A kit and method for detecting fecal calprotectin content

Abstract

This invention belongs to the field of biosensing and in vitro diagnostics technology, specifically relating to a kit and method for detecting fecal calprotectin levels. The method utilizes a sodium citrate reduction method to prepare gold nanoparticles (AuNPs) and binds them to nucleic acid aptamers that specifically recognize calprotectin. In the absence of a target, the aptamers protect AuNPs from salt ion-induced aggregation through electrostatic and steric hindrance. When calprotectin is present in the sample, the aptamers preferentially bind to the target and detach from the AuNP surface. Subsequently, under the influence of a certain concentration of salt ions, the unprotected AuNPs aggregate, and the solution color changes from red to blue. This invention enables sensitive quantitative detection of calprotectin using ultraviolet-visible absorption spectroscopy or the RGB channel of a smartphone image. This method eliminates the need for expensive antibodies and large, sophisticated instruments, offering advantages such as low cost, good stability, rapid response, and high specificity, making it suitable for primary care screening and point-of-care testing for inflammatory bowel disease.

Description

Technical Field

[0001] This invention belongs to the field of biosensing and in vitro diagnostic technology, specifically relating to a reagent kit and method for detecting fecal calprotectin content. Background Technology

[0002] Fecal calprotectin, a core biomarker for the management of inflammatory bowel disease, plays a crucial role in early clinical screening and management. Currently, enzyme-linked immunosorbent assay (ELISA) is commonly used clinically to detect calprotectin. While this method boasts high accuracy, it is cumbersome, time-consuming, and highly dependent on sophisticated equipment in central laboratories. In recent years, commercially available antibody-based immunochromatographic kits have partially alleviated the demand for point-of-care testing (POCT). However, traditional biological antibodies suffer from limitations such as high production costs, high inactivation rates (poor stability), and significant batch-to-batch variability, severely restricting the widespread adoption of calprotectin testing in primary healthcare institutions and home settings.

[0003] Nucleic acid aptamers, as single-chain oligonucleotides screened through exponential enrichment ligand system evolution technology, have become ideal alternative recognition elements to traditional antibodies due to their excellent chemical stability, low cost, ease of synthesis and modification.

[0004] Therefore, developing a portable, low-cost new calprotectin detection technology that combines nucleic acid aptamers has extremely important clinical and social value. Summary of the Invention

[0005] To address the technical challenges of existing calprotectin detection technologies, such as high cost, reliance on precision instruments, and poor antibody stability, this invention provides a kit and method for detecting fecal calprotectin content. This kit enables intelligent quantitative detection of fecal calprotectin content, is low-cost, and features high specificity, high sensitivity, and visualization.

[0006] Meanwhile, gold nanoparticles (AuNPs), with their significant localized surface plasmon resonance (LSPR) effect, can produce sensitive extinction spectral shifts (manifested as a change in solution color from red to blue) with changes in interparticle spacing, making them very suitable for building visualization detection platforms that do not require precision instruments.

[0007] The technical solution provided by this invention is as follows:

[0008] In a first aspect, the present invention provides a kit for detecting fecal calprotectin content.

[0009] This includes the detection system solution and the fecal sample processing buffer;

[0010] The detection system solution was obtained by incubating a mixture of nucleic acid aptamers and gold nanoparticles; the nucleotide sequence of the nucleic acid aptamers is shown in SEQ ID No. 1.

[0011] The fecal sample processing buffer is used to process the fecal samples to be tested.

[0012] Nucleotide sequence of the aptamer: 5'-GCGGTTTTCTTCGGCCCTTCGTGTCTTTTTGGCTGCTTTC-3'. (SEQ ID No. 1)

[0013] Furthermore, the gold nanoparticles are prepared by sodium citrate reduction method, with an average particle size of 12-14 nm and a negatively charged surface.

[0014] Furthermore, the concentration of nucleic acid aptamers was 10-100 nM; the final concentration of AuNPs was 9.37 nM.

[0015] Furthermore, the incubation conditions are as follows: incubation at 37°C for 1 hour.

[0016] Furthermore, the fecal sample is processed into a mixed solution containing NaCl, BSA, Tris-HCl, and Proclin 300.

[0017] Furthermore, in the fecal sample treatment, the concentration of Tris-HCl is 10-100 mM, the concentration of NaCl is 10-40 mM, the concentration of BSA is 1-2%, the concentration of Proclin 300 is 0.05-0.1%, and the pH is 7-9.

[0018] Furthermore, the volume ratio of the detection system solution to the fecal sample processing buffer is 1:1 – 1:1.2.

[0019] In a second aspect, the present invention provides a method for detecting fecal calprotectin content using the above-described reagent kit, characterized by comprising the following steps:

[0020] Add the fecal sample to be tested to the fecal sample processing buffer to obtain the test solution;

[0021] The solution to be tested is added to the detection system solution. After the reaction reaches equilibrium, the content of calprotectin in the fecal sample to be tested is detected by detecting changes in the optical signal.

[0022] Furthermore, the method of detecting the calprotectin content in the fecal sample by detecting changes in optical signals specifically involves:

[0023] A standard curve between optical signal and calprotectin concentration was established using an optical signal change detection method.

[0024] The content of calprotectin in the sample to be tested was detected based on the standard curve and optical signal.

[0025] Furthermore, the standard curve established using the optical signal change detection method is specifically any one of the following:

[0026] (A) The absorbance values ​​of the reaction solution at 523 nm, 650 nm and 800 nm were measured by ultraviolet-visible spectrophotometry. The absorbance ratio (A650-A800) / (A523-A800) was used as the quantitative signal to establish a standard curve with calprotectin concentration. The concentration x and absorbance y curve is y=0.00177x-0.12879.

[0027] (B) Images of the reaction solution were acquired using a smart device. The average gray values ​​of the red (R), green (G), and blue (B) channels in the central region of the image were extracted. The ratio of the blue to red channels (B / R) was used as a quantitative signal to establish a standard curve relating the concentration x to the concentration calprotectin concentration y. The curve for the concentration x to the ratio of the blue to red channels y is y = 2.46 * 10^6. -4 x+0.3579.

[0028] Compared with the prior art, the present invention has the following technical effects.

[0029] (1) Low cost and good stability: The use of nucleic acid aptamers to replace antibodies completely overcomes the shortcomings of traditional antibody-based calprotectin detection methods, such as easy inactivation, large batch-to-batch variability, and high cost. The system still maintains excellent detection efficiency after being stored at 4°C in the dark for 120 days.

[0030] (2) Simple operation and fast response: The entire detection process does not require complicated pretreatment and washing steps. After the sample is added, it only needs to be balanced for 10 minutes to read the results.

[0031] (3) Visualization and intelligent quantification: Not only can qualitative judgment be made by directly observing color changes with the naked eye, but also the image analysis of smartphones (RGB channel B / R ratio extraction) is innovatively introduced, which gets rid of the dependence on large spectrophotometers and greatly improves the practicality of point-of-care testing (POCT).

[0032] (4) High sensitivity and high specificity: The method has a wide linear range (50-1000 ng / mL) and has a strong anti-interference ability against common interfering substances in feces (such as BSA, glucose, lactoferrin, uric acid and hemoglobin). The actual fecal sample spike recovery rate is between 98% and 113%, and the accuracy is higher. Attached Figure Description

[0033] These and / or other aspects and advantages of the present invention will become apparent and readily understood from the following description of preferred embodiments taken in conjunction with the accompanying drawings, in which:

[0034] Figure 1 A schematic diagram illustrating the principle of this invention for detecting calprotectin based on a nano-gold-aptamer system is shown.

[0035] Figure 2 The morphology and particle size distribution of gold nanoparticles are shown. Figure A is a transmission electron microscope image of the gold nanoparticles (inset shows the prepared wine-red gold nanoparticle solution), and Figure B is a statistical distribution diagram of the gold nanoparticle size.

[0036] Figure 3 The linear response between UV-Vis absorption and calprotectin concentration is shown. Figure A shows the UV-Vis absorption spectrum measurement curves of calprotectin at different concentrations, and Figure B shows the standard curve of the absorption ratio y corresponding to (A650−A800) / (A523−A800) versus calprotectin concentration x. All experiments were performed in triplicate.

[0037] Figure 4 The linear relationship between the B / R channel ratio and calprotectin concentration based on smartphone digital image processing is shown. Figure A shows the visual detection process of calprotectin based on smartphone readings; Figure B shows the colors recorded by the phone after the reaction of protein concentrations from 0 to 1000 ng / mL, with the sample arrangement order from left to right and from top to bottom, corresponding to the increasing order of the concentration gradient; Figure C shows the extraction of B / R channel data from images taken with the phone and the plotting of a standard curve.

[0038] Figure 5 The results of repeatability and stability evaluation of the gold nanoparticle-aptamer detection system are shown in the figure. Figure A shows the UV-Vis absorption spectra of the system under different conditions after 10, 60, and 120 days of storage of the gold nanoparticle (AuNPs) group; Figure B shows the UV-Vis absorption spectra of the system under different conditions after 10, 60, and 120 days of storage under the 20 mM NaCl conditioned group; Figure C shows the UV-Vis absorption spectra of the system under different conditions after 10, 60, and 120 days of storage of the 400 ng / mL calprotectin detection group; Figure D shows the absorbance statistical analysis calculated based on the above spectral data.

[0039] Figure 6 The results of a visualized double-blind experiment based on a smartphone are shown. Figure A shows 20 positive samples with unknown concentrations of calprotectin in the upper half and 20 negative samples in the lower half. Figure B shows the statistical analysis and calculation of corresponding parameter values ​​for positive and negative samples based on the test results. Figure C shows the statistical results based on the B / R channel values, with the lines in the figures representing the critical values.

[0040] Figure 7 The kit of this invention is shown for actual sample testing and results. Figure A shows the basic operation procedure for actual fecal samples; Figure B shows the color record of actual samples and sample-added samples taken with a mobile phone; Figure C, numbered 1, 2, and 3, represents fecal samples from three different individuals. The sample-added groups consisted of three actual samples with randomly added amounts of standard calprotectin sample, corresponding to the following UV spectra: Sample 1: 200 ng / mL; Sample 2: 300 ng / mL; Sample 3: 400 ng / mL; Sample 4: 380 ng / mL; Sample 5: 500 ng / mL. Water was used as the control group. Figure D shows a statistical graph based on the absorbance ratio in Figure C; Figure E shows the statistical analysis of the B / R channel values ​​based on the photographed images, with the line representing the critical value. Three parallel experiments were performed simultaneously. Detailed Implementation

[0041] The technical solution of the present invention will be further described in detail below through embodiments and in conjunction with the accompanying drawings. In this specification, the same or similar reference numerals indicate the same or similar components. The following description of the embodiments of the present invention with reference to the accompanying drawings is intended to explain the overall inventive concept of the present invention and should not be construed as a limitation thereof.

[0042] It should be noted that the terms used in this application are generally those commonly used by those skilled in the art. If there is any inconsistency with commonly used terms, the terms used in this application shall prevail.

[0043] The materials used in the embodiments of this invention are as follows:

[0044] Reagents: Tetrachloroauric acid, sodium citrate, concentrated hydrochloric acid, concentrated nitric acid, sterile enzyme-free water, sodium chloride, bovine serum albumin;

[0045] Materials: Filter, flask, fecal sample collector, syringe;

[0046] Instruments: centrifuge, transmission electron microscope, ultraviolet spectrophotometer, smartphone.

[0047] Nucleic acid aptamers, single-stranded oligonucleotides screened through exponential enrichment ligand system evolution technology, offer advantages such as excellent chemical stability, low cost, and ease of synthesis and modification. Meanwhile, gold nanoparticles (AuNPs), with their significant localized surface plasmon resonance (LSPR) effect, exhibit a sensitive extinction spectral shift (manifested as a change in solution color from red to blue) with variations in interparticle spacing, making them ideal for constructing visualization detection platforms that do not require sophisticated instruments. Based on this, the applicant has developed the biosensor based on gold nanoparticles (AuNPs) and aptamers used in this application for detecting calprotectin levels, the principle of which is as follows: Figure 1 As shown. When calprotectin is absent in the system, the aptamer adsorbs onto the surface of AuNP, maintaining a stable dispersion in a high-salt environment (SALT). When a sample containing calprotectin is added, the aptamer specifically binds to the target protein, thereby detaching from the AuNP surface. The unprotected AuNP then aggregates under high-salt conditions, changing its color from red to blue / purple. This color change can be detected visually or with instruments, thus enabling the visual detection of calprotectin, an inflammatory marker.

[0048] Example 1: Preparation of gold nanoparticles and gold nanoparticle-aptamer detection system

[0049] (1) Synthesis of nano-gold (AuNPs): All glassware was cleaned by soaking in aqua regia. 99 mL of ultrapure water and 1 mL (100 mM) of tetrachloroauric acid trihydrate were added to a two-necked flask and heated to reflux. After boiling, 10 mL (38.8 mM) of sodium citrate solution was quickly added, and the solution changed from pale yellow to wine red within 1 minute. Heating was continued for 20 minutes and then cooled to room temperature.

[0050] (2) Aptamer treatment: The lyophilized powder of single-stranded DNA of 5'-GCGGTTTTCTTCGGCCCTTCGTGTCTTTTTGGCTGCTTTC-3' was centrifuged and then prepared into a 100 μM stock solution with sterile enzyme-free water.

[0051] (3) System construction: The aptamer was diluted and mixed with the AuNPs stock solution to make the final concentration of the aptamer 20 nM and the final concentration of AuNPs 9.37 nM. The mixture was incubated on a shaker at 37℃ for 1 h. The UV spectrum showed that the characteristic absorption peak shifted from 520 nm to 525 nm, proving that the nano-gold-aptamer system was successfully constructed.

[0052] Test Example 1: Transmission electron microscopy (TEM) morphology and particle size distribution of synthesized gold nanoparticles.

[0053] The gold nanoparticle solution was dropped onto a copper grid and allowed to dry at room temperature. The nanoparticles were then characterized using a TALOS transmission electron microscope. Experimental results ( Figure 2 The results show that the gold nanoparticles have a spherical nanostructure with a uniform particle size of approximately 13 nm.

[0054] Test Example 2: Quantitative Detection of Calprotectin

[0055] (1) Add 179 μL of the nano-gold-aptamer mixture obtained in Example 1 above, 20 μL of ultrapure water and 10 μL of calprotectin standard solutions of different concentrations to the colorimetric tube in sequence, so that the final protein concentrations in the system are 50, 100, 200, 300, 400, 500, 600, 750 and 1000 ng / mL, respectively.

[0056] (2) Add NaCl solution to make the final salt concentration 20 mM, and equilibrate at room temperature for 10 min.

[0057] (3) Ultraviolet spectrophotometry: Determine the ultraviolet spectrum of the reaction solution.

[0058] The absorbance values ​​of the reaction solution were measured at 523 nm, 650 nm, and 800 nm. The absorbance ratio (A650-A800) / (A523-A800) was used as the quantitative signal, and a graph was plotted with (A650-A800) / (A523-A800) as the ordinate. The curve of concentration x versus absorbance y is y = 0.00177x - 0.12879, as shown in the figure. Figure 3 As shown. See also Figure 3 As can be seen, with increasing protein concentration, the absorption peak at 523 nm decreases, while the absorption peak at 650 nm increases. A good linear relationship is observed in the range of 50-1000 ng / mL (R²=0.967).

[0059] Test Example 3: Smartphone Analysis and Detection of Calprotectin

[0060] (1) Add 179 μL of the nano-gold-aptamer mixture obtained in Example 1 above, 20 μL of ultrapure water and 10 μL of calprotectin standard solutions of different concentrations to the colorimetric tube in sequence, so that the final protein concentrations in the system are 50, 100, 200, 300, 400, 500, 600, 750 and 1000 ng / mL, respectively.

[0061] (2) Add NaCl solution to make the final salt concentration 20 mM, and equilibrate at room temperature for 10 min.

[0062] (3) Intelligent device analysis method: Place the colorimetric tube on a white background and take a picture with a smartphone. Import the image into image processing software and extract the gray values ​​of the red (R), green (G), and blue (B) channels in the central region. Plot the B / R ratio on the ordinate and the calprotectin concentration on the abscissa to establish a standard curve. The curve of concentration x versus the ratio of blue and red channels y is y = 2.46 * 10 -4 x + 0.3579. The linear relationship is excellent (R² = 0.988). The results are as follows... Figure 4 As shown.

[0063] Test Example 4: Repeatability and Stability Evaluation of the Gold Nanoparticle-Ampamer Detection System

[0064] (1) Long-term storage stability evaluation: The gold nanoparticle (AuNPs) solution synthesized in Example 1 was stored at 4°C in the dark for 10, 60, and 120 days, respectively. At the corresponding time points, the gold nanoparticle solution was taken out and incubated with aptamers according to the method in Example 1 to construct a detection system. 400 ng / mL of calprotectin standard and 20 mM NaCl were added for detection. The UV spectroscopy results showed that, regardless of whether it was the simple gold nanoparticle-aptamer system, the salt-treated group, or the detection group with added calprotectin, the peak shape and absorbance values ​​of the characteristic UV absorption spectra did not show significant fluctuations within 120 days. The results are as follows: Figure 5 As shown in A and B, this demonstrates that the core components of the detection reagent of the present invention possess excellent long-term stability, overcoming the technical challenges of easy inactivation and short shelf life in traditional antibody reagent kits.

[0065] (2) Intra-batch and inter-batch reproducibility evaluation: Different batches of the detection system obtained in Example 1 were synthesized over three consecutive days. Intra-batch evaluation: Multiple samples were randomly selected from the system synthesized on the same day, and gradient concentrations of calprotectin were added for detection; Inter-batch evaluation: Samples were randomly selected from different batches synthesized over three days, and a uniform concentration of calprotectin was added for detection. Results ( Figure 5 (C and D) indicate that the absorbance ratios of different concentrations detected within a batch highly conform to the trend of the standard curve, and the results of the three parallel experiments are almost identical; the results of the same concentrations detected between batches are also highly consistent. The method of this invention demonstrates excellent precision and reproducibility, and can meet the needs of large-scale production and standardized testing.

[0066] Test Example 5: Visualized Double-Blind Experiment Based on Smartphones for Verification

[0067] To further verify the reliability of the visualization detection model of the present invention in actual blind testing scenarios, a double-blind experiment was designed and carried out.

[0068] (1) Experimenter A and Experimenter B respectively used a blind method to process 20 groups of calprotectin positive samples with random concentrations, 20 groups of blank control samples, and 5 negative samples containing interfering substances (BSA, glucose, lactoferrin, uric acid, and hemoglobin).

[0069] (2) After completing the sample addition, salt addition, and equilibration for 10 minutes according to the steps in Test Example 2, photos were taken using smartphones to record the results, and the B / R channel values ​​of the images were extracted. (3) The experimental results showed that all 20 positive samples exhibited a characteristic color change visible to the naked eye (red to blue, 100% detection), while all negative samples and blank controls maintained their original red color (100% non-detection). (4) Statistical analysis showed that the sensitivity, specificity, accuracy, and precision of this detection model all reached 100%. The analysis based on the B / R channel values ​​further confirmed that there was a significant separation boundary (critical value) between positive and negative samples. The results are as follows: Figure 6 As shown in the figure, this embodiment powerfully demonstrates that the present invention can still provide absolutely reliable qualitative and quantitative analysis results without requiring professional personnel to know the sample background, which is very suitable for primary healthcare and home self-testing applications.

[0070] Test Example 6: Specificity of the kit and detection of actual stool samples

[0071] Take out solution A, solution B, and accessory C from the kit. Mix a human fecal sample the size of a soybean with solution B, shake for 30 seconds, filter using accessory C, add the filtrate to solution A, mix and let stand for 10 minutes, take a picture using a smartphone sample analysis program, and perform detection and analysis.

[0072] (1) Specificity evaluation: BSA, glucose, lactoferrin, uric acid, hemoglobin, and calprotectin, each at a concentration of 700 ng / mL, were added to the detection system obtained in Example 1. The results showed that only the calprotectin group showed significant color changes and spectral shifts, while the other interfering groups were no different from the blank control group, indicating that the method has extremely high specificity.

[0073] (2) Detection of actual fecal samples: Weigh 250 mg of frozen fecal sample and add it to buffer solution (containing 1% BSA, 100 mM Tris-HCl, 0.05% Proclin 300, 40 mM NaCl, pH 8.0), vortex for 10 min. After centrifugation at 10000 rpm for 10 min, take the supernatant and filter it through a 0.45 μm filter membrane, then dilute it at a ratio of 1:20. The method of this invention was used for detection, and a spiked recovery experiment was performed. The results showed that the average spiked recovery rate based on the intelligent device was good, confirming that this invention is fully applicable to the accurate quantitative detection of calprotectin in complex fecal matrices. In the spiked recovery determination, 100 ng / mL of actual fecal extract was used as the substrate, and calprotectin standards of different concentration gradients were added respectively. The average spiked recoveries calculated using the UV-Vis spectrophotometer detection method ranged from 98.71% to 112.83%; the average spiked recoveries calculated using the smart device (mobile phone image capture and B / R ratio extraction) analysis method also met the routine requirements in the field of clinical protein detection. Results are as follows... Figure 7 As shown, this fully demonstrates that the method of the present invention does not require expensive antibodies and large-scale precision instruments, and has the advantages of low cost, good stability, fast response, and high specificity, making it very suitable for primary care screening and home-based point testing of inflammatory bowel disease.

[0074] This invention provides a kit and method for detecting fecal calprotectin levels. The method utilizes gold nanoparticles to bind to nucleic acid aptamers that specifically recognize calprotectin, enabling rapid and sensitive detection of fecal calprotectin. This method eliminates the need for expensive antibodies and large, sophisticated instruments, offering advantages such as low cost, good stability, rapid response, and high specificity, making it ideal for primary care screening and point-of-care testing for inflammatory bowel disease.

[0075] While some embodiments of the present general inventive concept have been shown and described, those skilled in the art will understand that changes may be made to these embodiments without departing from the principles and spirit of the present general inventive concept, the scope of which is defined by the claims and their equivalents.

Claims

1. A kit for detecting the content of calprotectin in feces, characterized in that, comprising a detection system solution and a fecal sample processing buffer; The detection system solution is obtained by incubation after mixing of an aptamer and gold nanoparticles; the nucleotide sequence of the aptamer is shown in SEQ ID No. 1; The gold nanoparticles are prepared by sodium citrate reduction method, with an average particle size of 12-14 nm and a negative surface charge. In the detection system solution, the concentration of the aptamer is 10-100 nM, and the final concentration of AuNPs is 9.37 nM.

2. The kit of claim 1, wherein The incubation condition is incubation at 37℃ for 1 h.

3. The kit of claim 1, wherein The fecal sample processing is a mixed solution containing NaCl, BSA, Tris-HCl and Proclin300.

4. The kit of claim 1, wherein In the fecal sample processing, the concentration of Tris-HCl is 10-100 mM, the concentration of NaCl is 10-40 mM, the concentration of BSA is 1-2%, the concentration of Proclin300 is 0.05-0.1%, and the pH is 7-9.

5. The kit of claim 1, wherein The volume ratio of the detection system solution to the fecal sample processing buffer is 1:1-1:1.

2.

6. The kit of claim 1, wherein The kit comprises the following steps:

7. The kit of claim 1, wherein adding the fecal sample to be detected into the fecal sample processing buffer to obtain a solution to be detected; 8. A method for detecting the level of calprotectin in feces based on non-diagnostic purposes using the kit according to any one of claims 1 to 7, characterized in that, adding the solution to be detected into the detection system solution, and detecting the content of calprotectin in the fecal sample to be detected by detecting the change of optical signal after reaction equilibrium. The detection of the content of calprotectin in the fecal sample to be detected by detecting the change of the optical signal comprises the following steps: establishing a standard curve of optical signal and calprotectin concentration by using an optical signal change detection method; 9. The method of claim 8, wherein, detecting the content of calprotectin in the sample to be detected according to the standard curve and the optical signal. The establishment of the standard curve of optical signal and calprotectin concentration by using the optical signal change detection method comprises any one of the following: (A) measuring the absorbance values of the reaction solution at 523 nm, 650 nm and 800 nm by using ultraviolet-visible spectrophotometry, taking the absorbance ratio (A650-A800) / (A523-A800) as the quantitative signal, and establishing a standard curve of the absorbance and the calprotectin concentration, wherein the concentration x and the absorbance y curve is y=0.00177x-0.12879.

10. The method of claim 8, wherein, ​ ​ (B) The image of the reaction solution was collected by the intelligent device, the average gray values of the red (R), green (G), and blue (B) channels in the central region of the image were extracted, the blue-to-red channel ratio (B / R) was used as the quantitative signal, and the standard curve with the calprotectin concentration was established. The curve of the concentration x and the blue-to-red channel ratio y was y = 2.46*10 -4 x+0.3579.

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