Pioneering Role of Nanopore Single-Molecule Sensing in Environmental and Food Surveillance

Abstract

In recent years, environmental and food safety have garnered substantial focus due to their intimate connection with human health. Numerous biosensors have been developed for identifying deleterious compounds; however, these biosensors reveal certain limitations. Nanopore sensors, featuring nano-scaled pore size, have demonstrated outstanding performance in terms of rapidity, sensitivity, and selectivity as a single-molecule technique for environmental and food surveillance. In this review, we present a comprehensive overview of nanopore applications in these two fields. To elucidate the pioneering roles of nanopores, analytes are categorized into three distinct groups, including metal ions, synthetic contaminants, and biotoxins. Moreover, a variety of strategies are involved, such as the coalescence with ligand probes, the implementation of chemical reactions, the functionalization of nanopores, etc. These scientific studies showcase the versatility and diversity of the nanopore technique, paving the way for further developments of nanopore technology in environmental and food safety.

Keywords: additives; metal ions; nanopores; polymers; single-molecule sensing.

Scheme 1

The schematic illustration of nanopore single-molecule detection in environmental and food surveillance employing different detection strategies.

Figure 1

Nanopore sensing of Fe³⁺ with DTPMPA and a His-tagged α-HL M113K protein pore. Structures of (a) DTPMPA and (b) His-tagged M113K α-HL protein pore with the His-tag positions highlighted as gold spheres. (c) Schematic representation of the principle of Fe³⁺ detection. DTPMPA alone in the nanopore generated one major type of long-lived event. However, in the presence of Fe³⁺, a new type of event (attributed to the DTPMPA-Fe³⁺ complex) with a different dwell time was produced [45]. Copyright © 2024 Elsevier B.V. All rights reserved.

Figure 2

Schematics of the nanopore assay used for Cu²⁺ detection and real-time click reaction monitoring [47]. Copyright © 2019 American Chemical Society.

Figure 3

(a) Schematic illustration of the proposed label-free DNAzyme-based nanopore biosensor for Pb²⁺ detection. The target Pb²⁺ can trigger the DNAzyme catalysis cleavage reaction and release ssDNA products. Copyright © The Royal Society of Chemistry 2016. (b) Schematic representation of the enzymatic reaction-based nanopore detection of metal ions; without the target metal ions, the enzyme is inactive so that the current modulations are caused only by the peptide substrate, and with the analyte in the solution, the enzyme is activated and the cleavage of the peptide substrate by its corresponding enzyme produces new types of blockage events [51,52]. Copyright © The Royal Society of Chemistry 2019.

Figure 4

Schematic illustration of (a) Th⁴⁺ and (b) UO₂²⁺ in nanopores [56,57]. Copyright © 2018 American Chemical Society and 2017 American Chemical Society.

Figure 5

Schematic of the VOC nanopore sensor. (a) Formation of a lipid bilayer holding the nanopore by the general droplet contact method. (b) Modified agarose gel-based device for VOC absorption. (c) Formation of a rigid complex of the DNA aptamer and omethoate. (d) Free DNA aptamers translocate through the nanopore smoothly, whereas (e) the omethoate–DNA aptamer complexes are clogged due to the rigid secondary structure. Conceptual image of the DNA aptamer before (i), during (ii), and after (iii) translocation through the nanopore [62]. Copyright © The Royal Society of Chemistry 2017.

Figure 6

(a) A schematic of the new nanopore sensor for detecting paraquat (PQ), in which the CP[5]A acts as the recognition element introduced into the lumen of the (E111R/K147R)₇ αHL nanopore; (b) the current reduction when CP[5]A was the sole molecule; (c) the current reduction when CP[5]A was added to the cis side and PQ was added to the trans side; (d) the enlarged view of the part of the current in (c) [64]. Copyright © the Partner Organisations 2021.

Figure 7

Schematic illustration of the experimental setup (left) and the CD-mediated nanopore assay workflow for profiling PFAS molecules (right). (Left) A nanopore system. An α-HL protein nanopore is inserted into a lipid bilayer membrane suspended across the 200 μm aperture on a Delrin wall that divides a chamber into two compartments, which are cis and trans. A command voltage is applied on the trans side, and the cis side is grounded. An integrated current amplifier is used for measuring transient current blockages caused by the translocation of sample molecules after adding the sample into the cis side. (Right) PFAS molecule profiling. The samples are achieved by incubating water possibly containing PFAS with CD, followed by the nanopore measurement of current blockage events for PFAS profiling and quantification [67]. Copyright © 2024 The Authors.

Figure 8

The detection of additives in nanopore sensors. (a) Schematic illustration of the test setup with AeL nanopores for the identification of 15 steviol glycosides, highlighting the fragments of ionic current traces associated with SGs [70]. Copyright © 2024 American Chemical Society. (b) The schematic diagram of vitamin B1 identification obtained by MspA-NTA-Ni nanopores. MspA-NTA-Ni is a hetero-octameric MspA modified with an NTA-Ni adapter at its pore constriction. Vitamin B1 reversibly binds to and dissociates from the NTA-Ni adapter, generating nanopore events. The pyrimidine moiety of vitamin B1 offers a coordination site to the bound Ni²⁺ of the NTA-Ni adapter [74]. Copyright © 2024 American Chemical Society. (c) Schematic diagram showing the interaction of the DNA–melamine hybrid with αHL nanopores [75]. Copyright © The Royal Society of Chemistry 2018.

Figure 9

The detection of biotoxin in nanopore sensors. (a) Alphatoxin nanopore detection of mycotoxins. The typical ionic current, ionic current blockage events, and dwell time histograms of mycotoxin ochratoxin A (black), aflatoxin B₁ (red), and fumonisin B₁ (blue) in alphatoxin nanopores [82]. Copyright © 2023 by the authors. (b) Illustration of the principle for nanopore BoNT-B detection. After the cleavage of Lp-Sb2(1-93) by BoNT-B/Zn²⁺ (arrow), the short digested product Sb2(77-93)d would generate a distinct modulation of pore currents, as shown by downward transient blocks in the hypothetical trace, while the long product Lp-Sb2(1-76)d would not affect the nanopore current [84]. Copyright © 2014 American Chemical Society. (c) Schematic detection of cyclic heptapeptides microcystins in αHL nanopores [85]. Copyright © 2019 American Chemical Society.