A novel optical thrombin aptasensor based on magnetic nanoparticles and split DNAzyme

In this paper, we report a novel and sensitive optical sensing protocol for thrombin detection based on magnetic nanoparticles (MNPs) and thrombin aptamer, employing split HRP-mimicking DNAzyme halves as its sensing element, which can catalyze the H₂O₂-mediated oxidation of the colorless ABTS into a blue-green product. A single nucleotide containing the recognition element and sensing element is utilized in our protocol. The specific recognition of thrombin and its aptamer leads to the structure deformation of the DNA strands and causes the split of the DNAzyme halves. Therefore, the decrease of absorption spectra can be recorded by the UV–visible Spectrophotometer. DNA-coated MNPs are utilized to separate the interferential materials from the analyst, thus making this assay can be applied in the detection of thrombin in complex samples, such as human plasma. This original, sensitive and cost-effective assay showed favorable recognition for thrombin. The absorbance signals with the concentration of thrombin over a range from 0.5 to 20 nM and the detection limit of thrombin was 0.5 nM. The controlled experiments showed that thrombin signal was not interfered in the presence of other co-existence proteins.     

Aptamer Biosensor for Selective and Rapid Determination of Insulin

Insulin plays an important role in glucose metabolism and its detection in biological fluids is of interest. In this study, a triple-helix molecular switch was employed for the simple, sensitive, and rapid determination of insulin. The triple-helix molecular switch was composed of a target specific aptamer sequence flanked by two arm segments and a dual-labeled oligonucleotide acting as a signal transduction probe. This approach takes advantage of unique properties of aptamers and triple-helix molecular switches such as high affinity, selectivity, and stability. In the absence of insulin, the fluorescence of triple-helix molecular switch is on. Upon addition of insulin, the aptamer binds to its target, leading to the release of the signal transduction probe, folding of the signal transduction probe to a stem loop structure, and the quenching of the fluorescence. This sensor showed a high selectivity toward insulin and a limit of detection as low as 9.97 nM. The sensor was employed for the determination of insulin in biological samples. This platform may be generalizable for a variety of molecules.     

适配体纳米金比色分析技术研究进展

核酸适配体作为一种生物识别分子,其本质是一段单链DNA或RNA,折叠形成特定的二级、三级构象后与靶标分子以高亲和力高特异性结合。纳米金具有强烈的粒子间距光学效应,其在分散状态下呈红色,发生凝聚后变为蓝色。适配体可以通过共价偶联或静电吸附与纳米金结合,通过结合靶标来控制纳米金的颜色变化,从而提供了一种简单、快速、灵敏、可以肉眼观察的快速分析技术。综述了适配体纳米金比色分析技术的研究进展,通过对不同方法比较和讨论,提出了未来的研究方向。     

Label-free electrochemical aptasensor for the detection of lysozyme

This work reports the advantages of a label free electrochemical aptasensor for the detection of lysozyme. The biorecognition platform was obtained by the adsorption of the aptamer on the surface of a carbon paste electrode (CPE) previously blocked with mouse immunoglobulin under controlled-potential conditions. The recognition event was detected from the decrease in the guanine and adenine electro-oxidation signals produced as a consequence of the molecular interaction between the aptamer and lysozyme. The biosensing platform demonstrated to be highly selective even in the presence of large excess (9-fold) of bovine serum albumin, cytochrome C and myoglobin. The reproducibility for 10 repetitive determinations of 10.0 mg L⁻¹ lysozyme solution was 5.1% and 6.8% for guanine and adenine electro-oxidation signals, respectively. The detection limits of the aptasensor were 36.0 nmol L⁻¹ (if considering guanine signal) and 18.0 nmol L⁻¹ (if taking adenine oxidation current). This new sensing approach represents an interesting and promising alternative for the electrochemical quantification of lysozyme.     

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.     

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.     

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 based fluorescent probe for serum HER2-ECD detection: The clinical utility in breast cancer

HB5 aptamer-based probe has been developed for serum HER2-ECD test in auxiliary clinical diagnosis and treatment for HER2-positive breast cancer patients.     

Aptamer-Based Fluorescent Determination of Salmonella paratyphi A Using Phi29-DNA Polymerase-Assisted Cyclic Amplification

A rapid and ultrasensitive fluorescence aptasensor was developed for the detection of Salmonella paratyphi A based on aptamer and Phi29-DNA polymerase-assisted cyclic signal amplification. The method employed a designed arched probe, consisting of an aptamer and a primer, with a designed hairpin probe. The quenching groups and fluorescent groups were modified at the 3′ and 5′ ends of the hairpin probe, respectively. In the absence of the target, the primer was not released and the hairpin probe was not opened to produce fluorescence. The addition of target led to the release of the primer, which hybridized with the hairpin probe and triggered the chain-displacement polymerase reaction and produced a high fluorescence intensity. Under the optimized conditions, the linear range of this aptasensor was from 10² CFU·mL^?1 to 10⁸ CFU·mL^?1 with a detection limit of 10² CFU·mL^?1. Compared with other reported fluorescence detection methods, this approach has two advantages. First, this fluorescence aptasensor does not require nanomaterials as the quencher, which reduces the cost and saves time. Second, the chain-displacement polymerase reaction was used in this fluorescence aptasensor to amplify the signals, which further enhanced the sensitivity and lowered the detection limit. As this method was suitable for the detection of Salmonella paratyphi A in milk samples and potentially other bacteria, environmental monitoring and related food safety analysis should also be possible by this approach.