Recent advances in nanopore-based analysis for carbohydrates and glycoconjugates.

Saccharides are not only the basic constituents and nutrients of living organisms, but also participate in various life activities, and play important roles in cell recognition, immune regulation, development, cancer, etc. The analysis of carbohydrates and glycoconjugates is a necessary means to study their transformations and physiological roles in living organisms. Existing detection techniques can hardly meet the requirements for the analysis of carbohydrates and glycoconjugates in complex matrices as they are expensive, involve complex derivatization, and are time-consuming. Nanopore sensing technology, which is amplification-free and label-free, and is a high-throughput process, provides a new solution for the identification and sequencing of carbohydrates and glycoconjugates. This review highlights recent advances in novel nanopore-based single-molecule sensing technologies for the detection of carbohydrates and glycoconjugates and discusses the advantages and challenges of nanopore sensing technologies. Finally, current issues and future perspectives are discussed with the aim of improving the performance of nanopores in complex media diagnostic applications, as well as providing a new direction for the quantification of glycan chains and the study of glycan chain properties and functions.

Recent advances of nanopore technique in single cell analysis.

Single cells and their dynamic behavior are closely related to biological research. Monitoring their dynamic behavior is of great significance for disease prevention. How to achieve rapid and non-destructive monitoring of single cells is a major issue that needs to be solved urgently. As an emerging technology, nanopores have been proven to enable non-destructive and label-free detection of single cells. The structural properties of nanopores enable a high degree of sensitivity and accuracy during analysis. In this article, we summarize and classify the different types of solid-state nanopores that can be used for single-cell detection and illustrate their specific applications depending on the size of the analyte. In addition, their research progress in material transport and microenvironment monitoring is also highlighted. Finally, a brief summary of existing research challenges and future trends in nanopore single-cell analysis is tentatively provided.

Emerging Nanoparticles in Food: Sources, Application, and Safety.

Nanoparticles (NPs) are small-sized, with high surface activity and antibacterial and antioxidant properties. As a result, some NPs are used as functional ingredients in food additives, food packaging materials, nutrient delivery, nanopesticides, animal feeds, and fertilizers to improve the bioavailability, quality, and performance complement or upgrade. However, the widespread use of NPs in the industry increases the exposure risk of NPs to humans due to their migration from the environment to food. Nevertheless, some NPs, such as carbon dots, NPs found in various thermally processed foods, are also naturally produced from the food during food processing. Given their excellent ability to penetrate biopermeable barriers, the potential safety hazards of NPs on human health have attracted increased attention. Herein, three emerging NPs are introduced including carbon-based NPs (e.g., CNTs), nanoselenium NPs (SeNPs), and rare earth oxide NPs (e.g., CeO2 NPs). In addition, their applications in the food industry, absorption pathways into the human body, and potential risk mechanisms are discussed. Challenges and prospects for the use of NPs in food are also proposed.

Facile synthesis of boric acid-functionalized magnetic covalent organic frameworks and application to magnetic solid-phase extraction of trace endocrine disrupting compounds from meat samples.

A facile approach was proposed for the preparation of boric acid-functionalized core-shell structured magnetic covalent organic framework (COF) nanocomposites through employing the Fe3O4 nanoparticles as magnetic core, boric acid-functionalized COFs as the shell via sequential post-synthetic modification (denoted as Fe3O4@COF@BA). The synthesized nanocomposites showed large specific surface area, high magnetic responsiveness, and desirable chemical and thermal stability. Combined with HPLC-MS/MS, the as-prepared Fe3O4@COF@BA composite was applied as a sorbent for magnetic solid-phase extraction (MSPE) of endocrine disrupting compounds (EDCs) from meat samples. Under optimal conditions, the method displays low limits of detection (LODs, 0.08-0.72 µg kg-1) and good precision with relative standard deviations (RSD) lower than 5.4 %. The approach was successfully employed for the extraction and detection of EDCs in blank and spiked beef, chicken and pork samples with recovery ranging from 88.8 to 104.2 %.

Artificial clickase-triggered fluorescence "turn on" based on a click bio-conjugation strategy for the immunoassay of food allergenic protein.

Herein, based on an artificial clickase-catalyzed bio-conjugation strategy, we established a sensitive fluorescent clickase-linked immunosorbent assay (FCLISA) platform using an oligonucleotide-molecular beacon (Oligo-MB) hairpin structure as a fluorescence switch for detection of food allergenic protein. Firstly, a highly stable Cu(I)-containing nanocube was prepared for usage as an artificial clickase, which could catalyze the bio-conjugation of two short oligonucleotides (i.e., Oligo-A and Oligo-B labeled by a 5'-alkyne and a 3'-azide group, respectively) through clickase-catalyzed azide/alkyne cycloaddition reaction. Subsequently, the formed long-chain oligonucleotide (Oligo-A-B) could hybridize with Oligo-MB hairpin to open hairpin structure, leading to its fluorescence turn on. By using clickase as an alternative enzymatic label in conventional ELISAs, the established FCLISA showed high sensitivity and accuracy in detection of casein, achieving a limit of detection as low as 1.5 × 10-8 g/mL. Additionally, FCLISA has been challenged by detecting the casein in real samples, indicating a great potential in food safety assay.

High-efficiency enzyme-free catalyzed hairpin assembly-mediated homogeneous SERS and naked-eyes dual-mode assay for ultrasensitive and portable detection of mycotoxin.

We developed an aptamer recognition-trigged enzyme-free catalyzed hairpin assembly (CHA) assisted signal amplification homogeneous naked-eyes and surface-enhanced Raman scattering (SERS) dual-mode sensor for highly sensitive and portable detection of Aflatoxin B1 (AFB1) in food samples. The recognition of AFB1 by aptamer induced the generation of HP1-AFB1 complexes, which hybridized with Ag+-labeled hairpin DNA (HP2) and released Ag+, subsequently initiating the enzyme-free CHA reaction by a designed helper DNA (HP3) to form double-stranded DNA (HP2-HP3) and accompanied by the release of HP1-AFB1 complexes. The released HP1-AFB1 complexes were recognized by HP2 and HP3 again to trigger cascade recycling amplification and resulted in the generation of a larger number of free Ag+ and dsDNA. Then, methylene blue as Raman tag to intercalate into dsDNA and generating strong SERS signal with the assistance of Fe3O4@Au. Meanwhile, the free Ag+ induced the AuNPs aggregation and resulted in naked-eye distinguishable color transitions from red to black blue. Benefitting from the efficiently enzyme-free CHA assisted signal amplification and portably dual-mode detection system, this work successfully proposed a novel homogeneous biosensing strategy for highly sensitive and portable detection of AFB1. The SERS intensity and visualization signals were linearly correlated with the concentration of AFB1 ranging from 0.0156 to 31.2 ng mL-1 and 0.61-39 ng mL-1, and the limit of detections were 1.6 pg mL-1 and 152 pg mL-1, respectively. This strategy was successfully applied to real samples and provided an alternative approach for the highly sensitive detection of mycotoxins.

An aptamer-assisted biological nanopore biosensor for ultra-sensitive detection of ochratoxin A with a portable single-molecule measuring instrument.

Biological nanopore-based single-molecule detection technology has shown ultrahigh sensitivity to various target analyte. But the detection scope of interesting targets is limited due to the lack of effective signal conversion strategies. In addition, conventional nanopore detection instruments are cumbersome, resulting nanopore detection can only be performed in laboratory. Herein, a customizable nanopore current amplifier is constructed to lower the cost and increase the portability of the nanopore instrument, and then an immobilized aptamer-based signal conversion strategy is proposed for α-hemolysin (α-HL) nanopore to detect small molecules (ochratoxin A, OTA). The presence of OTA in sample would trigger the release of probe single-strand DNA (ssDNA) from magnetic beads, which could subsequently cause current blockage in nanopore. The results show that the signal frequency of probe ssDNA has a linear relationship with the OTA concentration in the range of 2 × 101～2 × 103 pmol/L. Compared to other methods, our sensing system has achieved an ultra-sensitive detection of OTA with the detection limit as low as 1.697 pmol/L. This strategy could broaden the scope of nanopore detection and have the potential for rapid and in-situ detection of other food contaminants in the future.

Low background interference SERS aptasensor for highly sensitive multiplex mycotoxin detection based on polystyrene microspheres-mediated controlled release of Raman reporters.

Mycotoxin contamination is a serious global issue in food safety. The accurate detection of mycotoxins in complex samples, particularly via a portable detection system that realizes multi-analyte detection, remains a great challenge. Here, a polystyrene (PS)-mediated surface-enhanced Raman scattering (SERS) aptasensor was designed for ultrasensitive and simultaneous detection of multiple mycotoxins in food samples. First, the novel Raman tags were prepared using PS as nanocontainer to encapsulate abundant Raman reporters. The PS nanocontainer with Raman reporters was then functionalized by aptamer as an effective target-recognition platform. Second, the single-strand binding protein modified magnetic nanoparticles (MNPs@SSB) were used as a magnetic capture substrate. When the target mycotoxin was present and recognized by its aptamer, the combination of PS-Apt and mycotoxins can hinder the binding between PS-Apt and MNPs@SSB. Consequently, after the magnetic separation and treatment by THF solution, fewer biological-silent reporter probes were released from PS, causing the reduction of SERS signal as a function of target mycotoxins concentration. Due to the high loading capacity of PS and the excellent signal conversion and amplification of aptamer-assisted SERS assay, the proposed strategy is reliable and ultrasensitive for the determination of multiple mycotoxins. The limit of detection was as low as 0.159 fg L-1, 2.015 fg L-1, and 1.561 fg L-1 for ZEN, OTA and AFB1, respectively. This strategy not only raises a new idea for designing novel Raman tag, but also broadens the application of SERS for multiple detection of trace target.

Recent advances of sensing strategies for the detection of ß-glucuronidase activity.

ß-Glucuronidase (ß-GLU), a kind of hydrolase, is widely distributed in mammalian tissues, body fluids, and microbiota. Abnormal changes of ß-GLU activity are often correlated with the occurrence of diseases and deterioration of water quality. Therefore, detection of ß-GLU activity is of great significance in biomedicine and environmental health such as cancer diagnosis and water monitoring. However, the conventional ß-GLU activity assay suffers from the limitations of low sensitivity, poor accuracy, and complex procedure. With the development of analytical chemistry, many advances have been made in the detection of ß-GLU activity in recent years. The sensors for ß-GLU activity detection which have the advantages of rapid and reliable detection have been attracting increased attentions. In this paper, the principles, performances, and limitations of these ß-GLU sensors, including colorimetric sensing, fluorescent sensing, electrochemical sensing for the determination of ß-GLU activity, have been summarized and discussed. Moreover, the challenges and research trends of ß-GLU activity assay are proposed.

Recent Progress on Single-Molecule Detection Technologies for Food Safety.

Rapid and sensitive detection technologies for food contaminants play vital roles in food safety. Due to the complexity of the food matrix and the trace amount distribution, traditional methods often suffer from unsatisfying accuracy, sensitivity, or specificity. In past decades, single-molecule detection (SMD) has emerged as a way to realize the rapid and ultrasensitive measurement with low sample consumption, showing a great potential in food contaminants detection. For instance, based on the nanopore technique, simple and effective methods for single-molecule analysis of food contaminants have been developed. To our knowledge, there has been a rare review that focuses on SMD techniques for food safety. The present review attempts to cover some typical SMD methods in food safety, including electrochemistry, optical spectrum, and atom force microscopy. Then, recent applications of these techniques for detecting food contaminants such as biotoxins, pesticides, heavy metals, and illegal additives are reviewed. Finally, existing research challenges and future trends of SMD in food safety are also tentatively proposed.

Bio-inspired Nanoenzyme Synthesis and Its Application in A Portable Immunoassay for Food Allergy Proteins.

Nanozymes as a cost-effective and robust enzyme mimic have attracted widespread attention in the development of novel analytical methods. Herein, a new nanozyme-enhanced surface-enhanced Raman scattering (SERS) immunoassay platform was successfully developed using a peroxidase-mimicking nanozyme to replace the natural enzymes as a catalytic label of the enzyme-linked immunosorbent assay for the detection of allergy proteins. In this platform, the peroxidase-mimicking nanozymes as a catalytic label could catalyze the oxidation of the Raman-inactive reporter [i.e., leucomalachite green (LMG)] to generate Raman-active malachite green (MG) with H2O2. Moreover, the produced MG Raman signal was further enhanced by the formed Raman "hot spot" through MG-induced gold nanoparticle aggregation, which could be recorded by a portable Raman spectrometer. On this basis, the established nanozyme-enhanced SERS immunoassay showed improved accuracy, high sensitivity, and good selectivity and was used for accurate quantification of α-lactalbumin (α-LA). With this method, α-LA could be detected with a limit of detection as low as 0.01 ng/mL. Moreover, the method was also verified by performing in food samples and showed satisfactory recoveries and high reliability. This study not only provides insight into the use of a nanozyme to establish new analytical methods but also broadens the applications of nanozymes in a food safety assay.

Crowding-Induced DNA Translocation through a Protein Nanopore.

A crowded cellular environment is highly associated with many significant biological processes. However, the effect of molecular crowding on the translocation behavior of DNA through a pore has not been explored. Here, we use nanopore single-molecule analytical technique to quantify the thermodynamics and kinetics of DNA transport under heterogeneous cosolute PEGs. The results demonstrate that the frequency of the translocation event exhibits a nonmonotonic dependence on the crowding agent size, while both the event frequency and translocation time increase monotonically with increasing crowder concentration. In the presence of PEGs, the rate of DNA capture into the nanopore elevates 118.27-fold, and at the same time the translocation velocity decreases from 20 to 120 µs/base. Interestingly, the impact of PEG 4k on the DNA-nanopore interaction is the most notable, with up to ΔΔG = 16.27 kJ mol-1 change in free energy and 764.50-fold increase in the binding constant at concentration of 40% (w/v). The molecular crowding effect will has broad applications in nanopore biosensing and nanopore DNA sequencing in which the strategy to capture analyte and to control the transport is urgently required.

Hybridization chain reaction (HCR) for amplifying nanopore signals.

Circulating tumor DNA (ctDNA) in the blood is an important biomarker for noninvasive diagnosis, assessment, prediction and treatment of cancer. However, sensing performance of solid nanopore is limited by the fast kinetics of small DNA targets and unmatched dimensions. Here, we combines hybridization chain reaction (HCR) with nanopore detection to translate the presence of a small DNA target to characteristic nanopore signals of a long nicked DNA polymer. The amplification of nanopore signals obtained by HCR not only overcomes the functional limitation of solid nanopore, but also significantly elevates both selectivity and signal-to-noise ratio, which allows to detect ctDNA at a detection limit of 2.8 fM (S/N = 3) and the single-base resolution. Furthermore, the proposed method can apply in detection of ctDNA of KRAS G12DM in serum sample.

Simultaneous High-Resolution Detection of Bioenergetic Molecules using Biomimetic-Receptor Nanopore.

A novel artificial receptor, heptakis-[6-deoxy-6-(2-hydroxy-3-trimethylammonion-propyl) amino]-beta-cyclomaltoheptaose, with similar functions of mitochondrial ADP/ATP carrier protein, was synthesized and harbored in the engineered α-HL (M113R)7 nanopore, forming a single-molecule biosensor for sensing bioenergetic molecules and their transformations. The strategy significantly elevates both selectivity and signal-to-noise, which enables simultaneous recognition and detection of ATP, ADP, and AMP by real-time single-molecule measurement.

Single-molecule transformation and analysis of glutathione oxidized and reduced in nanopore.

The determination of glutathione reduced (GSH) or oxidized (GSSG) in bulk solution has been reported previously. However, it is critically important to set up a simple and label-free method to recognize GSSG and GSH selectively and dynamically, especially at a single-molecule level. Here we report a novel nanopore method to recognize GSSG based on a newly synthesized per-6-quaternary ammonium-ß-cyclodextrin (p-QABCD), which is used as both the molecular adaptor of protein nanopore and the recognizing element of GSSG. Distinct current signature is observed upon GSSG binding in a mutant protein nanopore (M113R RL2)7 equipped with p-QABCD, while there is no signal for GSH. Thus GSSG in the mixture can be selectively detected in the concentration range of 6.00-90.0µM. Furthermore, the conversion between GSH and GSSG both in bulk solution and in nanochannel can be continuously monitored in real time and in situ. The label-free method provides a possibility to investigate enzymatic activity as well as its activators or inhibitors related to the transformation between GSH and GSSG.