阅读说明：本技术 微波快速简便合成氧化铬纳米颗粒的方法及其应用 (Method for quickly, simply and conveniently synthesizing chromium oxide nano-particles by microwave and application thereof ) 是由 黄伟涛 刘清玉 卜珍琦 姚清锋 丁学知 夏立秋 于 2021-08-10 设计创作，主要内容包括：本发明公开了微波快速简便合成氧化铬纳米颗粒的方法及其应用。本发明通过微波辅助加热Cr～(3+)和柠檬酸盐的混合溶液数分钟,开发了一种简便、快速、绿色、低成本且大规模的氧化铬纳米颗粒的合成方法。合成的Cr-(2)O-(3)NPs对各种分子表现出通用的荧光猝灭能力。利用DNA的特异性识别能力构建基于Cr-(2)O-(3)NPs的荧光传感系统来检测Hg～(2+),并设计用于实现简单逻辑门操作的分子逻辑计算系统和复杂的逻辑环路。本发明快速且大规模的制备铬基纳米材料提供新思路,并为广泛和深入探索铬基纳米材料的新特性(例如独特的光学性能或模拟酶)和多用途应用(传感、催化、逻辑计算和加密)提供新的机会。(The invention discloses a method for quickly, simply and conveniently synthesizing chromium oxide nano-particles by microwaves and application thereof. The invention heats Cr by microwave assistance 3+ And citrate for several minutes, a simple, rapid, green, low-cost and large-scale synthesis method of chromium oxide nanoparticles is developed. Synthesized Cr 2 O 3 NPs exhibit universal fluorescence quenching ability for a variety of molecules. Construction of Cr-based DNA Using the specific recognition ability of DNA 2 O 3 Fluorescence sensing system of NPs for detecting Hg 2+ And is designed to implement simple logic gatesA molecular logic computing system of operation and complex logic loops. The invention provides a new idea for preparing the chromium-based nano material rapidly and on a large scale, and provides a new opportunity for widely and deeply exploring new characteristics (such as unique optical performance or mimic enzyme) and multipurpose applications (sensing, catalysis, logic calculation and encryption) of the chromium-based nano material.)

1. The method for quickly, simply and conveniently synthesizing the chromium oxide nano-particles by microwave is characterized by comprising the following steps of:

(1) 20mM Cr was prepared³⁺Heating the aqueous solution for 1.5-2.5 minutes to obtain a heated aqueous solution;

(2) stopping heating and taking out the heated aqueous solution when the heated aqueous solution is boiled and a large amount of uniform bubbles appear; immediately adding sodium citrate water solution to obtain mixed solution; sodium citrate and the Cr³⁺Cr in aqueous solution³⁺In a ratio of 10: 1-1: 1;

(3) continuing to heat the mixed solution for 1-2 minutes, taking out and cooling to room temperature in the dark to obtain a mixture;

(4) centrifuging the mixture to obtain a blue-green precipitate;

(5) re-dispersing the precipitate with water to obtain purified chromium oxide nanoparticles (Cr)₂O₃ NPs)。

2. The microwave rapid and simple synthesis method of chromium oxide nanoparticles according to claim 1, characterized in that in the step (1) and the step (3): the heating is carried out by adopting a microwave oven at the power of 650-750W.

3. The microwave rapid and simple synthesis method of chromium oxide nanoparticles according to claim 1, characterized in that in the step (2): the concentration of the sodium citrate aqueous solution is 20mM-200 mM.

4. The microwave rapid and simple synthesis method of chromium oxide nanoparticles according to claim 1, characterized in that in the step (4): the rotation speed of the centrifugation is controlled to be 10,000-15,000RCF, and the time is controlled to be 15-25 minutes.

5. The microwave rapid and simple synthesis method of chromium oxide nanoparticles according to claim 1, characterized in that in the step (4): and washing the blue-green precipitate with ultrapure water for 1-3 times.

6. The microwave rapid and simple synthesis method of chromium oxide nanoparticles according to claim 1, characterized in that in the step (5): the final concentration of the dispersed chromium oxide nanoparticles was 200 mM.

7. The method for rapid and simple synthesis of chromium oxide nanoparticles according to claim 1 using microwaves as a starting material for preparing chromium oxide nanoparticles in Hg²⁺Application in fluorescence biosensing.

8. Use according to claim 7, characterized in that: the Hg is²⁺The fluorescence biological sensing adopts DNA-Cr₂O₃Detection of Hg by NPs complexes²⁺。

9. The use of the chromium oxide nanoparticles prepared by the method for rapid and simple synthesis of chromium oxide nanoparticles according to claim 1 in complex molecular logic calculations.

10. Use according to claim 9, characterized in that: the application adopts DNA-Cr₂O₃NPs sensing systems.

Technical Field

The invention relates to the technical field of preparation and application of chromium oxide nanoparticles, in particular to a method for quickly, simply and conveniently synthesizing chromium oxide nanoparticles by microwave and application thereof.

Background

The chromium-based nano material has potential application prospects in the fields of catalysis, high-grade colorants, electrochemical supercapacitors, biomedicine (anti-cancer, antibacterial and anti-diabetic drugs), environmental remediation and the like. Because of the highly sensitive and visible nature of fluorescence, nanomaterials (e.g., graphene or graphene quantum dots, gold nanoparticles or nanoclusters, MoS) are explored₂) In combination with biomolecules (e.g., DNA, polypeptides, antibodies) would facilitate the development of fluorescent biosensing and imaging methods for disease diagnosis and environmental monitoring, and the establishment of fluorescent molecular information techniques (molecular logic gates, molecular authentication, cryptography, etc.). For example, some fluorescent nanoquenchers (e.g., graphene, WS)₂、MoS₂、g-C₃N₄Nano-sheet, carbon nanotube) and biomolecular probes are widely used in the fields of biosensing, medical diagnosis, molecular logic computation (such as simple logic gate), and the like. Furthermore, by virtue of molecular recognition and the property of DNA to be readily fluorescently labeled (e.g., T-Hg)²⁺-T-bonding), some based on reduced graphene oxide, copper-gold nanoclusters and graphitic carbonitride (g-C) were developed₃N₄) Nano meterHigh-sensitivity Hg detection of nano sensing system of chip²⁺And simple molecular logic operation is realized.

The existing method for synthesizing the chromium-based nano material mainly comprises a biosynthesis method and a physical and chemical synthesis method. Synthesis of chromium oxide nanoparticles (Cr) by using plant extracts (e.g., garlic extract and fruit extract of H. theobica)₂O₃NPs) attracted the attention of researchers; however, the components of plant extracts are often complex, easily introduce unwanted impurities and difficult to purify. In addition, physicochemical synthesis methods include hydrothermal synthesis, sonochemistry, solid thermal decomposition, and laser vaporization, but these methods are time consuming (hours to tens of hours), labor intensive, hazardous (requiring vacuum, inert gas, or gamma ray radiation), and energy intensive (600 or 800 ℃ high temperature for several hours). Their practical and widespread use is limited by their manufacturing drawbacks, including complexity, cumbersome operations and harsh conditions. In addition, some nanomaterials (e.g., fluorescent nitrogen-doped carbon dots (NCDs), Au NBs @ Ag nanoparticles) detect Hg using colorimetric or fluorescent methods²⁺However, the detection limit is relatively high and the detection range is narrow.

Disclosure of Invention

The invention aims to solve the technical problem of providing a method for quickly, simply and conveniently synthesizing chromium oxide nano-particles by microwave and application thereof.

In order to solve the technical problems, the invention adopts the following technical scheme:

the method for quickly, simply and conveniently synthesizing the chromium oxide nano-particles by microwave and application thereof are provided, and comprise the following steps:

(1) 20mM Cr was prepared³⁺Heating the aqueous solution for 1.5-2.5 minutes to obtain a heated aqueous solution;

(2) stopping heating and taking out the heated aqueous solution when the heated aqueous solution is boiled and a large amount of uniform bubbles appear; immediately adding sodium citrate water solution to obtain mixed solution; sodium citrate and the Cr³⁺Cr in aqueous solution³⁺In a ratio of 10: 1-1: 1;

(3) continuing to heat the mixed solution for 1-2 minutes, taking out and cooling to room temperature in the dark to obtain a mixture;

(4) centrifuging the mixture to obtain a blue-green precipitate;

(5) re-dispersing the precipitate with water to obtain purified chromium oxide nanoparticles (Cr)₂O₃ NPs)。

Preferably, in the step (1) and the step (3): the heating is carried out by adopting a microwave oven at the power of 650-750W.

Preferably, in the step (2), the concentration of the sodium citrate aqueous solution is 20mM-200 mM.

Preferably, in the step (4): the rotation speed of the centrifugation is controlled to be 10,000-15,000RCF, and the time is controlled to be 15-25 minutes.

Preferably, in the step (4): and washing the blue-green precipitate with ultrapure water for 1-3 times.

Preferably, in the step (5): the final concentration of the dispersed chromium oxide nanoparticles was 200 mM.

The chromium oxide nano-particles prepared by the method for quickly, simply and conveniently synthesizing the chromium oxide nano-particles by microwaves are in Hg²⁺Application in fluorescence biosensing.

Preferably: the Hg is²⁺The fluorescence biological sensing adopts DNA-Cr₂O₃Detection of Hg by NPs complexes²⁺。

The chromium oxide nano-particles prepared by the method for quickly, simply and conveniently synthesizing the chromium oxide nano-particles by microwaves are applied to complex molecular logic calculation.

Preferably: the application adopts DNA-Cr₂O₃NPs sensing systems.

Compared with the prior art, the invention has the main beneficial technical effects that:

1. the invention develops the Cr with simplicity, convenience, rapidness, greenness, low cost and large scale₂O₃NPs synthesis method by microwave-assisted heating of Cr³⁺And citrate for several minutes to synthesize chromium oxide nanoparticles (Cr)₂O₃NPs). Cr prepared efficiently₂O₃NPs are uniformly dispersed and smooth-surfaced spherical particles (average)Diameter greater than 100 nm). Chemical composition analysis shows that the Cr is wrapped by the citrate₂O₃NPs have abundant surface functional groups.

2. Cr prepared by the method of the invention₂O₃NPs exhibit universal fluorescence quenching capabilities for a variety of molecules (e.g., dyes and dye-labeled DNA). Utilizing the specific recognition capability (T-Hg) of DNA²⁺-T binding) construction based on Cr₂O₃Fluorescence sensing system of NPs for detecting Hg²⁺The detection Limit (LOD) is 5.78nM and is lower than the allowable inorganic Hg of the United states environmental protection agency²⁺Is capable of sensitively detecting Hg at a maximum allowable level (10nM)²⁺(ii) a And to Hg²⁺Has high selectivity.

3. The invention discloses citrate-coated Cr with universal fluorescence quenching capability₂O₃NPs as sensing platform can be combined with biomolecules (DNA) for Hg²⁺Fluorescence biosensing and complex molecular logic calculations of (a): by the specific recognition capability of DNA (T-Hg)²⁺-T binding) construction of a Cr-based alloy₂O₃Fluorescence sensing system of NPs for detecting Hg²⁺AND molecular logic computing systems (YES, NOT, AND, OR) AND complex logic loops for implementing simple logic gate operations have been designed.

4. The application of the invention provides a new idea for preparing the chromium-based nano material rapidly and on a large scale, and provides a new opportunity for widely and deeply exploring new characteristics (such as unique optical performance or mimic enzyme) and multipurpose applications (sensing, catalysis, logic calculation and encryption) of the chromium-based nano material.

Drawings

FIG. 1.(A) heating of Cr by microwave³⁺And sodium citrate. Inserting the picture at the upper right corner: the color of the corresponding colloidal aqueous solution and its tyndall effect. (B) SEM and (C) TEM images confirmed that the prepared colloids were mainly regular spherical nanoparticles. Inserting the picture at the upper right corner: further magnified SEM and TEM images. Scale bars 500, 400 and 200 nm. (D, E) statistical alignment of spherical nanoparticle diameters obtained by processing SEM (D) and TEM (E) images using ImageJ softwareA block diagram. The curves are gaussian fit data.

FIG. 2.(A, B) Cr synthesized by microwave-assisted heating₂O₃XRD pattern and FT-IR spectrum of NPs. Among them, XRD peaks marked with asterisks at 21.42 °, 61.7 ° and 69.2 ° can be attributed to SiO₂A substrate. (C) Cr (chromium) component₂O₃EDS elemental analysis of NPs. (D-H) XPS survey (D) and Cr₂O₃The C1s (E), N1s (F), O1 s (G), Cr 2p (H) core spectra of NPs.

FIG. 3 (A) four different ratios (sodium citrate: Cr) under microwave heating³⁺1: 1. 2: 1. 5: 1. 10: 1) the tyndall effect of the reaction solution of (1). (B) The microwave heated reaction solution produced a blue-green centrifuge deposit (left), while the unheated reaction solution did not (right).

FIG. 4 (A-D) Low and high magnification SEM images demonstrating different synthesis ratios (sodium citrate: Cr)³⁺10: 1. 5: 1. 2: 1. 1: 1) the colloids obtained below were all regularly spherical nanoparticles. Scale bar: 100 and 400 nm. (E-H) statistical histogram of spherical nanoparticle diameters of SEM image was processed by using ImageJ software. The curves are gaussian fit data.

FIG. 5 (A) Cr₂O₃NPs (22mM) cause changes in the fluorescence emission spectra of three common fluorescent dyes (fluorescein sodium, rhodamine B, acridine orange). Right inset: addition of Cr₂O₃Fluorescence photographs of three fluorescent dye solutions before and after NPs. (B) Cr of different concentrations₂O₃Fluorescence quenching response of NPs (0, 0.5, 1.5, 3, 5, 10, 14, 22mM) to three dyes (fluorescein sodium, 0.5. mu.M; rhodamine B, 1.25. mu.M; acridine orange, 2. mu.M). (C) Cr (chromium) component₂O₃NPs (62mM) cause changes in fluorescence emission spectra of ssDNA having different base sequences (T33, A33, G33, C33 at a concentration of 100 nM). (D) The difference in fluorescence quenching rates of ssDNA and fluorescein sodium at different concentrations (0, 1, 2, 3, 4, 5, 10, 14, 18, 22, 26, 30, 33, 37, 40, 43, 46, 49, 52mM) of Cr2O3 NPs was compared.

FIG. 6 (A) addition of different concentrations of Hg²⁺(from top to bottom: 0, 0.21, 0.53, 1, 1.55,2. 4.2, 10.5, 20.8, 30.9, 40.8, 50.5, 60, 69.3, 78.4, 87.4, 96.2 μ M), T33-Cr₂O₃Fluorescence emission spectrum of NPs complex (100 nM: 30 mM). (B) T33-Cr₂O₃Fluorescence intensity Change at 520nM of NPs Complex (100 nM: 30mM) (F)₀-F)/F₀For Hg²⁺Dependence on concentration. (C-D) (F)₀-F)/F₀With Hg²⁺The concentration is in the range of 0.21-2 μ M and 4.2-50.5 μ M. (E) T33-Cr₂O₃NPs complex (100 nM: 30mM) vs Hg²⁺Measurement (Hg)²⁺And 5 μ M of other metal ions). Buffer solution: Tris-HAc, 5mM, pH 7.0.

Fig. 7 (a) a system model in which various components interact and exchange materials, energy, and information. (B) From the information aspect, the pair is based on T33-Cr₂O₃Hg of NPs²⁺The fluorescence sensing system performs boolean logic analysis. The red 2-bit binary digits represent the presence and level of matter and energy, respectively. 0 indicates the absence of the substance or low fluorescence, and 1 indicates the presence of the substance or high fluorescence. The fluorescence threshold was 150 (a.u.). (C) Fluorescence emission spectra of different combinations of molecular events. Cr (chromium) component₂O₃NPs，30mM；T33，100nM；Hg²⁺12 μ M; GSH, 24 μ M; buffer solution: 5mM Tris-HAc, pH 7.0.

FIG. 8 shows DNA-Cr₂O₃And the NPs sensing system is used for molecular logic calculation and operation. (A) Symbol and truth tables for the T33 YES substance gate and fluorescence gate. (B) (T33 AND Cr₂O₃NPs) substance (M) gate and T33 YES fluorescence (F) gate. (C) (GSH AND Hg)²⁺) The sum of the substances (Hg)²⁺NOT OR GSH) fluorescence gate. (D) Logic circuit sign and truth table for matter and fluorescence gates with three matter inputs and two outputs. (E) Complex logic circuit symbols (left) and logic processing capabilities (right radar plots) with four material inputs and two outputs (material and fluorescence). The orange column/dot of fluorescence (F) indicates a logical output of 1, and the gray column/dot indicates a logical output of 0. The blue circles represent logical thresholds. T33, 100 nM; cr (chromium) component₂O₃NPs，30mM；Hg²⁺,12μM；GSH，24μM；Buffer：5mM Tris-HAc， pH 7.0。

Detailed Description

The following examples are intended to illustrate the present invention in detail and should not be construed as limiting the scope of the present invention in any way. The instruments and devices referred to in the following examples are conventional instruments and devices unless otherwise specified; the related reagents are all conventional reagents in the market, if not specifically indicated; the test methods involved are conventional methods unless otherwise specified.

The sodium citrate dihydrate and chromium nitrate trihydrate (Cr (NO)₃)₃·9H₂O), fluorescein sodium, rhodamine B, acridine orange, Tris (hydroxymethyl) aminomethane (Tris), glacial acetic acid, Glutathione (GSH) and all the metal salts used were purchased from alatin reagent limited (shanghai, china) and all the products were analytically pure; the oligonucleotides were all synthesized by Sangon Biotechnology co, ltd. (shanghai, china), four oligonucleotides (T33, a33, C33 and G33) having carboxyfluorescein (FAM) -labels at the 5' end. The a33, T33 and C33 represent sequences comprising 33 consecutive a, T and C, respectively. The G33 represents the GGGGGGTGGGGGGGGGGGGGGGTGGGGGGTGGGGG sequence; the aqueous solutions were prepared using ultra pure water produced by a Milli-Q system (Millipore, 18.2 M.OMEGA.cm, Bedford, Mass.).

The ssDNA means single-stranded DNA; the standard recovery rate refers to the ratio of the result obtained by adding quantitative standard substances into a sample matrix without the measured substances and analyzing according to the processing steps of the sample to the theoretical value. The T-Hg²⁺the-T hairpin structure is thymine-Hg²⁺-thymine (T-Hg)²⁺-T) coordination phenomena, from Hg²⁺The substituted imino proton is bound to N3 of thymine and connects two thymine residues.

Example 1: chromium oxide nanoparticles (Cr)₂O₃NPs) preparation method

(1)Cr(NO₃)₃·9H₂O aqueous solution (20mM) was used in a Galanz HP3(S0) household microwave oven (China) at 700W powerHeat for about 2 minutes. When the solution boiled and a large number of uniform bubbles appeared, the heating was stopped and taken out, an aqueous solution of sodium citrate (20mM) was immediately added, the microwave heating of the mixed solution at 700W was continued for 1.5 minutes, taken out and cooled to room temperature in the dark. Subsequently, the mixture was centrifuged at 15,000RCF for 20 minutes, and the precipitate was blue-green after centrifugation, indicating that microwave heating may induce Cr³⁺And sodium citrate to form a colloid. And washing twice with ultrapure water to obtain the product. Finally, the blue-green product was redispersed in ultra pure water at a final concentration of 200mM to yield purified chromium oxide nanoparticles (Cr)₂O₃ NPs)。

(2) For detecting Cr₂O₃The absorption spectrum and fluorescence spectrum of NPs are obtained, and the prepared chromium oxide nano-particles (Cr) are taken₂O₃NPs) were placed on a clean silicon wafer or copper mesh and naturally dried, and then the purified Cr was observed with SU8010 field emission scanning electron microscope (SEM, hitachi, japan), Tecnai G2F 20 transmission electron microscope (TEM, FEI, usa) and Energy Dispersive Spectrometer (EDS)₂O₃NPs samples. SEM (at least 500 particles) and TEM (at least 300 particles) images were measured using Image J software and their particle size distributions were calculated by gaussian fitting.

Microscopic morphological and chemical composition analysis was used to characterize the blue-green colloids obtained. The resulting colloidal solution had a pronounced Tyndall effect, was off-white in color, had a broad absorption band in the 190-900nm range and a peak at 230nm (FIG. 1A). The SEM image in fig. 1B confirmed that the obtained colloid was mostly smooth and regular spherical particles with an average diameter of 123.68 ± 3.45nm (N ═ 500, R)²0.980, fig. 1D). The TEM image in fig. 1C also shows that the obtained colloid is monodisperse and uniform nanoparticles with a size range of 80-300nm and an average diameter of 140.06 ± 4.27nm (N-300, R)²0.986, fig. 1E), consistent with SEM results.

(3) Cr was measured by X-ray diffraction (XRD) system (Bruker D8, Germany), IS10 Fourier transform infrared (FT-IR) spectrometer (Nigla, USA), ESCALB 250xi X-ray photoelectron spectrometer (XPS, Sammer-Feishi science, USA)₂O₃Crystalline phase, surface functional groups and elemental composition of NPs.

XRD spectral analysis showed that the 2 theta values of 32.06 deg., 32.98 deg. and 75.26 deg. correspond to Cr respectively₂O₃Nanoparticles (Cr)₂O₃NPs) (111), (202) and (311) (fig. 2A, note that peaks at 21.42 °, 61.70 ° and 69.32 ° can be attributed to SiO₂A substrate). Cr (chromium) component₂O₃FT-IR analysis of the chemical functionality of the NPs revealed that Cr₂O₃NPs at 3415.10, 1386.32, 961.82, 552.51 and 518cm^-1There is a strong absorption band (fig. 2B). The characteristic bands of the distortion vibration of the hydroxyl group and the Cr-O are respectively positioned at 3405-3425cm^-1And 550 and 565cm^-1The absorption band is 1385-1395cm^-1The region may be a carboxylate ion and is located at 940-975cm^-1And 515-^-1The peaks of the regions correspond to Cr₂O₃Tensile vibration and bending vibration of NPs. EDS elemental analysis (FIG. 2C) attached to TEM revealed that Cr₂O₃The NPs contain C, O, Cr, Na and Cu elements, wherein Cu, C and Na can be attributed to copper mesh and sodium citrate covering the surfaces of the nanoparticles. Cr in FIG. 2D₂O₃XPS spectra of NPs showed several distinct peaks at 99.08, 285.08, 400.08, 532.08, 577.08 and 1071.08eV, demonstrating the presence of Si 2p, C1s (46.26%), O1 s (45.67%), N1s (3.01%), Cr 2p (5.06%) and Na 1s, with sodium citrate coated Cr₂O₃The elemental composition of the NPs was consistent (note that the presence of Si and N was related to the silicon wafer and organic nitrogen generated by microwave heating, respectively). High resolution nuclear power level spectra of C1s, O1 s, N1s, and Cr 2p were scanned, deconvoluted, and fitted to further identify elemental forms. The core spectrum of C1s (FIG. 2E) can be broken down into four distinct peaks, corresponding to the C-O/C-N (284.83eV), C-N (286.28eV), C-O (288.33eV) bonds, respectively; the N1s core spectrum in FIG. 2F shows two peaks at 399.83eV and 401.78eV, which are attributed to the C-N-C and N-H bonds, respectively; the O1 s core spectrum can be decomposed into C ═ O (531.48eV), -OH (533.03 eV) and-COOH (535.38eV) (fig. 2G). Cr (chromium) component₂O₃The Cr 2p core energy spectrum of NPs (FIG. 2H) shows that Cr₂O₃Three of the NPs existThe same chromium state includes Cr metal bond (574.28eV), CrO₃(589.28eV) and Cr₂O₃(576.38, 577.08, 579.83, 584.38, and 586.73 eV). According to Cr 2p signals, Cr in the prepared nano-particles₂O₃，CrO₃And the metal Cr in proportions of 90.19%, 5.41% and 4.40%, respectively. The above results show that Cr is heated by microwave³⁺And sodium citrate the resulting nanoparticles are citrate coated Cr₂O₃ NPs。

Example 2: sodium citrate and Cr (NO) in different proportions₃)₃·9H₂Effect of O on microwave heating induced colloid formation

Heating of sodium citrate (20, 40, 100, 200mM) and Cr (NO) with microwave detection using the method of example 1₃)₃·9H₂Influence of four different ratios (1: 1, 2: 1, 5: 1, 10: 1) of O (20 mM).

As shown in fig. 3:

under microwave heating, four different proportions (sodium citrate: Cr)³⁺1: 1. 2: 1. 5: 1. 10: 1) The reaction solution of (2) produces the tyndall effect.

As shown in fig. 4:

SEM images show that four different ratios of sodium citrate and chromium nitrate (10: 1, 5: 1, 2: 1, 1: 1) have no significant effect on the morphology of the resulting nanoparticles, with the majority of the nanoparticles still being regular spherical particles, but the average diameters of the four different ratios of nanoparticles being slightly different.

Example 3: detection of chromium oxide nanoparticles (Cr)₂O₃NPs) fluorescence quenching ability

Three common fluorescent dyes and four FAM-labeled ssDNA (T33, a33, C33, and G33) were selected to evaluate the chromium oxide nanoparticles (Cr) prepared in example 1₂O₃NPs) fluorescence quenching ability.

mu.L of fluorescein sodium (0.5. mu.M), rhodamine B (1.25. mu.M), acridine orange (2. mu.M), FAM-labeled ssDNA (T33, A33, C33 and G33; 100nM) solution was added to a 2-mm slit microfluorescent cuvette and subjected to different stimuliThe fluorescence emission spectra of the above solutions were measured at emission wavelengths (475, 550, 490, 485nm) in the ranges of 495-700 nm, 565-700nm and 510-700nm, respectively. Subsequently, Cr of different concentrations was added₂O₃NPs were added to the above solution, and the fluorescence emission spectrum of the mixture was measured under the same conditions.

The results show that with Cr₂O₃The fluorescence of the dyes was gradually quenched with increasing concentration of NPs (FIG. 5A), their quenching rates were 70.8%, 51% and 43%, respectively (FIG. 5B), and the fluorescence emission spectra of the three dyes (sodium fluorescein, red line; rhodamine B, green line; acridine orange, blue line) were at 500 to 700nm with Cr₂O₃The NPs absorption bands (black lines) overlap. This proves that Cr₂O₃NPs (citrate: Cr)³⁺1: 1) has wide fluorescence quenching capability and can be used as a potential quencher of various fluorescent molecules. However, Cr₂O₃NPs (citrate: Cr)³⁺10: 1) has certain enhancement effect on the fluorescence of the fluorescein sodium. This means that different proportions of Cr₂O₃NPs affect fluorescent dyes differently.

As shown in FIG. 5C, FAM fluorescently labeled A33, T33, C33 and G33 can all be Cr₂O₃NPs quench and follow Cr₂O₃The fluorescence quenching degree becomes more pronounced with a continuous increase in the concentration of NPs. Cr (chromium) component₂O₃The quenching efficiency of NPs on FAM-labeled DNA was lower than that of sodium fluorescein alone (fig. 5D), which may be due to steric hindrance caused by free single-stranded DNA molecules. And, Cr₂O₃Stern-Volmer constant (K) of NPs to T33 at different temperatures_SV) 0.0296L/mmol at 25 ℃, 0.0210L/mmol at 35 ℃ and 0.0192L/mmol at 45 ℃ decrease with increasing temperature, indicating Cr₂O₃A static quenching complex was formed between NPs and T33. Static quenching constant (K)_P) And binding constant K_A0.0218 and 0.0212L/mmol at 25 deg.C, 0.0166 and 0.0179L/mmol at 35 deg.C, and 0.0155 and 0.0224L/mmol at 45 deg.C, respectively.

Test example 1: DNA-Cr₂O₃Use of NPs complexes for detection of Hg²⁺

(1) The chromium oxide nanoparticles (Cr) prepared in example 1 were taken₂O₃NPs) by mixing 70. mu.L of 200mM Cr₂O₃NPs were added to 400. mu.L of T33 solution (100nM) to prepare T33-Cr₂O₃NPs complex. After 4 minutes of reaction, T33-Cr was measured₂O₃Initial fluorescence emission spectra of NPs complexes. Then, after adding metal ions at different concentrations and reacting for 4 minutes, T33-Cr was measured₂O₃Fluorescence emission spectra of mixtures of NPs with metal ions and the quenching rates were calculated.

Under the best conditions (T33: 100nM, Cr₂O₃NPs (neutral phosphorus complexes): 30mM, Tris-HAc: 5mM, pH 7.0), using T33-Cr₂O₃Detection of Hg by NPs complexes²⁺. As shown in FIGS. 6A and 6B, the Hg content is varied from 0 to 96.2. mu.M²⁺Increasing concentration of T33-Cr₂O₃Fluorescence intensity and quenching Rate (F) of NPs Complex (100 mM: 30mM)₀-F)/F₀And gradually decreases. FIGS. 6C and 6D show (F)₀-F)/F₀Value of Hg²⁺The concentration of the compound has two good linear relations between 0.21-2 mu M and 4.2-50.5 mu M. The two calibration equations are respectively 11.639x +3.116, and the correlation coefficient is R²0.983, and y 0.919x +34.067, R²0.989. According to the 3-sigma rule, the detection Limit (LOD) is 5.78nM, which is lower than the allowable inorganic Hg in drinking water by the United states environmental protection agency²⁺Maximum allowable level (10 nM).

With other previously reported Hg²⁺Compared with optical detection, the method provided by the invention has lower or equivalent detection limit. The results showed that T33-Cr₂O₃The NPs compound can sensitively detect Hg²⁺。

To further evaluate T33-Cr₂O₃Selectivity of NPs complexes to Hg using this method²⁺And other metal ions (Fe)³⁺、Mg²⁺、Cu²⁺，Pb²⁺，Na⁺，Ca²⁺，Al³⁺，K⁺，Mn²⁺，Cr³⁺，Zn²⁺， Ni²⁺，Cd²⁺，Ba²⁺，Be²⁺，Ag⁺And Co²⁺: 5 μ M each). As shown in FIG. 6E, only Hg²⁺Cause T33-Cr₂O₃NPs complexes gave a quenching rate of about 38.9%; while other interfering metal ions can cause fluorescence quenching of 6.6% or fluorescence recovery of 9.4% at most. The results showed that T33-Cr₂O₃NPs complex to Hg²⁺Has high selectivity.

(2) Filtering the pond water sample and the tap water sample of the Yue Wang pavilion of the Hunan university with a 0.22-mum filter membrane. 10mM Hg (NO)₃)₂The solution was added to tap and pond water samples to prepare 50mM and 1mM Hg (NO)₃)₂And (3) solution. Then, the reaction solution was stirred at T33-Cr₂O₃NPs complexes with 4. mu.L of 1mM Hg²⁺And 17 μ L of 50 μ M Hg²⁺After incubation for 4 minutes at room temperature, the fluorescence of the mixture was measured.

Tap and pond water samples were filtered using a 0.22- μm membrane to avoid sediment in the water sample adversely affecting the sensing system. The results are shown in Table 1, Hg in tap water at two spiked levels (2.00 and 10.00. mu.M)²⁺The recovery rates of the Hg in pond water are respectively 99.02 percent and 104.42 percent²⁺The recovery of (a) was 83.83% and 102.55%, respectively. The Relative Standard Deviation (RSD) of tap water and pond water was 2.00% to 3.53% and 1.68% to 3.43%, respectively. The result shows that the method has the function of detecting Hg in the actual water sample²⁺Has great potential.

TABLE 1 Hg in actual water samples²⁺Detection of

Wherein the content of the first and second substances,^ait was not detected.

Test example 2: DNA-Cr for molecular logic computation and manipulation₂O₃NPs sensing system

The chromium oxide nanoparticles (Cr) prepared in example 1 were taken₂O₃ NPs) by mixing 70. mu.L of 200mM Cr₂O₃NPs were added to 400. mu.L of T33 solution (100nM) to prepare T33-Cr₂O₃NPs complex. After 4 minutes of reaction, T33-Cr was measured₂O₃Initial fluorescence emission spectra of NPs complexes. Then, after adding metal ions at different concentrations and reacting for 4 minutes, T33-Cr was measured₂O₃Fluorescence emission spectra of mixtures of NPs with metal ions and the quenching rates were calculated. For performing the molecular logic operation, combinations of different input substances (T33, Cr)₂O₃ NPs、Hg²⁺GSH) was added to the buffer solution, and the fluorescence emission spectrum thereof was measured after 4 minutes of reaction.

As shown in FIG. 7, each component can interact and exchange materials, energy, and information in the system model (scenario 1A). In the above, Cr is used₂O₃The fluorescence quenching capability of NPs and the specific molecular recognition capability of ssDNA T33 construct a Cr-based molecular probe₂O₃Fluorescence sensing system of NPs-DNA for detecting Hg²⁺(scheme 1B). From the material and energy point of view, when FAM is labeled with T33 and Cr₂O₃When NPs are mixed, T33 can be rapidly adsorbed to Cr₂O₃NPs surface, resulting in fluorescence quenching (scheme 1Ba and C red lines). Addition of Hg²⁺Hg of mercury²⁺Possibly with Cr₂O₃T33 on NPs binds to form T-Hg²⁺A T hairpin structure, which results in a more compact interaction of the three components, leading to a stronger quenching (scheme 1Bb and C blue line). Furthermore, after Glutathione (GSH) was added, the GSH thiol groups were associated with Hg²⁺Competitive interaction of (A), T33-Cr₂O₃ NPs-Hg²⁺The interaction of the complex is disturbed, resulting in a restoration of fluorescence (scheme 1Bc and C green line).

From an information processing perspective, each component that is a fundamental event can be abstracted as a bit having two possible states (e.g., presence (logic 1) or absence (logic 0) at a material level, high (logic 1) or low (logic 0) at an energy level)The top event of the volume change, as a two-tier output (material and energy level) corresponding to information processing (e.g., logical computing operations). In a sensing system, the resulting event may be considered a substance output (M), and its logical output may be defined as when the target event does not exist as a logical 0 and exists as a logical 1, respectively. When only T33 is input as a substance, YES logic gates are formed in substance and energy as shown in fig. 8A. As shown in scheme 1Ba, T33 adsorbs to Cr₂O₃On the surface of the NPs, an AND gate T33-Cr is formed₂O₃NPs complex (T33. Cr)₂O₃NPs, intermediate event, point "·" represents AND boolean algebra, fig. 7B red gate symbol), resulting in fluorescence quenching. When reacting with Hg²⁺When mixed, T33. Cr₂O₃NPs (intermediate events) further with Hg²⁺Combined to form an AND gate T33-Cr₂O₃ NPs-Hg²⁺Compound (T33. Cr)₂O₃ NPs·Hg²⁺) Resulting in stronger quenching (scheme 1Bb, fig. 8D red gate symbol). When GSH is added, due to sulfhydryl of GSH and Hg²⁺Covalent bonding of (2), T33. Cr₂O₃ NPs·Hg²⁺Is disturbed to cause two main events (T33. Cr)₂O₃ NPs·Hg²⁺And GSH Hg²⁺Scheme 1Bc, fig. 8E) and cause the fluorescent moiety to recover. Therefore, based on Cr₂O₃The fluorescence sensing system of NPs-DNA can be self-assembled and is represented by (T33. Cr)₂O₃ NPs)·(Hg²⁺GSH) (fig. 8E red gate symbol).

Furthermore, the change in fluorescence in different combinations of the above molecular events can also be abstracted as a logical output of energy levels, with a logic 1 when fluorescence (F) ≧ 150(a.u.) fluorescence (F)<Logic 0 at 150 (a.u.). When Cr is present₂O₃When NPs and T33 are used as two inputs, the fluorescence output of the solution is1 as long as T33 is present, corresponding to the T33 YES fluorescence gate (fig. 8B). When mixing Hg²⁺And GSH as both inputs, when only Hg is present²⁺In the case of T33-Cr₂O₃The fluorescence output of the NPs complex was 0, corresponding to Hg²⁺NOT OR GSH fluorescence gate (fig. 8C). According to three input signals T33 and Cr₂O₃ NPs、Hg²⁺In the combination truth table of (1) AND (T33 AND NOT Cr) corresponding to the fluorescence output of the solution, provided that T33 is present AND the other three are NOT present at the same time₂O₃NPs)OR(T33 AND NOT Hg²⁺) A fluorescent gate (fig. 8D). If T33, Cr₂O₃ NPs，Hg²⁺AND GSH are four inputs, the entire fluorescence sensing system can implement more complex fluorescence logic functions (T33 AND NOT Cr)₂O₃ NPs)OR(T33 AND NOT Hg²⁺) OR (T33 AND GSH) (fig. 8E).

Using Cr₂O₃The fluorescence quenching ability of NPs AND the specific molecular recognition ability of ssDNA T33, molecular logic computational systems (YES, NOT, AND, OR) AND complex logic loops designed to enable simple logic gate manipulation. From the material and energy point of view, when FAM is labeled with T33 and Cr₂O₃When NPs are mixed, T33 can be rapidly adsorbed to Cr₂O₃NPs surface, resulting in fluorescence quenching. Addition of Hg²⁺Hg of mercury²⁺Possibly with Cr₂O₃T33 on NPs binds to form T-Hg²⁺-T hairpin structure, which results in a more compact interaction of the three components, resulting in a stronger quenching. Furthermore, after Glutathione (GSH) was added, the GSH thiol groups were associated with Hg²⁺Competitive interaction of (A), T33-Cr₂O₃ NPs-Hg²⁺The complex interaction is disturbed, resulting in fluorescence recovery.

In conclusion, the method only needs to utilize Galanz HP3(S0) household microwave oven (China) for microwave-assisted heating of Cr³⁺And the mixed solution of the Cr and the citrate is mixed for several minutes, and then the mixed solution is simply centrifuged and purified to synthesize the Cr₂O₃NPs; compared with other synthesis methods, the preparation method has the advantages of low cost, large scale, energy conservation and environmental protection, and does not use harsh conditions.

Prepared Cr₂O₃NPs have novel fluorescence quenching characteristics, can be used as a general fluorescence sensing platform and can be combined with DNA for Hg²⁺The detection range of (1) is 0.21-2 mu M and 4.2-50.5 mu M, the detection limit is 5.78nM, and other previous reportsHg of²⁺The proposed method has a lower or comparable detection limit compared to optical detection. Cr is analyzed comprehensively from the aspects of substance (molecular event), energy (fluorescence) and information flow₂O₃The NPs-ssDNA fluorescence sensing system may further perform from a single input to 4 inputs (Cr₂O₃ NP、T33、Hg²⁺GSH) corresponds to a molecular logic calculation of 2 outputs (substance AND fluorescence level) including simple logic gates (YES, NOT, AND, OR) AND complex logic circuits. In addition, no report about the fluorescence quenching characteristic of the chromium-based nano material and the application of the chromium-based nano material in fluorescence sensing and molecular logic is available at present.

The invention is explained in detail above with reference to the drawings and the embodiments; however, it will be understood by those skilled in the art that various changes in the specific parameters of the embodiments described above may be made or equivalents of the related materials and method steps may be substituted without departing from the spirit of the invention, thereby forming a plurality of specific embodiments, all of which are within the scope of the invention and will not be described in detail herein.