Universal CRISPR-Cas12a and Toehold RNA Cascade Reaction on Paper Substrate for Visual Salmonella Genome Detection.

Salmonella, the most prevalent food-borne pathogen, poses significant medical and economic threats. Swift and accurate on-site identification and serotyping of Salmonella is crucial to curb its spread and contamination. Here, a synthetic biology cascade reaction is presented on a paper substrate using CRISPR-Cas12a and recombinase polymerase amplification (RPA), enabling the programming of a standard toehold RNA switch for a genome of choice. This approach employs just one toehold RNA switch design to differentiate between two different Salmonella serotypes, i.e., S. Typhimurium and S. Enteritidis, without the need for reengineering the toehold RNA switch. The sensor exhibits high sensitivity, capable of visually detecting as few as 100 copies of the whole genome from a model Salmonella pathogen on a paper substrate. Furthermore, this robust assay is successfully applied to detect whole genomes in contaminated milk and lettuce samples, demonstrating its potential in real sample analysis. Due to its versatility and practical features, genomes from different organisms can be detected by merely changing a single RNA element in this universal cell-free cascade reaction.

Magnetic surfactant-modified clay for enhanced adsorption of mixtures of per- and polyfluoroalkyl substances (PFAS) in snowmelt: Improving practical applicability and efficiency.

The extensive use of per- and polyfluoroalkyl substances (PFAS) in many industrial and consumer contexts, along with their persistent nature and possible health hazards, has led to their recognition as a prevalent environmental issue. While various PFAS removal methods exist, adsorption remains a promising, cost-effective approach. This study evaluated the PFAS adsorption performance of a surfactant-modified clay by comparing it with commercial clay-based adsorbents. Furthermore, the impact of environmental factors, including pH, ionic strength, and natural organic matter, on PFAS adsorption by the modified clay (MC) was evaluated. After proving that the MC was regenerable and reusable, magnetic modified clay (MMC) was synthesized, characterized, and tested for removing a wide range of PFAS in pure water and snowmelt. The MMC was found to have similar adsorption performance as the MC and was able to remove > 90% of the PFAS spiked to the snowmelt. The superior and much better performance of the MMC than powdered activated carbon points to its potential use in removing PFAS from real water matrices at an industrial scale.

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.

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.

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.

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.

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.

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.

Switchable Fluorescence of Doxorubicin for Label-Free Imaging of Bioorthogonal Drug Release.

Monitoring the release and activation of prodrug formulations provides essential information about the outcome of a therapy. While the prodrug delivery can be confirmed by using different imaging techniques, confirming the release of active payload by using imaging is a challenge. Here, we have discovered that the switchable fluorescence of doxorubicin can validate drug release upon its uncaging reaction with a highly specific chemical partner. We have observed that the conjugation of doxorubicin with a trans-cyclooctene (TCO) diminishes its fluorescence at 595 nm. This quenched fluorescence of the doxorubicin prodrug is recovered upon its bond-cleaving reaction with tetrazine. Clinically assessed iron oxide nanoparticles were used to formulate a doxorubicin nanodrug. The release of doxorubicin from the nanodrug was studied under various experimental conditions. A fivefold increase in doxorubicin fluorescence is observed after complete release. The studies were carried out in vitro in MDA-MB-231 breast cancer cells. An increase in Dox signal was observed upon tetrazine administration. This switchable fluorescence mechanism of Dox could be employed for fundamental studies, that is, the reactivity of various tetrazine and TCO linker types under different experimental conditions. In addition, the system could be instrumental for translational research where the release and activation of doxorubicin prodrug payloads can be monitored by using optical imaging systems.

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.

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.

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.

Chemical and Biological Sensing Using Hybridization Chain Reaction.

Since the advent of its theoretical discovery more than 30 years ago, DNA nanotechnology has been used in a plethora of diverse applications in both the fundamental and applied sciences. The recent prominence of DNA-based technologies in the scientific community is largely due to the programmable features stored in its nucleobase composition and sequence, which allow it to assemble into highly advanced structures. DNA nanoassemblies are also highly controllable due to the precision of natural and artificial base-pairing, which can be manipulated by pH, temperature, metal ions, and solvent types. This programmability and molecular-level control have allowed scientists to create and utilize DNA nanostructures in one, two, and three dimensions (1D, 2D, and 3D). Initially, these 2D and 3D DNA lattices and shapes attracted a broad scientific audience because they are fundamentally captivating and structurally elegant; however, transforming these conceptual architectural blueprints into functional materials is essential for further advancements in the DNA nanotechnology field. Herein, the chemical and biological sensing applications of a 1D DNA self-assembly process known as hybridization chain reaction (HCR) are reviewed. HCR is a one-dimensional (1D) double stranded (ds) DNA assembly process initiated only in the presence of a specific short ssDNA (initiator) and two kinetically trapped DNA hairpin structures. HCR is considered an enzyme-free isothermal amplification process, which shows substantial promise and offers a wide range of applications for in situ chemical and biological sensing. Due to its modular nature, HCR can be programmed to activate only in the presence of highly specific biological and/or chemical stimuli. HCR can also be combined with different types of molecular reporters and detection approaches for various analytical readouts. While the long dsDNA HCR product may not be as structurally attractive as the 2D and 3D DNA networks, HCR is highly instrumental for applied biological, chemical, and environmental sciences, and has therefore been studied to foster a variety of objectives. In this review, we have focused on nucleic acid, protein, metabolite, and heavy metal ion detection using this 1D DNA nanotechnology via fluorescence, electrochemical, and nanoparticle-based methodologies.

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.

Controlled in-cell activation of RNA therapeutics using bond-cleaving bio-orthogonal chemistry.

Temporal control of siRNA activation is a major challenge for RNAi-based therapeutics. The majority of the reported siRNA delivery systems rely on environmental factors, such as differences in extracellular and intracellular redox potential, ATP concentration, or pH to activate an siRNA payload. However dynamic endogenous environments are far too complex to rely on for controllable siRNA release and can result in premature siRNA activation prior to reaching the intended biological target. In addition, there are uncertainties about timing, degree and rate of the siRNA activation with spontaneous release approaches. Herein we describe a bio-orthogonal chemistry approach to address this important challenge. With our approach we were able achieve two major goals: complete siRNA inactivation upon immobilization of the payload on the surface of iron oxide nanoparticles and controlled in-cell activation with the addition of a small non-toxic chemical trigger after sufficient cellular uptake of the nanoparticles was confirmed. We have demonstrated our in-cell activation approach using two siRNAs against green fluorescent protein (GFP) and cyclin dependent kinase 8 (CDK8) in GFP expressing MDA-MB-231 cell line. We anticipate that this methodology will potentially advance the clinical translation of RNAi-based therapeutics, as the described bio-orthogonal chemistry can be generalized for any siRNA of choice.

Universal sensor array for highly selective system identification using two-dimensional nanoparticles.

A typical lock-and-key sensing strategy, relying only on the most dominant interactions between the probe and target, could be too limiting. In reality, the information received upon sensing is much richer. Non-specific events due to various intermolecular forces contribute to the overall received information with different degrees, and when analyzed, could provide a much more powerful detection opportunity. Here, we have assembled a highly selective universal sensor array using water-soluble two-dimensional nanoparticles (nGO, MoS2 and WS2) and fluorescent DNA molecules. The array is composed of 12 fluorescently silent non-specific nanoreceptors (2D-nps) and used for the identification of three radically different systems; five proteins, three types of live breast cancer cells and a structure-switching event of a macromolecule. The data matrices for each system were processed using Partial Least Squares (PLS) discriminant analysis. In all of the systems, the sensor array was able to identify each object or event as separate clusters with 95% confidence and without any overlap. Out of 15 unknown entities with unknown protein concentrations tested, 14 of them were predicted successfully with correct concentration. 8 breast cancer cell samples out of 9 unknown entities from three cell types were predicted correctly. During the assembly of each nanoprobe, the intrinsic non-covalent interactions between unmodified 2D nanoparticles and ssDNAs were exploited. The unmodified 2D materials offer remarkable simplicity in the layout and the use of ssDNAs as probes provides limitless possibilities because the natural interaction of a ssDNA and 2D surface can be fine-tuned with the nucleobase composition, oligonucleotide length and type of 2D nanomaterial. Therefore, the approach described here can be advanced and fine-tuned indefinitely for meeting a particular sensing criterion. Though we have only studied three distinct elements, this approach is universal enough to be applied to a wide-range of systems.

Single-trigger dual-responsive nanoparticles for controllable and sequential prodrug activation.

Here we have developed a novel approach where two synergistically acting drugs were completely inactivated upon chemical immobilization on a nanoparticle template and activated in response to a chemical stimulus. The activation rate of each drug payload is controlled using a biologically inert bioorthogonal chemistry approach. By exploiting the subtle differences in the 'click-to-release' bioorthogonal reaction, we engineered a single delivery platform capable of releasing the payloads in a time-staggered manner in response to a single dose of a highly specific, yet reactive, small molecule. Incorporation of both di-axial, 'fast release', and di-equatorial, 'slow release', TCO linkers into our nanodrug assembly inhibited the activity of the drug molecules and enabled us to control the timing and activation of each payload. This single-trigger dual-responsive nanoparticle construct and its release kinetics were characterized using two molecular fluorescent probes and tested in vitro for efficient delivery of molecular payloads. In this manuscript we show that this approach was also successful in the treatment of triple negative BT-20 breast cancer cells. Our nanodrug loaded with the slow-releasing doxorubicin and fast-releasing PAC-1 prodrugs displayed a greater therapeutic response than the nanodrug which released both payloads simultaneously.

Low picomolar, instrument-free visual detection of mercury and silver ions using low-cost programmable nanoprobes.

The EPA's recommended maximum allowable level of inorganic mercury in drinking water is 2 ppb (10 nM). To our knowledge, the most sensitive colorimetric mercury sensor reported to date has a limit of detection (LOD) of 800 pM. Here, we report an instrument-free and highly practical colorimetric methodology, which enables detection of as low as 2 ppt (10 pM) of mercury and/or silver ions with the naked eye using a gold nanoprobe. Synthesis of the nanoprobe costs less than $1.42, which is enough to perform 200 tests in a microplate; less than a penny for each test. We have demonstrated the detection of inorganic mercury from water, soil and urine samples. The assay takes about four hours and the color change is observed within minutes after the addition of the last required element of the assay. The nanoprobe is highly programmable which allows for the detection of mercury and/or silver ions separately or simultaneously by changing only a single parameter of the assay. This highly sensitive approach for the visual detection relies on the combination of the signal amplification features of the hybridization chain reaction with the plasmonic properties of the gold nanoparticles. Considering that heavy metal ion contamination of natural resources is a major challenge and routine environmental monitoring is needed, yet time-consuming, this colorimetric approach may be instrumental for on-site heavy metal ion detection. Since the color transition can be measured in a variety of formats including using the naked eye, a simple UV-Vis spectrophotometer, or recording using mobile phone apps for future directions, our cost-efficient assay and method have the potential to be translated into the field.

Rapid Visual Screening and Programmable Subtype Classification of Ebola Virus Biomarkers.

The massive outbreaks of the highly transmissible and lethal Ebola virus disease were caused by infection with one of the Ebolavirus species. It is vital to develop cost-effective, highly sensitive and selective multitarget biosensing platforms that allow for both the detection and phenotyping. Here, a highly programmable, cost-efficient and multianalyte sensing approach is reported that enables visual detection and differentiation of conserved oligonucleotide regions of all Ebolavirus subtypes known to infect human primates. This approach enables the detection of as little as 400 amols (24 × 106 molecules) of target sequences with the naked eye. Furthermore, the detection assay can be used to classify four virus biomarkers using a single nanoprobe template. This can be achieved by using different combinations of short single stranded initiator molecules, referred to as programming units, which also enable the simultaneous and rapid identification of the four biomarkers in 16 different combinations. The results of 16 × 5 array studies illustrate that the system is extremely selective with no false-positive or false-negative. Finally, the target strands in liquid biopsy mimics prepared from urine specimens are also able to be identified and classified.