DNA magnetic nanoprobe, electrochemical biosensor and application and preparation method of DNA magnetic nanoprobe and electrochemical biosensor

Abstract

The invention belongs to the technical field of biomedical detection, and discloses a DNA magnetic nanoprobe, an electrochemical biosensor and application and a preparation method thereof, the DNA magnetic nanoprobe is obtained by connecting DNA modified with methylene blue and a Fe3O4 (at) Au composite nanomaterial through chemical bonds to obtain Fe3O4 (at) Au (at) MbDNA, and then freezing the Fe3O4 (at) Au (at) MbDNA at the temperature of-20 DEG C or below for more than 1 minute. When the DNA magnetic nanoprobe is applied to an electrochemical biosensor, an excellent electrochemical enhancement signal can be shown, so that the catalytic efficiency is remarkably improved, efficient signal amplification is realized, and the DNA magnetic nanoprobe can be used for detecting various biomarkers and is higher in universality.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical detection, and in particular relates to a DNA magnetic nanoprobe, an electrochemical biosensor and an application and preparation method thereof. Background Art

[0002] In recent years, with the increasing incidence of cancer and the demand for early screening, it is particularly important to develop highly sensitive, highly selective and rapid detection methods. Cancer markers refer to substances secreted by tumor cells or tumor tissues, which can usually be detected in blood, urine or other body fluids, such as alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA) and prostate-specific antigen (PSA) MicroRNA-21 (miRNA21). Traditional cancer marker detection methods mostly rely on immunological methods (such as enzyme-linked immunosorbent assay, ELISA) or molecular biology techniques (such as PCR). Although these methods have certain advantages in sensitivity and specificity, they also have defects such as long detection time, complex operation and high cost. In addition, sample processing and reaction conditions may affect the test results, resulting in false positive or false negative results.

[0003] Electrochemical analysis methods have gradually become a research hotspot for cancer marker detection due to their high sensitivity, rapid response, low cost, and suitability for rapid on-site detection. Such methods are usually based on the modification of electrode materials, signal amplification technology, and the development of new electrochemical sensors, which can achieve highly selective detection of cancer markers. For example, nanomaterials are used to increase the electrode surface area, thereby enhancing the signal response, or by introducing electrochemical selective molecular recognition elements, the recognition ability of target markers is improved, and the catalytic effect of nanozymes can be used to amplify electrical signals, thereby expressing highly sensitive analytical performance. The main challenge facing ultrasensitive sensors is not the sensitivity of the sensor or the affinity of the biorecognition species, but the mass transfer of analyte molecules to the sensor, that is, the rate at which the analyte reaches the electrode surface. For this problem, functionalized magnetic nanomaterials such as ferroferric oxide magnetic nanoparticles (Fe3O4) are usually used as detection probes to quickly enrich and separate the analyte and use the magnetism to adsorb it on the electrode surface with a magnet for electrochemical detection, so that the analyte quickly reaches the electrode surface. However, whether used to increase the surface area of ​​electrodes, capture molecules to be tested, or amplify catalytic signals, functionalized nanomaterials have the disadvantages of requiring a step-by-step process, being time-consuming and material-consuming, and having poor versatility. Summary of the invention

[0004] The object of the present invention is to provide a DNA magnetic nanoprobe which can be used in an electrochemical biosensor to enhance electrochemical signals.

[0005] The first aspect of the present invention provides a DNA magnetic nanoprobe, wherein the DNA magnetic nanoprobe is formed by chemically connecting DNA modified with methylene blue and Fe3O4@Au composite nanomaterial to obtain Fe3O4@Au@MbDNA, and the Fe3O4@Au@MbDNA is further frozen at a temperature below -20°C for more than 1 minute.

[0006] In some embodiments, the freezing temperature is -20°C to -25°C, and the freezing time is 1.0 min to 10 min.

[0007] In some embodiments, the freezing temperature is -20°C to -22°, and the freezing time is 1.0 min to 5 min, preferably 1.5 min to 5 min.

[0008] In some embodiments, the preparation method of the Fe3O4@Au composite nanomaterial comprises the following steps:

[0009] Step a, dissolving FeCl3·6H2O in ethylene glycol, adding sodium acetate and polyethylene glycol, stirring and reacting to obtain a mixture;

[0010] Step b, placing the obtained mixture in an autoclave for reaction to obtain Fe3O4 nanoparticles;

[0011] Step c, ultrasonically dispersing the Fe3O4 nanoparticles in water, adding HAuCl4 to stir and mix, and then adding NaBH4 solution to stir and react, so as to obtain the Fe3O4@Au composite nanomaterial.

[0012] In some of the embodiments, the usage ratio of the Fe3O4 nanoparticles to the HAuCl4 is 14 mg to 16 mg: 2 mg to 4 mg.

[0013] In some of the embodiments, the usage ratio of FeCl3·6H2O, sodium acetate and polyethylene glycol is 1.35:3-5:1-3.

[0014] In some embodiments, in step b, the reaction temperature is 190° C. to 220° C., and the reaction time is 7 h to 9 h.

[0015] In some of the embodiments, in the step c, the stirring temperature of the Fe3O4 nanoparticles and HAuCl4 is -1°C to 1°C, and the stirring time is 1h to 2h.

[0016] The second aspect of the present invention provides a method for preparing a DNA magnetic nanoprobe, the preparation method comprising the following steps:

[0017] S1. The Fe3O4@Au composite nanomaterial and DNA modified with methylene blue were allowed to react to obtain Fe3O4@Au@MbDNA precipitate;

[0018] S2. The obtained Fe3O4@Au@MbDNA precipitate is dissolved in PBS buffer to obtain a Fe3O4@Au@MbDNA solution, and then the Fe3O4@Au@MbDNA solution is frozen at a temperature below -20°C for more than 1 minute to obtain the DNA magnetic nanoprobe.

[0019] In some embodiments, the freezing treatment temperature is -20°C to -25°, and the freezing time is 1.0 min to 10 min; preferably, the freezing treatment temperature is -20°C to -22°, and the freezing time is 1.0 min to 5 min, and more preferably 1.5 min to 5 min.

[0020] In some embodiments, in the reaction system where the Fe3O4@Au composite nanomaterial reacts with DNA modified with methylene blue, the concentration of the Fe3O4@Au composite nanomaterial is 0.5 mg / mL to 1 mg / mL, and the concentration of the DNA modified with methylene blue in the reaction system is 8 μM to 12 μM; and / or,

[0021] In the reaction between the Fe3O4@Au composite nanomaterial and the DNA modified with methylene blue, the Fe3O4@Au@MbDNA precipitate is obtained by standing for 10 hours to 14 hours.

[0022] In some of the embodiments, the concentration of the Fe3O4@Au@MbDNA in PBS buffer is 0.01 μM to 0.1 mM.

[0023] The third aspect of the present invention provides a use of the DNA magnetic nanoprobe as described above in the preparation of an electrochemical sensor for quantitative detection of cancer markers.

[0024] In some embodiments, the cancer marker is miRNA; preferably, the cancer marker is miRNA21.

[0025] A fourth aspect of the present invention provides an electrochemical biosensor, which comprises an electrode and the DNA magnetic nanoprobe as described above bound to the electrode.

[0026] A fifth aspect of the present invention provides a use of the electrochemical biosensor as described above in the quantitative detection of cancer markers; preferably, the cancer marker is miRNA.

[0027] In the present invention, the inventors found that when the DNA magnetic nanoprobe obtained by freezing Fe3O4@Au@MbDNA at a temperature below -20°C for more than 1 minute is applied to an electrochemical biosensor, it can exhibit excellent electrochemical enhanced signals, thereby significantly improving the catalytic efficiency and achieving efficient signal amplification. It can be used for the detection of various biomarkers and has higher versatility. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the transmission electron microscope (TEM) morphology characterization picture of the prepared Fe3O4@Au; Figure 1 A is the TEM image of Fe3O4; Figure 1 B is the TEM image of Fe3O4@Au; Figure 1 C is the EDS scanning image of Fe3O4@Au composite nanomaterials; Figure 1 D is the element mapping image of Merge, Fe, O, and Au.

[0029] Figure 2 This is the hysteresis loop of the prepared Fe3O4 magnetic nanoparticles and Fe3O4@Au composite nanomaterials.

[0030] Figure 3 This is the characterization diagram of Fe3O4@Au@MbDNA magnetic nanoprobe. Figure 3 A and 3B are water and particle size and Zeta potential characterization diagrams of Fe3O4, Fe3O4@Au, and Fe3O4@Au@MbDNA, respectively. Figure C is the UV characterization spectrum of Fe3O4@Au@MbDNA.

[0031] Figure 4 This is the conformational verification of Fe3O4@Au@MbDNA magnetic nanoprobe under freezing induction. Figure 4 A is the fluorescence image of Fe3O4@Au@cy5DNA under freezing and room temperature conditions and the corresponding histogram in (4B). Scale bar, 10 μm. Figure 4 C is the SWV curve of the G-purine-rich DNA sequence on the gold electrode surface in 10 mM PBS (pH 7.4) solution under frozen and room temperature conditions.

[0032] Figure 5 Study on the freezing-induced Fe3O4@Au@MbDNA magnetic nanoprobe on the electrode surface. Figure 5 A is the SWV curve of the guanine G-rich DNA probe (Fe3O4@Au@GDNA) on the gold electrode surface under room temperature and freezing conditions (10mM PBS (pH7.4) solution). Figure 5B and 5C are the instantaneous impedance graphs of the electrode surface before and after adding 40 μL 500 nM cDNA (a sequence that binds to partial base complementary pairing of Mb-DNA) to Fe3O4@Au@MbDNA in 10 mM PBS at room temperature and frozen conditions, respectively. Figure 5 D is the SWV response curve of Fe3O4@Au@MbDNA under different temperature treatments.

[0033] Figure 6 A is the SWV curve of Fe3O4, Fe3O4@Au and Fe3O4@Au@MbDNA. Figure 6 B is the SWV response curve of Fe3O4@Au@MbDNA at room temperature and freezing conditions.

[0034] Figure 7 This is the characterization diagram of Fe3O4@Au@MbDNA@cDNA magnetic nanoprobe. Figure 7 A is a schematic diagram of the synthesis of probes for miRNA21 detection. Figure 7 B and 7C are the Zeta potential, water and particle size characterization diagrams of Fe3O4, Fe3O4@Au, Fe3O4@Au@MbDNA and Fe3O4@Au@MbDNA@cDNA, respectively. Figure 7 D is the UV characterization spectrum of Fe3O4@Au@MbDNA and Fe3O4@Au@MbDNA@cDNA.

[0035] Figure 8 This is the electrochemical signal response of the Probe used for miRNA21 detection on the electrode. Figure 8 A is the SWV response curve of Fe3O4@Au@MbDNA and Fe3O4@Au@MbDNA@cDNA(Probe). Figure 8 B is the SWV response curves of Fe3O4@Au@MbDNA and Fe3O4@Au@MbDNA@cDNA (Probe) at room temperature and freezing conditions, respectively.

[0036] Fig. 9 Schematic diagram of the Probe probe used for miRNA21 detection.

[0037] Fig.10 Schematic diagram of Fe3O4@Au@MbDNA magnetic nanoprobe on the electrode under room temperature and freezing conditions.

[0038] Fig.11 Verify the feasibility of using the Probe probe for electrochemical sensing of miRNA21 detection.

[0039] Fig.12 This is the optimization diagram of the freezing time during actual testing.

[0040] Fig.13 The electrochemical square wave voltammetry curve of the Fe3O4@Au@MbDNA magnetic nanoprobe obtained after being frozen on the electrode after incubation with different miRNA21 concentrations and the probe Probe.

[0041] Fig.14 The ΔCurrent corresponding to the logarithm of different miRNA21 concentrations is fitted with the linear relationship between different ΔCurrent and Log(miRNA concentration). The inset shows the ΔCurrent corresponding to different miRNA21 concentrations. DETAILED DESCRIPTION

[0042] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0043] The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0044] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0045] An embodiment of the present invention provides a DNA magnetic nanoprobe, wherein the DNA magnetic nanoprobe is obtained by chemically connecting DNA modified with methylene blue and a Fe3O4@Au composite nanomaterial to obtain Fe3O4@Au@MbDNA, and the Fe3O4@Au@MbDNA is then frozen at a temperature below -20°C for more than 1 minute.

[0046] The embodiment of the present invention further provides an electrochemical biosensor, which comprises an electrode and the above-mentioned DNA magnetic nanoprobe bound to the electrode, wherein the electrode and the magnetic nanoprobe are bound by magnetic attraction.

[0047] It can be understood that the DNA magnetic nanoprobe described in the present invention, the re-ordered arrangement of the DNA nanoprobe on the electrode surface caused by freezing promotes more Mb-DNA signal transmission on the electrode surface, realizes electrical signal amplification, and improves the sensitivity and selectivity of the electrochemical sensor. This may be because the specific stretching conformation of the DNA can be regulated by freezing treatment at a specific temperature, thereby regulating the molecular arrangement of the entire DNA magnetic nanoprobe on the electrode surface and making more methylene blue molecules attached to the electrode surface, so as to obtain a new DNA magnetic nanoprobe. When the DNA magnetic nanoprobe is used in an electrochemical biosensor, it has the following advantages:

[0048] (1) Compared with traditional signal enhancement strategies, this DNA magnetic nanoprobe saves time and cost in signal amplification, and is more efficient and versatile than other signal amplification technologies, avoiding defects such as unstable modification of nanomaterials on the electrode surface and slow detection rate during cyclic signal amplification.

[0049] (2) It has magnetic adsorption properties and can quickly reach the electrode surface and generate electrochemical signals. The probe can be designed to bind to its corresponding aptamer or nucleic acid sequence to identify and detect different markers to be tested, thereby improving versatility.

[0050] (3) The DNA magnetic nanoprobe has stable performance and is easy and fast to detect. The freezing-induced rearrangement of the DNA nanoprobe on the electrode surface promotes the transmission of more Mb-DNA signals on the electrode surface, achieves electrical signal amplification, improves the sensitivity and selectivity of electrochemical sensors, and promotes the development of sensors for analysis and bioassays.

[0051] (4) It has broad application prospects and can be used to design detection of various biomarkers and has the potential to be used in other fields.

[0052] The present invention is described below with reference to specific embodiments.

[0053] Table 1. Nucleic acid sequences and their modified forms in various examples

[0054]

[0055] Example 1 Preparation of Fe3O4@Au@MbDNA magnetic nanoprobe

[0056] This embodiment provides a method for preparing a Fe3O4@Au@MbDNA magnetic nanoprobe, and the specific steps are as follows:

[0057] (1) Dissolve 1.35 g of FeCl3·6H2O in 40 mL of ethylene glycol to form a transparent solution, then add sodium acetate (3.6 g) and polyethylene glycol (1.0 g), and stir the mixture continuously for 0.5 h.

[0058] (2) The mixture was sealed in a stainless steel autoclave with a polytetrafluoroethylene liner and reacted at 200°C for 8 hours. After cooling to room temperature, a black precipitate was obtained, which was then collected with a magnet. The prepared raw Fe3O4 nanoparticles were washed with ethanol three times and dried in vacuum at 60°C for 6 hours to obtain Fe3O4.

[0059] (3) The Fe3O4 (15 mg) obtained above was ultrasonically dispersed in 150 ml of distilled water. Then, 3 ml of HAuCl4 aqueous solution (6 mg / mL) was added to the Fe3O4 solution, and mechanical stirring was continued at 0°C for 1 h. Then, 0.9 mL of ice-cold NaBH4 solution (0.2 M) was slowly added to the mixture of Fe3O4 magnetic nanoparticles and HAuCl4 within 4 min. The reaction was stirred at 0°C for 15 min, and the color of the solution changed from light brown to dark purple. The prepared Fe3O4@Au composite nanomaterial was collected with a magnet and washed three times with ethanol. Finally, it was dried under vacuum conditions at 50°C. According to the above conventional method, Fe3O4@Au composite nanomaterial was prepared.

[0060] (4) The prepared Fe3O4@Au composite nanomaterial was dispersed in distilled water to a concentration of 1 mg / mL, and 800 μL Fe3O4@Au composite nanomaterial was added with 200 μL Mb-DNA (SEQ ID NO: 3) to a final concentration of 10 μM. The mixture was left undisturbed at 4°C for 12 hours to allow the Mb-DNA (thiol-terminated) to bind to the Fe3O4@Au composite nanomaterial through the Au-S bond.

[0061] (5) In order to remove excess reagents, the Mb-DNA modified Fe3O4@Au composite nanomaterials were separated by a magnet to obtain a Fe3O4@Au@MbDNA precipitate, which was washed several times with 10 mM phosphate buffer (pH 7.4). The prepared Fe3O4@Au@MbDNA precipitate was placed in a 10 mM PBS buffer to obtain a Fe3O4@Au@MbDNA solution for standby use; wherein the concentration of the Fe3O4@Au@MbDNA can be adjusted arbitrarily according to experimental needs. In this embodiment, the concentration of the Fe3O4@Au@MbDNA in the PBS buffer was 10 μM.

[0062] (6) The obtained Fe3O4@Au@MbDNA solution is placed in a temperature below -20°C for freezing treatment to obtain the DNA magnetic nanoprobe.

[0063] The structural characterization test of the Fe3O4@Au@MbDNA composite material prepared in Example 1 is as follows:

[0064] The Fe3O4@Au composite nanomaterial prepared in Example 1 was characterized by transmission electron microscopy. Figure 1 From the TEM image of A, it can be seen that the prepared Fe3O4 has a cloud-like spherical shape, a rough and uniform appearance, and an average size of 120nm. After the AuNPs are in situ reduced to the Fe3O4 surface, it can be observed that the AuNPs of uniform size are dispersed on the Fe3O4 surface ( Figure 1 B). The successful synthesis of Fe3O4@Au composite nanomaterials was further confirmed by analyzing the elemental composition and distribution of Fe3O4@Au. Figure 1 C and Figure 1 D)

[0065] The magnetic properties of the prepared Fe3O4 nanoparticles and Fe3O4@Au composite nanomaterials were analyzed by Quantum Design-PPMS 9T (USA). The saturation magnetization intensities were 41.0 and 19.6 emu / g, respectively. Figure 2 ). From the figure, although the saturation magnetization of the Fe3O4@Au composite nanomaterial is reduced, a rapid magnetic response can be obtained within 30 seconds using an external magnet. Therefore, the Fe3O4@Au composite nanomaterial can be used to quickly separate biomarkers from complex samples and enable subsequent DNA nanoprobes to quickly reach the electrode surface.

[0066] The particle size and zeta potential of Fe3O4, Fe3O4@Au, and Fe3O4@Au@MbDNA were characterized using a Malvern particle size analyzer. Figure 3 A, Dynamic light scattering was used to characterize the hydrated particle size of the composite material. After modification with Mb-DNA, the hydrated particle size of Fe3O4@Au increased from the original 190nm to 203nm. In addition, the zeta potential values ​​of Fe3O4, Fe3O4@Au, and Fe3O4@Au@MbDNA were approximately 15.9mV, -4mV, and -16.5( Figure 3 B), the results all indicate that Fe3O4@Au was successfully modified by Mb-DNA. In addition, the components in the probe were characterized by ultraviolet light ( Figure 3 C), by observing the ultraviolet absorption spectrum of Fe3O4@Au@MbDNA, it can be found that an obvious DNA characteristic absorption peak appears at 260nm, further indicating the successful synthesis of Fe3O4@Au@MbDNA magnetic nanoprobe.

[0067] Experimental Example 1 Conformational changes of Fe3O4@Au@MbDNA magnetic nanoprobe induced by freezing

[0068] By modifying the Fe3O4@Au surface with cy5-DNA (SEQ ID NO: 2) carrying a fluorescent label (the modification synthesis method is the same as that of Mb-DNA modification of Fe3O4@Au), we verified by confocal fluorescence imaging that under freezing conditions, the DNA modified on Fe3O4@Au has an extended and stretched conformation.

[0069] like Figure 4 As shown in A, in order to study the conformation of DNA on Fe3O4@Au composite nanomaterials at room temperature and frozen, the Fe3O4@Au modified with cy5-DNA was frozen at room temperature and at -20℃ for 5 minutes. It was found under an inverted fluorescence microscope that the fluorescence intensity of Fe3O4@Au@cy5DNA at room temperature was weak. It is speculated that this is because cy5DNA is folded and curled at room temperature. At this time, the cy5 fluorescent label is closer to the AuNPs on Fe3O4, resulting in fluorescence resonance transfer and partial quenching of inverted fluorescence. On the contrary, the fluorescence intensity of Fe3O4@Au@cy5DNA under freezing is significantly enhanced, as shown in Figure 1. Figure 4 B is Figure 4 A is the corresponding fluorescence intensity histogram obtained from the fluorescence image. At this time, it is speculated that freezing at -20℃ stretches the cy5-labeled DNA and makes it away from the AuNPs on Fe3O4, and no fluorescence quenching occurs. The results prove that freezing can transform the DNA conformation originally folded on the magnetic beads into a stretched state. Next, we applied the properties of frozen stretched DNA to the electrode to study the conformation of DNA on the electrode under freezing conditions. We first modified a single guanine-rich DNA on the gold electrode, such as Figure 4 As shown in Figure C, 0-0.8V SWV detection was performed under -20℃ freezing for 5min and room temperature (25℃). It can be seen in the figure that there is no obvious current signal under freezing conditions, while the electrochemical signal unique to guanine can be seen around 0.7V at room temperature. It is preliminarily confirmed that on the electrode surface, a single DNA presents a folded conformation, which makes G closer to the electrode surface. On the contrary, the frozen stretched DNA makes the G end farther away from the DNA, resulting in no obvious current signal.

[0070] Experimental Example 2 Study on the Freezing-Induced Fe3O4@Au@MbDNA Magnetic Nanoprobe on the Electrode Surface

[0071] To further explore the results of G-DNA modification on Fe3O4@Au, we magnetically adsorbed it on the electrode surface for SWV detection. Figure 5As shown in A, Fe3O4@Au@GDNA under freezing conditions (-20℃, 5min) has a higher peak current signal on the electrode. Therefore, it is speculated that the DNA modified on the magnetic beads has different conformations on the electrode surface, which may be caused by the different spatial conformational arrangements before and after freezing.

[0072] In the early stage of the experimental exploration, the electrochemical signal test of the same concentration of Fe3O4@Au@MbDNA (50μM) at three temperatures (-20℃, room temperature 25℃ and 4℃) was carried out. The test method is similar to the SWV test of the following method. The results are shown in Figure 5 As shown in D, the results show that -20℃ has a significant electrochemical signal enhancement effect compared with the other two groups of temperatures, but the electrochemical signals of the other two groups of 25℃ and 4℃ in the experiment are no different, so the room temperature 25℃ and -20℃ were selected for comparison in the subsequent verification experiment.

[0073] For further research, the transient frequency conversion impedance value of Fe3O4@Au@MbDNA after adding cDNA (sequence complementary to Mb-DNA) was measured on the electrode surface under freezing (-20℃, 5min) and room temperature (25℃). Figure 5 B and 5C, compared Figure 5 B. Under room temperature and freezing conditions, the impedance value near 100Hz increased significantly after adding cDNA. The impedance value changes after adding cDNA at room temperature. Therefore, we believe that on the electrode surface, the spacing of the Fe3O4@Au@MbDNA after freezing is more uniform and orderly due to the stretching of the DNA itself, and the conformation is a layer of tightly arranged Fe3O4@Au@MbDNA. At room temperature, the folded DNA is more flexible, so the arrangement on the electrode is in a stacked state. Therefore, when cDNA is added, the impedance value does not change. That is, it is more difficult for cDNA to enter the Mb-DNA on the magnetic beads for complementation under this conformation. However, under freezing conditions (-20℃, 5min), cDNA can more easily enter the monolayered magnetic beads on the tightly arranged Fe3O4@Au@MbDNA, so the impedance value increases.

[0074] The above research results were applied to actual SWV monitoring. Individual Fe3O4, Fe3O4@Au, and Fe3O4@Au@MbDNA were magnetically attached to the surface of a gold electrode for SWV analysis. Figure 6As shown in A, in 10 mM PBS, Fe3O4 and Fe3O4@Au have no obvious current signals, while the current signal of Fe3O4@Au@MbDNA gradually increases under the same conditions. This verifies that the current signal of this experiment is generated by the charged molecule Mb on the DNA magnetic nanoprobe, and there is no other signal interference.

[0075] Then, it was verified that the signal generated by Fe3O4@Au@MbDNA modified with methylene blue molecules in SWV detection was significantly enhanced under freezing conditions, such as Figure 6 As shown in B, the Fe3O4@Au@MbDNA after freezing (-20℃, 5min) has a stronger peak current signal on the gold electrode, which further verifies that the spacing of the Fe3O4@Au@MbDNA after freezing is more uniform and orderly due to the stretching of the DNA itself, and the conformation is a layer of tightly arranged Fe3O4@Au@MbDNA, which makes the signal molecules contact the electrode surface more fully and evenly.

[0076] Embodiment 2:

[0077] Based on Example 1, the present invention further develops a method for applying the freezing-induced Fe3O4@Au@MbDNA signal enhancement strategy to actual detection, and the specific steps are as follows:

[0078] (1) First, take the Fe3O4@Au@MbDNA magnetic nanoprobe (800 μL) prepared above and add 200 μL cDNA (a DNA sequence that can specifically recognize and complement miRAN21, SEQ ID NO: 4) to make the final concentration of cDNA 1 μM.

[0079] (2) Incubate at room temperature for 30 min, remove the supernatant by magnetic separation, redissolve the remaining precipitate in 1000 μL PBS, and place the prepared probe in PBS for later use.

[0080] The structural characterization test of Fe3O4@Au@MbDNA@cDNA prepared in Example 2 is as follows:

[0081] The particle size and zeta potential of Fe3O4, Fe3O4@Au, Fe3O4@Au@MbDNA and Fe3O4@Au@MbDNA@cDNA were characterized by Malvern particle size analyzer. Figure 7As shown in Figures B and 7C, the zeta potential and hydrated particle size of Fe3O4@Au@MbDNA@cDNA increased from -16.5mV to -20.6mV and from 203nm to 210nm, respectively, indicating that cDNA was successfully modified and complemented on the Fe3O4@Au@MbDNA nanoprobe. In addition, the components in the probe were characterized by ultraviolet light ( Figure 7 D), compared with the UV absorption spectrum of Fe3O4@Au@MbDNA, the characteristic absorption peak of Fe3O4@Au@MbDNA@cDNA at 260nm increased significantly, further indicating the successful synthesis of Fe3O4@Au@MbDNA@cDNA nanoprobe. Figure 7 A is a schematic diagram of the modification characterization of Fe3O4@Au@MbDNA@cDNA. For the convenience of naming, Fe3O4@Au@MbDNA@cDNA will be named Probe.

[0082] Embodiment 3:

[0083] The present invention provides an electrochemical sensing method for detecting miRNA21 based on Fe3O4@Au@MbDNA@cDNA (Probe), and the specific steps are as follows:

[0084] (1) The gold electrode was pretreated with 0.5 M sulfuric acid, and then an oxidation step (2 V, 5 s) and a reduction step (0.35 V, 10 s) were performed.

[0085] (2) Under acidic conditions (0.5 M sulfuric acid), further oxidation and reduction scans were performed in the range of 0.35 to 1.5 V (20 scans, scan rate 0.5 V / s, sampling interval 0.001 V).

[0086] (3) 100 μL miRNA21 (SEQ ID NO: 1) (1 nM) was added to 100 μL of the successfully prepared Probe, and the cDNA was complementary to part of miRNA21. The mixture was incubated at room temperature for 30 min, and then the free cDNA@miRNA21 complex was removed by magnetic separation, and the precipitate was dissolved with 200 μL 10 mM PBS (equivalent to the pre-enrichment process).

[0087] (4) The suspension was quickly frozen at -20°C. Then, 50 μL of the solution was carefully dropped onto the pre-treated gold electrode surface so that it completely covered the three-electrode area without overflowing.

[0088] (5) The suspension was placed at -20°C for different freezing times: 30 s, 60 s, 90 s, 120 s, 150 s, 180 s and 210 s. The suspension was then dropped onto a gold electrode with a magnet on the back for SWV detection.

[0089] (6) Finally, a magnet is placed on the back side of the gold electrode to pull the electroactive molecule-modified magnetic nanoprobe (in this case, methylene blue) toward the electrode interface for quantitative detection under square wave voltammetry (SWV).

[0090] Test Example 3

[0091] Application of Fe3O4@Au@MbDNA@cDNA(Probe) in electrochemical immunoassay for detection of miRNA21

[0092] In order to further verify the reliability of the Probe for subsequent detection of miRNA21, we also explored how the Probe affects the electrochemical signal on the electrode surface. Figure 8 As shown in A, after modifying cDNA, the signal of the Probe on the gold electrode decreases. We believe that this is due to the rigid DNA structure after complementation, which makes the distance between each molecule farther. In order to avoid that the conformation of the Probe under freezing will also enhance the electrical signal in subsequent detection, thereby affecting the signal enhancement effect of the Fe3O4@Au@MbDNA alone, the electrical signals generated by the Probe and Fe3O4@Au@MbDNA under freezing conditions are compared, as shown in Figure 1. Figure 8 As shown in B, the enhanced electrical signal of the probe after freezing does not affect Fe3O4@Au@MbDNA. This further proves the reliability of the Fe3O4@Au@MbDNA probe that can be modified into a probe for miRNA21 detection. Fig. 9 ) It can be seen that miRNA21 will be magnetically separated after binding to the cDNA on the Probe. The actual test solution after resuspension is a mixture of Fe3O4@Au@MbDNA probe and Fe3O4@Au@MbDNA@cDNA, so the above verification instructions are available.

[0093] In this work, we proposed a strategy to enhance electrical signals by freeze-induced ordered arrangement of DNA magnetic nanoprobes on electrode surfaces ( Fig.10), this strategy places Fe3O4@Au@MbDNA in -20℃ for rapid freezing for 2-3min, and then magnetically adsorbs it to the electrode surface for electrochemical detection. This process does not require additional signal labels or additional signal amplification methods. The signal enhancement is based on the orderly arrangement of DNA magnetic nanoprobes on the electrode surface, so that more Mb molecules are aggregated in a single layer close to the electrode surface, realizing an efficient, fast, green and enzyme-free signal amplification mode.

[0094] In order to verify the feasibility of using the Probe to detect miRNA21, miRNA21 was quantitatively detected by square wave voltammetry (SWV) in a 10 mM PBS solution. Fig.11 First, the initial signal line of Fe3O4@Au@MbDNA at room temperature. After adding cDNA to bind to it, the signal decreases due to the change in the arrangement of the probe conformation on the electrode surface (mentioned in the previous description). Finally, when adding 10fM miRNA21 for detection, the supernatant is separated by magnetic separation after the complementary binding of miRNA21 and cDNA, which makes the Fe3O4@Au@MbDNA in the test solution increase. Therefore, the probe with the final signal enhancement will be displayed only after adding miRNA21. Finally, the test solution is quickly frozen at -20℃, which causes the conformation of the electrode surface to change and the signal is amplified. Therefore, the above experimental results confirm the feasibility of this strategy for the successful detection of miRNA21. Finally, the difference between the signal baseline of the Probe and the signal amplification after freezing (ΔCurrent) is used as the final quantitative value.

[0095] The suspension was placed at -20°C for different freezing times for freezing treatment. After SWV detection, the generated ΔCurrent was plotted, and it was found that the highest value of ΔCurrent was found at 90s, and 90s was selected as the final optimized freezing time.

[0096] Subsequently, different concentrations of miRNA21( Fig.13 ), by fitting the linear relationship between ΔCurrent and the logarithm of different concentrations of miRNA21 ( Fig.14 ), the linear detection range of miRNA21 concentration was found to be 100aM~1nM, and the detection limit was 10aM.

[0097] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0098] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A DNA magnetic nanoprobe, characterized in that: The DNA magnetic nanoprobe is obtained by connecting DNA modified with methylene blue and Fe3O4@Au composite nanomaterials through chemical bonds to obtain Fe3O4@Au@MbDNA, and the Fe3O4@Au@MbDNA is then frozen at a temperature below -20°C for more than 1 minute.

2. The DNA magnetic nanoprobe according to claim 1, characterized in that The freezing temperature is -20°C to -25°C, and the freezing time is 1.0 min to 10 min.

3. The DNA magnetic nanoprobe according to claim 1, characterized in that The freezing temperature is -20°C to -22°C, and the freezing time is 1.0 min to 5 min.

4. A method for preparing a DNA magnetic nanoprobe, characterized in that: The preparation method comprises the following steps: S1. The Fe3O4@Au composite nanomaterial and DNA modified with methylene blue were allowed to react to obtain Fe3O4@Au@MbDNA precipitate; S2. The obtained Fe3O4@Au@MbDNA precipitate is dissolved in PBS buffer to obtain a Fe3O4@Au@MbDNA solution, and then the Fe3O4@Au@MbDNA solution is frozen at a temperature below -20°C for more than 1 minute to obtain the DNA magnetic nanoprobe.

5. The preparation method according to claim 4, characterized in that: The temperature of the freezing treatment is -20°C to -25°, and the freezing time is 1.0 min to 10 min; preferably, the temperature of the freezing treatment is -20°C to -22°, and the freezing time is 1.0 min to 5 min.

6. The preparation method according to claim 4, characterized in that: In the reaction system of step S1, the concentration of the Fe3O4@Au composite nanomaterial is 0.5 mg / mL to 1 mg / mL, and the concentration of the DNA modified with methylene blue in the reaction system is 8 μM to 12 μM; and / or, The Fe3O4@Au@MbDNA precipitate is obtained by standing for 10 to 14 hours.

7. The preparation method according to claim 4, characterized in that: The concentration of the Fe3O4@Au@MbDNA in PBS buffer is 0.01 μM to 0.1 mM.

8. Use of the DNA magnetic nanoprobe according to any one of claims 1 to 3 in the preparation of an electrochemical sensor for quantitative detection of cancer markers.

9. An electrochemical biosensor, characterized in that: The electrochemical biosensor comprises an electrode and the DNA magnetic nanoprobe according to any one of claims 1 to 3 bound to the electrode.

10. Use of the electrochemical biosensor according to claim 9 in the quantitative detection of cancer markers.

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