JP2024503238A - Optofluidic array for radical protein footprinting - Google Patents

Abstract

Systems and methods for in vivo and in vitro radical protein footprinting using optofluidic arrays are presented. These teachings may be used, for example, to study three-dimensional protein structure or biokinetics. Radical dosimetry including any specific standard is used. Real-time feedback based on internal standards provides comparability between different experiments, and in vivo and in vitro analysis results yield data representative of real biological conditions. [Selection diagram] Figure 3

Description

This application claims the benefit of U.S. Provisional Application No. 63/128,439, filed on December 21, 2020, and U.S. Nonprovisional Application No. 17/193,913, filed on March 5, 2021. It is also a continuation in part of PCT/US20/12430, filed on January 6, 2020, and a continuation in part of PCT/US19/57059, filed on October 18, 2019. It is also a continuation of the section. Both PCT/US20/12430 and PCT/US19/57059 are continuations-in-part of U.S. Nonprovisional Patent Application No. 16/316,006, filed on January 7, 2019, and are now U.S. Pat. No. 10,816,468. U.S. Non-Provisional Application No. 16/316,006 is based on U.S. Provisional Patent Application No. 62/747,247, filed on October 18, 2018, and U.S. Provisional Application No. 62/788, filed on January 4, 2019. Claim the benefit of No. 219. PCT/US19/57059 further directly claims the benefit of U.S. Provisional Patent Application No. 62/747,247, filed on October 18, 2018. Both PCT/US20/12430 and PCT/US19/57059 claim the benefit of US Provisional Patent Application No. 62/788,219, filed on January 4, 2019. All of the above patent applications are incorporated herein by reference.

This invention was made with government support under grants R43 GM137728, R43 GM125420, and R44 GM125420 awarded by the National Institute of General Medical Sciences. The Government has certain rights in this invention.

The present invention relates to an apparatus and methodology for conformational analysis of biomolecules by the process of radical protein footprinting. Some embodiments of the invention relate to identifying the tertiary and quaternary structure and associated conformations of biopharmaceuticals, using integrated optical and microfluidic chips to identify higher order biomolecular structures of proteins. Using improved equipment and methodology for flash photooxidation of processed proteins.

Any discussion of any research, publications, sales, or activities in this submission, including any documents submitted with this application, shall be construed as an admission that such work constitutes prior art. Shouldn't. Discussion of any activity, work, or publication herein is not an admission that such activity, work, or publication exists or is known in any particular jurisdiction.

A biosimilar is a therapeutic drug that is similar, but not identical, to an existing originator or reference product. Unlike small molecule drugs, biosimilars are not simply generic versions of the original product. Traditional generics are considered therapeutically and molecularly equivalent to their originator products. This is simply not the case with biosimilars, which are complex three-dimensional biomolecules whose heterogeneity and dependence on production within living cells are quite different from conventional medicines. The structure and functional activity of biopharmaceuticals are highly sensitive to the environment. The intended structure of a therapeutic agent is maintained by a delicate balance of factors including protein concentration, control of post-translational modifications, pH and co-solutes in the formulation, and production/purification scheme (Gabrielson, J.P.; Weiss IV , W.F., Technical decision-making with higher order structure data: starting a new dialogue; Journal of Pharmaceutical Sciences, 2015). Thus, the structure of biopharmaceuticals must be carefully maintained or undesirable and adverse pharmacological consequences may occur.

Adverse drug reactions (ADRs) of biopharmaceuticals are typically thought to be due to exaggerated pharmacological and facial reactions. The range of patient ADRs ranges from symptomatic irritation to morbidity and mortality. Although the pathogenesis of some ADRs may be due to the patient's pharmacogenomic susceptibility, many are thought to be due to the inherent properties of the therapeutic agent, which can lead to morbid and fatal consequences for the patient. , causing significant economic losses to the biopharmaceutical industry (Giezen, TJ; Schneider, CK, Safety assessment of biosimiles in Europe: a regulatory perspective; J. Generics and Biosimils Initiative Journal; 2014). Thus, the occurrence of catastrophic ADRs illustrates the need for improved assays for biopharmaceutical development and quality control.

To minimize ADRs and facilitate the development of biosimilars, the FDA, the Center for Drug Evaluation and Research, and the Center for Biologics Evaluation and Research are investigating protein higher order structures (HOS). ) has issued guidelines that emphasize the use of cutting-edge technology to assess quality considerations.
in demonstration bio-similarity of a therapeutic protein product to a reference
product;guidance for industry;USDepartment of Health and Human Services;J. Food and Drug Administration;Center for Drug Evaluation and Research;Center for Biologics Evaluation and Research Washington,DC;2015). HOS analysis involves the identification of the tertiary and quaternary structure and associated conformations of a given biomolecule. Such biomolecules include proteins and protein complexes that may or may not be considered biotherapeutic agents. Although various HOS assays exist today, their inability to reliably predict the efficacy and safety of biological therapies has been questioned, and there is a growing need for new and improved HOS assays. (Gabrielson, JP; Weiss IV, WF, Technical decision-making with higher order structure data: starting a new dialogue; Journal of pharmaceutical sciences; 2015).

A technique that addresses the unmet need for HOS analysis is radical protein footprinting, which relies on irreversible protein hydroxylation in combination with mass spectrometry (MS) (Hambly, DM; Gross, ML, Laser flash photolysis of hydrogen peroxide to oxidize protein solvent-accessible residues on the microsecond timescale;Journal of the American Society for Mass Spectrometry;2005).This process is based on hydroxyl radical protein foot-printing. A variety of techniques are used to perform HRPE. Perhaps the most widely used approach uses a single, high-flux, short pulse of UV light. It relies on fast photochemical oxidation of proteins (FPOP), which generates hydroxyl (OH) radicals from hydrogen peroxide (H ₂ O ₂ ). The reaction typically inserts a net oxygen atom into the amino acid. The OH radical is short-lived and, when generated by a short UV pulse, undergoes a conformational change by the labeled protein. Before the reaction between amino acids and radicals can be completed (Konermann, L.; Tong, X.; Pan, Y., Protein structure and dynamics studied by mass spectrometry:H/D exchange, hydroxyl radical labeling, and related approaches;Journal of mass spectrometry;2008).The mass spectra of the peptide products of enzymatic digestion show different levels of oxidation, characterized by peak shifts such as 16Da, 32Da, 48Da.This information It can be used to estimate which peptides are located outside the HOS, thus leading to a deeper understanding of the HOS.

Recently, Gross and coworkers demonstrated another radical protein footprinting technique based on the generation of trifluoromethyl (CF ₃ ) radicals formed by hydroxyl radical attack of aqueous sodium trifinate (Cheng, M. .et al.,Laser-Initiated Radical Tirfluoromethylation of Peptides and Proteins:Application to Mass-S
pectrometry-Based Protein Footprinting; Angewandte Chemie International Edition, 2017, 56(45):p.14007-14010). When proteins are labeled via the _CF3 radical, the mass spectra of their peptide products are shifted by +69 Da. When compared to OH radical attack, _CF3 radicals have been shown to be highly complementary in amino acid reaction kinetics, and when used in combination with OH radicals attack surface exposed amino acids compared to OH radicals alone. provides excellent coverage. As in the case of HRPF, _CF3 labeling information can be used to infer which peptides are located on the outside of the protein, thus leading to a deeper understanding of protein HOS.

A technical limitation of HRPF that adversely affects comparative studies is due to the reaction of OH radicals with non-analyte components in the sample, such as buffer components, initial solutes, and foreign biological materials. Variability in background removal ratios causes test-to-test nonreproducibility, which has limited comparative studies (Niu, B. et al.; Dosimetry determines the initial OH radical concentration
in fast photochemical oxidation of proteins (FPOP); Journal of the American Society for Mass Spectrometry; 2015). Although OH radicals are excellent probes of protein topography, they also react with many compounds included in analytical preparations. Since competition for free OH radicals exists between the analyte protein and the background scavenger, measure the effective concentration of radicals available to oxidize the target protein to ensure reproducible results. This is desirable. Similarly, variability in background subtraction also negatively impacts the reproducibility of _CF3 radical footprinting methods. In photochemistry, the effective radical concentration is measured using a radical dosimeter internal standard. Ideally, a dosimeter would have a simple relationship between effective radical concentration and dosimeter response, a simple, rapid and non-destructive means of measurement, and would be suitable for most proteins. Non-reactive.

Prior art teaches radical dosimetry performed using added peptide internal standards (Niu, B., et al., Dosimetry determines the initial OH radical concentration in fast photochemical oxidation of proteins (FPOP). J Am Soc Mass Spectrom,2015. 26(5):p.843-6;Niu,B.,et al.,Incorporation of a Reporter Peptide in FPOP Compensates for Adventitious Scavengers and Allows Time-Dependent Measurements. J Am Soc Mass (Xie, B.; Sharp , JS, Hydroxyl radical Dosimetry for High Flux Hydroxyl radical Protein Foot-printing Applications using a Simple Optical Detection Method. Analytical chemistry 2015, 87(21), 10719-23.). In peptide radical dosimetry, the labeled peptide and target protein are subsequently analyzed using LC-MS (selectively with proteolysis) to assess the relative ratio of oxidized peptide to oxidized target protein. If the desired peptide-to-protein oxidation ratio is not achieved, repeat the entire experiment, adjusting the concentration of H ₂ O ₂ as needed to increase or decrease the effective OH radical load. For adenine dosimetry, the effective change in adenine UV absorbance is determined upon completion of the labeling process, and the ratio of the achieved adenine UV absorbance change to the target adenine UV absorbance change is determined. The H ₂ O ₂ concentration is then varied according to the desired change in UV absorbance. Both approaches are performed after labeling is complete and do not allow real-time adjustment of the effective OH radical loading, consuming valuable sample and unnecessarily wasting researcher time.

US Patent No. 10,816,468 and International Application PCT/US18/34682 teach a system and method for performing real-time radical dosimetry as the biologic is labeled during the FPOP HRPF process. While creating a real-time means to adjust and compensate for variations in background subtraction, the systems and methods herein require the addition of an external internal standard dosimeter to the biological mixture. Under some conditions, external internal standards can induce artifactual changes in the conformation of biomolecules, so the desired goal of providing a simple means of characterizing the nascent conformation of biologics is It may not be suitable. The disclosures of US Pat. No. 10,816,468 and PCT/US18/34682 are incorporated herein by reference.

US Provisional Application No. 62/747,247 and PCT/US19/57059 describe devices and methodologies that allow commonly used biological buffer systems to be used as internal standards in radical dosimeters. The photometric properties of some commonly used biological buffers change in a predictable manner upon OH radical attack. Therefore, these buffers can be used as internal standards in radical dosimeters, eliminating the need for adding external reagents, and the solvation properties of these buffers also It is well established to stabilize the structure and therefore does not change the biological conformation.

The aforementioned techniques describe devices and methods for performing HRPF radical dosimetry while labeling proteins or biopharmaceuticals in vitro. However, applying the results of in vitro structural experiments to true in vivo behavior has been questioned (Mourao, MA; Hakim, JB; Schnell, S., Connecting the dots: the effects of macromolecular crowding on cell physiology. Biophysical Journal 2014, 107(12), 2761-2766). Due to the shortcomings of in vitro HRPF, there is recent interest and desire to extend the use of HRPF to intact whole cells in an in vivo manner. For example, US Pat. No. 10,851,335B2 describes methods and methodologies by which in vivo HRPF can be performed. Briefly, multiple fused silica capillaries and microfluidic fittings are used to support mixing of buffer _suspended cells with _H2O2 . As taught, H ₂ O ₂ is rapidly taken up by cells without causing cell destruction, inducing apoptosis, or promoting cell death. However, the taught systems and methods still present various drawbacks. U.S. provisional patent application Ser. describes an apparatus and methodology by which improvements are made. Furthermore, it is a means by which the sheath flow rate and subsequent intercell spacing can be evaluated and ultimately controlled to ensure effective irradiation of suspended cells during the in vivo labeling process. is taught.

The aforementioned techniques perform in vitro or in vivo FPOP HRPF to label proteins or biopharmaceuticals using fused silica capillaries as a means for transport, establishment of sheath flow, chemical processing, and initiation of photoradical reactions. , further describes devices and methods that provide an optical field for probing the photometric properties of proteins, cells, chemical reagents, and labeled products. Although useful for these early stage experiments, fused silica capillaries have some undesirable characteristics. 1) Fragile and prone to accidental damage, especially if the outer protective polyimide coating is removed. 2) Requires removal of the ultra violet (UV) opaque polyimide coating to allow hydroxyl group radical formation from H ₂ O ₂ and compatible UV photometric dosimetry. 3) Requires considerable care to ensure proper alignment to the optical components. 4) Requires expensive and cumbersome microfluidic fittings, e.g. to create and form the fluid circuits needed to facilitate in vivo FPOP. 5) Replacement is troublesome. It is not uncommon for small ID (<100 nm) capillaries to become accidentally clogged or damaged during high flux applications. Thus, previously taught systems and methods present various drawbacks.

Various embodiments of the present invention improve current in vitro radical protein footprinting methods by providing a means to replace fused silica capillaries with optofluidic chip components that support online mixing of target proteins and labeling reagents. systems and methods that address the above-mentioned shortcomings of. The disclosed system includes a serpentine photolysis cell that substantially increases radiation dose during photoinduced radical chemistry compared to a capillary configuration. Various embodiments include an in-line dosimetry cell for real-time photometric evaluation of effective radical production and adjustment of unwanted background removal using the photometric properties of an in vitro radical dosimeter internal standard.

Various embodiments of the present invention improve current methods of in vivo radical protein footprinting by providing a means to replace fused silica capillaries with optofluidic chip components that support online mixing of target cells and labeling reagents. Systems and methods that address the above-mentioned shortcomings are included. Various embodiments support the generation of microfluidic sheath flow to allow precise control of intercellular distance and substantially increase irradiance during photoinduced radical chemistry when compared to capillary configurations. Using a serpentine photolytic cell or the like, allow effective and consistent irradiation of the external and intracellular parts of each cell. Various embodiments include an in-line dosimetry cell for real-time photometric evaluation of effective radical production and adjustment of unwanted background removal using the photometric properties of an in-vivo radical dosimeter internal standard. Various embodiments also provide a means by which cell singulation and division can be assessed and reproducibly controlled, as well as a means to determine the time of arrival of cells to the HRPF photolysis zone.

Various embodiments of the invention include improved embodiments for performing in vitro or in vivo flash photo-oxidation of proteins that enable advanced radical protein footprinting methods. On-chip systems and methods for analyzing the higher-order structure of In some embodiments, the present invention establishes closed-loop control between the flash photolysis system and the dosimeter to control the flash photolysis system in response to measured changes in the photometric properties of the internal standard radical dosimeter. Provides an in-line radical dosimetry system that controls the irradiance of

In some embodiments, the invention establishes closed-loop control between an automated in-line microfluidic mixing system and a dosimeter to adjust labeled reagents in response to measured changes in the photometric properties of an internal standard radical dosimeter. including on-chip, in-line, in-vivo, or in-vitro radical dosimetry systems that control the concentration of

In some embodiments, the present invention establishes closed-loop control between the flash photolysis system and the dosimeter to control the flash photolysis system in response to measured changes in the photometric properties of the internal standard radical dosimeter. an on-chip, in-line, in-vivo, or in-vitro radical dosimetry system that controls the irradiance of the radicals generated by photolysis of the labeled reagent.

In some embodiments, using an on-chip in vivo in-line radical dosimetry system, the invention includes a method of producing labeled biomolecules for analysis, the method comprising: (1) (2) introducing the cells into the optical dosimetry zone; (3) nascent cells; (4) irradiating the cells with at least one UV irradiation in a photolysis zone; (5) determining changes in photometric properties for the cells after irradiation; and (6) adjusting the spectral irradiance of the UV light source according to changes in radical dosimetry light characteristics.

In some embodiments, using an on-chip in vitro in-line radical dosimetry system, the invention includes a method of producing labeled biomolecules for analysis, the method comprising (1)
) mixing the target protein(s) with a biological buffer, an internal standard radical dosimeter, and other labeling reagents; (2) introducing the mixture into an optical dosimetry zone; ) determining the nascent photometric properties of the mixture; (4) irradiating the mixture with at least one UV irradiation in an optical photolysis zone; (5) changing the photometric properties of the mixture after irradiation. and (6) adjusting the spectral irradiance of the UV light source according to the change in the radical dosimetry light characteristics.

In some embodiments, using an on-chip in vivo in-line radical dosimetry system, the invention includes a method of producing labeled biomolecules for analysis, the method comprising: (1) (2) introducing the cells into the optical dosimetry zone; (3) nascent cells; (4) irradiating the cells with at least one UV irradiation in a photolysis zone; (5) determining changes in photometric properties for the cells after irradiation; and (6) adjusting the concentration of the labeled reagent using an in-line microfluidic mixer according to changes in the radical dosimetry optical properties.

In some embodiments, using an on-chip in vitro in-line radical dosimetry system, the invention includes a method of producing labeled biomolecules for analysis, the method comprising: (1) target protein (singular (2) introducing the mixture into an optical dosimetry zone; (3) nascent phase of the mixture; (4) irradiating the mixture with at least one UV irradiation in a photolysis zone; (5) determining changes in photometric properties for the mixture after irradiation; and (6) adjusting the concentration of the labeled reagent using an in-line microfluidic mixer according to the change in radical dosimetry optical properties;

In some embodiments, using an on-chip in vivo in-line radical dosimetry system, the invention includes a method of producing labeled biomolecules for analysis, the method comprising: (1) (2) introducing the cells into the optical dosimetry zone; (3) exiting the dosimetry zone. (4) determining the elapsed time between successive cell arrivals by monitoring the intensity of the scattered light; (5) determining the elapsed time and net buffer flow rate; (6) adjusting the sheath flow and buffer flow parameters to achieve the desired cell isolation volume.

In some embodiments, using an on-chip in vivo in-line radical dosimetry system, the invention includes a method of producing labeled biomolecules for analysis, the method comprising: (1) (2) introducing the cells into the optical dosimetry zone; (3) exiting the dosimetry zone. Detecting cell arrival and presence by monitoring changes in the phase of light; (4) determining elapsed time between successive cell arrivals; (5) elapsed time and net buffer flow rate. and (6) adjusting the sheath flow and buffer flow parameters to achieve the desired cell isolation.

In some embodiments, using an on-chip in vivo in-line radical dosimetry system, the invention includes a method of producing labeled biomolecules for analysis, the method comprising: (1) (2) introducing the cells into the optical dosimetry zone; (3) exiting the dosimetry zone. detecting the arrival and presence of cells by monitoring the intensity of the scattered light; (4) determining the net flow rate for arriving cells; (5) interconnections extending from the photolysis zone and the dosimetry zone. (6) determining the transit time required for cells to move from the photolysis zone to the dosimetry zone; (7) determining the photolysis zone arrival time for the cells. (8) operating the photolysis system at a time when all subsequent cells arrive at the photolysis zone.

In some embodiments, using an on-chip in vivo in-line radical dosimetry system, the invention includes a method of producing labeled biomolecules for analysis, the method comprising: (1) (2) introducing the cells into the optical dosimetry zone; (3) exiting the dosimetry zone. (4) determining the net flow rate for arriving cells; (5) determining the amount of interconnection extending from the photolysis zone and the dosimetry zone; (6) determining the transit time required for the cells to move from the photolysis zone to the dosimetry zone; (7) determining the photolysis zone arrival time for the cells; and (8) ) operating the photolysis system for a time such that all subsequent cells arrive at the photolysis zone.

Following generation of the labeled biomolecule, other methods such as mass spectrometry or electrophoresis may be used to identify the labeled peptide and deduce information regarding the conformation of the biomolecule in vivo or in vitro. .

Various embodiments of the invention include an on-chip analysis system, the on-chip analysis system being a sample introduction system configured to provide a nascent biological entity to a photolysis zone; The target entities are isolated from each other in a focused sheath stream, a sample introduction system, a photolytic light source configured to generate light to generate radicals from a source of labeled reagent, and a sheath stream and light source. a photolysis zone configured to receive the biological entity from the photolysis zone and label the internal standard and label the biological compound of the biological entity in vivo; a dosimetry zone configured to detect the presence of the biological entity using a detector and detect oxidation of the internal standard using a fluorescence detector and a radical target for each of the biological entities; control logic configured to determine the concentration and adjust the operation of the photolysis zone to meet a target concentration of radicals and to receive a biological entity, including a labeled biological compound; a reservoir configured to.

Various embodiments of the present invention include on-chip methods for labeling biomolecules within nascent cells, which methods include introducing a sample mixture containing at least one cell into a photolytic zone and generating labeled radicals. introducing a source and a dosimeter internal standard into a photolysis zone of a flash photolysis system; and providing light to generate labeled radicals from the source of labeled radicals, the labeled radicals comprising: a step configured to label a biomolecule in at least one cell; and a radical dose configured to detect a photometric property of the dosimeter internal standard resulting from the reaction of the dosimeter internal standard with the labeled radical. waiting for an optional predetermined period of time until at least one cell reaches the dosimetry zone of the radical dosimeter, wherein the at least one cell is detected by light scattering in the dosimetry zone of the radical dosimeter; detectable within the dosimeter zone; and measuring photometric properties of the dosimeter internal standard using a radical dosimeter while the at least one cell is within the dosimetry zone; and measuring the dosimeter internal standard. determining that the target level of labeled radicals was not produced based on the photometric properties determined; and adjusting at least one of 1) to 4). and adjusting the concentration. 1) Amount of light supplied to the photolysis zone. 2) Concentration of labeled radical supply. 3) flow rate of at least one cell within the photolysis zone; or 4) time the supply of light to the photolysis zone. In the embodiments described above, the series of light pulses delivered to the photolysis zone has both periodicity and phase, and adjusting the phase of the periodic pulses can affect the timing of the pulses without changing the periodicity. Please note that changing the

The novel features of the invention are pointed out with particularity in the appended claims. A better understanding of the features and advantages of the invention may be obtained by reference to the following detailed description that describes exemplary embodiments in which the principles of the invention are utilized. Furthermore, the above objects and advantages of the present invention will become readily apparent to those skilled in the art from reading the following description of exemplary embodiments when considered in light of the accompanying drawings incorporating features of the present invention. Will. The components in the drawings are not necessarily to scale, emphasis instead being placed upon clearly illustrating the principles of the disclosure.

Any of the methods described herein can further utilize any one or more of the following, according to certain embodiments.

FIG. 1 shows an embodiment of a flash photolysis system. FIG. 2 shows an embodiment of a microfluidic system. FIG. 3 shows an embodiment of the components of the optofluidic chip in detail. FIG. 4 details the dosimetric zone photometric response plot for a mixture of adenine and hydrogen peroxide when irradiated with pulses of UV light at 1 second intervals. FIG. 5 shows an embodiment of a microfluidic system. FIG. 6 shows a preferred embodiment of an in-vivo optofluidic chip. FIG. 7 is a table listing flowing cells, fluorescence excitation, and emission wavelengths for several internal standard radical dosimeters. FIG. 8 shows an embodiment of the combined fluorescence and light scattering detector system of the present invention. FIG. 9 shows another embodiment of the combined fluorescence and light scattering detector of the present invention. FIG. 10 shows the fluorescence radical dosimetry response during irradiation of an in-vivo embodiment using CellRox® DeepRed and hydrogen peroxide in the present invention. FIG. 11 shows an exemplary process for identifying and controlling isolated volume or intercellular distance in the present invention. FIG. 12 shows an example process 1200 for cell arrival identification using coordinated adjustment of photolytic light triggering.

Device for the analysis of biomolecular conformation, catalyzed by on-chip in vivo or in vitro fast photooxidation with real-time monitoring and control of effective radical concentration, achieved by selective labeling of solvent-exposed molecular groups. and methods are provided. Further provided are devices and methods for the analysis of biomolecular conformation achieved by on-chip, in-line microfluidic mixing of labeled reagents with target proteins, cells, or other in-vivo embodiments. Furthermore, an apparatus and method for the analysis of biomolecular conformation achieved by an on-chip microfluidic channel that functions to generate a sheath flow to support fast photooxidation reactions in vivo is provided. Ru. Further provided are devices and methods for the analysis of biomolecular conformation achieved by on-chip in-line photochemically induced reactions using an integrated serpentine photolysis cell.

Device for the analysis of biomolecular conformation, catalyzed by on-chip in vivo or in vitro fast photooxidation with real-time monitoring and control of effective radical concentration, achieved by selective labeling of solvent-exposed molecular groups. and methods are provided. Furthermore, an apparatus and method for the analysis of biomolecular conformation achieved by on-chip in vivo fast photooxidation with real-time monitoring and control of the amount of isolated species in vivo, followed by flash photolysis is provided. Ru. The apparatus and methods are applicable to a variety of in vivo embodiments that are photometrically translucent or transparent, including, but not limited to, eukaryotic cells, prokaryotic cells, bacteria, intracellular viruses, virions, and virus-like particles. , unicellular organisms, eukaryotic tissues, and multicellular organisms. Although the present invention refers to cells for purposes of illustration, such references are not limiting and include all photometrically translucent or transparent in vivo biological or non-transparent cells. It is understood that it is essentially applicable to biological entities.

The equipment and methods are suitable for general protein biochemistry, diagnostic research and development, infectious disease research, biopharmaceutical research and development, antibody research and development, biosimilar development, therapeutic antibody research and development, small molecule drug research and development, It is applicable to various research fields such as the development of other therapeutic compounds and materials. In addition, the apparatus and method can be used for protein-ligand interaction analysis, protein-protein interaction analysis, protein-DNA interaction, protein-RNA interaction, protein fusion product analysis, protein conformation and conformational change analysis, cell-cell Applicable to various research analyzes such as interactions, virus-cell interactions, small drug molecule mode of action analysis, biopharmaceutical mode of action analysis, antibody-antigen analysis, protein epitope mapping, protein paratope mapping, and chemical reaction monitoring. It is.

The device enables stepwise introduction of target proteins or cells by manually pipetting the target proteins or cells into appropriate microcentrifuge tubes or microplates placed within the sample introduction assembly of the system. cells can be received for subsequent chemical labeling. Alternatively, the device can be combined with other separations and analyzers and can receive target proteins or target cells directly from other separations and analyzers, such as selective protein separation. , cell sorting, cell counting, and cell isolation from tissues.

This section describes a flash photolysis device with an on-chip in-line in-vivo or in-vitro radical dosimeter that uses the photometric properties of an internal standard dosimeter to evaluate and ultimately control the in-vivo or in-vivo effective radical concentration. Provide a general overview of Additionally, this section provides a general overview of the present invention, which uses the photometric properties of an in-vivo embodiment to assess isolated quantities and precisely control the separation of an in-vivo embodiment within a flowing stream of a buffer solution. provide. A detailed description of each subassembly is provided elsewhere herein. Additionally, a method of explaining the interaction of these subassemblies is provided to enable understanding of typical instrument operation.

Various embodiments of the invention include a flash photolysis system 100, as shown in FIG. Flash photolysis system 100 is configured for real-time radical dosimetry and includes several subassemblies. sample introduction system 101, photolysis zone 102, flash photolysis light source 103, radical dosimeter 104, control electronics 105, instrument controller 106, fluidic interconnect line 107 between sample introduction system 101 and photolysis zone 102, light Fluidic interconnect lines 108 between the decomposition cell and the radical dosimeter, optical communication interconnects 109 between the flash photolysis light source 103 and the photolysis zone 102, and electronics between the radical dosimeter 104 and control electronics 105. an interconnect 110, an electronic interconnect 111 between the sample introduction system 101 and the control electronics 105, an electronic interconnect 112 between the control electronics 105 and the instrument controller 106, and an electronic interconnect 112 between the control electronics and the flash photolysis system. An electronic interconnect 113 between is shown. The photolysis zone 102, flash photolysis light source 103, and radical dosimeter 104 together constitute a "flash photolysis system."

Photolysis zone 102 is typically included in a photolysis cell. Flash photolysis system 100
is configured to oxidize in vitro or in vivo sample cells in real time to achieve radical dosimetry. An object to be analyzed is introduced via sample introduction system 101 . Analytes can be presented using small volume microcentrifuge tubes or by using multi-well microtiter plates readily available from Eppendorf (Hamburg, Germany). The microfluidic circuit provides analyte aspiration, mixing with labeled reagents, cell hydrodynamic focusing using sheath flow devices for in vivo processing, photolysis and transport to the dosimetry zone, and transport of labeled products. and configured for precipitation. In some embodiments, sample introduction system 101 transfers nascent biological entities into a photolysis zone (e.g., photolysis zone 102, biological entities isolated from each other in a focused sheath flow). configured to provide. The sheath flow is typically configured to isolate biological entities from each other. For example, using appropriate conditions, biological entities are separated from each other by regular intervals.

Photooxidation occurs within the photolysis zone 102. In some embodiments, the photolysis zone is located within the optofluidic chip. In some embodiments, optofluidic chips are fabricated using various techniques such as lithography-assisted wet chemical etching, dry reactive ion etching, and laser ablation microstructuring to create microfluidic channels within a silicon or quartz substrate. Manufactured using technology. In some embodiments, optofluidic chips are manufactured by embossing fluidic channels into a plastic substrate, and the formed fluidic channels seal optically transparent windows in the areas where the plastic substrate is removed. The sample is transported into an optically transparent cell created by stopping the sample. Such optically transparent windows may be placed on only one side or on both sides of the photolysis and dosimetry cells disclosed herein. Exemplary plastic substrates include polycarbonate, polyethylene, polyether-ether-ketone, cyclic olefin polymers, cyclic olefin copolymers, polytetrafluoroethylene, Kalrez®, and polychlorotrifluoroethylene. but not limited to. The inner diameter of the fluidic and optical channels can range from 0.1 to 5.0 mm, but is not limited thereto. In some embodiments, the fluidic channels and optical channels can have different inner diameters to ideally address different requirements for fluid transport, fluid mixing, hydrodynamic focusing, and optical coupling. In some embodiments, the photolysis zone includes a serpentine array of channels that juxtapose multiple fluidic pathways in a countercurrent manner.

Photolysis zone 102 receives analyte from sample introduction system 101 via microfluidic pathway 107 . After processing, the irradiated analyte in the photolysis zone 102 is transferred to the radical dosimeter 104. Photolysis zone 102 is in optical communication with flash photolysis light source 103 . Flash photolysis light source 103 is an example of a photolysis light source configured to generate light for generating labeled radicals from a source of labeled reagent within photolysis zone 102 . In some embodiments, the photolytic zone 102 receives a sheath stream containing biological entities and receives light from a flash photolytic light source 103 to oxidize the dosimeter internal standard and generate biological entities in vivo. Configured to oxidize biological compounds of the entity. For example, proteins and peptides containing amino acids may be oxidized in photolysis zone 102.

Photolysis zone 102, flash photolysis light source 103, and radical dosimeter 104 constitute a flash photolysis system. A flash photolysis system consists of a plasma flash lamp or other suitable light source (e.g. excimer laser, solid state laser, or laser diode) and associated light collection/transmission to adapt the requirements of the light transmission means to the photolysis area. Contains optical components.

Radical dosimeter 104 is configured to receive an analyte to be labeled from photolysis zone 102, or in other embodiments, radical dosimeter 104 is configured to receive labeled analyte from photolysis zone 102 by using orthogonal optical paths. is incorporated into. Various photometric detection schemes may be used by radical dosimeters to monitor relevant photometric properties of dosimeter internal standards. In some embodiments, the dosimeter internal standard can be an external additive added into the biological sample. In some embodiments, the intrinsic photometric properties of the biological buffer system may serve as an intrinsic dosimeter internal standard. By "specific" or "buffer specific" is meant that the internal standard is one of the species that provides buffering properties. The buffer is a physiologically compatible buffer configured to maintain the analyte or cell at a physiological pH or ionic concentration, as appropriate. In some embodiments, the internal standard is configured to receive flash photolysis light in the photolysis zone 102 and, as a result, becomes fluorescent. For example, the dosimeter internal standard may increase fluorescence by a factor of at least 10x, 100x, or 1000x upon reaction with hydroxide radicals, in various embodiments.

Photometric detection schemes include, but are not limited to, fluorescence, photometric absorbance, refractive index detection, light scattering detection, and luminescence. In some embodiments, the photometric detection scheme includes a fluorescence detector that uses a UV light excitation source to generate ultraviolet (UV) fluorescence or luminescence. In some embodiments, the fluorescence detector uses a UV excitation source to generate visible fluorescence or luminescence. In some embodiments, the fluorescence detector uses a visible excitation source to generate visible fluorescence or luminescence. In some embodiments, the fluorescence detector also includes an integrating light scattering detector. In some embodiments, the fluorescence detector also includes an integrated photorefractive index detector.

Control electronics 105 provides direct current (DC) drive voltage derived from a laboratory alternating current (AC) power source to peripheral assemblies, provides analog and digital control signals to peripheral devices, and provides analog or digital control signals to peripheral devices. It functions to receive digital information from peripheral devices, provide ADC and digital to analog conversion functionality, provide data to equipment controller 106, and receive commands from equipment controller 106. In an exemplary embodiment, the control electronics assembly includes a motor controller that interfaces with a motor located within sample introduction system 101. Further, the control electronics assembly in such embodiments may include a universal serial bus (USB) hub for digital communication with the equipment controller 106.

Equipment controller 106 functions to provide process control for various equipment peripheral devices while receiving status and data information in digital form from these devices. In some embodiments, the instrument controller 106 is a software control having two main modules: a low-level multi-threaded module for instrument component control and a high-level user interface (UI) module. Run the program. In some embodiments, control electronics 105 includes an embedded microprocessor that controls low-level appliance control while communicating with a high-level UI control program of appliance controller 106 via a USB or wireless interface. provide component control;

Instrument controller 106, control electronics 105, and various interconnections together represent control logic circuitry configured to control flash photolysis system 100. The control logic circuit may be configured to perform any steps of the methods disclosed herein. For example, in some embodiments, the control theory circuit is configured to identify that a target concentration of labeled radicals has been generated for each biological entity. This target concentration is optionally selected to ensure that the protein or cellular component labeling reaction has sufficient labeling radical agent to approach the desired level of completion.

The control theory circuitry may further be configured to manage a feedback loop that includes adjusting conditions in the photolysis zone 102 to meet a target concentration of labeled radicals. Conditions in the photolysis zone 102 may include, for example, changing the concentration of the labeled radical source, changing the flow rate in the sample introduction system 101, changing the amount of light received from the photolysis light source 103, or changing the amount of light received from the photolysis light source 103. It may be adjusted by varying the time of exposure to the light source, varying the separation distance/amount of isolated cells, etc. By varying the conditions in the photolysis zone 102, the control theory circuitry provides feedback to the sample introduction system 101 and/or the photolysis light source 103 based on the analysis of the first analyte to improve the analysis of the second analyte. can be improved.

In some embodiments, the control theory circuit is configured to normalize quantification of oxidation and/or identified peptides from cells based on fluorescence signals from internal standards within the dosimetry zone. . This allows comparison of results from different experiments using different instances of flash photolysis system 100.

The control theory circuitry optionally further determines the times at which cells within the photolytic zone 102 receive photolytic light and/or the time intervals at which isolated cells enter the photolytic zone 102. configured to specify. These identifications may be based on detection of fluorescence, scattered light, or phase changes of light within the dosimetry zone and may be controlled by adjusting the flow rate and/or flow rate in the sample introduction system 101.

The control logic circuit is optionally further configured to control the flow of cells to the labeled cell reservoir and the analyzer. For example, control theory circuits control the flow of cells such that ruptured cells are diverted from specific vessels of labeled cell reservoirs, or that different oxidizing cells are placed in different compartments over a function of time. It may be configured to do so. In some embodiments, the control logic circuit is configured to control any aspect of the flash photolysis system 100 using the analyte signal from the analyzer.

Instrument controller 106 and control electronics 105 are optionally combined into a single device.
<Photochemical label for in vitro analysis>

FIG. 2 shows further details of a flash photolysis system 100 configured to support processing of in vitro analytes, along with a specific description of the microfluidic system 200 of the system. Sample/product microplate 201, sample inlet/product outlet port 202, combined photolysis and dosimetry cell 203, in-line microfluidic mixer 204, optofluidic chip 205, buffer syringe pump 206, labeled reagent syringe pump 207 , buffer reservoir 208, waste reservoir 209, sample storage loop 210, labeled reagent reservoir 211, reagent transfer line 212, buffer syringe pump four-way valve 213, in-line mixer gate valve 214, reagent waste line 215, labeled reagent syringe pump Four-way valve 216, reagent introduction line 217, buffer introduction line 218, buffer waste line 219, sample microwell 220, waste collection microwell 221, and product collection microwell 222 are shown.

The sample to be labeled is stored in a storage container, such as a sample/product microplate 201, such as a 96 or 384 well plate available from ThermoFisher (USA). The sample is introduced by suction through the sample inlet/product outlet port 202. The aspirated sample passes through the dosimetry and photolysis section 203 of the system and is then drawn through an in-line mixer 204. In-line mixer 204 and photolysis and dosimetry zone 203 are both formed within optofluidic chip 205.

Microfluidic system 200 includes two syringe pumps, a buffer syringe pump 206 and a labeled reagent pump 207. Syringe pump 206 is in communication with buffer reservoir 208, waste reservoir 209, and sample storage loop 210. Syringe pump 207 communicates with waste reservoir 209, reagent reservoir 211, and in-line mixer 204 via reagent transfer line 212.

Before the labeling process, the microfluidic circuit is primed. Syringe pump 206 is filled with buffer by switching its four-way valve 213 into communication with buffer reservoir 208 . The plunger is withdrawn to completely fill the syringe to its volume. The volume of the syringe pump 206 can range from, but is not limited to, 50 μL, 100 μL, 250 μL, 500 μL, 1.5 mL, 2.0 mL, and 2.5 mL, depending on the desired amount of sample to be labeled. Not done. Syringe pump 206 then primes the microfluidic system by switching valve 213 into communication with storage loop 210 and pumping enough volume to flush the system. During system flushing, buffer is directed to flow through the in-line mixer 204, photolysis and dosimetry zone 203, port 202, and finally dispensed into designated waste wells 221 of the microplate 201. be done. During this process, valve 214 remains closed to in-line mixer 204 so that buffer is directed through chip 205 and not out through mixer 204 and ultimately reagent transfer. It does not flow into line 212.

After priming the chip, the reagent delivery circuit is primed. Reagent pump 207 is completely filled with reagent drawn from reagent reservoir 211 by pulling back its plunger while valve 216 is connected to reagent introduction line 217. The capacity of the reagent pump 207 can range from, but is not limited to, 50 μL, 100 μL, 250 μL, 500 μL, 1.5 mL, 2.0 mL, and 2.5 mL depending on the desired amount of sample to be labeled. Not done. The reagent transfer line 212 is flushed by pumping with a reagent pump 207 having a four-way valve 216 configured to transfer the contents of the syringe 207 to the reagent transfer line 212, where the reagent transfer line 212 transfers the priming amount to It has a valve 214 configured to direct the reagent waste line 215 to the waste reservoir 209 . Valve 214 is positioned adjacent to in-line mixer 204 such that the interconnect volume from valve 214 to mixer 204 is minimized to a range of 0.25 to 1.5 μL. Further details of the fluid connections of chip 205 are provided in FIG.

The following steps outline an exemplary operational cascade reaction used by the embodiment described with respect to microfluidic system 200 to label an in vitro sample using a photoinduced radical reaction. Variations on this cascade reaction will be apparent to those skilled in the art, and such variations are to be construed as functionally equivalent and not inventively different from the examples provided herein. The sample/product microplate 201 is arranged such that separate microwells 220 are designated to contain the sample to be labeled, as well as empty microwells 221 and 222; Labeled products, waste buffers, or labeled reagents precipitated during fluidic priming of subsystems of microfluidic system 200 are selectively collected. For in vitro radical protein footprinting, microwells 220 contain proteins or other proteinaceous biological mixtures. After priming, buffer syringe pump 206 dispenses a predetermined amount of buffer by moving valve 213 into communication with sample loop 210 and, as a result, through optofluidic chip 205 and through inlet/outlet port 202. Pump buffer into designated waste microwells 221. The volume pumped here is slightly larger than the volume of sample to be labeled. For example, if a 100 mL sample is targeted for labeling, syringe pump 2
06 dispenses 120 mL of buffer into the waste microwell 221. Other different volume ratios can be used and the above examples are illustrative and not limiting.

Microplate 201 is moved such that ports 202 are immersed in microwells 220 containing the samples to be labeled. Buffer syringe pump 206 aspirates a predetermined amount of sample by withdrawing its plunger and fluidly communicating through valve 213, in-line mixer 204, photolytic dosimetry cell 203, and port 202. . The sample is drawn into the chip 205 through the photolytic dosimetry cell 203 and the in-line mixer 204 and finally into the sample storage loop 210. During this sample aspiration process, the in-line mixer gate valve 214 closes the mixer from drawing labeled reagent into the circuit.

Reagent reservoir 211 contains labeling reagents needed for the protein labeling process. In the case of HRPF, reservoir 211 contains, for example, aqueous hydrogen peroxide, typically at a concentration in the range of 20mM to 1000mM. For the trifluoromethyl footprint method, reagent reservoir 211 contains a mixture of hydrogen peroxide and aqueous sodium triflate. If primarily CF ₃ radical labeling is desired, H ₂ O ₂ and sodium triflate are mixed in a concentration ratio of 1:4, respectively, typically 5 mM H ₂ O ₂ and 20 mM sodium triflate are used. If simultaneous HRPF and _CF3 footprinting methods are desired, _H2O2 and triflinate are mixed in a 5:1 _ratio , respectively _, typically using 50mM _H2O2 and 10mM triflic acid. The above mixtures are intended to be illustrative and should not be construed as limiting the range; other ratios may function as functionally equivalent and absolute terms are May vary based on actual protein labeling conditions.

To begin the labeling process, pumps 206 and 207 begin dispensing their respective solutions into the microfluidic circuit. The relative pumping speeds of pumps 206 and 207 depend on the desired final concentration of labeled reagent entering the photolysis zone and the starting concentration of labeled reagent located within reservoir 211. Using hydroxyl radical labeling as an example, reagent reservoir 211 may contain a 1000 mM concentration of H ₂ O ₂ and the desired final concentration in the label is 100 mM. Under these conditions, the pumping speed of pump 207 is one-tenth the pumping speed of pump 206. The net flow rate is the sum of the pumping rates established by pumps 206 and 207.

FIG. 3 shows a further embodiment of an exemplary optofluidic array 300 to enable subsequent discussion. A detailed view of a photolysis cell 307 is shown including a reagent inlet port 301, a buffer inlet port 302, a mixer 303, a photolysis cell 304, a dosimetry cell 305, an inlet outlet port 306, and a fluidic channel 308. After establishing flow through the optofluidic chip 205, the photolysis light source 103 is activated to illuminate the photolysis area 304 at regular flash intervals. The flash frequency may range from 0.25Hz to 5Hz, but is not limited thereto. The flash frequency and net flow rate are such that the sample and labeled reagent mixture is illuminated by only one flash, and that each subsequent bolus of the illuminated mixture is divided by a predetermined isolated volume. established to ensure that Thus, the illuminated volume of the photolytic cell defines the required net flow rate and flash rate.

In capillary photoinduced radical footprinting, a photolytic light source is focused to illuminate a small volume of fluid located within the capillary lumen. Typical capillary internal diameters (ID) range from 50 to 250 μm, but are not limited thereto. Additionally, the irradiation area extends along the capillary axis defining an axial length of irradiation in the range of, but not limited to, 2-8 mm. In a typical application, a 100 μm ID capillary with an axial distance of 6 mm contains an illumination volume of 47 nL. If a 50 μL sample is desired for labeling, approximately 1,064 flashes are required, which requires many minutes to process and unnecessarily accelerates light source wear. Furthermore, under these conditions, only a small fraction of the photolyzed light is focused into the capillary lumen, since the refractive focusing limits of both excimer lasers and plasma sources result in beam waste that is larger than the inner diameter of the lumen. Ru. Thus, the previously taught capillary approach presents various drawbacks.

Optofluidic chip 205 includes a serpentine photolysis zone 304 as detailed by photolysis zone 307 . The fluid paths within the photolysis zone 307 are arranged in a folded or tortuous manner, with adjacent flow channels in close juxtaposition, and flow is established in a countercurrent manner. The serpentine photolysis zone can distribute the photolysis light over a wider incident area, overcoming refractive focus limitations and thus allowing better opto-fluidic matching between the photolysis light source 103 and the sample reagent mixture. Make it. The amount of flux of incident UV radiation required to effectively photolyze the labeled reagent depends on the UV source spectral irradiance and the labeled reagent extinction coefficient. For H ₂ O ₂ -based labels, a flow rate of at least 3 mJ/mm ² is typically required for UV wavelengths below 250 nm. For both OH and _CF3 radical labels, the flash photolysis light source 103 has sufficient spectral irradiance to allow an illuminated area of up to 16 ^mm2 . Under such conditions, fluidic channel 308 within photolysis cell 304 may be arranged to include an irradiation volume of approximately 6.4 mL. In this way, the illumination volume of the photolysis zone 304 is 136 times larger than the illumination volume of the 100 mm ID capillary system described above, and therefore the labeling proceeds in a fraction of the time required for capillary-style labeling. At the same time, it reduces the strain of the photolysis system by more than 99%.

The sample and labeled reagent mixture is illuminated by photolysis light source 103 in photolysis zone 304 . After exposure, radicals are rapidly formed, typically covalently modifying the sample within a few microseconds. Labeled samples, buffers, and modification reagents are pumped downstream into dosimetry zone 305. The contents of dosimetry zone 305 are probed using photometric means such as, but not limited to, photometric absorbance, fluorescence, light scattering, refractive index detection, and/or chemiluminescence. The real-time measurements made in the dosimetry zone 305 enable a feedback loop in which the photolysis conditions are adjusted to ensure that the desired amount of radical generation occurs and the desired amount of in vitro analyte label. It can be modified to During initial operation of the system, a baseline measurement of the photometric properties of the dosimetry zone containing the unirradiated sample and reagent mixture is taken. Once baseline measurements are made, photolysis proceeds and changes in photometric properties are identified. In the case of hydroxyl radical protein footprinting, OH radicals covalently label the sample and simultaneously attack an internal standard radical dosimeter, such as adenine, which is typically used. In this case, the photometric property evaluated is the UV photometric absorbance at 265 nm. Upon OH radical attack, adenine exhibits a decrease in UV 265 nm absorbance in a manner directly proportional to the effective OH radical yield. During the fine-tuning process, the effective OH radical yield is controlled by changing either the flash intensity or _{the H2O2} concentration in response to the difference between the estimated and target changes in the dosimetric signal. be done.

If the actual response of the internal standard radical dosimeter is less than the desired level, the flash energy of the photolysis light source 103 is increased until the desired level of dosimeter response is achieved. Alternatively, the relative pumping speed of reagent pump 207 is increased relative to buffer pump 206 to subsequently increase the effective concentration of labeled reagent after mixing with the sample within in-line mixer 204. If the actual response of the internal standard radical dosimeter is greater than the desired level, the flash energy of photolytic light source 103 is reduced until the desired level of dosimeter response is achieved. Alternatively, the relative pumping speed of reagent pump 207 is reduced relative to buffer pump 206 to subsequently reduce the effective concentration of labeled reagent after mixing with the sample in in-line mixer 204.

FIG. 4 shows a real-time output trace 400 of UV absorbance versus time obtained in the dosimetry zone 305 while generating hydroxyl group radicals in the photolysis zone 304, using a flash rate of 1 Hz and at 1 mM. 100 mM H ₂ O ₂ in the presence of aqueous adenine solution is used, which is a 1:1 (v/v) mixture of 2 mM aqueous adenine solution and 200 mM _{H 2} O ₂ in the microfluidic mixer 303. An unexposed baseline 401 is shown. A descending shoulder 402 is shown. The peak shift in the photometric response (minimum value 403) or in this case UV absorbance is shown. A rising shoulder 404 is shown. Re-established baseline 405, the minimum UV absorbance value achieved at time T1 406 is shown. The minimum UV absorbance value achieved at time T2 407 is shown. As will be apparent to those skilled in the art, other adenine/H ₂ O ₂ mixing ratios may be used to achieve the target concentration; the 1:1 mixing ratio is exemplary and limited. It means not. The baseline UV photometric absorbance measurement for the aforementioned mixture that has not been exposed to light is represented by the unexposed baseline 401. At the unexposed baseline 401, the baseline UV absorbance is approximately 55 milli-absorbance units (mAU), as specified against the background of the running buffer (in this case water). It is interpreted as Upon photoirradiation of this mixture within the photolysis zone 304, OH radicals are formed which subsequently oxidizes the adenine. Although the light irradiation time is not particularly limited, it is typically 10 μsec. Subsequently, the hydroxyl group radical reaction proceeds for about 1 to 5 μs, after which the reaction self-stops. As the irradiated mixture enters the flow dosimetry zone 305 from the photolysis zone 304, the photometric absorbance signal begins to decrease, as shown by the falling shoulder 402. The UV absorbance minimum of the labeled sample is interpreted to be approximately 33 mAU, as indicated by minimum value 403. Here, the effective change in adenine UV absorbance for the first irradiated bolus of the mixture is calculated by taking the difference between the unexposed baseline 401 and the minimum value 403 (55 mAU - 33 mAU), which is 22 mAU. It is identified as As the irradiated mixture flows out of the dosimetry zone 305, the measured UV absorbance returns to its baseline as indicated by the rising shoulder 404 and reaches the baseline 405 again. The identified change in UV absorbance upon light irradiation is compared to the target change for the labeling experiment. If the effective level of absorbance change deviates from the desired target, the flash energy and/or H ₂ O ₂ concentration is changed to adjust the OH radical yield using the control loop logic described above. Once the target level of internal standard dosimetric light property change is achieved, the light exposure/dosimeter response evaluation process is repeated until the desired amount of labeled sample is processed and subsequently collected.

During the labeling process, effective changes in dosimeter response may be selectively accessed for each flash cycle to determine the reproducibility and inherent quality of the overall labeling process. Under these conditions, the mean, standard deviation, and percent relative standard deviation (RSD) for the effective dosimeter response for each flash cycle are taken and compared to pre-established quality metrics. Typical RSD quality metrics are determined by the specific photochemical labeling conditions for the target protein and can be, but are not limited to, less than 5%. If the quality metrics are met, the labeling experiment is determined to be of acceptable quality. If the quality metrics are not met, the labeled experiment is flagged as questionable and the experimenter is appropriately alerted.

Each exposure bolus of mixture is divided into predetermined isolated volumes, where the predetermined isolated volumes include the unirradiated mixture that flows through the photolysis zone 304 between successive flashes. Isolated volume is essential to ensure that each bolus of the mixture is only photoirradiated once, and this prevents undesired backtracking of the irradiated sample back into the bolus of the next sample that is photoirradiated. By effectively acting as a buffer zone to reduce axial diffusion and adjusting laminar flow axial velocity differences. A useful isolation amount is one that ensures against undesired double irradiation while simultaneously reducing undesired dilution of the labeled product. An exemplary desired isolation volume target is taken to be an amount that dilutes the sample by about 10%.

The photometric response plot 400 shown in FIG. 4 can be used to calculate the effective isolated amount by identifying the difference in dosimeter response minima produced during OH radical attack of adenine. Other internal standard radical dosimeters, such as Tris or histidine buffers, exhibit increased photometric absorbance upon OH radical attack, in which case the photometric response plot 400 shows a local maximum. For this exemplary discussion, adenine is again used as the internal standard radical dosimeter. For adenine, two consecutively illuminated boluses of the mixture contain two respective effective dosimeter response minima, as shown by 406 and 407. The time difference between 406 (T1) and 407 (T2) may be calculated. For purposes of illustration, consider that the time difference between T1 and T2 is 1.25 seconds. For the aforementioned flash rate of 1 Hz, the flash interval is specified to be 1.0 seconds. In the adenine example, the isolated amount is determined by the net flow rate and the difference between the flash interval and the dosimeter minimum interval. In the adenine example, the net flow rate was 70 μL/min or 1.17 μL/sec. Therefore, the isolated volume is determined to be 1.17 μL/sec (1.25 seconds) = 0.29 μL. If the total exposure volume determined by the geometrical properties of the photolytic cell 304 is 3 μL, the isolated volume is considered to be approximately 10% of the irradiance, ensuring one flash exposure of each bolus. determined to be sufficient to If the isolated yield is less than the desired target, the net flow rate may be increased proportionally or the flash rate may be decreased proportionally to achieve the desired isolated yield. . If the isolated yield exceeds the desired target, the net flow rate may be proportionally decreased or the flash rate may be proportionally increased to achieve the desired isolated yield. . In one embodiment, the control theory logic described above is automatically executed by the equipment controller 106.

Once an appropriate dosimeter response and isolated amount is achieved, the sample mixture can be reliably labeled and the labeled product collected. During the label and product collection process, the instrument controller 106 records the time when the dosimeter and isolated volume goals are achieved and identifies the time of arrival of the appropriately labeled product at the outlet of the inlet exit port 202. . The arrival time is determined by the quotient of the amount transferred and the net flow rate from the dosimetry zone 305 to the inlet/outlet port 202. The outgoing contents of optofluidic chip 205 are dispensed through port 202 into a designated waste well 221 before the arrival of the appropriately labeled product. Under the control of the instrument controller 106 , once the appropriately labeled product reaches the outlet of the inlet/outlet port 202 , the microplate 201 is configured such that the labeled product is collected within the product collection microwell 222 . , are moved by the sample introduction system 101.
<In-vivo photochemical labeling>

The previous discussion described details of embodiments of the invention for labeling in vitro samples. Details regarding embodiments that support in vivo labeling reactions will now be described. FIG. 5 shows a microfluidic circuit 500 and an optofluidic chip 501 for on-chip in vivo photochemical labeling. Microplate 201, sample inlet product outlet port 202, cell-containing microplate well 220, waste receiving microwell 221, product collection microwell 221, in-vivo radical labeling optofluidic chip 501, sheath flow generator 502, in-line mixer 503, photolysis zone and dosimetry zone array 504, sample buffer microfluidic pumping system 505, sheath buffer microfluidic pumping system 506, reagent microfluidic pumping system 507, sheath flow buffer syringe pump 508, four-way microfluidic valve 509 , sheath buffer reservoir 510, waste reservoir 511, sheath buffer tee 512, sample buffer syringe pump 513, four-way microfluidic valve 514, waste reservoir 515, buffer reservoir 516, sample loop 517, Reagent syringe pump 518, four-way microfluidic valve 519, reagent reservoir 520, reagent transfer line 521, sheath inlet port 522, and in-line mixer reagent introduction port 523 are shown.

The optofluidic chip 501 includes three different functional areas: a sheath flow generator 502, a labeled reagent/sample in-line mixer 503, and a photolysis and dosimetry cell array 504. The optofluidic chip 501 is connected to three different microfluidic circuits: a buffer and sample fluid system 505, a sheath flow fluid system 506, and a reagent introduction fluid system 507. Before starting marking, all three fluid systems are primed. System 505 is primed by the action of buffer syringe pump 513 and valve 514. First valve 514 is configured to allow fluid communication between buffer reservoir 516 and syringe pump 513. Pump 513 is filled with running buffer drawn from buffer reservoir 516 . After filling, valve 514 is reconfigured to allow communication between syringe pump 513 and waste reservoir 515. The contents of syringe pump 513 are pumped into waste reservoir 515. This priming cycle continues until all the air in fluid system 505 is evacuated and the remnants of the previous buffer are flushed out. Under typical circumstances, the cycle is repeated three times, but is not limited to this. The syringe pump 513 is then refilled with running buffer and the valve 514 is configured to allow fluid communication between the syringe pump 513 and the optofluidic chip 501 via the sample loop 517. The running buffer is then pumped by a syringe pump 513 through a sample loop 517 into the optofluidic chip 501 and the buffer is pumped through a sheath flow generator 502, an in-line mixer 503, a photolysis and dosimetry zone array 504 through the inlet and outlet ports 202 into designated waste microwells 221 within the microplate 201.

After priming system 505, sheath flow fluid system 506 is primed. Valve 509 is configured to allow fluid communication between sheath buffer syringe pump 508 and sheath buffer reservoir 510. For in-vivo photochemical labeling, reservoirs 510 and 516 contain the same buffer. Sheath flow buffer is aspirated to fill sheath buffer syringe pump 508. After filling, valve 509 is reconfigured to allow fluid communication between sheath buffer syringe pump 508 and waste reservoir 511. Waste reservoir 511 may be shared with reagent introduction system 507. Additionally, reagent introduction system 507, microfluidic pumping system 506, and system 505 may optionally utilize the same waste buffer reservoir. Sheath buffer syringe pump 508 drains its contents into waste reservoir 511 . This priming cycle continues until all the air in fluid system 506 is evacuated and the remains of the previous buffer are flushed out. Under typical circumstances, the cycle is repeated three times, but is not limited to this. Sheath buffer syringe pump 508 is then refilled with sheath buffer, and valve 509 is configured to allow fluid communication between sheath buffer syringe pump 508 and optofluidic chip 501. The flow of sheath flow buffer is split by coupling tee 512, which establishes two streams of sheath flow buffer entering optofluidic chip 501 through port 522 of sheath flow generator 502. The sheath flow buffer then flows through the sheath flow generator 502, the in-line mixer 503, the photolysis and dosimetry zone array 504, and into the designated waste microwells 221 in the microplate 201 at the inlet/outlet ports 202. flows out through.

After priming system 505 and microfluidic pumping system 506, reagent fluid system 507 is primed. Valve 519 is configured to allow fluid communication between reagent reservoir 520 and reagent syringe pump 518. Reagent solution is aspirated to fill reagent syringe pump 518 . After filling, valve 519 is reconfigured to allow fluid communication between reagent syringe pump 518 and waste reservoir 511. Waste reservoir 511 may be shared with sheath buffer fluid system 506. Additionally, reagent fluid system 507, sheath buffer fluid system 506, and system 505 may optionally utilize the same waste buffer reservoir. Reagent syringe pump 518 pumps its contents into waste reservoir 511 . This priming cycle continues until all the air in fluid system 507 is evacuated and the remainder of the previous reagent solution is washed away. Under typical circumstances, the cycle is repeated three times, but is not limited to this.

Reagent syringe pump 518 is then refilled with reagent solution, and valve 519 is configured to allow fluid communication between reagent syringe pump 518 and optofluidic chip 501. Reagent solutions are pumped through reagent transfer line 521 and enter optofluidic chip 501 through reagent inlet port 523 of in-line mixer 503. Simultaneously with the latter, fluidic systems 506 and 505 pump their fluidic components into optofluidic chip 501, as described above. In this way, all fluid flow, including that from reagent fluid system 507, is directed down the chip and ultimately exits port 202 to waste microwell 221. The system is now ready for in vivo radical protein footprinting.

For in vivo radical protein footprinting, microwells 220 contain cells, tissues, or organisms suspended in a buffer. Buffer syringe pump 513 is connected to sample loop 517 via valve 514 while reagent microfluidic pump system 507 and sheath buffer syringe pump 508 remain stationary. Buffer syringe pump 513 is used to aspirate cells from microwells 220. Cells are aspirated to port 202 and flow through dosimetry zone array 504, in-line mixer 503, sheath flow generator 502, and finally into sample loop 517. Typically, a predetermined amount of cells suspended in a buffer is aspirated and stored within sample loop 517. In this way, cells also remain through each compartment and internal port 202 of the optofluidic chip 501. Once labeling begins, fluidic systems 507, 506, and 505 begin flowing simultaneously, ultimately pushing the cells stored in sample loop 517 into optofluidic chip 501, where they are exposed to labeling reagents. and then irradiated with light to begin the photoradical labeling process.

In order to ensure that the cells are isolated from each other alone and do not clump or aggregate together when they are irradiated with light, a sheath flow is established within the sheath flow generator 502 and a small volume of sheath flow buffer is added. Separate cells by aliquot size. Under typical conditions, but not limited to, the sheath buffer flow rate is established to be 10 times the buffer syringe flow rate. As discussed below, the flow rate ratio of fluidic systems 505 and 506 may be automatically adjusted to achieve the desired degree of cell isolation. Simultaneously with the pumping activity of fluidic systems 505 and 506, reagent fluidic system 507 delivers labeled reagents for mixing with flowing cells within in-line mixer 503. For in vitro labeling, the relative flow rate of reagent fluid system 507 to the net flow rate of sheath buffer syringe pump 508 and system 505 depends on the concentration of the label reagent and the effective radical yield as determined by the in-line radical dosimetry system. may be automatically adjusted depending on the desired level of.

FIG. 6 shows a detailed view of a preferred embodiment of an optofluidic chip 601. optofluidic chip 601, buffer syringe port 602, reagent inlet port 603, reagent mixer 604, sheath buffer inlet port 605, sheath flow generator 606, photolysis zone 607, dosimetry zone 608, and sample inlet/product outlet Port 609 is shown. In optofluidic chip 601, reagent mixer 604 is located upstream of sheath flow generator 606. Cells enter optofluidic chip 601 via buffer syringe port 602. At this point, they may not be isolated, but may enter as a dense clump of cells or other in-vivo embodiment. Reagent mixer 6
04, they are mixed with labeled reagents that enter buffer syringe port 602 via reagent inlet port 603. As known to those skilled in the art, a number of different in vivo labeling reagents may be used, but for exemplary purposes, we will discuss the use of hydrogen peroxide used in in vivo hydroxyl radical protein footprinting methods. explain. As described in Jones et al. As shown, hydrogen peroxide is quickly and easily taken up by cells or other in vitro entities and rapidly distributed to all cellular compartments. As discussed below, the final concentration of hydrogen peroxide used essentially specifies the effective hydroxyl radical yield as assessed by the change in the photometric properties of the internal standard radical dosimeter probed in the dosimetry zone 607. is determined in a closed-loop manner.

Prior to precipitation into microwells 220, the internal standard radical dosimeter is added to a population of cells or other in-vivo embodiment, and this mixture ensures that the internal standard radical dosimeter is taken up by all cells and the final cultured thoroughly to achieve uniform distribution between cells and within cell compartments. The cells are then sedimented to form a precipitate and the supernatant is removed. The cell pellet is then resuspended in a buffer and again subjected to a second sedimentation. This process is repeated until all extracellular internal standard radical dosimeters are removed. Once the extracellular internal standard radical dosimeter is exhausted, the cells are resuspended in buffer and finally transferred to well 220.

After passing through reagent mixer 604, the cell reagent mixture enters sheath flow generator 606. Sheath flow generator 606 includes a cavity for receiving a sheath flow buffer and a sheath flow buffer port 605, these components of generator 606 being shaped and shaped together to produce hydrodynamic focusing. Once placed, cells are isolated by sheath flow buffer. The relative flow rates of microfluidic systems 505, 506, and 507 are adjusted to establish the desired intercellular isolation volume while simultaneously achieving the target intracellular hydroxyl group radical yield. After passing through the sheath flow generator 606, the isolated cell population enters a photolysis zone 607 where it is illuminated with light that initiates an intracellular photolabeled radical reaction. After passing through the photolysis zone 607, the cells enter the dosimetry zone 608 where they are optically probed to assess the effective radical yield as well as the intercellular distance and subsequent partitioning volume. be done.

During in-vivo radical protein footprinting, the intracellular internal standard radical dosimeter response is measured photometrically as probed through the dosimetry zone 608. Typical photometric measurement means include, but are not limited to, photometric absorbance, photometric luminescence, and photometric fluorescence. Because it is desirable to detect photometric signals generated within single cells or other single in vivo embodiments, photometric fluorescence typically outperforms other previously mentioned methods in its dynamic range and analytical sensitivity. Therefore, it can be a preferred measurement method.

For in vivo radical protein footprinting methods, fluorescent internal standard radical dosimeters change fluorescence properties such as excitation and emission properties upon radical attack and final covalent modification. Such modifications may include changing the excitation and emission characteristics of the dosimeter such that the dosimeter increases or decreases fluorescence for a given excitation/emission wavelength setting. Since it is desirable to maximize the sensitivity of radical dosimeter signal detection, detecting the presence of acquired fluorescence due to radical attack allows detection of small signals in a dark or zero background. This is the preferred embodiment for this approach. Radical dosimeter fluorescence detectors are constructed using specific excitation and emission wavelengths for the covalently modified internal standard dosimeter. When the dosimeter is in its native state, no fluorescent signal is detected. Upon radical attack, the fluorescence properties of the dosimeter are changed to correspond to the selected excitation and emission wavelengths, and the detected fluorescence signal intensity is determined by the concentration of the covalently modified internal standard dosimeter. and, in turn, to the effective radical yield.

Figure 7 shows five different internal standard radical dosimeters with excitation and emission wavelength fluorescence signal signals shown in dose response manner when oxidized using hydroxyl group radical attack. shows. Table 700 lists cell distributions and fluorescence excitation and emission wavelengths for the following internal standard radical dosimeters that respond to hydroxyl group radical attack in a dose-response manner. Terephthalic acid, CellRox Green® Green, dichlorofluorescein, CellRox Orange, and CellRox Deep Red. The subcellular portion where each dosimeter is primarily distributed is also shown. CellROX® dyes are commercially available from ThermoFisher (USA).

FIG. 8 shows an embodiment of an internal standard radical dosimetry photodetector 800 of the present invention. Light source 801, focusing lens 802, excitation light 803, dosimetry zone 608, dichroic mirror 804, emitted light 805, collimator 806, interference notch filter 807, narrowband emitted light 808, fluorescence/emission light detector 809, scattered light 810 , a collimator 811, and a light scattering detector 812 are shown. Internal standard radical dosimetry photodetector 800 is a combined fluorescence and light scattering detector system. As will be clear to those skilled in the art, this combined system can be replaced by two separate (fluorescence and light scattering) detectors, so this embodiment is exemplary and ranges from It is not limited to. As biological elements enter the dosimetry zone 608 , they are illuminated by excitation light 803 from the light source 801 that is focused by a lens 802 and directed into the dosimetry zone 608 by a dichroic mirror 804 . Light source 801 can be a narrow bandwidth solid state UV source such as a UV light emitting diode (LED) available from Q-Photonics (Ann Arbor, MI) or a compact solid state laser available from Thorlabs (Newton, NJ). It may be. Alternatively, light source 801 may be a solid state visible light source such as a visible LED available from Q-Photonics (Ann Arbor, Mich.) or a compact solid state visible laser available from Thorlabs (Newton, NJ). Typical output powers can range from, but are not limited to, 0.1 to 10 mW, and typical bandwidths can range from, but are not limited to, from 1 to 15 nm. . The wavelength of the light source 801 is selected to be an appropriate choice for the excitation wavelength of the in-vivo internal standard radical dosimeter used. A focusing lens assembly 802 is used to focus the excitation light into a narrow beam on the order of, but not limited to, 1-200 μm at the center of the dosimetry area 608 to create a probe dosimetry area. Dichroic mirror 804 is selected to efficiently reflect excitation light but transmit emission light 805 resulting from the covalently modified internal standard dosimeter illuminated within dosimetry area 608 .

The emitted light 805 is finally collimated by a collimator 806 and then passes through an interference notch filter 807 to produce a narrowband emitted light 808 that is incident on a fluorescence emitted light detector 809 . Photodetector 809 may include a silicon photodiode, such as a S1336-8BQ silicon photodiode available from Hamamatsu (Hamamatsu, Japan). Alternatively, photodetector 809 may include a miniature photo-multiplier tube (PMT), such as the MicroPMT assembly H12400 available from Hamamatsu. The output current of photodetector 809 is processed by a current-to-voltage (IV) converter to provide a voltage proportional to the incident emission light intensity of narrowband emission light 808 . The output voltage of photodetector 809 is transmitted to control electronics 105, and an analog to digital converter (ADC) converts the digital signal to instrument controller 106, where fluorescence calculations are performed. generate.

When cells or other in-vivo embodiments enter the dosimetry zone 608, they are exposed to the excitation light 80
3 is elastically scattered. Due to the size difference between the incident excitation light wavelength (nm) and the in-vivo entity size (μm), the scattered light 810 that is perpendicular to the incident excitation light is preferentially detected. Scattered light 810 is collimated by collimator 811 and finally enters scattered light detector 812 . For a given excitation light intensity, the measured intensity of the scattered light is proportional to the size and number of biological entities located within the dosimetry area 618 whose volume is being probed. Since scattered light detection is performed in a direction perpendicular to the incident excitation light, background scattering is also measured. In the absence of biological entities, elastic and inelastic scattering of the contents of the probed volume, as well as elastic scattering from the radical dosimeter optical surface, is very low and therefore produces a very low background signal. . The light scattering signal is generated uniformly, independent of intrinsic fluorescence, so that the presence of cells or other in-vivo embodiments can be detected without an internal standard radical dosimeter signal.

Scattered light detector 812 may include a silicon photodiode, such as a S1336-8BQ silicon photodiode available from Hamamatsu (Hamamatsu, Japan). Alternatively, photodetector 812 may include a miniature photomultiplier tube (PMT), such as the MicroPMT assembly H12400 available from Hamamatsu. The output current of the scattered light detector 812 is processed by a current-to-voltage (IV) converter to provide a voltage proportional to the incident scattered light 810. The output voltage of photodetector 812 is transmitted to control electronics 105, and an analog-to-digital converter (ADC) generates a digital signal that is ultimately transmitted to instrument controller 106 where light scattering calculations are performed.

FIG. 9 shows another embodiment of an internal standard radical dosimetry photodetector 900 of the present invention. Fluorescence excitation light source 901, red diode laser interference pattern light source 902, light collection and transmission branch optical fiber 903, fiber output coupling optics 904, dosimetry cell 608, scattering light train 905, optical beam splitter 906, light scattering charge-coupled device camera 907 , fluorescence array 908, fluorescence collection optics 909, fluorescence selective interference filter 910, fluorescence detector 809, far-field interference fringe pattern 911, and extraction spatial frequency 912.

In the combined fluorescence and light scattering detector system 900, the light source may be exchanged to meet fluorescence internal standard radical dosimeter requirements, and light scattering detection images the interference pattern produced by the dosimetry cell. This is accomplished using a two-dimensional charged couple device (CCD) imaging camera. Fluorescence excitation source 901 and red diode interference pattern light source 902 are located remotely from optofluidic chip 601 and are coupled to dosimetry cell 608 using branch optical fiber 903 and necessary fiber optic coupling optics 904. The wavelength produced by light source 901 is selected to meet the excitation wavelength requirements of the internal standard radical dosimeter used. Red diode interference pattern light source 902 uses a 650 nm red diode laser to generate an optical interference pattern that is generated in the far optical field of dosimetry cell 608 . In such a configuration, light sources 901 and 902 can be mounted externally to the instrument and provide an easy means of replacing the light sources when performing radical dosimetry.

Coherent light from light sources 901 and 902 is homogenized within a single optical fiber 903 and directed into dosimetry cell 608 . A beam splitter 906 is used to split the light exiting the dosimetry cell 608 to produce two optical columns: a scattering column 905 and a fluorescent column 908. A light scattering array 905 is generated in the optical far field of the dosimetry cell 608. Since the red diode interference pattern light source 902 is a coherent light source, its modality interacts with the optical properties of the dosimetry cell 608 to provide far-field interference as imaged by the charge-coupled device two-dimensional camera 907. A striped pattern 911 is generated.

Fluorescent array 908 is collected and collimated by collection optics 909 , which directs the light to pass through interference filter 910 . The bandpass characteristics of interference filter 910 are selected to match the internal standard radical dosimeter emission spectrum. The optical bandwidth of the interference filter 910 may be 10 nm, but is not limited thereto. Interference filter 910 and fluorescence excitation source 901 are exchanged as a matched pair to accommodate multiple different internal standard radical dosimeters, such as those listed in table 700, so that interference filter 910 is user accessible and removable. is housed in a simple fitting. Fluorescent light is detected by fluorescence detector 809.

As cells enter the dosimetry cell 608, they elastically scatter the light of the red diode laser interference pattern light source 902, and such scattering changes the average degree of polarization of the light and, in turn, changes the refractive index and resultant phase of the light exiting the light. As the refractive index of the examination area changes, the imaged fringes 911 change positionally to the right or left depending on the change in the refractive index. By summing the intensity of each vertical pixel of the two-dimensional camera 907 for a given horizontal or x increment, the fringe pattern of the optical interference pattern 912 is represented as a spatial frequency distribution. Using a fast Fourier transform (FFT), the phase of the dominant spatial frequencies seen in the optical interference pattern 912 can be calculated at fast time intervals. As the cell enters the dosimetry cell 608, the accompanying light scattering changes the position of the stripes, which is then displayed as a change in phase. The phase change is proportional to the size and number of cells within the study area of dosimetry cell 608. As previously discussed, sheath flow generators 502 and 606 may function to isolate introduced cells or other in-vivo embodiments so that they are clearly separated and together No agglomeration, resulting in effective and consistent irradiation of each entity within the radical labeled photolysis zone. Controlled isolation of these biological entities is influenced by the different pumping speeds of sheath buffer syringe pump 508 and buffer syringe pump 513. Nominally, the pumping rate of sheath buffer syringe pump 508 is, for example, but not limited to, ten times the pumping rate of buffer syringe pump 513. As further described herein, effective isolation of each biological entity may be assessed by determining the time differences in the detected light scattering signals, which are shown in FIG. In a similar manner to the signal profile, it is maximum when an in-vivo entity is irradiated and minimum when this entity is absent. In this embodiment, a light scattering detector is used to assess the presence of in-vivo entities. In other embodiments, the light scattering detector is replaced with a refractive index detector that determines the change in phase of the detected light resulting from elastic scattering.

Once isolation of the desired cells or in vivo entities is achieved, radical labeling proceeds very similarly to that performed for in vitro experiments. The effective radical dose is estimated by monitoring the change in the fluorescence signal measured in the dosimetry zone 608 when the biological entity is irradiated in the photolysis zone 607.

FIG. 10 shows the radical dosimetry response 1000 using CellRox® DeepRed and a 100 mM concentration of hydrogen peroxide. a baseline fluorescence level 1001, a rising shoulder in the dosimeter fluorescence signal 1002, a maximum dosimeter fluorescence level 1003, a falling shoulder in the dosimeter signal 1004, a re-established baseline fluorescence level 1005, a maximum fluorescence level 1006 occurring at time T1, and The maximum fluorescence level 1007 occurring at time T2 is shown. As shown, the baseline fluorescence dosimeter signal 1001 rapidly transitions to a rising shoulder 1002 and peaks at fluorescence level 1003 during the pulse of the mixture. As the oxidized CellRox Deep Red exits the dosimetry area, the fluorescence signal decreases as shown by the falling shoulder 1004 and eventually re-establishes its low background fluorescence baseline at 1005.

Similar to in vitro radical labeling, between two consecutively exposed boluses of cells or two maximum fluorescence values for in vivo embodiments, as illustrated by 1006 (T1) and 1007 (T2). The time difference can be used to evaluate the amount of isolation and the intercellular separation distance produced by the sheath flow generator 606. Similar to in vitro systems, the reproducibility of all maximum fluorescence signals may be evaluated to determine the quality and reproducibility of the obtained in vivo radical labeling.

Once an appropriate dosimeter response and isolated amount of in vivo entity is achieved, the in vivo entity can be reliably labeled and the labeled in vivo product can be collected. During the labeling and product collection process, the instrument controller 106 records the time that the dosimeter and isolated volume goals are achieved and determines the time of arrival of the appropriately labeled product at the outlet of the inlet exit port 202. do. The arrival time is determined by the quotient of the volume transferred from the dosimetry zone 608 to the inlet/outlet port 202 and the net flow rate. Prior to the arrival of the appropriately labeled product, the effluent contents of optofluidic chip 601 are dispensed into designated waste wells 221 through inlet/outlet ports 202 . Under the control of the instrument controller 106 , once the appropriately labeled product reaches the outlet of the inlet/outlet port 202 , the microplate 201 is configured such that the labeled product is collected within the product collection microwell 222 . , are moved by the sample introduction system 101.
<Post-labeling analysis>

For both in vivo and in vitro radical footprinting experiments, post-labeling analysis typically follows and the collected sample is optionally analyzed using an analyzer. The analyzer is configured to perform chemical analysis of the sample. For example, the analyzer may be configured to identify oxidized proteins, peptides, carbohydrates, metals, nucleic acids, lipids, and/or amino acids in the photolysis zone. The analyzer can be, for example, a mass spectrometer, scintillator, electrophoresis device, chromatograph, and/or configured to separate and/or identify sample components based on radiation, mass, charge, size, or other properties. may also include any other devices that may be used. In some embodiments, the analyzer detects isotopic or radioisotopic labels within cells or other biological entities and selectively determines whether components containing such labels have been oxidized. configured to determine. In some embodiments, the analyzer is configured to measure the ratio of labeled/unlabeled concentrations of a particular component. In some embodiments, the analyzer is "in-line" with port 202 and is therefore configured to receive and analyze the sample in real time. For example, port 202 may include a capillary tube configured to flow sample directly into a mass spectrometer.
<Specific control of sheath flow>

Various embodiments of the present invention include methods for identifying and controlling intercellular isolation to enable reproducible in vivo radical dosimetry. For in-vivo radical dosimetry to be performed effectively, photometric measurements of an internal standard radical dosimeter must be performed when in-vivo entity(s) are present within the dosimetry zone to It should enable meaningful comparative measurements that represent differences in intracellular radical dosimeter responses.

FIG. 11 shows an exemplary method 1100 of identifying and controlling intercellular mass using the invention described herein, which method 1100 further enables detection of in vivo entities within a dosimetric area. Make it. Cell introduction step 1101, first cell detection step 1102, second cell detection step 1103, net flow rate identification step 1104, cell isolation amount identification step 1105, good isolation evaluation step 1106, poor isolation and sheath flow rate adjustment step 1107 , as well as an iterative cycle 1108.

In the cell introduction step 1101, a single tandem array of cells is formed and the dosimetry area 60
8. In a first cell detection step 1102, the signal from the scattered light detector 812 is monitored to detect the time of arrival of the first cell to the dosimetry area 608. When cells or other biological entities arrive within the dosimetry area 608, the intensity of the scattered excitation light 810 increases according to the size of the biological entities and the number of biological entities within the examination area. In the second cell detection step 1103, the process described above is used to detect the arrival time of the second cell to the dosimetry area 608. The net flow rate of the system is calculated by summing the pumping rates of syringe pump 508, syringe pump 513, and syringe pump 518 (1104). The flow rate may be reduced to such an extent that the time between detection of the first and second cells is long enough to reduce the probability that the two cells are in the dosimetry zone 608 at the same time. This achieves single cell isolation where conditions allow for irradiation and oxidation of one cell at a time.

In the cell isolation amount calculation step 1105, the intercellular isolation amount is determined by multiplying the arrival time difference between the first cell and the second cell by the net flow rate 1104. If the experimentally determined cell isolation amount deviates from the desired isolation amount (which is directly related to separation distance and separation time) by less than ±5% (or other predetermined limit), then the system proceeds to label additional cells without further adjustment in proceeding step 1006. Optionally, if the experimentally determined cell isolation amount deviates by more than ±5%, the pumping speed of the sheath flow syringe pump 508 is adjusted to the desired cell isolation target amount 1107 in a sheath flow rate adjustment step 1107. The identification process is repeated 1108 until the target cell isolation amount is achieved.

Various embodiments include systems and methods for detecting the presence of biological entities within dosimetry area 608. The detected times of entry and exit of biological entities within the dosimetry zone 608 may be used to identify data acquisition periods for photometrically determining the intracellular dosimeter internal standard radical response. . Upon entering the dosimetry zone 608, the biological entity causes a rapid increase in the intensity of the scattered light detected by the scattered light detector 812. Concordantly, when the in-vivo entity exits, the intensity of the scattered light detected by the scattered light detector 812 decreases rapidly. The time difference between the rise and fall of the scattered light intensity represents the period of existence of the dosimetric zone of an in-vivo entity, such as a cell. During the presence period, the light intensity values detected by the luminescent light detectors 809 are summed and/or integrated to determine the net dosimetric signal for the in-vivo entity of interest. By using this approach, photometric dosimetry detects signals arising from intracellular and/or extracellular photometric signals while eliminating any inherent background arising from extracellular fluid for regions devoid of in vivo entities. This may be done by rejecting the signal. In this way, the background signal for the extracellular fluid is, by experimental design, substantially lower than the background signal for the intracellular fluid, and measurements are only made when the in vivo entity(s) are present. Therefore, the majority of the measured photometric signal consists of those originating from intracellular components.
<Identification of in-vivo embodiment survival rate>

Some embodiments include systems and methods for determining the viability of an in-vivo entity. For a given biological entity, the scattered light intensity is proportional to the size and number of biological entities present within the dosimetry area 608 during the photometric evaluation. Using the methodology disclosed herein, means are described for effectively ensuring a constant arrival rate of biological entities within the dosimetry area 608. Under such circumstances, the number of in-vivo entities in each data acquisition period can be controlled to be constant, and the fluctuation of the measured scattered light becomes highly dependent on the change in in-vivo entity size. Changes in in vivo entity size may be due to inherent morphological variations or may be indicative of artifactual changes in cell/species morphology and may be indicative of cell destruction, cell death, or cell apoptosis. Since the unique goal of in vitro cultured HRPF is to assess the biomolecular complement HOS under viable conditions, it is desirable to detect and signal the presence of nonviable processes and/or conditions.

Embodiments of the invention described herein provide systems and methods for detecting potential harm to living but not destroyed parts of the body that may result from HRPF protocols. The present invention provides a means by which in-vivo HOS analysis can be performed for in-vivo entities that do not exist. For example, the presence of a destroyed cell or two or more cells within the dosimetry area 608 can be detected simultaneously based on the measured light scatter. Those entities detected under these conditions may be separated from entities that were not irradiated under these conditions and discarded.
<Controlled operation of flash photolysis light>

Various embodiments of the invention include systems and methods for operating flash photolysis light source 103 in synchronization with the arrival of biological entities to photolysis zone 102. In-vivo radical footprinting methods ideally ensure that the entity(s) mentioned above are irradiated in a reproducible manner and within the photolysis zone in order to photocatalytically induce reproducible intracellular radical loading. identifying and quantifying the presence of the in-vivo entity.

While FIG. 12 identifies the arrival of the biological entity to the photolysis zone 608, the flash photolysis light source 103 is precisely operated to flash upon the arrival of the biological entity to the photolysis zone 608. 12 illustrates an example method 1200 providing systems and methods. Cell introduction step 1201, first cell detection step 1202, determination of net flow rate 1203, determination of on-chip interconnection amount 1204, determination of transit time 1205, determination of photolysis zone arrival time 1206, light at cell arrival time 1207 Activation of the degrading action and continuation of the process with precipitation of appropriately labeled cells 1208 is shown.

In a cell introduction step 1201, a single tandem array of cells is formed as described elsewhere herein, and a first cell is introduced into the photolysis zone 102. In a detection step 1202, the output signal of the scattered light detector 812 is monitored to detect the time of arrival of the first cell to the dosimetry area 608. As cells or other biological entities enter the dosimetry area 608, the intensity of the scattered light increases according to the size of the biological entities and the number of biological entities within the examination area. This increase is detected by scattered light detector 812. In the net flow rate determination step 1203, the net flow rate of the system is calculated by summing the pumping rates of syringe pump 508, syringe pump 513, and syringe pump 518. In an interconnection identification step 1204, interconnections extending from the photolysis zone 102 and the dosimetry zone 608 are identified as a direct manifestation of the microfluidic system design, and are determined to be constant during in vivo radical protein footprinting. It remains as it is. In the transit time determination step 1205, the transit time required for the biological entity to travel from the photolysis zone 102 to the dosimetry zone 608 is calculated by dividing the amount of interconnection by the net flow rate. In the photolysis zone arrival time determination step 1206, the photolysis zone arrival time is calculated by determining the difference between the dosimetry zone arrival time and the transition time from the photolysis zone to the dosimetry zone. In operation step 1207, for subsequent cells or biological entities, the photolysis system is operated to flash at the identified photolysis zone arrival time or at regular intervals thereafter. In a continuing process step 1208, additional labeled cells are deposited into the labeled cell reservoir 221 until the target number of cells have been processed.

Although the teachings herein describe the particular utility of detection signals resulting from light scattering light detector 812 and fluorescence/emission light detector 809, these detectors may be combined with multiple undescribed combinations or means. It will be clear to those skilled in the art that it can be used for the purpose of detecting the arrival of a biological entity in the dosimetry zone 608 or for the purpose of predicting the arrival of a biological entity in the photolysis zone 102. It is. For example, the signal generated by the fluorescence/luminescence light detector 809 may be combined with the signal from the light scattering light detector 812 to improve the overall sensitivity for detecting the presence of biological entities within the dosimetry area. and can be summed in temporal coherence. As such, the methods described herein are intended to be illustrative and not limiting in scope.
<Radical dose measurement closed loop control>

Various embodiments of the invention include systems and methods for calibrating closed-loop controlled radical dosimetry systems. In these embodiments, the closed-loop controlled radical dosimetry system uses a calibration theory that is used to predict the required changes in optical flux or radical reagent concentration in response to measured radical dosimetric optical fluorescence changes. Contains circuit. The calibration function is determined empirically through multiple measurements in which known or controlled mixtures of support buffers, in-vivo entities, and radical dosimeters are used at various flux volumes or radical reagent concentration levels. For each separate controlled aliquot, a single flash is applied. In an exemplary embodiment, a software routine executed in either the low-level instrument control or the high-level user interface program (e.g., control electronics 105 and/or instrument controller 106) controls each flux or radical reagent concentration. Generate a lookup table or curve fit that describes the measured change in dosimetric light fluorescence at the applied flux volume and/or radical reagent concentration and the dosimeter fluorescence change measured for a single flash exposure. allows the generation of a mathematical expression or calibration function that describes the relationship between . In another embodiment, the look-up table and subsequent calibration function are manually generated by the user using fluorescence change values for each flash voltage value and/or radical reagent concentration.

During the in vivo radical protein footprinting process, background radical scavenging is assessed dosimetrically. The measured change in dosimetric photofluorescence is compared to a user-specified target change. If the measured dosimeter value deviates from the target value by more than ±10%, the applied flux amount or radical reagent concentration is changed to achieve the targeted change in measured dosimeter absorbance. . The above calibration function is used to predict the required changes in flux or radical reagent concentration.

It will be apparent to those skilled in the art that many more modifications than those already described are possible without departing from the inventive concept herein. Accordingly, the subject matter of the invention is not to be limited except in the spirit of the disclosure. Furthermore, in interpreting this disclosure, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms "comprises" and "comprising" should be construed as referring non-exclusively to an element, component, or step; Indicates that an element or step may be presented or utilized with, or combined with, other elements, components, or steps not explicitly referenced.

Although biological cells are used herein to describe various embodiments of the invention, in other embodiments, "cells" may refer to non-living cells in any example or claim. Biological entities, biological entities, viruses, multicellular organisms (e.g., fungi, spores, microorganisms, molds, algae, nematodes, amoebae, protozoa, gynoecium, yeast), or non-biological materials may be replaced by other entities such as

General structures and techniques, as well as more specific embodiments that may be used to accomplish different methods of carrying out more general goals, are described herein. Although only some embodiments are disclosed in detail above, other embodiments are possible and are considered to be included within this specification. This specification describes specific examples to achieve a more general goal that may be achieved in other ways. This disclosure is intended to be exemplary, and the claims are intended to cover any modifications or alternatives that may be foreseeable to those skilled in the art.

Logic circuits discussed herein may include electronic circuits, hardware, firmware, and/or software stored on non-transitory computer-readable media.

Only claims that use the term "means for" are intended to be construed under 35 USC 112, 6th paragraph. Furthermore, no limitations from the specification are intended to be read into any claims unless such limitations are expressly included in a claim. The computer described herein may be any type of computer, either a general purpose computer or some special purpose computer, such as a workstation or laboratory or manufacturing equipment. The computer may be an Intel (eg, Pentium or Core 2 duo, i3, etc.) or AMD-based computer running Windows 10, 8, 7, or Linux, or it may be a Macintosh computer. The computer may also be a handheld computer such as a PDA, cell phone, tablet, or laptop running any available operating system, including Android, Windows Mobile, iOS, and the like.

Copyright Notice: 37 C. F. R. Pursuant to Section 1.71(e), portions of this disclosure are subject to copyright protection and do not contain any material claimed to be copyrighted, such as source code listings, screenshots, user interfaces, or user instructions; Copyright protection is or may be available in any jurisdiction, including, but not limited to, any other aspects of this application. Copyright owners do not object to facsimile reproduction of either a patent document or patent disclosure because the patent document or patent disclosure appears in the Patent and Trademark Office's patent files or records. All other rights are reserved, and all other reproduction, distribution, creation of derivative works based on the Content, public display, and public performance of the Application or any portion thereof is prohibited by applicable copyright law. .

Claims (27)

An optofluidic chip for radical protein footprinting, comprising:
A substrate and
a first port, a second port, and a third port each defined within the substrate;
a mixing zone defined in the substrate and including a first cavity in fluid communication with both the second and third ports;
a photolysis zone defined in the substrate and including a second cavity in fluid communication with the mixing zone, further comprising a first optically transparent window sealed in the substrate and bounding one side of the second cavity; the photolysis zone, wherein the second cavity includes channels configured in a serpentine pattern, and the photolysis zone is transparent on both sides;
a dosimetry zone defined in the substrate and including a third cavity in fluid communication with the photolysis zone and further in fluid communication with the first port, the dosimetry zone being sealed to the substrate and on one side of the third cavity; the dosimetry area further comprising a second optically transparent window bounding the dosimetry area;
including an optofluidic chip. The optofluidic chip of claim 1, wherein the substrate comprises plastic. 3. The optofluidic chip of claim 1 or 2, wherein the substrate comprises quartz. 4. The optical fluid of claim 3, wherein the substrate includes an intermediate layer of silicon, an upper layer of quartz, and a lower layer of quartz, the intermediate layer being disposed between the upper layer and the lower layer. Chip. 4. The substrate of claim 3, wherein the substrate includes an intermediate layer of silicon, an upper layer of fused silica, and a lower layer of fused silica, and the intermediate layer is disposed between the upper layer and the lower layer. optofluidic chip. further comprising a sheath flow generator defined within the substrate and including a fourth cavity in fluid communication with both the mixing zone and the photolysis zone; 6. The fourth cavity includes two sheath inlet ports in fluid communication with a fourth cavity, the sheath inlet ports and the fourth cavity configured to create hydrodynamic focusing. optofluidic chip. The in-line mixer is disposed in fluid communication between the sheath flow generator and the photolysis zone, and the sheath flow generator is in fluid communication between the in-line mixer and the third port. , an optofluidic chip according to claim 6. The sheath flow generator is disposed in fluid communication between the photolysis zone and the in-line mixer, the in-line mixer being between the sheath flow generator and both the second and third ports. 7. The optofluidic chip of claim 6, wherein the optofluidic chip is in fluid communication. An optofluidic system for radical protein footprinting, comprising:
a chip including a substrate and a first port, a second port, and a third port each defined in the substrate, the chip further comprising:
a mixing zone defined in the substrate and including a first cavity in fluid communication with both the second and third ports;
a photolysis zone defined in the substrate and including a second cavity in fluid communication with the mixing zone, further comprising a first optically transparent window sealed in the substrate and bounding one side of the second cavity; the photolysis zone, wherein the second cavity includes a channel configured in a serpentine pattern;
a dosimetry zone defined in the substrate and including a third cavity in fluid communication with the photolysis zone and further in fluid communication with the first port, the dosimetry zone being sealed to the substrate and on one side of the third cavity; the dosimetry area further comprising a second optically transparent window bounding the dosimetry area;
a microfluidic system in fluid communication with both the second and third ports of the mixing zone;
optofluidic systems, including; 10. The system of claim 9, further comprising a reservoir configured to receive a biological entity including an oxidized biological compound. 11. The system of claim 9 or 10, further comprising a light source configured to provide light to the dosimetry area. 12. The system of claim 9, 10, or 11, further comprising a fluorescence photodetector configured to receive fluorescence emitted from within the dosimetry zone. 13. The system of claim 9-11 or 12, further comprising a light source configured to provide coherent light to the dosimetry area. 13 . The method of claim 13 , further comprising a scattered light detector configured to receive light scattered from within the dosimetry area in a direction perpendicular to a direction of the light from the light source. system described in. 5. The method further comprises a refractive index photodetector configured to receive light scattered from within the dosimetry zone in a direction perpendicular to the direction of the light from the light source. The system described in 13. The chip further includes a sheath flow generator defined in the substrate and including a fourth cavity in fluid communication with both the mixing zone and the photolysis zone, the sheath flow generator being defined in the substrate. and comprising two sheath inlet ports in fluid communication with the fourth cavity, the sheath inlet ports and the fourth cavity configured to create hydrodynamic focusing. or the system described in 15. A method for use in conjunction with an optofluidic array including a photolysis zone, a photolysis light source, a dosimetry zone, and a dosimetry light source, the method comprising:
introducing into the photolysis zone a mixture comprising a biological entity, a chemical compound, and a dosimeter internal standard;
providing a light pulse from the photolysis light source to the photolysis zone, the light pulse being one of a series of periodic light pulses to generate a concentration of radicals from the chemical compound; the radical is effective to react with the dosimeter internal standard;
providing light from the dosimetry light source to the dosimetry zone and detecting a change in light received from within the dosimetry zone after the light from the photolysis light source is provided to the photolysis zone; the change in the received light indicates that the biological entity is within the dosimetry zone, and measuring photometric properties of the dosimeter internal standard within the dosimetry zone;
determining that the measured photometric characteristic is less than a threshold;
identifying a change to be applied to the optofluidic array to bring the measured photometric property to the threshold, the change comprising: a change in the amount of light provided by the photolytic light source; changing the concentration of said compound in the mixture, changing the flow rate of said mixture through said photolysis zone, or adjusting the phase of said periodic pulse;
including methods. 18. The method of claim 17, wherein the photolytic light source comprises a plasma flash lamp, an excimer laser, a solid state laser, or a laser diode. 19. A method according to claim 17 or 18, wherein the dosimetric light source comprises a visible or UV light source. 20. The method of claim 17, 18, or 19, wherein the compound comprises hydrogen peroxide. 21. The method of claim 17-19 or 20, wherein the compound comprises triflinate. 22. A method according to claim 17-20 or 21, wherein the photometric property comprises UV absorbance. 23. A method according to claim 17-21 or 22, wherein the photometric property comprises UV or visible fluorescence. A method for sheath flow control in an optofluidic array including a dosimetry zone, a dosimetry light source, and a sheath flow generator, the method comprising:
introducing a mixture comprising a plurality of cells, a compound, and a dosimeter internal standard into the sheath flow generator, and further introducing a buffer solution into the sheath flow generator, the sheath flow generator comprising: effective for passing said mixture comprising said plurality of cells therethrough using mechanical focusing, said cells of said plurality of cells being passed individually with uniform spacing therebetween; , process and
receiving the mixture from the sheath flow generator into the dosimetry zone and providing light to the dosimetry zone from the dosimetry light source while monitoring light received from the dosimetry zone; the received light changes with periodicity as the cells individually pass through the dosimetry zone;
adjusting the flow rate of the mixture or the buffer into the sheath flow generator to control the periodicity;
including methods. 25. The method of claim 24, wherein the optofluidic array further includes a first pump that delivers the mixture to the sheath flow generator and a second pump that delivers the buffer to the sheath flow generator. . 26. The method of claim 25, wherein adjusting the flow rate of the mixture or the flow rate of the buffer to the sheath flow generator includes adjusting a differential pumping rate between the first and second pumps. Method. 26. The method of claim 25, wherein the pumping speed of the second pump is at least 10 times the pumping speed of the first pump.

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