College of Life Sciences, YanTai University, Yantai 264005, China; Key Laboratory of Agro-product Quality and Food Safety, Institute of Quality Standard &Testing Technology for Agro-Products, Chinses Academy of Agricultural Science, Beijing 100081, China.
Key Laboratory of Agro-product Quality and Food Safety, Institute of Quality Standard &Testing Technology for Agro-Products, Chinses Academy of Agricultural Science, Beijing 100081, China.
Sci Total Environ. 2020 May 15;717:137268. doi: 10.1016/j.scitotenv.2020.137268. Epub 2020 Feb 11.
Although the toxicity of triazophos is high and it has been pulled from the market in many countries; it is still widely used and frequently detected in agricultural products. While conventional analyses have been routinely used for the quantification and monitoring of triazophos residues, those for detecting low residual levels are deemed necessary. Therefore, we developed a novel and sensitive fluorometric signal amplification immunoassay employing bio-barcodes for the quantitative analysis of triazophos residues in foodstuffs and surface water. Herein, monoclonal antibodies (mAbs) attached to gold nanoparticles (AuNPs) were coated with DNA oligonucleotides (used as a signal generator), and a complementary fluorogenic RNA was used for signal amplification. The system generated detection signals through DNA-RNA hybridization and subsequent dissociation of fluorophores by Ribonuclease H (RNase H). It has to be noted that RNase H can only disintegrate the RNA in DNA-RNA duplex, but not cleave single or double-stranded DNA. Hence, with iterative cycles of DNA-RNA hybridization, sufficient strong signal was obtained for reliable detection of residues. Furthermore, this method enables quantitative detection of triazophos residues through fluorescence intensity measurements. The competitive immunoassay shows a wide linear range of 0.01-100 ng/mL with a limit of detection (LOD) of 0.0032 ng/mL. The assay substantially meets the demand for the low residue detection of triazophos residues in agricultural products and water samples. Accuracy (expressed as spiked recovery %) and coefficient of variation (CV) were ranged from 73.4% to 116% and 7.04% to 17.4%, respectively. The proposed bio-barcodes immunoassay has the advantages of being stable, reproducible, and reliable for residue detection. In sum, the present study provides a novel approach for detection of small molecules in various sample matrices.
尽管三唑磷毒性高，且已在许多国家被撤出市场，但它仍被广泛使用，并经常在农产品中被检测到。虽然常规分析已被常规用于三唑磷残留的定量和监测，但检测低残留水平的分析方法是必要的。因此，我们开发了一种新的基于生物条码的灵敏荧光信号放大免疫分析方法，用于定量分析食品和地表水的三唑磷残留。在此，金纳米粒子（AuNPs）上连接的单克隆抗体（mAbs）被 DNA 寡核苷酸覆盖（用作信号发生器），并使用互补的荧光 RNA 进行信号放大。该系统通过 DNA-RNA 杂交产生检测信号，随后核糖核酸酶 H（RNase H）将荧光团解离。需要注意的是，RNase H 只能将 RNA 从 DNA-RNA 双链体中分解，但不能切割单链或双链 DNA。因此，通过 DNA-RNA 杂交的迭代循环，可以获得足够强的信号，从而可靠地检测残留物。此外，该方法可以通过荧光强度测量来定量检测三唑磷残留。竞争免疫分析显示出 0.01-100ng/mL 的宽线性范围，检测限（LOD）为 0.0032ng/mL。该方法基本满足了农产品和水样中三唑磷残留低残留检测的需求。准确度（表示为加标回收率%）和变异系数（CV）分别在 73.4%至 116%和 7.04%至 17.4%的范围内。所提出的生物条码免疫分析具有稳定、可重现和可靠的残留检测优点。总之，本研究为各种样品基质中小分子的检测提供了一种新方法。
