Imprinted proteins for determination of ovalbumin.

A strategy to design imprinted proteins (IPs) able to detect a glycoprotein (ovalbumin, OVA) was proposed. Glucose oxidase (GOx) was used as a matrix for obtaining the binding cavities with high specificity towards the template protein. The effective method to purify the obtained IPs from the template molecules was developed based on a combination of dialysis and gel filtration. HRP was chosen as a model template to discover the optimal production conditions, and the optimal template concentration (100 µg⋅L-1) was chosen. The obtained imprinted proteins were characterized by the high adsorption selectivity towards the target protein (the imprinting factor towards OVA and HRP is 4.7). The developed method was transferred to the synthesis of the anti-OVA IPs. The binding properties of these IPs were estimated using the OVA conjugates with low- (FITC) and high- (HRP) molecular weight label molecules. The ability of the synthesized anti-OVA IPs as recognition elements in immunoassay was studied. Under the optimized experimental conditions, the proposed imprinted proteins exhibited a good linear response to OVA in the concentration range of 10-2000 ng⋅mL-1 with a detection limit of 6 ng⋅mL-1. The obtained recognition elements were tested for OVA determination in real samples of chicken egg white, and a sample of OVA-free cake spiked by OVA.

A novel electrochemical sensor for the detection of fipronil and its toxic metabolite fipronil sulfone using TiO2-polytriazine imide submicrostructured composite as an efficient electrocatalyst.

For the first time, a simple and sensitive electrochemical sensor based on a screen printed electrode (SPE) modified with titanium dioxide (TiO2) and polytriazine imide submicrostructured composite (TiO2-PTI) has been developed for the simultaneous detection of fipronil (FIP) and its toxic metabolite fipronil sulfone (FIP-S). The submicrostructured composite material based on TiO2 and PTI was obtained by simple hydrothermal treatment of the Ti peroxocomplexes in the presence of pristine. This carbon nitride allotrope has better crystallinity and conductivity than its graphitic analog. It was found that the TiO2-PTI submicrostructured composite enhanced the electrochemical sensing of the SPE electrode towards FIP and its metabolite FIP-S in 0.1 M Britton-Robinson buffer (pH 10) at the oxidation potentials of 0.82 V and 0.94 V, respectively. In addition, it showed good stability and reproducibility for the determination of both analytes. Under optimal conditions, the peak currents by square wave voltammetry were found to vary linearly with FIP and FIP-S concentrations in the range from 0.01 to 10 µM and from 10 to 50 µM, with a detection limit of 8.42 nM, 3.6 µg/kg for FIP and 9.72 nM, 4.04 µg/kg for FIP-S. This sensor was successfully used to detect FIP and FIP-S in eggs and water samples with good recoveries of 90%-106.6%.

Structural and biochemical insights into CRISPR RNA processing by the Cas5c ribonuclease SMU1763 from Streptococcus mutans.

The cariogenic pathogen Streptococcus mutans contains two CRISPR systems (type I-C and type II-A) with the Cas5c protein (SmuCas5c) involved in processing of long CRISPR RNA transcripts (pre-crRNA) containing repeats and spacers to mature crRNA guides. In this study, we determined the crystal structure of SmuCas5c at a resolution of 1.72 Å, which revealed the presence of an N-terminal modified RNA recognition motif and a C-terminal twisted ß-sheet domain with four bound sulphate molecules. Analysis of surface charge and residue conservation of the SmuCas5c structure suggested the location of an RNA-binding site in a shallow groove formed by the RNA recognition motif domain with several conserved positively charged residues (Arg39, Lys52, Arg109, Arg127, and Arg134). Purified SmuCas5c exhibited metal-independent ribonuclease activity against single-stranded pre-CRISPR RNAs containing a stem-loop structure with a seven-nucleotide stem and a pentaloop. We found SmuCas5c cleaves substrate RNA within the repeat sequence at a single cleavage site located at the 3'-base of the stem but shows significant tolerance to substrate sequence variations downstream of the cleavage site. Structure-based mutational analysis revealed that the conserved residues Tyr50, Lys120, and His121 comprise the SmuCas5c catalytic residues. In addition, site-directed mutagenesis of positively charged residues Lys52, Arg109, and Arg134 located near the catalytic triad had strong negative effects on the RNase activity of this protein, suggesting that these residues are involved in RNA binding. Taken together, our results reveal functional diversity of Cas5c ribonucleases and provide further insight into the molecular mechanisms of substrate selectivity and activity of these enzymes.

Recent advances in electrochemical monitoring of zearalenone in diverse matrices.

The current manuscript summarizes different electrochemical sensing systems developed within the last 5 years for the detection of zearalenone (ZEN) in diverse matrices such as food, feed, and biofluids. ZEN is one of the most prevalent non-steroidal mycotoxins that is often found in pre- and post-harvest crops. Crops contamination with ZEN and animal exposure to it via contaminated feed, is a global health and economic concern. The European Union has established various preventive programs to control ZEN contamination, and regulations on the maximum levels of ZEN in food and feed. Electrochemical (bio)sensors are a very promising alternative to sensitive but sophisticated and expensive chromatographic techniques. In the current review, recent developments towards electrochemical sensing of ZEN, sorted by type of transducer, their design, development, and approbation/validation are discussed, and the use of specialized electrochemical instrumentation is highlighted.

Homogenous FRET-based fluorescent immunoassay for deoxynivalenol detection by controlling the distance of donor-acceptor couple.

Semiconductor quantum dots (QDs) are one of the most popular luminescent labels that are widely used in food and medical analysis. Their unique optical properties establish QDs as excellent tools for highly sensitive biosensors based on Förster resonance energy transfer (FRET). To provide a convenient analytical system with long-term optical stability, a FRET pair consisting of QDs as energy donor and gold nanoparticles (GNs) as energy acceptor was developed. Careful selection of donor and acceptor properties allowed to achieve a large Förster distance of 12.9 nm and to use full-size specific antibody. As the immunoreagents pair, mycotoxins were bound to proteins and then to GNs, while QDs were conjugated with specific antibodies. FRET was observed as a result of the immunocomplex formation. Contributions of FRET and inner filter effect on the quenching were evaluated separately. The quenching effect in the donor-acceptor pair was compared for proteins with different sizes. The developed homogeneous FRET-based immunoassay for the detection of deoxynivalenol (DON) is an example of a fast method for high-throughput control of mycotoxins. The quenching effect of FRET was observed with a limit of detection of 28 µg kg-1 of DON in spiked wheat samples.

Novel multiplex capacitive sensor based on molecularly imprinted polymers: A promising tool for tracing specific amphetamine synthesis markers in sewage water.

The development of a sensing system for amphetamine (AMP), N-formyl amphetamine (NFA), and benzyl methyl ketone (BMK) in sewage is a strict requirement for enabling the on-site detection and tracing of the consumption of AMP, and the production and/or transportation of these target analytes. The present research is therefore devoted to the development of an on-site capacitive sensing system, based on molecularly imprinted polymers (MIPs) as recognition elements. To this end, the commercially available CapSenze capacitive sensor system was miniaturized by implementing an application-specific integrated circuit (ASIC), dedicated to the bias and read-out of the chemical sensor. MIPs towards AMP were purchased, whereas the ones towards NFA and BMK were synthesized in house. Gold transducers, consisting of six working electrodes with their corresponding reference electrodes and one common auxiliary electrode, were designed together with a flow cell to enable analyses. The applied water samples were filtered through a 20 micron filter before application in the sensors' flow cell. The limits of detection in filtered sewage water were determined to be 25 µM for NFA and BMK and 50 µM for AMP. The overall performance of the sensing system was tested by analysis of blind-coded sewage samples, provided by legal authorities. To the best of our knowledge, this is the first research presenting multiplex MIP-based detection of amphetamine synthesis markers using a capacitive sensor, miniaturized via ASIC technology. The presented technique is undoubtedly a potential solution for any analysis requiring constant reliable on-site monitoring of a substance of interest.

Capacitive sensing of an amphetamine drug precursor in aqueous samples: Application of novel molecularly imprinted polymers for benzyl methyl ketone detection.

Highly selective molecularly imprinted polymers (MIPs) towards benzyl methyl ketone (BMK) were synthesized for application as recognition elements in a capacitive sensor. A computational approach was employed to select the most appropriate monomers and cross-linkers. Using the selected compounds, different polymerization techniques and protocols were compared in order to study the effect on the MIP performance and characteristics. MIPs synthesized by bulk polymerization using itaconic acid and 1-vinylimidazole as monomers and p-divinylbenzene as cross-linker possess the highest affinity towards the target analyte. Prior to capacitive analysis, the developed particles were immobilized on the surface of gold transducers using tyramine as a linker. The validity of the developed sensor was checked by the BMK detection in spiked tap water and real water samples. A linear working range from 50 to 1000 µM was found while the limit of detection (LOD) was determined to be 1 µM in tap water. To the best of our knowledge, both the developed MIPs towards BMK and the electrochemical sensor for its detection have not been published or marketed to date.

Detection of toxins involved in foodborne diseases caused by Gram-positive bacteria.

Bacterial toxins are food safety hazards causing about 10% of all reported foodborne outbreaks in Europe. Pertinent to Gram-positive pathogens, the most relevant toxins are emetic toxin and diarrheal enterotoxins of Bacillus cereus, neurotoxins of Clostridium botulinum, enterotoxin of Clostridium perfringens, and a family of enterotoxins produced by Staphylococcus aureus and some other staphylococci. These toxins are the most important virulence factors of respective foodborne pathogens and a primary cause of the related foodborne diseases. They are proteins or peptides that differ from each other in their size, structure, toxicity, toxicological end points, solubility, and stability, types of food matrix to which they are mostly related to. These differences influence the characteristics of required detection methods. Therefore, detection of these toxins in food samples, or detection of toxin production capacity in the bacterial isolate, remains one of the cornerstones of microbial food analysis and an essential tool in understanding the relevant properties of these toxins. Advanced research has led into new insights of the incidence of toxins, mechanisms of their production, their physicochemical properties, and their toxicological mode of action and dose-response profile. This review focuses on biological, immunological, mass spectrometry, and molecular assays as the most commonly used detection and quantification methods for toxins of B. cereus, C. botulinum, C. perfringens, and S. aureus. Gathered and analyzed information provides a comprehensive blueprint of the existing knowledge on the principles of these assays, their application in food safety, limits of detection and quantification, matrices in which they are applicable, and type of information they provide to the user.

Recent developments in electrochemical detection of illicit drugs in diverse matrices.

Drug abuse is a global problem, requiring an interdisciplinary approach. Discovery, production, trafficking, and consumption of illicit drugs have been constantly growing, leading to heavy consequences for environment, human health, and society in general. Therefore, an urgent need for rapid, sensitive, portable and easy-to-operate detection methods for numerous drugs of interest in diverse matrices, from police samples, biological fluids and hair to sewage water has risen. Electrochemical sensors are promising alternatives to chromatography and spectrometry. Last decades, electrochemical sensing of illegal drugs has experienced a very significant growth, driven by improved transducers and signal amplifiers helping to improve the sensitivity and selectivity. The present review summarizes recent advances (last 10 years) in electrochemical detection of the most prevailing illicit drugs (such as cocaine, heroin, and (meth)amphetamine), their precursors and derivatives in different matrices. Various electrochemical sensors making use of different transducers with their (dis)advantages were discussed, and their sensitivity and applicability were critically compared. In those cases where natural or synthetic recognition elements were included in the sensing system to increase specificity, selected recognition elements, their immobilization, working conditions, and analytical performance were discussed. Finally, an outlook is presented with suggestions and recommendations for future developments.

Capacitive sensor based on molecularly imprinted polymers for detection of the insecticide imidacloprid in water.

This manuscript reports on the development of a capacitive sensor for the detection of imidacloprid (IMD) in water samples based on molecularly imprinted polymers (MIPs). MIPs used as recognition elements were synthesized via a photo-initiated emulsion polymerization. The particles were carefully washed using a methanol (MeOH) /acetic acid mixture to ensure complete template removal and were then dried. The average size of the obtained particles was less than 1 µm. The imprinting factor (IF) for IMD was 6 and the selectivity factor (α) for acetamiprid, clothianidin, thiacloprid and thiamethoxam were 14.8, 6.8, 7.1 and 8.2, respectively. The particles were immobilized on the surface of a gold electrode by electropolymerization. The immobilized electrode could be spontaneously regenerated using a mixture of MeOH/10 mM of phosphate buffer (pH = 7.2)/triethylamine before each measurement and could be reused for 32 times. This is the first-time that automated regeneration was introduced as part of a sensing platform for IMD detection. The developed sensor was validated by the analysis of artificially spiked water samples. Under the optimal conditions, the linearity was in the range of 5-100 µM, with a limit of detection (LOD) of 4.61 µM.

Silanized Luminescent Quantum Dots for the Simultaneous Multicolor Lateral Flow Immunoassay of Two Mycotoxins.

A critical point for the successful development of a fluorescent quantum dot (QD)-based immunoassay is maintaining the high fluorescence quantum yield of QDs during hydrophilization and bioconjugation. In this paper, we carefully designed CdSe/CdS and CdSe/CdS/ZnS core-shell heterostructures and extended them with silica coating of different surface composition allowing preservation of fluorescence quantum yield as high as 70% in aqueous media. The silanized QDs containing epoxy and carboxy surface groups were bioconjugated with monoclonal antibodies. The synthesized fluorescent conjugates were used in a multicolor lateral flow immunoassay for simultaneous determination of two mycotoxins. Zearalenone and deoxynivalenol were chosen as a proof of concept. Cutoff levels for the zearalenone and deoxynivalenol detection were adjusted to be at 40 and 400 µg kg-1, respectively, complying with the European Commission regulation. Validation of the developed test was performed by analysis of 34 naturally contaminated maize and wheat samples; as a confirmatory method, LC-MS/MS was used.

Immunochemical approaches for detection of aflatoxin B1 in herbal medicines.

INTRODUCTION: Aflatoxin B1 (AFB1) is a toxic low-molecular-weight secondary metabolite of Aspergillus flavus and A. parasiticus. AFB1 was classified as a Group I carcinogen by the World Health Organisation for Research on Cancer in 1993. AFB1 is an unavoidable natural contaminant of some herbal medicine, able to cause serious health issues for humans consuming the related medicine. OBJECTIVE: Therefore, this study aimed to develop an efficient fluorescence polarisation immunoassay (FPIA) and a rapid, low-cost, and easy-to-use membrane-based flow-through immunoassay (MBA) for determination of AFB1 in herbal medicine Origanum vulgare L., Rubus idaeus L., Urtica dioica L. and Sorbus aucuparia L. RESULTS: A cut-off level of the developed MBA was 0.8 ppb. Validation of the developed test was performed with blank and spiked samples. Using three naturally contaminated or three artificially spiked samples. The FPIA showed a linear working range of 8.6 to 64 ppb, and a half maximal inhibitory concentration (IC50 ) of 24 ppb. CONCLUSION: The results were in good correlation with the enzymelinked immunosorbent assay (ELISA) results (the IC50 0.1 ppb). Both the sample preparation and analysis are simple, cost-effective and easy to perform on-site in non-laboratory environments. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) was used as a confirmatory technique.

Development of a validated direct injection-liquid chromatographic tandem mass spectrometric method under negative electrospray ionization for quantitation of nine microcystins and nodularin-R in lake water.

Microcystins (MCs) are cyclic heptapeptide toxins produced by various cyanobacterial genera that are toxic to both animals and humans. In this study, a novel strategy was proposed for the quantitation of nine MCs and Nodularin-R (NOD) in lake water using UHPLC-MS/MS under negative ionization mode, in which only centrifugation was employed during sample preparation. As a result, limits of quantification (LOQ) ranging from 0.05 to 0.1 µg/L for all studied compounds were obtained in water samples, which were lower than the results obtained using positive ionization mode. Additionally, validation was performed by spiking three different levels of MCs at 0.05 or 0.1, 0.5, 1.0 µg/L (n = 6). Recoveries ranged from 88.6% to 101.8%, and intraday and interday variability were lower than 12% and 14%, respectively, for all targeted compounds. Furthermore, the proposed method was applied to investigate microcystins contamination in fifty lake water samples collected in different regions in China. As a result, MC-LR, MC-RR, MC-YR, MC-WR, MC-LW, MC-LA, MC-LY, and MC-HilR were detected in lake water samples at trace level ranging from 0.06 to 0.37 µg/L. The obtained results indicated that it was necessary to monitor the presence of MCs in lake water, especially during regular cyanobacterial blooms during warmer months.

Soft glass multi-channel capillaries as a platform for bioimprinting.

Multi-channel capillaries (MC) formed from thousands individual microcapillaries with diameters ranging 10-100 µm are of a great interest for their use as platforms for molecular imprinting due to their relatively large surface area, high mechanical stability and possibility of facile integration in sensor systems. The manuscript proposes a new format of immunoassay based on imprinted protein immobilized on a MC inner surface modified with poly-l-lysine. The combination of the environmentally friendly, easy-to-produce and cheap recognition element with the carrier allowing to increase the assay sensitivity makes the described technique a perspective alternative for the existing screening tests. Two bioimprinting approaches were described. The imprinted protein (ovalbumin, OVA) primarily prepared separately and later immobilized on a MC structure was compared to the imprinted OVA directly prepared on the MC surface. Detection of a food contaminant zearalenone was chosen as a proof-of-concept. In a case of the immobilization of the primarily prepared imprinted OVA the reached limit of detection (LOD) was 0.8 ng/mL, and for the in-situ imprinted OVA the LOD was 0.12 ng/mL. The sensitivity of the developed bioimprinted assay was comparable to the commercially available ELISA kits for ZEN detection. The OVA in-situ imprinted on the MC surface was tested for the detection of ZEN in artificially spiked wheat samples. The high recovery values (88-112%) and good repeatability (RSD of 8.5-9.6%) were demonstrated allowing to conclude that the IPs-based MC-ELISA is a promising tool for analysis of the mycotoxin in complex matrices.

Gel electrophoresis separation and origins of light emission in fluorophores prepared from citric acid and ethylenediamine.

We investigated light emission of hydrothermally treated citric acid and ethylenediamine (EDA) with various precursor ratios using gel-electrophoresis. We show that this relatively simple approach can deliver significant insights into the origins of photoluminescence. We found that products of the synthesis consist of both positively and negatively charged species and exhibit large dispersion in electrophoretic mobility (i.e. charge-to-size ratio). We observed that despite the large dispersion of the reaction products the blue light emission is confined to discrete bands clearly identifiable in the gel. We demonstrate clear evidence that this emission originates from the negatively charged light molecular fraction with the highest mobility which shows no excitation-dependent light emission. This molecular fluorophore exhibits spectral characteristics similar to previously reported 1,2,3,5-tetrahydro-5-oxo-imidazo[1,2-a]pyridine-7-carboxylic acid (IPCA). Secondary gel electrophoresis run performed on the bands extracted from the first run indicates that no further separation takes place. On the basis of our experimental results, we conclude that relatively stable binding exists between IPCA and EDA-derived product. Thus, the products of the reaction contain IPCA both in molecular form and in complexes with EDA-derived products. We conclude that excitation-dependent emission is related to the fluorophore binding to the positively charged EDA-derived products with a positive charge.

Silanization of quantum dots: Challenges and perspectives.

Here we report the facile strategies for synthesis of silica-coated quantum dots (QDs). Due to their small size, chemical and optical stability and high luminescence quantum yield these particles can be further applicable in chemical and biomedical analysis, and in vivo imaging. The article gives a structured and detailed overview of the silanization strategies and potential pitfalls and common technical issues in function of the stability of the obtaining QDs. Different types of QDs, In- and Cd-based, were compared in terms of their behaviour during the silica-encapsulation. To achieve that each silica nanoparticle contains only one single QD the silanization reaction was performed via a water-in-oil emulsion formation. We investigated a number of parameters which have an impact on the silica-coating process and on characteristics of the obtained nanoparticles, such as a nature of initial ligands on the QD surface, a silanization reagent, surfactant, ZnS shelling, etc. It was found that DDT is an optimal ligand for the silanization of InP-based quantum dots, but not for CdZnSeS-nanocomposites. GOPTES and CEST are the best silanization agents for covering InP-QDs, and the AOT/isooctane microemulsion is preferable for the silanization of CdZnSeS-composites.

Portable Multiplex Immunochromatographic Assay for Quantitation of Two Typical Algae Toxins Based on Dual-Color Fluorescence Microspheres.

A multiplex immunochromatographic assay (ICA) based on dual-color fluorescent microspheres (FMs) as a sensitive label was developed for the first time. Two typical algae toxins, microcystin-LR (MC-LR) and okadaic acid (OA), were chosen as proof-of concept targets to evaluate the feasibility of this ICA format. Commercial red- and green-colored FMs were selected to couple with monoclonal antibodies as fluorescent probes. The use of dual-wavelength FMs as labels guaranteed a lower consumption of material strips, lower sample volume, and shorter reaction time without increasing the length of ICA strips. Under optimal conditions, the multiplex FM-ICA could be completed in 20 min and reached limits of detection for the simultaneous determination of MC-LR and OA in fish samples, which were 0.074 and 2.42 µg/kg, respectively. The developed technique was validated using artificially spiked and naturally contaminated fish samples. Ultra-high-performance liquid chromatography-tandem mass spectrometry was used as confirmatory technique. In summary, this portable ICAs detection mode based on dual-wavelength FMs provided a reliable and sensitive on-site detection of multiple contaminants in food samples, which opens a new field for application of FMs in food safety.

Antibody immobilization strategy for the development of a capacitive immunosensor detecting zearalenone.

A highly sensitive flow-injection capacitive immunosensor was developed for detection of the mycotoxin zearalenone (ZEN). Different strategies for immobilization of an anti-ZEN antibody on the surface of a gold electrode, i.e. polytyramine or self-assembled monolayers (SAMs) of 3-mercaptopropionic acid (3-MPA) and lipoic acid (LA), were used and their performances were compared. The LA- and 3-MPA-based systems showed broad linear ranges for ZEN determination, i.e. from 0.010 nM to 10 nM and from 0.020 nM to 10 nM, respectively. Under optimal conditions, the LA-based immunosensor was capable of performing up till 13 regeneration-interaction cycles (with use of glycine HCl, pH 2.4) with a limit of detection (LOD) of 0.0060 nM, equivalent to 1.9 pg mL-1. It also demonstrated a good inter-assay precision (RSD < 10%). However, the tyramine-based capacitive immunosensor showed a bad repeatability (only 4 regeneration-interaction cycles were possible) and inter-assay precision (RSD > 15%) which did not allow sensitive and precise measurements. The LA-based method was compared with a direct ELISA. These results demonstrated that the label-free developed capacitive immunosensor had a better sensitivity and shorter analysis time in comparison with the direct microwell-plate format.

Bioimprinting for multiplex luminescent detection of deoxynivalenol and zearalenone.

A sensitive tool for simultaneous quantitative determination of two analytes in a single spot with the use of a bioimprinted protein is presented for the first time. BSA is chosen as a scaffold for generation of binding sites specific towards two compounds. A multiplex immunosorbent assay for screening of two cereal-born mycotoxins, deoxynivalenol and zearalenone, in wheat and maize is realized with the use of fluorescent silica coated quantum dots as labels. Water-soluble fluorescent nanostructures consist of core/shell Cd-QDs enrobed in silica shells to ensure their solubility. The mycotoxins are simultaneously detected by scanning the assay outcome at two different wavelengths, since two QD@SiO2 labelled conjugates fluorescent in different parts of the spectrum. The assay is combined with a rapid extraction technique. The limits of detection for the simultaneous determination were 100 and 700 µg kg-1 in both matrices for zearalenone and deoxynivalenol, respectively. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was used to confirm the obtained results.

Imprinted proteins as a receptor for detection of zearalenone.

A strategy to design an immunoassay based on imprinted proteins to detect a foodborne toxin zearalenone (ZEN) has been proposed. Proteins were used as scaffolds for generation of binding cavities with a high specificity against ZEN. Different proteins and different bioimprinting approaches were tested. The imprinted proteins primarily prepared in a tube and then immobilized on a microwell plate, and directly prepared in the microwell plate were compared for an application in immunosorbent detection of ZEN in naturally contaminated wheat and maize samples. The assay was combined with a rapid extraction technique. Specific interactions between the imprinted proteins and the target in a µg kg-1 linear range was achieved. As a confirmatory method, liquid chromatography coupled to tandem mass spectrometry was used. The obtained results allow using the developed immunoassay for the analysis of ZEN with a sensitivity matching the maximum permitted level for ZEN in unprocessed cereals established by the European Commission (100 µg kg-1).