Sensing of biomolecules and label-free discrimination of DNA containing a triple T–C/T–G mismatch pair with a fluorescence light-up probe, triazolylpyrene (TNDMBPy)

Cytopathic effects (CPEs) are a hallmark of infections. CPEs can be observed by phase contrast or fluorescence light microscopy, albeit at the cost of phototoxicity. We report that digital holo-tomographic microscopy (DHTM) reveals distinct patterns of virus infections in live cells with minimal perturbation. DHTM is label-free, and records the phase shift of low energy light passing through the specimen on a transparent surface. DHTM infers a 3-dimensional (3D) tomogram based on the refractive index (RI). By measuring RI and computing the refractive index gradient (RIG) values DHTM unveils on optical heterogeneity in cells upon virus infection. We find that vaccinia virus (VACV), herpes simplex virus (HSV) and rhinovirus (RV) infections progressively and distinctly increased RIG. VACV, but not HSV and RV infection induced oscillations of cell volume, while all three viruses altered cytoplasmic membrane dynamics, and induced apoptotic features akin to the chemical compound staurosporin, but with virus-specific signatures. In sum, we introduce DHTM for quantitative label-free microscopy in infection research, and uncover virus-type specific changes and CPE in living cells at minimal interference.

Download Full-text

Label-free fluorescence light-up detection of T4 polynucleotide kinase activity using the split-to-intact G-quadruplex strategy by ligation-triggered and toehold-mediated strand displacement release

The Analyst ◽

10.1039/c5an01032b ◽

2015 ◽

Vol 140 (16) ◽

pp. 5450-5453 ◽

Cited By ~ 11

Author(s):

Lu Zhou ◽

Xiaoqiang Shen ◽

Na Sun ◽

Kewei Wang ◽

Yuanyuan Zhang ◽

...

Keyword(s):

Kinase Activity ◽

Detection Method ◽

Label Free ◽

Strand Displacement ◽

Polynucleotide Kinase ◽

G Quadruplex ◽

Fluorescence Light ◽

T4 Polynucleotide Kinase

A label-free, fluorescence light-up detection method for T4 polynucleotide kinase activity has been developed using the split-to-intact G-quadruplex strategy.

Download Full-text

Label-free, mass-sensitive single-molecule imaging using interferometric scattering microscopy

Essays in Biochemistry ◽

10.1042/ebc20200023 ◽

2020 ◽

Author(s):

Nikolas Hundt

Keyword(s):

Single Molecule ◽

Cellular Systems ◽

Single Molecule Imaging ◽

Label Free ◽

Measurement Sensitivity ◽

Mass Distributions ◽

Quantitative Measurements ◽

Contrast Mechanism ◽

Cellular Components ◽

Scattering Signal

Abstract Single-molecule imaging has mostly been restricted to the use of fluorescence labelling as a contrast mechanism due to its superior ability to visualise molecules of interest on top of an overwhelming background of other molecules. Recently, interferometric scattering (iSCAT) microscopy has demonstrated the detection and imaging of single biomolecules based on light scattering without the need for fluorescent labels. Significant improvements in measurement sensitivity combined with a dependence of scattering signal on object size have led to the development of mass photometry, a technique that measures the mass of individual molecules and thereby determines mass distributions of biomolecule samples in solution. The experimental simplicity of mass photometry makes it a powerful tool to analyse biomolecular equilibria quantitatively with low sample consumption within minutes. When used for label-free imaging of reconstituted or cellular systems, the strict size-dependence of the iSCAT signal enables quantitative measurements of processes at size scales reaching from single-molecule observations during complex assembly up to mesoscopic dynamics of cellular components and extracellular protrusions. In this review, I would like to introduce the principles of this emerging imaging technology and discuss examples that show how mass-sensitive iSCAT can be used as a strong complement to other routine techniques in biochemistry.

Download Full-text

A microfluidic device with integrated impedance detection for λ-DNA

MRS Proceedings ◽

10.1557/proc-773-n6.5 ◽

2003 ◽

Vol 773 ◽

Cited By ~ 1

Author(s):

Myung-Il Park ◽

Jonging Hong ◽

Dae Sung Yoon ◽

Chong-Ook Park ◽

Geunbae Im

Keyword(s):

Microfluidic Device ◽

Electrochemical Impedance ◽

Direct Detection ◽

Full Potential ◽

Label Free ◽

Buffer Solution ◽

Detection Systems ◽

Significant Difference ◽

Detection Of Biomolecules ◽

Impedance Magnitude

AbstractThe large optical detection systems that are typically utilized at present may not be able to reach their full potential as portable analysis tools. Accurate, early, and fast diagnosis for many diseases requires the direct detection of biomolecules such as DNA, proteins, and cells. In this research, a glass microchip with integrated microelectrodes has been fabricated, and the performance of electrochemical impedance detection was investigated for the biomolecules. We have used label-free λ-DNA as a sample biomolecule. By changing the distance between microelectrodes, the significant difference between DW and the TE buffer solution is obtained from the impedance-frequency measurements. In addition, the comparison for the impedance magnitude of DW, the TE buffer, and λ-DNA at the same distance was analyzed.

Download Full-text