is performed with a plurality of lasers having the following wavelengths: 355 nm (UV), 405 nm (Violet), 488 nm (Blue), 561 nm (Yellow-Green), 638 nm (Red), and 808 nm (Infrared). In an embodiment, a method for calibrating a flow cytometer using lipid enveloped droplets further comprises generating a calibration report following step (c).

[020] In certain embodiments, methods of the present disclosure further comprise the step (g) generating an Antibody Bound per Cell (ABC) value or Number of Fluorescent Molecules per Droplet using: know n internal volume of each droplet; know n concentration of encapsulated analyte; and/or known number of fluorophores per reagent. In embodiments, methods of the present disclosure further comprise the step of (h) generating a calibration report to determine the sensitivity of detecting cellular structures between 30-2000 nm. In embodiments, methods of the present disclosure further comprise the step of (i) loading a test sample to detect a fluorescently labeled cellular structure with a diameter between 30 - 2000 nm.

Kits for calibrating a flow cytometer.

[021] An embodiment of the present disclosure is a kit for performing a method for calibrating a flow cytometer using lipid enveloped droplets comprising (a) loading at least a first reagent, a second reagent, and a third reagent, wherein the first reagent comprises a first known concentration of analytes encapsulated in lipid enveloped droplets, wherein the second reagent comprises a second known concentration of analytes encapsulated in lipid enveloped droplets; wherein the third reagent comprises a third known concentration of analytes encapsulated in lipid enveloped droplets; wherein the analytes have a fluorescent dye, into a flow' cytometer; (b) evaluating fluorescence sensitivity of the first, the second, and the third reagents, making a calibration curve allow ing a conversion of MFI values to MESF values; and optionally the calculation of ABC values, the kit comprising a system for calibrating a flow cytometer using lipid enveloped droplets comprising a first reagent, a second reagent, and a third reagent, wherein the first, second, and third reagents comprises a known concentration of analytes encapsulated in lipid enveloped droplets, wherein the analytes have a fluorescent dye; at least one vial to hold the first reagent; at least one vial to hold the second reagent; at least one vial to hold the third reagent; and instructions for using the kit. In some embodiments, at least two of the first reagent and the second reagent and the third reagent are held in the same vial.

[022] Embodiments of kits include using any one of the systems in clauses 1-13 in combination with any one of the methods in clauses 14-19.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows an embodiment of a presently disclosed method for calibrating a flow cytometer using lipid enveloped droplets.

FIG. 2 shows an embodiment of a presently disclosed method. More specifically, FIG. 2 show s good encapsulation of an antibody may via clear phase separation w here 100 is the clear media following separation, 102 is the line between the clear media and the cloudy encapsulated antibody, and 104 is the cloudy encapsulated antibody. FIG. 3 shows an embodiment of the present disclosure and demonstrates that both single and double emulsions were achieved and differentiable on a CytoFLEX VBR Analyzer.

FIG. 4 shows an embodiment of the present disclosure and demonstrates successful identification of double emulsions using a CytoFLEX VBR Analyzer.

DETAILED DESCRIPTION

[023] While the concepts of the present disclosure are illustrated and described in detail in the descriptions herein, results in the description are to be considered as examples and not restrictive in character; it being understood that only the illustrative embodiments are shown and described and that all changes and modifications that come within the spirit of the disclosure are desired to be protected.

[024] Unless defined otherwise, the scientific and technology nomenclatures have the same meaning as commonly understood by a person in the ordinary skill in the art pertaining to this disclosure.

[025] It w ill be understood by one of ordinary skill in the relevant arts that other suitable modifications and adaptations to the systems, methods, and kits described herein are readily apparent from the description of the disclosure contained herein in view of information known to the ordinarily skilled artisan, and may be made without departing from the scope of the disclosure or any embodiment thereof.

[026] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, preferred methods and materials are now described.

Definitions.

[027] As used herein, “g” represents gram; “L” represents liter; “mg” represents “milligram (10-3 gram);” “mL” or “cc” represents milliliter (10-3 liter). One “pL” equals to one microliter (10-6 liter). The unit of temperature used herein is degree Celsius (°C).

[028] The term “about” is used in conjunction with numeric values to include normal variations in measurements as expected by persons skilled in the art, and is understood to have the same meaning as “approximately” and to cover a typical margin of error, such as ±15%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the stated value. Whether or not modified by the term “about,” the claims include equivalents to the quantities. [029] It should be noted that, as used in this specification and the appended claims, the singular forms “a,” "an." and "the" include plural referents unless the content clearly dictates otherwise. For example, reference to “a method” includes having two or more methods that are either the same or different from each other. It should also be noted that the term “or” is generally employed in its sense including “and/or” unless the content clearly dictates otherwise. As used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[030] In the interest of brevity and conciseness, any ranges of values set forth in this specification contemplate all values within the range and are to be construed as support for claims reciting any sub-ranges having endpoints which are real number values within the specified range in question. By w ay of a hypothetical illustrative example, a disclosure in this specification of a range of from 1 to 5 shall be considered to support claims to any of the following ranges: 1-5; 1-4; 1-3; 1-2; 2-5; 2-4; 2-3; 3-5; 3-4; and 4- 5.

[031] The term “substantially” or “about” is utilized herein to represent the inherent degree of uncertainty that can be attributed to any quantitative comparison, value, measurement, or other representation. The term “substantially” or “about” is also utilized herein to represent the degree by which a quantitative representation can vary from a stated reference without resulting in a change in the basic function of the subject matter at issue.

[032] The term “comprise,” “comprises,” and “comprising” as used herein, specify the presence of the stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.

[033] The term “calibrate” or “calibrating” or “calibration” as used herein, specifies the use of the disclosed composition, methods, and kits to convert MFI to MESF value or calculate ABC.

General Description.

[034] An embodiment of the present disclosure is a system for calibrating a flow cytometer using lipid enveloped droplets comprising a first reagent, wherein the first reagent comprises a first known concentration of analytes encapsulated in lipid enveloped droplets, a second reagent, wherein the second reagent comprises a second know n concentration of the analytes encapsulated in lipid enveloped droplets and the second known concentration of the analytes is different than the first known concentration of the analytes; a third reagent, wherein the third reagent comprises a third known concentration of the analytes encapsulated in lipid enveloped droplets and the third know n concentration of the analytes is different than the first known concentration and second known concentration of the analytes; wherein the analytes have a fluorescent dye. In embodiments, the analytes encapsulated in lipid enveloped droplets comprise a fluorescent dye. a fluorescent dye conjugated to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), protein, antibody, antibody fragment, nanobody, a hydrophilic partner that can prevent the analyte from crossing the lipid envelop of the droplet, or a combination thereof. In embodiments, the analytes can include a single analyte, at least two analytes, at least three analytes, or at least four analytes. In certain embodiments, an analyte can be a hybrid of a fluorescent dye, DNA, RNA, protein, another hydrophilic partner, or antibody, e.g., a DNA-antibody conjugate. In certain embodiments, the analytes comprise a known concentration of a single analyte, a known concentration of more than one analyte, wherein all the analytes in the first reagent have the same concentration, or a known concentration of at least one analyte, at least two analytes, at least three analytes, or at least four analytes, wherein the analytes have different concentrations in the first reagent.

[035] In embodiments, the analytes encapsulated in lipid enveloped droplets comprise a fluorescent dye, protein-fluorescent dye conjugates, antibody-fluorescent dye conjugates, antibody fragment-fluorescent dye conjugates, nanobody-fluorescent dye conjugates, DNA-fluorescent dye conjugates, RNA-fluorescent dye conjugates, other hydrophilic partner -fluorescent dye conjugates, or a combination thereof. As discussed above, in embodiments, the analytes (e.g.. fluorescent dye, protein-fluorescent dye conjugates, antibody-fluorescent dye conjugates, antibody fragment-fluorescent dye conjugates, nanobody-fluorescent dye conjugates, DNA-fluorescent dye conjugates, RNA-fluorescent dye conjugates, other hydrophilic partner-fluorescent dye conjugates, can include a single analyte, at least two analytes, at least three analytes, or at least four analytes. In certain embodiments, the analytes comprise a known concentration of a single analyte, a known concentration of more than one analyte, wherein all the analytes in the first reagent have the same concentration, or a known concentration of at least one analyte, at least two analytes, at least three analytes, or at least four analysts, wherein the analytes have different concentrations in the first reagent. [036] Embodiments may further comprise at least a fourth reagent, wherein the fourth reagent comprises a fourth know n concentration of the analytes encapsulated in lipid enveloped droplets, and the fourth know n concentration of the analytes is different than the first known concentration, second known concentration, and the third known concentration of the analytes. Embodiments may further comprise at least a fifth reagent, wherein the fifth reagent comprises a fifth known concentration of the analytes encapsulated in lipid enveloped droplets, and the fifth known concentration of the analytes is different than the first known concentration, second known concentration, the third known, and the fourth know n concentration of the analytes.

[037] In embodiments, each of the reagents comprise the same analytes, e.g., each reagent includes a known concentration of an analyte, such as an antibody-fluorescent dye conjugate. In such embodiments, the at least two or more reagents can be provided individually at known concentrations of analytes or can be provided at a first known concentrated concentration and subsequent reagents (e.g., second reagent, third reagent, etc.) can be created via dilution of the first known concentrated concentration using appropriate buffers. In certain embodiments, the first reagent having a first known concentration of analyte and additional reagents having additional known concentrations have completely different analytes, or a population of analytes that are different between at least one reagent and a population of overlapping analytes w ith at least one reagent.

[038] In embodiments, the fluorescent dye comprises a small organic dye, a phycobiliprotein, quantum dots, a polymer dye, a fluorescent protein, a tandem dye, or a combination thereof. In embodiments, the fluorescent dye is at least one fluorescent dye, at least two fluorescent dyes, at least three fluorescent dyes, at least four fluorescent dyes, at least five fluorescent dyes, at least six fluorescent dyes, or at least seven fluorescent dyes. In certain embodiment, all the dyes are the same type of dye, e.g., a fluorescent protein, while exciting at the same or different wavelengths. In other embodiments, the at least two fluorescent dyes comprise more than one type of fluorescent dye, more than two types of fluorescent dyes, more than three types of fluorescent dyes, or more than four types of fluorescent dyes, wherein at least tw o of the fluorescent dyes excite at different wavelengths.

[039] In embodiments, the fluorescent dye is capable of excitation by a laser channel between 325 to 808 nm. In embodiments, the fluorescent dyes are capable of being excited by one or more lasers having the following wavelengths: 355 nm (UV), 405 nm (Violet), 488 nm (Blue), 561 nm (Yellow-Green), 638 nm (Red), 808 nm (Infrared), or any combination thereof.

[040] In embodiments, the lipid enveloped droplets are single-core double emulsions, single droplet double emulsion (DE), a double emulsion, a liposome, or a combination thereof. In embodiments, the lipid enveloped droplets are water-oil-water single droplet double emulsion. In embodiments, the lipid enveloped droplets are polymer-based vesicles. In embodiments, the lipid enveloped droplets are a combination of water-oil- water single droplet double emulsion and polymer particles. In certain embodiments, the lipid enveloped droplets are substantially free of single droplet single emulsion droplets or single droplet double emulsion droplets.

[041] In embodiments, the lipid enveloped droplets have a diameter of between about 1 pm and about 1000 pm, of between about 3 pm and about 500 pm, of between about 5 pm and about 100pm, of between about 7 pm and about 50 pm, between about 10 pm and about 30 pm, or between about 15 pm and about 22 pm. In embodiments, the lipid enveloped droplets have a diameter of between 1 nm and 1 pm, or any range therein. The present disclosure contemplates any range within the above-mentioned ranges. In certain embodiments, the lipid enveloped droplets have a diameter of greater than about 1 nm, greater than about 100 nm, greater than about 500 nm, greater than about 1 pm, greater than about 2 pm, greater than about 5 pm, greater than about 10 pm, greater than about 15 pm. greater than about 20 pm, greater than about 30 pm, greater than about 50 pm, or greater than about 100 pm. The present disclosure contemplates any range within the above-mentioned ranges. In certain embodiments, the lipid enveloped droplets have a diameter of less than about 100 pm, less than about 50 pm. less than about 30 pm. less than about 20 pm. less than about 15 pm. less than about 10 pm, less than about 5 pm, less than about 2 pm, less than about 1 pm, less than about 500 nm, or less than about 50 nm. The present disclosure contemplates any range within the above-mentioned ranges.

[042] An embodiment of the present disclosure includes a method for calibrating a flow cytometer using lipid enveloped droplets of any one of clauses 1-13. For example, an embodiment of the present disclosure is a method for calibrating a flow cytometer using lipid enveloped droplets comprising (a) loading a first reagent, wherein the first reagent comprises a first known concentration of analytes encapsulated in lipid enveloped droplets, wherein the analytes have a fluorescent dye, into a flow cytometer; (b) evaluating fluorescence sensitivity of at least one fluorescent channel of at least one laser in the flow cytometer (c) making a calibration curi e allowing a conversion of MFI values to MESF values; and optionally the calculation of ABC values. In an embodiment, the evaluation step (b) happens concurrently with the (c) determining step. In certain embodiments, the evaluation step (b) is complete before the (c) determining step begins. Regardless of whether the evaluation step (b) is concurrent with the (c) determining step or complete before the (c) determining step begins, in both scenarios, the method can cycle (b)-(c)-(b)-(c) until the determining step reaches a conclusion, e.g., the generation of an MESF value.

[043] An embodiment includes evaluating fluorescence sensitivity of at least one fluorescent channel of at least one laser in the flow cytometer based a single peak analysis. Embodiments of methods of the present disclosure further comprise (d) loading at least one additional reagent into a flow cytometer; (e) evaluating at least one of the following: evaluating fluorescence sensitivity⁷ of at least one fluorescent channel of at least one laser in the flow cytometer based on fluorescence intensity analysis; and (f) based on the evaluations in step (e), determining at least one of the following using the know n concentration of the first reagent and the at least on additional reagent: (i) making a calibration curve allowing a conversion of MFI values to MESF values; and optionally the calculation of ABC values.

[044] As discussed above, in an embodiment, the evaluation step (e) happens concurrently with the (f) determining step. In certain embodiments, the evaluation step (e) is complete before the (f) determining step begins. Regardless of whether the evaluation step (e) is concurrent with the (f) determining step or complete before the (f) determining step begins, in both scenarios, the method can cycle (e)-(f)-(e)-(f) until the determining step reaches a conclusion, e.g., the generation of an MESF value. In certain embodiments, the evaluation in step (b) or (e) is performed with a plurality of lasers having the following wavelengths: 355 nm (UV), 405 nm (Violet), 488 nm (Blue), 561 nm (Yellow-Green), 638 nm (Red), and 808 nm (Infrared). In certain embodiments, the evaluation step (b) or (e) is performed with at least two lasers, at least three lasers, at least four lasers, at least five lasers, at least six lasers, or at least seven lasers. In certain embodiments, the evaluation step (b) is performed with a different number of lasers than the evaluation step (e). In such embodiments, step (e) may use more or less lasers than step (b) depending on the determination step (c).

[045] In an embodiment, a method for calibrating a flow cytometer using lipid enveloped droplets further comprises generating a calibration report following step (c) or following step (f), or following the last determination step in the calibration method. In certain embodiments, each calibration with each known concentration of analyte in each reagent, e.g., first reagent, second reagent, etc. results in a calibration report. The calibration report may be displayed on the display screen of the flow cytometer. In embodiments, the calibration report is stored in memory' of the flow cytometer with relevant calibration parameters (e.g., reagent, analyte, analyte concentration, dyes, etc.) for future reference, use. or printing.

[046] In certain embodiments, methods of the present disclosure further comprise the step (g) generating an Antibody Bound per Cell (ABC) value or Number of Fluorescent Molecules per Droplet using: known internal volume of each droplet; known concentration of encapsulated analyte; and/or known number of fluorophores per reagent, or some combination thereof. In certain embodiments, the step of (g) generating an Antibody Bound per Cell (ABC) value or Number of Fluorescent Molecules per Droplet occurs after running the first reagent, after running the second reagent, after running the third reagent, after running the fourth reagent, after running the fifth reagent, or some combination thereof. In an embodiment, step (g) generates both an Antibody Bound per Cell (ABC) value or Number of Fluorescent Molecules per Droplet using one or more of the known internal volume of each droplet; known concentration of encapsulated analyte; the known number of fluorophores per reagent. [047] In embodiments, methods of the present disclosure further comprise the step of (h) generating a calibration report to determine the sensitivity of detecting cellular structures betw een 30-2000 nm. In embodiments, methods of the present disclosure further comprise the step of (i) loading a test sample to detect a fluorescently labeled cellular structure with a diameter between 30 - 2000 nm.

[048] An embodiment of the present disclosure is a kit for performing a method for calibrating a flow cytometer using lipid enveloped droplets comprising (a) loading a first reagent, wherein the first reagent comprises a first know n concentration of analytes encapsulated in lipid enveloped droplets, wherein the analytes have a fluorescent dye, into a flow cytometer; (b) evaluating at least one of the following: evaluating fluorescence sensitivity of at least one fluorescent channel of at least one laser in the flow cytometer based on a single peak analysis; and based on the evaluations in step (b), determining at least one of the following using the know n concentration of the first reagent: (i) an MESF value the kit comprising a system for calibrating a flow cytometer using lipid enveloped droplets comprising a first reagent, wherein the first reagent comprises a first known concentration of analytes encapsulated in lipid enveloped droplets, wherein the analytes have a fluorescent dye; at least one vial to hold the first reagent; and instructions for using the kit.

[049] In an embodiment, kits may further comprise at least a second vial or additional vials to hold the at least one additional reagent. In certain embodiments, the vials are each lOmls. In embodiments, a kit may include more than 1 vial, more than 2 vials, more than 3 vials, more than 4 vials, or more than 5 vials. In embodiments, a kit may include less than 5 vials, less than 4 vials, less than 3 vials, or less than 2 vials. The vials may be of appropriate size and the vials within the kit may be of different sizes. In certain embodiments, the vial arrives prefilled with known concentrations of reagents while in other embodiments, some vials are empty and designed for diluting a known concentration of reagent to create additional known concentrations of the same reagent. [050] Embodiments of kits include using any one of the systems in clauses 1-13 in combination with any one of the methods in clauses 14-19. For example, in an embodiment, a kit may include at least a second reagent, wherein the second reagent comprises a second known concentration of the analytes encapsulated in hpid enveloped droplets, and the second known concentration of the analytes is different than the first known concentration of the analytes. As an additional example, in an embodiment, the kit may include antibody -fluorescent dye conjugates encapsulated in the hpid enveloped droplets.

[051] Having described the aspects and implementations of the present disclosure, modifications and equivalents of the disclosed concepts may readily occur to one skilled in the art. However, it is intended that such modifications and equivalents be included within the scope of the claims which are appended hereto.

EXAMPLES

Example 1

[052] An embodiment of the presently disclosed method for calibrating a flow cytometer using hpid enveloped droplets is shown in FIG. 1. Referring to FIG. 1, which depicts an exemplary calibration curve based on the fluorescence sensitivity values of three reagents (Reagent 1, Reagent 2, and Reagent 3), each Reagent containing a different known concentration of Analyte A. The exemplary line connecting the three Reagent values can be used to directly calculate MESF for Analyte A based on the MFI. [053] Preparing Droplets: a 10 uM first reagent solution of antibody conjugated with dye is prepared. In addition, 10-fold serial dilutions (second reagent, third reagent, fourth reagent, etc.) of the 1 M solution of antibody conjugated with dye are prepared based on the desired calibration range, e.g., 1 nm, 10 nm, 100 nm and/or 1 pm. Then a Discover Xdrop DE20 microfluidic droplet generator (available from Samplix Inc., Bregnerodvej 96, 3460 Birkerod, Denmark) is used to generate droplets encapsulating the 10 uM solution and/or each of 10-fold serially diluted solutions. The droplets have a 15 pm internal diameter and 1.6 pL internal volume.

[054] Each droplet of an encapsulated 1 nM solution of antibody conjugated molecules will contain 6.02*10²³ x 1.6*10'¹² x 10'⁹ =1000 molecules of antibody.

[055] Data receptor density’ on the cell is in the range of 10,000 to 200,000. Accordingly, serial dilutions ranging from 1 nM to 1 pM solutions of antibody covers the entire working calibration range from 1,000 to 1,000,000 molecules.

[056] A 10-fold reduction in internal diameter of a lipid enveloped droplet, which can be controlled by during the generation process, results in a 1000-fold reduction in internal volume of each droplet. Accordingly, each droplet will have an antibody conjugate concentration necessary’ to calibrate a range of 10 to 10,000 molecules, which is suitable for nano-flow cytometer calibration.

[057] Calibration and Measurement: Referring to FIG. 1, a first reagent of antibody conjugated with dye having a known concentration (1 nM) is loaded into a flow cytometer and the sample is run. The MFI value of the droplet population is recorded.

[058] A second reagent of antibody conjugated with dye having a known concentration (10 nM) is loaded into a flow cytometer and the sample is run. The MFI value of the droplet population is recorded.

[059] A third reagent of antibody conjugated with dye having a known concentration (100 nM) is loaded into a flow cytometer and the sample is run. The MFI value of the droplet population is recorded. Referring to FIG. 1, a MFI-MRESF calibration curve is generated that allow to convert MFI to MESF values. In addition, an Antibody Bound per Cell (ABC) value can be calculated directly from the MFI of the cell population stained with the same antibody as used in the drop.

Example 2

[060] An embodiment for encapsulating fluorescent mAb follows.

[061] Preparing droplets: six 3-fold dilutions of SJ1D1 anti-CD13-PE and undiluted mAb at IpM were prepared using a Discover Xdrop DE20 microfluidic droplet generator (available from Samplix Inc., Bregnerodvej 96, 3460 Birkerod. Denmark). Good encapsulation may be identified by clear phase separation as shown in FIG. 2 where 100 is the clear media following separation, 102 is the line between the clear media and the cloudy encapsulated antibody, and 104 is the cloudy encapsulated antibody.

[062] Calibration and measurement: Encapsulated antibody was serially diluted as shown in Table 1 and the samples were run on a CytoFLEX VBR Analyzer with stock filters.

Table 1: Dilutions and Measurements

[063] ** indicates preps that failed and were unable to be run. The data from Table 1 is shown in the graph in FIG. 3. As shown in Table 1 and FIG. 3, both single and double emulsions were achieved and differentiable on a CytoFLEX VBR Analyzer. More specifically, using Tube 2 as an example, double emulsions are differentiable from single emulsions. FIG. 4 show additionally shows the ability to differentiate emulsions of this Example on a CytoFLEX VBR Analyzer.

CLAUSES

[064] The following numbered clauses define further example aspects and features of the present disclosure: A system for calibrating a flow cytometer using lipid enveloped droplets comprising: a first reagent, wherein the first reagent comprises a first know n concentration of analytes encapsulated in lipid enveloped droplets; a second reagent, wherein the second reagent comprises a second known concentration of the analytes encapsulated in lipid enveloped droplets and the second known concentration of the analytes is different than the first known concentration of the analytes; a third reagent, wherein the third reagent comprises a third known concentration of the analytes encapsulated in lipid enveloped droplets and the third known concentration of the analytes is different than the first known concentration and second known concentration of the analytes; wherein the analytes have a fluorescent dye. The system of clause 1, further comprising at least a fourth reagent, wherein the fourth reagent comprises a fourth known concentration of the analytes encapsulated in lipid enveloped droplets, and the fourth known concentration of the analytes is different than the first known concentration, second known concentration, and the third known concentration of the analytes, and/or at least a fifth reagent, wherein the fifth reagent comprises a fifth known concentration of the analytes encapsulated in lipid enveloped droplets, and the fifth known concentration of the analytes is different than the first known concentration, second known concentration, the third known, and the fourth known concentration of the analytes. The system of any one of clauses 1-2, wherein the analytes encapsulated in lipid enveloped droplets comprise a fluorescent dye, a fluorescent dye conjugated to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), protein, antibody, antibody fragment, nanobody, a hydrophilic partner that can prevent the analyte from crossing the lipid envelop of the droplet, or a combination thereof. The system of any one of clauses 1-3, wherein the analytes encapsulated in lipid enveloped droplets comprise a fluorescent dye, protein-fluorescent dye conjugates, antibody-fluorescent dye conjugates, antibody fragment-fluorescent dye conjugates, nanobody-fluorescent dye conjugates, DNA-fluorescent dye conjugates, RNA-fluorescent dye conjugates, hydrophilic partner-fluorescent dye conjugates, or a combination thereof. The system of any one of clauses 1-4, wherein the fluorescent dye comprises a small organic dye, a phycobiliprotein, quantum dots, a polymer dye, a fluorescent protein, a tandem dye, or a combination thereof. The system of any one of clauses 1-5, wherein the fluorescent dye is capable of excitation by a laser channel between 325 to 808 nm. The system of clause 6, wherein the fluorescent dyes are capable of being excited by lasers having the following wavelengths: 355 nm (UV), 405 nm (Violet), 488 nm (Blue), 561 nm (Yellow-Green), 638 nm (Red), and 808 nm (Infrared). The system of any one of clause 1-7, wherein the lipid enveloped droplets are single-core double emulsions, single droplet double emulsion (DE), a double emulsion, a liposome, or a combination thereof. The system of any one of clause 1-8, wherein the lipid enveloped droplets are water-oil -water single droplet double emulsion. The system of any one of clauses 1-9, wherein the lipid enveloped droplets are polymer-based vesicles. The system of any one of clauses 1-10, wherein the lipid enveloped droplets have a diameter of between about 1 pm and about 1000 pm, of between about 3 pm and about 500 pm, of between about 5 pm and about 100pm, of between about 7 pm and about 50 pm, between about 10 pm and about 30 pm, or between about 15 pm and about 22 pm. The system of any one of clauses 1-11, wherein the lipid enveloped droplets have a diameter of greater than about 2 pm, greater than about 5 pm, greater than about 10 pm, greater than about 15 pm, greater than about 20 pm, greater than about 30 pm, greater than about 50 pm, or greater than about 100 pm. The system of any one of clauses 1-12, wherein the lipid enveloped droplets have a diameter of less than about 100 pm, less than about 50 pm, less than about 30 pm, less than about 20 pm, less than about 15 pm, less than about 10 pm, or less than about 5 pm. A method for calibrating a flow cytometer using lipid enveloped droplets comprising: (a) loading the first reagent and the second reagent and the third reagent of any one of clauses 1-13 into a flow cytometer;

(b) evaluating fluorescence sensitivity of at least one fluorescent channel of at least one laser in the flow cytometer of the first reagent, the second reagent, and the third reagent

(c) based on the evaluations in step (i), determining a calibration curve;

(d) using the calibration curve to determine median fluorescent intensity (MFI) values, Molecules of Equivalent Soluble Fluorochrome (MESF) values, antibody per cell (ABC) values, or a combination thereof. The method of clauses 14, wherein the evaluation in step (b) is performed with a plurality of lasers having the following wavelengths: 355 nm (UV). 405 nm (Violet), 488 nm (Blue), 561 nm (Yellow-Green), 638 nm (Red), and 808 nm (Infrared). The method of any one of clauses 14-15, further comprising: generating a calibration report following step (c). The method of any one of clauses 14-16, wherein generating an Antibody- Bound per Cell (ABC) value or Number of Fluorescent Molecules (MESF) per Droplet comprises using: a. a known internal volume of each droplet; b. a known concentration of encapsulated analyte; and/or c. a known number of fluorophores per reagent. The method of any one of clauses 14-17, further comprising, (e) loading a test sample to detect a fluorescently labeled cellular structure with a diameter between 30 - 2000 nm. A kit for performing the method according to any one of clauses 14-19, the kit comprising: a system of any one of clauses 1-13; at least one vial to hold the first reagent, the second reagent, and the third reagent; and instructions for using the kit.

Claims

CLAIMS What is claimed is:

1. A system for calibrating a flow cytometer using lipid enveloped droplets comprising: a first reagent, wherein the first reagent comprises a first known concentration of analytes encapsulated in lipid enveloped droplets; a second reagent, wherein the second reagent comprises a second known concentration of the analytes encapsulated in lipid enveloped droplets and the second known concentration of the analytes is different than the first known concentration of the analytes; a third reagent, wherein the third reagent comprises a third known concentration of the analytes encapsulated in lipid enveloped droplets and the third known concentration of the analytes is different than the first known concentration and second know n concentration of the analytes; wherein the analytes have a fluorescent dye.

2. The system of claim 1, further comprising at least a fourth reagent, wherein the fourth reagent comprises a fourth known concentration of the analytes encapsulated in lipid enveloped droplets, and the fourth known concentration of the analytes is different than the first known concentration, second know n concentration, and the third known concentration of the analytes, and/or at least a fifth reagent, wherein the fifth reagent comprises a fifth known concentration of the analytes encapsulated in lipid enveloped droplets, and the fifth known concentration of the analytes is different than the first known concentration, second known concentration, the third known, and the fourth known concentration of the analytes.

3. The system of any one of claims 1-2, wherein the analytes encapsulated in lipid enveloped droplets comprise a fluorescent dye, a fluorescent dye conjugated to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), protein, antibody, antibody fragment, a hydrophilic partner that can prevent the analyte from crossing the lipid envelop of the droplet, a nanobody, protein-fluorescent dye conjugates, antibody-fluorescent dye conjugates, antibody fragment-fluorescent dye conjugates, nanobody-fluorescent dye conjugates. DNA- fluorescent dye conjugates, RNA-fluorescent dye conjugates, hydrophilic partner-fluorescent dye conjugates, or a combination thereof.

4. The system of any one of claims 1-3, wherein the fluorescent dye comprises a small organic dye, a phycobiliprotein, quantum dots, a polymer dye, a fluorescent protein, a tandem dye, or a combination thereof.

5. The system of any one of claims 1-4, wherein the fluorescent dye is capable of excitation by a laser channel between 325 to 808 nm.

6. The system of claim 5, wherein the fluorescent dyes are capable of being excited by lasers having the following wavelengths: 355 nm (UV), 405 nm (Violet), 488 nm (Blue), 561 nm (Yellow-Green), 638 nm (Red), and 808 nm (Infrared).

7. The system of any one of claims 1-6, wherein the lipid enveloped droplets are water-oil -water single droplet double emulsion.

8. The system of any one of claims 1-7. wherein the lipid enveloped droplets are polymer-based vesicles.

9. The system of any one of claims 1-9, wherein the lipid enveloped droplets have a diameter of between about 1 pm and about 1000 pm, of between about 3 pm and about 500 pm, of between about 5 pm and about 100pm, of between about 7 pm and about 50 pm, between about 10 pm and about 30 pm, or between about 15 pm and about 22 pm.

10. A method for calibrating a flow cytometer using lipid enveloped droplets comprising:

(a) loading the first reagent, the second reagent, and the third reagent of any one of claims 1-13 into a flow cytometer;

(b) evaluating fluorescence sensitivity of at least one fluorescent channel of the first reagent, the second reagent, and the third reagent; and

(c) based on the evaluations in step (b), determining a calibration curve; (d) using the calibration curve to determine median fluorescent intensity (MFI) values, Molecules of Equivalent Soluble Fluorochrome (MESF) values, antibody per cell (ABC) values, or a combination thereof.

11. The method of claim 10, wherein the evaluation in step (b) is performed with a plurality of lasers having the following wavelengths: 355 nm (UV). 405 nm (Violet), 488 nm (Blue). 561 nm (Yellow-Green). 638 nm (Red), and 808 nm (Infrared).

12. The method of any one of claims 10-11, further comprising: generating a calibration report following step (c) to determine the sensitivity of detecting cellular structures between 30-2000 nm.

13. The method of any one of claims 10-12, wherein generating an Antibody Bound per Cell (ABC) value or Number of Fluorescent Molecules (MESF) per Droplet comprises using: a. a known internal volume of each droplet; b. a known concentration of encapsulated analyte; and/or c. a know n number of fluorophores per reagent.

14. The method of any one of claims 10-13, further comprising, (e) loading a test sample to detect a fluorescently labeled cellular structure with a diameter between 30 - 2000 nm.

15. A kit for performing the method according to any one of claims 10-14, the kit comprising: a system of any one of claims 1-9; at least one vial to hold the first reagent, the second reagent, and the third reagent; and instructions for using the kit.