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. Both 2D-np and F-ssDNA components are cost-efficient and easy to synthesize, allowing for scaled-up data collection essential for machine learning modeling. The nanosensor library was subjected to various target groups, including proteins, breast cancer cells, and lethal-7 (let-7) miRNA mimics. We have demonstrated that AGONS could select the most essential nanosensors while achieving 100% predictive accuracy in all cases. With this approach, we demonstrate that machine learning can guide the design of nanosensor arrays with greater predictive accuracy while minimizing manpower, material cost, computational resources, instrumentation usage, and time. The biomarker-free detection attribute makes this approach readily available for biological targets without any detectable biomarker. We believe that AGONS can guide optical nanosensor array setups, opening broader opportunities through a biomarker-free detection approach for most challenging biological targets.

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. 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.

Regulation of the Peroxidase-Like Activity of nGO, MoS2 and WS2 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. To investigate the interference of a number of metal cations, we studied the effect of Ti2+ , Fe2+ , Ag+ , Hg2+ , Co2+ , Cu2+ , Ni2+ , Pb2+ , Ca2+ , Zn2+ and Mn2+ in a nanozyme assays of 2D-NPs using ABTS radical formation. Ti2+ , Co2+ , Cu2+ , Ni2+ , Ca2+ , Zn2+ and Mn2+ ions did not display any notable effect on the peroxidase-like activity of nGO, MoS2 and WS2 2D-NPs. However, Fe2+ , Ag+ , Hg2+ and Pb2+ ions' effects on the overall ABTS reaction were significant enough to be visualised by partial least square discriminant analysis (PLSDA). We report that, similar to that of many natural enzymes, the nanozyme activity of 2D-NPs is regulated by a number of metal cations allowing their identification and discrimination by using a statistical analysis tool.

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. 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 spectroscopy. We have observed that probe-full without any nick displayed trans-cleavage performance that was better than that of the form that contains a nick. Different experimental conditions of salt concentration, target concentration, and mismatch tolerance were examined to evaluate the probe performance. The activity of Cas12a was programmed for a dsDNA frame copied from a tobacco curly shoot virus (TCSV) or hepatitis B virus (HepBV) genome by using crRNA against TCSV or HepBV, respectively. While on-target activity offered detection of as little as 10 pM dsDNA target, off-target activity was not observed even at 1 nM control DNAs. This study demonstrates that trans-cleavage of Cas12a is not limited to ssDNA substrates, and Cas12a-based diagnostics can be extended to dsDNA substrates.

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

Two-dimensional MoS2 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 MoS2 2D-nps, offering low-nanomolar, label-free detection and identification in samples with unknown identity. The inhibition of the peroxidase-like activity of the MoS2 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 MoS2 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; MoS2 2D-nps are neither labeled with a signaling moiety nor modified with any ligands for signal readout. Only the intrinsic nanozyme activity of the MoS2 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 MoS2 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.

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. 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: WS2, MoS2, 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.

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. In addition, 2D-NAs assembled using WS2 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, ß-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.

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). This assay can detect the genomic targets directly in serum samples without an extraction step. The 4 h-long fluorometric assay was developed by combining three tiers of isothermal amplification processes and can be repurposed for any target of choice. This highly modular reaction setup is an untapped resource that can be incorporated into the front-runners of molecular diagnostics.

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. 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.

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. Hydrogels are usually made through chemical polymerization reactions. Whereas, DNA is a naturally occurring biopolymer which can assemble into highly ordered structures through noncovalent interactions. Here, we have employed a small molecule, cyanuric acid (CA), to assemble polyA-tailed DNA motif into a hydrogel. Encapsulation of a small molecule chemotherapeutic drug, a fluorescent molecule, two proteins and several nanoparticle formulations has been studied. Release of doxorubicin, small fluorescent molecule and fluorescently-labeled antibodies has been demonstrated.

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 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.