A noninvasive method for detecting bladder cancer through urine

Abstract

The present invention discloses a method for non-invasive detection of bladder cancer through urine. The present invention prepares a detection kit for bladder cancer by detecting the pressure signal generated by the catalytic reaction of exfoliated urine cells after incubation with nanomaterials. The present invention discovers that folate receptors are highly expressed on the surface of exfoliated tumor cell membranes in the urine of bladder cancer patients. Utilizing the characteristic of the high expression of folate receptors on the surface of exfoliated tumor cell membranes in the urine, it can be incubated and bound with nanomaterials and catalyze the decomposition of H2O2 to generate O2, thereby generating a pressure signal. And the size of the pressure signal is detected by the detection kit to determine the presence of bladder cancer tumor cells, so as to non-invasively detect bladder cancer. By detecting the pressure signal generated by the catalytic reaction of exfoliated urine cells after incubation with nanomaterials, the result judgment of the present invention is simple, rapid, clear, economical, and has a relatively high positive detection rate, providing a new non-invasive and highly sensitive detection method for the detection of bladder cancer.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a method for detecting bladder cancer, and in particular to a method for detecting bladder cancer based on pressure signals of urine exfoliated cells. Background Art

[0002] Bladder cancer is the fourth most common cancer in men, and ranks first in the incidence and mortality of urinary system tumors. It is one of the important causes of cancer-related deaths worldwide. In recent years, with the development of my country's social economy, changes in residents' lifestyles and dietary structure, and the acceleration of the aging process of the population, the incidence and mortality of bladder cancer in China have increased year by year. There are obvious gender and age differences in the bladder cancer population, with the elderly being the majority and more men than women, but the incidence rate in young and middle-aged people has gradually increased. More than 90% of bladder cancers are urothelial carcinomas, which can be divided into muscle invasive bladder cancer (MIBC) and non-muscle invasive bladder cancer (NMIBC) according to the degree of muscle invasion. About 5% of bladder cancer patients have distant metastasis when diagnosed, and the 5-year survival rate of patients with advanced or metastatic bladder cancer is less than 15%. Therefore, "early detection", "early diagnosis" and "early treatment" of bladder cancer are extremely important both for individual patients and for society; early diagnosis and early treatment of bladder cancer can not only significantly improve the prognosis of patients, but also reduce the socioeconomic burden. One of the biggest challenges in the treatment of bladder cancer is its high risk of recurrence and metastasis. Transurethral bladder tumor resection is currently the main diagnostic and treatment method for non-muscle invasive bladder cancer, accompanied by other adjuvant therapies such as intravenous chemotherapy. Adjuvant therapy can reduce the risk of recurrence and metastasis of bladder cancer to a certain extent, but it still cannot completely prevent the development of cancer. Therefore, regular monitoring of bladder cancer is necessary for early detection of tumor progression and prolonging patient survival. At present, the diagnosis and monitoring of bladder cancer are mainly based on imaging examinations, urine exfoliated cytology examinations and cystoscopic biopsy. Imaging examinations have poor sensitivity for most early bladder cancers, so they cannot be used as a method for detecting early tumors. Pathological biopsy under cystoscopic examination is not only invasive and costly, but also causes pain and discomfort to patients. Urine exfoliative cytology is an important auxiliary method for bladder cancer examination. Although it has high specificity, its sensitivity is low, about 21% to 50%, especially in low-grade bladder cancer. It is easy to miss very small bladder malignancies. Therefore, it is urgent to develop a bladder cancer detection method with less trauma, low cost, simple and fast, and high accuracy in clinical practice.

[0003] The applicant previously synthesized a metal nanomaterial with a gold nanostructure that has a pressure signal amplification effect (Sha Liu, Jian Chai, Shihao Sun, Lang Zhang, Jiayue Yang, Xu Fu, Jun Hai, Yu-Hong Jing, and Baodui Wang. ACS Applied Materials & Interfaces 2021 13 (39), 46451-46463), and the Ag-AgCl@Au-S-PFG-FA metal nanomaterial has a good photothermal conversion efficiency. It can absorb light energy and convert it into heat energy, and the surface of the Ag-AgCl@Au-S-PFG-FA metal nanomaterial carries folic acid. The present invention combines gold nanomaterials with bladder cancer detection, that is, metal nanomaterials are combined with tumor cells exfoliated from urine to detect the pressure signal generated by catalyzing H2O2 to generate O2 under laser irradiation conditions, thereby constructing a non-invasive detection method for bladder cancer that is less invasive, simple, fast, low-cost and highly accurate.

[0004] Up to now, there has been no report on the expression level of folate receptors on the membrane surface of tumor cells exfoliated in urine and the detection of bladder cancer through the pressure signals of tumor cells exfoliated in urine. The present invention studies the expression level of folate receptors on the membrane surface of tumor cells exfoliated in urine and the pressure signals they produce through clinical urine sample data. The results of the present invention can provide a new non-invasive detection method and detection kit for the clinical detection of bladder cancer. Summary of the invention

[0005] The purpose of the present invention is to provide a method for non-invasively detecting bladder cancer through urine, aiming to solve the problems of complex, low sensitivity, poor stability and high cost of the existing bladder cancer detection methods.

[0006] The technical solution of the present invention is as follows:

[0007] Studies have found that folate receptors are highly expressed on the membrane surface of tumor cells exfoliated in urine of bladder cancer patients. The present invention utilizes the characteristic of high expression of folate receptors on the membrane surface of tumor cells exfoliated in urine. After incubating the tumor cells exfoliated in urine with metal nanomaterials, the two can be combined through the recognition of folic acid and folate receptors, and the nanoparticles bound to the tumor cells are reacted to generate gas pressure. The presence of bladder cancer tumor cells can be determined by detecting the size of the pressure signal, thereby non-invasively detecting bladder cancer.

[0008] The present invention provides a method for non-invasively detecting bladder cancer through urine, and detects the presence of tumor cells by detecting the pressure signal generated by the catalytic reaction of urine exfoliated cells after incubation with nanomaterials to perform non-invasive detection of bladder cancer, which is specifically accomplished by the following detection kit and detection method:

[0009] The detection kit comprises a main box, a sample-laying groove is provided at the bottom of the main box, a sample-laying bottle is arranged in the sample-laying groove, a pressure gauge is provided on the upper part of the sample-laying bottle, the pressure gauge is fixed on the top of the main box, a pressure measuring probe is arranged below the pressure gauge, a laser transmitter is arranged on the left side of the main box, the laser probe of the laser transmitter is facing the sample-laying bottle, a temperature regulator is arranged on the right side of the main box, and a sample-injection window is arranged at the front end of the main box.

[0010] Detection method:

[0011] (1) Centrifuge the urine sample to be tested, and extract urine exfoliated cells by high-speed centrifugation; add the nanomaterial Ag-AgCl@Au-S-PFG-FA to the urine exfoliated cells and mix them evenly, and then incubate them in a 37°C constant temperature incubator for 0.8 to 1.2 hours; the surface of the Ag-AgCl@Au-S-PFG-FA metal nanomaterial carries folic acid, and the folic acid receptors on the surface of the tumor cell membrane exfoliated from the urine of bladder cancer patients are highly expressed. When the two are incubated, they can be combined together through the recognition of folic acid-folic acid receptors;

[0012] The speed of high-speed centrifugation is 3000 rpm, and the centrifugation time is 15 min. The concentration of the nanomaterial Ag-AgCl@Au-S-PFG-FA solution is 80~120 ug / ml, and the solvent is PBS. For the preparation method of the nanomaterial Ag-AgCl@Au-S-PFG-FA, please refer to the literature: Sha Liu, Jian Chai, Shihao Sun, Lang Zhang, Jiayue Yang, Xu Fu, Jun Hai, Yu-Hong Jing, and BaoduiWang.ACS Applied Materials&Interfaces 2021 13 (39), 46451-46463.

[0013] (2) After the incubation is completed, high-speed centrifugation is performed, the supernatant is discarded, and the urine exfoliated cells bound to the nanomaterials after incubation are extracted. The cells are washed with PBS and then high-speed centrifuged again. The supernatant is discarded and the urine exfoliated cells bound to the nanomaterials after washing are extracted; the nanomaterials not bound to the urine exfoliated cells are removed by two high-speed centrifugations and PBS washings; the speed of the high-speed centrifugation is 3000 rpm and the centrifugation time is 15 min.

[0014] (3) Turn on the pressure gauge, temperature regulator and laser transmitter in the main box to preheat, and adjust the temperature of the main box to 35~38℃.

[0015] (4) Add H2O2, NaOH solution and urine exfoliated cells combined with nanomaterials after incubation and washing into the sample bottle and mix well, then quickly cover the bottle with a sealing cap to seal it; the concentration of H2O2 is 30%, the pH of the NaOH solution is 12, and the volume ratio of the NaOH solution to H2O2 is 18:1~20:1; the NaOH solution provides a strong alkaline environment, and the catalytic effect of the Ag-AgCl@Au-S-PFG-FA metal nanomaterial on the decomposition of H2O2 to generate O2 is the best under the strong alkaline environment.

[0016] (5) The sealed sample bottle is quickly transferred to the sample groove inside the preheated main box through the sampling window at the front end of the main box, and the pressure probe of the pressure gauge is quickly inserted into the sample bottle through the sealed bottle cap to detect the pressure signal;

[0017] (6) Record the pressure signal value 10 minutes after the test. If it is higher than or equal to 5.6 kPa, it is judged as "bladder cancer"; if it is lower than 5.6 kPa, it is judged as "non-bladder cancer".

[0018] Detection mechanism: Ag-AgCl@Au-S-PFG-FA metal nanomaterials have good photothermal conversion efficiency. Under the irradiation of laser, they can absorb light energy and convert it into heat energy, and then play a role similar to catalase activity, catalyzing the decomposition of H2O2 to generate O2 and produce a pressure signal. The pressure signal is the gas pressure generated by the generated O2. Under the irradiation of laser, the nanomaterials combined with the surface of the tumor cell membrane shed in the urine of bladder cancer patients catalyze the decomposition of H2O2 to generate O2 and produce a pressure signal, converting the molecular signal recognized by the folic acid-folate receptor into an amplified pressure signal, and the presence of bladder cancer tumor cells is determined by detecting the size of the pressure signal.

[0019] Through a large number of studies, it was found that the expression of folate receptors on the membrane surface of urine exfoliated cells of patients without bladder cancer is low, so there are fewer nanomaterials incubated and bound, which catalyzes the decomposition of H2O2 to generate O2 and produces a small pressure signal, lower than 5.6kPa. However, the expression of folate receptors on the membrane surface of tumor cells exfoliated in urine of patients with bladder cancer is high, so there are more nanomaterials incubated and bound, which catalyzes the decomposition of H2O2 to generate O2 and produces a large pressure signal, higher than or equal to 5.6kPa, so as to detect bladder cancer non-invasively.

[0020] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0021] (1) The present invention found that folate receptors are highly expressed on the membrane surface of tumor cells shed in urine of bladder cancer patients. By utilizing the characteristics of high expression of folate receptors on the membrane surface of tumor cells shed in urine, nanomaterials can be incubated and combined to decompose H2O2 to generate pressure signals. The present invention is the first to propose a new auxiliary detection method and detection kit for non-invasive detection of bladder cancer through the pressure signals of tumor cells shed in urine.

[0022] (2) The result judgment is simple, fast, clear and economical. The present invention only needs to observe the pressure value displayed on the pressure gauge after the detection time is reached to make a preliminary judgment on whether bladder cancer is present. Compared with conventional methods for diagnosing and detecting bladder cancer such as imaging examination, urine exfoliative cytology examination and cystoscopic biopsy, it does not require the participation of doctors from the imaging department or pathology department. The detection personnel do not need professional knowledge of pathology, imaging and other disciplines to easily identify bladder cancer cells in urine, which can reduce the labor cost of bladder cancer diagnosis and treatment, and is particularly suitable for rapid screening.

[0023] (3) High positive detection rate. Through research, the present invention has concluded that the optimal cutoff value for detecting bladder cancer pressure is 5.6 kPa, that is, if the 10-minute pressure signal generated by the urine sample is higher than or equal to 5.6 kPa, it is judged as "bladder cancer", and if it is lower than 5.6 kPa, it is judged as "non-bladder cancer". Based on the above optimal pressure cutoff value for detecting bladder cancer, the positive detection rate of the present invention is 67.07%, while the positive detection rate of urine exfoliative cytology, which is currently commonly used for diagnosing bladder cancer, is only about 30%. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a schematic diagram of the structure of the detection kit of the present invention;

[0025] In the figure: 1-main box; 2-pressure gauge; 3-sampling groove; 4-laser transmitter; 5-temperature regulator; 6-injection window; 7-sampling glass pressure bottle; 8-pressure measuring probe of pressure gauge; 9-laser probe of laser transmitter.

[0026] Figure 2 This is an immunocytochemical experiment to analyze the expression of folate receptors on the surface of tumor cells shed in the urine of bladder cancer patients.

[0027] Figure 3 It is the test result of 10-minute pressure signal of positive control sample (bladder cancer patient) and normal urine sample (healthy person). DETAILED DESCRIPTION

[0028] The technical solution of the present invention is described in detail below in conjunction with example samples. The following examples are implemented on the premise of the technical solution of the present invention, and detailed implementation methods and specific operating processes are given, but the protection scope of the present invention is not limited to the following examples. Example

[0029] 1. Expression of folate receptors on the surface of tumor cells exfoliated in urine of bladder cancer patients

[0030] Preoperative urine samples from three patients who were initially diagnosed with bladder cancer were collected and centrifuged to collect urine sediments. Immunocytochemistry (ICC) was used to detect the expression level of folate receptors on the surface of tumor cell membranes in urine of bladder cancer patients, including the following steps:

[0031] Take urine sediment smear and fix it with 4% paraformaldehyde for 6 hours. Wash the slides 3 times with PBS, 5 minutes each time. Eliminate endogenous peroxidase activity: Incubate the smear with 3% H2O2 for 5 minutes. Wash the slides 3 times with PBS, 5 minutes each time.

[0032] Blocking: Incubate the specimen slides with 10% goat serum for 10 minutes. Wash the slides with PBS 3 times, 5 minutes each time.

[0033] Incubate with primary antibody: dilute rabbit anti-FOLR-1 (folate receptor-α) polyclonal antibody with antibody diluent at a ratio of 1:200, mix well, and drop onto the specimen smear. Then place the specimen smear in a moisturizing box and incubate it overnight at -4°C. Wash the slides with PBS 3 times, 5 minutes each time.

[0034] Incubate with secondary antibody: dilute the anti-rabbit biotinylated secondary antibody with antibody diluent at a ratio of 1:200 and add it to the specimen smear. Then place the specimen smear in a moisturizing box and incubate it at room temperature for 40 minutes. Wash the slides with PBS 3 times, 5 minutes each time. Add HRP-labeled streptavidin to the specimen smear and incubate it at room temperature for 40 minutes. Wash the slides with PBS 3 times, 5 minutes each time.

[0035] Color development: prepare fresh DAB color developing reagent and add it dropwise to the specimen smear. Allow to react for 3 to 10 minutes at room temperature. Observe the degree of color development under a microscope, terminate the reaction at an appropriate time, and rinse with tap water.

[0036] Counterstaining: Use hematoxylin for 2 minutes and rinse with tap water.

[0037] Sealing: Use neutral gum for sealing.

[0038] Microscopic examination: Observe, take photos and perform microscopic examination under an ordinary optical microscope. Figure 2 As shown, folate receptors are highly expressed on the surface of tumor cells shed in urine of bladder cancer patients.

[0039] 2. Detecting bladder cancer

[0040] like Figure 1As shown, the detection kit includes a main box 1, a sample-laying groove 3 is provided at the bottom end of the main box 1, a sample-laying bottle 7 is arranged in the sample-laying groove 3, the sample-laying bottle is a glass pressure bottle, a pressure gauge 2 is provided on the upper part of the sample-laying bottle 7, the pressure gauge 2 is fixed on the top of the main box 1, a pressure measuring probe 8 is arranged below the pressure gauge 2, and the pressure measuring probe 8 can be extended into the glass pressure bottle through the sealing bottle cap of the glass pressure bottle, a laser transmitter 4 is arranged on the left side of the main box 1, a laser probe 9 of the laser transmitter 4 is facing the sample-laying bottle 7, and the instrument specifications of the laser transmitter 4 are 808nm, 1.0W / cm 2 A temperature regulator 5 is provided on the right side of the main box 1, and a sample injection window 6 with a handle is provided on the front end of the main box 1.

[0041] Preparation and detection of clinical urine samples: Preoperative urine samples from 82 patients clinically diagnosed with bladder cancer and urine samples from 46 healthy people with normal physical examinations were collected, and urine samples were prepared and the pressure signals of urine exfoliated cells were detected using the detection kit provided by the present invention, including the following steps:

[0042] (1) Take 50 ml of urine sample to be tested and centrifuge it at high speed (3000 rpm, time 15 min) to extract urine exfoliated cells.

[0043] (2) Use 0.5 ml of 100 ug / ml PBS solution of nanomaterial Ag-AgCl@Au-S-PFG-FA to add to urine exfoliated cells and mix well, then incubate in a 37°C constant temperature incubator for 1 hour. The surface of Ag-AgCl@Au-S-PFG-FA metal nanomaterial carries folic acid, and the folic acid receptors on the surface of tumor cell membranes exfoliated from urine of bladder cancer patients are highly expressed. When the two are incubated, they are combined together through the recognition of folic acid-folic acid receptors.

[0044] (3) After incubation, centrifuge (speed: 3000 rpm, time: 15 min), discard the supernatant, and extract the urine exfoliated cells with nanomaterials after incubation. Wash with PBS twice and centrifuge again (speed: 3000 rpm, time: 15 min), discard the supernatant, and extract the urine exfoliated cells with nanomaterials after washing. Two high-speed centrifugations and PBS washings remove the nanomaterials that are not bound to the urine exfoliated cells.

[0045] (4) Add 950uL of NaOH solution with a pH of 12 to the urine exfoliated cells with nanomaterials after incubation and washing, and mix thoroughly to obtain a mixed solution.

[0046] (5) Add 50uL 30% H2O2 to a 1.5mL glass pressure bottle, then quickly drop the above mixture into the same glass pressure bottle and quickly cover it with a sealing cap to seal it.

[0047] (6) Turn on the pressure gauge, temperature regulator and laser transmitter in the main box to preheat, and adjust the temperature of the main box to 37°C;

[0048] (7) The sealed glass pressure bottle is quickly transferred to the position of the sample groove inside the preheated main box, and the pressure probe of the pressure gauge is quickly inserted into the glass pressure bottle through the sealed bottle cap to detect the pressure signal. Under the irradiation of the laser, the nanomaterials combined with the surface of the tumor cell membrane shed in the urine of bladder cancer patients catalyze the decomposition of H2O2 to generate O2 and generate a pressure signal. The presence of bladder cancer tumor cells can be determined by detecting the size of the pressure signal.

[0049] After the glass pressure bottle has tested a urine sample, it can be taken out from the sampling window at the front end of the main box and replaced with another new glass pressure bottle, thus ensuring the accuracy of the test results.

[0050] (8) Record the pressure signal value after 10 minutes of testing. If the pressure signal is higher than or equal to 5.6 kPa, it is judged as "bladder cancer", and if it is lower than 5.6 kPa, it is judged as "non-bladder cancer". Figure 3 As shown, the 10-minute pressure signal of the bladder cancer group was significantly higher than that of the healthy group, and the difference was statistically significant ( P <0.001).

[0051] Based on the above optimal pressure cutoff value for detecting bladder cancer, the present invention can detect 55 cases among 82 patients with newly diagnosed bladder cancer, with a positive detection rate of 67.07%, while the positive detection rate of urine exfoliative cytology, which is currently commonly used to diagnose bladder cancer, is only about 30%.

[0052] In summary, the present invention proposes for the first time a method for non-invasively detecting bladder cancer through urine. The high expression of folate receptors on the surface of tumor cell membranes in urine of bladder cancer patients not only proves its feasibility at the theoretical level, but also further verifies its reliability at the practical application level of clinical urine samples. In addition, the result judgment of the present invention is simple, fast, clear, and has a high positive detection rate, which is particularly suitable for clinical rapid screening of bladder cancer.

[0053] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by a person skilled in the art within the spirit and scope of the present invention shall also fall within the protection scope of the present invention.

Claims

1. Use of a detection kit in the preparation of a bladder cancer detection product, characterized in that: The detection kit comprises a main box, wherein a sample-laying groove is provided at the bottom of the main box, a sample-laying bottle is arranged in the sample-laying groove, a pressure gauge is provided on the upper part of the sample-laying bottle, a pressure measuring probe is provided below the pressure gauge, a laser transmitter is provided on the left side of the main box, a laser probe of the laser transmitter faces the sample-laying bottle, a temperature regulator is provided on the right side of the main box, and a sample-injection window is provided at the front end of the main box; How to use the test kit: (1) Add the nanomaterial Ag-AgCl@Au-S-PFG-FA to the urine exfoliated cells after high-speed centrifugation and mix well, then incubate in a 37°C constant temperature incubator for 0.8-1.2 hours; (2) After the incubation is completed, high-speed centrifugation is performed, the supernatant is discarded, and the urine exfoliated cells bound to the nanomaterials after incubation are extracted, and the urine exfoliated cells bound to the nanomaterials after washing are extracted again after washing with PBS. The nanomaterials that are not bound to the urine exfoliated cells are removed by two high-speed centrifugations and PBS washings; (3) Turn on the pressure gauge, temperature regulator and laser transmitter in the main box to preheat, and adjust the temperature of the main box to 35~38℃; (4) Add H2O2, NaOH solution and urine exfoliated cells combined with nanomaterials after incubation and washing into the sample bottle and mix well, and quickly cover the bottle with a sealing cap to seal; the pH of the NaOH solution is 12; the concentration of H2O2 is 30%; (5) The sealed sample bottle is quickly transferred to the sample groove inside the preheated main box through the sampling window at the front end of the main box, and the pressure probe of the pressure gauge is quickly inserted into the sample bottle through the sealed bottle cap to detect the pressure signal.

2. Use of the detection kit according to claim 1 in preparing a bladder cancer detection product, characterized in that: The pressure gauge (2) is fixed on the top of the main box (1).

3. Use of the detection kit according to claim 1 in preparing a bladder cancer detection product, characterized in that: In steps (1) and (2), the rotation speed of the high-speed centrifugation is 3000 rpm and the centrifugation time is 15 min.

4. Use of the detection kit according to claim 1 in preparing a bladder cancer detection product, characterized in that: In step (1), the concentration of the nanomaterial Ag-AgCl@Au-S-PFG-FA solution is 80-120 ug / ml, and the solvent is PBS.

Images