scholarly journals PCR-free, label-free detection of sequence-specific DNA with single-molecule sensitivity using in vitro N-hybrid system in microfluidic drops

2022 ◽  
Author(s):  
Yizhe Zhang ◽  
David A Weitz
Keyword(s):  
Single Molecule ◽  
High Specificity ◽  
Label Free ◽  
Hybrid Strategy ◽  
Single Molecule Level ◽  
Target Dna ◽  
Label Free Detection ◽  
Novel Method ◽  
Picoliter Droplet

We propose a novel method that can detect DNA with high specificity at the single-molecule level by employing the in vitro N-hybrid strategy realized in sub-picoliter microfluidic drops. It detects target DNA based on the specific interactions of the target-encoded proteins with their partner molecules, and achieves single-molecule sensitivity via signal-transduction and signal-amplification during gene-expression processes in a sub-picoliter droplet, therefore effectively avoiding complicated procedures in labeling-based methods or biases and artifacts in PCR-based methods.

scholarly journals Novel DNA Aptameric Sensors to Detect the Toxic Insecticide Fenitrothion

2021 ◽  
Vol 22 (19) ◽  
pp. 10846
Author(s):  
Kien Hong Trinh ◽  
Ulhas Sopanrao Kadam ◽  
Jinnan Song ◽  
Yuhan Cho ◽  
Chang Ho Kang ◽  
...  
Keyword(s):  
In Vitro Selection ◽  
High Specificity ◽  
Cross Reactivity ◽  
Dna Aptamers ◽  
Label Free ◽  
Wide Range ◽  
Label Free Detection ◽  
Detection Mode ◽  
Aptamer Sensor

Fenitrothion is an insecticide belonging to the organophosphate family of pesticides that is widely used around the world in agriculture and living environments. Today, it is one of the most hazardous chemicals that causes severe environmental pollution. However, detection of fenitrothion residues in the environment is considered a significant challenge due to the small molecule nature of the insecticide and lack of molecular recognition elements that can detect it with high specificity. We performed in vitro selection experiments using the SELEX process to isolate the DNA aptamers that can bind to fenitrothion. We found that newly discovered DNA aptamers have a strong ability to distinguish fenitrothion from other organophosphate insecticides (non-specific targets). Furthermore, we identified a fenitrothion-specific aptamer; FenA2, that can interact with Thioflavin T (ThT) to produce a label-free detection mode with a Kd of 33.57 nM (9.30 ppb) and LOD of 14 nM (3.88 ppb). Additionally, the FenA2 aptamer exhibited very low cross-reactivity with non-specific targets. This is the first report showing an aptamer sensor with a G4-quadruplex-like structure to detect fenitrothion. Moreover, these aptamers have the potential to be further developed into analytical tools for real-time detection of fenitrothion from a wide range of samples.

scholarly journals Biological Nanopores: Engineering on Demand

Life ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 27
Author(s):  
Ana Crnković ◽  
Marija Srnko ◽  
Gregor Anderluh
Keyword(s):  
Molecular Biology ◽  
Single Molecule ◽  
Enzymatic Reactions ◽  
Label Free ◽  
Natural Sources ◽  
On Demand ◽  
Label Free Detection ◽  
Long Read ◽  
Inorganic Molecules ◽  
Standard Molecular Biology

Nanopore-based sensing is a powerful technique for the detection of diverse organic and inorganic molecules, long-read sequencing of nucleic acids, and single-molecule analyses of enzymatic reactions. Selected from natural sources, protein-based nanopores enable rapid, label-free detection of analytes. Furthermore, these proteins are easy to produce, form pores with defined sizes, and can be easily manipulated with standard molecular biology techniques. The range of possible analytes can be extended by using externally added adapter molecules. Here, we provide an overview of current nanopore applications with a focus on engineering strategies and solutions.

Y-shaped probe for convenient and label-free detection of microRNA-21 in vitro

2016 ◽  
Vol 499 ◽  
pp. 8-14 ◽  
Author(s):  
Kui He ◽  
Rong Liao ◽  
Changqun Cai ◽  
Caishuang Liang ◽  
Chan Liu ◽  
...  
Keyword(s):  
Label Free ◽  
Label Free Detection

scholarly journals Bottom-up single-molecule strategy for understanding subunit function of tetrameric β-galactosidase

2018 ◽  
Vol 115 (33) ◽  
pp. 8346-8351 ◽  
Author(s):  
Xiang Li ◽  
Yu Jiang ◽  
Shaorong Chong ◽  
David R. Walt
Keyword(s):  
Single Molecule ◽  
Wild Type ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Single Molecule Level ◽  
Subunit Function ◽  
Enzyme Molecules ◽  
Individual Enzyme ◽  
Kinetics Of

In this paper, we report an example of the engineered expression of tetrameric β-galactosidase (β-gal) containing varying numbers of active monomers. Specifically, by combining wild-type and single-nucleotide polymorphism plasmids at varying ratios, tetrameric β-gal was expressed in vitro with one to four active monomers. The kinetics of individual enzyme molecules revealed four distinct populations, corresponding to the number of active monomers in the enzyme. Using single-molecule-level enzyme kinetics, we were able to measure an accurate in vitro mistranslation frequency (5.8 × 10−4 per base). In addition, we studied the kinetics of the mistranslated β-gal at the single-molecule level.

Enhancing the generating and collecting efficiency of single particle upconverting luminescence at low power excitation

Nanophotonics ◽  
2020 ◽  
Vol 9 (7) ◽  
pp. 1993-2000 ◽  
Author(s):  
Chenshuo Ma ◽  
Chunyan Shan ◽  
Kevin Park ◽  
Aaron T. Mok ◽  
Paul J. Antonick ◽  
...  
Keyword(s):  
Single Molecule ◽  
Single Particle ◽  
Excitation Power ◽  
Single Particle Tracking ◽  
Rare Earth Ions ◽  
High Excitation ◽  
Single Molecule Level ◽  
Collecting Efficiency ◽  
High Luminescence Intensity

AbstractUpconverting luminescent nanoparticles are photostable, nonblinking, and low chemically toxic fluorophores that are emerging as promising fluorescent probes at the single molecule level. High luminescence intensity upconversion nanoparticles (UCNPs) have previously been achieved by doping with high amounts of rare-earth ions using high excitation power (>2.5 MW/cm2). However, such particles are inadequate for in vitro live-cell imaging and single-particle tracking, as high excitation power can cause photodamage. Here, we compared UCNP luminescence intensities with different dopant concentrations and presented more efficient (about seven times) UCNPs at low excitation power by increasing the concentrations of Yb3+ and Tm3+ dopants (NaYF4: 60% Yb3+, 8% Tm3+) and adding a core-shell structure.

scholarly journals Impedimetric Immunosensor Utilizing Polyaniline/Gold Nanocomposite-Modified Screen-Printed Electrodes for Early Detection of Chronic Kidney Disease

Sensors ◽  
2019 ◽  
Vol 19 (18) ◽  
pp. 3990 ◽  
Author(s):  
Muhammad Omar Shaikh ◽  
Boyanagunta Srikanth ◽  
Pei-Yu Zhu ◽  
Cheng-Hsin Chuang
Keyword(s):  
Chronic Kidney Disease ◽  
Kidney Disease ◽  
Early Detection ◽  
Electrochemical Impedance ◽  
High Specificity ◽  
Homogeneous Distribution ◽  
Label Free ◽  
Gold Nanocrystals ◽  
Working Electrode ◽  
Label Free Detection

The presence of small amounts of human serum albumin (HSA) in urine or microalbuminuria (30–300 µg/mL) is a valuable clinical biomarker for the early detection of chronic kidney disease (CKD). Herein, we report on the development of an inexpensive and disposable immunosensor for the sensitive, specific, and label-free detection of HSA using electrochemical impedance spectroscopy (EIS). We have utilized a simple one-step screen-printing protocol to fabricate the carbon-based three-electrode system on flexible plastic substrates. To enable efficient antibody immobilization and improved sensitivity, the carbon working electrode was sequentially modified with electropolymerized polyaniline (PANI) and electrodeposited gold nanocrystals (AuNCs). The PANI matrix serves as an interconnected nanostructured scaffold for homogeneous distribution of AuNCs and the resulting PANI/AuNCs nanocomposite synergically improved the immunosensor response. The PANI/AuNCs-modified working electrode surface was characterized using scanning electron microscopy (SEM) and the electrochemical response at each step was analyzed using EIS in a ferri/ferrocyanide redox probe solution. The normalized impedance variation during immunosensing increased linearly with HSA concentration in the range of 3–300 µg/mL and a highly repeatable response was observed for each concentration. Furthermore, the immunosensor displayed high specificity when tested using spiked sample solutions containing different concentrations of actin protein and J82 cell lysate (a complex fluid containing a multitude of interfering proteins). Consequently, these experimental results confirm the feasibility of the proposed immunosensor for early diagnosis and prognosis of CKD at the point of care.

scholarly journals Single-Molecule Detection of DNA in a Nanochannel by High-Field Strength-Assisted Electrical Impedance Spectroscopy

Micromachines ◽  
2019 ◽  
Vol 10 (3) ◽  
pp. 189 ◽  
Author(s):  
Porpin Pungetmongkol ◽  
Takatoki Yamamoto
Keyword(s):  
Single Molecule ◽  
High Intensity ◽  
High Sensitivity ◽  
Electrical Impedance Spectroscopy ◽  
Random Coil ◽  
Label Free ◽  
Electrode Gap ◽  
Label Free Detection ◽  
Intensity Field ◽  
Nanogap Electrodes

Many researchers have fabricated micro and nanofluidic devices incorporating optical, chemical, and electrical detection systems with the aim of achieving on-chip analysis of macromolecules. The present study demonstrates a label-free detection of DNA using a nanofluidic device based on impedance measurements that is both sensitive and simple to operate. Using this device, the electrophoresis and dielectrophoresis effect on DNA conformation and the length dependence were examined. A low alternating voltage was applied to the nanogap electrodes to generate a high intensity field (>0.5 MV/m) under non-faradaic conditions. In addition, a 100 nm thick gold electrode was completely embedded in the substrate to allow direct measurements of a solution containing the sample passing through the gap, without any surface modification required. The high intensity field in this device produced a dielectrophoretic force that stretched the DNA molecule across the electrode gap at a specific frequency, based on back and forth movements between the electrodes with the DNA in a random coil conformation. The characteristics of 100 bp, 500 bp, 1 kbp, 5 kbp, 10 kbp, and 48 kbp λ DNA associated with various conformations were quantitatively analyzed with high resolution (on the femtomolar level). The sensitivity of this system was found to be more than about 10 orders of magnitude higher than that obtained from conventional linear alternating current (AC) impedance for the analysis of bio-polymers. This new high-sensitivity process is expected to be advantageous with regard to the study of complex macromolecules and nanoparticles.

Sign in / Sign up

Export Citation Format

Share Document