CN115974786A - 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 detection of DNA biosensors, and particularly discloses a photoelectrochemistry and electrochemistry dual-mode ctDNA sensor based on an ionic liquid functionalized lanthanide metal organic framework and a detection method thereof. The invention relates to an ionic liquid
Coordination into the MOFs structure and furtherSodium borohydride in-situ reduces chloroauric acid to introduce gold nanoparticles to the surface of the Nd-MOF nanosheet to obtain an AuNPs @ Nd-MOF composite material; the gold-sulfur bond is connected with Capture Probes (CPs), and is hybridized with target DNA (ctDNA) through DNA hybridization reaction, after hybridization, the impedance is increased, the transmission of photo-generated electrons is blocked, and the photocurrent is reduced, so that the quantitative detection of the ctDNA concentration is realized. On the other hand, ctDNA is hybridized with signal probes SPs with ferrocene, and the electrochemical signals of the SPs are measured by square wave voltammetry so as to achieve the aim of quantitatively analyzing the concentration of ctDNA. 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 detection of DNA biosensors, in particular to a photoelectrochemistry and electrochemistry dual-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, among other clinical uses. (Luciana Santos Pessoa, manoela Heringer, valeria Pereira Ferrer. Critical Reviews in Oncology/hematology. Volume 155, november 2020, 103109). Studies have shown that ctDNA can be detected in most early common cancers (e.g., triple negative breast cancer, TNBC). The presence of ctDNA detected after surgical resection also has significant prognostic value, since its presence and number after surgery or treatment reflects the persistent presence of micrometastatic residual lesions that cannot be detected clinically. Others have shown that detection of ctDNA can predict prognosis, as well as response to targeted therapy. ctDNA may therefore be an excellent marker for residual disease in TNBC patients and may be used to guide post-operative treatment decisions (Luca cavalone, adrianaaegular-macha, josianeLafleur, susieBrousse, mohammed aldamy, scientific reports. Volume 10, article number 14704 (2020)).
The high-sensitivity and high-selectivity biomarker determination has very important significance for the 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 determination works rely heavily on single mode readings, with the disadvantages of poor external interference rejection, high background and false signals caused by different operators, non-standard analysis processes, etc., thus affecting the accuracy of the analysis to a certain extent. (Hanmei Deng, yaqinchai, ruoYuan, yali Yuan, anal. Chem.2020,92,12,8364-8370).
[ summary of the invention ]
In view of the above-mentioned deficiencies of the prior art, it is an object of the present invention to provide a photoelectrochemical and electrochemical dual mode ctDNA sensor based on an ionic liquid functionalized lanthanide metal-organic framework and a detection method thereof. Compared with the reported sandwich type electrochemical biosensor for sensitively detecting the circulating tumor DNA (Honglizhao, zhenningniu, kaicha Chen, lijuan Chen, zhenxingWang, microchemical J outer. Volume 171, december 2021, 106783), the invention uses a photoelectrochemistry and electrochemistry dual-mode ctDNA sensor, based on two different response mechanisms and relatively independent signal transduction, effectively eliminates false positive and false negative signals in photoelectrochemistry detection, improves the accuracy and confidence coefficient of biological detection, and solves the problems of dependence on single-mode read signals, poor external anti-interference capability, different operators, instruments and non-standard test processes in the ctDNA concentration detection in the prior art.
The concept and principle of the present invention are illustrated as follows: auNPs @ Nd-MOF synthesized based on an ionic liquid functionalized lanthanide metal organic framework has excellent photoelectric conversion performance, the composite material is used as a photoelectric active material to construct a sensor, the sensor is connected with Capture Probes (CPs) through gold-sulfur bonds, the sensor is hybridized with target DNA (ctDNA) through DNA hybridization reaction, the impedance is increased after hybridization, and the photocurrent is reduced due to the blocking of photo-generated electron transmission, so that the quantitative detection of the ctDNA concentration is realized. On the other hand, ctDNA is hybridized with a ferrocene-carrying signal probe SPs, and the electrochemical signal of the SPs is measured by square wave voltammetry so as to achieve the aim of quantitative analysis of ctDNA concentration. By photoelectrochemistry (detection concentration range: 1fmol L) -1 -10nmol L -1 And detection limit: 0.63fmol L -1 ) And electrochemistry (detection concentration range: 1fmol L -1 -1nmol L -1 And detection limit: 0.30fmol L -1 ) And double-signal output provides mutual authentication of detection results in two detection modes, and the accuracy of ctDNA concentration detection is effectively improved.
In order to achieve the purpose, the invention adopts the following technical scheme:
the ctDNA sensor based on the ionic liquid functionalized lanthanide metal organic framework is prepared by the following steps:
(1) An ionic liquid functionalized lanthanide metal organic framework Ln-MOF is designed, wherein Ln refers to lanthanide metal elements such as La, ce, pr, nd and the like, and the Ln-MOF is prepared from BDBIm (Br) 2 Ionic liquid and Ln (OH) n Assembled and coordinated by a strong coordination bond; when Ln is Nd, the Nd-MOF is formed by BDBIm (Br) 2 Ionic liquid and neodymium hydroxide Nd (OH) 3 Assembled and coordinated by a strong coordination bond;
further, the Nd-MOF presents a two-dimensional sheet material with the length of 500 +/-100 nm, the width of 500 +/-100 nm and the thickness of 2 +/-0.5 nm;
further, the BDBDBIm (Br) 2 The structural formula of the ionic liquid is as follows:
further, the BDBDBIm (Br) 2 The synthetic route of the ionic liquid is as follows:
(2) In order to enhance the photoelectric conversion efficiency of Nd-MOF, sodium borohydride is used for reducing chloroauric acid in situ to introduce gold nanoparticles to Nd-MOF nanosheets, and the AuNPs @ Nd-MOF composite material is synthesized;
further, the average particle diameter of the gold nanoparticles is 3-5nm.
(3) Designing a capture probe DNA chain CPs, wherein the DNA chain CPs is connected with the AuNPs @ Nd-MOF composite material through a gold-sulfur bond, and the DNA sequence of the capture probe DNA chain CPs is as follows: 5'-GCATCATTCATTTGTTTCAAAAAA-3' - (CH) 2 ) 6 -SH, the DNA sequence of the target DNA strand ctDNA is: 5'-TGAAACAAATGAATGATGCACGTCATGG-3';
(4) Dripping the aqueous solution of the AuNPs @ Nd-MOF composite material prepared in the step (2) onto the surface of a glassy carbon electrode, and airing at room temperature to obtain an AuNPs @ Nd-MOF/GCE modified electrode;
(5) Dropwise adding a capture probe DNA chain CPs solution containing sulfydryl obtained by pretreatment on the AuNPs @ Nd-MOF/GCE modified electrode obtained in the step (4) for modification, connecting the sulfydryl of the DNA chain CPs with the AuNPs on the surface of the AuNPs @ Nd-MOF/GCE modified electrode through a gold-sulfur bond to obtain a CPs/AuNPs @ Nd-MOF/GCE modified electrode, and then sealing residual sites on the surface of the electrode by using 6-mercaptohexane-1-ol (MCH) to avoid nonspecific adsorption of DNA, so as to obtain an MCH/CPs/AuNPs @ Nd-MOF/GCE modified electrode; thus obtaining the ctDNA sensor.
In the step (4), the glassy carbon electrode is pretreated before use, and the pretreatment steps are as follows: 0.05 μm of Al is used 2 O 3 Polishing a Glass Carbon Electrode (GCE) with an effective diameter of 3mm with a suspension, rinsing the surface of the electrode with ultrapure water, and sequentially adding 10wt% of HNO 3 Ultrasonically cleaning the solution, absolute ethyl alcohol and ultrapure water, and airing for later use.
In the step (4), the concentration of the AuNPs @ Nd-MOF composite material is 10mg/mL, and the volume is 5-11 muL; preferably at a concentration of 10mg/mL and a volume of 8. Mu.L.
In the step (5), the CPs solution is pretreated to obtain a CPs solution containing sulfydryl: 200. Mu.L of 1. Mu. Mol L -1 CPs 5. Mu.L 50m mol L -1 TCEP reduces disulfide bond, heats at 95 deg.C for 5min, and naturally cools to room temperature.
In the step (5), the concentration of CPs is 0.1-2.0 mu mol L -1 Preferably 1. Mu. Mol L -1 (ii) a The modification temperature is 4 ℃; the modification time is 12-24h.
In step (5), the molar concentration of 6-mercaptohex-1-ol (MCH) may be greater than that of the solution of CPs, e.g., 1mmol L -1 (ii) a The modification temperature is room temperature; the modification time is 1-2h.
The ctDNA sensor based on the ionic liquid functionalized lanthanide metal organic framework is applied to preparation of a photoelectrochemical and/or electrochemical sensor for detecting ctDNA. The specific application can adopt a photoelectrochemical mode and/or an electrochemical mode for detection. The ctDNA concentration is preferably detected in a dual mode of photoelectrochemical mode and electrochemical mode. More preferably, when the 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: the ctDNA solution to be tested (1 fmol L) -1 -10nmol L -1 ) Drop-coated into the ctDNA sensor for reaction in a phosphate buffer solution (0.01 mol L) containing ascorbic acid -1 pH 7.0), detect the photoelectrochemical signal under the test condition of the constant potential of 0.1V; according to Δ I (μ a) =0.0369lgC ctDNA (nmol L -1 )+0.309(R 2 = 0.993), the ctDNA concentration is determined by the change in the photoelectrochemical signal.
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 chain SPs, wherein the DNA sequence of the signal probe DNA chain SPs is as follows: 5'-Fc-AAAAACCATGACGT-3', wherein: fc is ferrocene; the ctDNA solution (1 fmol L) to be tested -1 -1nmol L -1 ) Dropping 20. Mu.L of 1. Mu. Mol L into the ctDNA sensor for reaction -1 SPs solution was reacted onto the sensor in phosphate buffered solution (0.01 mol L) -1 pH 7.4) detecting electrochemical signals. According to the formula I (= 0.471 lgC) ctDNA (nmol L -1 )+5.539(R 2 = 0.992), the ctDNA concentration is determined by the change in electrochemical signal.
Further, the concentration of the signal DNA strand SPs is 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 to the MOFs structure, when the MOFs is excited by light, 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 the gold nanocrystals are threshold-limited grown in the MOFs pore structure, the gold nanoparticles have excellent conductivity and surface plasmon resonance effect, and are also favorable for improving the photocurrent response of the Nd-MOF. 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 double signal output, and effectively improves the accuracy of ctDNA concentration detection.
The dual-mode DNA sensor prepared by the method has the following advantages:
1. in the invention, the two-dimensional sheet Nd-MOF synthesized by the aid of the template has a plurality of unsaturated sites of metal ions on the surface, so that the carrier concentration can be increased, and the conductivity can be improved.
2. In the invention, naBH is used 4 In situ reduction of HAuCl 4 Au nano particles are introduced, an AuNPs @ Nd-MOF nano composite material is synthesized and used as a photosensitive element material, the separation of photo-generated electron-hole pairs is accelerated, and the photocurrent response performance is improved.
3. 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 provided by the invention adopts a labeling recovery method to carry out a recovery experiment, and the good reliability and the practical analysis capability of the sensor on the quantitative detection of human serum ctDNA are verified.
5. In the invention, the constructed dual-mode DNA sensor detects the ctDNA concentration by a photoelectrochemistry method and an electrochemistry method, outputs two relatively independent signals, effectively eliminates false positive and false negative signals in single-method detection, and improves the accuracy and the confidence degree of biological detection.
[ description of the drawings ]
FIG. 1 is BDBIm (Br) 2 Of ionic liquids 1 HNMR spectrogram.
FIG. 2 is BDBIm (Br) 2 Time-of-flight mass spectrum of ionic liquid.
FIG. 3 is BDBIm (Br) 2 Infrared spectrum of 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 (XRD) pattern of Nd-MOF and AuNPs @ Nd-MOF composite materials.
FIG. 7 is a morphology of Nd-MOF and AuNPs @ Nd-MOF composites.
FIG. 8 is EDS energy spectrum of AuNPs @ Nd-MOF composite.
FIG. 9 is an XPS energy spectrum of AuNPs @ Nd-MOF composite.
FIG. 10A is a graph of the photocurrent response of Nd-MOF and AuNPs @ Nd-MOF composites. Fig. 10B is a diagram of a photocurrent response mechanism.
FIG. 11 is a cyclic voltammetry test chart of a DNA sensor.
FIG. 12 is an AC impedance spectrum of the DNA sensor.
FIG. 13 is a DNA agarose gel electrophoresis image.
FIG. 14 is a current-time (i-t) plot of the stepwise modification process of the DNA sensor.
FIG. 15 is an atomic force microscope image before and after modification of the DNA sensor.
FIG. 16 is a graph of volume optimization of AuNPs @ Nd-MOF.
FIG. 17 is an AA concentration optimization plot.
FIG. 18 is a CPs concentration optimization graph.
Fig. 19 is a graph of ctDNA incubation time optimization.
FIG. 20 is a graph of SPs incubation time optimization.
FIGS. 21A and 21B are a photocurrent response graph and a linear relationship graph of MCH/CPs/AuNPs @ Nd-MOF/GCE with respect to ctDNA of different concentrations, respectively. FIGS. 21C and 21D are graphs of the square wave voltammetric current response and the linear relationship of SPs/ctDNA/MCH/CPs/AuNPs @ Nd-MOF/GCE to ctDNA of different concentrations, respectively.
FIG. 22 is a selectivity diagram of a photoelectrochemical DNA sensor.
FIG. 23 is a selectivity graph of an electrochemical DNA sensor.
FIG. 24 is a graph of the stability of AuNPs @ Nd-MOF composites.
FIG. 25 is a flowchart of the preparation of the DNA sensor.
[ detailed description of the invention ]
The following detailed description of the present invention is provided in connection with specific embodiments and 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)BDBDBIm(Br) 2 preparation of ionic liquid:
BDBIm (Br) was prepared in this example 2 The synthetic route of the ionic liquid is as follows:
the method comprises the following specific steps: (S) - (-) -1,1' -bi-2-naphthylamine (2.0450g, 7.19mmol) was added to 50mL of deionized water, 5 drops of 85wt% concentrated phosphoric acid were added, the mixture was stirred for 5min, and 40wt% aqueous glyoxal (5.2000g, 36mmol) and paraformaldehyde (1.0800g, 36mmol) and 50mL of 1,4-dioxane were added. The mixture was heated to 80 ℃ and ammonium chloride (1.9250g, 36mmol) was added. The solution was refluxed for 5h and cooled to room temperature. 50mL of saturated aqueous potassium carbonate solution was added, extracted with dichloromethane (4X 100 mL), the upper layer liquid was collected, the solvent was rotary evaporated, purified by column chromatography (eluent: dichloromethane: methanol =100:1, v/v), the product was collected, the solvent was rotary evaporated, dried in vacuo to give 1.2840g of product, yield: and 63 percent. Taking the product obtained in the first step(1.2000g, 3.12mmol, 1eq) and 5-bromomethyl isophthalic acid dimethyl ester (1.8800g, 6.55mmol, 2.1eq) were placed in a 250mL single-neck flask, dissolved by adding 140mL of acetonitrile, and the reaction was continued for 24 hours after heating to reflux. After the reaction is finished, pouring the reaction liquid into a proper amount of anhydrous ether, immediately generating a precipitate, performing suction filtration, washing with the anhydrous ether for 3 times, collecting a filter cake, and performing vacuum drying to obtain 0.6520g of a product, wherein the yield is as follows: 54 percent. The product obtained in the second step (600mg, 0.75mmol, 1eq) was placed in a 250mL single-neck flask, 100mL of absolute ethanol and 40mL of 20wt% hydrochloric acid were added, and the reaction was continued for 24 hours after heating to 85 ℃ under reflux. After the reaction was complete, the solvent was rotary evaporated and dried in vacuo to afford 552mg of product in yield: 92% of the product BDBDBIm (Br) 2 An ionic liquid.
(2) Preparing novel ionic liquid functionalized lanthanide metal organic framework Nd-MOF:
mixing NdCl 3 .6H 2 Dissolving O (35.9mg, 0.1mmol) and hexamethylenetetramine (168.2mg, 1.2mmol) in a mixed solution of 18ml deionized water and 2ml absolute ethyl alcohol, heating to 90 ℃, stirring for reaction for 1h, centrifuging to remove 15ml of solution, and obtaining 5ml Nd (OH) 3 White turbid solution. The cloudy solution was transferred to a 25ml reaction kettle and BDBIm (Br) was added 2 Heating ionic liquid (30mg, 0.04mmol) and 5ml of N, N-Dimethylformamide (DMF) to 120 ℃, keeping the temperature constant for 12h, reducing the temperature to room temperature at the speed of 5 ℃/h, centrifuging, washing and precipitating for 3 times by using DMF and absolute ethyl alcohol respectively, and drying in vacuum to obtain yellow solid, wherein the obtained solid is Nd-MOF (yield: 58%).
(3) Preparation of AuNPs @ Nd-MOF composite material:
Nd-MOF (8 mg) was weighed in a 25mL reaction flask, evacuated for 60min, and then 10mL of 20mmol L was injected thereinto under negative pressure -1 HAuCl 4 The solution is stirred vigorously for reaction for 3 hours in ice bath, after the reaction is finished, the solid is collected centrifugally and dispersed in ultrapure water, and 2mL of 0.01mol L is added dropwise into the ultrapure water under vigorous stirring -1 NaBH 4 And (3) immediately changing the color of the solution from yellow to black, stopping the reaction, then centrifugally collecting solids, respectively washing the solids for 3 times by using DMF (dimethyl formamide) and ultrapure water, and drying the solids in vacuum to obtain black solids, namely the AuNPs @ Nd-MOF composite material.
(4) Preparation of ctDNA sensor
Pretreating a glassy carbon electrode: 0.05 μm of Al is used 2 O 3 Polishing a Glass Carbon Electrode (GCE) with an effective diameter of 3mm with a suspension, rinsing the surface of the electrode with ultrapure water, and sequentially adding 10wt% of HNO 3 Ultrasonically cleaning the solution, absolute ethyl alcohol and ultrapure water, and airing for later use.
Before the capture probes CPs were immobilized on the electrode surface, 200. Mu.L of 1. Mu. Mol L was added -1 The CPs solution (the CPs solution was diluted with 1 XTE Buffer (pH 8.0)) was added to 5. Mu.L of 50mmol L -1 Tris (2-carboxyethyl) phosphine (TCEP solution in Tris-HCl (0.01 mol L) -1 pH7.4), shaking for 1h in dark, heating at 95 ℃ for 5min to obtain a capture probe CPs solution containing sulfydryl, and naturally cooling to room temperature for later use. AuNPs @ Nd-MOF nanocomposites (10.0 mg) were ultrasonically dispersed in ultrapure water (1 mL). The method comprises the following steps of (1) dripping an AuNPs @ Nd-MOF composite material (10.0 mg/mL,8.0 mu L) onto 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 of the extract was taken -1 Dropwise coating the CPs solution containing sulfydryl on the surface of the AuNPs @ Nd-MOF/GCE modified electrode, incubating for 12h at 4 ℃, and connecting the sulfydryl of the CPs with the AuNPs on the surface of the AuNPs @ Nd-MOF/GCE modified electrode through a gold-sulfur bond to obtain the CPs/AuNPs @ Nd-MOF/GCE modified electrode; then 20. Mu.L of 1mmol L of L is dripped on CPs/AuNPs @ Nd-MOF/GCE -1 Incubating 6-mercaptohexanol-1 (MCH) for 1h at room temperature to avoid non-specific adsorption of DNA, airing at room temperature for later use, and recording the modified electrode as MCH/CPs/AuNPs @ Nd-MOF/GCE to obtain the ctDNA sensor.
Example 2: ctDNA sensor for detecting ctDNA in photoelectrochemical and/or electrochemical modes
(1) Photoelectrochemical mode: adding 20 μ L of ctDNA (1 fmol L) with different concentrations dropwise onto MCH/CPs/AuNPs @ Nd-MOF/GCE -1 -10nmol L -1 ) And hybridizing at 37 ℃ for 50min to obtain the ctDNA/MCH/CPs/AuNPs @ Nd-MOF/GCE modified electrode. In the presence of ascorbic acid (AA, 0.1mol L) -1 ) Phosphoric acid buffer solution (0.01 mol L) -1 pH 7.0), constant potential of 0.1V.
(2) Electrochemical mode:adding 20 μ L of ctDNA (1 fmol L) with different concentrations dropwise on MCH/CPs/AuNPs @ Nd-MOF/GCE -1 -1nmol L -1 ) Hybridizing at 37 ℃ for 50min, and then, reacting at 37 ℃ with 20. Mu.L of 1.0. Mu. Mol L -1 Adding SPs to the surface of the ctDNA/CPs/AuNPs @ Nd-MOF/GCE modified electrode for hybridization for 50min to obtain a modified electrode which is marked as SPs/ctDNA/MCH/CPs/AuNPs @ Nd-MOF/GCE. In phosphate buffer solution (0.01 mol L) -1 pH 7.4) detecting electrochemical signals.
(3) Photoelectrochemical mode + electrochemical mode: adding 20 μ L of ctDNA (1 fmol L) with different concentrations dropwise onto MCH/CPs/AuNPs @ Nd-MOF/GCE -1 -1nmol L -1 ) Hybridizing at 37 deg.C for 50min in the presence of ascorbic acid (AA, 0.1mol L) -1 ) Phosphoric acid buffer solution (0.01 mol L) -1 Ph 7.0), detect the photoelectrochemical signal under the test condition of constant potential of 0.1V. After the electrode surface was washed, 20. Mu.L of 1.0. Mu. Mol/L was added under reaction conditions of 37 ℃ to the electrode -1 Adding SPs to the surface of the ctDNA/CPs/AuNPs @ Nd-MOF/GCE modified electrode for hybridization for 50min to obtain a modified electrode which is marked as SPs/ctDNA/MCH/CPs/AuNPs @ Nd-MOF/GCE. In a phosphate buffer solution (0.01 mol L) -1 pH 7.4) detecting electrochemical signals.
It should be noted that: actually, the solution to be detected containing ctDNA is detected in a dual mode of photoelectrochemical mode and electrochemical mode, and the solution to be detected is added only once. In order to avoid errors, the experimental results of the invention are that independent electrodes are prepared to respectively carry out signal detection in a photoelectrochemical mode and an electrochemical mode.
Each step was performed with phosphate buffer (0.01 mol L) -1 pH 7.4) to remove non-hybridized oligonucleotides.
All DNA oligonucleotides were synthesized by shanghai Sangon bioengineering technology services limited (china) and the sequences are as follows:
capture probes CPs:5'-GCATCATTCATTTGTTTCAAAAAA-3' - (CH) 2 ) 6 -SH
ctDNA：5′-TGAAACAAATGAATGATGCACGTCATGG-3′
Signal probes 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 is successfully prepared by the steps, and the preparation process is shown in figure 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, 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 an assay environment containing ascorbic acid (AA, 0.1mol L) -1 ) Phosphoric acid buffer solution (0.01 mol L) -1 pH 7.0), constant potential 0.1V.
Electrochemical test system: the electrochemical determination adopts a three-electrode system, 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 phosphate buffered saline (0.01 mol L) -1 pH 7.4), measured using Square Wave Voltammetry (SWV), with a measurement range of-0.2-0.6V, a potential increment of 4mV, an amplitude of 25mV, and a frequency of 15Hz.
The photoelectrochemistry and electrochemistry dual-mode ctDNA sensor based on the ionic liquid functionalized lanthanide metal organic framework prepared in the example 1 is subjected to characterization and performance test, and the results are shown in figures 1-24:
FIG. 1 is BDBIm (Br) 2 The nuclear magnetic resonance hydrogen spectrogram of the ionic liquid can be obtained by analyzing the chemical shift and the attribution of the corresponding hydrogen atoms in the molecular structure, and the ionic liquid is successfully synthesized.
FIG. 2 is BDBIm (Br) 2 Time-of-flight mass spectrum of ionic liquid. From the figure, M/z =742.8450 ([ M + H)] + ) The formula weight of the compound is consistent with that of the target compound, and the BDBDBIm (B) is proved to be successfully preparedr) 2 An ionic liquid.
FIG. 3 is BDBIm (Br) 2 Infrared spectrum of ionic liquid. 3426cm -1 3152cm and belongs to the stretching vibration peak of O-H in water molecules -1 The characteristic band of (A) is attributed to the carboxylic acid O-H stretching vibration peak, 1709cm -1 ，1545cm -1 And 1509cm -1 The characteristic peak of the ionic liquid belongs to the skeleton stretching vibration peak of naphthalene ring of the ionic liquid, 1370cm -1 And 1205cm -1 C = C and C = N skeleton extensional vibration peaks assigned to imidazole rings, respectively, in-plane flexural vibrations of = C-N-CH = and-CH = CH-N in imidazole rings were located at 820cm, respectively -1 And 743cm -1 ，656cm -1 And 610cm -1 The peak belongs to the out-of-plane bending vibration peak of the C-H framework. The infrared spectrum result shows that BDBDBIm (Br) is successfully prepared 2 An ionic liquid.
FIG. 4 is an infrared spectrum of Nd-MOF. 3416cm -1 The characteristic peak at (A) corresponds to the oscillation peak of O-H in water molecules, and moreover, in the expected region (1800-1680 cm) -1 ) No absorption band of the carboxylate group (COOH) was observed, which could be attributed to complete deprotonation of the carboxylate ligands. However, in the corresponding spectral region, in the range of 1642-1583cm -1 (asymmetric vibration) and 1447-1343cm -1 The characteristic band of carboxylate group exists at the (symmetrical vibration) position, so that the coordination of metal ions and carboxylate ligands can be well established, and the successful coordination of the metal ions and carboxylate radicals is confirmed. 1515cm -1 The characteristic peak of (A) 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 positioned at 838cm -1 And 760cm -1 ，655cm -1 The peak belongs to the out-of-plane bending vibration peak of the C-H framework. The metal ions are shown to be coordinated with carboxylate radicals, and Nd-MOF is 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, solid UV Diffuse Reflectance Spectroscopy (DRS) showed that Nd-MOF absorbed in the 200-620nm range. Then, calculating the forbidden bandwidth of the Nd-MOF by a Tauc plot method, wherein the calculation formula is as follows: (ah ν) 1/2 ＝A(hν-E g ) Wherein a is light absorptionThe result of the coefficient, h being the planck constant, ν being the frequency, a being the constant, and Eg being the semiconductor forbidden bandwidth is shown in fig. 5B, and the intersection of the tangent and the abscissa being the forbidden bandwidth of the semiconductor material, the forbidden bandwidth of the Nd-MOF obtained is 1.62eV.
FIG. 6 is an X-ray diffraction (XRD) pattern of Nd-MOF and AuNPs @ Nd-MOF composite materials. It can be seen that Nd-MOF has a better crystal structure (curve a). After Nd-MOF supports Au nanoparticles, diffraction peaks appearing at 38.2 degrees, 44.4 degrees, 64.6 degrees and 77.6 degrees respectively correspond to crystal faces (curve b) of (111), (200), (220) and (311) of metal Au, and are matched with JCPDS No-99-0056PDF card. AuNPs @ Nd-MOF has two diffraction peaks of Nd-MOF observed in the interpolated XRD pattern (partial magnified view of curve b), and it is noted that the diffraction peak intensity of AuNPs @ Nd-MOF at 48.1 deg. is very low due to the strong diffraction peak of Au nanoparticles, covering the diffraction peaks. The results of XRD characterization show that AuNPs @ Nd-MOF nanocomposite materials are successfully synthesized.
FIG. 7 is a morphology of Nd-MOF and AuNPs @ Nd-MOF composites. The shapes of Nd-MOF and AuNPs @ Nd-MOF are characterized by adopting a scanning electron microscope, a transmission electron microscope and an atomic force microscope. As shown in FIG. 7, A, B, C is an SEM, TEM and AFM image of a Nd-MOF material, respectively, from which it can be seen that the Nd-MOF exhibits a two-dimensional sheet material of 500 + -100 nm in length, 500 + -100 nm in width and 2 + -0.5 nm 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 surface of Nd-MOF and were relatively uniformly dispersed on the Nd-MOF two-dimensional nanoplatelets.
FIG. 8 is EDS energy spectrum of AuNPs @ Nd-MOF composite. 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 FIG. 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, so that the AuNPs @ Nd-MOF composite material is successfully prepared.
FIG. 9 is an XPS energy spectrum of AuNPs @ Nd-MOF composite. In the figure, (A) is an XPS energy spectrum of AuNPs @ Nd-MOF composite material. As shown in FIG. B978.6eV and 1000.0eV are assigned to Nd 3d 5/2 And Nd 3d 3/2 Indicating that Nd exists as Nd (III) in the composite; in FIG. C, 84.9eV and 88.5eV are Au respectively 0 4f 7/2 And Au 0 4f 5/2 The electron binding energies of (D), (E) and (F) indicate that 284.8eV, 288.8eV, 401.3eV and 532.4eV belong to the electron binding energies of C1s, N1 s and O1s respectively, and thus the successful preparation of AuNPs @ Nd-MOF composite material is demonstrated.
FIG. 10 shows a graph containing 0.1mol L -1 Phosphate buffer solution (0.01 mol L) of Ascorbic Acid (AA) -1 pH 7.0) were tested for photocurrent response plots of the Nd-MOF and AuNPs @ Nd-MOF composites, respectively. As shown in fig. 10A, curve a is the photocurrent response of the bare electrode, which is negligible. The photocurrent of the Nd-MOF was 966nA (curve B), and increased to 1.61 μ a (curve c) after loading the gold nanoparticles, probably because the gold nanoparticles had excellent conductivity, which could accelerate the electron transfer process, thereby effectively separating the photo-generated electron-hole pairs, and simultaneously, the surface plasmon resonance effect of the gold nanoparticles also helps to improve the photocurrent response of the Nd-MOF, and the mechanism diagram thereof is shown in fig. 10B.
FIG. 11 is a cyclic voltammetry test chart for a DNA sensor, at 5.0mmol.L -1 K 3 Fe(CN) 6 /K 4 Fe(CN) 6 For redox probes, the assembly process of the electrodes was characterized using Cyclic Voltammetry (CV). As shown in FIG. 11, the CV curve of the bare GCE has a good pair of Ks 3 Fe(CN) 6 /K 4 Fe(CN) 6 Redox peak (curve a). The peak current increased after loading AuNPs @ Nd-MOF due to its good electron conductivity (curve b). After modification with CPs, due to K 3 Fe(CN) 6 /K 4 Fe(CN) 6 The electrostatic repulsion between the probe and the negatively charged DNA phospho-scaffold resulted in a significant decrease in peak current (curve c). After MCH occlusion, the peak current drops slightly, probably because it has a blocking effect on electron transport (curve d). Subsequently, capture of ctDNA by CPs further enhanced electrostatic repulsion between the electrode interface and the probe, with a significant reduction in peak current (curve e). After hybridization of ctDNA with SPsA longer DNA double-stranded structure is formed on the electrode, so that the electronegativity of the electrode surface is further enhanced and the peak current is further reduced (curve f).
FIG. 12 is an AC impedance spectrum of a DNA sensor at 5.0mmol L -1 K 3 Fe(CN) 6 /K 4 Fe(CN) 6 0.1mol L as active probe -1 KCl is a supporting electrolyte, and Electrochemical Impedance Spectroscopy (EIS) is adopted to research the interface properties of various modified electrodes. The electrochemical alternating-current impedance spectrogram can detect the interface change of the modified electrode and consists of an arc part and a straight line part, wherein the low-frequency area of the straight line is a diffusion control area, and the high-frequency area of the arc part is an electron transfer limiting area. The larger the diameter of the arc in the high frequency region, the larger the charge transfer resistance (R) occurs on the surface of the corresponding modified electrode ct ) The larger the electron transport capacity, the weaker the electron transport capacity, and conversely, R ct The smaller the value, the smaller is [ Fe (CN) 6 ]] 3-/4- And the stronger the electron transfer rate between the electrodes. Solution resistance (R) in equivalent circuit of ZSIM Demo software s ) Warburg impedance (Zw), double layer capacitance (Cdl), and apparent charge transfer resistance (R) ct ) The relationship between them 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 to an equivalent circuit diagram of the ZSimDemo software. As can be seen in FIG. 12, R of the bare GCE ct The value is 63.1 omega (a), when AuNPs @ Nd-MOF is modified to the surface of the electrode, the charge transfer resistance is increased from 63.1 omega (a) to 1084.0 omega (b), which indicates that AuNPs @ Nd-MOF is successfully loaded to the surface of the electrode and hinders the charge transfer of the electrode interface. After immobilization of CPs on an AuNPs @ Nd-MOF electrode, R ct Value 1655.8 Ω (c), after active site blocking with MCH, R ct The value increased to 2220.1 Ω (d). This result demonstrates that CPs are efficiently immobilized to the aunps @ nd-MOF interface, thereby increasing steric hindrance of the electrode surface and inhibiting electron transfer on the electrode. Subsequently, ctDNA and SPs were drop-coated onto MCH/CPs/AuNPs @ Nd-MOF/GCE electrodes, R ct The values increased to 2959.6 Ω (e) and 3524.5 Ω (f), respectively. Successful fabrication of the DNA sensor is demonstrated by the gradual increase in charge transfer resistance.
FIG. 13 is a graph showing the DNA hybridization reaction process using agarose gel electrophoresis.
200. Mu.L of 10. Mu. Mol L -1 CPs solution 5. Mu.L 50mmol L -1 TCEP (the TCEP solution is Tris-HCl (0.01 mol L) -1 pH7.4), shaking in the dark for 1h, heating at 95 ℃ for 5min, and naturally cooling to room temperature for later use. Then, C-T: thiolated CPs (20. Mu.L, 10. Mu. Mol L) -1 ) With ctDNA (20. Mu.L, 10. Mu. Mol L) -1 ) After mixing, diluting the mixture until the concentration is 1 mu mol L -1 C-S: thiolated CPs (20. Mu.L, 10. Mu. Mol L) -1 ) With SPs (20. Mu.L, 10. Mu. Mol L) -1 ) Mixing and diluting to the concentration of 1 mu mol L -1 T-S: the ctDNA (20. Mu.L, 1. Mu. Mol L) -1 ) With SPs (20. Mu.L, 1. Mu. Mol L -1 ) Mixing and diluting to the concentration of 1 mu mol L -1 C-T-S: thiolated CPs (20. Mu.L, 10. Mu. Mol L) -1 ) With ctDNA (20. Mu.L, 10. Mu. Mol L) -1 )、SPs(20μL,10μmol L -1 ) After mixing, diluting the mixture until the concentration is 1 mu mol L -1 The above CPs solution, ctDNA solution, SPs solution were prepared with 1 XTE Buffer (pH 8.0), and the above mixture was diluted with 1 XTE Buffer (pH 8.0), 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 of agarose was weighed using an electronic analytical balance and placed in a conical flask, 25mL of 1 XTAE buffer was added, and the mixture was heated until the agarose was completely melted to prepare a 2% agarose gel solution, and 1. Mu.L of nucleic acid stain Ultra GelRed was added for staining. Cleaning the inner groove of the electrophoresis tank, drying in the air, putting the electrophoresis tank into a rubber plate, placing the inner groove in a horizontal position, and inserting a comb. Then, the agarose gel liquid cooled to about 60 ℃ is uniformly mixed and poured onto a glass plate of an inner groove to form a uniform gel layer, the agarose gel is kept still for about 30min at room temperature until the agarose gel is completely solidified, a comb is vertically pulled out slightly, the gel and the inner groove are placed into an electrophoresis tank, and 1 XTAE electrophoresis buffer solution is added until the agarose gel is submerged for about 1 cm. Add 6. Mu.L of 500bp DNA Ladder Marker to the leftmost lane;
mu.L of the DNA sample and 1. Mu.L of 6 XDNAloading Buffer were mixed on the spotting plate, and 6. Mu.L of the sample was taken with a 10. Mu.L pipette and added to each lane. And (5) switching in a power supply to carry out electrophoresis, and controlling the voltage to be 105V. And observing that the sample moves from the negative electrode to the positive electrode, and turning off the power supply when the 6 XDNA loading Buffer moves to a position 2/3 away from the lower part of the gel plate to stop electrophoresis. And (3) transferring the gel to a luminescence detection system, observing under ultraviolet light, displaying a fluorescence band when DNA exists, and storing a photo after exposure by using an imaging system.
As shown in FIG. 13, bands of CPs and ctDNA were observed, and the bands were not evident in the SPs lane because of the smaller molecular weight. Lanes C-T: the hybrid reaction of the CPs and the ctDNA generates a new band, the migration speed of the new band is slower than that of the CPs and the ctDNA, and the CPs and the ctDNA can be successfully hybridized; lanes C-S: CPs and SPs are mixed, and the bands are still CPs bands, which indicates that the CPs and the SPs can not generate hybridization reaction; lanes T-S: mixing ctDNA with SPs produced new bands, indicating that ctDNA can successfully hybridize to SPs; lanes C-T-S: representing the hybridization reaction process of CPs, ctDNA and SPs, a new band appeared, indicating the successful progress of the hybridization reaction. As is clear from comparison of the bands, a series of bands of different molecular weights appeared only in the presence of the target, indicating that the hybridization reaction occurred only in the presence of the target.
FIG. 14 is a step-by-step modification process of the DNA sensor that can also be characterized by a current-time (i-t) curve. Naked 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) in a medium containing 0.1mol L -1 Ascorbic Acid (AA) phosphate buffer solution (0.01 mol L) -1 pH 7.0) with 5W white Light (LED) as excitation source. In this photoelectrochemical sensing system, AA was chosen as the electron donor because it efficiently captures photogenerated holes, prevents recombination of electron-hole pairs and promotes generation of a photocurrent. The photocurrent of the bare GCE (curve a) was negligible, since there was almost no photocurrent response when a 0.10V potential was applied. After the AuNPs @ Nd-MOF nanocomposite is modified to the surface of GCE, the photocurrent is increased to 1.61 muA (curve b) because the AuNPs @ Nd-MOF nanocomposite has good photoelectric conversion performance. After CPs were immobilized at the AuNPs @ Nd-MOF/GCE interface, the photocurrent was reduced to 1.194. Mu.A (curve c) with 1mmol L -1 MCH blocks the active site and the photocurrent is reduced to 683.7nA (curve d). Subsequently, 1nmolL was added -1 After the ctDNA is dripped to the MCH/CPs/AuNPs @ Nd-MOF/GCE electrode sensing interface for hybridization reaction, the photoelectric current is further reduced to 374.8nA (curve e), and finally, 20 mu L of 1 mu mol L is dripped on the electrode -1 SPs were left to react for 1h with light, and the photocurrent was reduced to 236.7nA (curve f). The decrease in photocurrent is due to the poor size and conductivity of the biomolecules that prevent electron and mass transfer between the photo-electrically active material and the ascorbic acid probe, indicating successful fabrication of a photoelectrochemical DNA sensor.
FIG. 15 is a graph showing the surface morphology of AuNPs @ Nd-MOF/GCE (FIG. 15A, B) and after the ctDNA is immobilized at the electrode interface of MCH/CPs/AuNPs @ Nd-MOF/GCE (FIG. 15C, D) in order to further confirm the successful modification of ctDNA to the surface of AuNPs @ Nd-MOF/GCE by Atomic Force Microscope (AFM). The result shows that after the 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 the ctDNA is successfully immobilized on the surface of AuNPs @ Nd-MOF to construct a DNA sensing interface.
In order to improve the photoelectric performance of the photoelectrochemical DNA sensor, the influence of the dosage of Au NPs @ Nd-MOF on photocurrent is optimized. As shown in FIG. 16, the photocurrent signal value gradually increased with the increase of the volume of the Au NPs @ Nd-MOF (10 mg/ml) solution, and the maximum photocurrent response value was obtained when the volume of the Au NPs @ Nd-MOF was 8. Mu.L. Therefore, an optimal volume was chosen for the solution of 8. Mu. LAuNPs @ Nd-MOF.
FIG. 17 is an AA concentration optimized for improving the detection sensitivity of the DNA sensor. AA plays an important role in the photoelectric conversion process of PEC sensors. As shown in FIG. 17, the AA concentration was controlled to 0 to 0.25mol L -1 In the range, as the concentration value increases to 0.1mol L -1 When the photocurrent response of the AuNPs @ Nd-MOF composite material reaches the peak value; when the AA concentration value exceeds 0.1mol L -1 As time goes by, the photocurrent response gradually decreases. As AA concentration increases, more photogenerated holes can be captured to produce a higher photocurrent response; however, when the concentration of AA is too high, electron transfer is hindered, resulting in a decrease in photocurrent response value. Thus, the optimum concentration of ascorbic acid solution is 0.1mol L -1 。
The photocurrent is influenced by the fixed amount of the DNA chain on the surface of the electrode, and the CPs capture probe can effectively capture the ctDNA chain, so that the analysis sensitivity of the DNA sensor is influenced. As shown in FIG. 18, the concentration of the CPs capture probe exceeded 1. Mu. Mol L -1 And then, the change value of the photocurrent signal of the CPs/AuNPs @ Nd-MOF/GCE modified electrode is small. Thus, 1. Mu. Mol L was selected -1 Is the optimum concentration.
FIG. 19 shows the discrimination of 1.0nmolL by DNA sensor in consideration of the incubation time in the range of 10 to 60min -1 Influence of photocurrent response in ctDNA. As shown in fig. 19, as the incubation time increased, the difference in photocurrent before and after the sensor recognized ctDNA gradually increased; when the incubation time is 50min, the delta I value of the sensor reaches the maximum; further increasing the incubation time, the photocurrent difference value has no obvious change, so that 50min is selected as the optimal hybridization time of the CPs and the ctDNA to obtain the optimal photocurrent signal for the ctDNA detection.
FIG. 20 shows the discrimination of 1.0. Mu. MolL by DNA sensor in consideration of the incubation time in the range of 10 to 60min -1 The SWV current at SPs responds to the effects of the signal. As shown in fig. 20, as the incubation time increased, the SWV current response signals before and after the immunosensor recognized SPs gradually increased; 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 and SPs to obtain the optimal SWV current response signal for SPs detection.
FIG. 21A is the photocurrent response of MCH/CPs/AuNPs @ Nd-MOF/GCE to different concentrations of ctDNA. 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 for quantitative analysis of the ctDNA. As can be seen from FIG. 21B, under the optimal experimental conditions, photocurrent measurements were performed on ctDNA of different concentrations (a to h were sequentially 1fmol L) -1 、10fmol L -1 、100fmol L -1 、1pmol L -1 、10pmol L -1 、100pmol L -1 、1nmol L -1 、10nmol L -1 ctDNA), with increasing ctDNA concentration, photocurrent difference before and after hybridization of ctDNA (Δ I = I) 0 -I) increasing, the ratio of Δ I to ctDNA concentrationNumber lgC ctDNA In a good linear relationship, the linear equation can be expressed as: Δ I (μ a) =0.0369lgC ctDNA (nmol L -1 )+0.309(R 2 = 0.993), detection limit of 0.63fmol L -1 (S/N = 3), wherein I 0 And I is the photocurrent response value 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 the SWV current response of the electrochemical sensor at different concentrations of ctDNA. With increasing ctDNA concentration (1 fmol L from a to g in sequence) -1 、10fmol L -1 、100fmol L -1 、1pmol L -1 、10pmol L -1 、100pmol L -1 、1nmol L -1 ctDNA), the peak current response associated with the redox electroactive species also gradually increases. As shown in FIGS. 21C and D, which show the linear relationship between the peak current response and the logarithm of ctDNA concentration, the expressions of the regression equation are I (= 0.471 lgC), respectively ctDNA (nmol L -1 ) +5.539, linear response range of 1fmol L -1 -1nmolL -1 Value of correlation coefficient (R) 2 ) Is 0.992, and the detection limit is calculated to be 0.30fmolL -1 。
Table 1 is a table comparing analytical characteristics of the DNA sensor 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 highly sensitive detection of ctDNA. And the signals output in the two modes are relatively independent, so that false positive and false negative signals in the detection by a single method are effectively eliminated, and the accuracy and the confidence coefficient of biological detection are improved.
TABLE 1 comparison of analytical characteristics 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 non-complementary DNA (non-DNA), a DNA with one base mismatch (1 mis-DNA), a DNA with two base mismatches (2 mis-DNA), and a DNA with three base mismatches (3 mis-DNA) were selected as potential interferents to perform a selectivity experiment. The concentration of ctDNA was 1pmol L -1 The concentration of the interfering substance was controlled to be 100pmol L -1 Then (c) is performed. As shown in fig. 22: the difference of photocurrent response of ctDNA is much larger than that of other interferents, and the difference of photoelectrochemical response of ctDNA and the mixture of ctDNA and other interferents has no significant change. Meanwhile, in fig. 23: the electrochemical signal of ctDNA is much larger than other interferents, and no significant change in electrochemical signal of ctDNA and mixtures of ctDNA with other interferents was observed. The combination of the photoelectrochemical sensor and the electrochemical sensor to the ctDNA has good selectivity, and the photoelectrochemical sensor and the electrochemical sensor can be used as a potential tool for detecting the ctDNA in a complex sample.
In order to examine the long-term stability of the DNA sensor, twelve MCH/CPs/AuNPs @ Nd-MOF/GCE modified electrodes were soaked in phosphate buffer (0.01 mol L) -1 pH 7.4), stored at 4 ℃ for two weeks, and then 6 modified electrodes were taken each and used again in the measurement of 1.0pmol L in the photoelectrochemical mode and the electrochemical mode, respectively -1 The result of ctDNA is respectively taken as the average value, and the result shows that the photocurrent response value is only reduced by 4.07 percent compared with the photocurrent value before storage, and the electrochemical response value is only reduced by 5.04 percent compared with the electrochemical response value before storage, which indicates that the dual-mode sensor has good long-term stability. In addition, in order to evaluate the photoelectric stability of AuNPs @ Nd-MOF/GCE, the AuNPs @ Nd-MOF/GCE modified electrode is continuously excited for 20 times, as shown in FIG. 24, the photocurrent response has no obvious change, and the modified electrode is proved to have good photoelectric stability.
To investigate the reproducibility of the sensor, six ctDNA/MCH/C were prepared in parallelCarrying out photoelectrochemical detection on the Ps/AuNPs @ Nd-MOF/GCE modified electrode, and carrying out electrochemical detection on the six-branch SPs/ctDNA/MCH/CPs/AuNPs @ Nd-MOF/GCE modified electrode, wherein the concentration of ctDNA is 1.0pmol L -1 The Relative Standard Deviation (RSD) of the photocurrent response is 3.95%, and the RSD value of the electrochemical response is 3.67%, which indicates that the DNA sensor has good reproducibility and is important for clinical actual sample testing.
In order to further research the feasibility and the accuracy of the constructed DNA sensor in the detection application of the actual sample, a recovery experiment is carried out by adopting a standard recovery method. Diluting the human serum sample with 1 × TE Buffer solution 40 times, respectively adding ctDNA with different concentrations, including 1fmol L -1 、100fmol L -1 、10pmol L -1 、1nmol L -1 And the content of ctDNA in different samples is respectively and independently detected by a photoelectrochemical sensor and an electrochemical sensor. The results are summarized in Table 2, and Table 2 is a table of normalized recovery of ctDNA from serum of healthy persons by DNA sensors. The recovery rate is 93.48.52% -105.35%, RSD is less than 5%, and 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)
Claims (15)
1. Ionic liquid BDBDBDBIm (Br) 2 Characterized in that the ionic liquid BDBDBIm (Br) 2 The structural formula of (A) is as follows:
2. the ionic liquid BDBDBIm (Br) of claim 1 2 The application of the sensor in preparation of a ctDNA sensor.
3. The ctDNA sensor based on the ionic liquid functionalized lanthanide metal organic framework is characterized in that the preparation of the ctDNA sensor comprises the following steps:
(1) An ionic liquid functionalized lanthanide metal organic framework Ln-MOF is designed, wherein Ln refers to lanthanide metal elements, the Ln-MOF is a two-dimensional sheet material and is prepared from the ionic liquid BDBDBIm (Br) in claim 1 2 And Ln (OH) n Assembled and coordinated by a strong coordination bond;
(2) In order to enhance the photoelectric conversion efficiency of Nd-MOF, sodium borohydride is used for reducing chloroauric acid in situ to introduce gold nanoparticles to the surface of an Nd-MOF nanosheet, and an AuNPs @ Nd-MOF composite material is synthesized;
(3) Designing a capture probe DNA chain CPs, wherein the DNA chain CPs is connected with the AuNPs @ Nd-MOF composite material through a gold-sulfur bond, and the DNA sequence of the capture probe DNA chain CPs is as follows: 5'-GCATCATTCATTTGTTTCAAAAAA-3' - (CH) 2 ) 6 -SH, the DNA sequence of the target DNA strand ctDNA is: 5'-TGAAACAAATGAATGATGCACGTCATGG-3';
(4) Dripping the solution of the AuNPs @ Nd-MOF composite material prepared in the step (2) onto 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 pretreated capture probe DNA chain CPs solution containing sulfydryl on the AuNPs @ Nd-MOF/GCE modified electrode obtained in the step (4) for modification, and then sealing residual sites on the surface of the electrode by using 6-mercaptohexane-1-ol to obtain an MCH/CPs/AuNPs @ Nd-MOF/GCE modified electrode; thus obtaining the ctDNA sensor.
4. The ctDNA sensor based on an ionic liquid functionalized lanthanide metal organic framework as claimed in claim 3, characterized in that said lanthanide metal Ln is Nd and said ionic liquid functionalized lanthanide metal organic framework is Nd-MOF exhibiting 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 ionic liquid functionalized lanthanide metal organic framework-based ctDNA sensor of claim 4, wherein the BDBDBIm (Br) 2 The synthetic route of the ionic liquid is as follows:
6. the ctDNA sensor based on ionic liquid functionalized lanthanide metal-organic framework as claimed in any of claims 3-5, characterized in that in step (4), the glassy carbon electrode is pretreated before use, and the pretreatment steps are as follows: 0.05 μm Al was used 2 O 3 Polishing the glassy carbon electrode by suspension, rinsing the surface of the electrode by ultrapure water, and sequentially adding 10wt% of HNO 3 Ultrasonically cleaning the solution, absolute ethyl alcohol and ultrapure water, and airing for later use; and/or;
in the step (5), the CPs solution is pretreated to obtain a CPs solution containing sulfydryl: CPs were added to TCEP to reduce disulfide bonds, heated at 95 ℃ for 5min, and allowed to cool to room temperature.
7. The ctDNA sensor based on ionic liquid functionalized lanthanide metal-organic framework as claimed in any one of claims 3-5, wherein in step (4), the concentration of AuNPs @ Nd-MOF composite material is 10mg/mL, and the volume is 5-11 μ L; and/or;
in the step (5), the concentration of CPs is 0.1-2.0 [ mu ] molL -1 (ii) a The modification temperature is 4 ℃; the modification time is 12-24h; and/or;
in the step (5), the molar concentration of the 6-mercaptohexan-1-ol is greater than that of the CPs solution; the modification temperature is room temperature; the modification time is 1-2h.
8. The ctDNA sensor based on ionic liquid functionalized lanthanide metal-organic framework as claimed in claim 7,
in the step (5), the concentration of CPs is 1. Mu. MolL -1 (ii) a And/or;
in the step (5), the concentration of 6-mercaptohex-1-ol is 1mmol L -1 。
9. Use of a ctDNA sensor based on an ionic liquid functionalized lanthanide metal organic framework as described in any of claims 3-7 for the preparation of a photoelectrochemical and/or electrochemical sensor for the detection of ctDNA.
10. The use according to claim 9, characterized in that detection in a photoelectrochemical mode and/or in an electrochemical mode is used;
A. photoelectrochemical mode: dripping a ctDNA solution to be detected into the ctDNA sensor for reaction, and detecting an optical-electrochemical signal under the test condition that a constant potential is 0.1V in a phosphate buffer solution containing ascorbic acid; determining the concentration of ctDNA by a change in the photoelectrochemical signal;
the concentration of the ctDNA in the ctDNA solution to be detected is 1fmol L -1 -10nmolL -1 ；
The concentration of the phosphoric acid buffer solution is 0.01mol L -1 ，pH＝7.0；
B. Electrochemical mode: designing a signal probe DNA chain SPs, wherein the DNA sequence of the signal probe DNA chain 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, and dripping 1 mu molL -1 SPs solution reacts on the sensor, and electrochemical signals are detected in phosphate buffer solution; determining the concentration of ctDNA by a change in electrochemical signal;
the concentration of the ctDNA in the ctDNA solution to be detected is 1fmol L -1 -1nmolL -1 ；
The concentration of the phosphate buffer solution is 0.01mol L -1 ，pH＝7.4。
11. The use according to claim 10, wherein ctDNA concentration is detected in a dual mode of photoelectrochemical mode and electrochemical mode; the concentration of the ctDNA in the ctDNA solution to be detected is 1fmol L -1 -1nmolL -1 。
12. Use according to claim 10 or 11, wherein the ascorbic acid is present in a concentration of 0.0-0.25mol l -1 。
13. 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 detection in the photoelectrochemical mode: the hybridization temperature was 37 ℃; the hybridization time is 10-60min; and/or;
detection in electrochemical mode: the hybridization temperature was 37 ℃; the hybridization time is 10-60min.
15. Use according to claim 14, characterized in that detection in the photoelectrochemical mode: the hybridization time is 50min; and/or;
detection in electrochemical mode: the hybridization time was 50min.
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