CRISPR/Cas12a antifouling nanocomposite electrochemical biosensors enable amplification-free detection of Monkeypox virus in complex biological fluids.

The escalating global threat of infectious diseases, including monkeypox virus (MPXV), necessitates advancements in point-of-care diagnostics, moving beyond the constraints of conventional methods tethered to centralized laboratories. Here, we introduce multiple CRISPR RNA (crRNA)-based biosensors that can directly detect MPXV within 35 minutes without pre-amplification, leveraging the enhanced sensitivity and antifouling attributes of the BSA-based nanocomposite. Multiple crRNAs, strategically targeting diverse regions of the F3L gene of MPXV, are designed and combined to amplify Cas12a activation and its collateral cleavage of reporter probes. Notably, our electrochemical sensors exhibit the detection limit of 669 fM F3L gene without amplification, which is approximately a 15-fold improvement compared to fluorescence detection. This sensor also shows negligible changes in peak current after exposure to complex biological fluids, such as whole blood and serum, maintaining its sensitivity at 682 fM. This sensitivity is nearly identical to the conditions when only the F3L gene was present in PBS. In summary, our CRISPR-based electrochemical biosensors can be utilized as a high-performance diagnostic tool in resource-limited settings, representing a transformative leap forward in point-of-care testing. Beyond infectious diseases, the implications of this technology extend to various molecular diagnostics, establishing itself as a rapid, accurate, and versatile platform for detection of target analytes.

A CRISPR/Cas12 trans-cleavage reporter enabling label-free colorimetric detection of SARS-CoV-2 and its variants.

We present a label-free colorimetric CRISPR/Cas-based method enabling affordable molecular diagnostics for SARS-CoV-2. This technique utilizes 3,3'-diethylthiadicarbocyanine iodide (DISC2(5)) which exhibits a distinct color transition from purple to blue when it forms dimers by inserting into the duplex of the thymidine adenine (TA) repeat sequence. Loop-mediated isothermal amplification (LAMP) or recombinase polymerase amplification (RPA) was used to amplify target samples, which were subsequently subjected to the CRISPR/Cas12a system. The target amplicons would activate Cas12a to degrade nearby TA repeat sequences, preserving DISC2(5) in its free form to display purple as opposed to blue in the absence of the target. Based on this design approach, SARS-CoV-2 RNA was colorimetrically detected very sensitively down to 2 copies/µL, and delta and omicron variants of SARS-CoV-2 were also successfully identified. The practical diagnostic utility of this method was further validated by reliably identifying 179 clinical samples including 20 variant samples with 100% clinical sensitivity and specificity. This technique has the potential to become a promising CRISPR-based colorimetric platform for molecular diagnostics of a wide range of target pathogens.

Micrometer-thick and porous nanocomposite coating for electrochemical sensors with exceptional antifouling and electroconducting properties.

Development of coating technologies for electrochemical sensors that consistently exhibit antifouling activities in diverse and complex biological environments over extended time is vital for effective medical devices and diagnostics. Here, we describe a micrometer-thick, porous nanocomposite coating with both antifouling and electroconducting properties that enhances the sensitivity of electrochemical sensors. Nozzle printing of oil-in-water emulsion is used to create a 1 micrometer thick coating composed of cross-linked albumin with interconnected pores and gold nanowires. The layer resists biofouling and maintains rapid electron transfer kinetics for over one month when exposed directly to complex biological fluids, including serum and nasopharyngeal secretions. Compared to a thinner (nanometer thick) antifouling coating made with drop casting or a spin coating of the same thickness, the thick porous nanocomposite sensor exhibits sensitivities that are enhanced by 3.75- to 17-fold when three different target biomolecules are tested. As a result, emulsion-coated, multiplexed electrochemical sensors can carry out simultaneous detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid, antigen, and host antibody in clinical specimens with high sensitivity and specificity. This thick porous emulsion coating technology holds promise in addressing hurdles currently restricting the application of electrochemical sensors for point-of-care diagnostics, implantable devices, and other healthcare monitoring systems.

ANCA: artificial nucleic acid circuit with argonaute protein for one-step isothermal detection of antibiotic-resistant bacteria.

Endonucleases have recently widely used in molecular diagnostics. Here, we report a strategy to exploit the properties of Argonaute (Ago) proteins for molecular diagnostics by introducing an artificial nucleic acid circuit with Ago protein (ANCA) method. The ANCA is designed to perform a continuous autocatalytic reaction through cross-catalytic cleavage of the Ago protein, enabling one-step, amplification-free, and isothermal DNA detection. Using the ANCA method, carbapenemase-producing Klebsiella pneumoniae (CPKP) are successfully detected without DNA extraction and amplification steps. In addition, we demonstrate the detection of carbapenem-resistant bacteria in human urine and blood samples using the method. We also demonstrate the direct identification of CPKP swabbed from surfaces using the ANCA method in conjunction with a three-dimensional nanopillar structure. Finally, the ANCA method is applied to detect CPKP in rectal swab specimens from infected patients, achieving sensitivity and specificity of 100% and 100%, respectively. The developed method can contribute to simple, rapid and accurate diagnosis of CPKP, which can help prevent nosocomial infections.

Fully Automated Multiple Standard Addition on a Centrifugal Microfluidic System.

We herein describe a novel centrifugal microfluidic system to achieve multiple standard additions, which could minimize the effects of matrix interference and consequently lead to more accurate and reliable measurements of analyte concentrations in complex samples. The system leverages laser-irradiated ferrowax microvalves to automatically control fluid transfer on the disc without the need for external pumps or pressure systems, simplifying the procedures and eliminating the need for manual intervention. The disc incorporates metering chambers with rationally designed varying sizes, which could lead to the formation of six standard addition samples very rapidly in just 2.5 min. The final solutions are designed to contain a target component at gradually increasing concentrations but have an equal final volume containing the same amount of an analyte solution, thereby equalizing the matrix effect that is supposedly caused by the unknown components in the analyte solution. By utilizing this design principle, we were able to successfully quantify a model target component, salivary thiocyanate ions, that could be used as a biomarker for exposure to tobacco smoke. Our centrifugal microfluidic system holds great promise as a powerful analytical tool to achieve fully automated diagnostic microsystems involving a standard addition process.

Charge-shifting polyplex as a viral RNA extraction carrier for streamlined detection of infectious viruses.

The recent outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted the need for rapid, user-friendly nucleic acid testing that involves simple but efficient RNA extraction. Here, we present a charge-shifting polyplex as an RNA extraction carrier for advanced diagnosis of infectious viral diseases. The polyplex comprises poly(2-(dimethylamino) ethyl acrylate) (pDMAEA) electrostatically conjugated with RNA. The pDMAEA film can rapidly dissolve in the viral RNA solution, promoting immediate binding with RNA to form the polyplex, which enables the efficient capture of a substantial quantity of RNA. Subsequently, the captured RNA can be readily released by the quick hydrolysis of pDMAEA at the onset of quantitative reverse transcription-polymerase chain reaction (qRT-PCR), streamlining the entire process from RNA extraction to analysis. The developed method requires only 5 min of centrifugation and enables the detection of RNA in a one-pot setup. Moreover, the proposed method is fully compatible with high-speed qRT-PCR kits and can identify clinical samples within 1 h including the entire extraction to detection procedure. Indeed, the method successfully detected influenza viruses, SARS-CoV-2, and their delta and omicron variants in 260 clinical samples with a sensitivity of 99.4% and specificity of 98.9%. This rapid, user-friendly polyplex-based approach represents a significant breakthrough in molecular diagnostics.

Polyaniline-based 3D network structure promotes entrapment and detection of drug-resistant bacteria.

Sensitive and accurate capture, enrichment, and identification of drug-resistant bacteria on human skin are important for early-stage diagnosis and treatment of patients. Herein, we constructed a three-dimensional hierarchically structured polyaniline nanoweb (3D HPN) to capture, enrich, and detect drug-resistant bacteria on-site by rubbing infected skins. These unique hierarchical nanostructures enhance bacteria capture efficiency and help severely deform the surface of the bacteria entrapped on them. Therefore, 3D HPN significantly contributes to the effective and reliable recovery of drug-resistant bacteria from the infected skin and the prevention of potential secondary infection. The recovered bacteria were successfully identified by subsequent real-time polymerase chain reaction (PCR) analysis after the lysis process. The molecular analysis results based on a real-time PCR exhibit excellent sensitivity to detecting target bacteria of concentrations ranging from 102 to 107 CFU/mL without any fluorescent signal interruption. To confirm the field applicability of 3D HPN, it was tested with a drug-resistant model consisting of micropig skin similar to human skin and Klebsiella pneumoniae carbapenemase-producing carbapenem-resistant Enterobacteriaceae (KPC-CRE). The results show that the detection sensitivity of this assay is 102 CFU/mL. Therefore, 3D HPN can be extended to on-site pathogen detection systems, along with rapid molecular diagnostics through a simple method, to recover KPC-CRE from the skin.

Elution-free DNA detection using CRISPR/Cas9-mediated light-up aptamer transcription: Toward all-in-one DNA purification and detection tube.

Accurate and efficient detection of DNA is crucial for disease diagnosis and health monitoring. The traditional methods for DNA analysis involve multiple steps, including sample preparation, lysis, extraction, amplification, and detection. In this study, we present a one-step elution-free DNA analysis method based on the combination of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated light-up aptamer transcription (CLAT) assay and a DNA-capturing poly(2-dimethylaminomethyl styrene) (pDMAMS)-coated tube. The sample solution and lysis buffer are added to the pDMAMS-coated tube, and the DNA is efficiently captured on the surface via electrostatic interaction and directly detected by CLAT assay. The ability of the CRISPR/Cas9 system to specifically recognize DNA enables direct detection of DNA captured on the pDMAMS-coated tube. The combination of CLAT assay and pDMAMS-coated tube simplifies DNA detection in a single tube without the need for complicated extraction steps, improving sensitivity. Our platform demonstrated attomolar sensitivity in the detection of target DNA in cell lysate (0.92 aM), urine (7.7 aM), and plasma (94.6 aM) samples within 1 h. The practical applicability of this method was further demonstrated in experiments with tumor-bearing mice. We believe that this approach brings us closer to an all-in-one DNA purification and detection tube system and has potential applications in tissue and liquid biopsies, as well as various other DNA sensing applications.

Smartphone-Based SARS-CoV-2 and Variants Detection System using Colorimetric DNAzyme Reaction Triggered by Loop-Mediated Isothermal Amplification (LAMP) with Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR).

Coronavirus disease (COVID-19) has affected people for over two years. Moreover, the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has raised concerns regarding its accurate diagnosis. Here, we report a colorimetric DNAzyme reaction triggered by loop-mediated isothermal amplification (LAMP) with clustered regularly interspaced short palindromic repeats (CRISPR), referred to as DAMPR assay for detecting SARS-CoV-2 and variants genes with attomolar sensitivity within an hour. The CRISPR-associated protein 9 (Cas9) system eliminated false-positive signals of LAMP products, improving the accuracy of DAMPR assay. Further, we fabricated a portable DAMPR assay system using a three-dimensional printing technique and developed a machine learning (ML)-based smartphone application to routinely check diagnostic results of SARS-CoV-2 and variants. Among blind tests of 136 clinical samples, the proposed system successfully diagnosed COVID-19 patients with a clinical sensitivity and specificity of 100% each. More importantly, the D614G (variant-common), T478K (delta-specific), and A67V (omicron-specific) mutations of the SARS-CoV-2 S gene were detected selectively, enabling the diagnosis of 70 SARS-CoV-2 delta or omicron variant patients. The DAMPR assay system is expected to be employed for on-site, rapid, accurate detection of SARS-CoV-2 and its variants gene and employed in the diagnosis of various infectious diseases.

Palindromic hyperbranched rolling circle amplification enabling ultrasensitive microRNA detection.

We herein describe a palindromic hyperbranched rolling circle amplification (PH-RCA) reaction and its application for ultrasensitive detection of microRNAs (miRNAs). In this strategy, target miRNAs bind to a dumb-bell probe (DP) and initiate the RCA reactions, concomitantly converting the dumb-bell structure to the circular form, which then allows the annealing of the palindromic primers to promote an additional two RCA reactions. As a consequence of the RCA reactions promoted by both target miRNAs and palindromic primers, multiple long concatenated DNA strands would be produced. Importantly, the palindromic primers can also bind to numerous palindromic domains of the long linear single DNA strands, consequently promoting highly branched simultaneous extension reactions at multiple sites. By detecting the fluorescence signals resulting from the amplified DNA products, we successfully identified target miRNA under isothermal conditions with excellent specificity. The PH-RCA technique developed in this work would greatly advance the conventional RCA reaction and HRCA reaction by significantly enhancing the sensitivity and reducing the reaction time within 30 min.

Ligation-free isothermal nucleic acid amplification.

In this study, we uncover a ligation-free DNA extension method in two adjacent fragmented probes, which are hybridized to target RNA, for developing a ligation-free nucleic acid amplification reaction. In this reaction, DNA elongation occurs from a forward probe to a phosphorothioated-hairpin probe in the presence of target RNA regardless of ligation. The second DNA elongation then occurs simultaneously at the nick site of the phosphorothioated probe and the self-priming region. Therefore, the binding site of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) 12a is repeatedly amplified, inducing a fluorescence signal in the presence of CRISPR-Cas12a. This ligation-free isothermal gene amplification method enables the detection of target RNA with 49.2 fM sensitivity. Moreover, two types of mRNA detection are feasible, thus, demonstrating the potential of this method for cancer companion diagnostics. Notably, the proposed method also demonstrates efficacy when applied for the detection of mRNA extracted from human cells and tumor-bearing mouse tissue and urine samples. Hence, this newly developed ligation-free isothermal nucleic acid amplification system is expected to be widely used in a variety of gene detection platforms.

Ultrasensitive multiplexed miRNA detection based on a self-priming hairpin-triggered isothermal cascade reaction.

We herein describe an ultrasensitive isothermal strategy to detect miRNAs in a multiplexed manner by utilizing a self-priming hairpin-triggered cascade reaction and the adsorption properties of graphene oxide (GO). In principle, a self-priming hairpin probe (SHP) was designed to be opened through binding to the target miRNA and rearranged to serve as a primer. The following extension displaced the target miRNA to be recycled for opening another SHP and produced a double-stranded (ds) SHP with a longer stem region. The nicking enzyme recognition site within the ds SHP was then subjected to continuous repeated nicking and extension reactions, consequently producing a large amount of the trigger sequence. In the second reaction phase, the trigger also transformed another single-stranded (ss) target template probe (TTP) into ds TTP and simultaneously produced numerous target mimic strands (Target') in the same manner, which could activate the first reaction phase, mimicking the target miRNA. Since the ss portions of the two probes were all transformed to the ds forms (ds SHP and ds TTP), they are resistant to the adsorption by graphene oxide (GO) and then emitted intense fluorescence after the application of GO while the ss forms of the two probes produced a negligible fluorescence signal without the target miRNAs. Based on this unique design principle, we were able to simultaneously identify multiple target miRNAs very sensitively down to attomolar levels (42.63 aM for miRNA let-7a, 13.08 aM for miRNA-141, and 10.14 aM for miRNA-98) within 30 min.

Ferrowax microvalves for fully automated serial dilution on centrifugal microfluidic platforms.

We herein describe a centrifugal microfluidic system to accomplish a fully automated serial dilution. The liquid flow on the disc was regulated by utilizing ferrowax microvalves systematically integrated into the channels within specially designed metering structures. By opening the differently positioned microvalves through irradiation of IR laser to allow metering, the same amount of diluent was serially eluted to the dilution chamber from the same diluent chamber. After dilution, the diluted samples were automatically delivered to the respective final product chambers by appropriately opening or closing the microvalves in the connecting channels, followed by rotating the disc. Based on this unique design principle, six consecutive two-fold and 10-fold dilutions were successfully achieved, yielding excellent accuracy in a wide dynamic range up to six orders of magnitude. Very importantly, the overall serial dilution process, including the diluent addition, mixing, and product transfer steps, was completed very rapidly within 5 min, due to the minimized procedures enabled by the automated actuation of the ferrowax microvalves at the rationally designed positions. We expect our centrifugal microfluidic system would serve as a powerful elemental tool to realize fully automated diagnostic microsystems involving the serial dilution process.

CRISPR/Cas12a collateral cleavage activity for simple and rapid detection of protein/small molecule interaction.

To realize the full potential of the CRISPR/Cas system and expand its applicability up to the detection of molecular interactions, we herein describe a novel method to identify protein/small molecule interactions by utilizing the CRISPR/Cas12a collateral cleavage activity. This technique employs a single-stranded activator DNA modified with a specific small molecule, which would switch on the CRISPR/Cas12a collateral cleavage activity upon binding to crRNA within the CRISPR/Cas12a system. When the target protein binds to the small molecule on the activator DNA, the bound protein sterically hinders the access of the activator DNA to crRNA, thereby promoting less collateral cleavage activity of CRISPR/Cas12a. As a consequence, fewer reporter probes nearby are cleaved to produce accordingly reduced fluorescence signals in response to target protein. Based on this unique design principle, the two model protein/small molecule interactions, streptavidin/biotin and anti-digoxigenin/digoxigenin, were successfully determined down to 0.03 nM and 0.09 nM, respectively, with a fast and simple detection workflow (11 min). The practical applicability of this method was also verified by reliably detecting target streptavidin spiked in heterogeneous human serum. This work would provide great insight to construct novel strategies to identify protein/small molecule interaction by making the most of the CRISPR/Cas12a system beyond its superior capabilities in genome editing and molecular diagnostics.

Identification of thyroid hormone/thyroid hormone receptor interaction based on aptamer-assisted protein-induced fluorescence enhancement.

We herein describe a novel method to identify thyroid hormone (TH)/thyroid hormone receptor (TR) interaction, termed aptamer-assisted protein-induced fluorescence enhancement (AptPIFE). In this method, a detection probe consisting of an RNA strand incorporating TH-specific aptamer and a Cy3-labeled DNA strand holds TH in close proximity to Cy3. The corresponding TR then binds to the TH near Cy3, consequently stimulating Cy3 to emit a significantly enhanced fluorescence through PIFE phenomenon. Based on this simple yet efficient design principle, we successfully identified the interaction of TH with TR within 10 min, down to 0.37 pM with excellent specificity. The practical and robust applicability of this method was also successfully validated by properly screening TR antagonists and reliably quantifying TH present in real clinical serum samples from patients with hyperthyroidism and healthy volunteers.

Ultrasensitive nucleic acid detection based on phosphorothioated hairpin-assisted isothermal amplification.

Herein, we describe a phosphorothioated hairpin-assisted isothermal amplification (PHAmp) method for detection of a target nucleic acid. The hairpin probe (HP) is designed to contain a 5' phosphorothioate (PS)-modified overhang, a target recognition site, and a 3' self-priming (SP) region. Upon binding to the target nucleic acid, the HP opens and the SP region is rearranged to serve as a primer. The subsequent process of strand displacement DNA synthesis recycles the bound target to open another HP and produces an extended HP (EP) with a PS-DNA/DNA duplex at the end, which would be readily denatured due to its reduced thermal stability. The trigger then binds to the denatured 3' end of the EP and is extended, producing an intermediate double-stranded (ds) DNA product (IP). The trigger also binds to the denatured 3' end of the IP, and its extension produces the final dsDNA product along with concomitant displacement and recycling of EP. By monitoring the dsDNA products, the target nucleic acid can be identified down to 0.29 fM with a wide dynamic range from 1 nM to 1 fM yielding an excellent specificity to discriminate even a single base-mismatched target. The unique design principle could provide new insights into the development of novel isothermal amplification methods for nucleic acid detection.

A novel method to detect mutation in DNA by utilizing exponential amplification reaction triggered by the CRISPR-Cas9 system.

We, herein, describe a novel method to detect mutation in DNA by utilizing exponential amplification reaction (EXPAR) triggered by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9, called CRISPR-EXPAR. The CRISPR system consisting of two Cas9/sgRNA complexes was designed to cut out a specific mutation region within the target DNA, which would consequently promote EXPAR by continuously repeated extension and nicking reactions. As a consequence, a large number of final EXPAR products, which can be monitored through duplex-specific fluorescent staining, are produced. Based on this design principle, we successfully identified a model target mutation within the human epidermal growth factor receptor 2 (HER2) gene down to 437 aM with excellent specificity. The practical capability of this method was verified by reliably identifying the target mutation directly from the genomic DNA (gDNA) extracted from the lung cancer cell line, NCI-H1781 (H1781), and its universal applicability was further confirmed by identifying another EFGF L858R mutation. This technique could serve as a new isothermal platform to identify various mutations by rationally redesigning single guide RNA (sgRNA) according to the target mutation site.

Self-priming phosphorothioated hairpin-mediated isothermal amplification.

We herein describe a novel technology, termed self-priming phosphorothioated hairpin-mediated isothermal amplification (SP-HAMP), enabling target nucleic acid detection. Isothermal amplification strategies are a simple process that efficiently raises the amount of nucleic acid at a constant temperature, but still has lots of problems such as the requirement of multiple exogenous primers and enzymes, which trigger non-specific background signal and increase the complexity of procedures. The key component for overcoming the above-mentioned limitations is the designed hairpin probe (HP) consisting of self-priming region along the 3' stem and the 3' overhang and phosphorothioate modifications at the 5' overhang and the specific loop part. The HP was designed to open through binding to target nucleic acid. Upon opening of HP, its self-priming (SP) region is rearranged to form a smaller hairpin whose 3' end could serve as a primer. The following extension produces the extended HP and displaces the bound target nucleic acid, which is then recycled to open another HP. Due to the reduced stability caused by the specific two phosphorothioate (PS) modifications, the 3' end of EP1 is readily rearranged to form the foldback hairpin structure, which would promote the foldback extension to produce once more extended HP. Since the two PS modifications are always located at the same positions along the 5' stem within the further extended HPs, the foldback reaction followed by the extension would be continuously repeated, consequently producing a large number of the long hairpin concatamers. Based on this unique design principle, we successfully detected even a single copy of target DNA with outstanding discrimination capability under an isothermal condition by employing only a single HP without the requirement for the complicated multiple primers. In conclusion, the sophisticated design principle employed in this work would provide great insight for the development of self-operative isothermal amplifying system enabling short target nucleic acid detection such as microRNAs or any target which is less than 200 mer.

Ultrasensitive isothermal method to detect microRNA based on target-induced chain amplification reaction.

We herein describe an ultrasensitive isothermal method to detect microRNA (miRNA) by utilizing target-induced chain amplification reaction (CAR). The hairpin probe (HP) employed in this strategy is designed to be opened upon binding to target miRNA. The exponential amplification reaction (EXPAR) template (ET) then binds to the exposed stem of HP and DNA polymerase (DP) promotes the extension reactions for both HP and ET, consequently producing intermediate double-stranded DNA product (IP) and concomitantly recycling target miRNA to open another intact HP. The IPs would produce a large number of target-mimicking probes (TMPs) and trigger probes (TPs) through the continuously repeated nicking and extension reactions at the two separated nicking sites within the IP. TMP triggers another CAR cycle by binding to intact HP as target miRNA did while TP promotes conventional EXPAR by independently binding to free ET. As a consequence of these interconnected reaction systems, a large number of final double-stranded DNA products (FPs) are produced, which can be monitored by measuring the fluorescent signal produced from duplex-specific fluorescent dye. Based on this unique design principle, the target miRNA was successfully determined down to even a single copy with high selectivity against non-specific miRNAs. The practical applicability of this method was also verified by reliably detecting target miRNA included in the total RNA extracted from the human cancer cell.