DNA strand displacement-based sensor for the detection of miRNA-141 in serum samples

Isotype-specific agglutination-PCR (ISAP): A sensitive and multiplex method for measuring allergen-specific IgE

Development of a two-site immuno-PCR assay for hepatitis B surface antigen

Detection of very low amounts of illicit drugs such as cocaine in clinical fluids like serum continues to be important for many areas in the fight against drug trafficking. Herein, we constructed a label-free nanopore biosensor for rapid and highly sensitive detection of cocaine in human serum and saliva samples based on target-induced strand release strategy. In this bioassay, an aptamer for cocaine was prehybridized with a short complementary DNA. Owing to cocaine specific binding with aptamer, the short DNA strand was displaced from aptamer and translocation of this output DNA through α-hemolysin nanopore generated distinct spike-like current blockages. When plotted in double-logarithmic scale, a linear relationship between target cocaine concentration and output DNA event frequency was obtained in a wide concentration range from 50 nM to 100 μM of cocaine, with the limit of detection down to 50 nM. In addition, this aptamer-based sensor method was successfully applied for cocaine detection in complex biological fluids like human saliva and serum samples with great selectivity. Simple preparation, low cost, rapid, label-free, and real sample detection are the motivating factors for practical application of the proposed biosensor.

Developing a DNA aptamer-based approach for biosensing cystatin-c in serum: An alternative to antibody-based methods

Based on small molecule-linked DNA and the nicking endonuclease-assisted amplification (NEA) strategy, a novel electrochemical method for protein detection is proposed in this work. Specifically, the small molecule-linked DNA (probe 1) can be protected from exonuclease-catalyzed digestion upon binding to the protein target of the small molecule, so the DNA strand may hybridize with another DNA strand (probe 2) that is previously immobilized onto an electrode surface. Consequently, the NEA process is triggered, resulting in continuous removal of the DNA strands from the electrode surface, and the blocking effect against the electrochemical species [Fe(CN)(6)](3-/4-) becomes increasingly lower; thus, increased electrochemical waves can be achieved. Because the whole process is activated by the target protein, an electrochemical method for protein quantification is developed. Taking folate receptor (FR) as an example in this work, we can determine the protein in a linear range from 0.3 to 15 ng/mL with a detection limit of 0.19 ng/mL. Furthermore, because the method can be used for the assay of FR in serum samples and for the detection of other proteins such as streptavidin by simply changing the small molecule moiety of the DNA probes, this novel method is expected to have great potential applications in the future.

DNA covalently linked to graphene oxide for biotin–streptavidin interaction assay

目的 介绍巢式甲基化特异性聚合酶链反应(nMSP)法,探讨了最佳PCR扩增条件,并用该法分析新鲜癌组织、石蜡包埋组织及肿瘤患者血清中p16基因启动子的甲基化状态.方法将基因组DNA变性成为单链,用亚硫酸氢盐修饰单链DNA,将所有未甲基化的胞嘧啶被转变为尿嘧啶,而甲基化的胞嘧啶则不变.设计针对甲基化和非甲基化等位基因特异引物,进行巢式聚合酶链反应扩增,最后经凝胶电泳检测目的片段.结果在3种类型的标本中都检测出了p16基因启动子甲基化.用甲基化特异性聚合酶链反应法(MSP)和nMSP法分别检测34例非小细胞肺癌(NSCLC)患者血清,p16基因启动子甲基化阳性率分别为53%(18/34)和74%(25/34), nMSP法具有更高的灵敏度.结论筑巢式甲基化特异性聚合酶链反应是一种灵敏度高、特异性强的甲基化检测方法,可广泛应用于不同类型标本基因启动子甲基化分析。

DNA methyltransferase activity detection based on graphene quantum dots using fluorescence and fluorescence anisotropy

Ultrasensitive electrochemical paper-based biosensor for microRNA via strand displacement reaction and metal-organic frameworks

Target binding and DNA hybridization-induced gold nanoparticle aggregation for colorimetric detection of thrombin

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer (BC). Thus, early detection and accurate diagnosis of this cancer are crucial for improving the survival rate of patients. Specific microRNAs (miRNAs) have been implicated in the occurrence, proliferation, and metastasis of TNBC. Addressing this need, our study developed a biosensor platform for early and accurate TNBC diagnosis by integrating electrochemiluminescence (ECL) technology with a DNA sensing strategy. Specifically, synthesized positively charged carbon dots (CDs) were used to neutralize the electrostatic repulsion between DNA strands and facilitate the assembly of DNA triangular prisms (DNA TP-CDs). Hairpins were then incorporated into the DNA TP-CDs to form the final DNA crown structure. The early TNBC biomarker, microRNA-93-3p (miR-93-3p), allowed for the binding between the DNA Crown and the DNA track on the electrode and initiated the ECL signal. Subsequently, microRNA-210 (miR-210) unlocked the DNA tripedal walker, and its movement on the DNA Crown eventually quenched the ECL signal, enabling accurate TNBC diagnosis and tumor stage assessment. Our proposed biosensor had satisfactory sensing efficiency due to the ordered DNA track and rapid-moving DNA walker. The data revealed a good linear relationship between the ECL signals and the logarithm of miRNA concentrations, with miR-93-3p having a detection limit of 31.04 aM and miR-210 having a detection limit of 7.69 aM. The biosensor also showed satisfactory performance in serum samples and cells. Taken together, this study hopes to provide ideas and applications for clinical diagnosis as well as the personalized treatment of TNBC.

Design and analysis of self-priming extension DNA hairpin probe for miRNA detection based on a unified dynamic programming framework

MicroRNA (miRNA) is a potential biomarker for the early screening and diagnosis of cancers and is widely present in human blood, urine and saliva. Here, we report a microfluidics-assembled tool for miRNA detection based on the regulation of DNA locked and unlocked states and explore its application in complex samples. Microfluidic techniques are used to continuously assemble the locked-to-unlocked transforming system using a rapid one-step method. It only takes 2 min to produce enough locked-to-unlocked systems for a miRNA detection experiment. DNA molecules with a recognition sequence and a G-rich reporter sequence (G4m) are locked by attaching both ends to the surface of magnetic beads (MBs) in microchannels. The presence of the target miRNA can initiate the specific cleavage of one end of G4m by duplex-specific nuclease, resulting in the transition of G4m from a locked state to an unlocked state. This transition enables G4m to freely fold into a G-quadruplex, which can participate in the catalysis of ABTS oxidation and result in a turquoise color. During the whole process, the target miRNA remains intact and continuously initiate specific cleavage, facilitating signal amplification. Magnetic separation steps are employed to assist in miRNA enrichment and interference reduction. As a proof of concept, we quantified miRNA-21 using the locked-to-unlocked system. The assay allows specific detection of miRNA-21 in the range of 3.2-570 pM with a detection limit of 2.01 pM (S/N = 3). Furthermore, the locked-to-unlocked system is used to analyze miRNA-spiked urine, saliva and serum samples and shows robust performance in different matrices.

Stimulus-response click chemistry based aptamer-functionalized mesoporous silica nanoparticles for fluorescence detection of thrombin

Single-particle inductively coupled plasma mass spectrometry (SP-ICP-MS) has great potential for sensitive analysis of nucleic acids; however, it usually requires separation of target-induced nanoparticle reporters, and the sequence of probes on nanoparticle reporters has to be tuned for each target accordingly. Here, we developed a homogeneous multicomponent nucleic acid enzyme (MNAzyme) assay for universal nucleic acid detection. The two components of MNAzyme contain target recognition sites, substrate binding sites, and a catalytic core. Only in the presence of a specific nucleic acid target, the MNAzyme will assemble to trigger its nucleic acid enzyme activity and cleave its substrate (Linker DNA). The Linker DNA could link gold nanoparticle (AuNP) probes to form a larger assembled particle, while the cleavage of Linker DNA will disturb the linkage between probes, inducing a smaller assembled particle. The assembled particles with different sizes could be differentiated and sensitively detected in SP-ICP-MS, which also enables the tolerance of a complex matrix. By simply altering the sequences of the target recognition sites in MNAzyme, we applied the assay for two types of nucleic acids (long strand DNA and short strand RNA), malaria DNA and miRNA-10b. With increasing the target concentration, the signal intensity of each assembled particle decreases, but the frequency of assembled particle pulse increases. Both targets could be quantitatively detected from 0.1 to 25 pmol L-1 with high specificity in serum samples. The developed MNAzyme-SP-ICP-MS assay possesses simple operation in a homogeneous reaction, easy tunability for multiple types of nucleic acid targets, and good compatibility with clinic samples.