CN114487042A - Signal turnover type photoelectrochemical biosensor for detecting cancer marker and preparation method and application thereof - Google Patents
Signal turnover type photoelectrochemical biosensor for detecting cancer marker and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a preparation method of a signal reversal type photoelectrochemical biosensor for detecting a cancer marker, which comprises the steps of preparing an indium tin oxide/titanium dioxide electrode, preparing an indium tin oxide/titanium dioxide/single-chain auxiliary probe electrode, and preparing a conjugate Ab1‑Fe3O4@ Au-DNA preparation of conjugate Ab2AuNPs-DNA, construction of intermediate probes and preparation of sensors. The invention also provides a biosensor prepared by the method and application thereof. The biosensor for detecting the cancer marker is designed for photocurrent signal inversion based on the organic combination of a direct-contact photocurrent direction inversion strategy, a targeted induction three-dimensional dual-support DNA walker circulating signal amplification technology and the adsorption characteristic of a metal organic framework material, so that the biosensor can realize environment-friendly detection of the cancer marker and has the advantages of high sensitivity, low cost, high sensitivity, and the like,High selectivity, good reproducibility, high stability and the like.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a signal turnover type photoelectrochemical biosensor for detecting a cancer marker, and a preparation method and application thereof.
Background
Soluble CD146 (sCD 146) is closely related to the development of cancer, and has an important role in early diagnosis and treatment of cancer as a molecular marker. The advantages of the photoelectrochemistry biosensor for detecting the molecular marker are concerned, but most of the reported sensors are constructed based on a unidirectional signal response strategy, are not beneficial to eliminating the adverse effects of interferents in an actual sample on the detection anti-interference capability and sensitivity, may have false positive or false negative errors, and seriously limit the application and development of the sensors in the sCD146 analysis and detection.
The advantages of the signal inversion type photoelectrochemical biosensor having high selectivity and high sensitivity have recently been developed and popularized. The photocurrent direction reversal is used as an emerging signal response strategy, and the signal reversal mechanism and application research thereof still need to be further researched. As a core element of a photocurrent direction reversal system, a photoelectric active material directly affects the reversal efficiency, and two photoelectric active materials in the conventional reversal system usually require biological elements (such as antibodies, nucleic acids, and polypeptides) to be connected without direct contact, so that an electron transfer path is increased, and the photoelectric conversion efficiency is reduced. Meanwhile, the overturning systems constructed on the basis of cadmium sulfide, cadmium telluride, copper oxide and the like also limit the practical application of the overturning systems due to toxicity or poor light stability. Therefore, it is a significant research effort to develop a novel photocurrent direction reversal sensing platform with high performance.
Disclosure of Invention
In view of the above, the present invention provides a signal inversion type photoelectrochemical biosensor for detecting cancer markers, and a method for preparing the same and an application thereof, so as to solve the above problems.
Specifically, the invention provides a preparation method of a signal reversal type photoelectrochemical biosensor for detecting a cancer marker, which comprises the following steps:
preparing an indium tin oxide/titanium dioxide electrode:
carbonizing the metal organic framework material to obtain a titanium dioxide polyhedron; preparing a titanium dioxide polyhedron suspension by taking the titanium dioxide polyhedron as a solute; dripping the titanium dioxide polyhedral suspension onto an indium tin oxide electrode to prepare an indium tin oxide/titanium dioxide electrode;
preparing an indium tin oxide/titanium dioxide/single-stranded auxiliary probe electrode:
adsorbing a single-chain auxiliary DNA probe on the surface of the indium tin oxide/titanium dioxide electrode to seal an active site on the titanium dioxide polyhedron, and cleaning to obtain the indium tin oxide/titanium dioxide/single-chain auxiliary probe electrode;
preparation of conjugate Ab1-Fe3O4@Au-DNA：
By amination of Fe3O4Shaking the magnetic beads and the gold nanoparticles to ensure that the aminated Fe3O4The magnetic beads and the gold nanoparticles are combined to form Fe through covalent bonds3O4@ Au particles to obtain Fe3O4@ Au solution; in sequence towards the Fe3O4@ Au solution to which first target capture antibody (Ab) is added1) And a supporting DNA probe (Support DNA), and subjected to room-temperature shaking incubation and magnetic separation treatment, so that the Ab1Supporting DNA probe and the Fe3O4Binding of @ Au particles by covalent bond to produce Ab1-Fe3O4@ Au-DNA conjugate solution; wherein, the conjugate Ab1-Fe3O4@ Au-DNA can be expressed as: first target capture antibody Ab1Fe co-modified with supporting DNA probes3O4@ Au particle conjugates;
preparation of conjugate Ab2-AuNPs-DNA：
Adding a second target capture antibody (Ab) into a gold nanoparticle solution with the pH of 8-92) The reaction was performed, and a supporting DNA probe and a walking DNA probe (DNA walker) were added to form a mixed solution, and the mixed solution was reacted so that the Ab2The supporting DNA probe and the walking DNA probe are covalently bonded with the gold nanoparticle solutionGold nanoparticles are combined to obtain Ab2-AuNPs-DNA conjugate solution; wherein, the conjugate Ab2-AuNPs-DNA can be described as: second target Capture antibody Ab2A gold nanoparticle conjugate modified by the support probe and the walking probe;
constructing an intermediate probe:
different concentrations of cancer markers were first added to the Ab1-Fe3O4Incubation and magnetic separation in a solution of @ Au-DNA conjugate, adding the Ab2Carrying out incubation and magnetic separation treatment on the AuNPs-DNA conjugate solution, adding restriction endonuclease Nt.BsmAl for reaction and magnetic separation treatment, and obtaining supernatant, wherein the supernatant contains an intermediate probe for target induction of three-dimensional double-support DNA walker circulating signal amplification reaction;
preparing a sensor:
mixing and incubating the indium tin oxide/titanium dioxide/single-stranded auxiliary probe electrode and the intermediate probe in the supernatant to form a specific double strand with the single-stranded auxiliary DNA probe, releasing the single-stranded auxiliary DNA probe from the indium tin oxide/titanium dioxide/single-stranded auxiliary probe electrode, and exposing an active site on a titanium dioxide polyhedron in the indium tin oxide/titanium dioxide/single-stranded auxiliary probe electrode to obtain an indium tin oxide/titanium dioxide/single-stranded auxiliary probe/intermediate probe electrode with the active site; and adsorbing the carbon nitride quantum dots on the indium tin oxide/titanium dioxide/single-chain auxiliary probe/middle probe electrode to form an indium tin oxide/titanium dioxide/single-chain auxiliary probe/middle probe/carbon nitride quantum dot composite electrode, so that the titanium dioxide and the carbon nitride quantum dots are in direct contact to prepare the direct-contact signal turnover type biosensor.
Preferably, the metal organic framework material is MIL-125.
Based on the preparation method, the steps of preparing the indium tin oxide/titanium dioxide/single-chain auxiliary probe electrode comprise: annealing the single-stranded auxiliary DNA probe at 95 ℃ for 5 min, cooling to room temperature, dripping the single-stranded auxiliary DNA probe onto the surface of the indium tin oxide/titanium dioxide electrode for adsorption treatment, and cleaning to obtain the indium tin oxide/titanium dioxide/single-stranded auxiliary probe electrode.
Based on the preparation method, the cancer marker is sCD146, and different protein cancer markers can be selected by replacing corresponding antibodies.
Based on the preparation method, the carbon nitride quantum dots are synthesized in one step by a microwave method. Specifically, the preparation method of the carbon nitride quantum dot comprises the following steps: dissolving citric acid and urea serving as raw materials in ultrapure water, and performing microwave treatment to form a dark brown solid; cooling the dark brown solid to room temperature, and adding ultrapure water for dissolving to obtain a dark brown solution; and centrifuging the dark brown solution and taking the supernatant to obtain the carbon nitride quantum dots.
The invention also provides a signal reversal type photoelectrochemical biosensor for detecting the cancer marker, which is mainly prepared by the preparation method of the signal reversal type photoelectrochemical biosensor for detecting the cancer marker.
The invention also provides an application of the signal reversal type photoelectrochemical biosensor for detecting the cancer marker in the aspect of detecting the cancer marker.
Based on the above application, the biosensor for detecting cancer markers is placed in a Tril-HCl buffer solution containing 0.1M ascorbic acid with pH 7.4, and a bias voltage of-0.2V is applied to perform PEC detection, so as to detect photocurrent signals of the cancer markers at different concentrations; and constructing a linear equation between the concentration of the cancer marker and the photocurrent signal according to the detection result.
Based on the application, the time for adsorbing the single-chain auxiliary DNA probe to the surface of the indium tin oxide/titanium dioxide electrode is 30-120 min, and the time for adsorbing the carbon nitride quantum dot to the active site exposed by the titanium dioxide polyhedron in the indium tin oxide/titanium dioxide/single-chain auxiliary probe/middle probe electrode is 30-120 min. Preferably, the time for adsorbing the single-stranded auxiliary DNA probe on the surface of the indium tin oxide/titanium dioxide electrode is 90 min, and the time for adsorbing the carbon nitride quantum dot on the surface of the indium tin oxide/titanium dioxide/single-stranded auxiliary probe/middle probe electrode with an active site is 60 min.
Based on the application, when the concentration range of sCD146 is 10-5000000 fg/mL, the constructed linear regression equation isI=-174.34logC sCD146 -1314.00 (R2=0.9977), detection lower limit 2.1 fg/mL; wherein the content of the first and second substances,Iis a photocurrent signal detected by the biosensor for detecting a cancer marker,C sCD146 represents the concentration of sCD 146.
Therefore, the biosensor for detecting the cancer marker provided by the invention is a photoelectrochemical biosensor, belongs to a direct contact type titanium dioxide polyhedron// carbon nitride quantum dot environment-friendly photocurrent direction turning system, overcomes the problems of long electron transfer path or toxicity and the like of the existing photocurrent direction turning system, and simultaneously expands the application and development of a photocurrent direction turning strategy in the photoelectrochemical biosensor.
The biosensor for detecting the cancer marker is organically combined based on a direct contact type photocurrent direction reversal strategy, a targeted induction three-dimensional double-support DNA walker circulating signal amplification technology and the adsorption characteristic of a metal organic framework material, and photocurrent signal reversal design is carried out, so that the reading of photocurrent signals is amplified, the electronic transmission path between reversal materials is shortened, the photocurrent direction reversal efficiency is improved, the detection result of the biosensor is more accurate, and the detection sensitivity is improved; in addition, the biosensor provided by the invention can generate a photocurrent signal with opposite direction only when a target is identified based on a photocurrent direction turning strategy, so that different interference proteins have no obvious interference on the detection system; therefore, the biosensor provided by the invention can realize environment-friendly detection of the cancer marker, has the advantages of high sensitivity, high selectivity, good reproducibility, high stability and the like, can realize accurate detection of the cancer marker with low concentration, provides a new approach for photoelectrochemical biological analysis, and has good application prospect.
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Fig. 1 is a flowchart illustrating a method for manufacturing a signal inversion type photoelectrochemical sensor for detecting a cancer marker according to an embodiment of the present invention.
FIG. 2 is a representation diagram of a titanium dioxide polyhedron used in a biosensor according to an embodiment of the present invention.
Wherein, the A in the figure is the scanning electron micrograph of the MIL-125. FIG. B is a scanning electron micrograph of a titanium dioxide polyhedron. Panel C is an X-ray diffraction pattern of MIL-125 (a) and a titanium dioxide polyhedron (b). And the graph D is a titanium 2p X ray photoelectron spectrum of a titanium dioxide polyhedron. FIG. E is an oxygen 1s X ray photoelectron spectrum of a titanium dioxide polyhedron. FIG. F is a graph of isothermal adsorption and desorption of MIL-125 with nitrogen. And the graph G is a nitrogen isothermal adsorption and desorption curve chart of the titanium dioxide polyhedron. And the graph H is an ultraviolet-visible-near infrared absorption spectrum diagram of the titanium dioxide polyhedron.
FIG. 3 is a representation of the carbon nitride quantum dots used in the biosensor provided in example 1 of the present invention.
Wherein, the picture A in the figure is a transmission electron microscope picture of the carbon nitride quantum dots. And the graph B is an X-ray diffraction spectrum of the carbon nitride quantum dots. And the figure C is an ultraviolet-visible absorption spectrum diagram of the carbon nitride quantum dots. And the graph D is a Zeta potential diagram of the titanium dioxide polyhedron (a) and the carbon nitride quantum dot (b).
Fig. 4 is a mechanism verification diagram of the biosensor provided in example 1 of the present invention.
In the figure, a graph a is a photocurrent response curve of three electrodes (curve a represents an indium tin oxide/titanium dioxide electrode, curve b represents an indium tin oxide/carbon nitride quantum dot electrode, and curve c represents an indium tin oxide/titanium dioxide/carbon nitride quantum dot composite electrode). The graphs B-D are the Mott-Schottky curve diagrams of the indium tin oxide/titanium dioxide electrode, the indium tin oxide/carbon nitride quantum dot electrode and the indium tin oxide/titanium dioxide/carbon nitride quantum dot composite electrode in sequence. And the graph E and the graph F are Tauc graphs of the titanium dioxide polyhedron and the carbon nitride quantum dot in sequence.
FIG. 5 is a diagram illustrating steps of assembling a biosensor according to an embodiment of the present invention.
Wherein, a graph A in the graph is a photocurrent response graph of different modified electrodes. And the graph B is an alternating current impedance graph of different modified electrodes. Wherein the curves in each figure represent the following modified electrodes, respectively: curve a indium tin oxide/titanium dioxide, curve b indium tin oxide/titanium dioxide/single-chain auxiliary probe, curve c indium tin oxide/titanium dioxide/single-chain auxiliary probe/middle probe, and curve d indium tin oxide/titanium dioxide/single-chain auxiliary probe/middle probe/carbon nitride quantum dot composite electrode.
FIG. 6 is a diagram illustrating optimization of preparation conditions of a biosensor according to an embodiment of the present invention.
Wherein, the graph A is a relation graph of single-stranded auxiliary DNA probe adsorption time and photocurrent in the preparation process of the biosensor provided by the embodiment of the invention. Fig. B is a graph of the relationship between the adsorption time of the carbon nitride quantum dots on the titanium dioxide polyhedron and the photocurrent in the preparation process of the biosensor according to the first embodiment of the present invention.
Fig. 7 is a linear relationship diagram of the biosensor detecting sCD146 provided by the second embodiment of the present invention.
In the figure, a graph a is a photocurrent response graph of the biosensor detecting different concentrations of sCD 146. Graph B is a linear fit of the biosensor detected sCD146 concentration to the photocurrent response.
FIG. 8 is a diagram showing the results of a selective experiment using a detection method constructed using a biosensor provided in example two of the present invention.
FIG. 9 is a graph showing the results of a stability test of a detection method using a biosensor according to the second embodiment of the present invention.
Detailed Description
The technical solution of the present invention is further described in detail by the following embodiments.
The invention designs a direct contact titanium dioxide polyhedron// carbon nitride quantum dot novel overturning system mainly according to the overturning advantage of the photocurrent direction; the targeted induction three-dimensional double-support DNA walker circulation amplification technology and the metal organic framework material adsorption characteristic are combined to carry out photocurrent signal turnover design, so that an environment-friendly photoelectrochemical biosensor which is low in cost, high in selectivity and ultra-sensitive for detecting the cancer marker is constructed, the detection accuracy and sensitivity are verified by measuring the level of the cancer marker in serum, and a technical support is provided for researching the effect of the cancer marker in the occurrence and development of cancer.
The photoelectrochemical biosensor for detecting cancer markers provided by the invention, and the preparation method and application thereof are further explained by taking sCD146 as a cancer marker.
The embodiment of the invention adopts the following raw material reagents: sCD146 and antibody Ab thereof1And Ab2From Beijing Yinqiao Shenzhou science and technology, Inc.; bovine serum albumin, carcinoembryonic antigen (CEA), Prostate Specific Antigen (PSA), chloroauric acid are all from sigma aldrich (shanghai) trade ltd; bsmal endonuclease (Nt. Bsmal) was purchased from New England Biotechnology Inc.
Terephthalic acid, titanium isopropoxide, N-dimethylformamide, citric acid, urea, anhydrous methanol, trisodium citrate, ferric chloride hexahydrate, ethylene glycol, sodium acetate, 3-aminopropyltriethoxysilane, ascorbic acid, potassium ferricyanide and potassium ferrocyanide, sodium carbonate, sodium chloride are all analytically pure AR and are from national pharmaceutical group chemical agents, Inc.
Nucleic acid sequence (5 '→ 3'): walking DNA probe (DNA walker): SH- (CH)2)6-T60-AGT GTG CGA GAC GGT ATA TTT; support DNA probe (Support DNA): NH (NH)2-(CH2)6-TTT TAT ACC GTC TCG CAC ACT TTC GCT ACT CTG TTT; single-stranded helper DNA probe: AAA CAG AGT AGC GAA AGT GTG are provided.
The experimental instrument adopted by the embodiment of the invention comprises the following components:
all aqueous solutions were prepared using ultra pure water from a Milli-Q filtration system (millipore corp., USA). The uv-vis spectrum was tested using a DS5 uv-vis spectrophotometer (DS 5, uk). The transmission electron micrograph image was characterized by JEOL JEM 2100F (japan).
X-ray diffraction was obtained on an X-ray diffractometer (Bruker D8 Advance) using monochromated Cu ka radiation. X-ray photoelectron spectroscopy was obtained on a multi-functional imaging electron spectrometer (Thermo ESCALAB 250 XI).
Xenon lamp (PLS-SXE 300) with 420 nm cut-off filter was used as light source (wavelength)>420 nm), photoelectrochemical and electrochemical impedance spectroscopy measurements were performed by a CHI 660E electrochemical workstation with an indium tin oxide electrode (5.6 mm diameter) as the working electrode, a Saturated Calomel Electrode (SCE) as the reference electrode and a platinum wire as the counter electrode. EIS measurements at 5 mM (1:1) [ Fe (CN)6]3-/4-In a solution containing 0.1M KCl, with a frequency in the range of 0.1 Hz to 100 kHz and an amplitude of 5 mV. Electrochemical and photoelectrochemical tests adopted by the embodiment of the invention both use a three-electrode system: indium tin oxide (Kjeldahl is a photoelectric technology Co., Ltd.) conductive glass with a diameter of 5.6 mm is used as a working electrode, a saturated calomel electrode is used as a reference electrode, and a platinum wire is used as an auxiliary electrode.
Example 1
Referring to fig. 1, a signal inversion type photoelectrochemical biosensor for detecting cancer markers according to an embodiment of the present invention is mainly prepared by steps including preparing an ito/titania electrode, preparing an ito/titania/single-stranded auxiliary probe electrode, and preparing a conjugate Ab1-Fe3O4@ Au-DNA preparation of conjugate Ab2AuNPs-DNA, constructing an intermediate probe, preparing a sensor and the like.
First, prepare indium tin oxide/titanium dioxide electrode
Carbonizing the metal-organic framework material MIL-125 shown in FIG. 2A in a tubular furnace at 380 ℃ for 4 h to obtain a titanium dioxide polyhedron shown in FIG. 2B; dispersing the titanium dioxide polyhedron in ultrapure water to prepare a titanium dioxide polyhedron suspension with the concentration of 1 mg/mL; and dripping 25 mu L of the titanium dioxide polyhedral suspension liquid with the concentration of 1 mg/mL on a cleaned indium tin oxide working electrode to prepare the indium tin oxide/titanium dioxide electrode.
As can be seen from FIG. 2A, the MIL-125 polyhedron has a pie-like structure with an average dimension of about 600 nm and a depth of about 280 nm. After carbonizing it in air, the titanium dioxide polyhedrons shown in fig. 2B were obtained and maintained similar morphologies, but the average size of the titanium dioxide polyhedrons was reduced. The X-ray powder diffraction pattern shown in figure 2C further confirms the successful preparation of the titanium dioxide polyhedra. As can be seen from fig. 2C, curve a shows all diffraction peaks of MIL-125 polyhedron; after carbonization, the diffraction peak of the MIL-125 polyhedron completely disappears, as shown by the curve b, and corresponds to the crystal face of the anatase phase structure (JCPDS No. 73-1764) of the titanium dioxide, which indicates that the anatase type titanium dioxide is successfully synthesized.
And analyzing the element composition and chemical state of the titanium dioxide polyhedron by adopting X-ray photoelectron spectroscopy. As can be derived from FIG. 2D, the titanium 2p spectrum is attributed to titanium (IV) and titanium 2p3/2And Ti 2p1/2Wherein the binding energies are 458.8 and 464.7 eV, respectively. The O1 s spectrum shown in fig. 2E can be deconvoluted into three peaks at 530.02, 531.4 and 532.8 eV, corresponding to the metal oxide Ti-O, the fraction C = O or C-O and-O-H, respectively. Indicating the presence of a Ti-O-C bond and a-COOH group. In order to further explore the specific surface area and the porous structure of the titanium dioxide polyhedron, nitrogen adsorption and desorption isotherm tests were performed, and the result is shown in fig. 2E and 2F, where the BET specific surface area of the titanium dioxide polyhedron was calculated to be 102.3 m2/g。
In addition, the BJH pore size distribution curve in the inset of FIG. 2F shows that the average pore size of the titanium dioxide polyhedron is 5.1 nm, which is beneficial to the rapid mass transfer and adsorption of small molecules. The titanium dioxide polyhedron with high specific surface area and porous structure may have good photoelectrochemical properties and can provide enough active sites. Fig. 2H shows the uv-vis-nir absorption spectrum of the titanium dioxide polyhedra, with the maximum absorption band of the titanium dioxide polyhedra occurring at approximately 580 nm, significantly red-shifted compared to conventional titanium dioxide nanomaterials (380 nm), approximately due to their porous structure. The visible absorption of the titanium dioxide polyhedron is obviously enhanced, and the absorption range is wide, which shows that the titanium dioxide polyhedron is suitable for being used as a photoelectric active material under the irradiation of visible light (the wavelength is more than 420 nm), and is beneficial to the construction of a photoelectrochemical biosensor.
Secondly, preparing indium tin oxide/titanium dioxide/single-chain auxiliary probe electrode
Annealing a 10 mu M single-chain auxiliary DNA probe at 95 ℃ for 5 min, cooling to room temperature, dropwise adding 20 mu L of the annealed single-chain auxiliary DNA probe to the surface of the indium tin oxide/titanium dioxide electrode, combining the porous structure of a titanium dioxide polyhedron in the surface of the indium tin oxide/titanium dioxide electrode with electrostatic adsorption to the single-chain auxiliary probe, adsorbing the single-chain auxiliary DNA probe to the surface of the indium tin oxide/titanium dioxide electrode to seal active sites on the titanium dioxide polyhedron, and cleaning to obtain the indium tin oxide/titanium dioxide/single-chain auxiliary probe electrode after adsorbing for 30-120 min.
Third, Ab preparation1-Fe3O4@ Au-DNA conjugates
Fe3O4Nano-particles: dissolving 0.6 g of ferric chloride hexahydrate in 20 mL of ethylene glycol to form a clear solution, adding 1.5 g of sodium acetate, stirring vigorously for 30 min, transferring to a 50 mL reaction kettle, placing at 200 ℃, cooling to room temperature for 8 h, washing with anhydrous ethanol and ultrapure water for multiple times, carrying out magnetic separation, collecting solids, and carrying out freeze drying to obtain brownish black powder Fe3O4And (3) nanoparticles.
Au nanoparticles: first 150 mL of 1 mM HAuCl4Heating to bumping, adding 15 mL of 38.8 mM sodium citrate solution into the solution quickly, stirring vigorously for 10 min, placing the flask in an ice water bath to terminate the reaction quickly, collecting the solution and placing the solution at 4 ℃ for later use to obtain Au nanoparticles.
Fe3O4Preparation of @ Au solution: taking 30 mg of the Fe3O4 Dispersing the nanoparticles and 1 mL of 97% 3-aminopropyltriethoxysilane in 20 mL of absolute ethanol, performing ultrasonic treatment for 5 min, and shaking at room temperature for 6 h. Separated magnetically and redispersed in 15 mL of ultrapure water to give 1.5 mg/mL of aminated Fe3O4Nanoparticles, 1 mL of the above solution was taken, 10 mL of the Au nanoparticles prepared above and 1 mL of ultrapure water were added, and shaken for 3 h to aminated Fe3O4The nanometer magnetic beads and the Au nanometer particles are combined through covalent bonds, and Fe is obtained after magnetic separation3O4@ Au particles dispersed in 1 mL of water to obtain Fe3O4@ Au solution.
Ab1-Fe3O4Preparation of @ Au-DNA: take 30. mu.L Ab1Adding to 1 mL of said Fe3O4Reaction in @ Au solution at room temperature for 1 h, addition of 100. mu.L of supporting DNA probe (10. mu.M), shaking at room temperature for 12 h to allow Ab1The supporting DNA probe and the Fe3O4@ Au particles form Ab by covalent bond1-Fe3O4@ Au-DNA conjugate complex. After magnetic separation, the obtained Ab1-Fe3O4Re-dispersing the @ Au-DNA conjugate complex in PBS buffer to obtain Ab1-Fe3O4@ Au-DNA conjugate solution, stored for use.
Fourth, Ab preparation2-AuNPs-DNA conjugates
Adding 30 mu L of sodium carbonate solution into 1 mL of the Au nano-gold particles prepared above until the pH value of the solution is 8-9, and then adding 30 mu L of Ab2After reaction at room temperature for 1 hour, a mixed solution of 10. mu.L of the walking DNA probe and 100. mu.L of the supporting DNA probe was added thereto, and the mixture was allowed to stand at 4 ℃ for 18 hours to react Ab2The supporting DNA probe and the walking DNA probe are combined with the gold nanoparticle through covalent bonds to form Ab2-AuNPs-DNA conjugate complexes. Finally, the unbound nucleic acid chains and antibodies were removed by centrifugation at 15000 rpm for 30 min at 4 ℃ and Ab was obtained by dispersing in 1% BSA-containing PBS buffer (1% BSA and 1% PEG 20000-containing PBS buffer)2-AuNPs-DNA conjugate solution and stored at 4 ℃ protected from light.
Fifth, prepare the intermediate Probe
To 100. mu.L of the Ab1-Fe3O4@ Au-DNA conjugate solution was added with 10. mu.L of target sCD146, reacted at 37 ℃ for 90 min, then magnetic separation and washing were performed to remove unbound target and 100. mu.L of the Ab was added2AuNPs-DNA conjugate solution, shaking at 37 ℃ for 90 min, magnetic separation again and dispersion in 100. mu.L PBS solution. Then adding 5U restriction endonuclease Nt.BsmAl, reacting for 2h at 37 ℃, taking supernatant after magnetic separation, wherein the supernatant contains an intermediate probe for target induction of three-dimensional double-support DNA walker circulating signal amplification reaction.
Sixthly, preparing the sensor
The step of preparing the sensor comprises: preparing carbon nitride quantum dots, preparing indium tin oxide/titanium dioxide/single-chain auxiliary probe/middle probe electrode and preparing indium tin oxide/titanium dioxide/single-chain auxiliary probe/middle probe/carbon nitride quantum dot composite electrode.
5.1 the step of preparing the carbon nitride quantum dots comprises the following steps: adopting a microwave one-step method to synthesize the carbon nitride quantum dots, dissolving 0.5 g of citric acid and 0.5 g of urea in 25 mL of ultrapure water, carrying out microwave treatment on medium-high fire for 7 min, observing that the colorless liquid becomes a dark brown solid to form the carbon nitride quantum dots, cooling to room temperature, adding 5 mL of ultrapure water to dissolve to obtain a dark brown solution, centrifuging at 10000 rpm for 5 min, taking supernatant to obtain a carbon nitride quantum dot solution, and storing at 4 ℃ for later use.
The carbon nitride quantum dots prepared in the step are characterized in appearance and size by adopting a transmission electron microscope, as shown in fig. 3A, the carbon nitride quantum dots are observed to be in a quasi-spherical shape and uniformly dispersed in the graph 3A; analysis of the high resolution transmission image in the inset of FIG. 3A shows a face-to-face spacing of about 0.32 nm, which corresponds to a hexagon g-C3N4The (002) plane lattice plane thus shows that the carbon nitride quantum dots are successfully prepared by the step.
Fig. 3B tests the X-ray diffraction spectrum of the carbon nitride quantum dot, and the 2 θ value of the carbon nitride quantum dot shows a distinct characteristic peak at 27.1 °, which is the accumulation of conjugated aromatic rings on the (002) crystal face. In addition, fig. 3C further characterizes the uv-vis spectrum of the carbon nitride quantum dot, and it is observed that the carbon nitride quantum dot has a distinct absorption peak at 345 nm and a shoulder peak around 445 nm, which is basically consistent with the existing literature report.
Further, as can be seen from Zeta potential analysis fig. 3D: the surface of the titanium dioxide polyhedron is positively charged, and the potential is about +23.3 mV; the carbon nitride quantum dots are negatively charged and have a voltage of about-19.5 mV; thus, the titanium dioxide polyhedron and the carbon nitride quantum dots can be combined through electrostatic adsorption.
5.2 the step of preparing the indium tin oxide/titanium dioxide/single-stranded auxiliary probe/middle probe electrode comprises the following steps: and dripping the supernatant containing the intermediate probe for the target-induced three-dimensional double-support DNA walker circulating signal amplification reaction onto the indium tin oxide/titanium dioxide/single-chain auxiliary probe electrode, incubating for 90 min at room temperature to ensure that the intermediate probe is specifically combined with the single-chain auxiliary DNA probe on the indium tin oxide/titanium dioxide/single-chain auxiliary probe electrode, so that the single-chain auxiliary DNA probe is released from the titanium dioxide polyhedron to expose the active site on the titanium dioxide polyhedron, and obtaining the indium tin oxide/titanium dioxide/single-chain auxiliary probe/intermediate probe electrode with the titanium dioxide polyhedron active site.
5.3 the step of preparing the indium tin oxide/titanium dioxide/single-chain auxiliary probe/middle probe/carbon nitride quantum dot composite electrode comprises the following steps: cleaning the indium tin oxide/titanium dioxide/single-chain auxiliary probe/middle probe electrode; diluting the carbon nitride quantum dot solution prepared by the microwave one-step method by 10 times to obtain a diluted carbon nitride quantum dot solution; and (3) dripping 20 mu L of the diluted carbon nitride quantum dot solution on the cleaned indium tin oxide/titanium dioxide/single-chain auxiliary probe/middle probe electrode, adsorbing and incubating for 60 min at room temperature, so that the carbon nitride quantum dots are modified on the surface of the titanium dioxide polyhedron through the porous structure of the titanium dioxide polyhedron and the electrostatic adsorption effect, and forming an indium tin oxide/titanium dioxide/single-chain auxiliary probe/middle probe/carbon nitride quantum dot composite electrode, thereby preparing the direct contact type signal inversion sensor.
(1) Mechanism research of direct contact type signal turnover sensor
1) Test condition and method for detecting photocurrent signal
Respectively and directly dripping the prepared titanium dioxide polyhedron and carbon nitride quantum dot solution on an indium tin oxide electrode, and drying overnight to obtain an indium tin oxide/titanium dioxide electrode and an indium tin oxide/carbon nitride quantum dot electrode; and in addition, dropwise adding the carbon nitride quantum dot solution on the indium tin oxide/titanium dioxide electrode, and incubating for 30-120 min to obtain the indium tin oxide/titanium dioxide/carbon nitride quantum dot composite electrode. And then, by utilizing a three-electrode system of an electrochemical workstation, the prepared electrode is placed in a Tril-HCl buffer solution (pH 7.4 and 0.1M) containing 0.1M ascorbic acid to test an i-t curve to obtain a photocurrent signal. The results are shown in FIG. 4A.
As can be seen in fig. 4A: the anode photocurrent signal of the ito/titania electrode is 2.124 μ a in curve a, and the relatively weak anode photocurrent signal is shown in curve b. From curve c it can be seen that: after the indium tin oxide/titanium dioxide electrode is incubated with the carbon nitride quantum dot, the formed indium tin oxide/titanium dioxide/carbon nitride quantum dot composite modified electrode not only generates a relatively large cathode photocurrent-3.579 muA, but also has a direction opposite to that of the indium tin oxide/titanium dioxide electrode photocurrent shown by a curve a. Therefore, when the carbon nitride electronic point is directly contacted with the titanium dioxide polyhedron, the carbon nitride electronic point can be used as a signal turning agent to realize the conversion of the photocurrent direction.
2) The method comprises the steps of respectively placing the prepared indium tin oxide/titanium dioxide electrode, indium tin oxide/carbon nitride quantum dot electrode and indium tin oxide/titanium dioxide/carbon nitride quantum dot electrode in Tril-HCl buffer solution (pH 7.4, 0.1M) Na by utilizing an electrochemical workstation three-electrode system2SO4The impedance-voltage curve was tested in solution (pH 6.8, 0.2M) and then converted to a Mott-Schottky curve. The results are shown in FIGS. 4B to 4D.
As can be seen in fig. 4B and 4C: the ito/cntr electron dot electrode and ito/titania electrode both exhibit positive slopes, corresponding to n-type semiconductor characteristics, while the ito/titania/cntr quantum dot composite electrode exhibits negative slopes, corresponding to p-type semiconductor, consistent with the photocurrent signal study shown in fig. 4A.
3) According to the testing conditions and the method corresponding to the Tauc diagram, a DS5 ultraviolet-visible spectrophotometer (DS 5, UK) is used for testing ultraviolet-visible spectrums of the titanium dioxide polyhedral solution and the carbon nitride quantum dot solution respectively, and then the Tauc diagram is obtained through conversion. The results are shown in FIGS. 4E and 4F.
Referring to fig. 4E, the synthesized titanium dioxide polyhedron has a forbidden band of 2.52 eV, and its conduction band and valence band are-0.73V and 1.79V, respectively, relative to the common hydrogen electrode (NHE). From fig. 4F, it is shown that the carbon nitride quantum dot has a forbidden band of 2.55 eV, and its conduction band and valence band are-0.18V and 2.37V, respectively, relative to the ordinary hydrogen electrode NHE.
Therefore, it can be derived from fig. 4 that the mechanism of the photocurrent direction reversal of the present invention is: under visible light (wavelength greater than 420 nm) irradiation, the titanium dioxide polyhedron is excited, and photo-generated electrons/holes are respectively formed on a conduction band/a valence band. Then, the photo-generated electrons are transferred from the conduction band-0.73V of the titanium dioxide polyhedron to the indium tin oxide electrode, meanwhile, the oxidation potential of ascorbic acid serving as an electron donor is 0.15V, the ascorbic acid is positioned between the conduction band and the valence band of the titanium dioxide and can be oxidized by photo-generated holes of the titanium dioxide, so that more photo-generated electrons occupy the conduction band of the titanium dioxide polyhedron and are transferred to the indium tin oxide electrode, and a larger anode photocurrent is generated.
When the titanium dioxide polyhedron is decorated on the indium tin oxide electrode by the carbon nitride quantum adsorption to form the indium tin oxide/titanium dioxide/carbon nitride quantum dot composite decoration electrode, the titanium dioxide polyhedron and the carbon nitride quantum dot are easy to be excited, and corresponding photo-generated electrons/holes are respectively formed on a conduction band/a valence band, based on the energy level matching, the photo-generated electrons of the titanium dioxide polyhedron conduction band of-0.73V are transferred to the conduction band of-0.18V of the carbon nitride quantum for electrochemically reducing oxygen in the electrolyte, wherein the reduction potential of-0.046V of oxygen is taken as an electron acceptor; the photogenerated holes of the valence band 2.37V of the carbon nitride quantum dot are transferred to the conduction band 1.79V of the titanium dioxide polyhedron. In addition, some of the photo-generated electrons accompanying the conduction band of the carbon nitride quantum dots return to the valence band, resulting in recombination of photo-generated electron/hole pairs. In the process, the ascorbic acid effectively oxidizes and removes holes on a conduction band, inhibits the recombination of photogenerated electrons/hole pairs, and promotes the transfer of photogenerated electrons, so that the cathode photocurrent of the indium tin oxide/titanium dioxide/carbon nitride quantum dot composite modified electrode is enhanced. Therefore, based on the excellent matching horizontal energy band between the carbon nitride quantum dot and the titanium dioxide polyhedron, a titanium dioxide polyhedron// carbon nitride quantum dot photocurrent direction reversal system is formed, and the carbon nitride quantum dot can promote the separation and transfer of photo-generated electrons/holes of the titanium dioxide polyhedron and induce the photocurrent direction reversal of the titanium dioxide polyhedron.
(2) Characterization of photoelectrochemical biosensors
The test conditions are as follows: under a bias of-0.2V, the ITO/Titania electrode, ITO/Titania/SSB/ISB electrode, ITO/Titania/SSB/ISB/NSB electrode used in the examples of the present invention were placed in a Tril-HCl buffer solution (pH 7.4, 0.1M) containing 0.1M ascorbic acid for photoelectrochemical test, and placed in a 5 mM (1:1) [ Fe (CN)6]3-/4-Impedance measurements were performed in a solution containing 0.1M KCl with a frequency range of 0.1 Hz to 100 kHz and an amplitude of 5 mV. The test results are shown in fig. 5.
The photocurrent decayed with increasing illumination time due to the consumption of ascorbic acid. Therefore, the present invention displays the photoelectrochemical test results by using the maximum photocurrent values generated by the different modified electrodes. As can be seen in fig. 5A: the ito/titania electrode shown in curve a has a significant anode photocurrent of 2.124 pa. Curve b shows that the photocurrent of the indium tin oxide/titanium dioxide/single-chain auxiliary probe electrode modified by the single-chain auxiliary DNA probe is reduced, and the value is 1.010 μ a, because the single-chain auxiliary DNA probe adsorbed on the surface of the electrode has poor conductivity, the generation of photo-generated electrons is hindered, and thus the anode photocurrent signal is reduced.
Curve c shows: when the single-stranded auxiliary DNA probe and the intermediate probe are combined to form a double strand, the content of the single-stranded auxiliary DNA probe on the surface of the electrode is reduced, so that the anode photocurrent signal part of the indium tin oxide/titanium dioxide/single-stranded auxiliary probe/intermediate probe electrode is recovered (1.304 muA). Curve d shows: after the carbon nitride quantum dot solution is dripped for incubation, due to the introduction of the carbon nitride quantum dots and the energy level relationship between the titanium dioxide polyhedron and the carbon nitride quantum dots, the polarity of a photoelectric signal is converted, so that the indium tin oxide/titanium dioxide/single-chain auxiliary probe/middle probe/carbon nitride quantum dot composite electrode forms a larger cathode photocurrent-2.987 muA.
As can be seen from fig. 5B: curve a shows oxidationOf indium-tin/titanium dioxide electrodesR ctA value of 35.73 Ω; due to the poor conductivity of the nucleic acid strands, the immobilization of the single-stranded helper DNA probe on the ITO/Titania electrode surface effectively hindered [ Fe (CN)6]3−/4−The electron transfer process between the ion and the electrode leads to an increased resistance to charge transfer, so curve b shows that of the ITO/Titania/single-stranded helper probe electrodeR ctA value of 69.86 Ω; when the ITO/Titania/single-stranded auxiliary probe electrode is incubated with the nucleic acid chain intermediate probe released by the enzyme digestion, the single-stranded auxiliary probe and the intermediate probe form a double strand due to the base pairing relationship and are then washed away, so that curve c shows that a reduced amount of ITO/Titania/single-stranded auxiliary probe/intermediate probe electrode is obtainedR ctA value of 62.24 Ω; when the ITO/Titania/single-chain auxiliary probe/middle probe electrode is immersed in the carbon nitride quantum dot solution for incubation, the active sites on the titanium dioxide polyhedron are released after the single-chain auxiliary probe on the electrode is combined with the middle probe, so that the carbon nitride quantum dot can be directly fixed on the electrode by utilizing the aperture and electrostatic adsorption of the titanium dioxide polyhedron to promote the transfer of electrons, and therefore, the curve d shows that the ITO/Titania/single-chain auxiliary probe/middle probe/carbon nitride quantum dot electrode obtains a reduced oneR ctThe value is 55.88 omega. Each modified electrodeR ctThe expected change in value is substantially consistent with the results of the study shown in fig. 5A, indicating that a photoelectrochemical biosensor for detecting a cancer marker has been successfully prepared.
The signal reversal type photoelectrochemical biosensor for detecting the cancer marker provided by the embodiment of the invention has two main factors influencing the photocurrent signal: one is the adsorption time of the single-chain auxiliary probe in the step of preparing the indium tin oxide/titanium dioxide/single-chain auxiliary probe electrode, and the other is the adsorption time of the carbon nitride quantum dot in the preparation process of the indium tin oxide/titanium dioxide/carbon nitride quantum dot electrode in the step of preparing the sensor. These two factors are further analyzed below.
Effect of Single-Strand helper Probe adsorption time
The experimental conditions are as follows: basically, the method provided by the first embodiment above is used to prepare corresponding sensors according to different adsorption times of the single-stranded auxiliary probes, wherein in the step of "preparing indium tin oxide/titanium dioxide/single-stranded auxiliary probe electrode", the adsorption times of the single-stranded auxiliary probes are respectively 0, 30, 60, 90 and 120 min, and in the step of "preparing a sensor", the adsorption time of the carbon nitride quantum dots is 60 min, and other steps are the same as those in the first embodiment. The photoelectrochemical test was carried out using the prepared sensor described above with reference to the experimental conditions of the aforementioned section "(2) characterization of photoelectrochemical biosensor", and the test results are shown in fig. 6.
As can be seen in fig. 6: when the adsorption time of the single-stranded auxiliary DNA probe is 0-90 min, the photoelectric response of the indium tin oxide/titanium dioxide/single-stranded auxiliary probe electrode is gradually weakened, which is probably because the longer the adsorption reaction time of the single-stranded auxiliary DNA probe is, the more the single-stranded auxiliary DNA probes are fixed on the electrode, so that the resistance to the generation and transfer of the photo-generated electron-hole on the titanium dioxide polyhedron is enhanced, but experiments show that the trend of the photoelectric response weakening gradually becomes gentle after the adsorption time exceeds 90 min, which indicates that the quantity of the single-stranded auxiliary probes fixable on the indium tin oxide/titanium dioxide/single-stranded auxiliary probe electrode is basically saturated, and therefore, 90 min is preferably used as the adsorption reaction time of the single-stranded auxiliary DNA probe on the indium tin oxide/titanium dioxide electrode.
Influence of adsorption time of carbon nitride quantum dots
The experimental conditions are as follows: the adsorption time of the carbon nitride quantum dots in the step of preparing the sensor is 0, 30, 60, 90 and 120 min. The experimental set-up is shown in figure 7.
As can be seen in fig. 7: when the adsorption time of the carbon nitride quantum dots is 0-60 min, the cathode photocurrent generated by the indium tin oxide/titanium dioxide/carbon nitride quantum dot composite electrode is increased along with the extension of the adsorption time of the carbon nitride quantum dots, and the current response change almost tends to be smooth after 60 min. Therefore, 60 min is preferred as the optimal adsorption time of the carbon nitride quantum dots on the indium tin oxide/titanium dioxide electrode.
Example 2
This example provides the use of the biosensor for detecting a cancer marker provided in example 1 for detecting a cancer marker.
Specifically, in the preparation process of the biosensor provided in example 1, the adsorption reaction time of the single-stranded auxiliary probe on the ito/tio electrode is 90 min, and the adsorption time of the carbon nitride quantum dot on the ito/tio/single-stranded auxiliary probe/middle probe electrode is 60 min; placing the biosensor in 0.1M Tril-HCl buffer solution (pH 7.4, 0.1M) of ascorbic acid, applying-0.2V bias voltage, performing photoelectrochemical test, and detecting photocurrent signals of the sCD146 at the concentrations of 10, 100, 200, 1000, 10000, 50000, 100000, 1000000 and 5000000 fg/mL respectively; a linear equation between the concentration of sCD146 and the photocurrent signal was constructed from the detection results, as shown in fig. 8.
As can be seen in fig. 8: with the increase of the concentration of the sCD146 from 10 to 5000000 fg/mL, the cathode photocurrent generated by the polarity reversal of the indium tin oxide/titanium dioxide/single-chain auxiliary probe/middle probe/carbon nitride quantum dot electrode is increased, and has good linearity with the concentration of the sCD146 from the concentration range of 10 to 5000000 fg/mL, and the linear regression equation is thatI=-174.34logC sCD146 -1314.00 (R2= 0.9977); wherein the content of the first and second substances,Iis a photocurrent signal detected by the biosensor for detecting a cancer marker,C sCD146 represents the concentration of sCD 146.
Detection limit
Experimental method or condition the photocurrent signal of the blank solution was measured 10 times using the prepared sensor, and the limit of detection was calculated based on the limit of detection =3 σ/S (where σ is the standard deviation of the blank sample; S is the slope of the calibration curve). The lower limit of detection of the concentration of sCD146 was calculated to be 2.1 fg/mL.
Selectivity test
In the embodiment of the invention, PSA and CEA in serum are selected as interference proteins in an sCD146 selective experiment. The prepared sensors were used to test the photocurrent signals of the interfering protein solutions, respectively, and the test results are shown in fig. 9.
As can be seen in fig. 9: interferenceThe photocurrent response caused by the protein PSA and CEA solution is the same as that generated by the blank solution, and the magnitude of the current response value is similar. However, it was found experimentally that when the above-mentioned interfering proteins were mixed in the same concentration ratio of 100:1 as the target, respectively, that is, when the target was addedC PAS : C sCD146 500 ng/mL, 5 ng/mL,C CEA : C sCD146 500 ng/mL:5 ng/mL; all produced photocurrent responses in the same direction and similar magnitude as target sCD146, thus indicating that the method for detecting sCD146 by using the biosensor for detecting cancer markers provided by the embodiment of the present invention has good selectivity.
Reproducibility test
Five groups of biosensors are respectively prepared under the same experimental conditions, 200 fg/mL sCD146 solution is detected to obtain photocurrent signals, and the relative standard deviation of the five groups of photocurrents is calculated.
Five groups of biosensors are respectively prepared under the same experimental conditions, 200 fg/mL sCD146 solution is detected to obtain five groups of photocurrent signals, and the relative standard deviation is calculated to be 2.7%.
Stability test
After the biosensor provided by the embodiment of the invention is respectively placed in a refrigerator at 4 ℃ for 7 days, 14 days and 21 days, 10000fg/mL of sCD146 solution is sequentially tested to obtain a photocurrent signal. The test results are shown in fig. 9.
Experimental results as shown in fig. 9, the photocurrent of the biosensor maintained 99.7% of the initial signal for 3 weeks.
Therefore, the application of the signal inversion type sensor provided by the embodiment of the invention in the detection of the sCD146 has the following advantages:
1) ultra-sensitivity: the embodiment of the invention organically combines a direct contact type photocurrent direction turning strategy, a target induced three-dimensional dual-support DNA walker circulating signal amplification technology, the adsorption characteristic of a metal organic framework material and the like, thereby not only amplifying the reading of photocurrent signals, but also accelerating the electronic transfer path between turning materials, improving the photocurrent direction turning efficiency, enabling the detection of the sensor to be more accurate and improving the detection sensitivity. Under the optimal experimental conditions, the linear range of the sensor is 10-5000000 fg/mL through experiments, and the detection limit is as low as 2.1 fg/mL.
2) High selectivity: according to the embodiment of the invention, the sensor developed based on the photocurrent direction turning strategy can generate a photocurrent signal in the opposite direction only when a target is identified; different interference proteins have no obvious interference to the detection system.
3) Good reproducibility: five groups of biosensors were prepared under the same experimental conditions, respectively, to detect 200 fg/mL sCD146 with a relative standard deviation of 2.7%.
4) High stability: after the biosensor provided by the embodiment of the invention is respectively placed in a refrigerator at 4 ℃ for 7, 14 and 21 days, the photocurrent detected by the sCD146 still maintains about 99% of the initial photocurrent response.
Therefore, the photoelectrochemical biosensor for detecting the cancer marker provided by the embodiment of the invention integrates a photocurrent direction reversal strategy, a three-dimensional double-support DNA walker circulation amplification technology and metal organic framework material adsorption characteristics, carries out direct contact type photocurrent signal reversal design, develops an environment-friendly photoelectrochemical new method for detecting sCD146 with low cost, high selectivity and ultra-sensitivity, improves photocurrent direction reversal efficiency, solves the problem that the conventional photoelectrochemical biosensor cannot accurately and ultra-sensitively detect sCD146 due to the limitation of interferents and sensitivity, provides a novel technical strategy for the analysis and research of sCD146, and promotes the early diagnosis and prognosis evaluation of cancer.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention and not to limit it; although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art will understand that: modifications to the specific embodiments of the invention or equivalent substitutions for parts of the technical features may be made; without departing from the spirit of the present invention, it is intended to cover all aspects of the invention as defined by the appended claims.
Claims (9)
1. A method for preparing a signal inversion type photoelectrochemical biosensor for detecting a cancer marker, comprising the steps of:
preparing an indium tin oxide/titanium dioxide electrode:
carbonizing the metal organic framework material to obtain a titanium dioxide polyhedron; preparing a titanium dioxide polyhedron suspension by taking the titanium dioxide polyhedron as a solute; dripping the titanium dioxide polyhedral suspension onto an indium tin oxide electrode to prepare an indium tin oxide/titanium dioxide electrode;
preparing an indium tin oxide/titanium dioxide/single-stranded auxiliary probe electrode:
adsorbing a single-chain auxiliary DNA probe on the surface of the indium tin oxide/titanium dioxide electrode to seal an active site on the titanium dioxide polyhedron, and cleaning to obtain the indium tin oxide/titanium dioxide/single-chain auxiliary probe electrode;
preparation of conjugate Ab1-Fe3O4@Au-DNA：
By amination of Fe3O4Shaking the nanoparticles and the gold nanoparticles to make the aminated Fe3O4The nano particles and the gold nano particles are combined to form Fe through covalent bonds3O4@ Au particles to obtain Fe3O4@ Au solution; in sequence towards the Fe3O4Adding a first target capture antibody and a support DNA probe into the @ Au solution, and performing shaking incubation and magnetic separation treatment at room temperature to ensure that the first target capture antibody, the support DNA probe and the Fe are3O4Binding of @ Au particles by covalent bond to produce Ab1-Fe3O4@ Au-DNA conjugate solution; wherein, Ab for the first target capturing antibody1Represents;
preparation of conjugate Ab2-AuNPs-DNA：
Adding a second target capture antibody into a gold nanoparticle solution with the pH value of 8-9 for reaction, adding a support DNA probe and a walking DNA probe to form a mixed solution, and reacting the mixed solution to enable the second target capture antibody, the support DNA probe and the walking DNA probe to be combined with the gold nanoparticles in the gold nanoparticle solution through covalent bonds to obtain Ab2-AuNPs-DNA conjugate solution; wherein Ab for the second target capturing antibody2Represents;
constructing an intermediate probe:
different concentrations of cancer markers were first added to the Ab1-Fe3O4Incubation and magnetic separation treatment in @ Au-DNA conjugate solution, and addition of the Ab2Carrying out incubation and magnetic separation treatment on the AuNPs-DNA conjugate solution, adding restriction endonuclease Nt.BsmAl for reaction and magnetic separation treatment, and obtaining supernatant, wherein the supernatant contains an intermediate probe for target induction of three-dimensional double-support DNA walker circulating signal amplification reaction;
preparing a sensor:
mixing and incubating the indium tin oxide/titanium dioxide/single-stranded auxiliary probe electrode and the intermediate probe in the supernatant to form a specific double strand with the single-stranded auxiliary DNA probe, releasing the single-stranded auxiliary DNA probe from the indium tin oxide/titanium dioxide/single-stranded auxiliary probe electrode, and exposing an active site on a titanium dioxide polyhedron in the indium tin oxide/titanium dioxide/single-stranded auxiliary probe electrode to obtain an indium tin oxide/titanium dioxide/single-stranded auxiliary probe/intermediate probe electrode with the active site; and adsorbing the carbon nitride quantum dots on the indium tin oxide/titanium dioxide/single-chain auxiliary probe/middle probe electrode to form an indium tin oxide/titanium dioxide/single-chain auxiliary probe/middle probe/carbon nitride quantum dot composite electrode, so that the titanium dioxide and the carbon nitride quantum dots are in direct contact to prepare the direct-contact signal turnover type biosensor.
2. The method of claim 1, wherein the step of preparing the ITO/Titania/single-stranded auxiliary probe electrode comprises: annealing the single-stranded auxiliary DNA probe at 95 ℃ for 5 min, cooling to room temperature, dripping the single-stranded auxiliary DNA probe onto the surface of the indium tin oxide/titanium dioxide electrode for adsorption treatment, and cleaning to obtain the indium tin oxide/titanium dioxide/single-stranded auxiliary probe electrode.
3. The method of claim 1, wherein the cancer marker is sCD 146.
4. The method for preparing the biosensor according to claim 1, wherein the carbon nitride quantum dots are synthesized in one step by a microwave method.
5. A biosensor for detecting a cancer marker, which is obtained by the method for producing a biosensor for detecting a cancer marker according to any one of claims 1 to 4.
6. The use of the signal inversion type photoelectrochemical biosensor for detecting a cancer marker according to claim 5 for detecting a cancer marker.
7. The use of claim 6, which comprises placing the cancer marker-detecting biosensor in a Tril-HCl buffer solution containing 0.1M ascorbic acid at pH 7.4, and applying a bias voltage of-0.2V for photoelectrochemical detection to detect photocurrent signals of the cancer marker at different concentrations; and constructing a linear equation between the concentration of the cancer marker and the photocurrent signal according to the detection result.
8. The use according to claim 7, wherein the time for adsorbing the single-stranded auxiliary DNA probe onto the surface of the ITO/Titania electrode is 30-120 min, and the time for adsorbing the carbon nitride quantum dot onto the active site exposed by the titanium dioxide polyhedron in the ITO/Titania/single-stranded auxiliary probe/middle probe electrode is 30-120 min.
9. The use of claim 8, wherein when the concentration of sCD146 is in the range of 10-5000000 fg/mL, the linear regression equation is constructedI=-174.34logC sCD146 -1314.00 (R2=0.9977), detection lower limit 2.1 fg/mL; wherein the content of the first and second substances,Iis a photocurrent signal detected by the biosensor for detecting a cancer marker,C sCD146 represents the concentration of sCD 146.
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