CN115974786B - Photoelectrochemistry and electrochemistry dual-mode ctDNA sensor based on ionic liquid functionalized lanthanide metal organic framework - Google Patents
Photoelectrochemistry and electrochemistry dual-mode ctDNA sensor based on ionic liquid functionalized lanthanide metal organic framework Download PDFInfo
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Abstract
The invention relates to the technical field of DNA biosensor detection, and particularly discloses a photoelectrochemistry and electrochemistry double-mode ctDNA sensor based on an ionic liquid functionalized lanthanide metal organic framework and a detection method thereof. The invention uses ionic liquidCoordinating to MOFs structure, and further introducing gold nanoparticles to the surface of Nd-MOF nanosheets by in-situ reduction of chloroauric acid with sodium borohydride to obtain AuNPs@Nd-MOF composite material; the probe is connected with a Capture Probe (CPs) through a gold-sulfur bond, and hybridized with target DNA (ctDNA) through DNA hybridization reaction, the impedance is increased after hybridization, the photo-generated electron transmission is blocked, and the photocurrent is reduced, so that the quantitative detection of the ctDNA concentration is realized. On the other hand, ctDNA hybridizes with signal probe SPs with ferrocene, and the electrochemical signal of SPs is measured by square wave voltammetry to achieve the aim of quantitative analysis of ctDNA concentration. The invention provides mutual authentication of detection results in two detection modes through photoelectrochemistry and electrochemistry double-signal output, and effectively improves the accuracy of ctDNA concentration detection.
Description
[ Technical field ]
The invention relates to the technical field of DNA biosensor detection, in particular to a photoelectrochemistry and electrochemistry double-mode ctDNA sensor based on an ionic liquid functionalized lanthanide metal organic framework and a detection method thereof.
[ Background Art ]
CtDNA (circulating tumor DNA) is increasingly used as a biomarker to guide clinical decisions, leading to better diagnosis, assessment of optimal treatment and monitoring of disease, as well as other clinical use .(Luciana Santos Pessoa,ManoelaHeringer,Valéria Pereira Ferrer.Critical Reviews in Oncology/Hematology.Volume 155,November 2020,103109). studies, indicating that ctDNA can be detected in most early common cancers such as triple negative breast cancer TNBC. The presence of ctDNA detected after surgical excision is also of significant prognostic value, as its presence and number after surgery or treatment reflects the continued presence of clinically undetectable micrometastatic residual lesions. Others have shown that detection of ctDNA can predict prognosis and also predict response to targeted therapies. Thus, ctDNA may be an excellent marker for residual disease in TNBC patients and may be used to guide post-operative therapeutic decisions (Luca Cavallone,AdrianaAguilar-Mahecha,JosianeLafleur,SusieBrousse,MohammedAldamr y,Scientific Reports.volume 10,Article number:14704(2020)).
The biomarker assay with high sensitivity and high selectivity has great significance for diagnosis and prognosis of diseases. At present, excellent sensitivity has been achieved by using different detection techniques, such as Electrochemiluminescence (ECL), electrochemistry (EC), colorimetry (CL), photoelectrochemistry (PEC) and Fluorescence (FL), but most of the reported quantitative measurement works severely rely on single-mode readings, and have the disadvantages of poor external anti-interference capability, high background and error signals caused by different operators, nonstandard analysis processes and the like, thereby affecting the accuracy of analysis to a certain extent. (HANMEIDENG, YAQINCHAI, RUOYUAN, yali Yuan. Anal. Chem.2020,92,12,8364-8370).
[ Summary of the invention ]
In view of the above-mentioned shortcomings of the prior art, it is an object of the present invention to provide photoelectrochemical and electrochemical dual-mode ctDNA sensors based on ionic liquid functionalized lanthanide metal organic frameworks and detection methods thereof. Compared with the reported sandwich electrochemical biosensor for sensitively detecting the circulating tumor DNA, the sandwich electrochemical biosensor provided by the invention uses a photoelectrochemical and electrochemical dual-mode ctDNA sensor, effectively eliminates false positive and false negative signals in photoelectrochemical detection based on two different response mechanisms and relatively independent signal transduction, improves the accuracy and confidence of biological detection, and solves the problems of dependence on single-mode read-out signals, poor external anti-interference capability, different operators, instruments and nonstandard test processes in the detection of ctDNA concentration in the prior art.
The concept and principle of the invention are described as follows: auNPs@Nd-MOF synthesized based on ionic liquid functionalized lanthanide metal organic frameworks has excellent photoelectric conversion performance, the composite material is used as a photoelectric active material to construct a sensor, the sensor is connected with a Capture Probe (CPs) through a gold-sulfur bond, the sensor is hybridized with target DNA (ctDNA) through DNA hybridization reaction, impedance is increased after hybridization, photo-generated electron transmission is blocked, and photocurrent is reduced, so that the quantitative detection of ctDNA concentration is realized. On the other hand, ctDNA hybridizes with signal probe SPs with ferrocene, and the electrochemical signal of SPs is measured by square wave voltammetry to achieve the aim of quantitative analysis of ctDNA concentration. Through photoelectrochemistry (detection concentration range: 1fmolL -1-10nmol L-1, detection limit: 0.63fmol L -1) and electrochemistry (detection concentration range: 1fmol L -1-1nmol L-1, detection limit: 0.30fmol L -1) double-signal output, mutual authentication of detection results under two detection modes is provided, and accuracy of ctDNA concentration detection is effectively improved.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a ctDNA sensor based on an ionic liquid functionalized lanthanide metal organic framework, the preparation of the ctDNA sensor comprising the steps of:
(1) Designing an ionic liquid functionalized lanthanide metal organic framework Ln-MOF, wherein Ln refers to lanthanide metal elements such as La, ce, pr, nd and the like, and the Ln-MOF is formed by assembling and coordinating BDBDBDBIm (Br) 2 ionic liquid and Ln (OH) n through strong coordination bonds; when Ln is Nd, the Nd-MOF is formed by assembling and coordinating BDBDBIm (Br) 2 ionic liquid and neodymium hydroxide Nd (OH) 3 through strong coordination bonds;
Further, the Nd-MOF exhibits a two-dimensional sheet material having a length of 500.+ -.100 nm, a width of 500.+ -.100 nm, and a thickness of 2.+ -. 0.5 nm;
further, the structural formula of the BDBDBIm (Br) 2 ionic liquid is as follows:
Further, the synthetic route of the BDBDBIm (Br) 2 ionic liquid is as follows:
(2) In order to enhance the photoelectric conversion efficiency of Nd-MOF, gold nanoparticles are introduced into Nd-MOF nanosheets by sodium borohydride in-situ reduction chloroauric acid, and an AuNPs@Nd-MOF composite material is synthesized;
Further, the gold nanoparticles have an average particle diameter of 3-5nm.
(3) Designing a capture probe DNA strand CPs, wherein the DNA strand CPs are connected with the AuNPs@Nd-MOF composite material through gold-sulfur bonds, and the DNA sequence of the capture probe DNA strand CPs is as follows: 5'-GCATCATTCATTTGTTTCAAAAAA-3' - (CH 2)6 -SH), the DNA sequence of the target DNA chain ctDNA is 5'-TGAAACAAATGAATGATGCACGTCATGG-3';
(4) The aqueous solution of the AuNPs@Nd-MOF composite material prepared in the step (2) is dripped on the surface of a glassy carbon electrode, and the glassy carbon electrode is dried at room temperature to obtain an AuNPs@Nd-MOF/GCE modified electrode;
(5) Dropwise adding a thiol-containing capture probe DNA strand CPs solution obtained by pretreatment on the AuNPs@Nd-MOF/GCE modified electrode obtained in the step (4) for modification, wherein the thiol of the DNA strand CPs is connected with the AuNPs on the surface of the AuNPs@Nd-MOF/GCE modified electrode through gold sulfide bonds to obtain a CPs/AuNPs@Nd-MOF/GCE modified electrode, and then sealing the residual site on the surface of the electrode by using 6-mercaptohex-1-ol (MCH) to avoid DNA nonspecific adsorption, so as to obtain the MCH/CPs/AuNPs@Nd-MOF/GCE modified electrode; thus, a ctDNA sensor was obtained.
In the step (4), the glassy carbon electrode is pretreated before use, and the pretreatment steps are as follows: a glassy carbon electrode (Glassy carbon electrode, GCE) with an effective diameter of 3mm was polished with an Al 2O3 suspension of 0.05 μm, followed by rinsing the electrode surface with ultrapure water, followed by ultrasonic cleaning in 10wt% HNO 3 solution, absolute ethanol and ultrapure water, and air drying for use.
In the step (4), the concentration of the AuNPs@Nd-MOF composite material is 10mg/mL, and the volume is 5-11 mu L; the preferred concentration is 10mg/mL and the volume is 8. Mu.L.
In the step (5), the CPs solution is pretreated to obtain CPs solution containing sulfhydryl groups: 200. Mu.L of 1. Mu. Mol L -1 CPs was added to 5. Mu.L of 50 mmol L -1 TCEP to reduce disulfide bonds, heated at 95℃for 5min and cooled naturally to room temperature.
In step (5), the concentration of CPs is 0.1 to 2.0. Mu. Mol L -1, preferably 1. Mu. Mol L -1; the modification temperature is 4 ℃; the modification time is 12-24h.
In step (5), the molar concentration of 6-mercaptohex-1-ol (MCH) is greater than the molar concentration of the CPs solution, for example, 1mmol L -1; the modification temperature is room temperature; the modification time is 1-2h.
The application of the ctDNA sensor based on ionic liquid functionalized lanthanide metal organic framework in preparing photoelectrochemical and/or electrochemical sensors for detecting ctDNA. The detection can be performed in a photoelectrochemical mode and/or an electrochemical mode when the detection is applied in particular. The ctDNA concentration is preferably detected in both photoelectrochemical mode and electrochemical mode. More preferably, when ctDNA concentration is detected in a photoelectrochemical mode and an electrochemical mode, two independent electrodes are prepared for photoelectrochemical and electrochemical tests respectively.
A. Photoelectrochemical mode: dripping a ctDNA solution (1 fmol L -1-10nmol L-1) to be detected into the ctDNA sensor for reaction, and detecting a photoelectrochemical signal under the test condition that the constant potential is 0.1V, wherein the solution contains ascorbic acid and phosphate buffer solution (0.01 mol L -1 and pH 7.0); the concentration of ctDNA was determined by the change in photoelectrochemical signal according to Δi (μa) =0.0369 lgC ctDNA(nmol L-1)+0.309(R2 =0.993.
Further, the concentration of the ascorbic acid is 0.0 to 0.25mol L -1, preferably 0.1mol L -1.
Further, the hybridization temperature was 37 ℃; the hybridization time is 10-60min, preferably 50min.
B. Electrochemical mode: designing a signal probe DNA strand SPs, wherein the DNA sequence of the signal probe DNA strand SPs is as follows: 5'-Fc-AAAAACCATGACGT-3', wherein: fc is ferrocene; the ctDNA solution (1 fmol L -1-1nmol L-1) to be detected is dripped into the ctDNA sensor for reaction, then 20 mu L of 1 mu mol L -1 SPs solution is dripped onto the sensor for reaction, and an electrochemical signal is detected in a phosphate buffer solution (0.01 mol L -1, pH 7.4). The concentration of ctDNA was determined by the change in electrochemical signal according to I (μa) =0.471 lgC ctDNA(nmol L-1)+5.539(R2 =0.992.
Further, the concentration of the signal DNA strand SPs was 1. Mu. Mol L -1.
Further, the hybridization temperature was 37 ℃; the hybridization time is 10-60min, preferably 50min.
Compared with the prior art, the invention has the following beneficial technical effects:
The ionic liquid has excellent conductivity, the ionic liquid is coordinated into the MOFs structure, when the MOFs is excited by light, the photo-generated electrons can be quickly conducted to an electrode interface through the ionic liquid monomer, the separation of photo-generated electron-hole pairs is accelerated, the photoelectric conversion efficiency and the photocurrent response are improved, and the sensing performance of the photoelectric sensor is further improved. After gold nanocrystals are grown in the threshold of MOFs pore structure, the optical current response of Nd-MOFs is also facilitated to be improved due to the excellent conductivity and surface plasmon resonance effect of gold nanoparticles. The dual-mode DNA sensor prepared by the method has good biocompatibility, provides mutual authentication of detection results in two detection modes through photoelectrochemistry and electrochemistry dual-signal output, and effectively improves the accuracy of detecting ctDNA concentration.
The dual-mode DNA sensor prepared by the method has the following advantages:
1. in the invention, the surface of the two-dimensional flaky Nd-MOF synthesized by template assistance has a plurality of unsaturated sites of metal ions, so that the carrier concentration can be increased, and the conductivity is improved.
2. In the invention, naBH 4 is used for in-situ reduction of HAuCl 4 to introduce Au nano particles, an AuNPs@Nd-MOF nano composite material is synthesized, and the Au nano composite material is used as a photosensitive element material, so that the separation of photo-generated electron-hole pairs is accelerated, and the photocurrent response performance is improved.
3. In the invention, the constructed dual-mode DNA sensor is simple to prepare and has good selectivity, sensitivity, stability and repeatability.
4. The dual-mode DNA sensor prepared by the method adopts a labeled recovery method to carry out recovery experiments, and verifies the good reliability and practical analysis capability of the sensor for quantitative detection of human serum ctDNA.
5. In the invention, the constructed dual-mode DNA sensor detects ctDNA concentration through photoelectrochemistry and electrochemistry methods, outputs two relatively independent signals, effectively eliminates false positive and false negative signals in single-method detection, and improves accuracy and confidence of biological detection.
[ Description of the drawings ]
Fig. 1 is a 1 HNMR spectrum of BDBDBIm (Br) 2 ionic liquids.
Fig. 2 is a time-of-flight mass spectrum of BDBDBIm (Br) 2 ionic liquid.
Fig. 3 is an infrared spectrum of bddbim (Br) 2 ionic liquid.
FIG. 4 is an infrared spectrum of Nd-MOF.
FIG. 5A is a solid ultraviolet diffuse reflectance spectrum of Nd-MOF, and FIG. 5B is a Tauc plot of Nd-MOF.
FIG. 6 is an X-ray diffraction X-raydiffraction, XRD) pattern of Nd-MOF and AuNPs@Nd-MOF composites.
FIG. 7 is a topography of the Nd-MOF and AuNPs@Nd-MOF composite material.
FIG. 8 is an EDS energy spectrum of AuNPs@Nd-MOF composite material.
FIG. 9 is an XPS energy spectrum of AuNPs@Nd-MOF composite material.
Fig. 10A is a plot of photocurrent response of Nd-MOF and aunps@nd-MOF composites. Fig. 10B is a photocurrent response mechanism diagram.
FIG. 11 is a cyclic voltammogram of a DNA sensor.
Fig. 12 is an ac impedance spectrum of the DNA sensor.
FIG. 13 is a DNA agarose gel electrophoresis.
FIG. 14 is a current-time (i-t) graph of a stepwise modification process of a DNA sensor.
FIG. 15 is an atomic force microscope image of the DNA before and after sensor modification.
FIG. 16 is a plot of the volume optimization of AuNPs@Nd-MOF.
FIG. 17 is an AA concentration optimization chart.
FIG. 18 is a chart of CPs concentration optimization.
FIG. 19 is a graph of ctDNA incubation time optimizations.
FIG. 20 is a chart of SPs incubation time optimizations.
FIGS. 21A and 21B are graphs of photocurrent response and linear relationship of MCH/CPs/AuNPs@Nd-MOF/GCE to ctDNA of different concentrations, respectively. FIGS. 21C and 21D are graphs of square wave voltammetric current response, linear relationship plot of SPs/ctDNA/MCH/CPs/AuNPs@Nd-MOF/GCE versus ctDNA of different concentrations, respectively.
FIG. 22 is a selective diagram of a photoelectrochemical DNA sensor.
FIG. 23 is a selective diagram of an electrochemical DNA sensor.
FIG. 24 is a graph of the stability of AuNPs@Nd-MOF composites.
FIG. 25 is a flow chart of the preparation of a DNA sensor.
Detailed description of the preferred embodiments
The invention is described in further detail below in conjunction with the specific embodiments and the accompanying drawings, and should not be construed as limiting the scope of the invention as claimed.
Example 1: the preparation method of the ctDNA sensor based on the ionic liquid functionalized lanthanide metal organic framework comprises the following steps:
(1) Preparation of BDBDBIm (Br) 2 ionic liquid:
The synthetic route of the BDBDBIm (Br) 2 ionic liquid prepared in this example is as follows:
The method comprises the following specific steps: (S) - (-) -1,1' -bis-2-naphthylamine (2.0450 g,7.19 mmol) was added to 50mL of deionized water, 5 drops of 85wt% concentrated phosphoric acid were added, and the mixture was stirred for 5min, 40wt% aqueous glyoxal (5.2000 g,36 mmol) and paraformaldehyde (1.0800 g,36 mmol) and 50mL of 1, 4-dioxane were added. The mixture was heated to 80℃and ammonium chloride (1.9250 g,36 mmol) was added. The solution was refluxed for 5h and cooled to room temperature. 50mL of saturated aqueous potassium carbonate was added, extracted with dichloromethane (4X 100 mL), the supernatant collected, the solvent was rotary evaporated, and purified by column chromatography (eluent: dichloromethane: methanol=100:1, v/v), the product was collected, the solvent was rotary evaporated, and dried in vacuo to give 1.2840g of product in yield: 63%. The product obtained in the first step (1.2000 g,3.12mmol,1 eq) and dimethyl 5-bromomethyl isophthalate (1.8800 g,6.55mmol,2.1 eq) were placed in a 250mL single-neck flask, 140mL acetonitrile was added for dissolution, and the reaction was continued for 24h after heating to reflux. After the reaction, the reaction solution is poured into a proper amount of anhydrous diethyl ether, precipitation is generated immediately, suction filtration is carried out, the reaction solution is washed with the anhydrous diethyl ether for 3 times, a filter cake is collected, and vacuum drying is carried out to obtain 0.6520g of product, and the yield is: 54%. The product from the second step (600 mg,0.75mmol,1 eq) was placed in a 250mL single-neck flask, 100mL absolute ethanol, 40mL 20wt% hydrochloric acid was added, and the reaction was continued after heating to 85℃and refluxing. After the reaction was completed, the solvent was rotary evaporated and dried in vacuo to give 552mg of the product in the yield: 92%, namely BDBDBIm (Br) 2 ionic liquid.
(2) Preparation of novel ionic liquid functionalized lanthanide metal organic frameworks Nd-MOFs:
NdCl 3.6H2 O (35.9 mg,0.1 mmol) was dissolved in a mixed solution of 18ml deionized water and 2ml absolute ethanol, heated to 90℃and stirred for 1h, and 15ml of the solution was removed by centrifugation to give 5ml of Nd (OH) 3 as a white turbid solution. The turbid solution was transferred to a 25ml reaction kettle, BDBDBIm (Br) 2 ionic liquid (30 mg,0.04 mmol), 5ml N, N-Dimethylformamide (DMF) was added, heated to 120℃and kept at constant temperature for 12h, cooled to room temperature at a rate of 5℃per hour, centrifuged, the precipitate was washed 3 times with DMF, absolute ethanol, respectively, and dried in vacuo to give a yellow solid, the obtained solid was Nd-MOF (yield: 58%).
(3) Preparation of AuNPs@Nd-MOF composite material:
Weighing Nd-MOF (8 mg) in a 25mL reaction bottle, vacuumizing for 60min, injecting 10mL of 20mmol L -1HAuCl4 solution into the reaction bottle under negative pressure, stirring vigorously under ice bath for reaction for 3h, centrifuging to collect solid, dispersing the solid in ultrapure water, dropwise adding 2mL of 0.01mol L -1NaBH4 solution into the reaction bottle under vigorous stirring, immediately changing the color of the solution from yellow to black, stopping the reaction, centrifuging to collect solid, washing with DMF and ultrapure water for 3 times respectively, and vacuum drying to obtain black solid, thus obtaining the AuNPs@Nd-MOF composite material.
(4) Preparation of ctDNA sensor
Pretreatment of a glassy carbon electrode: a glassy carbon electrode (Glassy carbon electrode, GCE) with an effective diameter of 3mm was polished with an Al 2O3 suspension of 0.05 μm, followed by rinsing the electrode surface with ultrapure water, followed by ultrasonic cleaning in 10wt% HNO 3 solution, absolute ethanol and ultrapure water, and air drying for use.
Before the capture probe CPs were immobilized on the electrode surface, 200. Mu.L of 1. Mu. Mol L -1 CPs solution (CPs solution diluted with 1 XTE Buffer (pH 8.0)) was added to 5. Mu.L of 50mmol L -1 Tris (2-carboxyethyl) phosphine (TCEP solution diluted with Tris-HCl (0.01 mol L -1 pH 7.4)) to reduce disulfide bonds, followed by shaking for 1 hour in the absence of light, heating at 95℃for 5 minutes to obtain a thiol-containing capture probe CPs solution, and naturally cooling to room temperature for use. AuNPs@Nd-MOF nanocomposite (10.0 mg) was ultrasonically dispersed in ultrapure water (1 mL). Dripping AuNPs@Nd-MOF composite material (10.0 mg/mL,8.0 mu L) on the surface of a clean glassy carbon electrode, and airing at room temperature to obtain an AuNPs@Nd-MOF/GCE modified electrode; then 20 mu L of 1 mu mol L -1 of CPs solution containing sulfhydryl is dripped on the surface of the AuNPs@Nd-MOF/GCE modified electrode, and incubated for 12h at 4 ℃, and the sulfhydryl of the CPs is connected with the AuNPs on the surface of the AuNPs@Nd-MOF/GCE modified electrode through gold sulfide bond to obtain the CPs/AuNPs@Nd-MOF/GCE modified electrode; then 20 mu L of 1mmol L -1 of 6-mercaptohexan-1 alcohol (MCH) is dripped on CPs/AuNPs@Nd-MOF/GCE to incubate for 1h at room temperature so as to avoid nonspecific adsorption of DNA, the modified electrode is marked as MCH/CPs/AuNPs@Nd-MOF/GCE for standby at room temperature, and the ctDNA sensor is obtained.
Example 2: ctDNA sensor detects ctDNA in photoelectrochemical and/or electrochemical modes
(1) Photoelectrochemical mode: 20 mu L of ctDNA (1 fmol L -1-10nmol L-1) with different concentrations is dripped on MCH/CPs/AuNPs@Nd-MOF/GCE, and hybridization is carried out for 50min at 37 ℃ to obtain the ctDNA/MCH/CPs/AuNPs@Nd-MOF/GCE modified electrode. The photoelectrochemical signal was detected under test conditions with a constant potential of 0.1V in a phosphate buffer (0.01 mol L -1, pH 7.0) containing ascorbic acid (AA, 0.1mol L -1).
(2) Electrochemical mode: 20 mu L of ctDNA (1 fmol L -1-1nmol L-1) with different concentrations is dripped on the MCH/CPs/AuNPs@Nd-MOF/GCE, hybridization is carried out for 50min at 37 ℃, then 20 mu L of SPs with 1.0 mu mol L -1 is added on the surface of the ctDNA/CPs/AuNPs@Nd-MOF/GCE modified electrode for hybridization for 50min under the reaction condition of 37 ℃, and the modified electrode is recorded as SPs/ctDNA/MCH/CPs/AuNPs@Nd-MOF/GCE. The electrochemical signal was detected in phosphate buffer solution (0.01 mol L -1, pH 7.4).
(3) Photoelectrochemical mode + electrochemical mode: mu.L of ctDNA (1 fmol L -1-1nmol L-1) of various concentrations was dropped onto MCH/CPs/AuNPs@Nd-MOF/GCE, hybridized at 37℃for 50min, and the photoelectrochemical signal was detected under test conditions of constant potential of 0.1V in a phosphate buffer solution (0.01 mol L -1, pH 7.0) containing ascorbic acid (AA, 0.1mol L -1). After the electrode surface was cleaned, 20. Mu.L of 1.0. Mu. Mol L -1 of SPs was added to the ctDNA/CPs/AuNPs@Nd-MOF/GCE modified electrode surface and hybridized for 50min at 37℃to give the modified electrode designated SPs/ctDNA/MCH/CPs/AuNPs@Nd-MOF/GCE. The electrochemical signal was detected in phosphate buffer solution (0.01 mol L -1, pH 7.4).
It should be noted that: in practice, the solution containing ctDNA to be detected is detected in a dual mode by utilizing a photoelectrochemistry mode and an electrochemical mode, and the solution to be detected is only added with a sample once. In order to avoid errors, the experimental results of the invention are that independent electrodes are prepared to respectively detect signals in a photoelectrochemical mode and an electrochemical mode.
In each of the above steps, the electrode surface was washed with phosphate buffer (0.01 mol L -1, pH 7.4) to remove non-hybridized oligonucleotides.
All DNA oligonucleotides were synthesized by Sangon biotechnology service limited (china) in the Shanghai and the sequences were as follows:
capture probe CPs:5'-GCATCATTCATTTGTTTCAAAAAA-3' - (CH 2)6 -SH)
ctDNA：5′-TGAAACAAATGAATGATGCACGTCATGG-3′
Signal probe SPs:5'-Fc-AAAAACCATGACGT-3'
1misDNA：5′-TGAATCAAATGAATGATGCACGTCATGG-3′
2misDNA：5′-TGAATGAAATGAATGATGCACGTCATGG-3′
3misDNA：5′-TGAATGTAATGAATGATGCACGTCATGG-3′
non DNA：5′-TACAGAAAAGTGGTACTAAATTCTCTAA-3′
The DNA sensing platform was successfully prepared by the above steps, and the preparation process is shown in FIG. 25.
Measurement method
Photoelectrochemical test system: the excitation light source is a white light LED lamp (5W). The photocurrent measurement adopts a three-electrode system, wherein a working electrode is a ctDNA/MCH/CPs/AuNPs@Nd-MOF/GCE modified electrode, a platinum column electrode is a counter electrode, and a Saturated Calomel Electrode (SCE) is a reference electrode. All photoelectrochemical measurements were performed on a CHI660A electrochemical workstation in a phosphate buffer solution (0.01 mol L -1, pH 7.0) containing ascorbic acid (AA, 0.1mol L -1) at a constant potential of 0.1V.
Electrochemical test system: the electrochemical measurement adopts a three-electrode system, wherein the working electrode is SPs/ctDNA/MCH/CPs/AuNPs@Nd-MOF/GCE, the platinum column electrode is a counter electrode, and the Saturated Calomel Electrode (SCE) is a reference electrode. All electrochemical measurements were performed on a CHI660A electrochemical workstation in a phosphate buffer solution (0.01 mol L -1, pH 7.4) using Square Wave Voltammetry (SWV) in a range of-0.2-0.6V, with a potential increase of 4mV, an amplitude of 25mV, and a frequency of 15Hz.
The photoelectrochemistry and electrochemistry double-mode ctDNA sensor based on the ionic liquid functionalized lanthanide metal organic framework prepared in the embodiment 1 is characterized and tested for performance, and the results are shown in fig. 1-24:
FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of BDBDBIm (Br) 2 ionic liquid, and chemical displacement and attribution analysis of corresponding hydrogen atoms in a molecular structure can be obtained, so that the ionic liquid is successfully synthesized.
Fig. 2 is a time-of-flight mass spectrum of BDBDBIm (Br) 2 ionic liquid. From the figure, it can be seen that M/z= 742.8450 ([ M+H ] +), consistent with the formula weight of the target compound, and the BDBDBIm (Br) 2 ionic liquid is successfully prepared.
Fig. 3 is an infrared spectrum of bddbim (Br) 2 ionic liquid. 3426cm -1 is assigned to the stretching vibration peak of O-H in water molecules, 3152cm -1 is assigned to the stretching vibration peak of carboxylic acid O-H, 1709cm -1,1545cm-1 and 1509cm -1 are assigned to the stretching vibration peak of skeleton of naphthalene ring of the ionic liquid, 1370cm -1 and 1205cm -1 are assigned to the stretching vibration peaks of C=C and C=N skeleton of imidazole ring respectively, and the in-plane bending vibration of=C-N-CH=and-CH=CH-N in imidazole ring is respectively located at 820cm -1 and 743cm -1,656cm-1 and 610cm -1 is assigned to the out-of-plane bending vibration peak of C-H skeleton. The infrared spectrum result shows that BDBDBIm (Br) 2 ionic liquid is successfully prepared.
FIG. 4 is an infrared spectrum of Nd-MOF. The characteristic peak at 3416cm -1 corresponds to the oscillation peak of O-H in the water molecule, and furthermore, no absorption band of carboxylate groups (COOH) was observed in the expected region (1800-1680 cm -1), which can be attributed to complete deprotonation of the carboxylate ligands. However, in the corresponding spectral regions, the presence of characteristic bands of carboxylate groups at 1642-1583cm -1 (asymmetric vibration) and 1447-1343cm -1 (symmetric vibration) can well establish coordination of metal ions with carboxylate ligands, confirming successful coordination of metal ions with carboxylate groups. The characteristic peak of 1515cm -1 is attributed to the skeleton stretching vibration peak of the naphthalene ring of the ionic liquid, and the in-plane bending vibration of=c-N-ch=and-ch=ch-N in the imidazole ring is respectively located at 838cm -1 and 760cm -1,655cm-1 is attributed to the out-of-plane bending vibration peak of the C-H skeleton. Indicating that the metal ion has coordinated with carboxylate, nd-MOF was successfully prepared.
FIG. 5A is a solid ultraviolet diffuse reflectance spectrum of Nd-MOF, and FIG. 5B is a Tauc plot of Nd-MOF. As shown in FIG. 5A, the solid ultraviolet Diffuse reflectance spectrum (Diffuse REFLECTIVE SPECTRA, DRS) shows that Nd-MOF has absorption in the 200-620nm range. Then, the forbidden band width of Nd-MOF was calculated by Tauc plot method, the calculation formula is: (ah v) 1/2＝A(hν-Eg), wherein a is the absorbance coefficient, h is the planck constant, v is the frequency, a is the constant, eg is the semiconductor forbidden bandwidth, and as a result, as shown in fig. 5B, the intersection point of the tangent line and the abscissa is the forbidden bandwidth of the semiconductor material, and the forbidden bandwidth of the Nd-MOF is 1.62eV.
FIG. 6 is an X-ray diffraction (X-raydiffraction, XRD) pattern of Nd-MOF and AuNPs@Nd-MOF composites. From the figure, nd-MOF has a better crystal structure (curve a). After Nd-MOF is loaded with Au nano particles, diffraction peaks at 38.2 degrees, 44.4 degrees, 64.6 degrees and 77.6 degrees respectively correspond to (111), (200), (220) and (311) crystal faces (curve b) of metal Au, and are matched with JCPDS No-99-0056PDF card. Two diffraction peaks of AuNPs@Nd-MOF were observed in the interpolated XRD pattern (local magnified plot of curve b), notably the intensity of the diffraction peak at 48.1℃for AuNPs@Nd-MOF was very low, due to the Jiang Yanshe peak of Au nanoparticles, covering this diffraction peak. From XRD characterization results, auNPs@Nd-MOF nanocomposite materials are successfully synthesized.
FIG. 7 is a topography of the Nd-MOF and AuNPs@Nd-MOF composite material. The morphology of Nd-MOF and AuNPs@Nd-MOF was characterized using a scanning electron microscope, a transmission electron microscope and an atomic force microscope. As shown in FIG. 7, plot A, B, C is an SEM, TEM, and AFM image of an Nd-MOF material, respectively, showing by the plot that Nd-MOF exhibits a two-dimensional sheet material 500.+ -. 100nm in length, 500.+ -. 100nm in width, and 2.+ -. 0.5nm in thickness (FIG. A, B, C). From the TEM images (FIGS. 7D and E), we found that gold nanoparticles having an average size of about 3.9nm were formed on the Nd-MOF surface and relatively uniformly dispersed on the Nd-MOF two-dimensional nanoplatelets.
FIG. 8 is an EDS energy spectrum of AuNPs@Nd-MOF composite material. EDS (ENERGY DISPERSIVE spectrometer) energy spectrum analysis is carried out on the Au NPs@Nd-MOF composite material by using a high-angle annular dark field scanning transmission electron microscope (HAADF-STEM), the corresponding element diagram of the composite material is shown in figure 8, and the result shows that the Au, nd, C, N and O elements are uniformly distributed in the whole Au NPs@Nd-MOF composite material, and the AuNPs@Nd-MOF composite material is successfully prepared.
FIG. 9 is an XPS energy spectrum of AuNPs@Nd-MOF composite material. In the figure, (A) is an XPS energy spectrum of the AuNPs@Nd-MOF composite material. As can be seen from fig. (B), 978.6eV and 1000.0eV are respectively attributed to the electron binding energies of Nd 3d 5/2 and Nd 3d 3/2, indicating that Nd is present as Nd (III) in the complex; from graphs (D), (E) and (F), it can be seen that 84.9eV and 88.5eV in graph (C) are respectively the electron binding energies of Au 04f7/2 and Au 04f5/2, and 284.8eV, 288.8eV, 401.3eV and 532.4eV are respectively attributed to the electron binding energies of C1s, N1 s and O1s, which indicates that AuNPs@Nd-MOF composite material is successfully prepared.
FIG. 10 is a graph of photocurrent response of Nd-MOF and AuNPs@Nd-MOF composites, respectively, tested in a phosphate buffer solution (0.01 mol L -1, pH 7.0) containing 0.1mol L -1 of ascorbic acid (ascorbic acid, AA). As shown in fig. 10A, curve a is the photocurrent response of the bare electrode, which is negligible. The photocurrent of Nd-MOF was 966nA (curve B), and after gold nanoparticles were loaded, the photocurrent increased to 1.61 μa (curve c), probably because gold nanoparticles had excellent conductivity, and the electron transfer process could be accelerated, so that the photogenerated electron-hole pairs were effectively separated, and at the same time, the surface plasmon resonance effect of gold nanoparticles also helped to improve the photocurrent response of Nd-MOF, the mechanism diagram of which is shown in fig. 10B.
FIG. 11 is a Cyclic Voltammetry (CV) characterization of the electrode assembly process using 5.0mmol.L -1K3Fe(CN)6/K4Fe(CN)6 as redox probe for the DNA sensor. As shown in fig. 11, the CV curve of bare GCE has a pair of good K 3Fe(CN)6/K4Fe(CN)6 redox peaks (curve a). After loading aunps@nd-MOF, the peak current increases (curve b) due to its good electron conducting ability. After CPs modification, the peak current was significantly reduced due to electrostatic repulsion between the K 3Fe(CN)6/K4Fe(CN)6 probe and the negatively charged DNA phosphate scaffold (curve c). After MCH is closed, the peak current drops slightly, possibly because it has an impeding effect on electron transfer (curve d). Subsequently, the capture of ctDNA by CPs further enhanced the electrostatic repulsion between the electrode interface and the probe, with a significant decrease in peak current (curve e). After hybridization of ctDNA with SPs, a longer DNA double-stranded structure is formed on the electrode, so that electronegativity of the electrode surface is further enhanced, and peak current is further reduced (curve f).
FIG. 12 is an AC impedance spectrum of a DNA sensor, using 5.0 mmole L -1K3Fe(CN)6/K4Fe(CN)6 as an active probe and 0.1 mole L -1 KCl as a supporting electrolyte, and using electrochemical AC impedance spectroscopy (Electrochemical Impedance Spectroscopy, EIS) to study various modified electrode interface properties. The electrochemical alternating current impedance spectrogram can detect the interface change of the modified electrode, and the spectrogram consists of an arc and a straight line, wherein a straight line low-frequency region is a diffusion control region, and an arc high-frequency region is an electron transfer limiting region. The larger the arc diameter of the high frequency region, the larger the charge transfer resistance (R ct) generated corresponding to the surface of the modified electrode, and the weaker the electron transfer capability, whereas the smaller the R ct value, the stronger the [ Fe (CN) 6] 3-/4- and the electron transfer speed between the electrodes. The relationship between solution resistance (R s), warburg impedance (Zw), double layer capacitance (Cdl) and apparent charge transfer resistance (R ct) in the equivalent circuit of ZSimDemo software is shown in the inset of FIG. 12. The charge transfer resistance values of the modified electrodes of each preparation process can be obtained by fitting data through equivalent circuit diagrams of ZSimDemo software. As can be seen from fig. 12, the R ct value of the bare GCE was 63.1 Ω (a), and when aunps@nd-MOF was modified to the electrode surface, the charge transfer resistance increased from 63.1 Ω (a) to 1084.0 Ω (b), indicating that aunps@nd-MOF was successfully loaded to the electrode surface, impeding charge transfer at the electrode interface. After fixing CPs on AuNPs@Nd-MOF electrodes, the R ct value was 1655.8 Ω (c), and after blocking the active site with MCH, the R ct value increased to 2220.1 Ω (d). This result demonstrates that CPs are effectively immobilized to the aunps@nd-MOF interface, thereby increasing the steric hindrance of the electrode surface and inhibiting electron transfer on the electrode. Subsequently, ctDNA and SPs were applied dropwise to the MCH/CPs/AuNPs@Nd-MOF/GCE electrodes, and the R ct values were increased to 2959.6 Ω (e) and 3524.5 Ω (f), respectively. The successful preparation of the DNA sensor is demonstrated by a stepwise increase in the charge transfer resistance.
FIG. 13 is a schematic diagram showing the verification of DNA hybridization reaction by agarose gel electrophoresis.
200. Mu.L of 10. Mu. Mol L -1 CPs solution was added to 5. Mu.L of 50 mmol L -1 TCEP (TCEP solution diluted with Tris-HCl (0.01 mol L -1 pH 7.4)) and the solution was shaken for 1h in the absence of light, heated at 95℃for 5min and allowed to cool naturally to room temperature for further use. Then, C-T: sulfhydrylation of CPs (20. Mu.L, 10. Mu. Mol L -1) and ctDNA (20. Mu.L, 10. Mu. Mol L -1) were mixed and diluted to a concentration of 1. Mu. Mol L -1, C-S: sulfhydrylation of CPs (20. Mu.L, 10. Mu. Mol L -1) and SPs (20. Mu.L, 10. Mu. Mol L -1) were mixed and diluted to a concentration of 1. Mu. Mol L -1, T-S: ctDNA (20. Mu.L, 1. Mu. Mol L -1) was mixed with SPs (20. Mu.L, 1. Mu. Mol L -1) and diluted to a concentration of 1. Mu. Mol L -1, C-T-S: thiolated CPs (20. Mu.L, 10. Mu. Mol L -1) were mixed with ctDNA (20. Mu.L, 10. Mu. Mol L -1)、SPs(20μL,10μmol L-1) and diluted to a concentration of 1. Mu. Mol L -1, and the above CPs solution, ctDNA solution, SPs solution were each prepared with 1 XSTE Buffer (pH 8.0), and the above mixtures were each diluted with 1 XSTE Buffer (pH 8.0) and heated at 95℃for 5 minutes, naturally cooled to 37℃and incubated at 37℃for 1 hour to complete the hybridization reaction.
Agarose gel electrophoresis: 0.5g agarose was weighed using an electronic analytical balance and placed in a conical flask, 25mL of 1 XTAE buffer was added, heated until the agarose was completely melted, a 2% agarose gel was prepared, and 1. Mu.L of the nucleic acid stain Ultra GelRed was added for staining. Cleaning and airing the inner groove of the electrophoresis tank, putting the electrophoresis tank into a glue making plate, placing the inner groove in a horizontal position, and inserting a comb. And then uniformly mixing the agarose gel solution cooled to about 60 ℃ and pouring the mixture into an inner groove glass plate to form a uniform adhesive layer, standing the mixture at room temperature for about 30 minutes until the agarose gel is completely solidified, vertically and slightly pulling out a comb, placing the gel and an inner groove into an electrophoresis tank, and adding 1 xTAE electrophoresis buffer solution until the gel is about 1cm beyond the adhesive plate. mu.L of DNA LADDER MARKER bp was added to the leftmost lane;
mu.L of DNA sample and 1. Mu.L of 6X DNALoadding Buffer were mixed on a spotting plate, and 6. Mu.L of sample was taken with a 10. Mu.L pipette and added to each lane. And (5) connecting a power supply to carry out electrophoresis, and controlling the voltage at 105V. The sample was observed to move from negative to positive and the power was turned off when 6X DNA Loadding Buffer was moved 2/3 of the distance from the plate and the electrophoresis was stopped. The gel was transferred to a luminescence detection system, observed under uv light, and the DNA, when present, displayed a fluorescent band, and the photographs were saved after exposure using an imaging system.
As shown in FIG. 13, bands of CPs and ctDNA were observed, and SPs lanes were not visible due to the smaller molecular weight. C-T lane: the hybridization of CPs to ctDNA resulted in new bands that migrated at a slower rate than both, indicating that CPs hybridized successfully to ctDNA; lanes C-S: CPs are mixed with SPs, and the bands are still CPs bands, which indicates that CPs and SPs cannot undergo hybridization reaction; T-S lane: mixing ctDNA with SPs produced new bands, indicating that ctDNA can successfully hybridize with SPs; lanes C-T-S: representing the hybridization process of CPs, ctDNA and SPs, new bands appear, indicating successful hybridization. As is clear from a comparison of their bands, a series of bands of different molecular weights only occur in the presence of the target, indicating that hybridization only occurs in the presence of the target.
FIG. 14 is a graph showing that the stepwise modification process of a DNA sensor can also be characterized by a current-time (i-t) curve. Bare GCE(a),AuNPs@Nd-MOF/GCE(b),CPs/AuNPs@Nd-MOF/GCE(c),MCH/CPs/AuNPs@Nd-MOF/GCE(d),ctDNA/MCH/CPs/AuNPs@Nd-MOF/GCE(e) and SPs/ctDNA/MCH/CPs/aunps@nd-MOF/GCE (f) were measured in phosphate buffer solution (0.01 mol L -1, pH 7.0) containing 0.1mol L -1 Ascorbic Acid (AA) with 5W white Light (LED) as excitation source. In the photoelectrochemical sensing system, AA is selected as an electron donor because it can effectively capture photogenerated holes, prevent recombination of electron-hole pairs and promote generation of photocurrent. The photocurrent of the bare GCE (curve a) was negligible, since there was little photocurrent response when a potential of 0.10V was applied. After AuNPs@Nd-MOF nanocomposite is modified to the surface of GCE, the photocurrent is increased to 1.61 mu A (curve b) due to the good photoelectric conversion performance of the AuNPs@Nd-MOF nanocomposite. When CPs were immobilized on the AuNPs@Nd-MOF/GCE interface, the photocurrent was reduced to 1.194 μA (curve c), the active site was blocked with 1mmolL -1 MCH, and the photocurrent was reduced to 683.7nA (curve d). Subsequently, 1nmolL -1 ctDNA was dropped onto the MCH/CPs/AuNPs@Nd-MOF/GCE electrode sensing interface to undergo hybridization reaction, the photocurrent was further reduced to 374.8nA (curve e), and finally, 20. Mu.L of 1. Mu. Mol L -1 SPs was dropped onto the electrode to react for 1h in the absence of light, and the photocurrent was reduced to 236.7nA (curve f). The decrease in photocurrent is due to the poor size and conductivity of biomolecules, which can impede electron and mass transfer between the photoactive material and the ascorbic acid probe, indicating successful fabrication of photoelectrochemical DNA sensors.
FIG. 15 is a graph showing the surface morphology of AuNPs@Nd-MOF/GCE (FIG. 15A, B) and after fixing ctDNA at the MCH/CPs/AuNPs@Nd-MOF/GCE electrode interface (FIG. 15C, D) using an atomic force microscope (atomic force microscope, AFM) to further confirm that ctDNA was successfully modified to the AuNPs@Nd-MOF/GCE surface. The result shows that after ctDNA is modified on the surface of MCH/CPs/AuNPs@Nd-MOF/GCE, the particle morphology and size are obviously different from those of AuNPs@Nd-MOF/GCE, and the result shows that ctDNA is successfully immobilized on the surface of AuNPs@Nd-MOF, so that a DNA sensing interface is constructed.
In order to improve the photoelectric performance of the photoelectrochemical DNA sensor, the influence of the dosage of Au NPs@Nd-MOF on the photoelectric current is optimized. As shown in fig. 16, as the Au nps@nd-MOF (10 mg/ml) solution volume increases, the photocurrent signal value gradually increases, and the aunps@nd-MOF composite material obtains the maximum photocurrent response value when the aunps@nd-MOF volume is 8 μl. Thus, 8. Mu. LAuNPs@Nd-MOF solution was chosen as the optimal volume.
FIG. 17 is a graph of AA concentration optimized for improving detection sensitivity of a DNA sensor. AA plays an important role in the photoelectric conversion process of PEC sensors. As shown in FIG. 17, the AA concentration is controlled within the range of 0-0.25 mol L -1, and when the concentration value is increased to 0.1mol L -1, the photocurrent response of the AuNPs@Nd-MOF composite material reaches a peak value; when the AA concentration value exceeds 0.1mol L -1, the photocurrent response gradually decreases. As the AA concentration increases, more photo-generated holes can be trapped to produce a higher photocurrent response; however, if the AA concentration is too high, electron transfer is hindered, and the photocurrent response value is reduced. Therefore, the optimum concentration of the ascorbic acid solution is 0.1mol L -1.
The photocurrent is influenced by the fixed amount of the DNA strand on the surface of the electrode, and the CPs capture probe can effectively capture the ctDNA strand, so that the analysis sensitivity of the DNA sensor is influenced. As shown in FIG. 18, after the concentration of the CPs capture probe exceeds 1. Mu. Mol L -1, the change value of the photocurrent signal of the CPs/AuNPs@Nd-MOF/GCE modified electrode is small. Therefore, 1. Mu. Mol L -1 was selected as the optimal concentration.
FIG. 19 is a graph showing the effect of incubation time on photocurrent response when the DNA sensor recognizes 1.0nmolL -1 ctDNA in the range of 10 to 60 min. As shown in fig. 19, the difference in photocurrent before and after the sensor recognizes ctDNA gradually increases as the incubation time increases; when the incubation time is 50min, the delta I value of the sensor reaches the maximum; further increasing the incubation time, the difference of photocurrent is not changed obviously, so that 50min is selected as the optimal hybridization time of CPs and ctDNA to obtain the optimal photocurrent signal of ctDNA detection.
FIG. 20 is a graph showing the effect of incubation time on SWV current response signals when the DNA sensor recognizes 1.0. Mu. Mol L -1 SPs in the range of 10-60 min. As shown in fig. 20, the immunosensor recognizes that SWV current response signals before and after SPs gradually increase as incubation time increases; when the incubation time is 50min, the sensor SWV current response signal reaches the maximum; further increasing the incubation time, the SWV current response signal did not change significantly, so 50min was chosen as the optimal hybridization time of ctDNA with SPs to obtain the optimal SWV current response signal for SPs detection.
FIG. 21A is a plot of photocurrent response of MCH/CPs/AuNPs@Nd-MOF/GCE to ctDNA of different concentrations. The change of the photocurrent response can reflect the hybridization reaction process of ctDNA with different concentrations at the interface of the DNA sensor, thereby being used as the basis of the quantitative analysis of the ctDNA. As can be seen from fig. 21B, under the optimal experimental conditions, the photocurrent tests were performed on ctDNA with different concentrations (a to h are 1fmol L-1、10fmol L-1、100fmol L-1、1pmol L-1、10pmol L-1、100pmol L-1、1nmol L-1、10nmol L-1ctDNA),, with increasing ctDNA concentration, the photocurrent difference (Δi=i 0 -I) before and after hybridization of ctDNA gradually increases, Δi has a good linear relationship with the logarithm lgC ctDNA of ctDNA concentration, the linear equation can be expressed as Δi (μa) =0.0369 lgC ctDNA(nmol L-1)+0.309(R2 =0.993), the detection limit is 0.63fmol L -1 (S/n=3), where I 0 and I are photocurrent response values before and after the combination of MCH/CPs/aunps@nd-MOF/GCE and ctDNA, respectively.
Under the optimal experimental conditions, the electrochemical signal response performance of the DNA sensor to ctDNA with different concentrations is tested. Fig. 21C shows SWV current response of electrochemical sensors at different concentrations of ctDNA. As the ctDNA concentration gradually increases (a to g are also gradually increased by 1fmol L-1、10fmol L-1、100fmol L-1、1pmol L-1、10pmol L-1、100pmol L-1、1nmol L-1ctDNA), peak current responses related to redox electroactive substances in order, as shown in fig. 21C and D, linear relations of the peak current responses to the logarithm of ctDNA concentration are shown, the expression of the regression equation is I (μa) =0.471 lgC ctDNA(nmol L-1) +5.539, the linear response range is 1fmolL -1-1nmolL-1, the correlation coefficient value (R 2) is 0.992, and the calculated detection limit is 0.30fmolL -1, respectively.
Table 1 is a table comparing analytical properties of DNA sensors with other methods. As shown in Table 1, compared with other testing methods, the DNA sensor has a wider detection range and a lower detection limit in both the photoelectrochemical mode and the electrochemical mode, and can be used for specific and high-sensitivity detection of ctDNA. And the signals output in the two modes are relatively independent, so that false positive and false negative signals in single-method detection are effectively eliminated, and the accuracy and confidence of biological detection are improved.
Table 1 comparison of analytical properties of DNA sensors with other methods
Abbreviations:Surface-enhancedRaman spectroscopy(SERS);Fluorescence(FL);Electrochemical(EC);Photoelectrochemical(PEC);Chemiluminescent(CL);Lateral flow nucleic acidbiosensor(LFNAB).
To examine the selectivity of the DNA sensor, a selection experiment was performed using non-complementary DNA (non-DNA), one base mismatched DNA (1 mis-DNA), two base mismatched DNA (2 mis-DNA), and three base mismatched DNA (3 mis-DNA) as potential interferents. The concentration of ctDNA was 1pmol L -1, and the concentration of the interfering substance was 100pmol L -1. As shown in fig. 22: the difference in photocurrent response of ctDNA is much larger than that of other interferents, and the difference in photoelectrochemical response of ctDNA and mixtures of ctDNA and other interferents is not significantly changed. Meanwhile, in fig. 23: the electrochemical signal of ctDNA is much greater than other interferents and no significant change in the electrochemical signal of ctDNA and mixtures of ctDNA with other interferents was observed. The photoelectrochemical sensor and the electrochemical sensor are provided with good selectivity on the combination of ctDNA, and can be used as potential tools for detecting ctDNA in complex samples.
To examine the long-term stability of the DNA sensor, twelve MCH/CPs/AuNPs@Nd-MOF/GCE modified electrodes were immersed in a phosphate buffer (0.01 mol L -1, pH 7.4) and stored at 4℃for two weeks, then each of the 6 modified electrodes was used again for measuring 1.0pmol L -1 ctDNA in a photoelectrochemical mode and an electrochemical mode, and as a result, the average values thereof were taken, and it was found that the photocurrent response value was reduced by 4.07% only compared with the current value before storage, and the electrochemical response value was reduced by 5.04% only compared with the electrochemical response value before storage, indicating that the dual-mode sensor had good long-term stability. In addition, to evaluate the photoelectric stability of AuNPs@Nd-MOF/GCE, the AuNPs@Nd-MOF/GCE modified electrode was continuously excited 20 times, as shown in FIG. 24, the photocurrent response did not change significantly, indicating that the modified electrode had good photoelectric stability.
In order to examine the reproducibility of the sensor, six ctDNA/MCH/CPs/AuNPs@Nd-MOF/GCE modified electrodes are prepared in parallel for photoelectrochemical detection, six SPs/ctDNA/MCH/CPs/AuNPs@Nd-MOF/GCE modified electrodes are subjected to electrochemical detection, wherein the concentration of ctDNA is 1.0pmol L -1, the Relative Standard Deviation (RSD) of photocurrent response is 3.95%, and the RSD value of electrochemical response is 3.67%, which indicates that the DNA sensor has good reproducibility and is important for clinical practical sample testing.
In order to further investigate the feasibility and accuracy of the constructed DNA sensor in practical sample detection applications, recovery experiments were performed using a labeled recovery method. The serum sample of healthy people is diluted by 40 times by 1 XTE Buffer solution, ctDNA with different concentrations including 1fmol L -1、100fmol L-1、10pmol L-1、1nmol L-1 is added respectively, and the content of the ctDNA in different samples is detected respectively and independently by a photoelectrochemical sensor and an electrochemical sensor. The results are summarized in Table 2, and Table 2 is a table of the recovery of ctDNA from serum of healthy persons by the DNA sensor. The recovery rate is 93.48.52-105.35%, the RSD is less than 5%, and the good reliability and practical analysis capability of the dual-mode sensor for quantitative detection of human serum ctDNA are verified.
Table 2 detection of ctDNA in serum samples (n=3)
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Claims (15)
1. The ionic liquid BDBDBIm (Br) 2 is characterized in that the ionic liquid BDBDBDBIm (Br) 2 has the structural formula:
2. The use of the ionic liquid BDBDBIm (Br) 2 according to claim 1 in the preparation of a sensor for detecting ctDNA, wherein the DNA sequence of the ctDNA is:
5′-TGAAACAAATGAATGATGCACGTCATGG-3′。
3. the preparation method of the ctDNA sensor based on the ionic liquid functionalized lanthanide metal organic framework is characterized by comprising the following steps of:
(1) Designing an ionic liquid functionalized lanthanide metal organic framework Nd-MOF, wherein the Nd-MOF is a two-dimensional sheet material and is formed by assembling and coordinating ionic liquid BDBDBIm (Br) 2 and Nd (OH) 3 through strong coordination bonds;
(2) In order to enhance the photoelectric conversion efficiency of Nd-MOF, gold nanoparticles are introduced to the surface of an Nd-MOF nanosheet by sodium borohydride in-situ reduction chloroauric acid, and an AuNPs@Nd-MOF composite material is synthesized;
(3) Designing a capture probe DNA strand CPs, wherein the DNA strand CPs are connected with the AuNPs@Nd-MOF composite material through gold-sulfur bonds, and the DNA sequence of the capture probe DNA strand CPs is as follows: 5'-GCATCATTCATTTGTTTCAAAAAA-3' - (CH 2)6 -SH), the DNA sequence of the target DNA chain ctDNA is 5'-TGAAACAAATGAATGATGCACGTCATGG-3';
(4) Dripping the solution of the AuNPs@Nd-MOF composite material prepared in the step (2) on the surface of a glassy carbon electrode, and airing at room temperature to obtain an AuNPs@Nd-MOF/GCE modified electrode;
(5) Dropwise adding the capture probe DNA strand CPs solution containing the sulfhydryl group obtained by pretreatment on the AuNPs@Nd-MOF/GCE modified electrode obtained in the step (4) for modification, and then closing the residual site on the surface of the electrode by using 6-mercaptohexan-1-ol to obtain the MCH/CPs/AuNPs@Nd-MOF/GCE modified electrode; thus, a ctDNA sensor was obtained.
4. A ctDNA sensor based on an ionic liquid functionalized lanthanide metal organic framework according to claim 3, wherein the Nd-MOF exhibits a two-dimensional sheet material with a length of 500±100nm, a width of 500±100nm, and a thickness of 2±0.5 nm.
5. The ctDNA sensor based on ionic liquid functionalized lanthanide metal organic framework according to claim 4, wherein the synthetic route of BDBDBIm (Br) 2 ionic liquid is:
6. The ctDNA sensor based on ionic liquid functionalized lanthanide metal organic framework according to any one of claims 3 to 5, wherein in step (4), the glassy carbon electrode is subjected to pretreatment before use, and the pretreatment steps are as follows: polishing a glassy carbon electrode by using an Al 2O3 suspension with the thickness of 0.05 mu m, then flushing the surface of the electrode by ultrapure water, then sequentially carrying out ultrasonic cleaning in a HNO 3 solution with the weight percentage of 10 percent, and airing for later use; and/or;
In the step (5), the CPs solution is pretreated to obtain CPs solution containing sulfhydryl groups: CPs are added into TCEP to reduce disulfide bonds, heated for 5min at 95 ℃, and naturally cooled to room temperature.
7. The ionic liquid functionalized lanthanide metal organic framework-based ctDNA sensor according to any one of claims 3 to 5, wherein in step (4), the concentration of aunps@nd-MOF composite material is 10mg/mL, and the volume is 5 to 11 μl; and/or;
in the step (5), the concentration of CPs is 0.1-2.0 mu mol L -1; the modification temperature is 4 ℃; the modification time is 12-24 hours; and/or;
In the step (5), the molar concentration of the 6-mercaptohex-1-ol is greater than that of the CPs solution; the modification temperature is room temperature; the modification time is 1-2h.
8. The ionic liquid functionalized lanthanide metal organic framework-based ctDNA sensor according to claim 7,
In the step (5), the concentration of CPs is 1 mu mol L -1; and/or;
In step (5), the concentration of 6-mercaptohex-1-ol was 1mmol L -1.
9. Use of a ctDNA sensor based on an ionic liquid functionalized lanthanide metal organic framework according to any one of claims 3 to 7 for the preparation of a photoelectrochemical and/or electrochemical sensor for detecting ctDNA.
10. Use according to claim 9, wherein the specific use is detectable in photoelectrochemical mode and/or electrochemical mode;
A. Photoelectrochemical mode: dripping a ctDNA solution to be detected into the ctDNA sensor for reaction, and detecting a photoelectrochemical signal under the test condition that the constant potential is 0.1V in a phosphate buffer solution containing ascorbic acid; determining the concentration of ctDNA by a change in photoelectrochemical signal;
the concentration of ctDNA in the ctDNA solution to be detected is 1fmol L -1-10nmol L-1;
The concentration of the phosphate buffer solution is 0.01mol L -1, and the pH=7.0;
B. Electrochemical mode: designing a signal probe DNA strand SPs, wherein the DNA sequence of the signal probe DNA strand SPs is as follows: 5'-Fc-AAAAACCATGACGT-3', wherein: fc is ferrocene; dripping a ctDNA solution to be detected into the ctDNA sensor for reaction, dripping 1 mu mol L -1 SPs solution onto the sensor for reaction, and detecting an electrochemical signal in a phosphate buffer solution; determining the concentration of ctDNA by a change in electrochemical signal;
The concentration of ctDNA in the ctDNA solution to be detected is 1fmol L -1-1nmol L-1;
the phosphate buffer solution had a concentration of 0.01mol L -1, ph=7.4.
11. The use according to claim 10, wherein ctDNA concentration is detected in both photoelectrochemical and electrochemical modes; the concentration of ctDNA in the ctDNA solution to be detected is 1fmol L -1-1nmol L-1.
12. The use according to claim 10 or 11, characterized in that the concentration of ascorbic acid is 0.0-0.25mol L -1 and the concentration of ascorbic acid is not zero.
13. The use according to claim 12, characterized in that the concentration of ascorbic acid is 0.1mol L -1.
14. Use according to claim 10 or 11, characterized in that the detection in photoelectrochemical mode is: hybridization temperature was 37 ℃; hybridization time is 10-60min; and/or;
Detection in electrochemical mode: hybridization temperature was 37 ℃; hybridization time is 10-60min.
15. The use according to claim 14, wherein the photoelectrochemical mode is detected: hybridization time was 50min; and/or;
Detection in electrochemical mode: hybridization time was 50min.
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| Ionic liquid functionalized trapezoidal Zn-MOF nanosheets integrated with gold nanoparticles for photoelectrochemical immunosensing alpha-fetoprotein;Xinming Qin et al.;《Talanta》;20220611;第1-9页 * |
| 基于离子液体的生物电化学传感器;徐梦文等;《武汉大学学报( 理学版)》;20180228;第64卷(第1期);第17-27页 * |
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