A dual-channel colorimetric fluorescence composite probe for detecting trypsin, a detection method and an application thereof

Abstract

The present invention discloses a dual-channel colorimetric fluorescence composite probe for detecting trypsin, a detection method and an application thereof. The dual-channel colorimetric fluorescence composite probe comprises orange-red carbon quantum dots and cytochrome C with a volume ratio of 1.5:1. The dual-channel colorimetric fluorescence composite probe of the present invention can, on the one hand, conveniently and sensitively detect trypsin in samples including serum and urine samples, which is of great significance for studying the relationship between trypsin and the occurrence and development of diseases; on the other hand, the presence of a trypsin inhibitor can cause changes in the colorimetric fluorescence signal, thereby enabling the preliminary screening of novel trypsin inhibitors, providing a simple and rapid new method for the discovery of novel trypsin inhibitors. This detection method is expected to achieve the rapid diagnosis of trypsin-related diseases and the rapid screening of novel trypsin inhibitors.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and particularly relates to a dual-channel colorimetric fluorescence composite probe for detecting trypsin, a detection method and applications thereof. Background Art

[0002] Trypsin (Trpsin, EC3.4.21.4, Tps) is a common serine protease that exists in the digestive system and is secreted by the pancreas, and plays an important role in many biological processes. Trypsin can activate other proenzymes, such as procarboxypeptidase and chymotrypsinogen, and hydrolyze protein molecules into smaller peptides to facilitate their absorption. Pancreatic trypsin inhibitor (PSTI) regulates trypsin activity by inhibiting the secretion of trypsin activity by pancreatic acinar cells, otherwise pancreatic acinar cells will damage the pancreas and cause pancreatitis. Therefore, trypsin, as a specific biomarker, plays a crucial role in diagnosing some important biological diseases, such as pancreatic cancer, acute pancreatitis, cystic fibrosis, fetal meconium ileus, biliary cirrhosis, etc. In addition, pancreatic trypsin inhibitor can be used to treat pancreatitis, shock and disseminated intravascular coagulation caused by fibrosis, and has broad prospects in new drug research and development. Therefore, developing a simple and easy-to-operate method for determining trypsin activity and screening for pancreatic trypsin inhibitor is of great significance for clinical diagnosis and disease treatment.

[0003] Temler et al. (1974) first successfully determined trypsin by radioimmunoassay (RIA), but the labeling process of the RIA method is cumbersome and has radioactive hazards. So far, a variety of techniques have been used to detect trypsin, including gel electrophoresis, high performance liquid chromatography and enzyme-linked immunosorbent assay, etc. However, these methods require complex instruments and cumbersome procedures, are both expensive and time-consuming, and always require molecular labeling.

[0004] In recent years, many simple and rapid detection methods for trypsin have emerged, such as colorimetry, fluorescence method, electrochemistry, etc. Song et al. constructed an efficient biosensor based on a signal "on-off" peptide using a copper-based metal-organic framework (JUC-1000) for the determination of trypsin (TPN). Liu et al. based on ECL resonance energy transfer (ECL-RET), and the reduced ECL signal changes linearly with the concentration of trypsin. Colorimetry is the preferred method for detecting disease markers, viruses, pollutants, etc. because it is easy to observe, low-cost, and does not require instruments for qualitative analysis. At present, some people have used colorimetry to achieve the detection of various macromolecular disease markers. However, biological samples interfere greatly with colorimetry and the sensitivity is relatively low. Fluorescence analysis has become a popular analytical method for the detection of various disease markers in recent years due to its many advantages such as rapidity, simplicity, strong anti-interference ability, and sensitivity comparable to that of electrochemistry. Traditional sensing methods are mostly based on single-output signal channels and are inevitably limited by various inherent defects of the signal transduction system. For example, colorimetric sensing methods have the advantages of high cost-effectiveness, simple operation, and naked-eye signal reading in point-of-care testing (POCT), showing great potential in POCT, but always face the disadvantage of low detection sensitivity. Therefore, two or more methods may be needed to handle different requirements. At present, due to the advantages of high selectivity, precision, and mutual calibration, colorimetric fluorescence dual-channel sensors have attracted great attention. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the present invention provides a dual-channel colorimetric fluorescence composite probe for detecting trypsin, a detection method and an application thereof, which are expected to realize the rapid diagnosis of trypsin-related diseases and the rapid screening of new trypsin inhibitors.

[0006] The present invention is realized through the following technical solutions:

[0007] A dual-channel colorimetric fluorescence composite probe for detecting trypsin, comprising orange-red carbon quantum dots and cytochrome C with a volume ratio of 1.5:1;

[0008] The orange-red carbon quantum dots are synthesized in the following manner: Take rhodamine B and dissolve it in ultrapure water, then transfer the solution to a polytetrafluoroethylene autoclave, heat it in an oven at 160 °C for 2 h, take it out and cool it to room temperature to obtain an orange-red carbon quantum dot solution, centrifuge for 15 min to collect the supernatant; then dialyze the supernatant in a dialysis bag for 12 h; finally, freeze-dry to obtain orange-red carbon quantum dot powder.

[0009] A dual-sensing detection method for detecting trypsin based on the above dual-channel colorimetric fluorescence composite probe, comprising the following steps:

[0010] Step 1) Colorimetric detection: Add cytochrome C and trypsin samples with different concentrations into a cell culture plate, incubate at 37 °C for 1 h, then add 3,3’,5,5’-tetramethylbenzidine, and record the ultraviolet-visible absorption spectrum and the absorbance value at 652 nm.

[0011] Step 2) Fluorescence detection: Add cytochrome C and trypsin samples with different concentrations into a cell culture plate, incubate at 37 °C for 1 h, then add 3,3’,5,5’-tetramethylbenzidine and orange-red carbon quantum dots, and record the fluorescence emission spectrum and the fluorescence intensity value at 580 nm, with an excitation wavelength of 350 nm.

[0012] Preferably, the concentration of cytochrome C is 20 μg / mL; the concentration of 3,3’,5,5’-tetramethylbenzidine is 5 mmol / L.

[0013] Application of the above-mentioned dual-channel colorimetric fluorescence composite probe for detecting trypsin in the preparation of a trypsin detection kit.

[0014] A trypsin detection kit, comprising the above-mentioned dual-channel colorimetric fluorescence composite probe for detecting trypsin.

[0015] Preferably, it further comprises 3,3’,5,5’-tetramethylbenzidine with a concentration of 5 mmol / L.

[0016] The beneficial effects of the present invention are as follows:

[0017] The dual-channel colorimetric fluorescence composite probe for detecting trypsin of the present invention can, on the one hand, conveniently and sensitively detect trypsin in samples including serum and urine samples, which is of great significance for studying the relationship between trypsin and the occurrence and development of diseases; on the other hand, the change of colorimetric fluorescence signal caused by the presence of trypsin inhibitor can be used for the preliminary screening of new trypsin inhibitors, providing a simple and rapid new method for the discovery of new trypsin inhibitors. This detection method is expected to achieve the rapid diagnosis of trypsin-related diseases and the rapid screening of new trypsin inhibitors. Description of the Drawings

[0018] Figure 1 It is the transmission electron microscope characterization diagram of O-CDs in Example 1;

[0019] Figure 2 It is the ultraviolet spectrum (left) and fluorescence spectrum (right) of O-CDs in Example 1;

[0020] Figure 3 It is the standard curve diagram for detecting trypsin in serum by ultraviolet spectrometry in Example 3;

[0021] Figure 4Standard curve graph for detecting trypsin in serum by fluorescence spectrometry in Example 3;

[0022] Figure 5 Standard curve graph for detecting trypsin in urine by ultraviolet spectrometry in Example 4;

[0023] Figure 6 Standard curve graph for detecting trypsin in urine by fluorescence spectrometry in Example 4. Detailed implementation manners

[0024] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0025] Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art. The experimental methods without specific conditions indicated are all conventional methods in the art.

[0026] Unless otherwise specified, the reagents, equipment, etc. used in the following embodiments can all be obtained through commercial channels.

[0027] Example 1

[0028] A dual-channel colorimetric fluorescence composite probe for detecting trypsin, comprising orange-red carbon dots (O-CDs) and cytochrome C (CytC) with a volume ratio of 1.5:1.

[0029] Based on the above dual-channel colorimetric fluorescence composite probe, a dual-sensing detection method for detecting trypsin is as follows:

[0030] 1. Synthesis of orange-red carbon dots (O-CDs)

[0031] In a typical synthesis route, take rhodamine B (20 mg) and dissolve it in 10 mL of ultrapure water. Then transfer the solution to a polytetrafluoroethylene autoclave and heat it in an oven at 160 °C for 2 h. Take it out and cool it to room temperature to obtain the O-CDs solution. Centrifuge (16,500 rpm) for 15 min to collect the supernatant. Then dialyze the supernatant in a dialysis bag for 12 h. Finally, freeze-dry to obtain the O-CDs powder.

[0032] 2. Characterization of O-CDs

[0033] (1) Use a JEM-2100 type transmission electron microscope (JEOL Ltd, Japan) to characterize the microscopic morphology of the synthesized orange-red fluorescent carbon dots.

[0034] The transmission electron microscope image is as Figure 1 shown. The O-CDs have been successfully synthesized, are evenly distributed, are spherical in shape, have good dispersibility and clear crystal lattices, and the average particle size is 3.88 ± 0.45 nm.

[0035] (2) Using SpectraMax M 2e The optical properties of synthesized O-CDs were characterized by a multifunctional microplate reader (Molecular Devices, USA).

[0036] UV absorption spectrum Figure 2 As shown in the middle left figure, there are two characteristic absorption peaks at ~260nm and ~350nm, of which the absorption peak at 260nm may be attributed to sp 2 The π-π* transition of the structural domain and the absorption peak around 350nm may come from the n-π* transition of the CO bond.

[0037] Fluorescence spectra such as Figure 2 As shown in the middle right figure, when the excitation wavelength is 350nm, the emission wavelength of O-CDs appears at 580nm. Figure 2 As can be seen from the inserted figure, the O-CDs aqueous solution appears rose red and orange red under sunlight and ultraviolet light, respectively, and has obvious orange-red emission fluorescence.

[0038] (3) The carbon dots were measured by Fourier transform infrared spectroscopy (FT-IR) using an IRTracer-100 infrared spectrometer (Shimadzu, Japan). Rhodamine 6G was used as a reference substance, and the relative fluorescence quantum yield of O-CDs was calculated according to the following formula:

[0039]

[0040] Where: Ф s is the fluorescence quantum yield of carbon dots, Ф r is the fluorescence quantum yield of rhodamine 6G (0.95); OD s OD is the absorbance value of the carbon dot dispersion at an excitation wavelength of 365 nm. r is the absorbance value of Rhodamine 6G solution at an excitation wavelength of 365 nm; I s is the effective integrated fluorescence intensity of carbon dots at 500-700 nm, I r is the effective integrated fluorescence intensity of the Rhodamine 6G dispersion at 500-700 nm; η s is the refractive index of the carbon dot dispersion at 25°C, η r is the refractive index of Rhodamine 6G dispersion at 25°C.

[0041] According to the above formula, the relative fluorescence quantum efficiency of O-CDs is 22%.

[0042] 3. O-CDs&CytC dual sensing detection method

[0043] (1) The steps for detecting trypsin by colorimetry are as follows: Add 100 μL of CytC (20 μg / mL) and 100 μL of trypsin samples with different concentrations into a 96-well plate, incubate at 37 °C for 1 h, add 50 μL of 5 mM 3,3’,5,5’-tetramethylbenzidine (TMB), and record the ultraviolet-visible absorption spectrum and the absorbance value at 652 nm (n = 3).

[0044] (2) The steps for detecting trypsin by fluorescence are as follows: Add 80 μL of CytC (20 μg / mL) and 80 μL of trypsin samples with different concentrations into a 96-well plate, incubate at 37 °C for 1 h, add 40 μL of 5 mM TMB, and then add 50 μL of O-CDs, and record the fluorescence intensity value at 580 nm of the fluorescence emission spectrum (Ex = 350 nm, n = 3).

[0045] Optimization of detection conditions in Example 2

[0046] To obtain the best sensing performance for trypsin detection, the measurement parameters in this example were optimized, including the concentration of substrate CytC, incubation time, reaction pH, TMB concentration, and incubation temperature. Specifically as follows:

[0047] 1. Concentration of substrate CytC

[0048] The concentration of substrate CytC plays an important role in the determination of trypsin. The increase in the concentration of CytC leads to an increase in the heme peptide segments with oxidase activity, thus resulting in an increase in the detection signal. However, a high concentration of CytC will also lead to a higher blank signal. The concentration of CytC reaches the highest value for dual-mode detection at 20 μg / mL. As the concentration of CytC continues to increase, the detection signal decreases instead.

[0049] 2. Incubation time

[0050] The extension of the incubation time helps to enhance the detection signal of the system. When the incubation time of substrate CytC and Trp reaches 60 min, the detection signal hardly increases any more. In order to shorten the analysis cycle as much as possible and improve the detection efficiency, the optimal incubation time is set to 60 min.

[0051] 3. Reaction pH

[0052] The reaction pH has a very significant impact on the detection signal because it affects the activities of trypsin and the substrate. We investigated the signal response in the range of pH = 5.5 - 9.0. At pH = 7.5, the responses of the ultraviolet and fluorescence dual channels reach the maximum value. This is because the optimal pH range of trypsin is around 7.5. Therefore, in subsequent detections, we choose pH = 7.5 as the reaction pH.

[0053] 4. TMB concentration

[0054] Ultraviolet absorption at 652 nm and fluorescence ratio F / F at 580 nm 0 It gradually increases with the increase of TMB concentration. When the TMB concentration is 5 mmol / L, the dual-channel response of the system reaches the maximum value. Therefore, we choose the TMB concentration of 5 mmol / L.

[0055] 5. Incubation temperature

[0056] When the incubation temperature is 37 °C, the detection signals A652nm and F / F 0 have the maximum value.

[0057] In summary, we choose the CytC concentration of 20 μg / mL, the incubation time of 60 min, the reaction pH of 7.5, the incubation temperature of 37 °C, and the TMB concentration of 5 mmol / L as the subsequent reaction conditions.

[0058] Example 3 Detection of trypsin in human serum samples

[0059] 1. To further evaluate the performance of the composite probe and detection method of the present invention in detecting trypsin in biological samples, this example conducts the detection of trypsin in healthy human serum. By adding different concentrations of trypsin to the healthy human serum sample diluted 500 times, the dual-channel detection of trypsin in serum is achieved, specifically as follows:

[0060] (1) Human serum (clinically collected and reviewed by the First Hospital of Nanjing, ethical number KY20240902-KS-03) is diluted 500 times with Tris-HCl (50 mM containing 1 mM CaCl 2 , pH = 7.5), and different concentrations of trypsin are added to make the final concentration in the detection system 10 ng / mL, 500 ng / mL, 1 μg / mL, 2 μg / mL, 4 μg / mL, 6 μg / mL, 8 μg / mL.

[0061] (2) Ultraviolet detection: Add 100 μL of CytC (20 μg / mL) and 100 μL of the above-mentioned trypsin samples with different concentrations to a 96-well plate, incubate at 37 °C for 1 h, and then add 50 μL of 5 mM TMB. Use a SpectraMax M 2e multifunctional microplate reader to record the ultraviolet-visible absorption spectrum and the absorbance value at 652 nm (n = 3).

[0062] (3) Fluorescence detection: Add 80 μL of CytC (20 μg / mL) and 80 μL of trypsin with different concentrations to a 96-well plate, incubate at 37 °C for 1 h, and then add 40 μL of 5 mM TMB and 50 μL of O-CDs. Use a SpectraMax M2e A multifunctional microplate reader was used to record the fluorescence emission spectrum and the fluorescence intensity value at 580 nm, with an excitation wavelength of 350 nm (n = 3).

[0063] 2. Experimental results

[0064] As Figure 3 shown, the 652 nm ultraviolet absorption signal generated by the Trp-CytC / TMB detection system gradually increases with the increase of trypsin (Trp) concentration, and within the concentration range of 10 - 6000 ng / mL, there is a good linear relationship between the detection signal A652 nm and the trypsin concentration (Y = 0.0003498C Trp + 0.01060, R 2 = 0.9924).

[0065] As Figure 4 shown, the fluorescence signal of the Trp-CytC / TMB / O-CDs system gradually decreases with the increase of Trp concentration, and the fluorescence intensity linearly decreases within the trypsin concentration range of 10 - 8000 ng / mL (Y = -0.07653C Trp + 1508, R 2 = 0.9807). According to the 3σ / K rule, the LOD can reach 1 ng / mL.

[0066] 3. Since normal human serum does not contain trypsin, we evaluated the precision and accuracy of the ultraviolet and fluorescence dual channels by adding three different concentrations of trypsin, namely low concentration (100 ng / mL), medium concentration (3000 ng / mL), and high concentration (5000 / 7000 ng / mL), to diluted human serum.

[0067] The results showed that the accuracy of the dual-channel detection of trypsin at the three concentrations was greater than 90%, and the relative standard deviation (RSD) was less than 25%, indicating that the composite probe and detection method of the present invention can quickly, accurately, and specifically detect trypsin in actual samples.

[0068] Example 4 Detection of trypsin in human urine samples

[0069] To prove the potential application of the composite probe and detection method of the present invention in clinical diagnosis, this example further applied it to the determination of trypsin in human urine. The urine of healthy people was diluted 100 times with buffer, and different concentrations of trypsin were added for the detection of trypsin in urine samples. Specifically as follows:[[]]

[0070] (1) Human urine (clinically collected and reviewed by the First Hospital of Nanjing, with the ethics number KY20240902-KS-03) was diluted 100-fold with Tris-HCl (50 mM containing 1 mM CaCl 2 , pH = 7.5), and different concentrations of trypsin were added to make the final concentration in the detection system 10, 50, 250, 500, 1000, 1500, 2000 ng / mL.

[0071] (2) UV detection: 100 μL of CytC (20 μg / mL) and 100 μL of the above-mentioned trypsin samples with different concentrations were added to a 96-well plate, incubated at 37 °C for 1 h, and then 50 μL of 5 mM TMB was added. The UV-visible absorption spectrum and the absorbance value at 652 nm were recorded with a SpectraMax M 2e multi-functional microplate reader (n = 3).

[0072] (3) Fluorescence detection: 80 μL of CytC (20 μg / mL) and 80 μL of trypsin with different concentrations were added to a 96-well plate, incubated at 37 °C for 1 h, and then 40 μL of 5 mM TMB and 50 μL of O-CDs were added. The fluorescence emission spectrum and the fluorescence intensity value at 580 nm were recorded with a SpectraMax M 2e multi-functional microplate reader, and the excitation wavelength was 350 nm (n = 3).

[0073] 2. Experimental results

[0074] For the UV channel, as Figure 5 shown, there was a good linear relationship between the UV response at A652 nm and the trypsin concentration of 10 - 10000 ng / mL (Y = 0.0005661C Trp + 0.1569, R 2 = 0.9928).

[0075] For the detection of the fluorescence channel, as Figure 6 shown, there was a good linear relationship between the fluorescence response at F580 nm and the trypsin concentration of 10 - 2000 ng / mL (Y = -0.2864C Trp + 1656, R 2 = 0.9943).

[0076] 3. Since trypsin was not found in the urine of healthy people, the precision and accuracy were investigated by adding a certain amount of trypsin externally to human urine samples.

[0077] The results showed that the recovery rates of the dual channels were 94.12% - 102.26%, indicating the potential applicability of the composite probe and detection method of the present invention in clinical diagnosis.

[0078] The embodiments described above are only a part of the embodiments of the present invention, rather than all embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely represents selected embodiments of the present invention. The protection scope of the present invention shall be subject to the scope claimed in the claims. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without creative efforts shall fall within the protection scope of the present invention.

Claims

1. A dual-sensing detection method for trypsin based on a dual-channel colorimetric fluorescent composite probe, characterized in that: The dual-channel colorimetric fluorescent composite probe includes orange-red carbon quantum dots and cytochrome C in a volume ratio of 1.5:1; The orange-red carbon quantum dots are synthesized by: dissolving rhodamine B in ultrapure water, transferring the solution to a polytetrafluoroethylene autoclave, heating in an oven at 160° C. for 2 h, taking out and cooling to room temperature, centrifuging the obtained orange-red carbon quantum dot solution for 15 min to collect the supernatant; then dialyzing the supernatant in a dialysis bag for 12 h; and finally freeze-drying to obtain an orange-red carbon quantum dot powder; The dual sensing detection method comprises the following steps: Step 1) Colorimetric detection: Add cytochrome C and trypsin samples of different concentrations to the cell culture plate, incubate at 37°C for 1 h, then add 3,3',5,5'-tetramethylbenzidine, and record the UV-visible absorption spectrum and the absorbance value at 652 nm; The concentration of the cytochrome C is 20 μg / mL; the concentration of the 3,3',5,5'-tetramethylbenzidine is 5 mmol / L; Step 2) Fluorescence detection: Add cytochrome C and trypsin samples of different concentrations to the cell culture plate, incubate at 37°C for 1 h, then add 3,3',5,5'-tetramethylbenzidine and orange-red carbon quantum dots, record the fluorescence emission spectrum and the fluorescence intensity value at 580 nm, and the excitation wavelength is 350 nm; The concentration of the cytochrome C is 20 μg / mL; the concentration of the 3,3',5,5'-tetramethylbenzidine is 5 mmol / L.

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