Preparation method of a fluorescence-colorimetric dual-mode sensor based on a bifunctional MXene / Pt-Pd composite material

Abstract

The present invention belongs to the technical field of biosensing detection, and discloses a preparation method and application of a fluorescence-colorimetric dual-mode sensor based on a bifunctional MXene / Pt-Pd composite material. A bifunctional composite material with fluorescence quenching ability and peroxidase-like activity is prepared by in-situ growth of PtPd NPs on the surface of the MXene-Ti3C2 material. The DNAzyme-MBs system realizes the target recycling strategy while cleverly constructing fluorescence and colorimetric dual-mode channels. Utilizing the characteristic that double-strands are desorbed and adsorbed from the surface of the Ti3C2 / PtPd NPs material, the recovery of fluorescence in the fluorescence system and the color reaction in the colorimetric system are achieved. The quantitative analysis of Pb 2+ is carried out according to the enhancement of fluorescence intensity and absorbance in the dual-mode system. The dual-mode sensor constructed by the present invention is simple and convenient to operate, the detection results are sensitive and reliable, the detection limits are 12 pM and 72 pM respectively, and it has good anti-interference ability and practical application ability, providing a new biosensing platform for the analysis of actual samples.

Description

Technical Field

[0001] The present invention relates to a preparation method and application of a fluorescence-colorimetric dual-mode sensor based on a bifunctional MXene / Pt-Pd composite material. Background Art

[0002] In the process of human urbanization and industrialization, heavy metal pollution has become increasingly serious. The industrial pollutant lead is mainly released into the environment from sewage, sludge and waste gas. As one of the most harmful heavy metal pollutants, lead is non-biodegradable, carcinogenic and bioaccumulative. Lead exposure is associated with hypertension, kidney disease, decreased organ function and neurological diseases, and these diseases can affect the health of the next generation. Therefore, it is of great significance to monitor lead in the environment and food with high sensitivity and selectivity. Conventional detection methods mainly include atomic absorption spectrometry, inductively coupled plasma mass spectrometry, X-ray fluorescence spectrometry, atomic fluorescence spectrometry, etc. These methods perform excellently in detection accuracy and multi-element analysis, but they cannot avoid the problems of time-consuming, high cost and high operation difficulty. To overcome these drawbacks, alternative techniques for detecting heavy metal Pb 2+ have made great progress through different technologies such as electrochemistry, colorimetry, chemiluminescence, fluorescence, surface-enhanced Raman scattering, etc. Although these methods have acceptable sensitivity and fast response time for the detection of Pb 2+ , they stay in a single analysis mode and are vulnerable to the test environment. In contrast, the dual-mode analysis strategy can provide built-in cross-reference correction for environmental effects, making the detection results more accurate and reliable. Due to the high sensitivity of fluorescence and the visual analysis of colorimetry, the fluorescence / colorimetry method has become a widely used dual-mode sensing strategy for Pb 2+ analysis.

[0003] Although most of the currently developed fluorescence / colorimetry dual-mode strategies can obtain excellent analytical performance, colorimetric analysis fundamentally depends on gold nanoparticles or silver nanoparticles, etc. in Pb 2+Color changes caused by aggregation in the presence of [substances]. Such colorimetric experiments may be interfered by non-specific aggregation of metal nanoparticles. In recent years, nanozymes have been widely studied and applied in the fields of biocatalysis and immunoassay due to their good enzyme-like activity, durability, and high stability. For example, platinum nanoparticles (Pt NPs) have been widely used as catalysts, but the high cost and limited stability of pure platinum have always hindered its widespread development. It has been found that palladium (Pd) has almost the same structure as platinum, and Pd has lower cost and stronger oxygen affinity. Compared with Pt NPs, Pt-Pd bimetallic nanoparticles (PtPd NPs) exhibit a synergistic effect and show higher catalytic activity and stability. It has become a new type of nanozyme with excellent properties, which can catalyze the oxidation of peroxidase substrates to produce color changes.

[0004] MXene, as a new type of two-dimensional transition metal carbides and nitrides, has attracted much attention due to its excellent physicochemical properties. Due to its rich oxygen or hydroxyl-terminated surface and complete metal atomic layer, MXene can interact with most biomolecules through hydrogen bonds, van der Waals forces, electrostatic interactions, coordination bonds, etc., which makes it a unique nano-biological unit in the structure of biosensors. Compared with graphene oxide, MXene has obvious advantages in light absorption from the visible light to the near-infrared region. MXene can adsorb single-stranded DNA and effectively quench the fluorescence of fluorescent groups. Therefore, by utilizing the strong adsorption ability of MXene for single-stranded DNA and its advantages in visible light absorption, MXene can effectively quench the fluorescence of single-stranded DNA labeled with fluorophores, thereby significantly reducing the background signal.

[0005] Although the currently constructed dual-mode sensors have the advantages of accurate and reliable detection results, most dual-mode sensors rely on certain properties of two or even more complex nanomaterials, which leads to a complex and time-consuming preparation process for the sensors. Composite of multiple nanomaterials with special properties can obtain a new type of multifunctional material with all their properties, which can simplify the construction process of dual-mode sensors and improve the stability of the sensors. PtPd NPs are excellent candidates for peroxidase mimics. MXene, as a new type of two-dimensional nanomaterial with good reducibility and fluorescence quenching ability, has attracted wide attention. Therefore, it can also be used to in-situ reduce sodium chloropalladate (Na2PdCl4) and chloroplatinic acid (H2PtCl6) to prepare MXene / PtPd NPs composites, which simultaneously have peroxidase-like activity, the ability to adsorb single-stranded DNA, and fluorescence quenching characteristics.

[0006] In view of the problems existing in the existing detection technologies, such as low sensitivity, poor detection accuracy, unstable detection results, etc., the present invention constructs a new type of fluorescence colorimetric dual-mode sensor. First, DNAzyme is used for the target Pb2+ Specific recognition is carried out to enhance the detection specificity. Secondly, signal amplification is achieved through the DNAzyme-mediated target recycling strategy to improve the detection sensitivity. Then, the magnetic separation technology is used to ingeniously construct a fluorescence and colorimetric dual-mode channel, greatly reducing environmental interference, avoiding false positive signals, and improving the reliability of the sensor. Finally, a bifunctional MXene / bimetallic composite material is introduced into both dual-mode channels, which not only realizes the signal response caused by the target, but also simplifies the experimental operation. Based on the dual-mode sensing strategy of the present invention, while overcoming the problems existing in the above-mentioned prior art, it has the characteristics of being more sensitive, reliable, fast and stable, which is conducive to the popularization and application of the invention. Summary of the Invention

[0007] A preparation method of a fluorescence and colorimetric dual-mode sensor based on a bifunctional MXene / Pt-Pd composite material is carried out according to the following steps:

[0008] (1) Preparation of MXene / Pt-Pd composite material: The MAX phase material is etched with an etching solution to remove the A element therein, centrifuged and the pH is adjusted, the precipitate is collected, and a two-dimensional layered MXene material is obtained after vacuum drying. The mixture of MXene and pure water is made into a suspension by sufficient ultrasonic treatment, chloroplatinic acid and sodium palladium chloride are added thereto, and bimetallic nanoparticles are in-situ grown on the above-mentioned MXene material by a redox method, and then centrifuged at high speed and dried to constant weight to obtain the MXene / Pt-Pd composite material.

[0009] (2) Recognition of target Pb 2+ : S-DNA is fixed on the surface of magnetic beads (MBs) through the interaction between the amino group on the substrate strand S-DNA and the carboxyl group on the surface of the magnetic beads. Then, the enzyme strand E-DNA is added to form a double-stranded structure DNAzyme by base complementary pairing with S-DNA. After magnetic separation, the MBs@DNAzyme system is successfully constructed. The presence of target Pb 2+ activates the cleavage activity of DNAzyme, and the Pb 2+ cleavage site rA site of S-DNA is cleaved and single-stranded S-DNA-a is released. At the same time, the released single-stranded E-DNA serves as a walking strand and continues to bind to the next single-stranded S-DNA on the surface of MBs, and finally more single-stranded S-DNA-a is cleaved. After magnetic separation of the mixture, the supernatant is aspirated and the precipitate is retained.

[0010] (3) Construction of dual-mode channels: After magnetic separation of the above mixture, the supernatant and the precipitate are respectively used to construct fluorescence and colorimetric dual-mode channels. Fluorescence channel: Fluorescent group-labeled single-stranded S1 is added to the supernatant. After incubation, the sheared single-stranded S-DNA-a forms a double-stranded structure by base complementary pairing with single-stranded S1. MXene / Pt-Pd composite material is continuously added to the system. The double-stranded structure prevents single-stranded S1 from being adsorbed onto the material surface, resulting in enhanced fluorescence in the system. Colorimetric channel: After the precipitate is washed, only the other part of the sheared S-DNA, single-stranded S-DNA-b, remains on the surface of the magnetic beads. Single-stranded S2 is added to it. After incubation, the two form a double-stranded structure by base complementary pairing. MXene / Pt-Pd composite material, hydrogen peroxide (H2O2), and 3,3’,5,5’-tetramethylbenzidine (TMB) are continuously added to the system. After a short incubation, the color of the system changes and the absorbance increases. According to the enhancement of fluorescence intensity in the fluorescence channel and the increase in absorbance in the colorimetric channel after adding Pb 2+ a dual-mode quantitative analysis is achieved.

[0011] Based on magnetic separation technology and the fluorescence quenching ability and peroxidase-like activity of the multifunctional MXene / Pt-Pd composite material, the present invention constructs a dual-mode sensing strategy, which can further improve the detection sensitivity and stability, and the reliability is also further enhanced compared with single-mode sensors.

[0012] Further defined, in step (1), the etching solution is one or more of hydrofluoric acid, ammonium bifluoride, or a mixture of hydrochloric acid and lithium fluoride; the MAX phase is one of aluminum titanium carbide, aluminum molybdenum carbide, or silicon titanium carbide; the time of vacuum drying is between 12 and 48 h, and the temperature is between 37 and 100 °C.

[0013] Further defined, in step (2), the volume of the MBs is 1 - 10 μL, and the concentration is 5 - 10 mg / mL.

[0014] Further defined, in step (3), the fluorescent group is one of FAM, ROX, Cy3, or Cy5; the volume of the MXene / Pt-Pd composite material is 10 - 20 μL, and the concentration is 1 - 20 mg / mL; the volume of the H2O2 is 10 - 20 μL, and the concentration is 1 - 20 mg / mL; the volume of the TMB is 10 - 20 μL, and the concentration is 1 - 20 mg / mL.

[0015] Further defined, in steps (2) and (3), the concentration of the DNA strand is 0.5 - 2 μmol / L, the volume is 5 - 10 μL, and the incubation time is 0.5 - 2 h; the buffer solution is one or more of Tris-HCl, Buffer I, PBS, and HEPES solution.

[0016] Compared with the prior art, the present invention has the following remarkable advantages:

[0017] 1. The present invention utilizes the specific recognition of DNAzyme to Pb 2+ to ensure the selectivity of the sensor. At the same time, combined with magnetic separation technology, not only a fluorescence / colorimetric dual-mode channel is ingeniously constructed, but also the interference of background signals is reduced, improving the detection accuracy and reliability.

[0018] 2. The present invention adopts the DNAzyme target cycling strategy to achieve signal amplification and enhance the sensitivity of the dual-mode sensor.

[0019] 3. The present invention successfully prepares a novel bifunctional MXene / Pt-Pd composite material, and utilizes its fluorescence quenching ability and peroxidase-like activity to achieve fluorescence and colorimetric dual-mode detection, reducing experimental operations and also reducing the interference of the detection environment, improving the reliability of the sensor.

[0020] The above description is only an overview of the technical solution of the present invention. In order to be able to understand the technical means of the present invention more clearly and implement it in accordance with the content of the specification, the following is a detailed description of the preferred embodiments of the present invention. Brief Description of the Drawings

[0021] Figure 1 It is a schematic diagram for the preparation of a fluorescence-colorimetric dual-mode sensor based on a bifunctional MXene / Pt-Pd composite material.

[0022] Figure 2 It is the fluorescence (A) and colorimetric (B) detection results of the sensor constructed in Example 1 of the present invention before (dashed line) and after (solid line) adding Pb 2+ .

[0023] Figure 3 It is the fluorescence (A) and colorimetric (B) standard curves for the detection of lead ions by the dual-mode sensor constructed in Example 1 of the present invention.

[0024] Figure 4 It is the selectivity of the dual-mode sensor constructed in Example 1 of the present invention for lead ions in the presence of other interfering ions. (A) is the fluorescence channel, and (B) is the colorimetric channel. Detailed Embodiments

[0025] The following combines the drawings and embodiments to further describe in detail the specific embodiments of the present invention. The following embodiments are used to illustrate the present invention, but are not used to limit the scope of the present invention.

[0026] Example 1

[0027] Preparation method and application of a fluorescence-colorimetric dual-mode sensor based on a bifunctional MXene / Pt-Pd composite material, and the implementation method is as Figure 1 shown.

[0028] A preparation method of a fluorescence-colorimetric dual-mode sensor based on a bifunctional MXene / Pt-Pd composite material includes the following steps:

[0029] (1) Add 100 mg of titanium aluminum carbide powder (Ti3AlC2) to 200 mL of 40% hydrofluoric acid (HF), mix evenly, and stir at 35 °C and 400 rpm for 24 h. After the etching reaction ends, centrifuge at 8000 rpm for 5 min, repeatedly rinse with ultrapure water, and centrifuge until the pH of the supernatant ≥ 6. Collect the material and place it in a vacuum drying oven, dry at 60 °C for 12 h to obtain MXene-Ti3C2 nanosheets. Take 200 mg of MXene nanosheets and add them to 30 mL of ultrapure water, ultrasonicate thoroughly for 1 h to prepare a well-dispersed suspension. Add 1200 μL of 100 mmol / L Na2PdCl4 and 1200 μL of 100 mmol / L H2PtCl6 to the above suspension in sequence, and gently stir at 25 °C and 200 rpm for 1 h to complete the in-situ growth reaction. Centrifuge the mixture at 8000 rpm for 5 min, wash with ultrapure water, and centrifuge to collect the precipitate. Dry in an oven at 60 °C for 12 h to obtain the Ti3C2 / PtPd NPs composite material.

[0030] (2) Take 6 μL of 5 mg / mL carboxylated MBs and place them in a 200 μL centrifuge tube, and wash once with 50 μL of PBS buffer. At the same time, mix 10 μL of 1 μmol / L amino-modified substrate strand S-DNA and 10 μL of 0.5 μmol / L enzyme strand E-DNA evenly, and incubate at 37 °C for 1 h. Add the double-stranded mixture to the washed magnetic beads, place them at 37 °C, and oscillate and incubate overnight. Magnetically separate and wash 3 times with PBS buffer, remove the supernatant, and successfully construct the E-DNA / S-DNA@MBs system. Add 10 μL of a solution without Pb 2+ and a solution containing Pb 2+ to the system respectively, incubate at 37 °C for 1 h, after magnetic separation of the mixture, aspirate the supernatant and retain the precipitate.

[0031] (3) After the above magnetic separation, the supernatant and the precipitate are respectively used to construct a fluorescence and colorimetric dual-mode channel. Fluorescence channel: 10 μL of the supernatant is placed in a 200 μL centrifuge tube, 10 μL of 1 μmol / L single-stranded S1 is added thereto, incubated at 37 °C for 1 h, then 10 μL of 10 mg / mL Ti3C2 / PtPd NPs composite material is continuously added thereto, and oscillated and incubated at 37 °C for 30 min. The volume is supplemented to 200 μL with buffer solution, and fluorescence measurement is carried out. Colorimetric channel: The above precipitate is washed 3 times with PBS buffer solution, 10 μL of 1 μmol / L single-stranded S2 is added thereto, oscillated and incubated at 37 °C for 1 h, then 10 μL of 5 mg / mL Ti3C2 / PtPd NPs composite material, 10 μL of 5 mmol / L H2O2 and 10 μL of 15 mmol / L TMB are continuously added, the volume is supplemented to 100 μL with pure water, and incubated at 37 °C for 10 min for color development, and absorbance measurement is carried out.

[0032] (4) Establishment of the standard curve: 10 μL of Pb 2+ standard solutions with different concentrations are added to steps (2) and (3) to obtain sample detection solutions with different gradients. They are respectively detected by a fluorescence spectrophotometer and an ultraviolet spectrophotometer. The logarithm of the lead ion concentration is used as the abscissa, and the difference in FAM fluorescence intensity before and after spiking is used as the ordinate to establish the standard curve of the fluorescence channel. The logarithm of the lead ion concentration is used as the abscissa, and the difference in absorbance before and after spiking is used as the ordinate to establish the standard curve of the colorimetric channel.

[0033] As Figure 2 shown, the fluorescence (A) and colorimetric (B) detection results of the sensor constructed in Example 1 of the present invention before (dashed line) and after (solid line) adding Pb 2+ .

[0034] As Figure 3 shown, the fluorescence (A) and colorimetric (B) standard curves for detecting lead ions by the dual-mode sensor constructed in Example 1 of the present invention.

[0035] Example 2:

[0036] A preparation method and application of a fluorescence colorimetric dual-mode sensor based on a bifunctional MXene / Pt-Pd composite material. Its practical application includes the following steps:

[0037] In order to verify that the prepared dual-mode sensor has specific recognition for lead ions, a lead ion standard is added to the buffer solution so that the concentration of Pb 2+ in the sample is 100 nM; standard solutions of 8 other interfering metal ions are respectively prepared with the buffer solution, and the concentration of each is 10 μM, which is Pb 2+100 times the concentration. Using the detection system constructed in Example 1, the above 8 different interfering metal ion standard solutions and their mixed samples with Pb 2+ were detected, and the detection results are as Figure 4 shown, indicating that the method of the present invention has good selectivity for Pb 2+ .

[0038] Example 3:

[0039] A preparation method and application of a fluorescence-colorimetric dual-mode sensor based on a bifunctional MXene / Pt-Pd composite material. Its practical application includes the following steps:

[0040] (1) Actual sample treatment: Filter the lake water sample using a 0.22 μm filter membrane. The cereal sample is pretreated by microwave digestion method. First, weigh 0.5 g of cereal and add it to the tank of the microwave digestion furnace. At the same time, add 8 mL of HNO3 and 2 mL of H2O2. Set the reaction temperature, pressure, heating time, and holding time to 180 °C, 400 psi, 12 min, and 15 min respectively. After digestion, heat the tank containing the digestion solution on a hot plate to expel the acid in the digestion solution, and finally cool it at room temperature. Using the standard addition method, obtain the food extract.

[0041] (2) Sample detection: Take 10 μL of the food extract, measure the fluorescence and colorimetric signals according to steps (1), (2), and (3) of Example 1, and substitute them into the standard curve to obtain the concentration of Pb 2+ in the sample.

[0042] (3) When using cereal as the food sample for determination, based on the addition amount of 50 nmol / L, add Pb 2+ standard products with 0.02 times and 10 times the reference amount to the cereal respectively. Take 10 μL of the sample solution, measure the dual-mode signal according to steps (1), (2), and (3) of Example 1, and substitute it into the standard curve detected in Example 1 to obtain the concentration of Pb 2+ in the sample. Each sample is measured 3 times and the average value is taken. Calculate the recovery rate and RSD as shown in the following table:

[0043] Table 1

[0044]

[0045] It is verified that the prepared dual-mode sensor has the advantages of high sensitivity, good selectivity, and good reliability for the detection of lead ions. The detection of actual samples shows that the prepared sensor has very good practical application value.

[0046] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above-described exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic characteristics of the present invention. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be embraced within the present invention. Any reference signs in the claims should not be construed as limiting the claims involved.

[0047] In addition, it should be understood that although this specification is described in terms of embodiments, not every embodiment only contains an independent technical solution. This narrative manner of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A preparation method of a fluorescence-colorimetric dual-mode sensor based on a bifunctional MXene / Pt-Pd composite material, characterized in that, Including the following steps: (1) Etch the MAX phase material with an etching solution, and after vacuum drying, obtain a two-dimensional layered material MXene with the properties of adsorbing single-stranded DNA and fluorescence quenching. Add chloroplatinic acid and sodium chloropalladate, and in-situ grow bimetallic nanoparticles with peroxidase-like activity on the surface of MXene by redox method to prepare an MXene / Pt-Pd composite material. Compared with the MXene material with single properties, further in-situ generate Pt-Pd bimetallic nanoparticles on its surface to prepare a bifunctional composite material for dual-mode channels; (2) Fix the double-stranded DNAzyme formed by base complementary pairing of the DNA substrate strand S-DNA and the DNA enzyme strand E-DNA to the surface of magnetic beads MBs through the amidation reaction between amino and carboxyl groups, and add Pb to it. 2+ After incubation, the Pb 2+ cleavage site rA site of the DNA substrate strand S-DNA is cleaved and the DNA strand S-DNA-a is released. After magnetic separation of the mixture, the supernatant is aspirated and the precipitate is retained. At this time, a large amount of DNA strand S-DNA-a is contained in the supernatant, and only another DNA strand S-DNA-b after cleavage remains on the surface of the magnetic beads in the precipitate. Compared with the target recognition and signal amplification effects of the common DNAzyme-MBs structure, further use magnetic separation technology to construct a target-induced fluorescence and colorimetric dual-mode channel; (3) After magnetic separation of the above mixture, the supernatant and the precipitate are respectively used to construct fluorescence and colorimetric dual-mode channels. Fluorescence channel: Add a DNA strand S1 labeled with a fluorophore to the supernatant and incubate. The DNA strand S-DNA-a undergoes base complementary pairing with the DNA strand S1 labeled with a fluorophore to form a double-stranded structure. Then continue to add the MXene / Pt-Pd composite material thereto, oscillate and incubate, use a buffer solution to supplement the volume, and perform fluorescence measurement; Colorimetric channel: Wash the above precipitate with a buffer solution, add the DNA strand S2 thereto, oscillate and incubate. The DNA strand S-DNA-b on the surface of the magnetic beads forms a double-stranded structure with the DNA strand S2. Then continue to add the MXene / Pt-Pd composite material, hydrogen peroxide, and 3,3',5,5'-tetramethylbenzidine, supplement the volume with pure water and incubate, and then perform absorbance measurement. Different from ordinary single-mode and dual-mode sensing strategies, a dual-mode sensor is constructed using the properties of the bifunctional composite material.

2. The preparation method of a fluorescence-colorimetric dual-mode sensor based on a bifunctional MXene / Pt-Pd composite material according to claim 1, characterized in that, In step (1), the etching solution is one or more of hydrofluoric acid, ammonium bifluoride, or a mixture of hydrochloric acid and lithium fluoride; the MAX phase is one of aluminum titanium carbide, aluminum molybdenum carbide, or silicon titanium carbide; the time of vacuum drying is between 12 and 48 h, and the temperature is between 37 and 100 °C.

3. The preparation method of a fluorescence-colorimetric dual-mode sensor based on a bifunctional MXene / Pt-Pd composite material according to claim 1, wherein, In step (2), the volume of the MBs is 1 to 10 μL, and the concentration is 5 to 10 mg / mL.

4. The preparation method of a fluorescence-colorimetric dual-mode sensor based on a bifunctional MXene / Pt-Pd composite material according to claim 1, wherein, In step (3), the fluorophore is one of FAM, ROX, Cy3, or Cy5; the volume of the MXene / Pt-Pd composite material is 10 to 20 μL, and the concentration is 1 to 20 mg / mL; the volume of hydrogen peroxide is 10 to 20 μL, and the concentration is 1 to 20 mg / mL; the volume of 3,3',5,5'-tetramethylbenzidine is 10 to 20 μL, and the concentration is 1 to 20 mg / mL.

5. The preparation method of a fluorescence-colorimetric dual-mode sensor based on a bifunctional MXene / Pt-Pd composite material according to claim 1, wherein Further defined, in steps (2) and (3), the concentration of the DNA strand is 0.5 to 2 μmol / L, the volume is 5 to 10 μL, and the incubation time is 0.5 to 2 h; the buffer solution is one or more of Tris-HCl, Buffer I, PBS, and HEPES solution.

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