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A novel fluorescent biosensor based on dendritic DNA nanostructure in combination with ligase reaction for ultrasensitive detection of DNA methylation.
Zhang, S, Huang, J, Lu, J, Liu, M, Li, Y, Fang, L, Huang, H, Huang, J, Mo, F, Zheng, J
Journal of nanobiotechnology. 2019;(1):121
Abstract
BACKGROUND DNA methylation detection is indispensable for the diagnosis and prognosis of various diseases including malignancies. Hence, it is crucial to develop a simple, sensitive, and specific detection strategy. METHODS A novel fluorescent biosensor was developed based on a simple dual signal amplification strategy using functional dendritic DNA nanostructure and signal-enriching polystyrene microbeads in combination with ligase detection reaction (LDR). Dendritic DNA self-assembled from Y-DNA and X-DNA through enzyme-free DNA catalysis of a hairpin structure, which was prevented from unwinding at high temperature by adding psoralen. Then dendritic DNA polymer labeled with fluorescent dye Cy5 was ligated with reporter probe into a conjugate. Avidin-labeled polystyrene microbeads were specifically bound to biotin-labeled capture probe, and hybridized with target sequence and dendritic DNA. LDR was triggered by adding Taq ligase. When methylated cytosine existed, the capture probe and reporter probe labeled with fluorescent dye perfectly matched the target sequence, forming a stable duplex to generate a fluorescence signal. However, after bisulfite treatment, unmethylated cytosine was converted into uracil, resulting in a single base mismatch. No fluorescence signal was detected due to the absence of duplex. RESULTS The obtained dendritic DNA polymer had a large volume. This method was time-saving and low-cost. Under the optimal experimental conditions using avidin-labeled polystyrene microbeads, the fluorescence signal was amplified more obviously, and DNA methylation was quantified ultrasensitively and selectively. The detection range of this sensor was 10-15 to 10-7 M, and the limit of detection reached as low as 0.4 fM. The constructed biosensor was also successfully used to analyze actual samples. CONCLUSION This strategy has ultrasensitivity and high specificity for DNA methylation quantification, without requiring complex processes such as PCR and enzymatic digestion, which is thus of great value in tumor diagnosis and biomedical research.
2.
Quantum capacitance-limited MoS2 biosensors enable remote label-free enzyme measurements.
Le, ST, Guros, NB, Bruce, RC, Cardone, A, Amin, ND, Zhang, S, Klauda, JB, Pant, HC, Richter, CA, Balijepalli, A
Nanoscale. 2019;(33):15622-15632
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Abstract
We have demonstrated atomically thin, quantum capacitance-limited, field-effect transistors (FETs) that enable the detection of pH changes with 75-fold higher sensitivity (≈4.4 V per pH) over the Nernst value of 59 mV per pH at room temperature when used as a biosensor. The transistors, which are fabricated from monolayer films of MoS2, use a room temperature ionic liquid (RTIL) in place of a conventional oxide gate dielectric and exhibit very low intrinsic noise resulting in a pH resolution of 92 × 10-6 at 10 Hz. This high device performance, which is a function of the structure of our device, is achieved by remotely connecting the gate to a pH sensing element allowing the FETs to be reused. Because pH measurements are fundamentally important in biotechnology, the increased resolution demonstrated here will benefit numerous applications ranging from pharmaceutical manufacturing to clinical diagnostics. As an example, we experimentally quantified the function of the kinase Cdk5, an enzyme implicated in Alzheimer's disease, at concentrations that are 5-fold lower than physiological values, and with sufficient time-resolution to allow the estimation of both steady-state and kinetic parameters in a single experiment. The high sensitivity, increased resolution, and fast turnaround time of the measurements will allow the development of early diagnostic tools and novel therapeutics to detect and treat neurological conditions years before currently possible.