CN108896638B - Preparation method and application of immunosensor based on titanium dioxide doped graphene loaded sea cucumber-like gold-palladium core-shell nanoparticles - Google Patents
Preparation method and application of immunosensor based on titanium dioxide doped graphene loaded sea cucumber-like gold-palladium core-shell nanoparticles Download PDFInfo
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Abstract
The invention belongs to the technical field of novel functional materials, immunoassay and biosensing, and provides a preparation method and application of a titanium dioxide doped graphene-loaded sea cucumber-like gold-palladium core-shell nanoparticle immunosensor. The titanium dioxide-doped graphene-loaded sea cucumber-like gold-palladium core-shell nano particles are used as a signal amplification platform to construct a label-free electrochemical immunosensor, so that the quantitative detection of ovarian cancer markers is realized, and the method has the advantages of strong specificity, high sensitivity, low detection limit and the like. Has important scientific significance and application value for detecting ovarian diseases.
Description
Technical Field
The invention belongs to the technical field of novel nano materials, immunoassay and biosensing, and provides a preparation method of an immunosensor based on titanium dioxide doped graphene loaded sea cucumber-like gold-palladium core-shell nanoparticles, and application of an electrochemical immunosensor constructed by the method in detection of human epididymis protein 4 antigen.
Background
Ovarian cancer is a common malignant tumor in gynecology, and the mortality rate is the first of gynecological malignant tumors. Therefore, timely screening and detection in the early stages of ovarian cancer is an important approach to reduce mortality. However, early diagnosis is a difficult problem because early symptoms of ovarian cancer are atypical and easily cause missed diagnosis and misdiagnosis. Therefore, it is very important to develop a rapid, simple and sensitive method for detecting ovarian cancer markers. Human epididymis protein 4 is used as a new ovarian cancer tumor marker, has good sensitivity and high specificity, and can help early diagnosis and follow-up monitoring of ovarian cancer particularly at the asymptomatic expression stage of the early stage of the disease, so that the development of a high-sensitivity quantitative detection method of human epididymis protein 4 antigen is particularly important for early diagnosis of ovarian cancer.
The electrochemical immunosensor is a sensor which is based on the specific combination of antigen and antibody as a sensitive element, an electrode as a conversion element and detects signals by taking current or potential as characteristics, has the advantages of rapid detection, high sensitivity, simple operation, easy miniaturization and the like, and has important application value in the fields of clinical detection, environmental monitoring, biological monitoring and the like.
The invention prepares the immunosensor based on the titanium dioxide doped graphene loaded sea cucumber-like gold-palladium core-shell nanoparticles by utilizing a layer-by-layer self-assembly technology and taking the titanium dioxide doped graphene loaded sea cucumber-like gold-palladium core-shell nanoparticles as a signal amplification platform, realizes the quantitative detection of the human epididymis protein 4 antigen, has the advantages of high sensitivity, simple operation, high detection speed, wide detection range, low detection lower limit and the like, has good stability, reproducibility and selectivity, and provides a feasible detection means for the early diagnosis of ovarian cancer.
Disclosure of Invention
The invention provides an immunosensor based on titanium dioxide doped graphene loaded sea cucumber-like gold-palladium core-shell nanoparticles, which comprises: the device comprises a working electrode, a counter electrode and a reference electrode, wherein a substrate electrode of the working electrode is a glassy carbon electrode, the surface of the glassy carbon electrode is sequentially modified with titanium dioxide doped graphene loaded with sea cucumber-shaped gold-palladium core-shell nanoparticles, a human epididymis protein 4 antibody, bovine serum albumin and a human epididymis protein 4 antigen, the counter electrode is a platinum wire electrode, and the reference electrode is a saturated calomel electrode.
The invention aims to provide a titanium dioxide doped graphene loaded sea cucumber-like gold-palladium core-shell functional nano material, and a simple, quick and sensitive label-free electrochemical immunosensor is constructed.
The invention also aims to apply the unmarked electrochemical immunosensor constructed on the basis of titanium dioxide doped graphene loaded sea cucumber-like gold-palladium core-shell nanoparticles to the detection of ovarian cancer markers.
The technical scheme of the invention is as follows:
a preparation method of an immunosensor based on titanium dioxide doped graphene loaded sea cucumber-like gold-palladium core-shell nanoparticles is characterized by comprising the following steps:
(1) polishing a glassy carbon electrode with the diameter of 3.0 ~ 5.0.0 mm into a mirror surface by using aluminum oxide polishing powder with the diameter of 1.0 mu m, 0.3 mu m and 0.05 mu m in sequence, and ultrasonically cleaning the mirror surface in absolute ethyl alcohol;
(2) dropwise coating the titanium dioxide doped graphene loaded sea cucumber-like gold-palladium core-shell nanoparticle dispersion liquid of 6.0 muL and 0.5 ~ 4.0.0 mg/mL on the surface of the electrode, drying at room temperature, washing the surface of the electrode with ultrapure water, and drying;
(3) continuously dripping the human epididymis protein 4 antibody of 6.0 muL and 5.0 ~ 15.0.0 mug/mL on the surface of the electrode, standing in a refrigerator at 4 ℃ and drying;
(4) continuously dropwise adding bovine serum albumin solution of 3.0 muL and 1.0 ~ 2.0.0 wt% to the surface of the electrode to seal the nonspecific active sites on the surface of the electrode, washing the surface of the electrode with phosphate buffer solution with pH = 7.0, standing in a refrigerator at 4 ℃ and airing;
(5) dripping a series of human epididymis protein 4 antigen solutions with different concentrations, namely 6.0 muL and 50 fM ~ 80 nM, on the surface of an electrode, standing in a refrigerator at 4 ℃ for 30 ~ 40 min, washing the surface of the electrode with phosphate buffer solution with pH = 7.0, and drying in the refrigerator at 4 ℃ to prepare the working electrode of the amperometric immunosensor for detecting the human epididymis protein 4 antigen.
The preparation method of the immunosensor based on the titanium dioxide doped graphene loaded with the holothurian-shaped gold-palladium core-shell nanoparticles according to claim 1, wherein the preparation method of the titanium dioxide doped graphene comprises the following steps:
(1) preparation of graphene oxide
Placing 4.0 g of graphite and 1.0 g of sodium nitrate in a 2000 mL beaker, slowly adding 192 mL of concentrated sulfuric acid under magnetic stirring in an ice-water bath environment, then slowly adding 24.0 g of potassium permanganate and vigorously stirring, reacting for 1h in the ice-water bath, then transferring the mixture into a 35 ℃ oil bath kettle for reacting for 2 h, heating to 55 ℃, reacting for 1.5 h, then dropwise adding 160 mL of deionized water into the mixed solution and continuously stirring by using a glass rod, then adding 400 mL of deionized water, finally adding hydrogen peroxide (20.0 mL, 30%) into the mixed solution, reacting for 10min, standing overnight, taking out the lower layer for dialysis for one week, dispersing by ultrasonic waves (140W, 1 h), centrifuging (6000 rpm, 10 min), collecting the supernatant and freeze-drying for 24 h to obtain graphene oxide;
(2) preparation of aminated graphene Dispersion
Adding 100.0 mg of graphene oxide into 40.0 mL of ethylene glycol under ultrasonic treatment, performing ultrasonic treatment for 1h, adding 1.0mL of concentrated ammonia water into the solution to obtain a dark brown solution, transferring the mixed solution into a high-pressure reaction kettle, performing solvothermal reaction for 10 h at 180 ℃, filtering precipitates after the reaction, repeatedly washing the precipitates to be neutral by using distilled water, performing freeze drying for 24 h to obtain a flaky aminated graphene solid, dispersing the flaky aminated graphene solid into 5.0 mL of deionized water again to obtain an aminated graphene dispersion liquid, and storing the aminated graphene dispersion liquid at 4 ℃ for further use;
(3) preparation of titanium dioxide-doped graphene dispersion
Putting 10 mL of deionized water, 5mL of ethanol and 5 mg of sodium dodecyl sulfate into a 50 mL beaker, performing ultrasonic treatment until the deionized water, the ethanol and the sodium dodecyl sulfate are uniformly mixed, then dispersing 10 ~ 40 mg of prepared aminated graphene in a mixed solution, performing ultrasonic treatment for 15 min, then adding 0.05 ~ 0.5.5 mL of isopropyl titanate into a suspension, performing continuous ultrasonic treatment for 1h to stably and uniformly disperse the prepared aminated graphene, transferring the obtained suspension into a 20 mL autoclave with a polytetrafluoroethylene lining, reacting for 12 h at 130 ℃, centrifuging for 10min at 6000 r/min, thoroughly washing by using a mixture (volume ratio is 1: 1) of deionized water and ethanol, finally washing by using deionized water, dispersing in deionized water after washing, and storing at 4 ℃ for later use.
The preparation method of the immunosensor based on titanium dioxide-doped graphene-loaded holothurian-shaped aupd core-shell nanoparticles according to claim 1, wherein the preparation of the dispersion of the holothurian-shaped aupd core-shell nanoparticles comprises the following steps:
(1) preparation of gold nanorod Dispersion
Adding a chloroauric acid solution (250. mu.L, 10 mM) and a cetyltrimethylammonium bromide solution (7.5 mL, 100 mM) into a 20 mL flask, placing the flask in a 30 ℃ oil bath, magnetically stirring for 10min, then rapidly adding a freshly prepared ice-cold sodium borohydride solution (600. mu.L, 10 mM) into the solution with stirring, stopping stirring after 2 min, mixing and stirring the resulting brownish yellow solution and storing the solution at 30 ℃ for 3 h for later growth of gold nanorods, in the growth solution, adding a cetyltrimethylammonium bromide solution (40.0 mL, 100 mM), a chloroauric acid solution (1.7 mL, 10 mM) and a silver nitrate solution (250. mu.L, 10 mM) to the growth solution, slowly adding an ascorbic acid solution (270. mu.L, 100 mM) into the solution, finally adding a 200. mu.L ~ 500. mu.L of a gold seed crystal solution to the growth solution, standing the growth solution in a 30 ℃ water bath for 15 h, centrifuging, washing with precipitates, re-dispersing the obtained gold precipitates in a 20 mL dispersion tank, and storing the gold nanorods at 4 ℃ in ultrapure water;
(2) preparing a sea cucumber-shaped gold-palladium core-shell nanoparticle dispersion liquid
The chlorohexadecylpyridine solution was added to a 50 mL flask, and a sodium tetrachloropalladate solution (100. mu.L, 10 mM) and 1.0mL ~ 5.0.0 mL gold nanorods were sequentially added to the flask under a 65 ℃ oil bath, and then, a freshly prepared ascorbic acid solution (200. mu.L, 100 mM) was injected, magnetically stirred for 2 min and then stopped, the resulting solution was reacted for 30 min, centrifuged (8000 rpm, 5 min) to collect a black precipitate, and redispersed in water for further use.
The preparation method of the immunosensor based on the titanium dioxide doped graphene loaded sea cucumber-like gold-palladium core-shell nanoparticles according to claim 1, wherein the immunosensor is used for detecting human epididymis protein 4 antigen, and the detection steps are as follows:
(1) an electrochemical workstation is used for testing in a three-electrode system, a saturated calomel electrode is used as a reference electrode, a platinum wire electrode is used as an auxiliary electrode, the prepared sensor is used as a working electrode, and the test is carried out in 10 mL of phosphate buffer solution with the concentration of 50 mmol/L, pH 5.84.84 5.84 ~ 8.04.04;
(2) detecting the analyte by a time-current method, wherein the input voltage is-0.4V, the sampling interval is 0.1 s, and the running time is 400 s;
(3) and when the background current tends to be stable, injecting 10 mu L and 5 mol/L hydrogen peroxide solution into 10 mL and 50 mmol/L, pH = 7.4 phosphate buffer solution every 50 s, and recording the current change.
Advantageous results of the invention
(1) According to the invention, titanium dioxide doped graphene is used as a carrier, the aminated graphene has large specific surface area and electrical conductivity, more active sites can be provided for catalytic materials, the electron transfer efficiency on the surface of an electrode is accelerated, meanwhile, titanium dioxide nanoparticles have good biocompatibility and special catalytic property, the effect of amplifying signals is achieved, the hybridization of the titanium dioxide nanoparticles and the titanium dioxide nanoparticles can effectively increase the specific surface area of the surface of the electrode, the electron transfer efficiency on the surface of the electrode can be accelerated, the catalytic performance of the material is improved, and the method plays an important role in improving the sensitivity of an immunosensor.
(2) The sea cucumber-shaped gold-palladium core-shell nano particles with special shapes are used as a part of a signal amplification platform for the first time, so that the catalytic performance is further improved, and the sea cucumber-shaped gold-palladium core-shell nano particles are used for constructing an immunosensor.
(3) The electrochemical immunosensor realizes the aim of quantitative detection on the human epididymis protein 4 antigen, the linear detection range is 50 fM ~ 80 nM, and the lowest detection lower limit is 16.7 fM.
(4) The electrochemical immunosensor constructed by the method is simple to operate and rapid to detect, and can be used for rapid detection of actual samples.
Detailed Description
The invention will now be further illustrated by, but not limited to, the following specific embodiments
Embodiment 1. a preparation method of an immunosensor based on titanium dioxide doped graphene loaded sea cucumber-like gold-palladium core-shell nanoparticles, comprising the following operation steps:
(1) polishing a glassy carbon electrode with the diameter of 4.0 mm into a mirror surface by using alumina polishing powder with the diameter of 1.0 mu m, 0.3 mu m and 0.05 mu m in sequence, and ultrasonically cleaning the mirror surface in absolute ethyl alcohol;
(2) taking 6.0 muL and 1.0 mg/mL titanium dioxide doped graphene loaded sea cucumber-like gold-palladium core-shell nanoparticle dispersion liquid to be dripped on the surface of the electrode, drying at room temperature, washing the surface of the electrode with ultrapure water, and drying;
(3) continuously dripping the human epididymis protein 4 antibody of 6.0 muL and 10.0 mug/mL on the surface of the electrode, standing in a refrigerator at 4 ℃ and drying;
(4) continuously dropwise adding a bovine serum albumin solution of 3.0 muL and 1.0 wt% to the surface of the electrode to seal the non-specific active sites on the surface of the electrode, washing the surface of the electrode with a phosphate buffer solution with pH = 7.0, standing in a refrigerator at 4 ℃ and airing;
(5) dripping a series of human epididymis protein 4 antigen solutions with different concentrations, namely 6.0 muL and 50 fM ~ 80 nM, on the surface of an electrode, standing in a refrigerator with the temperature of 4 ℃ for 35 min, washing the surface of the electrode with phosphate buffer solution with the pH = 7.0, and drying in the refrigerator with the temperature of 4 ℃ to obtain the working electrode of the amperometric immunosensor for detecting the human epididymis protein 4 antigen.
Embodiment 2. a preparation method of an immunosensor based on titanium dioxide doped graphene loaded sea cucumber-like gold-palladium core-shell nanoparticles, comprising the following operation steps:
(1) polishing a glassy carbon electrode with the diameter of 4.0 mm into a mirror surface by using alumina polishing powder with the diameter of 1.0 mu m, 0.3 mu m and 0.05 mu m in sequence, and ultrasonically cleaning the mirror surface in absolute ethyl alcohol;
(2) taking titanium dioxide doped graphene loaded sea cucumber-like gold-palladium core-shell nanoparticle dispersion liquid of 6.0 muL and 2.0 mg/mL, dropwise coating the dispersion liquid on the surface of an electrode, drying the electrode at room temperature, washing the surface of the electrode with ultrapure water, and drying the electrode;
(3) continuously dripping the human epididymis protein 4 antibody of 6.0 muL and 10.0 mug/mL on the surface of the electrode, standing in a refrigerator at 4 ℃ and drying;
(4) continuously dropwise adding a bovine serum albumin solution of 3.0 muL and 1.0 wt% to the surface of the electrode to seal the non-specific active sites on the surface of the electrode, washing the surface of the electrode with a phosphate buffer solution with pH = 7.0, standing in a refrigerator at 4 ℃ and airing;
(5) dripping a series of human epididymis protein 4 antigen solutions with different concentrations, namely 6.0 muL and 50 fM ~ 80 nM, on the surface of an electrode, standing in a refrigerator with the temperature of 4 ℃ for 35 min, washing the surface of the electrode with phosphate buffer solution with the pH = 7.0, and drying in the refrigerator with the temperature of 4 ℃ to obtain the working electrode of the amperometric immunosensor for detecting the human epididymis protein 4 antigen.
Embodiment 3. a preparation method of an immunosensor based on titanium dioxide doped graphene loaded sea cucumber-like gold-palladium core-shell nanoparticles, comprising the following operation steps:
(1) polishing a glassy carbon electrode with the diameter of 4.0 mm into a mirror surface by using alumina polishing powder with the diameter of 1.0 mu m, 0.3 mu m and 0.05 mu m in sequence, and ultrasonically cleaning the mirror surface in absolute ethyl alcohol;
(2) taking 6.0 muL and 3.0 mg/mL titanium dioxide doped graphene loaded sea cucumber-like gold-palladium core-shell nanoparticle dispersion liquid to be dripped on the surface of the electrode, drying at room temperature, washing the surface of the electrode with ultrapure water, and drying;
(3) continuously dripping the human epididymis protein 4 antibody of 6.0 muL and 10.0 mug/mL on the surface of the electrode, standing in a refrigerator at 4 ℃ and drying;
(4) continuously dropwise adding a bovine serum albumin solution of 3.0 muL and 1.0 wt% to the surface of the electrode to seal the non-specific active sites on the surface of the electrode, washing the surface of the electrode with a phosphate buffer solution with pH = 7.0, standing in a refrigerator at 4 ℃ and airing;
(5) dripping a series of human epididymis protein 4 antigen solutions with different concentrations, namely 6.0 muL and 50 fM ~ 80 nM, on the surface of an electrode, standing in a refrigerator with the temperature of 4 ℃ for 35 min, washing the surface of the electrode with phosphate buffer solution with the pH = 7.0, and drying in the refrigerator with the temperature of 4 ℃ to obtain the working electrode of the amperometric immunosensor for detecting the human epididymis protein 4 antigen.
Embodiment 4. the method for preparing the immunosensor based on the titanium dioxide doped graphene loaded with the holothurian-shaped pd-au core-shell nanoparticles according to claim 1, wherein the preparation of the titanium dioxide doped graphene comprises the following steps:
(1) preparation of graphene oxide
Placing 4.0 g of graphite and 1.0 g of sodium nitrate in a 2000 mL beaker, slowly adding 192 mL of concentrated sulfuric acid under magnetic stirring in an ice-water bath environment, then slowly adding 24.0 g of potassium permanganate and vigorously stirring, reacting for 1h in the ice-water bath, then transferring the mixture into a 35 ℃ oil bath kettle for reacting for 2 h, heating to 55 ℃, reacting for 1.5 h, then dropwise adding 160 mL of deionized water into the mixed solution and continuously stirring by using a glass rod, then adding 400 mL of deionized water, finally adding hydrogen peroxide (20.0 mL, 30%) into the mixed solution, reacting for 10min, standing overnight, taking out the lower layer for dialysis for one week, dispersing by ultrasonic waves (140W, 1 h), centrifuging (6000 rpm, 10 min), collecting the supernatant and freeze-drying for 24 h to obtain graphene oxide;
(2) preparation of aminated graphene Dispersion
Adding 100.0 mg of graphene oxide into 40.0 mL of ethylene glycol under ultrasonic treatment, performing ultrasonic treatment for 1h, adding 1.0mL of concentrated ammonia water into the solution to obtain a dark brown solution, transferring the mixed solution into a high-pressure reaction kettle, performing solvothermal reaction for 10 h at 180 ℃, filtering precipitates after the reaction, repeatedly washing the precipitates to be neutral by using distilled water, performing freeze drying for 24 h to obtain a flaky aminated graphene solid, dispersing the flaky aminated graphene solid into 5.0 mL of deionized water again to obtain an aminated graphene dispersion liquid, and storing the aminated graphene dispersion liquid at 4 ℃ for further use;
(3) preparation of titanium dioxide-doped graphene dispersion
Putting 10 mL of deionized water, 5mL of ethanol and 5 mg of sodium dodecyl sulfate into a 50 mL beaker, performing ultrasonic treatment until the deionized water, the ethanol and the sodium dodecyl sulfate are uniformly mixed, then dispersing 15 mg of prepared aminated graphene in a mixed solution, performing ultrasonic treatment for 15 min, then adding 0.05 ~ 0.5.5 mL of isopropyl titanate into a suspension, performing continuous ultrasonic treatment for 1h to ensure that the prepared aminated graphene is stably and uniformly dispersed, transferring the obtained suspension into a 20 mL autoclave with a polytetrafluoroethylene lining, reacting for 12 h at 130 ℃, centrifuging for 10min at 6000 r/min, thoroughly washing by using a mixture (volume ratio of 1: 1) of deionized water and ethanol, finally washing by using deionized water, dispersing in deionized water, and storing at 4 ℃ for later use.
Embodiment 5. the method for preparing the immunosensor based on the titanium dioxide doped graphene loaded with the holothurian-shaped pd-au core-shell nanoparticles according to claim 1, wherein the preparation of the titanium dioxide doped graphene comprises the following steps:
(1) preparation of graphene oxide
Placing 4.0 g of graphite and 1.0 g of sodium nitrate in a 2000 mL beaker, slowly adding 192 mL of concentrated sulfuric acid under magnetic stirring in an ice-water bath environment, then slowly adding 24.0 g of potassium permanganate and vigorously stirring, reacting for 1h in the ice-water bath, then transferring the mixture into a 35 ℃ oil bath kettle for reacting for 2 h, heating to 55 ℃, reacting for 1.5 h, then dropwise adding 160 mL of deionized water into the mixed solution and continuously stirring by using a glass rod, then adding 400 mL of deionized water, finally adding hydrogen peroxide (20.0 mL, 30%) into the mixed solution, reacting for 10min, standing overnight, taking out the lower layer for dialysis for one week, dispersing by ultrasonic waves (140W, 1 h), centrifuging (6000 rpm, 10 min), collecting the supernatant and freeze-drying for 24 h to obtain graphene oxide;
(2) preparation of aminated graphene Dispersion
Adding 100.0 mg of graphene oxide into 40.0 mL of ethylene glycol under ultrasonic treatment, performing ultrasonic treatment for 1h, adding 1.0mL of concentrated ammonia water into the solution to obtain a dark brown solution, transferring the mixed solution into a high-pressure reaction kettle, performing solvothermal reaction for 10 h at 180 ℃, filtering precipitates after the reaction, repeatedly washing the precipitates to be neutral by using distilled water, performing freeze drying for 24 h to obtain a flaky aminated graphene solid, dispersing the flaky aminated graphene solid into 5.0 mL of deionized water again to obtain an aminated graphene dispersion liquid, and storing the aminated graphene dispersion liquid at 4 ℃ for further use;
(3) preparation of titanium dioxide-doped graphene dispersion
Putting 10 mL of deionized water, 5mL of ethanol and 5 mg of sodium dodecyl sulfate into a 50 mL beaker, performing ultrasonic treatment until the deionized water, the ethanol and the sodium dodecyl sulfate are uniformly mixed, then dispersing 25 mg of prepared aminated graphene in a mixed solution, performing ultrasonic treatment for 15 min, then adding 0.05 ~ 0.5.5 mL of isopropyl titanate into a suspension, performing continuous ultrasonic treatment for 1h to ensure that the prepared aminated graphene is stably and uniformly dispersed, transferring the obtained suspension into a 20 mL autoclave with a polytetrafluoroethylene lining, reacting for 12 h at 130 ℃, centrifuging for 10min at 6000 r/min, thoroughly washing by using a mixture (volume ratio of 1: 1) of deionized water and ethanol, finally washing by using deionized water, dispersing in deionized water, and storing at 4 ℃ for later use.
Embodiment 6. the method for preparing the immunosensor based on the titanium dioxide doped graphene loaded with the holothurian-shaped pd-au core-shell nanoparticles according to claim 1, wherein the preparation of the titanium dioxide doped graphene comprises the following steps:
(1) preparation of graphene oxide
Placing 4.0 g of graphite and 1.0 g of sodium nitrate in a 2000 mL beaker, slowly adding 192 mL of concentrated sulfuric acid under magnetic stirring in an ice-water bath environment, then slowly adding 24.0 g of potassium permanganate and vigorously stirring, reacting for 1h in the ice-water bath, then transferring the mixture into a 35 ℃ oil bath kettle for reacting for 2 h, heating to 55 ℃, reacting for 1.5 h, then dropwise adding 160 mL of deionized water into the mixed solution and continuously stirring by using a glass rod, then adding 400 mL of deionized water, finally adding hydrogen peroxide (20.0 mL, 30%) into the mixed solution, reacting for 10min, standing overnight, taking out the lower layer for dialysis for one week, dispersing by ultrasonic waves (140W, 1 h), centrifuging (6000 rpm, 10 min), collecting the supernatant and freeze-drying for 24 h to obtain graphene oxide;
(2) preparation of aminated graphene Dispersion
Adding 100.0 mg of graphene oxide into 40.0 mL of ethylene glycol under ultrasonic treatment, performing ultrasonic treatment for 1h, adding 1.0mL of concentrated ammonia water into the solution to obtain a dark brown solution, transferring the mixed solution into a high-pressure reaction kettle, performing solvothermal reaction for 10 h at 180 ℃, filtering precipitates after the reaction, repeatedly washing the precipitates to be neutral by using distilled water, performing freeze drying for 24 h to obtain a flaky aminated graphene solid, dispersing the flaky aminated graphene solid into 5.0 mL of deionized water again to obtain an aminated graphene dispersion liquid, and storing the aminated graphene dispersion liquid at 4 ℃ for further use;
(3) preparation of titanium dioxide-doped graphene dispersion
Putting 10 mL of deionized water, 5mL of ethanol and 5 mg of sodium dodecyl sulfate into a 50 mL beaker, performing ultrasonic treatment until the deionized water, the ethanol and the sodium dodecyl sulfate are uniformly mixed, dispersing 35 mg of prepared aminated graphene in a mixed solution, performing ultrasonic treatment for 15 min, then adding 0.05 ~ 0.5.5 mL of isopropyl titanate into a suspension, performing continuous ultrasonic treatment for 1h to ensure that the prepared aminated graphene is stably and uniformly dispersed, transferring the obtained suspension into a 20 mL autoclave with a polytetrafluoroethylene lining, reacting for 12 h at 130 ℃, centrifuging for 10min at 6000 r/min, thoroughly washing by using a mixture (volume ratio of 1: 1) of deionized water and ethanol, finally washing by using deionized water, dispersing in the deionized water, and storing at 4 ℃ for later use.
Embodiment 7. the method for preparing the immunosensor based on the titanium dioxide doped graphene loaded with the holothurian-shaped pd-au core-shell nanoparticles according to claim 1, wherein the preparation of the dispersion of the holothurian-shaped pd-au core-shell nanoparticles comprises the following steps:
(1) preparation of gold nanorod Dispersion
Adding chloroauric acid solution (250. mu.L, 10 mM) and cetyltrimethylammonium bromide solution (7.5 mL, 100 mM) into a 20 mL flask, placing in a 30 ℃ oil bath, magnetically stirring for 10min, then rapidly adding freshly prepared ice-cold sodium borohydride solution (600. mu.L, 10 mM) into the solution with stirring, stopping stirring after 2 min, obtaining a brownish yellow solution and storing at 30 ℃ for 3 h for later growth of gold nanorods, in the growth solution, adding cetyltrimethylammonium bromide solution (40.0 mL, 100 mM), chloroauric acid solution (1.7 mL, 10 mM) and silver nitrate solution (250. mu.L, 10 mM) to mix and stir uniformly, slowly adding ascorbic acid solution (270. mu.L, 100 mM) into the solution, and finally, adding 400. mu.L gold seed crystal to the growth solution, standing in a 30 ℃ water bath kettle, reacting for 15 h, centrifuging, washing with ultrapure water, re-dispersing the obtained precipitate in 20 mL of ultrapure water to obtain gold nanorod dispersion liquid, and storing in a refrigerator at 4 ℃;
(2) preparing a sea cucumber-shaped gold-palladium core-shell nanoparticle dispersion liquid
The chlorohexadecylpyridine solution was added to a 50 mL flask, and a sodium tetrachloropalladate solution (100. mu.L, 10 mM) and 1.5mL of gold nanorods were sequentially added to the flask under an oil bath at 65 ℃, and then, a freshly prepared ascorbic acid solution (200. mu.L, 100 mM) was injected, magnetically stirred for 2 min and stopped, the resulting solution was reacted for 30 min, centrifuged (8000 rpm, 5 min) to collect a black precipitate, and redispersed in water for further use.
Embodiment 8. the method for preparing the immunosensor based on the titanium dioxide doped graphene loaded with the holothurian-shaped pd-au core-shell nanoparticles according to claim 1, wherein the preparation of the dispersion of the holothurian-shaped pd-au core-shell nanoparticles comprises the following steps:
(1) preparation of gold nanorod Dispersion
Adding chloroauric acid solution (250. mu.L, 10 mM) and cetyltrimethylammonium bromide solution (7.5 mL, 100 mM) into a 20 mL flask, placing in a 30 ℃ oil bath, magnetically stirring for 10min, then rapidly adding freshly prepared ice-cold sodium borohydride solution (600. mu.L, 10 mM) into the solution with stirring, stopping stirring after 2 min, obtaining a brownish yellow solution and storing at 30 ℃ for 3 h for later growth of gold nanorods, in the growth solution, adding cetyltrimethylammonium bromide solution (40.0 mL, 100 mM), chloroauric acid solution (1.7 mL, 10 mM) and silver nitrate solution (250. mu.L, 10 mM) to mix and stir uniformly, slowly adding ascorbic acid solution (270. mu.L, 100 mM) into the solution, and finally, adding 400. mu.L gold seed crystal to the growth solution, standing in a 30 ℃ water bath kettle, reacting for 15 h, centrifuging, washing with ultrapure water, re-dispersing the obtained precipitate in 20 mL of ultrapure water to obtain gold nanorod dispersion liquid, and storing in a refrigerator at 4 ℃;
(2) preparing a sea cucumber-shaped gold-palladium core-shell nanoparticle dispersion liquid
The chlorohexadecylpyridine solution was added to a 50 mL flask, and a sodium tetrachloropalladate solution (100. mu.L, 10 mM) and 2.5 mL of gold nanorods were sequentially added to the flask under an oil bath at 65 ℃, and then, a freshly prepared ascorbic acid solution (200. mu.L, 100 mM) was injected, magnetically stirred for 2 min and stopped, the resulting solution was reacted for 30 min, centrifuged (8000 rpm, 5 min) to collect a black precipitate, and redispersed in water for further use.
Embodiment 9. the method for preparing the immunosensor based on the titanium dioxide doped graphene loaded with the holothurian-shaped pd-au core-shell nanoparticles according to claim 1, wherein the preparation of the dispersion of the holothurian-shaped pd-au core-shell nanoparticles comprises the following steps:
(1) preparation of gold nanorod Dispersion
Adding chloroauric acid solution (250. mu.L, 10 mM) and cetyltrimethylammonium bromide solution (7.5 mL, 100 mM) into a 20 mL flask, placing in a 30 ℃ oil bath, magnetically stirring for 10min, then rapidly adding freshly prepared ice-cold sodium borohydride solution (600. mu.L, 10 mM) into the solution with stirring, stopping stirring after 2 min, obtaining a brownish yellow solution and storing at 30 ℃ for 3 h for later growth of gold nanorods, in the growth solution, adding cetyltrimethylammonium bromide solution (40.0 mL, 100 mM), chloroauric acid solution (1.7 mL, 10 mM) and silver nitrate solution (250. mu.L, 10 mM) to mix and stir uniformly, slowly adding ascorbic acid solution (270. mu.L, 100 mM) into the solution, and finally, adding 400. mu.L gold seed crystal to the growth solution, standing in a 30 ℃ water bath kettle, reacting for 15 h, centrifuging, washing with ultrapure water, re-dispersing the obtained precipitate in 20 mL of ultrapure water to obtain gold nanorod dispersion liquid, and storing in a refrigerator at 4 ℃;
(2) preparing a sea cucumber-shaped gold-palladium core-shell nanoparticle dispersion liquid
The chlorohexadecylpyridine solution was added to a 50 mL flask, and a sodium tetrachloropalladate solution (100. mu.L, 10 mM) and 3.5 mL of gold nanorods were sequentially added to the flask under an oil bath at 65 ℃, and then, a freshly prepared ascorbic acid solution (200. mu.L, 100 mM) was injected, magnetically stirred for 2 min and stopped, the resulting solution was reacted for 30 min, centrifuged (8000 rpm, 5 min) to collect a black precipitate, and redispersed in water for further use.
Example 10. the preparation method of the immunosensor according to claim 1, for detecting human epididymis protein 4 antigen, comprising the following steps:
(1) testing in a three-electrode system by using an electrochemical workstation, taking a saturated calomel electrode as a reference electrode, taking a platinum wire electrode as an auxiliary electrode, taking the prepared sensor as a working electrode, and testing in 10 mL of phosphate buffer solution with the concentration of 50 mmol/L, pH = 6.6;
(2) detecting the analyte by a time-current method, wherein the input voltage is-0.4V, the sampling interval is 0.1 s, and the running time is 400 s;
(3) when the background current tends to be stable, 10 muL and 5 mol/L hydrogen peroxide solution is injected into 10 mL and 50 mmol/L, pH = 6.6 phosphate buffer solution every 50 s, and the current change is recorded;
(4) and drawing a working curve according to the linear relation between the obtained current intensity and the concentration of the human epididymis protein 4 antigen, wherein the linear range is 90 fmol/mL ~ 40 nmol/mL, and the detection limit is 30.0 fmol/mL.
Example 11 the constructed immunosensor is used for detecting human epididymis protein 4 antigen, and the detection steps are as follows:
(1) testing in a three-electrode system by using an electrochemical workstation, taking a saturated calomel electrode as a reference electrode, taking a platinum wire electrode as an auxiliary electrode, taking the prepared sensor as a working electrode, and testing in 10 mL of phosphate buffer solution with the concentration of 50 mmol/L, pH = 7.4;
(2) detecting the analyte by a time-current method, wherein the input voltage is-0.4V, the sampling interval is 0.1 s, and the running time is 400 s;
(3) when the background current tends to be stable, 10 muL and 5 mol/L hydrogen peroxide solution is injected into 10 mL and 50 mmol/L, pH = 7.4 phosphate buffer solution every 50 s, and the current change is recorded;
(4) according to the linear relation between the obtained current intensity and the concentration of the human epididymis protein 4 antigen, a working curve is drawn, the linear range is 50 fmol/mL ~ 80 nmol/mL, and the detection limit is 16.7 fmol/mL.
Example 12 the constructed immunosensor is used for detecting human epididymis protein 4 antigen, and the detection steps are as follows:
(1) testing in a three-electrode system by using an electrochemical workstation, taking a saturated calomel electrode as a reference electrode, taking a platinum wire electrode as an auxiliary electrode, taking the prepared sensor as a working electrode, and testing in 10 mL of phosphate buffer solution with the concentration of 50 mmol/L, pH = 7.7;
(2) detecting the analyte by a time-current method, wherein the input voltage is-0.4V, the sampling interval is 0.1 s, and the running time is 400 s;
(3) when the background current tends to be stable, 10 muL and 5 mol/L hydrogen peroxide solution is injected into 10 mL and 50 mmol/L, pH = 7.7 phosphate buffer solution every 50 s, and the current change is recorded;
(4) and drawing a working curve according to the linear relation between the obtained current intensity and the concentration of the human epididymis protein 4 antigen, wherein the linear range is 60 fmol/mL ~ 60 nmol/mL, and the detection limit is 20.0 fmol/mL.
Claims (2)
1. A preparation method of an immunosensor based on titanium dioxide doped graphene loaded sea cucumber-like gold-palladium core-shell nanoparticles is characterized by comprising the following steps:
(1) polishing a glassy carbon electrode with the diameter of 3.0 ~ 5.0.0 mm into a mirror surface by using aluminum oxide polishing powder with the diameter of 1.0 mu m, 0.3 mu m and 0.05 mu m in sequence, and ultrasonically cleaning the mirror surface in absolute ethyl alcohol;
(2) dropwise coating the titanium dioxide doped graphene loaded sea cucumber-like gold-palladium core-shell nanoparticle dispersion liquid of 6.0 muL and 0.5 ~ 4.0.0 mg/mL on the surface of the electrode, drying at room temperature, washing the surface of the electrode with ultrapure water, and drying;
(3) continuously dripping the human epididymis protein 4 antibody of 6.0 muL and 5.0 ~ 15.0.0 mug/mL on the surface of the electrode, standing in a refrigerator at 4 ℃ and drying;
(4) continuously dropwise adding bovine serum albumin solution of 3.0 muL and 1.0 ~ 2.0.0 wt% to the surface of the electrode to seal the nonspecific active sites on the surface of the electrode, washing the surface of the electrode with phosphate buffer solution with pH = 7.0, standing in a refrigerator at 4 ℃ and airing;
(5) dripping a series of human epididymis protein 4 antigen solutions with different concentrations, namely 6.0 muL and 50 fM ~ 80 nM, on the surface of an electrode, standing in a refrigerator at 4 ℃ for 30 ~ 40 min, washing the surface of the electrode with a phosphate buffer solution with pH = 7.0, and airing in the refrigerator at 4 ℃ to prepare a working electrode of the amperometric immunosensor for detecting the human epididymis protein 4 antigen;
the preparation method of the titanium dioxide doped graphene comprises the following steps:
Placing 4.0 g of graphite and 1.0 g of sodium nitrate in a 2000 mL beaker, slowly adding 192 mL of concentrated sulfuric acid under magnetic stirring in an ice-water bath environment, then slowly adding 24.0 g of potassium permanganate and vigorously stirring, reacting for 1h in the ice-water bath, then transferring the mixture into a 35 ℃ oil bath kettle for reacting for 2 h, heating to 55 ℃, reacting for 1.5 h, then dropwise adding 160 mL of deionized water into the mixed solution, continuously stirring with a glass rod, then adding 400 mL of deionized water, finally adding 20.0 mL and 30% of hydrogen peroxide into the mixed solution, reacting for 10min, standing overnight, taking the lower layer for dialysis for one week, dispersing for 1h by 140W ultrasonic waves, centrifuging at 6000 rpm for 10min, collecting supernatant, and freeze-drying for 24 h to obtain graphene oxide;
Adding 100.0 mg of graphene oxide into 40.0 mL of ethylene glycol under ultrasonic treatment, performing ultrasonic treatment for 1h, adding 1.0mL of concentrated ammonia water into the solution to obtain a dark brown solution, transferring the mixed solution into a high-pressure reaction kettle, performing solvothermal reaction for 10 h at 180 ℃, filtering precipitates after the reaction, repeatedly washing the precipitates to be neutral by using distilled water, performing freeze drying for 24 h to obtain a flaky aminated graphene solid, dispersing the flaky aminated graphene solid into 5.0 mL of deionized water again to obtain an aminated graphene dispersion liquid, and storing the aminated graphene dispersion liquid at 4 ℃ for further use;
Putting 10 mL of deionized water, 5mL of ethanol and 5 mg of sodium dodecyl sulfate into a 50 mL beaker, performing ultrasonic treatment until the deionized water, the ethanol and the sodium dodecyl sulfate are uniformly mixed, then dispersing 10 ~ 40 mg of prepared aminated graphene in a mixed solution, performing ultrasonic treatment for 15 min, then adding 0.05 ~ 0.5.5 mL of isopropyl titanate into a suspension, performing continuous ultrasonic treatment for 1h to ensure that the prepared aminated graphene is stably and uniformly dispersed, transferring the obtained suspension into a 20 mL autoclave with a polytetrafluoroethylene lining, reacting for 12 h at 130 ℃, centrifuging for 10min at 6000 rpm, thoroughly washing by using a mixture of deionized water and ethanol in a volume ratio of 1:1, finally washing by using deionized water, dispersing in the deionized water, and storing at 4 ℃ for later use;
the preparation method of the sea cucumber-shaped gold-palladium core-shell nanoparticle dispersion liquid comprises the following steps:
Adding 250 mu L of 10mM chloroauric acid solution and 7.5 mL of 100 mM hexadecyl trimethyl ammonium bromide solution into a 20 mL flask, placing the flask in a 30 ℃ oil bath kettle, magnetically stirring for 10min, then quickly adding 600 mu L of freshly prepared ice-cold 10mM sodium borohydride solution into the solution under stirring, stopping stirring after 2 min, storing the obtained brown yellow solution at 30 ℃ for 3 h for later-stage gold nanorod growth, adding 40.0 mL of 100 mM hexadecyl trimethyl ammonium bromide solution, 1.7 mL of 10mM chloroauric acid solution and 250 mu L of 10mM silver nitrate solution into the growth solution, mixing and stirring uniformly, slowly adding 270 mu L of 100 mM ascorbic acid solution into the growth solution, finally adding 200 mu L of ~ 500 mu L of gold seed crystal solution into the growth solution, standing the growth solution in a 30 ℃ water bath kettle for reaction for 15 h, centrifuging, washing, re-dispersing the obtained precipitate in 20 mL of ultrapure water to obtain a nanorod dispersion liquid, and storing the gold seed crystal dispersion liquid in a 4 ℃ ice bath kettle;
The chlorohexadecylpyridine solution was added to a 50 mL flask, 100. mu.L of a 10mM sodium tetrachloropalladate solution and 1.0mL of ~ 5.0.0 mL of gold nanorods were sequentially added to the flask under a 65 ℃ oil bath, then 200. mu.L of a freshly prepared 100 mM ascorbic acid solution was injected, magnetic stirring was stopped after 2 min, the resulting solution was reacted for 30 min, centrifuged at 8000 rpm for 5min to collect a black precipitate, and redispersed in water for further use.
2. The immunosensor prepared by the preparation method according to claim 1 and based on titanium dioxide doped graphene loaded with holothurian-shaped gold-palladium core-shell nanoparticles is used for detection of human epididymis protein 4 antigen, and the detection steps are as follows:
(1) an electrochemical workstation is used for testing in a three-electrode system, a saturated calomel electrode is used as a reference electrode, a platinum wire electrode is used as an auxiliary electrode, the prepared sensor is used as a working electrode, and the test is carried out in 10 mL of phosphate buffer solution with the concentration of 50 mmol/L, pH 5.84.84 5.84 ~ 8.04.04;
(2) detecting the analyte by a time-current method, wherein the input voltage is-0.4V, the sampling interval is 0.1 s, and the running time is 400 s;
(3) and when the background current tends to be stable, injecting 10 mu L and 5 mol/L hydrogen peroxide solution into 10 mL and 50 mmol/L, pH = 7.4 phosphate buffer solution every 50 s, and recording the current change.
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