Optofluidic arrays for radical protein footprinting

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 intrinsic standard is used. Real-time feedback based on internal standards provides comparability between different experiments, and in vivo and in vitro analytical results provide data representative of real biological conditions.

Description

[Technical field]

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 128,439, filed December 21, 2020, and is also a continuation-in-part of U.S. Nonprovisional Application No. 17 / 193,913, filed March 5, 2021, which is a continuation-in-part of PCT / US20 / 12430, filed January 6, 2020, and a continuation-in-part of PCT / US19 / 57059, filed October 18, 2019. Both PCT / US20 / 12430 and PCT / US19 / 57059 are continuations-in-part of U.S. Nonprovisional Application No. 16 / 316,006, filed January 7, 2019, now U.S. Patent No. 10,816,468. U.S. Nonprovisional Application No. 16 / 316,006 claims the benefit of U.S. Provisional Application No. 62 / 747,247, filed October 18, 2018, and No. 62 / 788,219, filed January 4, 2019. PCT / US19 / 57059 further claims the direct benefit of U.S. Provisional Application No. 62 / 747,247, filed October 18, 2018. Both PCT / US20 / 12430 and PCT / US19 / 57059 claim the benefit of U.S. Provisional Application No. 62 / 788,219, filed January 4, 2019. All of the above patent applications are incorporated herein by reference.

[0002] 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.

[0003] The present invention relates to an apparatus and methodology for higher order structure analysis of biomolecules by the process of radical protein footprinting. Some embodiments of the present invention relate to the identification of tertiary and quaternary structures and associated conformations of biopharmaceuticals, and use improved apparatus and methodology for flash photo-oxidation of proteins processed using integrated optics and microfluidic chips to identify the higher order biomolecular structure of proteins. [Background technology]

[0004] Discussion of any work, publication, sale, or activity in this submission, including any documents submitted with this application, should not be construed as an admission that such work constitutes prior art. Discussion of an activity, work, or publication herein is not an admission that such activity, work, or publication existed or was known in any particular jurisdiction.

[0005] Biosimilars are therapeutics that are similar but not identical to existing originator or reference products. Unlike in the case of small molecule drugs, biosimilars are not simply generic versions of the original product. Conventional generics are considered to be therapeutically and molecularly equivalent to their originator. This is simply not the case for biosimilars, which are complex three-dimensional biomolecules, whose heterogeneity and dependence on production in living cells are quite different from conventional drugs. The structure and functional activity of biopharmaceuticals are highly sensitive to the environment. The intended structure of the therapeutic is maintained by a delicate balance of factors including protein concentration, control of post-translational modifications, pH and cosolutes in the formulation, as well as production / purification schemes (Gabrielson, JP; Weiss IV, WF, 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 result.

[0006] Adverse drug reactions (ADRs) of biopharmaceuticals are typically exaggerated. ADRs in patients range from symptomatic irritation to morbidity and mortality. Although the etiology of some ADRs may be due to the pharmacogenomic susceptibility of patients, many are believed to be due to the inherent properties of therapeutic drugs, which lead to morbid and fatal outcomes for patients and significant economic losses for 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 devastating ADRs illustrates the need for improved analysis for the development and quality control of biopharmaceuticals.

[0007] To minimize ADRs and expedite the development of biosimilars, the FDA, the Center for Drug Evaluation and Research, and the Center for Biologics Evaluation and Research have issued guidelines that emphasize the use of state-of-the-art techniques for evaluating protein higher order structure (HOS) (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 biotherapeutics. Today, various HOS assays exist, but their inadequacy to reliably predict the efficacy and safety of biotherapeutics has been questioned, and an unmet need for new and improved HOS assays has been established (Gabrielson,JP;Weiss IV,WF,Technical decision-making with higher order structure data:starting a new dialogue;Journal of pharmaceutical sciences;2015).

[0008] A technique that addresses the unmet need for HOS analysis is radical protein footprinting, which relies on irreversible protein hydroxylation, coupled 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 has been termed hydroxyl radical protein foot-printing (HRPE). A variety of techniques are used to perform HRPE. Perhaps the most widely used approach is the fast photochemical oxidation of proteins, which uses a single, high-flux, short pulse of UV light to generate hydroxyl (OH) radicals from hydrogen peroxide (H2O2). The reaction of OH radicals with solvent-exposed amino acids typically results in the insertion of a net oxygen atom into the amino acid. OH radicals are short-lived, and when generated by short UV pulses, the reaction between the amino acid and the radical may be completed before conformational changes occur in the labeled protein (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). Mass spectra of peptide products of enzymatic digestion show various levels of oxidation characterized by peak shifts of 16 Da, 32 Da, 48 Da, etc.This information can be used to deduce which peptides are located outside the HOS, thus leading to a greater understanding of the HOS.

[0009] Recently, Gross and coworkers demonstrated another radical protein footprinting technique based on the generation of trifluoromethyl (CF3) radicals formed by hydroxyl radical attack of aqueous sodium triflate (Cheng, M. et al., Laser-Initiated Radical Tirfluoromethylation of Peptides and Proteins: Application to Mass-Spectroscopy, 2011, 1111-1123, 2011). (2017) spectrometry-Based Protein Footprinting; Angewandte Chemie International Edition, 2017, 56(45): p.14007-14010). When proteins are labeled via CF3 radicals, 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, they result in superior coverage of surface-exposed amino acids compared to OH radicals alone. As with HRPF, CF3 labeling information can be used to infer which peptides are located on the exterior of a protein, thus leading to a deeper understanding of protein HOS.

[0010] The technical limitations of HRPF that adversely affect comparative studies result from the reaction of OH radicals with non-analyte components in the sample, such as buffer components, initial solutes, and extraneous biological materials. The variability in the background removal ratio causes irreproducibility from test to test, 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). OH radicals are an excellent probe of protein topography, but they also react with many compounds present in analytical preparations. Since there is competition for free OH radicals between the analyte protein and background scavengers, it is desirable to measure the effective concentration of radicals available to oxidize the target protein to ensure reproducible results. Similarly, variability in background removal also adversely affects the reproducibility of the CF3 radical footprinting method. In photochemistry, the effective radical concentration is measured using a radical dosimeter internal standard. Ideally, the dosimeter would have a simple relationship between effective radical concentration and dosimeter response, a simple, rapid, non-destructive means of measurement, and be non-reactive to most proteins.

[0011] The prior art teaches radical dosimetry performed using an added peptide internal standard (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 Spectrom, 2016.) or using a UV-absorbing internal standard such as adenine added to a buffer and evaluated by post-labeling methods (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 (with selective proteolysis) to assess the relative ratio of oxidized peptide to oxidized target protein. If the desired peptide-to-protein oxidation ratio is not achieved, the entire experiment is repeated while adjusting the concentration of H2O2 according to the need to increase or decrease the effective OH radical load. For adenine dosimetry, the effective change in adenine UV absorbance is identified upon completion of the labeling process, and the ratio between the achieved adenine UV absorbance change and the target adenine UV absorbance change is determined. The H2O2 concentration is then varied according to the desired change in UV absorbance.Both approaches are performed after labeling is complete and do not allow for real-time tuning of the effective OH radical loading, consuming precious sample and unnecessarily wasting the researcher's time.

[0012] U.S. Patent No. 10,816,468 and International Application PCT / US18 / 34682 teach systems and methods for real-time radical dosimetry as biologics are labeled during the FPOP HRPF process. While creating a real-time means of adjusting and compensating for variations in background removal, the systems and methods herein require the addition of an external internal standard dosimeter to the biological mixture. Under some conditions, the external internal standard may cause artifactual changes in the higher order structure of the biomolecules, thus reducing the time required for the generation of the biologic. It may not meet the desired goal of providing a simple means of characterizing nascent conformation. The disclosures of U.S. Patent No. 10,816,468 and PCT / US18 / 34682 application are incorporated herein by reference.

[0013] US Provisional Application No. 62 / 747,247 and PCT / US19 / 57059 describe an apparatus and methodology that allows commonly used biological buffer systems to be used as internal standards in radical dosimeters. The photometric properties of some commonly used biological buffers are altered in a predictable manner by OH radical attack. Therefore, these buffers can be used as internal standards in radical dosimeters, eliminating the need to add external reagents, and the solvation properties of these buffers are well established to stabilize the nascent structure of biomolecules, so they do not alter biological conformation.

[0014] The aforementioned techniques describe devices and methods for performing HRPF radical dosimetry while labeling proteins or biopharmaceuticals in vitro. However, the act of applying the results of in vitro structural experiments to authentic 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, U.S. Patent No. 10,851,335 B2 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, H2O2 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. US Provisional Patent Application No. 62 / 788,219 and PCT / US2020 / 012430 describe an apparatus and methodology in which in vivo FPOP HRPF is improved by providing a means to perform intracellular radical dosimetry to evaluate and adjust intracellular cytoplasmic or organelle radical scavenging. In addition, it teaches a means by which the sheath flow rate and subsequent cell-to-cell spacing can be evaluated and ultimately controlled to ensure effective irradiation of suspended cells during the in vivo labeling process.

[0015] The aforementioned techniques describe apparatus and methods to perform in vitro or in vivo FPOP HRPF to label proteins or biopharmaceuticals using fused silica capillaries as a means for transport, establishing sheath flow, chemical processing, initiating photoradical reactions, and further providing an optical region for probing the photometric properties of proteins, cells, chemical reagents, and labeled products. Although useful for these early stage experiments, fused silica capillaries have several undesirable characteristics: 1) they are fragile and prone to accidental breakage, especially if the outer protective polyimide coating is removed; 2) they require removal of the ultraviolet (UV) opaque polyimide coating to allow for hydroxyl radical formation from H2O2 and compatible UV photometric dosimetry; 3) they require considerable care to ensure proper alignment to the optical components; 4) they require expensive and cumbersome microfluidic fittings to fabricate and form the fluidic circuits required to facilitate in vivo FPOP, for example; and 5) they are cumbersome to replace. It is not uncommon for small ID (<100 nm) capillaries to accidentally become clogged or break during high flux applications. Thus, previously taught systems and methods suffer from various shortcomings. Summary of the Invention [Problem to be solved by the invention]

[0016] Various embodiments of the present invention utilize a fused silica capillary to couple a target protein and a labeling reagent to the on-column. Systems and methods are included that address the above-mentioned shortcomings of current in vitro radical protein footprinting methods by providing a means to replace optofluidic chip components that support line mixing. The disclosed systems include a serpentine photolysis cell that substantially increases the dose during light-induced radical chemistry compared to capillary configurations. Various embodiments include an in-line dosimetry cell for real-time photometric assessment of effective radical production and adjustment for unwanted background removal using the photometric properties of an in vitro radical dosimeter internal standard.

[0017] Various embodiments of the present invention include systems and methods that address the above-mentioned shortcomings of current in vivo radical protein footprinting methods by providing a means to replace fused silica capillaries with optofluidic chip components that support on-line mixing of target cells and labeling reagents. Various embodiments support the generation of microfluidic sheath flows to allow precise control of cell-to-cell distances, allowing for effective and consistent irradiation of the exterior and intracellular portions of each cell, such as with a serpentine photolysis cell that substantially increases the dose during light-induced radical chemistry when compared to a capillary configuration. Various embodiments include an in-line dosimetry cell for real-time photometric assessment 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 splitting can be assessed and reproducibly controlled, as well as a means to identify the arrival time of cells into the HRPF photolysis zone.

[0018] Various embodiments of the present invention are directed to on-chip systems and methods for analyzing protein conformation, including improved embodiments for performing in vitro or in vivo flash photo-oxidation of proteins that enables advanced radical protein footprinting. In some embodiments, the present invention provides an in-line radical dosimetry system that establishes closed-loop control between the flash photolysis system and a dosimeter to control the irradiance of the flash photolysis system in response to measured changes in the photometric properties of an internal standard radical dosimeter.

[0019] In some embodiments, the invention includes an on-chip, in-line, in vivo, or ex vivo radical dosimetry system that establishes closed-loop control between an automated in-line microfluidic mixing system and a dosimeter to control the concentration of a labeled reagent in response to measured changes in the photometric properties of an internal standard radical dosimeter.

[0020] In some embodiments, the invention includes an on-chip, in-line, in vivo, or in vitro radical dosimetry system that establishes closed-loop control between a flash photolysis system and a dosimeter to control the irradiance of the flash photolysis system in response to measured changes in the photometric properties of an internal standard radical dosimeter, where radicals are generated by photolysis of a labeling reagent.

[0021] In some embodiments, using an on-chip in vivo in-line radical dosimetry system, the present invention includes a method for generating labeled biomolecules for analysis, the method including the steps of: (1) mixing cells with a biological buffer, an internal standard radical dosimeter that is ultimately taken up by the cells, and other labeling reagents; (2) introducing the cells into an optical dosimetry zone; (3) identifying nascent photometric characteristics of the cells; (4) irradiating the cells with at least one UV shot in a photolysis zone; (5) identifying changes in the photometric characteristics of the cells after photoirradiation; and (6) adjusting the spectral irradiance of a UV light source according to the changes in the radical dosimetry optical characteristics.

[0022] In some embodiments, using an on-chip ex vivo in-line radical dosimetry system, the present invention includes a method for producing labeled biomolecules for analysis, the method comprising: (2) 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 area; (3) determining a nascent photometric characteristic of the mixture; (4) irradiating the mixture with at least one UV shot in an optical photolysis area; (5) determining a change in the photometric characteristic of the mixture after irradiation; and (6) adjusting the spectral irradiance of the UV light source according to the change in the radical dosimetry optical characteristic.

[0023] In some embodiments, using an on-chip in vivo in-line radical dosimetry system, the present invention includes a method for generating labeled biomolecules for analysis, the method including the steps of: (1) mixing cells with a biological buffer, an internal standard radical dosimeter that is ultimately taken up by the cells, and other labeling reagents; (2) introducing the cells into an optical dosimetry zone; (3) identifying nascent photometric characteristics of the cells; (4) irradiating the cells with at least one UV exposure in a photolysis zone; (5) identifying changes in the photometric characteristics of the cells after photoirradiation; and (6) adjusting the concentration of the labeling reagent using an in-line microfluidic mixer according to the changes in the radical dosimetry optical characteristics.

[0024] In some embodiments, using an on-chip ex vivo in-line radical dosimetry system, the present invention includes a method for generating labeled biomolecules for analysis, the method including the steps of: (1) mixing a target protein(s) with a biological buffer, an internal standard radical dosimetry, and other labeling reagents; (2) introducing the mixture into an optical dosimetry zone; (3) determining nascent photometric properties of the mixture; (4) irradiating the mixture with at least one UV shot in a photolysis zone; (5) determining changes in photometric properties of the mixture after photoirradiation; and (6) adjusting the concentration of the labeled reagent using an in-line microfluidic mixer according to the changes in the radical dosimetry optical properties.

[0025] In some embodiments, using an on-chip in vivo in-line radical dosimetry system, the present invention includes a method for generating labeled biomolecules for analysis, the method including the steps of: (1) mixing cells with a biological buffer, a labeling reagent, and an internal standard radical dosimeter that is ultimately taken up by the cells; (2) introducing the cells into an optical dosimetry zone; (3) detecting the arrival and presence of the cells by monitoring the intensity of scattered light exiting the dosimetry zone; (4) determining the elapsed time between the arrival of successive cells; (5) determining the cell isolation volume per product of elapsed time and net buffer flow rate; and (6) adjusting sheath flow and buffer flow parameters to achieve the desired cell isolation volume.

[0026] In some embodiments, using an on-chip in vivo in-line radical dosimetry system, the present invention includes a method for generating labeled biomolecules for analysis, the method including the steps of: (1) mixing cells with a biological buffer, a labeling reagent, and an internal standard radical dosimeter that is ultimately taken up by the cells; (2) introducing the cells into an optical dosimetry zone; (3) detecting the arrival and presence of the cells by monitoring the change in phase of the light exiting the dosimetry zone; (4) determining the elapsed time between the arrival of successive cells; (5) determining the amount of cell isolation per product of the elapsed time and the net buffer flow rate; and (6) adjusting the sheath flow and buffer flow parameters to achieve the desired amount of cell isolation.

[0027] In some embodiments, using an on-chip in vivo in-line radical dosimetry system, the invention includes a method for generating labeled biomolecules for analysis, the method comprising the steps of: (1) mixing cells with a biological buffer, a labeling reagent, and an internal standard radical dosimeter that is ultimately taken up by the cells; (2) introducing the cells into an optical dosimetry zone; and (3) detecting the arrival and presence of the cells by monitoring the intensity of scattered light exiting the dosimetry zone. (4) determining a net flow rate for the arriving cells; (5) determining an interconnect volume extending from the photolysis zone and the dosimetry zone; (6) determining a transit time required for the cells to move from the photolysis zone to the dosimetry zone; (7) determining a photolysis zone arrival time for the cells; and (8) operating the photolysis system at a time such that all subsequent cells arrive at the photolysis zone.

[0028] In some embodiments, using an on-chip in vivo in-line radical dosimetry system, the present invention includes a method for generating labeled biomolecules for analysis, the method including: (1) mixing cells with a biological buffer, a labeling reagent, and an internal standard radical dosimeter that is ultimately taken up by the cells; (2) introducing the cells into an optical dosimetry zone; (3) detecting the arrival and presence of the cells by monitoring the phase exiting the dosimetry zone; (4) determining the net flow rate for the 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 at a time such that all subsequent cells arrive at the photolysis zone.

[0029] Following production of the labeled biomolecule, other methods such as mass spectrometry or electrophoresis may be used to identify the labeled peptides and to deduce information about the higher order structure of the biomolecule in vivo or in vitro.

[0030] Various embodiments of the invention include an on-chip analytical system including a sample introduction system configured to provide nascent biological entities to a photolysis zone, where the biological entities are isolated from one another in a focused sheath flow; a photolysis light source configured to generate light to generate radicals from a source of labeling reagent; a photolysis zone configured to receive the sheath flow and the light to label an internal standard and to label a biological compound of the biological entity in vivo; a dosimetry zone configured to receive the biological entities from the photolysis zone and detect the presence of the biological entity using a scattered light detector and detect oxidation of the internal standard using a fluorescence detector; control logic configured to identify a target concentration of radicals for each of the biological entities and adjust operation of the photolysis zone to meet the target concentration of radicals; and a reservoir configured to receive the biological entities including the labeled biological compounds.

[0031] Various embodiments of the invention include an on-chip method for labeling biomolecules in nascent cells, comprising the steps of: introducing a sample mixture including at least one cell into a photolysis zone; introducing a source of labeled radicals and a dosimeter internal standard into the photolysis zone of a flash photolysis system; providing light to generate labeled radicals from the source of labeled radicals, the labeled radicals configured to label the biomolecules in the at least one cell; and performing dosimetry with a radical dosimeter configured to detect a photometric property of the dosimeter internal standard resulting from a reaction between the dosimeter internal standard and the labeled radical. waiting, optionally for a predetermined time, until at least one cell arrives in the dosimetry zone of the radical dosimeter, where the at least one cell is detectable in the dosimetry zone of the radical dosimeter by light scattering; measuring a photometric characteristic of a dosimeter internal standard using the radical dosimeter while the at least one cell is in the dosimetry zone; determining that a target level of labeled radicals has not been generated based on the measured photometric characteristic of the dosimeter internal standard; and adjusting the concentration of labeled radicals in the photolysis zone by adjusting at least one of 1) to 4): 1) the amount of light provided to the photolysis zone; 2) the concentration of the provided labeled radicals; 3) the flow rate of at least one cell in the photolysis zone; or 4) the time of the provided light to the photolysis zone. In the above embodiment, the sequence of light pulses provided to the photolysis zone is adjusted to a predetermined value. Note that a pulse has both a periodicity and a phase, and adjusting the phase of a periodic pulse changes the timing of the pulse without changing the periodicity. [Brief description of the drawings]

[0032] The novel features of the present invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative 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 that incorporate features of the present invention. The components in the drawings are not necessarily to scale, emphasis instead being placed upon clearly illustrating the principles of the present disclosure.

[0033] Any of the methods described herein may, according to certain embodiments, further utilize any one or more of the following:

[0034] [Figure 1] FIG. 1 illustrates an embodiment of a flash photolysis system. [Diagram 2] FIG. 2 shows an embodiment of a microfluidic system. [Diagram 3] FIG. 3 shows in detail an embodiment of the components of an optofluidic chip. [Figure 4] FIG. 4 details the dosimetric area photometric response plot for a mixture of adenine and hydrogen peroxide when irradiated with pulses of UV light spaced 1 second apart. [Diagram 5] FIG. 5 shows an embodiment of a microfluidic system. [Figure 6] FIG. 6 shows a preferred embodiment of the in vivo optofluidic chip. [Figure 7] FIG. 7 is a table listing the flow cell, fluorescence excitation, and emission wavelengths for several internal standard radical dosimeters. [Figure 8] FIG. 8 shows an embodiment of a combined fluorescence and light scattering detector system of the present invention. [Figure 9] FIG. 9 shows another embodiment of a combined fluorescence and light scattering detector of the present invention. [Figure 10]FIG. 10 shows the fluorescent radical dosimetry response during irradiation of an in vivo embodiment of the present invention using CellRox® Deep Red and hydrogen peroxide. [Figure 11] FIG. 11 shows an exemplary process for specifying and controlling the isolation amount or intercellular distance in the present invention. [Figure 12] FIG. 12 shows an exemplary process 1200 for cell arrival identification using coordinated photolysis light triggering. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] Apparatus and methods are provided for the analysis of biomolecular conformation, achieved by selective labeling of solvent-exposed molecular groups, acting as a catalyst, via on-chip in vivo or in vitro fast photo-oxidation with real-time monitoring and control of effective radical concentration. Further, apparatus and methods are provided for the analysis of biomolecular conformation, achieved by on-chip in-line microfluidic mixing of labeling reagents with target proteins, cells, or other in vivo embodiments. Further, apparatus and methods are provided for the analysis of biomolecular conformation, achieved by on-chip microfluidic channels acting to generate sheath flow to support fast photo-oxidation reactions in vivo. Further, apparatus and methods are provided for the analysis of biomolecular conformation, achieved by on-chip in-line photochemically induced reactions using an integrated serpentine photolysis cell.

[0036] Catalyze by on-chip in vivo or in vitro fast photooxidation with real-time monitoring and control of effective radical concentrations, achieved by selective labeling of solvent-exposed molecular groups In accordance with the present invention, an apparatus and method for the analysis of biomolecular conformation is provided. Additionally, an apparatus and method for the analysis of biomolecular conformation is provided, which is achieved by on-chip in vivo fast photo-oxidation with real-time monitoring and control of in vivo species isolation amount, followed by flash photolysis. The apparatus and method 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, virus-like particles, unicellular organisms, eukaryotic tissues, and multicellular organisms. While the present invention refers to cells for purposes of illustration, such reference is not intended to be limiting, and such reference is understood to be applicable essentially to all photometrically translucent or transparent in vivo biological or non-biological entities.

[0037] The devices and methods are applicable to various research fields such as 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, and development of other therapeutic compounds and materials. Furthermore, the devices and methods are applicable to various research analyses such as protein-ligand interaction analysis, protein-protein interaction analysis, protein-DNA interaction, protein-RNA interaction, protein fusion product analysis, protein conformation and conformation change analysis, cell-cell interaction, virus-cell interaction, 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.

[0038] The device can accept cells for subsequent chemical labeling via stepwise introduction of target proteins or cells by manually pipetting the target proteins or cells into appropriate microcentrifuge tubes or microplates located in the sample introduction assembly of the system. Alternatively, the device can be combined with other separation and analysis instruments and can receive target proteins or cells directly from other separation and analysis instruments, including, but not limited to, instruments that perform selective protein separation, cell sorting, cell counting, and cell isolation from tissues.

[0039] This section provides a general overview of a flash photolysis device having an on-chip in-line in vivo or in vitro radical dosimeter that uses the photometric properties of an internal standard dosimeter to assess and ultimately control the in vivo or in vitro available radical concentration. Additionally, this section provides a general overview of the invention that uses the photometric properties of an in vivo embodiment to assess the isolation volume and precisely control the separation of the in vivo embodiment within a flowing stream of buffer. Detailed descriptions of each subassembly are provided elsewhere herein. Additionally, methods describing the interaction of these subassemblies are provided to enable understanding of typical instrument operation.

[0040] Various embodiments of the present invention include a flash photolysis system 100, as shown in Figure 1. The flash photolysis system 100 is configured for real-time radical dosimetry and includes several subassemblies. Shown are a sample introduction system 101, a photolysis zone 102, a flash photolysis source 103, a radical dosimeter 104, control electronics 105, an instrument controller 106, a fluid interconnection line 107 between the sample introduction system 101 and the photolysis zone 102, a fluid interconnection line 108 between the photolysis cell and the radical dosimeter, an optical communication interconnection 109 between the flash photolysis source 103 and the photolysis zone 102, an electronic interconnection 110 between the radical dosimeter 104 and the control electronics 105, an electronic interconnection 111 between the sample introduction system 101 and the control electronics 105, an electronic interconnection 112 between the control electronics 105 and the instrument controller 106, and an electronic interconnection 113 between the control electronics and the flash photolysis system. Photolysis zone 102, flash photolysis source 103, and radical dosimeter 104 together comprise a "flash photolysis system."

[0041] The photolysis zone 102 is typically contained in a photolysis cell. is configured to oxidize ex vivo or in vivo sample cells in real time to achieve radical dosimetry. Analytes are introduced via the sample introduction system 101. Analytes can be presented using small volume micro centrifuge tubes or by using multi-well microtiter plates readily available from Eppendorf (Hamburg, Germany). The microfluidic circuit is configured for analyte aspiration, mixing with labeling reagents, cell hydrodynamic focusing using a sheath flow device for in vivo processing, photolysis and transport to the dosimetry zone, and transport and precipitation of the labeled product. In some embodiments, the sample introduction system 101 is configured to provide nascent biological entities to the photolysis zone (e.g., photolysis zone 102, biological entities isolated from each other in the focused sheath flow). 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.

[0042] Photo-oxidation occurs within the photolysis zone 102. In some embodiments, the photolysis zone is located within an optofluidic chip. In some embodiments, the optofluidic chip is fabricated using a variety of techniques, such as lithographically supported wet chemical etching, dry reactive ion etching, and laser ablation microstructuring, to create microfluidic channels within a silicon or quartz substrate. In some embodiments, the optofluidic chip is fabricated by embossing fluidic channels within a plastic substrate, and the formed fluidic channels transport the sample within an optically transparent cell created by sealing optically transparent windows in the areas where the plastic substrate has been removed. Such optically transparent windows may be located on only one side or on both sides of the photolysis and dosimetry cells disclosed herein. Exemplary plastic substrates include, but are not limited to, polycarbonate, polyethylene, polyether-ether-ketone, cyclic olefin polymers, cyclic olefin copolymers, polytetrafluoroethylene, Kalrez®, and polychlorotrifluoroethylene. The inner diameters of the fluidic and optical channels may range from, but are not limited to, 0.1 to 5.0 mm. In some embodiments, the fluidic and optical channels can have different internal diameters to ideally address different requirements for fluid transport, fluid mixing, hydrodynamic focusing, and optical coupling, hi some embodiments, the photolysis zone comprises a serpentine array of channels that juxtaposes multiple fluidic paths in a countercurrent fashion.

[0043] The photolysis zone 102 receives the analyte from the sample introduction system 101 via the microfluidic pathway 107. After processing, the photoirradiated analyte in the photolysis zone 102 is transferred to the radical dosimeter 104. The photolysis zone 102 is in optical communication with the flash photolysis light source 103. The flash photolysis light source 103 is an example of a photolysis light source configured to generate light for generating labeled radicals from a labeled reagent source in the photolysis zone 102. In some embodiments, the photolysis zone 102 is configured to receive a sheath flow containing a biological entity and receive light from the flash photolysis light source 103 to oxidize a dosimeter internal standard and oxidize biological compounds of the biological entity in vivo. For example, proteins and peptides containing amino acids may be oxidized in the photolysis zone 102.

[0044] Photolysis zone 102, flash photolysis light source 103, and radical dosimeter 104 constitute a flash photolysis system. The flash photolysis system includes a plasma flash lamp or other suitable light source (e.g., an excimer laser, solid state laser, or laser diode) and associated light collection / transmission optics to match the requirements of the light transmission means to the photolysis zone.

[0045] The radical dosimeter 104 is configured to receive the labeled analyte from the photolysis zone 102, or in other embodiments, the radical dosimeter 104 uses an orthogonal optical path. The dosimeter internal standard is incorporated in the photolysis zone 102 by detecting the presence of a dosimeter internal standard. Various photometric detection schemes may be used by the radical dosimeter to monitor the relevant photometric properties of the dosimeter internal standard. In some embodiments, the dosimeter internal standard may be an external additive added in the biological sample. In some embodiments, the inherent photometric properties of the biological buffer system may serve as the inherent dosimeter internal standard. By "intrinsic" or "intrinsic to the buffer" it is meant that the internal standard is one of the chemical species that provides buffer properties. The buffer is a physiologically compatible buffer configured to maintain the analyte or cells 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 become fluorescent as a result. For example, the dosimeter internal standard may increase in fluorescence by a factor of at least 10-fold, 100-fold, or 1000-fold upon reaction with hydroxide radicals in various embodiments.

[0046] 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 an ultraviolet (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 integrated light scattering detector. In some embodiments, the fluorescence detector also includes an integrated optical refractive index detector.

[0047] The control electronics 105 functions to provide direct current (DC) drive voltages derived from a laboratory alternating current (AC) power source to the peripheral assemblies, provide analog and digital control signals to the peripherals, receive analog or digital information from the peripherals, provide ADC and digital to analog conversion functions, and provide data to and receive commands from the instrument controller 106. In a typical embodiment, the control electronics assembly includes a motor controller that interfaces with a motor disposed within the sample introduction system 101. Additionally, the control electronics assembly in such an embodiment may include a universal serial bus (USB) hub for digital communication with the instrument controller 106.

[0048] The instrument controller 106 functions to provide process control for the various instrument peripherals while receiving status and data information in digital form from these devices. In some embodiments, the instrument controller 106 executes a software control program having two main modules: a low-level multi-threaded module for instrument component control and a high-level user interface (UI) module. In some embodiments, the control electronics 105 includes an embedded microprocessor that provides low-level instrument component control while communicating with the instrument controller's 106 high-level UI control program via a USB or wireless interface.

[0049] The instrument controller 106, control electronics 105, and the various interconnections together represent a control logic circuit configured to control the flash photolysis system 100. This control logic circuit may be configured to perform any of the steps of the methods disclosed herein. For example, in some embodiments, the control logic circuit is configured to determine that a target concentration of labeled radicals has been generated for each biological entity. This target concentration is selected to ensure that the protein or cellular component labeling reaction has enough labeled radical reagent to approach a desired level of completion, as appropriate.

[0050] The control logic circuitry may be further configured to manage a feedback loop that includes adjusting the conditions in the photolysis zone 102 to meet a target concentration of labeled radicals. The conditions in the photolysis zone 102 may be adjusted, for example, by 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, changing the time that light is received from the photolysis light source, changing the separation distance / amount of isolated cells, etc. By varying the conditions in the photolysis zone 102, the control logic circuitry may provide 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.

[0051] In some embodiments, the control logic circuit is configured to normalize quantification of oxidized and / or identified peptides from cells based on the fluorescent signal from an internal standard within the dosimetry area, allowing comparison of results from different experiments using different instantiations of flash photolysis system 100.

[0052] The control logic circuitry is optionally further configured to determine the time at which cells receive photolytic light within the photolysis zone 102 and / or to determine the time interval at which isolated cells enter the photolysis zone 102. These determinations 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.

[0053] The control logic circuitry is optionally further configured to control the flow of cells to the labeled cell reservoir and the analyzer. For example, the control logic circuitry may be configured to control the flow of cells such that ruptured cells are diverted from a particular vessel of the labeled cell reservoir, or such that different oxidized cells are disposed in different compartments over a function of time. In some embodiments, the control logic circuitry is configured to control any aspect of the flash photolysis system 100 using the analyte signal from the analyzer.

[0054] The instrument controller 106 and control electronics 105 are optionally combined into a single device. <Photochemical labels for in vitro analysis>

[0055] 2 shows further details of a flash photolysis system 100 configured to support the processing of in vitro analytes, along with a specific description of the system's microfluidic system 200. Shown are a 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, labeling reagent syringe pump 207, buffer reservoir 208, waste reservoir 209, sample storage loop 210, labeling reagent reservoir 211, reagent transfer line 212, buffer syringe pump four-way valve 213, in-line mixer gate valve 214, reagent waste line 215, labeling reagent syringe pump four-way valve 216, reagent inlet line 217, buffer inlet line 218, buffer waste line 219, sample microwell 220, waste collection microwell 221, and product collection microwell 222.

[0056] 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 aspiration through a sample inlet / product outlet port 202. The aspirated sample passes through the dosimetry and photolysis section 203 of the system and is further drawn through an in-line mixer 204. The in-line mixer 204 and the photolysis and dosimetry section 203 are both formed in an optofluidic chip 205.

[0057] The microfluidic system 200 includes two syringe pumps, a buffer syringe pump 206 and a labeling reagent pump 207. The syringe pump 206 is in communication with a buffer reservoir 208, a waste reservoir 209, and a sample storage loop 210. The syringe pump 207 is in communication with the waste reservoir 209, a reagent reservoir 211, and the in-line mixer 204 via a reagent transfer line 212.

[0058] Prior to the labeling process, the microfluidic circuit is primed. The syringe pump 206 is filled with buffer by switching its four-way valve 213 to communicate with the buffer reservoir 208. Its 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. The syringe pump 206 then primes the microfluidic system by switching the valve 213 to communicate with the storage loop 210 and pumping a volume sufficient to flush the system. During the system flush, the buffer is directed to flow through the in-line mixer 204, the photolysis and dosimetry area 203, the port 202, and finally dispensed into a designated waste well 221 of the microplate 201. During this process, valve 214 remains closed to in-line mixer 204 so that the buffer is directed through tip 205 and does not exit through mixer 204 and ultimately flow into reagent transfer line 212.

[0059] After priming the chip, the reagent delivery circuit is primed. The reagent pump 207 is completely filled with reagent drawn from the reagent reservoir 211 by pulling back its plunger while the valve 216 connects it to the reagent inlet line 217. The capacity of the reagent pump 207 can range, 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 volume of sample to be labeled. The reagent transfer line 212 is flushed by pumping with the reagent pump 207 having a four-way valve 216 configured to transfer the contents of the syringe 207 to the reagent transfer line 212, which has a valve 214 configured to direct the priming volume to the waste reservoir 209 via the reagent waste line 215. Valve 214 is positioned adjacent to in-line mixer 204 such that the interconnect volume from valve 214 to mixer 204 is minimized to the range of 0.25 to 1.5 μL. Further details of the fluid connections of chip 205 are provided in FIG.

[0060] The following steps outline an exemplary operational cascade reaction used by the embodiments described with respect to the microfluidic system 200 to label in vitro samples using light-induced radical reactions. Variations of this cascade reaction will be apparent to those skilled in the art, and such variations will 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, which selectively collect the labeling product, waste buffer, or labeling reagent precipitated during fluidic priming of the subsystems of the microfluidic system 200. For in vitro radical protein footprinting, the microwells 220 contain proteins or other proteinaceous biological mixtures. After priming, the buffer syringe pump 206 dispenses a predetermined amount of buffer by moving the valve 213 into communication with the sample loop 210, thereby pumping the buffer through the optofluidic chip 205 and through the inlet / outlet port 202 to a designated waste microwell 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, the syringe pump 2 06 dispenses 120 mL of buffer into waste microwell 221. Other different volume ratios can be used, and the above examples are illustrative and not limiting in scope.

[0061] The microplate 201 is moved so that the port 202 is submerged in the microwell 220 containing the sample to be labeled. The buffer syringe pump 206 withdraws its plunger and aspirates a predetermined amount of sample by fluidly communicating through the valve 213, the in-line mixer 204, the photolysis dosimetry cell 203, and the port 202. The sample is drawn through the photolysis dosimetry cell 203 and the in-line mixer 204 into the chip 205 and finally into the sample storage loop 210. During this sample aspiration process, the in-line mixer gate valve 214 closes the mixer to prevent labeling reagent from being drawn into the circuit.

[0062] Reagent reservoir 211 contains the labeling reagents required for the protein labeling process. For HRPF, reservoir 211 contains aqueous hydrogen peroxide, for example, at a concentration typically ranging from 20 mM to 1000 mM. For trifluoromethyl footprinting, reagent reservoir 211 contains a mixture of hydrogen peroxide and aqueous sodium triflate. If primarily CF3 radical labeling is desired, H2O2 and sodium triflate are mixed in a concentration ratio of 1:4, respectively, typically using 5 mM H2O2 and 20 mM sodium triflate. If simultaneously HRPF and CF3 footprinting are desired, H2O2 and triflinate are mixed in a ratio of 5:1, respectively, typically using 50 mM H2O2 and 10 mM triflic acid. The above mixtures are intended to be exemplary and should not be construed as limiting in scope, other ratios may function as functional equivalents, and absolute conditions may vary based on actual protein labeling conditions.

[0063] To begin the labeling process, pumps 206 and 207 begin to dispense their respective solutions into the microfluidic circuit. The relative pumping speeds of pumps 206 and 207 depend on the desired final concentration of labeling reagent entering the photolysis zone and the starting concentration of labeling reagent located in reservoir 211. Using hydroxyl radical labeling as an example, reagent reservoir 211 may contain a 1000 mM concentration of H2O2, and the desired final concentration in the label is 100 mM. Under these conditions, the pumping speed of pump 207 is one tenth of the pumping speed of pump 206. The net flow rate is the sum of the pumping rates established by pumps 206 and 207.

[0064] FIG. 3 shows a further embodiment of an exemplary optofluidic array 300 that allows for the following description. A detailed view of the photolysis cell 307 is shown, including the reagent inlet port 301, the buffer inlet port 302, the mixer 303, the photolysis cell 304, the dosimetry cell 305, the inlet / outlet port 306, and the fluid channel 308. After establishing the flow through the optofluidic chip 205, the photolysis light source 103 is operated to illuminate the photolysis zone 304 at regular flash intervals. The flash frequency may be in the range of 0.25 Hz to 5 Hz, but is not limited thereto. The flash frequency and the net flow rate are established to ensure that the mixture of sample and labeling reagent is illuminated by only one flash, and that each subsequent bolus of the illuminated mixture is divided by a predetermined isolation volume. Thus, the illuminated volume of the photolysis cell defines the required net flow rate and flash rate.

[0065] In capillary-based 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 (IDs) range from, but are not limited to, 50-250 μm. Additionally, the illuminated area is determined by the capillary diameter, which defines an axial length of illumination, which ranges from, but is not limited to, 2-8 mm. extends along the tube axis. 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 the wear of the light source. Furthermore, under these conditions, only a small portion of the photolysis light is focused into the capillary lumen, since the refractive focusing limit of both the excimer laser and plasma light sources results in a beam wastage larger than the inner diameter of the lumen. Thus, the capillary approach taught previously results in various drawbacks.

[0066] The optofluidic chip 205 includes a serpentine photolysis zone 304 as detailed by the photolysis zone 307. The fluid pathway within the photolysis zone 307 is arranged in a folded or serpentine manner with adjacent flow channels in close juxtaposition and flow is established in a counter-current manner. The serpentine photolysis zone allows for better opto-fluidic alignment between the photolysis light source 103 and the sample reagent mixture since the photolysis light can be distributed over a wider incident area, overcoming the refractive focus limitation. The fluence required for incident UV irradiation to effectively photolyze the labeled reagent depends on the UV source spectral irradiance and the labeled reagent extinction coefficient. For H2O2-based labels, at least 3 mJ / mm for UV wavelengths below 250 nm is required. 2 For both OH and CF3 radical labeling, the flash photolysis source 103 is 16 mm 2 The photolysis cell 304 has sufficient spectral irradiance to allow an illumination area of ​​up to 100 mm ID. Under such conditions, the fluid channel 308 in the photolysis cell 304 can be arranged to contain an illumination volume of approximately 6.4 mL. Thus, the illumination volume of the photolysis zone 304 is 136 times larger than that of the 100 mm ID capillary system described above, and thus labeling proceeds in a fraction of the time required for capillary-style labeling, while reducing distortion of the photolysis system by over 99%.

[0067] The mixture of sample and labeling reagent is irradiated by the photolysis light source 103 in the photolysis zone 304. After exposure, radicals are rapidly formed, typically within a few microseconds, covalently modifying the sample. The labeled sample, buffer, and modification reagent are pumped downstream into the dosimetry zone 305. The contents of the 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. Real-time measurements made in the dosimetry zone 305 allow for a feedback loop in which photolysis conditions can be modified to ensure that a desired amount of radical production occurs, resulting in a desired amount of in vitro analyte labeling. During initial operation of the system, a baseline measurement of the photometric properties of the dosimetry zone containing the mixture of unirradiated sample and reagent is made. Once the baseline measurement is made, photolysis proceeds and changes in the 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 that is directly proportional to the effective OH radical yield. During the fine-tuning process, the effective OH radical yield is controlled by modifying either the flash intensity or the H2O2 concentration in response to the difference between the evaluated and target changes in the dosimetry signal.

[0068] 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 the reagent pump 207 is increased with respect to the buffer pump 206, which then increases the effective concentration of the labeled reagent after mixing with the sample in the in-line mixer 204. If the actual response of the internal standard radical dosimeter is greater than the desired level, the flash energy of the photolysis light source 103 is decreased until the desired level of dosimeter response is achieved. Alternatively, the relative pumping speed of the reagent pump 207 is increased with respect to the buffer pump 206, which then increases the effective concentration of the labeled reagent after mixing with the sample in the in-line mixer 204. Reduces effective concentration.

[0069] FIG. 4 shows a real-time output trace 400 of UV absorbance versus time obtained in the dosimetry zone 305 while generating hydroxyl radicals in the photolysis zone 304, using a 1 Hz flash rate and 100 mM H2O2 in the presence of 1 mM aqueous adenine, which was mixed 1:1 (v / v) with 2 mM aqueous adenine and 200 mM H2O2 in a microfluidic mixer 303. The unexposed baseline 401 is shown. The falling shoulder 402 is shown. The peak shift (minimum 403) in the photometric response, or in this case UV absorbance, is shown. The rising shoulder 404 is shown. The 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 one skilled in the art, other adenine / H2O2 mixing ratios may be used to achieve the target concentration, and the 1:1 mixing ratio is meant to be exemplary and not limiting in scope. A baseline UV photometric absorbance measurement for the aforementioned mixture that has not been exposed to light is represented by the unexposed baseline 401. In the unexposed baseline 401, the baseline UV absorbance is interpreted to be approximately 55 milli-absorbance units (mAU) as determined against the background of the running buffer (water in this case). Irradiation of the mixture in the photolysis zone 304 results in the formation of OH radicals, which subsequently oxidize the adenine. The exposure time is not particularly limited, but is typically 10 μs. The hydroxyl radical reaction then proceeds for approximately another 1-5 μs, after which the reaction self-terminates. As the irradiated mixture leaves the photolysis zone 304 and enters the flow dosimetry zone 305, the photometric absorbance signal begins to decrease, as shown by the falling shoulder 402. The UV absorbance minimum of the labeled sample is taken to be approximately 33 mAU, as shown by minimum 403. Here, the effective change in adenine UV absorbance for the first illuminated bolus of mixture is calculated by taking the difference between the unexposed baseline 401 and minimum 403 (55 mAU-33 mAU) and is determined to be 22 mAU.As the irradiated mixture flows out of the dosimetry area 305, the measured UV absorbance returns to its baseline as indicated by a rising shoulder 404, and reaches baseline 405 again. The determined 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 H2O2 concentration are modified to adjust the OH radical yield using the control loop logic previously described. Once the target level of internal standard dosimetry light characteristic change is achieved, the light irradiation / dosimeter response evaluation process is repeated until the desired amount of labeled sample has been processed and subsequently collected.

[0070] During the labeling process, the effective change in the dosimeter response may be selectively accessed for each flash cycle to identify the reproducibility and inherent quality of the entire labeling process. Under these conditions, the average, 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. A typical RSD quality metric is determined by the specific photochemical labeling conditions for the target protein and may be, but is not limited to, less than 5%. If the quality metric is achieved, the labeling experiment is determined to be of acceptable quality. If the quality metric is not achieved, the labeling experiment is flagged as suspect and the experimenter is appropriately alerted.

[0071] Each exposed bolus of mixture is divided into a predetermined isolated volume, which includes the unirradiated mixture that flows through the photolysis zone 304 between successive flashes. The isolated volume is essential to ensure that each bolus of mixture is only illuminated once, by effectively acting as a buffer zone to mitigate undesired retrograde axial diffusion of the illuminated sample back to the bolus of the next sample to be illuminated, and by adjusting for laminar flow axial velocity differences. A useful isolated volume is one that prevents undesired release of labeled products. An exemplary desired isolation volume target is taken to be an amount that dilutes the sample by about 10%.

[0072] The photometric response plot 400 shown in FIG. 4 can be used to calculate the effective isolated dose by identifying the difference in dosimeter response minima generated 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 exhibits a local maximum. For this exemplary discussion, adenine is again used as the internal standard radical dosimeter. For adenine, two successively light-irradiated 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) can be calculated. For illustrative purposes, consider the time difference between T1 and T2 to be 1.25 seconds. For the 1 Hz flash rate mentioned above, the flash interval is identified to be 1.0 seconds. In the adenine example, the isolated dose is identified by the net flow rate and the difference between the flash interval and the dosimeter minimum interval. In the example of adenine, the net flow rate was 70 μL / min or 1.17 μL / sec. Thus, 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 geometric characteristics of the photolysis cell 304, is 3 μL, the isolated volume is deemed to be approximately 10% of the irradiance volume and determined to be sufficient to ensure one flash exposure of each bolus. If the isolated volume is less than the desired target, the net flow rate may be proportionally increased or the flash rate may be proportionally decreased to achieve the desired isolated volume. If the isolated volume 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 volume. In one embodiment, the control theory logic described above is automatically executed by the instrument controller 106.

[0073] Once the appropriate dosimeter response and isolation volume are achieved, the sample mixture can be reliably labeled and the labeled product collected. During the labeling and product collection process, the instrument controller 106 records the time when the dosimeter and isolation volume targets are achieved and identifies the arrival time of the properly labeled product at the outlet of the inlet-outlet port 202. The arrival time is identified by the quotient of the transfer volume and the net flow rate from the dosimetry area 305 to the inlet-outlet port 202. Prior to the arrival of the properly labeled product, the outgoing contents of the optofluidic chip 205 are dispensed through the port 202 into a designated waste well 221. Under the control of the instrument controller 106, once the properly labeled product reaches the outlet of the inlet-outlet port 202, the microplate 201 is moved by the sample introduction system 101 such that the labeled product is collected in the product collection microwell 222. <In vivo photochemical labeling>

[0074] The previous discussion detailed embodiments of the invention for labeling in vitro samples. We now provide details on embodiments that support in vivo labeling reactions. Figure 5 shows a microfluidic circuit 500 and 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 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.

[0075] The optofluidic chip 501 includes three different functional areas: a sheath flow generator 502, a labeling 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 fluidic system 505, a sheath flow fluidic system 506, and a reagent introduction fluidic system 507. Before starting the labeling, all three fluidic systems are primed. The system 505 is primed by the action of a buffer syringe pump 513 and a valve 514. A first valve 514 is configured to allow fluid communication between a buffer reservoir 516 and the syringe pump 513. The pump 513 is filled with running buffer aspirated from the buffer reservoir 516. After filling, the valve 514 is reconfigured to allow communication between the syringe pump 513 and a waste reservoir 515. The contents of the syringe pump 513 are pumped into the waste reservoir 515. This priming cycle continues until all air in the fluidic system 505 is evacuated and remnants of the previous buffer are flushed out. Under typical circumstances, but not limited to, the cycle is repeated three times. 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 opto-fluidic chip 501 via the sample loop 517. The running buffer is then pumped by the syringe pump 513 through the sample loop 517 and into the opto-fluidic chip 501, where the buffer flows through the sheath flow generator 502, the in-line mixer 503, the photolysis and dosimetry area array 504, and out through the inlet / outlet port 202 into a designated waste micro-well 221 in the microplate 201.

[0076] 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 selectively utilize the same waste buffer reservoir. Sheath buffer syringe pump 508 drains its contents into waste reservoir 511. This priming cycle continues until all air in the fluidic system 506 is evacuated and remnants of the previous buffer are flushed out. Under typical circumstances, but not limited to, the cycle is repeated three times. The sheath buffer syringe pump 508 is then refilled with sheath buffer, and the valve 509 is configured to allow fluid communication between the sheath buffer syringe pump 508 and the opto-fluidic chip 501. The sheath flow buffer stream is split by the connecting tee 512, which establishes two streams of sheath flow buffer that enter the opto-fluidic chip 501 through port 522 of the 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 area array 504, and exits via the inlet-outlet port 202 to a designated waste micro-well 221 in the microplate 201.

[0077] 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 expels its contents into waste reservoir 511. This priming cycle continues until all air within fluid system 507 has been expelled and remnants of the previous reagent solution have been flushed out. Under typical circumstances, but not limited to, the cycle is repeated three times.

[0078] The reagent syringe pump 518 is then refilled with the reagent solution, and the valve 519 is configured to allow fluid communication between the reagent syringe pump 518 and the opto-fluidic chip 501. The reagent solution is pumped through the reagent transfer line 521 and enters the opto-fluidic chip 501 through the reagent inlet port 523 of the in-line mixer 503. Simultaneously with the latter, the fluidic systems 506 and 505 pump their fluidic components into the opto-fluidic chip 501, as previously described. In this way, all fluid flow, including that from the reagent fluidic system 507, is directed down the chip, eventually exiting port 202 towards the waste microwell 221. The system is now ready for in vivo radical protein footprinting.

[0079] For in vivo radical protein footprinting, the microwell 220 contains cells, tissues, or organisms suspended in a buffer solution. While the reagent microfluidic pump system 507 and the sheath buffer syringe pump 508 remain stationary, the buffer syringe pump 513 is connected to the sample loop 517 via the valve 514. The buffer syringe pump 513 is used to aspirate the cells from the microwell 220. The cells are aspirated up to the port 202, through the dosimetry area array 504, the in-line mixer 503, the sheath flow generator 502, and finally into the sample loop 517. Typically, a predetermined amount of cells suspended in a buffer solution is aspirated and stored in the sample loop 517. In this way, the cells also leave through each compartment of the optofluidic chip 501 and the internal port 202. When labeling begins, fluidic systems 507, 506, and 505 begin to flow simultaneously, eventually pushing the cells stored in sample loop 517 into optofluidic chip 501, where the cells are mixed with the labeling reagent and then illuminated with light to initiate the photoradical labeling process.

[0080] To ensure that the cells are singly isolated from one another and do not clump or aggregate together when they are photoirradiated, a sheath flow is established in the sheath flow generator 502 to separate the cells by small aliquots of sheath flow buffer. Under typical conditions, the sheath buffer flow rate is established to be 10 times the buffer syringe flow rate, but is not limited thereto. As described below, the flow rate ratio of the fluidic systems 505 and 506 may be automatically adjusted to achieve the desired degree of cell isolation. Simultaneous with the pumping activity of the fluidic systems 505 and 506, the reagent fluidic system 507 delivers a labeling reagent to mix with the flowing cells in the in-line mixer 503. For ex vivo labeling, the relative flow rate of the reagent fluidic system 507 to the net flow rate of the sheath buffer syringe pump 508 and system 505 may be automatically adjusted depending on the concentration of the labeling reagent and the desired level of effective radical yield as determined by the in-line radical dosimetry system.

[0081] FIG. 6 shows a detailed view of a preferred embodiment of an optofluidic chip 601. Shown are the 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. In the optofluidic chip 601, the reagent mixer 604 is located upstream of the sheath flow generator 606. Cells enter the optofluidic chip 601 via the buffer syringe port 602. At this point, they are not isolated and may enter as a tightly clumped population of cells or other in vivo embodiments. The reagent mixer 604 is located upstream of the sheath flow generator 606. Cells enter the optofluidic chip 601 via the buffer syringe port 602. At this point, they are not isolated and may enter as a tightly clumped population of cells or other in vivo embodiments. Upon entering the buffer solution 604, they are mixed with the labeling reagent entering the buffer syringe port 602 via the reagent inlet port 603. As known to those skilled in the art, several different in vivo labeling reagents may be used, but for illustrative purposes, the use of hydrogen peroxide used for in vivo hydroxyl radical protein footprinting is described. As described in Jones et al. (Espino, JA; Mali, VS; Jones, LM, In Cell Footprinting Coupled with Mass Spectrometry for the Structural Analysis of Proteins in Live Cells. Analytical chemistry 2015,87(15),7971-7978.), hydrogen peroxide is quickly and easily taken up by cells or other in vivo entities and is rapidly dispensed to all cellular compartments. As described below, the final concentration of hydrogen peroxide used is determined in a closed-loop manner, essentially by determining the effective hydroxyl radical yield, which is evaluated by the change in the photometric characteristics of the internal standard radical dosimeter probed in the dosimetry area 607.

[0082] Prior to sedimentation into the microwell 220, the internal standard radical dosimeter is added to a population of cells or other in vivo embodiment, and the mixture is incubated sufficiently so that the internal standard radical dosimeter is taken up by all cells, eventually achieving a uniform distribution between cells and within the cellular compartment. The cells are then sedimented to form a pellet, and the supernatant is removed. The cell pellet is then resuspended in a buffer solution and sedimented again for a second time. This process is repeated until all the extracellular internal standard radical dosimeter is removed. Once the extracellular internal standard radical dosimeter is gone, the cells are resuspended in a buffer solution and finally transferred to the well 220.

[0083] After passing through the reagent mixer 604, the cell-reagent mixture enters the sheath flow generator 606. The sheath flow generator 606 includes a cavity for receiving a sheath flow buffer and a sheath flow buffer port 605, and these components of the generator 606 are shaped and arranged together to generate hydrodynamic focusing, and the cells are isolated by the sheath flow buffer. The relative flow rates of the microfluidic systems 505, 506, and 507 are adjusted to establish a desired intercellular isolation volume while simultaneously achieving a targeted intracellular hydroxyl radical yield. After passing through the sheath flow generator 606, the isolated cell population enters the photolysis zone 607, where it is irradiated with light to initiate 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 intercellular partitioning volume.

[0084] During in vivo radical protein footprinting, the intracellular internal standard radical dosimeter response is measured photometrically as it is probed through the dosimetric area 608. Exemplary 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 a single cell or other single in vivo embodiment, photometric fluorescence may be the preferred measurement means since its dynamic range and analytical sensitivity are typically superior to the other aforementioned methods.

[0085] For in vivo radical protein footprinting, the fluorescent internal standard radical dosimeter changes its fluorescence properties, such as excitation and emission properties, upon radical attack and eventual covalent modification. Such modifications may include changing the excitation and emission properties of the dosimeter so that the dosimeter undergoes an increase or decrease in 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 is the preferred embodiment for this approach, as it allows for detection of a small signal in a dark or zero background. The radical dosimeter fluorescence detector is configured using specific excitation and emission wavelengths for the covalently modified internal standard dosimeter. When the dosimeter is in its native state, no fluorescence 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 increased or decreased by the amount of the covalently modified dosimeter. It is proportional to the concentration of the decorated internal standard dosimeter and, in turn, to the available radical yield.

[0086] FIG. 7 shows five different internal standard radical dosimeters and shows the fluorescence signal signals of the excitation and emission wavelengths shown in a dose response manner when oxidized using hydroxyl radical attack. Table 700 lists the cellular distribution and fluorescence excitation and emission wavelengths for the following internal standard radical dosimeters that respond to hydroxyl radical attack in a dose response manner: terephthalic acid, CellRox Green® Green, dichlorofluorescein, CellRox Orange, and CellRox Deep Red. The intracellular portion where each dosimeter is primarily distributed is also shown. CellROX® dyes are commercially available from ThermoFisher (USA).

[0087] FIG. 8 shows an embodiment of the internal standard radical dosimetry photodetector 800 of the present invention. Shown are light source 801, focusing lens 802, excitation light 803, dosimetry area 608, dichroic mirror 804, emission light 805, collimator 806, interference notch filter 807, narrowband emission light 808, fluorescence / luminescence photodetector 809, scattered light 810, collimator 811, and light scatter detector 812. The internal standard radical dosimetry photodetector 800 is a combined fluorescence and light scatter detector system. As will be apparent to those skilled in the art, this combined system can be replaced by two separate (fluorescence and light scatter) detectors, so this embodiment is illustrative and not limiting in scope. As the in vivo elements enter the dosimetry area 608, they are illuminated by excitation light 803 from light source 801, which is focused by lens 802 and directed by dichroic mirror 804 to the dosimetry area 608. The light source 801 may 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). Alternatively, the light source 801 may be a solid-state visible light source, such as a visible LED available from Q-Photonics (Ann Arbor, MI), or a compact solid-state visible laser available from Thorlabs (Newton, NJ). A typical output power may be in the range of, but is not limited to, 0.1-10 mW, and a typical bandwidth may be in the range of, but is not limited to, 1-15 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 being 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 in the center of the dosimetry area 608, to generate the scan dosimetry area. Dichroic mirror 804 is selected to efficiently reflect the excitation light but transmit the emission light 805 resulting from the covalently modified internal standard dosimeter illuminated within dosimetry area 608 .

[0088] The luminescence light 805 is finally collimated by a collimator 806 and then passes through an interference notch filter 807 to generate a narrowband luminescence light 808 that is incident on a fluorescence emission photodetector 809. The photodetector 809 may include a silicon photodiode, such as an S1336-8BQ silicon photodiode available from Hamamatsu (Hamamatsu, Japan). Alternatively, the photodetector 809 may include a miniature photo-multiplier tube (PMT), such as a MicroPMT assembly H12400 available from Hamamatsu. The output current of the photodetector 809 is processed by a current-to-voltage (IV) converter to provide a voltage proportional to the incident luminescence light intensity of the narrowband luminescence light 808. The output voltage of the photodetector 809 is transmitted to the control electronics 105, where an analog to digital converter (ADC) generates a digital signal that is ultimately transmitted to the instrument controller 106 where the fluorescence calculations are performed.

[0089] As cells or other in vivo embodiments enter the dosimetry area 608, they are exposed to the excitation light 80 3 elastically scatters the nuclei 810. Due to the size difference between the incident excitation light wavelength (nm) and the biological entity size (μm), the scattered light 810 is preferentially detected perpendicular to the incident excitation light. The scattered light 810 is collimated by a collimator 811 and finally enters the 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 probing the volume. Since the 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, the elastic and inelastic scattering of the contents of the probed volume, as well as the elastic scattering from the radical dosimeter optical surfaces, is very low, thus generating a very low background signal. The light scattering signal is generated uniformly, independent of intrinsic fluorescence, thus allowing the presence of cells or other biological entities to be detected without an internal standard radical dosimeter signal.

[0090] The scattered light detector 812 may comprise a silicon photodiode, such as an S1336-8BQ silicon photodiode available from Hamamatsu (Hamamatsu, Japan). Alternatively, the photodetector 812 may comprise a miniature photomultiplier tube (PMT), such as a 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 the photodetector 812 is transmitted to the control electronics 105, and an analog-to-digital converter (ADC) generates a digital signal that is ultimately transmitted to the instrument controller 106 where light scattering calculations are performed.

[0091] 9 shows another embodiment of an internal standard radical dosimetry photodetector 900 of the present invention. Shown are a fluorescence excitation light source 901, a red diode laser interference pattern light source 902, a light collection and delivery bifurcated optical fiber 903, fiber output coupling optics 904, a dosimetry cell 608, a scattered light column 905, an optical beam splitter 906, a light scattering charge-coupled device camera 907, a fluorescence column 908, fluorescence collection optics 909, a fluorescence selective interference filter 910, a fluorescence detector 809, a far-field interference fringe pattern 911, and an extracted spatial frequency 912.

[0092] In the combined fluorescence and light scattering detector system 900, the light source may be replaced to match the fluorescence internal standard radical dosimeter requirements, and light scattering detection is accomplished using a two-dimensional charged couple device (CCD) imaging camera that images the interference pattern generated by the dosimetry cell. The fluorescence excitation source 901 and red diode interference pattern light source 902 are located remotely from the optofluidic chip 601 and coupled to the dosimetry cell 608 using a bifurcated optical fiber 903 and the necessary fiber optic coupling optics 904. The wavelength generated by the light source 901 is selected to match the excitation wavelength requirements of the internal standard radical dosimeter used. The red diode interference pattern light source 902 uses a 650 nm red diode laser to generate the optical interference pattern that is generated in the far optical field of the dosimetry cell 608. In such a configuration, the light sources 901 and 902 can be mounted external to the instrument, providing an easy means of replacing the light sources when performing radical dosimetry.

[0093] Coherent light from light sources 901 and 902 is homogenized in a single optical fiber 903 and directed to enter the dosimetry cell 608. A beam splitter 906 is used to split the light exiting the dosimetry cell 608 to generate two optical trains: a scattering train 905 and a fluorescence train 908. The light scattering train 905 is generated in the optical far field of the dosimetry cell 608. Because 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 generate a far-field interference fringe pattern 911 as imaged by a charge-coupled device two-dimensional camera 907.

[0094] The fluorescent column 908 is collected and collimated by collection optics 909, which direct the light through an interference filter 910. The bandpass characteristics of the interference filter 910 are selected to match the internal standard radical dosimeter emission spectrum. The optical bandwidth of the interference filter 910 can be, but is not limited to, 10 nm. The interference filter 910 and the fluorescence excitation source 901 are interchanged as a matched pair to accommodate a number of different internal standard radical dosimeters, such as those listed in table 700, so that the interference filter 910 is housed in a user accessible and easy to remove fixture. The fluorescent emission light is detected by the fluorescence detector 809.

[0095] 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, which in turn changes the refractive index and resultant phase of the light exiting the interrogation region. As the refractive index of the interrogation region changes, the imaged fringes 911 shift positionally to the right or left in response to the change in 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 cells enter the dosimetry cell 608, the accompanying light scattering changes the position of the fringes, which is then displayed as a change in phase. The phase change is proportional to the size and number of cells in the interrogation region of the dosimetry cell 608. As mentioned above, the sheath flow generators 502 and 606 may function to isolate the introduced cells or other in vivo entities so that they are clearly separated and not clumped together, resulting in effective and consistent irradiation of each entity in the radical labeling photolysis zone. The controlled isolation of these in vivo entities is affected by the different pumping speeds of the sheath buffer syringe pump 508 and the buffer syringe pump 513. Nominally, the pumping speed of the sheath buffer syringe pump 508 is, for example, but not limited to, 10 times the pumping speed of the buffer syringe pump 513. As described further herein, the effective isolation of each in vivo entity may be assessed by identifying a time difference in the detected light scattering signal, which is maximum when the in vivo entity is illuminated and minimum when the entity is not present, in a manner similar to the signal profile shown in FIG. 4. In this embodiment, a light scattering detector is used to assess the presence of the in vivo entity. 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.

[0096] Once isolation of the desired cell or bioentity is achieved, radical labeling proceeds very similarly to that performed for in vitro experiments: the effective radical dose is assessed by monitoring the change in the fluorescent signal measured in the dosimetry zone 608 as the bioentity is irradiated in the photolysis zone 607.

[0097] FIG. 10 shows a radical dosimetry response 1000 using CellRox® Deep Red and hydrogen peroxide at a concentration of 100 mM. Shown are the baseline fluorescence level 1001, the rising shoulder of the dosimeter fluorescence signal 1002, the maximum dosimeter fluorescence level 1003, the falling shoulder of the dosimeter signal 1004, the re-established baseline fluorescence level 1005, the maximum fluorescence level 1006 occurring at time T1, and the maximum fluorescence level 1007 occurring at time T2. As shown, the baseline fluorescence dosimeter signal 1001 transitions rapidly to the rising shoulder 1002 and reaches a maximum at the fluorescence level 1003 during the mixture being pulsed. As the oxidized CellRox Deep Red exits the dosimetry zone, the fluorescence signal decreases as shown by the falling shoulder 1004, and eventually re-establishes its low background fluorescence baseline at 1005.

[0098] Similar to in vitro radical labeling, the time difference between two maximum fluorescence values ​​for two successively exposed boluses of cells or in vivo embodiments, as illustrated by 1006 (T1) and 1007 (T2), may be used to assess the isolation volume, and inter-cell separation distance generated by the sheath flow generator 606. As with the in vitro system, the reproducibility of all maximum fluorescence signals may be assessed to judge the quality and reproducibility of the in vivo radical labeling obtained.

[0099] Once the appropriate dosimeter response and bioentity isolation volume are achieved, the bioentity can be reliably labeled and the labeled bioentity product can be collected. During the labeling and product collection process, the instrument controller 106 records the time when the dosimeter and isolation volume targets are achieved and identifies the arrival time of the properly labeled product at the outlet of the inlet-outlet port 202. The arrival time is identified by the quotient of the transfer volume from the dosimeter area 608 to the inlet-outlet port 202 and the net flow rate. Prior to the arrival of the properly labeled product, the effluent contents of the optofluidic chip 601 are dispensed through the inlet-outlet port 202 into a designated waste well 221. Under the control of the instrument controller 106, once the properly labeled product reaches the outlet of the inlet-outlet port 202, the microplate 201 is moved by the sample introduction system 101 so that the labeled product is collected in the product collection microwell 222. <Post-labeling analysis>

[0100] For both in vivo and in vitro radical footprinting experiments, post-labeling analysis is typically performed followed by the collected sample being optionally analyzed using an analyzer. The analyzer is configured to perform a chemical analysis of the sample. For example, the analyzer may be configured to identify proteins, peptides, carbohydrates, metals, nucleic acids, lipids, and / or amino acids that have been oxidized in the photolysis zone. The analyzer may include, for example, a mass spectrometer, a scintillator, an electrophoretic device, a chromatograph, and / or any other device configured to separate and / or identify sample components based on radiation, mass, charge, size, or other properties. In some embodiments, the analyzer is configured to detect isotope or radioisotope labels in cells or other biological entities and optionally determine whether components containing such labels have been oxidized. In some embodiments, the analyzer is configured to measure the ratio of labeled / unlabeled concentrations of certain components. In some embodiments, the analyzer is "in-line" with the port 202 and is thus configured to receive and analyze samples in real time. For example, the port 202 may include a capillary tube configured to direct the sample into a mass spectrometer. <Specific control of sheath flow>

[0101] Various embodiments of the present invention include methods to identify and control 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 should be performed when an in vivo entity or entities are present within the dosimetry area to enable meaningful comparative measurements that represent differences in intracellular radical dosimeter response between cells.

[0102] 11 illustrates an exemplary method 1100 for identifying and controlling cell-to-cell isolation volume using the invention described herein, which further enables detection of in vivo entities within the dosimetry area. Shown are cell introduction step 1101, first cell detection step 1102, second cell detection step 1103, net flow rate identification step 1104, cell isolation volume identification step 1105, good isolation evaluation step 1106, bad isolation and sheath flow rate adjustment step 1107, and repeat cycle 1108.

[0103] In the cell introduction step 1101, a single vertical 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 arrival time of the first cell into the dosimetry area 608. When a cell or other in vivo biological entity arrives within the dosimetry area 608, the intensity of the scattered excitation light 810 increases according to the size of the in vivo entity and the number of in vivo entities within the interrogation area. In a second cell detection step 1103, the process described above is used to detect the arrival time of the second cell into the dosimetry area 608. The net flow rate of the system is calculated by summing the pumping rates of the syringe pump 508, the syringe pump 513, and the syringe pump 518 (1104). The flow rate can be reduced to an extent that the time between the detection of the first cell and the second cell is long enough to reduce the probability that the two cells are in the dosimetry area 608 at the same time. This achieves single cell isolation where conditions allow for irradiation and oxidation of one cell at a time.

[0104] In a cell isolation volume calculation step 1105, the cell isolation volume 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 volume deviates by less than ±5% (or other predetermined limit) from the desired isolation volume (which is directly related to the separation distance and separation time), then the system proceeds to label additional cells without further adjustments in a proceed step 1006. Optionally, if the experimentally determined cell isolation volume deviates by more than ±5%, the pumping speed of the sheath flow syringe pump 508 is altered to achieve the desired cell isolation target volume 1107 in a sheath flow rate adjustment step 1107, and the determination process is repeated (1108) until the target cell isolation volume is achieved.

[0105] Various embodiments include systems and methods for detecting the presence of a biological entity within the dosimetry zone 608. The detected times of entry and exit of the biological entity within the dosimetry zone 608 may be used to identify a data acquisition period for photometrically identifying the intracellular dosimeter internal standard radical response. Upon entry into the dosimetry zone 608, the biological entity causes a rapid rise in the intensity of the scattered light detected by the scattered light detector 812. Correspondingly, upon exit of the biological entity, the intensity of the scattered light detected by the scattered light detector 812 drops sharply. The time difference between the rise and fall in the scattered light intensity represents the dosimetry zone residence period of the biological entity, e.g., a cell. During the residence period, the light intensity values ​​detected by the luminescence light detector 809 are summed and / or integrated to identify a net dosimetry signal for the biological entity of interest. By using this approach, photometric dosimetry may be performed by detecting signals arising from intracellular and / or extracellular photometric signals while rejecting any inherent background signal arising from extracellular fluid for areas lacking biological entities. In this way, the background signal for extracellular fluid is, by experimental design, substantially lower than that of intracellular fluid, and since measurements are only made when biological entity(ies) are present, the majority of the measured photometric signal will consist of that arising from intracellular components. <Determining in vivo embodiment viability>

[0106] Some embodiments include systems and methods for determining the viability of a biological entity. For a given biological entity, the scattered light intensity is proportional to the size and number of the biological entity present within the dosimetry area 608 during photometric evaluation. Using the methodology disclosed herein, means are described to effectively ensure a constant arrival rate of biological entities within the dosimetry area 608. Under such circumstances, the number of biological entities for each data acquisition period can be controlled to be constant, and the measured scattered light variation becomes highly dependent on the change in biological entity size. The change in biological entity size may be due to inherent morphological variation or may indicate an artifactual change in cell / species morphology, which may indicate cell destruction, cell death, or cell apoptosis. Since the inherent goal of in vivo culture HRPF is to evaluate biomolecular complement HOS under viable conditions, non-viable processes and / or conditions may be evaluated. It is desirable to detect and signal the presence of

[0107] The embodiments of the invention described herein provide systems and methods for detecting potential harm to in vivo moieties that may result from HRPF protocols, and provide a means by which in vivo HOS analysis can be performed on viable but undestroyed in vivo entities. For example, the presence of a destroyed cell or two or more cells within the dosimetry area 608 can be detected simultaneously based on measured light scattering. Those entities detected under these circumstances may be separated and discarded from those entities that were not irradiated under these conditions. <Controlled operation of flash photolysis light>

[0108] Various embodiments of the present invention include systems and methods for operating a flash photolysis light source 103 in synchronization with the arrival of an in vivo entity in the photolysis zone 102. In vivo radical footprinting ideally involves identifying and quantifying the presence of an in vivo entity within the photolysis zone in order to reliably irradiate said entity(ies) in a reproducible manner and photocatalytically induce a reproducible intracellular radical load.

[0109] 12 illustrates an exemplary method 1200 that provides a system and method for determining the arrival of a biological entity at the photolysis zone 608 while the flash photolysis light source 103 is precisely operated to flash upon the arrival of the biological entity at the photolysis zone 608. Shown is the continuation of the process with a cell introduction step 1201, a first cell detection step 1202, determining the net flow rate 1203, determining the amount of on-chip interconnect 1204, determining the transit time 1205, determining the photolysis zone arrival time 1206, activating the photolysis operation at the cell arrival time 1207, and precipitation of appropriately labeled cells 1208.

[0110] In cell introduction step 1201, a single column array of cells is formed as described elsewhere herein and a first cell is introduced into the photolysis zone 102. In detection step 1202, the output signal of the scattered light detector 812 is monitored to detect the arrival time of the first cell into the dosimetry zone 608. Upon entry of a cell or other biological entity into the dosimetry zone 608, the intensity of the scattered light increases according to the size of the biological entity and the number of biological entities in the interrogation region. This increase is detected by the scattered light detector 812. In net flow rate determination step 1203, the net flow rate of the system is calculated by summing the pumping speeds of the syringe pump 508, the syringe pump 513, and the syringe pump 518. In interconnect volume determination step 1204, the interconnect volume extending from the photolysis zone 102 and the dosimetry zone 608 is determined and remains constant during the in vivo radical protein footprinting method as a direct manifestation of the microfluidic system design. In a transit time determination step 1205, the transit time required for the biological entity to move from the photolysis zone 102 to the dosimetry zone 608 is calculated by dividing the interconnect volume by the net flow rate. In a 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 an action step 1207, for subsequent cells or biological entities, the photolysis system is operated to flash at the determined 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 has been processed.

[0111] While the teachings herein describe the particular utility of the detection signals arising from the light scattering detector 812 and the fluorescence / luminescence detector 809, it will be apparent to one of ordinary skill in the art that these detectors may be used for the purposes of detecting the arrival of an in vivo entity at the dosimetry zone 608 or for the purposes of predicting the arrival of an in vivo entity at the photolysis zone 102 in a number of undescribed combinations or means. For example, the signal generated by the fluorescence / luminescence detector 809 may be used to detect the arrival of an in vivo entity at the dosimetry zone 608 or to predict the arrival of an in vivo entity at the photolysis zone 102. The signal may be summed in temporal coherence with the signal from the light scatter detector 812 to improve the overall sensitivity for detecting the presence of biological entities within the dosimetry area. Thus, the methods described herein are intended to be illustrative and not limiting in scope. <Closed-loop control of radical dose measurement>

[0112] Various embodiments of the present invention include systems and methods for calibrating a closed loop controlled radical dosimetry system. In these embodiments, the closed loop controlled radical dosimetry system includes a calibration logic circuit that is used to predict the required change in optical fluence or radical reagent concentration in response to the measured radical dosimetry light fluorescence change. The calibration function is empirically identified through multiple measurements in which known or controlled mixtures of support buffer, biosubstance, and radical dosimeter are processed with a single flash of light for each separate controlled aliquot at various fluence or radical reagent concentration levels. In an exemplary embodiment, a software routine executed in either the low level instrument control or high level user interface program (e.g., control electronics 105 and / or instrument controller 106) generates a lookup table or curve fit that describes the measured change in dosimetry light fluorescence at each fluence or radical reagent concentration, allowing the generation of a mathematical expression or calibration function that describes the relationship between the applied fluence and / or radical reagent concentration and the measured dosimeter fluorescence change for a single flash of light exposure. In another embodiment, the look-up table and subsequent calibration functions are generated manually by a user using fluorescence change values ​​for each light flash voltage value and / or radical reagent concentration.

[0113] During the in vivo radical protein footprinting treatment, background radical scavenging is assessed dosimetry. The measured change in dosimetric light fluorescence is compared to a user-specified target change. If the measured dosimeter value deviates from the target value by more than ±10%, the applied fluence or radical reagent concentration is altered to achieve the targeted change in measured dosimeter absorbance. The calibration function described above is used to predict the required change in fluence or radical reagent concentration.

[0114] It will be apparent to those skilled in the art that many more modifications beyond those already described are possible without departing from the inventive concept herein. Thus, the subject matter of the present invention should not be limited except in the spirit of the disclosure. Moreover, 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 interpreted as non-exclusively referring to elements, components, or steps, indicating that the referenced element, component, or step may be present or utilized with or combined with other elements, components, or steps not expressly referenced.

[0115] Although biological cells are used herein to describe various embodiments of the invention, in other embodiments, "cells" may be replaced in any example or claim by other entities, such as non-biological entities, biological entities, viruses, multicellular organisms (e.g., fungi, spores, microorganisms, molds, algae, nematodes, amoebas, protozoa, trichoplax, yeasts), or non-biological materials.

[0116] Described herein are general structures and techniques, as well as more specific embodiments that can be used to achieve different ways of carrying out a more general goal. Although only a few embodiments have been disclosed in detail above, other embodiments are possible and are considered to be encompassed within this specification. This specification describes specific examples for achieving a more general goal that may be achieved in another way. This disclosure is intended to be exemplary, and should not be construed as limiting the scope of the claims. The range is intended to encompass any modifications or alternatives that may be predictable to one skilled in the art.

[0117] The logic circuitry discussed herein may include electronic circuitry, hardware, firmware, and / or software stored on a non-transitory computer-readable medium.

[0118] Only claims using the term "means for" are intended to be interpreted under 35 USC 112, paragraph 6. Moreover, limitations from the specification are not intended to be read into any claim unless those limitations are expressly included in the claim. The computers 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 computers may be Intel (e.g., Pentium or Core 2 duo, i3, etc.) or AMD-based computers running Windows 10, 8, 7, or Linux, or may be Macintosh computers. The computers may also be handheld computers such as PDAs, cell phones, tablets, or laptops running any available operating system, including Android, Windows Mobile, iOS, etc.

[0119] COPYRIGHT NOTICE: In accordance with 37 CFR 1.71(e), portions of this disclosure are subject to copyright protection, including material for which copyright protection is claimed (such as, but not limited to, source code listings, screen shots, user interface, or user instructions, or any other aspect of this application in which copyright protection is or may be available in any jurisdiction). The copyright owner has no objection to the facsimile reproduction of any of the patent document or patent disclosure as it appears in the Patent and Trademark Office 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 are prohibited by applicable copyright law.

Claims

1. An optofluidic chip for radical protein footprinting, comprising: A substrate; a first port, a second port, and a third port defined respectively within the substrate; a mixing zone including a first cavity defined within the substrate and in fluid communication with both the second and third ports; a photolysis zone including a second cavity defined within the substrate and in fluid communication with the mixing zone, the photolysis zone further including a first optically transparent window sealed to the substrate and bounding one side of the second cavity, the second cavity including channels arranged in a serpentine pattern, the photolysis zone being transparent on both sides; a dosimetry area including a third cavity defined within the substrate, in fluid communication with the photolysis area and in further fluid communication with the first port, the dosimetry area further including a second optically transparent window sealed to the substrate and bounding one side of the third cavity; An optofluidic chip comprising:

2. The optofluidic chip of claim 1 , wherein the substrate comprises plastic.

3. The optofluidic chip of claim 1 , wherein the substrate comprises quartz.

4. The optofluidic chip of claim 3 , wherein the substrate includes a middle layer of silicon, a top layer of quartz, and a bottom layer of quartz, the middle layer being disposed between the top layer and the bottom layer.

5. The optofluidic chip of claim 3 , wherein the substrate includes a middle layer of silicon, an upper layer of fused silica, and a lower layer of fused silica, the middle layer being disposed between the upper layer and the lower layer.

6. a sheath flow generator including a fourth cavity defined in the substrate and in fluid communication with both the mixing zone and the photolysis zone, the sheath flow generator including two sheath inlet ports defined in the substrate and in fluid communication with the fourth cavity, the sheath inlet ports The optofluidic chip of claim 1 , wherein the first cavity and the fourth cavity are configured to generate hydrodynamic focusing.

7. The optofluidic chip of claim 6 , wherein the mixing zone 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 mixing zone and the third port.

8. The optofluidic chip of claim 6 , wherein the sheath flow generator is disposed in fluid communication between the photolysis zone and the mixing zone, and the mixing zone is in fluid communication between the sheath flow generator and both the second and third ports.

9. 1. An optofluidic system for radical protein footprinting comprising: a chip including a substrate and a first port, a second port, and a third port defined within the substrate, the chip further comprising: a mixing zone including a first cavity defined within the substrate and in fluid communication with both the second and third ports; a photolysis zone including a second cavity defined within the substrate and in fluid communication with the mixing zone, the photolysis zone further including a first optically transparent window sealed to the substrate and bounding one side of the second cavity, the second cavity including channels arranged in a serpentine pattern; a dosimetry area including a third cavity defined within the substrate, in fluid communication with the photolysis area and in further fluid communication with the first port, the dosimetry area further including a second optically transparent window sealed to the substrate and bounding one side of the third cavity; a microfluidic system in fluid communication with both the second and third ports of the mixing zone; 1. An optofluidic system comprising:

10. 10. The system of claim 9, further comprising a reservoir configured to receive a biological entity comprising the 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. 12. The system of claim 9, 10, or 11, further comprising a fluorescent light detector configured to receive fluorescent light emitted from within the dosimetry area.

13. 13. The system of claim 9, further comprising a light source configured to provide coherent light to the dosimetry area.

14. 14. The system of claim 13, further comprising a scattered light detector configured to receive light scattered from within the dosimetry area in a direction orthogonal to the direction of the light from the light source.

15. 14. The system of claim 13, further comprising a refractive index photodetector configured to receive light scattered from within the dosimetry area in a direction orthogonal to the direction of the light from the light source.

16. The chip further includes a sheath flow generator including a fourth cavity defined in the substrate and in fluid communication with both the mixing zone and the photolysis zone, the sheath flow generator including two sheath inlet ports defined in the substrate and in fluid communication with the fourth cavity, the sheath inlet ports and the fourth cavity configured to generate hydrodynamic focusing. The system according to claim 9 to 14 or 15.

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