Ratio-type fluorescent probe and application thereof in distinguishing and detecting aflatoxin B1 and aflatoxin B2a

Abstract

The invention discloses a ratio type fluorescent probe and application thereof in distinguishing and detecting aflatoxin B1 and aflatoxin B2a, and belongs to the field of fluorescent probe detection. The probe is assembled by taking serum albumin (HSA) as a subject and dicyanoisophorone dye (MDM) as an object. Based on the combination principle difference that an MDM derivative and aflatoxin B1 tend to coexist in HSA and the MDM derivative and aflatoxin B2a tend to be replaced, the ratio-type fluorescent probe MDM-coated HSA disclosed by the invention can realize distinguished detection of B1 and B2a by utilizing spectral response sensitivity difference and fluorescence color signals. The probe can be used for quantitatively detecting aflatoxin B2a in a real food sample due to high sensitivity of B2a, a colorimetric signal generated by ratiometric fluorescence of the probe can be used for on-site qualitative rapid detection, and dual-wavelength response can be used for in-situ differentiated imaging of B1 and B2a in a biological system.

Description

Technical Field

[0001] This invention relates to the field of fluorescent probes, and more particularly to a ratiometric fluorescent probe and its application in distinguishing between aflatoxin B1 and aflatoxin B2a. Background Technology

[0002] The World Health Organization (WHO) has clearly identified mycotoxins as significant foodborne pathogens, with aflatoxin B1 (AFB1) being the most toxic and the second leading cause of cancer risk globally. If food is stored for extended periods without strict light protection, AFB1 will undergo photodegradation under ultraviolet radiation, transforming into AFB2a. This substance has a highly similar molecular structure to AFB1, and although its toxicity is lower than AFB1, it still exhibits hepatotoxicity, teratogenicity, and carcinogenicity. Currently, existing analytical methods almost entirely focus on AFB1, neglecting the assessment of AFB2a. Given the different toxicity and phototransformation characteristics of these two substances, detecting AFB1 and AFB2a in actual food samples is crucial for hazard control and disease prevention.

[0003] In recent years, fluorescence analysis has developed rapidly in the detection field due to its advantages such as high sensitivity and rapid response. Among them, ratiometric fluorescent probes, with their self-calibration function and significant fluorescence color change, are particularly suitable for naked-eye observation and can be combined with tools such as mobile phones and test strips for portable on-site detection, achieving significant progress in aflatoxin detection. For example, Chinese Patent 202410922880.1 discloses the application of a copper-gold nanocluster / carbon quantum dot ratiometric fluorescent probe in the detection of aflatoxin. Although it can achieve convenient and accurate detection of aflatoxin based on the aflatoxin fluorescence detection system of nanocluster aggregation fluorescence quenching, the degradation of aflatoxin AFB1 to AFB2a under ultraviolet light during long-term food storage, with high structural similarity, leads to false negatives or the omission of cases where both toxins coexist. Currently, whether using ratiometric fluorescence sensing or other fluorescence colorimetric strategies, it is still difficult to accurately distinguish between them when achieving rapid on-site detection or in-situ imaging. This problem has become a bottleneck in current technology. Therefore, there is an urgent need to develop ratiometric fluorescent probes that can distinguish between aflatoxin B1 and aflatoxin B2a. Summary of the Invention

[0004] In view of this, the present invention provides a ratiometric fluorescent probe to solve the problem of the difficulty in accurately distinguishing and detecting aflatoxin B1 and aflatoxin B2a.

[0005] This invention provides a ratiometric fluorescent probe assembled with serum albumin as the host and dicyanoisophorone dye as the guest.

[0006] Preferably, the dicyanoisophorone dye has the following structural formula:

[0007] .

[0008] Preferably, serum albumin is human serum albumin.

[0009] Preferably, the molar ratio of dicyanoisophorone dye to serum albumin is 1:1.

[0010] Another aspect of the present invention provides the application of a ratiometric fluorescent probe in the quantitative differentiation and detection of aflatoxin B1 and aflatoxin B2a.

[0011] Preferably, the fluorescence intensity of the ratiometric fluorescent probe is fitted to the concentration of aflatoxin B1 as y = 2.2exp(-x / 0.022) + 11.9exp(-x / 1.85) + 2.0; and the fluorescence intensity of the ratiometric fluorescent probe is fitted to the concentration of aflatoxin B2a as y = 14.2exp(-x / 0.039) + 3.3exp(-x / 0.67) + 0.066.

[0012] Another aspect of the present invention provides the application of a ratiometric fluorescent probe in the preparation of a test strip for the qualitative and rapid differentiation and detection of aflatoxin B1 and aflatoxin B2a.

[0013] Another aspect of the present invention provides an in situ imaging application of a ratiometric fluorescent probe in distinguishing and detecting aflatoxin B1 and aflatoxin B2a in biological systems.

[0014] In summary, this invention provides a ratiometric fluorescent probe, assembled from human serum albumin as the main component and dicyanoisophorone dye as the guest component, forming a supramolecular fluorescent probe. Based on the principle that MDM derivatives tend to coexist with AFB1 but tend to be substituted with AFB2a, the difference in spectral response sensitivity and fluorescence color signal can be used to distinguish and detect AFB1 and AFB2a. Therefore, the ratiometric fluorescent probe MDM@HSA of this invention can be used for quantitative detection of AFB1 and AFB2a in food samples, rapid on-site qualitative detection, and in-situ imaging differentiation of the two in biological systems (zebrafish larvae). Attached Figure Description

[0015] Figure 1 The image shows the fluorescence response of the fluorescent probe MDM@HSA prepared in Example 3 to AFB1 and AFB2a, where 1a and 1b represent the fluorescence spectra of MDM@HSA (10 μM) after the addition of different concentrations of AFB1 and AFB2a, respectively. ex = 365 nm. 1c and 1d represent I0 when different concentrations of AFB1 and AFB2a are added to the probe, respectively. 435 and I 675Values. 1e and 1f are the ratio signals (I0 and If, respectively). 675 / I 435 Standard curves for AFB1 and AFB2a concentrations.

[0016] Figure 2 The results of the selectivity and anti-interference test of the fluorescent probe MDM@HSA prepared in Example 3 against AFB1 and AFB2a are shown. 2a and 2b represent the fluorescence spectra and I values ​​of the probe solution (10 μM) with aflatoxin B1, aflatoxin B2a, citrinin, patulin, T-2 toxin, fumonisin, and ergoconazole B2 (10 μM) added, respectively. 435 / I 675 Ratio; λ ex =365 nm. 2c shows the ratio signal changes of the probe before and after the addition of AFB1 and AFB2a in the presence of different interfering substances using a bar chart. The names of the interfering substances are listed on the right, and the concentration of each interfering substance is 100 μM.

[0017] Figure 3 This document describes the on-site detection performance of the fluorescent probe MDM@HSA prepared in Example 3 and its application in the fabrication of a POCT device. 3a shows fluorescence photographs after adding different concentrations of AFB1 and AFB2a to the probe solution, using a 365nm handheld UV lamp as the light source. 3b shows the color difference (ΔE*) calculated after pairing the color patches in 3a. 3c displays a radar chart showing the changes in ΔE* values ​​caused by AFB1 and AFB2a at the test concentrations. 3d, 3e, and 3f show the fitting of AFB1 and AFB2a concentrations using ΔE*, hue, and the red-blue-grayscale ratio (R / B), respectively. 3g is a schematic diagram of the 3D printing equipment and the on-site detection effect based on the test paper.

[0018] Figure 4 The results of the application verification of the fluorescent probe MDM@HSA prepared in Example 3 in the detection of AFB2a in real food are shown. Among them, 4 c-1 to c-4 represent the change of fluorescence color of the test paper after adding AFB2a to the food extract; 4 d-1 to d-4 represent the relationship between the R / G ratio and the concentration of added AFB2a.

[0019] Figure 5 The results of applying the fluorescent probe MDM@HSA prepared in Example 3 to distinguish and detect AFB1 and AFB2a in zebrafish larvae are shown in Figure 5a. Figure 5a represents confocal fluorescence imaging of AFB1 and AFB2a in zebrafish larvae using MDM@HSA. The blue channel λ... ex The wavelength is 405 nm, and the λ of the red channel is... exThe value is 640 nm; the scale bar represents 100 μm; 5b is the merged image of each group and the image after selecting the intestinal region and subtracting the background; 5c is the integrated optical density of the three selected intestinal regions. Detailed Implementation

[0020] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0021] Human serum albumin (A9731) was purchased from Sigma-Aldrich, and aflatoxin B1 (A832707) was purchased from Maclean's. The aflatoxin B2a standard sample was provided by Guangzhou Institute of Quality Supervision and Inspection. Other chemical reagents were obtained from legitimate companies.

[0022] Example 1: MDM, a dicyanoisophorone skeleton derivative, was prepared according to the following method. Its structural formula is shown in Formula 1.

[0023] MDM: 2-(3,5,5-trimethyl-2-cyclohexene-1-yl)malononitrile (500 mg, 2.7 mmol) and 4-(diethylamino)-2-hydroxybenzaldehyde (1.04 g, 5.4 mmol) were dissolved in 15 mL of ethanol, and 2–5 drops of piperidine were added. The mixture was heated at 80°C for 12 h. The organic solvent was removed by rotary evaporation, and the resulting purple solid was purified on a silica gel column using eluent (DCM / PE) to give 334 mg of MDM, with a yield of 39%. The characterization data are as follows: 1H NMR (600 MHz, DMSO) δ 7.53 (d, J = 9.0 Hz, 1H), 7.47 (d, J = 15.9 Hz, 1H), 7.05 (d, J = 15.9 Hz, 1H), 6.64 (s, 1H), 6.26 (dd, J = 8.9, 2.1 Hz, 1H), 6.16 (d, J = 2.2 Hz, 1H), 3.36 (q, J = 7.0 Hz, 4H), 2.56 (s, 2H), 1.12 (t, J = 7.0 Hz, 6H), 1.02 (s, 6H). 13C NMR (101 MHz, DMSO) δ 169.91, 159.10, 151.06, 145.44, 135.32, 129.84, 122.87, 119.40, 115.43, 114.62, 111.48, 105.16, 97.59, 71.35, 44.43, 42.79, 38.72, 32.09, 27.93, 13.12.

[0024]

[0025] Formula 1

[0026] Example 2: Based on the dicyanoisophorone skeleton derivative MJL, it was prepared according to the following method, and its structural formula is shown in Formula 2.

[0027] MJL: 2-(3,5,5-trimethyl-2-cyclohexene-1-ethylenediamide)malonitrile (500 mg, 2.7 mmol) and 8-hydroxyjulonidine-9-carboxaldehyde (1.173 g, 5.4 mmol) were dissolved in 15 mL of ethanol, followed by the addition of 2–5 drops of piperidine. The mixture was heated at 80°C for 12 hours. The organic solvent was removed by rotary evaporation, and the resulting red solid was purified onto a silica gel column by elution with DCM / PE. 375 mg of MJL was obtained in a yield of 36%. Its 1H NMR characterization data are as follows: 1H NMR (600 MHz, d6-DMSO) δ: 8.71 (s, 1H), 7.59 (d, J = 15.6 Hz, 1H), 7.24 (s, 1H), 6.98 (d, J = 15.6 Hz, 1H), 6.64 (s, 1H), 3.25 - 3.15 (m, 4H), 2.63 (t, J = 6.2 Hz, 2H), 2.58 (d, J = 6.5 Hz, 2H), 2.56 (d, J = 13.7 Hz, 4H), 1.88 - 1.82 (m, 4H), 1.02 (s, 6H). 13C NMR (101 MHz, d6-DMSO) δ: 169.54, 158.83, 153.71, 146.60, 135.65, 125.01, 122.36, 119.05, 115.03, 112.27, 107.61, 70.45, 49.94, 49.24, 42.73, 39.97, 32.08, 27.26, 21.96, 21.56, 21.14.

[0028]

[0029] Formula 2

[0030] Example 3: A ratiometric fluorescent probe MDM@HSA was prepared by mixing a dicyanoisophorone backbone derivative MDM and human serum albumin HSA at a molar ratio of 1:1. Specifically, 2 μL of 10 mM MDM stock solution (solvent: DMSO) and 4 μL of 5 mM HSA stock solution (solvent: PBS buffer, 1 mM, pH ~7.4) were transferred into 2 mL of PBS buffer (1 mM, pH ~7.4) using a micropipette. The mixture was then vortexed (2000 rpm, 200 s) and allowed to stand for 5 min before being lyophilized for later use.

[0031] Example 4: The difference between Example 4 and Example 3 is that a ratiometric fluorescent probe is obtained by mixing dicyanoisophorone backbone derivative MJL and human serum albumin HSA in a molar ratio of 1:1 to obtain fluorescent probe powder, while the other steps are the same.

[0032] Comparative Example 1: The difference between Comparative Example 1 and Example 3 is that the same molar amount of chalcone derivative NC-3 is used instead of dicyanoisophorone skeleton derivative MDM.

[0033]

[0034] Comparative Example 2: The difference between Comparative Example 2 and Example 3 is that the triphenylamine derivative Me-TPA-P-C10 is replaced with the dicyanoisophorone skeleton derivative MDM in an equal molar amount.

[0035]

[0036] (1) Detection performance of fluorescent probes prepared in Examples 3 and 4 and Comparative Examples 1 and 2 for AFB1 and AFB2a

[0037] Isoflurane dyes MDM and MJL, as well as chalcone derivative NC-3 and triphenylamine derivative Me-TPA-P-C10, were used as guest molecules and assembled with human serum albumin to form supramolecular fluorescent probes. Their detection performance for AFB1 and AFB2a was compared. The results in Table 1 show that among the various guest molecules examined, only MDM@HSA can effectively detect and distinguish AFB1 and AFB2a based on spectral response. The other probes, after introducing the two analytes, showed similar response results within their own fluorescence emission range, exhibiting either almost unchanged fluorescence intensity or synchronous enhancement.

[0038] Table 1. Detection performance of isophorone dyes and other guest dyes on AFB1 and AFB2a.

[0039]

[0040] Note I a :I 435 fluorescence intensity at λ em The ratio of maximum fluorescence intensity. b After adding the analyte AFB1, I 435 fluorescence intensity at λ em The ratio of maximum fluorescence intensity. c After adding the analyte AFB2a, I 435 fluorescence intensity at λ em The ratio of maximum fluorescence intensity.

[0041] (2) Study the fluorescence differential response of the fluorescent probe MDM@HSA to AFB1 and AFB2a.

[0042] Several groups of lyophilized MDM@HSA powder were reconstituted into 2 mL of PBS buffer to obtain a 10 μM probe solution. 4 μL of different concentrations of AFB1 and AFB2a stock solutions were added to prepare test samples with AFB1 and AFB2a concentrations ranging from 0 to 10 μM. These samples were then vortexed (30–120 s), allowed to stand for 30 s, and then subjected to spectral detection. Figure 1 As shown, the addition of (0–10 μM) AFB1 and (0–10 μM) AFB2a to (10 μM) MDM@HSA solution resulted in new fluorescent bands appearing in the short wavelength region (400–500 nm), while the initial band of MDM@HSA (600–800 nm) showed a different response. Figure 1 a and 1 b). To visually demonstrate the fluorescence intensity at the two peak wavelengths (I... 675 and I 435 The response trends of the two analytes (AFB1, AFB2a) were analyzed using radar plots, and the following findings were made: 675 There was no significant response to increases in AFB1 concentration, with only slight fluctuations around 4000. Figure 1 The red dot in c), while the other three symbols ( Figure 1 The circles in d show a gradual change trend. Based on the sensitivity difference, MDM@HSA can effectively distinguish AFB1 and AFB2a at all test concentrations. Quantitative detection can be achieved by fitting the concentration correlation ratio signal to a standard curve. The specific fitting relationship involves standard curves that conform to the following equations: AFB1: y = 2.2exp(-x / 0.022) +11.9exp(-x / 1.85) + 2.0; AFB2a: y = 14.2exp(-x / 0.039) + 3.3exp(-x / 0.67) +0.066). Figure 1As shown in e and 1f, the limits of detection (LOD) of this probe for AFB1 and AFB2a are 20 nM (6.24 ppb) and 4.2 nM (1.3 ppb), respectively, both lower than the maximum residue limit of AFB1 (20 ppb) specified in Chinese standard GB 2761-2017 for corn and peanut-related foods, and also lower than the maximum permissible limit of AFB1 (20 ppb) set by the US Food and Drug Administration for interstate food and feed transportation. This demonstrates that the MDM@HSA probe prepared in Example 3 of this invention can produce differentiated ratio fluorescence responses to AFB1 and AFB2a, thereby enabling single-probe differentiation and detection of AFB1 and AFB2a.

[0043] (3) Study the selectivity of the fluorescent probe MDM@HSA for AFB1 and AFB2a.

[0044] like Figure 2 Results a and 2b show that MDM@HSA exhibits excellent selectivity for seven mycotoxins, including AFB2a (aflatoxin B1, aflatoxin B2a, citrinin, patulin, T-2 toxin, fumonisin, and ergoconazole B2), and also shows good selectivity for most ions (K+). + Na + Mg 2+ Ca 2+ NH 4+ HPO4 2− SO3 2− SO4 2− HCO3 − NO2 − NO3 − Amino acids (Ala, Arg, His, Lys, Trp, Pro, Ser, Thr), vitamins, and sugars all have anti-interference effects. Figure 2 c and 2 d).

[0045] (4) Application of the fluorescent probe MDM@HSA in the preparation of test strips to distinguish between AFB1 and AFB2a: When the same concentrations of the two analytes (AFB1 and AFB2a) are added, their fluorescence colors show significant differences. The color difference of MDM@HSA against AFB2a at different experimental concentration gradients (1-10 μM) is much greater than 3.5 (μM). Figure 3(a-3c) These are color changes perceptible to the naked eye for untrained personnel, while AFB1 only shows a small color difference (< 3.5) at 0.1 μM. When the two analytes are added at the same concentration, their fluorescence colors show significant differences. This phenomenon is particularly pronounced in samples containing higher concentrations (10 μM) of the analyte (AFB1 is purplish-red, AFB2a is blue). Quantitative detection can be achieved by establishing linear fitting standard curves between the RGB, Lab, and Hue-SB coordinates and the concentrations of AFB1 and AFB2a. Figure 3 The standard curve (d-3f) conforms to the following equation for linear fitting: y = a + bx. Figure 3 AFB1 in d: y = -0.1+60.7x; AFB2a: y = 30.3+60.8x. Figure 3 In equation e, AFB1: y = 385.8-39.6x; AFB2a: y = 347.4-67.1x. Therefore, 3D printing devices and laboratory-prepared test strips are used for rapid qualitative or semi-quantitative screening of AFB1 and AFB2a. Figure 3 g).

[0046] (5) Validation and application of the fluorescent probe MDM@HSA in the detection of AFB2a in food extracts: Four corn-related foods (corn kernels, corn starch, corn silk tea, and corn oil) were selected for spiked recovery experiments. Specifically, the lyophilized powder of MDM@HSA was reconstituted into 2 mL of food extract solution (solvent: PBS buffer) to obtain a test solution with a probe concentration of 10 μM. Six parallel groups were prepared, with 5 test solutions in each group. Three groups were used as standard groups, and 4 μL of AFB2a stock solution (concentrations of 0.1, 0.15, 0.2, and 0.25 mM) was added to the four test solutions in each group to obtain standard spiked samples (AFB2a concentrations of 0.2, 0.3, 0.4, and 0.5 μM). The ratio signal (IL) obtained from the fluorescence spectrum of the standard spiked samples was measured. 675 / I 435 I 435 / I 675 A standard curve for quantitative analysis can be obtained by fitting the AFB2a concentration. The other three groups are test groups. 4 μL of AFB2a stock solution (concentrations of 0.1, 0.15, and 0.25 mM) was added to each of the five samples in each group to obtain spiked test samples (AFB2a concentrations of 0.2, 0.3, and 0.5 μM). The ratio signals obtained after measuring the fluorescence spectra were substituted into the standard curve, and the corresponding AFB2a concentrations (c) were read. detectedThe calculated spiked response rate and relative standard deviation (RSD) results are shown in Table 2. The response rates of all AFB2a-spiked samples ranged from 96.4% to 110.5%, with a relative standard deviation (RSD) of less than 3.8%. This response rate range is slightly better than that obtained by liquid chromatography-mass spectrometry (70.9%-96.1%), indicating that the probe obtained in this invention has good ability to quantitatively detect AFB2a in food extracts. In addition to spectral analysis, when the test paper is immersed in a food extract containing increasing concentrations of AFB2a, it exhibits a fluorescent colorimetric signal ranging from red to purple-white and then to blue. Figure 4 c-1 to c-4). Based on the regular changes in the R / B ratio, semi-quantitative analysis of AFB2a in actual food extracts can be achieved in rapid on-site detection. Figure 4 (d-1 to d-4).

[0047] Table 2 Comparison of AFB2a content detection results in real samples using the probe of this invention and liquid chromatography-mass spectrometry (LC-MS) combined method.

[0048]

[0049] The sample preparation for LC-MS detection followed the "National Food Safety Standard - Determination of Aflatoxin B and G Substances in Food" (GB 5009.22-2016).

[0050] (6) Validation of the application of the fluorescent probe MDM@HSA in distinguishing and detecting AFB1 and AFB2a in zebrafish larvae. For example... Figure 5 As shown in Figure a, the MDM@HSA probe enables in-situ tracking of AFB1 and AFB2a in dual-channel imaging mode. When zebrafish larvae were co-cultured with AFB1 or AFB2a, respectively, fluorescence signals were observed primarily in the intestinal region via the blue channel (430-530 nm). By combining the signal changes of the red channel (600-700 nm), a differential response was achieved: in the intestinal region, the fluorescence quenching effect induced by AFB2a was significantly stronger than that of AFB1. By displaying the ratio image of the red / blue dual channels, we were able to semi-quantitatively analyze the presence of AFB1 and AFB2a (the ratio decrease for AFB1 was 1.0–1.5, while that for AFB2a was 0.06–0.08). Figure 5 A composite image of the bright field and two fluorescent channels (b) shows that AFB1 and AFB2a, two aflatoxins, aggregate in the intestinal region. Furthermore, the integrated density of selected regions in the image was measured using ImageJ software to verify the differential detection capability of the probe. Figure 5c shows that, compared to AFB2a, AFB1 induces a moderate increase (blue channel) and a decrease (red channel) in both channels. This result is consistent with the spectral test results and fully demonstrates the potential of MDM@HSA to distinguish between AFB1 and AFB2a in a living environment.

[0051] In summary, this invention provides a ratiometric fluorescent probe, assembled from human serum albumin as the host and dicyanoisophorone dye as the guest, to form a supramolecular fluorescent probe. Utilizing the difference in binding affinity between MDM@HSA and AFB1 and AFB2a, a differential red fluorescence response is generated during the competitive binding process. Simultaneously, blue light is generated based on the coumarin backbone of aflatoxin as a reference, ultimately enabling ratiometric differentiation and detection. Further molecular docking simulations also verified that the MDM derivative tends to coexist with AFB1, while tending to be replaced by AFB2a, thus achieving ratiometric differentiation detection based on the red light difference response. Therefore, the ratiometric fluorescent probe MDM@HSA of this invention can be used for quantitative detection of AFB1 and AFB2a in real food samples, rapid on-site qualitative detection, and in-situ imaging differentiation of the two in biological systems (zebrafish larvae).

Claims

1. A ratiometric fluorescent probe, characterized in that, The probe is assembled using serum albumin as the host and dicyanoisophorone dye as the guest.

2. The ratiometric fluorescent probe according to claim 1, characterized in that, The structural formula of the dicyanoisophorone dye is: 。 3. The ratiometric fluorescent probe according to claim 2, characterized in that, The serum albumin is human serum albumin.

4. The ratiometric fluorescent probe according to claim 3, characterized in that, The molar ratio of the dicyanoisophorone dye to serum albumin is 1:

1.

5. The application of the ratiometric fluorescent probe according to any one of claims 1 to 4 in the quantitative differentiation and detection of aflatoxin B1 and aflatoxin B2a.

6. The application of the ratiometric fluorescent probe according to claim 5 in the quantitative differentiation and detection of aflatoxin B1 and aflatoxin B2a, characterized in that, The fitting relationship between the fluorescence intensity of the ratiometric fluorescent probe and the concentration of aflatoxin B1 is y = 2.2exp(-x / 0.022) + 11.9exp(-x / 1.85) + 2.0; and the fitting relationship between the fluorescence intensity of the ratiometric fluorescent probe and the concentration of aflatoxin B2a is y = 14.2exp(-x / 0.039) + 3.3exp(-x / 0.67) + 0.

066.

7. The application of the ratiometric fluorescent probe according to any one of claims 1 to 4 in the preparation of a test strip for the qualitative and rapid differentiation and detection of aflatoxin B1 and aflatoxin B2a.

8. The ratiometric fluorescent probe according to any one of claims 1 to 4 is used for in situ imaging in biological systems to differentiate and detect aflatoxin B1 and aflatoxin B2a.

Images