Competitive Aptasensor Research Articles

A highly sensitive competitive aptasensor for AFB1 detection based on an exonuclease-assisted target recycling amplification strategy.

Aflatoxin B1 (AFB1) is a typical mycotoxin found in agricultural products, and poses a huge threat to both humans and animals. Accurate and rapid measurement of AFB1 is essential for environmental analysis and food safety. Based on molecular docking simulation design and exonuclease-assisted target recycling amplification, we designed a competitive fluorescence aptasensor to detect AFB1 rapidly and sensitively. According to the molecular docking simulations, a complementary strand (cDNA) was designed by searching for potential binding sites of the aptamer, which had the lowest binding energy. Magnetic beads modified with biotin-Apt were used as the capture probe, while FAM-labeled cDNA acted as the reporter probe. By using EXO I for target recycling amplification, this aptasensor was highly sensitive and selective for AFB1. The detection limit of the suggested aptasensor under optimal conditions was 0.36 ng mL-1 (S/N = 3) in the range of 1-1000 ng mL-1 (R2 = 0.991). The developed aptasensor was successfully used to analyze AFB1 in oil samples.

A highly selective and sensitive competitive aptasensor capable of quantifying thrombin elevated with side effects in severe COVID-19

To protect critical injury from blood clots with side effects in severe COVID-19, a highly selective and sensitive biosensor was developed for the quantification of trace levels of thrombin using the combination of a DNA aptamer (TBA) of thrombin and a complementary strand of TBA. TBA rapidly binds with thrombin, whereas it slowly binds with the complementary strand to form a double stranded DNA (dsDNA). SFC green intercalated into dsDNA cannot emit light in 1,1′-oxalyldiimidazole chemiluminescence (ODI-CL) reaction because high-energy intermediates formed from ODI-CL reaction cannot transfer energy to SFC trapped in dsDNA. However, SFC freely existing with the formation of G-quadruplex from the reaction of thrombin and TBA emits bright chemiluminescence because the high-energy intermediates can transfer energy to SFC (or camel) in solution. Thus, the brightness of light emitted in ODI-CL reaction was proportionally enhanced with the increase of thrombin in a sample due to the increase of G-quadruplex and reduction of dsDNA. The limit of detection (LOD) of the label free aptasensor operated with good linear calibration curve (10–320 mU/ml) was as low as 3 mU/ml (or 43 pM). Also, the biosensor was quantified trace levels of thrombin with good accuracy, precision, and reliability.

Competitive smartphone-based portable electrochemical aptasensor system based on an MXene/cDNA-MB probe for the determination of Microcystin-LR

A competitive smartphone-based portable electrochemical aptamer sensing system was developed for MC-LR detection. The platform was composed with a smartphone-controlled portable electrochemical system, a smartphone with custom application and a screen-printed carbon electrode (SPCE). The portable electrochemical system consisted a homemade ARM STM32 with chronoamperometry (CA), cyclic voltammetry (CV) and square wave voltammetry (SWV) modules that had a similar performance to commercial electrochemical workstations. The control of the portable electrochemical system, reception and visualization of the assay results were implemented by a smartphone. SPCE was modified by in situ electrodeposition with gold nanoparticles (AuNPs) and reduced graphene oxide (rGO). Subsequently, the aptamer was incubated onto the SPCE through Au-S bonds. The complementary DNA labeled with methylene blue (cDNA-MB) was conjugated onto the surface of MXene to form the probe for signal amplification. The competitive aptasensor was accomplished when the probe was complemented with the aptamer. The fabricated system exhibited the outstanding capability for the determination of MC-LR over a range of 0.0001–5 nM with a limit of detection of 4 × 10−5 nM. Furthermore, the system demonstrated acceptable stability, reproducibility, specificity and high recovery rate. The smartphone-based competitive aptasensor system exhibited the merits of portability, low-cost and fast-response to satisfy the demand for on-site detection of MC-LR.

Competitive electrochemiluminescence aptasensor based on the Ru(II) derivative utilizing intramolecular ECL emission for E2 detection

One competitive aptasensor based on novel intramolecular ECL emission system was investigated for ultrasensitive detection of E2. It should be noted that the Ru(II) derivative for the mechanism of intramolecular ECL could improve electron transfer efficiency and reduce energy consumption. The Ru(II) derivatives were prepared by optimizing distribution ratio of Ru(dcbpy)32+ and carbohydrazide for acquiring the ECL signal maximum. Stimulated by In3+/In+ redox reversible electron pair in the InVO4/β-AgVO3 heterostructures, the InVO4/β-AgVO3 heterostructures were designed as co-reaction accelerator to expedite ECL emission reaction between CON4H6• and Ru(dcbpy)33+. Taking advantage of associative competition between cDNA and E2 with aptamer, the ECL signal realized further amplification. The specific binding of the aptamer to E2, was stronger than that to the cDNA by hybridization. A mass of Ru(II) derivatives could adsorb and embed into the hybridized double-stranded DNA to immobilize on the electrode surface and generate satisfactory ECL signal. The specific binding of the aptamer to E2 decreased the cDNA content on electrode surface, which contributed to lessen the adsorption amount of Ru(dcbpy)32+ and distinctly affected the ECL signal. Under optimal conditions, the proposed biosensor provided an admirable linearity to the level of E2 between 0.001 nmol/L and 100 nmol/L with a low detection limit of 0.27 pmol/L. The proposed aptasensor not only stimulates more interest in the mechanism of intramolecular ECL but also has towardly development potential for constructing competitive strategies.

Competitive aptasensor for the ultrasensitive multiplexed detection of cancer biomarkers by fluorescent nanoparticle counting.

Cancer biomarker quantification in human serum is of great importance for accurate patient diagnosis and informed clinical management. To date, ultrasensitive multiplexed detection of proteins without amplification is still a major challenge. Herein, we proposed a competitive aptasensor strategy for ultrasensitive multiplexed cancer biomarker detection by fluorescent nanoparticle (FNP) counting. The sequences are designed such that the binding abilities of linker DNA (L-DNA) with DNA-functionalized FNPs (DNA-FNPs) and aptamer are comparable. As long as one target binds with one molecule of aptamer, a signalling FNP forms a sandwich-structured nanocomposite, which was subsequently observed and enumerated with a fluorescence microscope. This 1 : 1 target-to-signal FNP production assured an improved sensitivity, benefiting from the reasonably good brightness and photostability of FNPs. For both singleplexed and multiplexed detection, this proposed strategy achieved an approximately 1000-fold improved limit of detection than the conventional method with the detection volume of 3.2 μL. Notably, the results for carcinoembryonic antigen (CEA) detection obtained directly from 9 human serum samples (colorectal/lung/healthy individuals) were consistent with that obtained by ELISA, showing potential application in clinical diagnosis.

Competitive aptasensor with gold nanoparticle dimers and magnetite nanoparticles for SERS-based determination of thrombin.

It is known that the intensity of surface-enhanced Raman scattering (SERS) of monomeric gold nanoparticles (GNPs) is insufficient for ultrasensitive analysis. The authors describe dimeric GNPs for use in a competitive SERS and aptamer based assay for thrombin. The reagent 1,2-bis(4-pyridyl) ethylene serves as both the coupling agent and the Raman reporter on the GNP dimers. In the presence of thrombin, the hybridization of two aptamers, one attached to the GNP dimers, the other to magnetic nanoparticles, is competitively prevented. This method takes advantage of the unique "hot spots" of the GNP dimers to amplify the Raman signal. This results in an ultra-sensitive thrombin assay when compared to assays using GNP monomers. The limit of detection is as low as 1 fM of thrombin. The Raman intensity, best measured at 1612cm-1, increases linearly in the 1 fM to 10nM thrombin concentration range. The method was applied to the determinaiton of thrombin in spiked simulated body fluid and human serum. Graphical abstract This method takes advantage of the unique "hot spots" of the gold nanoparticle dimers to amplify the Raman signal. The dimers are linked to the magnetic nanoparticles via an aptamer. The use of both competitive displacementand magnetic separation greatly improves the sensitivity of the thrombin assay.

Tetrahedral DNA probe coupling with hybridization chain reaction for competitive thrombin aptasensor

A novel competitive aptasensor for thrombin detection is developed by using a tetrahedral DNA (T-DNA) probe and hybridization chain reaction (HCR) signal amplification. Sulfur and nitrogen co-doped reduced graphene oxide (SN-rGO) is firstly prepared by a simple reflux method and used for supporting substrate of biosensor. Then, T-DNA probe is modified on the electrode by Au-S bond and a competition is happened between target thrombin and the complementary DNA (cDNA) of aptamer. The aptamer binding to thrombin forms an aptamer-target conjugate and make the cDNA remained, and subsequently hybridizes with the vertical domain of T-DNA. Finally, the cDNAs trigger HCR, which results in a great current response by the catalysis of horseradish peroxidase to the hydrogen peroxide + hydroquinone system. For thrombin detection, the proposed biosensor shows a wide linearity range of 10–13–10−8M and a low detection limit of 11.6fM (S/N = 3), which is hopeful to apply in biotechnology and clinical diagnosis.

Fluorescent competitive aptasensor for detection of aflatoxin B1.

Aflatoxin B1 (AFB1 ) is one of the most commonly found mycotoxins in food commodities, particularly cereals, oilseeds, spices and tree nuts. In the past decade, aptamers have come into limelight and emerged as a new biosensing element replacing antibodies in various detection formats. Herein we report a faster, more sensitive, high throughput method for the detection of AFB1 using AFB1 -specific aptamers. The assay format was based on a competitive reaction of the fluorescent tagged aptamer specific to AFB1 with the aflatoxin conjugate. Under optimal conditions, a linear range of detection (50ng to 50pg) was achieved with a limit of detection (LOD) of 10pg/mL in the buffer system. Results of inter- and intra-assay revealed that the assay was repeatable with standard deviation in acceptable range. The assay was also validated in food samples such as dried red chilies, groundnut and whole pepper with recovery in the range of 92 to 102% at 10ng/mL and 100pg/mL levels. The aptasensor assay was also compared with standard analytical method of HPLC and was found to be more sensitive. This detection technique has the potential to be developed into a biosensor platform for AFB1 detection.

Sensitive quantitation of Ochratoxin A in cocoa beans using differential pulse voltammetry based aptasensor

In this work, we propose for the first time a sensitive Ochratoxin A (OTA) detection in cocoa beans using competitive aptasensor by differential pulse voltammetry (DPV). In the proposed method, biotin labeled and free OTA competed to bind with immobilized aptamer onto the surface of a screen printed carbon electrode (SPCE), and percentage binding was calculated. The detection was performed after adding avidin-ALP to perform avidin–biotin reaction; the signal was generated through a suitable substrate 1-naphthyl phosphate (1-NP), for alkaline phosphatase (ALP). The cocoa samples were extracted and purified using molecular imprinted polymer (MIP) columns specifically designed for OTA. The developed aptasensor showed a good linearity in the range 0.15–5ng/mL with the limit of detection (LOD) 0.07ng/mL and 3.7% relative standard deviation (RSD). The aptasensor displayed good recovery values in the range 82.1–85% with 3.87% RSD, thus, demonstrated the efficiency of proposed aptasensor for such matrices.

Label-free and sensitive electrochemiluminescence aptasensor for the determination of 17β-estradiol based on a competitive assay with cDNA amplification

A label-free competitive electrochemiluminescence (ECL) aptasensor for the determination of 17β-estradiol (E2) was developed based on the immobilization of thiol-capped E2 aptamers onto a gold electrode. A complementary DNA (cDNA) was designed to be used as a detection probe to bind with any unbound E2 aptamer. Tris(2,2′-bipyridyl)ruthenium(II) (Ru(bpy)32+) was used as an ECL indicator and was electrostatically bound to the E2 aptamer; it competes with the cDNA for aptamer binding sites. With less E2, increased amounts of cDNA can bind to the E2 aptamer and adsorb more Ru(bpy)32+, greatly enhancing the ECL intensity. With more E2, the aptamer will capture E2 and bind less cDNA with an accompanying decrease in Ru(bpy)32+ and a decrease in ECL intensity. Thus, a competitive aptasensor based on the specific binding of the E2 aptamer to E2 and cDNA was achieved. The biosensor for E2 possesses a widely linear detection range from 0.01 to 10 nmol L−1 with a detection limit of 1.1 × 10−12 mol L−1 (S/N = 3). The application of the present protocol was demonstrated by determining E2 in human serum, human urine, and tap water samples with recoveries of 89.8–100.0%, 90.0–103.5% and 89.5–95.0%, respectively. The proposed method provides a powerful tool for the rapid and sensitive detection of small molecules in biological and environmental samples.

An ultrasensitive HRP labeled competitive aptasensor for oxytetracycline detection based on grapheme oxide–polyaniline composites as the signal amplifiers

In this work, we explored an amplification strategy which was based on graphene oxide–polyaniline (GO–PANI) and horseradish peroxidase (HRP) to construct the competitive aptasensor for ultrasensitive detection of oxytetracycline (OTC). In the protocol, the GO–PANI film was immobilized on the surface of the electrodes. Then, gold nanoparticles (AuNPs) were electrodeposited on the electrode surface using a constant potential stripping technique. The selected aptamer which had high affinity and specificity for OTC was used as a capture probe and labeled with HRP. The linear response to OTC concentration of the developed aptasensor was in the range of 4.0 × 10−6 mg L−1 to 1.0 mg L−1. The detection limit (LOD) of 2.3 × 10−6 mg L−1 was obtained (S/N = 3). In addition to good repeatability and stability, the proposed aptamer sensor also showed the advantages of low background current and high sensitivity to examine OTC in real samples.

Development of an automated flow-based electrochemical aptasensor for on-line detection of Ochratoxin A

The development of a fully automated flow electrochemical aptasensor based on the use of a selective capture aptamer and modified magnetic beads (MBs) is reported. The developed flow-based aptasensor is demonstrated under continuous/stopped flow conditions for the detection of ochratoxin A (OTA) employing direct and indirect competitive strategies. In the direct format, biotin labeled and free OTA competed to bind with immobilized aptamer onto the surface of a screen printed carbon electrode (SPCE) placed in a flow cell. In the indirect assay, immobilized OTA and free OTA in solution competed to bind with aptamer in solution. The current response obtained from the conversion of the substrate 1-naphtyl phosphate by ALP decreased proportionally to the concentration of OTA in the sample. A lower limit of detection (0.05 μg/L) was obtained with the indirect flow-based aptasensor. Finally, the flow-based aptasensor was validated with real beer samples, and the good recovery values demonstrated the efficiency of the competitive aptasensor.