Biological sample collection card and biological sample detection kit

Abstract

The present invention belongs to the field of biological detection, and specifically relates to a biological sample collection card and a biological sample detection kit, wherein the biological sample collection card is a test paper pretreated with an inorganic salt solution, which can maintain a high elution rate while maintaining the stability of the NAD+ content in the blood sample, thereby realizing the dry blood spot method detection of NAD+ concentration. It can be used for the collection, transportation, analysis, identification and / or storage of blood samples.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection, and specifically relates to a biological sample collection card and a biological sample detection kit. The biological sample collection card can be used for the collection, transportation, analysis, identification and / or storage of biological samples. Background Art

[0002] The biological sample collection card is a plant fiber or synthetic fiber paper, which is specially designed and can be used for the collection, transportation and storage of various biological fluid samples (including but not limited to blood, semen, saliva, cell culture fluid, microorganisms, plant tissue fluid and virus culture fluid) at room temperature. The collection card uses a special treatment method to rupture the cell membrane in the biological sample, denature the protein, and adsorb and fix the marker to be tested. In 1962, Robert Guthrie published a method for screening neonatal phenylketonuria, which first introduced the dried blood spot sampling technology (DBS). Relying on the 903 and FTA patented blood collection filter paper, it has achieved great success in the field of medical diagnosis and forensic identification. The dried blood spots of this type of blood collection filter paper can be used for analysis methods such as time-resolved immunofluorescence (TRFIA), tandem mass spectrometry (LC-MS / MS), enzyme chemistry, and fluorescence chemistry. It is an important means of screening and researching neonatal inherited metabolic diseases.

[0003] Nicotinamide adenine dinucleotide (NAD+) is an important energy metabolite in the human body. It is an important electron transport medium for cellular glycolysis, tricarboxylic acid cycle and fatty acid oxidation, and is involved in almost all energy metabolism and transport processes in the human body. At the same time, a large number of studies have shown that with the continuous increase of age, the NAD+ level in body cells will drop significantly, and the slowdown of cellular energy metabolism will cause the body to gradually show an aging state, and even eventually develop a series of aging-related diseases. At the same time, many unhealthy lifestyles such as work pressure, excessive drinking, lack of sleep, etc. will also cause a significant decrease in the body's NAD+ level.

[0004] Tandem mass spectrometry (LC-MS / MS) can be used to detect the concentration of NAD+ in the blood samples of subjects after taking NAD+ precursor supplements (such as vitamin B3, NR, NMN, etc.) to reflect the absorption of the supplements. It can also reflect the energy metabolism level of the subjects by detecting changes in NAD+ levels. This type of detection is of great reference significance for evaluating the overall health status of the subjects. At present, various commercially available blood collection filter papers cannot meet the detection needs of NAD+, and are prone to sample NAD+ degradation, low elution rate and large detection errors. Therefore, it is urgent to develop a collection card that is more suitable for biological samples containing NAD+. Summary of the invention

[0005] The purpose of the present invention is to provide a biological sample collection card specially developed for NAD+ detection, which uses dry blood spot sampling technology to achieve quantitative detection of NAD+ in blood and other various biological samples.

[0006] The specific technical solutions are as follows:

[0007] A biological sample collection card is a test paper pretreated with an inorganic salt solution.

[0008] In some embodiments, the pretreatment refers to soaking the test paper in an inorganic salt solution and then drying it.

[0009] In some embodiments, the inorganic salt is selected from any one or more of ammonium sulfate, sodium sulfate, magnesium sulfate, sodium chloride, ammonium chloride or sodium dihydrogen phosphate, preferably any one of ammonium sulfate, magnesium chloride or ammonium chloride.

[0010] In some specific embodiments, the test paper is a water-absorbent test paper, preferably any one selected from PET test paper, quantitative filter paper, FTA test paper or 903 test paper.

[0011] In some embodiments, the biological sample contains a metabolic small molecule, preferably NAD or a precursor molecule thereof, and the NAD+ precursor is selected from one or more of tryptophan, quinolinic acid, nicotinic acid (NA), nicotinamide (NAM), nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), or a food or pharmaceutically acceptable salt, derivative or prodrug thereof, further preferably nicotinamide mononucleotide (NMN).

[0012] In some embodiments, the biological sample is a blood sample and / or a cell sample, wherein the blood sample is a whole blood sample.

[0013] Another object of the present invention is to provide the use of the biological sample collection card as described above in the collection, transportation, analysis, identification and / or storage of blood samples.

[0014] Another object of the present invention is to provide a kit for biological sample detection, wherein the kit contains the biological sample collection card as described above.

[0015] The present invention also provides a method for detecting a biological sample, wherein the method uses the biological sample collection card as described above, or the reagent kit as described above.

[0016] The detection method comprises:

[0017] (1) Immersing the biological sample in the biological sample collection card and then drying it to obtain a sampled card;

[0018] (2) Elute the sampled card and test the eluate.

[0019] In some embodiments, the detection method has any one or more of the following features:

[0020] 1) The detection method is high performance liquid chromatography tandem mass spectrometry (LC-MS / MS);

[0021] 2) The LC-MS / MS method contains an internal standard, which is an isotope-labeled internal standard of the molecule to be measured, preferably β-NAD-13C5;

[0022] 3) The biological sample is a blood sample and / or a cell sample, wherein the blood sample is a whole blood sample;

[0023] 4) The biological sample contains metabolic small molecules, preferably NAD or its precursor molecules, and the NAD+ precursor is selected from one or more of tryptophan, quinolinic acid, nicotinic acid (NA), nicotinamide (NAM), nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), or their food or pharmaceutically acceptable salts, derivatives or prodrugs, and more preferably nicotinamide mononucleotide (NMN).

[0024] The beneficial effects of the present invention are:

[0025] The biological sample collection card of the present invention can maintain the stability of NAD+ content in blood samples while maintaining a high elution rate, thereby realizing the dry blood spot method detection of NAD+ concentration. The existing biological sample collection cards such as 903 test paper and FTA test paper used for dry blood spot sampling cannot achieve this function, and there is a certain error in the detection result of NAD+ concentration.

[0026] In addition, the preparation requirements of the biological sample collection card are low and the operation is simple. The detection results of NAD or its precursor molecules are not affected by the pH of the inorganic salt solution. The concentration only needs to ensure that no crystallization occurs after the test paper is soaked and dried.

[0027] Biological sample collection cards use a lower amount of blood sample, have a simple collection method, and do not require freezing, making them more suitable for the collection, transportation, testing and storage of large quantities of samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It shows the comparison of NAD+ content in dried blood spots made with different test strips;

[0029] Figure 2 It was shown that biological sample collection cards pretreated with inorganic salt solutions increased the preservation rate of NAD+;

[0030] Figure 3It shows the comparison of NAD+ content in dried blood spots made with 903 test strips pretreated with different inorganic salt solutions;

[0031] Figure 4 It showed that the NAD+ stability of dried blood spots made using biological sample collection cards lasted up to 14 days;

[0032] Figure 5 The dried blood spots made using the biological specimen collection card showed 7-day stability of NAD+;

[0033] Figure 6 A comparison of biological sample collection cards pretreated with different concentrations of inorganic salt solutions is shown;

[0034] Figure 7 A comparison of biological sample collection cards pre-treated with salt solutions of different pH values ​​is shown. DETAILED DESCRIPTION

[0035] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0036] One of the purposes of the present invention is to provide a biological sample collection card, wherein the biological sample collection card is a test paper pretreated with an inorganic salt solution.

[0037] In some embodiments, the pretreatment refers to soaking the test paper in an inorganic salt solution and then drying it. The inorganic salt solution can be used in any concentration and any pH. In some embodiments, the concentration is 0.2-1.2 mol / L. In some embodiments, the pH is 2.5-10.5.

[0038] In some embodiments, the inorganic salt is selected from sulfates, hydrochlorides, and phosphates, for example, selected from any one or more of ammonium sulfate, sodium sulfate, magnesium sulfate, sodium chloride, ammonium chloride, or sodium dihydrogen phosphate.

[0039] In some specific embodiments, the inorganic salt is preferably any one of ammonium sulfate, magnesium chloride or ammonium chloride.

[0040] In some embodiments, the test paper includes but is not limited to any one of PET test paper, quantitative filter paper, FTA test paper or 903 test paper. Any test paper with water absorption function can be applied, including various materials such as synthetic fiber materials and plant fiber materials.

[0041] The "PET test paper" is made of polyester fiber material with low fiber density. "PET" is specifically polyethylene terephthalate, with an irregular fiber mesh structure on the surface and a textile density of 66 g / m 2 .

[0042] During the manufacturing process of "quantitative filter paper", the pulp is treated with hydrochloric acid and hydrofluoric acid, and then washed with distilled water to remove most of the impurities in the paper fibers. Therefore, there is very little residual ash after burning, which has almost no effect on the analysis results, making it suitable for precise quantitative analysis.

[0043] "FTA Test Paper" (Flinders Technology Associates) is a special filter paper. It is soaked with a patented formula of strong denaturants and chelating agents, and contains special chemicals on the fiber matrix. When cells are captured, they are automatically lysed and combined with nucleic acids to maintain the integrity of the DNA in the sample, protect nucleic acids from degradation, damage from nucleases, oxidants and ultraviolet rays, and prevent the growth of bacteria and other microorganisms. It is mostly used for criminal investigation DNA collection and detection.

[0044] "903 test paper" can also be called "903 protein preservation card", which means that whatman 903 sample collection paper is made of 100% pure cotton wool without any wet strength additives, and is used for the collection and transfer of human fluid specimens.

[0045] In some embodiments, the biological sample to be tested contains metabolic small molecules, preferably NAD or its precursor molecules.

[0046] The term "NAD+ precursor" is any small molecule that results in an increase in NAD+, and the small molecule can exist in reduced or non-reduced form.

[0047] NAD+ is a cofactor that plays a core role in metabolism, and NAD+ levels decrease with age. NAD+ can help repair DNA, regulate immune cell conduction, provide cell energy and reduce aging. During the in vitro preparation of immune cells, adding NAD+ treatment can provide sufficient energy for immune cell expansion, improve immune cell expansion and reduce cell aging indicators. NAD+ precursors are compounds that are converted into NAD+ through chemical conversion.

[0048] In some embodiments, the NAD+ precursor is selected from one or more of tryptophan, quinolinic acid, nicotinic acid (NA), nicotinamide (NAM), nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), or a food or pharmaceutically acceptable salt, derivative or prodrug thereof; preferably nicotinamide mononucleotide (NMN). As a direct precursor of NAD+, NMN can be easily converted directly into NAD+ by NMNAT enzyme and easily absorbed by cells.

[0049] In some embodiments, the biological sample is a blood sample and / or a cell sample, wherein the blood sample is a whole blood sample, and the cell sample is a human cell, including nerve cells, white blood cells, red blood cells, platelets, macrophages, epithelial cells, cardiomyocytes, stem cells, etc. For example, PBMC can be used.

[0050] The term "whole blood" refers to the mixture formed by collecting blood from the human body into a blood collection bag, which includes all components of blood cells and plasma.

[0051] The term "PBMC" stands for peripheral blood mononuclear cells (PBMC), which are cells with a single nucleus in peripheral blood, including lymphocytes and monocytes.

[0052] A second object of the present invention is to provide the use of the biological sample collection card as described above in the collection, transportation, analysis, identification and / or storage of blood samples.

[0053] A third object of the present invention is to provide a kit for biological sample detection, wherein the kit contains the biological sample collection card as described above.

[0054] A fourth object of the present invention is to provide a method for detecting a biological sample, wherein the method uses the biological sample collection card as described above, or the reagent kit as described above.

[0055] In some embodiments, a certain volume of biological sample is first immersed in the biological sample collection card, for example, the biological sample is dropped on the biological sample collection card.

[0056] Then, the collection card (sampled card) with the collected biological sample can be dried for easy storage and transportation to the testing site. The drying method is, for example, natural drying at room temperature.

[0057] When testing is required, the sampled card can be eluted and the eluate can be tested. To facilitate testing, a solution containing an internal standard can be used as an eluent. The elution method can be oscillation, ultrasound, etc.

[0058] In some embodiments, the detection method is high performance liquid chromatography tandem mass spectrometry (LC-MS / MS). High performance liquid chromatography-tandem mass spectrometry technology uses a liquid chromatography system to separate the substance to be tested and then connects it to a mass spectrometer. The substance to be tested is ionized and converted into an electrical signal. After computer data processing, it is analyzed according to the mass spectrum peak. It has the excellent separation ability of liquid chromatography and the high sensitivity and high selectivity of mass spectrometry.

[0059] When using the LC-MS / MS method, the internal standard method is used, and the internal standard is an isotope-labeled compound of the molecule to be tested. An isotope internal standard of known concentration is added to a certain amount of a standard reference substance and a sample to be tested, and the concentration of the molecule to be tested in the sample to be tested is calculated based on the relationship between the peak area, mass or concentration of the molecule to be tested, the reference substance and the internal standard in the sample to be tested:

[0060] F = (A s / m s ) / (A r / m r )

[0061] m i = (F*A i ) / (A s / m s )

[0062] Where F is the correction factor, A s , A r , A i is the peak area of ​​internal standard, reference substance and analyte, m s 、m r 、m i is the mass or concentration of the molecule to be tested in the internal standard, reference substance and sample to be tested. This calculation process can usually be completed automatically on the LC-MS / MS supporting software.

[0063] During sample processing, samples are usually diluted or concentrated. The concentration of the molecule to be tested in the biological sample can be calculated by the concentration of the molecule to be tested and the dilution factor detected by LC-MS / MS:

[0064] D = V a / V i

[0065] m a = m i *D

[0066] Where D is the dilution factor, V a , V i are the volume of biological sample and the volume of the test fluid after sample processing, m a 、m i It is the mass or concentration of the molecule to be tested in the biological sample and the test solution.

[0067] In some specific embodiments, the molecule to be detected is NAD+, and the internal standard is β-NAD- 13 C5. The calculation method of NAD+ content in biological samples is:

[0068] Dried blood spot sample testing: m sample = m report*(V s +V SAS ) / V sample

[0069] Whole blood sample testing: m sample = m report *(V s +V SAS +V sample ) / V sample

[0070] Where D is the dilution factor, m sample 、m report is the NAD+ concentration in biological samples and the NAD+ concentration detected by LC-MS / MS, V s 、V SAS 、V sample are the volumes of internal standard solution, saturated ammonium sulfate solution and biological sample, respectively.

[0071] In some embodiments, the biological sample is a blood sample and / or a cell sample, wherein the blood sample is a whole blood sample. According to the method disclosed herein, a very small amount of blood can be collected for detection, such as 10 μL of fingertip blood. A quantitative capillary can be used to quantitatively collect fingertip blood.

[0072] In some embodiments, the biological sample contains metabolized small molecules.

[0073] In some specific embodiments, the metabolic small molecule is preferably NAD or a precursor molecule thereof, and the NAD+ precursor is selected from one or more of tryptophan, quinolinic acid, nicotinic acid (NA), nicotinamide (NAM), nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), or a food or pharmaceutically acceptable salt, derivative or prodrug thereof, and is further preferably nicotinamide mononucleotide (NMN).

[0074] The present invention will be described below in the form of specific examples. It should be understood that these examples are merely illustrative and are not intended to limit the scope of the present invention. The methods and materials used in the examples are, unless otherwise stated, materials and methods conventional in the art.

[0075] 1. Preparation and pretreatment of dried blood spots

[0076] 1.1 Preparation method of dried blood spots

[0077] Use a pipette to take 10 μL of the collected whole blood sample and drop it on the biological sample collection card. Place the dried blood spot card at room temperature to dry for 2-4 hours. After the dried blood spot is completely dry, transfer it to the sealing tape and store it at room temperature for later use.

[0078] 1.2 Pretreatment of dried blood spot samples

[0079] Use a sampler to remove the complete dried blood spot filter paper, put it into a 2 mL EP tube, and add 300 μL of internal standard solution (β-NAD- 13 C5), shake evenly to make the internal standard solution immerse the dried blood spot filter paper; ultrasonicate in an ice water bath for 20 min; after ultrasonicate, add 1200 μL of saturated ammonium sulfate solution and shake evenly or ultrasonicate for 1 min; after ultrasonicate, put the EP tube into a centrifuge and centrifuge at 10000 rpm for 3 min; take the supernatant and filter it and put it into a sample bottle for later use.

[0080] 2. Pretreatment of whole blood samples

[0081] Add 1190 μL of saturated ammonium sulfate solution to a 1.5 mL centrifuge tube, add 10 μL of whole blood sample from the same donor and shake evenly, and freeze the EP tube in liquid nitrogen for 2 min;

[0082] Take out the frozen EP tube and place it in an ice box to thaw. After thawing, add 300 μL of internal standard solution (β-NAD- 13 C5), shake evenly; put the EP tube into an ice water bath for ultrasonication for 10 min; immediately after ultrasonication, put the EP tube into a centrifuge and centrifuge at 10,000 rpm for 3 min; take the supernatant after centrifugation, filter it, and put it into a sample bottle for later use.

[0083] Example 1 Comparison of dried blood spots made with different test strips

[0084] PET test paper (Labconco Replacement Filter Paper 7544810), quantitative filter paper (Titan, rapid quantitative filter paper 7 cm), FTA test paper (Whatman FTA standard card) and 903 test paper (Whatman903 protein storage card) were used to prepare dried blood spots from whole blood samples according to method 1.1, and the samples were pre-treated according to method 1.2 and injected into LC-MS / MS to detect the NAD+ content in the samples. Whole blood samples from the same donor were processed according to method 2, and the obtained NAD+ content was used as a reference.

[0085] After testing several dried blood spots, it was found that ( Figure 1 ), NAD+ in the dried blood spots on the quantitative filter paper, 903 test paper, and FTA test paper has been degraded, and the NAD+ content in the PET test paper is close to the NAD+ content in the fresh blood sample. The possible reason is that the PET filter paper has the smallest thickness, the blood diffuses quickly, the drying time is short, and the NAD+ enzymatic hydrolysis time before the dried blood spots are dried is even shorter. Therefore, ensuring the stability of NAD+ before the dried blood spots are dried is a necessary condition for detecting the NAD+ content in dried blood spots.

[0086] Example 2 Comparison of biological sample collection cards after inorganic salt pretreatment

[0087] 2.1 Test paper preparation: prepare 0.6 mol / L ammonium sulfate solution, soak PET test paper, quantitative filter paper and 903 test paper respectively, lay them flat and dry for later use.

[0088] The blood samples were made into dried blood spots using the above test paper according to method 1.1, and the samples were pre-treated according to method 1.2, and then injected into LC-MS / MS to detect the NAD+ content in the samples. The whole blood samples of the same donor were processed according to method 2, and the obtained NAD+ content was used as a reference.

[0089] The NAD+ test on dried blood spots revealed that ( Figure 2 ), the NAD+ content in the dried blood spots made by PET test paper pretreated with ammonium sulfate solution is close to the test results of fresh blood with untreated PET test paper, and the NAD+ content in the dried blood spots made by the treated 903 test paper and quantitative filter paper is significantly higher than that of the untreated test paper. The results show that the salt solution pretreatment can effectively ensure the stability of NAD+ in the process of dried blood spot preparation and improve the NAD+ preservation rate in blood samples.

[0090] Example 3 Comparison of biological sample collection cards pretreated with different inorganic salt solutions

[0091] 3.1 Preparation of biological sample collection card: prepare 0.6 mol / L ammonium sulfate solution, sodium sulfate solution, sodium chloride solution, magnesium sulfate solution, ammonium chloride solution, and sodium dihydrogen phosphate solution respectively, soak 903 test paper, lay it flat and dry it for later use.

[0092] Blood samples were prepared into dried blood spots using the above test strips according to method 1.1, and untreated 903 test strips were used as the control group. After sample pretreatment according to method 1.2, the samples were injected into LC-MS / MS to detect the NAD+ content in the samples. Whole blood samples from the same donor were processed according to method 2, and the obtained NAD+ content was used as a reference.

[0093] Comparison of NAD+ content in dried blood spots of biological sample collection cards treated with different salts revealed that ( Figure 3 ), biological sample collection cards pretreated with different salts can ensure the stability of NAD+ in dried blood spots to a certain extent, among which ammonium sulfate, magnesium chloride and ammonium chloride have the best effects.

[0094] Example 4 Stability of dried blood spots prepared using biological sample collection cards

[0095] PET test paper and 903 test paper were used to verify the stability of the dried blood spots on the biological sample collection card.

[0096] 4.1 Preparation of biological sample collection card: Prepare 0.6 mol / L ammonium sulfate solution, soak the PET test paper, lay it flat and dry it for later use.

[0097] Blood samples were prepared into dried blood spots using the above test strips according to method 1.1, and untreated PET test strips were used as the control group. After sample pretreatment according to method 1.2, the samples were injected into LC-MS / MS to detect the NAD+ content in the samples. Whole blood samples from the same donor were processed according to method 2, and the obtained NAD+ content was used as a reference.

[0098] After 14 days of testing on dried blood spots, it was found that ( Figure 4 ), the NAD+ content of the pretreated biological sample collection card changed by less than 10% in 14 days, and the NAD+ content of the dried blood spot test was about 96.63% of the fresh blood test result at 14 days, while the NAD+ content of the dried blood spot made of the untreated PET test paper dropped to 84.62% in 7 days and 73.79% in 14 days. The results show that the biological sample collection card pretreated with inorganic salt solution can effectively ensure the stability of NAD+ in the dried blood spot and prolong the room temperature storage time of the dried blood spot sample.

[0099] 4.2 Preparation of biological sample collection card: Prepare 0.6 mol / L ammonium sulfate solution, soak 903 test paper, lay it flat and dry it for later use.

[0100] Blood samples were prepared into dried blood spots using the above test strips according to method 1.1, and untreated 903 test strips were used as the control group. After sample pretreatment according to method 1.2, the samples were injected into LC-MS / MS to detect the NAD+ content in the samples. Whole blood samples from the same donor were processed according to method 2, and the obtained NAD+ content was used as a reference.

[0101] After 7 days of testing on dried blood spots, it was found that ( Figure 5 ), the pretreated biological sample collection card was stored at room temperature for 7 days without obvious degradation of NAD+, which can ensure the stability of NAD+ in the dried blood spots for 7 days.

[0102] Example 5 Comparison of biological sample collection cards pretreated with salt solutions of different concentrations

[0103] 5.1 Preparation of biological sample collection cards: prepare 0.2, 0.4, 0.6, and 1.2 mol / L ammonium sulfate solutions respectively, soak the PET test paper, lay it flat and dry it for later use.

[0104] Blood samples were prepared into dried blood spots using the above test strips according to method 1.1, and untreated PET test strips were used as the control group. After sample pretreatment according to method 1.2, the samples were injected into LC-MS / MS to detect the NAD+ content in the samples. Whole blood samples from the same donor were processed according to method 2, and the obtained NAD+ content was used as a reference.

[0105] After testing the dried blood spots stored for 1 day and 7 days, it was found that ( Figure 6 ), the NAD+ content of dried blood spots made from biological sample collection cards pretreated with different salt concentrations was stable for 7 days, and pretreatment of biological sample collection cards with salt solutions of different concentrations could extend the room temperature storage time of dried blood spot samples.

[0106] Example 6 Comparison of biological sample collection cards pretreated with salt solutions of different pH values

[0107] 6.1 Preparation of biological sample collection cards: Prepare 0.6 mol / L ammonium sulfate solutions with pH values ​​of 2.5, 4.5, 6.5, 8.5, and 10.5 respectively, soak the PET test paper, lay it flat and dry it for later use.

[0108] Blood samples were prepared into dried blood spots using the above test strips according to method 1.1, and untreated PET test strips were used as the control group. After sample pretreatment according to method 1.2, the samples were injected into LC-MS / MS to detect the NAD+ content in the samples. Whole blood samples from the same donor were processed according to method 2, and the obtained NAD+ content was used as a reference.

[0109] After testing the dried blood spots stored for 1 day and 7 days, it was found that ( Figure 7 ), the NAD+ content of the dried blood spots made from PET filter paper pretreated with salt solutions of different pH values ​​did not show obvious degradation for 7 days, which can extend the room temperature storage time of the dried blood spot samples.

[0110] In summary, the dried blood spot samples prepared using the biological sample collection card of the present invention can be stored at room temperature for 14 days, maintaining NAD+ stability, and meeting the needs of long-distance sample transportation and actual sample testing.

Claims

1. A biological sample collection card for detecting NAD+, characterized in that: The biological sample collection card is a test paper pretreated with an inorganic salt solution, the biological sample contains NAD+ or its precursor molecule, the NAD+ precursor is selected from any one or more of tryptophan, quinolinic acid, nicotinic acid (NA), nicotinamide (NAM), nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), or any one or more of their food or pharmaceutically acceptable salts, derivatives or prodrugs, and the inorganic salt is selected from any one or more of ammonium sulfate, sodium sulfate, magnesium sulfate, sodium chloride, ammonium chloride or sodium dihydrogen phosphate.

2. The biological sample collection card according to claim 1, characterized in that: The pretreatment refers to soaking the test paper in an inorganic salt solution and then drying it.

3. The biological sample collection card according to claim 1 or 2, characterized in that: The test paper is a water-absorbent test paper.

4. The biological sample collection card according to claim 3, characterized in that: The water-absorbent test paper is selected from any one of PET test paper, quantitative filter paper, FTA test paper or 903 test paper.

5. The biological sample collection card according to claim 1 or 2, characterized in that: The biological sample is a blood sample.

6. Use of the biological sample collection card according to any one of claims 1 to 5 in the collection, transportation, analysis, identification and / or storage of biological samples.

7. A kit for detecting NAD+ in a biological sample, characterized in that: The kit contains the biological sample collection card according to any one of claims 1 to 5.

8. A method for detecting NAD+ in a biological sample, characterized in that: The detection method uses the biological sample collection card according to any one of claims 1 to 5, or the kit according to claim 7.

9. The detection method according to claim 8, characterized in that: The detection method comprises the following steps: (1) Immersing a certain volume of biological sample into the biological sample collection card and then drying it to obtain a sampled card; (2) Elute the sampled card and test the eluate; The steps include any of the following features: 1) The detection method is high performance liquid chromatography tandem mass spectrometry (LC-MS / MS); 2) The LC-MS / MS method contains an internal standard, which is an isotope-labeled internal standard β-NAD- 13 C5; 3) The biological sample is a blood sample; 4) The biological sample contains metabolic small molecules selected from NAD or its precursor molecules, and the NAD+ precursor is selected from tryptophan, quinolinic acid, nicotinic acid (NA), nicotinamide (NAM), nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), or any one or more of their food or pharmaceutically acceptable salts, derivatives or prodrugs.

Images