A novel dual probe-based method for mutation detection using isothermal amplification.

Cost efficient and rapid detection tools to detect mutations especially those linked to drug-resistance are important to address concerns of the rising multi-drug resistance infections. Here we integrated dual probes, namely a calibrator probe and an indicator probe, into isothermal amplification detection system. These two probes are designed to bind distinct regions on the same amplicon to determine the presence or absence of mutation.

Recombinase amplified CRISPR enhanced chain reaction (RACECAR) for viral genome detection.

We have developed a 'recombinase amplified CRISPR enhanced chain reaction' (RACECAR) assay that can detect as little as 40 copies of hepatitis B virus (HBV) genome using a benchtop spectrofluorometer. The limit of detection was determined to be 3 copies of HBV genome. The specificity of RACECAR was confirmed against hepatitis A virus (HAV).

Algorithmically Guided Optical Nanosensor Selector (AGONS): Guiding Data Acquisition, Processing, and Discrimination for Biological Sampling.

Here, we report a biomarker-free detection of various biological targets through a programmed machine learning algorithm and an automated computational selection process termed algorithmically guided optical nanosensor selector (AGONS). The optical data processed/used by algorithms are obtained through a nanosensor array selected from a library of nanosensors through AGONS. The nanosensors are assembled using two-dimensional nanoparticles (2D-nps) and fluorescently labeled single-stranded DNAs (F-ssDNAs) with random sequences.

Recognition of DNA Target Formulations by CRISPR-Cas12a Using a dsDNA Reporter.

CRISPR-Cas12a is a powerful platform for DNA-based diagnostics. The detection scheme relies on unselective shredding of a fluorescent ssDNA reporter upon target DNA recognition. To extend the reporter library beyond ssDNAs, we discovered a fluorescent reporter type using a dsDNA template.

Machine-Learning Single-Stranded DNA Nanoparticles for Bacterial Analysis.

A two-dimensional nanoparticle-single-stranded DNA (ssDNA) array has been assembled for the detection of bacterial species using machine-learning (ML) algorithms. Out of 60 unknowns prepared from bacterial lysates, 54 unknowns were predicted correctly. Furthermore, the nanosensor array, supported by ML algorithms, was able to distinguish wild-type from its mutant by a single gene difference.

Reprogrammable Gel Electrophoresis Detection Assay Using CRISPR-Cas12a and Hybridization Chain Reaction.

Hybridization chain reaction (HCR) is a DNA-based target-induced cascade reaction. Due to its unique enzyme-free amplification feature, HCR is often employed for sensing applications. Much like DNA nanostructures that have been designed to respond to a specific stimulus, HCR employs nucleic acids that reconfigure and assemble in the presence of a specific trigger.

Regulation of the Peroxidase-Like Activity of nGO, MoS and WS Nanozymes by Using Metal Cations.

Two dimensional nanoparticles (2D-NPs) along with other nanoscale materials have been deemed to be the next generation of artificial enzymes (nanozymes). The low-cost bulk-scale production, ease of storage and modification of such nanomaterials have given nanozymes an advantage over traditional enzymes. Many studies have been aimed at developing methods to increase the performance of these nanozymes, and also identify interfering agents.

Small molecule-induced DNA hydrogel with encapsulation and release properties.

Hydrogels are networks of polymers that can be used for packaging different payload types. They are proven to be versatile materials for various biomedical applications. Implanted hydrogels with encapsulated drugs have been shown to release the therapeutic payloads at disease sites.

Probing CRISPR-Cas12a Nuclease Activity Using Double-Stranded DNA-Templated Fluorescent Substrates.

The CRISPR-Cas12a nuclease shreds short single-stranded DNA (ssDNA) substrates indiscriminately through trans-cleavage upon activation with a specific target DNA. This shredding activity offered the potential for development of ssDNA-templated probes with fluorescent dye (F) and quencher (Q) labels. However, the formulations of double-stranded DNA (dsDNA)-templated fluorescent probes have not been reported possibly due to unknown (or limited) activity of Cas12a against short dsDNAs.

Masking the Peroxidase-Like Activity of the Molybdenum Disulfide Nanozyme Enables Label-Free Lipase Detection.

Two-dimensional MoS nanoparticles (2D-nps) exhibit artificial enzyme properties that can be regulated at bio-nanointerfaces. We discovered that protein lipase is able to tune the peroxidase-like activity of MoS 2D-nps, offering low-nanomolar, label-free detection and identification in samples with unknown identity. The inhibition of the peroxidase-like activity of the MoS 2D-nps was demonstrated to be concentration dependent, and as low as 5 nm lipase was detected with this approach.

Systematic Investigation of Two-Dimensional DNA Nanoassemblies for Construction of a Nonspecific Sensor Array.

We have performed a systematic study to analyze the effect of ssDNA length, nucleobase composition, and the type of two-dimensional nanoparticles (2D-nps) on the desorption response of 36 two-dimensional nanoassemblies (2D-NAs) against several proteins. The studies were performed using fluorescently labeled polyA, polyC, and polyT with 23, 18, 12, and 7 nucleotide-long sequences. The results suggest that the ssDNAs with polyC and longer sequences are more resistant to desorption, compared to their counterparts.

Homologous miRNA Analyses Using a Combinatorial Nanosensor Array with Two-Dimensional Nanoparticles.

A novel combinatorial nanosensor array for miRNA analyses was assembled using the intrinsic noncovalent interactions of unmodified two-dimensional nanoparticles. Discrimination of nine miRNA analogues with as little as a single nucleotide difference was demonstrated under 2 h. All nine targets were identified simultaneously with 95% confidence.