A ratiometric fluorescent probe and a preparation method and application thereof

Abstract

The present application relates to the technical field of fluorescent probe, in particular to a ratio fluorescent probe and its preparation method and application. The ratio fluorescent probe is prepared from isothiocyanate dye modified semiconductor polymer quantum dots, and has strong fluorescence emission peaks at 530 nm and 581 nm, respectively. The isothiocyanate dye is rhodamine B isothiocyanate, the semiconductor polymer quantum dots are composed of polystyrene maleic anhydride copolymer and poly[(9,9-dioctylfluorenyl-2,7-diyl)-alt-(benzo[2,1,3]thiadiazole-4,8-diyl)], and the particle size of the ratio fluorescent probe is 10-200 nm. The detection limit of the ratio fluorescent probe for tyramine is 1.023 μM, which is lower than the allowable limit of tyramine residue, and meets the detection requirements of freshness and safety of aquatic products. The ratio fluorescent probe can be used for real-time / onsite visual quantitative detection of tyramine, and makes the detection process portable, fast and low-cost.

Description

Technical Field

[0001] This invention relates to the field of fluorescent probe technology, specifically to a ratiometric fluorescent probe, its preparation method, and its application. Background Technology

[0002] Biogenic amines exert biological activity that impacts human health, serving as essential substances for maintaining normal physiological functions. They regulate body temperature, blood pressure, digestion, and influence the formation of physiological structures in cells and other tissues. Among biogenic amines, tyramine's physiological toxicity is second only to histamine. Tyramine in the human body is metabolized via the monoamine oxidase pathway, and monoamine oxidase inhibitors are commonly used to treat depression and Parkinson's disease. However, individuals receiving monoamine oxidase inhibitor treatment face a higher risk of tyramine poisoning when consuming foods containing tyramine due to their reduced tyramine metabolism. Tyramine poisoning, also known as the "cheese reaction," manifests as migraines, rapid heart rate, nausea, vomiting, respiratory disturbances, and elevated blood sugar. Tyramine negatively impacts human health and food quality; therefore, strictly controlling tyramine content during food production and distribution has significant social and economic value.

[0003] Therefore, there is an urgent need to establish a fluorescence detection method for quantitative and intuitive detection of tyramine, which is crucial for ensuring food safety and dietary health. Summary of the Invention

[0004] To address the above technical problems, this invention provides a ratiometric fluorescent probe, its preparation method, and its applications. This ratiometric fluorescent probe enables real-time / on-site visual quantitative detection of tyramine, exhibiting high sensitivity, a detection limit of 1.023 μM, and a portable, rapid, and low-cost detection process.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides a ratiometric fluorescent probe, which is prepared by modifying semiconductor polymer quantum dots (Pdots) with isothiocyanate dye to obtain nanoparticles, which have strong fluorescence emission peaks at 530 nm and 581 nm, respectively; wherein, the isothiocyanate dye is rhodamine B isothiocyanate; the semiconductor polymer quantum dots are composed of polystyrene maleic anhydride copolymer and poly[(9,9-dioctylfluorenyl-2,7-diyl)-alt-(benzo[2,1,3]thiadiazole-4,8-diyl)].

[0007] In this invention, rhodamine B isothiocyanate (RBI) is used as an isothiocyanate to modify semiconductor polymer quantum dots composed of polystyrene-maleic anhydride copolymer (PSMA) and poly[(9,9-dioctylfluorenyl-2,7-diyl)-alt-(benzo[2,1,3]thiadiazole-4,8-diyl)] (PF8BT) to obtain the PF8BT-RBI ratiometric fluorescent probe. The fluorescence of rhodamine B isothiocyanate and poly[(9,9-dioctylfluorenyl-2,7-diyl)-alt-(benzo[2,1,3]thiadiazole-4,8-diyl)] serves as the fluorescence detection signal for tyramine. RBI acts as the energy acceptor, and PF8BT acts as the energy donor. Based on the fluorescence resonance energy transfer (FRET) mechanism, the PF8BT-RBI ratiometric fluorescent probe is constructed. After absorbing energy, the PF8BT energy donor transfers it to the RBI energy acceptor, and the RBI energy acceptor emits fluorescence. The concentration of tyramine, the analyte, is varied, causing changes in the fluorescence intensity of the PF8BT energy donor and the RBI energy acceptor. The ratio of these fluorescence intensities is linearly related to the concentration of the analyte, enabling quantitative detection of tyramine. Simultaneously, changes in the fluorescence intensity of PF8BT and RBI result in changes in the fluorescence color of the probe. When a tyramine-containing substance is added to the PF8BT-RBI ratiometric fluorescent probe, the solution changes from orange-yellow to yellow-green under 350nm UV excitation, achieving visual detection of tyramine. The ratiometric fluorescent probe of this invention has a detection limit of 1.023 μM for tyramine, lower than the permissible limit for tyramine residue, and can accurately detect tyramine concentrations in the range of 0-250 μM. It allows for real-time / on-site visual quantitative detection of tyramine, making the detection process portable, rapid, and cost-effective, meeting the requirements for detecting the freshness and safety of aquatic products.

[0008] Secondly, the present invention also provides a method for preparing the above-mentioned ratiometric fluorescent probe, comprising:

[0009] Step 1: Mix the dimethyl sulfoxide solution of rhodamine isothiocyanate B, the tetrahydrofuran solution of polystyrene-maleic anhydride copolymer, and the tetrahydrofuran solution of poly[(9,9-dioctylfluorenyl-2,7-diyl)-alt-(benzo[2,1,3]thiadiazole-4,8-diyl)], and dilute to volume with tetrahydrofuran to obtain solution A;

[0010] Step 2: Under the conditions of 0℃ ice water bath and ultrasound, the solution A is injected into ultrapure water in one go, and ultrasound is continued for 1-2 minutes. Then, gas is introduced into the solution to purge it while heating to remove tetrahydrofuran. The remaining liquid is collected, diluted with ultrapure water, filtered, and the resulting filtrate is the ratio fluorescent probe.

[0011] In step 1, the mass ratio of rhodamine isothiocyanate B, polystyrene-maleic anhydride copolymer, and poly[(9,9-dioctylfluorenyl-2,7-diyl)-alt-(benzo[2,1,3]thiadiazole-4,8-diyl)] in solution A is 0.005-0.05:0.1-1:1.

[0012] In step 2, the volume ratio of solution A to ultrapure water is 2-5:10;

[0013] In step 2, the temperature for removing tetrahydrofuran by purging with gas is 95-110℃, and the holding time is 25-30 minutes.

[0014] The ratiometric fluorescent probe prepared by the method described above has a particle size of 10-200 nm.

[0015] This invention uses Pdots as the nanostructure of a ratiometric fluorescence sensor, embedding fluorescent dyes into Pdots via a reprecipitation method. The structures of Rhodamine isothiocyanate B, polystyrene-maleic anhydride copolymer, and poly[(9,9-dioctylfluorenyl-2,7-diyl)-alt-(benzo[2,1,3]thiadiazole-4,8-diyl)] are shown in formula (I).

[0016]

[0017] All three substances mentioned above are commercially available, eliminating the need for complex preparation processes.

[0018] In step 1 above, the dimethyl sulfoxide solution of rhodamine B thiocyanate, the polystyrene maleic anhydride copolymer, and the tetrahydrofuran solution of poly[(9,9-dioctylfluorenyl-2,7-diyl)-alt-(benzo[2,1,3]thiadiazole-4,8-diyl)] do not have a specific order of addition; the goal is to achieve a uniform mixing of all materials.

[0019] In step 2 above, solution A is injected into ultrapure water in one go, emphasizing rapid injection without interruption. "Rapid" refers to the fastest speed within the experimental conditions and equipment capabilities.

[0020] Exemplarily, according to experimental practices, the present invention provides a method for preparing a ratiometric fluorescent probe, comprising:

[0021] Step 1: Add the dimethyl sulfoxide solution of rhodamine isothiocyanate B and the tetrahydrofuran solution of polystyrene-maleic anhydride copolymer to the tetrahydrofuran solution of poly[(9,9-dioctylfluorenyl-2,7-diyl)-alt-(benzo[2,1,3]thiadiazole-4,8-diyl)] in sequence and mix them. Then, make up to volume with tetrahydrofuran to obtain solution A.

[0022] Step 2: Under the conditions of 0℃ ice water bath and ultrasound, the solution A is injected into ultrapure water in one go, and ultrasound is continued for 1-2 minutes. Then, gas is introduced into the solution to purge it while heating to remove tetrahydrofuran. The remaining liquid is collected, diluted with ultrapure water, filtered, and the resulting filtrate is the ratio fluorescent probe.

[0023] In step 1, the mass ratio of rhodamine isothiocyanate B, polystyrene-maleic anhydride copolymer, and poly[(9,9-dioctylfluorenyl-2,7-diyl)-alt-(benzo[2,1,3]thiadiazole-4,8-diyl)] in solution A is 0.005-0.05:0.1-1:1.

[0024] In step 2, the volume ratio of solution A to ultrapure water is 2-5:10;

[0025] In step 2, the temperature for removing tetrahydrofuran during gas purging and heating is 95-110℃, and the holding time is 25-30 minutes.

[0026] In this invention, Rhodamine B isothiocyanate (RBI) acts as the energy acceptor, producing a fluorescence emission peak at 581±5 nm. Poly[(9,9-dioctylfluorenyl-2,7-diyl)-alt-(benzo[2,1,3]thiadiazole-4,8-diyl)] (PF8BT) acts as the energy donor, producing a fluorescence emission peak at 530±5 nm. A PF8BT-RBI ratiometric fluorescent probe is constructed based on the fluorescence resonance energy transfer (FRET) mechanism. The PF8BT energy donor absorbs energy and transfers it to the RBI energy acceptor, which then emits fluorescence. The fluorescence intensity of the PF8BT donor and the RBI acceptor changes according to the concentration of the analyte tyramine. The linear relationship between the fluorescence intensity ratio of PF8BT and RBI and the concentration of the analyte allows for the quantitative detection of tyramine concentration in the analyte.

[0027] Preferably, the concentration of the dimethyl sulfoxide solution of Rhodamine B isothiocyanate is 1-2 mg / mL; the concentration of the tetrahydrofuran solution of the polystyrene-maleic anhydride copolymer is 1-2 mg / mL; and the concentration of the tetrahydrofuran solution of poly[(9,9-dioctylfluorenyl-2,7-diyl)-alt-(benzo[2,1,3]thiadiazole-4,8-diyl)] is 1-2 mg / mL.

[0028] Preferably, the mass ratio of Rhodamine isothiocyanate B, polystyrene-maleic anhydride copolymer, and poly[(9,9-dioctylfluorenyl-2,7-diyl)-alt-(benzo[2,1,3]thiadiazole-4,8-diyl)] in solution A is 0.02:0.8:1.

[0029] Excessive amounts of Rhodamine B isothiocyanate will increase the free fluorescent dye in the solution, thereby reducing the effective reaction; while insufficient amounts of Rhodamine B isothiocyanate will reduce the fluorescence intensity of Rhodamine B isothiocyanate, thereby reducing the compatibility of instrument errors and the accuracy of detection results.

[0030] Preferably, the remaining liquid is collected and diluted to 10 mL with ultrapure water.

[0031] Preferably, the gas in step 2 is any one or a combination of nitrogen, argon, or helium.

[0032] Preferably, the filtration is performed using a 0.22μm aqueous phase filter.

[0033] In this invention, the tetrahydrofuran in the solvent is completely evaporated, and the content of the remaining dimethyl sulfoxide is ≤2‰, which will not affect the filtration performance of the aqueous filter head and meets the usage requirements.

[0034] Thirdly, the present invention also provides the application of the ratiometric fluorescent probe described above or the ratiometric fluorescent probe prepared by the above method in the detection of tyramine.

[0035] Fourthly, the present invention also provides a method for detecting tyramine using the ratio fluorescent probe, comprising: mixing the ratio fluorescent probe with the tyramine to be tested in a certain proportion to form a reaction system, reacting at 25-30°C for 10-20 min, and then performing spectral detection using a fluorescence spectrometer, thereby detecting tyramine by means of the fluorescence intensity change of the ratio fluorescent probe at 30 nm and 581 nm.

[0036] Preferably, the reaction system preparation method includes: adding 50 μL of ratiometric fluorescent probe, 30 μL of tyramine to be tested and an appropriate amount of buffer to prepare a 1 mL reaction system; the buffer is PBS buffer with pH 7-7.5.

[0037] Preferably, a fluorescence spectrometer is used for spectral detection, and the fluorescence intensity I at the fluorescence emission peak at 530 nm is recorded. 530 fluorescence intensity I at 581 nm 581 Calculate I 530 / I 581 The ratio of fluorescence emission peak value is used to obtain the fluorescence emission peak value. The calibration curve is obtained by using the functional relationship between the fluorescence emission peak value and the tyramine concentration. The concentration of the tyramine to be tested is calculated using the calibration curve.

[0038] The ratiometric fluorescent probe of this invention, in the absence of tyramine, allows the PF8BT energy donor to transfer energy to the RBI energy acceptor upon excitation, causing the RBI energy acceptor to emit fluorescence. In the presence of tyramine, the isothiocyanate group of the RBI energy acceptor undergoes an addition reaction with the amino group, further leading to intramolecular charge transfer in the RBI, thereby quenching the RBI fluorescence. When the PF8BT energy donor is excited, it emits fluorescence. Depending on the tyramine concentration, the fluorescence intensity at the two emission wavelengths of PF8BT and RBI changes, enabling ratiometric fluorescence detection of tyramine content.

[0039] Specifically, the PF8BT energy donor and RBI energy acceptor are highly sensitive to changes in spatial location.

[0040] When the PF8BT energy donor and the RBI energy acceptor are close enough, the fluorescence emission spectrum of the PF8BT energy donor and the ultraviolet absorption spectrum of the RBI energy acceptor overlap. As a result, the chromophore of the PF8BT energy donor will transfer energy to the chromophore of the RBI energy acceptor with high efficiency, which will weaken or disappear the emission fluorescence of the PF8BT energy donor. At this time, the fluorescence intensity of the RBI energy acceptor is detected at 581 nm.

[0041] When the relative spatial positions of the PF8BT energy donor and RBI energy acceptor are altered—for example, when tyramine, the analyte, is added to the ratiometric fluorescent probe—an addition reaction occurs between the amino group of tyramine and the isothiocyanate group of the RBI, causing fluorescence quenching of the RBI. This alters the relative spatial positions of the PF8BT energy donor and the RBI energy acceptor, preventing fluorescence resonance energy transfer. In this case, fluorescence at 530 nm is detected in the PF8BT energy acceptor. Changes in the concentration of the analyte tyramine cause changes in the fluorescence intensity of the PF8BT donor and the RBI acceptor. By observing the linear relationship between the fluorescence intensity ratio of PF8BT and RBI and the concentration of the analyte, the concentration of tyramine in the analyte can be detected.

[0042] Preferably, the conditions for the fluorescence test include: a fluorescence excitation wavelength of 460-485 nm and a fluorescence emission spectrum observation range of 520-600 nm.

[0043] Fifthly, the present invention also provides the application of the above-described ratio fluorescent probe method for detecting tyramine in aquaculture water, aquatic products, and meat. Attached Figure Description

[0044] Figure 1 Images and particle size distribution of the PF8BT-RBI ratio fluorescent probe with a RBI to F8BT mass ratio of 2% as described in this invention, obtained under a transmission electron microscope. Figure 1 a is the particle size distribution diagram. Figure 1 b is the image under a transmission electron microscope;

[0045] Figure 2 The difference in fluorescence emission peak ratio of PF8BT-RBI ratio fluorescent probe solutions with different RBI to F8BT mass ratios;

[0046] Figure 3 The fluorescence emission spectra of fluorescent probes prepared by nanoprecipitation method by mixing different concentrations of fluorescein isothiocyanate into PFO precursor solutions were obtained. Figure 3 a), and its normalized fluorescence emission spectrum ( Figure 3 b);

[0047] Figure 4 Fourier transform infrared spectra of PF8BT-RBI and PPVK-FITC ratiometric fluorescent probes before and after the addition of tyramine (where, Figure 4 A is the Fourier transform infrared spectrum of PF8BT-RBI. Figure 4 B is the PPVK-FITC Fourier transform infrared spectrum;

[0048] Figure 5 Fluorescence lifetime plots of PF8BT-RBI and PPVK-FITC ratiometric fluorescent probes (where, Figure 5 C represents the fluorescence lifetime of PF8BT, PF8BT-RBI, and PF8BT-RBI+tyramine. Figure 5 D represents the fluorescence lifetime of PVK, PPVK-FITC, and PPVK-FITC+Tyr.

[0049] Figure 6 The zeta potential of PF8BT-RBI and PPVK-FITC ratio fluorescent probes in the detection of tyramine;

[0050] Figure 7 The absolute quantum yield of the PF8BT-RBI and PPVK-FITC ratio fluorescent probes;

[0051] Figure 8 The fluorescence emission spectra of PF8BT-RB ratio fluorescent probe solutions at different tyramine concentrations are shown.

[0052] Figure 9 Fluorescence images of PF8BT-RBI ratio fluorescent probe solutions at different concentrations of tyramine standard solution;

[0053] Figure 10 The linear range of fluorescence emission peak ratios of the PF8BT-RBI ratio fluorescent probe solution with different tyramine standard solution concentrations is given.

[0054] Figure 11The fluorescence emission spectra of the PPVK-FITC ratio fluorescent probe solutions at different tyramine concentrations are shown.

[0055] Figure 12 Fluorescence images of PPVK-FITC ratio fluorescent probe solutions with different tyramine standard solution concentrations;

[0056] Figure 13 The linear range of fluorescence emission peak ratio values ​​of PF8BT-RBI ratio fluorescent probe solutions with different tyramine standard solution concentrations is given.

[0057] Figure 14 Different interfering substances were added to the ratiometric fluorescent probe of this invention (1,9: blank, 2: cadaverine, 3: 1,4-diaminobutane dihydrochloride, 4: histamine, 5: tryptophan hydrochloride, 6: spermine, 7: β-phenylethylamine, 8,16: tyramine, 10: Na). + 11:K + ,12:Mg 2+ ,13:Fe 3+ ,14:Cr 3+ 15: Zn 2+ ), the ratio of fluorescence emission peaks of the ratio fluorescent probe solution. Detailed Implementation

[0058] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0059] Example 1

[0060] Preparation of PF8BT-RBI ratiometric fluorescent probe

[0061] Take 1 mL of tetrahydrofuran solution (1 mg / mL) of poly[(9,9-dioctylfluorenyl-2,7-diyl)-alt-(benzo[2,1,3]thiadiazole-4,8-diyl)] (F8BT), add 0.005-0.05 mL of dimethyl sulfoxide solution (1 mg / mL) of rhodamine isothiocyanate B (RBI) and 0.8 mL of tetrahydrofuran solution (1 mg / mL) of polystyrene-maleic anhydride copolymer (PSMA) and mix. Finally, make up to 2 mL with tetrahydrofuran to obtain solution A with RBI to F8BT mass ratios of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, and 5%.

[0062] The solutions A of the above concentrations were injected into 10 mL of ultrapure water in an ultrasonic environment. The solution was continuously sonicated in an ice-water bath at 0 °C for 1 min, and then nitrogen gas was introduced to purge the solution to evaporate the solvent. The solution was then heated to 100 °C in a silicone oil bath and reacted for 30 min. The remaining liquid was collected and then diluted to 10 mL with ultrapure water. The solution was then filtered through a 0.22 μm aqueous phase filter to obtain a series of PF8BT-RBI ratiometric fluorescent probes with different mass concentrations of Rhodamine isothiocyanate B. The probes were stored in a 4 °C refrigerator for later use.

[0063] Figure 1 Images and particle size distribution of the PF8BT-RBI ratio fluorescent probe with a RBI to F8BT mass ratio of 2% as described in this invention, obtained under a transmission electron microscope. Figure 1 a is the particle size distribution diagram. Figure 1 b is the image under a transmission electron microscope.

[0064] from Figure 1 It can be seen that the PF8BT-RBI ratio fluorescent probe with a mass ratio of 2% RBI to F8BT consists of spherical particles of 10-200 nm with an average particle size of 48.1 nm.

[0065] Example 2

[0066] Screening for the optimal PF8BT-RBI ratio fluorescent probe

[0067] (1) Preparation of standard stock solution

[0068] Weigh out tyramine standard and dissolve it in PBS buffer to prepare a standard stock solution with a mass concentration of 5 mM. The PBS buffer is 20 mM with a pH of 7.5.

[0069] (2) Analysis of the numerical difference in fluorescence emission peak ratio of ratio fluorescent probe solutions at different concentrations of Rhodamine B isothiocyanate

[0070] Take 0.05 mL of each of the series of PF8BT-RBI ratio fluorescent probes prepared in Example 1, add 0.03 mL of standard stock solution, and dilute to 1 mL with PBS buffer to obtain a series of PF8BT-RBI-tyramine solutions, wherein the concentration of tyramine is 150 μM.

[0071] Take 0.05 mL of each of the series of PF8BT-RBI ratio fluorescent probes prepared in Example 1, and dilute to 1 mL with PBS buffer to obtain a series of PF8BT-RBI-tyramine blank solutions.

[0072] The fluorescence emission spectra of PF8BT-RBI-tyramine solution and PF8BT-RBI-tyramine blank solution were detected using a fluorescence spectrophotometer. The fluorescence intensity I at the fluorescence emission peak of PF8BT-RBI-tyramine solution and PF8BT-RBI-tyramine blank solution at 530 nm was recorded respectively. 530 fluorescence intensity I at 581 nm 581 Based on the formula (II) for calculating the fluorescence emission peak ratio, the fluorescence emission peak ratio of PF8BT-RBI-tyramine solution and PF8BT-RBI-tyramine blank solution was calculated.

[0073] R = I 530 / I 581 Formula (II).

[0074] Where R: fluorescence emission peak ratio;

[0075] I 530 The fluorescence intensity at 530 nm is the fluorescence emission spectrum.

[0076] I 581 The fluorescence intensity at 581 nm in the fluorescence emission spectrum is given.

[0077] Then, using the formula (III) for the numerical difference of fluorescence emission peak ratio, the numerical difference of fluorescence emission peak ratio of the standard substance tyramine solution is calculated.

[0078] R 0-T =R0-R T Formula (III).

[0079] Among them, R 0-T This represents the numerical difference in the ratio of fluorescence emission peak values.

[0080] R0: The ratio of fluorescence emission peak values ​​of the PF8BT-RBI-tyramine blank solution;

[0081] R T The ratio of fluorescence emission peaks in PF8BT-RBI-tyramine solution.

[0082] Plotting different RBI to F8BT mass ratios in PF8BT-RBI-tyramine solutions on the x-axis, R... 0-T Plot a bar chart for the vertical axis, such as Figure 2 As shown.

[0083] Figure 2 The difference in fluorescence emission peak ratio of the fluorescent probe solution with different RBI to F8BT ratios in this invention is shown.

[0084] from Figure 2It is known that the ratio of RBI to F8BT prepared at a mass ratio of 2% is the most sensitive for tyramine detection. Therefore, a mass ratio of 2% RBI to F8BT is selected as the optimal ratio for preparing PF8BT-RBI ratio fluorescent probe solution A.

[0085] Example 3:

[0086] Preparation of PF8BT-RBI ratiometric fluorescent probe

[0087] Take 1 mL of tetrahydrofuran solution (1 mg / mL) of poly[(9,9-dioctylfluorenyl-2,7-diyl)-alt-(benzo[2,1,3]thiadiazole-4,8-diyl)](F8BT), add 0.02 mL of dimethyl sulfoxide solution (1 mg / mL) of rhodamine isothiocyanate B (RBI) and 0.8 mL of tetrahydrofuran solution (1 mg / mL) of polystyrene maleic anhydride copolymer (PSMA) and mix. Finally, make up to 2 mL with tetrahydrofuran to obtain solution A.

[0088] The above solution A was injected into 10 mL of ultrapure water in an ultrasonic environment. The solution was continuously sonicated in an ice-water bath at 0 °C for 1 min. Nitrogen gas was then introduced to purge the solution to evaporate the solvent. The solution was then heated to 100 °C in a silicone oil bath and reacted for 30 min. The remaining liquid was collected and then diluted to 10 mL with ultrapure water. The solution was then filtered through a 0.22 μm aqueous phase filter to obtain the PF8BT-RBI ratiometric fluorescent probe, which was stored in a 4 °C refrigerator for later use.

[0089] Comparative Example 1

[0090] PPVK-FITC ratiometric fluorescent probe

[0091] In Example 3, F8BT was replaced with poly(N-vinylcarbazole) (PPVK), and RBI was replaced with fluorescein isothiocyanate (FITC). The preparation method was the same as in Example 3, and a PPVK-FITC ratio fluorescent probe was obtained and stored in a 4°C refrigerator for later use.

[0092] Comparative Example 2

[0093] PFO-FITC ratiometric fluorescent probe

[0094] In Example 1, F8BT was replaced with poly(9,9-dioctylfluorene) (PFO), and RBI was replaced with fluorescein isothiocyanate (FITC). The preparation method was the same as in Comparative Example 1. A series of PFO-FITC ratio fluorescent probes with a PFO to FITC mass ratio of 0-12% were obtained and stored in a 4°C refrigerator for later use.

[0095] Verification Example 1

[0096] 1. The ratiometric fluorescent probe prepared in Comparative Example 2 was subjected to fluorescence emission spectroscopy analysis. The results are as follows: Figure 3 As shown;

[0097] Figure 3 The fluorescence emission spectra of fluorescent probes prepared by nanoprecipitation method by mixing different concentrations of fluorescein isothiocyanate into PFO precursor solutions were obtained. Figure 3 a), and its normalized fluorescence emission spectrum ( Figure 3 b);

[0098] from Figure 3 It is known that the fluorescence emission spectrum of PFO-FITC only has fluorescence emission peaks belonging to PFO and does not produce emission peaks belonging to FITC. Therefore, there is no fluorescence resonance energy transfer between PFO and FITC, so the combination of PFO-FITC cannot be used to construct a ratiometric fluorescent probe.

[0099] 2. The ratiometric fluorescent probes prepared in Example 3 and Comparative Example 1 were characterized by Fourier transform infrared spectroscopy before and after the addition of tyramine. The results are as follows: Figure 4 As shown.

[0100] Figure 4 Fourier transform infrared spectra of PF8BT-RBI and PPVK-FITC ratiometric fluorescent probes before and after the addition of tyramine (where, Figure 4 A is the Fourier transform infrared spectrum of PF8BT-RBI. Figure 4 B is the PPVK-FITC Fourier transform infrared spectrum;

[0101] from Figure 4 As can be seen from A, the N=C=S stretching vibration can be found at 2050.94 cm⁻¹ in the Fourier transform infrared spectrum of PF8BT-RBI. -1 The absorption and splitting of [something] almost disappeared after the addition of tyramine. From [something] Figure 4 As can be seen from B, the N=C=S stretching vibration at 2057.24 cm⁻¹ can also be found in the Fourier transform infrared spectrum of PPVK-FITC. -1 The absorption split at [location] almost disappeared after the addition of tyramine. In the Fourier transform infrared spectrum, the peak corresponding to the C=S stretching vibration appeared at 1586.36 cm⁻¹. -1 and 1589.81cm -1 No change was observed. The Fourier transform infrared spectra of PF8BT-RBI and PPVK-FITC showed the same trend for the isothiocyanate groups, which further indicates that tyramine and isothiocyanate groups reacted.

[0102] 3. Fluorescence lifetime was used to investigate the establishment of a ratiometric fluorescence mechanism using PF8BT-RBI and PPVK-FITC ratiometric fluorescent probes. The results are as follows: Figure 5 As shown.

[0103] Figure 5 Fluorescence lifetime plots of PF8BT-RBI and PPVK-FITC ratiometric fluorescent probes (where, Figure 5 C represents the fluorescence lifetime of PF8BT, PF8BT-RBI, and PF8BT-RBI+tyramine. Figure 5 D represents the fluorescence lifetime of PVK, PPVK-FITC, and PPVK-FITC+Tyr.

[0104] from Figure 5 As shown in C, the fluorescence lifetime of PF8BT is 2.77 ns, which becomes 2.21 ns after the addition of RBI. However, the fluorescence lifetime of PF8BT becomes 2.14 ns after the addition of tyramine, which does not cause a substantial change in fluorescence lifetime.

[0105] from Figure 5 As shown in D, the fluorescence lifetime of PPVK is 6.43 ns, which becomes 4.07 ns after mixing with FITC, and then becomes 1444.06 ns after adding tyramine.

[0106] The aforementioned changes in fluorescence lifetime indicate that the fluorescence quenching between PF8BT, PPVK and RBI, FITC is based on dynamic quenching via fluorescence resonance energy transfer (FRET).

[0107] 4. The zeta potentials of PF8BT-RBI and PPVK-FITC ratiometric fluorescent probes in the detection of tyramine are shown in the following results. Figure 6 As shown.

[0108] Figure 6 The zeta potential of PF8BT-RBI and PPVK-FITC ratiometric fluorescent probes in the detection of tyramine.

[0109] from Figure 6 It was found that the potentials of PF8BT-RBI and PPVK-FITC ratiometric fluorescent probes increased after the addition of tyramine. Specifically, the potentials of PF8BT-RBI and PPVK-FITC increased by 1.96 mV and 3.77 mV, respectively, after the addition of tyramine. This is likely due to the reaction between tyramine and isothiocyanate groups, which suppresses the electronegativity of the ratiometric fluorescent probe surface.

[0110] 5. The absolute quantum yields of PF8BT-RBI and PPVK-FITC ratiometric fluorescent probes are shown in the following results. Figure 7 As shown.

[0111] Figure 7The absolute quantum yield of the PF8BT-RBI and PPVK-FITC ratio fluorescent probes;

[0112] from Figure 7 The absolute quantum yields of PF8BT, PPVK, PF8BT-RBI, and PPVK-FITC were 3.61%, 1.48%, 9.45%, and 8.83%, respectively. The addition of isothiocyanate dyes RBI and FITC significantly improved the quantum yield of the ratiometric fluorescent probes.

[0113] Verification Example 2

[0114] Example 3: Performance comparison of ratiometric fluorescent probes in Comparative Example 1

[0115] 1. PF8BT-RBI ratiometric fluorescent probe of Example 3

[0116] (1) Plotting the standard curve

[0117] Take 0.05 mL of the PF8BT-RBI ratiometric fluorescent probe prepared in Example 3, add different volumes of 5 mM tyramine standard stock solution, and dilute to 1 mL with PBS buffer solution to obtain homogeneous solutions with tyramine detection concentrations of 0 μM, 50 μM, 100 μM, 150 μM, 200 μM, and 250 μM. After reacting at 25-30℃ for 10-20 min, perform fluorescence spectroscopy detection. Detect the fluorescence emission spectra of the above homogeneous solutions at a fluorescence excitation wavelength of 460-485 nm and a fluorescence emission spectrum observation range of 520-600 nm.

[0118] Figure 8 The fluorescence emission spectra of PF8BT-RBI ratio fluorescent probe solutions at different tyramine concentrations are shown.

[0119] from Figure 8 It can be seen that the fluorescence emission peak of the PF8BT-RBI ratiometric fluorescent probe at 581 nm originates from RBI, and RBI concentration-dependent quenching occurs after the addition of tyramine. The fluorescence emission peak of the PF8BT-RBI ratiometric fluorescent probe at 530 nm originates from PF8BT, and the fluorescence emission peak of PF8BT is enhanced after the addition of tyramine. The increase or decrease in the fluorescence intensity of PF8BT and RBI leads to changes in fluorescence color. Observing the change in fluorescence color of the solution under a 350 nm UV lamp, after the addition of the PF8BT-RBI ratiometric fluorescent probe to the tyramine-containing substance, the homogeneous solution changes from orange-yellow to yellow-green under 350 nm UV excitation. Figure 9 As shown, the color illustrations are available in the substantive examination materials.

[0120] Fluorescence intensity I recorded at 530 nm 530 fluorescence intensity I at 581 nm 581The fluorescence emission peak ratio values ​​of the homogeneous solutions at each concentration were calculated according to equation (II). A standard curve equation was plotted with the concentration of the tyramine standard solution as the abscissa and the fluorescence emission peak ratio values ​​as the ordinate, relating the fluorescence emission peak ratio values ​​to the tyramine concentration.

[0121] y = 0.0041x + 0.3949

[0122] Among them, R 2 It is 0.9991;

[0123] x represents the concentration of the tyramine standard solution;

[0124] y represents the fluorescence emission peak ratio.

[0125] Figure 10 The linear range of fluorescence emission peak ratios of the PF8BT-RBI ratio fluorescent probe solution with different tyramine standard solution concentrations is given.

[0126] from Figure 10 It is known that the PF8BT-RBI ratiometric fluorescent probe of the present invention has a sensitive response to tyramine, and the fluorescence emission peak ratio increases with the increase of tyramine concentration. It can accurately detect tyramine in the concentration range of 0-250 μM, and shows a linear relationship in the range of 20-150 μM.

[0127] 2. Comparative Example 1: PPVK-FITC ratiometric fluorescent probe

[0128] (1) Plotting the standard curve

[0129] Take 0.05 mL of the PPVK-FITC ratiometric fluorescent probe prepared in Comparative Example 1, add different volumes of 5 mM tyramine standard stock solution, and dilute to 1 mL with PBS buffer solution to obtain homogeneous solutions with tyramine detection concentrations of 0 μM, 100 μM, 150 μM, 200 μM, 250 μM, 300 μM, 350 μM, 400 μM, 450 μM, and 500 μM. After reacting at 25-30℃ for 10-20 min, perform fluorescence spectroscopy detection. Detect the fluorescence emission spectra of the above homogeneous solutions at a fluorescence excitation wavelength of 320-470 nm and a fluorescence emission spectrum observation range of 425-510 nm.

[0130] Figure 11 The fluorescence emission spectra of the PPVK-FITC ratio fluorescent probe solutions at different tyramine concentrations are shown.

[0131] from Figure 11It was found that the addition of tyramine to the PPVK-FITC ratiometric fluorescent probe did not cause a change in the FITC fluorescence intensity at 517 nm, while the fluorescence intensity of PPVK at 377 nm decreased in a concentration-dependent manner due to the addition of tyramine. After adding tyramine, the color change of the solution fluorescence under UV light was observed; the color of PPVK-1FITC under UV excitation changed from deep blue to light blue, and then gradually extinguished. Figure 12 As shown, the color illustrations are available in the substantive examination materials.

[0132] Figure 12 Fluorescence images of PPVK-FITC ratio fluorescent probe solutions at different tyramine standard solution concentrations.

[0133] Record the fluorescence emission spectra of the homogeneous solutions at each of the above concentrations, and the fluorescence intensity I at 517 nm. 517 Fluorescence intensity I at 377nm 377 And calculate the fluorescence emission peak ratio value I. 377 / I 517 With the concentration of the tyramine standard solution on the x-axis and the fluorescence emission peak ratio on the y-axis, the standard curve equation between the fluorescence emission peak ratio and the tyramine concentration is as follows:

[0134] y = -0.0051x + 6.8857

[0135] Among them, R 2 It is 0.9904;

[0136] x represents the concentration of the tyramine standard solution;

[0137] y represents the fluorescence emission peak ratio.

[0138] Figure 13 The linear range of fluorescence emission peak ratios of the PF8BT-RBI ratio fluorescent probe solution with different tyramine standard solution concentrations is given.

[0139] Based on the calibration curves of PF8BT-RBI and PPVK-FITC, the limit of detection (LOD) for tyramine was determined using the classic 3σ / s formula. The LODs for tyramine of PF8BT-RBI and PPVK-FITC were 1.023 μM and 4.312 μM, respectively. Comparing the detection range, calibration curves, and detection limits, it is evident that the PF8BT-RBI ratiometric fluorescent probe prepared in Example 3 of this invention exhibits better detection performance.

[0140] Verification Example 3

[0141] 1. To investigate the effects of different biogenic amines and metal ions on the specific emissivity of the PF8BT-RBI ratiometric fluorescent probe.

[0142] Other amines and metal ions present in seafood can interfere with the detection of tyramine. Therefore, it is necessary to study the influence of different biogenic amines and metal ions on the sensor. The selectivity of the PF8BT-RBI ratiometric fluorescent probe prepared in Example 3 was studied, using seven amines and six metal ions (chloride salt solutions) in the selectivity experiment. The results are as follows: Figure 14 As shown.

[0143] Figure 14 The ratio of fluorescence emission peaks of the PF8BT-RBI ratioic fluorescent probe of the present invention with the addition of different interfering substances;

[0144] Figure 14 In the table, 1 and 9 are blanks, 2 is cadaverine, 3 is 1,4-diaminobutane dihydrochloride, 4 is histamine, 5 is tryptophan hydrochloride, 6 is spermine, 7 is β-phenylethylamine, 8 and 16 are tyramine, and 10 is Na+. + 11 is K + 12 is Mg 2+ 13 is Fe 3+ 14 is Cr 3+ 15 is Zn 2+ .from Figure 14 It can be seen that the PPF8BT-RBI ratiometric fluorescent probe reacts to seven biogenic amines to varying degrees, exhibiting the strongest sensitivity to tyramine; the reaction to metal ions is low, indicating that the PF8BT-RBI ratiometric fluorescent probe is very stable in metal salt solutions, and metal ions do not interfere with the detection of tyramine.

[0145] 2. The spiked recovery rate of the PF8BT-RBI ratiometric fluorescent probe prepared in Example 3 was used to detect the test sample.

[0146] (1) Sample collection and processing

[0147] 1) Tuna sample test solution

[0148] Refer to GB5009.208-2016. For the sample extraction solution, dissolve 12.1 g of tris(hydroxymethyl)aminomethane in 950 mL of 70% ethanol, then adjust the pH to 8.5 with hydrochloric acid, and finally bring the volume to 1000 mL with 70% ethanol.

[0149] For sample processing, dissolve tuna (4 ± 0.02 g) in a 50 mL centrifuge tube, then add 10 mL of sample extraction solution, vortex vigorously for 3 min at 4000 rpm. -1 Centrifuge for 3 minutes and retain the supernatant.

[0150] For the sample dilution solution, tris(hydroxymethyl)aminomethane (12.1 g) was dissolved in water (950 mL), then the pH was adjusted to 7.0 with hydrochloric acid, and finally the volume was brought up to 1000 mL with water.

[0151] Dissolve the sample processing solution (0.6 mL) in the sample diluent (1.4 mL) to obtain the test solution.

[0152] 2) Aquaculture water samples

[0153] The tyramine concentration in the aquaculture water was adjusted to a linear range of 0-250 μM, and the sample was obtained by filtering it through a water phase filter (0.22 μm).

[0154] (2) Sample determination

[0155] The concentration of tyramine in the cultured water sample and tuna sample was detected using the PF8BT-RBI ratiometric fluorescent probe prepared in Example 3. Since the tyramine content in the sample was not actually detected, a spiked recovery experiment was performed. Tyramine standards of 20.0 μM, 50.0 μM, and 100.0 μM were added to the cultured water sample and tuna sample, respectively. The recovery rate of the spiked tyramine in the cultured water sample and tuna sample was determined using the PF8BT-RBI ratiometric fluorescent probe. The results are shown in Table 1.

[0156] Table 1 Spike Recovery Test

[0157]

[0158] a) Standard deviation b) Relative standard deviation (RSD%) (SD / mean) × 100%

[0159] Table 1 shows that the average recoveries of the PF8BT-RBI ratiometric fluorescent probe prepared according to this invention for detecting aquaculture water samples ranged from 85.65% to 92.07%, indicating good recovery rates that are beneficial for calibrating standard detection curves. When detecting tuna samples, the average recoveries ranged from 100.75% to 115.71%, with RSDs ranging from 4.6% to 9.3%. This indicates that the accuracy of using PF8BT-RBI for determining tyramine in test samples is more acceptable.

[0160] In summary, the PF8BT-RBI ratiometric fluorescent probe of this invention exhibits good recovery, high accuracy, and high precision. This method can be used for the detection of tyramine in real samples, demonstrating superior performance in both quantitative and visual detection.

[0161] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A ratiometric fluorescent probe, characterized in that, The ratiometric fluorescent probe is prepared by modifying semiconductor polymer quantum dots with isothiocyanate dye to obtain nanoparticles, which have strong fluorescence emission peaks at 530 nm and 581 nm, respectively; wherein, the isothiocyanate dye is Rhodamine B isothiocyanate; the semiconductor polymer quantum dots are composed of polystyrene maleic anhydride copolymer and poly[(9,9-dioctylfluorenyl-2,7-diyl)-alt-(benzo[2,1,3]thiadiazole-4,8-diyl)].

2. A method for preparing the ratiometric fluorescent probe according to claim 1, characterized in that, Includes the following steps: Step 1: Mix the dimethyl sulfoxide solution of rhodamine isothiocyanate B, the tetrahydrofuran solution of polystyrene-maleic anhydride copolymer, and the tetrahydrofuran solution of poly[(9,9-dioctylfluorenyl-2,7-diyl)-alt-(benzo[2,1,3]thiadiazole-4,8-diyl)], and dilute to volume with tetrahydrofuran to obtain solution A; Step 2: Under the conditions of 0℃ ice water bath and ultrasound, the solution A is injected into ultrapure water in one go, and ultrasound is continued for 1-2 minutes. Then, gas is introduced into the solution to purge it while heating to remove tetrahydrofuran. The remaining liquid is collected, diluted with ultrapure water, filtered, and the resulting filtrate is the ratio fluorescent probe. In step 1, the mass ratio of rhodamine isothiocyanate B, polystyrene-maleic anhydride copolymer, and poly[(9,9-dioctylfluorenyl-2,7-diyl)-alt-(benzo[2,1,3]thiadiazole-4,8-diyl)] in solution A is 0.005-0.05:0.1-1:

1. In step 2, the volume ratio of solution A to ultrapure water is 2-5:10; In step 2, the temperature for removing tetrahydrofuran by purging with gas is 95-110 ℃, and the holding time is 25-30 min. The ratiometric fluorescent probe prepared by the method described above has a particle size of 10-200 nm.

3. The method for preparing the ratiometric fluorescent probe according to claim 2, characterized in that, The concentration of the dimethyl sulfoxide solution of Rhodamine B isothiocyanate was 1-2 mg / mL; and / or The tetrahydrofuran solution concentration of the polystyrene-maleic anhydride copolymer is 1-2 mg / mL; and / or The concentration of the tetrahydrofuran solution of the poly[(9,9-dioctylfluorenyl-2,7-diyl)-alt-(benzo[2,1,3]thiadiazole-4,8-diyl)] is 1-2 mg / mL.

4. The method for preparing the ratiometric fluorescent probe according to claim 3, characterized in that, The mass ratio of Rhodamine isothiocyanate B, polystyrene-maleic anhydride copolymer, and poly[(9,9-dioctylfluorenyl-2,7-diyl)-alt-(benzo[2,1,3]thiadiazole-4,8-diyl)] in solution A is 0.02:0.8:

1.

5. The method for preparing the ratiometric fluorescent probe according to claim 2, characterized in that, The remaining liquid is collected and diluted to 10 mL with ultrapure water; and / or In step 2, the gas is any one or a combination of nitrogen, argon, or helium; and / or The filtration is performed using a 0.22 μm water phase filter.

6. The ratiometric fluorescent probe of claim 1 or the ratiometric fluorescent probe prepared by any one of claims 2-5 is used in the detection of tyramine.

7. A method for detecting tyramine using a ratiometric fluorescent probe as described in claim 1, characterized in that, The ratiometric fluorescent probe was mixed with the tyramine to be tested in a certain proportion to prepare a reaction system. After reacting at 25-30 °C for 10-20 min, the system was subjected to spectral detection using a fluorescence spectrometer. Tyramine was detected by measuring the fluorescence intensity changes of the ratiometric fluorescent probe at 530 nm and 581 nm.

8. The method for detecting tyramine using a ratiometric fluorescent probe according to claim 7, characterized in that, The methods for preparing the reaction system include: Add 50 μL of ratiometric fluorescent probe, 30 μL of tyramine to be tested, and an appropriate amount of buffer to prepare a 1 mL reaction system; the buffer is PBS buffer, pH 7-7.

5.

9. The method for detecting tyramine using a ratiometric fluorescent probe according to claim 7, characterized in that, Spectroscopic detection was performed using a fluorescence spectrometer, and the fluorescence intensity I at the fluorescence emission peak of 530 nm was recorded. 530 fluorescence intensity I at 581 nm 581 Calculate I 530 / I 581 The ratio of fluorescence emission peak value is used to obtain the fluorescence emission peak value. Then, the calibration curve is obtained by using the functional relationship between the fluorescence emission peak value and the tyramine concentration. The concentration of the tyramine to be tested is calculated using the calibration curve.

10. The method for detecting tyramine using a ratiometric fluorescent probe according to any one of claims 7-9 is used for detecting tyramine in aquaculture water, aquatic products, and meat.

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