Sandwich enzyme-linked aptamer-based assay for the detection of Trichomonas vaginalis.

Trichomoniasis is the most prevalent curable, non-viral sexually transmitted infection (STI), with an estimated 156 million new infections in 2020. It can potentially result in adverse birth outcomes as well as infertility in men, whilst it also increases the risk of acquiring HIV and contracting other vaginal infections. It is mostly prevalent among women in low-income countries and especially in Africa and the Americas. This STI is caused by Trichomonas vaginalis (TV) and a robust, cost-effective, sensitive, specific and rapid diagnostic test is urgently required. We report the screening of 6 full-length and 4 truncated aptamers previously selected in our group for use in a microplate-based sandwich assay. The combination of dual aptamers comprising a short 14-mer truncated capture aptamer (termed A1_14mer) and a full-length non-truncated reporter aptamer (A6) was elucidated to be the optimum pair for a sensitive sandwich enzyme-linked aptamer assay (ELAA) for the detection of TV achieving a detection limit of 3.02 × 104 TV cells/mL. The results obtained with the A1_14mer-A6 ELAA correlate excellently with wet-mount microscopy for the detection of TV in clinical specimens, cervicovaginal lavages and vaginal swabs, highlighting the potential clinical application of this assay for cost-effective population screening and subsequent prevention of the onset of complications associated with undiagnosed and untreated TV.

DNA Meets Protein─Development, Characterization, and Application of Aptamers against Peanut Allergen.

Allergen detection methods support food labeling and quality assessment at the allergen component level of allergen preparations used for allergy diagnosis and immunotherapy (AIT). Commonly applied enzyme-linked immunosorbent assay (ELISA) requires animal antibodies but potentially shows batch variations. We developed synthetic aptamers as alternative binders in allergen detection to meet the replacement, reduction, and refinement (3R) principle on animal protection in science. ssDNA aptamers were specifically selected against the major peanut allergen Ara h 1 and identified by next-generation sequencing. Application in various detection systems (ELISA-like assays, western blot, and surface plasmon resonance) was demonstrated. The ELISA-like assay comprised a sensitivity of 10 ng/mL Ara h 1, comparable to published antibody-based ELISA, and allowed Ara h 1 detection in various peanut flours, similar to those used in peanut AIT as well as in processed food. This ELISA-like aptamer-based assay proofs antibody-free allergen detection for food labeling or quality assessment of diagnostic and therapeutic allergen products.

Characterization of an Aptamer Targeting Neu5Gc, as an Endogenous Pathogenic Factor Derived from Red Meat.

N-glycolylneuraminic acid (Neu5Gc), a sialic acid predominantly found in the non-neurohumoral fluids of hind-mouthed animals, is incapable of synthesizing Neu5Gc due to a deletion in the CMAH exon of the gene encoding human CMP-Neu5Gc hydroxylase. But consumption of animal-derived foods that contain Neu5Gc, such as red meat, can instigate an immune response in humans, as Neu5Gc is recognized as a foreign substance by the human immune system. This recognition leads to the production of anti-Neu5Gc antibodies, subsequently resulting in chronic inflammation. When Neu5Gc is consumed excessively or frequently, it may contribute to the development of heart disease and cancer. This makes Neu5Gc, an endogenous pathogenic factor derived from red meat, a new hot topic in red meat safety research. In this study, aptamers obtained by the magnetic bead SELEX technique were subjected to homology and secondary structure prediction analysis as well as affinity determination. The result indicated that the aptamer 2B.N2A9 exhibited a robust binding affinity, with an affinity constant (Ka) of 1.87 × 108 L/mol. This aptamer demonstrated optimal binding specificity within a pH range of 5.4 to 7.4. Molecular docking analysis further revealed that aptamer 2B.N2A9 formed stable binding interactions with the target Neu5Gc at specific sites, namely G-14, C-15, G-13, G-58, G-60, and C-59. An Enzyme-Linked Oligonucleotide Sorbent Assay (ELOSA) methodology was established to detect the endogenous pathogenic factor Neu5Gc present in red meat. This method demonstrated a limit of detection (LOD) of 0.71 ng/mL, along with an average recovery rate of 92.23%. The aptamer obtained in this study exhibited favorable binding properties to Neu5Gc. The assay was relatively convenient and demonstrated good sensitivity. Further investigation into the distribution of Neu5Gc in various red meats is of public health significance and scientific potential. A practical detection method should be provided to guide red meat diets and ensure the nutrition and safety of meat products.

Zeolitic Imidazolate Framework-8 Composite-Based Enzyme-Linked Aptamer Assay for the Sensitive Detection of Deoxynivalenol.

The mycotoxin deoxynivalenol (DON) is a prevalent contaminant in cereals that threatens the health of both humans and animals and causes economic losses due to crop contamination. The rapid and sensitive detection of DON is essential for food safety. Herein, a colorimetric biosensor based on horseradish peroxidase- and gold nanoparticle-encapsulated zeolitic imidazolate framework-8 (HRP&Au@ZIF-8) was developed for the sensitive screening of DON. The synthesized HRP&Au@ZIF-8 probes not only held great potential for signal amplification but also exhibited stable catalytic activity even under extreme conditions, which endowed the biosensor with both good sensitivity and stability. Under the optimized conditions, qualitative measurement of DON can be achieved through visual inspection, and quantitative evaluation can be performed via absorbance measurements at a characteristic wavelength of 450 nm. The proposed method has demonstrated high sensitivity with a linear detection range of 1-200 ng/mL and a detection limit of 0.5068 ng/mL. It also presented good selectivity and reliability. Furthermore, DON in spiked cereal samples has been quantified successfully using this method. This novel approach demonstrates significant potential for the facile and expeditious detection of DON in cereal products and brings us one step closer to enhancing food safety.

Enzyme-Linked Aptamer Kits for Rapid, Visual, and Sensitive Determination of Lactoferrin in Dairy Products.

Lactoferrin (Lf), as a popular nutritional fortification in dairy products, has the ability regulate the body's immune system and function as a broad-spectrum antibacterial, which is of great significance to the growth and development of infants and children. Herein, an indirect competitive enzyme-linked aptamer assay (ELAA) kit was established for rapid, sensitive, and visual determination of Lf in dairy products. In the construction, the Lf aptamer was conjugated with horseradish peroxidase (HRP) as the recognition probe and aptamer complementary strand (cDNA) were anchored onto the microplate as the capture probe. The recognition probes were first mixed with a sample solution and specifically bound with the contained Lf, then added into the microplate in which the free recognition probes in the mixture were captured by the capture probe. After washing, the remaining complex of cDNA/Aptamer/HRP in the microplate was conducted with a chromogenic reaction through HRP, efficiently catalyzing the substrate 3, 3', 5, 5'-tetramethylbenzidine (TMB), therefore the color shade would directly reflect Lf concentration. Under the optimization conditions, a good linear relationship (R2, 0.9901) was obtained in the wide range of 25-500 nM with the detection limit of 14.01 nM and a good specificity, as well as the reliable recoveries. Furthermore, the ELAA kits achieved the Lf determination with an accuracy of 79.71~116.99% in eleven samples, which consisted of three kinds of dairy products: including goat milk powder, cow milk powder, and nutrition drop. Moreover, the results were also validated by the high-performance capillary electrophoresis (HPCE) method. The ELAA kit provides a simple and convenient determination for Lf in dairy products, and it is highly expected to be commercialized.

A high-affinity aptamer with base-appended base-modified DNA bound to isolated authentic SARS-CoV-2 strains wild-type and B.1.617.2 (delta variant).

Simple, highly sensitive detection technologies for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are crucial for the effective implementation of public health policies. We used the systematic evolution of ligands by exponential enrichment with a modified DNA library, including a base-appended base (uracil with a guanine base at its fifth position), to create an aptamer with a high affinity for the receptor-binding domain (RBD) of the SARS-CoV-2 spike glycoprotein. The aptamer had a dissociation constant of 1.2 and < 1 nM for the RBD and spike trimer, respectively. Furthermore, enzyme-linked aptamer assays confirmed that the aptamer binds to isolated authentic SARS-CoV-2 wild-type and B.1.617.2 (delta variant). The binding signal was larger that of commercially available anti-SARS-CoV-2 RBD antibody. Thus, this aptamer as a sensing element will enable the highly sensitive detection of SARS-CoV-2.

Selection, identification, and application of DNA aptamers against bovine pregnancy-associated glycoproteins 4.

The bovine pregnancy-associated glycoproteins (bPAGs) have been widely used as robust markers for early diagnosis of pregnancy in the cattle. The current immune recognition methods for detecting bPAGs are limited and, to a certain extent, are associated with high costs and poor stability of the antibody. Aptamers that are more stable and easily synthesized than antibodies might serve as suitable candidates for the development of rapid detection methods. This paper describes selection and characterization of bPAG4 aptamers and theirs applicability to detect bPAG4 in the serum. In this work, the recombinant bovine pregnancy-associated glycoproteins 4 (bPAG4) with a relative molecular mass of about 48 kDa was successfully expressed in human embryonic kidney 293 (HEK 293) cells. Subsequently, the ssDNA aptamers were selected by systematic evolution of ligands by exponential enrichment (SELEX) using magnetic beads (MB) coated with bPAG4 as target. After 9 rounds of selection, three aptamers with high affinity to bPAG4 (Kd = 11.7~40.2 nM) were identified. The selected aptamers were successfully used in enzyme-linked aptamer assay (ELAA) to detect bPAG4 at a detection limit of 0.09 ng/mL. Meanwhile, it has been successfully applied for the detection of bPAG4 in serum samples. This work demonstrated that the selected aptamers could be used as promising affinity probes in the development of inexpensive, simple, and sensitive analysis methods for detecting bPAGs. Graphical abstract.

Evaluation of Staphylococcus aureus DNA aptamer by enzyme-linked aptamer assay and isothermal titration calorimetry.

To monitor the specificity of Staphylococcus aureus aptamer (SA-31) against its target cell, we used enzyme-linked aptamer assay. In the presence of target cell, horseradish peroxidase-conjugated streptavidin bound to biotin-labeled SA-31 showed specific binding to S  aureus among 3 different bacteria with limit of detection of 103 colony-forming unit per milliliter. The apparent Ka was 1.39 µM-1  ± 0.3 µM-1 . The binding of SA-31 to membrane proteins extracted from cell surface was characterized using isothermal titration calorimetry, and the effect of changes in binding temperature and salt concentrations of binding buffer was evaluated based on thermodynamic parameters (Ka , ΔH, and ΔG). Since binding of aptamer to its targets solely depends on its 3-dimensional structure under experimental conditions used in selection process, the change in temperature and ion concentration changed the affinity of SA-31 to its target on surface of bacteria. At 4°C, SA-31 did not show an affinity to its target with poor heat change upon injection of membrane fraction to aptamer solution. However, the apparent association constants of SA-31 slightly varied from Ka  = 1.56 µM-1  ± 0.69 µM-1 at 25°C to Ka  = 1.03 µM-1  ± 0.9 µM-1 at 37°C. At spontaneously occurring exothermic binding reactions, affinities of S aureus aptamer to its target were also 9.44 µM-1  ± 0.38 µM-1 at 50mM, 1.60 µM-1  ± 0.11 µM-1 at 137mM, and 3.28 µM-1  ± 0.46 µM-1 at 200 mM of salt concentration. In this study, it was demonstrated that enzyme-linked aptamer assay and isothermal titration calorimetry were useful tools for studying the fundamental binding mechanism between a DNA aptamer and its target on the outer surface of S aureus.

Screening and identification of a DNA aptamer to concanavalin A and its application in food analysis.

Herein, a novel aptamer that targets concanavalin A (Con A), a plant lectin, is isolated using systematic evolution of ligands by an exponential enrichment (SELEX) technique. Nine rounds of SELEX screening over an agarose spin column have resulted in enrichment of eight sequences having high affinity to Con A. The highest affinity sequence was selected as a potent aptamer and characterized it in detail. The evolved Con A aptamer (Con A-aptabody) is a 41 nt ssDNA that binds the Con A specifically with a dissociation constant of 172.7 ± 29.7 nM. In silico analyses predict the Con A-aptabody to form G-quadruplex due to its G-rich sequence (GGAAGGCGGAGGG). A detection method developed using Con A-aptabody is found to have a detection range of 10-750 ng/mL with limits of detection and quantification being 13.22 and 44.09 ng/mL, respectively. The utility of the method is demonstrated by analyzing jack bean (Canavalia ensiformis), kidney bean (Phaseolus vulgaris), wheat (Triticum spp.), mung bean (Vigna radiata), and lentil (Lens culinaris) for their Con A contents. Hence, the developed Con A-aptabody provides a useful substitute to Con A-antibody for food analysis and related applications.

A direct competitive assay-based aptasensor for sensitive determination of tetracycline residue in honey.

Tetracycline (TC) is a common antibacterial agent used for prevention and control of animal diseases. The increasing concern about TC residue in food demands high-performing analytical techniques for food quality assessment. Biosensors represent a promising tool for food safety analysis as they can fulfill some demand that the conventional methods do not attain. In this study, a novel colorimetric aptasensor was developed for sensitive detection of TC in honey. The aptasensor was based on a modified direct competitive enzyme-linked aptamer assay (dc-ELAA) scheme utilizing a 76 mer single-stranded DNA (ssDNA) aptamer selected by Systematic Evolution of Ligands by Exponential Enrichment (SELEX). The optimized aptasensor showed a good limit of detection (LOD of 0.0978 ng/mL), a wide linear range (0.1-1000 ng/mL) toward TC in honey, with good recoveries (92.09-109.7%) in TC-spiked honey, and was compared with an indirect competitive assay-based aptasensor and validated with a standard ELISA. The biosensor based on dc-ELAA with good limit of detection and simplicity can be applied for high-throughput detection of TC in food.

Selection of aptamers for fluorescent detection of alpha-methylacyl-CoA racemase by single-bead SELEX.

This paper first reports DNA aptamers and a fluorescent enzyme-linked aptamer assay (ELAA) targeting alpha-methylacyl-CoA racemase (AMACR), an emerging prostate cancer biomarker. The aptamers were in vitro selected using a new single-bead SELEX approach, which was rapid and consumed only ca. 45 ng AMACR. Before SELEX, silane chemistry was used to prepare epoxide-functionalized glass microbeads (EGBs, 500 µm in size and manipulated by tweezers) for AMACR coating. Recombinant AMACR was also prepared. During SELEX, the ligand evolution was assured by a differential real-time quantitative PCR assay. After SELEX, the aptamers were identified by the alignment analysis and 2nd structure prediction from the selected, cloned sequences. The circular dichroism (CD) analysis revealed that the aptamers formed stable B-form, stem-loop conformations. The fluorescent ELAA method confirmed the nM-level affinity and high specificity of the aptamers against AMACR. Finally, an aptamer-based fluorescent AMACR assay was demonstrated. The assay featured a wide dynamic range (from 10(-1) to 10(3) nM of AMACR), a low detection limit of 0.44 nM (19.5 ng/mL), and high AMACR specificity and is promising for clinical AMACR diagnostics.

Development of an indirect competitive assay-based aptasensor for highly sensitive detection of tetracycline residue in honey.

Tetracycline (TC) is widely used for prevention and control of animal diseases for its broad spectrum antimicrobial activity and low cost, but its abuse can seriously affect human health and may result in trade loss. Thus there is an imperative need to develop high-performing analytical technique for TC detection. In this study, we developed a biosensor based on an indirect competitive enzyme-linked aptamer assay (ic-ELAA). A 76mer single-stranded DNA (ssDNA) aptamer, selected by Systematic Evolution of Ligands by Exponential Enrichment (SELEX), was applied for the recognition and detection of TC in honey. The limit of detection was 9.6×10(-3) ng/mL with a linear working range from 0.01 to 100 ng/mL toward TC in honey, and a mean recovery rate of 93.23% in TC-spiked honey was obtained. This aptasensor can be applied to detect TC residue in food with high sensitivity and simplicity, and it is prospective to develop useful ELAA Kits for TC determination in food.

Selection of DNA aptamers that bind to influenza A viruses with high affinity and broad subtype specificity.

Many cases of influenza are reported worldwide every year. The influenza virus often acquires new antigenicity, which is known as antigenic shift; this results in the emergence of new virus strains, for which preexisting immunity is not found in the population resulting in influenza pandemics. In the event a new strain emerges, diagnostic tools must be developed rapidly to detect the novel influenza strain. The generation of high affinity antibodies is costly and takes time; therefore, an alternative detection system, aptamer detection, provides a viable alternative to antibodies as a diagnostic tool. In this study, we developed DNA aptamers that bind to HA1 proteins of multiple influenza A virus subtypes by the SELEX procedure. To evaluate the binding properties of these aptamers using colorimetric methods, we developed a novel aptamer-based sandwich detection method employing our newly identified aptamers. This novel sandwich enzyme-linked aptamer assay successfully detected the H5N1, H1N1, and H3N2 subtypes of influenza A virus with almost equal sensitivities. These findings suggest that our aptamers are attractive candidates for use as simple and sensitive diagnostic tools that need sandwich system for detecting the influenza A virus with broad subtype specificities.

Tailing DNA aptamers with a functional protein by two-step enzymatic reaction.

An efficient, quantitative synthetic strategy for aptamer-enzyme conjugates was developed by using a two-step enzymatic reaction. Terminal deoxynucleotidyl transferase (TdT) was used to first incorporate a Z-Gln-Gly (QG) modified nucleotide which can act as a glutamine donor for a subsequent enzymatic reaction, to the 3'-OH of a DNA aptamer. Microbial transglutaminase (MTG) then catalyzed the cross-linking between the Z-QG modified aptamers and an enzyme tagged with an MTG-reactive lysine containing peptide. The use of a Z-QG modified dideoxynucleotide (Z-QG-ddUTP) or a deoxyuridine triphosphate (Z-QG-dUTP) in the TdT reaction enables the controlled introduction of a single or multiple MTG reactive residues. This leads to the preparation of enzyme-aptamer and (enzyme)n-aptamer conjugates with different detection limits of thrombin, a model analyte, in a sandwich enzyme-linked aptamer assay (ELAA). Since the combination of two enzymatic reactions yields high site-specificity and requires only short peptide substrates, the methodology should be useful for the labeling of DNA/RNA aptamers with proteins.