Inflammation marker protein detection method

Abstract

The invention discloses a detection method of inflammatory marker protein. According to the method, the micro-fluidic chip with a sample loading area, a buffer treatment area, a sample channel and a detection area is prepared on filter paper through wax printing; chitosan is preset in the sample loading area and is used for treating a sample, and bromothymol blue is fixed in the detection area and is used as a pH indicator. During detection, a sample is driven by a phosphate buffer solution with the pH value of 6.0 to flow to a detection area through capillary action, the C-reactive protein and the indicator are subjected to non-specific binding, so that the color of the detection area is changed from yellow to green, the length of a green developing signal is in direct proportion to the concentration of the CRP, and quantitative detection can be realized by measuring the length. The method does not need an antibody, is low in cost, is simple and convenient to operate, can complete detection within 15 minutes, has the detection limit of 1.56 ng / ml and the linear range of 5-25 ng / ml, and is suitable for rapid screening and field detection of basic medical institutions.

Description

Technical Field

[0001] This invention belongs to the field of biodetection technology, specifically relating to a rapid protein detection method. More particularly, it relates to a method and dedicated chip for the quantitative detection of the inflammatory marker protein C-reactive protein (CRP) based on paper-based microfluidic technology and pH-dependent color reaction. Background Technology

[0002] C-reactive protein (CRP) is an acute-phase reactive protein synthesized by the liver and has important clinical diagnostic value. CRP levels rise rapidly within hours of inflammation, infection, or tissue damage, making it a commonly used non-specific inflammatory marker in clinical practice. Elevated CRP levels can indicate inflammatory responses triggered by bacterial infection, autoimmune diseases, or trauma, and are particularly useful in differentiating between bacterial and viral infections; the former often leads to a significant increase in CRP, while the latter only shows a slight increase. Furthermore, for inflammatory diseases such as rheumatoid arthritis and vasculitis, CRP testing can effectively assess their activity and treatment efficacy. Additionally, in postoperative monitoring, observing dynamic changes in postoperative CRP levels can help doctors determine the presence of infection or complications.

[0003] Currently, there are various methods for detecting CRP. Immunoturbidimetry is the most commonly used method, which uses the antigen-antibody reaction to form turbidity, thereby quantitatively detecting CRP concentration. Enzyme-linked immunosorbent assay (ELISA) has high sensitivity, but the operation is relatively complex. Point-of-care testing (POCT) of CRP mainly uses colloidal gold or fluorescence immunochromatography, which greatly shortens the detection time and is very suitable for emergency scenarios or primary healthcare. High-sensitivity C-reactive protein (hs-CRP) is mainly used for the detection of very low concentrations of C-reactive protein and can be used to assess low-level chronic inflammation, especially for predicting cardiovascular disease risk. Semi-quantitative C-reactive protein test strips are currently available, with a cutoff value of 1-3-10 ng / ml, based on the colloidal gold method using lateral flow chromatography. Paper-based microfluidics involves constructing microchannels on specially treated paper, driving liquid detection through capillary action. Currently, there are no observed applications of paper-based microfluidics in CRP detection. Summary of the Invention

[0004] To address the aforementioned problems in the prior art, this invention discloses a method for detecting inflammatory marker proteins. By constructing a special fluid channel, we convert the concentration of CRP into a colorimetric length signal, thereby achieving its quantitative detection.

[0005] This invention includes the following technical solutions: A method for detecting inflammatory marker proteins, based on a paper-based microfluidic chip, comprising the following steps: S1: Provide a paper-based microfluidic chip, wherein the chip defines a sample loading area, a detection area, and a hydrophilic channel connecting the two by forming a hydrophobic barrier by printing wax on filter paper; S2: Chitosan solution is pre-placed in the sample loading area, and bromothymol blue, a pH indicator, is fixed in the detection area; S3: Apply the sample to be tested to the sample loading area; S4: Add pH 6.0 phosphate buffer to the sample loading area, and use capillary action to drive the liquid through the hydrophilic channel to the detection area; S5: Observe the length of the green color signal generated in the detection area due to the non-specific binding of pH indicator to C-reactive protein, and realize the quantitative detection of C-reactive protein based on this length.

[0006] Furthermore, in the above method, the filter paper is Whatman No.1 filter paper.

[0007] Furthermore, in the above method, the chip printed with wax is heated at 150°C for 3 minutes to melt the wax and form a hydrophobic barrier.

[0008] Furthermore, in the above method, the concentration of the chitosan solution is 1%.

[0009] Furthermore, in the above method, the concentration of the bromothymol blue solution is 0.5 g / L.

[0010] Furthermore, the detection limit of the above method is 1.56 ng / ml, and the linear range is 5-25 ng / ml.

[0011] This invention also discloses a paper-based microfluidic chip for implementing the above-mentioned detection method, characterized in that the chip comprises: A sample loading area; One testing area; A sample channel connecting the sample loading area and the detection area; And a buffer processing area located between the sample loading area and the sample channel.

[0012] Furthermore, in the aforementioned paper-based microfluidic chip, the sample loading area and buffer processing area are circular regions, and the detection area is a rectangular strip region.

[0013] The present invention also discloses the use of the above-mentioned paper-based microfluidic chip in the preparation of diagnostic devices or kits for rapid detection of C-reactive protein.

[0014] The present invention also discloses a detection system, comprising: The aforementioned paper-based microfluidic chip; And a tool for measuring the length of the colorimetric signal in the detection area, the tool being a ruler or a mobile terminal with image recognition software installed.

[0015] Compared with the prior art, the present invention has the following outstanding advantages: 1. Innovative Principle, Breakthrough in Cost: This invention abandons the traditional immunochromatographic principle that relies on expensive antibodies. It innovatively utilizes the physicochemical principle that C-reactive protein non-specifically binds to bromothymol blue under a specific acidic buffer environment, causing a change in the ratio of its protonated (HIn, yellow) to deprotonated (In⁻, blue) states, producing an intermediate color (green). This fundamental change in principle eliminates dependence on core biological raw materials (antibodies), significantly reducing production costs and improving reagent stability for long-term storage.

[0016] 2. Ingenious Quantification Method and Simple Equipment: By converting the concentration information of the analyte into a visible colorimetric length signal, intuitive quantification on a paper-based chip is achieved. This method does not rely on expensive optical detection equipment; only a ruler or a mobile phone with a camera is needed to complete the measurement, greatly reducing the threshold for detection and equipment costs, making it particularly suitable for environments with limited resources.

[0017] 3. Integration and Convenience: By integrating sample processing, reaction and detection onto a low-cost chip using paper-based microfluidic technology, the operation steps are extremely simplified. Non-professionals can complete the operation after simple training, realizing true "sample in, result out" and meeting the core needs of point-of-care testing (POCT). Attached Figure Description

[0018] Figure 1 Design of paper-based microfluidics; Figure 2 Schematic diagram of paper-based microfluidics; Figure 3 : The standard curve of CRP. Detailed Implementation

[0019] Step 1: Design and fabrication of paper-based microfluidics Design as Figure 1 The paper-based microfluidic pattern shown has the following regions: Region 1 is a sample loading area with a radius of 8 mm; Region 2 is a 3 mm x 10 mm rectangle, which is the sample channel; Region 3 is a buffer processing area with a radius of 8 mm; and Region 4 is a 2 mm x 30 mm rectangle, which is the detection area.

[0020] The pattern was printed onto Whatman No.1 filter paper using a wax printer. The pattern was then placed in an oven and baked at 150°C for 3 minutes. The choice of Whatman No.1 filter paper as the substrate material was proven in practice, as its low porosity, small pore size, and uniform distribution are beneficial for blood cell filtration. The oven treatment aims to melt the wax, forming a hydrophobic barrier, thus creating hydrophilic channels in the unprinted wax areas, without affecting the subsequent fixation of reagents in the hydrophilic regions.

[0021] Step 2: Reagent fixation (1) Measure 99 ml of deionized water and add 1 ml of glacial acetic acid to obtain 1% acetic acid. Weigh 1 g of chitosan powder and add it to the above 100 ml solution, stir well to obtain 1% chitosan solution. Take 3 μL of the above 1% chitosan solution and add it dropwise to area 1.

[0022] A 1% chitosan solution is used to treat samples, primarily to precipitate red blood cells.

[0023] Bromothymol blue is an acid-base indicator with a color change range of pH 6.0 (yellow) to 7.6 (blue). Phosphate buffer (0.2M pH 6.0) is a buffer solution to counteract potential pH interference from the sample itself.

[0024] CRP is a non-specific binding agent, primarily causing bromothymol blue to transition from yellow (HIn) to blue (In⁻), forming an intermediate green color.

[0025] (2) Prepare 0.2M pH 6.0 phosphate buffer and add 3 μL to region 3.

[0026] (3) Weigh 0.1 g of bromothymol blue, dissolve it in 50% ethanol, and dilute it to 200 ml. Add 2 μL (0.5 g / L) of bromothymol blue solution to area 4.

[0027] (4) All reagents should be allowed to air dry. (Along) Figure 1 Fold along the dotted line and seal the sides with tape to form the following structure ( Figure 2 ) Step 3: Detection Steps (1) Add 3 μL of CRP-containing sample to region 1.

[0028] (2) Prepare a 0.01M pH 6.0 phosphate buffer solution. Add an appropriate amount of the aforementioned phosphate buffer solution to area 1 and let it stand at room temperature for 15 minutes. The sample diffuses to the detection area through capillary action. The detection area changes from yellow to green.

[0029] (3) Measure the length of the green signal with a ruler or mobile phone.

[0030] (4) Establish a standard curve and obtain the concentration value of the sample to be tested based on the standard curve. The above method is not immunochromatography and does not use antibodies. It has advantages such as low price, easy production and easy storage. CRP is a non-specific binding agent, which mainly causes bromothymol blue to change from yellow (HIn) to blue (In⁻) to form an intermediate green color. It is a pH-dependent color reaction.

[0031] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] Example 1 (1) Measure 99 ml of deionized water and add 1 ml of glacial acetic acid (Shanghai test) to obtain 1% acetic acid. Weigh 1 g of chitosan (Merck) powder and add it to the above 100 ml solution, stir well to obtain 1% chitosan solution. Take 3 μL of the above 1% chitosan solution and add it dropwise to area 1.

[0033] (2) Prepare 0.2M pH 6.0 phosphate buffer and add 3 μL to region 3.

[0034] (3) Weigh 0.1 g of bromothymol blue (Sigma-Aldrich), dissolve it in 50% ethanol, and dilute it to 200 ml. Add 2 μL (0.5 g / L) of bromothymol blue solution to area 4.

[0035] (4) All reagents should be allowed to air dry. (Along) Figure 1 Fold along the dotted line and seal the sides with tape. Step 3: Detection process (room temperature) (1) Take 10 µg of recombinant human CRP (acbam) and dilute it to 10 ml with 0.01 M pH 7.4 phosphate buffer to obtain a CRP solution of 1 µg / ml. Take 100 µl of the above solution and dilute it to 1 ml with 0.01 M pH 7.4 phosphate buffer to obtain a CRP solution of 100 ng / ml. Take 50, 100, 150, 200, and 250 µl of the above solution and dilute it to 1 ml with 0.01 M pH 7.4 phosphate buffer to obtain CRP standard solutions of 5 ng / ml, 10 ng / ml, 15 ng / ml, 20 ng / ml, and 25 ng / ml, respectively, for subsequent establishment of standard curves.

[0036] Prepare a 0.01M pH 6.0 phosphate buffer solution and add 3 μL of CRP-containing standard solution to region 1. Establish a standard curve by taking five concentration gradients: 5 ng / ml, 10 ng / ml, 15 ng / ml, 20 ng / ml, and 25 ng / ml.

[0037] (2) Add 3 μL of the above CRP standard solution to area 1, and then add 10 μL of the above phosphate buffer to area 1. Let it stand at room temperature for 15 minutes. The sample diffuses to the detection area through capillary action, and the detection area changes from yellow to green.

[0038] (3) Measure the length of the green signal with a ruler.

[0039] (4) Input the concentration values ​​and signal values ​​into Excel, and establish a standard curve through linear fitting. Figure 3 ) Its linear range is 5-25 ng / ml, and its detection limit is 1.56 ng / ml.

[0040] (5) Take 10 μl of serum sample, 10 μl of 100 ng / ml CRP standard solution, and add 80 μl of 0.01 M pH 7.4 phosphate buffer solution. Take 3 μl of the above solution and perform the above steps, calculate the concentration according to the standard curve, and perform three parallel determinations.

[0041] Take 10 μl of serum sample, add 10 μl of 200 ng / ml CRP standard solution, and add 80 μl of 0.01 M pH 7.4 phosphate buffer solution. Take 3 μl of the above solution and perform the same procedure as described above, calculating the concentration according to the standard curve. Perform the determination in triplicate. The results are shown in Table 1.

[0042] Conclusion: As shown in Table 1, the recovery rate of CRP obtained by the standard addition method is between 98.00% and 98.06%, close to 100%; meanwhile, the relative standard deviations (RSDs) of the three parallel determinations are only 1.13% and 2.04%, respectively. These data fully demonstrate that the detection method provided by this invention has excellent accuracy and good repeatability (precision), and can achieve reliable quantitative analysis of C-reactive protein.

[0043] Example 2 Clinical serum sample testing Objective: To verify the accuracy and anti-interference ability of the method described in this invention for detecting actual clinical serum samples.

[0044] step: 1. Paper-based chip preparation: Same as steps (1) to (4) in Example 1.

[0045] 2. Clinical sample testing: Three anonymous clinical serum samples were collected, and their C-reactive protein (CRP) concentrations were determined using a standard clinical laboratory method (immunoturbidimetric assay) as reference values.

[0046] Following the detection steps of Example 1, 3 μL of the above serum sample was taken and added to area 1 (sample loading area).

[0047] Then, 10 μL of 0.01M pH 6.0 phosphate buffer was added to region 1.

[0048] After standing at room temperature for 15 minutes, the sample diffuses to the detection area through capillary action, and the detection area changes from yellow to varying shades of green.

[0049] The length of the green signal in three parallel experiments for each sample was precisely measured with a ruler, and the average value was taken.

[0050] Substituting the average signal length into the standard curve established in Example 1 ( Figure 3 The concentration determined by the method of the present invention is calculated.

[0051] 3. Results and Analysis: The results of this invention were compared with the reference values ​​of the standard method, and the relative error was calculated. The results are shown in the table below.

[0052] Conclusion: As shown in Table 2, the relative errors of the method of this invention for measuring three clinical serum samples of different concentrations were all within ±5% compared with the reference values ​​of the standard method. This indicates that the paper-based microfluidic detection method established in this invention has good accuracy and resistance to serum matrix interference, and is suitable for rapid detection of actual clinical samples.

[0053] Comparative Example Chitosan solution was not pre-placed in the sample loading area. Objective: To investigate the necessity of pre-positioning chitosan solution in the sample loading area for processing complex biological samples (simulated whole blood samples).

[0054] step: 1. Fabrication of comparative sample chips: The chip design and fabrication process is exactly the same as in Example 1, except that: no 1% chitosan solution is added to region 1 (sample loading area), and it is simply allowed to air dry.

[0055] 2. Sample preparation and testing: To simulate interference from whole blood samples, 100 μL of healthy human serum was taken and a small amount of fresh anticoagulated whole blood was added (to make the solution slightly turbid).

[0056] CRP standard was added to the simulated sample to achieve a concentration of approximately 15 ng / ml.

[0057] Take 3 μL of this simulated sample and add it to region 1 of the comparative chip.

[0058] The subsequent steps were exactly the same as in Example 1: 10 μL of 0.01M pH 6.0 phosphate buffer was added, and the mixture was allowed to stand at room temperature for 15 minutes. The phenomenon was then observed.

[0059] 3. Results and Analysis: Flow phenomenon: In the comparative chip, the capillary flow velocity of the liquid in the sample channel (region 2) is significantly slower than that of the normal chip in Example 1, and the flow front is irregular.

[0060] Color development phenomenon: After the liquid reaches the detection area (area 4), the color development signal is blurred, and the boundary between the green band and the yellow background is unclear, making it impossible to accurately measure the length with a ruler.

[0061] Conclusion: Due to the lack of chitosan's precipitation and filtration effects on solid components such as erythrocytes, impurities in the sample interfered with the normal capillary flow and colorimetric reaction of the liquid within the microfluidic channel, leading to detection failure. This comparative example strongly demonstrates that pre-positioning chitosan solution in the sample loading area is a key and indispensable technical feature enabling this method to reliably detect complex samples (such as whole blood or hyperlipidemic serum).

[0062] In summary, through systematic verification of the embodiments, this invention demonstrates excellent technical performance: Example 1 confirms that the method exhibits good linearity in the range of 5-25 ng / ml (detection limit as low as 1.56 ng / ml), and proves its high accuracy (recovery rate ~98%) and high precision (RSD < 2.1%) through the standard addition method. Example 2 further shows that the detection results of this method on actual clinical serum samples are highly consistent with the standard method (relative error < ± 5%), possessing reliable resistance to matrix interference and practical application potential. The comparative examples, on the other hand, demonstrate from the opposite perspective the crucial role of pre-placed chitosan in the sample loading area for processing complex samples and ensuring detection success, highlighting the rigor and necessity of the overall scheme design.

[0063] It is worth noting that the above description of the embodiments focuses on illustrating the technical solution of the present invention, rather than precisely defining its scope of protection. Those skilled in the art should understand that appropriate adjustments and optimizations can be made based on the technical details disclosed in the embodiments of the present invention, or equivalent substitutions can be implemented for individual or even all technical elements. Such adjustments and substitutions will not deviate from the core essence of the technical solution of the present invention and should be included within the technical protection scope of the embodiments of the present invention. In short, the protection of the present invention should not be limited to the concrete presentation of the above embodiments, but broadly covers all equivalent changes and improvements that do not depart from its basic concept. In summary, the protection definition of the present invention should be based on the statement of the claims, and the above embodiments are only used as a reference guide for understanding the present invention.

Claims

1. A method for detecting inflammatory marker proteins, characterized in that, Based on a paper-based microfluidic chip, the method includes the following steps: S1: Provide a paper-based microfluidic chip, wherein the chip defines a sample loading area, a detection area, and a hydrophilic channel connecting the two by forming a hydrophobic barrier by printing wax on filter paper; S2: Chitosan solution is pre-placed in the sample loading area, and bromothymol blue, a pH indicator, is fixed in the detection area; S3: Apply the sample to be tested to the sample loading area; S4: Add pH 6.0 phosphate buffer to the sample loading area, and use capillary action to drive the liquid through the hydrophilic channel to the detection area; S5: Observe the length of the green color signal generated in the detection area due to the non-specific binding of pH indicator to C-reactive protein, and realize the quantitative detection of C-reactive protein based on this length.

2. The detection method according to claim 1, characterized in that, The filter paper is Whatman No.1 filter paper.

3. The detection method according to claim 1, characterized in that, The chip printed with wax is heated at 150°C for 3 minutes to melt the wax and form a hydrophobic barrier.

4. The detection method according to claim 1, characterized in that, The concentration of the chitosan solution is 1%.

5. The detection method according to claim 1, characterized in that, The concentration of the bromothymol blue solution is 0.5 g / L.

6. The detection method according to claim 1, characterized in that, The detection limit of the method is 1.56 ng / ml, and the linear range is 5-25 ng / ml.

7. A paper-based microfluidic chip for implementing the detection method according to any one of claims 1-6, characterized in that, The chip includes: One sample loading area (1); One detection area (4); A sample channel (2) connecting the sample loading area (1) and the detection area (4); And a buffer processing area (3) located between the sample loading area (1) and the sample channel (2).

8. The paper-based microfluidic chip according to claim 7, characterized in that, The sample loading area (1) and the buffer processing area (3) are circular areas, and the detection area (4) is a rectangular strip area.

9. Use of the paper-based microfluidic chip according to claim 7 or 8 in the preparation of diagnostic devices or kits for rapid detection of C-reactive protein.

10. A detection system, characterized in that, include: The paper-based microfluidic chip according to claim 7 or 8; And a tool for measuring the length of the colorimetric signal in the detection area, the tool being a ruler or a mobile terminal with image recognition software installed.

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