Joshi Sweccha, Segarra-Fas Anna, Peters Jeroen, Zuilhof Han, van Beek Teris A, Nielen Michel W F
Laboratory of Organic Chemistry, Wageningen University, Dreijenplein 8, 6703 HB Wageningen, The Netherlands.
RIKILT Wageningen UR, P.O. Box 230, 6700 AE Wageningen, The Netherlands.
Analyst. 2016 Feb 21;141(4):1307-18. doi: 10.1039/c5an02512e.
A 6-plex competitive inhibition immunoassay for mycotoxins in barley was developed on a prototype portable nanostructured imaging surface plasmon resonance (iSPR) instrument, also referred to as imaging nanoplasmonics. As a benchmark for the prototype nanoplasmonics instrument, first a double 3-plex assay was developed for the detection of deoxynivalenol (DON), zearalenone (ZEA), T-2 toxin (T-2), ochratoxin A (OTA), fumonisin B1 (FB1) and aflatoxin B1 (AFB1) using a well-established benchtop SPR instrument and two biosensor chips. To this end, ovalbumin (OVA) conjugates of mycotoxins were immobilized on the chip via amine coupling. The SPR response was then recorded upon injection of a mixture of antibodies at a fixed concentration and the sample (or matrix-matched standard) over a chip with immobilized mycotoxin-OVA conjugates. The chips were regenerated after each sample using 10 mM HCl and 20 mM NaOH and could be used for at least 60 cycles. The limits of detection in barley (in μg kg(-1)) were determined to be 26 for DON, 6 for ZEA, 0.6 for T-2, 3 for OTA, 2 for FB1 and 0.6 for AFB1. Preliminary in-house validation showed that DON, T-2, ZEA and FB1 can be detected at the European Union regulatory limits, while for OTA and AFB1 sensitivities should be improved. Furthermore, measurement of naturally contaminated barley showed that the assay can be used as a semi-quantitative screening method for mycotoxins prior to liquid chromatography tandem mass spectrometry (LC-MS/MS). Finally, using the same bio-reagents the assay was transferred to a 6-plex format in the nanoplasmonics instrument and subsequently the two assays were compared. Although less sensitive, the 6-plex portable iSPR assay still allowed detection of DON, T-2, ZEA and FB1 at relevant levels. Therefore, the prototype iSPR shows potential for future applications in rapid in-field and at-line screening of multiple mycotoxins.
在一台原型便携式纳米结构成像表面等离子体共振（iSPR）仪器（也称为成像纳米等离子体技术）上开发了一种用于检测大麦中霉菌毒素的6重竞争抑制免疫分析方法。作为该原型纳米等离子体仪器的基准，首先使用一台成熟的台式SPR仪器和两个生物传感器芯片开发了一种双重3重分析方法，用于检测脱氧雪腐镰刀菌烯醇（DON）、玉米赤霉烯酮（ZEA）、T-2毒素（T-2）、赭曲霉毒素A（OTA）伏马菌素B1（FB1）和黄曲霉毒素B1（AFB1）。为此，通过胺偶联将霉菌毒素的卵清蛋白（OVA）缀合物固定在芯片上。然后在固定有霉菌毒素-OVA缀合物的芯片上注入固定浓度的抗体混合物和样品（或基质匹配标准品）后记录SPR响应。每个样品使用10 mM HCl和20 mM NaOH再生芯片，并且可以使用至少60个循环。大麦中霉菌毒素的检测限（以μg kg⁻¹计）确定为：DON为26、ZEA为6、T-2为0.6、OTA为3、FB1为2、AFB1为0.6。初步的内部验证表明，DON、T-2、ZEA和FB1可以在欧盟监管限值下被检测到，而对于OTA和AFB1，灵敏度需要提高。此外，对天然污染大麦的测量表明，该分析方法可作为液相色谱串联质谱（LC-MS/MS）之前霉菌毒素的半定量筛选方法。最后，使用相同的生物试剂，将该分析方法转移到纳米等离子体仪器的6重格式中，随后对这两种分析方法进行了比较。尽管灵敏度较低，但6重便携式iSPR分析方法仍然能够检测到相关水平的DON、T-2、ZEA和FB1。因此，该原型iSPR在未来用于多种霉菌毒素的快速现场和在线筛选方面显示出潜力。
