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Rapid detection method special for pesticide residues of dichlorvos in fresh fruits and vegetables
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A technology for dichlorvos and pesticide residues, applied in the field of rapid detection of pesticide residues, which can solve problems such as difficult special detection
Pending Publication Date: 2021-12-17
DALIAN UNIV OF TECH
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Organophosphorus and carbamate pesticides have a certain inhibitory ability to AChE enzymes. This kind of detection method based on the principle of enzyme inhibition is still difficult to realize the special detection of dichlorvos pesticide residues in fruits and vegetables.
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Embodiment 1
[0027] Embodiment 1: Synthesis of fluorescent probe substrate formula-1
[0030] Synthesis of intermediate b: Weigh 2.00g fluorescein a, 1.71gCs 2 CO 3 , add 10mL DMF to dissolve, stir at room temperature, add dropwise 0.75g (0.75mL) CH 3 I. After the reaction, 5% sodiumhydroxide solution was added, extracted with DCM, the organic phase was washed with saturated brine, and the solvent was evaporated to obtain 1.55 g of intermediate b, with a yield of 71.4%. 1 HNMR (500MHz, CDCl 3 ) δ 8.24 (d, J =7.9Hz,1H),7.74(t, J =7.2Hz,1H),7.67(t, J =7.6Hz,1H),7.31(d, J =7.5Hz,1H),6.97(d, J =2.3Hz,1H),6.87(dd, J =18.1,9.3Hz,1H),6.74(dd, J =8.9,2.3Hz,1H),6.54(d, J =9.7Hz, 1H), 6.47(s, 1H), 3.92(s, 2H), 3.64(s, 1H).
[0031] Synthesis of intermediate c: take intermediate b0.800g, add 20mLCH 3 OH, 5mL of 10% NaOH solution, stirred at room temperature for 6h for hydrolysis. After methanol was distilled off, it was neutralized with 10 mL (3...
Embodiment 2
[0033] Embodiment 2: Extraction method of pesticide residues in fruits and vegetables:
[0034] For the edible parts of vegetables and fruits, weigh 0.3g of the sample into a 2mL centrifuge tube, add 1.5mL of extract (ethyl acetate / acetone=9:1), and extract in a vibrating mixer for 5min. After centrifugation, take 100 μL of the supernatant to a 2 mL centrifuge tube and blow dry. Add 0.6mL LPBS (100mM, pH = 7.4) to dissolve, to be tested.
[0036] 1. Take 6µL of the 0.6mL extractant to be tested (or PBS solution spiked with pesticides, or PBS solution) prepared in the pretreatment, and then dilute 100 times with PBS (100mM, pH=7.4) to 600µL;
[0037] 2. Add 5 µL of enzyme agent (extracted from duck blood, AChE protein content 2.3 mg / ml) to 300 µL of the diluted extract, and mix in a constant temperature mixer at 37 o C, pre-incubation reaction 7min;
[0038] 3. Take 300 µL of the pre-incubation solution, add it to a cuvette, and then add 30 µL of DMSO solution containing a fluorescent probe (not specifically referred to as the fluorescent probe of Formula-1), and put it in a constant temperature mixer at 37 o C, shock reaction for 12 minutes;
[0039] 4. Add 200µL Terminator B to the cuvette, mix well, and test;
[0040] 5. Start the fluorescencedetector and link with the mobile APP; according to the different fluorescent probes, set the corre...
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Abstract
The invention discloses a rapid detection method special for pesticide residues of dichlorvos in fresh fruits and vegetables, and belongs to the technical field of rapid detection of pesticide residues. Based on the specific inhibition effect of organophosphorus pesticides and carbamate pesticides on the activity of acetylcholin esterase (AChE, derived from duck blood extraction), the inhibition degree of AChE is detected by using an ultrahigh-sensitivity fluorescein fluorescent probe. When the addition amount of the dichlorvos in the detection system is the same, the weakened fluorescence intensity (I dichlorvos) of the dichlorvos is 100 times or more lower than that of other pesticides (I other pesticides), thereby showing that even if the content of the dichlorvos is 100 times lower than that of other pesticides, the residual quantity of the dichlorvos can be quantitatively detected. Through diluting a pesticide residue extraction liquid by 100 times, the influence of other pesticide residues on AChE is reduced, the pesticide residue range of specific detection of dichlorvos in fruits and vegetables is 0.02-1.2 mg / kg, the error rate R2 of a detection standard curve is greater than 0.99, and the standard additionrecovery rate of the fresh fruits and vegetables reaches 71-86%.
Description
technical field [0001] The invention belongs to the field of rapid detection of pesticide residues, and in particular relates to an ultrahigh-sensitive fluorescent probe substrate of AChE enzyme and an application thereof. Background technique [0002] Dichlorvos (DDVP, dichlorvos) is a kind of organophosphorus insecticide. It has a wide insecticidal spectrum and rapid drug effect. It has stomach poisoning, contact killing and fumigation effects. A, Armyworm, Cabbage Spodoptera, Spodoptera litura, Red Spider, Great Ape Leafworm, etc., have strong and fast knockdown power against pests, and are often used to control pests when vegetables and fruits are approaching harvest. If dichlorvos is used near the time of fruit and vegetable picking, or if it is used in excessive amounts, it is easy to cause excessive residues and cause harm to the human body. In recent years, the market supervision department has reported that dichlorvos residues in apples, pears, celery, tomatoes and...
Claims
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