Modular Point-of-Need Tropane Alkaloid Detection at\nRegulatory Levels: Combining Solid–Liquid Extraction from Buckwheat\nwith a Paper-Immobilized Liquid-Phase Microextraction and Immuno-Detection\nin Interconnectable 3D-Printed Devices

Contamination with\ntropane alkaloids in cereals is expected to\nincrease globally. However, current identification tools (e.g., liquid\nchromatography–mass spectrometry) for tropane alkaloids are\ntime-consuming and expensive. Furthermore, their miniaturized alternatives\nlack sensitivity and robustness. Therefore, there is a pressing need\nfor inexpensive and effective screening methods. Here, an on-site\napplicable modular workflow for tropane alkaloid detection in buckwheat\nis presented. The modular workflow combines paper microfluidics and\ninterconnectable 3D-printed sample preparation tools and was evaluated\nfor different tropane alkaloids, including atropine and scopolamine.\nFurthermore, integration with an indirect competitive lateral flow\nimmunoassay (icLFIA) for atropine detection at relevant levels was\ndemonstrated. In the modular workflow, to minimize matrix coextraction,\ntropane alkaloids were extracted from the milled buckwheat cereals\nby a mixture of alkaline aqueous and immiscible organic solvents (extraction\nrecoveries: 66–79%). The tropane alkaloids were subsequently\nconcentrated with a newly developed paper-immobilized liquid-phase\nmicroextraction (PI-LPME, extraction recoveries: 34–60%, concentration\nfactor to immobilized solution in paper: 60–108×). After\nthe PI-LPME, with an integrated 3D-printed setup, the tropane alkaloids\nwere directly eluted (elution recoveries: 83–93%) and detected\nwith the icLFIA. Digital read-out of the icLFIA, by employing a hand-held\nreader, enabled semiquantification of atropine (IC<sub>50</sub> =\n0.56 ng mL<sup>–1</sup> in standard solutions). The modular\nworkflow was validated by analyzing 24 blank and spiked buckwheat\ncereal samples with 5 and 10 μg kg<sup>–1</sup> atropine.\nA cutoff value was established with an estimated false negative rate\nof 1% and estimated false positive rate of 0.68%. Therefore, the modular\nworkflow can aid in fast, inexpensive, and on-site atropine detection\nby nonexperts, and when integrated with a scopolamine-specific icLFIA\nexpanded toward scopolamine detection. Moreover, the developed sample\nextraction and concentration method (PI-LPME) is suitable for the\nanalysis of many other compounds with pH-dependent polarity.