Disclosure of Invention
Aiming at the problems, the invention provides a preparation method and application of an MIL-101 biosensor for oral cancer in saliva, wherein ZrO is mixed in MIL-101 2 The particles are coated, and then oral cancer marker recognition protein named anti-Cyfra-21-1 is covalently bondedBond Assembly to ZrO 2 In @ MOFs, the oxidation current of the sensor is changed due to the anti-Cyfra-21-1 antibody generation reaction of the electrode sensor modified by biomarker proteins Cyfra-21-1 and MIL-101 of oral cancer, and whether oral cancer cells exist is judged by detecting whether the current is changed. On the basis, the surface morphology and particle properties of the sensor are mainly researched by using a Scanning Electron Microscope (SEM), X-ray powder diffraction (XRD) and conversion infrared spectrum characterization (FI-IR), and the identification mechanism of the sensor is clarified by analyzing in combination with the reproducibility and stability of the sensor. Thereby obtaining the MIL-101 immune electrode biosensor which has excellent biocompatibility, high stability and excellent oral cancer markers in saliva. Provides a new strategy for designing and obtaining a non-invasive human body type and painless body type external oral cancer detection biosensor, and provides important theoretical significance and application value for in vitro diagnosis.
In order to achieve the purpose, the invention adopts the following technical scheme:
a preparation method of an MIL-101 biosensor for oral cancer in saliva comprises the following steps:
step 1, MIL-101 preparation: dissolving 2.4g of chromium nitrate nonahydrate, 1.5g of 1, 4-terephthalic acid, 0.67g of hexadecyl trimethyl ammonium bromide and 0.25mL of hydrofluoric acid in 30mLN, N-dimethylformamide, stirring at room temperature for reaction, and obtaining MIL-101 after the reaction is finished;
step 2, activation of MIL-101: adding N, N-dimethylformamide into the MIL-101 obtained in the step 1, stirring at a constant temperature in a water bath kettle, centrifuging the mixed solution, washing the obtained solid with hot ethanol, an ammonium fluoride solution and deionized water in sequence, and finally drying the solid to obtain activated MIL-101;
step 3, performing APTES modification on the activated MIL-101: ultrasonically oscillating activated MIL-101 and APTES with absolute ethyl alcohol, heating and stirring in a water bath kettle, centrifuging a mixed solution, washing an obtained solid with the absolute ethyl alcohol, and drying in vacuum to obtain modified MIL-101;
step 4, preparing nano zirconia: 0.5g of sodium dodecyl sulfate is dissolved in 50mL of deionized water,heating in water bath until the precursor is completely dissolved, adding ammonium bicarbonate solution, stirring, adding zirconium salt solution, stirring, performing hydrothermal reaction, cleaning the obtained precursor with hot ethanol, and naturally air drying to obtain mesoporous ZrO 2 The precursor of (2); then heating to 400 ℃ at the heating rate of 10 ℃/min, and calcining for 4h to obtain nano zirconia;
step 5, preparing the MIL-101 immune electrode biosensor: sanding the surface of the electrode with 1000-mesh and 2000-mesh sandpaper, and then using Al 2 O 3 Polishing the electrode surface with polishing powder until the electrode surface becomes mirror surface, and sequentially polishing with HNO 3 The solution, absolute ethyl alcohol and secondary distilled water are used for washing the surface of the electrode for 5-8 min respectively, and modified 0.2g of MIL-101 and 0.4g of nano-zirconia are taken to be ultrasonically dispersed in 10mL of deionized water to form ZrO 2 @ MOFs solution, 2. Mu.L anti-Cyfra-21-1 was added dropwise to 0.5mL ZrO 2 And @ MOFs solution, fully stirring to form suspension, dripping the suspension on the surface of an electrode, dripping Nafion solution with the volume concentration of 0.5% on the surface of the electrode after the electrode is dried, and naturally drying at room temperature to obtain the MIL-101 immune electrode sensor.
Further, the stirring time in the step 1 is 30-40 min; the reaction temperature is 220-240 ℃, and the reaction time is 8-10 h.
Further, the volume of the N, N-dimethylformamide in the step 2 is 15-20 mL.
Further, the temperature of the water bath kettle in the step 2 is 40-45 ℃; the stirring time is 50-60 min.
Further, the temperature of the hot ethanol in the step 2 is 70-80 ℃, the concentration of the ammonium fluoride solution is 1-2 mol/L, and the temperature of the deionized water is 80-100 ℃; the drying temperature is 130-150 ℃, and the drying time is 8-10 h.
Further, the heating and stirring temperature in the step 3 is 80-90 ℃, and the heating and stirring time is 10-12 hours; the washing times are 3-5 times, the vacuum drying temperature is 100-120 ℃, and the vacuum drying time is 8-10 h.
Further, the temperature of the hydrothermal reaction in the step 4 is 120-140 ℃, the reaction time is 7 hours, and the temperature of the hot ethanol in the step 4 is 40 ℃.
Further, the concentration of the sodium bicarbonate solution in the step 4 is 0.3-0.5 mol/L, and the concentration of the zirconium salt solution is 0.3mol/L.
Further, HNO in the step 5 3 The concentration of the solution was 6mol/L.
Use of MIL-101 biosensor against oral cancer in saliva, oral cancer was detected based on Cyfra-21-1 protein in saliva.
Compared with the prior art, the invention has the following advantages:
1. several techniques have been developed for the diagnosis and detection of oral cancer, including: the laser microscopic capture cutting technology, the visual auxiliary equipment, the living body detection technology and the like are invasive, can cause harm to human bodies, and consume a large amount of manpower, financial resources and time. However, the invention provides a new strategy for designing and obtaining the MOFs immune electrode biosensor based on the oral cancer marker in saliva, realizing the non-invasive human body type and painless type in-vitro oral cancer detection biosensor, and providing important theoretical significance and application value for in-vitro diagnosis.
2. Aiming at the in vitro detection of oral cancer, the invention provides a first assembly mode to be adopted based on novel Metal Organic Frameworks (MOFs) and ZrO is adopted 2 The MOFs nanoparticles are used as a substrate, the surface of the MOFs nanoparticles is firstly modified, and then the MOFs nanoparticles are combined with protein to carry out self-assembly, so that an electrode sensor with excellent biocompatibility, high stability and excellent performance is expected to be obtained.
Detailed Description
Example 1
A preparation method of an MIL-101 biosensor for oral cancer in saliva comprises the following steps:
step 1, preparation of MIL-101: dissolving 2.4g of chromium nitrate nonahydrate, 1.5g of 1, 4-terephthalic acid, 0.67g of hexadecyl trimethyl ammonium bromide and 0.25mL of hydrofluoric acid in 30mLN and N-dimethylformamide, stirring for 30min at room temperature, adding the mixed solution into a 40mL stainless steel high-pressure reaction kettle, reacting for 8h at 220 ℃, and obtaining MIL-101 after the reaction is finished;
step 2, activation of MIL-101: moving the MIL-101 obtained in the step 1 to a beaker, adding 15mL of N, N-dimethylformamide into the beaker, stirring the mixture for 1h at a constant temperature in a water bath kettle at 40 ℃, centrifuging the mixed solution, washing the obtained solid with hot ethanol at 78 ℃, 1mol/L of ammonium fluoride solution and deionized water at 100 ℃ in sequence, and finally placing the solid in a vacuum drying oven at 150 ℃ for drying for 8h to obtain activated MIL-101;
step 3, performing APTES modification on the activated MIL-101: ultrasonically oscillating 0.2g of activated MIL-101, 0.175mL of APTES and 30mL of absolute ethyl alcohol for 5min, then heating and stirring in a water bath kettle at 85 ℃ for 12h, centrifuging the mixed solution, then washing the obtained solid with absolute ethyl alcohol for three times, and drying in a vacuum drying oven at 100 ℃ for 8h to obtain modified MIL-101;
step 4, preparing nano zirconia: 0.5g of sodium dodecyl sulfate is dissolved in 50mL of deionized water,heating in water bath until the materials are completely dissolved, adding 0.4mol/L ammonium bicarbonate solution, stirring uniformly, adding 0.3mol/L zirconium salt solution, stirring fully, placing in a high-pressure kettle at 120 ℃ for hydrothermal reaction for 7 hours, after the reaction is finished, cleaning the obtained precursor with hot ethanol, and naturally drying to obtain the mesoporous ZrO 2 The precursor of (2); then heating to 400 ℃ at the heating rate of 10 ℃/min, and calcining for 4h to obtain nano zirconia;
step 5, preparing an MIL-101 immune electrode biosensor: sanding the surface of the electrode with 1000-mesh and 2000-mesh sandpaper, and then using Al 2 O 3 Polishing the electrode surface by polishing powder until the electrode surface becomes a mirror surface, and then using 6mol/L HNO 3 Washing the electrode surface with the solution, absolute ethyl alcohol and secondary distilled water for 5min respectively, and ultrasonically dispersing the modified 0.2g of MIL-101 and 0.4g of nano-zirconia in 10mL of deionized water to form ZrO 2 @ MOFs solution, 2. Mu.L anti-Cyfra-21-1 was added dropwise to 0.5mL ZrO 2 And @ MOFs solution, fully stirring to form suspension, dripping the suspension on the surface of an electrode, dripping Nafion solution with the volume concentration of 0.5% on the surface of the electrode after the electrode is dried, and naturally drying at room temperature to obtain the MIL-101 biosensor.
The crystal structure of MIL-101 was determined by X-ray diffraction (XRD): the prepared MIL-101 powder sample was poured onto a slide, which was then placed on a sample holder and the assay was started. The scanning range is set to be 3 degrees to 50 degrees, the step size is set to be 0.02 degree, the scanning speed is set to be 8 degrees/min, then an X-ray diffractometer is started to carry out detection, the result is shown in figure 1, wherein figure 1a is an X-ray powder diffraction spectrum of MIL-101, and the X-ray powder diffraction spectrum is compared with a standard spectrum (shown in figure 1 b) of MIL-101, and the crystal characteristic diffraction peaks at 8.7 degrees and 17.78 degrees are basically consistent.
Scanning electron microscope analysis: dipping a fine rod slightly to obtain a certain amount of sample powder, adhering the sample powder to a conductive adhesive, adhering the conductive adhesive to a sample frame, spraying gold firstly because the sample has weaker conductivity, and then observing the microscopic morphology with different magnifications under a Scanning Electron Microscope (SEM), wherein the prepared MIL-101 crystal has higher crystallinity, regular appearance, uniform size and about 100nm of size as shown in figure 2.
Transform infrared spectrogram analysis: using KBr as background, mix MIL-101 and modified MIL-101 with KBr at a ratio of 1 -1 The absorption peak is due to-OH absorption by MIL-101; and 1668cm -1 、1555cm -1 And 1402cm -1 The absorption peak at (b) is a vibrational peak due to-COOH. FIG. 3b shows the infrared spectrum of APTES-MIL-101, which is 1030-1360 cm in comparison with the spectrum of MIL-101 -1 The change of the spectrum is due to C-NH in APTES 2 Resulting from a bond, and C-NH 2 Is beneficial to better combining APTES-MIL-101 with anti-Cyfra 21-1. Therefore, APTES-MIL-101 has the vibration peak of MIL-101 and the vibration peak of APTES, and the APTES modified MOFs material successfully prepared by the method can synergistically play the roles of the two substances.
Example 2
Weighing 8.0g NaCl, 0.2g KCl and 2.89g NaHPO 4 ·12H 2 O and 0.2gKH 2 PO 4 Dissolving in 1000mL of secondary distilled water, and continuously adjusting the pH value in the dissolving process to stabilize the pH value to about 7. After the preparation, the solution was placed in a refrigerator at-4 ℃ for use, and the PBS solution was used to dilute the recognition protein and serve as an electrolyte.
10mL of Cyfra-21-1 diluted with PBS was added to the PBS solution at room temperature, and the mixture was thoroughly stirred to prepare an electrolyte solution. The MIL-101 biosensor is used as a working electrode, a calomel electrode is used as a counter electrode, and a platinum electrode is used as an auxiliary electrode, so that a three-electrode system is formed.
As shown in fig. 4, which is a cyclic voltammogram of MIL-101, it can be seen from fig. 4 that the MIL-101 biosensor prepared according to the present invention has conductivity and good electrochemical properties.
As shown in FIG. 5, the MIL-101 biosensor has differential pulse voltammetry curves of pure PBS solution and PBS solution added with Cyfra-21-1 recognition protein. Wherein, the curve A is the curve in pure PBS solution, and the curve B is the curve of the recognition protein in the addition of Cyfra-21-1. From the comparison of the two curves, the electrode has obvious oxidation reduction peak in the electrolyte added with Cyfre-21-1 recognition protein. And in the electrolyte without adding the labeled protein, no obvious redox peak exists, which indicates that the MIL-101 biosensor can realize the detection of oral cancer in the PBS solution.
Example 3
A preparation method of an MIL-101 biosensor for oral cancer in saliva comprises the following steps:
step 1, preparation of MIL-101: dissolving 2.4g of chromium nitrate nonahydrate, 1.5g of 1, 4-terephthalic acid, 0.67g of hexadecyl trimethyl ammonium bromide and 0.25mL of hydrofluoric acid in 30mLN and N-dimethylformamide, stirring at room temperature for 35min, adding the mixed solution into a 40mL stainless steel high-pressure reaction kettle, reacting at 230 ℃ for 10h, and obtaining MIL-101 after the reaction is finished;
step 2, activation of MIL-101: transferring the MIL-101 obtained in the step 1 to a beaker, adding 18mL of N, N-dimethylformamide into the beaker, stirring the mixture at a constant temperature of 50mu n in a water bath kettle at 42 ℃, centrifuging the mixed solution, washing the obtained solid with hot ethanol at 70 ℃, 1.5mol/L of ammonium fluoride solution and deionized water at 80 ℃ in sequence, finally placing the solid in a vacuum drying box at 130 ℃, and drying the solid for 10 hours to obtain activated MIL-101;
step 3, performing APTES modification on the activated MIL-101: ultrasonically oscillating 0.2g of activated MIL-101, 0.175mL of APTES and 30mL of absolute ethyl alcohol for 5min, heating and stirring in a water bath kettle at 80 ℃ for 10h, centrifuging the mixed solution, washing the obtained solid with absolute ethyl alcohol for 4 times, and drying in a vacuum drying oven at 110 ℃ for 9h to obtain modified MIL-101;
step 4, preparing nano zirconia: dissolving 0.5g of sodium dodecyl sulfate in 50mL of deionized water, heating in a water bath until the sodium dodecyl sulfate is completely dissolved, adding 0.3mol/L ammonium bicarbonate solution, uniformly stirring, adding 0.3mol/L zirconium salt solution, fully stirring, placing in a high-pressure kettle at 130 ℃ for hydrothermal reaction for 7 hours, after the reaction is finished, cleaning the obtained precursor by using hot ethanol, and naturally air-drying to obtain the mesoporous ZrO 2 A precursor of (a); then heating to 400 ℃ at the heating rate of 10 ℃/min, and calcining for 4h to obtain the nano oxygenZirconium is treated;
step 5, preparing an MIL-101 immune electrode biosensor: sanding the surface of the electrode with 1000-mesh and 2000-mesh sandpaper, and then using Al 2 O 3 Polishing the electrode surface by polishing powder until the electrode surface becomes a mirror surface, and sequentially using 6mol/L HNO 3 Washing the electrode surface with the solution, absolute ethyl alcohol and secondary distilled water for 6min respectively, and ultrasonically dispersing the modified 0.2g of MIL-101 and 0.4g of nano-zirconia in 10mL of deionized water to form ZrO 2 @ MOFs solution, 2. Mu.L of anti-Cyfra-21-1 was added dropwise to 0.5mL of ZrO 2 And @ MOFs solution, fully stirring to form suspension, dripping the suspension on the surface of an electrode, dripping Nafion solution with the volume concentration of 0.5% on the surface of the electrode after the electrode is dried, and naturally drying at room temperature to obtain the MIL-101 biosensor.
Example 4
A preparation method of an MIL-101 biosensor for oral cancer in saliva comprises the following steps:
step 1, MIL-101 preparation: dissolving 2.4g of chromium nitrate nonahydrate, 1.5g of 1, 4-terephthalic acid, 0.67g of hexadecyl trimethyl ammonium bromide and 0.25mL of hydrofluoric acid in 30mLN and N-dimethylformamide, stirring for 40min at room temperature, adding the mixed solution into a 40mL stainless steel high-pressure reaction kettle, reacting for 9h at 240 ℃, and obtaining MIL-101 after the reaction is finished;
step 2, activation of MIL-101: moving the MIL-101 obtained in the step 1 to a beaker, adding 18mL of N, N-dimethylformamide into the beaker, stirring the mixture for 55min at a constant temperature in a water bath kettle at 45 ℃, centrifuging the mixed solution, washing the obtained solid with hot ethanol at 80 ℃, 2mol/L ammonium fluoride solution and deionized water at 90 ℃ in sequence, and finally placing the solid in a vacuum drying oven at 140 ℃ for drying for 9h to obtain activated MIL-101;
step 3, performing APTES modification on the activated MIL-101: ultrasonically oscillating 0.2g of activated MIL-101, 0.175mL of APTES and 30mL of absolute ethyl alcohol for 5min, heating and stirring in a 90 ℃ water bath kettle for 9h, centrifuging the mixed solution, washing the obtained solid with absolute ethyl alcohol for 5 times, and drying in a 120 ℃ vacuum drying oven for 10h to obtain modified MIL-101;
step 4, preparing nano zirconia: dissolving 0.5g of sodium dodecyl sulfate in 50mL of deionized water, heating in a water bath until the sodium dodecyl sulfate is completely dissolved, adding 0.5mol/L ammonium bicarbonate solution, uniformly stirring, adding 0.3mol/L zirconium salt solution, fully stirring, placing in a high-pressure kettle at 140 ℃ for hydrothermal reaction for 7 hours, after the reaction is finished, cleaning the obtained precursor by using hot ethanol, and naturally air-drying to obtain the mesoporous ZrO 2 A precursor of (a); then heating to 400 ℃ at the heating rate of 10 ℃/min, and calcining for 4h to obtain nano zirconia;
step 5, preparing an MIL-101 immune electrode biosensor: sanding the surface of the electrode with 1000-mesh and 2000-mesh sandpaper, and then using Al 2 O 3 Polishing the electrode surface by polishing powder until the electrode surface becomes a mirror surface, and then using 6mol/L HNO 3 The solution, absolute ethyl alcohol and secondary distilled water are used for washing the surface of the electrode for 8min respectively, and modified 0.2g of MIL-101 and 0.4g of nano-zirconia are taken to be ultrasonically dispersed in 10mL of deionized water to form ZrO 2 @ MOFs solution, 2. Mu.L anti-Cyfra-21-1 was added dropwise to 0.5mL ZrO 2 And @ MOFs solution, fully stirring to form suspension, dripping the suspension on the surface of an electrode, dripping Nafion solution with the volume concentration of 0.5% on the surface of the electrode after the electrode is dried, and naturally drying at room temperature to obtain the MIL-101 biosensor.