Search

Salmonella typhimurium and Listeria monocytogenes are relevant foodborne bacterial pathogens which may cause serious intoxications and infectious diseases in humans. In this study, a sensitive immunochromatographic analysis (ICA) for the simultaneous detection of these two pathogens was developed. For this, test strips containing two test zones with specific monoclonal antibodies (MAb) against lipopolysaccharides of S. typhimurium and L. monocytogenes and one control zone with secondary antibodies were designed, and the double-assay conditions were optimized to ensure high analytical parameters. Prussian blue nanoparticles (PBNPs) were used as nanozyme labels and were conjugated with specific MAbs to perform a sandwich format of the ICA. Peroxidase-mimic properties of PBNPs allowed for the catalytic amplification of the colorimetric signal on test strips, enhancing the assay sensitivity. The limits of detection (LODs) of Salmonella and Listeria cells were 2 × 10 2 and 7 × 10 3 cells/mL, respectively. LODs were 100-fold less than those achieved due to the ICA based on the traditional gold label. The developed double ICA was approbated for the detection of bacteria in cow milk samples, which were processed by simple dilution by buffer before the assay. For S. typhimurium and L. monocytogenes , the recoveries from milk were 86.3 ± 9.8 and 118.2 ± 10.5% and correlated well with those estimated by the enzyme-linked immunosorbent assay as a reference method. The proposed approach was characterized by high specificity: no cross-reactivity with other bacteria strains was observed. The assay satisfies the requirements for rapid tests: a full cycle from sample acquisition to result assessment in less than half an hour. The developed ICA has a high application potential for the multiplex detection of other foodborne pathogens., Competing Interests: The authors declare no conflicts of interest.

A promising and sought-after class of nanozymes for various applications is Pt-containing nanozymes, primarily Au@Pt, due to their easy preparation and remarkable catalytic properties. This study aimed to explore the freeze-thaw method for functionalizing Pt-containing nanozymes with oligonucleotides featuring a polyadenine anchor. Spherical gold nanoparticles ([Au]NPs) were synthesized and subsequently used as seeds to produce urchin-like Au@Pt nanoparticles ([Au@Pt]NPs) with an average diameter of 29.8 nm. The nanoparticles were conjugated with a series of non-thiolated DNA oligonucleotides, each consisting of three parts: a 5'-polyadenine anchor (A n , with n = 3, 5, 7, 10; triple-branched A 3 , or triple-branched A 5 ), a random sequence of 23 nucleotides, and a linear polyT block consisting of seven deoxythymine residues. The resulting conjugates were characterized using transmission electron microscopy, spectroscopy, dynamic light scattering, and emission detection of the fluorescent label at the 3'-end of each oligonucleotide. The stability of the conjugates was found to depend on the type of oligonucleotide, with decreased stability in the row of [Au@Pt]NP conjugates with A 7 > A 5 > 3A 3 > 3A 5 > A 10 > A 3 anchors. These [Au@Pt]NP-oligonucleotide conjugates were further evaluated using lateral flow test strips to assess fluorescein-specific binding and peroxidase-like catalytic activity. Conjugates with A 3 , A 5 , A 7 , and 3A 3 anchors showed the highest levels of signals of bound labels on test strips, exceeding conjugates in sensitivity by up to nine times. These findings hold significant potential for broad application in bioanalytical systems.

Adulteration of meat products is a serious problem in the modern society. Consumption of falsified meat products can be hazardous to health and/or lead to violating religious dietary principles. To identify such products, rapid and simple test systems for point-of-need detection are in demand along with complex laboratory methods. This study presents the first double lateral flow (immunochromatographic) test system, which allows simultaneous revealing two prevalent types of falsifications-undeclared addition of pork and chicken components to meat products. In the proposed test system, porcine myoglobin (MG) and chicken immunoglobulin Y (IgY) were used as specific biomarkers recognizable by antibodies. Within the optimization of the analysis, the concentrations of the immune reagents and regimes of their application on the working membrane were selected, which provided minimal limits of detection (LODs) for both analytes. The developed test system enables the detection of MG and IgY with the LODs of 10 and 12 ng/mL, respectively, which accords to addition of 0.1% of the undeclared meat compounds. The applicability of the test system to control the composition of raw meat mixtures and cooked food products was confirmed. The developed approach can be considered as a promising tool for monitoring composition of meat products., Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-024-05944-y., Competing Interests: Conflict of interestThe authors declare no conflict of interest., (© Association of Food Scientists & Technologists (India) 2024. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

Cheap chicken meat is often used as an undeclared substitute in meat products. In this study, two formats of the immunochromatographic assay (ICA) of immunoglobulins of class Y (IgY) as a biomarker for chicken authentication were developed. In both competitive ICA (cICA) and sandwich ICA (sICA), gold nanoparticles (GNP) were conjugated with anti-species antibodies. A simple procedure of sample preparation, which took only 30 min, was proposed. Test systems demonstrated high sensitivity and rapidity: visual limits of detection of IgY and assay durations were 12/14 ng/mL and 10/15 min for cICA and sICA, respectively. The absence of cross-reactivity with the mammalian species confirmed the high specificity of the test systems. Good applicability of the assays was confirmed for the detection of chicken in raw meat mixtures: as low as 3% and 0.2% (w/w) of chicken could be revealed in beef and pork by cICA and sICA, respectively. The influence of heat processing of meat-based products on immune recognition and, consequently, the analytical performance of the test systems was revealed. It was shown that sICA is preferable for the detection of IgY even in thermally processed meat. The proposed ICAs can be recommended for rapid on-site control of meat products' composition., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Fluorescence induced by the excitation of a fluorophore with plane-polarized light has a different polarization depending on the size of the fluorophore-containing reagent and the rate of its rotation. Based on this effect, many analytical systems have been implemented in which an analyte contained in a sample and labeled with a fluorophore (usually fluorescein) competes to bind to antibodies. Replacing antibodies in such assays with aptamers, low-cost and stable oligonucleotide receptors, is complicated because binding a fluorophore to them causes a less significant change in the polarization of emissions. This work proposes and characterizes the compounds of the reaction medium that improve analyte binding and reduce the mobility of the aptamer-fluorophore complex, providing a higher analytical signal and a lower detection limit. This study was conducted on aflatoxin B1 (AFB1), a ubiquitous toxicant contaminating foods of plant origins. Eight aptamers specific to AFB1 with the same binding site and different regions stabilizing their structures were compared for affinity, based on which the aptamer with 38 nucleotides in length was selected. The polymers that interact reversibly with oligonucleotides, such as poly-L-lysine and polyethylene glycol, were tested. It was found that they provide the desired reduction in the depolarization of emitted light as well as high concentrations of magnesium cations. In the selected optimal medium, AFB1 detection reached a limit of 1 ng/mL, which was 12 times lower than in the tris buffer commonly used for anti-AFB1 aptamers. The assay time was 30 min. This method is suitable for controlling almond samples according to the maximum permissible levels of their contamination by AFB1. The proposed approach could be applied to improve other aptamer-based analytical systems.

Fluoroquinolone antibiotics are used to cure and protect bees and apiaries from infections. Consequently, they may contaminate honey and other products of beekeeping. In this study, a highly sensitive immunoenzyme assay (EIA) was for the first time developed for the determination of a fluoroquinolone flumequine (FLU) in honey. The EIA was carried out in an indirect competitive format with colorimetric detection. The analysis was characterized by a low limit of detection of 30 pg mL -1 . The polyclonal antibodies used showed no cross-reactivity with 24 other (fluoro)quinolones; the assay was highly specific only toward FLU. Different coating FLU-protein conjugates were tested to achieve the most sensitive competitive immunodetection. A highly simplified and rapid (3-5 min) sample preparation was proposed based on the 100-300 times dilution of honey by a buffer. The developed EIA has been tested to detect FLU in honey of different origins, namely acacia, flower, buckwheat, chestnut, and linden honey. It has been demonstrated that 76.2-115.9% of FLU could be determined by the assay. The versatility, simplicity, and rapidity of the EIA enable us to propose this method as an effective tool to control the contamination of honey.

CRISPR/Cas12a is a potent biosensing tool known for its high specificity in DNA analysis. Cas12a recognizes the target DNA and acquires nuclease activity toward single-stranded DNA (ssDNA) probes. We present a straightforward and versatile approach to transforming common Cas12a-cleavable DNA probes into enhancing tools for fluorescence anisotropy (FA) measurements. Our study involved investigating 13 ssDNA probes with linear and hairpin structures, each featuring fluorescein at one end and a rotation-slowing tool (anchor) at the other. All anchors induced FA changes compared to fluorescein, ranging from 24 to 110 mr. Significant FA increases (up to 180 mr) were obtained by adding divalent metal salts (Mg 2+ , Ca 2+ , Ba 2+ ), which influenced the rigidity and compactness of the DNA probes. The specific Cas12a-based recognition of double-stranded DNA (dsDNA) fragments of the bacterial phytopathogen Erwinia amylovora allowed us to determine the optimal set (probe structure, anchor, concentration of divalent ion) for FA-based detection. The best sensitivity was obtained using a hairpin structure with dC10 in the loop and streptavidin located near the fluorescein at the stem in the presence of 100 mM Mg 2+ . The detection limit of the dsDNA target was equal to 0.8 pM, which was eight times more sensitive compared to the common fluorescence-based method. The enhancing set ensured detection of single cells of E. amylovora per reaction in an analysis based on CRISPR/Cas12a with recombinase polymerase amplification. Our approach is universal and easy to implement. Combining FA with Cas12a offers enhanced sensitivity and signal reliability and could be applied to different DNA and RNA analytes.

In this study, we developed a sensitive immunochromatographic analysis (ICA) of the Salmonella typhimurium bacterial pathogen contaminating food products and causing foodborne illness. The ICA of S. typhimurium was performed using Au@Pt nanozyme as a label ensuring both colorimetric detection and catalytic amplification of the analytical signal due to nanozyme peroxidase-mimic properties. The enhanced ICA enabled the detection of S. typhimurium cells with the visual limit of detection (LOD) of 2 × 10 2 CFU/mL, which outperformed the LOD in the ICA with traditional gold nanoparticles by two orders of magnitude. The assay duration was 15 min. The specificity of the developed assay was tested using cells from various Salmonella species as well as other foodborne pathogens; it was shown that the test system detected only S. typhimurium . The applicability of ICA for the determination of Salmonella in food was confirmed in several samples of milk with different fat content, as well as chicken meat. For these real samples, simple pretreatment procedures were proposed. Recoveries of Salmonella in foodstuffs were from 74.8 to 94.5%. Due to rapidity and sensitivity, the proposed test system is a promising tool for the point-of-care control of the Salmonella contamination of different food products on the whole farm-to-table chain.

In this study, a fluorescence resonance energy transfer (FRET)-based aptasensor for the detection of aflatoxin B1 (AFB1) was designed using a carboxyfluorescein (FAM)-labeled aptamer and short complementary DNA (cDNA) labeled with low molecular quencher RTQ1. The sensing principle was based on the detection of restored FAM-aptamer fluorescence due to the ligand-induced displacement of cDNA in the presence of AFB1, leading to the destruction of the aptamer/cDNA duplex and preventing the convergence of FAM and RTQ1 at the effective FRET distance. Under optimal sensing conditions, a linear correlation was obtained between the fluorescence intensity of the FAM-aptamer and the AFB1 concentration in the range of 2.5-208.3 ng/mL with the detection limit of the assay equal to 0.2 ng/mL. The assay time was 30 min. The proposed FRET aptasensor has been successfully validated by analyzing white wine and corn flour samples, with recovery ranging from 76.7% to 91.9% and 84.0% to 86.5%, respectively. This work demonstrates the possibilities of labeled cDNA as an effective and easily accessible tool for sensitive AFB1 detection. The homogeneous FRET aptasensor is an appropriate choice for contaminant screening in complex matrices.

The analytical applications of antibodies are often associated with their immobilization on different carriers, which is accompanied by a loss of antigen-binding activity for a sufficient proportion of the bound antibodies. In contrast to data on plain carriers, minimal data are available on the properties of antibodies on the surfaces of nanoparticles. Protein antigens have been predominantly investigated, for which space restrictions do not allow them to occupy all active sites of immobilized antibodies. This study considered a low-molecular-weight compound, fluorescein, as an antigen. Spherical gold nanoparticles with five different sizes, two differently charged forms of fluorescein, and three different levels of surface coverage by immobilized antibodies were tested. For gold nanoparticles with diameters from 14 to 35.5 nm with monolayers of immobilized antibodies, the percentage of molecules capable of binding carboxyfluorescein varied from 6% to 17%. The binding of aminofluorescein was more efficient; for gold nanoparticles with an average diameter of 21 nm, the percentage of active binding sites for the immobilized antibodies reached 27% compared with 13% for the carboxyfluorescein case. A fourfold reduction in the coverage of the nanoparticles' surface compared with that of the monolayer did not lead to reliable changes in the percentage of active binding sites. The obtained data demonstrate that an antigen's binding to immobilized antibodies is limited even for small antigens and depends on the size of the nanoparticles and the electrostatic repulsion near their surface.

This study was aimed at the sensitive immunodetection of porcine myoglobin (MG) as a species-specific biomarker in meat products. The enhanced lateral flow immunoassay (LFIA) was created in the sandwich format using monoclonal antibodies (Mab) with specificity to porcine MG and labeled by Prussian blue nanoparticles (PBNPs) as peroxidase-mimicking nanozymes. Signal amplification was provided by the colored product of oxidation catalyzed by the PBNPs. Several Mab-PBNP conjugates with different antibody loads were synthesized; the one that provided the best analytical characteristics of the LFIA was selected. Advanced optimization of the test system was carried out. As a result, the visual limit of detection (LOD) of MG was 1.5 ng/mL. Involvement of the catalytic nanozyme properties allowed the LOD to be decreased by ~9 times in comparison to the LFIA based on gold nanomarkers, and by ~27 times compared to the LFIA based on PBNP coloration. The assay time was 30 min, including catalytic enhancement. A simple technique of meat sample pre-treatment aimed at effective MG extraction and matrix disposal was proposed. The specificity of the LFIA towards the pork meat was demonstrated. The applicability of the created test system was shown by testing extracts obtained from finished meat products.

The toxic effects of antimony pose risks to human health. Therefore, simple analytical techniques for its widescale monitoring in water sources are in demand. In this study, a sensitive microplate apta-enzyme assay for Sb 3+ detection was developed. The biotinylated aptamer A 10 was hybridized with its complementary biotinylated oligonucleotide T 10 and then immobilized on the surface of polysterene microplate wells. Streptavidin labeled with horseradish peroxidase (HRP) bound to the biotin of a complementary complex and transformed the 3,3',5,5'-tetramethylbenzidine substrate, generating an optical signal. Sb 3+ presenting in the sample bounded to an A 10 aptamer, thus releasing T 10 , preventing streptavidin-HRP binding and, as a result, reducing the optical signal. This effect allowed for the detection of Sb 3+ with a working range from 0.09 to 2.3 µg/mL and detection limit of 42 ng/mL. It was established that the presence of Ag + at the stage of A 10 /T 10 complex formation promoted dehybridization of the aptamer A 10 and the formation of the A 10 /Sb 3+ complex. The working range of the Ag + -enhanced microplate apta-enzyme assay for Sb 3+ was determined to be 8-135 ng/mL, with a detection limit of 1.9 ng/mL. The proposed enhanced approach demonstrated excellent selectivity against other cations/anions, and its practical applicability was confirmed through an analysis of drinking and spring water samples with recoveries of Sb 3+ in the range of 109.0-126.2% and 99.6-106.1%, respectively.

Lateral flow immunoassay (LFIA) has found a broad application for testing in point-of-care (POC) settings. LFIA is performed using test strips-fully integrated multimembrane assemblies containing all reagents for assay performance. Migration of liquid sample along the test strip initiates the formation of labeled immunocomplexes, which are detected visually or instrumentally. The tradeoff of LFIA's rapidity and user-friendliness is its relatively low sensitivity (high limit of detection), which restricts its applicability for detecting low-abundant targets. An increase in LFIA's sensitivity has attracted many efforts and is often considered one of the primary directions in developing immunochemical POC assays. Post-assay enhancements based on chemical reactions facilitate high sensitivity. In this critical review, we explain the performance of post-assay chemical enhancements, discuss their advantages, limitations, compared limit of detection (LOD) improvements, and required time for the enhancement procedures. We raise concerns about the performance of enhanced LFIA and discuss the bottlenecks in the existing experiments. Finally, we suggest the experimental workflow for step-by-step development and validation of enhanced LFIA. This review summarizes the state-of-art of LFIA with chemical enhancement, offers ways to overcome existing limitations, and discusses future outlooks for highly sensitive testing in POC conditions.

Pathogen identification requires nucleic acid diagnosis with simple equipment and fast manipulation. Our work established an all-in-one strategy assay with excellent sensitivity and high specificity, Transcription-Amplified Cas14a1-Activated Signal Biosensor (TACAS), for the fluorescence-based bacterial RNA detection. The DNA as a promoter probe and a reporter probe directly ligated via SplintR ligase once specifically hybridized to the single-stranded target RNA sequence, with the ligation product transcribed into Cas14a1 RNA activators by T7 RNA polymerase. This forming sustained isothermal one-pot ligation-transcription cascade produced RNA activators constantly and enabled Cas14a1/sgRNA complex to generate fluorescence signal, thus leading to a sensitive detection limit of 1.52 CFU mL -1 E. coli within 2 h of incubation time. TACAS was applied in contrived E. coli infected fish and milk samples, and a significant signal differentiation between positive (infected) and negative (uninfected) samples was reached. Meanwhile, E. coli colonization and transmit time in vivo were explored and the TACAS assay promoted the understanding of the infection mechanisms of the E. coli infection, demonstrating an excellent detection capability., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

CRISPR/Cas12-based biosensors are emerging tools for diagnostics. However, their application of heterogeneous formats needs the efficient detection of Cas12 activity. We investigated DNA probes attached to the microplate surface and cleaved by Cas12a. Single-stranded (ss) DNA probes (19 variants) and combined probes with double-stranded (ds) and ssDNA parts (eight variants) were compared. The cleavage efficiency of dsDNA-probes demonstrated a bell-shaped dependence on their length, with a cleavage maximum of 50%. On the other hand, the cleavage efficiency of ssDNA probes increased monotonously, reaching 70%. The most effective ssDNA probes were integrated with fluorescein, antibodies, and peroxidase conjugates as reporters for fluorescent, lateral flow, and chemiluminescent detection. Long ssDNA probes (120-145 nt) proved the best for detecting Cas12a trans-activity for all of the tested variants. We proposed a test system for the detection of the nucleocapsid (N) gene of SARS-CoV-2 based on Cas12 and the ssDNA-probe attached to the microplate surface; its fluorescent limit of detection was 0.86 nM. Being united with pre-amplification using recombinase polymerase, the system reached a detection limit of 0.01 fM, thus confirming the effectiveness of the chosen ssDNA probe for Cas12-based biosensors.

Reliable detection of specific antibodies against pathogens by lateral flow immunoassay (LFIA) greatly depends on the composition of the detectable complex and the order of its assembly. We compared three LFIA formats for revealing anti-SARS-CoV-2 antibodies in sera with the following detected complexes in the analytical zone of the strip: antigen-antibodies-labeled immunoglobulin-binding protein (Scheme A); antigen-antibodies-labeled antigen (Scheme B); and immunoglobulin-binding protein-antibodies-labeled antigen (Scheme C). The lowest detection limit was observed for Scheme C, and was equal to 10 ng/mL of specific humanized monoclonal antibodies. When working with pooled positive sera, Scheme C had a detection limit 15 times lower than Scheme B and 255 times lower than Scheme A. Due to the high sensitivity of Scheme C, its application for the panel of human sera (n = 22) demonstrated 100% diagnostic specificity and sensitivity. These consistent results be useful for designing the format of LFIA serodiagnosis for other diseases.

Biosensors based on endonuclease Cas12 provide high specificity in pathogen detection. Sensitive detection using Cas12-based assays can be achieved using trans-cleaved DNA probes attached to simply separated carriers, such as magnetic particles (MPs). The aim of this work was to compare polyA, polyC, and polyT single-stranded (ss) DNA with different lengths (from 10 to 145 nt) as trans-target probes were immobilized on streptavidin-covered MPs. Each ssDNA probe was labeled using fluorescein (5') and biotin (3'). To compare the probes, we used guide RNAs that were programmed for the recognition of two bacterial pathogens: Dickeya solani (causing blackleg and soft rot) and Erwinia amylovora (causing fire blight). The Cas12 was activated by targeting double-stranded DNA fragments of D. solani or E. amylovora and cleaved the MP-ssDNA conjugates. The considered probes demonstrated basically different dependencies in terms of cleavage efficiency. PolyC was the most effective probe when compared to polyA or polyT probes of the same length. The minimal acceptable length for the cleavage follows the row: polyC < polyT < polyA. The efficiencies of polyC and polyT probes with optimal length were proven for the DNA targets' detection of D. solani and E. amylovora . The regularities found can be used in Cas12a-based detection of viruses, bacteria, and other DNA/RNA-containing analytes.

Antibiotic usage has become very widespread in aquaculture, and the abuse or overuse of antibiotics has led to the evolution of antibiotic-resistance bacteria, which has adverse effects on aquatic products and ecosystems. Moreover, this evolution can potentially cause harm to human health. Thus, there is an urgent need for diagnostic tools for antibiotic-resistant microorganisms. Herein, we proposed a signal-off Cas14a1-based platform (SOCP) for the detection of methicillin-resistant Staphylococcus aureus (MRSA). In this SOCP, we have designed single-stranded DNA (ssDNA) that not only can activate the trans-cleavage ability of dual Cas14a1-sgRNA complex but also can be used as the primers for the amplified methicilin-resistant gene (mecA). When MRSA is present, the primers can be transformed into products with amplification, leading to the signal decrease of trans-cleavage activity of Cas14a1. The SOCP showed high specificity and fair sensitivity for mecA gene and MRSA. In the detection of real samples, this platform also showed consistent results compared with qPCR. The SOCP could provide an alternative tool for the diagnosis of antibiotic-resistant bacteria in aquaculture, food industry and other fields., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Finding optimal conditions for competitive lateral flow immunoassay is a controversial task. The content of specific antibodies labeled by nanoparticles should be simultaneously high to reach intense signals and low to register an influence on the signals for minimal concentrations of the target analyte. We propose to use two kinds of complexes of gold nanoparticles in the assay, with antigen-protein conjugates and with specific antibodies. The first complex interacts both with immobilized antibodies in the test zone and with antibodies on the surface of the second complex. In this assay, the coloration is enhanced by the binding of two-colored preparations in the test zone, whereas the antigen in the sample inhibits both the binding of the first conjugate with the immobilized antibodies and with the second conjugate. This approach is realized for the detection of insecticide imidacloprid (IMD), an important toxic contaminant connected with the recent global death of bees. The proposed technique expands the working range of the assay, that is, in accordance with its theoretical analysis. The reliable change of coloration intensity is achieved for a 2.3-times-lower concentration of the analyte. The limit of IMD detection is 0.13 ng/mL for tested solutions and 1.2 µg/kg for initial honey samples. The combination of two conjugates doubles the coloration in the absence of the analyte. The developed lateral flow immunoassay is applicable for five-fold-diluted honey samples without extraction, does not require additional stages (all reagents are pre-applied to the test strip), and is implemented in 10 min.

A scheme of modular competitive immunochromatography with an analyte-independent test strip and changeable specific immunoreactants has been proposed. Native (detected) and biotinylated antigens interact with specific antibodies during their preincubation in solution, that is, without the immobilization of reagents. After this, the detectable complexes on the test strip are formed by the use of streptavidin (which binds biotin with high affinity), anti-species antibodies, and immunoglobulin-binding streptococcal protein G. The technique was successfully applied for the detection of neomycin in honey. The visual and instrumental detection limits were 0.3 and 0.014 mg/kg, respectively, and the degree of neomycin revealed in honey samples varied from 85% to 113%. The efficiency of the modular technique with the use of the same test strip for different analytes was confirmed for streptomycin detection. The proposed approach excludes the necessity of finding the condition of immobilization for each new specific immunoreactant and transferring the assay to other analytes by a simple choice of concentrations for preincubated specific antibodies and the hapten-biotin conjugate.

Sequence-specific endonuclease Cas12-based biosensors have rapidly evolved as a strong tool to detect nucleic acids. Magnetic particles (MPs) with attached DNA structures could be used as a universal platform to manipulate the DNA-cleavage activity of Cas12. Here, we propose nanostructures of trans- and cis-DNA targets immobilized on the MPs. The main advantage of the nanostructures is a rigid double-stranded DNA adaptor that distances the cleavage site from the MP surface to ensure maximum Cas12 activity. Adaptors with different lengths were compared by detecting the cleavage by fluorescence and gel electrophoresis of the released DNA fragments. The length-dependent effects for cleavage on the MPs' surface were found both for cis- and trans-targets. For trans-DNA targets with a cleavable 15-dT tail, the results showed that the optimal range of the adaptor length was 120-300 bp. For cis-targets, we varied the length and location of the adaptor (at the PAM or spacer ends) to estimate the effect of the MP's surface on the PAM-recognition process or R-loop formation. The sequential arrangement of an adaptor, PAM, and a spacer was preferred and required the minimum adaptor length of 3 bp. Thus, with cis-cleavage, the cleavage site can be located closer to the surface of the MPs than with trans-cleavage. The findings provide solutions for efficient Cas12-based biosensors using surface-attached DNA structures.

Surface-enhanced Raman scattering (SERS) is considered an efficient technique providing high sensitivity and fingerprint specificity for the detection of pesticide residues. Recent developments in SERS-based detection aim to create flexible plasmonic substrates that meet the requirements for non-destructive analysis of contaminants on curved surfaces by simply wrapping or wiping. Herein, we reported a flexible SERS substrate based on cellulose fiber (CF) modified with silver nanostructures (AgNS). A silver film was fabricated on the membrane surface with an in situ silver mirror reaction leading to the formation of a AgNS-CF substrate. Then, the substrate was decorated through in situ synthesis of raspberry-like silver nanostructures (rAgNS). The SERS performance of the prepared substrate was tested using 4-mercaptobenzoic acid (4-MBA) as a Raman probe and compared with that of the CF-based plasmonic substrates. The sensitivity of the rAgNS/AgNS-CF substrate was evaluated by determining the detection limit of 4-MBA and an analytical enhancement factor, which were 10 nM and ~10 7 , respectively. Further, the proposed flexible rAgNS/AgNS-CF substrate was applied for SERS detection of malathion. The detection limit for malathion reached 0.15 mg/L, which meets the requirements about its maximum residue level in food. Thus, the characteristics of the rAgNS/AgNS-CF substrate demonstrate the potential of its application as a label-free and ready-to-use sensing platform for the SERS detection of trace hazardous substances.

Changes in the limits of detection (LODs) for a multiplex lateral flow immunoassay (LFIA) caused by different locations of the binding zone on the test strips were studied. Due to the non-equilibrium conditions of the immune reactions in LFIAs, their analytical parameters are susceptible to the binding constants of antigen-antibody reactions and assay duration. Consequently, the integration of several tests into one multiplex assay can cause a significant worsening of the sensitivity. In this study, we propose a simple methodology for the determination of the best arrangement of binding zones, which takes into account the binding constants for immunoreagents. LFIAs of four mycotoxins, namely, aflatoxin B1, deoxynivalenol, T-2 toxin, and ochratoxin A, were integrated into a multiplex test strip. An enzyme-linked immunosorbent assay was applied to determine the equilibrium and kinetic constants of the immunoreactants for each analyte. It was found that the arrangement of binding zones with a descending order of the equilibrium association constants was optimal and provided both lower detection limits and a more uniform coloration. The selected position of the binding zones allowed decreasing the LODs down to 2 and 27 times for ochratoxin A and deoxynivalenol, respectively. The proposed approach can be applied to multiplex LFIAs for different analytes.

Isothermal amplifications allow for the highly sensitive detection of nucleic acids, bypassing the use of instrumental thermal cycling. This work aimed to carry out an experimental comparison of the four most promising techniques: recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP) coupled with lateral flow test or coupled with additional amplification based on CRISPR/Cas12a resulting from the fluorescence of the Cas12a-cleaved probe. To compare the four amplification techniques, we chose the bacterial phytopathogen Erwinia amylovora (causative agent of fire blight), which has a quarantine significance in many countries and possesses a serious threat to agriculture. Three genes were chosen as the targets and primers were selected for each one (two for RPA and six for LAMP). They were functionalized by labels (biotin, fluorescein) at the 5' ends for amplicons recognition by LFT. As a result, we developed LAMP-LFT, LAMP-CRISPR/Cas, RPA-LFT, and RPA-CRISPR/Cas for E. amylovora detection. The detection limit was 10 4 CFU/mL for LAMP-LFT, 10 3 CFU/mL for LAMP-CRISPR/Cas, and 10 2 CFU/mL for RPA-LFT and RPA-CRISPR/Cas. The results of four developed test systems were verified by qPCR on a panel of real samples. The developed assays based on RPA, LAMP, CRISPR/Cas12a, and LFT are rapid (30-55 min), user-friendly, and highly sensitive for E. amylovora detection. All proposed detection methods can be applied to fire blight diagnosis and effective management of this disease.

The work considers a combination of three enhancing approaches for immunochromatographic assay (ICA) and the integration of their impacts into changes of the limit of detection (LOD). Human fatty acid binding protein (FABP), an early biomarker of acute myocardial infarction, was the target analyte. Starting from the common ICA protocol with an LOD equal to 11.2 ng/mL, three approaches were realized: (1) replacement of spherical gold nanoparticles with gold nanoflowers having a branched surface (20-fold lowering the LOD); (2) enhanced labeling of immune complexes via nanoparticle aggregates (15-fold lowering); (3) in-situ growth of bound nanoparticles by reduction of gold salts (3-fold lowering). Single and combined implementations of these approaches have been studied. It has been shown that the LOD decrease for combined approaches is close to the multiplied contribution of each of them. The final LOD for FABP was 0.05 ng/mL, which is 220 times lower than the LOD for the common ICA protocol. The efficiency of the enhanced ICA with three combined approaches was confirmed by testing human serum samples for FABP presence and content. The development presents a new efficient technique for rapid sensitive detection of FABP for medical diagnostics. Moreover, the demonstrated multiple enhancements could be applied for various demanded analytes.

Assessment of the composition of meat-containing products is the task in demand due to their frequent deviations from declared recipes. The paper presents the developed test system for immunochromatographic determination of total meat content. The assay is based on the simultaneous use of monoclonal antibodies, which specifically interacts with mammalian skeletal troponin I, and polyclonal antibodies, which specifically detect bird immunoglobulin Y. To integrate the detection of both types of meat by the same test strip, the antibodies are mixed in the analytical zone of the test strip and in complex with a gold nanoparticle label. The chosen ratios of the antibodies for both mixtures provide the same contribution of different types of mammalian and bird raw materials of muscle tissues to the label binding. The test system demonstrates suitability for products containing beef, pork, rabbit, lamb, chicken, and turkey meat. The minimal detectable content of meat in samples is 0.1%. The samples for the testing are diluted 100 times, thus eliminating matrix effects, and providing high reproducibility of the color intensity for extracts of different compositions. The obtained results allow the recommendation of the developed test system for rapid on-site control of meat products.

The paper presents development and characterization of a new bioanalytical test system for rapid detection of lipopolysaccharide (LPS) and whole cells of Francisella tularensis , a causative agent of tularemia, in water samples. Gold nanoparticles (AuNPs) coated by the obtained anti-LPS monoclonal antibodies were used for the assay. Their contact with antigen in tested samples leads to aggregation with a shift of absorption spectra from red to blue. Photometric measurements at 530 nm indicated the analyte presence. Three preparations of AuNPs with different diameters were compared, and the AuNPs having average diameter of 34 nm were found to be optimal. The assay is implemented in 20 min and is characterized by detection limits equal to 40 ng/mL for LPS and 3 × 10 4 CFU/mL for whole cells of F. tularensis . Thus, the proposed simple one-step assay integrates sensitivity comparable with other immunoassay of microorganisms and rapidity. Selectivity of the assay for different strains of F. tularensis was tested and the possibility to choose its variants with the use of different antibodies to distinguish virulent and non-virulent strains or to detect both kinds of F. tularensis was found. The test system has been successfully implemented to reveal the analyte in natural and tap water samples without the loss of sensitivity.

In this study, a lateral flow immunoassay (LFIA) was developed to detect okadaic acid (OA) belonging to the diarrheic shellfish poisoning group of aquatic toxins. Newly obtained anti-OA monoclonal antibodies and bimetallic core@shell Au@Pt nanoparticles were used in the indirect format of the LFIA. Peroxidase-mimicking nanozyme properties of Au@Pt enabled using them to enhance band coloration on the test strips and, consequently, for increasing the LFIA sensitivity. The instrumental limit of detection (LOD), the working range of detectable concentrations, and the visual cutoff of the assay were 0.5, 0.8-6.8, and 10 ng/mL, respectively. The assay duration was 20 min. The rapid and simple sample preparation procedure was applied for seawater, river water, and fish samples. The total duration of the sample pretreatment and LFIA was 25/40 min for water/fish samples, ensuring testing rapidity. The developed test system provides sensitive control of raw materials and food products and can be used to detect OA at all stages of the food industry «from sea to fork» chains.

The use of a large amount of toxic synthetic materials leads to an increase in the pollution of environmental objects. Phthalates are compounds structurally related to esters of phthalic acid that are widely used in the manufacturing of synthetic packaging materials as plasticizers. Their danger is conditioned by leaching into the environment and penetrating into living organisms with negative consequences and effects on various organs and tissues. This work presents the first development of lateral flow immunoassay to detect dibutyl phthalate, one of the most common representatives of the phthalates group. To form a test zone, a hapten-protein conjugate was synthesized, and gold nanoparticles conjugated with antibodies to dibutyl phthalate were used as a detecting conjugate. The work includes the preparation of immunoreagents, selectivity investigation, and the study of the characteristics of the medium providing a reliable optical signal. Under the selected conditions for the analysis, the detection limit was 33.4 ng/mL, and the working range of the determined concentrations was from 42.4 to 1500 ng/mL. Time of the assay-15 min. The developed technique was successfully applied to detect dibutyl phthalate in natural waters with recovery rates from 75 to 115%.

Aquatic toxins are a group of toxic compounds produced by several types of freshwater and marine algae and cyanobacteria and transported through the food chains of water bodies. Potential contamination of aquaculture products (raw and processed fish and seafood) with aquatic toxins requires the use of efficient screening methods for their control. In this study, a multiplex immunochromatographic test system for the simultaneous detection of three aquatic toxins-phycotoxins domoic acid (DA) and okadaic acid (OA), and cyanotoxin microcystin-LR (MC-LR)-is for the first time developed. For this, a competitive indirect immunochromatographic analysis (ICA) based on gold-labeled secondary antibodies was carried out. The LODs/cutoffs/working ranges of the ICA were 0.05/0.3/0.07-0.29, 1.3/100/3.2-58.2, and 0.1/2.0/0.2-1.1 ng/mL for MC-LR, DA, and OA, respectively. The assay duration was 18 min. The developed test system was used to analyze water samples from natural sources (salt and fresh water) and fish samples. For sample preparation of water, simple dilution with a buffer was proposed; for fish samples, methanol-water extraction was utilized. It was demonstrated that the triple LFIA specifically detected target aquatic toxins with recoveries of 85.0-121.5%. The developed multiplex LFIA can be considered a promising analytical solution for the rapid, easy, and sensitive control of water and food safety., (© 2022. Springer-Verlag GmbH Germany, part of Springer Nature.)

In this investigation, a double immunochromatographic analysis (ICA) of two relevant phycotoxins, domoic acid (DA) and okadaic acid (OA), was developed for the first time. The ICA was performed in the indirect competitive format using gold nanoparticles conjugated with anti-species antibodies. Under optimal conditions, the instrumental detection limits/cutoffs for simultaneous detection of DA and OA were 1.2/100 and 0.1/2.5 ng/mL, respectively. The time of the assay was 18 min. The ICA was applied to test seawater and a large panel of seafood, including mussels, tiger shrimps, octopuses, whelks, crabs, and scallops. The proposed simple sample preparation method for seafood takes only 20 min. For seawater, a dilution by buffer was implemented. The assay recoveries varied from 80.8% to 124.5%. The competitive potential of the proposed technique as a tool to control natural water and seafood samples is determined by its simplicity, rapidity, and sensitivity.

Magnetic beads (MBs) are often considered as an effective carrier in heterogeneous assays due to the simplicity of separation and washing, and the ability to increase and control the surface area. However, the effect of the MBs surface on the analytical parameters is poorly characterized and is often postulated from intuitive considerations. Herein, experimental evaluation through the comparison of MBs and microwell plate was carried out using the miRNA-141 (biomarker for cancer) as a target, the detection of which was performed by chemiluminescent assay with a homogeneous mismatched catalytic hairpin assembly (mCHA) reaction. The mCHA reaction produced double-stranded (ds) DNA labeled at one end with fluorescein (Flu) for capture with anti-Flu antibodies immobilized on a solid carrier, on the other end with biotin for recognition by streptavidin-polyperoxidase conjugate. The conditions of immobilization of anti-Flu antibody on MBs (a diameter of 440 nm) performed using a carbodiimide method were optimized by varying the antibody concentration in the reaction solution. It was shown that the dependence of chemiluminescent signal as a function of the concentration of anti-FluAb-MBs conjugates had a bell-shaped character. The maximum chemiluminescence was produced at the concentration of the conjugates of 2 × 10 9 particles/mL, with a surface area of 65 mm 2 . The identical surface area was used upon the assay performance with polystyrene microplates. Comparison of MBs- and microplate-assays for miRNA-141 determination showed that the obtained calibration curves and their detection limit values were the same and did not depend on the used carrier. The results showed that the choice of a carrier for heterogeneous assays should be guided by the convenience of the assay performance, not its surface area., (Copyright © 2022 Elsevier B.V. All rights reserved.)

CRISPR-Cas12-based biosensors are a promising tool for the detection of nucleic acids. After dsDNA-target-activated Cas12 cleaves the ssDNA probe, a lateral flow test (LFT) is applied for rapid, simple, and out-of-laboratory detection of the cleaved probe. However, most of the existing approaches of LFT detection have disadvantages related to inverted test/control zones in which the assay result depends not only on the cleavage of the probe but also on the second factor: the binding of the non-cleaved probe in the control zone. We proposed a novel platform for the detection of trans-cleaved DNA using a universal DNA-IgG probe and LFT with the sequential direct location of test and control zones. The advantage of the platform consists of the assay result depending only on the cleaved probe. For this, we designed a composite probe that comprise two parts: the DNA part (biotinylated dsDNA connected to ssDNA with fluorescein) (FAM), and the antibody part (mouse anti-FAM IgG). The Cas12, with guide RNA, was activated by the dsDNA-target. The activated Cas12 cleaved the probe, releasing the ssDNA-FAM-IgG reporter that was detected by the LFT. The sandwich LFT was proposed with anti-mouse IgG adsorbed in the test zone and on the surface of gold nanoparticles. We called the platform with direct location zones and direct analyte-signal dependence the DNA-Immunoglobulin Reporter Endonuclease Cleavage Test (DIRECT 2 ). Therefore, this proof-of-concept study demonstrated that the combination of the proposed DNA-IgG probe and direct LFT opens new opportunities for CRISPR-Cas12 activity detection and its bioanalytical applications., (Copyright © 2022 Elsevier B.V. All rights reserved.)

A lateral flow immunoassay (LFIA) of phycotoxin domoic acid (DA) contaminating seawater and marine organisms was developed in this investigation. Nine clones of monoclonal antibodies against DA were produced and characterized. The test system was implemented in the indirect competitive format, where gold nanoparticles as a marker were conjugated with secondary antibodies. The developed test system allows for the detection of DA with a cutoff of 60 ng mL -1 and an instrumental detection limit of 1.4 ng mL -1 within 15 min. The LFIA was applied to detect DA in seawater, mussels, shrimps, and octopuses. A simple method of seafood sample preparation was proposed. The entire analytical cycle, from obtaining a sample to the estimation of final results, takes only 30 min. The assay recoveries ranged from 88.5% to 124%. The developed analytical method is a promising solution for rapid on-site monitoring of marine toxicants in water and food throughout the farm-to-fork chain.

The presence of pathogen-specific antibodies in the blood is widely controlled by a serodiagnostic technique based on the lateral flow immunoassay (LFIA). However, its common one-stage format with an antigen immobilized in the binding zone of a test strip and a nanodispersed label conjugated with immunoglobulin-binding proteins is associated with risks of very low analytical signals. In this study, the first stage of the immunochromatographic serodiagnosis was carried out in its traditional format using a conjugate of gold nanoparticles with staphylococcal immunoglobulin-binding protein A and an antigen immobilized on a working membrane. At the second stage, a labeled immunoglobulin-binding protein was added, which enhanced the coloration of the bound immune complexes. The use of two separated steps, binding of specific antibodies, and further coloration of the formed complexes, allowed for a significant reduction of the influence of non-specific immunoglobulins on the assay results. The proposed approach was applied for the serodiagnosis using a recombinant RBD protein of SARS-CoV-2. As a result, an increase in the intensity of test zone coloration by more than two orders of magnitude was demonstrated, which enabled the significant reduction of false-negative results. The diagnostic sensitivity of the LFIA was 62.5% for the common format and 100% for the enhanced format. Moreover, the diagnostic specificity of both variants was 100%.

In this investigation, a new approach for developing a sensitive lateral flow immunoassay (LFIA) was proposed for the detection of the hazardous marine toxin okadaic acid (OA). It is based on the indirect format with anti-species antibodies labeled by gold nanoparticles (AuNPs) and cascade signal amplification. The latter is performed by first passing a mixture of anti-OA antibodies and a tested sample along the immunochromatographic test strip and then performing several cycles of the interaction of anti-species antibodies conjugated with AuNPs with free antibodies, which bind to anti-species antibodies but are not specific to the target analyte. As a result, branched aggregates are formed, due to which the colorimetric signal intensification occurs. The developed test system enabled the detection of OA with an instrumental detection limit of 30 pg/mL and a cutoff of 1 ng/mL, which exceeds these characteristics in the LFIA without amplification by 7 and 2 times, respectively. The OA recoveries from seawater, fish, and seafood varied from 76.9% to 126%. The test system may be required for point-of-care monitoring of samples for phycotoxin contamination; the developed principle of signal amplification can be used in cases where highly sensitive detection of trace amounts of a contaminant is required.

A new bioanalytical labeling system based on alloyed quantum dots' (QDs) photoluminescence quenching caused by an enzymatic reaction has been developed and tested for the first time. The catalytic role of the enzyme provides high sensitivity and the possibility of varying detecting time to improve assay sensitivity. Alloyed luminescent QDs were chosen in view of their small size (5-7 nm) and the high sensitivity of their optical properties to physicochemical interactions. Here, we described the synthesis of alloyed luminescent QDs and demonstrated the possibility of using them as a luminescent turn-off substrate for enzymatic assay. Synthesized alloyed QDs were found to be a sensitive turn-off substrate for glucose oxidase in homogeneous and heterogeneous assay models. CdZnSeS and CdZnSeS/ZnS QDs covered with dihydrolipoic acid and 2-mercaptoethanol were tested. A glucose oxidase limit of detection of 6.6 nM for the heterogenous high-throughput model assay was reached., (© 2022. Springer-Verlag GmbH Germany, part of Springer Nature.)

A new scheme of reagents interaction for lateral flow immunoassay (LFIA) is proposed, which combines the features of competitive and sandwich assay and provides highly sensitive detection of low-molecular-weight analytes. Namely, the antigen in the sample interferes with the formation of the antibody (on the membrane)-hapten-protein-antibody (on the nanoparticle-marker) complex, competing with hapten-protein conjugate in both reactions. The proposed scheme was modelled using COPASI software, with a prediction of limit of detection (LOD) decrease by one order of magnitude compared to the standard competitive LFIA. This feature was experimentally confirmed for the detection of chloramphenicol (CAP) in honey. When tested in spiked honey, the visual LOD was 50 ng/mL for the common scheme and 5 ng/mL for the proposed scheme. Instrumental LOD was 300 pg/mL (1.2 µg/kg in conversion per sample weight of honey) in the standard scheme and 20 pg/mL (80 ng/kg in conversion per sample weight of honey) in the proposed scheme.

Short oligonucleotides are widely used for the construction of aptamer-based sensors and logical bioelements to modulate aptamer-ligand binding. However, relationships between the parameters (length, location of the complementary region) of oligonucleotides and their influence on aptamer-ligand interactions remain unclear. Here, we addressed this task by comparing the effects of short complementary oligonucleotides (ssDNAs) on the structure and ligand-binding ability of an aptamer and identifying ssDNAs' features that determine these effects. Within this, the interactions between the OTA-specific G-quadruplex aptamer 1.12.2 (5'-GATCGGGTGTGGGTGGCGTAAAGGGA GCATCGGACA-3') and 21 single-stranded DNA (ssDNA) oligonucleotides complementary to different regions of the aptamer were studied. Two sets of aptamer-ssDNA dissociation constants were obtained in the absence and in the presence of OTA by isothermal calorimetry and fluorescence anisotropy, respectively. In both sets, the binding constants depend on the number of hydrogen bonds formed in the aptamer-ssDNA complex. The ssDNAs' having more than 23 hydrogen bonds with the aptamer have a lower aptamer dissociation constant than for aptamer-OTA interactions. The ssDNAs' having less than 18 hydrogen bonds did not affect the aptamer-OTA affinity. The location of ssDNA's complementary site in the aptamer affeced the kinetics of the interaction and retention of OTA-binding in aptamer-ssDNA complexes. The location of the ssDNA site in the aptamer G-quadruplex led to its unfolding. In the presence of OTA, the unfolding process was longer and takes from 20 to 70 min. The refolding in the presence of OTA was possible and depends on the length and location of the ssDNA's complementary site. The location of the ssDNA site in the tail region led to its rapid displacement and wasn't affecting the G-qaudruplex's integrity. It makes the tail region more perspective for the development of ssDNA-based tools using this aptamer.

The growing interest in the development of new platforms for the application of Raman spectroscopy techniques in biosensor technologies is driven by the potential of these techniques in identifying chemical compounds, as well as structural and functional features of biomolecules. The effect of Raman scattering is a result of inelastic light scattering processes, which lead to the emission of scattered light with a different frequency associated with molecular vibrations of the identified molecule. Spontaneous Raman scattering is usually weak, resulting in complexities with the separation of weak inelastically scattered light and intense Rayleigh scattering. These limitations have led to the development of various techniques for enhancing Raman scattering, including resonance Raman spectroscopy (RRS) and nonlinear Raman spectroscopy (coherent anti-Stokes Raman spectroscopy and stimulated Raman spectroscopy). Furthermore, the discovery of the phenomenon of enhanced Raman scattering near metallic nanostructures gave impetus to the development of the surface-enhanced Raman spectroscopy (SERS) as well as its combination with resonance Raman spectroscopy and nonlinear Raman spectroscopic techniques. The combination of nonlinear and resonant optical effects with metal substrates or nanoparticles can be used to increase speed, spatial resolution, and signal amplification in Raman spectroscopy, making these techniques promising for the analysis and characterization of biological samples. This review provides the main provisions of the listed Raman techniques and the advantages and limitations present when applied to life sciences research. The recent advances in SERS and SERS-combined techniques are summarized, such as SERRS, SE-CARS, and SE-SRS for bioimaging and the biosensing of molecules, which form the basis for potential future applications of these techniques in biosensor technology. In addition, an overview is given of the main tools for success in the development of biosensors based on Raman spectroscopy techniques, which can be achieved by choosing one or a combination of the following approaches: (i) fabrication of a reproducible SERS substrate, (ii) synthesis of the SERS nanotag, and (iii) implementation of new platforms for on-site testing.

The current COVID-19 pandemic has increased the demand for pathogen detection methods that combine low detection limits with rapid results. Despite the significant progress in methods and devices for nucleic acid amplification, immunochemical methods are still preferred for mass testing without specialized laboratories and highly qualified personnel. The most widely used immunoassays are microplate enzyme-linked immunosorbent assay (ELISA) with photometric detection and lateral flow immunoassay (LFIA) with visual results assessment. However, the disadvantage of ELISA is its considerable duration, and that of LFIA is its low sensitivity. In this study, the modified LFIA of a specific antigen of the causative agent of COVID-19, spike receptor-binding domain, was developed and characterized. This modified LFIA includes the use of gold nanoparticles with immobilized antibodies and 4-mercaptobenzoic acid as surface-enhanced Raman scattering (SERS) nanotag and registration of the nanotag binding by SERS spectrometry. To enhance the sensitivity of LFIA-SERS analysis, we determined the optimal compositions of SERS nanotags and membranes used in LFIA. For benchmark comparison, ELISA and conventional colorimetric LFIA were used with the same immune reagents. The proposed method combines a low detection limit of 0.1 ng/mL (at 0.4 ng/mL for ELISA and 1 ng/mL for qualitative LFIA) with a short assay time equal to 20 min (at 3.5 h for ELISA and 15 min for LFIA). The results obtained demonstrate the promise of using the SERS effects in membrane immuno-analytical systems.

The detection limit of lateral flow immunoassay (LFIA) is largely determined by the properties of the label used. We compared four nanoparticle labels differing in their chemical composition and colour: (1) gold nanoparticles (Au NPs), red; (2) Au-core/Pt-shell nanoparticles (Au@Pt NPs), black; (3) latex nanoparticles (LPs), green; and (4) magnetic nanoparticles (MPs), brown. The comparison was carried out using one target analyte- Erwinia amylovora , the causal bacterial agent of fire blight. All nanoparticles were conjugated with antibodies through methods that provide maximum functional coverage like physical adsorption (Au NPs, Au@Pt NPs) and covalent bonding (LPs, MPs). All conjugates demonstrated the same ability to bind with E. amylovora through enzyme-linked immunosorbent assay where optical properties of the nanoparticles do not determine the registered signal. However, half-maximal binding was achieved at different numbers of nanoparticles because they differ in size. All conjugates based on four nanoparticle labels were used for lateral flow assays. As a result, Au@Pt NPs provided the minimal detection limit that corresponded to 10 3 CFU/mL. Au NPs and LPs detected 10 4 CFU/mL, and MPs detected 10 5 CFU/mL. The results highlight that simply choosing a coloured label can significantly affect the detection limit of LFIA.

Antibody-nanoparticle conjugates are widely used analytical reagents. An informative parameter reflecting the conjugates' properties is the number of antibodies per nanoparticle that retain their antigen-binding ability. Estimation of this parameter is characterized by a lack of simple, reproducible methods. The proposed method is based on the registration of fluorescence of tryptophan residues contained in proteins and combines sequential measurements of first the immobilized antibody number and then the bound protein antigen number. Requirements for the measurement procedure have been determined to ensure reliable and accurate results. Using the developed technique, preparations of spherical gold nanoparticles obtained by the most common method of citrate reduction of gold salts (the Turkevich-Frens method) and varying in average diameter from 15 to 55 nm have been characterized. It was shown that the number of antibodies (immunoglobulins G) bound by one nanoparticle ranged from 30 to 194 during adsorptive unoriented monolayer immobilization. C-reactive protein was considered as the model antigen. The percentage of antibody valences that retained their antigen-binding properties in the conjugate increased from 17 to 34% with an increase in the diameter of gold nanoparticles. The proposed method and the results of the study provide tools to assess the capabilities of the preparations of gold nanoparticles and their conjugates as well as the expediency of seeking the best techniques for various practical purposes.

Verifying the authenticity of food products is essential due to the recent increase in counterfeit meat-containing food products. The existing methods of detection have a number of disadvantages. Therefore, simple, cheap, and sensitive methods for detecting various types of meat are required. In this study, we propose a rapid full-cycle technique to control the chicken or pig adulteration of meat products, including 3 min of crude DNA extraction, 20 min of recombinase polymerase amplification (RPA) at 39 °C, and 10 min of lateral flow assay (LFA) detection. The cytochrome B gene was used in the developed RPA-based test for chicken and pig identification. The selected primers provided specific RPA without DNA nuclease and an additional oligonucleotide probe. As a result, RPA-LFA, based on designed fluorescein- and biotin-labeled primers, detected up to 0.2 pg total DNA per μL, which provided up to 0.001% w / w identification of the target meat component in the composite meat. The RPA-LFA of the chicken and pig meat identification was successfully applied to processed meat products and to meat after heating. The results were confirmed by real-time PCR. Ultimately, the developed analysis is specific and enables the detection of pork and chicken impurities with high accuracy in raw and processed meat mixtures. The proposed rapid full-cycle technique could be adopted for the authentication of other meat products.

Rapid, sensitive, and timely diagnostics are essential for protecting plants from pathogens. Commonly, PCR techniques are used in laboratories for highly sensitive detection of DNA/RNA from viral, viroid, bacterial, and fungal pathogens of plants. However, using PCR-based methods for in-field diagnostics is a challenge and sometimes nearly impossible. With the advent of isothermal amplification methods, which provide amplification of nucleic acids at a certain temperature and do not require thermocyclic equipment, going beyond the laboratory has become a reality for molecular diagnostics. The amplification stage ceases to be limited by time and instruments. Challenges to solve involve finding suitable approaches for rapid and user-friendly plant preparation and detection of amplicons after amplification. Here, we summarize approaches for in-field diagnostics of phytopathogens based on different types of isothermal amplification and discuss their advantages and disadvantages. In this review, we consider a combination of isothermal amplification methods with extraction and detection methods compatible with in-field phytodiagnostics. Molecular diagnostics in out-of-lab conditions are of particular importance for protecting against viral, bacterial, and fungal phytopathogens in order to quickly prevent and control the spread of disease. We believe that the development of rapid, sensitive, and equipment-free nucleic acid detection methods is the future of phytodiagnostics, and its benefits are already visible.

The combination of recombinase polymerase amplification (RPA) and lateral flow test (LFT) is a strong diagnostic tool for rapid pathogen detection in resource-limited conditions. Here, we compared two methods generating labeled RPA amplicons following their detection by LFT: (1) the basic one with primers modified with different tags at the terminals and (2) the nuclease-dependent one with the primers and labeled oligonucleotide probe for nuclease digestion that was recommended for the high specificity of the assay. Using both methods, we developed an RPA-LFT assay for the detection of worldwide distributed phytopathogen-alfalfa mosaic virus (AMV). A forward primer modified with fluorescein and a reverse primer with biotin and fluorescein-labeled oligonucleotide probe were designed and verified by RPA. Both labeling approaches and their related assays were characterized using the in vitro-transcribed mRNA of AMV and reverse transcription reaction. The results demonstrated that the RPA-LFT assay based on primers-labeling detected 10 3 copies of RNA in reaction during 30 min and had a half-maximal binding concentration 22 times lower than probe-dependent RPA-LFT. The developed RPA-LFT was successfully applied for the detection of AMV-infected plants. The results can be the main reason for choosing simple labeling with primers for RPA-LFT for the detection of other pathogens.

Many studies have found that gold nanoparticles with branched surfaces (nanoflowers) are markers for immunosensors that provide higher sensitivity gains than the commonly used spherical gold nanoparticles. Although the analytical characteristics of nanoparticle-using systems vary significantly depending on their size and shape, the question of choosing the best gold nanoflowers remains open. This work presents a comparative study of a panel of 33 preparations of gold nanoflowers formed by varying several parameters: the size of spherical nanoparticles-nuclei, the concentrations of nuclei, and tetrachloroauric acid during growth. The sizes of the resulting particles, their sorption capacity under antibody immobilization, mobility along membranes for lateral flow assays, and the effects of these parameters on the limits of detection of lateral flow immunoassay are characterized. The optimality of preparations obtained by growing a 0.2% v / v solution of nuclei with a diameter of 10 or 20 nm with tetrachloroauric acid at a concentration of 0.12 mM was shown. With their use, lateral flow immune tests were developed to determine markers of acute myocardial infarction-fatty acids binding protein and troponins I and T. The use of gold nanoflowers obtained under the proposed protocols led to significant gains in the limits of detection-3 to 10 times under visual detection and over 100 times under instrumental detection-compared to spherical gold nanoparticles. The significant increase under instrumental detection is due to the label's low nonspecific binding.

In this study, we developed a lateral flow immunoassay (LFIA) for the detection of pork additives in meat products. LFIA of porcine immunoglobulins (IgG) as a molecular biomarker was carried out in a sandwich format for species identification. Gold nanoparticles as a nano-dispersed label were conjugated to secondary antibodies specific to anti-porcine IgG. The test system was characterized by high specificity, which was confirmed by the absence of cross-reactivity with any other species tested. A short technique of sample preparation was proposed aimed at the effective extraction of IgG from meat samples. The developed LFIA enabled the detection of a pork ingredient at a level as low as 0.063% (w/w) in raw meat within 35 min including sample preparation. A large panel of real meat samples was analyzed by the LFIA. The results showed that porcine IgG can be reliably recognized both in raw meat products and processed meat foodstuffs., (Copyright © 2021 Elsevier Ltd. All rights reserved.)