Homogeneous immunoassay for cyclopiazonic acid based upon mimotopes and upconversion-resonance energy transfer.

Strains of Penicillium spp. are used for fungi-ripened cheeses and Aspergillus spp. routinely contaminate maize and other crops. Some of these strains can produce toxic secondary metabolites (mycotoxins), including the neurotoxin α-cyclopiazonic acid (CPA). In this work, we developed a homogeneous upconversion-resonance energy transfer (UC-RET) immunoassay for the detection of CPA using a novel epitope mimicking peptide, or mimotope, selected by phage display. CPA-specific antibody was used to isolate mimotopes from a cyclic 7-mer peptide library in consecutive selection rounds. Enrichment of antibody binding phages was achieved, and the analysis of individual phage clones revealed four different mimotope peptide sequences. The mimotope sequence, ACNWWDLTLC, performed best in phage-based immunoassays, surface plasmon resonance binding analyses, and UC-RET-based immunoassays. To develop a homogeneous assay, upconversion nanoparticles (UCNP, type NaYF4:Yb3+, Er3+) were used as energy donors and coated with streptavidin to anchor the synthetic biotinylated mimotope. Alexa Fluor 555, used as an energy acceptor, was conjugated to the anti-CPA antibody fragment. The homogeneous single-step immunoassay could detect CPA in just 5 min and enabled a limit of detection (LOD) of 30 pg mL-1 (1.5 µg kg-1) and an IC50 value of 0.36 ng mL-1. No significant cross-reactivity was observed with other co-produced mycotoxins. Finally, we applied the novel method for the detection of CPA in spiked maize samples using high-performance liquid chromatography coupled to a diode array detector (HPLC-DAD) as a reference method.

Biosensing Tacrolimus in Human Whole Blood by Using a Drug Receptor Fused to the Emerald Green Fluorescent Protein.

Tacrolimus (FK506) is an immunosuppressant drug (ISD) used to prevent organ rejection after transplantation that exhibits a narrow therapeutic window and is subject to wide inter- and intra-individual pharmacokinetic fluctuations requiring careful monitoring. The immunosuppressive capacity of FK506 arises from the formation of a complex with immunophilin FKBP1A. This paper describes the use of FKBP1A as an alternative to common antibodies for biosensing purposes. Bioassays use recombinant FKBP1A fused to the emerald green fluorescent protein (FKBP1A-EmGFP). Samples containing the immunosuppressant are incubated with the recombinant protein, and free FKBP1A-EmGFP is captured by magnetic beads functionalized with FK506 to generate a fluorescence signal. Recombinant receptor-drug interaction is evaluated by using a quartz crystal microbalance and nuclear magnetic resonance. The limit of detection (3 ng mL-1) and dynamic range thus obtained (5-70 ng mL-1) fulfill therapeutic requirements. The assay is selective for other ISD usually coadministered with FK506 and allows the drug to be determined in human whole blood samples from organ transplant patients with results comparing favorably with those of an external laboratory.

Molecular super-gluing: a straightforward tool for antibody labelling and its application to mycotoxin biosensing.

Mycotoxins are low molecular weight toxic compounds, which can cause severe health problems in animals and humans. Immunoassays allow rapid, simple and cost-effective screening of mycotoxins. Sandwich assays with a direct readout provide great improvement in terms of selectivity and sensitivity, compared to the widely used competitive assay formats, for the analysis of low molecular weight molecules. In this work, we report a non-competitive fluorescence anti-immune complex (IC) immunoassay, based on the specific recognition of HT-2 toxin with a pair of recombinant antibody fragments, namely antigen-binding fragment (Fab) (anti-HT-2 (10) Fab) and single-chain variable fragment (scFv) (anti-IC HT-2 (10) scFv). The SpyTag and SpyCatcher glue proteins were applied for the first time as a bioconjugation tool for the analysis of mycotoxins. To this aim, a SpyTag-mScarlet-I (fluorescent protein) and scFv-SpyCatcher fusion proteins were constructed, produced and fused in situ during the assay by spontaneous Tag-Catcher binding. The assay showed an excellent sensitivity with an EC50 of 4.8 ± 0.4 ng mL-1 and a dynamic range from 1.7 ± 0.3 to 13 ± 2 ng mL-1, an inter-day reproducibility of 8.5% and a high selectivity towards HT-2 toxin without cross-reactivity with other Fusarium toxins. The bioassay was applied to the analysis of the toxin in an oat reference material and in oat samples, with a LOD of 0.6 µg kg-1, and the results were validated by analysing a certificate reference material and by HPLC-MS/MS.

Recombinant antibodies and their use for food immunoanalysis.

Antibodies are widely employed as biorecognition elements for the detection of a plethora of compounds including food and environmental contaminants, biomarkers, or illicit drugs. They are also applied in therapeutics for the treatment of several disorders. Recent recommendations from the EU on animal protection and the replacement of animal-derived antibodies by non-animal-derived ones have raised a great controversy in the scientific community. The application of recombinant antibodies is expected to achieve a high growth rate in the years to come thanks to their versatility and beneficial characteristics in comparison to monoclonal and polyclonal antibodies, such as stability in harsh conditions, small size, relatively low production costs, and batch-to-batch reproducibility. This review describes the characteristics, advantages, and disadvantages of recombinant antibodies including antigen-binding fragments (Fab), single-chain fragment variable (scFv), and single-domain antibodies (VHH) and their application in food analysis with especial emphasis on the analysis of biotoxins, antibiotics, pesticides, and foodborne pathogens. Although the wide application of recombinant antibodies has been hampered by a number of challenges, this review demonstrates their potential for the sensitive, selective, and rapid detection of food contaminants.

Recombinant Peptide Mimetic NanoLuc Tracer for Sensitive Immunodetection of Mycophenolic Acid.

Mycophenolic acid (MPA) is an immunosuppressant drug commonly used to prevent organ rejection in transplanted patients. MPA monitoring is of great interest due to its small therapeutic window. In this work, a phage-displayed peptide library was used to select cyclic peptides that bind to the MPA-specific recombinant antibody fragment (Fab) and mimic the behavior of MPA. After biopanning, several phage-displayed peptides were isolated and tested to confirm their epitope-mimicking nature in phage-based competitive immunoassays. After identifying the best MPA mimetic (ACEGLYAHWC with a disulfide constrained loop), several immunoassay approaches were tested, and a recombinant fusion protein containing the peptide sequence with a bioluminescent enzyme, NanoLuc, was developed. The recombinant fusion enabled its direct use as the tracer in competitive immunoassays without the need for secondary antibodies or further labeling. A bioluminescent sensor, using streptavidin-coupled magnetic beads for the immobilization of the biotinylated Fab antibody, enabled the detection of MPA with a detection limit of 0.26 ng mL-1 and an IC50 of 2.9 ± 0.5 ng mL-1. The biosensor showed good selectivity toward MPA and was applied to the analysis of the immunosuppressive drug in clinical samples, of both healthy and MPA-treated patients, followed by validation by liquid chromatography coupled to diode array detection.

Biosensing based on upconversion nanoparticles for food quality and safety applications.

Food safety and quality regulations inevitably call for sensitive and accurate analytical methods to detect harmful contaminants in food and to ensure safe food for the consumer. Both novel and well-established biorecognition elements, together with different transduction schemes, enable the simple and rapid analysis of various food contaminants. Upconversion nanoparticles (UCNPs) are inorganic nanocrystals that convert near-infrared light into shorter wavelength emission. This unique photophysical feature, along with narrow emission bandwidths and large anti-Stokes shift, render UCNPs excellent optical labels for biosensing because they can be detected without optical background interferences from the sample matrix. In this review, we show how this exciting technique has evolved into biosensing platforms for food quality and safety monitoring and highlight recent applications in the field.

Competitive upconversion-linked immunoassay using peptide mimetics for the detection of the mycotoxin zearalenone.

Due to increasing food safety standards, the analysis of mycotoxins has become essential in the food industry. In this work, we have developed a competitive upconversion-linked immunosorbent assay (ULISA) for the analysis of zearalenone (ZEA), one of the most frequently encountered mycotoxins in food worldwide. Instead of a toxin-conjugate conventionally used in competitive immunoassays, we designed a ZEA mimicking peptide extended by a biotin-linker and confirmed its excellent suitability to mimic ZEA by nuclear magnetic resonance (NMR) and surface plasmon resonance (SPR) analysis. Upconversion nanoparticles (UCNP, type NaYF4:Yb,Tm) served as background-free optical label for the detection of the peptide mimetic in the competitive ULISA. Streptavidin-conjugated UCNPs were prepared by click reaction using an alkyne-PEG-neridronate linker. The UCNP conjugate clearly outperformed conventional labels such as enzymes or fluorescent dyes. With a limit of detection of 20 pg mL-1 (63 pM), the competitive ULISA is well applicable to the detection of ZEA at the levels set by the European legislation. Moreover, the ULISA is specific for ZEA and its metabolites (α- and ß-zearalenol) without significant cross-reactivity with other related mycotoxins. We detected ZEA in spiked and naturally contaminated maize samples using liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) as a reference method to demonstrate food analysis in real samples.

Hierarchically Imprinted Polymer for Peptide Tag Recognition Based on an Oriented Surface Epitope Approach.

FLAG tag (DYKDDDDK) is a short peptide commonly used for the purification of recombinant proteins. The high price of the affinity columns and their limited reusability are a shortcoming for their widespread use in biotechnology applications. Molecularly imprinted polymers (MIPs) can circumvent some of the limitations of bioaffinity columns for such applications, including long-term stability, reusability, and cost. We report herein the synthesis of MIPs selective to the FLAG tag by hierarchical imprinting. Using the epitope imprinting approach, a 5-amino acid peptide DYKDC was selected as a template and was covalently immobilized on the surface of microporous silica beads, previously functionalized with different aminosilanes, namely, 3-(2-aminoethylamino)propyldimethoxymethylsilane, AEAPMS, and N-(2-aminoethyl)-2,2,4-trimethyl-1-aza-2-silacyclopentane, AETAZS. We investigated the effect of the type of silane on the production of homogeneous silane-grafted layers with the highest extent of silanol condensation as possible using 29Si CP/MAS NMR. We observed that the right orientation of the imprinted cavities can substantially improve analyte recoveries from the MIP. After template and silica removal, the DYKDC-MIPs were used as sorbents for solid-phase extraction (molecularly imprinted solid-phase extraction) of the FLAG peptide, showing that the polymer prepared with AETAZS-bound silica beads contained binding sites more selective to the tag (RMIP-AZA = 87.4% vs RNIP-AZA = 4.1%, n = 3, RSD ≤ 4.2%) than those prepared using AEAPMS (RMIP-DM = 73.4% vs RNIP-DM = 23.2%, n = 3, RSD ≤ 4.0%) as a functionalization agent. An extensive computational molecular modeling study was also conducted, shedding some light on the interaction mechanism between the FLAG peptide and the imprinted template in the binding cavities.

Bioluminescent detection of zearalenone using recombinant peptidomimetic Gaussia luciferase fusion protein.

The development of a bioluminescent immunosensor is reported for the determination of zearalenone (ZEA) based on a peptide mimetic identified by phage display. The peptide mimetic GW, with a peptide sequence GWWGPYGEIELL, was used to create recombinant fusion proteins with the bioluminescent Gaussia luciferase (GLuc) that were directly used as tracers for toxin detection in a competitive immunoassay without the need for secondary antibodies or further labeling. The bioluminescent sensor, based on protein G-coupled magnetic beads for antibody immobilization, enabled determination of ZEA with a detection limit of 4.2 ng mL-1 (corresponding to 420 µg kg-1 in food samples) and an IC50 value of 11.0 ng mL-1. The sensor performance was evaluated in spiked maize and wheat samples, with recoveries ranging from 87 to 106% (RSD < 20%, n = 3). Finally, the developed method was applied to the analysis of a naturally contaminated reference matrix material and good agreement with the reported concentrations was obtained.Graphical abstract.

Phage Display in the Quest for New Selective Recognition Elements for Biosensors.

Phages are bacterial viruses that have gained a significant role in biotechnology owing to their widely studied biology and many advantageous characteristics. Perhaps the best-known application of phages is phage display that refers to the expression of foreign peptides or proteins outside the phage virion as a fusion with one of the phage coat proteins. In 2018, one half of the Nobel prize in chemistry was awarded jointly to George P. Smith and Sir Gregory P. Winter "for the phage display of peptides and antibodies." The outstanding technology has evolved and developed considerably since its first description in 1985, and today phage display is commonly used in a wide variety of disciplines, including drug discovery, enzyme optimization, biomolecular interaction studies, as well as biosensor development. A cornerstone of all biosensors, regardless of the sensor platform or transduction scheme used, is a sensitive and selective bioreceptor, or a recognition element, that can provide specific binding to the target analyte. Many environmentally or pharmacologically interesting target analytes might not have naturally appropriate binding partners for biosensor development, but phage display can facilitate the production of novel receptors beyond known biomolecular interactions, or against toxic or nonimmunogenic targets, making the technology a valuable tool in the quest of new recognition elements for biosensor development.

Development and comparison of mimotope-based immunoassays for the analysis of fumonisin B1.

Mycotoxins can be found as natural contaminants in many foods and feeds, and owing to their toxic effects, it is essential to detect them before they enter the food chain. An interesting approach for the analysis of mycotoxins by competitive immunoassays is the use of epitope-mimicking peptides, or mimotopes, which can replace the toxin conjugates traditionally used in such assays. Mimotopes can be selected from phage-displayed peptide libraries even without any prior knowledge of the antibody-antigen interaction, and after identifying the target specific clones, individual clones can be efficiently amplified in bacteria and used directly in the immunoassay. Following such approach, we have previously selected and identified a dodecapeptide which functions as a mimotope for the mycotoxin fumonisin B1. In this work, we present the development and comparison of various immunoassays based on this mimotope, named A2, which has been used in the phage-displayed format in which it was selected, but also as a fluorescent recombinant fusion protein or as a synthetic peptide. The highest sensitivity was obtained with a magnetic bead-based assay using the synthetic peptide and enzymatic detection which provided a detection limit of 0.029 ng mL-1. Analysis of the binding kinetics by surface plasmon resonance (SPR) further reinforced the suitability of the synthetic peptide for the competitive immunoassays, as this mimotope showed a slightly lower affinity for the target antibody in comparison with the recombinant fusion protein. Graphical abstract.

Tag-Specific Affinity Purification of Recombinant Proteins by Using Molecularly Imprinted Polymers.

Epitope tagging is widely used to fuse a known epitope to proteins for which no affinity receptor is available by using recombinant DNA technology. One example is FLAG epitope (DYKDDDDK), which provides better purity and recoveries than the favorite poly histidine tag. However, purification requires using anti-FLAG antibody resins, the high cost and nonreusability of which restrict widespread use. One cost-effective solution is provided by the use of bioinspired anti-FLAG molecularly imprinted polymers (MIPs). This work describes the development of MIPs, based on the epitope approach, synthesized from the tetrapeptide DYKD as template that affords purification of FLAG-derived recombinant proteins. Polymer was optimized by using a combinatorial approach to select the functional monomer(s) and cross-linker(s), resulting in the best specific affinity toward FLAG and the peptide DYKD. The imprinted resin obtained was used to purify mCherry proteins tagged with either FLAG or DYKD epitopes from crude cell lysates. Both mCherry variants were highly efficiently purified ( R ≥ 95%, RSD ≤ 15%, n = 3) and impurities were removed. Unlike existing antibody-based resins, the proposed tag-imprinting strategy provides a general method for meeting the growing demand for efficient, inexpensive, and versatile materials for tagged proteins purification.

Homogeneous Quenching Immunoassay for Fumonisin B1 Based on Gold Nanoparticles and an Epitope-Mimicking Yellow Fluorescent Protein.

Homogeneous immunoassays represent an attractive alternative to traditional heterogeneous assays due to their simplicity, sensitivity, and speed. On the basis of a previously identified epitope-mimicking peptide, or mimotope, we developed a homogeneous fluorescence quenching immunoassay based on gold nanoparticles (AuNPs) and a recombinant epitope-mimicking fusion protein for the detection of mycotoxin fumonisin B1 (FB1). The fumonisin mimotope was cloned as a fusion protein with a yellow fluorescent protein that could be used directly as the tracer for FB1 detection without the need of labeling or a secondary antibody. Furthermore, owing to the fluorescence quenching ability of AuNPs, a homogeneous immunoassay could be performed in a single step without washing steps to separate the unbound tracer. The homogeneous quenching assay showed negligible matrix effects in 5% wheat extract and high sensitivity for FB1 detection, with a dynamic range from 7.3 to 22.6 ng mL-1, a detection limit of 1.1 ng mL-1, and IC50 value of 12.9 ng mL-1, which was significantly lower than the IC50 value of the previously reported assay using the synthetic counterpart of the same mimotope in a microarray format. The homogeneous assay was demonstrated to be specific for fumonisins B1 and B2, as no significant cross-reactivity with other mycotoxins was observed, and acceptable recoveries (86% for FB1 2000 µg kg-1 and 103% for FB1 4000 µg kg-1), with relative standard deviation less than 6.5%, were reported from spiked wheat samples, proving that the method could provide a valuable tool for simple analysis of mycotoxin-contaminated food samples.

Optical Biosensors for Label-Free Detection of Small Molecules.

Label-free optical biosensors are an intriguing option for the analyses of many analytes, as they offer several advantages such as high sensitivity, direct and real-time measurement in addition to multiplexing capabilities. However, development of label-free optical biosensors for small molecules can be challenging as most of them are not naturally chromogenic or fluorescent, and in some cases, the sensor response is related to the size of the analyte. To overcome some of the limitations associated with the analysis of biologically, pharmacologically, or environmentally relevant compounds of low molecular weight, recent advances in the field have improved the detection of these analytes using outstanding methodology, instrumentation, recognition elements, or immobilization strategies. In this review, we aim to introduce some of the latest developments in the field of label-free optical biosensors with the focus on applications with novel innovations to overcome the challenges related to small molecule detection. Optical label-free methods with different transduction schemes, including evanescent wave and optical fiber sensors, surface plasmon resonance, surface-enhanced Raman spectroscopy, and interferometry, using various biorecognition elements, such as antibodies, aptamers, enzymes, and bioinspired molecularly imprinted polymers, are reviewed.