A method for detecting thiourea using carbon nanodots-gold nanoparticles as fluorescent probes

Abstract

The present invention discloses a method for detecting thiourea using carbon nanodots and gold nanoparticles as fluorescent probes, which belongs to the field of analytical detection. The technical solution of the present invention is: fluorescent carbon nanodots are mixed with gold nanoparticles in advance, and the fluorescence of the carbon nanodots will be quenched based on the fluorescence resonance energy transfer effect; then, if thiourea is present in the sample to be tested mixed with the probe, the thiourea will trigger the aggregation of gold nanoparticles through coordination and decombinate the carbon nanodots from the gold nanoparticles, and finally restore the quenched fluorescence of the carbon nanodots. Under the optimized detection conditions, the relative fluorescence intensity of the carbon nanodot-gold nanoparticle probe shows a good linear relationship with the thiourea concentration in the range of 0.05-1μM, and the detection limit (S / N=3) is 0.0362μM. The detection method established by the present invention is not only convenient, but also has excellent selectivity and sensitivity, and can be effectively applied to the detection of thiourea in tap water, lake water and fruit and vegetable juice.

Description

Technical Field

[0001] The invention relates to a method for detecting thiourea by using carbon nanodots-gold nanoparticles as fluorescent probes, and belongs to the technical field of analysis and detection. Background Art

[0002] Thiourea, a sulfur-containing organic compound, is widely used in numerous applications. However, its illegal misuse can leave residues in soil, river water, and even fruit, posing a threat to human health and environmental safety. Thiourea is classified as a Group 3 carcinogen on the list of carcinogens published by the World Health Organization's International Agency for Research on Cancer (IARC); it also appears on the list of carcinogens published by the National Institute of Toxicology, part of the U.S. Department of Health and Human Services, as a reasonably suspected human carcinogen. In aqueous solutions and soil, thiourea and its derivatives can inhibit nitrification. Therefore, developing a convenient and efficient method for monitoring thiourea is crucial.

[0003] Several methods have been proposed for thiourea detection, such as electrochemical method, colorimetric method, chemiluminescence method, high performance liquid chromatography, Raman spectroscopy and fluorescence spectroscopy. Regarding the electrochemical method, RAHMAN MM et al. used a glassy carbon electrode coated with silver oxide / titanium dioxide / zirconium dioxide ternary co-doped nanoparticles (Ag2O / TiO2 / ZrO2 / GCE) to establish a linear calibration curve between current and thiourea concentration. The linear working range of the thiourea sensor spans from 0.1nM to 0.01mM, and R 2 =0.9996 and a detection limit of 91.70±4.59 pM. Although this electrochemical method, which utilizes doped nanostructured materials to prepare electrochemical sensing probes, exhibits excellent thiourea detection capabilities, the probe preparation process is relatively complex. Based on the catalytic activity of thiourea and its derivatives in the reaction of hydrogen peroxide and bromopyrogallol red, Dikunets MA et al. developed a detection method for thiourea and its derivatives, combining the separation capabilities of high-performance liquid chromatography with post-column catalytic detection. However, this method has low sensitivity, with a detection limit of 50 μM for thiourea, and requires expensive laboratory equipment.

[0004] Among the numerous methods, fluorescence spectroscopy based on fluorescent probes has gradually become widely used in the field of analytical detection due to its advantages such as fast response, high sensitivity, low sample requirement, and visualization. Carbon nanodots, as an emerging fluorescent material, have a very broad application prospect due to their ability to undergo specialized surface modification according to application requirements. In addition, their advantages such as low cost, good hydrophilicity, and good biocompatibility are also worthy of attention. The characteristic of gold nanoparticles is that they have an extremely high extinction coefficient in the visible light band, which makes their absorption spectrum well compatible with the emission spectrum of fluorescent materials such as carbon nanodots. Therefore, gold nanoparticles often appear as fluorescence quenchers in fluorescent probe systems based on fluorescence resonance energy transfer or the inner filter effect. Summary of the Invention

[0005] Technical issues:

[0006] The present invention addresses the technical problem of sensitively detecting thiourea using a carbon nanodot-gold nanoparticle fluorescent probe. By addressing the shortcomings of existing technologies and combining the fluorescence quenching phenomenon caused by fluorescence resonance energy transfer between carbon nanodots and gold nanoparticles, and the ability of thiourea to induce gold nanoparticle aggregation and restore fluorescence in the detection system, the present invention develops a method for sensitively detecting thiourea using a carbon nanodot-gold nanoparticle fluorescent probe. The carbon nanodot-gold nanoparticle fluorescent probe has the ability to recognize thiourea, and the relative fluorescence intensity of the carbon nanodot-gold nanoparticle fluorescent probe exhibits a good linear relationship with thiourea concentration within a certain range. This method combines the advantages of good selectivity and high sensitivity, demonstrating good thiourea detection capabilities in real-world samples such as tap water, lake water, and fruit and vegetable juices.

[0007] The technical solutions adopted by the present invention to achieve the above-mentioned objectives are as follows.

[0008] The present invention provides a method for detecting thiourea using carbon nanodots-gold nanoparticles as a fluorescent probe, the detection method comprising the following steps:

[0009] (1) mixing a carbon nanodot solution, a gold nanoparticle solution, and a phosphate buffer solution and oscillating them to obtain a carbon nanodot-gold nanoparticle fluorescent probe solution; then adding a series of thiourea standard solutions of known concentrations to the probe solution, mixing them to obtain standard samples;

[0010] (2) After reacting at room temperature for a period of time, the fluorescence spectrum of each standard sample is collected and the corresponding fluorescence intensity is measured;

[0011] (3) A detection model was constructed by linearly correlating the concentration of the thiourea standard solution with the corresponding fluorescence intensity;

[0012] (4) According to step (1), a sample to be tested is prepared by replacing the thiourea solution of known concentration with a sample to be tested containing thiourea, and according to step (2), the relative intensity of the fluorescence emission peak of the sample to be tested is obtained, and then the concentration of thiourea in the sample to be tested is calculated according to the detection model in step (3).

[0013] As one of the preferred embodiments of the present invention, carbon nanodots are prepared by a microwave method using citric acid and urea as precursors. If the absorption value of the carbon nanodot solution at 351nm in the ultraviolet-visible absorption spectrum is 0.12, the corresponding concentration is set as 1 unit, and the concentration of the carbon nanodots in the standard sample and the sample to be tested is 0.1-0.5 units.

[0014] As one of the preferred embodiments of the present invention, gold nanoparticles are prepared by reducing chloroauric acid with sodium citrate, and the concentration of gold nanoparticles in the standard sample and the test sample is 0.5-3 nM.

[0015] As one of the preferred solutions of the embodiment of the present invention, the phosphate buffer solution used to prepare the standard sample and the test sample has a pH value of 7.0, a concentration of 0.02M, and a volume accounting for 50-90% of the total volume of the sample.

[0016] As one of the preferred embodiments of the present invention, the volume ratio of the carbon nanodot solution, the gold nanoparticle solution, the phosphate buffer solution, and the thiourea standard solution is 1:(1-2):(5-8):1; specifically, 1:1.2:6.8:1 can be selected.

[0017] As one of the preferred solutions of the embodiment of the present invention, the concentration of the gold nanoparticle solution is 10 nM.

[0018] As one of the preferred embodiments of the present invention, the reaction time in step (2) is 2-20 minutes.

[0019] As one of the preferred embodiments of the present invention, the excitation light wavelength when collecting the sample fluorescence spectrum in step (2) is 405 nm, and the fluorescence emission peak in step (3) is taken from 505 nm in the fluorescence spectrum.

[0020] As a preferred embodiment of the present invention, the sample to be tested in step (4) comprises ultrapure water, tap water, river water, and fruit and vegetable juice. The ultrapure water sample does not require pretreatment; the tap water and river water samples are filtered through a 0.22 μm filter membrane before use; and the fruit and vegetable juice sample is carrot compound fruit and vegetable juice (Weiquan brand). After unsealing, the juice is centrifuged at 7000 rpm for 10 minutes to remove the pulp, and the upper liquid is collected for later use.

[0021] The present invention provides a method for detecting thiourea in a water sample, comprising the following steps:

[0022] 1) mixing a carbon nanodot solution, gold nanoparticles, a phosphate buffer solution, and a water sample and oscillating the mixture to obtain a carbon nanodot-gold nanoparticle fluorescent probe solution; then adding a series of thiourea standard solutions of known concentrations to the probe solution and mixing to obtain standard samples;

[0023] 2) After reacting for a period of time at room temperature, the fluorescence spectrum of each standard sample is collected and the corresponding fluorescence intensity is measured;

[0024] 3) A detection model was constructed by linearly correlating the concentration of the thiourea standard solution with the corresponding fluorescence intensity;

[0025] 4) Substituting the thiourea solution of known concentration with the thiourea sample to prepare a test sample according to step 1), obtaining the relative intensity of the fluorescence emission peak of the test sample according to step 2), and then calculating the concentration of thiourea in the test sample according to the detection model in step 3).

[0026] In one embodiment of the present invention, when testing a water sample, the concentration of AuNPs in the standard sample in step 1) is 1.2 nM.

[0027] In one embodiment of the present invention, the volume ratio of the carbon nanodot solution, the gold nanoparticle solution, the phosphate buffer solution, the water sample, and the thiourea standard solution is 1:(1-2):(5-8):1:1.

[0028] In one embodiment of the present invention, the water sample includes tap water, lake water, river water, etc.

[0029] The present invention provides a method for detecting thiourea in fruit and vegetable juice, comprising the following steps:

[0030] A) mixing a carbon nanodot solution, gold nanoparticles, phosphate buffer solution, and fruit and vegetable juice and shaking to obtain a carbon nanodot-gold nanoparticle fluorescent probe solution; then adding a series of thiourea standard solutions of known concentrations to the probe solution and mixing to obtain standard samples;

[0031] B) After reacting for a period of time at room temperature, the fluorescence spectrum of each standard sample is collected and the corresponding fluorescence intensity is measured;

[0032] C) constructing a detection model by linearly correlating the concentration of the thiourea standard solution with the corresponding fluorescence intensity;

[0033] D) Substituting the thiourea solution of known concentration with the thiourea sample to prepare a test sample according to step A), obtaining the relative intensity of the fluorescence emission peak of the test sample according to step B), and then calculating the concentration of thiourea in the test sample according to the detection model in step C).

[0034] In one embodiment of the present invention, when testing fruit and vegetable juice, the concentration of AuNPs in the standard sample in step A) is 2.5 nM.

[0035] In one embodiment of the present invention, the volume ratio of the carbon nanodot solution, the gold nanoparticle solution, the phosphate buffer solution, the fruit and vegetable juice, and the thiourea standard solution is 1:(1-2):(5-8):1:1.

[0036] Beneficial effects:

[0037] The present invention combines the fluorescence quenching phenomenon caused by the fluorescence resonance energy transfer effect between carbon nanodots and gold nanoparticles with the ability of the probe to recover fluorescence after thiourea-induced gold nanoparticle aggregation to develop a carbon nanodot-gold nanoparticle fluorescent probe that can be used for sensitive detection of thiourea. In a pH = 7.0 (0.02M PBS) environment, the relative fluorescence intensity of the fluorescent probe shows a good linear relationship with the thiourea concentration in the range of 0.05-1μM, with a detection limit (S / N = 3) of 0.0362μM. The detection method established by the present invention has the advantages of good selectivity and high sensitivity, and has good detection capabilities for thiourea in actual samples such as tap water, lake water, and fruit and vegetable juices. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Schematic diagram of the carbon nanodot-gold nanoparticle fluorescent probe for detecting thiourea.

[0039] Figure 2 (A) HRTEM image and (B) particle size distribution of carbon nanodots; (C) HRTEM image and (D) particle size distribution of gold nanoparticles.

[0040] Figure 3 Fourier transform infrared spectrum of carbon nanodots.

[0041] Figure 4 X-ray photoelectron spectra of carbon nanodots: (A) full spectrum, (B) high-resolution C1s spectrum, (C) high-resolution N1s spectrum, and (D) high-resolution O1s spectrum.

[0042] Figure 5 (A) Multi-wavelength excitation fluorescence spectra of carbon nanodots when the excitation light wavelength is in the range of 365-420 nm; (B) UV-visible absorption spectra of carbon nanodots, carbon nanodots + thiourea, carbon nanodots + gold nanoparticles, and carbon nanodots + gold nanoparticles + thiourea; (C) Zeta potential of carbon nanodots, gold nanoparticles, and carbon nanodots + gold nanoparticles; (D) Fluorescence attenuation graph of carbon nanodots before and after the addition of gold nanoparticles.

[0043] Figure 6(A) Fluorescence emission spectrum of carbon nanodots (λex = 405 nm) and UV-visible absorption spectrum of gold nanoparticles; (B) Fluorescence emission spectra of carbon nanodots, carbon nanodots + thiourea, carbon nanodots + gold nanoparticles, and carbon nanodots + gold nanoparticles + thiourea (λex = 405 nm); HRTEM images of (C) carbon nanodots + gold nanoparticles and (D) carbon nanodots + gold nanoparticles + thiourea.

[0044] Figure 7 (A) Fluorescence emission spectra of the carbon nanodot-gold nanoparticle probe when thiourea concentrations are different in the system; (B) Relationship between the relative fluorescence intensity of the carbon nanodot-gold nanoparticle probe at 505 nm and the thiourea concentration in the thiourea concentration range of 0-10 μM (the inset shows the linear fitting result of the 0.05-1 μM part, y represents the relative fluorescence emission intensity at 505 nm, and x represents the thiourea concentration).

[0045] Figure 8 (A) UV-visible absorption spectrum, fluorescence excitation spectrum, and emission spectrum of BCDs; (B) UV-visible absorption spectrum, fluorescence excitation spectrum, and emission spectrum of GCDs; (C) fluorescence emission spectra (λex = 346 nm) of BCDs, BCDs+thiourea, BCDs+gold nanoparticles, and BCDs+gold nanoparticles+thiourea; (D) fluorescence emission spectra (λex = 390 nm) of GCDs, GCDs+thiourea, GCDs+gold nanoparticles, and GCDs+gold nanoparticles+thiourea.

[0046] Figure 9 Fluorescence emission spectra of BCDs (λex=346 nm), fluorescence emission spectra of GCDs (λex=390 nm) and UV-visible absorption spectra of gold nanoparticles.

[0047] Figure 10 (A) The relationship between the fluorescence quenching rate of carbon nanodots and the gold nanoparticle concentration; (B) The relationship between the fluorescence enhancement rate of the carbon nanodot-gold nanoparticle system and the gold nanoparticle concentration when the gold nanoparticle concentration is in the range of 0.5-3 nM.

[0048] Figure 11 Figure 3 shows the relative intensity of the fluorescence emission peak of carbon nanodots under different pH conditions within the pH range of 3 to 12, (A) the fluorescence quenching rate of gold nanoparticles on carbon nanodots, (B) the fluorescence enhancement rate of thiourea on the carbon nanodot-gold nanoparticle system (C 金纳米颗粒 =1.2nM, C 硫脲 =0.1 μM).

[0049] Figure 12The changes in the relative fluorescence intensity of the carbon nanodot-gold nanoparticle probe and the carbon nanodot-gold nanoparticle + thiourea system at 505 nm within 1-20 minutes.

[0050] Figure 13 (A) Fluorescence enhancement rate of carbon nanodot-gold nanoparticle probe after adding thiourea or other interfering substances; (B) relative intensity of the fluorescence emission peak at 505 nm in the system in the presence of thiourea and interfering substances (the concentration of thiourea and other nitrogen-rich substances is 2 μM, and the concentration of common amino acids, glucose, ascorbic acid, and common ions is 10 μM).

[0051] Figure 14 (A) The relationship between the relative fluorescence intensity of the carbon nanodot-gold nanoparticle probe and the thiourea concentration in tap water samples (the inset shows the linear fitting results in the range of 0.1-1 μM); (B) The relationship between the relative fluorescence intensity of the carbon nanodot-gold nanoparticle system and the thiourea concentration in river water samples (the inset shows the linear fitting results in the range of 0.1-1 μM); (C) The relationship between the relative fluorescence intensity of the carbon nanodot-gold nanoparticle system and the thiourea concentration in fruit and vegetable juice samples (the inset shows the linear fitting results in the range of 0.1-1 μM).

[0052] Figure 15 Flowchart for the detection of thiourea based on carbon nanodot-gold nanoparticle fluorescent probe. DETAILED DESCRIPTION

[0053] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.

[0054] Example 1: Preparation and characterization of carbon nanodot-gold nanoparticle fluorescent probe

[0055] The carbon nanodot-gold nanoparticle fluorescent probe consists of two parts: carbon nanodots and gold nanoparticles:

[0056] (1) Preparation of carbon nanodots:

[0057] First, 1g of citric acid and 1g of urea were weighed into a beaker and added with 10mL of ultrapure water. The mixture was stirred to fully dissolve. The solution was then microwaved at 700W for 3 minutes. After cooling to room temperature, 20mL of ultrapure water was added to fully dissolve the product and filtered. Finally, the desired carbon nanodots were separated from the filtrate by silica gel column chromatography using a methanol / dichloromethane mixture (1:8, v / v) as the eluent. The carbon nanodots were then diluted to a UV-Vis absorption value of 0.12 at 351nm using a rotary evaporator. The concentration of the carbon nanodot solution was then set to 1 unit. The carbon nanodot solution was stored at 4°C for subsequent use.

[0058] (2) Preparation of gold nanoparticles:

[0059] First, heat 200 mL of a 1 mM aqueous solution of chloroauric acid in an oil bath to boiling. Then, while vigorously stirring, quickly add 20 mL of an 11 mg / mL aqueous solution of trisodium citrate. The color of the solution will rapidly change from light yellow to purple-black and stabilize to wine red after continued constant stirring for 30 minutes. After the product cools to room temperature, store it at 4°C in the dark until ready for use.

[0060] (3) Preparation of carbon nanodot-gold nanoparticle fluorescent probe:

[0061] 0.3 mL of carbon nanodot solution (concentration of 1 unit), 0.36 mL of gold nanoparticle solution (10 nM), and 2.04 mL of phosphate buffer solution (0.02 M, pH = 7.0) were mixed and shaken to obtain a carbon nanodot-gold nanoparticle fluorescent probe solution.

[0062] The morphology of the prepared carbon nanodots and gold nanoparticles was characterized using high-resolution transmission electron microscopy (HRTEM). Figure 2 As shown in Figure A, the prepared carbon nanodots are spherical and well dispersed. The particle size obtained from HRTEM images is mainly distributed in the range of 12.6-17.4 nm, with an average particle size of 15.5 nm. Figure 2 C is the HRTEM image of gold nanoparticles, which also appear as monodispersed spherical particles with particle sizes concentrated in the range of 12.2-15.5 nm and an average particle size of 13.5 nM.

[0063] The functional group information on the surface of carbon nanodots can be obtained by analyzing the characterization results of Fourier transform infrared (FT-IR) spectroscopy and X-ray photoelectron spectroscopy (XPS). Figure 3 The FT-IR spectrum of carbon nanodots is shown in Figure 2, where the wavelengths at 3202, 1718, and 1190 cm -1The peaks at 3430 and 1404 cm correspond to the stretching vibrations of OH, C=O and CO, respectively. -1 The absorption peak at corresponds to the stretching vibration of NH and CN. This information indicates that there are hydrophilic groups -COOH and -NH2 on the surface of carbon nanodots, so carbon nanodots have good dispersibility in water. Figure 4 As shown in A, there are three main peaks in the XPS spectrum of carbon nanodots. The peaks at 283.82, 398.88 and 530.63 eV correspond to C1s, N1s and O1s respectively. Based on the XPS spectrum, we know that the atomic ratio of the three elements C:N:O is about 55.54:18.36:26.10, which indicates that nitrogen has been successfully doped into carbon nanodots. The narrow scan spectrum of C1s ( Figure 4 The peaks at 284.82, 286.58 and 288.48 eV in B) correspond to the aromatic rings CC / C=C, CN / CO and C=O, respectively; the narrow scan spectrum of N1s ( Figure 4 The peaks at 399.89 and 402.25 eV in C) indicate the presence of CN and NH, respectively; the narrow scan spectrum of O1s ( Figure 4 D) contains two peaks at 531.63 and 533.22 eV, attributed to CO and C=O, respectively. The surface functional group information of the carbon nanodots obtained by XPS characterization is consistent with the characterization results of FT-IR, both indicating the successful doping of nitrogen and the presence of amino groups.

[0064] The characterization results of Zeta potential also confirmed that the surface of carbon nanodots is rich in amino groups, such as Figure 5 As shown in Figure C, the surface is rich in amino groups, which makes the Zeta potential of carbon nanodots positive (ζ=+14.8mV); while the Zeta potential of gold nanoparticles is measured to be negative (ζ=-30.3mV), that is, the surface electrical properties of gold nanoparticles and carbon nanodots are opposite, which is presumably because their surfaces are rich in carboxyl groups.

[0065] Figure 5 A is the multi-wavelength excitation fluorescence spectrum of carbon nanodots when the excitation light wavelength is in the range of 365-420nm. It can be seen that the fluorescence emission peak of carbon nanodots red-shifts with the change of excitation light wavelength. When the excitation light wavelength is 405nm, the fluorescence emission with the maximum relative fluorescence intensity is obtained, and the emission peak is at 505nm. From the ultraviolet-visible (UV-Vis) spectrum of carbon nanodots and gold nanoparticles ( Figure 5 B) It can be seen that the carbon nanodots have an absorption peak at 351nm. The absorption peak of gold nanoparticles is located at 520nm.

[0066] The concentration calculation of the gold nanoparticle solution involved in the present invention is as follows: the extinction coefficient of AuNPs with a particle size of about 13 nm at 520 nm is 2.7×10 8(M·cm) -1 Therefore, the concentration of AuNPs can be quantified by measuring the absorbance of the product at 520 nm and combining it with the Lambert-Beer's law.

[0067] Example 2: Detection of thiourea in ultrapure water using carbon nanodot-gold nanoparticle fluorescent probe

[0068] First, 0.3 mL of carbon nanodot solution (concentration of 1 unit), 0.36 mL of gold nanoparticle solution (10 nM) and 2.04 mL of phosphate buffer solution (0.02 M, pH = 7.0) were mixed and shaken to form a probe solution; then 0.3 mL of thiourea solution of different concentrations (0, 0.2, 0.5, 0.7, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 100 μM) was added to the probe solution to obtain the test sample (wherein the concentration of gold nanoparticles was 1.2 nM); and the blank control group was added to the probe solution with an equal amount of ultrapure water and shaken again. After incubation at room temperature for 10 minutes, the fluorescence spectrum of each sample under 405 nm excitation was measured, as shown in FIG. Figure 7 As shown in A.

[0069] Figure 7 Figure B shows the relationship between the fluorescence intensity of the carbon nanodot-gold nanoparticle probe at 505 nm and the thiourea concentration (0-10 μM). It can be seen that the fluorescence intensity of the probe first increases and then tends to saturation with the increase of thiourea concentration. The fluorescence intensity y and the thiourea concentration x show a linear correlation in the range of 0.05-1 μM. The linear fitting equation is y=27270.85928x+30253.68326, R 2 =0.9958, limit of detection LOD=0.0362 μM (S / N=3).

[0070] The detection mechanism of thiourea sensing by carbon nanodot-gold nanoparticle fluorescent probe is as follows Figure 1 As shown in Figure 2, the sensing mechanism is divided into two parts: gold nanoparticles quench the fluorescence of carbon nanodots and thiourea restores the fluorescence of carbon nanodots quenched by gold nanoparticles. Figure 6 As shown in Figure B. However, when the gold nanoparticles were removed from the system, the relative fluorescence intensity of the carbon nanodots remained basically the same before and after the addition of thiourea, indicating that there was no direct interaction between the carbon nanodots and thiourea to cause changes in fluorescence performance.

[0071] Depend on Figure 6As shown in Figure 1, there is significant overlap between the fluorescence emission spectrum of carbon nanodots and the UV-Vis absorption spectrum of gold nanoparticles, so the mechanism by which gold nanoparticles quench the fluorescence of carbon nanodots can be preliminarily classified as between IFE and FRET. Proving that the fluorescence quenching mechanism is FRET generally requires the following three conditions: first, the emission spectrum of the donor and the absorption spectrum of the acceptor must partially overlap, and experimental results show that the carbon nanodots and gold nanoparticles used in this method meet this condition; second, the distance between the donor and the acceptor must be sufficiently small (generally less than 10nm), which is confirmed by the HRTEM images of carbon nanodots and gold nanoparticles; and third, the fluorescence lifetime of the donor will decay. Figure 5 D shows the fluorescence decay of carbon nanodots before and after the addition of gold nanoparticles. The average fluorescence lifetime of the carbon nanodots is 7.93 ns, but after the addition of gold nanoparticles, the average fluorescence lifetime of the system decreases to 7.44 ns. Based on this analysis, it can be concluded that the fluorescence quenching mechanism of gold nanoparticles on carbon nanodots in this method is primarily FRET.

[0072] In order to further explore the interaction mechanism between carbon nanodots and gold nanoparticles in the probe system, this work measured the Zeta potential of carbon nanodots, gold nanoparticles and their mixture. The results are as follows Figure 5 As shown in C. Since the surface of carbon nanodots is rich in -NH2, its Zeta potential is positive (ζ=+14.8mV); while the Zeta potential of gold nanoparticles is negative (ζ=-30.3mV). The opposite electrical properties of the two enable electrostatic interaction between them. After the addition of carbon nanodots, the Zeta potential of the system also rises to -12.4mV. Since sulfur atoms have a strong binding affinity with gold nanoparticles, the coordination interaction between gold nanoparticles and thiourea is stronger than the electrostatic interaction between carbon nanodots and gold nanoparticles, so that the addition of thiourea will trigger the aggregation of gold nanoparticles and thus destroy the conditions for the FRET effect between carbon nanodots and gold nanoparticles, and ultimately restore the quenched fluorescence of carbon nanodots. Observe the UV-Vis absorption spectrum of the reaction system ( Figure 5 B) It can be found that the carbon nanodots have an absorption peak at 351nm, and after the addition of gold nanoparticles, the system will have a characteristic absorption peak belonging to the gold nanoparticles at 524nm in addition to the absorption peak of the carbon nanodots. The addition of the test substance thiourea does not change the absorption peak of the carbon nanodots, but it will significantly reduce the characteristic absorption peak of the carbon nanodot-gold nanoparticle system at 524nm, and a new absorption peak will appear at 680nm. The appearance of the new absorption peak at 680nm indicates that the gold nanoparticles have aggregated. This aggregation phenomenon can be more intuitively reflected in the HRTEM images of the carbon nanodot-gold nanoparticle system before and after the addition of thiourea, as shown in Figure 6 C and 6D.

[0073] Comparative Example 1: Exploring the effects of different carbon dots

[0074] Referring to Example 1, except that urea was replaced with 1 mL of diethylenetriamine, all other procedures remained unchanged. The resulting blue-light-emitting carbon dots, designated BCDs, were diluted to a UV-Vis absorption spectrum with an absorbance of 0.12 at 359 nm. The concentration of BCDs at this point was defined as 1 unit.

[0075] Green-emitting carbon dots (GCDs) were prepared by a hydrothermal method. First, 0.4 g of 3,5-diaminobenzoic acid was added to 40 mL of ethanol and stirred until completely dissolved. The solution was then transferred to a 100 mL Teflon-sealed autoclave and heated in an oven set at 180°C for 12 hours. After the reaction, the product, which had cooled to room temperature, was filtered to remove dark brown insoluble matter, and the filtrate was concentrated to one-fourth its original volume using a rotary evaporator. Finally, the desired GCDs were purified by silica gel column chromatography using a methanol / dichloromethane mixture (4:1, v / v) as the mobile phase. After the solvent was exchanged back to ultrapure water using a rotary evaporator, the GCDs solution was diluted to an absorbance of 0.12 at 374 nm on the UV-Vis absorption spectrum. The concentration of GCDs at this point was determined to be 1 unit.

[0076] like Figure 8 As shown in A, the UV-Vis absorption spectrum of BCDs has an absorption peak at 359 nm, and its fluorescence emission peak is located at 451 nm. When the excitation light wavelength is 346 nm, the fluorescence emission with the maximum relative fluorescence intensity can be obtained. Figure 8 Figure B shows the spectrum of GCDs, with a UV-Vis absorption band at 374 nm. The fluorescence emission peak of GCDs is at 490 nm, corresponding to an optimal excitation wavelength of 390 nm.

[0077] Referring to Example 2, other conditions were kept unchanged, only the concentration of thiourea solution was fixed at 100 μM and equal amounts of BCDs and GCDs solutions were used instead of carbon nanodot solution to construct BCDs-gold nanoparticle probes and GCDs-gold nanoparticle probes. The thiourea response results of these two probes were as follows: Figure 8 C and 8D. Figure 6 From the thiourea response results of the carbon nanodot-gold nanoparticle probe shown in B, it can be found that when the characteristic absorption peak intensity of each fluorescence signal source, the gold nanoparticle concentration and the thiourea concentration are kept consistent, the probe constructed with carbon nanodots produces better results, whether for the fluorescence quenching process caused by gold nanoparticles or the fluorescence recovery process caused by thiourea. Figure 9This indicates that although the fluorescence emission spectra of BCDs and GCDs overlap with the UV-Vis absorption spectrum of gold nanoparticles, the degree of overlap is not as great as that of carbon nanodots. This indicates that the FRET efficiency between carbon nanodots and gold nanoparticles is higher, leading to a higher degree of fluorescence quenching of carbon nanodots when adding the same concentration of gold nanoparticles. Therefore, to achieve the same fluorescence recovery space, a higher concentration of gold nanoparticles is required when constructing probes using BCDs and GCDs. However, a higher concentration of quencher is not conducive to lowering the detection limit.

[0078] The above results show that carbon nanodots prepared by microwave method using citric acid and urea as precursors have greater advantages in the quantitative detection of thiourea. Not all carbon dots can be used as the fluorescent signal source of the probe to obtain the detection results described in Example 2.

[0079] Example 3: Exploring the Effect of Gold Nanoparticle Concentration on Thiourea Detection

[0080] The test sample configuration method refers to Example 2, and the carbon nanodot concentration (consistent with Example 2) and TU concentration (10 -7 M) remains unchanged, and only the concentration of gold nanoparticles in the test sample is changed (0.5-3nM) to explore the effect of gold nanoparticle concentration on the thiourea detection ability of the carbon nanodot-gold nanoparticle fluorescent probe. The fluorescence quenching rate is defined as (F0-F) / F0 and the fluorescence enhancement rate is defined as (F'-F) / F, where F0 is the relative fluorescence intensity of the carbon nanodots, and F and F' are the relative fluorescence intensities of the carbon nanodot-gold nanoparticle probe before and after the addition of thiourea. Figure 10 As shown in A, the fluorescence quenching rate of the carbon nanodot-gold nanoparticle system increases with the increase of gold nanoparticle concentration, but the quenching rate tends to be saturated when the gold nanoparticle concentration is greater than 2nM. Figure 10 B shows that when the concentration of gold nanoparticles is 1.2 nM, the fluorescence enhancement rate of thiourea on the carbon nanodot-gold nanoparticle system is the highest, so the preferred concentration of gold nanoparticles is 1.2 nM.

[0081] Example 4: Exploring the influence of the pH value of the detection environment on thiourea detection

[0082] The sample preparation method refers to Example 2. The samples with pH values ​​of 6.0 and 7.0 were prepared using phosphate buffer, and the samples under other pH conditions were directly adjusted using HCl and NaOH. Figure 11 As shown in A, the fluorescence intensity of the carbon nanodots used fluctuates slightly under the conditions of pH = 3.0 to 8.0, but begins to drop significantly when pH > 8.0. Figure 11 B. Figure 11C shows that the fluorescence quenching rate of gold nanoparticles on carbon nanodots increases first and then decreases with increasing pH, reaching a maximum fluorescence quenching rate of approximately 75% at pH 8.0. The fluorescence enhancement rate of thiourea on the carbon nanodot-gold nanoparticle probe also shows a general trend of increasing first and then decreasing within the pH range of 4.0 to 12.0, with optimal fluorescence recovery at pH 7.0. Since there is no significant difference in the fluorescence intensity of carbon nanodots or the fluorescence quenching rate of gold nanoparticles on carbon nanodots at pH 7.0 and 8.0, and the fluorescence recovery effect directly reflects the probe's ability to detect thiourea, a pH of 7.0 is the preferred detection environment.

[0083] Example 5: Investigate the effect of reaction time on thiourea detection.

[0084] The sample configuration method refers to Example 2, Figure 12 The relative fluorescence intensity at 505 nm of the carbon nanodot-gold nanoparticle probe and the carbon nanodot-gold nanoparticle + thiourea system changes over 20 minutes. Both interactions reach stability after 2 minutes. To ensure the stability of the test results, a 10-minute sample reaction time is preferred.

[0085] Example 6: Investigating the selectivity and anti-interference ability of the carbon nanodot-gold nanoparticle fluorescent probe.

[0086] Select other nitrogen-rich substances (allantoin, urea, melamine, dicyandiamide, biuret, guanidine hydrochloride), common amino acids (phenylalanine, alanine, glycine, tyrosine, serine, aspartic acid, valine, histidine) and other coexisting substances such as ions (glucose, ascorbic acid, Ca 2+ , K + 、Cu 2+ 、Fe 3+ Mg 2+ 、Zn 2+ 、Mn 2+ 、SO4 2- 、NO 3- ) was used as an interfering substance for the experiment. The concentration of thiourea and other nitrogen-rich substances was 2 μM, while the concentration of common amino acids, glucose, ascorbic acid, and common ions was 10 μM. The sample preparation method was similar to that in Example 2. In the selective experiment, the thiourea solution was replaced by different interfering substance solutions, while in the anti-interference experiment, different interfering substances and thiourea were added simultaneously. Figure 13 As shown in A, under the above concentration conditions, melamine alone can also make the carbon nanodot-gold nanoparticle probe show a relatively significant fluorescence recovery, but it is still lower than the fluorescence recovery ability of thiourea. Figure 11 B shows that, except Cu 2+ with Fe 3+In addition, when there are nitrogen-rich substances with the same concentration as thiourea, amino acids with a concentration 5 times that of thiourea, or other ions in the detection system, the thiourea detection ability of the probe will not be affected. 2+ with Fe 3+ All of them have a certain inhibitory effect on the fluorescence recovery ability of thiourea. Perhaps we can try to find masking agents to eliminate their negative effects in practical applications. The above experimental results show that the carbon nanodot-gold nanoparticle probe has excellent selectivity and anti-interference ability for thiourea.

[0087] Example 7: Thiourea detection of carbon nanodot-gold nanoparticle fluorescent probe in actual samples

[0088] The actual samples selected in this embodiment are tap water, lake water and fruit and vegetable juice. The tap water samples were collected from the laboratory, and the lake water samples were from the campus lake of Jiangnan University. Both water samples were filtered with a 0.22μm filter membrane before use. For water samples, first, 0.3mL of carbon nanodot solution (concentration of 1 unit), 0.3mL of tap water or lake water sample, 0.3mL of thiourea solution of different concentrations and 1.74mL of phosphate buffer solution (0.02M, pH=7.0) were mixed evenly, and then 0.36mL of gold nanoparticles (10nM) were added and shaken again to obtain a test sample (the concentration of AuNPs in the test sample was 1.2nM). After incubation at room temperature for 10 minutes, the fluorescence spectrum was collected under 405nm excitation.

[0089] The fruit and vegetable juice samples used were carrot compound fruit and vegetable juice (Weiquan brand), purchased from a local supermarket in Wuxi. Before use, the pulp was removed by centrifugation at 7000 rpm for 10 minutes, and the supernatant liquid was collected for later use. For the analysis of fruit and vegetable juice samples, the concentration of AuNPs in the CNDs-AuNPs probe needed to be increased: 0.3 mL of CNDs solution (concentration of 1 unit), 0.3 mL of fruit and vegetable juice sample, 0.3 mL of TU solution of different concentrations, and 1.6 mL of phosphate buffer solution (0.02 M, pH = 7.0) were mixed evenly. Then, 0.5 mL of AuNPs (15 nM) was added and shaken again to obtain the test sample (the concentration of AuNPs in the test sample was 2.5 nM). After incubation at room temperature for 10 minutes, fluorescence spectra were collected under 405 nm excitation.

[0090] The linear detection range and detection limit of the carbon nanodot-gold nanoparticle fluorescent probe in various actual sample environments are shown in Table 1. The linear range for all three samples was 0.1-1 μM, and the calculated detection limits (S / N = 3) were 0.025 μM, 0.017 μM, and 0.056 μM, respectively.

[0091] Table 1 Thiourea detection ability of carbon nanodot-gold nanoparticle fluorescent probe in different actual samples

[0092]

[0093]

[0094] After establishing the detection curve, TU spike recovery experiments were conducted in real samples. The results are shown in Table 2. The spiked thiourea recoveries in real samples ranged from 95.8% to 103%, with relative standard deviations ranging from 0.633% to 8.025%. These results demonstrate that the CNDs-AuNPs fluorescent probe has broad application prospects in monitoring thiourea content in real samples.

[0095] Table 2 Recovery results of thiourea spiked in actual samples

[0096]

[0097] The above embodiments illustrate the basic principles, main features, and advantages of the present invention. However, those skilled in the art should understand that the present invention is not limited to the above embodiments. Various changes and improvements are possible without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be defined by the claims.

Claims

1. A method for detecting thiourea using carbon nanodots-gold nanoparticles as fluorescent probes, characterized in that: The following steps are included: (1) A carbon nanodot solution, a gold nanoparticle solution, and a phosphate buffer solution are mixed and shaken to obtain a carbon nanodot-gold nanoparticle fluorescent probe solution; a series of thiourea standard solutions of known concentrations are then added to the probe solution and mixed to obtain standard samples; wherein the carbon nanodots are prepared by microwave method using citric acid and urea as precursors; (2) After reacting at room temperature for a period of time, the fluorescence spectrum of each standard sample is collected and the corresponding fluorescence intensity is measured; (3) A detection model was constructed by linearly correlating the concentration of the thiourea standard solution with the corresponding fluorescence intensity; (4) preparing a sample to be tested by replacing the thiourea solution of known concentration with a sample of the sample to be tested according to step (1), obtaining the relative intensity of the fluorescence emission peak of the sample to be tested according to step (2), and then calculating the concentration of thiourea in the sample to be tested according to the detection model in step (3); The concentration of the gold nanoparticles in the standard sample in step (1) is 0.5-3 nM; the concentration of the gold nanoparticles in the test sample in step (4) is 0.5-3 nM; If the concentration corresponding to the absorption value of the carbon nanodot solution at 351 nm in the UV-visible absorption spectrum is 0.12, then the concentration of the carbon nanodots in the standard sample and the sample to be tested is 0.1-0.5 units.

2. The method according to claim 1, characterized in that The volume ratio of the carbon nanodot solution, the gold nanoparticle solution, the phosphate buffer solution and the thiourea standard solution is 1:(1-2):(5-8):

1.

3. The method according to claim 1, characterized in that The reaction time in step (2) is 2-20 min.

4. The method according to claim 1, wherein The excitation light wavelength when collecting the sample fluorescence spectrum in step (2) is 405 nm, and the fluorescence emission peak in step (3) is taken from 505 nm in the fluorescence spectrum.

5. A method for detecting thiourea in a water sample, characterized in that: The following steps are included: 1) A carbon nanodot solution, gold nanoparticles, phosphate buffer solution, and a water sample are mixed and shaken to obtain a carbon nanodot-gold nanoparticle fluorescent probe solution; a series of thiourea standard solutions of known concentrations are then added to the probe solution and mixed to obtain standard samples; wherein the carbon nanodots are prepared using citric acid and urea as precursors by microwave method; 2) After reacting for a period of time at room temperature, the fluorescence spectrum of each standard sample is collected and the corresponding fluorescence intensity is measured; 3) A detection model was constructed by linearly correlating the concentration of the thiourea standard solution with the corresponding fluorescence intensity; 4) Substituting the thiourea solution of known concentration for the thiourea sample according to step 1) to prepare a test sample, obtaining the relative intensity of the fluorescence emission peak of the test sample according to step 2), and then calculating the concentration of thiourea in the test sample according to the detection model in step 3); The concentration of the gold nanoparticles in the standard sample in step (1) is 0.5-3 nM; the concentration of the gold nanoparticles in the test sample in step (4) is 0.5-3 nM; If the concentration corresponding to the absorption value of the carbon nanodot solution at 351 nm in the UV-visible absorption spectrum is 0.12, then the concentration of the carbon nanodots in the standard sample and the sample to be tested is 0.1-0.5 units.

6. The method according to claim 5, characterized in that When testing water samples, the concentration of AuNPs in the standard sample in step 1) is 1.2 nM.

7. The method according to claim 5, characterized in that The volume ratio of the carbon nanodot solution, the gold nanoparticle solution, the phosphate buffer solution, the water sample and the thiourea standard solution is 1:(1-2):(5-8):1:

1.

8. A method for detecting thiourea in fruit and vegetable juice, characterized in that: The following steps are included: A) mixing a carbon nanodot solution, gold nanoparticles, phosphate buffer solution, and fruit and vegetable juice and vortexing to obtain a carbon nanodot-gold nanoparticle fluorescent probe solution; then adding a series of thiourea standard solutions of known concentrations to the probe solution and mixing to obtain standard samples; wherein the carbon nanodots are prepared using citric acid and urea as precursors by microwave method; B) After reacting for a period of time at room temperature, the fluorescence spectrum of each standard sample is collected and the corresponding fluorescence intensity is measured; C) constructing a detection model by linearly correlating the concentration of the thiourea standard solution with the corresponding fluorescence intensity; D) preparing a test sample according to step A) by replacing the thiourea solution of known concentration with the thiourea sample to be tested, obtaining the relative intensity of the fluorescence emission peak of the test sample according to step B), and then calculating the concentration of thiourea in the test sample according to the detection model in step C); The concentration of the gold nanoparticles in the standard sample in step (1) is 0.5-3 nM; the concentration of the gold nanoparticles in the test sample in step (4) is 0.5-3 nM; If the concentration corresponding to the absorption value of the carbon nanodot solution at 351 nm in the UV-visible absorption spectrum is 0.12, then the concentration of the carbon nanodots in the standard sample and the sample to be tested is 0.1-0.5 units.

9. The method according to claim 8, characterized in that Step A) The concentration of AuNPs in the standard sample is 2.5 nM.

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