Compound stabilizer for blood sampling card

Abstract

The invention belongs to the technical field of biomarker sampling and stabilization, and particularly relates to a compound stabilizer for a blood sampling card, which comprises the following components: a cell lysis agent, which is selected from guanidine, sodium dodecyl sulfate, quaternary ammonium salt or polyquaternary ammonium salt and has a concentration of 1-5%; the NAD < + > metabolic enzyme inhibitor is selected from nicotinamide riboside, apigenin or ABT-888, and the concentration of the NAD < + > metabolic enzyme inhibitor is 0.1-1.0 mM; the NAD < + > degradation product is selected from adenosine diphosphate ribose, adenosine monophosphate, nicotinamide mononucleotide or nicotinamide, and the concentration of the NAD < + > degradation product is 0.1-1.0 mM; the pH stabilizer is selected from citric acid or EDTA (Ethylene Diamine Tetraacetic Acid), and the concentration of the pH stabilizer is The materials used in the formula are all common chemical reagents in the market, are low in toxicity, green and safe, and are simple in preparation and smearing process and suitable for industrial production.

Description

Technical Field

[0001] This invention belongs to the field of biomarker sampling and stabilization technology, specifically a composite stabilizer for blood collection cards. Background Technology

[0002] NAD+ (nicotinamide adenine dinucleotide) is an indispensable coenzyme in the human body, deeply involved in almost all energy metabolism and transport processes, including glycolysis and the tricarboxylic acid cycle. Numerous studies have confirmed that its levels are not only closely linked to human health and the aging process, but also exhibit significant fluctuations in various diseases such as atherosclerosis, arthritis, and hypertension. Therefore, accurately measuring the NAD+ content in organisms is of great significance for current scientific research and clinical monitoring.

[0003] Home blood sampling is a minimally invasive, convenient, and efficient blood collection method. With its advantages of high accessibility, low cost, rapid results, and intuitive interpretation, it is particularly suitable for telemedicine scenarios and personal health self-monitoring, and shows great application potential in disease prevention, chronic disease management, and other fields.

[0004] However, home blood sampling testing technology still faces challenges in the preservation of specific biomarkers, particularly in NAD+ detection. Traditional Whatman 903DBS cards (dried blood spot cards) experience extremely rapid NAD+ degradation when stored at room temperature (25°C). Although QIAcard FTA DMPK-B cards can maintain NAD+ stability above 85% for up to a week (Analytical and Bioanalytical Chemistry, 2023), after 7 days, the concentration loss exceeds 25% (Revvity Omics, Pittsburgh, PA15275; 2.). In contrast, the NAD+ half-life of untreated DBS cards can be as short as 3-5 days (Journal of Mass Spectrometry, 2021). This is mainly because residual enzymes remain active after blood drying, and changes in environmental humidity accelerate NAD+ degradation.

[0005] Furthermore, existing related technical solutions also have significant limitations. For example, the invention patent with publication number CN118837152B, entitled "A biological sample collection card and a biological sample detection kit," uses an inorganic salt formula that significantly inhibits enzyme activity, causing high-throughput detection methods such as enzyme-linked immunosorbent assay (ELISA) that rely on enzyme-catalyzed reactions to fail to function properly. On the other hand, the technical solutions of other related patents on the market are often only suitable for low-throughput detection methods such as LC-MS / MS, and are not compatible with high-throughput methods such as ELISA that can simultaneously detect a large number of samples. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing a composite stabilizer for blood collection cards, thereby solving the technical problems mentioned in the background art.

[0007] To address the above technical problems, the following technical solution is adopted: A composite stabilizer for blood collection cards, comprising the following components:

[0008] Cell lysis agent, selected from guanidines, sodium dodecyl sulfate, quaternary ammonium salts or polyquaternary ammonium salts, at a concentration of 1-5%;

[0009] NAD+ metabolic enzyme inhibitors, selected from nicotinamide riboside, apigenin or ABT-888, at a concentration of 0.1-1.0 mM;

[0010] NAD+ degradation products, selected from adenosine diphosphate ribose, adenosine monophosphate, nicotinamide mononucleotide or nicotinamide, at a concentration of 0.1-1.0 mM;

[0011] pH stabilizer, selected from citric acid or EDTA, at a concentration of 40-60 mM.

[0012] Preferably, the cell lysis agent is sodium dodecyl sulfonate.

[0013] Preferably, the NAD+ metabolic enzyme inhibitor is apigenin or nicotinamide riboside.

[0014] Preferably, the NAD+ degradation product is adenosine diphosphate ribose or nicotinamide.

[0015] Preferably, the pH stabilizer is a combination of citric acid and EDTA.

[0016] As a preferred embodiment, a blood collection card is prepared by impregnating cellulose paper with a composite stabilizer and then drying it in an electric heating drying oven at 60-105°C for 4 hours.

[0017] Preferably, the thickness of the cellulose paper is 0.3-0.5 mm.

[0018] As a preferred embodiment, a method for preparing a dried blood spot sample includes the following steps:

[0019] Use a pipette to add 10 μL of whole blood sample onto the blood collection card as described in claim 6 or 7;

[0020] After drying and shaping at room temperature, the blood collection cards are stored in a sealed bag.

[0021] As a preferred embodiment, a method for extracting NAD+ from dried blood spot samples includes the following steps:

[0022] Samples were taken from the dried blood spot sample prepared according to claim 8 using a paper punch.

[0023] After sampling, the dried blood spots were transferred to centrifuge tubes, lysis buffer was added, the mixture was vortexed, and the supernatant was extracted.

[0024] The supernatant and neutralization solution were mixed before testing.

[0025] Preferably, the lysis buffer is a mixture of 10% acetic acid and erythrocyte lysis buffer at a volume ratio of 9:1.

[0026] The beneficial effects of this invention are:

[0027] 1. An innovatively designed composite stabilizer system consisting of cell lysing agent, NAD+ metabolic enzyme inhibitor, NAD+ degradation products, and pH stabilizer extends the half-life of NAD+ at room temperature from 3 days (traditional DBS card) to 10 days (after 10 days of storage at room temperature). The deviation of WST-8 assay is ≤5% (compared to ≥30% deviation of DMPK-B card) after 10 days of storage at room temperature.

[0028] 2. Compared with untreated blood collection cards on the market, the present invention significantly improves the extraction rate. Using blood collection cards treated by the present invention, the extraction rate of NAD+ in a single blood spot can be increased from 80±5% to 98±2%.

[0029] 3. The blood collection card processed by this invention can achieve an NAD+ degradation rate of <10% within 10 days at room temperature (27±3℃).

[0030] 4. The materials used in the formula of this invention are all common chemical reagents on the market, with low toxicity, green and safe, and the preparation and application process is simple, suitable for industrial production, and easy to store and transport. Attached Figure Description

[0031] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] In the attached diagram:

[0033] Figure 1 This is a chromatogram of NAD+ alkaline degradation;

[0034] Figure 2 This is a chromatogram of NAD+ acid degradation;

[0035] Figure 3 This is a comparison chart of NAD+ content on day 5 for single-component stabilizers;

[0036] Figure 4 This is a graph showing the test results of the compound formulation;

[0037] Figure 5 This is a comparison chart of the extraction rates of dried blood spots before and after pretreatment;

[0038] Figure 6 These are comparison charts showing the extraction effects of different extracts;

[0039] Figure 7 This is a graph showing the changes in NAD+ content 1-10 days before and after pretreatment;

[0040] Figure 8 This is a Pearson's correlation graph of the detection data from the same sample using HPLC and enzymatic cycling methods;

[0041] Figure 9 This is a graph showing the effect of different pH stabilizers on NAD+ in blood spots. Detailed Implementation

[0042] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.

[0043] To further illustrate the technical means and effects of the present invention, the following description, in conjunction with preferred embodiments of the present invention, further explains the technical solution of the present invention. However, the present invention is not limited to the scope of the embodiments. The sources of the active ingredients in the products involved in the following embodiments and comparative examples are as follows (only the active ingredients are shown; the necessary excipients contained in other commercially available raw materials are not described in detail).

[0044] Specific implementation examples are given below.

[0045] This invention provides a composite stabilizer for blood collection cards, comprising a cell lysis agent, an NAD+ metabolic enzyme inhibitor, NAD+ degradation products, and a pH stabilizer, achieving efficient protection of NAD+ through a synergistic mechanism. In the specific implementation process, cellulose paper of a specific thickness (0.3-0.5 mm) is first immersed in the composite stabilizer solution, and then dried in an electrically heated drying oven at 60-105°C for 4 hours to obtain a pretreated blood collection card. This blood collection card can significantly prolong the half-life of NAD+ at room temperature and improve its extraction rate and detection accuracy.

[0046] Cellulose paper of a specific thickness (0.3-0.5 mm) is selected as the carrier, which has higher adsorption capacity and uniformity compared to traditional blood collection cards. After the surface of the cellulose paper is treated with a composite stabilizer solution, a uniform stabilizer coating is formed. This coating is tightly bonded to the cellulose paper through physical adsorption and chemical bonding, ensuring the durability of the stabilizer on the blood collection card.

[0047] The compound stabilizer formulation consists of a cell lysis module, an NAD+ metabolic enzyme inhibitor, a degradation product blocking module, and a pH adjustment module. Specifically, the cell lysis agent (e.g., sodium dodecyl sulfate) releases NAD+ by disrupting the cell membrane; the NAD+ metabolic enzyme inhibitor (e.g., apigenin, NR) specifically inhibits CD38 and PARP enzyme activity; the NAD+ degradation products (e.g., ADPR, NAM) protect NAD+ by competitively blocking degradation pathways; and the pH stabilizer (e.g., citric acid, EDTA) binds to metal ions and provides an acidic environment, further stabilizing NAD+. The components work synergistically to achieve highly efficient protection.

[0048] EDTA in the pH stabilizer can chelate metal ions, preventing the catalytic degradation of NAD+ by metal ions. In experiments using a rusty metal punch for sampling, the NAD+ content on untreated blood collection cards decreased significantly, while pretreated blood collection cards exhibited excellent resistance to metal ion interference.

[0049] Use a pipette to collect 10 μL of whole blood sample and vertically drop it onto a pre-treated blood collection card. After the blood spot has completely dried and solidified at room temperature, store it in a sealed bag. During this process, the compound stabilizer formulation rapidly lyses blood cells through the cell lysis module, releasing NAD+, and inhibits its degradation through the metabolic enzyme inhibitor and degradation product blocking modules.

[0050] The dried blood spot sample was completely removed using a paper punch and transferred to a centrifuge tube. Lysis buffer (e.g., 10% acetic acid) was added, and the sample was vortexed for 15 minutes. The supernatant was then added to a neutralization solution, and the sample was analyzed using both HPLC and enzymatic cycling methods. The HPLC instrument separated NAD+ and its degradation products using a chromatographic column. The detection wavelength was set to 260 nm, the flow rate to 1.00 mL / min, and the column temperature to 30℃. The NAD+ content was detected using the WST-8 method with a microplate reader. Pearson's correlation coefficient was calculated using data analysis software to verify the consistency between the two methods.

[0051] The lysis buffer can be an organic acid or an inorganic acid, such as formic acid, acetic acid, perchloric acid, hydrochloric acid, trichloroacetic acid, etc.

[0052] Pretreated and untreated blood collection cards were stored at room temperature (27±3℃), and NAD+ content was measured at 1, 3, 5, 7, and 10 days. The results showed that the NAD+ content of the pretreated blood collection cards remained stable (88.9%) over 10 days, while that of the untreated blood collection cards decreased significantly to 53.5%.

[0053] 1. Preparation and treatment of dried blood spots

[0054] 1.1 Preparation of dried blood spots:

[0055] Use a pipette to take 10 μL of well-mixed whole blood sample and drop it vertically onto the blood collection card. Allow the blood spot to dry and solidify completely at room temperature, then place it in a sealed bag and store it at room temperature for later use.

[0056] 1.2 NAD+ extraction from dried blood spots:

[0057] Use an 8mm paper punch to completely remove the dried blood spot, transfer it to a 2.5mL centrifuge tube, add 120μL of lysis buffer (10% acetic acid), vortex at 1000rpm for 15min, take 40μL of supernatant, and add 40μL of neutralization solution.

[0058] 2. Whole blood sample processing

[0059] Take 10 μL of well-mixed whole blood sample with a pipette and place it in a 0.5 mL centrifuge tube. Add 120 μL of lysis buffer and vortex at 1000 rpm for 15 min. Take 40 μL of the supernatant and add 40 μL of neutralization solution.

[0060] 3. NAD+ Quantification Methods

[0061] 3.1 HPLC method:

[0062] Instrument Model: SHIMADZU LCMS-2020

[0063] Column: Inertsil ODS-35μm 4.6mm×250mm

[0064] Detection wavelength 260nm

[0065] Flow rate: 1.00 mL / min

[0066] Column temperature: 30℃

[0067] Injection volume: 20 μL

[0068] Analysis time: 15 min.

[0069] The gradient table for NAD+ quantification is shown in Table 1 below.

[0070] Time (min) A (methanol) B (10mM ammonium formate aqueous solution) 0-3 1 99 4-5 5 95 6-10 10 90 12-15 100 0

[0071] 3.2 Enzyme cycling method

[0072] Using a self-developed kit (WST-8 method), take 80 μL of the supernatant after extraction and neutralization from step 1.2, 40 μL of working solution C, and 40 μL of working solution D, incubate at 37℃ for 15 min, and measure the absorbance at 450 nm using a microplate reader.

[0073] Example 1

[0074] NAD+ forced degradation experiment

[0075] NAD+ standard degradation:

[0076] Take four portions of NAD+ standard (10±2 mg), and add 5M HCl, 0.05M NaOH, and 3% H2O2 respectively to perform acid, alkali, and oxidative degradation tests. Add one portion to ultrapure water as a control group. After dissolution, take 10 μL and add it to 990 μL of ultrapure water and mix well. Take 20 μL and inject it into HPLC (high performance liquid chromatograph).

[0077] like Figure 1 , Figure 2 The NAD+ ions shown produced significant degradation products in 5M HCl and 0.05M NaOH solutions, but did not degrade in ultrapure water and 3% H2O2. The solutions after degradation with 5M HCl and 0.05M NaOH were analyzed by LC-MS (liquid chromatography-mass spectrometry). It is speculated that the degradation products are as follows: Figure 1 Figure 2 ;

[0078] Among them, ADPR (ribose-adenosine diphosphate), AMP (adenosine monophosphate), NAM (nicotinamide), and NAD+ (nicotinamide adenine dinucleotide);

[0079] NMN (nicotinamide mononucleotide), AMP (adenosine monophosphate), NAD+ (nicotinamide adenine dinucleotide).

[0080] The main degradation products of NAD+ under strong acid and alkaline conditions are ADPR (ribose diphosphate), AMP (adenosine monophosphate), NMN (nicotinamide mononucleotide), NAM (nicotinamide), and adenosine-5-amine.

[0081] Example 2

[0082] To screen the optimal stabilizer formulation, single-component stabilizer tests were first conducted, and then composite formulation optimization was carried out based on the single-component results.

[0083] Single-component stabilizer test:

[0084] Different stabilizer components (cell lysing agent, NAD+ metabolic enzyme inhibitor, NAD+ degradation product) were prepared to a concentration of 50 mM. The blood collection cards were immersed in different stabilizers, dried at 105°C for 4 hours, and then stored in dry self-sealing bags for later use.

[0085] Stabilizers for single-component blood collection cards - Table 2 is as follows:

[0086]

[0087] Several blood spots were prepared as described above, stored at room temperature for 5 days, and then extracted according to method 1.2. The NAD+ content was detected by HPLC. Figure 3 As shown.

[0088] Composite formulation stabilizer test

[0089] Based on the above tests, take one or two effective components from the three groups, mix them, soak the blood collection card, and vacuum dry at 105℃ for 4 hours to prepare several blood spots for later use.

[0090] The compound formulation is shown in Table 3 below:

[0091]

[0092]

[0093] Test results of compound formulations, such as Figure 4 As shown, the composite formulation test results showed that formulation 1 (sodium dodecyl sulfate + 5-iodoisoquinoline + CD38 inhibitor 78c + ADPR + NAM) and formulation 4 (sodium dodecyl sulfate + guanidine isothiocyanate + NR + ADPR) had the best stabilizing effect. After 7 days, the NAD+ content in dried blood spots was maintained at 88.9% and 90.2%, respectively, which was significantly higher than the 53.5% of the blank control group, indicating that the synergistic effect of multiple components can effectively inhibit NAD+ degradation.

[0094] Example 3

[0095] The effect of blood collection card material on NAD+ extraction rate of dried blood spots:

[0096] After soaking in the compound formulation 1 from Example 2 using Whatman 903 protein saver, German MAHEREY-NAGEL 0.42mm cellulose filter paper, and DMPK cellulose cotton, the samples were dried at 105°C for 4 hours. Dried blood spots were prepared according to 1.1. Immediately after adding blood samples, they were placed in resealable bags with a desiccant and dried at a low temperature (4°C) for 4 hours to eliminate the risk of NAD+ degradation in the blood. The extraction rates of dried blood spots before and after pretreatment were compared as follows: Figure 5 As shown.

[0097] Effects of different extraction solutions on NAD+ extraction rate of dried blood spots:

[0098] Four blood samples were collected to prepare dried blood spots, which were immediately placed in resealable bags with desiccant and dried at 4°C for 4 hours. NAD+ was extracted using 10% acetic acid, erythrocyte lysis buffer, SDS lysis buffer, and 10% acetic acid + erythrocyte lysis buffer (9:1), respectively. Figure 6As shown, the extraction efficiency of the extracts was compared. The extraction rate of 10% acetic acid + erythrocyte lysis buffer (9:1) was the highest (average 94.9%), and its reproducibility was better than that of 10% acetic acid alone.

[0099] Example 4

[0100] Stabilizing effect of stabilizers and data from HPLC method compared with enzymatic cycling method;

[0101] Stabilizing effect of stabilizers on NAD+ in blood spots:

[0102] Blood collection cards were pretreated using Formula 1 to prepare dried blood spots. Samples were taken at 1, 3, 5, 7, and 10 days according to a 1.2 extraction ratio. The NAD+ content was analyzed using both enzymatic cycling and HPLC methods. Each sample was tested three times, and the average value was taken. Table 4 shows the NAD+ content of dried blood spots from pretreated and untreated blood collection cards after 10 days.

[0103]

[0104] Changes in NAD+ levels 1-10 days before and after pretreatment, such as Figure 7 As shown.

[0105] The NAD+ content in the pretreated group remained stable (88.9%) over 10 days, while it decreased significantly to 53.5% in the untreated group.

[0106] Comparison of HPLC detection results with enzymatic cycling method:

[0107] The NAD+ content of the same sample was detected using both enzymatic cycling and HPLC methods. Pearson's correlation coefficients were calculated. The Pearson's correlation plots of the HPLC and enzymatic cycling data for the same sample are shown below. Figure 8 As shown, the results of HPLC and enzymatic cycling methods were in good agreement, with a high correlation between the two sets of data (R = 0.992) and an average difference of only 2.3%.

[0108] Example 5

[0109] Effect of stabilizer pH on NAD+ stability

[0110] Blood collection cards were soaked in stabilizers with different pH values, and dried blood spots were prepared according to 1.1. Samples were taken on days 1 and 3 according to 1.2 to detect NAD+ content. The effect of different pH stabilizers on NAD+ in blood spots was investigated. Figure 9 As shown, NAD+ is significantly degraded when pH>7 (only 80.2±8% of the original amount after 3 days), while NAD+ has better stability when pH<7 (still 93.2±3% after 3 days).

[0111] Example 6

[0112] The effect of metal ions on NAD+ content in blood spots:

[0113] Using plastic scissors and a rusty metal paper punch, samples were taken from the blood spots on the untreated blood collection cards.

[0114] The effects of sampling with plastic scissors and rusty metal punches on NAD+ in blood spots are shown in Table 5 below:

[0115]

[0116] Sampling with a rusty punch causes rapid degradation of NAD+ in dried blood spots and interferes with enzyme cycling.

[0117] The above experiment was repeated using blood collection cards pretreated with Formula 1, and the results are shown in Table 6:

[0118]

[0119]

[0120] After applying the stabilizer, the NAD+ content in the dried blood spots was significantly higher than that in the untreated ones, proving that the stabilizer can effectively eliminate the interference of metal ions.

[0121] In the description of this invention, preferred embodiments of the invention have been described in detail above. However, the invention is not limited to the specific details of the above embodiments. Within the scope of the technical concept of the invention, various simple modifications can be made to the technical solution of the invention, and these simple modifications all fall within the protection scope of the invention.

[0122] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable way without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A composite stabilizer for blood collection cards, characterized in that, Includes the following components: Cell lysis agent, selected from guanidines, sodium dodecyl sulfate, quaternary ammonium salts or polyquaternary ammonium salts, at a concentration of 1-5%; NAD+ metabolic enzyme inhibitors, selected from nicotinamide riboside, apigenin or ABT-888, at a concentration of 0.1-1.0 mM; NAD+ degradation products, selected from adenosine diphosphate ribose, adenosine monophosphate, nicotinamide mononucleotide or nicotinamide, at a concentration of 0.1-1.0 mM; pH stabilizer, selected from citric acid or EDTA, at a concentration of 40-60 mM.

2. The composite stabilizer for blood collection cards according to claim 1, characterized in that: The cell lysis agent is sodium dodecyl sulfonate.

3. The composite stabilizer for blood collection cards according to claim 1, characterized in that: The NAD+ metabolic enzyme inhibitor is apigenin or nicotinamide riboside.

4. The composite stabilizer for blood collection cards according to claim 1, characterized in that: The NAD+ degradation products are adenosine diphosphate ribose or nicotinamide.

5. A composite stabilizer for blood collection cards according to claim 1, characterized in that: The pH stabilizer is a combination of citric acid and EDTA.

6. A blood collection card, characterized in that, The blood collection card is prepared by soaking cellulose paper in the composite stabilizer described in any one of claims 1 to 5, and then drying it in an electric heating drying oven at 60-105°C for 4 hours.

7. A blood collection card according to claim 6, characterized in that: The thickness of the cellulose paper is 0.3-0.5 mm.

8. A method for preparing a dried blood spot sample, characterized in that, Includes the following steps: Use a pipette to add 10 μL of whole blood sample onto the blood collection card as described in claim 6 or 7; After drying and shaping at room temperature, the blood collection cards are stored in a sealed bag.

9. A method for extracting NAD+ from dried blood spot samples, characterized in that, Includes the following steps: Samples were taken from the dried blood spot sample prepared according to claim 8 using a paper punch. After sampling, the dried blood spots were transferred to centrifuge tubes, lysis buffer was added, the mixture was vortexed, and the supernatant was extracted. The supernatant and neutralization solution were mixed before testing.

10. The extraction method according to claim 9, characterized in that, The lysis buffer is a mixture of 10% acid and erythrocyte lysis buffer at a volume ratio of 9:1.

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