The 3′–5′ exonuclease enzyme plays a dominant role in multiple pivotal physiological activities, such as DNA replication and repair processes. In this study, we designed a sensitive graphene oxide (GO)-based probe for the detection of exonuclease enzymatic activity. In the absence of Exo III, the strong π–π interaction between the fluorophore-tagged DNA and GO causes the efficient fluorescence quenching via a fluorescence resonance energy transfer (FRET). In contrast, in the presence of Exo III, the fluorophore-tagged 3′-hydroxyl termini of the DNA probe was digested by Exo III to set the fluorophore free from adsorption when GO was introduced, causing an inefficient fluorescence quenching. As a result, the fluorescence intensity of the sensor was found to be proportional to the concentration of Exo III; towards the detection of Exo III, this simple GO-based probe demonstrated a highly sensitive and selective linear response in the low detection range from 0.01 U mL −1 to 0.5 U mL −1 and with the limit of detection (LOD) of 0.001 U mL −1 . Compared with other fluorescent probes, this assay exhibited superior sensitivity and selectivity in both buffer and fetal bovine serum samples, in addition to being cost effective and having a simple setup.
more »
« less
A Bioluminescent Protein‐Graphene Oxide Donor‐Quencher Pair in DNA Hybridization Assays
Despite fluorescent quenching with graphene oxide (GO) having shown great success in various applications ‐ bioluminescent quenching has not yet been demonstrated using GO as a quencher. To explore the ability of GO to quench bioluminescence, we used
- Award ID(s):
- 1841419
- PAR ID:
- 10383366
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- ChemPlusChem
- Volume:
- 87
- Issue:
- 12
- ISSN:
- 2192-6506
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
null (Ed.)Abstract Small RNAs are non-coding RNAs that play important roles in the lives of both animals and plants. They are 21- to 24-nt in length and ∼10 nm in size. Their small size and high diversity have made it challenging to develop detection methods that have sufficient resolution and specificity to multiplex and quantify. We created a method, sRNA-PAINT, for the detection of small RNAs with 20 nm resolution by combining the super-resolution method, DNA-based points accumulation in nanoscale topography (DNA-PAINT), and the specificity of locked nucleic acid (LNA) probes for the in situ detection of multiple small RNAs. The method relies on designing probes to target small RNAs that combine DNA oligonucleotides (oligos) for PAINT with LNA-containing oligos for hybridization; therefore, we developed an online tool called ‘Vetting & Analysis of RNA for in situ Hybridization probes’ (VARNISH) for probe design. Our method utilizes advances in DNA-PAINT methodologies, including qPAINT for quantification, and Exchange-PAINT for multiplexing. We demonstrated these capabilities of sRNA-PAINT by detecting and quantifying small RNAs in different cell layers of early developmental stage maize anthers that are important for male sexual reproduction.more » « less
-
Double-stranded (ds) biosensors are homogeneous oligonucleotide probes for detection of nucleic acid sequences in biochemical assays and live cell imaging. Locked nucleic acid (LNA) modification can be incorporated in the biosensors to enhance the binding affinity, specificity, and resistance to nuclease degradation. However, LNA monomers in the quencher sequence can also prevent the target-fluorophore probe binding, which reduces the signal of the dsLNA biosensor. This study investigates the influence of LNA modification on dsLNA biosensors by altering the position and amount of LNA monomers present in the quencher sequence. We characterize the fluorophore–quencher interaction, target detection, and specificity of the biosensor in free solution and evaluate the performance of the dsLNA biosensor in 2D monolayers and 3D spheroids. The data indicate that a large amount of LNA monomers in the quencher sequence can enhance the specificity of the biosensor, but prevents effective target binding. Together, our results provide guidelines for improving the performance of dsLNA biosensors in nucleic acid detection and gene expression analysis in live cells.more » « less
-
This paper reports an integrated dual-modality microfluidic sensor chip, consisting of a patterned periodic array of nanoposts coated with gold (Au) and graphene oxide (GO), to detect target biomarker molecules in a limited sample volume. The device generates both electrochemical and surface plasmon resonance (SPR) signals from a single sensing area of Au–GO nanoposts. The Au–GO nanoposts are functionalized with specific receptor molecules, serving as a spatially well-defined nanostructured working electrode for electrochemical sensing, as well as a nanostructured plasmonic crystal for SPR-based sensing via the excitation of surface plasmon polaritons. High sensitivity of the electrochemical measurement originates from the presence of the nanoposts on the surface of the working electrode where radial diffusion of redox species occurs. Complementarily, the SPR detection allows convenient tracking of dynamic antigen–antibody interactions, to describe the association and dissociation phases occurring at the sensor surface. The soft-lithographically formed nanoposts provide high reproducibility of the sensor response to epidermal growth factor receptor ( ErbB2 ) molecules even at a femtomolar level. Sensitivities of the electrochemical measurements to ErbB2 are found to be 20.47 μA μM −1 cm −2 in a range from 1 fM to 0.1 μM, and those of the SPR measurements to be 1.35 nm μM −1 in a range from 10 pM to 1 nM, and 0.80 nm μM −1 in a range from 1 nM to 0.1 μM. The integrated dual-modality sensor offers higher sensitivity (through higher surface area and diffusions from nanoposts for electrochemical measurements), as well as the dynamic measurements of antigen–antibody bindings (through the SPR measurement), while operating simultaneously in a same sensing area using the same sample volume.more » « less
-
more » « less
Fast and accurate detection of nucleic acids is key for pathogen identification. Methods for DNA detection generally rely on fluorescent or colorimetric readout. The development of label-free assays decreases costs and test complexity. We present a novel method combining a one-pot isothermal generation of DNA nanoballs with their detection by electrical impedance. We modified loop-mediated isothermal amplification by using compaction oligonucleotides that self-assemble the amplified target into nanoballs. Next, we use capillary-driven flow to passively pass these nanoballs through a microfluidic impedance cytometer, thus enabling a fully compact system with no moving parts. The movement of individual nanoballs is detected by a change in impedance providing a quantized readout. This approach is flexible for the detection of DNA/RNA of numerous targets (severe acute respiratory syndrome coronavirus 2, HIV, β-lactamase gene, etc.), and we anticipate that its integration into a standalone device would provide an inexpensive (<$5), sensitive (10 target copies), and rapid test (<1 hour).
