Measuring radiation-induced damage to a biopolymer

Abstract

A process for measuring radiation-induced damage to a biopolymer includes providing a known biopolymer of known length or composition; dissolving the biopolymer in a medium to create a sample solution; irradiating the sample solution with ionizing radiation; preparing the biopolymer for analysis; assembling a measurement chamber including two electrolyte chambers separated by a dielectric membrane containing a nanopore; infusing the irradiated biopolymer into the measurement chamber; applying a voltage across the membrane to cause the biopolymer to translocate through the nanopore; measuring the ionic current through the nanopore; and analyzing the measured ionic current to determine the extent of radiation-induced damage to the biopolymer.

Description

MEASURING RADIATION-INDUCED DAMAGE TO A BIOPOLYMER STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0001] This invention was made with United States Government support from the National Institute of Standards and Technology (NIST), an agency of the United States Department of Commerce. The Government has certain rights in this invention. CROSS-REFERENCE TO RELATED APPLICATION

[0002] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 621,891 (filed January 17, 2024), which is herein incorporated by reference in its entirety. BACKGROUND

[0003] The present invention generally relates to the field of measuring radiation- induced damage to a biopolymer, and more particularly to techniques for determining the relative biological effectiveness of ionizing radiation.

[0004] The ability to accurately measure the biological effects of ionizing radiation is crucial in various fields, including radiation therapy, radiation protection, and space exploration. Current methods for assessing the relative biological effectiveness (RBE) of ionizing radiation often rely on macroscopic endpoints, such as cell survival or tissue damage. These methods can be time-consuming, costly, and may not adequately reflect the underlying molecular damage caused by different types of radiation. Furthermore, traditional RBE measurements lack the resolution to quantify damage at the single-molecule level, hindering our ability to fully understand the mechanisms of radiation-induced biological effects. This limitation is particularly problematic in radiation therapy, where precise knowledge of RBE is essential for optimizing treatment plans and minimizing damage to healthy tissue.

[0005] Conventional methods for determining the biological effects of radiation often involve laborious cell culture and analysis techniques. These approaches typically require large sample sizes, long incubation periods, and subjective interpretation of results. Moreover, macroscopic measurements of radiation damage may not accurately reflect the heterogeneous distribution of energy deposition at the molecular level. For instance, two different types of radiation may produce the same overall cell survival rate, yet have vastly different effects on specific biomolecules, such as DNA. This information gap limits our understanding of the molecular events that initiate radiation damage and drive downstream biological effects.Therefore, the development of rapid, sensitive, and quantitative methods for measuring radiation damage at the molecular level is critical for advancing radiation research and improving the efficacy and safety of radiation-based therapies.

[0006] Existing techniques also suffer from a lack of standardization and reproducibility, making it challenging to compare results across different laboratories and experimental conditions. For example, variations in cell culture media, incubation temperature, and assay protocols can significantly influence the outcome of traditional RBE measurements. Moreover, macroscopic endpoints may not be sensitive enough to detect subtle but important differences in the biological effects of different types of radiation, particularly at low doses. This lack of sensitivity hinders our ability to accurately assess the risks associated with environmental radiation exposure and develop effective mitigation strategies. Therefore, there is a need for a robust, standardized method for quantifying radiation-induced damage that can be readily implemented across diverse research settings.

[0007] It is therefore an objective of the present invention to provide a method and apparatus for directly measuring radiation-induced damage to biopolymers at the single- molecule level, thereby overcoming the above-mentioned disadvantages of the prior art at least in part. Accordingly, a process and apparatus for rapidly, accurately, and quantitatively measuring the relative biological effectiveness of ionizing radiation using nanopore technology would be advantageous and would be favorably received in the art. BRIEF DESCRIPTION

[0008] One aspect of the present invention relates to an apparatus for measuring radiation-induced damage to a biopolymer. It may be provided that the apparatus comprises a dielectric membrane separating two electrolyte chambers. One advantage of this arrangement is that it electrically isolates the two electrolyte chambers, preventing electrical interference between the chambers while permitting measurement of ionic current flow through the nanopore. This isolation is crucial for obtaining accurate and sensitive measurements of biopolymer translocation events. Another advantage is that it creates defined electrolyte environments in each chamber, enabling precise control of experimental conditions, such as pH, salt concentration, and electric field strength. This control is essential for optimizing biopolymer translocation and ensuring reproducible results.

[0009] The apparatus further comprises a nanopore in the dielectric membrane, the nanopore having, e.g., a diameter of 2 nm to 20 nm and a length, e.g., of 5 nm to 500 nm. A nanopore is a nanoscale hole or channel that allows the passage of single molecules. Oneadvantage of incorporating a nanopore within the dielectric membrane is that it creates a confined space through which biopolymers can be driven by an applied electric field. This confinement enables single-molecule detection and analysis, providing high-resolution information about the biopolymer's size, shape, and charge. The specified diameter and length range of the nanopore ensures selective translocation of biopolymers while maintaining sufficient signal-to-noise ratio for accurate measurement of ionic current blockades. This selectivity is critical for distinguishing between different biopolymer species and detecting subtle changes in their properties caused by radiation damage.

[0010] The apparatus further includes a positive electrode in a cis electrolyte chamber and a negative electrode in a trans electrolyte chamber. An electrode is an electrical conductor used to establish an electrical connection with a nonmetallic part of a circuit, such as an electrolyte. One advantage of using electrodes in the electrolyte chambers is to establish an electric field across the membrane. This electric field drives the translocation of biopolymers through the nanopore, enabling measurement of their physical properties. The placement of positive and negative electrodes in separate chambers allows for controlled manipulation of the electric field direction and strength, optimizing biopolymer capture and translocation.

[0011] The apparatus includes a voltage source electrically connected to the positive and negative electrodes. A voltage source is a two-terminal device that maintains a fixed voltage difference between its terminals. One advantage of including a voltage source is that it provides the electrical potential necessary to drive biopolymer translocation through the nanopore. This controlled application of voltage ensures consistent and reproducible measurement of ionic current blockades.

[0012] The apparatus includes a transimpedance amplifier electrically connected to the positive and negative electrodes. A transimpedance amplifier is a device that converts current to voltage, with high precision. One advantage of a transimpedance amplifier is that it amplifies the small ionic current signals generated by biopolymer translocation through the nanopore, enabling their detection and measurement. This amplification improves signal-to- noise ratio, enhancing the sensitivity and accuracy of the RBE measurement. The direct electrical connection of the amplifier to the electrodes minimizes signal loss and distortion, further enhancing measurement precision.

[0013] One aspect of the present invention relates to a process for measuring radiation-induced damage to a biopolymer. Measuring may be understood as quantifying or determining the extent or magnitude of something. Radiation-induced damage may beunderstood as alterations or modifications in the structure or properties of a molecule caused by exposure to ionizing radiation. A biopolymer may be understood as a large molecule, such as DNA, RNA, or protein, composed of repeating subunits.

[0014] It may be provided that the process comprises providing a known biopolymer of known length or composition. Providing a known biopolymer of known length or composition establishes a controlled starting material for the RBE measurement. This knowledge of the biopolymer's initial state is essential for accurately quantifying any subsequent changes induced by radiation exposure. Using a purified sample minimizes confounding factors arising from impurities or variations in molecular size, enhancing the accuracy and reproducibility of the measurement. Knowing the biopolymer's length or composition enables precise calculation of radiation-induced damage, such as the number of strand breaks per unit length or the frequency of specific chemical modifications.

[0015] The process includes dissolving the biopolymer in a medium to create a sample solution. Dissolving the biopolymer in a medium creates a homogenous sample solution suitable for irradiation and subsequent analysis. This ensures uniform exposure of the biopolymer to ionizing radiation, minimizing variations in radiation dose and enhancing the accuracy of the RBE measurement. The choice of medium can be tailored to mimic specific biological or environmental conditions, enabling investigation of radiation effects under controlled experimental settings.

[0016] The process further comprises irradiating the sample solution with ionizing radiation. Irradiating the sample solution introduces controlled radiation damage to the biopolymer, mimicking the effects of radiation exposure in biological systems. The type and dose of ionizing radiation can be precisely controlled, enabling investigation of RBE across a wide range of radiation conditions relevant to radiation therapy, environmental exposure, and space exploration.

[0017] The process includes preparing the biopolymer for analysis. Preparing the biopolymer ensures compatibility with the nanopore measurement platform, enabling sensitive and accurate detection of radiation-induced damage. This preparation step may involve dilution, buffer exchange, or addition of reagents to optimize biopolymer translocation through the nanopore and enhance the signal-to-noise ratio of the ionic current measurement.

[0018] The process includes assembling a measurement chamber, the chamber comprising two electrolyte chambers separated by a dielectric membrane containing a nanopore. Assembling a measurement chamber creates the platform for single-molecule analysis of the irradiated biopolymer. The two electrolyte chambers, separated by a dielectricmembrane containing a nanopore, establish the electrical circuit for measuring ionic current blockades caused by biopolymer translocation. This arrangement allows for precise control of experimental conditions and isolation of individual translocation events.

[0019] The process includes infusing the irradiated biopolymer into the measurement chamber. Infusing the irradiated biopolymer introduces the sample into the nanopore measurement system. This step places the biopolymer in proximity to the nanopore, enabling its capture and translocation by the applied electric field.

[0020] The process includes applying a voltage across the membrane to cause the biopolymer to translocate through the nanopore. Applying a voltage initiates biopolymer translocation through the nanopore, creating transient blockades in the ionic current flow. The controlled application of voltage allows for precise manipulation of biopolymer movement, optimizing capture rate and translocation speed. This control is crucial for accurate and efficient measurement of radiation-induced damage.

[0021] The process further comprises measuring the ionic current through the nanopore. Measuring the ionic current provides a real-time signal of biopolymer translocation through the nanopore. Transient changes in ionic current, or blockades, reflect the physical properties of the translocating biopolymer, including its size, shape, and charge. One advantage of this is that it translates the molecular properties of the biopolymer into an electrical signal, providing the fundamental data for RBE determination.

[0022] The process comprises analyzing the measured ionic current to determine the extent of radiation-induced damage to the biopolymer. Analyzing the measured ionic current converts the raw electrical signals into meaningful information about radiation-induced damage. This step utilizes signal processing and data analysis techniques to quantify changes in the biopolymer's properties, such as the frequency of strand breaks, or the occurrence of chemical modifications. One advantage of this arrangement is that it generates quantitative data on the extent of radiation damage, enabling precise determination of RBE for different types of radiation. This information is essential for optimizing radiation therapies, assessing environmental risks, and advancing our understanding of radiation's biological effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The following description cannot be considered limiting in any way. Various objectives, features, and advantages of the disclosed subject matter can be more fully appreciated with reference to the following detailed description of the disclosed subject matterwhen considered in connection with the following drawings, in which like reference numerals identify like elements.

[0024] FIG. 1 shows, according to some embodiments, nanopore-based single- molecule dosimetry. a, An aqueous solution of 2.5 kbp DNA is irradiated with a60Co calibrated gamma source. b, Single-strand breaks in the sugar-phosphate backbone accumulateuntil two breaks occur close enough (here shown as ^^^^ = 3 bases) to compromise the stabilityof the molecule and cause a double-strand break. c, Irradiated DNA sample is quantified post- exposure with a glass nanopipette. d, Histograms of the concentrations of the irradiated 2.5 kbpand unirradiated internal standards of 5 kbp and 10 kbp DNA as a function of dose ( ^^^^0 = 1bp). Absolute size and concentration are calculated from the ECD and capture frequencies by calibrating against these two internal standards.

[0025] FIG.2 shows, according to some embodiments, molecular standards as an internal calibration and ruler. a, Ionic current versus time showing resistive pulses of the 5 kbp and 10 kbp DNA internal standards and 1.0 Gy gamma irradiated 2.5 kbp DNA. b, Ionic current of an identical DNA mixture in which the 2.5 kbp DNA has been irradiated at 15.0 Gy. Characteristic current events for the irradiated length, 2.5 kbp (left), and the two internal standards, 5 kbp (center) and 10 kbp (right), are shown for 1.0 Gy and 7.5 Gy enlarged in the boxes. The ECD is the area, A, shaded in orange or blue. c, Without calibration, by visual inspection, the 5 kbp 1.0 Gy events are closer in magnitude to the 2.5 kbp events in the 7.5 Gyrun than are to the corresponding 5 kbp events (here, ^^^^0 = 1pC ). d, This is corrected byaligning the internal standard peaks horizontally (here, ^^^^0 = 1bp ). The concentration iscalculated by comparing the average integral of the 5 kbp and 10 kbp peaks, to the known concentration at which they were added. After alignment, the final 1.0 Gy and 7.5 Gy histograms show a decreasing concentration of intact 2.5 kbp DNA with dose. The integrated area of the 2.5 kbp peak is taken as its measured concentration for each dose.

[0026] FIG. 3 shows, according to some embodiments, DNA dose response. The intact DNA concentration (blue circles and error bars) versus dose shows that the response is roughly Gaussian. At very low dose, one expects such behavior since bimolecular radical decaywill be negligible. However, the best fit (red, dashed line) at low dose, ≤ 3 Gy, to a Gaussianmodel does not work well at high doses, overestimating the loss of intact DNA. A best fit to the whole range of data (not shown) similarly does not fit well (overestimating intact DNA at intermediate doses and underestimating at high doses), as the shape of the curve is not actually Gaussian in ^^^^ but an elongated Gaussian. Including bimolecular decay yields a two parametermodel and a highquality fit to the data (orange, dotted line). The inset shows the measured concentration of three key molecules. The concentration of 2.5 kbp DNA decreases with increasing dose. Error bars are a scaled average of the standard deviation between the measured concentrations of the 5 kbp and 10 kbp internal standards (that were fixed between runs). The scaling is based on the length of the dataset and the number of 2.5 kbp events recorded at a particular dose.

[0027] FIG. 4 shows, according to some embodiments, fragment concentrationbias. a, A comprehensive molecular ladder of ≤ 10kbp DNA run through a pipette preparedidentically to those used in the primary experiments. Separate ladders containing100bp, 250bp, 600bp, 1.5kbp, 2.5kbp, 5kbp, and 10 kbp fragments (red), and300bp, 900bp, 2kbp, 5kbp, and 10 kbp fragments (blue) were recorded and aligned asdescribed in the Methods to minimize overlap. b, Molecular size distribution for all captured molecules after exposure to 15.0 Gy of radiation. Fragments between 100 bp and 2.5 kbp arevisible below the 2.5 kbp peak indicated with the red arrow ( ^^^^0 = 1bp ). c, The probability,^^^^^^^^, of successfully capturing a DNA fragment of a given size as calculated from the ladder shown in a. For nanopipettes of this size, ^^^^^^^^is relatively flat in the 2 to 10 kbp range, anddecreases logarithmically for fragments ≤ 2kbp in size. Error bars are the standard deviationof the best-fit parameters of a logarithmic fit (black, dotted line) to the capture rates of the ladder run through three identically prepared pipettes. d, The ratio of nucleotides in fragments(the difference between a "fragment" and the intact Gaussian fitted peak being > 2 standarddeviations) to nucleotides in the intact 2.5 kbp DNA after correcting for the deflated ^^^^^^^^of small molecules. After correction, the fragment-to-intact ratio is monotonically increasing with dose.

[0028] FIG. 5 shows, according to some embodiments, post-irradiation DNA concentration. The DNA concentration for each of the eight irradiated samples (at doses from 0 Gy to 15 Gy ). We obtain the concentration with UV light absorbance. Error bars show plus / minus one standard deviation over 3 runs. The concentration is constant within the measurement error.

[0029] FIG. 6 shows, according to some embodiments, ionic current time series. From top to bottom, the left panels show the ionic current versus time for each of the doses. The right panels show the corresponding histogram for each of these real-time traces. This data highlights the variability of individual capillaries, from the baseline current to the positions of the different peaks. The dual internal molecular standards of this work correct for this variability and enable quantitative detection of nucleic acid analytes.

[0030] FIG. 7 shows, according to some embodiments, DNA damage labeling schemes.

[0031] FIG.8 shows, according to some embodiments, a DNA molecule containing two lesions on different bases. Site 1 is labeled with a small tag (e.g.22-mer hairpin nucleotide) and Site 2 is labeled with a large tag (e.g. a 44-mer containing two hairpins). As the DNA is driven through the pore under an applied electric field, the ionic current measured is approximatly proportional to the non-occluded volume. In this example, double-stranded DNA, DNA+label1 and DNA+label2, each result in distinct time-resolved ionic currents. This figures illustrates DNA damage detection, including detection and characterization of DNA- lesion size-selective labels.

[0032] FIG.9 shows, according to some embodiments, a measurement chamber for measuring relative biological effectiveness (RBE) of ionizing radiation.

[0033] FIG. 10 shows, according to some embodiments, DNA translocation through a nanopore in the measurement chamber shown in FIG.9.

[0034] FIG. 11 shows, according to some embodiments, a process for measuring relative biological effectiveness (RBE) of ionizing radiation.

[0035] FIG. 12 shows, according to some embodiments, a process for measuring relative biological effectiveness (RBE) of ionizing radiation.

[0036] FIG.13 shows, according to some embodiments, (a) DNA translocation, (b) time series data for ionic current from DNA translocation, and (c) equivalent charge deficit involved in measuring relative biological effectiveness (RBE) of ionizing radiation.

[0037] FIG. 14 shows, according to some embodiments, quartz nanopipette fabrication.

[0038] FIG. 15 shows, according to some embodiments, equivalent charge deficit proportional to length of DNA molecule.

[0039] FIG.16 shows, according to some embodiments, extracting molecular size from ionic current.

[0040] FIG. 17 shows, according to some embodiments, elucidation of radiation- induced fragmentation of DNA.

[0041] FIG. 18 shows, according to some embodiments, 2D histograms revealing discrete populations.

[0042] FIG. 19 shows, according to some embodiments, elucidation of radiation- induced DNA damage.

[0043] FIG.20 shows, according to some embodiments, high-LET vs. low-LET or proton vs. gamma radiation.

[0044] FIG. 21 shows, according to some embodiments, a dose response without initial concentration.

[0045] FIG. 22 shows, according to some embodiments, data for a supercoiled DNA conformation in pore.

[0046] FIG. 23 shows, according to some embodiments, decoding data with structure.

[0047] FIG. 24 shows, according to some embodiments, results of irradiation of supercoiled DNA.

[0048] FIG. 25 shows, according to some embodiments, decoding data with structure. DETAILED DESCRIPTION

[0049] A detailed description of one or more embodiments is presented herein by way of exemplification and not limitation.

[0050] Conventional methods for measuring the biological effects of ionizing radiation often rely on time-consuming and costly macroscopic endpoints, such as cell survival assays. These assays provide limited information about the underlying molecular damage and may not accurately reflect the heterogeneous distribution of energy deposition at the single- molecule level. Traditional RBE measurements lack the resolution and sensitivity to quantify damage to individual biomolecules, hindering our ability to understand the specific mechanisms of radiation action. Existing methods suffer from poor standardization, making it difficult to compare results across different laboratories and experimental conditions.

[0051] The process for measuring radiation-induced damage to a biopolymer overcomes these limitations by providing a rapid, sensitive, and quantitative method for assessing radiation damage at the single-molecule level. It has been discovered that a process for measuring radiation-induced damage to a biopolymer can accurately determine the extent of radiation damage at the molecular level, enabling precise RBE measurements. One advantage is that it provides high-resolution information about the type and extent of damage to individual biomolecules, such as DNA or proteins, enabling a deeper understanding of radiation's biological effects. This detailed molecular information is not readily accessible using conventional cell-based assays, which typically measure macroscopic endpoints, such as cell survival or growth inhibition. The nanopore-based measurement is rapid and requiresminimal sample preparation, significantly reducing the time and cost associated with traditional RBE measurements. This enhanced efficiency is especially advantageous in high-throughput applications, such as screening for radioprotective compounds or evaluating the effectiveness of radiation therapies. The process is adaptable to a wide range of biopolymers, experimental conditions, and radiation types, making it a versatile tool for radiation research. This flexibility contrasts with traditional RBE assays, which are often specific to particular cell types or radiation sources. The method provides objective, quantitative data, improving reproducibility and facilitating comparison of results across different studies and laboratories. This standardization is lacking in traditional RBE assays, which can be influenced by subjective factors, such as the interpretation of microscopic images or the scoring of tissue damage. The process requires significantly less material, and thus, it may allow for clinical and emergency applications for determining, in particular, the extent of radiation-induced damage from X- rays, CT scans and other low dose exposures. The process, by directly detecting radiation- induced damage to biopolymers provides a rapid and accurate method of assessing the cumulative damage of low dose exposures.

[0052] In an embodiment, an apparatus for measuring radiation-induced damage to a biopolymer comprises a dielectric membrane separating two electrolyte chambers. In an embodiment, the biopolymer is a nucleic acid. In an embodiment, the nucleic acid is DNA. In an embodiment, the DNA has a length of 2,500 base pairs. In an embodiment, the biopolymer is a protein. In an embodiment, the protein is a peptide having a length of 10 amino acids. In an embodiment, the biopolymer is a polysaccharide. In an embodiment, the dielectric membrane comprises silicon nitride. In an embodiment, the nanopore has a diameter of 5 nm. In an embodiment, the apparatus further comprises an analog-to-digital converter electrically connected to the transimpedance amplifier.

[0053] With reference to FIG. 10 and FIG. 11, the apparatus for measuring radiation-induced damage to a biopolymer includes a dielectric membrane (100) separating two electrolyte chambers (102, 101). The dielectric membrane serves as a barrier between the two electrolyte chambers, preventing the bulk flow of electrolyte while permitting the passage of ions through the nanopore. This separation is for establishing distinct electrochemical environments in each chamber and enabling precise control of the electric field that drives biopolymer translocation. The membrane may be fabricated from various materials, such as silicon nitride, glass, or polymers, chosen for their electrical insulation properties, mechanical stability, and compatibility with the electrolyte solutions. For example, silicon nitride is arobust material suitable for high-voltage applications and is compatible with a wide range of electrolytes.

[0054] The apparatus further incorporates a nanopore (107) within the dielectric membrane. The nanopore is a nanoscale channel that allows single biopolymers to pass through the membrane under an applied electric field. As a biopolymer translocates through the nanopore, it transiently blocks the flow of ionic current, generating a measurable signal that reflects the biopolymer’s physical properties. The nanopore's diameter and length are critical parameters that determine its sensitivity and selectivity. Nanopores may be fabricated using various techniques, including laser pore formation, focused ion beam milling, electron beam lithography, or chemical etching. Biological nanopores, such as those formed by membrane proteins, can also be employed. The nanopore diameter may range from 2 nm to 20 nm, and its length may range from 5 nm to 500 nm, or even greater, chosen to optimize the detection of specific biopolymer species or types of radiation damage. A 5 nm diameter nanopore, for example, is suitable for analyzing DNA, while larger nanopores may be necessary for studying proteins or polysaccharides.

[0055] Within the apparatus, a positive electrode (104) resides in the cis electrolyte chamber, and a negative electrode (103) is positioned in the trans electrolyte chamber. These electrodes establish the electric field across the membrane, driving the translocation of charged biopolymers through the nanopore. The electrodes may be fabricated from conductive materials, such as platinum, gold, or silver, chosen for their electrochemical stability and low noise characteristics. Gold electrodes are commonly used in biological applications due to their stable potential and biocompatibility. The electrodes are electrically connected to a voltage source, enabling precise control of the applied electric field.

[0056] The apparatus includes a voltage source (106) electrically connected to both the positive and negative electrodes. The voltage source provides the electrical potential difference necessary to drive biopolymer translocation through the nanopore. The applied voltage creates an electric field across the membrane, which exerts a force on charged biopolymers, causing them to move toward the electrode of opposite polarity. The voltage source may be a battery, a power supply, or any other device capable of generating a stable voltage. The magnitude of the applied voltage is a parameter that affects translocation speed and capture rate, typically ranging from millivolts to hundreds of millivolts depending on the nanopore dimensions and the biopolymer's charge.

[0057] The apparatus incorporates a transimpedance amplifier (105) electrically connected to both the positive and negative electrodes. The transimpedance amplifier convertsthe small ionic current signals generated by biopolymer translocation into measurable voltage signals. These amplified voltage signals provide a real-time record of biopolymer translocation events, enabling analysis of the biopolymer's properties, such as its size, shape, and charge. The transimpedance amplifier's gain and bandwidth are chosen to optimize the detection of the translocation events. Commercially available transimpedance amplifiers or custom-designed circuits may be employed.

[0058] This specific implementation of the apparatus elements achieves significant technical advantages. The use of a dielectric membrane with a nanopore enables single- molecule analysis of radiation-induced damage, providing high-resolution information not readily accessible using conventional methods. Precise control of the applied voltage and electrolyte conditions optimizes biopolymer translocation and ensures reproducible measurements. The transimpedance amplifier enhances measurement sensitivity by converting the small ionic current signals into measurable voltage outputs. The apparatus’s compact design and minimal sample requirements make it suitable for a broad range of applications, including clinical diagnostics, environmental monitoring, and space research.

[0059] The biopolymer (108) may be a nucleic acid. Nucleic acids, such as DNA and RNA, are essential biomolecules that carry genetic information and play critical roles in cellular processes. Measuring radiation-induced damage to nucleic acids can provide insights into the mechanisms of radiation-induced mutagenesis, carcinogenesis, and cell death. Different types of nucleic acids, including genomic DNA, mitochondrial DNA, messenger RNA, and transfer RNA, can be analyzed using the present invention to investigate the effects of radiation on diverse biological processes. For instance, analyzing damage to genomic DNA can reveal the frequency and distribution of radiation-induced mutations, while studying damage to mitochondrial DNA can shed light on radiation's impact on cellular energy production.

[0060] The nucleic acid may be DNA. DNA, or deoxyribonucleic acid, is the primary carrier of genetic information in most organisms. Measuring radiation-induced damage to DNA is essential for understanding the effects of radiation on heredity, development, and disease. Analyzing radiation-induced damage to DNA, such as double- strand breaks, single-strand breaks, and base modifications, is crucial for developing effective strategies for cancer treatment, radiation protection, and environmental remediation. The DNA may be prepared from various sources, such as cells, tissues, or biological fluids, using standard DNA extraction and purification methods.

[0061] The length of the DNA can be any length. In an embodiment, a 2,500 base pair DNA molecule is a suitable model system for studying radiation-induced damage. This length is representative of many genes and other functional DNA elements. Analyzing a DNA molecule of defined length simplifies data interpretation and allows for precise quantification of radiation-induced damage, such as the number of strand breaks per unit length. Other DNA lengths can also be analyzed, and the length of DNA may be chosen based on the specific research question or application. For instance, longer DNA molecules may be necessary for studying the effects of radiation on chromosome structure, while shorter DNA fragments may be more suitable for investigating specific DNA repair pathways.

[0062] The biopolymer may be a protein. Proteins are essential biomolecules that perform diverse functions in living organisms, including catalyzing biochemical reactions, transporting molecules, and providing structural support. Measuring radiation-induced damage to proteins provides insights into radiation's effects on cellular function, metabolism, and signaling. Various proteins, including enzymes, structural proteins, and regulatory proteins, can be analyzed to investigate the impact of radiation on different biological processes. For instance, analyzing radiation-induced damage to enzymes can reveal the extent of enzyme inactivation, while studying damage to structural proteins can shed light on radiation's effects on tissue integrity.

[0063] The protein may be a peptide can be any length, e.g., a length of 10 amino acids. A peptide with a length of 10 amino acids can be model system for investigating radiation-induced damage. This length is representative of many bioactive peptides and provides sufficient structural complexity for studying various types of radiation damage. Peptides of varying lengths and compositions can be analyzed, chosen based on the specific application or research question. Analyzing shorter peptides allows for precise identification of radiation-induced modifications to specific amino acid residues, while longer peptides or full-length proteins can be used to investigate radiation's effects on protein folding and function.

[0064] The biopolymer may be a polysaccharide. Polysaccharides are complex carbohydrates that play important structural and functional roles in living organisms, including energy storage, cellular signaling, and immune responses. Measuring radiation-induced damage to polysaccharides provides insights into radiation's effects on plant cell walls, bacterial capsules, and other biological systems. Various polysaccharides, including cellulose, starch, and chitin, can be analyzed using the present invention to investigate the impact of radiation on different biological processes. For instance, measuring radiation-induced damageto cellulose can reveal the extent of cellulose degradation, while studying damage to chitin can shed light on radiation's effects on insect exoskeletons or fungal cell walls.

[0065] The dielectric membrane may comprise silicon nitride. Silicon nitride is a durable, chemically inert material well-suited for fabricating nanopore membranes. Its high mechanical strength and resistance to chemical degradation ensures long-term stability of the nanopore, reducing the need for frequent membrane replacements. Silicon nitride is compatible with a wide range of electrolytes, enabling versatile application of the apparatus in various experimental settings. Other materials, such as silicon dioxide, aluminum oxide, or polymers, can also be used. The choice of membrane material may be based on factors such as nanopore fabrication method, experimental conditions, and desired measurement sensitivity.

[0066] The nanopore may have a diameter effective for translocation of the biopolymer, e.g., a diameter of 5 nm. A 5 nm nanopore is suitable for analyzing many biopolymers, including DNA, RNA, and small proteins. This diameter provides a balance between high sensitivity for detecting small changes in biopolymer properties and sufficient throughput for efficient analysis. Nanopores with other diameters may be used, chosen based on the specific biopolymer being analyzed and the type of radiation damage being investigated. For instance, larger nanopores may be necessary for analyzing larger biomolecules or complexes, while smaller nanopores may enhance the sensitivity for detecting subtle changes in biopolymer structure or conformation.

[0067] The apparatus may comprise an analog-to-digital converter (208) electrically connected to the transimpedance amplifier. An analog-to-digital converter transforms the analog voltage signals from the transimpedance amplifier into digital data suitable for computer processing and analysis. This digitization enables precise quantification of the translocation events, facilitating automated data analysis and reducing potential errors associated with manual data interpretation. Various analog-to-digital converters with different resolutions and sampling rates may be employed. The analog-to-digital converter may be integrated into the measurement system or may be a separate, stand-alone device connected to the transimpedance amplifier.

[0068] The dielectric membrane (100) is a component of the apparatus, serving as a physical barrier between the two electrolyte chambers while allowing the passage of ions through the nanopore. It is typically made of a thin, insulating material, such as silicon nitride, silicon dioxide, aluminum oxide, or a polymer like polyethylene terephthalate (PET). The choice of material depends upon factors such as mechanical strength, chemical inertness, and compatibility with the electrolyte solutions and the biopolymer being analyzed. Themembrane's thickness can range from a few nanometers to several micrometers, specifically from 10 nm to 100 nm, and more specifically from 20 nm to 50 nm. Thinner membranes generally provide higher signal-to-noise ratios for nanopore measurements, but they are also more fragile and susceptible to damage. The membrane's shape and size are determined by the design of the measurement chamber. It can be circular, square, or any other shape that fits the chamber. Its area can range from a few square micrometers to several square millimeters, chosen to accommodate the nanopore and provide sufficient surface area for biopolymer capture. The membrane is positioned between the two electrolyte chambers and securely sealed to prevent leakage or mixing of the electrolyte solutions. This separation is essential for establishing distinct electrochemical environments in each chamber and generating a stable ionic current through the nanopore.

[0069] The cis electrolyte chamber (102) houses the positive electrode and contains an electrolyte solution that facilitates ionic current flow through the nanopore. The chamber is one of two electrolyte chambers included in the measurement device. It may have a volume from 1 microliter to 1 milliliter, specifically from 10 microliters to 100 microliters, and more specifically from 20 microliters to 50 microliters. The chamber’s shape can vary depending on the overall apparatus design, including cylindrical, rectangular, or other configurations suitable for accommodating the electrodes and the membrane. The chamber is made of a non- conductive material, like polycarbonate or glass, which minimizes electrical interference. The chamber is one component of the measurement chamber, together with the dielectric membrane and a trans electrolyte chamber (101). It contains the positive electrode (104) and is filled with an electrolyte solution chosen to ensure biopolymer stability and facilitate translocation through the nanopore. The chamber is sealed against the dielectric membrane, which contains a nanopore, preventing electrolyte leakage between the two chambers. The chamber’s physical dimensions and configuration are designed to minimize electrical noise and interference, enhancing the sensitivity of the nanopore measurement. This chamber allows control of electrolyte conditions, and maintains a stable electrical environment, critical for repeatable measurements.

[0070] The trans electrolyte chamber (101) is the other electrolyte-containing component included in the apparatus. It houses the negative electrode (103) and contains the same or a different electrolyte solution as the cis electrolyte chamber (102). Its volume may range from 1 microliter to 1 milliliter, specifically from 10 microliters to 100 microliters, and more specifically from 20 microliters to 50 microliters. Its shape, like that of the first chamber, varies with apparatus design and may be cylindrical, rectangular, or any other suitableconfiguration. Similar to the first chamber, the second chamber is made of electrically non- conductive material to minimize interference. It is sealed against the dielectric membrane (100), which physically separates the chamber from the cis electrolyte chamber, preventing electrolyte leakage and maintaining distinct electrochemical environments in each chamber. This chamber contains the negative electrode (103), establishing the electric field necessary to drive biopolymer translocation through the nanopore (107). The chamber’s physical dimensions and construction minimize electrical noise, enhancing measurement sensitivity. This chamber may accommodate different electrolyte solutions, enabling precise control of experimental conditions and optimization of biopolymer translocation. This adaptability makes the apparatus suitable for analyzing a broad range of biopolymers under different experimental settings. One advantage of the trans electrolyte chamber is its electrical isolation, enhancing measurement precision. Its small volume minimizes sample use, and its material compatibility with various electrolytes and biopolymers provides versatility. The chamber’s ability to accommodate different electrolyte solutions expands the range of biopolymers and conditions that can be analyzed.

[0071] The nanopore (107) is a nanoscale channel in the dielectric membrane (100) that separates the two electrolyte chambers (102, 101). It is a critical component of the apparatus, providing a means for analyzing single biomolecules based on their translocation characteristics. The nanopore's diameter can range from 2 nm to 20 nm, specifically from 3 nm to 10 nm, and more specifically from 4 nm to 6 nm. This size range is suitable for analyzing many biopolymers, including DNA, RNA, and proteins. The nanopore’s length can range from 5 nm to 500 nm, specifically from 10 nm to 100 nm, and more specifically from 20 nm to 50 nm, which length is sufficient to generate measurable ionic current blockades as biomolecules translocate through the pore. Nanopores can be fabricated using a variety of techniques, including focused ion beam milling, electron beam lithography, and chemical etching. Biological nanopores, formed by membrane proteins, can also be employed. The nanopore is positioned within the dielectric membrane that separates the two electrolyte chambers, creating a conductive pathway for ions between the chambers. When a voltage is applied across the membrane, an electric field is established that drives the movement of ions and charged biomolecules through the nanopore. As a biopolymer translocates through the nanopore, it partially blocks the flow of ions, creating a measurable change in the ionic current. This change in current reflects the biopolymer’s physical properties, including its size, shape, and charge. One advantage of employing a nanopore is that it enables high-resolution, single-molecule analysis of biopolymers. This approach provides higher sensitivity and more detailedinformation about radiation-induced damage. The nanopore’s dimensions are chosen to optimize detection of specific biopolymers. Fabricating the nanopore in a robust dielectric material, such as silicon nitride, enhances device stability. The nanopore’s precise dimensions provide high reproducibility and reliable performance for single-molecule measurements, improving the accuracy and consistency of RBE determinations. The nanopore's position within the membrane separating the electrolyte chambers ensures electrical isolation, minimizing background noise, and enhancing sensitivity.

[0072] The positive electrode (104) is positioned within the cis electrolyte chamber (102). It is made of a conductive material, such as platinum, gold, or silver, and serves to apply a positive voltage to the electrolyte solution in the first chamber. Its surface area can range from 0.1 mm2to 10 mm2, specifically from 0.5 mm2to 5 mm2, and more specifically from 1 mm2to 2 mm2, optimized to minimize noise while ensuring efficient current flow. The shape of the electrode can be cylindrical, planar, or any other configuration that fits within the chamber, and its dimensions may be chosen to maximize surface area and enhance current flow while avoiding interference with the nanopore measurement. The electrode is electrically connected to the positive terminal of the voltage source (106), which applies the voltage necessary to drive biopolymer translocation through the nanopore. The electrode is immersed in the electrolyte solution in the first chamber and is sealed to prevent leakage or contamination.

[0073] The negative electrode (103) resides in the trans electrolyte chamber (101) and complements the positive electrode (104) in establishing the electric field that drives biopolymer translocation. It is made of same or a different conductive material, such as platinum, gold, or silver, and applies a negative voltage to the trans electrolyte chamber. Its surface area can range from 0.1 mm2to 10 mm2, specifically from 0.5 mm2to 5 mm2, and more specifically from 1 mm2to 2 mm2, to ensure efficient current flow. Its shape and dimensions may vary to suit the chamber’s geometry, such as a cylindrical or planar electrode. The negative electrode is electrically connected to the negative terminal of the voltage source, completing the electrical circuit. The electrode is immersed in the electrolyte solution in the second chamber and is sealed to prevent leakage. This arrangement creates the electrical field across the dielectric membrane, essential for biopolymer translocation. The use of a conductive, low-noise material minimizes interference. Controlling the electrode’s dimensions optimizes current flow while minimizing noise. The secure sealing and positioning of the negative electrode within the electrolyte chamber prevent electrolyte leakage and maintain a stable environment, enhancing the reproducibility of nanopore measurements. The electricalconnection to the voltage source enables precise voltage modulation and electric field control for optimizing biopolymer translocation and measurement sensitivity.

[0074] The voltage source (106) provides the electrical potential difference between the two electrodes (104, 103), generating the electric field that drives biopolymer translocation through the nanopore (107). It can be a battery, a DC power supply, or any other device capable of delivering a stable voltage. The voltage output can range from millivolts to hundreds of volts, specifically from 10 mV to 500 mV, and more specifically from 50 mV to 200 mV, depending on the nanopore's characteristics, the biopolymer being analyzed, and the desired translocation speed. The voltage source is electrically connected to the positive electrode (104) in the cis electrolyte chamber (102) and the negative electrode (103) in the trans electrolyte chamber (101), completing the electrical circuit. The voltage source may include features for precise voltage control and monitoring, such as a digital display or computer interface, enabling accurate and reproducible application of the electric field. One advantage of this is that it allows for dynamic control of the applied voltage during an experiment, optimizing biopolymer capture and translocation rates.

[0075] The transimpedance amplifier (105) converts the small ionic current signals generated by biopolymer translocation through the nanopore (107) into measurable voltage signals. These current signals, typically in the picoampere to nanoampere range, are too small for direct detection and measurement. The amplifier converts the current signal to a voltage signal, with the output voltage proportional to the input current. The amplifier’s gain, typically in the range of 106to 109V / A, amplifies the small current signals, enabling their detection and measurement. The amplifier’s bandwidth can be from 1 kHz to 1 MHz, specifically from 10 kHz to 100 kHz, and more specifically from 20 kHz to 50 kHz. It is electrically connected to both the positive (104) and negative (103) electrodes, measuring the ionic current flowing through the nanopore. The amplifier may include features for filtering noise and other artifacts that can interfere with nanopore measurements, such as a low-pass filter to remove high- frequency noise, improving the signal-to-noise ratio of the measurement. It can include an offset adjustment to zero the baseline current in the absence of a translocating biopolymer. The transimpedance amplifier’s output is connected to an analog-to-digital converter (208), which digitizes the analog voltage signal for subsequent computer processing and analysis. This arrangement enhances nanopore measurement sensitivity by converting low ionic currents into measurable voltages. Precise gain control and noise filtering improve measurement accuracy. The ability to adjust the amplifier’s bandwidth optimizes the detection of biopolymer translocation events. The offset adjustment feature enhances measurement precision by zeroingthe baseline current. The transimpedance amplifier’s compatibility with different types of nanopores and measurement chambers makes it a versatile component for various single- molecule analysis experiments.

[0076] The analog-to-digital converter (208) transforms the analog voltage signals from the transimpedance amplifier (105) into digital data for computer analysis. The analog voltage signal, which represents the ionic current through the nanopore, is a continuous signal. The analog-to-digital converter samples the analog signal at discrete time intervals and converts each sample into a digital value. The resolution of the converter, typically in the range of 8 bits to 24 bits, determines the precision of the digital representation of the analog signal. The sampling rate, which can range from 1 kHz to 1 MHz, specifically from 10 kHz to 100 kHz, and more specifically from 20 kHz to 50 kHz, dictates the time resolution of the measurement. The converter is electrically connected to the output of the transimpedance amplifier and may be integrated into the amplifier circuit or may be a separate, standalone device. The digital data from the converter is transferred to a computer for storage, processing, and analysis using algorithms for detecting translocation events, collecting salient features of the current signature, and determining the extent of radiation-induced damage. This digitization of the ionic current signal enables quantitative analysis of biopolymer translocation events using algorithms implemented on a digital computer. The converter’s resolution and sampling rate are chosen to capture translocation events accurately. Integration of the analog-to-digital conversion within the apparatus streamlines the RBE measurement process, and the digitized format of the ionic current data makes this apparatus adaptable to different data acquisition and analysis platforms, improving experimental flexibility and efficiency. Its compatibility with different transimpedance amplifiers and computer systems expands the apparatus’s versatility.

[0077] The known biopolymer (108) serves as the analyte in the RBE measurement process. It is a molecule of known length or composition, carefully chosen based on the experimental goals. The biopolymer may be a nucleic acid, such as DNA or RNA, a protein, or a polysaccharide, all of which are essential biomolecules with diverse biological functions. It is contemplated that biopolymer (108) can be polymers of natural origin like plasmids or mitochondrial DNA or synthetic constructs. Nucleic acids, the carriers of genetic information, include DNA and RNA. Proteins are structural components, enzymes, and signaling molecules, and polysaccharides play essential roles in energy storage and cellular recognition. The biopolymer is purified to remove contaminants that could interfere with the measurement, using techniques such as chromatography, electrophoresis, or filtration. The length or composition of the known biopolymer is determined using methods such as gel electrophoresis,mass spectrometry, or sequencing. This knowledge of the biopolymer’s initial state enables precise quantification of any changes induced by radiation. The known biopolymer is dissolved in a suitable medium to create a sample solution for irradiation and nanopore analysis. The concentration of the biopolymer in the sample solution is carefully controlled to optimize the nanopore measurement.

[0078] The medium (210) is the solvent in which the known biopolymer (108) is dissolved to create the sample solution for irradiation and nanopore analysis. The medium is chosen based on its compatibility with the biopolymer, the irradiation process, and the nanopore measurement. It may be water, a buffer solution, or any other solvent that maintains the biopolymer’s stability and does not interfere with the measurement. Water, a readily available solvent compatible with most biopolymers, simplifies sample preparation. Buffer solutions may be used to maintain a specific pH, ionic strength, or other experimental conditions during irradiation and nanopore analysis. The concentration of the biopolymer in the medium is carefully controlled to optimize the nanopore measurement.

[0079] Ionizing radiation is electromagnetic or particulate radiation with enough energy to remove tightly bound electrons from atoms or molecules, creating ions. Exposure to ionizing radiation can cause a range of damage to biopolymers, depending on the type and dose of radiation and the chemical and structural properties of the biomolecule. Ionizing radiation includes electromagnetic radiation, such as gamma rays and X-rays, and particulate radiation, such as alpha particles, beta particles, neutrons, protons, and ions. Gamma rays are high-energy photons emitted from radioactive isotopes, like cobalt-60 or cesium-137, while X-rays are generated by X-ray tubes or linear accelerators. The dose of ionizing radiation may range from a few grays to thousands of grays, specifically from 0.1 Gy to 100 Gy, and more specifically from 1 Gy to 50 Gy, chosen to induce measurable damage to the biopolymer without causing complete degradation. The dose rate is contemplated to be various ranges, e.g., a low rate dose as well as FLASH doses.

[0080] The voltage (216) is an electrical potential difference applied across the dielectric membrane (100). It is generated by the voltage source (106) and applied to the electrodes (104, 103) positioned in the two electrolyte chambers (102, 101). The applied voltage establishes the electric field that drives the translocation of charged biopolymers through the nanopore (107). The magnitude of the voltage may range from millivolts to hundreds of volts, specifically from 10 mV to 500 mV, and more specifically from 50 mV to 200 mV. The polarity of the voltage is chosen such that the biopolymer is driven towards and through the nanopore. The voltage's strength is optimized based on the nanopore'scharacteristics, the biopolymer’s charge, and desired translocation speed. A higher voltage generally increases translocation speed but can also increase noise and reduce measurement accuracy. The voltage is applied and maintained throughout the measurement process, ensuring consistent translocation and accurate detection of radiation-induced changes. Precise control of the applied voltage enhances the sensitivity and reproducibility of the measurement by optimizing biopolymer translocation, ensuring consistent electric field strength, and enabling accurate detection of changes in the biopolymer’s properties caused by radiation. The adaptability of the voltage to different experimental setups and biopolymers enhances versatility. Its precise control and stable application during measurement improve accuracy and reproducibility.

[0081] The ionic current (217) is the flow of ions through the nanopore (107) in the dielectric membrane (100). It is driven by the applied voltage (216) and measured using a transimpedance amplifier (105) connected to the electrodes (104, 103) in the two electrolyte chambers (102, 101). The ionic current is sensitive to the presence of biopolymers translocating through the nanopore. As a biopolymer enters and passes through the nanopore, it partially blocks the flow of ions, causing a transient reduction in the ionic current. The magnitude and duration of this current reduction, or resistive pulse, reflect the biopolymer's physical properties, such as its length, volume, charge, and conformation. The ionic current is continuously monitored during the nanopore measurement, providing real-time information about the translocation events. The ionic current data is amplified by the transimpedance amplifier and then digitized by an analog-to-digital converter (208) for subsequent computer processing and analysis. Algorithms may be used to detect and count translocation events, measure the duration and amplitude of resistive pulses, and quantify changes in biopolymer properties caused by radiation exposure. This information may then be used to calculate RBE values. One advantage of using ionic current is that it provides a sensitive measure of biopolymer translocation. It translates molecular properties into an electrical signal. This signal can be amplified, digitized, and analyzed to quantify radiation-induced damage. Continuous current monitoring allows real-time detection of translocation events, and its quantitative nature improves measurement precision. Its compatibility with different nanopore materials and biopolymers enhances versatility, expanding applicability across diverse experimental setups.

[0082] The size selective tag can enhance detection of specific radiation-induced modifications to the biopolymer (108). Some types of radiation damage, like single-strand breaks or chemical modifications of bases, may not significantly affect the biopolymer’soverall length, making detection difficult by measuring changes in ECD alone. The size- selective tag is a molecule of known size that attaches specifically to the site of these radiation- induced modifications. The tag's size is chosen to be readily detectable by nanopore analysis, typically larger than the biopolymer’s monomeric subunits but smaller than the intact biopolymer. The tag may be attached via chemical or enzymatic methods, chosen based on the biopolymer and the nature of the targeted modification. For instance, DNA damage may be tagged using base-excision repair enzymes, like DNA glycosylase, which recognize specific damaged bases and create a nick in the DNA backbone. An oxime-modified tag can then be attached to the nick. The tag alters the biopolymer's effective length or charge at the damaged site, generating a distinct signal during nanopore translocation. One advantage of this tagging approach is that it increases sensitivity for particular radiation-induced damage. The tag's known size allows for precise identification of modification sites, and its specific attachment enhances measurement precision by reducing background signals. The ability to use a variety of tag types and attachment methods, tailored to the biopolymer and targeted modification, expands versatility, accommodating various research needs.

[0083] The ionizing radiation source can be an X-ray source that generates ionizing radiation, e.g., in the form of x-rays. X-rays are a form of electromagnetic radiation with wavelengths ranging from 0.01 nm to 10 nm. X-rays are generated by accelerating electrons to high energies and then directing them at a target material, typically tungsten or molybdenum. The interaction of the electrons with the target material produces X-rays, which can then be used to irradiate the biopolymer sample. The energy and dose of the X-rays can be controlled by adjusting the voltage and current of the electron beam. Dosimetry standards are employed to calibrate the X-ray source and ensure accurate dose delivery. X-ray sources may include X- ray tubes, linear accelerators, or synchrotron radiation facilities. The X-ray source’s energy spectrum and dose rate are chosen to induce the desired types and levels of damage to the biopolymer. Using an X-ray source for irradiation delivers a precisely controlled dose relevant to medical imaging and radiotherapy. The source's tunable energy spectrum allows simulation of different radiation environments, and its well-characterized dose rate makes this approach suitable for RBE studies.

[0084] The apparatus may further include a signal processing unit, such as a computer, for analyzing the digitized ionic current data. This unit may employ algorithms for detecting and counting translocations, collecting salient features of the current signatures, and determining the extent of radiation-induced damage. The signal processing unit enhances measurement automation and reduces human error, improving objectivity. Various algorithmsmay be used, such as threshold analysis, peak detection, and statistical methods for data interpretation. The choice of algorithm depends upon the specific biopolymer and the types of radiation-induced modifications being investigated. The flexibility of this approach allows for adaptation to diverse applications and research needs.

[0085] Alternative nanopore configurations, such as solid-state nanopores fabricated in materials like silicon nitride or graphene, may also be employed. These solid-state nanopores offer advantages in terms of stability and reproducibility, enabling high-throughput measurements. The nanopore dimensions can be precisely controlled during fabrication, ensuring consistent performance across different devices and experiments. This precision allows for more controlled analysis of changes in translocation dynamics, such as duration of passage, number of steps and current steps.

[0086] Different types of biopolymers, including nucleic acids (DNA and RNA), proteins, and polysaccharides, may be analyzed using the present invention. The choice of biopolymer depends upon the specific biological effects of radiation under investigation. DNA, the primary carrier of genetic information, is a critical target for radiation-induced damage. Analyzing DNA damage provides insights into the mechanisms of radiation-induced mutagenesis and carcinogenesis. Proteins, which perform diverse functions in cells, are also susceptible to radiation damage. Analyzing protein damage informs radiation’s effects on cellular function and metabolism. Polysaccharides, key structural components in various biological systems, such as plant cell walls and bacterial capsules, are another relevant target. Analyzing polysaccharide damage elucidates radiation's broader biological effects beyond DNA and proteins. The versatility of the nanopore-based method allows for analysis of various biopolymers, facilitating comprehensive investigation of radiation effects in diverse biological systems.

[0087] Different sources of ionizing radiation, such as gamma rays, X-rays, alpha particles, beta particles, or ion beams, may be employed, depending on the research application. Gamma rays, emitted by radioactive isotopes like cobalt-60, provide a homogenous radiation field for uniform sample irradiation. X-rays, produced by X-ray tubes, are commonly employed in medical imaging and radiation therapy, providing insights into the biological effects of clinically relevant radiation exposures. It should be appreciated that "high LET radiation" refers to radiation with a high linear energy transfer (LET), meaning it deposits a large amount of energy in a very small area when passing through matter, making it significantly more damaging to biological tissue compared to low LET radiation like X-rays or gamma rays. Examples of high LET radiation include alpha particles and neutrons. That is,alpha particles, helium nuclei emitted by certain radioactive isotopes, are high-LET radiation that causes dense ionization tracks, useful for simulating the effects of high-LET radiation encountered in space or during targeted alpha-particle therapy. Beta particles, high-energy electrons emitted by radioactive decay, represent another form of ionizing radiation. Ion beams, generated by particle accelerators, provide a highly controlled and versatile radiation source, enabling precise delivery of defined ion species and energies to the biopolymer sample. The choice of radiation source depends on factors such as the desired LET, dose rate, and penetration depth of the radiation, as well as the specific biopolymer and experimental goals. The method's adaptability to different radiation sources enables investigation of RBE across a wide range of radiation types and energies, informing radiation protection strategies and therapeutic applications.

[0088] The electrolyte solutions in the two chambers may be different to optimize biopolymer translocation and measurement sensitivity. For example, a higher salt concentration in one chamber may be used to enhance biopolymer capture, while a lower salt concentration in the other chamber facilitates translocation through the nanopore. The pH of the electrolyte solutions can also be adjusted to optimize biopolymer stability and minimize aggregation. The use of different electrolyte solutions allows for tailoring experimental conditions to the specific biopolymer being analyzed.

[0089] The apparatus may include additional components for automated data acquisition and analysis. For instance, a computer interfaced with the analog-to-digital converter may be employed to automatically record and store the digitized ionic current data. Specialized software or custom-written scripts may be used to analyze the data, enabling automated detection and counting of translocation events, collection of salient features, generation of histograms, and calculation of RBE values. This automation increases throughput and reduces human error, while improving measurement consistency and objectivity. The software may also include features for data visualization, statistical analysis, and reporting, enhancing data interpretation and dissemination.

[0090] The measurement device may be integrated with other analytical instruments, such as a mass spectrometer or a fluorescence microscope. This integration enables multi-modal analysis of radiation-induced damage, providing a more comprehensive understanding of radiation’s biological effects. For example, coupling the nanopore measurement device with a mass spectrometer could allow for identification and quantification of radiation-induced chemical modifications to biopolymers, while integration with a fluorescence microscope could enable visualization of radiation-induced changes inbiopolymer localization or conformation within cells. This integration broadens the range of applications and provides more detailed information for assessing radiation effects.

[0091] In an embodiment, a process for measuring radiation-induced damage to a biopolymer comprises providing a known biopolymer of known length or composition, dissolving the biopolymer in a medium to create a sample solution, irradiating the sample solution with ionizing radiation, preparing the biopolymer for analysis, assembling a measurement chamber comprising two electrolyte chambers separated by a dielectric membrane containing a nanopore, infusing the irradiated biopolymer into the measurement chamber, applying a voltage across the membrane to cause the biopolymer to translocate through the nanopore, measuring the ionic current through the nanopore, and analyzing the measured ionic current to determine the extent of radiation-induced damage to the biopolymer. In an embodiment, the biopolymer is a nucleic acid. In an embodiment, the nucleic acid is DNA. In an embodiment, the DNA has a length of 2,500 base pairs. In an embodiment, the biopolymer is a protein. In an embodiment, the protein is a peptide having a length of 10 amino acids. In an embodiment, the biopolymer is a polysaccharide. In an embodiment, the process further comprises adding a size selective tag to the irradiated biopolymer. In an embodiment, the ionizing radiation comprises photons. In an embodiment, the photons are generated by an x-ray source. In an embodiment, the analyzing comprises detecting and counting the number of translocations through the nanopore. In an embodiment, the analyzing comprises collecting salient features of the current signature as the molecule passes through the pore, the salient features comprising the magnitude of the current step, duration of the passage, number of discrete current steps within the resistive pulse, and the integrated area between the current level with no molecule present and the current level while the molecule is driven through the pore. In an embodiment, the analyzing comprises producing a histogram of one or more salient features for quantitative analysis. In an embodiment, the salient feature is equivalent charge deficit (ECD). In an embodiment, the salient feature is number of current levels observed. In an embodiment, the determining comprises identifying scission reactions. In an embodiment, the determining comprises identifying internal bond-breaking reactions. In an embodiment, the process further comprises employing wavelet or other joint time-frequency analysis to identify spectral signatures of damage. In an embodiment, the process further comprises building a dose-response curve by plotting integrated peaks against radiation dose. In an embodiment, the process further comprises quantifying RBE ratios by generating a collection of dose-response curves with varying conditions.

[0092] The process for measuring radiation-induced damage to a biopolymer begins by providing a known biopolymer (108) of known length or composition. Providing a known biopolymer with known characteristics establishes a controlled starting point for the RBE measurement. This knowledge of the biopolymer's initial state enables precise measurement of any radiation-induced changes. Known biopolymers of various lengths and compositions can be obtained from commercial sources or prepared using standard biochemical techniques. For instance, DNA fragments of defined length can be generated by PCR amplification or enzymatic digestion, while proteins can be purified using chromatography or electrophoresis. Knowing the biopolymer’s length allows for accurate calculation of radiation- induced damage per unit length, enabling precise RBE quantification.

[0093] The process includes dissolving the biopolymer in a medium (210) to create a sample solution. Dissolving the biopolymer creates a homogenous sample solution suitable for irradiation. This ensures uniform exposure of the biopolymer to the radiation field, enhancing measurement accuracy. The medium may be water, a buffer solution, or any other solvent compatible with the biopolymer and the subsequent analysis steps. The choice of medium can be tailored to mimic specific biological or environmental conditions relevant to the research question. For instance, a buffered solution may be used to maintain a stable pH during irradiation, while a cell culture medium may be employed to investigate radiation effects in a more biologically relevant context.

[0094] The process comprises irradiating the sample solution with ionizing radiation. Irradiating the sample solution introduces controlled radiation damage to the biopolymer. The type and dose of ionizing radiation can be precisely selected based on the research question or application. Various sources of ionizing radiation, such as gamma rays, X-rays, or particle beams, can be used. The radiation dose can be calibrated using dosimetry standards to ensure accurate RBE measurements. For example, a cobalt-60 source may be used to deliver a specific gamma radiation dose, while a linear accelerator may be employed to generate a defined X-ray dose. Precise control of radiation dose is critical for building accurate dose-response curves and determining RBE.

[0095] The process further includes preparing the biopolymer for analysis. Preparing the biopolymer ensures compatibility with the nanopore measurement platform. This step may involve dilution, buffer exchange, or addition of reagents to optimize biopolymer translocation through the nanopore. For instance, the irradiated biopolymer may be diluted in a high-salt buffer to facilitate its capture and translocation, or an enzyme may be added to cleave the biopolymer into smaller fragments suitable for nanopore analysis. Optimizingsample preparation maximizes the number of detectable translocation events, enhancing the accuracy and statistical significance of the RBE measurement.

[0096] The process includes assembling a measurement chamber. The measurement chamber comprises two electrolyte chambers (102, 101) separated by a dielectric membrane (100) containing a nanopore (107). Assembling the measurement chamber creates the platform for single-molecule analysis of the irradiated biopolymer. The two electrolyte chambers, separated by a membrane with a nanopore, establish the electrical circuit for measuring ionic current changes during biopolymer translocation. The membrane typically comprises an insulating material, such as silicon nitride or glass, and the nanopore may be fabricated using various techniques, including focused ion beam milling or electron beam lithography. This arrangement electrically isolates the chambers, enabling sensitive detection of ionic current blockades.

[0097] The process includes infusing the irradiated biopolymer into the measurement chamber. Infusing the sample places the irradiated biopolymer in proximity to the nanopore, enabling capture and translocation. The infusion may be achieved by directly injecting the sample solution into one of the electrolyte chambers, or by replacing the electrolyte solution in one chamber with the sample.

[0098] The process comprises applying a voltage (216) across the membrane. Applying a voltage across the membrane generates an electric field, driving the translocation of the charged biopolymer through the nanopore. The magnitude and polarity of the applied voltage are optimized to control the biopolymer's translocation speed and capture rate, ensuring efficient and accurate measurement of radiation-induced damage. The voltage may be applied using a voltage source connected to electrodes in each chamber. The magnitude of the applied voltage is typically in the range of millivolts to hundreds of millivolts.

[0099] The process further includes measuring the ionic current (217) through the nanopore. Measuring the ionic current provides a real-time signal of biopolymer translocation events. Transient changes in ionic current, or blockades, reflect the physical properties of the translocating biopolymer, including size, shape, and charge. The ionic current may be measured using a transimpedance amplifier connected to electrodes in each chamber. This amplifier converts the small ionic current signals into measurable voltage outputs, which can be digitized and recorded for analysis.

[0100] The process comprises analyzing the measured ionic current to determine the extent of radiation-induced damage to the biopolymer. Analyzing the measured ionic current converts the raw electrical signals into meaningful information about radiation damage.This analysis may involve algorithms that detect and count translocation events, measure the magnitude and duration of current blockades, and quantify changes in the biopolymer’s properties caused by radiation exposure. This information provides a basis for quantifying the relative biological effectiveness of the radiation.

[0101] This implementation provides a significant improvement in the art. Providing a known biopolymer and controlling irradiation dose enhances the accuracy and reproducibility of the RBE measurement. Preparing the biopolymer for analysis ensures compatibility with the nanopore platform. Assembling the measurement chamber with a nanopore creates the single-molecule analysis platform. Applying a voltage across the membrane drives biopolymer translocation through the nanopore, enabling measurement of its properties. Measuring the ionic current translates the biopolymer’s properties into an electrical signal, providing the raw data for RBE determination. Analyzing the measured current transforms the raw data into quantitative information on radiation-induced damage, enabling precise RBE calculations. The process is rapid, low material intensive and can be employed for clinical or emergency applications, where other technologies would be inadequate, particularly for low dose exposures where standard methods cannot directly measure RBE. The process also is easily standardized, because data on the biopolymer can be directly obtained by use of a computer algorithm operating on stored data acquired by electronic measurements.

[0102] The biopolymer may be a nucleic acid. Nucleic acids, encompassing both DNA and RNA, are critical biomolecules that carry genetic information. Measuring radiation- induced damage to nucleic acids is essential for understanding the effects of radiation on heredity and disease. This approach allows for the analysis of radiation-induced mutations, DNA repair processes, and other molecular events relevant to radiation biology and therapy. Different types of nucleic acids, including genomic DNA, mitochondrial DNA, and RNA, can be analyzed to investigate the effects of radiation on diverse cellular processes.

[0103] The nucleic acid may be DNA. DNA, the primary genetic material in most organisms, serves as a crucial target for radiation damage. Measuring radiation-induced damage to DNA is vital for understanding the mechanisms of radiation-induced mutagenesis, carcinogenesis, and cell death. Analyzing DNA damage, including double-strand breaks, single-strand breaks, and base modifications, is essential for developing effective strategies for radiation therapy, radiation protection, and environmental remediation. DNA may be isolated from a variety of sources, such as cells, tissues, or biological fluids.

[0104] The DNA may have an arbitrary length, known or unknown, e.g., a length of 2,500 base pairs. A DNA molecule of 2,500 base pairs serves as a model system for studyingradiation-induced damage. This defined length facilitates precise quantification of DNA damage, such as strand breaks per unit length. Different DNA lengths can be analyzed, chosen based on the specific research question or the limitations of the nanopore measurement platform. Shorter DNA fragments may be more suitable for analyzing specific types of damage, such as base modifications, while longer DNA molecules are necessary for studying chromosome-level effects.

[0105] The biopolymer may be a protein. Proteins are essential biomolecules that perform diverse functions in living organisms, acting as enzymes, structural components, and signaling molecules. Measuring radiation-induced damage to proteins provides insights into radiation's impact on cellular function, metabolism, and signaling pathways. Analyzing protein damage, including protein oxidation, crosslinking, and fragmentation, is critical for understanding the molecular mechanisms of radiation toxicity and developing strategies for radiation protection. Proteins may be isolated from diverse sources, such as cells, tissues, or purified protein preparations.

[0106] The protein may be a peptide of a length of 10 amino acids. A peptide with a defined length provides a standardized model system for investigating radiation-induced damage to proteins. This length is typical of many bioactive peptides. Shorter or longer peptides can also be analyzed, with peptide length chosen based on the specific application. Analyzing shorter peptides allows for precise detection of amino acid modifications, while longer peptides or full-length proteins may be employed for assessing radiation-induced changes in protein folding and function.

[0107] The biopolymer may be a polysaccharide. Polysaccharides are complex carbohydrates that play diverse structural and functional roles in living organisms, contributing to energy storage, cellular recognition, and immune responses. Measuring radiation-induced damage to polysaccharides provides valuable insights into radiation's effects on plant cell walls, bacterial capsules, and other biological systems. Analyzing polysaccharide damage, such as chain scission and crosslinking, is essential for understanding radiation-induced changes in the properties and functions of these important biomolecules. Various polysaccharides, including cellulose, starch, and chitin, may be analyzed, depending on the research focus.

[0108] The process may comprise adding a size selective tag to the irradiated biopolymer. Adding a size selective tag to the irradiated biopolymer enhances the detection and characterization of radiation-induced damage. The tag may be a fluorescent molecule, a radioactive isotope, or any other marker that allows for specific detection of the damagedbiopolymer. This tagging approach improves sensitivity and specificity, enabling precise measurement of even low levels of radiation-induced damage. Different tagging strategies may be employed, chosen based on the type of biopolymer, the nature of the radiation damage, and the available detection methods. For example, DNA damage can be tagged using fluorescently labeled nucleotides, while protein damage may be tagged using antibodies specific to modified amino acid residues.

[0109] The ionizing radiation may comprise photons. Photons, such as gamma rays and X-rays, represent a common type of ionizing radiation encountered in medical, industrial, and environmental settings. Analyzing the effects of photon radiation is essential for understanding the risks associated with radiation exposure and developing strategies for radiation protection and therapy. Photons may be generated by various sources, including radioactive isotopes, X-ray tubes, and synchrotrons. The energy and dose of the photon radiation can be precisely controlled, enabling investigation of RBE across a range of relevant exposure conditions.

[0110] The photons may be generated by an x-ray source. X-rays are a form of ionizing radiation widely employed in medical imaging and radiation therapy. Analyzing the biological effects of X-rays is crucial for optimizing X-ray-based diagnostic and therapeutic procedures and minimizing potential risks to patients and medical personnel. Various X-ray sources with different energy spectra and dose rates may be employed. For instance, low- energy X-rays may be used for diagnostic imaging, while high-energy X-rays are typically employed in radiation therapy. Precise control of the X-ray dose and energy spectrum is essential for accurate RBE determination.

[0111] Analyzing the measured ionic current may comprise detecting and counting the number of translocations through the nanopore. This direct measurement of translocation events provides a quantitative measure of the number of intact biopolymers in the irradiated sample. The number of translocations observed is inversely proportional to the extent of radiation-induced damage, enabling direct assessment of RBE. One advantage of this analysis method is that it is objective and automated, minimizing potential biases associated with subjective interpretation of results. Various algorithms may be employed for detecting and counting translocations, based on features of the ionic current signal, such as the magnitude and duration of current blockades.

[0112] Analyzing the measured ionic current may comprise collecting salient features of the current signature as the molecule passes through the pore. These features reflect the physical properties of the translocating molecule. The magnitude of the current step isproportional to the volume of the biopolymer passing through the nanopore, providing information about the biopolymer’s size. The duration of the passage is inversely proportional to the biopolymer’s speed of translocation, reflecting its charge and conformation. The number of discrete current steps within the resistive pulse may indicate the presence of radiation- induced modifications, such as strand breaks or chemical adducts, that alter the biopolymer's effective length or charge. The integrated area between the current levels provides a measure of the total charge carried by the biopolymer, reflecting its length and charge density. These salient features can be extracted from the ionic current data using various signal processing and data analysis techniques. This detailed analysis enables precise identification and quantification of radiation-induced damage to the biopolymer, providing a foundation for accurate RBE determination.

[0113] The specific implementations of these process steps provide significant technical advantages. Using nucleic acids, especially DNA, as the biopolymer allows for precise measurement of radiation-induced damage to this critical genetic material. Analyzing a 2,500 base-pair DNA molecule provides a standardized model system for RBE studies. Analyzing a peptide with a specific length and sequence provides a model system for investigating radiation effects on proteins. Analyzing damage to polysaccharides extends the applicability of the method to other important biomolecules. Adding a size-selective tag improves measurement sensitivity and specificity. Using photons, generated by an X-ray source, provides a controlled radiation exposure relevant to medical and industrial applications. Counting the number of translocations provides a direct measure of intact biopolymers, while collecting salient features of the current signature allows for detailed analysis of radiation- induced damage. These implementations significantly enhance the accuracy, sensitivity, and versatility of the process.

[0114] Before irradiating the sample, the sample may be conditioned to mimic an in vivo environment. This conditioning may involve dissolving the biopolymer in a medium, such as water or a buffer solution, and adding electrolytes, free radical scavengers, or free radical enhancers. Electrolytes, such as phosphate buffered saline, provide a physiologically relevant ionic environment. Free radical scavengers, like tris(hydroxymethyl)aminomethane, protect the biopolymer from radiation-induced damage caused by reactive oxygen species. Free radical enhancers, such as N2O(g), increase the amount of radiation-induced damage to mimic in vivo conditions more accurately. This process of conditioning optimizes the experimental conditions to mimic the cellular environment, providing useful information on the efficacy of the method for clinical or diagnostic application. The sample solution may be distributed intoa matrix, such as an agarose gel, prior to irradiation. This spatial segregation of the sample can be useful for analyzing radiation effects in a heterogeneous environment, mimicking the distribution of biomolecules within cells or tissues. The use of a gel matrix allows for controlled retrieval of the sample after irradiation, enhancing measurement accuracy and minimizing potential artifacts arising from sample handling. Retrieving the sample from the matrix may involve physical or chemical methods, such as cutting out sections of the gel containing the irradiated sample or dissolving the gel matrix to release the biopolymer. The retrieved sample is then prepared for analysis using standard techniques, such as dilution or buffer exchange.

[0115] Size-selective tagging of the irradiated molecule can be accomplished using a variety of techniques, such as reacting the biopolymer with a base-specific enzyme, like DNA glycosylase, to create reactive sites for attaching a tag. The tag may be a DNA molecule of known size, a polymer, or any other molecule that modifies the biopolymer's physical properties in a size-dependent manner. The tag may be attached directly to the damaged site or may be linked to a molecule that recognizes and binds to the damaged site. The choice of tagging method may depend upon factors such as the type of biopolymer, the nature of the radiation damage, and the desired resolution of the analysis.

[0116] Adding internal standards improves sizing accuracy and quantitation of bond-breaking processes. A known biopolymer may be added as an internal standard to allow for direct quantitative analysis of the irradiated sample. Two or more discretely sized internal standards can enable both size-selective and quantitative analysis, providing detailed information about the distribution of radiation-induced damage to the biopolymer. For example, in DNA analysis, two internal standards of distinct sizes, such as a 5,000 base-pair DNA and a 10,000 base-pair DNA, can be used to calibrate the nanopore measurement and determine the size distribution of irradiated DNA fragments.

[0117] The measurement chamber may comprise two electrolyte chambers separated by a dielectric membrane, such as silicon nitride, containing a single nanopore. Silicon nitride is a durable and chemically inert material suitable for fabricating nanopore membranes. Other dielectric materials, such as silicon dioxide, aluminum oxide, or polymers, can also be used. The nanopore's diameter and length are carefully chosen to optimize detection of the specific biopolymer being analyzed. For DNA analysis, a nanopore with a diameter, e.g., of 5 nm and a length of 12 nm may be employed.

[0118] Infusing the irradiated biopolymer may involve replacing the electrolyte solution in one of the chambers with the prepared biopolymer solution. Alternatively, thebiopolymer may be directly injected into the chamber. The polarity of the applied voltage is chosen such that the biopolymer is driven towards and through the nanopore. For DNA, the molecule typically migrates from the negative electrode to the positive electrode.

[0119] Applying a voltage across the membrane generates an electric field, which drives the translocation of charged biopolymers through the nanopore. The magnitude of the applied voltage is chosen to optimize the capture rate and translocation speed of the biopolymer. For DNA analysis, a voltage of 300 mV may be appropriate.

[0120] Measuring the ionic current through the nanopore may be accomplished using a transimpedance amplifier electrically connected to the electrodes in each chamber. The amplifier converts the small ionic current signals into measurable voltage signals, improving sensitivity. The measured ionic current data is digitized using an analog-to-digital converter, and the data is stored for subsequent analysis. This digital data can be analyzed using algorithms that detect and count the number of biopolymer translocations, collect salient features of the current signature, and quantify changes in the biopolymer's properties caused by radiation exposure.

[0121] Analysis of the digitized ionic current may be carried out using a threshold analysis algorithm to identify and count translocation events. This algorithm identifies transient changes in the ionic current that exceed a predefined threshold, indicating the passage of a biopolymer through the nanopore.

[0122] Salient features of the current signature may include the magnitude of the current step, the duration of the biopolymer's passage through the nanopore, the number of discrete current steps within the resistive pulse, and the integrated area between the current level with no molecule present and the current level while the molecule is passing through the nanopore. These features provide detailed information about the biopolymer’s size, shape, charge, and conformation. This information can be used to quantify RBE ratios for different types of radiation. It can be appreciated that "salient features” refer to those characteristics of the current signature that are most significant for distinguishing between different biopolymers or for determining specific properties of the biopolymer, such as its size, shape, or charge.

[0123] The data may be further analyzed by constructing histograms of one or more salient features, such as the equivalent charge deficit (ECD) or the number of current levels observed. A histogram of ECD values can reveal the size distribution of the irradiated biopolymers, providing a measure of radiation-induced fragmentation. A histogram of the number of observed current levels can indicate the presence of internal bond-breaking reactions without necessarily altering the biopolymer's overall size. These histograms can bequantitatively analyzed to determine the extent of radiation-induced damage. Further analysis may involve signal processing techniques, such as wavelet or other joint time-frequency analyses, to identify spectral signatures of damage, providing insights into changes in the biopolymer's physical properties caused by radiation.

[0124] Dose-response curves may be generated by plotting integrated peak areas against the corresponding radiation doses. Integrated peak areas provide a measure of the total amount of each biopolymer species present in the irradiated sample. Plotting the change in integrated peaks resulting from irradiation against the dose would result in dose-response curves. These curves reveal the relationship between radiation dose and the extent of damage to the biopolymer.

[0125] A collection of dose-response curves may be generated with varying conditions, such as different biopolymers, different radiation types, or different electrolyte conditions. This comprehensive analysis allows for quantification of RBE ratios, which compare the biological effectiveness of different types of radiation. RBE ratios are essential for optimizing radiation therapies, assessing radiation risks, and developing radiation protection strategies. Tissue sensitivities may be quantified by analyzing the dose-response curves for different cell types or tissues. This information may provide insights into the relative susceptibility of different tissues to radiation damage, informing radiation treatment planning and minimizing harmful side effects. The dose-response curves can be further used to inform kinetic modeling of radiation-induced damage processes. This modeling can provide valuable insights into the molecular mechanisms of radiation action and enable prediction of radiation effects under different exposure scenarios.

[0126] The process begins by providing a known biopolymer (108) of known length or composition. This step establishes a well-defined starting material for the RBE measurement. The biopolymer may be a nucleic acid, such as DNA or RNA, a protein, or a polysaccharide. Nucleic acids are essential for storing and transmitting genetic information. Proteins perform diverse structural and functional roles in cells. Polysaccharides contribute to energy storage and cellular recognition. The biopolymer is purified using techniques such as chromatography, electrophoresis, or filtration to remove contaminants that could interfere with the nanopore measurement. The biopolymer's length or composition are determined using methods like gel electrophoresis, mass spectrometry, or sequencing. Knowing these characteristics allows for precise quantification of radiation-induced changes. The length of the biopolymer may range from a few hundred base pairs or amino acids to tens of thousands, specifically from 1,000 to 5,000 base pairs or amino acids, and more specifically from 2,000to 3,000 base pairs or amino acids. The composition of the biopolymer may be a specific sequence of nucleotides or amino acids, or a defined mixture of monomers in the case of polysaccharides. For instance, a 2,500 base pair double-stranded DNA molecule, a peptide with a specific amino acid sequence, or a purified cellulose sample could be provided. The quantity of biopolymer provided depends on the sensitivity of the nanopore measurement and the desired throughput of the experiment.

[0127] The next step involves dissolving the biopolymer (108) in a medium (210) to create a sample solution. This step is essential for ensuring uniform exposure of the biopolymer to ionizing radiation and for preparing the sample for nanopore analysis. The medium is chosen based on its compatibility with the biopolymer and the subsequent analysis steps. It may be water, a buffer solution, or any other solvent that maintains the biopolymer’s stability and does not interfere with the nanopore measurement. Water is a simple and cost- effective medium suitable for many biopolymers. Buffer solutions, such as phosphate-buffered saline or Tris buffer, are employed to maintain a specific pH or ionic strength, protecting the biopolymer from degradation or aggregation. The concentration of the biopolymer in the medium is carefully controlled to optimize nanopore measurement performance. A concentration too low may result in insufficient translocation events for accurate analysis, while a concentration too high may lead to nanopore clogging or aggregation of the biopolymer. The sample solution may be prepared in a variety of containers, such as Eppendorf tubes, microcentrifuge tubes, or glass vials, chosen based on the sample volume and the compatibility of the container material with the medium and biopolymer. The volume of the sample solution can range from microliters to milliliters, specifically from 10 µL to 100 µL, and more specifically from 25 µL to 75 µL, depending on the experimental setup and the amount of biopolymer needed for analysis. The dissolution process may involve gentle mixing, vortexing, or sonication to ensure complete dissolution of the biopolymer and a homogenous sample solution. This controlled dissolution process yields several advantages.

[0128] The next step, irradiating the sample solution with ionizing radiation, introduces controlled damage to the biopolymer (108). The type and dose of radiation are chosen based on the research question and the biopolymer's radiation sensitivity. Ionizing radiation may include electromagnetic radiation, such as gamma rays or X-rays, or particle radiation, such as alpha particles, beta particles, or neutrons. Gamma rays, emitted by radioactive sources like cobalt-60, are commonly used for their high penetration depth and uniform dose distribution. X-rays, generated by X-ray tubes or linear accelerators, offer greater control over energy and dose rate. Particle radiation, generated by specialized sources oraccelerators, provides high-LET radiation useful for studying specific damage mechanisms. The radiation dose, the amount of energy absorbed by the sample per unit mass, can range from a few grays to thousands of grays, specifically from 0.1 Gy to 100 Gy, and more specifically, from 1 Gy to 50 Gy. Dosimetry standards, such as thermoluminescent dosimeters or ionization chambers, are used to calibrate the radiation source and ensure accurate dose delivery. The irradiation time, which can range from seconds to hours, is chosen based on the desired dose and the dose rate of the radiation source. The sample solution may be irradiated in various containers, including Eppendorf tubes, glass vials, or specialized irradiation chambers, which container is chosen for its compatibility with the radiation source and the sample medium. During irradiation, the sample may be maintained at a specific temperature, such as room temperature or a controlled temperature within an incubator or water bath, to minimize variations in radiation sensitivity. This controlled irradiation process is essential for accurate and reproducible RBE measurements.

[0129] Precise control over radiation type and dose allows for investigation of RBE across a wide range of conditions. The use of calibrated radiation sources and dosimetry standards ensures accurate dose delivery. Selecting appropriate irradiation containers and controlling temperature minimize variations in radiation sensitivity. This controlled approach contrasts with traditional methods, where variations in radiation exposure can introduce significant uncertainties in RBE measurements. The ability to precisely control radiation dose and other experimental conditions is particularly crucial for studying low-dose effects, where subtle changes in biopolymer properties may be masked by experimental variability. The flexibility in radiation type expands applicability, enabling studies of different radiation damage mechanisms relevant to diverse research questions. This approach offers advantages in terms of accuracy, reproducibility, and versatility.

[0130] Following irradiation, the biopolymer (108) is prepared for analysis. This step ensures compatibility with the nanopore measurement platform and optimizes detection of radiation-induced damage. Preparation may involve dilution, buffer exchange, or addition of reagents to facilitate biopolymer translocation through the nanopore (107) and enhance the signal-to-noise ratio of the ionic current measurement. Dilution adjusts the biopolymer concentration to a level suitable for nanopore analysis, typically in the nanomolar to micromolar range, specifically from 1 nM to 100 nM, and more specifically, from 10 nM to 50 nM, minimizing nanopore clogging or biopolymer aggregation while ensuring a sufficient number of translocation events for accurate analysis. Buffer exchange replaces the irradiation medium (210) with an electrolyte solution compatible with the nanopore measurement.Commonly used electrolytes include lithium chloride, potassium chloride, or sodium chloride, at concentrations ranging from 0.1 M to 4 M, specifically from 0.5 M to 2 M, and more specifically, from 0.75 to 1.25 M. The electrolyte solution's ionic strength and pH are chosen based on the biopolymer being analyzed and the characteristics of the nanopore. Reagents, such as surfactants or enzymes, may be added to improve biopolymer translocation through the nanopore or to enhance detection of specific radiation-induced modifications. Surfactants can reduce biopolymer aggregation and improve capture rate, while enzymes can cleave the biopolymer at specific sites, facilitating translocation of longer biopolymers through the nanopore. Preparation steps are carried out using standard laboratory techniques, such as pipetting, centrifugation, and filtration, ensuring minimal sample loss and maintaining sample integrity. Careful sample preparation maximizes the number of detectable translocation events, improving measurement sensitivity. Buffer exchange optimizes biopolymer stability and translocation dynamics. Addition of reagents enhances detection of specific modifications, providing more comprehensive information about the effects of radiation exposure, critical for accurate RBE determination. This controlled preparation improves reproducibility, minimizes variability, and reduces background noise. This approach offers advantages over conventional methods, where variations in sample preparation can introduce inconsistencies in results. The flexibility to tailor preparation steps to specific biopolymers and nanopore platforms makes this method adaptable to a broad range of applications and experimental conditions. The inclusion of optional reagents expands the range of modifications detectable. These carefully optimized preparation steps ensure high-quality data for RBE determination.

[0131] The process includes assembling a measurement chamber. The measurement chamber forms the core of the nanopore analysis platform, providing a controlled environment for measuring ionic current changes as biopolymers translocate through a nanopore. The chamber comprises two electrolyte chambers (102, 101) separated by a dielectric membrane (100) containing a nanopore (107). The electrolyte chambers are typically made of a non-conductive material, such as polycarbonate or glass, to minimize electrical interference. Their volumes range from 1 µL to 1 mL, specifically from 10 µL to 100 µL, and more specifically, from 25 µL to 75 µL, chosen to minimize sample consumption while ensuring sufficient volume for stable measurements. The dielectric membrane, made of a material like silicon nitride, separates the two chambers, preventing bulk flow of electrolyte while allowing ionic current to pass through the nanopore. The membrane’s thickness ranges from a few nanometers to several micrometers, specifically from 10 nm to 100 nm, and more specifically, from 20 nm to 50 nm. The nanopore, a nanoscale channel in the membrane, has adiameter from 2 nm to 20 nm, specifically from 3 nm to 10 nm, and more specifically, from 4 nm to 6 nm, and a length from 5 nm to 500 nm, specifically from 10 nm to 100 nm, and more specifically, from 20 nm to 50 nm. These dimensions are tailored to the biopolymer being analyzed. The chamber includes electrodes (104, 103) positioned in each electrolyte chamber, connected to a voltage source (106) to establish the electric field that drives biopolymer translocation. The electrodes are typically made of conductive materials, such as platinum or gold, and a transimpedance amplifier (105) is connected to the electrodes to measure the ionic current.

[0132] The process continues with infusing the irradiated biopolymer (108) into the measurement chamber. This step introduces the prepared sample into the nanopore analysis platform, enabling detection of radiation-induced damage at the single-molecule level. The prepared biopolymer solution, containing the irradiated biopolymer in a suitable electrolyte, is introduced into one of the electrolyte chambers (102 or 101), typically the cis chamber (102). This infusion may be achieved by directly injecting the sample into the chamber using a micropipette or by replacing the electrolyte solution in the chamber with the prepared biopolymer solution. The volume of sample infused ranges from 1 µL to 1 mL, specifically from 10 µL to 100 µL, and more specifically, from 25 to 75 µL, depending upon the chamber’s volume and the desired biopolymer concentration. The concentration of the biopolymer in the chamber is carefully controlled to optimize nanopore measurement. A concentration too low may result in infrequent translocation events, reducing measurement sensitivity, while a concentration too high can lead to nanopore clogging or biopolymer aggregation. The electrolyte conditions in both the cis and trans chambers (101) are carefully chosen to facilitate biopolymer capture and translocation through the nanopore (107).

[0133] This controlled infusion process ensures that the irradiated biopolymer reaches the nanopore, maximizing the probability of translocation events. Optimizing the biopolymer concentration improves measurement sensitivity while preventing artifacts due to clogging or aggregation. Tailoring the electrolyte conditions in the two chambers enables controlled biopolymer translocation and maximizes the signal-to-noise ratio. This precise control is critical for accurate and reproducible measurement of radiation-induced damage, ensuring that the observed signal changes are due to the effects of radiation and not variations in sample handling or experimental conditions. This approach provides an advantage over conventional methods that rely on bulk measurements, where individual biomolecules may not be uniformly exposed to the analytical technique, potentially masking subtle changes caused by radiation damage. The small sample volumes required for nanopore analysis minimizematerial consumption, and the controlled infusion process further enhances efficiency by maximizing the number of translocation events per unit volume of sample. This enhanced efficiency is essential for high-throughput analysis of radiation-induced damage.

[0134] The next step, applying a voltage (216) across the membrane (100), establishes the electric field that drives biopolymer (108) translocation through the nanopore (107). The voltage is generated by the voltage source (106) and applied to the electrodes (104, 103) positioned in the two electrolyte chambers (102, 101). The voltage’s magnitude may range from millivolts to hundreds of volts, specifically from 10 mV to 500 mV, and more specifically, from 50 mV to 200 mV. The polarity is chosen such that the biopolymer is driven from the cis chamber (102) towards and through the nanopore into the trans chamber (101). For negatively charged biopolymers, like DNA, a positive voltage is applied to the trans chamber and a negative voltage to the cis chamber. The magnitude of the voltage is optimized based on the nanopore's dimensions, the biopolymer's charge and size, and the desired translocation speed. Higher voltages generally increase translocation speed but may also increase noise and reduce measurement resolution. The voltage is applied and maintained throughout the duration of the nanopore measurement, ensuring a consistent electric field and controlled biopolymer translocation. Precise control of the applied voltage is achieved using a voltage source with adjustable output and monitoring capabilities. This control ensures that each biopolymer molecule experiences a similar electric field during translocation, minimizing variability in the measurements. The magnitude and polarity of the applied voltage are optimized to balance translocation speed, capture rate, and measurement resolution. This optimization is critical for accurately detecting and quantifying radiation-induced changes in biopolymer properties.

[0135] Applying a voltage across the membrane provides several advantages. It initiates and controls biopolymer translocation through the nanopore. The optimized voltage maximizes capture rate and measurement resolution. Precise control of the electric field using a stable voltage source improves the reproducibility of the measurements, ensuring that the observed changes in ionic current are due to the biopolymer's properties and not to variations in the applied voltage. The method’s adaptability to different nanopore dimensions, biopolymer types, and desired translocation speeds enhances its versatility for various applications. This controlled application of voltage offers significant improvements over traditional RBE assays that do not provide direct control over molecular translocation. The ability to fine-tune the voltage based on the specific biopolymer and nanopore characteristicsis particularly valuable for analyzing complex biopolymer mixtures or for studying subtle changes in biopolymer properties induced by low-dose radiation exposure.

[0136] The process involves measuring the ionic current (217) through the nanopore (107). This step captures the signal generated by biopolymer (108) translocation, providing the raw data for analyzing radiation-induced damage and determining RBE. As the biopolymer translocates through the nanopore, it partially blocks the flow of ions, causing a transient change in the ionic current. The magnitude and duration of this change, known as a resistive pulse, reflect the biopolymer's physical properties, including its length, volume, charge, and conformation. Changes in these properties due to radiation exposure alter the characteristics of the resistive pulse, enabling detection and quantification of radiation-induced damage. The ionic current is measured using a transimpedance amplifier (105) connected to the electrodes (104, 103) positioned in the electrolyte chambers (102, 101). The amplifier converts the small ionic current signal, typically in the picoampere to nanoampere range, into a measurable voltage signal. The amplifier’s gain and bandwidth are chosen to optimize the signal-to-noise ratio for the specific biopolymer and nanopore being used. The amplified voltage signal is then digitized using an analog-to-digital converter (208) for subsequent computer processing. The sampling rate and resolution of the analog-to-digital converter are selected to accurately capture the transient changes in ionic current associated with biopolymer translocation. The digitized current data is recorded as a function of time, generating a time series that reflects the sequence of translocation events. Measuring the ionic current through the nanopore provides several key advantages. It translates the biopolymer’s physical properties into a measurable electrical signal. This signal can be amplified and digitized for quantitative analysis. Continuous monitoring of the ionic current enables real-time detection of translocation events. The method's high sensitivity allows detection of even subtle changes in ionic current, reflecting small changes in biopolymer properties caused by radiation. The quantitative nature of the measurement facilitates precise characterization of radiation-induced damage and accurate RBE determination. This approach offers significant improvements over traditional RBE assays, which often rely on indirect measurements of radiation effects. The ability to measure ionic current in real time enables detailed analysis of biopolymer translocation dynamics, including the detection of transient intermediates or conformational changes that may occur during translocation. The high sensitivity of the nanopore measurement allows for detection of even low levels of radiation-induced damage, providing valuable information for assessing radiation risks and developing protective strategies.

[0137] The final step in the process is analyzing the measured ionic current (217) to determine the extent of radiation-induced damage to the biopolymer (108). This step involves processing and interpreting the digitized ionic current data to quantify changes in the biopolymer’s properties caused by radiation exposure. The analysis may include several steps, such as detecting and counting individual translocation events, measuring the magnitude and duration of resistive pulses, and quantifying changes in the biopolymer's length, volume, charge, or conformation. Algorithms are employed to analyze the digitized ionic current data. These algorithms identify and characterize individual translocation events based on the transient changes in ionic current. One common approach is threshold analysis, where a translocation event is detected when the ionic current drops below a predefined threshold value for a specified duration. More sophisticated algorithms may incorporate additional features of the resistive pulse, such as its shape or area, to improve detection accuracy and reduce noise. Once translocation events are identified, their characteristics, including the magnitude and duration of the resistive pulse, are extracted. These data provide quantitative information about the biopolymer’s properties, enabling precise detection of radiation-induced changes. The analysis may also include statistical methods for comparing the properties of irradiated and unirradiated biopolymers, quantifying the extent of damage, and determining RBE values. The results of the analysis may be presented in various forms, such as histograms, scatter plots, or dose-response curves. These visualizations facilitate data interpretation and provide insights into the relationship between radiation dose and biopolymer damage. This analysis step converts the raw ionic current data into meaningful information about radiation-induced damage. Using algorithms for event detection and feature extraction ensures objectivity and reproducibility, minimizing human error and bias. The quantitative nature of the analysis enables precise determination of RBE values. The flexibility of this approach allows the analysis to be tailored to the specific biopolymer and type of radiation damage under investigation, enhancing its versatility for diverse applications. This detailed, quantitative analysis of radiation-induced damage provides significant advantages over conventional RBE assays, which often rely on subjective interpretation of macroscopic endpoints. The single- molecule resolution of the nanopore measurement, coupled with advanced data analysis techniques, enables detection of subtle changes in biopolymer properties, providing a more complete and nuanced understanding of radiation’s biological effects. The high-throughput capability of nanopore technology, combined with automated data analysis, facilitates rapid and efficient RBE determination.

[0138] In an embodiment, the analyzing comprises detecting and counting the number of translocations through the nanopore. In an embodiment, the analyzing comprises collecting salient features of the current signature as the molecule passes through the pore, the salient features comprising the magnitude of the current step, duration of the passage, number of discrete current steps within the resistive pulse, and the integrated area between the current level with no molecule present and the current level while the molecule is driven through the pore. In an embodiment, the analyzing comprises producing a histogram of one or more salient features for quantitative analysis. In an embodiment, the salient feature is equivalent charge deficit (ECD). In an embodiment, the salient feature is the number of current levels observed.

[0139] Analyzing the measured ionic current may comprise detecting and counting the number of translocations through the nanopore. Detecting and counting translocations provides a direct measure of the number of intact biopolymers remaining after irradiation. This quantitative data is used to assess the extent of radiation-induced fragmentation, providing a basis for RBE determination. Various algorithms can be used to detect and count translocations based on characteristic features of the ionic current signal, such as the magnitude and duration of transient current blockades. One such algorithm employs a threshold analysis, in which current blockades exceeding a predefined amplitude and duration are counted as translocation events. The threshold values are chosen to minimize false positives arising from noise or other artifacts while ensuring accurate detection of true translocation events. This automated analysis reduces potential biases associated with manual data interpretation and facilitates high- throughput analysis of large datasets.

[0140] Analyzing the measured ionic current may also involve collecting a set of salient features of the current signature. These features reflect the physical properties of the translocating biopolymer, enabling detailed analysis of radiation-induced changes. The magnitude of each current step, for example, is proportional to the volume of the biopolymer excluded from the nanopore, providing information about the biopolymer’s size. The duration of each translocation event is inversely proportional to the biopolymer's translocation speed, reflecting its length, charge, and conformation. The number of discrete current steps observed within a single resistive pulse may indicate internal bond-breaking reactions or the presence of radiation-induced modifications that alter the biopolymer’s effective length or charge. The integrated area of each resistive pulse, also known as the equivalent charge deficit (ECD), provides a precise measure of the biopolymer’s length, reflecting radiation-induced fragmentation. These salient features may be extracted from the digitized ionic current data using a combination of digital filtering, thresholding, and peak detection algorithms.

[0141] Analyzing the measured ionic current may involve generating a histogram of one or more salient features. Generating histograms provides a visual representation of the distribution of the salient features, allowing for quantitative assessment of radiation-induced changes in the biopolymer population. The histograms may be generated using standard statistical software or custom-designed algorithms. One advantage of histogram analysis is that it reveals subtle changes in the biopolymer population that may not be apparent from simple inspection of the raw data. For instance, a shift in the mean value of ECD indicates radiation-induced fragmentation, while an increase in the width of the ECD distribution may reflect increased heterogeneity in the size of the irradiated biopolymers.

[0142] The salient feature may comprise the equivalent charge deficit (ECD). The ECD is the integrated area of each resistive pulse, which is proportional to the biopolymer’s length and charge. Generating a histogram of ECD values provides a measure of radiation- induced changes in the length distribution of the biopolymer population. This analysis is particularly useful for detecting double-strand breaks in DNA, a critical form of radiation damage that can lead to cell death or mutations. The ECD is calculated by integrating the area of each resistive pulse, which represents the transient reduction in ionic current caused by the biopolymer’s passage through the nanopore. This measurement is highly sensitive to changes in the biopolymer’s length, enabling precise quantification of radiation-induced fragmentation.

[0143] Alternatively, the salient feature may comprise the number of current levels observed. The number of current levels observed within a single resistive pulse is a measure of radiation-induced changes in the biopolymer’s effective length or charge. A single current level typically corresponds to an intact biopolymer translocating through the nanopore, while multiple levels indicate the presence of radiation-induced modifications that alter the biopolymer’s physical properties. For instance, a DNA molecule with a single radiation- induced break may translocate through the nanopore in two discrete steps, generating two distinct current levels within the resistive pulse. A histogram of the number of observed current levels is thus useful in characterizing radiation-induced structural changes, such as bond- breaking reactions and formation of chemical adducts. This analysis provides complementary information to ECD measurements, enabling comprehensive assessment of radiation-induced damage.

[0144] The use of these analytical techniques improves the sensitivity and specificity of the RBE measurements. Detecting and counting the number of translocations provides a direct measure of intact biopolymers, while collecting salient features, including ECD and the number of current levels, allows for precise characterization of radiation-induceddamage. Generating histograms and analyzing ECD values provides a visual and quantitative measure of the size distribution of irradiated biopolymers. These methods enable comprehensive characterization of radiation damage at the single-molecule level, improving the accuracy and precision of RBE measurements.

[0145] In an embodiment, the determining comprises identifying scission reactions. In an embodiment, the determining comprises identifying internal bond-breaking reactions. In an embodiment, the process further comprises employing wavelet or other joint time-frequency analysis to identify spectral signatures of damage. In an embodiment, the process further comprises building a dose-response curve by plotting integrated peaks against radiation dose. In an embodiment, the process further comprises quantifying RBE ratios by generating a collection of dose-response curves with varying conditions.

[0146] Determining the extent of radiation-induced damage may comprise identifying scission reactions. Scission reactions involve the breaking of chemical bonds within the biopolymer, leading to fragmentation. Quantifying scission reactions is essential for understanding the mechanisms of radiation-induced damage and for accurately determining RBE. Scission reactions may be identified by analyzing changes in the biopolymer’s length or molecular weight distribution. In nanopore analysis, scission reactions are detected as a decrease in the average equivalent charge deficit (ECD), reflecting a reduction in the biopolymer’s effective length. This information provides a direct measure of the extent of fragmentation caused by radiation.

[0147] Determining the extent of radiation-induced damage may comprise identifying internal bond-breaking reactions. Internal bond-breaking reactions refer to damage that occurs within the biopolymer without causing fragmentation. These reactions may involve the breaking of hydrogen bonds, disulfide bonds, or other non-covalent interactions that maintain the biopolymer’s structure and function. Identifying these internal bond-breaking reactions can reveal subtle changes in biopolymer conformation or stability that may not be detectable by simply measuring fragmentation. Internal bond-breaking may be identified by analyzing changes in the biopolymer's translocation dynamics through the nanopore. Radiation-induced modifications, such as base damage or protein crosslinking, can alter the biopolymer’s effective charge or flexibility, affecting its translocation speed and the number of discrete current steps observed within a resistive pulse. These changes in translocation dynamics can be detected and quantified using signal processing and data analysis techniques, providing insights into the nature and extent of internal bond-breaking.

[0148] The process may further comprise employing wavelet or other joint time- frequency analysis to identify spectral signatures of damage. Wavelet analysis and other joint time-frequency methods provide a powerful tool for analyzing the complex signals generated by biopolymer translocation through a nanopore. These methods decompose the signal into its constituent frequencies, revealing subtle changes in the signal’s spectral characteristics caused by radiation-induced damage. Spectral signatures, such as shifts in peak frequencies or changes in the relative amplitudes of different frequency components, can provide detailed information about changes in the biopolymer's physical properties, such as its flexibility or charge distribution. These changes provide unique insights into the nature and extent of radiation- induced damage at the molecular level, enhancing the accuracy and sensitivity of RBE measurements. One advantage of wavelet analysis is its ability to analyze signals with both time and frequency resolution, enabling the detection of transient changes in the biopolymer’s translocation dynamics. Other joint time-frequency methods, such as short-time Fourier transform and Wigner-Ville distribution, can also be employed.

[0149] The process may further comprise building a dose-response curve by plotting integrated peaks against radiation dose. Building a dose-response curve establishes the quantitative relationship between radiation dose and the extent of damage to the biopolymer. This curve is essential for determining RBE values, which compare the effectiveness of different types of radiation in causing biological damage. The dose-response curve may be generated by plotting the values of one or more salient features extracted from the nanopore data, such as the average ECD or the number of current levels observed, against the corresponding radiation doses. Integrated peaks, obtained from histograms of the salient features, provide a quantitative measure of the number of biopolymers exhibiting a particular type of damage. By plotting integrated peaks against dose, a clear dose-response relationship can be established. This relationship may be linear, sigmoidal, or other mathematical function, depending on the nature of the radiation damage and the properties of the biopolymer. Fitting the dose-response data to an appropriate mathematical model can provide quantitative parameters, such as the slope and intercept of the curve, which may be used to compare the effectiveness of different types of radiation or to predict radiation effects at different doses.

[0150] The process may further comprise quantifying RBE ratios by generating a collection of dose-response curves with varying conditions. Quantifying RBE ratios enables direct comparison of the biological effectiveness of different types of radiation. This is critical for optimizing radiation therapies, designing radiation protection strategies, and assessing the risks associated with radiation exposure in various settings. A collection of dose-responsecurves may be generated by irradiating the same biopolymer with different types of radiation at varying doses and then measuring the resulting damage using the nanopore-based method. RBE ratios are calculated by comparing the doses of different types of radiation required to produce the same level of biological damage. By generating multiple dose-response curves under varying conditions, RBE ratios can be determined for a wide range of radiation types and experimental settings. This detailed information enables comprehensive evaluation and optimization of radiation applications, enhancing their efficacy and safety.

[0151] In an embodiment, a process for measuring radiation-induced damage to a biopolymer includes: providing a known biopolymer of known length and composition; dissolving the biopolymer in deionized water to create a sample solution; adding electrolytes, free radical scavengers, and free radical enhancers to the sample solution; distributing the sample solution into a gel matrix; irradiating the sample solution with ionizing radiation from a cobalt-60 source; retrieving the sample solution from the gel matrix; preparing the biopolymer for analysis by diluting the sample and adding lithium chloride; labeling the irradiated molecule with a size-selective DNA tag using DNA glycosylase coupled with an oxime-modified label; adding two discretely sized biopolymer internal standards for size selective and quantitative analysis; assembling a measurement chamber comprising two electrolyte chambers separated by a silicon nitride membrane containing a nanopore; infusing the irradiated biopolymer into the measurement chamber; applying a voltage across the membrane to cause the biopolymer to translocate through the nanopore; measuring the ionic current generated by the translocation; digitizing the measured ionic current; analyzing the digitized ionic current by detecting and counting translocations using a threshold analysis algorithm; collecting the magnitude of the current step, duration of the passage, number of discrete current steps, and the integrated area of the current steps; producing histograms of equivalent charge deficit (ECD) and number of current levels observed to quantify scission reactions and internal bond-breaking reactions, respectively; employing signal analysis using wavelet and joint time-frequency methods to identify spectral signatures of damage; and generating dose-response curves by plotting integrated peaks against varying doses of ionizing radiation to quantify RBE ratios.

[0152] The process for measuring radiation-induced damage to a biopolymer begins by providing a known biopolymer (108) of known length and composition. Providing a known biopolymer establishes a controlled starting material for the experiment. This knowledge of the biopolymer’s length and composition allows for accurate quantification of radiation-induced damage, such as the number of strand breaks per unit length. Thebiopolymer may be a nucleic acid, protein, or polysaccharide. Nucleic acids, like DNA and RNA, are essential for storing and transmitting genetic information. Proteins perform diverse functions in cells, acting as enzymes, structural components, and signaling molecules. Polysaccharides play critical roles in energy storage, cell signaling, and immune responses. For instance, a 2,500 base pair DNA molecule, a peptide with a defined sequence of amino acids, or a specific polysaccharide like cellulose could be selected as the biopolymer.

[0153] The process involves dissolving the biopolymer in deionized water to create a sample solution. Deionized water is free of impurities that could interfere with the RBE measurement. Other media, such as buffer solutions or cell culture media, may be used to mimic specific biological or environmental conditions. Dissolving the biopolymer in a suitable medium ensures homogenous distribution of the biopolymer molecules, facilitating uniform exposure to radiation. This controlled environment maximizes the accuracy and consistency of RBE measurements by reducing potential artifacts due to variations in radiation dose or sample composition. One example is dissolving 2,500 base-pair double-strand DNA in deionized water in an Eppendorf tube.

[0154] Electrolytes, free radical scavengers, and free radical enhancers are added to the sample solution. Electrolytes, such as phosphate-buffered saline, mimic physiological conditions. Free radical scavengers, like tris(hydroxymethyl)aminomethane, protect biomolecules from indirect radiation damage. Free radical enhancers, such as nitrous oxide (N2O), amplify radiation-induced damage to reflect in vivo conditions. The specific choice and concentration of these additives are determined by the type of biopolymer and the experimental goals.

[0155] Distributing the sample solution into a gel matrix, such as agarose, prior to irradiation creates a heterogeneous environment. This can mimic the spatial distribution of biomolecules within cells or tissues. Other matrices, such as polyacrylamide gels or biological tissues, can also be used. The gel matrix may be segmented into defined volumes for precise control of radiation dose and subsequent analysis.

[0156] Irradiating the sample with ionizing radiation from a cobalt-60 source delivers a controlled dose of gamma radiation. Cobalt-60 is a commonly used gamma radiation source with a well-characterized energy spectrum. Other sources, such as x-ray generators or particle beams, may also be employed, depending on the research focus. The radiation dose is calibrated using dosimetry standards.

[0157] Retrieving the sample from the gel matrix after irradiation allows for controlled collection of the irradiated biopolymer. Segments of the gel may be excised, or thegel matrix dissolved to release the biopolymer. This ensures accurate measurement of radiation-induced damage by preventing cross-contamination or sample loss.

[0158] Diluting the sample and adding lithium chloride prepares the biopolymer for analysis. Lithium chloride is a commonly used electrolyte in nanopore experiments that facilitates DNA translocation through the nanopore. Other electrolytes, such as potassium chloride or sodium chloride, may be employed. The dilution factor and electrolyte concentration are chosen to optimize nanopore measurement performance.

[0159] Labeling the irradiated molecule with a size-selective DNA tag using DNA glycosylase and an oxime-modified label allows for the targeted detection and analysis of specific types of radiation-induced damage. DNA glycosylase is an enzyme that recognizes and excises damaged bases in DNA. The oxime-modified label attaches to the site of damage, creating a size-selective tag that can be detected by nanopore analysis. Other enzymes or tagging strategies may be used, selected to detect specific types of radiation damage. The size of the tag is chosen to be easily distinguishable by nanopore analysis from both the intact biopolymer and smaller fragments resulting from radiation-induced scission.

[0160] Adding two discretely sized biopolymer internal standards to the sample further enhances the analysis. The internal standards are biopolymers of known length and composition. These standards are not irradiated and serve as calibration markers for nanopore measurement, enabling accurate size determination of the irradiated biopolymers. Different biopolymers, such as DNA or proteins, with lengths specifically chosen to be readily distinguishable from both the intact biopolymer and smaller radiation-induced fragments may be used as internal standards. For DNA analysis, two internal standards of known length, such as a 5,000 base-pair DNA molecule and a 10,000 base-pair DNA molecule, may be added.

[0161] Assembling a measurement chamber with two electrolyte chambers separated by a silicon nitride membrane containing a nanopore prepares the system for single- molecule analysis. Silicon nitride is a durable material suitable for nanopore fabrication. Other materials, such as glass or polymers, may also be employed. The nanopore's diameter and length are chosen based on the size of the biopolymer being analyzed. The two electrolyte chambers are filled with an electrolyte solution, such as lithium chloride or potassium chloride, to facilitate ionic current flow through the nanopore.

[0162] Infusing the irradiated biopolymer into one of the electrolyte chambers introduces the sample to the nanopore measurement platform. The chamber is assembled so that applying a voltage across the membrane drives the biopolymer towards and through thenanopore. The polarity of the applied voltage is selected to ensure biopolymer translocation in the desired direction.

[0163] Applying a voltage across the membrane establishes an electric field that drives biopolymer translocation through the nanopore. The magnitude of the applied voltage is chosen to optimize biopolymer capture and translocation rates, maximizing throughput and measurement precision.

[0164] Measuring the ionic current through the nanopore provides a real-time signal of biopolymer translocation events. Transient blockades in the ionic current reflect changes in the nanopore's effective cross-sectional area due to biopolymer translocation. The ionic current is measured using a transimpedance amplifier, which converts the small ionic current signals into measurable voltage outputs.

[0165] The measured ionic current is digitized using an analog-to-digital converter. This digitization enables precise quantification of the ionic current changes associated with biopolymer translocation, facilitating automated data analysis. The resolution and sampling rate of the analog-to-digital converter are chosen based on the desired signal-to-noise ratio and the temporal resolution needed to capture translocation events.

[0166] The digitized current is analyzed using a threshold analysis algorithm to detect and count translocations. The algorithm identifies transient reductions in the ionic current that exceed a predefined threshold, indicating biopolymer passage through the nanopore. The threshold value is chosen to minimize false-positive detection due to noise while ensuring that genuine translocations are captured.

[0167] Collecting the magnitude of the current step, duration of the passage, number of discrete current steps, and the integrated area of the current steps provides quantitative information about biopolymer properties. The magnitude of the current step reflects the biopolymer's size and conformation. The duration of passage represents its speed of translocation. The number of discrete steps may indicate internal bond-breaking events or conformational changes. The integrated area, also known as the equivalent charge deficit (ECD), provides a precise measure of biopolymer length, reflecting radiation-induced fragmentation.

[0168] Producing histograms of ECD and the number of current levels observed transforms the raw data into a form for quantitative analysis. The ECD histogram reveals the size distribution of the irradiated biopolymers, quantifying fragmentation. The histogram of current levels shows the presence of internal bond-breaking without requiring fragmentation.

[0169] Signal analysis using wavelet and joint time-frequency methods provides spectral signatures of damage, indicating radiation-induced changes in the biopolymer's physical properties. These signatures may include shifts in peak frequencies or changes in the relative amplitudes of different frequency components, indicative of altered flexibility, charge distribution, or other properties.

[0170] Dose-response curves are generated by plotting integrated peak areas of histograms against radiation dose. This analysis establishes a quantitative relationship between radiation dose and the level of damage. Plotting a collection of such curves generated under varying conditions, such as different biopolymers or radiation types, enables quantification of RBE ratios and tissue sensitivities. These ratios and sensitivities inform treatment planning and risk assessment.

[0171] These implementations yield numerous advantages. Using known biopolymers and deionized water ensures sample homogeneity and minimizes interference. Adding electrolytes, scavengers, and enhancers mimics in vivo conditions. The agarose gel matrix creates a heterogeneous environment. Cobalt-60 irradiation delivers a precise gamma ray dose. Retrieving the sample from the gel allows controlled collection of irradiated material. Diluting the sample and adding lithium chloride prepares the biopolymer for nanopore analysis. DNA tags enable detection of specific radiation-induced modifications. Internal standards ensure accurate size determination, and the silicon nitride membrane provides a stable platform for nanopore measurements. Applying a voltage across the membrane and measuring the resulting ionic current enables detection of single-molecule translocation events. Digitizing the current and using a threshold analysis algorithm facilitates automated event detection. Collecting salient features and generating histograms allow for quantitative analysis of radiation-induced changes. Signal analysis reveals spectral signatures of damage, and dose- response curves enable quantification of RBE and tissue sensitivities. This process enables precise, high-resolution analysis of radiation-induced damage at the single-molecule level, offering advantages over traditional cell-based assays in terms of speed, sensitivity, and quantitative information. The process is well-suited for low dose exposure analysis, where other techniques are inadequate.

[0172] In an embodiment, the biopolymer is a nucleic acid. In an embodiment, the nucleic acid is a double-strand DNA, e.g., a 2,500 base-pair double-strand DNA. In an embodiment, the biopolymer is a peptide. In an embodiment, the peptide is Asp-Arg-Val-Tyr- Ile-His-Pro-Phe-His-Leu. In an embodiment, the biopolymer is a polysaccharide. In an embodiment, the medium is water. In an embodiment, the gel matrix is agarose. In anembodiment, the nanopore is fabricated in silicon nitride. In an embodiment, the two discretely sized biopolymer internal standards are a 5,000 base-pair DNA and a 10,000 base-pair DNA.

[0173] The biopolymer may be a nucleic acid. Nucleic acids, such as DNA and RNA, are critical targets for radiation-induced damage due to their role in storing and transmitting genetic information. Measuring damage to nucleic acids provides insights into the mechanisms of radiation-induced mutagenesis, carcinogenesis, and cell death. Various types of nucleic acids, including genomic DNA, mitochondrial DNA, and different RNA species, can be analyzed to investigate radiation's effects on diverse cellular processes.

[0174] The nucleic acid may be a double-strand DNA. A DNA molecule of defined length facilitates precise quantification of radiation-induced damage, such as the number of strand breaks per unit length. 2,500 base pairs is a suitable length for analysis using nanopore technology, providing a balance between sufficient resolution for detecting individual damage events and efficient throughput for analyzing a statistically significant number of molecules. Other DNA lengths can also be analyzed, depending on the specific goals of the experiment and the capabilities of the nanopore measurement system. Using a double-stranded DNA molecule allows for detection of both single-strand and double-strand breaks, providing comprehensive information about the extent of radiation-induced damage.

[0175] The biopolymer may be a peptide. Peptides are short chains of amino acids that can serve as model systems for studying radiation-induced damage to proteins. Analyzing peptide damage provides valuable information about the effects of radiation on protein structure and function. Peptides of varying lengths and sequences can be employed, depending on the research focus.

[0176] The peptide may be Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu. This specific peptide sequence can be used as a model system for studying radiation-induced damage. Its defined sequence enables precise identification and quantification of amino acid modifications caused by radiation. Other peptides with different sequences and lengths can also be analyzed to explore the effects of radiation on specific amino acid residues or protein domains.

[0177] The biopolymer may be a polysaccharide. Polysaccharides are complex carbohydrates with essential structural and functional roles in living organisms. Analyzing radiation-induced damage to polysaccharides provides insights into the effects of radiation on plant cell walls, bacterial capsules, and other biological structures. Various polysaccharides can be studied using this process, such as cellulose, starch, chitin, or pectin, depending on theresearch focus. The selection of a particular polysaccharide may be based on its biological relevance or its suitability for nanopore analysis.

[0178] The medium may be water. Water is a simple and readily available solvent suitable for dissolving many biopolymers. Its use simplifies sample preparation and minimizes potential interference with the nanopore measurement. Other media, such as buffer solutions, may be employed where necessary to maintain a stable pH or other experimental conditions during irradiation or analysis.

[0179] The gel matrix may be agarose. Agarose is a commonly used gel matrix for electrophoretic separation and analysis of biomolecules. Its porous structure allows for diffusion of small molecules while impeding the movement of larger biopolymers, enabling controlled retrieval of the irradiated sample. Other gel matrices, such as polyacrylamide or specialized gels designed for specific applications, can also be used. The choice of gel matrix depends on the size and properties of the biopolymer being analyzed.

[0180] The nanopore may be fabricated in silicon nitride. Silicon nitride is a durable, chemically inert material well-suited for nanopore fabrication due to its high mechanical strength, thermal stability, and resistance to chemical degradation. These properties minimize nanopore clogging and ensure consistent performance over multiple experiments. Other materials, like silicon dioxide, aluminum oxide, or polymers may be employed, chosen based on factors such as fabrication methods, biopolymer compatibility, and desired pore dimensions.

[0181] The two discretely sized internal biopolymer standards may be a 5,000 base- pair DNA and a 10,000 base-pair DNA. These DNA molecules of defined length are suitable internal standards for DNA damage analysis using nanopore technology. Their known lengths enable calibration of the nanopore measurement, ensuring accurate size determination of irradiated DNA fragments. Other DNA molecules or biopolymers of different sizes and types may be used, chosen based on the size and type of the primary biopolymer being analyzed. The internal standards should be readily distinguishable in size from both the intact biopolymer and smaller radiation-induced fragments, enabling accurate quantification of radiation damage.

[0182] The use of a nucleic acid, particularly a defined-length double-strand DNA molecule, provides a standardized model system for accurately quantifying radiation-induced damage. Analyzing a peptide with a specific sequence allows for investigation of radiation effects on protein structure and function. Polysaccharides extend the applicability of the method to other essential biomolecules. Water as the medium simplifies sample preparation, and agarose gels enable controlled sample retrieval after irradiation. Fabricating the nanoporein silicon nitride enhances measurement stability. Adding two discretely sized DNA standards enables precise size determination of irradiated DNA fragments. These specific implementations contribute to the accuracy, sensitivity, and versatility of the process.

[0183] In an embodiment, the providing a known biopolymer step comprises selecting a known biopolymer of known length. In an embodiment, the dissolving comprises dissolving the biopolymer in water in an Eppendorf tube. In an embodiment, the adding step comprises adding phosphate buffered saline, tris(hydroxymethyl)aminomethane, and a selected gas, e.g., N2O(g). In an embodiment, the distributing step comprises distributing the sample solution into an agarose gel. In an embodiment, the irradiating comprises irradiating the sample with a gamma radiation field at a dose rate of 1.373 Gy / min. In an embodiment, the retrieving step comprises extracting the sample from segments of the agarose gel matrix. In an embodiment, the labeling comprises reacting the irradiated DNA with a base-specific DNA glycosylase to create reactive sites for attaching the DNA tag. In an embodiment, the applying step comprises applying a voltage of 300 mV. In an embodiment, the digitizing step comprises using an analog-to-digital converter.

[0184] Providing a known biopolymer may comprise selecting a known biopolymer of known length. Selecting a biopolymer of known length improves the accuracy and consistency of radiation damage measurements. This knowledge of the biopolymer's initial length enables precise calculation of radiation-induced changes in length, such as fragmentation or conformational changes. The length of the biopolymer is chosen based on the specific research question and the limitations of the nanopore measurement platform. For instance, shorter biopolymers may be preferred for analyzing specific modifications, while longer biopolymers may be necessary for studying larger-scale structural changes. A 2,500 base-pair DNA molecule, for example, provides a suitable model system for studying radiation- induced DNA damage.

[0185] Dissolving the biopolymer may comprise dissolving the biopolymer in water in an Eppendorf tube. An Eppendorf tube is a small, disposable plastic tube commonly used in laboratories for handling and storing small volumes of liquids. Dissolving the biopolymer in water within an Eppendorf tube simplifies sample handling and minimizes the risk of contamination. Other containers, such as microcentrifuge tubes or glass vials, can also be used. The choice of container depends on the volume of sample being prepared and the compatibility of the container material with the biopolymer and the chosen solvent. Dissolving the biopolymer in a controlled volume within a suitable container ensures accuratemeasurement of concentration and minimizes sample loss, improving the precision and reproducibility of the RBE determination.

[0186] Adding electrolytes, free radical scavengers, and free radical enhancers may comprise adding phosphate-buffered saline (PBS), tris(hydroxymethyl)aminomethane (Tris), and nitrous oxide (N2O(g)). PBS is a commonly used buffer solution that maintains a stable pH and provides a physiologically relevant ionic environment. Tris is a free radical scavenger that protects the biopolymer from radiation-induced damage caused by reactive oxygen species. N2O(g) is a free radical enhancer that amplifies the effects of radiation, allowing for more sensitive detection of radiation-induced modifications. Other electrolytes, scavengers, and enhancers may be employed, chosen based on the specific biopolymer being analyzed, the type of radiation used, and the experimental goals. The choice and concentration of these reagents are optimized to mimic in vivo conditions and enhance the accuracy of the RBE measurement.

[0187] Distributing the sample solution may comprise distributing the sample solution into an agarose gel. Agarose is a biocompatible material with a porous structure, enabling uniform diffusion of small molecules while hindering the movement of larger biomolecules. This property allows for controlled distribution of the sample solution, minimizing variations in radiation dose and facilitating retrieval of the irradiated sample. Other matrices, such as polyacrylamide gels, may also be used. The choice of matrix may depend on the size and properties of the biopolymer being analyzed and the method of sample retrieval.

[0188] Irradiating the sample may comprise irradiating the sample with a gamma radiation field at a dose rate of 1.373 Gy / min. Gamma radiation is a form of ionizing radiation commonly employed in RBE studies. A dose rate of 1.373 Gy / min, delivered from a calibrated source like a cobalt-60 irradiator, allows for precise control of the radiation dose delivered to the sample. Other radiation sources and dose rates may be employed, based on the research question and the sensitivity of the biopolymer to radiation damage. Precise control over radiation dose and exposure time ensures accurate measurement of RBE.

[0189] Retrieving the sample may comprise extracting the sample from segments of the agarose gel matrix. This controlled retrieval method minimizes cross-contamination and ensures accurate measurement of the irradiated sample. Segments of the gel containing the irradiated sample may be excised using a scalpel or other cutting tool. The biopolymer may then be extracted from the gel segments using standard biochemical techniques, such as centrifugation or filtration. Alternative retrieval methods, such as dissolving the gel matrix, can also be employed, depending on the nature of the gel and the properties of the biopolymer.

[0190] Labeling may comprise reacting the irradiated DNA with a base-specific DNA glycosylase to create reactive sites for attaching the DNA tag. DNA glycosylase is an enzyme that recognizes and removes damaged or modified bases in DNA, creating an abasic site. This abasic site may then be used as a reactive site for attaching a size-selective DNA tag. The choice of DNA glycosylase and tagging strategy depends upon the specific type of DNA damage targeted for analysis. Other enzymes or chemical methods may be employed to introduce tags into the irradiated DNA.

[0191] Applying a voltage may comprise applying a voltage of 300 mV. This voltage may be suitable for driving DNA translocation through a nanopore of specific dimensions. The specific voltage used is chosen based on the nanopore characteristics, the properties of the electrolyte solution, and the desired translocation speed of the biopolymer. Applying a controlled voltage ensures consistent and reproducible nanopore measurements.

[0192] Digitizing may comprise using an analog-to-digital converter. This converter transforms the analog voltage signals from the transimpedance amplifier into digital data suitable for computer processing and analysis. The resolution and sampling rate of the converter are chosen based on the desired signal-to-noise ratio and the temporal resolution needed to resolve translocation events accurately.

[0193] These process steps enhance the precision and efficiency of the RBE measurement. Selecting a biopolymer of known length improves quantification accuracy. Dissolving the biopolymer in water in an Eppendorf tube ensures sample homogeneity and simplifies handling. Adding PBS, Tris, and N2O(g) mimics physiological conditions and controls radiation-induced damage. Distributing the sample into an agarose gel allows for controlled irradiation in a heterogeneous environment. Irradiating with a defined gamma radiation field from a cobalt-60 source enables accurate dose delivery. Extracting the sample from gel segments ensures precise measurement of irradiated material. Reacting the DNA with DNA glycosylase enables targeted labeling of damaged bases. Applying a controlled voltage drives translocation efficiently, and digitizing the current enables quantitative data analysis. These refined process steps improve the accuracy, sensitivity, and reproducibility of the RBE measurement.

[0194] FIG. 1 illustrates nanopore-based single-molecule biodosimetry. The process begins with the provision of a known biopolymer sample (108). The biopolymer may be a nucleic acid, such as DNA or RNA, a protein, or a polysaccharide. The sample is prepared in a controlled environment to maintain its integrity and minimize contamination. The known length or composition of the biopolymer is essential for accurate quantification of anyradiation-induced changes. The sample is then dissolved in a suitable medium (210), such as water, to create a sample solution. Electrolytes, free radical scavengers, or free radical enhancers may be added to mimic physiological conditions and control the effects of radiation. The sample solution may be distributed into a matrix, such as an agarose gel, to simulate a heterogeneous biological environment. The sample is irradiated with ionizing radiation, which may comprise photons, such as gamma rays or X-rays, or particles, such as electrons or protons. The radiation source may be a cobalt-60 irradiator, an X-ray generator, or a particle accelerator. The radiation dose is carefully controlled and calibrated using dosimetry standards. Following irradiation, the sample is retrieved from the matrix and prepared for analysis. Preparation steps may include dilution, buffer exchange, or addition of reagents to optimize compatibility with the nanopore measurement system. The sample is analyzed using a nanopore measurement device, which includes two electrolyte chambers (102, 101) separated by a dielectric membrane (100) containing a nanopore (107). The nanopore has a diameter from 1 nanometer to 20 nanometers, and specifically, from 2 nanometers to 10 nanometers, and more specifically, from 4 nanometers to 6 nanometers. The nanopore’s length can be from 1 nanometer to 500 nanometers, specifically from 5 nanometers to 100 nanometers and more specifically, from 10 nanometers to 20 nanometers. A voltage is applied across the membrane using a voltage source (106) connected to electrodes (104, 103) in each chamber. This voltage establishes an electric field that drives the translocation of the biopolymer through the nanopore. As the biopolymer translocates, it causes a transient blockade in the ionic current flowing through the nanopore. This current is measured using a transimpedance amplifier (105) and digitized using an analog- to-digital converter (208). The digitized current is analyzed using algorithms that detect and count the translocations, and measure the magnitude and duration of the current blockades. These data are used to determine the extent of radiation-induced damage to the biopolymer, such as the number of strand breaks in DNA or changes in protein conformation. The results of the analysis are plotted against radiation dose to generate a dose-response curve. A collection of dose-response curves generated under varying conditions, such as different biopolymer types or different radiation sources, allows for determination of RBE ratios. These ratios provide a quantitative measure of the relative effectiveness of different types of radiation in causing biological damage. This approach leverages the single-molecule sensitivity of nanopore technology to directly measure radiation-induced damage, overcoming limitations of traditional macroscopic assays. Precise control of experimental conditions and the use of known biopolymers enhance measurement accuracy. Employing a range of radiation doses and sources enables comprehensive RBE determination. The process provides valuableinsights into the mechanisms of radiation action at the molecular level. The illustrated process significantly improves prior art methods by enabling rapid, accurate, and quantitative RBE measurements, using less material than conventional techniques. The controlled sample preparation and irradiation conditions minimize variability and enhance reproducibility. The nanopore-based measurement platform allows for single-molecule analysis, providing detailed information about the nature and extent of radiation-induced damage. The use of digital data acquisition and automated analysis streamlines the process and improves objectivity. The ability to generate dose-response curves and quantify RBE ratios under varying conditions makes this method a versatile tool for radiation research and applications.

[0195] FIG. 2 illustrates the use of molecular standards as an internal calibration and ruler in nanopore-based single-molecule dosimetry. Accurate measurement of radiation- induced damage to biopolymers requires precise determination of the biopolymer’s length or molecular weight. Nanopore technology provides a means for measuring biopolymer length based on the duration and magnitude of ionic current blockades caused by the biopolymer’s passage through a nanopore. However, variations in nanopore dimensions and experimental conditions can affect the measured ionic current signals, making it challenging to compare results across different experiments or laboratories. To address this challenge, internal molecular standards are employed as an internal calibration and ruler. These standards are biopolymers of known length, which are added to the irradiated sample prior to nanopore analysis. By measuring the translocation characteristics of the internal standards, the nanopore measurement can be calibrated, and variations in nanopore properties or experimental conditions can be corrected. The figure shows representative ionic current traces for a mixture of irradiated and unirradiated DNA molecules. The top panel (a) displays the ionic current versus time for a sample of 2.5 kbp DNA irradiated at a dose of 1.0 Gy, along with unirradiated internal standards of 5 kbp and 10 kbp DNA. Panel (b) shows a similar experiment where the 2.5 kbp DNA was irradiated at a higher dose of 15.0 Gy. The characteristic current events for each DNA species are highlighted in the insets. The area shaded in orange or blue represents the equivalent charge deficit (ECD), which is a measure of the biopolymer’s length. The ECD is calculated by integrating the area of the resistive pulse, which is the transient reduction in ionic current caused by the biopolymer’s passage through the nanopore. As shown in the figure, the ECD is proportional to the biopolymer’s length. Shorter DNA molecules, resulting from radiation-induced fragmentation, exhibit smaller ECD values compared to longer, intact DNA molecules. The internal standards serve as both a calibration and a ruler for the nanopore measurement. The known lengths of the internal standards enable precise determination of therelationship between ECD and biopolymer length. By aligning the ECD peaks for the internal standards across different experiments, variations in nanopore properties or experimental conditions can be corrected, allowing for accurate comparison of biopolymer length distributions. This calibration process is used for quantifying radiation-induced damage and determining RBE ratios. In addition, the known spacing between the internal standard peaks in the ECD histograms can be used to create a molecular ruler, enabling accurate estimation of the lengths of irradiated biopolymer fragments. The choice of internal standards may depend on the size range of the biopolymer being analyzed and on the resolution of the nanopore measurement. For DNA damage analysis, DNA molecules of defined length provide ideal calibration standards and molecular rulers. In the present example, 5 kbp and 10 kbp DNA molecules serve as suitable internal standards for analyzing radiation-induced damage to a 2.5 kbp DNA molecule. The time series data collected in these experiments can be analyzed using algorithms that detect and count individual translocation events, extract salient features of the current signatures, and generate histograms of ECD values. These analyses provide a quantitative measure of radiation-induced damage, enabling precise determination of RBE.

[0196] Variations in nanopore fabrication and experimental conditions can lead to significant differences in the measured ionic current signals, making it challenging to compare results across different experiments. To address this challenge, the internal molecular standards are used to calibrate the nanopore measurement and correct for these variations. As shown in panel (c), the ECD peaks for the internal standards may vary significantly between different nanopore measurements. However, by aligning the internal standard peaks, as illustrated in panel (d), the variations can be corrected, and the ECD histograms can be accurately compared. This calibration process enables precise quantification of radiation- induced damage and determination of RBE values. The internal standards also function as a molecular ruler, allowing for estimation of the lengths of irradiated biopolymer fragments. The specific choice of internal standards may depend on the size and type of the biopolymer being analyzed and on the resolution of the nanopore measurement platform. For instance, 5 kbp and 10 kbp DNA molecules are appropriate standards for assessing radiation damage to a 2.5 kbp DNA. The use of internal standards improves the accuracy and reproducibility of nanopore- based RBE measurements, enabling reliable comparison of data across different laboratories and experimental conditions. The calibrated ECD histograms are useful for accurately quantifying radiation-induced damage and establishing dose-response relationships.

[0197] FIG.3 shows a DNA dose-response curve generated using nanopore-based single-molecule dosimetry. The curve plots the intact DNA concentration against radiationdose, providing a quantitative measure of radiation-induced DNA damage. The experimental data points (blue circles) are obtained by measuring the concentration of intact 2.5 kbp DNA molecules after exposure to varying doses of gamma radiation, from 0 Gy to 15 Gy. The concentration of intact DNA decreases with increasing radiation dose, reflecting the accumulation of radiation-induced damage, primarily double-strand breaks. The error bars represent the standard deviation of the measurements, which reflect variations in nanopore performance and sample preparation. Two internal standards, 5 kbp and 10 kbp DNA, were included in the samples to control for variations in nanopore measurement, and their measured concentrations are relatively constant across all radiation doses, indicating good measurement accuracy and reproducibility. The dose-response curve is fitted to two different mathematical models. The red, dashed line represents a fit to a simple Gaussian decay model, which assumes a linear relationship between radiation dose and DNA damage. This model is appropriate at low doses, where the probability of multiple damage events within a single DNA molecule is relatively small. However, at higher doses, where multiple damage events become more likely, the simple Gaussian model deviates significantly from the experimental data. The green, curve represents a fit to a modified Gaussian decay model that incorporates bimolecular radical decay. This model assumes that DNA damage is caused primarily by reactive oxygen species generated by water radiolysis, and that the concentration of these reactive species decreases with increasing radiation dose due to bimolecular reactions. The modified Gaussian model provides a better fit to the experimental data across the entire dose range, especially at the higher radiation doses where radical interactions are more significant. The inset displays representative ECD histograms for the different radiation doses, showing a decrease in the concentration of intact 2.5 kbp DNA (indicated by the peak centered at approximately log10(L / L0) = 3.4) with increasing radiation dose. The inset also shows the relatively constant concentrations of the two internal standards (5 kbp and 10 kbp DNA), indicating consistent nanopore performance. The dose-response curve demonstrates the quantitative relationship between radiation dose and the fraction of intact DNA molecules, enabling accurate calculation of RBE values. The modified Gaussian model that incorporates bimolecular radical decay provides a more accurate description of this relationship compared to the simple Gaussian model, especially at higher doses. The use of internal molecular standards enables precise measurement of DNA length and concentration, correcting for variations in nanopore performance and experimental conditions. This precise control of experimental conditions enhances the accuracy and reproducibility of the RBE determination. The dose-response curve provides valuable information for optimizing radiation therapy treatments, assessing the risksassociated with radiation exposure, and developing strategies for radiation protection. The curve's shape can reveal information about the mechanisms of radiation-induced DNA damage, informing the development of more effective radiation therapies and countermeasures. For example, the elongated Gaussian shape of the dose-response curve observed in this study indicates that double-strand breaks in DNA are caused primarily by two nearby single-strand breaks, consistent with the model of indirect DNA damage via reactive oxygen species.

[0198] FIG.4 illustrates the fragment concentration bias in nanopore-based single- molecule dosimetry. Nanopore technology provides a means for measuring the length of biopolymers based on their translocation characteristics through a nanopore. However, the probability of capturing and detecting a biopolymer molecule during a nanopore measurement can be affected by its length, with smaller molecules having a lower capture probability compared to larger molecules. This length-dependent capture bias can distort the measured biopolymer length distribution and lead to inaccurate quantification of radiation-induced damage. To address this bias, a calibration process is employed using a ladder of DNA fragments of known lengths. By measuring the capture probabilities of the DNA fragments of varying sizes, a correction factor can be determined and applied to correct the measured biopolymer length distributions. Panel (a) presents the raw ECD histograms for the DNA ladder, including fragments of 100, 250, 300, 600, 900, 2,000, 5,000, and 10,000 base pairs in length. The histograms were generated using three identically prepared nanopipettes, and they reveal the length-dependent capture bias, with smaller fragments exhibiting lower peak heights compared to larger fragments. Panel (b) shows an example of a DNA molecule exposed to 15 Gy of gamma radiation. The peak corresponding to the intact 2.5 kbp DNA is reduced, while a distribution of smaller fragments appears at lower ECD values. Panel (c) plots the capture probability, P, of DNA fragments of varying sizes relative to the capture probability of the 10 kbp DNA fragment. The data are fitted to a logarithmic function, which provides a measure of the capture bias as a function of DNA length. The curve shows that for DNA fragments larger than 2 kbp, the capture probability is relatively constant, but for smaller fragments, the capture probability decreases substantially. Panel (d) plots the corrected ratio of nucleotides in smaller DNA fragments to nucleotides in the intact 2.5 kbp DNA as a function of radiation dose. The data show that, after correcting for the capture bias, the ratio of fragments to intact DNA increases monotonically with dose, reflecting accumulation of radiation-induced damage. The fragment concentration bias correction method shown in FIG.4 enables accurate quantification of radiation-induced damage by correcting for the length-dependent capture bias in nanopore measurements. The DNA ladder provides a calibration standard for determining the captureprobability of fragments of varying sizes. The logarithmic fit to the capture probability data allows for correction of the measured biopolymer length distributions. The corrected ratio of fragment concentration provides a quantitative measure of radiation-induced damage that is not distorted by the capture bias. This method improves the accuracy and reproducibility of RBE measurements, allowing for reliable comparison of results across different experiments or laboratories. The corrected ratio of fragment concentration also enables detection of subtle changes in the biopolymer length distribution that may not be apparent from the raw nanopore data, providing detailed insights into the mechanisms of radiation-induced damage. The use of a DNA ladder with fragments of varying lengths is essential for establishing an accurate correction factor for capture bias, and the logarithmic fit function provides a convenient means for interpolating and extrapolating the capture probability data to cover a wide range of biopolymer lengths.

[0199] FIG. 7 illustrates DNA damage labeling schemes for enhancing the detection and characterization of specific types of radiation-induced modifications. Nanopore- based single-molecule dosimetry measures radiation-induced damage to biopolymers by analyzing changes in their translocation characteristics through a nanopore. However, some types of radiation damage, such as chemical modifications to DNA bases or single-strand breaks, may not significantly alter the biopolymer’s overall length, making them difficult to detect by simply measuring changes in ECD. To address this challenge, labeling schemes are employed that introduce size-selective tags at the sites of these specific types of damage. These tags enhance their detectability by nanopore analysis. The figure illustrates two different types of damage labeling schemes for DNA: (1) direct detection of double-strand breaks, and (2) base excision repair (BER) pathway-mediated labeling of other types of damage. Double- strand breaks, which involve the complete severing of the DNA double helix, are readily detectable by nanopore analysis as a reduction in the ECD. The BER pathway is a cellular DNA repair mechanism that recognizes and removes damaged or modified bases, creating a single-strand break at the site of damage. The BER pathway can be exploited to introduce size- selective tags at the sites of various types of DNA damage, enhancing their detection by nanopore analysis. The labeling scheme involves the use of enzymes, such as DNA glycosylase and AP endonuclease, which recognize and cleave the DNA backbone at the damaged site. A size-selective tag, such as a short DNA molecule or a polymer, is attached to the site of the break, creating a labeled DNA molecule that can be analyzed by nanopore technology. The size of the tag is chosen such that it is easily distinguishable by nanopore analysis from both the intact DNA and smaller fragments. The tag also introduces a fixed charge at the site ofdamage, altering the diameter of the molecule at the damaged site. As the labeled DNA molecule translocates through the nanopore, the presence of the tag causes a characteristic change in the ionic current signal, which can be detected and analyzed to determine the location and type of damage. The sensitivity of nanopore-based DNA damage detection can be enhanced by using multiple tags of different sizes. The use of multiple tags increases the complexity of signal decoding, but it also provides more detailed information about the types and locations of radiation-induced modifications. One advantage of these labeling methods is their ability to target specific types of radiation damage that may not be readily detectable by simply measuring changes in ECD. The introduction of size-selective tags at the sites of damage enhances their detectability by nanopore analysis. The use of multiple tags with differing sizes allows for simultaneous detection of multiple types of damage. These labeling techniques, coupled with nanopore analysis, provide detailed molecular information about radiation's effects on biopolymers, informing the development of more effective radiation therapies and protection strategies. The ability to label specific DNA lesions, including those induced by base-excision repair mechanisms, with size-specific labels, allows for their detection by nanopore analysis. Each labeled DNA lesion can be identified by measuring time- dependent resistive pulses as the labeled molecules are driven through a nanopore.

[0200] FIG. 8 provides a schematic illustration of DNA damage detection using nanopore technology, including the detection and characterization of DNA-lesion size- selective labels. Nanopore-based single-molecule dosimetry measures radiation-induced damage to biopolymers by analyzing changes in their translocation characteristics through a nanopore. While double-strand breaks in DNA are readily detectable as a reduction in the molecule's ECD, other types of damage, such as single-strand breaks or chemical modifications to DNA bases, may not significantly alter the overall length of the DNA molecule and thus may be difficult to detect directly. To overcome this limitation, size-selective labels may be employed that specifically bind to the damaged sites, increasing their detectability by nanopore analysis. The figure depicts a DNA molecule containing two distinct lesions at different locations. Lesion 1 is labeled with a relatively small tag, such as a 22-mer hairpin nucleotide, while Lesion 2 is labeled with a larger tag, such as a 44-mer containing two hairpins. As the labeled DNA molecule is driven through the nanopore under an applied electric field, changes in the ionic current are measured. The magnitude of the ionic current blockade is approximately proportional to the volume of the nanopore occupied by the DNA molecule. The presence of the size-selective tags at the sites of damage alters the effective diameter of the DNA molecule at those locations, creating distinct ionic current signatures. The figure shows representativeionic current traces for double-stranded DNA, DNA with Label 1, and DNA with Label 2. The distinct current signatures enable identification and localization of different types of DNA lesions. The use of size-selective tags improves the sensitivity and specificity of nanopore- based single-molecule dosimetry for detecting and characterizing various types of radiation- induced DNA damage. The tags enhance the detectability of lesions that may not cause significant changes in the DNA's overall length. By using tags of different sizes, multiple types of lesions can be distinguished and quantified simultaneously. The figure shows how distinct ionic current signatures are generated by the tags, enabling identification and localization of damage. This information provides detailed insights into the molecular mechanisms of radiation-induced DNA damage.

[0201] FIG. 9 depicts a measurement chamber (100) for analyzing biopolymers using nanopore technology. Nanopore-based single-molecule analysis measures the properties of individual biomolecules, such as DNA, RNA, or proteins, by analyzing their translocation characteristics through a nanopore. As a biomolecule passes through the nanopore, it causes a transient blockade in the ionic current flowing through the pore. The magnitude and duration of this blockade reflect the biomolecule's physical properties, enabling determination of its size, shape, and charge. The measurement chamber shown in FIG. 9 provides a controlled environment for nanopore-based single-molecule analysis, enabling precise measurement of ionic current and biopolymer properties. The chamber comprises two electrolyte compartments, referred to as the cis chamber (102) and trans chamber (101), separated by a dielectric membrane (100). The membrane typically comprises an insulating material, such as silicon nitride or glass, and contains a single nanopore (107). The nanopore’s dimensions, including its diameter and length, are carefully chosen based on the size and properties of the biomolecules being analyzed. For DNA analysis, a nanopore with a diameter of 5 nm and a length of 12 nm may be employed. Electrodes (103, 104), typically made of a conductive material such as platinum or gold, are placed in each chamber to apply a voltage across the membrane and measure the ionic current. A voltage source (106) is connected to the electrodes, and a transimpedance amplifier (105) is used to amplify the small ionic current signals generated by biomolecule translocation, which are converted into a measurable voltage. The amplified voltage signal is then recorded and analyzed using a computer. The measurement chamber provides a controlled environment for nanopore-based single-molecule analysis. The separation of the two chambers by the dielectric membrane enables precise control of the electrolyte conditions in each chamber and prevents electrical interference between them. The nanopore’s dimensions are chosen to optimize detection of specific biomolecules, and thevoltage source and transimpedance amplifier enable precise control and sensitive measurement of ionic current. The chamber’s design minimizes noise and other artifacts, improving measurement accuracy. The measurement chamber allows for single-molecule analysis of biopolymers in a controlled environment, improving throughput and reproducibility. The chamber shown in FIG. 9 is particularly suitable for studying radiation-induced damage to biopolymers. By analyzing the changes in translocation characteristics caused by radiation exposure, the RBE of different types of radiation can be determined. The chamber's design enables efficient and accurate measurements while providing valuable information about the effects of radiation on biomolecules at the single-molecule level. The use of separate chambers enables tailoring of the electrolyte conditions on each side of the membrane, including optimizing parameters like pH and salt concentration for specific biopolymers or analysis conditions. This versatility makes the apparatus useful in a wide range of experiments.

[0202] FIG. 10 illustrates DNA translocation through a nanopore in the measurement chamber shown in FIG. 9. Nanopore-based single-molecule analysis measures the properties of individual biomolecules, such as DNA, by analyzing their translocation characteristics through a nanopore. As a DNA molecule (108) passes through the nanopore (107), it causes a transient reduction in the ionic current flowing through the pore. The magnitude and duration of this current reduction, or blockade, reflects the DNA molecule's physical properties, including its length, enabling precise measurement of biopolymer length and detection of changes due to radiation damage. The figure shows a DNA molecule translocating through a nanopore in a dielectric membrane (100) separating two electrolyte chambers (101, 102). The DNA molecule is driven through the nanopore by an applied electric field established by electrodes (103, 104) placed in each chamber and connected to a voltage source (106). A transimpedance amplifier (105) is used to convert the ionic current through the nanopore into a measurable voltage signal. As the DNA molecule enters and passes through the nanopore, it partially blocks the flow of ionic current, resulting in a transient reduction in the measured current. The duration and magnitude of this current blockade reflect the DNA molecule’s length. Longer DNA molecules spend more time translocating through the pore and produce larger current blockades compared to shorter DNA molecules. Analyzing the magnitude and duration of ionic current changes provide information on the length of the translocating DNA molecule. Nanopore analysis enables single-molecule DNA length measurements, providing high-resolution data useful for characterizing radiation-induced DNA damage. By measuring changes in DNA length distributions after exposure to varying radiation doses, the biological effectiveness of the radiation can be determined. This capabilitymakes nanopore technology a valuable tool for RBE studies. The specific dimensions of the nanopore are carefully chosen based on the size range of the DNA molecules being analyzed. For instance, a nanopore with a diameter of 5 nm and a length of 12 nm may be employed for analyzing DNA fragments with lengths ranging from a few hundred to several thousand base pairs. The magnitude and polarity of the applied voltage, typically in the range of millivolts to hundreds of millivolts, are chosen to control the translocation speed of the DNA molecule. The ionic current through the nanopore is converted into a voltage signal by the transimpedance amplifier, which also filters and amplifies the signal. This amplified voltage signal is then recorded and analyzed to determine the characteristics of the DNA translocation events.

[0203] FIG. 11 provides a simplified process flow diagram for measuring the relative biological effectiveness (RBE) of ionizing radiation using a biopolymer sample. The process focuses on utilizing nanopore technology to detect and quantify radiation-induced damage to individual biomolecules, offering advantages over traditional methods that rely on macroscopic endpoints, such as cell survival or tissue damage. The simplified process flow diagram outlines the key steps involved in the RBE measurement process, starting with sample collection and preparation, followed by irradiation, nanopore analysis, and data interpretation. The diagram highlights the streamlined nature of the process, emphasizing its efficiency and ease of implementation compared to traditional RBE measurement techniques. The first step in the process is selecting a suitable biopolymer sample. The biopolymer may be a nucleic acid, such as DNA or RNA, a protein, or a polysaccharide. The sample should be purified to a known length or composition to enable accurate quantification of radiation-induced damage. The next step is preparing and irradiating the sample. This involves dissolving the biopolymer in a suitable medium, such as water or a buffer solution, and adding any necessary reagents, such as electrolytes or free radical scavengers. The sample solution is then exposed to a controlled dose of ionizing radiation, with the radiation type and dose chosen based on the specific research question. After irradiation, the sample is collected from the matrix, if one was used, and the electrolyte is adjusted for measurement. This adjustment may involve dilution, buffer exchange, or addition of reagents to optimize the nanopore measurement. The prepared sample is then introduced into the nanopore measurement chamber. A voltage bias is applied across the membrane containing the nanopore, causing the biopolymer molecules to translocate through the pore. As the biomolecules translocate, changes in ionic current through the nanopore are measured, providing information about their physical properties. The final step is measuring the ionic current and analyzing the resulting data. The measured ionic current is digitized, and the digitized data is analyzed using algorithms that detect and quantify changesin the biopolymer’s properties caused by radiation exposure. These changes may include biopolymer fragmentation or modifications to its structure or conformation. The analysis results are then used to determine RBE values. The simplified process flow diagram highlights some steps involved in the nanopore-based RBE measurement process. The diagram emphasizes the efficiency of the method and its suitability for high-throughput analysis. The use of a biopolymer sample enables direct measurement of radiation-induced damage at the molecular level. The optional preparation and irradiation steps allow for customization of the experiment to mimic specific in vivo conditions. The nanopore measurement provides single- molecule resolution, enhancing sensitivity and accuracy. The application of a voltage bias drives biopolymer translocation, while measurement of the ionic current provides the raw data for analysis. The analysis of the ionic current data enables quantification of radiation-induced damage and determination of RBE values. This streamlined process makes nanopore technology a versatile tool for radiation research, providing rapid, quantitative, and high- resolution information about radiation’s biological effects. The flow diagram provides a simplified overview of the nanopore-based RBE measurement process, focusing on the key steps involved. The diagram emphasizes the method’s efficiency and its adaptability to various experimental conditions.

[0204] FIG. 12 shows a more detailed process flow diagram for measuring the relative biological effectiveness (RBE) of ionizing radiation using nanopore technology. The diagram illustrates the key steps involved in the measurement process, starting with sample selection and preparation, followed by irradiation, sample processing, nanopore analysis, data analysis, and RBE determination. Compared to the simplified diagram in FIG.11, this diagram provides additional detail about the sample preparation, labeling, and data analysis steps, highlighting the flexibility and versatility of the method for analyzing different types of radiation-induced damage. The process begins with selecting a biopolymer sample, which may be a nucleic acid, protein, or polysaccharide. The selected biopolymer is then prepared for irradiation by dissolving it in a suitable medium and adding any necessary reagents, such as electrolytes or free radical scavengers. The sample is then irradiated with a controlled dose of ionizing radiation. After irradiation, the sample may be collected from a matrix, if used, and then dispersed in an electrolyte solution for nanopore analysis. Optionally, a size-selective label may be added to the irradiated sample to enhance the detection of specific types of radiation-induced damage. The labeled sample is then measured using a nanopore sensor, which detects changes in ionic current as the biopolymer translocates through the nanopore. The measured ionic current data is then analyzed and counted to determine the number andproperties of translocating molecules. This analysis may involve detecting and counting individual translocation events, measuring the magnitude and duration of current blockades, and quantifying changes in the biopolymer’s properties caused by radiation exposure. Finally, the data is converted into a dose-response curve, and RBE values are determined by comparing the dose-response curves for different types of radiation. The additional detail in this flow diagram, compared to FIG.11, highlights the steps involved in sample preparation, labeling, and data analysis. The optional step of adding a size-selective label enables detection of various types of radiation-induced damage, including those that may not alter the biopolymer's overall length. The data analysis step now explicitly mentions converting molecular counts to a dose- response curve, and the generation of multiple dose-response curves enables calculation of RBE ratios and determination of tissue sensitivities. This expanded flow diagram underscores the versatility and precision of the nanopore-based RBE measurement method, particularly its adaptability to different types of radiation-induced damage and its use in quantifying biological effectiveness and tissue-specific responses.

[0205] FIG. 13 illustrates the principles of nanopore-based single-molecule analysis for measuring radiation-induced damage to biopolymers, such as DNA. The figure shows (a) a schematic of DNA translocation through a nanopore, (b) a representative ionic current time series, and (c) a close-up view of a single translocation event, highlighting the equivalent charge deficit (ECD). Nanopore technology provides analyzing individual biomolecules by measuring changes in ionic current as they pass through a nanoscale pore. As a biomolecule translocates through the nanopore, it partially blocks the flow of ionic current, creating a measurable signal that reflects the biomolecule’s physical properties. Panel (a) depicts a DNA molecule being driven through a nanopore by an applied electric field provided by voltage (V). The translocation is driven by electrophoretic and electroosmotic forces, represented by Ftrs and Fcis respectively. As the negatively charged DNA molecule moves towards the positive electrode, it enters and passes through the nanopore, which is a small channel in a membrane separating two electrolyte chambers. The duration of the DNA’s passage through the nanopore depends on its length: longer DNA molecules take longer to translocate than shorter DNA molecules. Panel (b) shows a representative ionic current trace measured during DNA translocation. Each downward spike in the current represents a single DNA molecule passing through the nanopore. The amplitude of the spike, or blockade depth, reflects the volume of the nanopore occupied by the DNA, and thus provides information about DNA length and conformation. The time between spikes corresponds to the time interval between translocation events, related to the DNA concentration in the sample. Panel (c)presents a magnified view of a single DNA translocation event. The resistive pulse is the transient reduction in ionic current caused by the DNA molecule blocking the nanopore. The ECD is the integrated area of the resistive pulse and provides a precise measure of the DNA molecule’s length. Radiation-induced damage, such as double-strand breaks, reduces the length of DNA molecules, causing a decrease in the ECD. By analyzing changes in ECD histograms, the extent of radiation-induced DNA damage may be quantified and used to determine RBE values. The nanopore measurement is highly sensitive and capable of detecting single-molecule events. This single-molecule resolution allows for precise measurement of biopolymer properties and accurate detection of even subtle changes caused by radiation exposure. The quantitative nature of the ECD measurement enables accurate calculation of RBE values and provides detailed insights into the effects of radiation on biomolecules at the molecular level.

[0206] FIG. 14 shows the process for fabricating quartz nanopipettes used in nanopore-based single-molecule analysis. Nanopipettes are small, hollow glass structures with nanoscale tips that are employed in a variety of applications, including single-molecule sensing and manipulation. In nanopore-based analysis, nanopipettes serve as a platform for measuring the properties of biomolecules, such as DNA, by analyzing their translocation characteristics through a nanopore located at the nanopipette's tip. The figure depicts the steps involved in fabricating quartz nanopipettes using a laser-assisted pipette puller, such as the Sutter P-2000. The fabrication process begins with a quartz capillary, which is a small glass tube with a defined inner and outer diameter, typically 1.0 mm and 0.5 mm, respectively. The capillary is cleaned and placed in the puller, which uses a laser to heat a localized region of the capillary. The capillary is then pulled apart, creating two nanopipettes with tapered tips. The fabrication parameters, such as laser power, pull strength, and delay time, are carefully controlled to achieve the desired tip dimensions and taper profile. The figure includes scanning electron micrographs (SEMs) of nanopipette tips, showing the nanoscale dimensions of the tip and the internal taper geometry. The tip diameter can be tuned from tens to hundreds of nanometers, depending on the application. After fabrication, the nanopipettes are filled with an electrolyte solution and used in nanopore-based analysis. The nanopore can be formed at the nanopipette’s tip using various techniques, such as laser-induced breakdown or focused ion beam milling. The fabricated nanopipettes can be employed in nanopore-based single-molecule dosimetry to measure the length of biopolymers, such as DNA, based on their translocation characteristics through a nanopore at the tip. The use of quartz nanopipettes in these measurements provides several advantages, including high sensitivity, reproducibility, and control over experimentalconditions. The fabrication process involves heating a localized region of the quartz capillary using a laser. The capillary is then pulled apart, creating nanopipettes with tapered tips. The dimensions of the tip, including its diameter and taper profile, are determined by the parameters of the pulling process, which can be carefully controlled to produce nanopipettes suitable for various nanopore-based single-molecule analysis experiments. The figure illustrates the fabrication process and the resulting nanopipette structure using both photographs and SEMs. The precision of the laser-assisted pulling method enables fabrication of nanopipettes with well-defined tip dimensions and taper profiles, improving measurement accuracy and consistency. The choice of quartz as the material ensures high mechanical strength and thermal stability, minimizing drift during measurements. The figure illustrates the fabrication of nanopipettes using a laser puller, demonstrating the method’s ability to create nanoscale tips suitable for single-molecule analysis. The precision of this method, coupled with the high quality of quartz material, provides a robust platform for nanopore-based measurements of radiation-induced damage to biopolymers.

[0207] Traditional methods for assessing the biological effects of ionizing radiation rely on macroscopic endpoints, such as cell survival or tissue damage, which provide limited information about the underlying molecular mechanisms of radiation action. These methods often require large sample sizes, long incubation periods, and can be influenced by variations in experimental conditions, leading to inconsistencies and difficulties in comparing results across different laboratories. Existing techniques may not be sensitive enough to detect subtle but important differences in biological effectiveness, especially at low radiation doses.

[0208] The processes and apparatus described herein distinguish themselves by providing a rapid, sensitive, and quantitative method for measuring radiation-induced damage at the single-molecule level. The process employs nanopore technology, in which changes in ionic current through a nanopore are measured as individual biomolecules translocate through the pore. This approach enables direct measurement of radiation-induced changes in biopolymer length, conformation, and other physical properties, offering advantages over traditional macroscopic assays that measure indirect endpoints like cell survival. The process involves preparing a known biopolymer sample of known length or composition, dissolving it in a suitable medium, irradiating the sample with a controlled dose of ionizing radiation, and then analyzing the irradiated sample using a nanopore measurement device. The device includes two electrolyte chambers separated by a dielectric membrane containing a nanopore. A voltage is applied across the membrane, driving the biopolymer through the nanopore, and changes in ionic current through the nanopore are measured and analyzed to determine theextent of radiation-induced damage. This approach involves single-molecule techniques to overcome limitations of traditional methods, and the analysis directly probes the irradiated biopolymer using nanopore technology, enabling a mechanistic understanding of radiation effects. Internal molecular standards provide a calibration tool for quantifying damage and ensuring reproducibility. The analysis provides single-molecule measurements, enhancing sensitivity, and its adaptability to various biopolymers, radiation types, and experimental conditions provides versatility. The generation of dose-response curves and the ability to quantify RBE ratios allow for direct comparison of the biological effectiveness of different types of radiation, informing treatment planning and risk assessment. The process’ rapidity and low material requirements provide clinical utility, as for diagnostic and emergency purposes and for low dose exposures. The standardized assay, based on well-known technology and employing automated quantitative measurement, should prove highly reproducible.

[0209] The articles and processes herein are illustrated further by the following Example, which is non-limiting. EXAMPLE

[0210] SINGLE-MOLECULE BIODOSIMETRY

[0211] Inferring characteristics of radiation exposure using biological molecules is extremely challenging. Current methods, in particular, lack a clear connection between dose and molecular response. Here, we demonstrate that resistive-pulse nanopore sensors enable single-molecule biodosimetry by quantifying the frequency of double-strand DNA scissions versus gamma radiation dose. The resulting response curve shows an elongated Gaussian behavior, reminiscent of cell survival rates versus dose. We demonstrate that the competition of radical damage of DNA-i.e., single-strand lesions that lead to breakage - with bimolecular radical loss captures the form of the response. Our sensors and protocol provide a foundation for numerous technological advances. These include rapid dosimetry for triage in emergency situations and ex vivo monitoring of radiotherapy effectiveness in order to tailor treatment to patient- and tumor-specific response.

[0212] Understanding the biological consequences and damage mechanisms of ionizing radiation is central to cancer therapy. Quantifying radiation exposure, in particular, enables posttherapy assessment of the delivered dose and will be important in other scenarios, such as radiological accidents or nuclear conflicts. Moreover, beyond the acute effects of high-dose exposure ( > 2 Gy ), the accumulated impact of low-dose exposure ( ≈ 100^^^^ Gy ), suchas from medical imaging or working in mines, is a poorly understood factor in assessing health. Current tools are often inadequate for these medical and emergency applications. For instance, the "gold standard" for retrospective dosimetry - dicentric chromosomal analysis-requires > 48 h of preparation time for cell culturing after sample collection to produce actionable data. Other chromosomal counting assays and proteomic tools have similar issues. Leveraging advances in biotechnology and nanoscience is a promising route to overcome such challenges and provide tools to quantitatively assess damage and mechanisms, as well as to measure the exposure of individuals rapidly and accurately under a broad range of conditions.

[0213] In this regard, the most well-studied biomarker for assessing radiation damage is DNA. It has long been known that radiation-induced damage to genomic DNA can lead to cell death, with as little as one double-strand break (DSB) being sufficient in certain circumstances. In living systems, cell death can be mitigated by the action of DNA repair enzymes. However, in a buffer containing DNA, in addition to the lack of molecular packing (as in cells), radiation damage proceeds unperturbed by repair enzymes. This yields a more straightforward picture of the damage mechanisms and rates. For DNA, damage includes both direct and indirect lesions on the bases or sugars. In practice, irradiation deposits the most energy in water-the most abundant species by mass. This initiates a cascade of reactivehydrolysis products, including ∙OH, e− ∙ ∙ ∙− +aq , H , HO2 , O2 , H3O , H2, and H2O2. Of these,∙OH, e− , O ∙− and H O2 are particularly damaging ∙aq 2 2 to DNA, with OH specifically associatedwith strand scission. H-abstraction on the ribose occurs in 10% to 20% of all∙OH reactionswith DNA, with ≈ 20% of these abstraction reactions leading to strand-break reactions.

[0214] Nanopore sensors emerged as a tool to study the properties of DNA and other biopolymers in 1996. The core sensing principle is similar to other biophysical research tools, such as the Coulter counter: A thin membrane (with a narrow pore) partitions an electrolyte solution, where a voltage drop drives an ionic current from one side to the other. When the analyte is driven through the pore - via fluid pressure for a Coulter counter or electrophoretically for DNA - a resistive pulse occurs in the current. The capture process itself is complex. The electric fields and polymer dynamics, as well as the interplay between electrophoretic and electroosmotic forces with the effective charge of the molecule, all play a role. The molecular volumes of nanopores (rather than cellular volumes of Coulter counters) allow the system to measure single molecules. In particular, the magnitude of the resistive pulse is proportional to the volume of the pore occupied by the translocating molecule. For long,uniformly-charged molecules, such as DNA, integrating the pulse yields a value proportional to the molecule's length.

[0215] Since introduction in 2001, solid-state nanopores are a workhorse of single- biomolecule sensing. A common approach is to fabricate nanopores in ultrathin silicon nitride membranes, which enables sampling above megahertz frequencies and therefore maximizes discrimination of sublevel blockades within individual translocations. Yet, these pores are highly susceptible to fouling. Other geometries and materials, such as conical pores in quartz glasses, are similarly effective for sensing. On-demand laser pulled capillaries, for instance, provide a simple, deployable alternative to more difficult to fabricate pores. Yet, like other solid--state pores, they have highly variable pore characteristics (surfaces, taper, etc.). Despite this, we employ pulled quartz nanopipettes to demonstrate single-molecule biodosimetry. We will provide a method that can tolerate large pore-to-pore variation and enable precise, quantitative measurement of DNA damage, while being simple to use.

[0216] Herein, single-molecule biodosimetry employs nanopores to quantify double strand breaks from an irradiated sample. Here, we expose a series of aqueous solutions containing 2.5 kbp DNA, FIG.1a, to gamma radiation with doses up to 15 Gy, which results in double-strand breaks, FIG.1b. In order to extract quantitative information, two longer DNA molecules (here, 5 kbp and 10 kbp ) are added post-irradiation as simultaneous concentration and molecular-length standards.

[0217] During measurement, DNA is electrophoretically driven into the mouth of a glass nanopipette, FIG.1c. The resulting current blockades - the resistive pulses - are analyzed and compiled into a histogram of molecular length, FIG. 1d. The equivalent charge deficit (ECD) - the total area of a resistive pulse (shaded in FIG. 2a,b) - yields information on the molecular size for a single nanopore.

[0218] Yet, it is not feasible to use the same pore for every dose, as there can be both cross contamination of samples and pore fouling (typically after less than two hours, depending on analyte concentration and pore size). These are common limitations for solid- state nanopores, and we must calibrate pore-to-pore variations between a series of pores prepared identically. Indeed, as seen in FIG.2c, there is a large variability in the uncalibrated ECD peaks for each dose. We introduce two internal standards, which provides a correction for both size and concentration simultaneously, see FIG. 2c,d. Moreover, it also yields quantitative data about uncertainty.

[0219] The resulting dose-response curve agrees well with a model of double- strand breaks originating from two nearby single-strand lesions. Regardless of whether thoselesions form directly from radiation damage or indirectly from reaction of the DNA with radicals, they deplete the initial intact DNA concentration Φ0according to a universal form in the small ^^^^Λlimit,Here, ^̃^^^ is the effective length (in number of base pairs) of the DNA and ^^^^Λis the lesion probability, i.e., the likelihood of individual single-strand lesions. The effective length, ^̃^^^, is proportional to the actual length of DNA and, unlike ^^^^Λ, independent of dose. While Eq. (1) is general, we consider only indirect damage via radical reactions, as the mass fraction of DNA is very small.

[0220] To lowest order in ^^^^, the probability to generate a lesion should growlinearly with the energy deposited per unit mass, or dose, i.e., ^^^^Λ ∼ ^^^^. A reaction model whereradicals damage nucleotides and are also subject to (effective) unimolecular decay gives such a linear dependence provided the initial radical concentration formed by water radiolysis itselfis linearly dependent on ^^^^. FIG. 3 suggests Gaussian decay of Φ with ^^^^. Yet, a lowdose ( ≤ 3Gy) fit (the red, dotted line in FIG. 3), where this model should be valid, gives significantly less intact DNA than expected at large ^^^^. This indicates that another important reaction or other process must be present.

[0221] Bimolecular decay of radicals, i.e., two radicals combine to form an inert species, may be such a reaction. If this loss mechanism is present, the intact DNA still follows Eq. (1) but with a defect probabilitywhere ^^^^ is the rate constant for DNA damage, ^^^^ the bimolecular radical decay rate, ^^^^ the unimolecular radical decay rate, ^^^^0the initial nucleotide concentration during irradiation, and^^^^0 = ^^^^^^^^ is the initial radical concentration. In the latter, ^^^^, a constant of proportionalityrelating ^^^^0to dose, ^^^^, is the radiation chemical yield.40This model has several parameters. Yet, as a fit to experimental data, it has only two effective parameters,

[0222] With K and Γ, the experimental data is fit extraordinarily well (green, dashed line in FIG.3). We note that these two parameters are correlated, with a decrease in one generally occurring with an increase in the other (i.e., КΓ is roughly constant since the initialdecay is Gaussian). The best fit to the data (without accounting for uncertainty) is K = 1.23and Γ = 0.155 Gy−1. Including uncertainty, the fit is in FIG. 3, with the main line the averageintact concentration from the joint distribution of K and Γ, and the shaded region given by plusand minus one standard deviation. Treating the two parameters independently gives K = 1.3 ±0.4 and Γ = (0.16 ± 0.05)Gy−1, yet this does not capture their correlations.

[0223] Extraction of any of the fundamental parameters in Eq. (3) (i.e., ^̃^^^, ^^^^,^^^^,^^^^,or ^^^^ ) requires either tuning various quantities (such as concentrations) or independently measuring or estimating some subset of those parameters. For instance, the ratio of ^^^^ (SSB generation rate) to ^^^^ (bimolecular radical decay) depends only on ^̃^^^ and K, see Eq. (3). Prior measurements provide the length scale, ^^^^, for which SSBs on opposite strands lead to a DSB,which is linearly related to ^̃^^^ via ^̃^^^ = (2^^^^ − 1)^^^^ (see the Methods). Those measurements give^^^^ from about 3 bp to 16 bp for high and low ionic strengths, respectively, or ^̃^^^ between 3^^^^ and31L. Taking ^̃^^^ = 10^^^^, inline with the ionic conditions here, gives ^^^^ / ^^^^ = 0.017 ± 0.005.

[0224] The value of ^^^^ allows us to go one step further and quantify ^^^^ (unimolecular radical decay). Prior measurements of the ^^^^ for∙OH give, in conventional units, 2.56 to 5.5molecules per 100 eV of deposited energy. Taking ^^^^ = 2.56, which is relevant for theconditions here, gives ^^^^ = (3.4 ± 0.4)^^^^^^^^0. The radical scavenging rate is thus of the sameorder as ^^^^^^^^0, the total SSB rate. This suggests that the dominant (effective) unimolecular decay route is to create DNA damage that does not lead to breakage. Future experiments (e.g., versus ^^^^0and ^^^^ ) can unambiguously demonstrate this molecular mechanism.

[0225] Measuring universal, reproducible trends with multiple solid-state nanopores, even pores that are nominally identical, has generally proven elusive. Pore-to-pore variability in diameter, internal taper, and surface charge and roughness, among other factors, has a significant impact on characteristic molecular signatures, such as ECD. We have taken a two-pronged approach to overcome the challenges in getting quantitative trends. Firstly, weemploy relatively large nanopipettes (i.e., ≈ 12 nm to 17 nm ), which leads to a number ofadvantages over reliance upon smaller pores in addition to ease of use: (i) Large pores frequently enable collection of large data sets (approximately 10 k events before pore fouling); (ii) They are more robust, filling the narrow sensing region of the tip completely a larger percentage of the time and giving a higher yield of pores with very low baseline noise ( ^^^^^^^^^^^^^^^^≈ 4.4pA ); and (iii) They have a flat capture probability across the size range of the three target molecules as shown in FIG. 4c. Incidentally, the nanopipettes also enable recovering the sample, as opposed to traditional solid-state pores where one would have to pipette out the sample and risk contamination. Secondly, we correct for the analyte size and capture-ratefluctuations that result from the natural pore-to-pore variability by using the fixed sizes and concentrations of the two internal molecular standards, which allows us to infer the concentration of all molecules in our target size range, from 2.5 kbp to 10 kbp , for our nanopipettes.

[0226] This approach enables low uncertainty, molecule-by-molecule measurement of the intact DNA concentration. Yet, using larger pores and optimizing the standards around them is not without trade off: We lose resolution of the concentration of small fragments. FIG.4a shows the concentration across a wide range of molecular sizes, where the weight on the smaller fragments is smaller than expected. There are two primary reasons for this. For small molecules, it is not possible to fully distinguish capture and translocation, partial capture, and system noise. Also, there is a complicated interplay of physical processes on the capture of molecules diffusing near the terminal aperture. Generally speaking, for negatively charged surfaces, the electro-osmotic force on the exterior of the tip of the nanopipette, mediated through fluidic drag interactions, creates a spherical region of pre-concentrated DNA around the pore mouth. Competition occurs between many molecules fluctuating in and out of the high electric field immediately inside the vestibule of the nanopipette, ultimately giving single-file capture. Overall, this latter factor dominates and leads to a lower number of small molecule (relative to the pore) translocations than their concentrations would indicate.

[0227] In other words, the correction of capture events needs to be non-linear. DNA standards with a size of the same order as our target DNA permits a linear correction for the larger size molecules but under weights small fragment events. As a preliminary estimate, we measure the bias, FIG.4c, of a ladder of known fragment concentrations below the intact DNA size. This is done with three nanopipettes identically prepared to those in the primary experiments. We note that this provides an average correction factor rather than a pore-by-pore calibration as was done for the primary experiments. Consequently, the error bars are larger for the small fragments. Despite this lack of optimization, the corrected ratio of measured DNA fragments (in nucleotides) to intact 2.5 kbp DNA (in nucleotides) monotonically increases with dose, see FIG.4d. In some scenarios, such as emergency exposure, the initial concentration of undamaged DNA in a sample will not be known. Under such conditions, a ratiometric approach-i.e., measuring the concentration of fragments relative to intact DNA - may enable determination of the dose to the needed accuracy by employing ratio-dose response data like that in FIG.4d. To apply the ratiometric approach rigorously, one would first need to overcome many deficits in the theory of nanopore capture physics. We anticipate that ratiometric data canbe significantly improved using pores optimized for quantitative measurement of molecules on the order of the fragment size.

[0228] Post-irradiation DNA concentration

[0229] We prepare DNA solutions such that, after irradiation and dilution, the concentration of total nucleic acid is equivalent 3nM(4.875ng / ^^^^L)2.5kbp DNA. We confirm this after irradiation with UV-vis spectroscopy, FIG.5.

[0230] Ionic current signals

[0231] FIG.6 shows the raw ionic current data (left panels) from each of the eight runs used to measure the intact DNA concentration as a function of dose, as well as direct uncorrected histograms (right panels). Each run begins with identically-prepared DNA solution from a single stock solution. We collect each time-series with a unique nanopipette both to prevent cross contamination and to prevent fouling. We then use the Nanolyzer software package to extract and tabulate events (based on ECD). This data highlights a variety of different capture rates that appear uncorrelated to dose, as well as the variability in pore characteristics.

[0232] Sample preparation

[0233] 2.5 kbp DNA fragments were exchanged from TE buffer ( 10 mM tris(hydroxymethyl)aminomethane, 2 mM ethylenediaminetetraacetic acid) to 20 mM sodiumphosphate ( pH = 7.4 ) using 1 kDa cut-off mini dialysis tubes ( Cy tiva) in order to minimizethe effects of radical scavenging. The molecular concentration of DNA in the irradiated solution was 10 nM .5 kbp and 10 kbp DNA were added post irradiation as internal standards to calibrate for absolute DNA size and concentration. This accounts for any variation in nanopipette geometry between runs. For irradiation, 300^^^^ L of solution was placed into eight 0.5 mL DNA LoBind tubes. Each solution was then exposed to a gamma-ray field from a Gammacell 22060Co irradiator for the time required to achieve the desired dose. The dose rate was 1.373 Gy / min and the irradiation temperature was 23∘C. The uncertainty in absorbed dose in water is bounded by ±1.6% for this irradiation geometry.

[0234] Nanopipette measurement

[0235] Laser pulling process

[0236] With reference to FIG. 14, we constructed the pulled nanopipette pores as follows. We first clean 7.5 cm quartz capillaries (O.D.1.0 mm , I.D.0.50 mm ) with an internal filament (Sutter instruments) by sonicating them upright in acetone for 15 minutes. They are then dried with compressed air and baked in a 70∘C oven for 20 min . The pulling process usesa Sutter P-2000G laser-assisted pipette puller set to following parameters: HEAT = 575, FIL =0, VEL = 25, DEL = 180, and PUL = 225. After pulling, we secure the nanopipetteshorizontally and fill them with 4 M LiCl and 10 mM TE buffer using 20^^^^ L microloader pipette tips. If an air bubble was visible near the nanopipette tip, then we refill the nanopipette in the same manner until the air bubble is removed.

[0237] Nanopipettes were filled with a solution of 4 M LiCl and 10 mM TE buffer at pH 7.4. The exterior of the nanopipette was dipped into a 3 nM solution of the irradiated 2.5 kbp DNA, along with aliquots of the 5 kbp and 10 kbp internal standards (added at 0.3 nM concentration) in identical 4 M LiCl and 10 mM TE buffer. Each measurement was performed by a separate nanopipette to avoid cross contamination. Ionic current was monitored with an Axopatch 200B amplifier (Molecular Devices, San Jose, CA) sampling at 500 kHz , with a low pass bessel filter of 10 kHz . The ionic current data was analyzed using a threshold algorithm set at 5^^^^, where ^^^^ is the standard deviation of the ionic current to identify DNA-based resistive pulses. The signals were then decoded with a modified CUSUM-based algorithm as implemented in the Nanolyzer software package (Northern Nanopore, Ottawa, Canada).

[0238] Modeling-We consider a model for the DNA damage process that has two overall components. The first (combinatorial) component is how individually damaged nucleotides lead to double-strand DNA breaks (therefore reducing the intact DNA concentration we measure). The second is a reaction model for the damage of individual nucleotides. This reaction model provides the input - the defect probability, ^^^^Λ-into the first component. Otherwise, the two components are independent.

[0239] Component 1

[0240] When individual nucleotides are damaged along the double-helix, some fraction of those will result in a double-strand break. The damaged nucleotides-in particular, single-strand breaks - on opposing strands will likely lead to a double-strand break when theyare within ^^^^ − 1 base pairs of each other (i.e., for ^^^^ = 1 this means that the two breaks have tobe on a nucleotide pair). At room temperature and under normal salt conditions, ^^^^ should be of the order of 5 base pairs. That is the scale when the base pairs in between two single-strand breaks can unravel and the double helix breaks into two fragment helices.

[00241] The model thus needs to find the likelihood of two defects being within ^^^^ −1 base pairs of each other when every individual nucleotide has a probability ^^^^Λof being damaged. For a double-helix of length ^^^^ base pairs, a configuration of damaged (" 0 ") and pristine (" 1 ") nucleotides will occur with probability^^^^where ^^^^^^^^ = 0 or 1 for the nucleotide on the top strand at position ^^^^ and ^^^^^^^^ = 0 or 1 for thebottom strand. Summing Eq. (4) over all configurations is equal to 1 and thus covers the configuration space.

[0242] Up to the resolution of detection, every original double-strand of DNA that has one or more double-strand breaks will reduce the intact DNA concentration. Thus, we need to sum Eq. (4) over all configurations that do not have any double-strand breaks, i.e., allconfigurations that do not have a damaged nucleotide on the top strand within ^^^^ − 1 base pairsof a damaged nucleotide on the bottom strand. When ^^^^ = 1 and only damaged nucleotides in apair lead to a double-strand break, the sum should be over (^^^^^^^^ , ^^^^^^^^) = (1,0), (0,1), and (1,1) foreach ^^^^. This sum can be done sequentially, resulting in a total intact DNA helix probability, ^^^^, of

[0243] The approximate equality is from a Taylor expansion and is accurate forsmall ^^^^Λ. As a side note, exp (−^^^^^^^^2Λ) upper bounds (1 − ^^^^2Λ)^^^^ and this approximation can notexplain the higher intact DNA concentrations that we observe at high doses.

[0244] Beyond this simple case, though, the sum over intact configurations willlead to a transfer matrix. However, the net effect of ^^^^ > 1 is only to introduce an effectivelength scale ^̃^^^, leading to ^^^^ ≈ ^^^^−^̃^^^^^^^Λ2 (6)for small ^^^^Λ, which gives Eq. (1) when converted to a concentration. For the purposes here, this is all that is needed, since the fitting of experimental data employs aggregate parameters and thus cannot distinguish changes in ^̃^^^ versus other parameters (see component 2 below).

[0245] In order to provide estimates of those other parameters, though, it is important to have a ballpark estimate of ^̃^^^. When ^^^^Λis small, the relationship of ^̃^^^ and ^^^^ is as follows: When any given nucleotide on one strand is damaged (with probability ^^^^Λ), then the likelihood that there is damaged nucleotide on the opposing strand that will lead to a DSB issimply (2^^^^ − 1)^^^^Λ. The overall probability, neglecting end effects, that there is damage onopposing strands within ^^^^ of each other is then ^^^^^^^^Λ ⋅ (2^^^^ − 1)^^^^Λ, giving an effective length of^̃^^^ = (2^^^^ − 1)^^^^. Considering ^^^^ ≈ 5, we expect ^̃^^^ ≈ 10 ⋅ ^^^^, which is the value we use to estimateother fundamental parameters.

[0246] We note that the effective length absorbs not only the physical effect of how single-strand breaks lead to double-strand breaks but also measurement effects. When analyzing real data, ^̃^^^ will also be impacted by measurement resolution. For instance, if the DNA breaks near its end, one will not be able to resolve the broken DNA from its intact form. Thus, one should subtract off some length (e.g., below about 100 base pairs for the measurements and analyte here). We work with a quite long sequence of DNA- 2.5 kbp -and we do not expect the latter to be significant.

[0247] Component 2

[0248] The likelihood of a gamma photon directly interacting with a nucleotide is roughly in proportion to the ratio of the mass of DNA in solution to the mass of water (here, about 0.002% ). Therefore, under our conditions, radicals mediate almost all of the DNA damage. As the irradiated sample equilibrates, every∙OH radical will react.∙OH can abstract hydrogen from the sugar of the nucleotide creating a SSB, or any of the bases which can propagate to a SSB or terminate in a host of non-scission terminating modifications. All other hydroxyl radicals have to react with a radical scavenger, which includes other radicals produced upstream in radiolysis reactions.

[0249] Giving these considerations, we consider the reactionswhere ^^^^ is the total nucleotide concentration, ^^^^ the radical concentration, and Λ the total defect concentration. Defects occur via a bimolecular reaction of nucleotides and radicals with rate ^^^^. Radicals decay via a unimolecular process with rate ^^^^ and a bimolecular process with rate ^^^^, both resulting in annihilation of the radicals (i.e., an inert chemical species, ∅ ). All reactions are taken to be irreversible since they all involve a substantial dissipation of energy. We emphasize that the unimolecular decay process does not influence the model fit to experimental data since it does not increase the number of effective (fit) parameters. However, it does influence the extraction of basic parameters and their interpretation. We also note that the reaction model, Eq. (7), does not consider any particular radical species or damage product. The underlying phenomenology is that the exact microscopic processes are unimportant to understand the emergence of double-strand breaks and the details are all absorbed by effective rate parameters.

[0250] The reactions above give rise to a set of rate equations,^^^^^^^^ ^^^^^^^^= −^^^^^^^^^^^^ (10)^^^^Λ ^^^^^^^^= ^^^^^^^^^^^^ (11)^^^^^^^^ =−^^^^^^^^^^^^ − ^^^^^^^^ − 2^^^^^^ 2^^^^^^^^^^ (12)

[0251] In addition to these rate equations, we need the initial concentrations. The initial total nucleotide concentration during irradiation is ^^^^0 = 2^^^^Π0, (13)where ^^^^ is the length of the DNA and Π0is its initial concentration of DNA during irradiation( Π0 = 10nM here). The initial radical concentration is taken to be linearly proportional to theradiation dose according to the well-known ^^^^-value, ^^^^0 = ^^^^^^^^. (14)

[0252] We assume that immediately after gamma irradiation the solution contains a uniform distribution of hydroxyl radicals and all nucleotides are equally likely to be damaged.Finally, the initial defect concentration is zero, Λ0 = 0.

[0253] When there are a lot of nucleotides compared to radicals (i.e., ^^^^0 ≫ ^^^^0 ) orthe radical loss is fast compared to defect generation, then the depletion of nucleotides will besmall, i.e., ^^^^0 ≫ Λ(^^^^ → ∞). Under these conditions, we can approximate the rate equations as^^^^^^^^ ^^^^^^^^≈ −^^^^^^^^^^^^0 (15)^^^^Λ ^^^^^^^^≈ ^^^^^^^^^^^^0 (16)^^^^^^^^ ^^^≈ −^^^^^^^^^^^^ − ^ 2^^^^^0 ^^^^^^^ − 2^^^^^^^^ (17)

[0254] Replacing ^^^^ with ^^^^0on the right hand side of these equations will give a larger defect concentration at all times since ^^^^ decreases monotonically with time. For theexperimental results we present in this work, ^^^^Λ < 0.01 for all conditions (i.e., all radiationdoses). The magnitude of this probability indicates that replacing ^^^^ with ^^^^0is a good approximation. We note that ^^^^ is an effective unimolecular decay parameter, since one decaychannel is to react with nucleotides in a way that does not cause an SSB (i.e., ^^^^ ∝ ^^^^0 ).

[0255] The approximate set of equations, Eq. (15) through Eq. (17), can be solved analytically.

[00256] The solution for the radical concentration, taking ^^^^ = ^^^^^^^^0 + ^^^^, isand the defect concentration is

[0257] After all radicals react, the defect probability is Λ(^^^^ → ∞)^^^^ 2^^^^^^^^ =lo 0^^^^ 2^^g�1 + � . (20)0^^ ^^^^ This is the main quantity of interest, Eq. (2), and is the input to the model of double-strand breaks, Eq. (1).

[0258] Finally, the fitting to Eq. (1) is done with the two effective fit parameters, Eq. (3), as we describe in the main text. Errors on the fit parameters are found via a boot- strapping calculation where the intact DNA concentrations are chosen according to a Gaussian distribution around their experimentally observed value with standard deviation given by the experimental error, and dose is taken to uniformly have a 1.6% error. The prefactor-the intact DNA concentration, Φ0, when there is no irradiation - is taken as a known constant in each calculation within the boot-strapping process (i.e., Φ0is taken as its value in the realization of noise). Errors for fundamental quantities come from the joint distribution of fit parameters.

[0259] We demonstrate single-molecule biodosimetry using quartz nanopipettes. Our results give the first quantitative, direct connection between dose and DNA response. Calibration against internal molecular standards, as we introduced here as a parallel to DNA electrophoresis ladders, is used for the acquisition of fully quantitative results and will be critical to the adoption of nanopores in applications outside of specialized academic labs. The DNA dose response curve exhibits an elongated Gaussian shape - one similar to cell death response curves - that strongly indicate double-strand DNA breakage proceeds with three key characteristics: (i) indirect single-strand breakage by reaction with radicals, (ii) nearby SSBs on opposing strands generate a DSB, and (iii) damage competes with bimolecular radical decay in the relevant dose range for clinical and emergency response applications. This agrees with prior results that suggest reaction with radicals, rather than direct damage with high energy particles, is the dominant mechanism of DNA damage in near physiological conditions.

[0260] Nanopore-based sensors thus show promise for a wide range of applications, including rapid dosimetry after accidental, imaging, or therapeutic radiation exposures. For instance, DNA damage due to different types of external beam radiation (x-rays, electrons, protons, etc.) or radiopharmaceuticals that emit many types of short-range radiation (e.g., soft x-rays, auger electrons, ^^^^ particles, ^^^^ particles), could be measured and characterized. We believe this data would provide quantitative information on the sensitivity of DNA to a rangeof different energies, linear energy transfer, dose rates, and other properties of radiation. Such information would be a fundamental building block to new models and dose calculations that improve how we use radiation to treat cancer.

[0261] Equivalent charge deficit (ECD) to measure the length of a DNA molecule

[0262] FIG.15 illustrates the using equivalent charge deficit (ECD) to measure the length of a DNA molecule translocating through a nanopore. Nanopore-based single-molecule analysis measures the properties of individual biomolecules, such as DNA, by analyzing changes in ionic current as they pass through a nanopore. As a DNA molecule translocates through the nanopore, it partially blocks the flow of ions, creating a transient reduction in the measured current. The duration and magnitude of this current reduction, or blockade, reflect the DNA molecule’s physical properties, enabling length determination and detection of radiation-induced damage. The figure depicts a DNA molecule translocating through a nanopore at the tip of a quartz nanopipette. The nanopipette is filled with an electrolyte solution, and a voltage is applied between the nanopipette and a bulk electrolyte solution containing the DNA molecules. The applied voltage generates an electric field that drives the DNA molecules towards and through the nanopore. As a DNA molecule enters and passes through the nanopore, it partially blocks the flow of ionic current, creating a characteristic “resistive pulse” in the measured current. The ECD, which is the time integral of the resistive pulse, is proportional to the DNA molecule’s length. The figure shows a representative ionic current trace, with each downward spike corresponding to a DNA translocation event. The shaded area of one of the spikes represents the ECD for that event. Radiation-induced damage to DNA, such as double-strand breaks, can cause fragmentation of the DNA molecules, resulting in shorter fragments that exhibit smaller ECD values. By analyzing changes in ECD histograms after exposure to varying radiation doses, the biological effects of the radiation can be quantified, and RBE values can be determined. Nanopore technology's single-molecule sensitivity enables detection of even small changes in DNA length caused by radiation, providing a precise and quantitative method for measuring RBE. The duration and magnitude of resistive pulses associated with biopolymer translocations depend upon the characteristics of both the nanopore and the biopolymer being analyzed. Variations in nanopore geometry or surface charge can affect the ionic current signals, as can variations in the biopolymer’s length, charge, or conformation. To minimize these effects, nanopipettes with well-defined tip dimensions are fabricated using laser-assisted pulling techniques, biopolymers with known lengths or compositions are used, and electrolyte conditions, such as salt concentration and pH, are carefully controlled. The ECD is calculated by integrating the area of the resistive pulse.This integration provides a precise measure of the charge deficit caused by the DNA molecule blocking the nanopore, reflecting its length. The figure shows a representative ionic current trace, with each downward spike corresponding to a DNA translocation event. The area of each spike, or resistive pulse, is proportional to the DNA’s length. Longer DNA molecules generate larger resistive pulses and thus have higher ECD values compared to shorter DNA molecules. By analyzing changes in the distribution of ECD values, radiation-induced DNA damage can be quantified and RBE values may be determined. The experimental conditions, such as the applied voltage, the electrolyte concentration, and the dimensions of the nanopore, are carefully controlled to optimize the signal-to-noise ratio and ensure accurate ECD measurements. The use of quartz nanopipettes enhances sensitivity by minimizing noise and drift. This sensitivity enables detection of even subtle changes in DNA length caused by radiation, and the quantitative ECD measurement provides valuable information about the effects of radiation on biomolecules. This information is useful for a range of applications, including radiation therapy treatment planning and development of radioprotective countermeasures.

[0263] Extracting molecular size from ionic current

[0264] FIG. 16 further illustrates extracting molecular size from ionic current in nanopore-based single-molecule analysis. The process involves driving a biopolymer, such as DNA, through a nanopore under an applied electric field and measuring changes in the ionic current flowing through the pore. As the biopolymer translocates through the nanopore, it partially blocks the flow of ions, causing a transient reduction in current. The magnitude and duration of this current blockade, or resistive pulse, reflect the biopolymer’s properties, including length, enabling size determination. The figure shows a schematic of a DNA molecule translocating through a nanopore, along with a representative ionic current trace and a corresponding ECD histogram. The schematic shows a voltage (V) being applied across a membrane containing a nanopore. Electrophoretic and electroosmotic forces (represented by arrows) act on the DNA molecule, driving it through the nanopore. As the DNA molecule enters and passes through the nanopore, it partially blocks the flow of ionic current, generating a resistive pulse. The magnitude of the pulse, or blockade depth, provides information about the DNA’s size and conformation. The integrated charge, or ECD (represented by the shaded area in the trace), gives the length of a molecule translocating pore. The ionic current trace shows individual DNA translocation events as transient downward spikes. The ECD, proportional to DNA length, is calculated for each event. The ECD histogram plots the frequency of observed ECD values, providing information about the DNA’s length distribution in the sample. The inset graph shows an ECD histogram for three different DNA lengths: 200,2,500, and 10,000 base pairs. The histogram peaks occur at distinct ECD values, reflecting the different DNA lengths. Radiation-induced DNA damage causes fragmentation, resulting in shorter DNA fragments and a shift in the ECD histogram towards lower ECD values. By analyzing changes in the ECD histogram after exposure to varying radiation doses, one can quantify DNA damage and determine RBE values. The nanopore measurements are performed in a controlled environment, with precise control of experimental conditions, such as applied voltage, electrolyte concentration, and temperature, which ensures high sensitivity and reproducibility. The use of quartz nanopipettes minimizes noise and drift. This stability enables the detection of even subtle changes in ECD caused by radiation-induced DNA damage. The precise length determination of DNA molecules by ECD is particularly valuable for studying the effects of radiation at the molecular level. The figure illustrates how nanopore technology is used to measure the length of individual DNA molecules. As the DNA molecule translocates through the nanopore, it causes a transient blockade in the ionic current. The magnitude and duration of this blockade, or resistive pulse, depend on the length of the translocating molecule. The ECD, which is calculated by integrating the area of the resistive pulse, is proportional to the DNA length. The figure shows a representative ionic current trace with multiple resistive pulses, and the inset shows the corresponding ECD histogram. The histogram displays the frequency of occurrence of different ECD values. The peaks in the histogram occur at ECD values corresponding to the lengths of the DNA molecules in the sample. The process is applicable to a wide range of biomolecules, and it is particularly well-suited for analyzing DNA damage caused by radiation. The high-resolution single-molecule measurements enable quantification of damage, RBE determination, and provide insights into radiation's biological effects at the molecular level.

[0265] Radiation-induced fragmentation of a 2.5 kbp DNA molecule

[0266] FIG.17 depicts radiation-induced fragmentation of a 2.5 kbp DNA molecule analyzed using nanopore technology. Nanopore-based single-molecule dosimetry quantifies radiation-induced damage to biopolymers, such as DNA, by measuring changes in their translocation characteristics through a nanopore. As a DNA molecule passes through the nanopore, it causes a transient blockade in ionic current. The magnitude and duration of this blockade, or resistive pulse, are related to the DNA's length and conformation. Radiation- induced damage, such as double-strand breaks, fragments DNA molecules, resulting in shorter fragments with altered translocation characteristics. The figure shows ionic current traces, a 2D histogram (average blockade versus log10(ECD)), and corresponding ECD histograms for a 2.5 kbp DNA molecule exposed to varying doses of gamma radiation. The left panels displayrepresentative ionic current traces for the DNA molecule irradiated at 0 Gy, 10 Gy, and 50 Gy. At 0 Gy, the DNA molecules are mostly intact, and the ionic current trace exhibits relatively long blockades, indicative of long DNA molecules passing through the nanopore. As the radiation dose increases, double-strand breaks accumulate and fragment DNA, resulting in shorter DNA molecules that exhibit shorter blockade durations in the ionic current traces, as seen in the 10 Gy and 50 Gy panels. The middle panel presents 2D histograms of average blockade depth versus log10(ECD) for the same DNA molecule irradiated at the three different doses. Average blockade depth, calculated as the average magnitude of the resistive pulse, provides information on the size and conformation of the translocating DNA. ECD, determined by integrating the area under each resistive pulse, reflects the DNA’s length. At 0 Gy, the 2D histogram shows a distinct cluster of events corresponding to intact 2.5 kbp DNA. As the radiation dose increases, the cluster spreads downwards and to the left, indicative of DNA fragmentation. The right panel shows the ECD histograms for the three radiation doses. The histograms reveal a decrease in the concentration of intact 2.5 kbp DNA (indicated by the arrow) and an increase in the concentration of shorter DNA fragments with increasing dose. This dose-dependent fragmentation provides a means for quantifying radiation-induced damage and determining RBE. This figure demonstrates how nanopore technology provides a sensitive and quantitative method for measuring radiation-induced DNA damage and determining RBE. The ionic current traces and ECD histograms reveal the extent of DNA fragmentation caused by varying doses of gamma radiation. The 2D histograms demonstrate how fragmentation affects both the blockade depth and ECD of the DNA molecules. The dose- dependent shift in the ECD histograms is consistent with an accumulation of radiation-induced double-strand breaks, providing direct information on the biological effectiveness of the radiation. The method is applicable to a wide range of biomolecules, making it a versatile tool for analyzing radiation effects on various cellular components. This data is valuable for assessing radiation risks, optimizing radiation therapies, and developing radiation protection strategies.

[0267] 2D histograms and corresponding ECD histograms

[0268] FIG.18 shows 2D histograms and corresponding ECD histograms for a 2.5 kbp DNA molecule exposed to 0 Gy and 50 Gy of gamma radiation. Nanopore-based single- molecule dosimetry measures radiation-induced damage to biopolymers by analyzing changes in their translocation characteristics through a nanopore. As a biopolymer, such as DNA, passes through a nanopore, it causes a transient blockade in the ionic current flowing through the pore. The magnitude and duration of this current blockade, or resistive pulse, reflect the biopolymer’sphysical properties, including its length and conformation. Radiation can induce damage to DNA, such as double-strand breaks, leading to fragmentation and changes in translocation characteristics. The 2D histograms plot the average blockade depth (a measure of the DNA's size and conformation) versus log10(ECD) (a measure of DNA length), providing a comprehensive view of how radiation affects both properties. The ECD histograms provide the distribution of DNA lengths in the sample, revealing the extent of radiation-induced fragmentation. The left panels show data for a 2.5 kbp DNA molecule exposed to 0 Gy of radiation (control). The 2D histogram exhibits a well-defined cluster of events, indicating a relatively homogenous population of intact DNA molecules. The corresponding ECD histogram shows a sharp peak at an ECD value corresponding to 2.5 kbp, further confirming that the DNA is predominantly intact. The right panels present data for the same DNA molecule irradiated at 50 Gy. The 2D histogram shows a significant spread of events downwards and to the left, reflecting the heterogeneous mixture of DNA fragments of varying lengths produced by radiation-induced fragmentation. The ECD histogram reveals a substantial decrease in the peak corresponding to the intact 2.5 kbp DNA and a broad distribution of shorter DNA fragments at lower ECD values, further demonstrating fragmentation. The data show how radiation-induced DNA damage is measured and quantified using nanopore technology. The 2D histograms provide a visual representation of the changes in DNA length and conformation caused by radiation, while the ECD histograms enable quantification of these changes. The dose-dependent shift in the distribution of events in the 2D histograms and ECD histograms provides a quantitative measure of radiation-induced damage and enables RBE determination. The method's sensitivity is evident in its ability to detect fragmentation at the single-molecule level. The use of nanopore technology provides several advantages for RBE measurements, including high sensitivity, single-molecule resolution, and the ability to analyze a wide range of biopolymers. The quantitative nature of the measurements ensures accuracy and reproducibility, and the 2D histograms provide a comprehensive view of radiation's impact on biopolymer properties. The method is applicable to a variety of radiation types and biomolecules, and it can be employed for measuring RBE in diverse experimental settings. The detailed information obtained from nanopore analysis allows researchers to study the molecular mechanisms of radiation damage, informing the development of more effective radiation therapies and protection strategies. The method's high throughput capabilities enable rapid screening of large numbers of molecules, and its adaptability to various experimental conditions enhances versatility. The use of 2D histograms provides a means of visualizing and quantifying the effects of radiation on both DNA length and conformation.

[0269] Radiation-induced DNA damage analysis

[0270] FIG. 19 shows results of radiation-induced DNA damage analysis using nanopore-based single-molecule dosimetry. The figure presents ionic current traces and ECD histograms for a DNA molecule exposed to varying doses of gamma radiation. The left panels display representative ionic current traces for the DNA molecule irradiated at 0 Gy, 10 Gy, and 50 Gy. At 0 Gy, the trace shows long current blockades corresponding to intact DNA molecules passing through the nanopore. As the radiation dose increases, the blockades become shorter and more frequent, reflecting fragmentation of the DNA molecules due to double-strand breaks. The right panel shows ECD histograms for the DNA samples at 0 Gy, 10 Gy, and 50 Gy. The histograms reveal the distribution of DNA fragment lengths following radiation exposure. At 0 Gy, the histogram shows a distinct peak representing the intact DNA molecules. With increasing dose, the peak height decreases as intact DNA is fragmented, and smaller DNA fragments becomes more prevalent as reflected by a broadening of the peak and an increase in the frequency of shorter DNA fragments. These results reveal the sensitivity of nanopore-based single-molecule dosimetry to radiation-induced DNA damage. The data demonstrate how radiation dose correlates with a decrease in intact DNA molecules and a corresponding increase in DNA fragmentation. The ECD histograms provide quantitative information about the fragment length distributions, enabling precise measurement of DNA damage. The method is applicable to a wide range of DNA lengths and radiation types, and it can be adapted to study other biomolecules, such as RNA and proteins. The method allows for direct measurement of radiation-induced DNA damage at the single-molecule level. This high-resolution approach provides advantages over conventional cell-based assays, including speed, sensitivity, and the ability to quantify specific types of DNA damage, such as double-strand breaks. The technique's high throughput allows for analysis of large numbers of molecules, ensuring statistical significance, while its ability to be automated reduces operator bias and enhances reproducibility.

[0271] Effects of high-linear energy transfer (LET) proton radiation and low-LET gamma radiation on DNA

[0272] FIG. 20 compares the effects of high-linear energy transfer (LET) proton radiation and low-LET gamma radiation on DNA. Linear energy transfer (LET) refers to the amount of energy deposited by radiation per unit length of travel through a material. High-LET radiation, such as protons or alpha particles, deposits energy more densely than low-LET radiation, such as gamma rays or X-rays. This difference in energy deposition patterns can lead to variations in biological effectiveness, with high-LET radiation generally causing moresevere damage per unit dose than low-LET radiation. The figure presents ECD histograms and a graph comparing the intact DNA fraction for DNA exposed to varying doses of cobalt-60 gamma rays and high-LET protons. The left panels show the ECD histograms for the DNA samples at different doses of the respective types of radiation. For the gamma radiation experiments, samples were irradiated at 0 Gy, 10 Gy, and 50 Gy. For high-LET proton radiation, DNA was irradiated at 0 Gy, 2 Gy, 5 Gy, and 20 Gy. The right panel plots the percentage of intact DNA remaining after irradiation as a function of radiation dose for both gamma rays and protons. The data are fitted to an exponential decay model for gamma rays, and to a model incorporating bimolecular decay of radicals for the proton experiments. The graph clearly shows that high-LET proton radiation induces DNA damage and reduces the concentration of intact DNA more effectively than low-LET gamma radiation. This difference reflects the greater biological effectiveness of high-LET radiation. The comparison of high- LET and low-LET radiation presented in FIG. 20 demonstrates how nanopore-based single- molecule dosimetry can be used to quantify the differences in biological effectiveness between different types of radiation. The ECD histograms and intact DNA fractions reveal the greater effectiveness of high-LET radiation, such as protons, in causing DNA damage compared to low-LET radiation, such as gamma rays. This information is valuable for determining the RBE of different radiation types. The quantitative nature of the method enables precise measurement of radiation damage and accurate RBE calculations. The method is applicable to a wide range of biomolecules and radiation types, and it is particularly useful for assessing damage in heterogeneous environments, such as cells or tissues. The nanopore-based approach provides significant advantages over traditional macroscopic assays by delivering high resolution measurements.

[0273] Dose response without initial concentration

[0274] FIG.21 shows how nanopore-based single-molecule dosimetry can be used to determine radiation dose even when the initial concentration of undamaged DNA is unknown. Traditional methods for measuring radiation-induced DNA damage often require knowledge of the initial DNA concentration to accurately quantify the extent of damage. However, in some situations, such as emergency radiation exposure scenarios, the initial DNA concentration may be unknown. Nanopore technology provides a means for determining radiation dose without prior knowledge of the initial DNA concentration by measuring the ratio of damaged DNA fragments to intact DNA. The figure presents bar graphs and a line graph illustrating this approach. The left panel displays two bar graphs showing the nucleoside concentration as a function of radiation dose for a 2.5 kbp DNA molecule. The top graphrepresents the total nucleoside concentration, which remains relatively constant across all radiation doses, ranging from 0 Gy to 15 Gy. The bottom graph shows the nucleoside concentrations for DNA fragments both larger than and smaller than 2.5 kbp. The concentration of fragments smaller than 2.5 kbp increases with increasing dose, while the concentration of longer fragments (representing intact or partially damaged 2.5 kbp DNA) decreases with increasing radiation dose. The right panel plots the corrected ratio of the concentration of nucleotides in fragments smaller than 2.5 kbp to the concentration of nucleotides in intact 2.5 kbp DNA as a function of radiation dose. The correction factor accounts for the lower capture probability of smaller DNA fragments in nanopore measurements. The graph demonstrates a clear dose-dependent increase in the corrected fragment concentration ratio, allowing for radiation dose estimation without prior knowledge of initial DNA concentration. This capability is advantageous in situations where traditional methods for quantifying DNA damage are not applicable due to an unknown initial DNA concentration. The method involves analyzing the distribution of DNA fragment lengths after radiation exposure. Nanopore technology measures DNA length based on the molecule’s translocation characteristics through a nanopore. Radiation-induced damage, like double- strand breaks, fragments DNA, shifting the distribution towards shorter lengths. By measuring the relative abundance of different fragment lengths, the extent of radiation-induced damage is quantified, and radiation dose may be estimated even without knowledge of initial concentration. The figure demonstrates how the ratio of shorter DNA fragments to intact DNA increases with radiation dose, allowing dose estimation. This approach is applicable in emergency radiation exposure scenarios or other situations where the initial DNA concentration is unknown. The method provides a rapid, sensitive, and quantitative measure of radiation damage. The method shown enhances traditional assays by enabling dose determination without prior knowledge of DNA concentration. The single-molecule resolution of nanopore measurements is valuable for analyzing complex mixtures of DNA fragments produced by radiation. The quantitative nature of nanopore data enables precise quantification of DNA damage. This information is valuable for assessing radiation exposure in various scenarios, including radiation therapy and environmental monitoring.

[0275] Supercoiled DNA within a nanopore

[0276] FIG. 22 depicts the conformation of supercoiled DNA within a nanopore. DNA exists in various conformations, including linear, circular, and supercoiled. Supercoiled DNA is a highly compact form of DNA that is twisted upon itself, and is commonly found in bacterial plasmids and some viruses. Nanopore-based single-molecule analysis provides ameans for distinguishing between different DNA conformations based on their translocation characteristics through a nanopore. The figure illustrates the difference in average blockage depth between supercoiled DNA and other DNA conformations. Blockage depth refers to the magnitude of the ionic current blockade caused by a molecule passing through the nanopore. Supercoiled DNA, due to its compact structure, exhibits a greater average blockage depth than either circular or linear DNA. The figure includes a schematic of a supercoiled DNA molecule and a corresponding nanopore event trace. The molecule's twisted shape, with its superhelix axis and branch points, is shown. As the DNA molecule passes through the nanopore, the ionic current is partially blocked, generating a measurable signal. The magnitude and duration of this signal depend upon the DNA’s length, conformation, and interactions with the nanopore walls. Supercoiled DNA, due to its compact and branched structure, produces a deeper and more complex blockade signal compared to linear or circular DNA. The trace shows how the actual current fluctuates as the DNA translocates, and the fitted current highlights the overall shape of the event. The ability to differentiate between DNA conformations by nanopore analysis is valuable in diverse applications. In nanopore-based single-molecule dosimetry, determining the conformation of DNA molecules in irradiated samples is used to assess structural damage beyond fragmentation. Changes in DNA conformation, such as the relaxation of supercoiling or the formation of knots or other complex structures, provide additional information on radiation-induced DNA modifications, including double-strand scission reactions, single- strand scission reactions, and base-damage. This information improves RBE determination and understanding the biological effects of radiation. Nanopore-based analysis of DNA conformation provides high-resolution information on the molecule’s structure and topology, and its ability to distinguish between different DNA conformations makes it a useful technique for studying radiation-induced DNA damage. The method is sensitive enough to detect changes in supercoiling or the formation of other complex structures, providing a comprehensive view of radiation's effects on biomolecules.

[0277] Analysis of DNA molecules with structure

[0278] FIG.23 depicts the use of nanopore technology to analyze DNA molecules with structure. Nanopore-based single-molecule dosimetry quantifies radiation-induced damage to biopolymers, such as DNA, by analyzing changes in their translocation characteristics through a nanopore. The figure presents ionic current traces and ECD histograms for linear and structured DNA molecules at different radiation doses. The left panel displays ionic current traces for linear DNA (top) and structured DNA (bottom) at 0 Gy. The structured DNA molecule exhibits more complex translocation dynamics compared to thelinear DNA, reflected in the current trace as multiple current levels or “steps” during translocation. This complex behavior reflects the presence of internal structures or modifications, such as hairpins or protein binding sites, that transiently interact with the nanopore during passage. The middle traces shows similar experiments for structured DNA irradiated at 5 Gy. The right panel displays ECD histograms for structured DNA at 0 Gy, 1 Gy, 2 Gy, and 5 Gy of gamma radiation. The histograms reveal the distribution of ECD values for the structured DNA at different radiation doses. The ECD histograms demonstrate how radiation-induced DNA damage can be detected as a reduction in the peak corresponding to the intact DNA. While the ECD provides information about the overall length of the DNA molecule, it does not directly reveal the presence of internal structures or modifications. The use of wavelet or other joint time-frequency methods allows identification of spectral signatures of damage, such as changes in DNA flexibility or the presence of lesions or bound proteins. These signatures provide insights into the type and extent of radiation-induced DNA modifications. The data shown in FIG.23 illustrate how nanopore technology analyzes DNA molecules with complex structures. The ionic current traces and ECD histograms demonstrate how the technique differentiates between linear and structured DNA, detects radiation-induced damage as a reduction in the peak corresponding to intact DNA in the ECD histogram, and identifies spectral signatures of damage using joint time-frequency methods. The method provides a high-resolution, single-molecule approach for quantifying radiation-induced damage and measuring RBE. The method's ability to detect subtle changes in DNA structure and conformation caused by radiation damage is essential for studying radiation's biological effects and developing effective countermeasures. The high-throughput nature of nanopore analysis enables statistically significant measurements, enhancing the method’s reliability and applicability to a wide range of experimental conditions and research questions. The use of joint time-frequency methods enhances the detection and characterization of DNA lesions beyond simple measurement of fragmentation.

[0279] Supercoiled DNA with varying doses of gamma radiation

[0280] FIG.24 presents data illustrating the results of irradiating supercoiled DNA with varying doses of gamma radiation and analyzing the irradiated samples using nanopore technology. Nanopore-based single-molecule dosimetry measures radiation-induced damage to biopolymers, such as DNA, by analyzing changes in their translocation characteristics through a nanopore. Supercoiled DNA, a highly compact form of DNA commonly found in bacterial plasmids and some viruses, exhibits distinct translocation behavior compared to linear or circular DNA. Radiation-induced damage, such as double-strand breaks, can alter thesupercoiling of DNA, resulting in changes in its translocation properties. The figure shows 2D histograms and ECD histograms for a supercoiled DNA molecule (ΦX174) exposed to 0 Gy, 1 Gy, and 5 Gy of gamma radiation. The 2D histograms plot average blockage (a measure of DNA size and conformation) versus translocation time, providing information on how radiation affects both properties. The ECD histograms, while providing information on overall DNA length, may not capture changes in DNA topology, such as the relaxation of supercoiling. The figure demonstrates how nanopore-based single-molecule dosimetry can be used to study the effects of radiation on supercoiled DNA. Supercoiled DNA should have a greater blockage than relaxed circular plasmids, which should have a greater blockage than linear DNA. As radiation dose increases, more events occur with smaller blockage and a greater variety of sizes. The top left panel displays the 2D histogram for the unirradiated supercoiled DNA (0 Gy), showing a distinct cluster of events corresponding to the intact supercoiled conformation. The cluster’s tight distribution indicates a homogenous population of molecules with similar translocation characteristics. The top right panel shows the ECD histogram for the 0 Gy sample, revealing a single peak corresponding to the intact supercoiled DNA. The middle and bottom panels present similar data for the DNA samples irradiated at 1 Gy and 5 Gy, respectively. As the radiation dose increases, the 2D histograms show a broadening of the event clusters, indicating greater heterogeneity in the DNA conformations, due to accumulation of single- strand DNA lesions. This broadening suggests that radiation causes relaxation of supercoiling and the formation of more diverse DNA structures. The ECD histograms for the irradiated samples show a decrease in the peak corresponding to intact supercoiled DNA and a broadening of the distribution, consistent with the formation of a more heterogeneous mixture of DNA structures. These data illustrate how nanopore measurements provide information on both DNA length (ECD) and conformation (blockage depth and translocation time). The results presented in FIG. 24 show that radiation exposure alters the conformation and translocation behavior of supercoiled DNA. The dose-dependent changes observed in the 2D histograms and ECD histograms are consistent with the accumulation of radiation-induced DNA damage, which causes a shift in the ECD distribution towards smaller ECD values. The method's single- molecule sensitivity is essential for detecting subtle changes in DNA conformation that may not be apparent from ensemble measurements. The nanopore-based analysis allows for quantification of these changes, providing valuable insights into the molecular mechanisms of radiation-induced DNA damage. The method’s adaptability to different biopolymers, experimental conditions, and radiation types is an advantage over traditional RBE assays. Thedetailed information obtained from these single-molecule measurements enables accurate assessment of radiation risks, optimization of radiation therapies, and development of radioprotective countermeasures.

[0281] Decoding data with “structure”

[0282] FIG. 25 illustrates the concept of decoding nanopore data with structure using joint time-frequency analysis. Nanopore-based single-molecule dosimetry measures radiation-induced damage to biopolymers, such as DNA, by analyzing their translocation characteristics through a nanopore. While ECD provides information about the biopolymer’s overall length, it does not capture subtle structural features or modifications that may be induced by radiation. Joint time-frequency analysis, such as wavelet analysis, provides a method for extracting additional structural information from nanopore data by analyzing changes in the ionic current signal’s frequency content over time. The left panel of FIG. 25 shows a 2D histogram of average blockage versus log10(ECD) for a structured DNA molecule, which exhibits more complex translocation dynamics compared to linear DNA. The right panel displays a similar 2D histogram where the x-axis represents translocation time. Structured DNA, which may contain internal loops, hairpins, or other structural modifications, generates distinct ionic current signatures as it translocates through the nanopore. These features appear as multiple current levels during the translocation event. The left panel emphasizes that the ECD provides information on length but not structure because the ECD is calculated by integrating the total charge deficit during a translocation event, providing a measure of the biopolymer's overall length. However, it does not directly capture information about changes in structure. The right panel shows how translocation time reveals structural details because translocation time is affected by factors such as DNA length, conformation, and interactions with the nanopore. Analyzing changes in translocation time provides insights into radiation- induced changes in DNA structure and conformation, which is important for improving RBE measurements. The use of 2D histograms enhances analysis of DNA molecules with “structure” by providing additional information about their translocation dynamics. The data shown in FIG.25 demonstrate how nanopore technology, combined with joint time-frequency analysis, can be used to extract detailed structural information from nanopore data, enhancing the sensitivity and accuracy of radiation-induced damage measurements. This enhanced sensitivity is particularly valuable for detecting and characterizing subtle changes in DNA structure that may not be reflected in ECD measurements. The method's high throughput enables statistically significant analyses, and its applicability to various biopolymers makes it a versatile tool for radiation research. The data reveal how radiation-induced changes in DNAtopology, such as the relaxation of supercoiling, affect the molecule’s translocation time. This information is valuable for a complete understanding of radiation's effects on biomolecules and development of radiation countermeasures. The method is particularly valuable in that it complements other techniques, like ECD histogram analysis, by providing detailed information on structural changes.

[0283] The processes described herein may be embodied in, and fully automated via, software code modules executed by a computing system that includes one or more general purpose computers or processors. The code modules may be stored in any type of non- transitory computer-readable medium or other computer storage device. Some or all the methods may alternatively be embodied in specialized computer hardware. In addition, the components referred to herein may be implemented in hardware, software, firmware, or a combination thereof.

[0284] Many other variations than those described herein will be apparent from this disclosure. For example, depending on the embodiment, certain acts, events, or functions of any of the algorithms described herein can be performed in a different sequence, can be added, merged, or left out altogether (e.g., not all described acts or events are necessary for the practice of the algorithms). Moreover, in certain embodiments, acts or events can be performed concurrently, e.g., through multi-threaded processing, interrupt processing, or multiple processors or processor cores or on other parallel architectures, rather than sequentially. In addition, different tasks or processes can be performed by different machines or computing systems that can function together.

[0285] Any logical blocks, modules, and algorithm elements described or used in connection with the embodiments disclosed herein can be implemented as electronic hardware, computer software, or combinations of both. To clearly illustrate this interchangeability of hardware and software, various illustrative components, blocks, modules, and elements have been described above generally in terms of their functionality. Whether such functionality is implemented as hardware or software depends upon the particular application and design constraints imposed on the overall system. The described functionality can be implemented in varying ways for each particular application, but such implementation decisions should not be interpreted as causing a departure from the scope of the disclosure.

[0286] The various illustrative logical blocks and modules described or used in connection with the embodiments disclosed herein can be implemented or performed by a machine, such as a processing unit or processor, a digital signal processor (DSP), an application specific integrated circuit (ASIC), a field programmable gate array (FPGA) or otherprogrammable logic device, discrete gate or transistor logic, discrete hardware components, or any combination thereof designed to perform the functions described herein. A processor can be a microprocessor, but in the alternative, the processor can be a controller, microcontroller, or state machine, combinations of the same, or the like. A processor can include electrical circuitry configured to process computer-executable instructions. In another embodiment, a processor includes an FPGA or other programmable device that performs logic operations without processing computer-executable instructions. A processor can also be implemented as a combination of computing devices, e.g., a combination of a DSP and a microprocessor, a plurality of microprocessors, one or more microprocessors in conjunction with a DSP core, or any other such configuration. Although described herein primarily with respect to digital technology, a processor may also include primarily analog components. For example, some or all of the signal processing algorithms described herein may be implemented in analog circuitry or mixed analog and digital circuitry. A computing environment can include any type of computer system, including, but not limited to, a computer system based on a microprocessor, a mainframe computer, a digital signal processor, a portable computing device, a device controller, or a computational engine within an appliance, to name a few.

[0287] The elements of a method, process, or algorithm described in connection with the embodiments disclosed herein can be embodied directly in hardware, in a software module stored in one or more memory devices and executed by one or more processors, or in a combination of the two. A software module can reside in RAM memory, flash memory, ROM memory, EPROM memory, EEPROM memory, registers, hard disk, a removable disk, a CD-ROM, or any other form of non-transitory computer-readable storage medium, media, or physical computer storage known in the art. An example storage medium can be coupled to the processor such that the processor can read information from, and write information to, the storage medium. In the alternative, the storage medium can be integral to the processor. The storage medium can be volatile or nonvolatile.

[0288] While one or more embodiments have been shown and described, modifications and substitutions may be made thereto without departing from the spirit and scope of the invention. Accordingly, it is to be understood that the present invention has been described by way of illustrations and not limitation. Embodiments herein can be used independently or can be combined.

[0289] All ranges disclosed herein are inclusive of the endpoints, and the endpoints are independently combinable with each other. The ranges are continuous and thus contain every value and subset thereof in the range. Unless otherwise stated or contextuallyinapplicable, all percentages, when expressing a quantity, are weight percentages. The suffix (s) as used herein is intended to include both the singular and the plural of the term that it modifies, thereby including at least one of that term (e.g., the colorant(s) includes at least one colorants). Option, optional, or optionally means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event occurs and instances where it does not. As used herein, combination is inclusive of blends, mixtures, alloys, reaction products, collection of elements, and the like.

[0290] As used herein, a combination thereof refers to a combination comprising at least one of the named constituents, components, compounds, or elements, optionally together with one or more of the same class of constituents, components, compounds, or elements.

[0291] All references are incorporated herein by reference.

[0292] The use of the terms “a,” “an,” and “the” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. It can further be noted that the terms first, second, primary, secondary, and the like herein do not denote any order, quantity, or importance, but rather are used to distinguish one element from another. It will also be understood that, although the terms first, second, etc. are, in some instances, used herein to describe various elements, these elements should not be limited by these terms. For example, a first current could be termed a second current, and, similarly, a second current could be termed a first current, without departing from the scope of the various described embodiments. The first current and the second current are both currents, but they are not the same condition unless explicitly stated as such.

[0293] The modifier about used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context (e.g., it includes the degree of error associated with measurement of the particular quantity). The conjunction or is used to link objects of a list or alternatives and is not disjunctive; rather the elements can be used separately or can be combined together under appropriate circumstances. / / PARTS LIST / / dielectric membrane 100 cis electrolyte chamber 102 trans electrolyte chamber 101 nanopore 107 positive electrode 104 negative electrode 103 voltage source 106 transimpedance amplifier 105 analog-to-digital converter 208 known biopolymer 108 medium 210 ionizing radiation voltage 216 ionic current 217 size selective tag x-ray source measuring radiation-induced damage to a biopolymer

Claims

What is claimed is:

1. A process for measuring radiation-induced damage to a biopolymer, the process comprising: providing a known biopolymer (108) of known length or composition; dissolving the biopolymer in a medium (210) to create a sample solution; irradiating the sample solution with ionizing radiation; preparing the biopolymer for analysis; assembling a measurement chamber, the chamber comprising two electrolyte chambers (102, 101) separated by a dielectric membrane (100) containing a nanopore (107); infusing the irradiated biopolymer into the measurement chamber; applying a voltage (216) across the membrane to cause the biopolymer to translocate through the nanopore; measuring the ionic current (217) through the nanopore; and analyzing the measured ionic current to determine the extent of radiation-induced damage to the biopolymer.

2. The process of claim 1, wherein the biopolymer is a nucleic acid.

3. The process of claim 2, wherein the nucleic acid is DNA.

4. The process of claim 3, wherein the DNA has a length of 2,500 base pairs.

5. The process of claim 1, wherein the biopolymer is a protein.

6. The process of claim 5, wherein the protein is a peptide having a length of 10 amino acids.

7. The process of claim 1,wherein the biopolymer is a polysaccharide.

8. The process of claim 1, further comprising adding a size selective tag to the irradiated biopolymer.

9. The process of claim 1, wherein the ionizing radiation comprises photons.

10. The process of claim 9, wherein the photons are generated by an x-ray source.

11. The process of claim 1, wherein the analyzing comprises detecting and counting the number of translocations through the nanopore.

12. The process of claim 1, wherein the analyzing comprises collecting salient features of the current signature as the molecule passes through the pore, the salient features comprising the magnitude of the current step, duration of the passage, number of discrete current steps within the resistive pulse, and the integrated area between the current level with no molecule present and the current level while the molecule is driven through the pore.

13. The process of claim 1, wherein the analyzing comprises producing a histogram of one or more salient features for quantitative analysis.

14. The process of claim 13, wherein the salient feature is equivalent charge deficit (ECD).

15. The process of claim 13, wherein the salient feature is number of current levels observed.

16. The process of claim 1, wherein the determining comprises identifying scission reactions.

17. The process of claim 1, wherein the determining comprises identifying internal bond-breaking reactions.

18. The process of claim 1, further comprising employing wavelet or other joint time- frequency analysis to identify spectral signatures of damage.

19. The process of claim 1, further comprising building a dose-response curve by plotting integrated peaks against radiation dose.

20. The process of claim 1, further comprising quantifying RBE ratios by generating a collection of dose-response curves with varying conditions.

21. A process for measuring radiation-induced damage to a biopolymer, the process comprising: providing a known biopolymer of known length and composition; dissolving the biopolymer in deionized water to create a sample solution; adding electrolytes, free radical scavengers, and free radical enhancers to the sample solution; distributing the sample solution into a gel matrix; irradiating the sample solution with ionizing radiation from a cobalt-60 source; retrieving the sample solution from the gel matrix; preparing the biopolymer for analysis by diluting the sample and adding lithium chloride; labeling the irradiated molecule with a size-selective DNA tag using DNA glycosylase coupled with an oxime-modified label; adding two discretely sized biopolymer internal standards for size selective and quantitative analysis; assembling a measurement chamber comprising two electrolyte chambers separated by a silicon nitride membrane containing a nanopore; infusing the irradiated biopolymer into the measurement chamber; applying a voltage across the membrane to cause the biopolymer to translocate through the nanopore; measuring the ionic current generated by the translocation; digitizing the measured ionic current; analyzing the digitized ionic current by detecting and counting translocations using a threshold analysis algorithm; collecting the magnitude of the current step, duration of the passage, number of discrete current steps, and the integrated area of the current steps; producing histograms of equivalent charge deficit (ECD) and number of current levels observed to quantify scission reactions and internal bond-breaking reactions, respectively; employing signal analysis using wavelet and joint time-frequency methods to identify spectral signatures of damage;and generating dose-response curves by plotting integrated peaks against varying doses of ionizing radiation to quantify RBE ratios.

22. The process of claim 21, wherein the biopolymer is a nucleic acid.

23. The process of claim 22, wherein the nucleic acid is a 2,500 base-pair double-strand DNA.

24. The process of claim 21, wherein the biopolymer is a peptide.

25. The process of claim 24, wherein the peptide is Asp-Arg-Val-Tyr-Ile-His-Pro-Phe- His-Leu.

26. The process of claim 21, wherein the biopolymer is a polysaccharide.

27. The process of claim 21, wherein the medium is water.

28. The process of claim 21, wherein the gel matrix is agarose.

29. The process of claim 21, wherein the nanopore is fabricated in silicon nitride.

30. The process of claim 21, wherein the two discretely sized biopolymer internal standards are a 5,000 base-pair DNA and a 10,000 base-pair DNA.

31. The process of claim 21, wherein the providing comprises selecting a known biopolymer of known length.

32. The process of claim 21, wherein the dissolving comprises dissolving the biopolymer in water in an Eppendorf tube.

33. The process of claim 21, wherein the adding comprises adding phosphate buffered saline, tris(hydroxymethyl)aminomethane, and N2O(g).

34. The process of claim 21, wherein the distributing comprises distributing the sample solution into an agarose gel.

35. The process of claim 21, wherein the irradiating comprises irradiating the sample with a gamma radiation field at a dose rate from 0 Gy / min to 100 Gy / min.

36. The process of claim 21, wherein the retrieving comprises extracting the sample from segments of the agarose gel matrix.

37. The process of claim 21, wherein the labeling comprises reacting the irradiated DNA with a base-specific DNA glycosylase to create reactive sites for attaching the DNA tag.

38. The process of claim 21, wherein the applying comprises applying a voltage of 300 mV.

39. The process of claim 21, wherein the digitizing comprises using an analog-to-digital converter.

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