Method for generating a DNA solution free of cells and subcellular bodies for quantification in microdroplets

Abstract

The present development is a method for quantifying total DNA in a biological fluid from an individual using a microdroplet of sample, wherein the method comprises depositing a volume of 0.1-100 µL of a biological fluid on a solid support; subjecting the biological fluid to a DNA release process; incubating; and quantifying total DNA. The described method provides a simple, fast and economical alternative for releasing DNA from a sample and quantifying same directly and completely in biological samples using a minimal sample volume, and enables fast and effective detection of diseases related to the amount of total DNA in a biological sample, including disorders associated with physical and mental stress and conditions associated with biological age.

Description

[0001] METHOD FOR GENERATING A CELL- AND SUBCELLULAR BODY-FREE DNA SOLUTION FOR QUANTIFICATION IN MICRODROPLETS

[0002] TECHNICAL FIELD

[0003] The present development is related to the field of cellular and molecular biology, particularly with methods for releasing DNA from cells, subcellular bodies adding to their presence in a liquid (free and circulating DNA) and quantifying DNA, and more particularly with methods for releasing and quantifying total DNA in a microdroplet of biological fluid.

[0004] BACKGROUND

[0005] The total amount of DNA in a biological sample is a key diagnostic marker for identifying numerous diseases in humans and animals, and can even be related to an individual's mental health and biological age. Chronic stress, in both its physical and mental forms, has been identified as a contributing factor in the development of a variety of diseases, including, but not limited to, cardiovascular and psychological disorders. The lack of specific and reliable biomarkers for assessing stress, especially mental stress, is a significant deficiency in current healthcare. However, in recent years, several authors have proposed circulating cell-free DNA (cfDNA) as one of the potential biomarkers for obtaining an accurate diagnosis of this condition (Hummel, EM et al. 2018. Cell-free DNA release under psychosocial and physical stress conditions. Translational Psychiatry, 8(1): 236).

[0006] In healthy individuals, circulating cell-free DNA (cfDNA) originates from blood cells, endothelial cells, and hepatocytes (Moss, J., et al. 2018. Comprehensive human cell-type methylation atlas reveals origins of circulating cell-free DNA in health and disease. Nature Communications; 9(1): 5068). However, during the stress or disease response, the body also releases subcellular byproducts such as microvesicles, which can also contain DNA. These microvesicles, which originate from the cell membrane, play a crucial role in intercellular communication and can transport proteins, lipids, and genetic material between cells. Their increased circulation during episodes of stress or disease suggests their involvement in the propagation of stress signals and the modulation of the immune response, which could exacerbate the inflammatory state and contribute to the pathogenesis of various diseases.

[0007] The study of unbound DNA (lcDNA) as a biomarker offers an opportunity to develop a method for the early detection of psychological and physical stress, which would allow for the prevention of its adverse effects on human health. Early identification of stress can help prevent cardiovascular disease, emotional and mental disorders, thus improving people's quality of life.

[0008] Although there are currently different methodologies for quantifying DNA from various tissues and biological fluids, there is still a need to formulate alternative strategies that allow overcoming obstacles that limit its routine application in diagnostic procedures, such as the cost of the reagents used, the need for removal of contaminants, the invasive nature of the sample collection methods and the time required to obtain results.

[0009] One alternative to overcome these drawbacks is found in US patent 8088576, which describes a device for isolating DNA using a genetic material binding step from a sample, preferably a blood sample. Specifically, the method is based on releasing DNA from biological structures present in the sample using dry lysis reagents. This DNA is then isolated using DNA-binding substrates, but the method does not aim to isolate cell bodies or subcellular components, nor to obtain a solution containing total DNA.

[0010] Ghatak and colleagues developed a method for extracting genomic DNA from biological samples such as saliva, hair, urine, and blood from human subjects. According to the authors, this cost-effective method allows for the extraction of DNA, which can then be used for subsequent processes and analyses, such as PCR amplification and digestion of the resulting products. In general terms, the method described in this study involves treating the sample with proteinase K and a lysis buffer containing EDTA, SDS, and Tris-HCl; organic extraction with phenol-chloroform (except for the urine sample); and DNA precipitation with absolute ethanol or isopropanol and sodium acetate (Ghatak S., et al. 2013. A simple method of genomic DNA extraction from human samples for PCR-RFLP analysis. J Biomol Tech.; 24(4): 224-31).

[0011] In contrast, the method described in this disclosure provides a simple, rapid, and cost-effective alternative for releasing DNA from a sample and directly quantifying it in total in biological samples from a minimal volume. This allows for the rapid and effective detection of diseases related to the total amount of DNA in a biological sample, including, among others, disorders associated with physical and mental stress, and conditions associated with biological age. This development enables a broader and more accessible application of DNA quantification in clinical and research settings, improving the accuracy and effectiveness of disease diagnosis and facilitating its use in daily practice.

[0012] BRIEF DESCRIPTION

[0013] The present development corresponds to a method for quantifying total DNA in a biological fluid where the method comprises depositing a volume between 0.1 pL and 100 pL of a biological fluid on a solid support; subjecting the biological fluid to a DNA release process; and quantifying the total DNA.

[0014] BRIEF DESCRIPTION OF THE FIGURES

[0015] FIG. 1 General scheme of the method for extraction and quantification of total DNA.

[0016] FIG. 2 Fluorometry estimation of total DNA released from skin fibroblasts exposed to ultraviolet (UV) radiation.

[0017] FIG. 3 Spectrophotometric estimation of total DNA in individuals of different ages. The three asterisks (***) indicate a high level of significance (p < 0.001) determined by the Mann-Whitney test.

[0018] FIG. 4A Spectrophotometric estimation of total DNA in a 24-year-old individual under stress conditions. The three asterisks (***) indicate a high level of significance (p < 0.001) determined by the Mann-Whitney test.

[0019] FIG. 4B Spectrophotometric estimation of total DNA in a 39-year-old individual under stress conditions. The three asterisks (***) indicate a high level of significance (p < 0.001) determined by the Mann-Whitney test.

[0020] FIG. 5 Total DNA estimation by spectrophotometry in medical students before (PRE) and after (POST) their hospital stay. The three asterisks (***) indicate a high level of significance (p < 0.001) determined by the Mann-Whitney test.

[0021] FIG. 6 Estimation of total DNA by fluorometry of individuals subjected to periods of stress.

[0022] DETAILED DESCRIPTION

[0023] For the purpose of interpreting the terms used throughout this document, their usual meaning in the technical field should be considered, unless a specific definition is included or the context clearly indicates otherwise. Additionally, terms used in the singular form will also include the plural form.

[0024] Method for quantifying total DNA from a biological sample

[0025] The present development is directed to a method for quantifying the total DNA of cells, subcellular and extracellular components of a biological fluid, which comprises the following steps:

[0026] (a) depositing 0.1 pL to 100 pL of a biological fluid onto a solid support;

[0027] (b) subjecting the biological fluid from (a) to a DNA release process;

[0028] (c) quantifying the total DNA present in the product of step (b); wherein the quantification of total DNA is performed by spectrophotometry, fluorometry or other methods of quantifying nucleosides, nucleotides or analogues.

[0029] In some variations of the method, the biological fluid is selected from either a natural or a synthetic biological fluid. In specific variations, the natural biological fluid corresponds to a sample of blood, urine, saliva, sweat, tears, cerebrospinal fluid, or mucus from an individual, whether human or animal, resuspended scrapings, or cell cultures.In other specific modalities, synthetic biological fluid corresponds to a biomanufactured product with ingredients of biotechnological origin or for the purpose of a biotechnological application, such as synthetic blood based on recombinant hemoglobin and perfluorocarbons; synthetic amniotic fluid composed of isotonic solutions enriched with growth factors; synthetic cerebrospinal fluid based on balanced electrolytes and neuroprotective agents; synthetic plasma for the preservation of organs and tissues; bioinks for 3D printing of tissues composed of hydrogel and encapsulated cells; and synthetic cell culture media enriched with serum and specific differentiation factors.

[0030] For the purposes of the present invention, the "solid support" on which the biological fluid is deposited corresponds to an inert surface or container made of an organic or inorganic material that does not interfere with the release and subsequent quantification of the DNA present in the sample. In some embodiments, the solid support is made of a material selected from glass, paper, silicon, nitrocellulose, gold, silver, polystyrene, polypropylene, graphene, among others. In one particular embodiment, the solid support is made of polypropylene.

[0031] The DNA release process involves cell lysis through techniques known in the field, which can be mechanical, physical, and / or chemical. In some methods, these mechanical processes involve disrupting cellular structures containing DNA by agitating the sample in the presence of spherical particles, also known as beads. In other methods, physical DNA release processes are selected from thermal shock and sonication. In still other methods, DNA release involves chemical processes, such as cell lysis using a lysis buffer, a technique generally known in the field. This chemical cell lysis process involves the use of detergents that disintegrate cellular and nuclear membranes at the molecular level.In certain modalities, these chemical methods involve enzymatic treatments that allow the digestion of proteins that could interfere with DNA extraction. In certain modalities, the lysis buffer is selected from various options specific to DNA extraction. One modality uses a lysis buffer containing SDS, EDTA, and Tris-HCl. Sodium dodecyl sulfate (SDS) is an anionic detergent that solubilizes cell and nuclear membranes, allowing the release of their contents. EDTA (ethylenediaminetetraacetic acid) is added to chelate divalent ions, such as magnesium, which are essential cofactors for nucleases that could degrade DNA. Tris-HCl acts as a buffer to maintain a stable pH during the lysis process.

[0032] Additionally, some formulations include proteinase K, an enzyme that digests contaminating proteins, especially nucleoproteins, facilitating the release of high-purity DNA. Proteinase K is effective in degrading proteins present in the cell, resulting in a DNA solution less contaminated by proteins.

[0033] The DNA release process can include a precipitation step, where certain agents that bind specifically to DNA are used to facilitate its separation from the other cellular components. A classic example is the use of sodium acetate along with alcohols such as ethanol or isopropanol. These agents promote the binding of DNA molecules, allowing them to clump together and precipitate out of solution. The precipitated DNA can be collected by centrifugation, forming a pellet that can be washed and resuspended in a suitable buffer for further analysis.

[0034] In some methods, the DNA release process involves incubating the biological fluid for 10 seconds to 40 minutes. In specific methods, the incubation lasts between 1 and 20 minutes. In more specific methods, the incubation lasts between 2 and 20 minutes, 1 and 15 minutes, 2 and 15 minutes, 2 and 10 minutes, or 2 and 5 minutes.

[0035] In some formulations, incubation is carried out at a temperature between 15 and 40 °C. In specific formulations, the incubation temperature is 20 to 40 °C. In more specific formulations, the incubation temperature is 22 to 40 °C, 20 to 35 °C, 22 to 30 °C, 22 to 35 °C, 25 to 35 °C, 25 to 30 °C, or 30 to 35 °C. The quantification of total DNA in step (c) is performed using methods known in the art, including, but not limited to, spectrophotometry, fluorometry, or other methods for quantifying nucleosides, nucleotides, or analogues. In specific formulations, DNA quantification is performed by spectrophotometry or fluorometry.

[0036] For the detection and quantification of DNA using fluorophores, molecules that bind specifically to DNA and fluoresce when excited by a light source are employed. A common example is ethidium bromide, an intercalating agent that inserts itself between the bases of double-stranded DNA. Upon binding to DNA, ethidium bromide emits intense orange fluorescence under ultraviolet light, allowing the visualization and quantification of DNA in agarose gels.

[0037] Another example is the use of fluorophores such as SYBR Green, a DNA-binding dye widely used in real-time PCR assays. SYBR Green preferentially binds to the double strand of DNA and fluoresces green when excited. The intensity of the fluorescence is proportional to the amount of DNA present, allowing for the precise detection and quantification of DNA during real-time amplification.

[0038] The detection and quantification of DNA using spectrophotometry utilizes principles of light absorption at specific wavelengths. DNA exhibits a characteristic absorption peak at 260 nm, allowing its concentration in a solution to be measured. Using a spectrophotometer, the amount of DNA can be determined by measuring the absorbance of the sample at this wavelength. Furthermore, the absorbance ratio at 260 / 280 nm is used to assess DNA purity, as protein and other contaminants also absorb light, but at different wavelengths.

[0039] Diagnostic use

[0040] The DNA quantification method described here facilitates the diagnosis of diseases and disorders associated with the total amount of DNA in a biological fluid sample from an individual. In some applications, this method allows for the identification of individuals at risk for various diseases and disorders, including proliferative diseases such as breast cancer, cervical cancer, lung cancer, colorectal cancer, prostate cancer, ovarian cancer, leukemia, and melanoma; pulmonary diseases such as chronic obstructive pulmonary disease (COPD), chronic bronchitis, and emphysema; autoimmune diseases such as lupus and multiple sclerosis; psychiatric disorders such as anxiety and depression; cardiovascular diseases such as coronary artery disease and myocardial infarction; and metabolic diseases such as metabolic syndrome and diabetes; among others.

[0041] In other modalities, the method disclosed here is useful for diagnosing stress in an organism, including but not limited to cellular stress, physical stress, and psychological stress.

[0042] In other modalities, the method disclosed here allows the determination of an individual's biological age.

[0043] EXAMPLES

[0044] The technological development is presented in detail through the following examples, which are provided for illustrative purposes only and not to limit its scope.

[0045] Example 1. Evaluation of the method for DNA release and quantification from fibroblasts treated with UV light

[0046] In the first application, human skin fibroblasts cultured in a synthetic biological fluid containing cell nutrients (DMEM with 10% fetal bovine serum) were used under optimal conditions to maintain viability and avoid stress. These fibroblasts were exposed to ultraviolet (UV) radiation emitted by a BS-02 UVR system (Purifier Logic Class II, KS, USA) for 10 to 30 minutes, with the plate lid in place. To estimate the amount of DNA released using the method described here, a measurement was performed using fluorometry. The fluid used is a biosynthesized cell culture medium enriched with factors released by the cells related to stress or survival.

[0047] The cells were subjected to different doses of UV radiation (exposures of 10, 20 and 30 minutes equivalent to 18 mJ / cm²). 2 , 36 mJ / cm 2 and 54 mJ / cm 2(respectively) a significant increase in extracellular DNA was observed, quantified using the described method. First, 10 µl of the medium from the different conditions were taken and placed on a polypropylene plastic surface, then 10 µl of lysis buffer were added. The mixtures were homogenized and subsequently incubated for 5 minutes, then 5 µl were taken for the quantification step by spectrophotometry. The 5 µl were mixed with the fluorimetric measurement standard (5 µl in 95 µl), incubated for 30 minutes, and the measurement was performed with a Qubit 4 Fluorometer to detect cell-free DNA.

[0048] This extracellular DNA, present in subcellular bodies such as microvesicles, organelles, dead cells that float in the medium and release their DNA by the described method, and other structures, corresponds to an increase in the amount of cellular DNA due to exposure to UVR radiation, which increases cellular damage. This cellular damage can be effectively and accurately identified in total numbers using the described method (FIG. 2).

[0049] Example 2. Determination of biological age

[0050] In a second application, the method from Example 1 was used to release the cellular and subcellular DNA content from a microdroplet of blood containing only 10 pL, with the aim of quickly determining a human's biological age. For this study, 10 individuals aged 21 to 24 years (young adults) were compared to 10 older adults aged 39 to 52 years. The estimation method was spectrophotometry.

[0051] To ensure that the results reflected only the effects of biological age in each group of individuals evaluated, several control measures were implemented. Prior to the study, a comprehensive survey was conducted, and informed consent was obtained from all participants. In this survey, individuals provided detailed information about their general health status, exercise, sleep duration, and diet, including the absence of chronic or acute illnesses such as diabetes, hypertension, cardiovascular disease, or recent infections. Furthermore, it was verified that none of the participants were undergoing medical treatment that could affect the biological age assessment, such as immunosuppressant or hormone therapies. This data collection allowed for the selection of individuals who, according to their self-reported health, were in optimal condition, thus reducing the influence of external factors on the study results.

[0052] Significant differences were observed between the groups, corresponding to an increase in the amount of circulating DNA with age (Fig. 3). These differences were quantified using a non-parametric Mann-Whitney statistical analysis, comparing the two conditions or age groups. The results indicated that older individuals had significantly higher levels of circulating DNA compared to younger adults. This increase in the amount of circulating DNA with age could be related to cellular aging and the deterioration of DNA repair mechanisms, which is reflected in the greater amount of genetic material released into the bloodstream and subsequently in the greater amount of DNA released into cells by the method described.

[0053] The described method offers the unique advantage of generating a solution containing total DNA, derived from subcellular components, circulating DNA, and cellular DNA, which can be used in applications such as biological age estimation. The solution obtained through this method for aged individuals contains a wealth of factors related to cellular damage and stress, enabling biological age estimation. The method's ability to efficiently release and quantify total DNA facilitates its application in aging studies and in assessing the overall health status of individuals. Furthermore, the correlation between extracellular DNA levels and biological age provides an innovative tool for investigating aging processes and developing strategies to mitigate their effects.This methodology stands out not only for its accuracy and speed, but also for its potential to be used in various clinical and research contexts, where the determination of biological age is crucial.

[0054] Example 3. Analysis of the effects of stress on the amount of lcDNA

[0055] Effects of stress on individuals of different ages. In a third application, the method from Example 1 was used to release DNA present in cell and subcellular bodies from two individuals, one 24 years old and the other 39 years old, from a 10 pL microdroplet of blood, following the described method. The methodology used for estimation was spectrophotometry.

[0056] It was determined that both individuals, when subjected to stressful conditions, released a greater amount of lcDNA (Fig. 4). In the pre-conducted survey, detailed data were collected on various stress-related factors, including amount of sleep, workload, and perceived worry or emotional stress. These factors were considered to assess the participants' stress levels. The results suggest that stress, whether due to lack of sleep, overwork, or personal worries, can contribute to the release of greater amounts of lcDNA into the bloodstream. This phenomenon could be related to stress-induced cell damage or cell death, which underscores the importance of considering life context and environmental conditions when interpreting lcDNA levels as indicators of health or disease.This response directly correlates with their health status in relation to stress.

[0057] The method allowed for the detection of differences in DNA release between the two individuals, suggesting that stress has a significant impact on the amount of extracellular DNA released and on the presence of subcellular bodies. This approach not only facilitates the accurate quantification of extracellular DNA under stress conditions but also provides a valuable tool for assessing the impact of stress on cellular health. The ability of this method to measure specific stress responses in individuals of different ages highlights its potential for applications in clinical research and in the development of strategies to manage and mitigate the effects of stress on health.

[0058] Effects of physical and psychological stress on individuals of the same age

[0059] In a subsequent application, 10 medical students participated in a study where a 10-pl blood sample was collected and processed using the method in Example 1, before and after their hospital stay. This period in the hospital is known to be highly stressful due to the amount of worry, lack of rest, sleep deprivation, and constant pressure from the medical team and patients. The method allows for the release of DNA from cells and subcellular bodies, providing sufficient capacity to estimate changes in cellular physiology after 24 hours of stress.

[0060] Compared to other more expensive and less accessible methods, such as the quantification of proinflammatory cytokines or cortisol levels, this method stands out for its ease and accuracy in detecting these changes. The results showed a significant change in extracellular DNA levels associated with post-shift stress, reflecting an alteration in cellular physiology from the cellular level down to the subcellular level (FIG. 5). This tool represents an important advance for monitoring stress responses in clinical settings and could be fundamental for developing interventions that mitigate the negative health effects of stress.

[0061] Example 4. Analysis of the effect of periods of cellular stress on the total amount of DNA

[0062] To understand the dynamics of total DNA present in a sample and its association with periods of cellular stress, samples were taken from three individuals: a healthy 25-year-old man, a healthy 40-year-old man (both with healthy lifestyles, exercising three times a week, and abstaining from alcohol except for work-related activities), and a 28-year-old woman with symptoms of depression. The samples were obtained from a six-day follow-up of each individual.

[0063] The analysis showed that individuals who reduced their stress levels also exhibited a decrease in total DNA levels (FIG. 6). However, the 28-year-old woman consistently had higher total DNA levels than the other two individuals. She was subsequently diagnosed with persistent worry, though not clinical depression, and with polycystic ovary syndrome.

[0064] This example demonstrates that individuals with healthy habits have lower total DNA levels compared to those with health conditions, whose levels remain high for their age and circumstances. This method allows for early detection before clinical symptoms develop, representing a significant advantage over conventional blood tests. The ability to detect and quantify total DNA provides a valuable tool for preventative health monitoring and stress management, facilitating earlier and more effective interventions.

Claims

CLAIMS 1. A method for quantifying the total DNA of cells, subcellular and extracellular components of a biological fluid, comprising the following steps: (a) depositing 0.1 pL to 100 pL of a biological fluid onto a solid support; (b) subjecting the biological fluid from (a) to a DNA release process; and (c) quantifying the total DNA present in the incubated mixture of (c); wherein the quantification of total DNA is performed by spectrophotometry, fluorometry or other methods of quantifying nucleosides, nucleotides or analogues.

2. The method according to Claim 1, wherein the biological fluid corresponds to a sample of blood, urine, saliva, sweat, tears, cerebrospinal fluid or mucus, resuspended scrapings, cell cultures or a biosynthetic product.

3. The method according to Claim 1, wherein the solid support is selected from glass, paper, silicon, nitrocellulose, gold, silver, polystyrene, polypropylene, graphene, organic or inorganic biomaterial polymers.

4. The method according to any of Claims 1 to 3, wherein the DNA release process corresponds to cell lysis with a lysis buffer.

5. The method according to Claim 1, wherein the incubation of step (c) is carried out at a temperature between 22 and 30 °C.

6. Use of the method according to Claim 1 for the diagnosis of cellular stress, physical stress or psychological stress.

7. Use of the method according to Claim 1 for the diagnosis of proliferative diseases, pulmonary diseases, autoimmune diseases, psychiatric disorders, cardiovascular diseases, and metabolic diseases.

8. Use of the method according to Claim 1 for the determination of biological age.

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