Two-channel colorimetric fluorescent composite probe for detecting trypsin as well as detection method and application of two-channel colorimetric fluorescent composite probe

Abstract

The invention discloses a two-channel colorimetric fluorescent composite probe for detecting trypsin as well as a detection method and application. The two-channel colorimetric fluorescent composite probe comprises orange red carbon quantum dots and cytochrome C in a volume ratio of 1.5: 1. On one hand, the two-channel colorimetric fluorescent composite probe disclosed by the invention can conveniently and sensitively realize detection of trypsin in serum and urine samples, and has important significance for researching the relationship between the trypsin and the occurrence and development of diseases; on the other hand, the existence of the trypsin inhibitor can cause the change of a colorimetric fluorescence signal, so that the preliminary screening of the novel trypsin inhibitor is carried out, and a simple, convenient and rapid new method is provided for the discovery of the novel trypsin inhibitor. The detection method is expected to realize rapid diagnosis of trypsin-related diseases and rapid screening of novel trypsin inhibitors.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and in particular relates to a dual-channel colorimetric fluorescent composite probe for detecting trypsin, a detection method and an application 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, playing an important role in many biological processes. Trypsin can activate other proenzymes, such as procarboxypeptidase and chymotrypsinogen, to hydrolyze protein molecules into smaller peptides to promote their absorption. Trypsin inhibitor (PSTI) regulates trypsin activity by inhibiting the secretion of trypsin activity by pancreatic acinar cells, which otherwise damage the pancreas and lead to pancreatitis. Therefore, trypsin, as a specific biomarker, plays a vital role in the diagnosis of some important biological diseases, such as pancreatic cancer, acute pancreatitis, cystic fibrosis, fetal fecal ileus, biliary cirrhosis, etc. In addition, trypsin inhibitors can be used to treat pancreatitis, shock and disseminated vascular coagulation caused by fibrosis, and have broad prospects in the development of new drugs. Therefore, the development of a simple and easy-to-operate method for trypsin activity determination and trypsin inhibitor screening is of great significance for clinical diagnosis and disease treatment.

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

[0004] In recent years, many simple and rapid methods for the detection of trypsin, such as colorimetry, fluorescence, and electrochemistry, have emerged. Song et al. used a copper-based metal organic framework (JUC-1000) to construct an efficient biosensor based on a signal "on-off" peptide for the determination of trypsin (TPN). Liu et al. used ECL resonance energy transfer (ECL-RET) to reduce the ECL signal 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 detect a variety of macromolecular disease markers. However, biological samples have a large interference with colorimetry and low sensitivity. 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, and sensitivity comparable to electrochemical methods. Traditional sensing methods are mostly based on a single output signal channel, which is inevitably limited by various inherent defects of the signal transduction system. For example, colorimetric sensing methods have shown great potential in POCT (point-of-care testing) due to their advantages of high cost-effectiveness, simple operation, and signal reading by naked eye, but they always face the disadvantage of low detection sensitivity. Therefore, two or more methods may be needed to handle different needs. At present, dual-channel sensors of colorimetric fluorescence have attracted great attention due to the advantages of high selectivity, accuracy, and mutual calibration. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a dual-channel colorimetric fluorescent composite probe for detecting trypsin, as well as a detection method and application, which is expected to achieve rapid diagnosis of trypsin-related diseases and rapid screening of new trypsin inhibitors.

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

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

[0008] The orange-red carbon quantum dots are synthesized by: dissolving rhodamine B in ultrapure water, transferring the solution to a polytetrafluoroethylene autoclave, heating the solution in an oven at 160° C. for 2 h, taking the solution out and cooling it to room temperature, and 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 the solution to obtain an orange-red carbon quantum dot powder.

[0009] The dual-sensor detection method for detecting trypsin based on the above dual-channel colorimetric fluorescent composite probe comprises the following steps:

[0010] Step 1) Colorimetric detection: cytochrome C and trypsin samples of different concentrations were added to the cell culture plate, incubated at 37°C for 1 h, and then 3,3',5,5'-tetramethylbenzidine was added, and the UV-visible absorption spectrum and the absorbance value at 652 nm were recorded;

[0011] Step 2) Fluorescence detection: Cytochrome C and trypsin samples of different concentrations were added to the cell culture plate, incubated at 37°C for 1 h, and then 3,3',5,5'-tetramethylbenzidine and orange-red carbon quantum dots were added, and the fluorescence emission spectrum and the fluorescence intensity value at 580 nm were recorded, 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 dual-channel colorimetric fluorescent composite probe for detecting trypsin in the preparation of a trypsin detection kit.

[0014] A trypsin detection kit comprises the above-mentioned dual-channel colorimetric fluorescent composite probe for detecting trypsin.

[0015] Preferably, the mixture further comprises 3,3',5,5'-tetramethylbenzidine at 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, 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 inhibitors can be used to conduct preliminary screening of new trypsin inhibitors, providing a simple and fast new method for the discovery of new trypsin inhibitors. This detection method is expected to achieve rapid diagnosis of trypsin-related diseases and rapid screening of new trypsin inhibitors. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 TEM characterization image of O-CDs in Example 1;

[0019] Figure 2 The UV spectrum (left) and fluorescence spectrum (right) of O-CDs in Example 1;

[0020] Figure 3 The standard curve diagram of the detection of trypsin in serum by ultraviolet spectroscopy in Example 3;

[0021] Figure 4This is a standard curve diagram for detecting trypsin in serum using fluorescence spectroscopy in Example 3;

[0022] Figure 5 The standard curve diagram of the detection of trypsin in urine by ultraviolet spectroscopy in Example 4;

[0023] Figure 6 This is a standard curve diagram for detecting trypsin in urine using fluorescence spectroscopy in Example 4. DETAILED DESCRIPTION

[0024] The present invention is 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 examples are all conventional means well known to those skilled in the art, and the experimental methods without specifying the specific conditions are all conventional methods in the art.

[0026] Unless otherwise specified, the reagents, equipment, etc. used in the following examples can be obtained from commercial sources.

[0027] Example 1

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

[0029] The dual-sensor detection method for detecting trypsin based on the above dual-channel colorimetric fluorescent composite probe has the following specific steps:

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

[0031] In a typical synthesis route, Rhodamine B (20 mg) was dissolved in 10 mL of ultrapure water, and the solution was transferred to a polytetrafluoroethylene autoclave, heated in an oven at 160°C for 2 h, removed and cooled to room temperature, and the resulting O-CDs solution was centrifuged (16,500 rpm) for 15 min to collect the supernatant. The supernatant was then dialyzed in a dialysis bag for 12 h. Finally, the O-CDs powder was freeze-dried.

[0032] 2. Characterization of O-CDs

[0033] (1) The microstructure of the synthesized orange-red fluorescent carbon dots was characterized using a JEM-2100 transmission electron microscope (JEOL Ltd, Japan).

[0034] Transmission electron microscopy images Figure 1 As shown, O-CDs have been successfully synthesized and are evenly distributed in a spherical shape with good dispersion and clear lattice, and the average particle size is 3.88±0.45nm.

[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 colorimetric detection of trypsin were as follows: 100 μL of CytC (20 μg / mL) and 100 μL of trypsin samples of different concentrations were added to a 96-well plate, incubated at 37°C for 1 h, 50 μL of 5 mM 3,3',5,5'-tetramethylbenzidine (TMB) was added, and the UV-visible absorption spectrum and the absorbance value at 652 nm were recorded (n = 3).

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

[0045] Example 2 Optimization of detection conditions

[0046] In order to obtain the best sensing performance for trypsin detection, the present embodiment optimized the assay parameters, including the concentration of substrate CytC, incubation time, reaction pH, TMB concentration and incubation temperature. Specifically, they are as follows:

[0047] 1. Substrate CytC concentration

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

[0049] 2. Incubation time

[0050] Prolonging the incubation time helps to enhance the detection signal of the system. When the incubation time of the substrates CytC and Trp reaches 60 minutes, the detection signal is almost no longer enhanced. In order to shorten the analysis cycle as much as possible and improve the efficiency of the detection, the optimal incubation time is set to 60 minutes.

[0051] 3. Reaction pH

[0052] The reaction pH has a very significant effect on the detection signal because it affects the activity of trypsin and substrate. We examined the signal response in the range of pH = 5.5 to 9.0. At pH 7.5, the responses of both UV and fluorescence channels reached their maximum values. This is because the optimal pH range for trypsin is around 7.5. Therefore, in subsequent tests, we chose pH = 7.5 as the reaction pH.

[0053] 4. TMB concentration

[0054] The ultraviolet absorption at 652nm and the fluorescence ratio F / F0 at 580nm gradually increased with the increase of TMB concentration. When the TMB concentration was 5mmol / L, the dual-channel response of the system reached the maximum value, so we chose the TMB concentration of 5mmol / L.

[0055] 5. Incubation temperature

[0056] When the incubation temperature was 37°C, the detection signal A652nm and F / F0 had maximum values.

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

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

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

[0060] (1) Human serum (clinically collected and reviewed by Nanjing First Hospital, ethics number KY20240902-KS-03) was diluted 500 times with Tris-HCl (50 mM containing 1 mM CaCl2, pH = 7.5), and different concentrations of trypsin were 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, and 8 μg / mL.

[0061] (2) UV detection: 100 μL of CytC (20 μg / mL) and 100 μL of the above trypsin samples of different concentrations were added to a 96-well plate and incubated at 37°C for 1 h. Then, 50 μL of 5 mM TMB was added and the plate was analyzed using the SpectraMax M 2e The UV-visible absorption spectrum and the absorbance value at 652 nm were recorded by a multifunctional microplate reader (n=3).

[0062] (3) Fluorescence detection: 80 μL of CytC (20 μg / mL) and 80 μL of trypsin at different concentrations were added to a 96-well plate and incubated at 37°C for 1 h. Then, 40 μL of 5 mM TMB and 50 μL of O-CDs were added and the plate was analyzed using a SpectraMax M 2eThe fluorescence emission spectrum and the fluorescence intensity value at 580 nm were recorded by a multifunctional microplate reader, and the excitation wavelength was 350 nm (n=3).

[0063] 2. Experimental results

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

[0065] like Figure 4 As shown in Figure 2, the fluorescence signal of the Trp-CytC / TMB / O-CDs system gradually decreased with the increase of Trp concentration, and the fluorescence intensity decreased linearly in the trypsin concentration range of 10 to 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 UV 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), into diluted human serum.

[0067] The results showed that the accuracy of dual-channel detection of trypsin at 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 detect trypsin quickly, accurately and specifically in actual samples.

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

[0069] In order to demonstrate the potential application of the composite probe and detection method of the present invention in clinical diagnosis, this example further applies it to the determination of trypsin in human urine. The urine of healthy people was diluted 100 times with a buffer solution, and trypsin of different concentrations was added to detect trypsin in urine samples. The details are as follows:

[0070] (1) Human urine (clinically collected and reviewed by Nanjing First Hospital, ethics number KY20240902-KS-03) was diluted 100-fold with Tris-HCl (50 mM containing 1 mM CaCl2, 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, and 2000 ng / mL.

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

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

[0073] 2. Experimental results

[0074] For the UV channel, we can observe Figure 5 As shown in Figure 2, the UV response at A652 nm has a good linear relationship with the trypsin concentration of 10 to 10000 ng / mL (Y = 0.0005661C Trp +0.1569, R 2 =0.9928).

[0075] For the detection of fluorescent channels, such as Figure 6 As shown in Figure 2, the fluorescence response at F580 nm has a good linear relationship with the trypsin concentration of 10 to 2000 ng / mL (Y = -0.2864C Trp +1656, R 2 =0.9943).

[0076] 3. Since trypsin is not found in the urine of healthy people, a certain amount of trypsin was added to the human urine sample to investigate the precision and accuracy.

[0077] The results showed that the recovery rate of the dual channels was 94.12% to 102.26%, which indicates that the composite probe and detection method of the present invention have potential applicability in clinical diagnosis.

[0078] The embodiments described above are only some 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 invention claimed for protection, but merely represents selected embodiments of the present invention. The scope of protection of the present invention shall be subject to the scope required by the claims. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work shall fall within the scope of protection of the present invention.

Claims

1. A dual-channel colorimetric fluorescent composite probe for detecting trypsin, characterized in that: It 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 the solution in an oven at 160° C. for 2 h, taking the solution out and cooling it to room temperature, and 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 the solution to obtain an orange-red carbon quantum dot powder.

2. A dual-sensing detection method for detecting trypsin based on the dual-channel colorimetric fluorescent composite probe according to claim 1, characterized in that: The following steps are involved: Step 1) Colorimetric detection: cytochrome C and trypsin samples of different concentrations were added to the cell culture plate, incubated at 37°C for 1 h, and then 3,3',5,5'-tetramethylbenzidine was added, and the UV-visible absorption spectrum and the absorbance value at 652 nm were recorded; Step 2) Fluorescence detection: Cytochrome C and trypsin samples of different concentrations were added to the cell culture plate, incubated at 37°C for 1 h, and then 3,3',5,5'-tetramethylbenzidine and orange-red carbon quantum dots were added, and the fluorescence emission spectrum and the fluorescence intensity value at 580 nm were recorded, with an excitation wavelength of 350 nm.

3. The dual sensing detection method according to claim 2, characterized in that: The concentration of the cytochrome C is 20 μg / mL; the concentration of the 3,3',5,5'-tetramethylbenzidine is 5 mmol / L.

4. Use of the dual-channel colorimetric fluorescent composite probe for detecting trypsin as claimed in claim 1 in preparing a trypsin detection kit.

5. A trypsin detection kit, characterized in that: It comprises the dual-channel colorimetric fluorescent composite probe for detecting trypsin as described in claim 1.

6. A trypsin detection kit according to claim 5, characterized in that: 3,3',5,5'-tetramethylbenzidine was also included at a concentration of 5 mmol / L.

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