Biology subjects

Korensky, G.

Publications and source records attributed to Korensky, G..

3 recordsLinked to original sources

bioRxiv2020

Magnetic Bead-Quantum Dot (MB-Qdot) CRISPR Assay for Instrument-Free Viral DNA Detection

We have developed a novel detection system which couples CRISPR-Cas recognition of target sequences, Cas mediated nucleic acid probe cleavage, and quantum dots as highly sensitive reporter molecules for instrument-free detection of viral nucleic acid targets. After target recognition and Cas mediated cleavage of biotinylated ssDNA probe molecules, the probe molecules are bound to magnetic particles. A complimentary ssDNA oligonucleotide quantum dot conjugate is then added, which only hybridizes to un-cleaved probes on the magnetic beads. After separation of hybridized from unhybridized quantum dot conjugates by magnetic sequestration, the signal is measured fluorometrically to provide a signal proportional to the cleaved probes and therefore the amount of target nucleic acid. To demonstrate the power of this assay, a 250 bp DNA target sequence matching a portion of the African swine fever virus (ASFV) genome is used and a detection limit of ~0.5 nM is achieved without target amplification using a simple portable ultraviolet flashlight. The positive samples are readily confirmed by visual inspection, completely avoiding the need for complicated devices and instruments. This work establishes the feasibility of a simple, instrument free assay for rapid nucleic acid screening in both hospitals and point-of-care settings.

biochemistry↗

bioRxiv2020

Single Chlamydomonas reinhardi (C. reinhardtii) cell separation from bacterial cells and auto-fluorescence tracking with a nano-sieve device

A planar, transparent, and adaptable nano-sieve device is developed for efficient microalgae/bacteria separation. In our strategy, a sacrificial layer is applied in the dual photolithography patterning to achieve a one-dimensional channel with a very low aspect ratio (1:10,000). Microalgae/bacteria mixture is then introduced into the deformable polydimethylsiloxane (PDMS) nano-channel. The hydrodynamic deformation of the nano-channel is regulated to allow the bacteria cells to pass through while leaving the microalgae cells trapped in the device. At a flow rate of 4 l/min, ~100% of the microalgae cells are trapped in the device. Additionally, this device is capable of immobilizing single cells in a transparent channel for auto-fluorescence tracking. These microalgae cells demonstrate minimal photo-bleaching over 250 s laser exposure and can be used to monitor hazardous compounds in the sample with a continuous flow fashion. Our method will be valuable to purify microalgae samples containing contaminations and study single cell heterogeneity.

bioengineering↗

bioRxiv2019

High-throughput and all-solution phase African Swine Fever Virus (ASFV) detection using CRISPR-Cas12a and fluorescence based point-of-care system

Here we report the development of a high throughput, all-solution phase, and isothermal detection system to detect African Swine Fever Virus (ASFV). CRISPR-Cas12a programmed with a CRISPR RNA (crRNA) is used to detect ASFV target DNA. Upon ASFV DNA binding, the Cas12a/crRNA/ASFV DNA complex becomes activated and degrades a fluorescent single stranded DNA (ssDNA) reporter present in the assay. We combine this powerful CRISPR-Cas assay with fluorescence-based point-of-care (POC) system we developed for rapid and accurate virus detection. Without nucleic acid amplification, a detection limit of 1 pM is achieved within 2 hrs. In addition, the ternary Cas12a/crRNA/ASFV DNA complex is highly stable at physiological temperature and continues to cleave the ssDNA reporter even after 24 hrs of incubation, resulting in an improvement of the detection limit to 100 fM. We show that this system is very specific and can differentiate nucleic acid targets with closely matched sequences. The high sensitivity and selectivity of our system enables the detection of ASFV in femtomolar range. Importantly, this system features a disposable cartridge and a sensitive custom designed fluorometer, enabling compact, multiplexing, and simple ASFV detection, intended for low resource settings.

bioengineering↗