Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
  • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
• • G—PHYSICS
  • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
  • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
  • G16B30/00—ICT specially adapted for sequence analysis involving nucleotides or amino acids
• • G—PHYSICS
  • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
  • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
  • G16H50/00—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics
  • G16H50/20—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics for computer-aided diagnosis, e.g. based on medical expert systems

Definitions

• the present disclosure provides methods, compositions, and systems related to the detection of cancer DNA.
• the present disclosure provides methods, compositions, and systems related to the electrochemical detection and/or quantification of cancer DNA in biological samples for diagnosis, detection, prognosis, monitoring, and evaluation of disease (e.g., cancer) status.
• disease e.g., cancer
• Imaging tracks tumor size, but requires large, expensive instrumentation, operated, and interpreted by highly trained individuals. Imaging is classically a lagging indicator, slow to indicate changes. To effectively treat patients with methods personalized to their response, an accessible tool is needed to quickly determine treatment efficacy and improve long-term outcomes.
• Embodiments of the present disclosure include methods for determining cancer status in a subject.
• the methods comprise measuring cancer cell free DNA (cfDNA) in a first biological sample and a second biological sample, wherein the first and the second biological sample are obtained from the subject separated by a period of time and classifying the cancer as progressive, regressive, or unchanged based on a change in the amount, quantity, concentration and/or level of cancer cfDNA in the second biological sample as compared to the first biological sample.
• cfDNA cancer cell free DNA
• measuring cancer cfDNA comprises: adding an acidifying agent to a biological sample to form an acidic sample; contacting an electrode with the acidic sample; and detecting an electrochemical signal indicating the cancer cfDNA.
• the acidic sample is at a pH of less than about 4. In some embodiments, the acidic sample is at a pH from about 1 to about 3. In some embodiments, the acidic sample is at a pH of about 2. In some embodiments, the acidic sample comprises an acidifying agent.
• the cancer is progressive when the cancer cfDNA in the second biological sample is increased compared to the first biological sample. In some embodiments, the cancer is regressive when the cancer cfDNA in the second biological sample is decreased compared to the first biological sample.
• the period of time is at least one week. In some embodiments, the period of time is at least one month. In some embodiments, the period of time is less than 6 months.
• the methods further comprise treating, or changing the treatment of, the subject based on the cancer progression.
• the first biological sample predates the start of a treatment regimen and the second biological sample postdates the start of the treatment regimen.
• the methods further comprise determining the clinical benefit of the treatment regimen based on if the cancer status was progressive, regressive, or unchanged.
• the treatment regimen comprises surgery, administration of a chemotherapeutic agent, immunotherapy, radiotherapy, or combinations thereof.
• the treatment regimen comprises immunotherapy.
• the methods further comprise treating the subject based on the clinical benefit of the treatment regimen.
• Embodiments of the present disclosure also include methods for detecting cancer in a subject.
• the cancer is an advanced stage cancer.
• the methods comprise: measuring cancer cell free DNA (cfDNA) in a biological sample obtained from the subject and identifying the subject as having cancer based on: the cancer cfDNA as compared to: control, non-cancerous cfDNA; a cutoff value; or a cutoff value for a ratio of cancer cfDNA to total cfDNA.
• cfDNA cancer cell free DNA
• measuring cfDNA comprises: adding an acidifying agent to a biological sample to form an acidic sample; contacting an electrode with the acidic sample; and detecting an electrochemical signal indicating the cancer cfDNA.
• detecting the electrochemical signal can include performing at least one of voltammetry, cyclic voltammetry, square wave voltammetry, differential pulse voltammetry, amperometry, chronoamperometry, potentiometry, chronopotentiometry, coulometry, chronocoulometry, conductometry, and impedometry.
• the electrochemical signal is a measure of the cancer cfDNA adsorbed to the electrode. In some embodiments, the electrochemical signal is a change in peak current magnitude following contacting the electrode with the acidic sample.
• the acidic sample is at a pH of less than about 4. In some embodiments, the acidic sample is at a pH from about Ito about 3. In some embodiments, the acidic sample is at a pH of about 2. In some embodiments, the acidic sample comprises an acidifying agent.
• the methods further comprise: extracting cfDNA from the biological sample prior to forming an acidified sample, prior to measuring the cancer cfDNA, or both.
• the methods further comprise: extracting cfDNA from the biological sample, adjusting it to the desired concentration prior to measuring the cancer cfDNA.
• the biological sample is a whole blood sample, a plasma sample, a serum sample, and a urine sample.
• Embodiments of the present disclosure also include methods for detecting cancer DNA in a sample.
• the methods comprise: contacting an electrode with an acidic sample comprising or suspected of comprising cancer DNA and measuring an electrochemical signal indicating the cancer DNA.
• the acidic sample is at a pH of less than about 4. In some embodiments, the acidic sample is at a pH from about 1 to about 3. In some embodiments, the acidic sample is at a pH of about 2. In some embodiments, the acidic sample comprises an acidifying agent.
• the acidic sample comprises a biological sample.
• the methods comprise adding the acidifying agent to the biological sample, thereby forming the acidic sample, prior to contacting the acidic sample with the electrode.
• detecting the electrochemical signal can include performing at least one of voltammetry, cyclic voltammetry, square wave voltammetry, differential pulse voltammetry, amperometry, chronoamperometry, potentiometry, chronopotentiometry, coulometry, chronocoulometry, conductometry, and impedometry.
• the electrochemical signal is a measure of the cancer DNA adsorbed to the electrode.
• the electrochemical signal comprises a change in peak current magnitude following contacting the electrode with the acidic sample.
• the electrode is a gold electrode.
• the acidic sample comprises less than 100 pg/uL DNA. In some embodiments, the acidic sample comprises less than 10 pg/uL DNA.
• the subject has cancer when the cancer cfDNA in the biological sample is greater than the control and/or greater than the cutoff value. In some embodiments, the subject has cancer when the ratio of cancer cfDNA to total cfDNA is greater than the cutoff value.
• the methods further comprise administering, or changing, a treatment regimen to the subject identified as having cancer.
• the treatment regimen comprises surgery, administration of a chemotherapeutic agent, immunotherapy, radiotherapy, or combinations thereof.
• Embodiments of the present disclosure also include systems for carrying out the disclosed methods.
• the systems comprise: an electrochemical detection system comprising a gold electrode and an acidic sample comprising cancer DNA or a sample comprising cancer DNA and an acidifying agent.
• the electrochemical detection system further comprises one or more of: a working electrode, a counter electrode, a reference electrode, a sample reservoir, a potentiostat and a data acquisition unit.
• the system further comprises a sample purification system.
• the sample purification system comprises filters, chromatography columns, chromatography beads, microfluidic devices, or combinations thereof.
• Embodiments of the present disclosure also include compositions comprising cancer DNA and a buffer or acidifying agent.
• the composition is at acidic pH.
• the composition has a pH of less than about 4.
• the composition has a pH from about 1 to about 3.
• the composition has a pH of about 2.
• FIG. 1 shows relative peak current values for cfDNA individual samples for healthy subjects (Normal), patients with colorectal cancer (CRC), and patients with non-small-cell lung cancer (NSCLC) following incubation of the cfDNA with the electrode at acidic (pH 2) and neutral (7.2).
• FIGS. 2A-2E show relative peak current values for cfDNA individual samples for healthy subjects (Normal), patients with colorectal cancer (CRC), and patients with non-small- cell lung cancer (NSCLC).
• FIG. 2C shows the large separation between peak current values for cancer samples versus normal samples, red bars. Box and whisker plots corresponding to the relative peak current values in the bar graphs of FIGS. 2A and 3B show each individual measurement for the TFEs (FIG. 2D) and the SPEs (FIG. 2E).
• the p-values on the TFEs for Norm. vs. CRC and Norm. vs. NSCLC are 4.2E" 4 and 1.5E” 4 respectively.
• the p- values on the SPEs are 2.6E" 5 and 4.8E" 4 respectively.
• FIGS. 3A-3C show relative peak current values for clinical cfDNA samples (10 pg/pL) obtained from patients with lymphoma (FIG. 3A, Patient 1), NSCLC (FIG. 3B, Patient 2) and NSCLC (FIG. 3C, Patient 3) at two different timepoints ranging from before diagnosis to after treatment.
• the p-values indicate the statistical significance between the %ir values between T 1 and T2 for each patient.
• FIGS. 4A and 4B show adsorption of clinical cfDNA pooled patient samples for healthy individuals (Norm., grey) and patients with colorectal (CRC, orange) or non-small-cell lung cancer (NSCLC, green).
• FIG. 4A is a graph of the relative peak current values as a function of cfDNA concentration (0.1 - 10 pg/pL).
• FIG. 4B shows the difference in relative peak current magnitude values between healthy and cancer cfDNA plotted as a function of concentration.
• FIGS. 5A and 5B show cfDNA signals differentiate cancer patients responding to treatment (responders or cancer regression) and those patients in which the cancer is progressing (progressors or cancer progression) from two different sample sets: Scripps patient samples (FIG. 5A) and Drammen Hospital samples (FIG. 5B).
• ctDNA circulating tumor DNA
• cfDNA circulating free DNA
• each intervening number there between with the same degree of precision is explicitly contemplated.
• the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.
• the term “derived from” as used herein refers to cells or a biological sample (e.g., blood, tissue, bodily fluids, plants, etc.) and indicates that the cells or the biological sample were obtained from the stated source at some point in time.
• the term includes directly obtained from, isolated and cultured, or obtained, frozen, and thawed.
• the term “derived from” may also refer to a component or fragment of a cell obtained from a tissue or cell, including, but not limited to, a protein, a nucleic acid, a membrane or fragment of a membrane, and the like.
• Controls as used herein generally refers to a reagent whose purpose is to evaluate the performance of a measurement system in order to assure that it continues to produce results within permissible boundaries (e.g., boundaries ranging from measures appropriate for a research use assay on one end to analytic boundaries established by quality specifications for a commercial assay on the other end).
• permissible boundaries e.g., boundaries ranging from measures appropriate for a research use assay on one end to analytic boundaries established by quality specifications for a commercial assay on the other end.
• a control should be indicative of patient results and optionally should somehow assess the impact of error on the measurement (e.g., error due to reagent stability, calibrator variability, instrument variability, and the like).
• “Dynamic range” as used herein refers to range over which an assay readout is proportional to the amount of target molecule or analyte (e.g., cancer DNA) in the sample being analyzed.
• the dynamic range can be the range of linearity of the standard curve.
• Reference level refers to an assay cutoff value that is used to assess diagnostic, prognostic, or therapeutic efficacy and that has been linked or is associated herein with various clinical parameters (e.g., presence of disease, stage of disease, severity of disease, progression, non-progression, or improvement of disease, etc.).
• reference levels may vary depending on the nature of the assay used and that assays can be compared and standardized. It further is well within the ordinary skill of one in the art to adapt the disclosure herein for other assays to obtain specific reference levels for those other assays based on the description provided by this disclosure. Whereas the precise value of the reference level may vary between assays, the findings as described herein should be generally applicable and capable of being extrapolated to other assays.
• sample is used in its broadest sense. In one sense, it is meant to include a specimen obtained from any source, including biological samples. Biological samples may be obtained from animals (including humans) and encompass fluids, solids, tissues, and gases. Such examples are not however to be construed as limiting the sample types.
• a sample is a fluid sample such as a liquid sample.
• liquid samples that may be assayed include bodily fluids (e.g., blood, serum, plasma, saliva, urine, ocular fluid, semen, sputum, sweat, tears, pleural effusions, ascites, thin needle aspirates and spinal fluid.
• Viscous liquid, semisolid, or solid specimens may be used to create liquid solutions, eluates, suspensions, or extracts that can be samples.
• throat or genital swabs may be suspended in a liquid solution to make a sample.
• Samples can comprise biological materials, such as cells, microbes, organelles, and biochemical complexes.
• Liquid samples can be made from solid, semisolid, or highly viscous materials, such as fecal matter, tissues, organs, biological fluids, or other samples that are not fluid in nature.
• solid or semisolid samples can be mixed with an appropriate solution, such as a buffer, a diluent, and/or extraction buffer.
• the sample can be macerated, frozen and thawed, or otherwise extracted to form a fluid sample. Residual particulates may be removed or reduced using conventional methods, such as filtration or centrifugation.
• “Test sample,” “sample from a subject,” “biological sample,” and “patient sample” as used interchangeably herein may be a sample of blood, such as whole blood (including for example, capillary blood, venous blood, dried blood spot, etc.), tissue, urine, serum, plasma, amniotic fluid, an anal sample (such as an anal swab specimen), lower respiratory specimens such as, but not limited to, sputum, endotracheal aspirate or bronchoalveolar lavage, nasal mucus, cerebrospinal fluid, placental cells or tissue, endothelial cells, leukocytes, or monocytes.
• blood such as whole blood (including for example, capillary blood, venous blood, dried blood spot, etc.), tissue, urine, serum, plasma, amniotic fluid, an anal sample (such as an anal swab specimen), lower respiratory specimens such as, but not limited to, sputum, endotracheal aspirate or bron
• the sample can be used directly as obtained from a patient or can be pre-treated, such as by filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, and the like, to modify the character of the sample in some manner as discussed herein or otherwise as is known in the art.
• a variety of cell types, tissue, or bodily fluid may be utilized to obtain a sample.
• Such cell types, tissues, and fluid may include sections of tissues such as biopsy and autopsy samples, oropharyngeal specimens, nasopharyngeal specimens, nasal mucus specimens, frozen sections taken for histologic purposes, blood (such as whole blood, dried blood spots, etc.), plasma, serum, red blood cells, platelets, an anal sample (such as an anal swab specimen), interstitial fluid, cerebrospinal fluid, etc.
• Cell types and tissues may also include lymph fluid, cerebrospinal fluid, or any fluid collected by aspiration.
• a tissue or cell type may be provided by removing a sample of cells from a human and a non-human animal, but can also be accomplished by using previously isolated cells (e.g., isolated by another person, at another time, and/or for another purpose). Archival tissues, such as those having treatment or outcome history, may also be used. Protein or nucleotide isolation and/or purification may not be necessary.
• a “subject” or “patient” may be human or non-human and may include, for example, animal strains or species used as “model systems” for research purposes, such a mouse models, prokaryotic models (e.g., bacteria), archea, and single-celled eukaryotes(e.g., yeast).
• subject may include either adults or juveniles (e.g., children).
• patient may mean any living organism, preferably a mammal (e.g., humans and non-humans) that may benefit from the uses of compositions and methods contemplated herein.
• mammals include, but are not limited to, any member of the Mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like.
• non-mammals include, but are not limited to, birds, fish, and the like.
• the subject is a human.
• the term “treat,” “treating,” or “treatment” are each used interchangeably herein to describe reversing, alleviating, or inhibiting the progress of a disease and/or injury, or one or more symptoms of such disease, to which such term applies.
• the term also refers to preventing a disease, and includes preventing the onset of a disease, or preventing the symptoms associated with a disease (e.g., viral infection).
• a treatment may be either performed in an acute or chronic way.
• the term also refers to reducing the severity of a disease or symptoms associated with such disease prior to affliction with the disease.
• Such prevention or reduction of the severity of a disease prior to affliction refers to administration of a treatment to a subject that is not at the time of administration afflicted with the disease.
• embodiments of the present disclosure include methods for detecting cancer DNA (e.g., the presence or determining an amount, quantity, concentration and/or level of cancer DNA (e.g., cancer cfDNA, ctDNA)) in a sample.
• the methods comprise contacting an electrode with an acidic sample comprising or suspected of comprising cancer DNA and measuring an electrochemical signal indicating the cancer DNA, e.g., the presence, amount, quantity, concentration and/or level of the cancer DNA.
• the electrochemical signal increases with increasing presence, amount, quantity, concentration and/or level of the cancer DNA.
• the electrochemical signal decreases with decreasing presence, amount, quantity, concentration and/or level of the cancer DNA.
• the acidic sample is at a pH of less than about 4.
• the acidic sample may be at a pH less that about 3.5, less than about 3, less than about 2.5, less than about 2, less than about 1.5, less than about 1.
• the acidic sample may be at a pH from about 1 to about 4, from about 1 to about 3, from about 1 to about 2, from about 2 to about 4, from about
• the acidic sample is at a pH from about 1 to about 3.
• the acidic sample comprises an acidifying agent.
• the acidifying agent is any agent which acts to lower the pH of the comprising or suspected of comprising cancer DNA, such as an inorganic or organic acid.
• Suitable acidifying agents include, for example, organic acids such as ascorbic acid (vitamin C), salicylic acid, acetyl salicylic acid, acetic acid or a salt or a derivative thereof, ammonium or aluminum salts, phenol, inorganic acids such as hydrochloric acid, nitric acid or a salt or a derivative thereof.
• the acidifying agent may be present as a dissolved salt or in a liquid form.
• the acidifying agents may be part of a buffer or buffering system.
• the acidic sample comprises or is derived from a biological sample.
• the acidifying agent may be added as a dissolved salt, in a liquid form, in a buffer solution, or as a solid (e.g., powder or granulate).
• the acidic sample may be derived from a biological sample by other methods known in the art to adjust pH values, including but not limited to dialysis and column exchange.
• compositions comprising cancer DNA and a buffer or acidifying agent, wherein the composition is at an acidic pH.
• the composition is at a pH of less than about 4.
• the composition may be at a pH less that about 3.5, less than about 3, less than about 2.5, less than about 2, less than about 1.5, less than about 1.
• the composition may be at a pH from about 1 to about 4, from about 1 to about 3, from about 1 to about 2, from about 2 to about 4, from about 2 to about 3, from about
• the composition is at a pH from about 1 to about 3.
• measuring the electrochemical signal can include performing at least one of voltammetry, amperometry, potentiometry, coulometry, conductometry, impedometry, or other methods known in the art.
• measuring the electrochemical signal can include performing at least one of cyclic voltammetry, square wave voltammetry, differential pulse voltammetry, chronoamperometry, chronopotentiometry, and chronocoulometry.
• measuring the electrochemical signal can include performing at least one of differential pulse voltammetry and cyclic voltammetry.
• the electrochemical signal is a measure of the presence, amount, quantity, concentration and/or level of cancer vs normal DNA adsorbed to the electrode. Any electrochemical signal altered by the adsorption of the cancer DNA to the electrode or the presence of cancer DNA in the sample may be measured and correlated to the amount or relative amount of cancer DNA.
• standard or control values may be used to determine the relationship between the cancer DNA (e.g., the amount, quantity, concentration and/or level) to an electrochemical signal. These standard or control values may be determined prior to conducting the disclosed methods and may include determination of dynamic range, limit of detection, limit of quantitation, linearity, sensitivity, and other calibration values.
• the electrochemical signal comprises a change in peak current magnitude following contacting the electrode with the acidic sample. In some embodiments, the electrochemical signal comprises a change in potential at peak following contacting the electrode with the acidic sample.
• the correlation between electrochemical signal and cancer DNA may be dependent on the state of the sample (e.g., source, purification state, presence of interfering components, and the like), the quantity of sample, the type, size, or configuration of the electrode, and the methods used to measure the electrochemical signal.
• state of the sample e.g., source, purification state, presence of interfering components, and the like
• the quantity of sample e.g., the type, size, or configuration of the electrode, and the methods used to measure the electrochemical signal.
• the electrode may comprise any material which confers differential adsorption of cancer DNA compared to non-cancer DNA under acidic conditions.
• the electrode comprises a metal (e.g., gold, platinum, palladium, silver, copper) or metal alloy.
• the electrode is a gold electrode.
• the electrode may be any size or form which facilitates contacting with the acidic sample.
• the electrode may be a screen-printed electrode, thin film or thick-film electrode.
• the electrode may be a thin-film electrode.
• the electrode may be modified to have a higher reactive surface, increase the mass transfer rate, and/or the electrocatalytic activity (e.g., with the use of nanoparticles, methods of forming electrode surface (e.g., by varying temperature at which the electrodes are screen-printed or type of ink used), type of substrate, thickness of film, and the like).
• the acidic sample comprises less than 300 pg/uL DNA (e.g., less than 250 pg/uL, less than 200 pg/uL, less than 150 pg/uL, less than 100 pg/uL, less than 90 pg/uL, less than 80 pg/uL, less than 70 pg/uL, less than 60 pg/uL, less than 50 pg/uL, less than 40 pg/uL, less than 30 pg/uL, less than 20 pg/uL, less than 10 pg/uL, less than 5 pg/uL, less than 2 pg/uL, less than 1 pg/uL, less than 0.1 pg/uL).
• DNA e.g., less than 250 pg/uL, less than 200 pg/uL, less than 150 pg/uL, less than 100 pg/uL, less than 90 pg/uL, less than 80
• the acidic sample comprises less than 10 pg/uL DNA.
• the acidic sample may comprise 0.1 - 10 pg/pL DNA, e.g., about 0. 1 pg/pL DNA, about 0.5 pg/pL DNA, about 1 pg/pL DNA, about 1.5 pg/pL DNA, about 2 pg/pL DNA, about 2.5 pg/pL DNA, about 3 pg/pL DNA, about 3.5 pg/pL DNA, about 4 pg/pL DNA, about 4.5 pg/pL DNA, about 5 pg/pL DNA, about 5.5 pg/pL DNA, about 6 pg/pL DNA, about 6.5 pg/pL DNA, about 7 pg/pL DNA, about 7.5 pg/pL DNA, about 8 pg/pL DNA, about 8.5 pg/pL DNA, about 9 pg/pL DNA, about 9.5 pg/pL DNA,
• Embodiments of the present disclosure also include methods for determining cancer status and diagnosing cancer in a subject.
• the methods comprise measuring cancer cell free DNA (cfDNA) (e.g., an amount, quantity, concentration and/or level of cancer cfDNA) in one or more biological samples.
• cfDNA cancer cell free DNA
• the term “cell free DNA” or “cell-free DNA” or “cfDNA” refers to single and/or double-stranded deoxyribose nucleic acids (DNA) found free of cells, generally found in biological fluids including plasma, serum, urine, and the like.
• the sample comprises less than 300 pg/uL DNA (e.g., less than 250 pg/uL, 200 pg/uL, 150 pg/uL, 100 pg/uL, 90 pg/uL, 80 pg/uL, 70 pg/uL, 60 pg/uL, 50 pg/uL, 40 pg/uL, 30 pg/uL, 20 pg/uL, 10 pg/uL, 5 pg/uL, 2 pg/uL, 1 pg/uL, 0.1 pg/uL).
• the acidic sample comprises less than 10 pg/uL DNA.
• the methods described herein measure total cancer cfDNA (e.g., the amount, quantity, concentration and/or level of total cancer cfDNA). For example, in some embodiments, the methods do not select any specific cfDNA sequences based for analysis. Rather, the methods query the cancer or non-cancer of the entire cfDNA in the sample, not a specific viral sequence, mutation, SNP, indel, or signature. Thus, in some embodiments, the methods do not comprise sequencing the cfDNA or associating the cancer status with any particular cfDNA sequence. In some embodiments, the methods do not comprise identifying the type or location of the methyl groups in the cancer cfDNA.
• the methods further comprise obtaining the biological sample(s) from the subject.
• the biological sample(s) are each individually selected from a whole blood sample, a plasma sample, a serum sample, and a urine sample.
• the sample(s) can be obtained using techniques known to those skilled in the art, and the sample(s) may be used directly as obtained from the source or following a pretreatment to modify the character of the sample.
• Such pretreatment may include, for example, preparing plasma from blood, diluting viscous fluids, filtration, precipitation, dilution, distillation, mixing, concentration, inactivation of interfering components, the addition of reagents, lysing, and the like.
• the methods further comprise isolating nucleic acids, preferably DNA, from the sample prior to measuring the amount, quantity, concentration and/or level of cancer cfDNA.
• the methods further comprise extracting cfDNA from a sample prior to measuring the amount, quantity, concentration and/or level of cancer cfDNA.
• cfDNA extraction may include the use of one or more of chromatography or affinity columns or magnetic beads, phenol-chloroform-based methods, and filtration-based methods.
• the methods further comprise analyzing the cfDNA and, if necessary, adjusting the concentration of the cfDNA prior to measuring the amount, quantity, concentration and/or level of cancer cfDNA.
• analyzing the cfDNA is done in a manner which measures the total cfDNA (cancer and non-cancer related cfDNA).
• analyzing the cfDNA is done in a manner which measures the non-cancer related cfDNA.
• the methods comprise measuring cancer cfDNA in a first biological sample and a second biological sample taken from the subject, wherein the second biological sample is separated from the first biological sample by a period of time.
• the period of time between taking the first biological sample and the second biological sample may be at least four hours, at least one day, at least one week, at least one month, at least two months, at least three months, at least four months, at least five months, at least six months, at least nine months, at least twelve months, at least eighteen months, at least two years, or more.
• the period of time between taking the first biological sample and the second biological sample is at least one week. In some embodiments, the period of time between taking the first biological sample and the second biological sample is at least one month.
• the period of time between taking the first biological sample and the second biological sample may be less than one year, less than nine months, less than six months, less than five months, less than four months, less than three months, less than two months, or less than one month. In some embodiments, the period of time is less than about six months. The period of time may be less than the time necessary to detect changes in cancer or tumor burden by an imaging technique.
• a second or subsequent biological sample is taken from the subject at the time of another bioassay which assesses change in cancer status. The bioassay may be used to confirm the results of the methods disclosed herein and/or assist in ruling out pseudo-progression (false positive apparent growth on imaging). For example, a second biological sample taken from the subject may be taken at the same time as imaging analysis of the cancer.
• the methods comprise classifying the cancer as progressive, regressive, or unchanged based on change of the cancer cfDNA (e.g., the amount, quantity, concentration and/or level of cancer cfDNA) in the second, or subsequent, biological sample as compared to the first biological sample.
• the cancer is progressive, indicating an increased cancer burden, when the cancer cfDNA in the second biological sample is increased compared to the first biological sample.
• the cancer is regressive, indicating a decrease in cancer burden, when the cancer cfDNA in the second biological sample is decreased compared to the first biological sample.
• the methods may evaluate the change in cancer status, e.g., the progression or regression of the cancer, over time.
• the first biological sample predates the start of a treatment regimen and the second biological sample postdates the start of the treatment regimen.
• the methods may evaluate the progression or regression of the cancer after different interventions or treatment regimens (e.g., surgery, administration of a chemotherapeutic agent, immunotherapy, radiotherapy, or combinations thereof).
• the methods may evaluate the growth of the cancer after different interventions or treatment regimens.
• the methods may allow short- or long-term longitudinal evaluation of single or multiple interventions or treatment regimens.
• the methods may further comprise measuring cancer cell free DNA (cfDNA) (e.g., an amount, quantity, concentration and/or level of cancer cfDNA) in at least one additional biological sample separated from an immediately preceding, or any preceding, biological sample by a period of time.
• cfDNA cancer cell free DNA
• any number of biological samples taken at different timepoints during the duration of a cancer or during or after interventions or treatment regimens may be used in the disclosed methods with the cancer status or change in cancer status being determined between each sample or from the first sample to the most recent sample based on the change of the cancer cfDNA.
• the methods further comprise determining a growth rate of the cancer based on the cancer cell free DNA (cfDNA) in two or more biological samples or a change in slope of the cancer cell free DNA (cfDNA) in three or more biological samples.
• the disclosed methods may evaluate the growth rate of the cancer over time before and/or after the start or change of a treatment regimen .
• the growth rate may be used to detect hyper progressive cancer, which is generally defined as a growth rate of over double the pre-treatment level.
• each of the samples may be separated by any period of time from weeks to years.
• each sample is separated from the immediately preceding sample by at least one week, at least one month, at least two months, at least three months, at least four months, at least five months, at least six months, at least nine months, at least twelve months, at least eighteen months, at least two years, or more.
• each sample is separated from the immediately preceding sample by less than one year, less than nine months, less than six months, less than five months, less than four months, less than three months, less than two months, or less than one month.
• the biological samples may be examined at the time or near the time of acquisition.
• biological samples may be taken at the desired timepoints and analyzed simultaneously at a single point in time.
• one or more biological samples may be taken and stored prior to analysis at a later time point.
• the methods comprise identifying a subject as having or at risk of having a cancer based on the cancer cfDNA in a biological sample.
• the cancer may be identified by comparing the amount, quantity, concentration and/or level of cancer cfDNA in a biological sample to a control or reference, non-cancerous cfDNA amount, quantity, concentration, and/or level or previous samples from the same person. Further, the cancer may be identified based on a cutoff value for a ratio of the amount, quantity, concentration and/or level of cancer cfDNA to total cfDNA in the biological sample. For example, when the ratio of cancer cfDNA to total cfDNA is greater than a cutoff value, the subject may have cancer.
• Total cfDNA may be measured using any method known in the art, including a coordinating electrochemical method.
• the cancer may be identified by comparing the amount, quantity, concentration and/or level of cancer cfDNA in a biological sample to a cutoff value for the amount, quantity, concentration and/or level of cancer cfDNA. For example, when the amount, quantity, concentration and/or level of cancer cfDNA in the biological sample is greater than the control and/or greater than the cutoff value, the subject may have cancer.
• measuring an amount, quantity, concentration and/or level of cancer cfDNA comprises: adding an acidifying agent to a biological sample to form an acidic sample; contacting an electrode with the acidic sample and detecting an electrochemical signal indicating the amount, quantity, concentration and/or level of cancer cfDNA.
• measuring an amount, quantity, concentration and/or level of cancer cfDNA comprises: adding an acidifying agent to a biological sample to form an acidic sample; contacting an electrode with the acidic sample and detecting an electrochemical signal indicating the amount, quantity, concentration and/or level of cancer cfDNA.
• the electrochemical signal comprises a change in peak current magnitude following contacting the electrode with the acidic sample.
• a progressive cancer is characterized by a decreased peak current magnitude of a subsequent biological sample compared to a previous biological sample (e.g., second biological sample compared to a first biological sample).
• a regressive cancer is characterized by an increased peak current magnitude of a subsequent biological sample compared to a previous biological sample (e.g., second biological sample compared to a first biological sample).
• the methods described herein are not limited by type of cancer.
• the cancer may include carcinoma, sarcoma, lymphoma, leukemia, melanoma, mesothelioma, multiple myeloma, or seminoma.
• the cancer may be a cancer of the bladder, blood, bone, brain, breast, cervix, colon/rectum, endometrium, head and neck, kidney, liver, lung, lymph nodes, muscle tissue, ovary, pancreas, prostate, skin, spleen, stomach, testicle, thyroid, or uterus.
• the cancer comprises a solid tumor.
• the cancer is metastatic cancer.
• the cancer is invasive and/or metastatic cancer (e.g., stage III cancer or stage IV cancer).
• the cancer is a lymphoma.
• the cancer is lung cancer or colorectal cancer.
• the cancer is invasive and/or metastatic (advanced stage) cancer (e.g., stage II cancer, stage III cancer or stage IV cancer).
• the cancer is an early stage cancer (e.g., stage 0 cancer, stage I cancer), and/or is not invasive and/or metastatic cancer.
• the methods disclosed herein further comprise treating the subject.
• the methods described herein may be integrated into a treatment regimen for a subject.
• the biological sample(s) is analyzed by the methods described herein and the subject is treated based on the results (e.g., commence a new treatment, continue existing treatment, change in treatment (e.g., change in intervention type, dose, timing, etc.), or stop treatment.
• the treatment comprises administration of an anti-cancer agent or chemotherapeutic.
• Anti-cancer agent refers to any small molecule or other drug used in cancer treatment or prevention, whether cytostatic, cytotoxic, kinase inhibitor, or other MOA.
• Chemotherapeutics include, but are not limited to, cyclophosphamide, methotrexate, 5 -fluorouracil, doxorubicin, docetaxel, daunorubicin, bleomycin, vinblastine, dacarbazine, cisplatin, paclitaxel, raloxifene hydrochloride, tamoxifen citrate, abemacicilib,
• alpelisib anastrozole, pamidronate, anastrozole, exemestane, capecitabine, epirubicin hydrochloride, eribulin mesylate, toremifene, fiilvestrant, letrozole, gemcitabine, goserelin, ixabe
• the second therapy includes immunotherapy.
• Immunotherapies include chimeric antigen receptor (CAR) T-cell or T-cell transfer therapies, NK cell therapy, TIL cell therapy, other cellular therapy, cytokine therapy, immunomodulators, cancer vaccines, or administration of antibodies (e.g., monoclonal antibodies).
• the immunotherapy comprises administration of antibodies.
• the antibodies may target antigens either specifically expressed by tumor cells or antigens shared with normal cells.
• the immunotherapy may comprise an antibody targeting, for example, CD20, CD33, CD52, CD30, HER (also referred to as erbB or EGFR), VEGF, CTLA-4 (also referred to as CD 152), epithelial cell adhesion molecule (EpCAM, also referred to as CD326), and PD- 1 /PD-L 1.
• an antibody targeting for example, CD20, CD33, CD52, CD30, HER (also referred to as erbB or EGFR), VEGF, CTLA-4 (also referred to as CD 152), epithelial cell adhesion molecule (EpCAM, also referred to as CD326), and PD- 1 /PD-L 1.
• Suitable antibodies include, but are not limited to, rituximab, blinatumomab, trastuzumab, gemtuzumab, alemtuzumab, ibritumomab, tositumomab, bevacizumab, cetuximab, panitumumab, ofatumumab, ipilimumab, brentuximab, pertuzumab, and the like).
• the additional therapeutic agent may comprise anti-PD-l/PD-Ll antibodies, including, but not limited to, pembrolizumab, nivolumab, cemiplimab, atezolizumab, avelumab, durvalumab, and ipilimumab.
• the antibodies may also be linked to a chemotherapeutic agent.
• the antibody is an antibody-drug conjugate.
• the treating comprises active surveillance.
• the methods described herein find use in classifying a patient as suitable for active surveillance.
• the subject is monitored with additional screenings or tests for changes in overall health or changes directly related to cancer progression.
• the methods comprise collecting or receiving a series of samples over a time period from the subject and detecting the cancer cfDNA (e.g. the amount, quantity, concentration and/or level) of cancer cfDNA in each of the series of samples and comparing any measurable change in the cancer cfDNA over the period of time.
• each of the series of samples may be used for diagnosing or detecting cancer status or progression, as described in the methods herein.
• the treatment may be administered to a subject by a variety of methods.
• administration may be by various routes known to those skilled in the art, including without limitation oral, inhalation, intravenous, intramuscular, topical, subcutaneous, systemic, and/or intraperitoneal administration to a subject in need thereof.
• the treatment may be administered by parenteral administration (including, but not limited to, subcutaneous, intramuscular, intravenous, intraperitoneal, intracardiac and intraarticular injections).
• the disclosure also provides a systems for measuring cancer DNA (e.g., the concentration, level, quantity, or amount of cancer DNA) in a sample, for example, as described in the above disclosed methods.
• the systems may comprise an electrochemical detection system comprising a gold electrode and an acidic sample comprising cancer DNA or a sample comprising cancer DNA and an acidifying agent.
• the system further comprises the electrochemical detection system further comprises one or more of: a working electrode, a counter electrode, a reference electrode a sample reservoir, and a data acquisition unit.
• a working electrode e.g., a single electrode, an electrode array, a microfluidic device, an electrochemical sensor, or point-of -care device.
• the sample reservoir may be configured to receive any type of sample, as described above, or may further comprise reagents/components (e.g., acidifying agent, purification reagent, buffers, and the like) used to process the sample prior to analysis.
• reagents/components e.g., acidifying agent, purification reagent, buffers, and the like
• the sample reservoir is integrated with one or more of the electrodes.
• the system may further comprise a sample receiving area in which the sample is initially provided prior to being loaded into the sample reservoir.
• the system may further comprise a sample purification system.
• the purification system may comprise any of: chromatography columns or media, affinity columns or media, beads or particles, filters, and the like useful in purification of the sample (e.g., to remove contaminating materials or purify DNA).
• the system may further a flow management or dispensing device (e.g., a pump). The flow management or dispensing device may transfer the sample from the sample receiving area to the sample reservoir or to the electrodes.
• the data acquisition unit may include a power supply, a potentiostat, a bipotentiostat, a galvanostat, an impedance analyzer, one or more processors (e.g., one or more computers or computer systems), and/or a computer-readable medium to perform any or all of: measuring the electrochemical signal, detecting the presence or determining an amount, quantity, concentration and/or level of cancer DNA, comparing the results between biological samples, comparing the results to cutoff values or reference values, classifying the cancer, or diagnosing the patient as having cancer.
• the data acquisition unit may be configured to communicate with the other components of the system or the data acquisition unit via wired or wireless communications.
• the system comprises an indicator or display configured to show the electrochemical signal or the presence, amount, quantity, concentration and/or level of cancer DNA.
• the system comprises an indicator or display to show when the cancer DNA (e.g., cfDNA) (e.g., amount, quantity, concentration and/or level of cancer DNA (e.g., cfDNA)) is lower or higher than a reference amount, quantity, concentration and/or level.
• the indicator or display is further configured to show the ratio of the amount, quantity, concentration and/or level of cancer DNA (e.g., cfDNA) to the amount, quantity, concentration and/or level of total DNA (e.g., total cfDNA) and/or when the ratio is lower or higher than a cutoff value.
• cancer DNA e.g., cfDNA
• total DNA e.g., total cfDNA
• the indicator or display may use any manner of visual or audible means to display the result including but not limited to text, graphs, charts, heat maps, other image based methods, color indications, beeping, and the like.
• the indicator or display may be configured to transmit the results to another device connected wirelessly or integral to the present system.
• the indicator or display may transmit the result to a clinical device, a patient record, a patient device, a data storage repository, or similar.
• the systems optionally may include disposable/consumable components that are utilized for the analysis or sample preparation.
• the system or kit may further contain additional containers or devices for use with the methods disclosed herein.
• kits that any or all of the components of the systems.
• the kits include an electrochemical detection system comprising a gold electrode, an acidic sample comprising cancer DNA or a sample comprising cancer DNA and an acidifying agent, a working electrode, a counter electrode, a sample reservoir, a data acquisition unit, a sample purification system and/or a flow management or dispensing device (e.g., a pump).
• the kits also include calibration and/or control samples.
• kits Individual member components of the kits may be physically packaged together or separately.
• the components of the kits may be provided in bulk packages (e.g., multi-use packages) or single-use packages.
• the kits provided herein are in suitable packaging.
• suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like.
• the DPV baseline measurement was conducted using a Palmsens benchtop potentiostat.
• the electrochemical window for the measurements was -0.5-0.5 V.
• the experimental parameters were E-step (0.01 V), E pulse (0.05 V), t pulse (0.05 s) and the scan rate (0.1 V/s).
• the baseline measurement was conducted using 50 pL of 2.5 mM
• the working cfDNA samples were obtained by diluting the appropriate amount of cfDNA from each pool for each working concentration.
• the acidic samples were diluted in buffer to achieve pH 2.
• the neutral samples were diluted in 0. 1 M phosphate buffered saline solution.
• a 10 pL droplet of the cfDNA sample solution was then drop casted on the working electrode surface and was left to incubate for 10 minutes. The sample droplet was then gently rinsed from the electrode surface using approximately 5 drops of 0. 1 M PBS.
• the DPV sample measurement was conducted using a Palmsens benchtop potentiostat.
• the electrochemical window for the measurements was -0.5-0.5 V.
• the experimental parameters were E-step (0.01 V), E pulse (0.05 V), t pulse (0.05 s) and the scan rate (0.1 V/s).
• the baseline measurement was conducted using 50 pL of 2.5 mM solution.
• the relative peak current is defined as the change in the peak current magnitude of the baseline DPV after incubating cfDNA sample on the electrode surface and performing a second sample measurement DPV.
• the relative peak current (% ir) was calculated using peak current values for the baseline and sample DPV measurements by the following equation:
• SPEs screen-printed gold electrodes
• TFEs thin-film gold electrodes
• Electrode characterization was performed using a variety of experimental techniques including optical profilometry (OP), electrochemical characterization (CVs) and scanning electron microscopy (SEM) to determine the surface roughness, electroactive surface area and the surface morphology of the commercial electrodes. Evaluation of the electrodes began with OP experiments to record a digital map of the roughness profiles for each electrode, while the electroactive surface areas were calculated using the Cottrell equation by performing a series of CVs at increasing scan rates. Data indicating the surface roughness, electroactive surface area, experimental throughput, and adsorption characteristics of patient cfDNA samples is shown for both the SPEs and the TFEs, Table 1. SEM images of the SPEs and the TFEs were recorded to observe differences in the surface morphology of each commercial electrode.
• OP optical profilometry
• CVs electrochemical characterization
• SEM scanning electron microscopy
• cfDNA samples extracted from plasma using standard PCI extraction methods (25:24: 1 Phenol:Chloroform:Isoamyl) obtained from the University of Colorado Biorepository.
• cfDNA was obtained from 15 different subjects, 6 were from healthy individuals (Normal), 5 were from patients with colorectal (CRC) cancer and 4 were from patients with non-small-cell lung cancer (NSCLC).
• the relative adsorption of the patient cfDNA samples (each diluted to a working concentration of 1 pg/pL) were recorded on both the SPEs (FIG. 2A) and TFEs (FIGS. 2B and 2C) .
• FIGS. 2A-2C the normal, CRC and NSCLC cfDNA samples are shown in grey (left), orange (middle), and green (right) respectively.
• the normal cfDNA samples were from each of the six different healthy individuals, FIGS. 2A-C (P1-P6, grey).
• the CRC cfDNA samples were from each of five different patient cfDNA samples diagnosed with CRC cancer, FIGS. 2A-C (P1-P5, orange).
• the NSCLC cfDNA samples were from each of four different patient cfDNA samples diagnosed with NSCLC cancer, FIGS. 2A-C (P1-P4, green). DNA was tested for quality and quantity as described in Example 1.
• Cancer burden of a diagnosed patient is expected to vary from time of diagnosis and throughout various stages of treatment reflecting progression and/or remission. Accordingly, the electrochemical signal derived from the cfDNA of a recovered patient in remission would more closely resemble the signal obtained from a healthy individual. Conversely, increased cancer burden resulting from ineffective therapy would yield an electrochemical response in the opposite direction. In this regard, the ability to monitor the efficacy of a patient specific cancer treatment would require longitudinal patient specific sample analysis. Sets of clinical cfDNA patient samples were obtained before and after cancer treatment and were electrochemically measured using SPEs, FIGS. 3A-3C.
• a plasma sample from a blood donor directly prior to diagnosis with aggressive B cell lymphoma was compared to a longitudinal sample from patient 1 after finishing treatment (1 year later).
• the samples were extracted using UltraPrep DNA extraction and normalized by AccuBlue High Sensitivity dsDNA kit concentration measurement (Biotium).
• the second set of plasma samples was obtained from a patient with NSCLC before the start of pembrolizumab and at day 34 after undergoing treatment (patient 2) where later imaging at 6 months determined the patient responded positively to treatment.
• Patient 2 samples were extracted using QIAamp MinElute cfDNA kit (Qiagen) and the concentration was measured using high sensitivity dsDNA Qubit kit (ThermoFisher).
• the third set of cfDNA was extracted using UltraPrep DNA extraction from urine obtained from PrecisionMed and normalized by Agilent 2100 BioAnalyzer.
• the patient had NSCLC.
• the first specimen was obtained June 30 th , 2020, at the time of therapy initiation; this was 3 weeks after the diagnosis.
• the second specimen was obtained July 29 th , 2021, after the patient progressed following treatment with nivolumab and ipilimumab.
• Table 2 provides information on the cfDNA samples from all three patients, including the timepoints for sample collection and the expected cancer burden based upon treatment efficacy.
• the lower signal obtained from cfDNA of cancer patients correlates to T1 (high expected cancer burden, Table 2) and the higher signal obtained from cfDNA of healthy subjects correlates to T2 (decreased cancer burden; or even remission, Table 2).
• the cfDNA specimens used in patient 2 were isolated from their urine, illustrating application to a variety of biofluids. The opposite effect was observed for patient 3, where the relative peak current was decreased when comparing T1 and T2. The cancer treatment for patient 3 was ineffective, and the sample acquired at T2 was correlated to a progressive disease profile. As a result, the lower relative peak current observed at T2 compared with T1 is in agreement with the expected adsorption of cfDNA for a high cancer burden.
• the difference in adsorption between the normal and cancer cfDNA samples was calculated by taking the average of the relative peak current values for the normal cfDNA pool and subtracting the average of the values for both the CRC and NSCLC pools and was plotted as a function of concentration, FIG. 4B.
• the difference in adsorption between the cfDNA of healthy individuals and those with cancer (e.g., difference in average %ir values) at each concentration was quantified, and the p-values representing the statistical differences at each concentration are displayed in Table 3.
• FIG. 4A To identify the optimal cfDNA concentration with respect to the largest difference in adsorption between cfDNA from healthy individuals and those with either CRC or NSCLC cancer a range of concentrations (0.1 - 10 pg/pL) were investigated, FIG. 4A. Within the 0.1- 2 pg/pL range there is not a statistically significant separation between the relative peak current values for the normal and cancer cfDNA sample pools. However, at concentrations above 2 pg/pL statistically significant separation between the normal and cancer cfDNA pools was observed.
• FIG. 4B shows the difference in relative peak current (%ir) between the normal and an average of both cancer cfDNA pools as a function of increasing concentration.
• the commercial gold screen-printed electrodes (SPE; C223BT model) with a gold working electrode diameter of 1.6 mm, gold counter electrode and a silver reference electrode were obtained from Dropsens and were stored at room temperature protected from light and humidity.
• the gold SPEs were fabricated on a ceramic substrate (33 x 10 x 0.5mm).
• Evaluation of the adsorption DNA samples to the electrode surface was performed using a series of differential pulse voltammetry (DPV) experiments conducted using a Palmsens benchtop potentiostat. The electrochemical window for the measurements was -0.5- 0.5 V.
• the experimental parameters were E-step (0.01 V), E pulse (0.05 V), t pulse (0.05 s) and the scan rate (0.1 V/s).
• the SPEs were stratified based on the baseline signal - ranking from highest to lowest peak current magnitude. All patient timepoints were exposed to the entire range of peak current magnitudes, for example alternate between patient timepoints for each electrode across the baseline spectrum of peak current magnitudes.

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