A carbon-nitrogen fluorescent quantum dot tumor cell detection kit and a method of using the same

Abstract

The application discloses a kind of carbon-nitrogen fluorescent quantum dot tumor cell detection kit and its use method, it is characterized in that, including A liquid and B liquid;Wherein, the A liquid is carbon-nitrogen fluorescent quantum dot solution, the B liquid is buffer solution;The carbon-nitrogen fluorescent quantum dot solution is composed of carbon-nitrogen fluorescent quantum dot and solvent.Its use process, at least includes the following steps: step S10, provides reagent kit A liquid, B liquid, patient pleural fluid, urine sample;Step S20, high-speed centrifugation, after B liquid resuspension, add A liquid dyeing;Step S30, high-speed centrifugation, B liquid resuspension;Step S40, spectrophotometer detects fluorescence intensity.The detection of tumor cells in pleural fluid and urine tissue fluid of the application has high specificity and high sensitivity, and simple operation, cost is small.Not only can improve the diagnosis level of doctor to clinical tumor patient, also can provide help for prognosis evaluation of tumor, drug screening, individualized treatment.

Description

Technical Field

[0001] This invention relates to the field of tumor cell detection technology, and in particular to a carbon-nitrogen fluorescent quantum dot tumor cell detection kit and its method of use. Background Technology

[0002] In primary and metastatic tumors of the chest and urinary system, tumor cells spontaneously detach and are released into tissue fluids such as pleural effusion or urine, serving as a real-time marker for tumor lesion detection and widely used in clinical tumor diagnosis. Accurate and sensitive detection of detached tumor cells in pleural effusion and urine has significant potential clinical value in understanding tumor development mechanisms, early diagnosis, pathological classification, metastasis, treatment, prognosis assessment, evaluation of antitumor drug resistance, and individualized treatment planning.

[0003] With the development of biochemical testing technology, the accuracy of detecting tumor lesions by examining exfoliated cells in pleural effusion and urine has been continuously improving. However, diagnosis often relies on the doctor's subjective experience, which can easily lead to misdiagnosis and missed diagnoses. Currently, commonly used laboratory tests for pleural effusion and urine include hematoxylin and eosin (HE) staining and microscopic examination of tumor cells, flow cytometry DNA analysis, and chromosome examination. Among these, HE staining and microscopic examination suffers from high false negative rates, low accuracy and sensitivity, poor stability, and the inability to sort tumor cells or perform further analysis. Flow cytometry DNA analysis and chromosome examination are complex and costly, limiting their large-scale clinical application.

[0004] Therefore, those skilled in the art are dedicated to developing a tumor cell detection kit that is highly specific and sensitive to tumor cells in pleural effusion and urinary tissue fluid, and is simple to operate and low in cost. Summary of the Invention

[0005] In view of the above-mentioned deficiencies of the prior art, the technical problem to be solved by the present invention is how to improve the specificity and sensitivity of tumor cell detection, while reducing the complexity and cost of its operation.

[0006] To achieve the above objectives, this invention provides a novel application of carbon-nitrogen fluorescent quantum dots in the preparation of tumor cell detection products. The tumor cell detection products should contain at least carbon-nitrogen fluorescent quantum dots.

[0007] The tumor cell detection product can be a tumor cell detection kit based on carbon-nitrogen fluorescent quantum dots, including solution A and solution B, wherein solution A is a carbon-nitrogen fluorescent quantum dot solution and solution B is a buffer solution; the carbon-nitrogen fluorescent quantum dot solution is composed of carbon-nitrogen fluorescent quantum dots and a solvent.

[0008] Furthermore, solution B is phosphate-buffered saline (PBS).

[0009] Further, the solvent is dimethyl sulfoxide (DMSO).

[0010] Further, the concentration of the carbon-nitrogen fluorescent quantum dots in the carbon-nitrogen fluorescent quantum dot solution is 0.01-50 μg / mL.

[0011] Further, the carbon-nitrogen fluorescent quantum dots are N-doped graphene quantum dots, C3N4 quantum dots, C2N quantum dots or C3N quantum dots, and the size of the carbon-nitrogen fluorescent quantum dots is 1-100 nm.

[0012] Further, the carbon-nitrogen fluorescent quantum dots have tumor targeting property.

[0013] Further, the tumor types that can be detected by the kit with high sensitivity include, but are not limited to, intraocular malignant tumor, liver cancer, lung cancer, gastric cancer, bladder cancer, breast cancer, ovarian cancer, thyroid cancer, melanoma and colon cancer.

[0014] Meanwhile, the application also provides a use method of the tumor cell detection kit based on carbon-nitrogen fluorescent quantum dots, which comprises the following steps:

[0015] Step one: collect the pleural effusion or urine sample of a patient by using a puncture needle and store it in a sterile centrifuge tube;

[0016] Step two: centrifuge the sample obtained in the step one, discard the supernatant and obtain the precipitate of the sample;

[0017] Step three: add the B solution, resuspend the precipitate obtained in the step two and obtain the resuspension of the precipitate;

[0018] Step four: add the A solution, mix the resuspension obtained in the step three uniformly and incubate;

[0019] Step five: centrifuge the resuspension after incubation in the step four, discard the supernatant and obtain the precipitate after incubation of the sample;

[0020] Step six: add the B solution, resuspend the precipitate after incubation in the step five and obtain the uniformly mixed resuspension;

[0021] Step seven: use a fluorescence spectrophotometer to detect the fluorescence intensity of the resuspension obtained in the step six;

[0022] Step eight: perform semi-quantitative analysis on the sample according to the fluorescence intensity obtained in the step seven.

[0023] Further, the volume of the pleural effusion or urine sample in the step one is 1-10 mL, and the storage temperature of the sample is 0-25°C.

[0024] Further, the centrifugal speed in the step two and the step five is 1000-2000 revolutions per minute, and the centrifugal time is 1-60 minutes.

[0025] Further, the liquid adding amount of the A liquid in the step four is 0.01-10 mL.

[0026] Further, the liquid adding amount of the B liquid in the step three and the step six is 0.1-10 mL.

[0027] Further, the incubation temperature in the step four is 4-40 DEG C, and the incubation time is 10-480 minutes.

[0028] Further, the excitation wavelength of the sample resuspension liquid in the step seven is 400-800 nm.

[0029] Further, in the step eight, the fluorescence intensity background of normal pleural effusion or urine is less than 5000.

[0030] Technical effect

[0031] The application provides a tumor cell high-sensitivity detection kit based on tumor-targeting carbon-nitrogen fluorescent quantum dots. Compared with the traditional mode, the application has the advantages that:

[0032] (1) The carbon-nitrogen fluorescent quantum dots in the application have tumor targeting, can respond to the microenvironment of tumor cells, recognize tumor cells hidden in pleural effusion and urine, improve the accuracy of tumor cell detection, have good light stability, and ensure the repeatability of detection.

[0033] (2) The application overcomes the problems of low detection accuracy, low sensitivity, poor stability, complex operation, high cost and the like in the prior art, improves the diagnosis level of doctors on clinical tumor patients, and provides help for the prognosis evaluation, drug screening and individualized treatment of tumors.

[0034] The concept, specific structure and generated technical effects of the application will be further described below with reference to the drawings, so as to fully understand the purposes, features and effects of the application. DETAILED DESCRIPTION

[0035] Figure 1 is the use flow diagram of the tumor cell detection kit based on carbon-nitrogen fluorescent quantum dots provided by the application;

[0036] Figure 2 is the bright light and fluorescence contrast diagram of the carbon-nitrogen fluorescent quantum dots capturing tumor cells in the pleural effusion sample in example 1 of the application;

[0037] Figure 3 is the corresponding relationship between the tumor cell reference concentration and the fluorescence reference value in example 1 of the application.

[0038] Figure 4 is a bright light and fluorescence control diagram of tumor cells captured by carbon-nitrogen fluorescent quantum dots in urine samples in Embodiment 4 of the present application;

[0039] Figure 5 is the sensitivity of tumor cells captured by carbon-nitrogen fluorescent quantum dots in pleural effusion samples and urine samples in Embodiments 1 and 4 of the present application. DETAILED DESCRIPTION

[0040] The technical content of the present application is made clearer and easier to understand by introducing a plurality of preferred embodiments of the present application with reference to the accompanying drawings of the specification. The present application can be embodied in many different forms, and the protection scope of the present application is not limited to the embodiments mentioned in the text.

[0041] Figure 1 is a schematic diagram of the use process of the tumor cell detection kit based on carbon-nitrogen fluorescent quantum dots disclosed in the present application, and the operation steps thereof at least include:

[0042] Step S10, providing reagent kit A liquid, B liquid, patient pleural effusion and urine sample;

[0043] Step S20, high-speed centrifugation, after B liquid is resuspended, A liquid is added for staining;

[0044] Step S30, high-speed centrifugation, B liquid is resuspended;

[0045] Step S40, detecting fluorescence intensity by spectrophotometer.

[0046] Specifically, step S10 includes step S11: providing reagent kit A liquid and B liquid, and step S12: providing patient pleural effusion and urine sample. In step S11, the reagent kit A liquid is a solution of 0.01-50 μg carbon-nitrogen fluorescent quantum dots dissolved in 1 mL DMSO, wherein the carbon-nitrogen fluorescent quantum dots are N-doped graphene quantum dots, C3N4 quantum dots, C2N quantum dots or C3N quantum dots; the size of the carbon-nitrogen fluorescent quantum dots is 1-100 nm; and the reagent kit B liquid is PBS buffer. In step S12, the patient's pleural effusion or urine sample is collected by a puncture needle, the sample volume is 1-10 mL, and the sample is stored in a 50 mL centrifuge tube at 0-25℃.

[0047] Step S20 includes step S21: high-speed centrifugation, step S22: B liquid resuspension, and step S23: adding A liquid staining. In step S21, the sample is centrifuged at a speed of 1000-2000 rpm for 1-60 minutes. In step S22, the supernatant in the centrifuge tube is discarded, 0.1-10 mL of reagent kit B liquid is added, and the precipitate at the bottom of the centrifuge tube is resuspended. In step S23, 0.01-10 mL of reagent kit A liquid is added, mixed uniformly with the resuspension, incubated at a temperature of 4-40°C for 10-480 minutes, and the tumor cells in the sample are stained.

[0048] Step S30 includes step S31: high-speed centrifugation and step S32: B liquid resuspension. In step S31, the suspension after A liquid incubation is centrifuged at a speed of 1000-2000 rpm for 1-60 minutes. In step S32, the supernatant in the centrifuge tube is discarded, reagent kit B liquid is added, and the precipitate at the bottom of the centrifuge tube is resuspended. The amount of B liquid added is 0.1-10 mL.

[0049] Step S40 includes step S41: detecting the fluorescence intensity of the sample and step S42: semi-quantitative analysis. In step S41, a fluorescence spectrophotometer is used, the excitation wavelength and emission wavelength are set according to the size of the carbon-nitrogen fluorescent quantum dots, and the fluorescence intensity of the sample is read. In step S42, the fluorescence intensity of the pleural effusion or urine sample is semi-quantitatively analyzed according to the corresponding relationship between the tumor cell reference concentration and the fluorescence reference value.

[0050] Example 1: Detection of pleural effusion samples from patients with ocular choroidal melanoma liver metastasis

[0051] A tumor cell detection kit based on carbon-nitrogen fluorescent quantum dots is used, including A liquid and B liquid. The A liquid is an N-doped graphene quantum dot solution, the solvent is DMSO, the concentration of carbon-nitrogen fluorescent quantum dots is 1 μg / mL, the size of carbon-nitrogen fluorescent quantum dots is 50 nm, and the B liquid is a PBS buffer solution. The kit is used to detect pleural effusion samples from patients with ocular choroidal melanoma liver metastasis.

[0052] A pleural effusion sample (10 mL) was collected from the patient using a puncture needle and stored in a 50 mL centrifuge tube at room temperature (25°C). The sample was centrifuged at 1000 rpm for 20 minutes, and the supernatant was discarded, yielding the precipitate. 10 mL of solution B was added to the centrifuge tube to resuspend the precipitate. Then, 1 mL of solution A was added, and the mixture was thoroughly incubated at 25°C for 60 minutes. After 60 minutes, the sample was centrifuged at 1000 rpm for 20 minutes, and the supernatant was discarded. 1 mL of solution B was added to obtain the resuspension. The pleural effusion sample was then analyzed using a fluorescence spectrophotometer with an excitation wavelength of 400 nm and an emission wavelength of 530 nm.

[0053] Figure 2 This is a comparison image showing the bright and fluorescent contrast of carbon-nitrogen fluorescent quantum dots capturing tumor cells in a patient's pleural effusion sample. The image shows the dashed red circle (…). Figure 2 The cells within the dashed circle on the left side of the image are tumor cells from the patient's pleural effusion, while the cells outside the dashed red circle are exfoliated epithelial cells from the pleural effusion. Under the green fluorescent protein (GFP) channel, the tumor cells clearly show bright green fluorescence. Figure 2 The brighter portion (on the right side) showed fluorescence, while the epithelial cells showed no fluorescence. The fluorescence intensity of the patient's pleural effusion sample was measured at 6300. (Reference) Figure 3 The correspondence between the reference concentration of tumor cells and the reference value of fluorescence was studied. The tumor cell count in the pleural effusion of the patient was 10-100 cells / mL.

[0054] Example 2: Comparison of incubation temperature and time in testing

[0055] Based on Example 1, with the concentration and size of carbon-nitrogen fluorescent quantum dots and the sample volume of solutions A and B remaining constant, the effects of incubation temperature and time on detection were compared. The detection results are as follows:

[0056] Incubation temperature Incubation time Fluorescence intensity 4 degrees 480 minutes 6724 25 degrees 60 minutes 6300 40 degrees 10 minutes 5658

[0057] Example 3 compares the concentration of carbon-nitrogen fluorescent quantum dots and the volume of solutions A and B in the detection.

[0058] Based on Example 1, under the conditions of carbon-nitrogen fluorescent quantum dots with a size of 50 nm, an incubation temperature of 25 degrees Celsius, and an incubation time of 60 minutes, the effects of the concentration of carbon-nitrogen fluorescent quantum dots and the amount of solution A and solution B added on the detection were compared.

[0059] (1) Compare the concentrations of carbon-nitrogen fluorescent quantum dots

[0060] Under the conditions of 1 mL of solution A, 10 mL of solution B, and a carbon-nitrogen fluorescent quantum dot size of 50 nm, the concentrations of carbon-nitrogen fluorescent quantum dots were compared: 50 μg / mL, 1 μg / mL, and 0.02 μg / mL. The detection results are as follows:

[0061] Concentration of carbon-nitrogen fluorescent quantum dots Fluorescence intensity 50 μg / mL 17300 1 μg / mL 6300 0.02 μg / mL 4210

[0062] (2) Compare the volumes of liquids A and B added.

[0063] Under the conditions of a carbon-nitrogen fluorescent quantum dot concentration of 1 μg / mL, a solution A:solution B (v / v) ratio of 1:10, and a carbon-nitrogen fluorescent quantum dot size of 50 nm, the reaction volumes of 0.1 mL, 1 mL, and 10 mL were compared, and the detection results are as follows:

[0064] The reaction volume had no significant effect on the fluorescence results, and the fluorescence intensity was distributed between 6000 and 6500.

[0065] Example 4: Urine sample testing from patients with ureteral epithelial carcinoma

[0066] The tumor cell detection kit based on carbon-nitrogen fluorescent quantum dots includes solutions A and B. Solution A is a C3N quantum dot solution in DMSO with a C3N quantum dot concentration of 1 μg / mL and a C3N quantum dot size of 50 nm. Solution B is a PBS buffer used to detect urine samples from patients with ureteral epithelial carcinoma.

[0067] Collect 10 mL of midstream urine and store it in a 50 mL centrifuge tube at room temperature (25°C). Centrifuge the urine sample at 1500 rpm for 20 minutes, discarding the supernatant to obtain the urine precipitate. Add 10 mL of solution B to the centrifuge tube to resuspend the precipitate at the bottom of the tube. Then add 1 mL of solution A, mix well, and incubate at 25°C for 60 minutes. After 60 minutes, centrifuge at 1500 rpm for 20 minutes, discarding the supernatant and adding 1 mL of solution B to obtain the resuspension. Set the detection conditions of the fluorescence spectrophotometer to an excitation wavelength of 400 nm and an emission wavelength of 530 nm to detect the patient's urine sample.

[0068] Figure 4 This is a comparison image of the bright light and fluorescence of C3N quantum dots capturing tumor cells in a patient's urine sample. The image shows the dotted red circle (…). Figure 4 The cells within the dashed circle on the left are tumor cells from the patient's urine, while the cells outside the dashed red circle are exfoliated epithelial cells from the patient's urinary tract. Under the green fluorescent protein (GFP) channel, the tumor cells clearly show bright green fluorescence. Figure 4The fluorescence intensity of the urine sample of the patient was 8400. Referring to Figure 3 The tumor cell reference concentration corresponds to the fluorescence reference value, and the count of tumor cells in the urine of the patient is 100-1000 cells / mL.

[0069] In Examples 1 and 4, the sensitivity of carbon-nitrogen fluorescent quantum dots in capturing tumor cells in the pleural fluid sample and the urine sample is shown in the table as follows. Figure 5 As can be seen from the table, the sensitivity of carbon-nitrogen fluorescent quantum dots in capturing tumor cells in the urine sample of the patient is more than 72%; and the sensitivity of carbon-nitrogen fluorescent quantum dots in capturing tumor cells in the pleural fluid sample of the patient is 100%.

[0070] The above describes preferred embodiments of the present application in detail. It should be understood that those skilled in the art can make many modifications and changes without creative labor based on the concept of the present application. Therefore, any technical solution that can be obtained by logical analysis, reasoning or limited experiments based on the concept of the present application and the prior art should be within the protection scope defined by the claims.

Claims

1. A tumor cell detection kit based on carbon-nitrogen fluorescent quantum dots, characterized in that, It includes solution A and solution B; wherein solution A is a carbon-nitrogen fluorescent quantum dot solution and solution B is a buffer solution; the carbon-nitrogen fluorescent quantum dot solution is composed of carbon-nitrogen fluorescent quantum dots and a solvent; the carbon-nitrogen fluorescent quantum dots are C2N quantum dots or C3N quantum dots, and the size of the carbon-nitrogen fluorescent quantum dots is 1-100 nm; Solution B is phosphate-buffered saline (PBS); the solvent is dimethyl sulfoxide (DMSO); the concentration of carbon-nitrogen fluorescent quantum dots in the carbon-nitrogen fluorescent quantum dot solution is 0.01–50 μg / mL; The method of using the tumor cell detection kit includes the following steps: Step 1: Preserve the pleural effusion or urine sample in a sterile centrifuge tube; Step 2: Centrifuge the sample obtained in Step 1, discard the supernatant, and obtain the precipitate of the sample; Step 3: Add the B solution to resuspend the precipitate obtained in Step 2 to obtain a resuspension of the precipitate; Step 4: Add the A solution and mix it evenly with the resuspended solution obtained in Step 3, then incubate. Step 5: Centrifuge the resuspended solution after incubation in Step 4, discard the supernatant, and obtain the precipitate of the sample after incubation; Step 6: Add the B solution to resuspend the precipitate after incubation in step 5 to obtain a uniformly mixed resuspension; Step 7: Use a fluorescence spectrophotometer to detect the fluorescence intensity of the resuspension obtained in Step 6; Step 8: Perform semi-quantitative analysis on the sample based on the fluorescence intensity obtained in Step 7.

2. The tumor cell detection kit based on carbon-nitrogen fluorescent quantum dots as described in claim 1, characterized in that, The volume of the pleural fluid or urine sample in step one is 1 to 10 mL, and the sample is stored at a temperature of 0°C to 25°C.

3. The tumor cell detection kit based on carbon-nitrogen fluorescent quantum dots as described in claim 1, characterized in that, In steps two and five, the centrifugation speed is 1000-2000 rpm, and the centrifugation time is 1-60 minutes.

4. The tumor cell detection kit based on carbon-nitrogen fluorescent quantum dots as described in claim 1, characterized in that, In step four, the amount of solution A added is 0.01 to 1 mL.

5. The tumor cell detection kit based on carbon-nitrogen fluorescent quantum dots as described in claim 1, characterized in that, The amount of solution B added in steps three and six is ​​0.1 to 10 mL.

6. The tumor cell detection kit based on carbon-nitrogen fluorescent quantum dots as described in claim 1, characterized in that, The incubation temperature in step four is 4℃ to 40℃, and the incubation time is 10 to 480 minutes.

7. The tumor cell detection kit based on carbon-nitrogen fluorescent quantum dots as described in claim 1, characterized in that, In step seven, the excitation wavelength of the resuspended sample to be tested is 400–800 nm.

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