Yanli Wen

A DNA nanostructure-based biomolecular probe carrier platform for electrochemical biosensing.

A critical challenge in surface-based biomolecular detection is the reduced accessibility of target molecules to probes arranged on a heterogeneous surface compared to probe–target recognition in homogeneous solution.[1–5] To improve the recognition abilities of such heterogeneous surface probes, much effort has been devoted to control the surface chemistry, conformation, and packing density of the probe molecules as well as the size and geometry of the surface.[6–11] Here, we devise a new concept to achieve improved probe–target recognition properties by introducing a probe bearing a 3D DNA nanostructure-based chip platform. DNA nanotechnology has attracted intense interest because the unparalleled self-recognition properties of DNA offer flexibility and convenience for the ‘bottom-up’ construction of exquisite nanostructures with high controllability and precision.[12–20] Our strategy to design and construct 3D nanostructured recognition probes on a surface provides a significantly enhanced spatial positioning range and accessibility of the probes on a surface over previously reported linear or stem-loop probe structures.[2,7]

DNA Nanostructure-Decorated Surfaces for Enhanced Aptamer-Target Binding and Electrochemical Cocaine Sensors

The sensitivity of aptamer-based electrochemical sensors is often limited by restricted target accessibility and surface-induced perturbation of the aptamer structure, which arise from imperfect packing of probes on the heterogeneous and locally crowded surface. In this study, we have developed an ultrasensitive and highly selective electrochemical aptamer-based cocaine sensor (EACS), based on a DNA nanotechnology-based sensing platform. We have found that the electrode surface decorated with an aptamer probe-pendant tetrahedral DNA nanostructure greatly facilitates cocaine-induced fusion of the split anticocaine aptamer. This novel design leads to a sensitive cocaine sensor with a remarkably low detection limit of 33 nM. It is also important that the tetrahedra-decorated surface is protein-resistant, which not only suits the enzyme-based signal amplification scheme employed in this work, but ensures high selectivity of this sensor when deployed in sera or other adulterated samples.

Target-Responsive, DNA Nanostructure-Based E-DNA Sensor for microRNA Analysis

Because of the short size and low abundance of microRNAs, it is challenging to develop fast, inexpensive, and simple biosensors to detect them. In this work, we have demonstrated a new generation (the third generation) of E-DNA sensor for the sensitive and specific detection of microRNAs. Our third generation of E-DNA sensor can sensitively detect microRNA target (microRNA-141) as low as 1 fM. The excellent specificity has been demonstrated by its differential ability to the highly similar microRNA analogues. In our design, the use of DNA tetrahedron ensures the stem-loop structure in well controlled density with improved reactivity. The regulation of the thermodynamic stability of the stem-loop structure decreases the background signal and increases the specificity as well. The enzymes attached bring the electrocatalytic signal to amplify the detection. The combination of these effects improves the sensitivity of the E-DNA sensor and makes it suitable to the microRNA detection. Finally, our third generation of E-DNA sensor is generalizable to the detection of other micro RNA targets (for example, microRNA-21).

DNA Nanostructure-Based Universal Microarray Platform for High-Efficiency Multiplex Bioanalysis in Biofluids

Microarrays of biomolecules have greatly promoted the development of the fields of genomics, proteomics, and clinical assays because of their remarkably parallel and high-throughput assay capability. Immobilization strategies for biomolecules on a solid support surface play a crucial role in the fabrication of high-performance biological microarrays. In this study, rationally designed DNA tetrahedra carrying three amino groups and one single-stranded DNA extension were synthesized by the self-assembly of four oligonucleotides, followed by high-performance liquid chromatography purification. We fabricated DNA tetrahedron-based microarrays by covalently coupling the DNA tetrahedron onto glass substrates. After their biorecognition capability was evaluated, DNA tetrahedron microarrays were utilized for the analysis of different types of bioactive molecules. The gap hybridization strategy, the sandwich configuration, and the engineering aptamer strategy were employed for the assay of miRNA biomarkers, protein cancer biomarkers, and small molecules, respectively. The arrays showed good capability to anchor capture biomolecules for improving biorecognition. Addressable and high-throughput analysis with improved sensitivity and specificity had been achieved. The limit of detection for let-7a miRNA, prostate specific antigen, and cocaine were 10 fM, 40 pg/mL, and 100 nM, respectively. More importantly, we demonstrated that the microarray platform worked well with clinical serum samples and showed good relativity with conventional chemical luminescent immunoassay. We have developed a novel approach for the fabrication of DNA tetrahedron-based microarrays and a universal DNA tetrahedron-based microarray platform for the detection of different types of bioactive molecules. The microarray platform shows great potential for clinical diagnosis.

DNA nanostructure-based ultrasensitive electrochemical microRNA biosensor

MicroRNAs (miRNAs) are key regulators of a wide range of cellular processes, and have been identified as promising cancer biomarkers due to their stable presence in serum. As an surface-based electrochemical biosensors which offer great opportunities for low-cost, point-of-care tests (POCTs) of disease-associated miRNAs. Nevertheless, the sensitivity of miRNA sensors is often limited by mass transport and the surface crowding effect at the water-electrode interface. Here, we present a protocol as well as guidelines for ultrasensitive detection of miRNA with DNA nanostructure-based electrochemical miRNA biosensor. By employing the three-dimensional DNA nanostructure-based interfacial engineering approach, we can directly detect as few as attomolar (<1000 copies) miRNAs with high single-base discrimination ability. Since this ultrasensitive electrochemical miRNA sensor (EMRS) is highly reproducible and essentially free of prior target labeling and PCR amplification, it can conveniently and reliably analyze miRNA expression levels in clinical samples from esophageal squamous cell carcinoma (ESCC) patients.

Development of mercury (II) ion biosensors based on mercury-specific oligonucleotide probes

Mercury (II) ion (Hg(2+)) contamination can be accumulated along the food chain and cause serious threat to the public health. Plenty of research effort thus has been devoted to the development of fast, sensitive and selective biosensors for monitoring Hg(2+). Thymine was demonstrated to specifically combine with Hg(2+) and form a thymine-Hg(2+)-thymine (T-Hg(2+)-T) structure, with binding constant even higher than T-A Watson-Crick pair in DNA duplex. Recently, various novel Hg(2+) biosensors have been developed based on T-rich Mercury-Specific Oligonucleotide (MSO) probes, and exhibited advanced selectivity and excellent sensitivity for Hg(2+) detection. In this review, we explained recent development of MSO-based Hg(2+) biosensors mainly in 3 groups: fluorescent biosensors, colorimetric biosensors and electrochemical biosensors.

Analysis of telomerase activity based on a spired DNA tetrahedron TS primer

The development of sensitive telomerase biosensors is hindered by the restricted accessibility of telomere strand (TS) primer and the limited enzyme reaction space, which is mainly confined by the vertical distance. In this work, we designed an electrochemical telomerase biosensor based on a spired DNA tetrahedron TS primer (STTS). By adding a rigid dsDNA spire onto the top of the DNA tetrahedron, we successfully regulated the distance between the TS primer and the surface, and thus greatly facilitated the telomerase elongation on surface. The signal-to-noise ratio was 2 times higher than TSP without the spire structure. The limit of detection was calculated to be lower than 10 HeLa cells, which is at least 2 magnitudes lower than other surface extension-based electrochemical telomerase sensors without amplification. The practicability of STTS sensor was also demonstrated by analysing various other cell lines including cancer cells, stem cells of high telomerase activity and somatic cells of low telomerase activity.

A Sensitive and Label-Free Pb(II) Fluorescence Sensor Based on a DNAzyme Controlled G-Quadruplex/Thioflavin T Conformation

Pb(II) can cause serious damaging effects to human health, and thus, the study of Pb2+ detection methods to sensitively and selectively monitor Pb(II) pollution has significant importance. In this work, we have developed a label-free fluorescence sensing strategy based on a Pb(II) DNAzyme cleavage and the ThT/G-quadruplex complex. In the presence of Pb(II), a G-rich tail was cut and released from the substrate strand, which then would form a G-quadruplex structure by combination with ThT dye. The fluorescence signal increase was then measured for sensitive Pb(II) quantification with a limit of detection of 0.06 nM. Our sensor also demonstrated high selectivity against six different metal ions, which is very important for the analysis of complex samples.

Multifunctional Yolk–Shell Nanostructure as a Superquencher for Fluorescent Analysis of Potassium Ion Using Guanine-Rich Oligonucleotides

The excellent performance of a biosensor generally depends on the high signal-to-noise ratio, and the superquencher plays a dominant role in fluorescent sensors. Novel nanoquenchers exhibited high quenching efficiency in various fluorescent assays of biological/chemical molecules. Here, we developed a novel nano-biosensor using Fe3O4@C yolk-shell nanoparticles (YSNPs) and studied their quenching effect. We found Fe3O4@C YSNP was a superquencher and exhibited an ultrastrong quenching ability, up to almost 100% quenching efficiency, toward fluorophores. Also, Fe3O4@C YSNPs possessed the most superior fluorescence restoration efficiency, due to biomolecular recognition event, compared to the other nanoquenchers, including bare Fe3O4 NPs, graphene oxide (GO), and single-wall carbon nanotubes (SWCNTs). On the basis of that, a fluorescent sensing platform for potassium-ion (K+) analysis with guanine (G)-rich oligonucleotides was designed. As a result, Fe3O4@C YSNP-based fluorescent sensors demonstrated excellent performance, with an ultrahigh sensitivity of a detection limit as low as 1.3 μM, as well as a wide dynamic range from 50 μM to 10 mM. The proposed method is fast, simple, and cost-effective, suggesting the great potential for practical applications in biomedical detection and clinical diagnosis.