Competitive Enzyme-linked Aptamer Assay Research Articles

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.

Development of an ELAA kit to detect neomycin in milk

Antibiotics used in livestock production offer various benefits as an antimicrobial agent, growth promoter, and feed effective improvement. However, the abuse of antibiotics leads to antibiotic resistance which may seriously threaten human and animal welfare, and growing levels of antibiotics or antibiotic-resistant bacteria in the environment increase the numbers of drug-resistant infection outbreaks. Therefore, many detection methods have been being developed to quickly assess antibiotic content and its residues in foods. Among many analytical methods, the aptamer-based biosensor has considerable attention for its outstanding advantages such as high specificity, high sensitivity, and good selectivity. We use the ELAA (Enzyme-Linked Aptamer Assay) method - a variant of ELISA - which has a high affinity with neomycin. Firstly, we investigated different buffers to create the Neo-BSA complex. As result, 2-(N-morpholino) ethanesulfonic acid (MES) buffer pH 7 was found with the best results. Next, to help the Neo-BSA complex be fixed well on polystyrene wells, we surveyed various buffers and found the coating buffer (50mM Bicarbonate buffer, pH 9.6) rated as the most suitable for this process. In addition, the quality of the kit is also assessed through competitive ELAA reaction components. Therefore, we have investigated and optimized conditions such as aptamer concentration 25 nM in PBS buffer, and the biotinized aptamers did not need heat treatment prior to joining the reaction. From the results, we have successfully developed a calibration curve for antibiotic residue in milk using the ELAA technique, linear range 0,1 ng/mL and 100 ng/mL. Then, we initially surveyed 20 milk samples found that the ELAA method was consistent with the results from LC-MS/MS was obtained showing no difference between the two methods. We continued to test the samples to determine the kit’s sensitivity and specificity. The results showed that the kit has a specificity and sensitivity of 100%. Finally, LOD and LOQ value had xavg = 0.448; SD = 0.22, LOD = xavg + 3SD = 1.11 (ng / ml); LOQ = x tb + 10SD = 2.65 (ng / mL). We will continue to optimize the kit before being brought to the market.

Amplified colorimetric detection of tetracycline based on an enzyme-linked aptamer assay with multivalent HRP-mimicking DNAzyme.

Tetracycline (TC) is widely used to treat bacterial infections in humans and animals due to its low price and good antibacterial properties. The abuse of tetracycline has led to TC residues in daily food that could seriously affect human health. Thus, it is imperative to develop highly sensitive and selective methods for TC detection. In this work, we developed a colorimetric method for TC detection based on an enzyme-linked aptamer assay (ELAA) with multivalent HRP-mimicking DNAzyme. An aptamer was used as an alternative recognition element in the enzyme-linked immunosorbent assay (ELISA). Multivalent HRP-mimicking DNAzyme, assembled via hybridization chain reactions (HCR), was used for catalytic substrate color rendering in ELAA. The multivalent HRP-mimicking DNAzyme exhibited enhanced catalytic capacity and improved the detection sensitivity greatly. The limit of detection was 8.1 × 10-2 ng mL-1 with a linear range from 1.0 × 10-2 ng mL-1 to 1.0 × 104 ng mL-1 toward TC in buffer. To challenge the practical application capability of this strategy, the detection of TC in milk samples was also investigated and showed similar linear relationships. Due to the introduction of an aptamer, this ELAA strategy shows high selectivity towards TC and has potential for the detection of a wide spectrum of analytes.

Selection and Identification of Novel Aptamers Specific for Clenbuterol Based on ssDNA Library Immobilized SELEX and Gold Nanoparticles Biosensor.

We describe a multiple combined strategy to discover novel aptamers specific for clenbuterol (CBL). An immobilized ssDNA library was used for the selection of specific aptamers using the systematic evolution of ligands by exponential enrichment (SELEX). Progress was monitored using real-time quantitative PCR (Q-PCR), and the enriched library was sequenced by high-throughput sequencing. Candidate aptamers were picked and preliminarily identified using a gold nanoparticles (AuNPs) biosensor. Bioactive aptamers were characterized for affinity, circular dichroism (CD), specificity and sensitivity. The Q-PCR amplification curve increased and the retention rate was about 1% at the eighth round. Use of the AuNPs biosensor and CD analyses determined that six aptamers had binding activity. Affinity analysis showed that aptamer 47 had the highest affinity (Kd = 42.17 ± 8.98 nM) with no cross reactivity to CBL analogs. Indirect competitive enzyme linked aptamer assay (IC-ELAA) based on a 5′-biotin aptamer 47 indicated the limit of detection (LOD) was 0.18 ± 0.02 ng/L (n = 3), and it was used to detect pork samples with a mean recovery of 83.33–97.03%. This is the first report of a universal strategy including library fixation, Q-PCR monitoring, high-throughput sequencing, and AuNPs biosensor identification to select aptamers specific for small molecules.

A Highly Sensitive Aptasensor for Sulfamethazine Detection Using an Enzyme-Linked Aptamer Assay

To detect sulfamethazine (SMZ) residues in edible animal products, a colorimetric aptasensor that is based on indirect competitive enzyme-linked aptamer assay (ELAA) is developed in this study. This ELAA uses SMZ-specific aptamers as recognition elements and enzyme as the signal readout element. The detection sensitivity and the specificity of the ELAA were investigated. Our results showed that the indirect competitive ELAA had a ultrasensitivity with a low detection limit of 0.05 ng mL−1. In addition, the proposed indirect competitive ELAA exhibited an 50% inhibition concentration value of 0.64 ng mL−1 for SMZ, and recoveries up to 80.5–92.3% with coefficients of variation of less than 10%. Furthermore, when the ELAA was used for incurred samples by detecting SMZ in chicken muscles, the results obtained by the ELAA methods showed a good agreement with those obtained by established TRFIA and HPLC methods. These results demonstrate that the proposed indirect competitive ELAA can be efficiently used with good accuracy and precision, providing a simple method for the detection of SMZ residue in animal tissues.

Graphene oxide-assisted non-immobilized SELEX of okdaic acid aptamer and the analytical application of aptasensor.

Okadaic acid (OA) is a low-molecular-weight marine toxin from shellfish that causes abdominal pain, vomiting and diarrhea, i.e., diarrheic shellfish poisoning. In this study, a ssDNA aptamer that specifically binds to OA with high affinity was obtained via Systematic Evolution of Ligands by Exponential Enrichment (SELEX) assisted by graphene oxide (GO). This aptamer was then applied to fabricate a novel direct competitive enzyme-linked aptamer assay (ELAA). At the optimized conditions, this ELAA method showed a low detection limit (LOD of 0.01 ng/mL), wide linear range (from 0.025 to 10 ng/mL), good recovery rate (92.86–103.34% in OA-spiked clam samples) and repeatability (RSD of 2.28–4.53%). The proposed method can be used to detect OA in seafood products with high sensitivity and can potentially be adapted for the determination of other small molecular analytes.

Magnetic-nanobead-based competitive enzyme-linked aptamer assay for the analysis of oxytetracycline in food.

This study presents a novel analytical method for the detection of oxytetracycline (OTC) in complex food matrices based on a direct competitive enzyme-linked aptamer assay and magnetic separation technology. In this protocol, free OTC competed with horseradish peroxidase labeled OTC (OTC-HRP) for binding to the OTC aptamer immobilized on magnetic beads. The parameters that can affect the response, such as avidin concentration, aptamer concentration, OTC-HRP concentration, incubation temperature, incubation time, blocking agent, and binding buffer, were optimized. Under the optimal conditions, the linear range for the OTC concentration detection is 0.5-100 ng mL(-1), with a concentration of OTC needed to obtain 50 % of the maximum signal of 14.47 ng mL(-1). The limit of detection and the limit of quantitation were 0.88 and 3.40 ng mL(-1), respectively. There was no obvious cross-reactivity with most of the tetracycline pesticides. The recovery rates ranged from 71.0 to 91.2 % for the food samples, including chicken, milk, and honey, and the relative standard deviation was less than 15.0 %. The proposed method was applied to measure OTC in real samples, and was validated using high-performance liquid chromatography. This method has the advantages of magnetic separation and the concentration effect of magnetic nanoparticles, the specificity of the aptamer, and the high-throughput of microtiter plates; it offers a promising approach for the screening of OTC because it is simple, rapid, highly sensitive, and has low cost.

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 76mer 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 0.0978ng/mL), a wide linear range (0.1–1000ng/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.

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 9.6×10−3ng/mL with a linear working range from 0.01 to 100ng/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.

Enzyme Linked Aptamer Assay: Based on a Competition Format for Sensitive Detection of Antibodies to Mycoplasma bovis in Serum

Mycoplasma bovis (M. bovis) is a major, but often overlooked, pathogen that causes respiratory disease, mastitis, and arthritis in cattle. It has been widespread in China since 2008. In this study, single-stranded DNA (ssDNA) aptamers with high affinity and specificity against the P48 protein of M. bovis were selected using microplates as the matrix. Of nine candidates, aptamer WKB-14 showed the best affinity in an indirect enzyme-linked aptamer assay (ELAA) and good specificity by dot blotting. To the best of our knowledge, this is the first time that an aptamer has been used in a competitive ELAA for the serological detection of M. bovis. The percent inhibition (PI) cutoff value of the indirect competitive ELAA (ic-ELAA) was 40%, assessed using 20 negative sera. In a comparative study of different detection methods, ic-ELAA with dc-ELISA and dot blotting had a higher positive detection rate than the other two commercial indirect ELISA kits.

ADVANTAGEOUS SENSITIVITY IN THE DNA HOMOLOG OF THE RNA DOPAMINE APTAMER

A competitive enzyme-linked aptamer assay for DA was performed by using two aptamers, individually; one is a 57 mer-RNA aptamer and the other is its homolog DNA aptamer. The difference between the RNA aptamer and the DNA aptamer are based on their particular nucleotides. It is known that the lack of a hydroxyl group in the 2' position of DNA is related with its chemical and biological stability. Thus, the use of the DNA homolog of the RNA aptamer could improve the affinity toward DA due to stability and finally, lower the detection limit. In this paper, we report advantageous sensitivity and specificity of its homolog DNA aptamer assay as compared to the RNA aptamer assay. Both aptamer assays were performed with 0.01 µg mL−1 of each aptamer and 1.205 × 10−8 M DA-HRP conjugate using the optimized method. A dose-response curve was constructed, and the limit of detection for the DA was determined as 6.3 × 10−8 M for RNA aptamer assay, and 3.2 × 10−12 M for the homolog DNA aptamer assay, respectively. These results demonstrated that the assay sensitivity was more than 104 times improved with the DNA homolog of the RNA aptamer compared to its original RNA aptamer obtained through SELEX process. Also these results confirmed that the DNA homolog of the RNA aptamer can maintained the binding site and retained a function in both structure. Thus, the switching to the DNA version of RNA aptamer is possible to bind more stably and still able to bind to dopamine.

An indirect competitive assay-based aptasensor for detection of oxytetracycline in milk

Oxytetracycline (OTC) is a common antibacterial agent used for the control of animal diseases. OTC abuse can seriously affect human health; therefore, we developed a biosensor using single-stranded DNA (ssDNA) aptamers for the detection of OTC. The binding probe aptamers for OTC were selected by a Systematic Evolution of Ligands by the exponential enrichment (SELEX) process and identified by the enzyme-linked aptamer assay (ELAA). Among the selected 5 aptamers, aptamer OTC3 showed the strongest affinity (Kd=4.7nM) and highest specificity for OTC compared to structurally similar antibiotics, tetracycline and chlortetracycline. OTC was detected using indirect competitive ELAA. The limit of detection and quantitation with aptamer OTC3 were 12.3 and 49.8µg/L, respectively, in milk and showed recovery rates of more than 90% in OTC-spiked milk. This biosensor method with high sensitivity and specificity based on indirect competitive ELAA can be applied to OTC detection in food products on-site because of the simplicity of detection.

Sensitivity and Selectivity on Aptamer-Based Assay: The Determination of Tetracycline Residue in Bovine Milk

A competitive enzyme-linked aptamer assay (ELAA) to detect tetracycline in milk was performed by using two different aptamers individually; one is 76 mer-DNA aptamer and the other is 57 mer-RNA aptamer. The best optimum condition was obtained without monovalent ion, Na+ and also by adding no Mg2+ ion in the assay buffer, along with RT incubation. The optimized ELAA showed a good sensitivity (LOD of 2.10 × 10−8 M) with a wide dynamic range (3.16 × 10−8 M ~ 3.16 × 10−4 M). In addition, the average R.S.D. across all data points of the curve was less than 2.5% with good recoveries (~101.8%) from the milk media. Thus, this method provides a good tool to monitor tetracycline in milk from MRLs' point of view. However, this ELAA method was not superior to the ELISA method in terms of specificity. This paper describes that it does not always give better sensitivity and specificity in assays even though aptamers have several advantages over antibodies and have been known to be good binders for binding assays.

Evaluation of hepatogenic differentiation potential of menstrual blood derived stem cells

Ochratoxin A (OTA) is one of the most important mycotoxins because of its high toxicity to both humans and animals and its occurrence in a number of basic foods and agro-products. The need to develop high-performing methods for OTA analysis able to improve the traditional ones is evident. In this work, through in vitro SELEX (Systematic Evolution of Ligands by EXponential enrichment) two aptamers, designated H8 and H12 were produced that bind with nanomolar affinity with Ochratoxin A (OTA). Two strategies were investigated by using an indirect and a direct competitive Enzyme-Linked Aptamer Assay (ELAA) and were compared to the classical competitive Enzyme-Linked Immunosorbent Assay (ELISA) for the determination of OTA in spiked red wine samples. The limit of detection attained (1 ng/mL), the midpoint value obtained (5 ng/mL) and the analysis time needed (125 min) for the real sample analysis validate the direct competitive ELAA as useful screening tool for routine use in the control of OTA level in wine.

Development of direct competitive enzyme-linked aptamer assay for determination of dopamine in serum

A rapid and cost-effective screening method based on a competitive enzyme-linked aptamer assay (ELAA) for dopamine (DA) in serum has been optimized and validated. In this paper, we report advantageous sensitivity and specificity of aptamer assays as compared to the existing antibody based-immunoassays. The RNA aptamer (67 mer) was immobilized via site-directed immobilization with biotin both at the 3′-end on aptamer and at neutravidin plate. Various factors such as incubation temperature, divalent ion – Mg 2+ ion and treatment of serum solution were evaluated for the performance of ELAA. The aptamer was incubated for 1 h at 4 °C in the assay buffer containing 5 mM Mg 2+ ion, and serum was diluted (1:9, serum:assay buffer) and filtrated through a 3 kDa dialysis membrane to extract the proteins present in the serum. Assay was performed with 0.01 μg mL −1 of aptamer and 1.205 × 10 −7 M DA-HRP conjugate using the optimized method. A dose–response curve was constructed, and the limit of detection and a dynamic range for the DA were determined as 1.0 × 10 −12 M and four orders (1.0 × 10 −7 M to 5.0 × 10 −11 M) of magnitude, respectively. The correlation diagram of the absorbance obtained both in buffer and in serum has shown a good agreement with the correlation coefficient ( R 2 = 0.9872): Abs. (in serum) = 0.9612 × Abs. (in buffer) − 0.0556. The cross-reactivity evaluation demonstrated that norepinephrine showed some cross-reactivity (3.68%) whereas 3-methoxytyramine, epinephrine, homovanillic acid and 3,4-dihydroxyphenylacetic acid showed almost no cross-reactivity (<1%). Percent recoveries of DA in serum were quite satisfactory (∼95%). This paper describes usefulness of the aptamer assay in monitoring DA in human serum.

Enzyme-Linked Aptamer Assays (ELAAs), based on a competition format for a rapid and sensitive detection of Ochratoxin A in wine

Ochratoxin A (OTA) is one of the most important mycotoxins because of its high toxicity to both humans and animals and its occurrence in a number of basic foods and agro-products. The need to develop high-performing methods for OTA analysis able to improve the traditional ones is evident. In this work, through in vitro SELEX (Systematic Evolution of Ligands by EXponential enrichment) two aptamers, designated H8 and H12 were produced that bind with nanomolar affinity with Ochratoxin A (OTA). Two strategies were investigated by using an indirect and a direct competitive Enzyme-Linked Aptamer Assay (ELAA) and were compared to the classical competitive Enzyme-Linked Immunosorbent Assay (ELISA) for the determination of OTA in spiked red wine samples. The limit of detection attained (1 ng/mL), the midpoint value obtained (5 ng/mL) and the analysis time needed (125 min) for the real sample analysis validate the direct competitive ELAA as useful screening tool for routine use in the control of OTA level in wine.