Hybridization chain reaction (HCR) is a DNAbased target-induced cascade reaction. Due to its unique enzymefree 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. Despite its standalone capabilities, HCR is highly modular; therefore, it can be advanced and repurposed when coupled with latest discoveries. To this effect, we have developed a gel electrophoresis-based detection approach which combines the signal amplification feature of HCR with the programmability and sensitivity of the CRISPR-Cas12a system. By incorporating CRISPR-Cas12a, we have achieved greater sensitivity and reversed the signal output from TURN OFF to TURN ON. CRISPR-Cas12a also enabled us to rapidly reprogram the assay for the detection of both ssDNA and dsDNA target sequences by replacing a single reaction component in the detection kit. Detection of conserved, both ssDNA and dsDNA, regions of tobacco curly shoot virus (TCSV) and hepatitis B virus (HepBV) genomes is demonstrated with this methodology. This low-cost gel electrophoresis assay can detect as little as 1.5 fmol of the target without any additional target amplification steps and is about 100-fold more sensitive than HCR-alone approach.

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. The developed nanotechnology offered identification and quantification of unknown targets with unknown concentration. Discrimination of target mixtures from low-to-high ratios was demonstrated. The DNA and RNA analogues of targets were identified using the combinatorial sensory approach. Identification of a target in a complex biological matrix prepared with human urine was demonstrated. The nanosensor array was put together using 15 nanoassemblies (2D-NAs) constructed using three two-dimensional nanoparticles (2D-nps: WS, MoS, and nanographene oxide (nGO)) and five rationally designed fluorescently labeled 15-nt-long ssDNAs (probes). In this approach, each target has only a small yet varying degree of complementarity with each of the five probes adsorbed on the 2D-np surface. The probes in each 2D-NA are desorbed from the surface by each target with a different degree that was recorded with fluorescence recovery measurements. The fluorescence data set was processed by partial least squares discriminant analysis (PLSDA), and each target was discriminated successfully. This new approach has a number of advantages over the classical bind-and-release model, typically used for 2D-np based biosensors, and opens greater detection opportunities with 2D-nps.

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. In addition, 2D-NAs assembled using WS were least susceptible to desorption by the proteins tested, whereas nGO 2D-NAs were the most susceptible nanoassemblies. Later, the results of these systematic studies were used to construct a sensor array for discrimination of seven model proteins (BSA, lipase, alkaline phosphatase, acid phosphatase, protease, β-galactosidase, and Cytochrome c). Neither the ssDNAs nor the 2D-nps have any specific interaction with the proteins tested. Only the displacement of the ssDNAs from the 2D-np surface was measured upon the disruption of the existing forces within 2D-NAs. A customized sensor array with five 2D-NAs was developed as a result of a careful screening/filtering process. The sensor array was tested against 200 nM of protein targets, and each protein was discriminated successfully. The results suggest that the systematic studies performed using various ssDNAs and 2D-nps enabled the construction of a sensor array without a bind-and-release sensing mechanism. The studies also demonstrate the significance of systematic investigations in the construction of two-dimensional DNA nanoassemblies for functional studies.

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 Escherichia coli from its mutant by a single gene difference. In addition, the nanosensor array was able to distinguish untreated wild-type E. coli from those treated with antimicrobial drugs. This work demonstrates the potential of nanoparticle–ssDNA arrays and ML algorithms for the discrimination and identification of complex biological matrixes.

The CRISPR-Cas12a nuclease shreds short single-stranded DNA (ssDNA) substrates indiscriminately through transcleavage 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. The ssDNA probes have been shown to be powerful for diagnostic applications; however, limiting the probe selections to short ssDNAs could be restrictive from an application and probe diversification standpoint. Here, we report a dsDNA substrate (probe-full) for probing Cas12a trans-cleavage activity upon target detection. A diverse set of Cas12a substrates with alternating dsDNA character were designed and studied using fluorescence

Small molecule cyanuric acid is used to assemble a novel DNA hydrogel which is programmed to encapsulate and release a variety of compounds including drug molecules.

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. The results were compared with those obtained with several other proteins that did not display any significant interference with the nanozyme behavior of the MoS 2D-nps. This unique response of lipase was characterized and exploited for the successful identification of lipase in six unknown samples by using qualitative visual inspection and a quantitative statistical analysis method. The developed methodology in this approach is noteworthy for many aspects; MoS 2D-nps are neither labeled with a signaling moiety nor modified with any ligands for signal readout. Only the intrinsic nanozyme activity of the MoS 2D-nps is exploited for this detection approach. No analytical equipment is necessary for the visual detection of lipase. The synthesis of the water-soluble MoS 2D-nps is low costing and can be performed in bulk scale. Exploring the properties of 2D-nps and their interactions with biological materials reveals highly interesting yet instrumental features that offer the development of novel bioanalytical approaches.

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. In this design, the fluorescence of the dsDNA reporter is quenched via contact-quenching mechanism. Upon detection, the quenched fluorescence recovers with the activation Cas12a complex. Here, we compared the probing performance of two dsDNA reporters with two ssDNA reporters. The rate of the Cas12a trans-cleavage reaction was studied using one of the dsDNA reporters under different settings. The detection of different sizes of dsDNA or ssDNA targets was studied systematically under three different temperatures. Lower thresholds for ssDNA and dsDNA target size were identified. The mismatch tolerance and target specificity were examined for both ssDNA and dsDNA targets, separately. The probing performance of the dsDNA reporter was evaluated in a random DNA pool with and without target strands. We report that dsDNA can serve as a tunable fluorescence reporter template expanding the toolbox for Cas12a-based diagnostics.