Artificial biomimetic receptors, such as aptamers and molecular-imprinted polymers, show antibody-like properties which are due to molecular recognition phenomena characterized by high affinity and ...selectivity. These binding features have made them suitable in all those application fields in which selective recognition is required. Thus, it is not surprising that they are finding applications in affinity CE as well. Recently, a variety of ACE methods have shown themselves to be suitable tools to provide a detailed quantitative characterization of the thermodynamic and kinetic aspects of binding. At the same time, affinity CE can exploit the peculiarities of these binding interactions to set up CE-based analytical tools for the separation and the determination of specific target molecules in microscale formats. This review will provide a detailed description of affinity CE methods recently reported in the literature and related to these topics.
Patulin is a water-soluble mycotoxin produced by several species of fungi. Governmental bodies have placed it under scrutiny for its potential negative health effects, and maximum residue limits are ...fixed in specific food matrices to protect consumers' health. Confirmatory analysis of patulin in complex food matrices can be a difficult task, and sample clean-up treatments are frequently necessary before instrumental analyses. With the aim of simplifying the clean-up step, we prepared a 256-member combinatorial polymeric library based on 16 functional monomers, four cross-linkers and four different porogenic solvents. The library was screened for the binding towards patulin in different media (acetonitrile and citrate buffer at pH 3.2), with the goal of identifying polymer formulations with good binding properties towards the target compound. As a proof of concept, a methacrylic acid-co-pentaerithrytole tetraacrylate polymer prepared in chloroform was successfully used as a solid-phase extraction material for the clean-up and extraction of patulin from apple juice. Clean chromatographic patterns and acceptable recoveries were obtained for juice spiked with patulin at concentration levels of 25 (64 ± 12%), 50 (83 ± 5.6%) and 100 μg L
(76 ± 4.5%). The within-day and between-day reproducibility evaluated at a concentration level of 25 μg L
were 5.6 and 7.6%, respectively.
Molecularly imprinted polymers, MIPs, are man-made receptors mimicking the thermodynamic and kinetic binding behaviour of natural antibodies. Therefore, it is not surprising that many researchers ...have thought about MIPs as artificial receptors in immunoassay-like analytical applications, where the general machinery of the assay is maintained, but the molecular recognition is no longer assured by an antibody but by an artificial receptor. However, the number of papers devoted explicitly to applications of MIPs in the immunoassay field is quite limited if compared to the huge number of papers covering the multifaceted molecular imprinting technology. For this reason, this critical review wants to give a general view of MIP-based immunoassays, trying to highlight the critical points that have so far prevented a wider application of molecular imprinting technology in the immunoassay field and, possibly, try to suggest strategies to overcome them.
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•MIPs can mimic natural antibodies in immunoassay.•Innovative approaches have improved the binding properties of MIPs.•MIP-based immunoassays have passed by the proof-of-the-concept level to practical applications.•Several issues related to the development of robust assays still remain to be explored.
A multiplex Lateral Flow Immunoassay was developed based on the use of a single Test line and multicolour gold nanoparticles (GNPs) as signal reporters. Red and blue GNPs were linked to antibodies ...directed towards two different analytes and included in a typical lateral flow immunoassay configuration, in which the Test line was formed by the mixture of two antigens. As a result of the immunoreactions occurring at the Test zone, diverse combinations of red and blue GNPs labels were captured. Therefore, the Test line assumed different colours depending on which – and how much – analyte is present in the sample. The multiplexing capability of the ‘colour-encoded assay’ is illustrated by the simultaneous detection of aflatoxin B1 (AFB1) and type-B fumonisins (FMs) in wheat and food products that made with wheat. Reproducible detection of AFB1 and FMs contamination in raw and processed food was achieved with visual cut-off levels at 1 ng mL−1 and 50 ng mL−1, respectively. The contaminant was identified based on the colour of the label according with a specific colour code. Furthermore, strips images were acquired by means of a common smartphone and analysed through RGB data analysis providing semi-quantitative detection of the two mycotoxins.
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•A novel lateral flow assay using dual colour gold nanoparticles is described.•Aflatoxins and fumonisins were simultaneously detected in cereal-based food.•A single Test line was fabricated that was sensitive to both mycotoxins.•A colour code allowed for detecting mycotoxins and discriminating among the two Smartphone-based detection provided quantification through RGB data analysis.
To accurately diagnose COVID-19 infection and its time-dependent progression, the rapid, sensitive, and noninvasive determination of immunoglobulins A specific to SARS-CoV-2 (IgA) in saliva and serum ...is needed to complement tests that detect immunoglobulins G and M. We have developed a dual optical/chemiluminescence format of a lateral flow immunoassay (LFIA) immunosensor for IgA in serum and saliva. A recombinant nucleocapsid antigen specifically captures SARS-CoV-2 antibodies in patient specimens. A labelled anti-human IgA reveals the bound IgA fraction. A dual colorimetric and chemiluminescence detection enables the affordable and ultrasensitive determination of IgA to SARS-CoV-2. Specifically, a simple smartphone-camera-based device measures the colour signal provided by nanogold-labelled anti-human IgA. For the ultrasensitive chemiluminescence transduction, we used a contact imaging portable device based on cooled CCD, and measured the light signal resulting from the reaction of the HRP-labelled anti-human IgA with a H2O2/luminol/enhancers substrate. A total of 25 serum and 9 saliva samples from infected and/or recovered individuals were analysed by the colorimetric LFIA, which was sensitive and reproducible enough for the semi-quantification of IgA in subjects with a strong serological response and in the early stage of COVID-19 infection. Switching to CL detection, the same immunosensor exhibited higher detection capability, revealing the presence of salivary IgA in infected individuals. For the patients included in the study (n = 4), the level of salivary IgA correlated with the time elapsed from diagnosis and with the severity of the disease. This IgA-LFIA immunosensor could be useful for noninvasively monitoring early immune responses to COVID-19 and for investigating the diagnostic/prognostic utility of salivary IgA in the context of large-scale screening to assess the efficacy of SARS-CoV-2 vaccines.
•A LFIA for the point-of-care detection of IgA specific to SARS-CoV2 in serum and saliva was developed.•GNP and HRP-mediated optical/chemiluminescence signals were detected by a smartphone CMOS and a portable CCD, respectively.•The one-step colorimetric and two-step CL IgA-LFIA enabled the detection of anti-SARS-CoV-2 IgA in 15 min.•Salivary IgA were identified in four COVID-19 patients by the CL IgA-LFIA after two weeks from diagnosis.
Food contamination from natural or anthropogenic sources poses severe risks to human health. It is now largely accepted that continuous exposure to low doses of toxic chemicals can be related to ...several chronic diseases, including some type of cancer and serious hormonal dysfunctions.
Contemporary analytical methods have the sensitivity required for contamination detection and quantification, but direct application of these methods on food samples can be rarely performed. In fact, the matrix introduces severe disturbances, and analysis can be performed only after some clean-up and preconcentration steps. Current sample pre-treatment methods, mostly based on the solid phase extraction technique, are very fast and inexpensive but show a lack of selectivity, while methods based on immunoaffinity extraction are very selective but expensive and not suitable for harsh environments. Thus, inexpensive, rapid and selective clean-up methods, relaying on “intelligent” materials are needed. Recent years have seen a significant increase of the “molecularly imprinted solid phase extraction” (MISPE) technique in the food contaminant analysis. In fact, this technique seems to be particularly suitable for extractive applications where analyte selectivity in the presence of very complex and structured matrices represents the main problem. In this review, several applications of MISPE in food contamination analysis will be discussed, with particular emphasis on the extraction of pesticides, drugs residua, mycotoxins and environmental contaminants.
Stable and efficient conjugates between antibodies and gold nanoparticles (GNP-Ab) are sought to develop highly sensitive and robust biosensors with applications in medicine, toxicology, food safety ...controls, and targeted drug delivery. Several strategies have been proposed for directing the antibody attachment to GNPs thus preserving antibody activity, including covalently coupling the antibody to a polymer grafted on GNP surface and exploiting the high affinity of bioreceptors as mediators for the binding. Both approaches also allow for shielding GNPs with a protective layer that guarantees the robustness of the conjugate. Notwithstanding, antibodies freely adsorb to GNP with high binding efficiency. The nonspecific adsorption is far more simple, fast, and inexpensive than any mediated coupling. Therefore, it is preferred for most applications, although it is considered to produce GNP-Ab with a limited activity. In this work, we compared three strategies for producing GNP-Ab, such as (i) covalent coupling mediated by a chemical layer, (ii) affinity-based binding mediated by a biomolecular layer composed of Staphylococcal protein A, and (iii) direct attachment via adsorption. The so-prepared GNP-Ab were employed as probes in a colorimetric lateral flow immunoassay (LFIA) for measuring salivary cortisol as a model biosensor that relies on the use of active GNP-Ab conjugates. Unexpectedly, the biosensors fabricated using the three probes were completely comparable in terms of their ability to measure salivary cortisol. Furthermore, we observed that the sensitivity of the LFIA primarily depended on the amount of the antibody bound to GNPs rather than on the method by which it was bound. The probes prepared using both the direct adsorption approach and mediated coupling via the biochemical mediator enabled development of point-of-care devices for the fast, sensitive, and reliable measurement of human salivary cortisol.
Immunoassays have gained considerable attention in safety assurance for food, feed and agricultural products. Generally, immunoassays are presented either in a competitive or non-competitive, ...sandwich-type format, and the former is extensively employed for low-molecular-weight contaminants, which usually bear one accessible epitope. Theoretically, non-competitive, sandwich-type immunoassays have higher sensitivity, precision and linearity. However, the analyte to be measured in such a format must be large enough to have at least two epitopes to be captured. It is not feasible to detect low-molecular-weight contaminants through conventional non-competitive sandwich-type immunoassay. Consequently, there is a trend to develop new types of sensitive non-competitive immunoassays for low-molecular-weight contaminants.
This article reviews the progress in non-competitive immunoassays for low molecular weight contaminants in food, feed and agricultural products, including the principles, applications and suggested perspectives for this field.
Anti-metatype antibody-based immunoassays are the most promising method, but dissociation of the antibody-hapten complex might be a challenge, and therefore more in-depth research should be focused on preparation of new formats of the antibody-hapten complex. Meanwhile, strategies for direct non-competitive detection or aimed at the simultaneous detection of different targets would be especially desirable besides focusing on improving the sensitivity and specificity of the detection.
•Non-competitive immunoassays are favored due to sensitivity, specificity and linearity.•Use of non-competitive immunoassays to detect small molecules in Agri-food products.•Future trends to detect low-molecular-weight hazards by non-competitive immunoassays.
A high affinity and selectivity DNA aptamer for aflatoxin B
(AFB
) was designed through Genetic Algorithm (GA) based in silico maturation (ISM) strategy. The sequence of a known AFB
aptamer (Patent: ...PCT/CA2010/001292, Apt1) applied as a probe in many aptasensors was modified using seven GA rounds to generate an initial library and three different generations of ss DNA oligonucleotides as new candidate aptamers. Molecular docking methodology was used to screen and analyze the best aptamer-AFB
complexes. Also, a new pipeline was proposed to faithfully predict the tertiary structure of all single stranded DNA sequences. By the second generation, aptamer Apt1 sequence was optimized in the local search space and five aptamers including F20, g12, C52, C32 and H1 were identified as the best aptamers for AFB
. The selected aptamers were applied as probes in an unmodified gold nanoparticles-based aptasensor to evaluate their binding affinity to AFB
and their selectivity against other mycotoxins (aflatoxins B
, G
, G
, M
, ochratoxin A and zearalenone). In addition, a novel direct fluorescent anisotropy aptamer assay was developed to confirm the binding interaction of the selected aptamers over AFB
The ISM allowed the identification of an aptamer, F20, with up to 9.4 and 2 fold improvement in affinity and selectivity compared to the parent aptamer, respectively.
Exposure to mycotoxins, which may contaminate food and feed commodities, represents a serious health risk for consumers. Ochratoxin A (OTA) is one of the most abundant and toxic mycotoxins, thus ...specific regulations for fixing its maximum admissible levels in foodstuff have been established. Lateral Flow ImmunoAssay (LFIA)-based devices have been proposed as screening tools to avoid OTA contamination along the whole food chain. We report a portable, user-friendly smartphone-based biosensor for the detection and quantification of OTA in wine and instant coffee, which combines the LFIA approach with chemiluminescence (CL) detection. The device employs the smartphone camera as a light detector and uses low-cost, disposable analytical cartridges containing the LFIA strip and all the necessary reagents. The analysis can be carried out at the point of need by non-specialized operators through simple manual operations. The biosensor allows OTA quantitative detection in wine and coffee samples up to 25 μg L−1 and with limits of detection of 0.3 and 0.1 μg L−1, respectively, which are below the European law-fixed limits. These results demonstrate that the developed device can be used for routine monitoring of OTA contamination, enabling rapid and reliable identification of positive samples requiring confirmatory analysis.
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•Smartphone-based biosensor developed by chemiluminescence Lateral Flow Immunoassay.•Ochratoxin A quantitative detection below EU law limits in wine and coffee samples.•Ready-to-use self-contained analytical cartridge for rapid on-field application.•The biosensor enables screening for OTA contamination along the whole food chain.
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