Dual lateral flow test for simple and rapid simultaneous immunodetection of bisphenol A and dimethyl phthalate, two priority plastic related environmental contaminants.

Contamination of the environment by technogenic endocrine disrupting compounds (EDCs) becomes serious threat to public health. To effectively prevent this threat, it is necessary to improve analytical methods for EDCs to ensure mass, fast and productive monitoring. In the given work, a dual lateral flow test (LFT) is developed in the first time for simultaneous immunodetection of bisphenol A and dimethyl phthalate, priority EDCs releasing from plastic and belonging to different chemical classes. It combines integrated detection of two EDCs by one analytical system with rapidity and simplicity of LFTs allowing for off-lab testing without additional reagents and devices. Gold nanoparticles differing in shape and color (red gold nanospheres and blue gold nanoflowers) are applied as markers to simplify interpretation of the obtained results. Under the optimal conditions chosen for efficient control of the both analytes, the detection limits of bisphenol A and dimethyl phthalate are 0.67 ng/mL and 2.22 ng/mL, respectively. Time of the assay is 15 min. The proposed dual LFT has confirmed its practical applicability by analyzing natural water samples with recovery of bisphenol A and dimethyl phthalate in the ranges of 90.4-107.0% and 86.8-118.0%, respectively.

Prussian-Blue-Nanozyme-Enhanced Simultaneous Immunochromatographic Control of Two Relevant Bacterial Pathogens in Milk.

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 × 102 and 7 × 103 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.

Freeze-Driven Adsorption of Oligonucleotides with polyA-Anchors on Au@Pt Nanozyme.

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 (An, with n = 3, 5, 7, 10; triple-branched A3, or triple-branched A5), 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 A7 > A5 > 3A3 > 3A5 > A10 > A3 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 A3, A5, A7, and 3A3 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.

Polycations as Aptamer-Binding Modulators for Sensitive Fluorescence Anisotropy Assay of Aflatoxin B1.

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.

Double lateral flow immunosensing of undeclared pork and chicken components of meat products.

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.

Comparison of competitive and sandwich immunochromatographic analysis in the authentication of chicken in meat products.

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.

Sensitive immunoenzyme assay for the detection of antibiotic flumequine in honey.

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.

A Fluorescence Resonance Energy Transfer Aptasensor for Aflatoxin B1 Based on Ligand-Induced ssDNA Displacement.

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.

Sensitive Immunochromatographic Determination of Salmonella typhimurium in Food Products Using Au@Pt Nanozyme.

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 × 102 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.

Ability of Antibodies Immobilized on Gold Nanoparticles to Bind Small Antigen Fluorescein.

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.

DNA Probes for Cas12a-Based Assay with Fluorescence Anisotropy Enhanced Due to Anchors and Salts.

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 (Mg2+, Ca2+, Ba2+), 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 Mg2+. 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.

Sensitive Silver-Enhanced Microplate Apta-Enzyme Assay of Sb3+ Ions in Drinking and Natural Waters.

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 Sb3+ detection was developed. The biotinylated aptamer A10 was hybridized with its complementary biotinylated oligonucleotide T10 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. Sb3+ presenting in the sample bounded to an A10 aptamer, thus releasing T10, preventing streptavidin-HRP binding and, as a result, reducing the optical signal. This effect allowed for the detection of Sb3+ 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 A10/T10 complex formation promoted dehybridization of the aptamer A10 and the formation of the A10/Sb3+ complex. The working range of the Ag+-enhanced microplate apta-enzyme assay for Sb3+ 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 Sb3+ in the range of 109.0-126.2% and 99.6-106.1%, respectively.

Post-Assay Chemical Enhancement for Highly Sensitive Lateral Flow Immunoassays: A Critical Review.

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.

A Critical Study on DNA Probes Attached to Microplate for CRISPR/Cas12 Trans-Cleavage Activity.

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.

Comparison of Single-Stranded DNA Probes Conjugated with Magnetic Particles for Trans-Cleavage in Cas12a-Based Biosensors.

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.

Comparison of Three Lateral Flow Immunoassay Formats for the Detection of Antibodies against the SARS-CoV-2 Antigen.

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.

A Sensitive Fluorescence Polarization Immunoassay for the Rapid Detection of Okadaic Acid in Environmental Waters.

In this study, a homogeneous fluorescence polarization immunoassay (FPIA) for the detection of hazardous aquatic toxin okadaic acid (OA) contaminating environmental waters was for the first time developed. A conjugate of the analyte with a fluorophore based on a fluorescein derivative (tracer) was synthesized, and its interaction with specific anti-OA monoclonal antibodies (MAbs) was tested. A MAbs-tracer pair demonstrated highly affine immune binding (KD = 0.8 nM). Under optimal conditions, the limit of OA detection in the FPIA was 0.08 ng/mL (0.1 nM), and the working range of detectable concentrations was 0.4-72.5 ng/mL (0.5-90 nM). The developed FPIA was approbated for the determination of OA in real matrices: river water and seawater samples. No matrix effect of water was observed; therefore, no sample preparation was required before analysis. Due to this factor, the entire analytical procedure took less than 10 min. Using a compact portable fluorescence polarization analyzer enables the on-site testing of water samples. The developed analysis is very fast, easy to operate, and sensitive and can be extended to the determination of other aquatic toxins or low-molecular-weight water or food contaminants.

Modular Set of Reagents in Lateral Flow Immunoassay: Application for Antibiotic Neomycin Detection in Honey.

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.

Enhanced Lateral Flow Immunoassay with Double Competition and Two Kinds of Nanoparticles Conjugates for Control of Insecticide Imidacloprid in Honey.

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.

Engineering of DNA Structures Attached to Magnetic Particles for Effective Trans- and Cis-Cleavage in Cas12-Based Biosensors.

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.