Dye-sensitized up-conversion fluorescent probe, preparation method and application

Abstract

The invention discloses a dye-sensitized up-conversion fluorescent probe, a preparation method and application, the fluorescent probe is formed by compounding a near-infrared cyanine dye and up-conversion nanoparticles including 20% of beta-NaYF4: Yb, 10% of Er2% atNaYF4: Yb and 10% of Nd, and the structural formula of the near-infrared cyanine dye is shown in the specification. The fluorescent probe with high pigment interference resistance is realized, is respectively used for efficient detection of copper ions and glyphosate, and has important significance in the fields of environmental health risk assessment, food safety guarantee and precise chemical analysis.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of fluorescence detection, and particularly relates to a dye-sensitized upconversion fluorescence probe, a preparation method and application. BACKGROUND

[0002] Copper ions are effective catalysts for many chemical reactions. In chemical production, copper ion catalyzed reactions can significantly improve reaction rate and selectivity, and reduce production cost. However, as a heavy metal, the ionic form of copper can enter the human body through various ways (such as contaminated water sources, food chain enrichment, etc.), and pose a threat to health. Excessive copper intake can interfere with the absorption and metabolic balance of essential trace elements (such as zinc and iron) in the human body. More seriously, copper ions can accumulate in organs such as the liver, causing oxidative stress damage, leading to abnormal liver function and even cirrhosis. Long-term exposure to high copper environment is also associated with nervous system damage (such as Wilson's disease), kidney dysfunction, and potential carcinogenic risk. Therefore, accurate monitoring of copper ions in environmental media (especially water sources, soil and agricultural products) is of great importance to protect human health.

[0003] Glyphosate, as the most widely used organic phosphorus herbicide in the world, has raised widespread concerns about its ecological and health risks. Residual glyphosate can be transmitted through the food chain and bioaccumulated, ultimately threatening human health. Toxicology studies have shown that glyphosate not only has reproductive and developmental toxicity, teratogenicity and neurotoxicity, but is also listed as a 2A class (very likely to cause cancer in humans) carcinogen by the International Agency for Research on Cancer (IARC). Therefore, it is urgent to develop a simple, rapid and accurate method for detecting glyphosate.

[0004] Endogenous colored compounds (such as curcumin, beta-carotene, lycopene and anthocyanins) commonly present in food matrices often cause serious optical interference to probes based on fluorescence detection due to their strong absorption characteristics in the ultraviolet-visible spectral region, posing a key challenge. Traditional strategies attempt to selectively remove pigments through pretreatment, but the types of pigments in plant-derived samples are diverse and their physicochemical properties (such as fat-soluble chlorophyll and water-soluble anthocyanins) differ significantly, making it difficult to establish a universal purification scheme.

[0005] Therefore, in view of the above technical problems, it is necessary to provide a dye-sensitized upconversion fluorescence probe, a preparation method and application. SUMMARY

[0006] The purpose of the present application is to provide a dye-sensitized upconversion fluorescence probe, a preparation method and application, which realizes a fluorescence probe with high anti-pigment interference capability, and is respectively used for efficient detection of copper ions and glyphosate, which has important significance in the fields of environmental health risk assessment, food safety protection and precise chemical analysis.

[0007] In order to achieve the above object, a specific embodiment of the present application provides the technical scheme as follows.

[0008] The dye-sensitized upconversion fluorescence probe is composed of a near-infrared cyanine dye and beta-NaYF4:Yb20%, Er2% @ NaYF4:Yb10%, Nd10% upconversion nanoparticles, wherein the structural formula of the near-infrared cyanine dye is as follows: .

[0009] In one or more embodiments of the present application, the preparation method of the dye-sensitized upconversion fluorescence probe comprises the following steps.

[0010] The cyclodextrin aqueous solution is added to the water / ethanol mixed solvent with the upconversion nanoparticles, ultrasonic dispersion is performed, stirring is conducted at room temperature, centrifugation is performed, and the obtained product is washed with water and then dispersed in ultrapure water again to obtain an UNs@CD dispersion liquid;

[0011] The near-infrared cyanine dye is dissolved in N,N'-dimethylformamide, and then added to the UNs@CD dispersion liquid for mixing and stirring, centrifugation is performed, and the obtained product is washed with water and then dispersed in ultrapure water again to obtain a dye-sensitized rare earth upconversion nanoprobe.

[0012] In one or more embodiments of the present application, the alpha-cyclodextrin is an amphiphilic alpha-cyclodextrin. At this time, since the alpha-cyclodextrin has a cavity in the center of the ring structure, the internal -CH- and the oxygen atom combined with the glucoside are hydrophobic, the ligand of the upconversion nanoparticle is assembled with the ring structure to form a structure form with the ligand facing the inside of the ring. The hydrophobic near-infrared cyanine dye is assembled to the inside of the ring structure, thereby forming a composite assembly of an upconversion nanoparticle / alpha-cyclodextrin / near-infrared cyanine dye.

[0013] In one or more embodiments of the present application, the content of the alpha-cyclodextrin in the alpha-cyclodextrin aqueous solution is 5-15 mg / mL.

[0014] In one or more embodiments of the present application, the ratio of water to ethanol in the water / ethanol mixed solvent is v / v: 1: (1-5).

[0015] In one or more embodiments of the present application, the addition amount of the upconversion nanoparticles in the water / ethanol mixed solvent is 0.5-3 mg / mL.

[0016] In one or more embodiments of the present application, the preparation of the near-infrared cyanine dye is as follows.

[0017] The intermediate is prepared by condensation reaction of 4,4'-dithiodibenzoic acid and 6-mercapto-purine under the action of a condensing agent and an alkali;

[0018] The intermediate is reduced to prepare a purine derivative containing a mercapto group.

[0019] Under a protective atmosphere and in the absence of light, IR783 is reacted with a thiol-containing purine derivative under alkaline conditions at room temperature to obtain anthocyanin derivatives. Preferably, the protective atmosphere is a nitrogen or argon atmosphere. Preferably, the alkaline conditions are in the presence of K2CO3 or Na2CO3. Preferably, the molar ratio of base to purine derivative is 1:1.

[0020] In one or more embodiments of the present invention, the condensing agent is selected from 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate; and / or, the base is selected from N,N-diisopropylethylamine; and / or, the reducing agent used for reduction is selected from tris(2-carboxyethyl)phosphine.

[0021] In one or more embodiments of the present invention, a dye-sensitized upconversion fluorescent probe is used in Cu... 2+ Detection or Cu 2+ Application in combined detection with glyphosate. Preferably, the concentration of near-infrared cyanine dye in the detection system is no greater than 3 μM. Of course, higher concentrations are also feasible for detection.

[0022] The operation of the fluorescent probe for recognizing copper ions in this invention is as follows: the near-infrared anthocyanin dye probe can "turn-off" the detection of copper ions, specifically meaning that the fluorescent probe is not affected by the K+ of metal ions. + Ca 2+ Mn 2+ Ni 2+ Zn 2+ Pb 2+ Ba 2+ Hg 2+ Cr 3+ and Fe 3+ Interference. Only with the addition of Cu 2+ The upconversion intensity will only decrease when this occurs. This allows for highly interference-resistant, highly sensitive, and visual identification of copper ions.

[0023] Furthermore, in CyPh-UNs@CD-Cu 2+ (Refers to the introduction of Cu into the dye-sensitized upconversion nanoprobe system) 2+ Adding glyphosate to the sample restores the upconversion luminescence signal, enabling "turn-on" detection of glyphosate. The fluorescent probe is unaffected by other organophosphorus pesticides such as triazophos, glufosinate, methyl parathion, chlorpyrifos, dichlorvos, trichlorfon, fenitrothion, and dimethoate. Only when glyphosate is added does the pre-quenched CyPh-UNs@CD-Cu... 2+ The system then showed a clear recovery of the luminescence signal. It can achieve highly selective and sensitive identification of glyphosate.

[0024] In one or more embodiments of the present invention, Cu2+ The detection is introducing Cu 2+ into the detection solution comprising the dye-sensitized upconversion fluorescence probe; the upconversion luminescence intensity decreases; and / or, Cu 2+ The combined detection is introducing Cu 2+ into the detection solution comprising the dye-sensitized upconversion fluorescence probe; the upconversion luminescence intensity decreases; and then introducing glyphosate into the detection solution, the upconversion luminescence intensity increases again.

[0025] Further, since the signal intensity of the probe in the application is linearly related to the concentration of the to-be-detected substance, the optical equipment of a terminal device such as a smart phone or a computer can be used to collect the light signal, so as to obtain the RGB value by combining the RGB analysis platform of the device, and then the corresponding concentration value can be matched according to the RGB value, so that on-site rapid screening can be realized.

[0026] Compared with the prior art, the dye-sensitized upconversion fluorescence probe, the preparation method and the application have the following advantages:

[0027] 1. The energy transfer effect of the dye-sensitized upconversion nanoparticle can effectively improve the detection sensitivity of copper ions and glyphosate, and the detection limit of copper ions and glyphosate is 0.19 mu M, which is one order of magnitude lower than that of the traditional fluorescence detection method.

[0028] 2. The dye-sensitized upconversion nanoprobe provided by the application has high selectivity and sensitivity to copper ions and is not interfered by common metal ions such as K + , Ca 2+ , Mn 2+ , Ni 2+ , Zn 2+ , Pb 2+ , Ba 2+ , Hg 2+ , Cr 3+ and Fe 3+ .

[0029] 3. The dye-sensitized upconversion nanoprobe provided by the application has high selectivity and sensitivity to glyphosate ions and is not interfered by common organophosphorus pesticides such as triazophos, glufosinate-ammonium, methyl parathion, chlorpyrifos, dichlorvos, nematode, sulfotep and dimethoate.

[0030] 4. The dye-sensitized upconversion nanoprobe provided by the application has a response time of about 5s to copper ions and glyphosate, which is significantly faster than most reported fluorescence probes.

[0031] 5、The detection method of copper ions and glyphosate provided by the application can realize accurate detection in four typical dark matrix samples such as mulberry, grape, purple potato and purple cabbage, is not affected by food pigment matrix, provides a new method for designing biological probes with high selectivity and high sensitivity, and has potential application prospects in monitoring. BRIEF DESCRIPTION OF DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments described in the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0033] Figure 1 The upconversion spectrum of CyPh-UNs@CD containing different concentrations under 808nm excitation of the application;

[0034] Figure 2 The upconversion response of CyPh-UNs@CD (1mg / mL) in the application and the relationship between Cu 2+ concentration;

[0035] Figure 3 The upconversion spectrum of CyPh-UNs@CD (1mg / mL) when different metal ions (5μM) are added;

[0036] Figure 4 The competition experiment column chart between Cu 2+ and various metal ions;

[0037] Figure 5 The upconversion luminescence spectrum of CyPh-UNs@CD-Cu 2+ added with different organophosphorus pesticides (5μM);

[0038] Figure 6 The competition experiment column chart between glyphosate and various pesticides;

[0039] Figure 7 The upconversion response of CyPh-UNs@CD-Cu 2+ under 808nm excitation and the relationship between glyphosate concentration;

[0040] Figure 8 The linear fitting graph of the upconversion intensity (655nm) and the glyphosate concentration;

[0041] Figure 9 The linear fitting graph of the R / G value of the solution and the glyphosate concentration. DETAILED DESCRIPTION

[0042] In order to make the technical scheme in the present disclosure better understood by the person skilled in the art, the technical scheme in the present disclosure will be clearly and completely described below in combination with the disclosed embodiments of the present disclosure. Obviously, the described embodiments are only some of the embodiments of the present disclosure, but not all the embodiments. Based on the embodiments in the present disclosure, all other embodiments obtained by the person skilled in the art without creative labor should fall within the protection scope of the present disclosure.

[0043] Group one

[0044] Embodiment 1

[0045] This embodiment is used to illustrate the preparation of near-infrared cyanine dye CyPh

[0046] (1) Synthesis of thiol purine derivative

[0047]

[0048] Dissolve 6-chloropurine (1.1 g, 7.1 mmol) and 80% hydrazine hydrate (1.65 mL, 28.4 mmol) in anhydrous ethanol (20 mL), stir and reflux for 8 h. After cooling to room temperature, remove the solvent by rotary evaporation, then wash the remaining solid with ethanol and ice water three times to obtain 6-hydrazine purine, which is placed for standby. Dissolve 4-mercaptobenzoic acid (1.0 g, 6.5 mmol) in 20 mL of ethanol, and add I2 saturated solution in ethanol dropwise until the mixture turns light yellow. The solid after centrifugation is washed twice with ethanol to obtain white solid 4,4'-dithioalkylbenzoic acid. Dissolve 4,4'-dithioalkylbenzoic acid (1.0 g, 3.4 mmol) and 6-hydrazine purine (0.98 g, 6.8 mmol) in acetonitrile. Then add HATU (3.1 g, 8.0 mmol) and DIEA (0.11 mL, 13.6 mmol) respectively, and stir the mixture at 80°C for 8 hours. Remove the solvent by rotary evaporation, and wash the remaining solid with ethanol twice. Add the solid after centrifugation and tris-HCl solution [pH 7.5: DMF (1:1)] to tris-HCl solution [pH 7.5: DMF (1:1)], and stir at room temperature for 4 h. Remove the solvent by vacuum distillation, and wash the residual solid with ethanol to obtain a light yellow solid (1.16 g, 59.7%). MS (TOF): m / z = 286.4, calculated M for C 12 H 10 M for C r =2 86.3, found: m / z = 286.4.

[0049] (2) Synthesis of cyanine dye CyPh

[0050]

[0051] The IR783 and the thiol purine derivative prepared in the previous step were stirred in a mixed solution of K2CO3 and N,N-dimethylformamide under nitrogen protection at room temperature for 12 h in the dark. The solvent was removed under reduced pressure, and the filtered solid was purified by column chromatography to obtain the near-infrared cyanine dye.

[0052] 1 HNMR (400 MHz, DMSO-d6) δ: 8.705 (d, J = 4 Hz, 1H), 8.10 (d, J = 8 Hz, 2H), 7.96 (s, 1H), 7.905 (d, J = 4 Hz 2H), 7.67 (d, J = 16 Hz, 2H), 7.45-7.42 (m, 2H), 7.34 (s, 2H), 7.32 (d, J = 8 Hz 2H), 7.27 (d, J = 8 Hz 2H), 7.215 (d, J = 4 Hz 2H), 7.165 (d, J = 4 Hz 2H), 7.08 (d, J = 8 Hz, 1H), 6.82 (d, J = 16 Hz, 1H), 6.64 (d, J = 8 Hz, 1H), 6.585 (d, J = 12 Hz, 1H), 5.89 (d, J = 8 Hz, 1H), 5.72 (d, J = 16 Hz, 1H), 5.37 (d, J = 16 Hz, 1H), 4.88 (s, 2H), 2.74 (s, 2H), 2.335 (d, J = 4 Hz, 2H), 1.59 (d, J = 8 Hz, 6H), 1.25 (d, J = 8 Hz, 4H), 1.145 (d, J = 12 Hz, 6H), 1.03 (s, 4H). 13 CNMR (100 MHz, DMSO-d6,) δ: 173.87, 172.47, 142.46, 140.23, 138.96, 137.46, 134.08, 133.52, 131.95, 131.19, 130.96, 130.39, 130.20, 128.91, 128.30, 127.94, 127.75, 127.71, 126.68, 125.52, 123.90, 122.76, 113.84, 112.28, 101.70, 55.98, 51.19, 48.27, 44.38, 29.99, 29.95, 27.55, 22.15, 21.56, 20.01, 19.84, 14.49, 14.29, 14.12. MS (TOF): m / z = 977.1. Found: m / z = 977.1. 50 H 55 N8O7S3 Calculated M r = 977.2, Found: m / z = 977.1.

[0053] Example 2

[0054] This example is directed to the preparation of rare earth upconversion nanocrystals β-NaYF4:Yb20%, Er2% @ NaYF4:Yb10%, Nd10% (UNs)

[0055] (1) Synthesis of β-NaYF4:Yb20%, Er2%

[0056] A total of 1 mmol of rare earth chlorides (a mixture of YCl3, YbCl3and ErCl3in a molar ratio of 78:20:2) was dissolved in 5 mL of methanol, which was then transferred into a mixture containing 7 mL of oleic acid and 15 mL of 1-octadecene. The mixture was heated to 140 °C for 40 min to form a clear and transparent solution. After the system was cooled to 50 °C, a solution containing NaOH (2.5 mmol) and NH4F (4.0 mmol) in 5 mL of methanol was added, and the temperature was maintained for 30 min with stirring. Then, the system was heated to 110 °C to remove methanol and water. Subsequently, the reaction mixture was heated to 325 °C and reacted under an argon atmosphere for 1.5 h. After the system was cooled, ethanol was added, and the precipitated product was separated by centrifugation. After being washed with anhydrous ethanol and cyclohexane three times, the purified nanoparticles were dispersed in cyclohexane for storage.

[0057] (2) Synthesis of UNs

[0058] A total of 1 mmol of rare earth chlorides (a mixture of YCl3, YbCl3and NdCl3in a molar ratio of 80:10:10) was mixed with 7 mL of oleic acid and 15 mL of 1-octadecene. The mixture solution was heated to 140 °C to obtain a transparent solution. After the system was naturally cooled to 50 °C, a 10 mL cyclohexane dispersion containing NaYF4:Yb, Er nanocrystals, a methanol (5 mL) solution containing NaOH (2.5 mmol) and NH4F (4.0 mmol) was added in sequence, and the temperature was maintained at 50 °C for 30 min with stirring. The remaining steps were the same as those described above in (1), and UNs with an oleic acid ligand, i.e., OA-UNs, were obtained.

[0059] Example 3

[0060] This example is directed to the construction of a dye-sensitized upconversion nanoprobe system

[0061] A 4 mL aqueous solution of a-cyclodextrin (a-CD) (10 mg / mL) was added to 5 mL of UNs (10 mg) containing oleic acid ligand in water / ethanol (1 / 1, v / v) and ultrasonically dispersed for 5 min. Then it was stirred overnight at room temperature and centrifuged at 10000 r / min for 15 min. After washing twice with water, it was re-dispersed in ultrapure water to obtain UNs@CD. CyPh was dissolved in DMF and added to the UNs@CD mixture and stirred for 12 h. Then excess CyPh was removed by centrifugation at 10000 r / min for 15 min. After washing twice with water, it was re-dispersed in ultrapure water to obtain CyPh-UNs@CD.

[0062] The upconversion luminescence of the dye-sensitized nanosystem containing different concentrations of CyPh was tested under 808 nm laser pumping. As shown in Fig. 8, with the increase of the loading capacity of CyPh, the upconversion emission first increased and then decreased. When the concentration of CyPh reached 3.0 μM, the luminescence intensity of the system reached a maximum, which was 45 times higher than that of UNs@CD. This significant luminescence enhancement effect was attributed to the "antenna effect" of CyPh molecules, which could efficiently capture the excitation light energy due to its broad near-infrared absorption characteristics. Figure 1

[0063] Example 4

[0064] This example is used to illustrate the selectivity and anti-interference of the nanoprobe for copper ions.

[0065] When Cu 2+ (0-5 μM) was introduced into the CyPh-UNs@CD system with a CyPh concentration of 3.0 μM in Example 3, it was found that the upconversion luminescence signal showed a concentration-dependent quenching phenomenon, as shown in Fig. 9. With the increase of the concentration of Cu Figure 2 2+ , the upconversion signal was gradually quenched. When 3.0 μM of Cu 2+ was added, the intensity was reduced to the lowest, and the signal was reduced by 91.4%, indicating that it had excellent quenching response ability.

[0066] The selectivity experiment showed that under the interference of common metal ions (all at a concentration of 5.0 μM) such as K + , Ca 2+ , Mn 2+ , Ni 2+ , Zn 2+ , Pb 2+ , Ba 2+ , Hg 2+ , Cr 3+ and Fe 3+ , the upconversion spectrum of the CyPh-UNs@CD system did not change significantly, while only Cu 2+ ​​show specific response, such as Figure 3 As shown in the figure, the Cu 2+ signal curve is 3.0 μM. Anti-interference experiments further confirmed that the upconversion intensity remained unchanged in the presence of other metal ions, but was significantly quenched after the addition of Cu 2+ Figure 4 , where Blank refers to the response of the CyPh-UNs@CD system before and after the introduction of Cu 2+ , and the response comparison), which fully proves that the composite system has a high selective recognition ability for Cu 2+ .

[0067] Example 5

[0068] This example further illustrates the selectivity and anti-interference of the nanoprobe system CyPh-UNs@CD-Cu 2+ for glyphosate.

[0069] To the CyPh-UNs@CD-Cu 2+ system in Example 4 (Cu 2+ concentration 3.0 μM, CyPh concentration 3.0 μM), 9 common organophosphorus pesticides were added for selective spectral analysis, including triazophos, glufosinate-ammonium, methyl parathion, chlorpyrifos, dichlorvos, nematode, sulfotep, dimethoate and glyphosate (concentration 5.0 μM), as shown in Figure 5 The experimental results show that only when glyphosate is added, the pre-quenched CyPh-UNs@CD-Cu 2+ system shows obvious recovery of luminescence signal. In contrast, the signal changes caused by other pesticides are negligible, which confirms the high selectivity of the probe for glyphosate. In order to further explore the competitive recognition ability of the composite nanoprobe for glyphosate, glyphosate was added in the presence of 30 μM other pesticides, and an anti-interference experiment was carried out. As shown in Figure 6 , even in the presence of high concentrations of other interfering substances, the probe can still specifically recognize glyphosate and trigger signal recovery, while the control system has no response. This shows that Cu 2+ in CyPh-UNs@CD-Cu 2+ can achieve upconversion signal activation by competing with glyphosate, which can be used for high specificity and strong anti-interference detection of glyphosate.

[0070] Example 6

[0071] This example illustrates the dye-sensitized upconversion nanoprobe CyPh-UNs@CD-Cu 2+ To the CyPh-UNs@CD-Cu 2+ system in Example 4, Cu 2+ ​The response of glyphosate at concentrations of 3.0 μM and 3.0 μM CyPh

[0072] like Figure 7 As shown, the upconversion luminescence intensity at 655 nm gradually increases with increasing glyphosate concentration. Linear regression analysis revealed a significant positive correlation between the signal intensity at 655 nm and glyphosate concentration within the concentration range of 0 to 3.0 μM (R0). 2 =0.991), with a detection limit as low as 19.01 nM, such as Figure 8 As shown, this indicates that the probe successfully achieved ultrasensitive detection of glyphosate under near-infrared excitation through a dye-sensitized energy transfer mechanism.

[0073] In contrast, to evaluate the rationality of the sensing platform design, a system based on the purely organic dye CyPh-Cu was constructed. 2+ (Cu) 2+ A glyphosate detection system (3.0 μM concentration of glyphosate and 3.0 μM concentration of CyPh) was developed. As expected, the detection limit of this dye system was 0.28 μM, which is 14.7 times lower than the sensitivity of the composite system. This significant difference in sensitivity is attributed to the multi-level energy transfer process induced by dye sensitization, which can effectively enhance the signal response intensity.

[0074] Example 7

[0075] This embodiment is used to illustrate the detection in the presence of plant pigments.

[0076] Spiked recovery experiments were conducted on four typical dark-colored crops: grapes, mulberries, purple sweet potatoes, and purple cabbage. Mulberries, grapes, purple sweet potatoes, and purple cabbage were cut into small pieces and dried in an oven. Then, they were added to beakers containing PBS buffer, sonicated for 10 min, and filtered. The four buffer solutions were then diluted 10-fold sequentially and set aside. Before analysis, different concentrations of glyphosate standard samples (0-3 μM) were incorporated into the diluted samples, and the samples were analyzed using a dye-sensitized upconversion probe. The recovery rate was calculated using a standard calibration curve. Each experiment was repeated five times to calculate the average detected glyphosate concentration. The experimental results showed that in the near-infrared detection channel (655 nm), the system achieved recoveries of 97.0%-104.0% in the tested matrices, with an RSD below 2.73% (n=5), meeting the accuracy requirements of pesticide residue analysis standards.

[0077] Example 8

[0078] This embodiment illustrates a glyphosate-assisted smartphone sensing system.

[0079] The RGB color recognition technology of a smart phone is combined with a nano composite sensing system. The smart program is used for capturing CyPh-UNs@CD-Cu 2+ RGB values of different concentrations of glyphosate, so as to realize on-site quantitative detection. Under the excitation of 808nm, after the addition of glyphosate, the color of the solution changes from colorless to red. By measuring the RGB values of the probe solution added with different concentrations of glyphosate, it is found that in the concentration range of 0-3μM, there is a stable linear relationship between the concentration of glyphosate and the R / G value, as shown in Figure 9 The detection limit is 0.42μM. The method combines the color response of the nano probe with intelligent analysis, not only shows high precision in sample detection, but also is simple to operate, and provides a convenient scheme for accurate detection of glyphosate.

[0080] In the detection process, CyPh first specifically coordinates with Cu 2+ , so that the fluorescence of CyPh is quenched, and the fluorescence resonance energy transfer (FRET) channel between CyPh and UNs is blocked, so that the upconversion luminescence signal of UNs is quenched, so that the detection of Cu 2+ is realized. Subsequently, glyphosate is added, which competes with CyPh to bind Cu 2+ , and Cu 2+ is taken out of the CyPh-Cu 2+ complex, so that the quenching state of CyPh is released, the FRET process is restored, so that the upconversion luminescence signal of UNs is restored, so that the detection of glyphosate is realized. Combined with the RGB analysis platform of a smart phone, the probe system can realize on-site rapid screening, and provides an efficient and convenient solution for pesticide residue monitoring. In addition, the probe generates upconversion red light emission by using near-infrared light excitation, which effectively avoids the common pigment internal filter effect in traditional fluorescence detection. The unique detection mechanism and fluorescent probe provided by the application lay a key foundation for the detection of Cu 2+ and glyphosate, and show broad prospects in agricultural monitoring applications.

[0081] Group two

[0082] Example 11

[0083] The difference between this embodiment and example 3 is that the concentration of the alpha-cyclodextrin (alpha-CD) aqueous solution is 5mg / mL.

[0084] In this embodiment, the upconversion luminescence of the CyPh-UNs@CD prepared was tested under 808 nm laser pumping, using dye-sensitized nanosystems containing different concentrations of CyPh. As the CyPh loading capacity increased, the upconversion emission first increased and then decreased. When the CyPh concentration reached 1.0 μM, the luminescence intensity of the system reached its maximum, representing a 10-fold increase compared to the luminescence of UNs@CD.

[0085] Selectivity experiments showed that introducing K at a concentration of 2.0 μM into the CyPh-UNs@CD system... + Ca 2+ Mn 2 + Ni 2+ Zn 2+ Pb 2+ Ba 2+ Hg 2+ Cr 3+ and Fe 3+ When faced with common metal ions, the upconversion spectra of the CyPh-UNs@CD system did not change significantly, except for Cu. 2+ It exhibits a specific quenching response.

[0086] Furthermore, in CyPh-UNs@CD-Cu 2+ System (Cu) 2+ In a 1.0 μM solution, 2.0 μM concentrations of triazophos, glufosinate, methyl parathion, chlorpyrifos, dichlorvos, trichlorfon, fenitrothion, dimethoate, and glyphosate were added respectively. Experiments showed that only the addition of glyphosate quenched CyPh-UNs@CD-Cu. 2+ Only then did the system show a clear recovery of the luminous signal.

[0087] In CyPh-UNs@CD-Cu 2+ System (Cu) 2+ Further validation at a concentration of 1.0 μM showed that, within the concentration range of 0 to 1.0 μM, the upconversion luminescence intensity at 655 nm gradually increased with increasing glyphosate concentration. Linear regression analysis revealed a significant positive correlation between the signal intensity at 655 nm and the glyphosate concentration, with a detection limit of 20 μM. Furthermore, experiments demonstrated that the detection of glyphosate was unaffected by interference from pigments in grapes, mulberries, purple sweet potatoes, and purple cabbage.

[0088] Example 12

[0089] The only difference between this embodiment and Example 3 is that the concentration of the α-cyclodextrin (α-CD) aqueous solution is 15 mg / mL.

[0090] The CyPh-UNs@CD prepared in this example was tested for upconversion luminescence of dye-sensitized nanosystem containing different concentrations of CyPh under 808 nm laser pumping. With the increase of CyPh loading capacity, the upconversion emission first increased and then decreased. When the concentration of CyPh reached 3.0 μM, the luminescence intensity of the system reached the maximum, which was 45 times higher than that of UNs@CD.

[0091] The selective experiment showed that when the above CyPh-UNs@CD system was introduced into K + , Ca 2+ , Mn 2 + , Ni 2+ , Zn 2+ , Pb 2+ , Ba 2+ , Hg 2+ , Cr 3+ and Fe 3+ , etc. common metal ions, the upconversion spectrum of the CyPh-UNs@CD system did not change significantly, and only Cu 2+ showed specific quenching response.

[0092] Further, in the CyPh-UNs@CD-Cu 2+ system (Cu 2+ concentration 3.0 μM), triazole phosphorus, glufosinate ammonium, methyl parathion, chlorpyrifos, dichlorvos, dimetilan, sulfotep, dimethoate and glyphosate were added with a concentration of 5.0 μM, respectively. The experiment showed that only when glyphosate was added, the quenched CyPh-UNs@CD-Cu 2+ system showed obvious recovery of luminescence signal.

[0093] Further verification in the CyPh-UNs@CD-Cu 2+ system (Cu 2+ concentration 3.0 μM) showed that within the concentration range of 0 to 3.0 μM, with the increase of glyphosate concentration, the upconversion luminescence intensity at 655 nm showed a gradually increasing trend. Through linear regression analysis, there was a significant positive correlation between the signal intensity at 655 nm and the concentration of glyphosate, and the detection limit was 19.01 nM. At the same time, further experiments showed that the detection of glyphosate was not interfered by plant pigments such as grape, mulberry, purple potato and purple cabbage.

[0094] Example 13

[0095] The difference between this example and Example 3 is only that the ratio of water and ethanol in the water / ethanol mixed solvent is v / v: 1:2.

[0096] The CyPh-UNs@CD prepared in this example was tested for upconversion luminescence of dye-sensitized nanosystem containing different concentrations of CyPh under 808 nm laser pumping. With the increase of CyPh loading capacity, the upconversion emission first increased and then decreased. When the concentration of CyPh reached 2.0 μM, the luminescence intensity of the system reached the maximum, which was 24 times higher than that of UNs@CD.

[0097] The selective experiment showed that when the above CyPh-UNs@CD system was introduced into K + , Ca 2+ , Mn 2 + , Ni 2+ , Zn 2+ , Pb 2+ , Ba 2+ , Hg 2+ , Cr 3+ and Fe 3+ , etc. common metal ions, the upconversion spectrum of the CyPh-UNs@CD system did not change significantly, and only Cu 2+ showed specific quenching response.

[0098] Further, in the CyPh-UNs@CD-Cu 2+ system (Cu 2+ concentration 2.0 μM), 4.0 μM of triazole phosphorus, glufosinate ammonium, methyl parathion, chlorpyrifos, dichlorvos, nematode, sulfotep, dimethoate and glyphosate were added respectively, and the experiment showed that only when glyphosate was added, the quenched CyPh-UNs@CD-Cu 2+ system showed obvious recovery of luminescent signal.

[0099] Further verification in the CyPh-UNs@CD-Cu 2+ system (Cu 2+ concentration 2.0 μM) showed that in the concentration range of 0 to 2.0 μM, with the increase of glyphosate concentration, the upconversion luminescence intensity at 655 nm showed a gradually increasing trend. Through linear regression analysis, there was a significant positive correlation between the signal intensity at 655 nm and the concentration of glyphosate, and the detection limit was 1.5 μM. At the same time, further experiments showed that the detection of glyphosate was not interfered by plant pigments such as grape, mulberry, purple potato and purple cabbage.

[0100] Example 14

[0101] The difference between this example and Example 3 is only that the ratio of water and ethanol in the water / ethanol mixed solvent is v / v: 1:5.

[0102] The CyPh-UNs@CD prepared in this example was tested for upconversion luminescence of dye-sensitized nanosystem containing different concentrations of CyPh under laser pumping at 808 nm. With the increase of CyPh loading capacity, the upconversion emission first increased and then decreased. When the concentration of CyPh reached 0.5 μM, the luminescence intensity of the system reached the maximum, which was 7 times higher than that of UNs@CD.

[0103] The selective experiment showed that when the above CyPh-UNs@CD system was introduced into 1.0 μM of K + , Ca 2+ , Mn 2 + , Ni 2+ , Zn 2+ , Pb 2+ , Ba 2+ , Hg 2+ , Cr 3+ and Fe 3+ , etc. common metal ions, the upconversion spectrum of the CyPh-UNs@CD system did not change significantly, and only Cu 2+ showed specific quenching response.

[0104] Further, in the CyPh-UNs@CD-Cu 2+ system (Cu 2+ concentration 0.5 μM), 1.0 μM of triazole phosphorus, glufosinate ammonium, methyl parathion, chlorpyrifos, dichlorvos, dimetan, sulfotep, Rogor and glyphosate were added respectively, and the experiment showed that only when glyphosate was added, the quenched CyPh-UNs@CD-Cu 2+ system showed obvious recovery of luminescent signal.

[0105] Further verification in the CyPh-UNs@CD-Cu 2+ system (Cu 2+ concentration 0.5 μM) showed that in the concentration range of 0 to 0.5 μM, with the increase of glyphosate concentration, the upconversion luminescence intensity at 655 nm showed a gradually increasing trend. Through linear regression analysis, there was a significant positive correlation between the signal intensity at 655 nm and the concentration of glyphosate, and the detection limit was 33 μM. At the same time, further experiments showed that the detection of glyphosate was not interfered by plant pigments such as grape, mulberry, purple potato and purple cabbage.

[0106] Example 15

[0107] The difference between this example and Example 3 is that the amount of UNs containing oleic acid ligand in the water / ethanol mixed solvent is 2.5 mg.

[0108] The CyPh-UNs@CD prepared in this example was tested for upconversion luminescence of dye-sensitized nanosystem containing different concentrations of CyPh under 808 nm laser pumping. With the increase of CyPh loading capacity, the upconversion emission first increased and then decreased. When the concentration of CyPh reached 1.5 μM, the luminescence intensity of the system reached the maximum, which was 22.5 times higher than that of UNs@CD.

[0109] The selective experiment showed that when the above CyPh-UNs@CD system was introduced into K + , Ca 2+ , Mn 2 + , Ni 2+ , Zn 2+ , Pb 2+ , Ba 2+ , Hg 2+ , Cr 3+ and Fe 3+ , etc. at a concentration of 3.0 μM, the upconversion spectrum of the CyPh-UNs@CD system did not change significantly, and only Cu 2+ showed specific quenching response.

[0110] Further, in the CyPh-UNs@CD-Cu 2+ system (Cu 2+ concentration 1.5 μM), triazole phosphorus, glufosinate ammonium, methyl parathion, chlorpyrifos, dichlorvos, nematode, phosalone, and glyphosate were added at a concentration of 3.0 μM, respectively. The experiment showed that only when glyphosate was added, the quenched CyPh-UNs@CD-Cu 2+ system showed obvious recovery of luminescent signal.

[0111] Further verification in the CyPh-UNs@CD-Cu 2+ system (Cu 2+ concentration 1.5 μM) showed that within the concentration range of 0 to 1.5 μM, with the increase of glyphosate concentration, the upconversion luminescence intensity at 655 nm showed a gradually increasing trend. Through linear regression analysis, there was a significant positive correlation between the signal intensity at 655 nm and the concentration of glyphosate, and the detection limit was 23.43 nM. At the same time, further experiments showed that the detection of glyphosate was not interfered by plant pigments such as grape, mulberry, purple potato and purple cabbage.

[0112] Example 16

[0113] The difference between this example and Example 3 is that the amount of UNs containing oleic acid ligand in the water / ethanol mixed solvent is 15 mg.

[0114] In this embodiment, the upconversion luminescence of the CyPh-UNs@CD prepared was tested under 808 nm laser pumping, using dye-sensitized nanosystems containing different concentrations of CyPh. As the CyPh loading capacity increased, the upconversion emission first increased and then decreased. When the CyPh concentration reached 0.6 μM, the luminescence intensity of the system reached its maximum, representing a 6-fold increase compared to the luminescence of UNs@CD.

[0115] Selectivity experiments showed that introducing K at a concentration of 1.2 μM into the CyPh-UNs@CD system effectively reduced K+ concentration. + Ca 2+ Mn 2 + Ni 2+ Zn 2+ Pb 2+ Ba 2+ Hg 2+ Cr 3+ and Fe 3+ When faced with common metal ions, the upconversion spectra of the CyPh-UNs@CD system did not change significantly, except for Cu. 2+ It exhibits a specific quenching response.

[0116] Furthermore, in CyPh-UNs@CD-Cu 2+ System (Cu) 2+ In a solution containing 0.6 μM triazophos, glufosinate, methyl parathion, chlorpyrifos, dichlorvos, trichlorfon, fenitrothion, dimethoate, and glyphosate, each at a concentration of 1.2 μM, experiments showed that only the addition of glyphosate quenched CyPh-UNs@CD-Cu. 2+ Only then did the system show a clear recovery of the luminous signal.

[0117] In CyPh-UNs@CD-Cu 2+ System (Cu) 2+ Further validation at a concentration of 0.6 μM showed that, within the concentration range of 0 to 0.6 μM, the upconversion luminescence intensity at 655 nm gradually increased with increasing glyphosate concentration. Linear regression analysis revealed a significant positive correlation between the signal intensity at 655 nm and the glyphosate concentration, with a detection limit of 29 μM. Furthermore, experiments demonstrated that the detection of glyphosate was unaffected by interference from pigments in grapes, mulberries, purple sweet potatoes, and purple cabbage.

[0118] It will be apparent to those skilled in the art that the disclosure is not limited to the details of the above-exemplified embodiments and that the disclosure can be implemented in other particular forms without departing from the spirit or essential characteristics of the disclosure. The presently disclosed embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the disclosure being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. No feature of the claims is to be construed as limiting the claims to the exact nature of the features described therein.

[0119] Furthermore, it should be understood that although the description is made on the basis of the embodiments, not every embodiment contains only one independent technical solution, and the description of the specification is only for the sake of clarity, and those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that those skilled in the art can understand.

Claims

1. A dye-sensitized upconversion fluorescent probe, which is composed of near-infrared cyanine dye and β-NaYF4:Yb20%,Er2% @NaYF4:Yb10%,Nd10% upconversion nanoparticles, wherein the structural formula of the near-infrared cyanine dye is: .

2. The method for preparing the dye-sensitized upconversion fluorescence probe according to claim 1, comprising: adding a cyclodextrin aqueous solution into a water / ethanol mixed solvent with added upconversion nanoparticles, ultrasonic dispersion, stirring at room temperature, centrifugation, washing with water and then re-dispersing in ultrapure water to obtain a UNs@CD dispersion; dissolving a near-infrared cyanine dye in N,N'-dimethylformamide, adding into the UNs@CD dispersion, mixing and stirring, centrifugation, washing with water and then re-dispersing in ultrapure water to obtain the dye-sensitized rare earth upconversion nanoprobes.

3. The method for preparing the dye-sensitized upconversion fluorescent probe according to claim 2, characterized in that, The cyclodextrin is an amphiphilic α-cyclodextrin.

4. The method for preparing the dye-sensitized upconversion fluorescent probe according to claim 2, characterized in that, The content of α-cyclodextrin in the cyclodextrin aqueous solution is 5-15 mg / mL.

5. The method for preparing the dye-sensitized upconversion fluorescent probe according to claim 2, characterized in that, The ratio of water and ethanol in the water / ethanol mixed solvent is v / v: 1: (1-5).

6. The method for preparing the dye-sensitized upconversion fluorescent probe according to claim 2, characterized in that, The added amount of upconversion nanoparticles in the water / ethanol mixed solvent is 0.5-3 mg / mL.

7. The method for preparing the dye-sensitized upconversion fluorescent probe according to claim 2, characterized in that, The preparation of the near-infrared cyanine dye is: condensation of 4,4'-dithiodibenzoic acid with 6-mercapto-purine in the presence of a condensing agent and a base to obtain an intermediate; reduction of the intermediate to obtain a purine derivative containing a mercapto group; reaction of IR783 with the purine derivative containing a mercapto group in basic conditions at room temperature under a protective atmosphere and in the dark to obtain the cyanine derivative.

8. The method for preparing the dye-sensitized upconversion fluorescent probe according to claim 7, characterized in that, The condensing agent is selected from 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate; and / or, the base is selected from N,N-diisopropylethylamine; and / or, the reducing agent used in the reduction is selected from tris(2-carboxyethyl)phosphine.

9. The dye-sensitized upconversion fluorescence probe according to claim 1 for use in the detection of Cu 2+ or Cu 2+ and glyphosate in combination.

10. Use according to claim 9, characterized in that, The Cu 2+ is detected by introducing Cu 2+ into the detection solution containing the dye-sensitized upconversion fluorescent probe; and / or, the Cu 2+ and glyphosate are jointly detected by introducing Cu 2+ into the detection solution containing the dye-sensitized upconversion fluorescent probe, and the upconversion luminescence intensity is reduced, and then the glyphosate is introduced again, and the upconversion luminescence intensity is increased again.

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