Analysis of DNA complexes with small solutes by CE with LIF detection

In this article, we describe the analysis of aptamers for Hg²⁺ ions through CE with LIF (CE‐LIF) detection using 2% poly(ethylene oxide) solutions containing OliGreen (fluorophore). In the presence of an EOF, DNA strands migrating against the EOF were detected at the cathode end. Four DNA strands – T₃₃, T₅C₂₈, T₅C₅T₂₃, and T₁₅C₅T₁₃ – could not be separated through CE‐LIF in the absence of Hg²⁺. At 0.3 mM Hg²⁺, however, all four were partially separated within 20 min, with SDs of the migration times all being less than 2.5%. From the CE, fluorescence, and ellipticity data, we concluded that the conformations of these four DNA strands all changed from random‐coil to folded structures as a result of T–Hg²⁺–T bonding. In addition, we found that this CE approach provided different electropherograms patterns for T₇, T₁₅, and T₃₃ in the absence and presence of Hg²⁺, indicating various interactions of the DNA strands with Hg²⁺. Using this simple, high‐resolution CE approach, we also demonstrated that adenosine triphosphate has a stronger interaction with the adenosine triphosphate aptamer than with either the platelet‐derived growth factor aptamer or T₃₃. This CE approach holds great potential for screening aptamers for small solutes, studying the catalytic activity of DNAzymes, and evaluating the biological functions of microRNA.     

Simple and Fast Picomolar Detection of Ochratoxin A Using a Reusable Label Free Aptasensor Built with a Layer‐by‐layer Procedure

A label free impedimetric aptasensor for simple, fast and reusable picomolar detections of Ochratoxin A (OTA) in grape juices was designed. Two main factors were observed to affect the accurate detections of the toxin: i‐lateral interactions between self‐assembled aptamers ii‐ adsorption of large molecules present in complex matrixes like grape juices. Lateral interactions between aptamers were minimized by constructing the aptasensor in a Layer‐by‐Layer procedure. The interferences associated to the unspecific and irreversible adsorption of large molecules present in grape juice, were reduced by submitting samples to ultrafiltration prior to analysis. With this protocol, a 0.12 pM limit of detection and 0.24 pM limit of quantification in spiked grape juices were achieved after only 5–7 mins of interaction with the samples. The presented aptasensor can be recovered after a simple immersion in hot water (90 °C) for ten minutes.     

The Use of Xenonucleic Acids Significantly Reduces the In Vivo Drift of Electrochemical Aptamer-Based Sensors

Electrochemical aptamer-based sensors support the high-frequency, real-time monitoring of molecules-of-interest in vivo. Achieving this requires methods for correcting the sensor drift seen during in vivo placements. While this correction ensures EAB sensor measurements remain accurate, as drift progresses it reduces the signal-to-noise ratio and precision. Here, we show that enzymatic cleavage of the sensor's target-recognizing DNA aptamer is a major source of this signal loss. To demonstrate this, we deployed a tobramycin-detecting EAB sensor analog fabricated with the DNase-resistant “xenonucleic acid” 2’O-methyl-RNA in a live rat. In contrast to the sensor employing the equivalent DNA aptamer, the 2’O-methyl-RNA aptamer sensor lost very little signal and had improved signal-to-noise. We further characterized the EAB sensor drift using unstructured DNA or 2’O-methyl-RNA oligonucleotides. While the two devices drift similarly in vitro in whole blood, the in vivo drift of the 2’O-methyl-RNA-employing device is less compared to the DNA-employing device. Studies of the electron transfer kinetics suggested that the greater drift of the latter sensor arises due to enzymatic DNA degradation. These findings, coupled with advances in the selection of aptamers employing XNA, suggest a means of improving EAB sensor stability when they are used to perform molecular monitoring in the living body.     

Switchable Fluorescent Light-Up Aptamers Based on Riboswitch Architectures

Fluorescent light-up RNA aptamers (FLAPs) such as Spinach or Mango can bind small fluorogens and activate their fluorescence. Here, we adopt a switching mechanism otherwise found in riboswitches and use it to engineer switchable FLAPs that can be activated or repressed by trigger oligonucleotides or small metabolites. The fluorophore binding pocket of the FLAPs comprises guanine (G) quadruplexes, whose critical nucleotides can be sequestered by corresponding anti-FLAP sequences, leading to an inactive conformation and thus preventing association with the fluorophore. We modified the FLAPs with designed toehold hairpins that carry either an anti-FLAP or an anti-anti-FLAP sequence within the loop region. The addition of an input RNA molecule triggers a toehold-mediated strand invasion process that refolds the FLAP into an active or inactive configuration. Several of our designs display close-to-zero leak signals and correspondingly high ON/OFF fluorescence ratios. We also modified purine aptamers to sequester a partial anti-FLAP or an anti-anti-FLAP sequence to control the formation of the fluorogen-binding conformation, resulting in FLAPs whose fluorescence is activated or deactivated in the presence of guanine or adenine. We demonstrate that switching modules can be easily combined to generate FLAPs whose fluorescence depends on several inputs with different types of input logic.     

An Aptamer-Based Competitive Fluorescence Quenching Assay for IgE

A simple competitive fluorescence quenching assay based on aptamer was developed for IgE detection. Two DNA probes were used. One is 5′-end fluorescein-labeled IgE aptamer; the other is 3′-end DABCYL-labeled short DNA, which would hybridize with IgE aptamer to quench the fluorescence. In the presence of IgE, the aptamer-IgE complex formed is strong enough to prevent the short DNA probes hybridizing with the bounded aptamer probes, which results in the less decrease of fluorescence intensity. The signal change was found to be proportional to the concentration of IgE from 0.35 to 35 nM with a detection limit of 0.17 nM.     

光学适配体传感器检测赭曲霉毒素A的研究进展

赭曲霉毒素A(Ochratoxin A,OTA)是真菌产生的次级代谢产物,性质稳定,不易去除,人体摄入后将产生严重的健康危害。数十年来,核酸适配体不断发展,成为生物传感器的重要识别元件之一,适体传感器被广泛用于生物、医药、疾病等分析检测。本文总结了用于检测OTA的经典方法和基于核酸适配体的生物传感器方法,并主要从光学适配体传感器方面阐述了近年用于检测赭曲霉毒素A的适配体传感器,并对其进行了总结和展望。     

Electrogenerated chemiluminescence aptamer-based method for the determination of thrombin incorporating quenching of tris(2,2-bipyridine)ruthenium by ferrocene

A novel electrogenerated chemiluminescence (ECL) aptamer-based biosensing method for the determination of thrombin was developed on basis of a structure-switching ECL-dequenching mechanism. A thiolated ss-DNA capture probe, composing of a ss-DNA sequence to adopt two distinct structures-a DNA double strand with a complementary DNA sequence tagged with a ECL signal producer tris(2,2^′-bipyridyl)ruthenium derivative and a DNA duplex with a thrombin-binding aptamer tagged with a ECL-quencher ferrocene (FcDNA), was self-assembled onto surface of a gold electrode. In the presence of thrombin, the aptamer sequence prefers to form the aptamer-thrombin complex and the switch of the binding partners occurs in conjunction with the generation of a strong ECL signal owing to the dissociation of FcDNA. The integrated ECL intensity versus the concentration of thrombin was linear in the range from 2.0 × 10⁻¹⁰ M to 2.0 × 10⁻⁷ M. The detection limit was 6 × 10⁻¹¹ M. The relative standard derivation for 2.0 × 10⁻⁹ M was 5.7% (n = 7). This work demonstrates that the sensitivity and specificity of ECL aptamer-based method for proteins can be greatly improved using quenching ECL signal producer by a chemical quencher such as ferrocene.     

Dual-color upconversion fluorescence and aptamer-functionalized magnetic nanoparticles-based bioassay for the simultaneous detection of Salmonella Typhimurium and Staphylococcus aureus

A sensitive luminescent bioassay for the simultaneous detection of Salmonella Typhimurium and Staphylococcus aureus was developed using aptamer-conjugated magnetic nanoparticles (MNPs) for both recognition and concentration elements and using upconversion nanoparticles (UCNPs) as highly sensitive dual-color labels. The bioassay system was fabricated by immobilizing aptamer 1 and aptamer 2 onto the surface of MNPs, which were employed to capture and concentrate S. Typhimurium and S. aureus. NaY_0.78F₄:Yb_0.2,Tm_0.02 UCNPs modified aptamer 1 and NaY_0.28F₄:Yb_0.70,Er_0.02 UCNPs modified aptamer 2 further were bond onto the captured bacteria surface to form sandwich-type complexes. Under optimal conditions, the correlation between the concentration of S. Typhimurium and the luminescent signal was found to be linear within the range of 10¹–10⁵ cfu mL⁻¹ (R² = 0.9964), and the signal was in the range of 10¹–10⁵ cfu mL⁻¹ (R² = 0.9936) for S. aureus. The limits of detection of the developed method were found to be 5 and 8 cfu mL⁻¹ for S. Typhimurium and S. aureus, respectively. The ability of the bioassay to detect S. Typhimurium and S. aureus in real water samples was also investigated, and the results were compared to the experimental results from the plate-counting methods. Improved by the magnetic separation and concentration effect of MNPs, the high sensitivity of UCNPs, and the different emission lines of Yb/Er- and Yb/Tm-doped NaYF₄ UCNPs excited by a 980 nm laser, the present method performs with both high sensitivity and selectivity for the two different types of bacteria.     

Highly sensitive determination of recombinant human erythropoietin-α in aptamer-based affinity probe capillary electrophoresis with laser-induced fluorescence detection

Recombinant human erythropoietin-α (rHuEPO-α) has been widely used in clinic for anemia treatment. The detection and quantification of rHuEPO-α is essential for monitoring this widespread recombinant glycoprotein pharmaceutical. In this paper, we developed a new affinity probe capillary electrophoresis/laser-induced fluorescence (APCE/LIF) method for the detection of rHuEPO-α by using a specific single-stranded DNA aptamer probe for the first time. In this method, the complex of aptamer-rHuEPO-α and the free aptamer can be well separated and identified by their migration and fluorescence intensity after systematic optimization. The existence of sodium cation in the sample buffer and running buffer played a critical role for stabilizing complex and enhancing the separation efficiency, additionally, suitable high voltage and sample buffer additives were also important for improving the peak height of the complex. Under the optimized conditions, the method was successfully applied for the quantification of rHuEPO-α in physiological buffer, artificial urine and human serum. The linear range for rHuEPO-α was from 0.2 to 100 nM and the limit of detection was 0.2 nM (i.e. 7.4 ng/mL). Further binding experiments using fluorescein isothiocyanate-labeled rHuEPO-α (F-rHuEPO-α) and N-deglycosylated F-rHuEPO-α demonstrated that the oligosaccharides moiety was of importance in the specific interaction between rHuEPO-α and its aptamer.     

以聚硫堇为电化学探针的非标记型核酸适配体传感器

采用电聚合法制备了聚硫堇氧化还原电化学探针, 以金纳米粒子为固定核酸适配体的载体构建了非标记型核酸适配体传感器. 用电化学阻抗谱对传感器的组装过程进行了监测, 用循环伏安法和差分脉冲伏安法考察了传感器的电化学行为. 结果表明, 该传感器对凝血酶的检测在1.0 pg/mL～500 ng/mL范围内呈良好的线性关系, 相关系数为0.998, 检出限为0.38 pg/mL. 该传感器制备简单、 灵敏度高且抗干扰能力强.