Sample processing fluid for 25-oh-vd detection, preparation thereof and microfluidic detection method

Abstract

This invention belongs to the field of biomedical detection technology, and discloses a sample processing solution for 25-OH-VD detection, its preparation, and a microfluidic detection method. The sample processing solution comprises two components: a release agent and a neutralizing agent. The release agent contains guanidine isothiocyanate, SDS, DTT, etc., which can efficiently dissociate serum-bound 25-OH-VD. The neutralizing agent contains BSA and ProClin 300, which can precisely adjust the immunoassay-appropriate neutral pH. Both components are pre-stored in the chamber of a disc-type microfluidic chip, and sample pretreatment is automatically completed by centrifugation at 800-1500 rpm, combined with chemiluminescence detection. The reagent physicochemical parameters are adapted to the microchannel, with a release efficiency ≥95% and intra-assay CV <1.1%. Comparison with the LC-MS / MS gold standard yields R0. 2 =0.934, with sample deviations all within ±20%. This invention overcomes the shortcomings of traditional reagent microfluidic adaptation and inability to achieve fully automated dissociation and neutralization, and is suitable for POCT detection of 25-OH-VD.

Description

Technical Field

[0001] This invention belongs to the field of biomedical detection technology, specifically relating to a sample processing solution for 25-OH-VD detection, its preparation and microfluidic detection method, which is suitable for 25-OH-VD chemiluminescence detection on a disc-type microfluidic platform. Background Technology

[0002] Vitamin D is an essential fat-soluble vitamin for the human body, and its levels are closely related to osteoporosis, immune regulation, cardiovascular disease, and the development of various tumors. 25-hydroxyvitamin D (25-OH-VD) is the main circulating form of vitamin D in the body, and its serum concentration is the recognized gold standard for evaluating vitamin D nutritional status. In recent years, with the rapid development of point-of-care testing (POCT) technology, integrating the detection of 25-hydroxyvitamin D into miniaturized and automated microfluidic platforms has become an important development direction in the field of in vitro diagnostics.

[0003] Disc-based microfluidic technology, with its advantages of low sample consumption (only a few microliters of whole blood or serum), rapid reaction speed, and ability to process multiple indicators in parallel, has shown great potential in the POCT field. However, approximately 85%–90% of 25-hydroxyvitamin D in human blood is tightly bound to vitamin D-binding protein (VDBP), making it difficult to detect directly using conventional immunoassay methods. Sample pretreatment is necessary to fully dissociate and release 25-hydroxyvitamin D from the binding protein, while simultaneously adjusting the pH of the reaction system to a neutral range suitable for the immunoreaction. The automation and miniaturization of this sample pretreatment step is a key technological bottleneck in achieving fully automated "sample in, result out" detection.

[0004] In existing technologies, sample release agents for 25-hydroxyvitamin D often use organic solvents (such as acetonitrile and methanol) or strong acid and strong base conditions. Although these methods can achieve dissociation and release, they have the following shortcomings: First, the release agent components are incompatible with the subsequent immune reaction system, requiring additional neutralization or dilution steps. Second, traditional sample processing solutions are mostly designed for open reaction containers (such as test tubes and microplates), and they have significant deficiencies in terms of flow characteristics in microfluidic chip microchannels, compatibility with chip materials, and stability during pre-storage within the chip. Specifically, this manifests as: viscosity being too high or too low, resulting in uncontrollable flow in microchannels; excessive surface tension, easily forming bubbles or residues within the channels; and poor compatibility with chip materials such as COC (Cycloolefin Copolymer) and PMMA (Polymethyl methacrylate, commonly known as acrylic or plexiglass), which may lead to chip swelling or reagent adsorption upon contact.

[0005] Furthermore, current technologies lack sample processing solutions specifically designed for disc-type microfluidic platforms that integrate release and neutralization functions. Pre-storing the release agent and neutralizing agent in a suitable form within a specific chamber of the disc-type microfluidic chip, enabling automatic sample release and neutralization during chip centrifugation, is a core technical challenge for achieving fully automated detection. Summary of the Invention

[0006] The purpose of this invention is to overcome the technical defects of traditional reagent microchannels, such as poor fluid performance, inability to automatically complete dissociation and neutralization on the chip, low release efficiency, and insufficient detection accuracy. It provides a sample processing solution for 25-OH-VD detection, its preparation, and a microfluidic detection method, which is adapted to a disc-type microfluidic platform to achieve fully automated chemiluminescence POCT detection of 25-OH-VD.

[0007] To achieve the above objectives, the first aspect of the present invention provides a sample processing solution for the detection of 25-OH-VD, comprising two independent components: a releasing agent and a neutralizing agent. The releasing agent enables the rapid and complete release of 25-OH-VD from the binding protein, with a release efficiency of over 95%. The neutralizing agent precisely adjusts the pH of the reaction system to a suitable neutral range for the immune response. The two components work together to complete sample pretreatment in an integrated manner within a disc-type microfluidic chip, achieving full automation. The releasing agent is a liquid reagent comprising, by weight percentage, the following components: guanidine isothiocyanate 1.0%–2.0%, sodium dodecyl sulfate (SDS) 0.03%–0.08%, dithiothreitol (DTT) 0.03%–0.08%, EDTA-2Na 0.01%–0.04%, bovine serum albumin (BSA) 0.5%–1.5%, with the balance being 10 mM phosphate-buffered saline (PBS) at pH 7.4. Guanidine isothiocyanate denatures vitamin D-binding proteins, releasing 25-OH-VD; SDS assists in protein denaturation, improving release efficiency; DTT breaks disulfide bonds in the protein, promoting the unfolding of the binding protein structure; PBS maintains pH stability in the reaction system; EDTA-2Na chelates metal ions, protecting released vitamin D; and BSA protects released 25-hydroxyvitamin D from degradation.

[0008] The neutralizing agent is a liquid reagent comprising the following components by weight percentage: bovine serum albumin 0.5%–1.5%, ProClin 300 0.03%–0.08%, with the balance being 10 mM phosphate buffer at pH 7.4; PBS provides a stable pH environment; BSA protects released 25-hydroxyvitamin D and antibodies in subsequent immune responses; ProClin 300 inhibits microbial growth, ensuring the stability of the reagent during pre-storage within the chip.

[0009] Furthermore, the release agent has a pH of 7.0–8.0, a viscosity (25°C) of 1.0–5.0 cP, a surface tension (25°C) of 30–50 mN / m, and a density (25°C) of 1.00–1.10 g / mL.

[0010] Furthermore, the pH of the releasing agent is preferably 7.4, the viscosity is preferably 1.5 to 3.0 cP, and the surface tension is preferably 35 to 45 mN / m.

[0011] This invention limits the physicochemical parameters of the release agent to the aforementioned range, allowing for better adaptation to microchannels and ensuring good flowability and controllability of the release agent within the microchannels (width 100–500 μm, depth 50–200 μm) of the disc-type microfluidic chip. Excessive viscosity can lead to slow or even blocked liquid flow in the microchannels; conversely, excessively low viscosity may result in excessively rapid flow and inadequate mixing under centrifugal force. Maintaining surface tension within a suitable range effectively prevents the formation of bubbles or liquid residue due to poor wettability within the microchannels.

[0012] Furthermore, the neutralizing agent has a pH of 6.5–7.5, a viscosity (25°C) of 1.0–4.0 cP, and a surface tension (25°C) of 35–55 mN / m.

[0013] Furthermore, the neutralizing agent preferably has a pH of 7.4, a viscosity of 1.5 to 2.5 cP, and a surface tension of 40 to 50 mN / m.

[0014] The viscosity and surface tension of the neutralizing agent are limited to the above range, which can ensure that the neutralizing liquid flows smoothly in the subsequent microchannels, mixes thoroughly with the dissociated sample, stabilizes the pH of the neutralization system, and avoids channel adsorption and liquid residue on the wall, thus ensuring the consistency of the reaction on the board.

[0015] To achieve the above objectives, a second aspect of the present invention provides a method for preparing the sample processing solution, comprising the following steps: (1) Preparation of release agent: Accurately weigh guanidine isothiocyanate, sodium dodecyl sulfate, dithiothreitol and EDTA-2Na, dissolve in 10mM phosphate buffer at pH 7.4, stir to dissolve, then add bovine serum albumin, continue stirring until all components are completely dissolved; adjust the pH of the system, filter to remove bacteria, measure viscosity and surface tension, and pre-store in the release chamber of the disc microfluidic chip; (2) Neutralizing agent preparation: Accurately weigh bovine serum albumin and ProClin 300, dissolve them in 10mM phosphate buffer at pH 7.4, stir until completely dissolved; adjust the pH of the system, filter to remove bacteria, measure the viscosity and surface tension, and pre-store in the neutralization chamber of the disc microfluidic chip.

[0016] Furthermore, the pH values ​​of the system adjusted in steps (1) and (2) are determined by micro-titration with 0.1 mol / L dilute hydrochloric acid or 0.1 mol / L sodium hydroxide solution to avoid drastic local pH fluctuations that could damage the stability of bovine serum albumin, and to avoid introducing interfering ions that could affect the subsequent chemiluminescent immunoassay.

[0017] This invention conducted release efficiency evaluation experiments and clinical sample comparison experiments, confirming that the release agent can achieve a 25-OH-VD dissociation efficiency of over 95%, and the neutralizing agent can accurately stabilize the pH value of the reaction system within the neutral range suitable for chemiluminescent immunoassay. The physicochemical parameters of the processed liquid in this invention are matched with the microchannels of a disc-type microfluidic chip, eliminating fluid defects such as bubbles and liquid residue, with an intra-batch detection coefficient of variation of less than 1.1%. Forty clinical serum samples with concentrations ranging from 5 to 150 ng / mL were selected and compared using liquid chromatography-tandem mass spectrometry (LC-MS / MS) as the gold standard. Linear regression results showed a correlation coefficient R between the two detection methods. 2 =0.934, and the detection deviation of all samples ranged from -17.2% to +19.0%, all falling within the clinically acceptable error range of ±20%. This proves that the detection accuracy of the present invention meets the requirements of clinical diagnosis and has the comprehensive advantages of high dissociation efficiency, good compatibility with microfluidic platforms, and excellent detection repeatability and storage stability.

[0018] To achieve the above objectives, a third aspect of the present invention also provides the application of the sample processing solution in 25-OH-VD microfluidic chemiluminescence detection.

[0019] To achieve the above objectives, a fourth aspect of the present invention also provides a 25-OH-VD disc-type microfluidic chemiluminescence detection method, using the sample processing solution described in the present invention, comprising the following steps: (1) Add the serum to be tested into the inlet of the disc microfluidic chip and centrifuge at a speed of 800-1500 rpm to make the serum to be tested flow through the release chamber of the pre-stored release agent and the neutralization chamber of the pre-stored neutralizing agent in sequence to complete the dissociation of 25-OH-VD from the binding protein and the neutralization of the pH of the reaction system. (2) The concentration of 25-OH-VD was determined by chemiluminescence immunoassay on a microfluidic chip to obtain the serum concentration of 25-OH-VD.

[0020] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a customized two-component sample processing solution for disc-type microfluidic platforms. By precisely defining the viscosity, surface tension, and other physicochemical parameters of the release agent and neutralizing agent, it perfectly adapts to microchannel structures with widths of 100–500 μm and depths of 50–200 μm. This fundamentally avoids common industry problems such as uncontrolled liquid flow, channel bubbles, reagent residue, and chip swelling and adsorption. The pre-filled and sealed reagents can be stably stored in the chip chamber for extended periods. The formulation system does not contain high-concentration organic solvents, strong acids, or strong alkalis, and will not interfere with subsequent immunoassays. The neutralized system can be directly used for chemiluminescence detection without additional dilution. The invention features a highly efficient release agent, maintaining a stable release efficiency of over 95% for bound 25-OH-VD across the entire clinical concentration range of 5–150 ng / mL. The accompanying on-chip centrifugation mode automatically completes the entire pretreatment process, including dissociation and neutralization, eliminating the need for manual liquid transfer and achieving truly automated "sample in, result out" POCT testing. The methodology exhibits excellent performance, with intra-batch coefficients of variation for all concentration standards below 1.1%, demonstrating excellent repeatability. A comparison of 40 serum samples using liquid chromatography-tandem mass spectrometry (LC-MS / MS) as the gold standard showed a high correlation coefficient (R0) between the two detection methods. 2 =0.934, and the detection deviation of all samples was controlled within the clinically acceptable range of ±20%, and the detection accuracy fully met the clinical diagnostic requirements for vitamin D nutritional status. Attached Figure Description

[0021] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0022] Figure 1 This is a bar chart comparing the theoretical and measured concentrations of the standard solutions at various concentrations in Example 2.

[0023] Figure 2 This is a box plot of the release efficiency of standard solutions of various concentrations in Example 2.

[0024] Figure 3 This is a bar chart showing the intra-batch coefficient of variation for the standard solutions of various concentrations in Example 2.

[0025] Figure 4 This is a scatter plot comparing the detection results of the detection method of the present invention and the LC-MS / MS reference method in Example 3 with linear regression.

[0026] Figure 5 This is a bar chart showing the percentage of relative deviation in the detection of 40 serum samples in Example 3. Detailed Implementation

[0027] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that these embodiments are only for explaining the present invention and do not constitute any limitation on the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0028] Unless otherwise specified, the experimental methods used in this invention are all conventional methods, and the experimental equipment, materials, reagents, etc. used can all be purchased from chemical companies.

[0029] The sample processing solution of this invention is applicable to various disc-type microfluidic detection platforms. The disc-type microfluidic chip used in this embodiment is a self-made chip in our laboratory. The 25-OH-VD standard and clinical serum samples used in the experiment are all from cooperating hospitals.

[0030] Example 1 This embodiment provides a sample processing solution for the detection of 25-OH-VD, comprising two independent components: a releasing agent and a neutralizing agent; The release agent is a liquid reagent comprising, by weight percentage, the following components: guanidine isothiocyanate 1.5%, sodium dodecyl sulfate (SDS) 0.05%, dithiothreitol (DTT) 0.05%, EDTA-2Na 0.02%, bovine serum albumin (BSA) 1.0%, with the balance being 10 mM phosphate-buffered saline (PBS) at pH 7.4. The neutralizing agent is a liquid reagent comprising, by weight percentage, the following components: bovine serum albumin (BSA) 1.0%, ProClin 300 0.05%, and the balance being 10 mM phosphate buffer at pH 7.4.

[0031] Its preparation method includes the following steps: (1) Preparation of release agent: Accurately weigh guanidine isothiocyanate, sodium dodecyl sulfate, dithiothreitol and EDTA-2Na, add to phosphate buffer, stir to dissolve, then add bovine serum albumin, stir until all components are completely dissolved; use 0.1 mol / L sodium hydroxide solution for microtitration to adjust the pH of the system to 7.4; filter through a 0.22 μm aqueous filter membrane for sterilization; at 25℃, its viscosity is 2.2 cP, surface tension is 40 mN / m and density is 1.1 g / mL, and it is pre-stored in the release chamber of a self-made disc-type microfluidic chip (microchannel width 300 μm, depth 100 μm); (2) Neutralizing agent preparation: Accurately weigh bovine serum albumin, dissolve it in 10 mM phosphate buffer, add ProClin 300, and after complete dissolution, use 0.1 mol / L sodium hydroxide solution for micro-titration to adjust the pH of the system to 7.4; filter it through a 0.22 μm aqueous filter membrane for sterilization; at 25℃, its viscosity is 2.0 cP and its surface tension is 45 mN / m, and it is pre-stored in the neutralization chamber of the self-made disc-type microfluidic chip.

[0032] Example 2: Release Efficiency Evaluation Experiment The sample processing solution prepared in Example 1 was used to carry out this experiment.

[0033] 25-OH-VD standard solutions with theoretical concentrations of 5, 25, 50, 100, and 150 ng / mL were prepared using 25-OH-VD standard, with six replicates for each concentration group. Each concentration standard solution was mixed with a releasing agent at a 1:1 volume ratio, incubated at room temperature, and then a neutralizing agent was added to adjust the pH to 7.4. The 25-OH-VD concentration was determined using chemiluminescent immunoassay, and the release efficiency and intra-assay coefficient of variation were calculated. The release efficiency was calculated using the formula: Release efficiency (%) = (Measured concentration / Theoretical concentration) × 100%. The experimental results are shown below. Figures 1-3 As shown.

[0034] Depend on Figure 1 As can be seen, the mean measured concentration of the 5 ng / mL group was 4.86 ng / mL, with a standard deviation of 0.96; the mean measured concentration of the 25 ng / mL group was 24.38 ng / mL, with a standard deviation of 0.86; the mean measured concentration of the 50 ng / mL group was 48.80 ng / mL, with a standard deviation of 0.70; the mean measured concentration of the 100 ng / mL group was 96.88 ng / mL, with a standard deviation of 0.74; and the mean measured concentration of the 150 ng / mL group was 144.95 ng / mL, with a standard deviation of 0.62. The measured concentrations were highly consistent with the theoretical concentrations, and the standard deviations of each group were small, indicating low detection fluctuations. This demonstrates that the sample processing solution of this invention can fully dissociate and release 25-OH-VD, resulting in a stable detection signal response.

[0035] Depend on Figure 2 As can be seen, the release efficiency was 97.2% for the 5 ng / mL concentration group, 97.5% for the 25 ng / mL concentration group, 97.6% for the 50 ng / mL concentration group, 96.9% for the 100 ng / mL concentration group, and 96.6% for the 150 ng / mL concentration group. The release efficiency reached over 95% for all concentration groups across the entire clinical concentration range, indicating that the sample processing solution of this invention possesses excellent target analyte release capability.

[0036] Depend on Figure 3 As can be seen, the coefficient of variation (CV) for the 5 ng / mL concentration group was 1.0%; for the 25 ng / mL concentration group, it was 0.9%; for the 50 ng / mL concentration group, it was 0.7%; for the 100 ng / mL concentration group, it was 0.8%; and for the 150 ng / mL concentration group, it was 0.6%. The intra-batch CV for each concentration group was less than 1.1%, indicating that the entire detection system exhibits good intra-batch repeatability after samples are treated with the sample processing solution of this invention.

[0037] Example 3: Clinical Sample Comparison Trial Forty clinical serum samples (concentration range 5-150 ng / mL) were collected. A disc-type microfluidic chip pre-stored with the sample processing solution from Example 1, combined with chemiluminescence, was used as the experimental group. Liquid chromatography-tandem mass spectrometry (LC-MS / MS) served as the control group for 25-OH-VD concentration detection. The consistency between the detection method of this invention and the reference method was evaluated, including the following steps: (1) 40 serum samples were added to the inlet of the disc microfluidic chip and centrifuged at a speed of 800-1500 rpm. The serum samples flowed through the release chamber and the neutralization chamber in sequence. The dissociation of 25-OH-VD from the binding protein and the pH neutralization of the reaction system were completed inside the disc microfluidic chip. (2) The concentration of 25-OH-VD in 40 serum samples was determined by chemiluminescence immunoassay on a microfluidic chip, and the results of each sample were recorded. (3) The concentration of 25-OH-VD in the above 40 serum samples was determined by liquid chromatography-tandem mass spectrometry reference method. The relative deviation of the detection method of the present invention relative to the reference method was calculated, and the detection consistency of the two methods was evaluated.

[0038] Experimental results are as follows Figure 4 , Figure 5 As shown: Through linear regression analysis, the fitted linear regression equation is y = 1.038x - 2.152, and the correlation coefficient R between the two test methods is... 2 =0.934, such as Figure 4 As shown, the detection method of this invention has a good correlation with the LC-MS / MS reference method. Figure 5As shown, the relative deviation percentage of the 40 serum samples ranged from -17.2% to +19.0%. The relative deviation of all samples fell within the clinically acceptable ±20% error range, with no significant systematic bias. This meets the clinical performance acceptance standard for testing, indicating that the detection method of this invention can be adapted to real clinical serum samples. By using a disc-type microfluidic chip, the sample pretreatment and detection of 25-OH-VD can be achieved, and the detection accuracy meets the requirements for clinical vitamin D level screening.

[0039] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them; those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions will not cause the essence of the corresponding technical solutions to deviate from the protection scope of the present invention.

Claims

1. A sample processing solution for the detection of 25-OH-VD, characterized in that: It includes two independent components: a releasing agent and a neutralizing agent; The releasing agent comprises, by weight percentage, the following components: guanidine isothiocyanate 1.0%–2.0%, sodium dodecyl sulfate 0.03%–0.08%, dithiothreitol 0.03%–0.08%, EDTA-2Na 0.01%–0.04%, bovine serum albumin 0.5%–1.5%, with the balance being 10 mM phosphate buffer at pH 7.4; The neutralizing agent comprises, by weight percentage, the following components: bovine serum albumin 0.5%–1.5%, ProClin 300 0.03%–0.08%, and the balance being 10 mM phosphate buffer at pH 7.

4.

2. The sample processing solution according to claim 1, characterized in that: The releasing agent has a pH of 7.0–8.0, a viscosity of 1.0–5.0 cP, and a surface tension of 30–50 mN / m.

3. The sample processing solution according to claim 2, characterized in that: The releasing agent has a pH of 7.4, a viscosity of 1.5–3.0 cP, and a surface tension of 35–45 mN / m.

4. The sample processing solution according to claim 1, characterized in that: The neutralizing agent has a pH of 6.5–7.5, a viscosity of 1.0–4.0 cP, and a surface tension of 35–55 mN / m.

5. The sample processing solution according to claim 4, characterized in that: The neutralizing agent has a pH of 7.4, a viscosity of 1.5–2.5 cP, and a surface tension of 40–50 mN / m.

6. The method for preparing the sample processing solution according to any one of claims 1 to 5, characterized in that, Includes the following steps: (1) Preparation of release agent: Accurately weigh guanidine isothiocyanate, sodium dodecyl sulfate, dithiothreitol and EDTA-2Na, dissolve in 10mM phosphate buffer at pH 7.4, stir to dissolve, then add bovine serum albumin, continue stirring until all components are completely dissolved; adjust the pH of the system, filter to remove bacteria, measure viscosity and surface tension, and pre-store in the release chamber of the disc microfluidic chip; (2) Neutralizing agent preparation: Accurately weigh bovine serum albumin and ProClin 300, dissolve them in 10mM phosphate buffer at pH 7.4, stir until completely dissolved; adjust the pH of the system, filter to remove bacteria, measure the viscosity and surface tension, and pre-store in the neutralization chamber of the disc microfluidic chip.

7. The application of the sample processing solution according to any one of claims 1 to 5 in 25-OH-VD microfluidic chemiluminescence detection.

8. A 25-OH-VD disk-based microfluidic chemiluminescence detection method, characterized in that, Using the sample processing solution according to any one of claims 1 to 5, the steps include: (1) Add the serum to be tested into the inlet of the disc microfluidic chip and centrifuge at a speed of 800-1500 rpm to make the serum to be tested flow through the release chamber of the pre-stored release agent and the neutralization chamber of the pre-stored neutralizing agent in sequence to complete the dissociation of 25-OH-VD from the binding protein and the neutralization of the pH of the reaction system. (2) The concentration of 25-OH-VD was determined by chemiluminescence immunoassay to obtain the serum concentration of 25-OH-VD.

Images