JP7574195B2 - In vivo radical dosimetry and protein footprinting of hydroxyl radicals in vivo - Google Patents

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. Provisional Application No. 62/788,219, filed January 4, 2019, the disclosure of which is incorporated herein by reference.

The present invention relates to an apparatus and method for higher order structure analysis of biomolecules by the process of hydroxyl radical protein footprinting. Some embodiments of the present invention relate to determining the tertiary and quaternary structure and associated conformations of biopharmaceuticals using improved apparatus and methods for in vivo flash photo-oxidation of proteins in cells, tissues or organisms to determine their higher order biomolecular structure.

RELATED ART: Discussion of a work, publication, sale, or activity anywhere in this filing, including any documents filed with this application, is not 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.

Biosimilars are therapeutics that are similar but not identical to existing innovator or reference products. Unlike in the case of small molecule drugs, biosimilars are not simply generic versions of the originator. Conventional generic drugs are considered to be therapeutically and molecularly equivalent to their originator counterparts. This is not true for biosimilars, which are complex three-dimensional biomolecules that are quite different from conventional drugs due to their heterogeneity and reliance on production in living cells. The structure and functional activity of biopharmaceuticals are highly sensitive to their environment (Non-Patent Document 1). The intended structure of a therapeutic is maintained by a delicate balance of multiple factors - including protein concentration, control of post-translational modifications, pH and coeluents in the formulation, and manufacturing/purification procedures (Non-Patent Document 1). Thus, the structure of a biopharmaceutical must be carefully maintained because, if unchecked, undesirable and adverse pharmacological consequences may occur.

Adverse drug reactions (ADRs) of biopharmaceuticals are generally due to excessive pharmacological or immunological effects. ADRs in patients range from symptomatic irritation to morbidity and mortality. While the etiology of some ADRs is due to the pharmacogenetic susceptibility of the patient, many are due to the intrinsic properties of the therapeutic drug, resulting in morbid and fatal outcomes for the patient and significant economic losses for the biopharmaceutical industry (Non-Patent Document 2). Thus, the occurrence of catastrophic ADRs illustrates the need for improved analytical methods for the development and quality control of biopharmaceuticals.

To minimize ADRs and facilitate the development of biosimilars, the FDA, the Center for Drug Evaluation and Research, and the Center for Biologics Evaluation and Research have issued guidelines emphasizing the use of state-of-the-art techniques to evaluate protein higher-order structure (HOS) (Non-Patent Document 3). HOS analysis involves the determination 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. Currently, various HOS analyses exist, but they are problematic in their inadequacy in reliably predicting the efficacy and safety of biotherapeutics, and it has become clear that new and improved HOS analyses are needed but have not been realized (Non-Patent Document 4).

A technique that answers the unfulfilled need for HOS analysis is irreversible protein hydroxylation coupled with mass spectrometry (MS) (5). This method has been termed hydroxyl radical protein footprinting (HRPF). Various techniques have been used to perform HRPF. Perhaps the most widely used method relies on fast photochemical protein oxidation (FPOP), which uses a single short, high-fluence 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 single oxygen atom into the amino acid. _OH radicals are short-lived, and when generated by a short UV pulse, the reaction of the radical with the amino acid may be completed before the conformational change caused by the labeled protein occurs (6). Mass spectra of peptide products from enzymatic digestion show various levels of oxidation suggested by peak shifts of 16 Da, 32 Da, 48 Da, etc. This information can be used to improve our understanding of the HOS by inferring which peptides lie outside the HOS.

The technical limitations of FPOP HRPF that adversely affect comparative studies are due to the reaction of OH radicals with non-analyte components in the sample, such as buffer components, initial solutes, and foreign organisms. Variations in the rate of background removal lead to low reproducibility between tests, limiting comparative studies (Non-Patent Document 7). While OH radicals are excellent probes of protein topography, they also react with many compounds present in the analytical formulation. Since there is competition between the analyte protein and background scavengers for free OH radicals, to obtain reproducible results it is desirable to measure the effective concentration of radicals available to oxidize the target protein. In photochemistry, the effective radical concentration is measured by using an internal standard in the radical dosimeter. 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 insensitive to most proteins.

Prior art teaches radical dosimetry performed with spiked peptide internal standards (Non-Patent Document 8) or UV-absorbing internal standards such as adenine (Non-Patent Document 9) that are added to the buffer and evaluated after labeling. In peptide radical dosimetry, the labeled peptide and target protein are then analyzed using LC-MS (with optional proteolysis) to evaluate the relative ratio of oxidized peptide to target protein. If the desired peptide and _protein oxidation ratio is not obtained, the entire experiment is repeated to adjust the concentration of _H2O2 depending on whether the effective OH radical load is increased or decreased. In adenine dosimetry, the effective change in adenine UV absorbance is determined upon completion of the labeling process, and the ratio of the achieved adenine UV absorbance to the target adenine UV absorbance change is determined. The _H2O2 concentration is then changed depending on the desired change in UV absorbance. Both of these methods are performed after labeling is completed and do _not allow for real-time adjustment of the effective OH radical load, consuming valuable samples and wasting the researcher's time.

U.S. Patent Nos. 5,393,633 and 5,433,363 teach systems and methods for performing radical dosimetry in real time as organisms are labeled during the FPOP HRPF method. While establishing a real-time means for adjusting and compensating for variations in background removal, the systems and methods taught in these applications require the addition of an external internal standard dosimeter to the biological mixture. Under some conditions, the external internal standard may induce artificial changes in the conformation of biomolecules, which in such cases contradicts the desired objective of providing a facile means for characterizing the nascent conformation of organisms. The disclosures of U.S. Patent Nos. 5,393,633 and 5,433,363 are incorporated herein by reference.

Patent document 3 describes an apparatus and method by which commonly used biological buffer systems can be employed as internal standards in radical dosimeters. The optical properties of commonly used biological buffers change in a predictable manner upon attack by OH radicals. Therefore, these buffers can be used as internal standards in radical dosimeters, eliminating the need to add external reagents. Furthermore, the solvation properties of these buffers are established to stabilize the initial configuration of biomolecules, and therefore do not alter biological higher-order structures.

The aforementioned techniques describe devices and means for performing dosimetry of HRPF radicals while labeling proteins and biopharmaceuticals in vitro. However, the practice of applying the results of in vitro structural experiments to real in vivo behavior has been questioned (Non-Patent Document 10). Due to the shortcomings of in vitro HRPF, there has been recent interest and desire to extend the use of HRPF to whole cells in vivo. For example, Patent Document 4 describes means and methods by which in vivo _HRPF can be performed. In brief, multiple fused _{silica capillaries and microfluidic couplings are used to assist in mixing cells suspended in a buffer with H2O2. As taught, H2O2 is rapidly} taken up by cells without causing cell destruction, inducing apoptosis, or hastening cell death. However, the taught systems and methods still present various shortcomings.

U.S. Provisional Patent Application No. 62/511,571 International Patent Application No. PCT/US2018/34682 U.S. Provisional Patent Application No. 62/747,247 US Patent Application Publication No. 2018/0079998 US Patent Application Publication No. 2014/0030751

Gabrielson, J. P.; Weiss IV, W. F., Technical decision-making with higher order structure data: starting a new dialogue; Journal of pharmaceutical sciences, 2015 Giezen, T. J.; Schneider, C. K., Safety assessment of biosimilars in Europe: a regulatory perspective; Generics and Biosimilars Initiative Journal; 2014 Quality considerations in demonstrating bio-similarity of a therapeutic protein product to a reference product; guidance for industry; U.S. Department of Health and Human Services; Food and Drug Administration; Center for Drug Evaluation and Research; Center for Biologics Evaluation and Research Washington, DC ; 2015 Gabrielson, J. P.; Weiss IV, W. F., Technical decision-making with higher order structure data: starting a new dialogue; Journal of pharmaceutical sciences; 2015 Hambly, D. M.; Gross, M. L., 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 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 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 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 Permits Time-Dependent Measurements.J Am Soc Mass Spectrom, 2016. Xie, B.; Sharp, J. S., 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. Mourao, M. A.; Hakim, J. B.; Schnell, S., Connecting the dots: the effects of macromolecular crowding on cell physiology. Biophysical Journal 2014, 107 (12), 2761-2766

Various embodiments of the present invention include systems and methods that address the shortcomings of current ex vivo HRPF, as described above, by providing a means by which cell separation and compartmentalization may be assessed and reproducibly controlled by performing real-time in vivo measurements of effective hydroxyl radical concentrations and adjusting for unwanted background removal by using the photometric properties of an in vivo radical dosimeter internal standard, as well as a means to determine the time of cell arrival at the HRPF photolysis region.

Various embodiments of the present invention relate to systems and methods for protein conformation analysis, including improved embodiments that allow for advanced protein footprinting with hydroxyl radicals by performing in vivo flash photooxidation of proteins. In some embodiments, the present invention provides a controlled in vivo radical dosimetry system in which closed-loop control is established between the flash photolysis system and the dosimeter, controlling the dose of the flash photolysis system in response to measured changes in the photometric properties of an internal standard radical dosimeter within the cells.

In some embodiments, the invention includes a controlled in vivo radical dosimetry system in which a closed-loop control is established between an automatically controlled microfluidic mixing system and a dosimeter _that controls the concentration of _H2O2 in response to measured changes in the photometric properties of an internal standard radical dosimeter within a cell.

In some embodiments, the invention includes a controlled in vivo radical dosimetry system in which a closed loop control is established between the flash photolysis system and a dosimeter that controls the dose of the flash photolysis system in response to measured changes in the photometric properties of _an intracellular internal standard radical dosimeter in which OH radicals are generated by photolysis of intracellular _H2O2 .

In some embodiments, the invention includes a controlled in vivo radical dosimetry system in which closed-loop control is established between the flash photolysis system and a dosimeter that controls the dose of the flash photolysis system in response to measured changes in the photometric properties of an internal standard radical dosimeter within a cell such that OH radicals are generated from water using a photocatalytic metal oxide outside the cell.

In some embodiments, the invention includes a controlled in vivo radical dosimetry system in which closed-loop control is established between the flash photolysis system and a dosimeter that controls the dose of the flash photolysis system in response to measured changes in the photometric properties of an internal standard radical dosimeter within the cell such that OH radicals are generated from water using a photocatalytic metal oxide within the cell.

In some embodiments using a controlled in vivo radical dosimeter system, the invention includes a method for preparing labeled biomolecules for analysis, the method comprising the steps of: (1) mixing cells with a biological buffer, an internal standard radical dosimeter that is ultimately taken up by the cells, and any other necessary labeling reagents; (2) introducing the cells into a light dosimetry region; (3) determining initial photometric properties of the cells; (4) exposing the cells to at least one burst of UV light in a photolysis region; (5) measuring changes in the photometric properties of the cells after exposure to light; and (6) adjusting the spectral irradiance of the UV source light in response to changes in the photometric properties of the radical dosimeter.

In some embodiments using a controlled in vivo radical dosimeter system, the invention includes a method for preparing labeled biomolecules for analysis, comprising the steps of: (1) mixing cells with a biological buffer, an internal standard radical dosimeter that is ultimately taken up by the cells, and any other necessary labeling reagents, (2) introducing the cells into a light dosimetry region, (3) determining initial optical properties of the cells, (4) exposing the cells to at least one burst of UV radiation within a photolysis region, (5) measuring the change in the photometric properties of the cells after exposure to light, and (6) adjusting the concentration of _H2O2 using a controlled microfluidic mixer in response to the change in the photometric properties of the radical dosimeter _.

In some embodiments using a controlled in vivo radical dosimeter system, the invention includes a method for preparing labeled biomolecules for analysis, the method comprising the steps of: (1) mixing cells with a biological buffer, an internal standard radical dosimeter that is ultimately taken up by the cells, and a metal oxide photocatalyst; (2) introducing the cells into a light dosimetry region; (3) determining initial photonic properties of the cells; (4) exposing the cells to at least one burst of UV light in a photolysis region; (5) measuring changes in the photometric properties of the cells after exposure to light; and (6) adjusting the spectral irradiance of the UV source light in response to changes in the photometric properties of the radical dosimeter.

In some embodiments using a controlled in vivo radical dosimeter system, the invention includes a method for preparing labeled biomolecules for analysis, the method comprising the steps of: (1) mixing cells with a biological buffer and an internal standard radical dosimeter that is ultimately taken up by the cells; (2) introducing the cells into a light dosimetry region; (3) detecting the arrival and presence of the cells by monitoring the intensity of scattered light exiting the dosimetry region; (4) determining the elapsed time between the arrival of successive cells; (5) determining the cell separation volume per elapsed time times the net buffer flow rate; and (6) adjusting sheath flow and buffer flow parameters to achieve the desired cell separation volume.

In some embodiments using a controlled in vivo radical dosimeter system, the invention includes a method for preparing a labeled biomolecule for analysis, the method comprising the steps of: (1) mixing cells with a biological buffer and an internal standard radical dosimeter that is ultimately taken up by the cells; (2) introducing the cells into a light dosimetry region; (3) detecting the arrival and presence of the cells by monitoring the intensity of scattered light exiting the dosimetry region; (4) determining the net flow rate of the arriving cells; (5) determining the interconnected volume extending from the photolysis region and the light dosimetry region; (6) determining the transit time required for the cells to travel from the photolysis region to the light dosimetry region; (7) determining the photolysis region arrival time of the cells; and (8) triggering the photolysis system when all subsequent cells arrive at the photolysis region.

After preparation of the labeled biomolecule, other methods such as mass spectrometry and electrophoresis can be used to identify the labeled peptide and infer information about the higher order structure of the biomolecule in vivo.

Various embodiments of the present invention include an analytical system comprising a sample introduction system configured to provide intact organisms separated from one another in a focused sheath flow to a photolysis region; a photolysis light source configured to generate light to generate hydroxyl radicals from a hydroxyl radical source; a photolysis region configured to receive the sheath flow and the light to oxidize an internal standard and oxidize a biological compound of the organism within the living organism; a dosimetry region configured to receive the organisms from the photolysis region and detect the presence of the organisms using a scattered light detector and detect oxidation of the internal standard using a fluorescence detector; control logic configured to determine that a target concentration of hydroxyl radicals has been generated for each of the organisms and to adjust operation of the photolysis region to meet the target concentration of hydroxyl radicals; and a container configured to receive the organisms containing the oxidized biological compounds.

Various embodiments of the present invention include a method of oxidizing a biomolecule in an intact cell. The method includes the steps of: introducing a sample mixture containing at least one cell into a photolysis region; introducing a hydroxyl radical source and a dosimeter internal standard into the photolysis region of a flash photolysis system; providing light to generate hydroxyl radicals from the hydroxyl radical source that oxidize a biomolecule in the at least one cell; and detecting the at least one cell at any point before reaching a dosimeter region of a radical dosimeter that is detectable in the dosimeter region by light scattering, the dosimeter region being configured to detect a photometric characteristic of the dosimeter internal standard resulting from a reaction between the dosimeter internal standard and the hydroxyl radical. the step of waiting a predetermined time; the step of measuring a photometric characteristic of the dosimeter internal standard using the radical dosimeter while the at least one cell is in the dosimeter region; the step of determining that a target level of hydroxyl radicals has not been generated based on the measured photometric characteristic of the dosimeter internal standard; and the step of adjusting the concentration of hydroxyl radicals in the photolysis region by adjusting at least one of: 1) the amount of light provided to the photolysis region; 2) the concentration of the hydroxyl radical source; 3) the flow rate of the at least one cell in the photolysis region; or 4) the time for which the light is provided to the photolysis region.

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 present invention are utilized. Furthermore, the above objects and advantages of the present invention will become readily apparent to those skilled in the art from the following description of the illustrative embodiments when viewed in light of the accompanying drawings incorporating 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.

Any of the methods described herein may, according to certain embodiments, further utilize any one or more of the following:
1 shows an embodiment of a flash photolysis system 100 configured for real-time radical dosimetry and comprised of multiple subassemblies. Shown are a sample introduction system 101, a photolysis region 102, a flash photolysis light 103, a radical dosimeter 104, control electronics 105, an instrument controller 106, fluidic interconnect lines 107 between the sample introduction system 101 and the photolysis region 102, fluidic interconnect lines 108 between the photolysis cell and the radical dosimeter, optical interconnect lines 109 between the flash photolysis system 103 and the photolysis region 102, electronic interconnect lines 110 between the radical dosimeter 104 and the control electronics 105, electronic interconnect lines 111 between the sample introduction system 101 and the control electronics 105, electronic interconnect lines 112 between the control electronics 105 and the instrument controller 106, and electronic interconnect lines 113 between the control electronics and the flash photolysis system. Additionally, the photolysis region 102, the flash photolysis light 103, and the radical dosimeter 104 are included in a “flash photolysis system.” The photolysis region 102 is usually included in a photolysis cell. 1 illustrates sample movement between various components of the flash photolysis system 100 shown in FIG. 1 according to various embodiments, including introduction of cells, sample processing, and collection of labeled cells. Shown are cells suspended in buffer 201, cell suspension syringe pump 202, microfluidic mixer 203, hydrogen peroxide reservoir 204, hydrogen peroxide syringe pump 205, sheath buffer reservoir 206, sheath buffer syringe pump 207, hydrodynamic focusing mixer 208, photolysis region 209, dosimetry region 210, labeled cell reservoir 211, and analyzer 212. A method 300 for in vivo cell labeling according to various embodiments of the present invention is shown, including steps 301 adding an internal standard, 302 washing the cells, 303 introducing the labeled cell(s), 304 measuring the non-irradiated cells, 305 irradiating the cells to initiate the in vivo labeling process, 306 measuring the irradiated cells, 307 analyzing the change in the optical properties of the sample after irradiation to evaluate the effective OH radical concentration, 308 deciding how to treat the labeled cells after achieving the desired effective OH radical concentration, 309 good result step of further treating the cells based on the desired level of effective OH radical concentration, or 310 adjusting the irradiation conditions of the subsequent cells. As a result of the adjustment step 310, one or more additional cells are irradiated after adjusting the flash spectrum irradiance and/or _H2O2 concentration _. Included is a table 400 of exemplary in vitro fluorescent dosimeter internal standards that can be used with the present invention. Each example is listed with its respective maximum excitation and emission wavelengths, as well as the typical intracellular compartment in which the fluorescent internal standard dosimeter accumulates. These similar compounds can be used individually or in combination. 1 shows further details of components of the inventive fluorescence and light scattered light detection system, e.g., a flash photolysis system, according to various embodiments of the invention, which may include a photolysis region 102, a flash photolysis light 103, and/or a radical dosimeter 104, as shown in FIG. 1. Shown are an ultraviolet narrow band light source 501, e.g., an ultraviolet light emitting diode or an ultraviolet diode laser, a focusing lens 502, an excitation light 503, a dichroic mirror 504, a dosimetry region 210, emission light 506 from fluorescence generated within the dosimetry region 210, a collimating lens 507, an interference notch filter 508 that selectively transmits emission light, a fluorescence/emission light photodetector 509, scattered excitation light 510 from the dosimetry region 210, a collimating lens 511, and a scattered light photodetector 512. 6 shows a method 600 for establishing a consistent cell separation volume according to various embodiments of the present invention. It includes a cell introduction step 601, in which one or more first cells to be labeled are introduced into a sheath flow configured to separate individual cells or groups of cells, a first cell detection step 602, in which the arrival of the first cell (or set of cells) introduced in step 601 into the photolysis region 102 is detected, a second cell detection step 603, in which the arrival of a second cell within the photolysis region 102 is detected, a net flow rate determination step 604, in which a net flow rate (which depends on the pumping speeds of the sheath flow, hydrogen peroxide, and cell buffer syringe pumps) is determined, and the cell separation volume is determined as the product of the difference in cell arrival times and the net flow rate. 6 shows a step 605 of determining the amount of cell separation that is to be achieved; a decision step 606 of determining whether the desired cell separation volume has been achieved (the cell separation volume being a measure of how well each cell or group of cells has been separated within the photolysis region 102); an adjustment step 607 that is performed if the desired cell separation volume has not been achieved; a step 608 in which the flow rate (e.g., of the syringe pumps 202, 205 and/or 207) is adjusted to more readily reach the separation volume and the previous steps are repeated; and a success step 606 in which the desired amount of separation has been achieved and the cell labeling and analysis process can proceed. 1 illustrates how flash photolysis activity of a photolysis system according to various embodiments can be coordinated with the arrival of cells within the photolysis region. Shown are an introduction step 701 in which a single isolated cell or group of cells to be labeled is introduced using a focused sheath flow; a detection step 702 in which the arrival of a first cell is detected in the photolysis region 102; a net flow rate determination step 703 in which a net flow rate is determined based on the sheath flow, the hydrogen peroxide, and the pump speed of the cell buffer syringe pump; an interconnect volume determination step 704 in which a volume between the photolysis region 102 and the dosimetry region 210 (of the radical dosimeter 104) is determined; a transit time determination step 705 in which a cell transit time from the photolysis region 102 to the dosimetry region 210 is determined; an arrival time determination step 706 in which an arrival time of the first cell at the photolysis region 102 is determined; a trigger step 707 in which a flash photolysis light 103 is issued to provide light to the photolysis region 102 at the arrival time determined in step 706; and a continuing step 708 in which the process is repeated for additional cells.

Apparatus and methods are provided for the analysis of biomolecular conformation achieved by selective labeling of solvent-exposed molecular groups catalyzed by rapid in vivo photooxidation with real-time monitoring and control of available OH radical concentration. Additionally, apparatus and methods are provided for the analysis of biomolecular conformation achieved by rapid in vivo photooxidation with real-time monitoring and control of in vivo species dissociation and subsequent flash photolysis. The apparatus and methods of the present invention can be applied 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 to essentially any photoelectrically translucent or transparent in vivo living or non-living body.

The device and method can be applied to various research fields such as general protein biochemistry, diagnostic research, 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 device and method can be applied 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, mechanism of action analysis of small molecule drugs, mechanism of action analysis of biopharmaceuticals, antibody-antigen analysis, protein epitope mapping, protein paratope mapping, and chemical reaction monitoring.

The device can receive cells for subsequent chemical labeling, which can be done stepwise by manually pipetting the cells into appropriate microcentrifuge tubes or microplates placed in the sample introduction assembly of the system. Alternatively, the device can be coupled to and receive cells directly from other separation and analysis devices, such as devices for selective cell sorting, cell counting, and cell isolation from tissue.

This section provides a general overview of a flash photolysis apparatus with a controlled in vivo radical dosimeter that uses the photometric properties of an internal standard dosimeter to assess and ultimately control the available OH radical concentration in vivo. In addition, this section provides an overview of the invention that uses the photometric properties of an in vivo embodiment to assess the sequestration volume and control the precise triggering of a flash photolysis system such that the in vivo embodiment arrives at the photolysis region of the system. A detailed description of each subassembly is provided elsewhere herein. In addition, a method is provided to describe the interaction of these subassemblies to understand the operation of a typical apparatus.

Various embodiments of the present invention include a flash photolysis system 100, as shown in FIG. 1. The flash photolysis system 100 is configured to oxidize sample cells in real time to achieve in vivo radical dosimetry. In vivo analytes (i.e., cells) are introduced via a sample introduction system 101. Cells in suspension can be presented using small volume microcentrifuge tubes or multi-well microtiter _plates readily available from Eppendorf (Hamburg, Germany). A microfluidic circuit is configured for aspirating cells, mixing with _H2O2 , hydrodynamic focusing of cells using a sheath flow device, transporting to a photolysis and dosimetry area, and transporting and depositing labeled cells. In some embodiments, the sample introduction system 101 is configured to provide intact organisms to the photolysis area (e.g., photolysis area 102), where the organisms are separated from each other in the focused sheath flow. The sheath flow is typically configured to separate the organisms from each other. For example, the organisms are separated from one another at regular intervals using appropriate conditions.

Photo-oxidation occurs within the photolysis region 102. In some embodiments, the photolysis region 102 is comprised of fused silica capillaries available from Polymicro Technologies-Molex (Phoenix, AZ, USA). Typical capillary inner diameters range from 50 micrometers to 5 millimeters. Typical wall thicknesses are 50-2000 micrometers. In some embodiments, optofluidic chips are fabricated using a variety of techniques, such as wet chemical etching by lithography, dry reactive ion etching, and microstructuring by laser ablation to form microfluidic channels in a quartz substrate. In some embodiments, optofluidic chips are fabricated by embossing fluidic channels in a plastic substrate, and the formed fluidic channels transport the sample to optically transparent cells created by sealing optically transparent windows in areas where the plastic substrate has been removed. Exemplary plastic substrates include, but are not limited to, polycarbonate, polyethylene, polyetheretherketone, cyclic olefin polymers, cyclic olefin copolymers, polytetrafluorene, Kalrez®, polychlorotrifluoroethylene, and the like. The inner diameters of the fluidic and optical channels range from, but are not limited to, 0.1-5.0 mm. In some embodiments, the fluidic and optical channels can have different inner diameters to ideally suit different requirements for fluid transport, fluid mixing, hydrodynamic focusing, and optical coupling. Additionally, the optofluidic chip can include optical waveguiding structures such as integrated optical fibers, monolithic waveguides, liquid core waveguides, or evanescent waveguiding means using metal oxides, rare earth metals, or grating structures. In another embodiment, at least one sample contacting surface of the photolysis cell is coated with a photocatalytic metal oxide such as _TiO2 . In some photocatalytic metal oxide formulations, long UV (wavelength >300 nm) or visible light can be used to initiate photolysis. In these embodiments, the capillaries and optofluidic chips can be fabricated using various types of glasses such as BK-7 or Borofloat® 33 (Schott AG, Germany) instead of fused silica or quartz. In another embodiment, a quartz or glass optofluidic cell is equipped with a resonant structure to support resonance and/or multi-pass collision of incident photons with suspended cells, dissolved reactants such as _H2O2 , suspended metal oxide nanoparticles, or immobilized _metal oxide films on at least one sample contact surface.

The photolysis region 102 receives the suspended cells from the sample introduction system 101 via the microfluidic pathway 107. After processing, the light-irradiated cells in the photolysis region 102 are transferred to the radical dosimeter 104. The photolysis region 102 is in optical communication with the flash photolysis light 103. The flash photolysis light 103 is an example of a photolysis light source configured to generate light for generating hydroxyl radicals in the photolysis region 102 from a source of hydroxyl radicals. In some embodiments, the photolysis region 102 is configured to receive a sheath flow containing the organism and to receive light from the flash photolysis light 103 to oxidize a dosimeter internal standard to oxidize a biological compound of the organism in vivo. For example, a peptide consisting of amino acids may be oxidized in the photolysis region 102.

The photolysis region 102, the flash photolysis light 103, and the radical dosimeter 104 constitute a flash photolysis system. A flash photolysis system consists of a plasma flash lamp or other suitable light source, such as an excimer laser, solid-state laser, or laser diode, and associated collection/transmission optics tailored to the requirements of the light delivery means to the photolysis region.

The radical dosimeter 104 is configured to receive the fluid and suspended cells from the photolysis region 102, or in an alternative embodiment, the radical dosimeter 104 is integrated into the photolysis region 102 by employing an orthogonal light path. Various photometric detection methods may be employed 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 spiked into the biological sample. In some embodiments, the inherent photometric properties of a biological buffer system once incorporated into the cells can serve as an inherent dosimeter internal standard. Intrinsic or inherent to the buffer means that the internal standard is one of the chemical species that provides the buffer properties. The buffer is optionally a physiologically compatible buffer configured to maintain the cells at a physiological pH or ion concentration. In some embodiments, the internal standard is configured to become fluorescent as a result of the flash photolysis light received in the photolysis region 102. For example, the dosimeter's internal standard, in various embodiments, can increase in fluorescence by a factor of at least 10-fold, 100-fold, or 1000-fold upon reaction with hydroxyl radicals.

Photometric detection methods include, but are not limited to, fluorescence, absorbance, refractive index detection, light scattering detection, and luminescence. In some embodiments, the photometric detection method comprises a fluorescence detector employing an ultraviolet (UV) light excitation source to generate UV fluorescence or luminescence. In some embodiments, the fluorescence detector employs a UV excitation source to generate visible fluorescence or luminescence. In some embodiments, the fluorescence detector employs a visible excitation source to generate visible fluorescence or luminescence. In some embodiments, the fluorescence detector also includes an integrated light scattering detector.

The control electronics 105 provides the peripheral assemblies with direct current (DC) drive voltages derived from the laboratory alternating current (AC) power source; provides analog and digital control signals to the peripheral devices; receives analog or digital information from the peripheral devices; provides ADC and digital-to-analog conversion (DAC) functions; and provides data to and receives commands from the instrument controller 106. In a typical embodiment, the control electronics assembly comprises a motor controller that interfaces with a motor disposed within the sample introduction and collection system 101. Additionally, the control electronics assembly of such an embodiment may include a universal serial bus (USB) hub for digital communication with the instrument controller 106.

The appliance controller 106 functions to provide process control for the various appliance peripherals while receiving status and data information in digital form from these devices. In some embodiments, the appliance controller 106 executes a software control program that has two main modules: a low-level multi-threaded module for appliance 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 appliance component control while communicating with the appliance 106 high-level UI control program via a USB or wireless interface.

The equipment controller 106, control electronics 105, and various interconnections together represent control logic configured to control the flash photolysis system 100. This control logic can be configured to perform any of the steps of the methods disclosed herein. For example, in some embodiments, the control logic is configured to determine that a target concentration of hydroxyl radicals has been generated for each organism. This target concentration is arbitrarily selected to ensure that the oxidation reaction of the cellular components has enough oxidant to approach a desired level of completion.

The control logic may further be configured to manage a feedback loop that includes adjusting the conditions in the photolysis region 102 to meet a target concentration of hydroxyl radicals. The conditions in the photolysis region 102 may be adjusted, for example, by changing the concentration of the hydroxyl 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 of receiving light from the photolysis light source, changing the separation distance/volume of the separated cells, and/or the like. By altering the conditions in the photolysis region 102, the control logic may provide feedback to the sample introduction system 101 and/or the photolysis light source 103 based on the analysis of the first cell to improve the analysis of the second cell.

In some embodiments, the control logic is configured to normalize quantification of oxidized and/or identified peptides from cells based on a fluorescent signal from an internal standard in the dosimetry region 210. This allows results from different experiments using different instances of the flash photolysis system 100 to be compared.

The control logic is optionally further configured to determine the time at which the photolysis light is received by cells in the photolysis region 102 and/or to determine the period during which isolated cells enter the photolysis region 102. These determinations may be based on detection of fluorescent and/or scattered light in the dosimetry region 210 and may be controlled by adjusting the flow rate and/or volume in the sample introduction system 101.

The control logic is optionally further configured to control the flow of cells to the labeled cell container 211 and the analyzer 212. For example, the control logic may be configured to control the flow of cells such that ruptured cells are removed from a particular container of the labeled cell container 211, or such that different oxidized cells are placed in different compartments over a function of time. In some embodiments, the control logic is configured to use the analyte signal from the analyzer 212 to control any aspect of the flash photolysis system 100.

The appliance controller 106 and control electronics 105 are optionally combined into a single device.

2 shows further details of the flash photolysis system 100 of FIG. 1. The flash photolysis system 100 is used for introducing cells, processing the cells, and collecting the processed cells. The cells to be processed are suspended/placed in a buffer and stored in the container 201. In some embodiments, the cells are pre-incubated with a buffer containing an internal standard for exogenous and/or endogenous radical dosimeters. This incubation causes the internal standard to enter (be absorbed) inside the cells and/or to be adsorbed on the surface of the cells. In some embodiments, the buffer contains a compound that essentially acts as an endogenous in vivo internal standard for performing radical dosimeters. Under the control of a microprocessor (e.g., control electronics 105 and/or instrument controller 106), a syringe pump 202 aspirates the cells from the container 201 and pumps them into the mixer 203. Items 201-208 and 211 are part of the optional sample introduction system 101.

_H2O2 is stored in a container 204. Under microprocessor control, a syringe pump 205 _{aspirates H2O2} from the container 204 and pumps the aspirated _H2O2 to the mixer 203. _The aqueous concentration of _H2O2 in the container 204 is selected to achieve an effective desired final concentration _{of H2O2 at} a desired net flow rate for the pumps 202 and 205. Exemplary _{H2O2 concentrations} range from, but are not limited to, 5-200 mM. Exemplary net flow rates after the mixer ₂₀₃ range from, but are not limited to, 5-100 _{uL /} min. _H2O2 is an example of a source of hydroxyl radicals and is added to the cells (or other organisms) by the sample introduction system 101.

The sheath buffer is stored in a container 206. Under the control of the microprocessor, a syringe pump ₂₀₇ aspirates the sheath buffer from the container 206 and pumps the sheath buffer to a hydrodynamic focusing mixer 208. The cells mixed with _H2O2 are pumped from the mixer 203 to the hydrodynamic focusing mixer 208 by the combined pumping action of pumps 202 and 205. The sheath buffer then acts to hydrodynamically focus the cells, creating a single file array of cells, each separated by a volume of sheath buffer. The single file array of cells is created by contracting the stream from the mixer 203 such that the diameter of the stream is similar to the diameter of the cells. Exemplary sheath buffer flow rates range from 20 to 1000 uL/min, but are not limited thereto.

After in-line cell separation by the combined pumping of syringe pumps 202, 205, and 207, the cells are transported to the photolysis region 102 where they can be illuminated by flash photolysis light 103. After flowing through the photolysis region 102, the cells can be pumped to a dosimetry region 210 that is part of the radical dosimeter 104, where photometric properties of the cells can be measured. The dosimetry region 210 is configured to detect oxidation of an internal standard, for example, using a fluorescence detector. The dosimetry region 210 is also optionally configured to detect the presence of cells (or other organisms) in the dosimetry region 210 using a detector configured to detect light scattering. This scattered light detector is also optionally configured to determine whether the cells are intact. For example, the scattered light detector and associated instrument control logic may be configured to distinguish between intact cells, disrupted cells, and cells that are attached together. In some embodiments, the radical dosimeter 104 includes both the dosimetry region 210 and the photolysis region 102. The dosimetry region 210 includes the photolysis region 102. The optical properties measured can include, but are not limited to, optical fluorescence and/or optical absorbance. After exiting the dosimetry region 210, the suspended cells are optionally pumped and collected into a labeled cell container 211. The real-time measurements made in the dosimetry region 210 _enable a feedback loop in which the photolysis conditions can be modified to ensure that a desired amount of _H2O2 production occurs and a desired amount of in vitro or in vivo analyte oxidation occurs.

During initial operation of the system, a baseline measurement of the optical properties of the cells is optionally performed. The baseline measurement is performed on cells introduced without photolysis. Once the baseline measurement is performed, photolysis proceeds and the photometric properties of the light-exposed cells are evaluated as the light-exposed cells (one or more) enter the dosimetry region. In some embodiments, the labeled cell container 211 includes one or more fluid storage compartments. For example, the labeled cell container 221 may include multiple compartments or wells in which oxidized biological compounds can be selectively placed. In some embodiments, different aliquots of the output of the dosimetry region are placed in different compartments based on the fluorescence and/or light scattering measurements made in the dosimetry region 210. In one compartment, non-light exposed cells that flowed through the system during the baseline measurement process are collected. In another compartment, light-exposed intact cells are collected. In another compartment, cells that were found to be intact may be placed. Light-exposed cells (e.g., oxidized cells) may be placed in different compartments as a function of time to perform time-based experiments such as kinetic studies. A compartment may be a "sample well," a "length of capillary," a "channel," etc.

The sample collected from the labeled cell container 211 is optionally analyzed using an analyzer 212. The analyzer 212 is configured to perform a chemical analysis of the sample. For example, the analyzer 212 may be configured to identify peptides, carbohydrates, metals, nucleic acids, lipids, and/or amino acids that have been oxidized in the photolysis region 102. The analyzer 212 may include, for example, a mass spectrometer, a scintillator, an electrophoretic device, a chromatograph, and/or other devices configured to separate and/or identify sample components based on radioactivity, mass, charge, size, or other chemical properties. In some embodiments, the analyzer 212 is configured to detect isotope or radioisotope labels in cells or other organisms, and optionally identify whether components containing such labels have been oxidized. In some embodiments, the analyzer 212 is configured to measure the ratio of oxidized/non-oxidized concentrations of certain components. In some embodiments, the analyzer 212 is in communication with the labeled cell container 211 and is therefore configured to receive and analyze samples in real time. For example, the labeled cell container 211 may include a capillary configured to provide a sample directly to the input of a mass spectrometer.

Figure 3 illustrates a method 300 for in vivo cell labeling according to various embodiments of the present invention.

In optional add internal standard step 301, the dosimeter internal standard is added to the cells or the like to be analyzed. The internal standard may be inherent in the buffer into which the cells are mixed or added. The cells may be left in contact with the internal standard for a period of time so that the internal standard has time to be absorbed into the interior of the cells and/or adsorbed to the surface of the cells. After this period of time, the internal standard may be washed from the cells in wash cells step 302. Steps 301 and 302 leave the internal standard absorbed or adsorbed to the cells while leaving substantially no internal standard in the surrounding liquid. These two steps improve the accuracy of the measurement of the internal standard by reducing the background of internal standard not bound to the cells. Step 301 is optional, for example, if the cells already contain the dosimeter internal standard.

In the introduction step 303, one or more first cells are introduced into the flash photolysis system using the sample introduction system 101. As described elsewhere, the introduced cells may be loaded with a dosimeter internal standard. For example, the cells may be placed in a buffer solution that contains an endogenous internal standard that is adsorbed to the surface of the cells or absorbed into the interior of the _cells . After the cells receive the internal standard, they may be placed in a solution that has minimal components that can be oxidized by _H2O2 . This allows the internal standard to associate with the cells but not with the surrounding solution.

In an optional measurement step 304, the first introduced cell(s) containing the endogenous or exogenous dosimetric internal standard are measured in an unirradiated state (i.e., not yet oxidized using photolysis) to assess its initial photometric properties. This initial assessment serves as a baseline measurement against which subsequent measurements of the photometric properties are compared.

In an irradiation step 305, the introduced cell (or cells) is irradiated in the photolysis region 102. The irradiated cell can be a first introduced cell or a second introduced cell. In a measurement step 306, one or more photometric properties of the irradiated cell(s) are measured using the radical dosimeter 104. The measurements made in step 306 can be compared to the measurements made in step ₃₀₄ to determine the effective _H2O2 concentration and the extent to which oxidation of biomolecules has occurred due to photolysis. In embodiments where the dosimeter internal standard is primarily associated with the cells (absorbed & adsorbed), the measured photolysis-induced oxidation represents a quantitative measurement of the in vivo oxidation of the cells' biomolecules.

In evaluation step 307, the measured change in the photometric property is used as a surrogate to access the effective OH radical concentration, thereby assessing the completeness of the oxidation reaction with the target (in vivo) biomolecule. In decision step 308, it is determined whether the target OH radical concentration has been achieved. If the determination is true, the method proceeds to a good result step 309, in which the photolysis conditions are considered satisfactory and the oxidation process is continued with additional cells, with subsequent irradiated cells finally being collected and analyzed as shown in FIG. 2. The good result step 309 optionally includes analyzing the cells with the analyzer 212 and using the results from the analyzer 212 to determine properties of the cells, such as kinetics, three-dimensional protein structure, molecular interaction kites, and/or the like. If the determination is false, the method proceeds to an adjustment step 310, in which the fluence and/or _H2O2 concentration of the photolysis system are varied and the method of FIG. 3 is repeated for additional cells, evaluating the new level of OH radical yield resulting _from the adjusted parameters. In some embodiments, method 300 is operated manually by a user. In some embodiments, all or parts of method 300 are automated under microprocessor control using the instrument controller 106 or control electronics 105. The method may be repeated until a desired level of OH radical yield and/or oxidation of biomolecules is achieved. The measuring step 304 may or may not be performed in every cycle of the method.

Various embodiments of the invention include in vivo radical dosimetry using intracellular fluorescent indicators of oxidation reactions. Technical limitations of fast photochemical oxidation of proteins (FPOP) hydroxyl radical protein footprinting (HRPF) arise from the reaction of OH radicals with background or nonanalyte components in the sample, such as buffer components, extraneous proteins, cellular structures, and initial solutes. The variability in the degree of background scavenging leads to a lack of reproducibility between tests, limiting 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). While OH radicals are an excellent probe of protein topography, they also react with many compounds present in analytical formulations. There is competition for free OH radicals between target proteins and background scavengers. Therefore, to obtain reproducible results, it is desirable to measure the concentration of hydroxyl radicals available to oxidize the target protein and adjust the total hydroxyl radical production accordingly. In vivo HRPF allows for optional radical dosimetry by monitoring only changes in the available OH radical load generated within cells, and in some cases preferentially within specific organelles.

In photochemistry, the effective radical concentration is measured using a radical dosimeter such as the Radical Dosimeter 104. Ideally, the dosimeter would have a simple relationship between effective radical concentration and dosimeter response, a simple, rapid and non-destructive means of measurement, and be non-reactive to most proteins. In one approach, the use of radical dosimeters for the evaluation of background scavenging in in vitro systems involves measuring the absorbance change of adenine to determine the free OH radical concentration. This method involves an offline method of collecting photoirradiated adenine and associated analyte proteins, where the flow is diverted from a capillary photolysis cell and directed to an offline UV detector. This method consumes a significant amount of product (several microliters) and requires a lot of time to transport the sample and generate a sufficient volume to perform UV absorbance measurements. Other methods provide real-time in vitro radical dosimetry as living organisms are labeled in the HRPF process of FPOP. In these approaches, photometric detection methods are applied to the analyte flow to detect changes in the optical properties of the dosimeter internal standard. In particular, the use of a dosimeter internal standard, in which adenine is added to the analytical sample as an exogenous or endogenous component, is described. Furthermore, these approaches include methods in which the labeling parameters can be changed in real time to achieve a desired level of effective _{OH radical} concentration and associated labeling efficiency, and methods in which the effective OH radical yield can be controlled by varying the fluence and/or spectral irradiance of the plasma flash lamp source in addition to dithering the H2O2 concentration. See, for example, U.S. Patent Nos. 5,333,433, 5,333,433 and 5,333,433.

The above approaches teach in vitro methods for performing offline and inline closed-loop radical dosimetry to improve the reproducibility of in vitro HRPF, but do not address the requirements of in vivo HRPF. To effectively perform in vivo HRPF, it is preferable to photoirradiate each cell only once with a brief UV exposure, with a typical pulse width on the order of 10-20,000 nanoseconds. Furthermore, for inline in vivo radical dosimetry to be most useful, the photometry should be performed in such a way that a single cell or a fixed amount of cells is consistently probed to allow comparative measurements. Thus, the cell stream should be created in a precise manner that provides a predictable amount of separation volume to separate each cell in a line, and allows for a precise and predictable temporal delivery of each cell or a given number of cells to the photolysis and dosimetry area. It is also desirable to separate irradiated and non-irradiated cells during both measurement and collection.

In vivo HRPF benefits greatly from the assessment of effective intracellular OH radical concentrations. Thus, in vivo radical dosimetry is beneficial when the dosimeter internal standard (endogenous or exogenous) is present only (or mainly) within the cell and not in the extracellular fluid. Depending on the cell type, the intracellular volume is 10-100 nL. Typical working concentrations of internal standard radical dosimeters are around 1-10 mM. Therefore, the effective amount of dosimeter in the cell is in the range of 10 picomolar to 1 nanomolar, making detection by optical measurement extremely difficult. Under these constraints, spectrophotometric detection is likely to be insufficient in analytical sensitivity to accurately capture the change in absorbance of the intracellular radical dosimeter upon introduction of OH radicals. This is because spectrophotometry works by detecting the change in the light transmitted through the sample due to the incident light. At solute concentrations below 1 μM, the resulting optical change in the transmitted light becomes immeasurable and practically insignificant compared to the original intensity of the incident light.

Fluorescence detection, unlike absorption detection, has the ability to detect small changes in light against a dark background, since detection light is only generated when the fluorophore is illuminated with excitation light of the appropriate wavelength that is prohibited from entering the fluorescence detector. Thus, fluorescence detection is 1,000 to 10,000 times more sensitive than absorption methods. With 10,000 times the sensitivity, it has been applied to imaging and optical measurement of single cells. When measuring radical dose using an internal standard, an ideal fluorescent dosimeter would have a simple relationship between effective radical concentration and fluorescence response, be insensitive to cellular components and biomolecular complements, and not fluoresce unless attacked by oxidative chemicals. There is a large literature describing fluorescent moieties that can be used for dosimetry in vivo, but the majority of these compounds have intrinsic fluorescence. Under these conditions, the detection of effective OH radical concentration using a true fluorescent radical dosimeter would depend on detecting the loss of fluorescence due to OH radical attack. Thus, these fluorescent probes have limited sensitivity, similar to the absorbance of a photometer, because they are detected by looking for small changes in light intensity against a large background.

In various embodiments of the present invention, an endogenous or exogenous intracellular radical dosimeter internal standard can be used to assess the effective concentration of generated OH radicals by comparing the difference in the fluorescence signal of irradiated and non-irradiated cells. During protocol development, measurements of dosimeter fluorescence change are compared to empirical values of in vivo HRPF to determine the ideal effective OH radical concentration for subsequent experiments. Once an index of change in dosimeter fluorescence signal is established, the variability of measurements of dosimeter fluorescence change in subsequent experiments performed with similar cells and microfluidic conditions can be used to monitor changes in background removal. Once background scavenging is assessed, a correction can be applied to correct for run-to-run variability, as shown in Figure 3. In some methods, the light dose can be changed in proportion to the change in background removal. The dose can be increased to compensate for increased scavenging or decreased to compensate for decreased scavenging. In some methods, the _{concentration} of _H2O2 can be adjusted proportionally to accommodate variations in intracellular removal. Alternatively, the measured abundance of an oxidized species in two or more different runs, as detected by mass spectrometry or other detection methods such as isoelectric focusing, can be normalized between runs by multiplying the response by a normalization factor obtained from the ratio of the changes in the fluorescent signal of the different runs.

Exemplary exogenous fluorescent radical dosimeter internal standards are shown in Table 400 of FIG. 4. The internal standards listed in FIG. 4 are intended to be illustrative and not limiting in scope, and other internal standards will be apparent to those of skill in the art in light of the teachings presented herein. The dosimeter internal standards listed in FIG. 4 exhibit low intrinsic fluorescence and are converted to fluorescent species upon oxidative attack, the conversion process being dependent on the kinetics of oxidative attack, which in turn is dependent on the yield or concentration of the oxidative species. Furthermore, these internal standards are readily taken up by the cells when added to cell suspensions or nutrient buffers of cultured cell lines. Prior to in vivo HRPF, cells are separated from the original nutrient buffer by a process of dialysis, buffer exchange, or centrifugation, and then resuspended in a dosimeter-free buffer. In the in vivo HRPF process, cells are mixed _{with H2O2} immediately prior to photolysis. _{H2O2 is} rapidly and easily taken up by cells and, when irradiated with UV light as described herein, is photolyzed into OH radicals that rapidly attack intracellular components, including the fluorescent radical dosimeter. Once oxidized, the radical dosimeter shown in Figure 4 becomes brilliantly fluorescent when irradiated with the appropriate excitation wavelength as shown, with the net change in measured fluorescence depending on the effective yield of OH radicals. Fluorescence emission wavelengths for various properties are also listed.

In some in vivo HRPF studies, it is desirable to analyze the biological complement and biophysiology of cellular organelles, such as the nucleus when studying DNA replication or DNA-mRNA transcription, or the mitochondria when studying cellular energetics and respiration. Under these circumstances, it is useful and possible to use an internal standard in an in vivo dosimeter that preferentially compartmentalizes to said organelles. One such in vivo dosimeter is ThermoFocus CellROX® Green (ThermoFisher, USA). CellROX Green selectively localizes to the nucleus and mitochondria of eukaryotic cells.

As shown in FIG. 5, some embodiments include an integrated photometric fluorescence and light scattering detector 500 for in vivo HRPF radical dosimetry. FIG. 5 shows the key optical components for an integrated photometric fluorescence and light scattering detector 500 for use in in vivo HRPF radical dosimetry. This integrated device provides at least two basic functions, but is not limited to: 1) providing a system that can generate and detect fluorescent signals for intracellular, internal standard, fluorescent radical dosimeters; and 2) providing a system that detects the entry of in vivo organisms into the dosimeter area 210, counts the number of in vivo organisms that enter the dosimeter area 210 within a specified time period, determines the size of the in vivo organisms, and determines the residence time of a single in vivo organism within the dosimeter area 210.

The theory of operation of the fluorescence detector is as follows: Excitation light 503 is provided by a high flux, narrow bandwidth solid-state UV source 501, such as a UV light emitting diode (LED) available from Q-Photonics (Ann Arbor, MI), or a miniature solid-state laser available from Thorlabs (Newton, NJ). Typical power is 0.1-10 mW, with a typical bandwidth of 1-15 nm, but not limited thereto. The wavelength of the UV source 501 is chosen to be an appropriate choice for the excitation wavelength of the in vivo standard radical dosimeter employed. A focusing lens assembly 502 is used to focus the excitation light into a narrow beam waste on the order of 1-20 μm within the center of the dosimetry region 210, creating the probed dosimetry region. The excitation light is directed by a dichroic mirror 504 to strike the dosimetry region 210, which may contain the in vivo organism. The dichroic mirror 504 preferentially reflects light at the excitation wavelength while being transparent to light at longer wavelengths such as the fluorescent emission light 506. The fluorescent emission light 506 is collimated using a collimator 507. After collimation, the emission light can be optionally filtered by a notch filter 508 to selectively transmit light at the appropriate emission wavelength. The notch filter 508 may be necessary because the collected emission light may consist of both the fluorescent light as well as a small amount of the original excitation light transmitted through the dichroic mirror 504 caused by back scattering of the excitation light incident on the optical surface of the cell or the probed contents. The emission light is ultimately directed to impinge on a fluorescence/emission light detector 509, which serves to measure the intensity of the fluorescence/emission light. The light detector 509 may consist of a UV-responsive silicon photodiode such as an S1336-8BQ silicon photodiode available from Hamamatsu (Hamamatsu, Japan). Alternatively, the photodetector 509 may consist of a miniature photomultiplier tube (PMT), such as the Micro PMT assembly H12400 available from Hamamatsu. The output current of the photodetector 509 is processed by a current-to-voltage (I-V) converter to obtain a voltage proportional to the incident luminescence light intensity 506. The output voltage of the photodetector 509 is transmitted to the control electronics 105, where an analog-to-digital converter (ADC) creates a digital signal that is ultimately transmitted to the instrument controller 106 to perform the fluorescence calculations.

The theory of operation of the light scattering detector is as follows: Excitation light 503 from a UV source 501 is focused by a lens 502 and reflected by a dichroic mirror 504 into the dosimetry region 210. Upon incidence on an in vivo organism located within the probe region of the dosimetry region 210, the incident excitation light 503 is elastically scattered. Due to the size difference between the incident excitation wavelength (nm) and the in vivo organism size (μm), the scattered light 510 is preferentially detected orthogonally to the incident excitation light. The scattered light 510 is collimated by a collimator 511 and finally impinges on the scattered light detector 512. For a given excitation light intensity, the measured intensity of the scattered light is proportional to the size and number of in vivo organisms located within the probed volume of the dosimetry region 210. Because the detection of scattered light is performed orthogonal to the incident excitation light, the measured background scattering in the absence of in vivo organisms is due to elastic and inelastic scattering from the contents of the probed volume, as well as elastic scattering from the optical surfaces of the radical dosimeter 104, producing a very low background signal.

The scattered light detector 512 may consist of a UV-responsive silicon photodiode, such as the S1336-8BQ silicon photodiode available from Hamamatsu (Hamamatsu, Japan). Alternatively, the photodetector may consist of a miniature photomultiplier tube (PMT), such as the Micro PMT assembly H12400 available from Hamamatsu. The output current of the scattered light detector 512 is processed by a current-to-voltage (I-V) converter to obtain a voltage proportional to the incident scattered light 506. The output voltage of the photodetector 509) is transmitted to the control electronics 105, where an analog-to-digital converter (ADC) creates a digital signal that is ultimately transmitted to the instrument controller 106, where the light scattering calculations are performed.

Various embodiments of the present invention include methods for determining and controlling the amount of intercellular sequestration to enable reproducible in vivo radical dosimetry. To effectively perform in vivo HRPF radical dosimetry, photometric measurements of the internal standard radical dosimeter must be performed when the in vivo organism(s) are present within the dosimetry volume to enable meaningful comparative measurements that represent differences in intercellular and intracellular radical dosimeter responses.

6 illustrates a method 600 for determining and controlling cell-to-cell separation volume using the invention described herein, which further enables detection of in vivo organisms in the dosimetry region. In cell introduction step 610, a line array of cells is formed and introduced into mixer 203, from which it flows into dosimetry region 210, as described elsewhere herein. In first cell detection step 602, the signal from Scatter photodetector 512 is monitored to detect the arrival time of the first cell into dosimetry region 210. Upon entry of a cell or other in vivo organism into the dosimetry region, the intensity of the scattered excitation light 510 increases according to the size and number of in vivo organisms in the probed region. In second cell detection step 603, the arrival time of the second cell into dosimetry region 210 is detected using the process described above. The net flow rate of the system is calculated 604 by summing the pumping rates of syringe pump 202, syringe pump 205, and syringe pump 207. The flow rate can be reduced until the time between detection of the first cell is long enough to reduce the probability that two cells are simultaneously present in the dosimetry region 210. This achieves single cell isolation, a condition that allows irradiation and oxidation of one cell at a time.

In a calculate cell separation volume step 605, the cell-to-cell separation volume is determined by multiplying the arrival time difference between the first cell and the second cell by the net flow rate 604. If the empirically determined cell separation volume deviates by less than ±5% (or other predetermined limit) from the desired separation volume (which is directly related to the separation distance and separation time), the system proceeds to label additional cells without further adjustments in a continue step 606. The proceed step 606 optionally includes the method shown in FIG. 3. Optionally, if the empirically determined cell separation volume deviates by more than ±5%, the pump speed of the sheath flow syringe pump 207 is changed in an adjust sheath flow rate step 607 to achieve the desired cell separation target volume 607, and the determination process repeats 608 until the target cell separation volume is achieved.

Various embodiments include systems and methods for detecting the presence of an in vivo organism within the dosimetry region 210. The detected times of entry and exit of the in vivo organism within the dosimetry region can be used to determine a data acquisition period for photometrically determining the intracellular dosimeter internal standard radical response. Upon entry into the dosimetry region 210, the in vivo organism causes a sudden increase in the intensity of the scattered light detected by the scattered light detector 512. Concomitantly, upon exit of the in vivo organism, there is a sudden decrease in the intensity of the scattered light detected by the scattered light detector 512. The time difference between the increase and decrease in the scattered light intensity represents the residence period of the in vivo organism, e.g., a cell, in the dosimetry region. During the residence period, the light intensity values detected by the luminescence light detector 509 are summed and/or integrated to determine the net dosimetry signal of the in vivo organism of interest. By adopting this approach, optical dosimetry can be performed by detecting signals arising from intracellular and/or extracellular optical metrology signals, while rejecting the inherent background signal arising from extracellular fluids for areas where no endogenous organisms are present. Thus, the background of the extracellular fluid is substantially lower than the background of the intracellular fluids by experimental design, and since measurements are only performed in the presence of endogenous organisms, the majority of the measured photometric signal is due to intracellular components.

Some embodiments include systems and methods for determining the viability of in vivo organisms. For a given in vivo organism, the scattered light intensity is proportional to the size and number of in vivo organisms present in the dosimetry region 210 during photometric evaluation. Using the methods disclosed herein, means are described that effectively ensure a constant arrival rate of in vivo organisms in the dosimetry region. Under such circumstances, the number of in vivo organisms in each data acquisition period can be constantly controlled, and the measured scattered light fluctuations will be highly dependent on changes in in vivo organism size. Changes in in vivo organism size may be due to intrinsic morphological changes or may indicate artifactual changes in cell/species morphology, which may indicate cell destruction, cell death, or cell apoptosis. Since the original purpose of in vivo HRPF is to evaluate the HOS of biomolecular complements under viable conditions, it may be desirable to detect and signal the presence of non-viable processes and/or conditions. The embodiments of the invention described herein provide systems and methods for detecting potential harm to in vivo sites that may result from HRPF protocols, and as such, provide a means by which in vivo HOS analysis may be performed on viable, undisrupted in vivo organisms. For example, disrupted cells, or the simultaneous presence of multiple cells within the dosimetry region 210, may be detected based on measured light scattering. Organisms detected under these circumstances may be separated and discarded from organisms that were not irradiated under these circumstances.

Various embodiments of the present invention include systems and methods that emit flash photolysis light 103 in synchronization with the arrival of an in vivo organism in the photolysis region 102. In vivo HRPF ideally involves determining and quantifying the presence of the in vivo organism in the photolysis region to reliably irradiate the organism(s) in a reproducible manner and photocatalytically create a reproducible intracellular OH radical load.

FIG. 7 illustrates a method 700 for determining the arrival of an in vivo organism in the photolysis region 102, while providing a system and method for precisely flashing the photolysis light 103 in response to the arrival of an in vivo organism in the photolysis region. In a cell introduction step 701, a single-file array of cells is formed as described elsewhere herein, and a first cell is introduced into the photolysis region 102. In a detection step 702, the output signal of the scattered light detector 512 is monitored to detect the arrival time of the first cell in the dosimetry region 102. The entry of a cell or other in vivo organism into the dosimetry region 102 causes an increase in the intensity of the scattered light depending on the size and number of in vivo organisms in the probed region. This increase is detected by the scattered light detector 512. In a net flow determination step 703, the net flow rate of the system is calculated by summing the pump speeds of the syringe pump 202, the syringe pump 205, and the syringe pump 207. In a determine interconnect volume step 704, as a direct manifestation of the microfluidic system design, the interconnect volume extending from the photolysis region and the dosimetry region is determined and held constant during the in vivo HRPF process. In a determine transit time step 705, the transit time required for the in vivo organism to move from the photolysis region 102 to the dosimetry region 210 is calculated by dividing the interconnect volume by the net flow rate. In a determine photolysis region arrival time step 706, the photolysis region arrival time is calculated by determining the difference between the dose measurement region arrival time and the transit time from the photolysis region to the dose measurement region. In a trigger step 707, for subsequent cells or in vivo organisms, the photolysis system is triggered to flush at the determined photolysis region arrival time or at a consistent interval thereafter. In a continue processing step 708, processing proceeds and additional labeled cells are deposited in the labeled cell container 211 until the target number of cells is processed.

While the teachings herein describe the particular utility of the detection signals resulting from the light scattering detector 512 and the fluorescence/luminescence detector 509, it will be apparent to one of ordinary skill in the art that the aforementioned detectors may be used to detect the arrival of an in vivo organism in the dosimetry region 210 or to predict the arrival of an in vivo organism in the photolysis region 102 in multiple undescribed combinations or means. For example, the signal generated by the fluorescence/luminescence detector 509 may be summed in temporal coherence with the signal from the light scattering detector 512 to improve the overall sensitivity for detecting the presence of an in vivo organism in the dosimetry region. Thus, the methods described herein are intended to be illustrative and not limiting in scope.

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 calibration logic that is used to predict the required light fluence or hydrogen peroxide concentration change in response to the measured photometric fluorescence change of the radical dosimeter. The calibration function is empirically determined through multiple measurements of known or controlled mixtures of supporting buffer, in vivo organisms, and radical dosimeter treated with a single light flash for each different control aliquot at various fluence or _H2O2 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 dosimetric light fluorescence at each fluence or _H2O2 concentration, allowing the creation of a mathematical equation, or calibration function, that describes the relationship between the applied fluence and/or _H2O2 concentration for a single flash exposure and the measured dosimeter fluorescence change. In another embodiment, the look-up table and _subsequent calibration function are manually generated by a user using the fluorescence change values for each flash voltage value and/or _H2O2 concentration.

During in vivo HRPF treatment, background hydroxyl radical scavenging is assessed by the dosimeter. The measured change in the dosimeter's photometric fluorescence is compared to a user-specified target change. If the dosimeter reading deviates from the target by more than ±10%, the applied fluence or _H2O2 concentration is changed to achieve the _target change in the dosimeter's measured absorbance. The calibration functions _described above are used to predict the required fluence or _H2O2 concentration change.

Various embodiments of the invention include post- _analytical normalization of the abundance of the labeled products. In these embodiments, the spectral irradiance and/or _H2O2 concentration are altered to adjust for undesired changes in background scavenging of OH radicals, and as such represent a pre-analytical or data pre-processing method of correction. It is also possible to perform post-analytical or data processing scavenging correction on the acquired HRPF data. In the post-analytical correction, the response is normalized by multiplying the measured abundance of the oxidized species in the experimental test, detected by other detection modalities such as mass spectrometry or isoelectric focusing, by a normalization factor obtained from the ratio of the dosimeter fluorescence change determined between the experimental test and the reference test. Specifically, the normalization factor is the ratio of the measured dosimeter fluorescence change in the experimental test divided by the measured dosimeter fluorescence change in the reference test. Alternatively, the normalization factor can be the ratio of the measured dosimeter fluorescence change in the reference test divided by the experimental test. In this way, for example, the ion current of a mass spectrometry (MS) measurement of a given protein or a single or tandem MS measurement of a peptide can be adjusted by multiplying said ion current value by the determined normalization factor. The application of pre-analytical and post-analytical normalization schemes are not mutually exclusive and can be employed alternatively or in tandem to achieve a higher level of compensation than would otherwise be achievable. In one embodiment, post-analytical normalization is applied to data obtained from in vivo HRPF experiments performed under the control of pre-analytical scavenging correction.

An in vivo radical dosimetry system for hydroxyl radical protein footprinting is disclosed. However, it should be apparent to one skilled in the art that many modifications beyond those already described are possible without departing from the inventive concepts herein. The subject matter of the present invention should not therefore be limited except in accordance with 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 "having" and "comprising" should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced element, component, or step may be presented, utilized, or combined with other elements, components, or steps not expressly referenced.

Although biological cells are used herein to describe various embodiments of the invention, in alternative embodiments, in any example or claim, "cell" may be replaced by other organisms, such as non-living organisms, organisms, viruses, multicellular organisms (e.g., fungi, spores, nanobees, molds, algae, nematodes, amoebas, protozoa, Trichoplax adhaerens or yeast), or non-biological materials.

Described herein are general structures and techniques, as well as more specific embodiments that can be used to achieve various ways of accomplishing the more general goals. Although only some embodiments have been disclosed in detail above, other embodiments are possible and the inventors intend to include these within this specification. Described herein are specific examples for accomplishing more general goals that can be achieved in other ways. This disclosure is intended to be illustrative, and the claims are intended to cover any modifications or alternatives predictable to those of ordinary skill in the art.

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

Also, the inventors intend that only those claims using the word "means" be interpreted under paragraph 6 of 35 USC 112. Moreover, no limitations from this specification are 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, whether a general purpose computer or some special purpose computer such as a workstation or laboratory or manufacturing equipment. The computers may be Intel (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.

COPYRIGHT NOTICE. In accordance with 37 CFR 1.71(e), applicants note that portions of this disclosure contain material that is subject to copyright protection (e.g., source code listings, screen shots, user interfaces, user instructions, or other aspects of this submission that are or may be subject to copyright protection in any jurisdiction). The copyright owner has no objection to anyone reproducing by facsimile the patent document or the patent disclosure as it appears in the Patent and Trademark Office patent file or records. All other rights are reserved, and the copying, distribution, creation of derivative works based on the contents, public display, and public performance of this application or any portion thereof are prohibited by applicable copyright law.

Claims (35)

a sample introduction system configured to provide untreated biological matter separated from one another in the focused sheath flow to a photolysis region;
a photolysis light source configured to generate light to generate hydroxyl radicals from a hydroxyl radical source;
a photolysis region configured to receive the sheath flow and light to oxidize an internal standard and to oxidize in vivo a biological compound of the organism;
a dosimetry region configured to receive the biological material from the photolysis region and detect the oxidation of the internal standard using a fluorescence detector;
control logic configured to determine when a target concentration of hydroxyl radicals has been generated for each of the organisms and to adjust operation of the photolysis region to meet the target concentration of hydroxyl radicals;
a container configured to receive the biological material containing the oxidized biological compounds;
An analysis system comprising: The system of claim 1, further comprising an analysis device configured to identify peptides or amino acids oxidized in the photolysis region. The system of claim 2, wherein the analytical device is configured to separate peptides based on mass, charge, or size. The system of claim 1, 2 or 3, wherein the sample introduction system is configured to separate biological organisms from one another using a sheath flow. The system according to any one of claims 1 to 4, wherein the sample introduction system is configured to add the hydroxyl radical source to the organism. A system according to any one of claims 1 to 5,
the internal standard is present in a buffer in which the organism is mixed;
the internal standard is adapted to be absorbed inside the organism; and/or
The internal standard is configured to be adsorbed to a surface of the biological body.
system. The system according to any one of claims 1 to ₆ , wherein the source of hydroxyl radicals is _H2O2 . A system according to any one of claims 1 to 7, wherein the dosimetry region is further configured to detect the presence of the biological body using a scattered light detector. The system of claim 8, wherein the scattered light detector is also configured to determine whether the biological organism is untreated. A system according to any one of claims 1 to 9, wherein the control logic is configured to determine the time at which the light is received by the organism in the photolysis region based on detection of fluorescent or scattered light in the dosimetry region. The system of any one of claims 1 to 10, wherein the control logic is configured to adjust the operation of the photolysis region by varying the concentration of the hydroxyl radical source, varying the amount of light received from the photolysis light source, and/or varying the time that the light is received from the photolysis light source. A system according to any one of claims 1 to 11, wherein the control logic is configured to determine a period of time during which an isolated number of the organisms enter the photolysis region. The system of any one of claims 1 to 12, wherein the control logic is configured to control the flow of the organisms such that the ruptured organisms are released from the container. The system according to any one of claims 1 to 13, wherein the fluorescence of the internal standard is configured to increase by at least 10 times upon reaction with the hydroxyl radical. A system according to any one of claims 2 to 14, wherein the control logic is configured to normalize the results of the quantification of the peptide based on a fluorescent signal from the internal standard within the dosimetry region. A system according to any one of claims 1 to 15, wherein the control logic is configured to provide feedback to the sample introduction system or the photolysis light source based on an analysis of a first one of the organisms to improve an analysis of a second one of the organisms. 1. A method for oxidizing a biomolecule in an intact cell, comprising:
introducing a sample mixture comprising at least one cell into a photolysis region and introducing a hydroxyl radical source and a dosimeter internal standard into the photolysis region of a flash photolysis system;
providing light to generate hydroxyl radicals from the hydroxyl radical source that oxidize biomolecules in the at least one cell;
waiting a time until the at least one cell detectable in a dosimeter area of a radical dosimeter by light scattering reaches a dosimeter area of a radical dosimeter configured to detect a photometric characteristic of the dosimeter internal standard resulting from a reaction of the dosimeter internal standard with the hydroxyl radical;
measuring a photometric characteristic of the dosimeter internal standard with the radical dosimeter while the at least one cell is within the dosimeter region;
determining that a target level of hydroxyl radicals has not been generated based on a measured photometric characteristic of the dosimeter internal standard;
adjusting the concentration of hydroxyl radicals in the photolysis region by adjusting at least one of: 1) the amount of light provided to the photolysis region; 2) the concentration of the hydroxyl radical source; 3) the flow rate of the at least one cell in the photolysis region; or 4) the time for which the light is provided to the photolysis region;
The method includes: 20. The method of claim 17,
introducing at least a second cell into the photolysis area;
providing light to generate hydroxyl radicals from the hydroxyl radical source at the controlled concentration that oxidize biomolecules in the second cells;
waiting for a time for the second cell to reach a dosimetric region of a radical dosimeter;
measuring a photometric characteristic of the dosimeter internal standard with the radical dosimeter while the second cell is within the dosimeter region;
determining that a target level of hydroxyl radicals was produced while the second cell was within the dosimeter volume based on the measured photometric characteristic of the dosimeter internal standard;
A step of collecting the second cells and performing an in vivo three-dimensional structural analysis of the biomolecules of the second cells based on the oxidation of the biomolecules in the second cells or on the surface of the second cells;
The method further comprises: 19. The method of claim 17 or 18,
placing said at least one cell in a buffer containing said dosimeter internal standard for a time sufficient for said dosimeter internal standard to migrate through the interior of said at least one cell;
prior to introducing the sample mixture containing the at least one cell into the photolysis region of the flash photolysis system, removing the at least one cell from an extracellular solution surrounding the at least one cell by washing the at least one cell;
The method further comprises: 20. The method of claim 19, wherein the dosimeter internal standard is inherent to the buffer solution. The method of any one of claims 17 to 20, wherein the dosimeter internal standard comprises at least one of terephthalic acid, dichlorofluorescein, CellROX® Green, CellROX® Orange, or CellROX® Deep Red. A method according to any one of claims 17 to 21, wherein the dosimeter internal standard is configured to be absorbed inside the at least one cell. The method of any one of claims 17 to 22, wherein the at least one cell is part of an intact multicellular organism. The method according to any one of claims 17 to 23, wherein the at least one cell is an isolated cell. A method according to any one of claims 17 to 24, comprising the steps of:
the at least one cell is a portion of a plurality of cells introduced into the photolysis region;
each of the plurality of cells is separated from adjacent cells of the plurality of cells by an identical separation volume;
method. 24. The method of claim 23 , wherein the intact multi-cellular organism comprises at least one cell, an intact virus, a virus attached to an intact cell, or a virus attached to a multi-cellular organism. The method of claim 17 or 18, wherein the photometric property is fluorescence or luminescence. A method according to any one of claims 17 to 27, wherein the dosimetry region is configured to simultaneously detect both a fluorescent signal and a scattered light signal from the at least one cell. A method according to any one of claims 17 to 28, wherein the concentration of the hydroxyl radicals is adjusted by varying the amount of light provided. A method according to any one of claims 17 to 29, further comprising a step of identifying the at least one intracellular amino acid oxidized by the hydroxyl radical in the photolysis region. The method of any one of claims 17, 18, or 27-30, further comprising normalizing the analysis of the oxidation of the at least one cell based on the photometric properties of the dosimeter internal standard. A method according to any one of claims 17 to 31, comprising
the at least one cell comprises a plurality of cells;
The sample mixture is introduced into a sheath flow configured to place each of the plurality of cells in a line separated by approximately the same distance from each other.
method. The method according to any of claims 17 to 32, wherein the light scattering signal in the dosimetry area is used to determine the time at which the light generates the hydroxyl radicals. The method of claim 18, further comprising determining that the second cell is untreated based on a scattering signal detected within the radical dosimeter. The system of claim 2 or 3, wherein the analytical device is configured to detect oxidation of an isotope-labeled nucleic acid, amino acid, lipid, carbohydrate, or peptide.

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