Double Magnetic Separation-assisted Fluorescence Method for Sensitive Detection of Ochratoxin A

A double magnetic separation-assisted fluorescence method was developed to rapidly detect ochratoxin A(OTA). The OTA aptamer functionalized magnetic nanomaterial(Fe3O4-Aptanier) and complementary DNA conjugated nitrogen-doped graphene quantum dots(NGQDs-cDNA) were used in this assay. Aptamer could hybridize with cDNA, which induced tlie NGQDs-cDNA to bind onto Fe3O4-Aptamer, and resulted in the fluorescence quenching of NGQDs. After the addition of OTA, the NGQDs-cDNA could release into the solution, and resulted in the recovery of fluorescence signal of NGQDs consequently. By utilizing the magnetic separation, the unbonded NGQDs-cDNA and residual Fe3O4-Aptamer were removed, which significantly increased the fluorescence signal intensity. OTA could be detected in the linear range of 10 nmol/L to 2000 nmol/L, with a limit of detection as 0.66 mnol/L. The advantages of this method include simple operation, good selectivity and high sensitivity, and this method can be used for the rapid detection of ochratoxin A in wheat and com.     

磁珠负载的适配体修饰纳米金探针用于血清中细菌内毒素检测

生物传感检测血清中的细菌内毒素具有重要的应用价值,但其开发过程受到血清所含物质复杂性的限制。本文开发了一种基于磁珠-纳米金-适配体(MB-AuNPs-APT)的生物探针比色传感器。通过生物探针捕获内毒素来影响其过氧化物酶活性,进而影响H_2O_2催化3,3′,5,5′-四甲基联苯胺(TMB)产生氧化TMB的量,最终达到比色分析内毒素的目的。分析了该方法对血清中内毒素检测的可行性,结果表明,基于MB-AuNPs-APT生物探针比色传感法的检测限为0.402ng/mL,线性范围为0.1～100 ng/mL。在50%血清样品中此生物传感器检测内毒素的回收率为99.59%～112.00%,展示了该生物探针在血清中检测内毒素的可靠性。     

Label-free fluorescent strategy for sensitive detection of tetracycline based on triple-helix molecular switch and G-quadruplex

In this assay, a label-free fluorescent sensing platform based on triple-helix molecular switch (THMS) and G-quadruplex was developed for the detection of tetracycline. We demonstrated this approach by using THMS, which consists of a central section with a shortened 8-mer aptamer sequence with high affinity to tetracycline and flanked by two arm segments. G-rich oligonucleotide can specifically bind to thioflavin T (ThT) as a signal transduction probe (STP). In the absence of tetracycline, THMS remains stable, the fluorescence of background is low. By the addition of target tetracycline, the aptamer-target binding results in the formation of a structured aptamer-target complex, which disassembles the THMS and releases the STP. The free STP self-assembles into G-quadruplex and specifically binds to ThT which generates a obvious fluorescence enhancement. Using the triple-helix molecular switch, the developed aptamer-based fluorescent sensing platform showed a linear relationship with the concentration of tetracycline ranging from 0.2 to 20.0 nmol/L. The detection limit of tetracycline was determined to be 970.0 pmol/L. The assay avoids complicated modifications or chemical labeling, making it simple and cost-effective. So, it is expected that this aptamer-based fluorescent assay could be extensively applied in the field of food safety inspection.     

An Aptamer‐Based Protein Biosensor by Detecting the Amplified Impedance Signal

A label‐free electrochemical impedance based protein biosensor was introduced by using aptamer as recognition tool. Our sensing protocol utilizes the affinity interaction between the thrombin and the self‐assembled DNA aptamer on gold electrode. This specific interaction increases the electrode interfacial electronic transfer resistance. The resistance signal is then “amplified” by using guanidine hydrochloride to denature the captured thrombin for increasing the hydrated radius of the thrombin, consequently blocking the electron transfer from solution to electrode. The sensor sensitivity is improved using this strategy and as low as 1.0×10^?14 mol L^?1 thrombin (enzymatic activity 10 U/mg) can be detected out.     

Highly Uniform DNA Monolayers Generated by Freezing-Directed Assembly on Gold Surfaces Enable Robust Electrochemical Sensing in Whole Blood

Assembling DNA on solid surfaces is fundamental to surface-based DNA technology. However, precise control over DNA conformation and organization at solid–liquid interfaces remains a challenge, resulting in limited stability and sensitivity in biosensing applications. We herein communicate a simple and robust method for creating highly uniform DNA monolayers on gold surfaces by a freeze-thawing process. Using Raman spectroscopy, fluorescent imaging, and square wave voltammetry, we demonstrate that thiolated DNA is concentrated and immobilized on gold surfaces with an upright conformation. Moreover, our results reveal that the freezing-induced DNA surfaces are more uniform, leading to improved DNA stability and target recognition. Lastly, we demonstrate the successful detection of a model drug in undiluted whole blood while mitigating the effects of biofouling. Our work not only provides a simple approach to tailor the DNA-gold surface for biosensors but also sheds light on the unique behavior of DNA oligonucleotides upon freezing on the liquid-solid interface.     

Aptamers: molecular tools for analytical applications

Aptamers are artificial nucleic acid ligands, specifically generated against certain targets, such as amino acids, drugs, proteins or other molecules. In nature they exist as a nucleic acid based genetic regulatory element called a riboswitch. For generation of artificial ligands, they are isolated from combinatorial libraries of synthetic nucleic acid by exponential enrichment, via an in vitro iterative process of adsorption, recovery and reamplification known as systematic evolution of ligands by exponential enrichment (SELEX). Thanks to their unique characteristics and chemical structure, aptamers offer themselves as ideal candidates for use in analytical devices and techniques. Recent progress in the aptamer selection and incorporation of aptamers into molecular beacon structures will ensure the application of aptamers for functional and quantitative proteomics and high-throughput screening for drug discovery, as well as in various analytical applications. The properties of aptamers as well as recent developments in improved, time-efficient methods for their selection and stabilization are outlined. The use of these powerful molecular tools for analysis and the advantages they offer over existing affinity biocomponents are discussed. Finally the evolving use of aptamers in specific analytical applications such as chromatography, ELISA-type assays, biosensors and affinity PCR as well as current avenues of research and future perspectives conclude this review.     

Competitive affinity capillary electrophoresis assay based on a "hybrid" pre-incubation/on-capillary mixing format using an enantioselective aptamer as affinity ligand

In this paper, we describe an aptamer-based competitive affinity CE (ACE) assay involving (i) the pre-incubation of the target (D-arginine) and the specific ligand (anti-D-arginine-L-RNA aptamer) before (ii) the competition with the labeled target (dansylated D-arginine) through an on-capillary mixing strategy. The effects of some critical operating parameters such as the applied voltage and the sample-aptamer mixture plug length on the assay sensitivity were investigated. The ACE assay appeared particularly dependent on the plug length of the pre-incubated sample-aptamer solution. It was shown that this "hybrid" strategy significantly improved the assay sensitivity relative to that obtained with a "full" on-capillary mixing approach.     

Deep Learning-Enhanced Potentiometric Aptasensing with Magneto-Controlled Sensors

Bioelectronic sensors that report charge changes of a biomolecule upon target binding enable direct and sensitive analyte detection but remain a major challenge for potentiometric measurement, mainly due to Debye Length limitations and the need for molecular-level platforms. Here, we report on a magneto-controlled potentiometric method to directly and sensitively measure the target-binding induced charge change of DNA aptamers assembled on magnetic beads using a polymeric membrane potentiometric ion sensor. The potentiometric responses of the negatively charged aptamer, serving as a receptor and reporter, were dynamically controlled and modulated by applying a magnetic field. Based on a potentiometric array, this non-equilibrium measurement technique combined with deep learning algorithms allows for rapidly and reliably classifying and quantifying diverse small molecules using antibiotics as models. This potentiometric strategy opens new modalities for sensing applications.     

Aptamer sensor for cocaine using minor groove binder based energy transfer

We report on an optical aptamer sensor for cocaine detection. The cocaine sensitive fluorescein isothiocyanate (FITC)-labeled aptamer underwent a conformational change from a partial single-stranded DNA with a short hairpin to a double-stranded T-junction in the presence of the target. The DNA minor groove binder Hoechst 33342 selectively bound to the double-stranded T-junction, bringing the dye within the Förster radius of FITC, and therefore initiating minor groove binder based energy transfer (MBET), and reporting on the presence of cocaine. The sensor showed a detection limit of 0.2 μM. The sensor was also implemented on a carboxy-functionalized polydimethylsiloxane (PDMS) surface by covalently immobilizing DNA aptamers. The ability of surface-bound cocaine detection is crucial for the development of microfluidic sensors.     

Gold Nanoparticle-Based Fluorescence Resonance Energy Transfer Aptasensor for Ochratoxin A Detection

In this paper, a sensitive and specific fluorescence resonance energy transfer (FRET) aptasensor for the detection of Ochratoxin A (OTA) was developed based on a dye-tagged ssDNA hybridized with aptamer-conjugated Au nanoparticles (Au NPs). The binding between the aptamer-Au NPs conjugate and the dye-labeled ssDNA leads to the fluorescence quenching of FAM due to its close proximity. The addition of OTA results in fluorescence recovery, attributed to the formation of a quadruplex-OTA complex, which detaches from the surface of Au NPs. Under optimal conditions, the relative fluorescence intensity (ΔI) is proportional to the concentration of the OTA in the range of 5 × 10^?12 to 5 × 10^?9 g/mL, with a detection limit of 2 × 10^?12 g/mL. The proposed method was successfully applied to measure the concentration of OTA in naturally contaminated maize samples and validated using a commercially available enzyme-linked immunosorbent assay (ELISA) method. This work demonstrates that the combination of an aptamer that has a high binding affinity for the analyte with highly sensitive Au NPs that undergo FRET is a promising approach for the detection of small molecule toxins.