CN112858434B - Cysteine protease inhibitor B detection device and preparation method and application thereof - Google Patents
Cysteine protease inhibitor B detection device and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a cysteine protease inhibitor B detection device, a preparation method and application thereof, and solves the technical problems that the concentration detection range and the detection change interval of the existing saliva detection method of cysteine protease inhibitor B are limited, and the concentration reduction cannot be sensitively and specifically detected. The invention also provides a preparation method and application thereof. The invention can be used in the field of detection of cystatin B.
Description
Technical Field
The invention relates to a saliva component detection device and a preparation method and application thereof, in particular to a cysteine protease inhibitor B detection device and a preparation method and application thereof.
Background
In recent years, the examination of biological fluids for the detection of oral cancer biomarkers has been a revolutionary change over traditional detection methods. According to research, the content reduction of a common protein 'Cystatin B (CSTB)' in saliva is an important target for predicting oral squamous cell carcinoma, so that timely finding of concentration change in the saliva is helpful for accurately predicting the disease, metastasis and prognosis states of oral squamous cell patients. The detection of the change of the concentration of the CSTB in the saliva of the oral cavity is an effective method for early detection of the occurrence or recurrence of the oral cancer, and is beneficial to the diagnosis and treatment of a clinician.
However, CSTB has low saliva concentration and is not easy to detect by detection. The conventional methods comprise enzyme-linked immunosorbent assay, enzyme-amplified lanthanide luminescence, radioimmunoassay, immunopolyase chain reaction analysis, matrix-assisted laser desorption, selective reaction monitoring mass spectrometry and the like, and the concentration detection range and the detection change interval of the CSTB protein are limited, so that the concentration reduction cannot be sensitively and specifically detected.
Disclosure of Invention
The invention provides a detection device for a cysteine protease inhibitor B, which can sensitively and specifically detect the reduction of the concentration of CSTB, and a preparation method and application thereof, aiming at the technical problems that the concentration detection range and the detection change interval of the existing CSTB saliva detection method are limited, and the reduction of the concentration cannot be sensitively and specifically detected.
The cysteine protease inhibitor B detection device is a polyethylene terephthalate film provided with asymmetric nanopores, and the asymmetric nanopores are asymmetrically and continuously tapered.
Preferably, the asymmetric nanopore is provided with a large opening end and a small opening end, the diameter of the large opening end is 400nm-650nm, and the diameter of the small opening end is 5nm-35 nm.
The invention also provides a preparation method of the cysteine protease inhibitor B detection device, which comprises the following steps: (1) bombarding the polyethylene terephthalate film by heavy ions to form a bombarded rail; (2) irradiating the polyethylene terephthalate film obtained in the step (1) after heavy ion bombardment by using an ultraviolet lamp; (3) carrying out asymmetric optical nanopore etching on the polyethylene terephthalate membrane obtained in the step (2) to obtain an asymmetric nanochannel membrane; (4) soaking the membrane obtained in the step (3) in an aqueous solution of N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride and N-hydroxysulfonyl succinimide sodium salt at room temperature to activate carboxyl groups on the membrane; (5) washing the membrane obtained in the step (4) with distilled water, soaking in an aqueous solution of a cystatin B monoclonal antibody to react the antibody, and finally fixing the antibody on the inner surface of the membrane, namely obtaining the detection device of the cystatin B;
preferably, in the step (3), the etching solution used for etching is a sodium hydroxide solution, and the stop solution is formic acid and potassium chloride.
Preferably, in the step (3), the membrane is clamped in an etching device, the etching solution is added to one side of the anode of the membrane, the stop solution is added to one side of the cathode of the membrane, the current change is monitored, then the etching solution on one side of the anode is sucked out and cleaned by the stop solution, and finally a new stop solution is added to obtain the asymmetric nanochannel membrane.
The invention also provides application of the cysteine protease inhibitor B detection device in preparation of oral squamous cell carcinoma detection equipment.
Preferably, the application of the cystatin B detection device provided by the invention in preparing an oral squamous cell carcinoma detection device comprises the following steps: (A) bombarding the polyethylene terephthalate film by heavy ions to form a bombarded rail; (B) irradiating the polyethylene terephthalate film obtained in the step (A) after the heavy ion bombardment by using an ultraviolet lamp; (C) performing asymmetric optical nanopore etching on the polyethylene terephthalate membrane obtained in the step (B) to obtain an asymmetric nanochannel membrane; (D) soaking the membrane obtained in the step (C) with an aqueous solution of N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride and N-hydroxysulfonyl succinimide sodium salt at room temperature to activate carboxyl groups on the membrane; (E) washing the membrane obtained in the step (D) with distilled water, soaking in an aqueous solution of a cystatin B monoclonal antibody to react the antibody, and finally fixing the antibody on the inner surface of the membrane, namely obtaining the detection device of the cystatin B; (F) dripping cystatin B solutions with different standard concentration gradients on the surface of the nano-channel, and then testing the current levels on two sides of the nano-channel; (G) and centrifuging the saliva to obtain a supernatant, dripping the supernatant on the nanometer pore canal, and detecting the current on two sides of the membrane.
The invention has the following beneficial effects:
the invention can effectively detect the concentration change condition of the specific antigen CSTB in the mouth, and the asymmetric nano-channel of the invention determines the detection range to be 10 based on the continuous change of the CSTB concentration -8 -10 -12 g/mL. Within this range, when the antigen concentration is from 10 -8 g/mL is gradually reduced to 10 -12 At g/mL, the ion current dropped from-5.5 nA to-2.6 nA at-2.0V, and stable and regular changes were observed. In addition to the voltage-current (I-V) curve, the current change rate ((I) measured at-2.0V 0 -I)/I 0 ) The rate of change of current increased from 0.13 to 1.06 as the CSTB concentration increased. At the same time at 10 -9 -10 -12 A good linear relationship (R) is observed in the g/mL range 2 0.95256). When the asymmetric nano-channel is used for detecting the concentration of CSTB in saliva, the current change rate calculation result of normal people is 1.28-2.00; the results for patients with oral squamous cell carcinoma were: 0.60 to 1.27; the results for patients with oral squamous cell carcinoma with metastasis were: below 0.60.
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FIGS. 1A, 1B and 1C are electron microscope topographies of the asymmetric nanochannel cross-section and large and small aperture ends of the present invention, wherein FIG. 1A is the cross-section; FIG. 1B is a large mouth end; FIG. 1C is a small mouth end;
FIG. 2 is a graph of current (square) at both sides of an asymmetric nanopore membrane after the end of etching and current (circular) at both sides of an asymmetric nanopore functionalized with a CSTB specific monoclonal antibody according to the present invention;
FIGS. 3A, 3B and 3C are diagrams of the functionalized nanochannel for detecting CSTB of various concentrations in accordance with the present invention, wherein FIG. 3A is a graph of I-V curves detected by CSTB of various concentrations; FIG. 3B is a diagram showing the absolute value of the current corresponding to the-2V voltage in the I-V curve; FIG. 3C is a normalized CSTB concentration versus current change rate regression curve for functionalized nanochannels;
FIG. 4 shows saliva test results of healthy people, oral squamous cell carcinoma people and oral squamous cell carcinoma metastasis-associated people in the present invention.
FIGS. 5A, 5B, 5C and 5D show the detection sensitivity of the conventional detection method, wherein FIG. 5A shows the change of OD value with increasing CSTB concentration in ELISA detection; FIG. 5B is a regression curve of the change in the concentration of CSTB and the OD value in ELISA; FIG. 5C shows the sensitivity of ELISA detection; FIG. 5D shows the sensitivity of the Dotblot detection.
Detailed Description
The present invention will be further described with reference to the following examples.
Example 1
(1) Constructing an asymmetric nanopore: the asymmetric nanopore system is constructed by performing experiments by using a polyethylene terephthalate (PET) membrane which forms a bombardment track after heavy ion bombardment. The film thickness was 12 μm and the trajectory after bombardment was the full length of the asymmetric nanopore. The front and back sides of the film were each irradiated with an ultraviolet lamp for 1 hour. Followed by asymmetric optical nanopore etching on the membrane: the etching device, the etching solution (9M sodium hydroxide solution) and the stopping solution (1M formic acid +1M potassium chloride) are preheated on a heating table at 30 ℃ for 30 minutes, and the operation is carried out on the heating table. The film was clamped in an etching apparatus and the current on both sides of the film was monitored for real-time changes at 1V using a picometer. And adding an etching solution to one side of the anode of the membrane, and adding a stopping solution to one side of the cathode of the membrane. When the monitoring current reaches (5-7) x 10 -9 And A, sucking the etching solution on the side of the positive electrode out, washing the etching solution for three times by using the stopping solution, and finally adding new stopping solution for stopping for 30 minutes. And obtaining the asymmetric nano-pore system.
(2) Asymmetric nanopore detection standard: and soaking the etched membrane in pure water for over 12 hours to obtain the asymmetric nanochannel membrane. As shown in fig. 1, the cross section of the nanopore is observed by a scanning electron microscope or a laser confocal microscope, and the whole section of the nanopore presents an asymmetric and continuous cone shape; the size of the big opening end is about 400nm-650nm, and the diameter of the small opening end is 5nm-35 nm. The method comprises the following steps of (1) monitoring the change of current on two sides by using a phosphate buffer as electrolyte of a positive electrode and a negative electrode and using a Peachmeter under the voltage of-2V to +2V, wherein the detection current range is about: the current of the nanopore system at-2V is- (3-5) x 10 -9 A; under the voltage of +2V, the nanopore system is electrifiedThe flow is + (0.5-1.5). times.10 -9 A. As shown by the square curve in fig. 2.
(3) Preparing a functionalized nano pore channel: the CSTB monoclonal antibody was immobilized to the inner surface of the nanochannel by a two-step chemical reaction. First, the etched film was soaked with an aqueous solution of N- (3-Dimethylaminopropyl) -N '-ethylcarbodiimide hydrochloride (N- (3-Dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride: EDC) (15mg/mL) and N-hydroxysulfonyl succinimide sodium salt (N-hydroxysuccinimide sodium salt) (3mg/mL) at room temperature for 1h to activate the carboxyl groups on the film. Washing the membrane with distilled water, and soaking at 1 × 10 -6 g/ml of an aqueous solution of CSTB monoclonal antibody for 2h, the antibody was reacted and finally immobilized on the inner surface of the membrane. The detection current range is about: the current of the nanopore system at-2V is- (1-3) x 10 -9 A; under the voltage of +2V, the current of the nanopore system is plus (0.2-1.0) multiplied by 10 -9 A. As shown in the circular curve of fig. 2.
(4) Using different standard concentration gradients (10) -9 g/ml,10 -10 g/ml,10 -11 g/ml,10 -12 g/ml,) on the surface of the nanochannel, and then the current levels on both sides of the functionalized nanochannel were tested, as shown in fig. 3A, 3B; the rate of change of current means: (I-I) 0 )/I 0 (I-current level corresponding to-2V Voltage after binding CSTB; I 0 Current corresponding to-2V voltage after antibody modification) to obtain a regression curve of the rate of change of current versus CSTB concentration, as shown in fig. 3C.
Example 2
Taking the saliva in the oral cavity more than 2h after the patient finishes eating: ordering a patient to rinse with normal saline, taking 2ml of saliva, and centrifuging at 1500rcf and 4 ℃ for 5 minutes to obtain a supernatant; and centrifuging the supernatant for 5 minutes at 15000rcf and 4 ℃ to obtain the supernatant, and dropwise adding the supernatant on the functionalized nano pore channel for detecting the current on two sides of the membrane.
As shown in fig. 4, the current change rate calculation result of the normal population is 1.28-2.00; the results for patients with oral squamous cell carcinoma were: 0.60 to 1.27; the results for patients with oral squamous cell carcinoma with metastasis were: below 0.60.
Comparative example
As shown in FIG. 5, the detection concentration of the detection result obtained by the enzyme-linked immunosorbent assay (ELISA) kit using the same CSTB monoclonal antibody ranged from about 10 -9 -10 -7 g/mL, minimum concentration of 6X 10 using ELISA -9 g/mL. The Dot blot experiment result shows that the detection concentration range is more than 10 -8 g/mL. The detection sensitivity of the asymmetric nano-channel of the method can be accurate to 10 -12 g/mL。
However, the above description is only exemplary of the present invention, and the scope of the present invention should not be limited thereby, and the replacement of the equivalent components or the equivalent changes and modifications made according to the protection scope of the present invention should be covered by the claims of the present invention.
Claims (7)
1. A preparation method of a cysteine protease inhibitor B detection device is characterized by comprising the following steps:
(1) bombarding the polyethylene terephthalate film by heavy ions to form a bombarded rail;
(2) irradiating the polyethylene terephthalate film obtained in the step (1) after heavy ion bombardment by using an ultraviolet lamp;
(3) carrying out asymmetric optical nanopore etching on the polyethylene terephthalate membrane obtained in the step (2) to obtain an asymmetric nanochannel membrane;
(4) soaking the membrane obtained in the step (3) in an aqueous solution of N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride and N-hydroxysulfonyl succinimide sodium salt at room temperature to activate carboxyl groups on the membrane;
(5) and (3) washing the membrane obtained in the step (4) with distilled water, soaking the membrane in an aqueous solution of the cystatin B monoclonal antibody to react the antibody, and finally fixing the antibody on the inner surface of the membrane, namely the detection device of the cystatin B.
2. The method for preparing a cystatin B detection device according to claim 1, wherein in the step (3), the etching solution used for etching is a sodium hydroxide solution, and the blocking solution is formic acid and potassium chloride.
3. The method for preparing a cystatin B detection device according to claim 2, wherein in the step (3), the membrane is held in an etching device, the etching solution is added to a positive electrode side of the membrane, the blocking solution is added to a negative electrode side of the membrane, a current change is monitored, the etching solution on the positive electrode side is sucked out and washed with the blocking solution, and finally a new blocking solution is added to obtain the asymmetric nanochannel membrane.
4. The cysteine protease inhibitor B detection device according to the production method of a cysteine protease inhibitor B detection device according to claim 1, wherein the cysteine protease inhibitor B detection device is a polyethylene terephthalate film provided with asymmetric nanopores, and the asymmetric nanopores are asymmetrically and continuously tapered.
5. The cystatin B detection device according to claim 4, wherein the asymmetric nanopore is provided with a large-mouthed end and a small-mouthed end, the large-mouthed end having a diameter of 400nm to 650nm, and the small-mouthed end having a diameter of 5nm to 35 nm.
6. Use of the cystatin B detection device according to claim 4 for the preparation of an oral squamous cell carcinoma detection apparatus.
7. Use of the cystatin B detection device according to claim 6 for the manufacture of an oral squamous cell carcinoma detection apparatus, characterized by comprising the steps of:
(A) bombarding the polyethylene terephthalate film by heavy ions to form a bombarded rail;
(B) irradiating the polyethylene terephthalate film obtained in the step (A) after heavy ion bombardment by using an ultraviolet lamp;
(C) performing asymmetric optical nanopore etching on the polyethylene terephthalate membrane obtained in the step (B) to obtain an asymmetric nanochannel membrane;
(D) soaking the membrane obtained in the step (C) with an aqueous solution of N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride and N-hydroxysulfonyl succinimide sodium salt at room temperature to activate carboxyl groups on the membrane;
(E) washing the membrane obtained in the step (D) with distilled water, soaking in an aqueous solution of a cystatin B monoclonal antibody to react the antibody, and finally fixing the antibody on the inner surface of the membrane, namely obtaining the detection device of the cystatin B;
(F) dripping cystatin B solutions with different standard concentration gradients on the surface of the nano-channel, and then testing the current levels on two sides of the nano-channel;
(G) and centrifuging the saliva to obtain a supernatant, dripping the supernatant on the nanometer pore canal, and detecting the current on two sides of the membrane.
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| CN105136875A (en) * | 2015-07-31 | 2015-12-09 | 江苏巨珩新材料科技有限公司 | Method utilizing nano-channel sensor modified by nucleic acid probe having super sandwich structure to high-sensitively and high-specifically detect Zn2+ |
| CN112179955A (en) * | 2020-09-27 | 2021-01-05 | 西北工业大学 | Method for preparing pH-responsive nanofluid diode based on nano holes modified by polyarginine and polyglutamic acid layer by layer |
| CN112179954A (en) * | 2020-09-27 | 2021-01-05 | 西北工业大学 | Method for preparing nano-fluid diode responding to pH and temperature based on gelatin modified solid-state nano-pores |
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