Amplification-Free COVID-19 Detection by Digital Droplet REVEALR.

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, exposed a pressing need for new public health tools for pathogen detection, disease diagnosis, and viral genotyping. REVEALR (RNA-encoded viral nucleic acid analyte reporter) is an isothermal DNAzyme-based point-of-care diagnostic that functions with a detection limit of ∼10 copies/µL when coupled with a preamplification step and can be utilized for viral genotyping of SARS-CoV-2 variants of concern through base pair mismatch recognition in a competitive binding format. Here, we describe an advanced REVEALR platform, termed digital droplet REVEALR (ddREVEALR), that can achieve direct viral detection and absolute sample quantitation utilizing a signal amplification strategy that relies on chemical modifications, DNAzyme multiplexing, and volume compression. Using an AI-assisted image-based readout, ddREVEALR was found to achieve 95% positive predictive agreement from a set of 20 nasal pharyngeal swabs collected at UCI Medical Center in Orange, California. We propose that the combination of amplification-free and protein-free analysis makes ddREVEALR a promising application for direct viral RNA detection of clinical samples.

Detection of Large Genomic RNA via DNAzyme-Mediated RNA Cleavage and Rolling Circle Amplification: SARS-CoV-2 as a Model.

A new method for the detection of genomic RNA combines RNA cleavage by the 10-23 DNAzyme and use of the cleavage fragments as primers to initiate rolling circle amplification (RCA). 230 different 10-23 DNAzyme variants were screened to identify those that target accessible RNA sites within the highly structured RNA transcripts of SARS-CoV-2. A total of 28 DNAzymes were identified with >20 % cleavage, 5 with >40 % cleavage and one with >60 % in 10 min. The cleavage fragments from these reactions were then screened for coupling to an RCA reaction, leading to the identification of several cleavage fragments that could efficiently initiate RCA. Using a newly developed quasi-exponential RCA method with a detection limit of 500 aM of RNA, 14 RT-PCR positive and 15 RT-PCR negative patient saliva samples were evaluated for SARS-CoV-2 genomic RNA, achieving a clinical sensitivity of 86 % and specificity of 100 % for detection of the virus in <2.5 h.

Colorimetric detection of viral RNA fragments based on an integrated logic-operated three-dimensional DNA walker.

Timely and accurate detection of virus is crucial for preventing spread of disease and early treatment of the infected cases. Herein we design an integrated logic-operated three-dimensional DNA walker for colorimetric detection of viral RNA fragments, by taking SARS-CoV-2 as an example. The DNA walker is composed of small amounts of dually-blocked walking strands and large amounts of dual-stem-loop track strands on gold nanoparticles. The walking strand contains a swing arm domain and a DNAzyme domain blocked at both sides of catalytic core, while the track strand contains a substrate domain located at the peripheral larger loop. Only the presence of both ORF1ab and N RNA fragments can fully de-block the walking strand, which then continuously hybridizes with track strands and cleaves them by DNAzyme-catalyzed hydrolysis. As the cleavage of track strands from long-stranded, double stem-loop structure to short-stranded, linear sequence, the DNA walker shows much lowered stability due to decreased negative charge density and diminished steric repulsion, which then gets aggregated at high salt concentration, accompanied by a visible color change. The colorimetric DNA walker detects RNA fragments down to 1 nM, responds dual viral genes in a "AND" logic way, and shows high specificity to target sequence. It can further detect large nucleic acids containing ORF1ab and N sequences, and reach 200 copies/mL detection limit by coupling a simple upstream amplification of sample. The method may provide a convenient way for reliable detection of viral RNA.

Toward a Home Test for COVID-19 Diagnosis: DNA Machine for Amplification-Free SARS-CoV-2 Detection in Clinical Samples.

Nucleic acid-based detection of RNA viruses requires an annealing procedure to obtain RNA/probe or RNA/primer complexes for unwinding stable structures of folded viral RNA. In this study, we designed a protein-enzyme-free nano-construction, named four-armed DNA machine (4DNM), that requires neither an amplification stage nor a high-temperature annealing step for SARS-CoV-2 detection. It uses a binary deoxyribozyme (BiDz) sensor incorporated in a DNA nanostructure equipped with a total of four RNA-binding arms. Additional arms were found to improve the limit of detection at least 10-fold. The sensor distinguished SARS-CoV-2 from other respiratory viruses and correctly identified five positive and six negative clinical samples verified by quantitative polymerase chain reaction (RT-qPCR). The strategy reported here can be used for the detection of long natural RNA and can become a basis for a point-of-care or home diagnostic test.

REVEALR-Based Genotyping of SARS-CoV-2 Variants of Concern in Clinical Samples.

The SARS-CoV-2 virus has evolved into new strains that increase viral transmissibility and reduce vaccine protection. The rapid circulation of these more harmful strains across the globe has created a pressing need for alternative public health screening tools. REVEALR (RNA-encoded viral nucleic acid analytic reporter), a rapid and highly sensitive DNAzyme-based detection system, functions with perfect accuracy against patient-derived clinical samples. Here, we design REVEALR into a novel genotyping assay that detects single-base mismatches corresponding to each of the major SARS-CoV-2 strains found in the United States. Of 34 sequence-verified patient samples collected in early, mid, and late 2021 at the UCI Medical Center in Orange, California, REVEALR identified the correct variant [Wuhan-Hu-1, alpha (B.1.1.7), gamma (P.1), epsilon (B.1.427/9), delta (B.1.617.2), and omicron (B.1.1.529)] with 100% accuracy. The assay, which is programmable and amenable to multiplexing, offers an important new approach to personalized diagnostics.

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.

Systematic mapping of DNAzymes research from 1995 to 2019.

DNAzymes (catalytic DNA) have gained significant diagnostic and therapeutic applications with increasing research output over the years. Functional oligonucleotides are used as molecular recognition elements within biosensors for detection of analytes and viral infections such as SARS-CoV-2. DNAzymes are also applied for silencing and regulating cancer specific genes. However, there has not been any report on systematic analysis to track research status, reveal hotspots, and map knowledge in this field. Therefore, in the present study, research articles on DNAzymes from 1995 to 2019 were extracted from Web of Science (SCI-Expanded) after which, 1037 articles were imported into Rstudio (version 3.6.2) and analysed accordingly. The highest number of articles was published in 2019 (n = 138), while the least was in 1995 (n = 1). The articles were published across 216 journals by 2344 authors with 2337 multi-author and 7 single authors. The most prolific authors were Li Y (n = 47), Liu J (n = 46), Wang L (n = 33), Willner I (n = 33) and Zhang L (n = 33). The top three most productive countries were China (n = 2018), USA (n = 447) and Canada (n = 251). The most productive institutions were Hunan University, China (n = 141), University of Illinois, USA (n = 139) and Fuzhou University, China (n = 101). Despite the increasing interest in this field, international collaborations between institutions were very low which requires immediate attention to mitigate challenges such as limited funding, access to facilities, and existing knowledge gap.

Sensitive fluorescence detection of pathogens based on target nucleic acid sequence-triggered transcription.

Accurate identification of mutant pathogens derived from genetic polymorphisms is highly desired in clinical diagnosis. However, current detection methods based on Watson-Crick hybridization suffers from false positives due to the cross-reactivity of wild-type sequences. In this study, we developed an accurate identification of mutant pathogens by combining programmable DNAzyme and target nucleic acid sequence-triggered transcription. Single nucleotide variants (SNVs) are the most plentiful type of mutations in the genome. High specificity to discriminate SNV was first achieved by rational design of dual-hairpin DNA structure and DNAzyme's capability of site-specific cleavage. T7 RNA polymerase-mediated transcription amplification was introduced to exponentially increase the sensitivity by encompassing T7 promoter sequence into the dual-hairpin DNA structure. The design of this biosensor is fast and straightforward without many computational steps, and the highly sensitive biosensor can detect not only SNVs but also occasional insertions and large deletions in the genome. We showed that the assay could rapidly detect COVID-19 variant and methicillin-resistant Staphylococcus aureus (MRSA), and the limit of detection is 0.96 copy/µL. The modular design of functional DNA enables this biosensor be easily reconfigured and is useful diagnosis of emerging infectious diseases caused by mutant pathogens.

Target DNA- and pH-responsive DNA hydrogel-based capillary assay for the optical detection of short SARS-CoV-2 cDNA.

DNA is recognized as a powerful biomarker for clinical diagnostics because its specific sequences are closely related to the cause and development of diseases. However, achieving rapid, low-cost, and sensitive detection of short-length target DNA still remains a considerable challenge. Herein, we successfully combine the catalytic hairpin assembly (CHA) technique with capillary action to develop a new and cost-effective method, a target DNA- and pH-responsive DNA hydrogel-based capillary assay, for the naked eye detection of 24 nt short single-stranded target DNA. Upon contact of target DNA, three individual hairpin DNAs hybridize with each other to sufficiently amplify Y-shaped DNA nanostructures (Y-DNA) until they are completely consumed via CHA cycling reactions. Each arm of the resultant Y-DNA contains sticky ends with i-motif DNA structure-forming sequences that can be self-assembled in an acidic environment (pH 5.0) to form target DNA- and pH-responsive DNA hydrogels by means of i-motif DNA-driven crosslinking. When inserting a capillary tube in the resultant solution, the liquid level inside clearly reduces due to the decrease in capillary force induced by the gels. In this way, the developed assay demonstrates sensitive and quantitative detection, with a detection limit of approximately 10 pM of 24 nt short complementary DNA (cDNA) targeting SARS-CoV-2 RNA genes at room temperature within 1 h. The assay is further shown to successfully detect target cDNA in serum, and it is also applied to detect several types of target sequences. Requiring no analytic equipment, precise temperature control, or enzymatic reactions, the developed DNA hydrogel-based capillary assay has potential as a promising naked eye detection platform for target DNA in resource-limited clinical settings.

Tetrahedron-Based Constitutional Dynamic Network for COVID-19 or Other Coronaviruses Diagnostics and Its Logic Gate Applications.

Considering the large-scale outbreak of the coronavirus, it is essential to develop a versatile sensing system for different coronaviruses diagnostics, such as COVID-19, severe acute respiratory syndrome-related coronavirus (SARS-CoV), and bat SARS-like coronavirus (Bat-SL-CoVZC45). In this work, a tetrahedron-based constitutional dynamic network was built as the sensing platform for coronavirus detection. Four different DNA probes were used to construct the tetrahedron structure. DNAzyme and the fluorophore modified substrate strand were used to generate different fluorescence signals, which can be used to distinguish different coronaviruses. The coronavirus biosensor shows a high sensitivity for COVID-19, Bat-SL-CoVZC45, and SARS-CoV detection, with detection limits of 2.5, 3.1, and 2.9 fM, respectively. Also, the platform is robust, and the possible interference from clinical samples was negligible. Using different coronaviruses as inputs, we have fabricated several concatenated logic gates, such as "AND-OR", "INHIBIT-AND", "AND-AND-AND", and "AND-INHIBIT". Importantly, our logic system can also be used to identify SARS-CoV-2 Delta and Lambda variants in the logic operations. Due to the unique advantages of high sensitivity and selectivity, multiple logic biocomputing capabilities, and multireadout mode, this flexible sensing system provides a versatile sensing strategy for intelligent diagnostics of different coronaviruses with low false-negative rates.

Collaborative Equilibrium Coupling of Catalytic DNA Nanostructures Enables Programmable Detection of SARS-CoV-2.

Accessible and adaptable nucleic acid diagnostics remains a critical challenge in managing the evolving COVID-19 pandemic. Here, an integrated molecular nanotechnology that enables direct and programmable detection of SARS-CoV-2 RNA targets in native patient specimens is reported. Termed synergistic coupling of responsive equilibrium in enzymatic network (SCREEN), the technology leverages tunable, catalytic molecular nanostructures to establish an interconnected, collaborative architecture. SCREEN mimics the extraordinary organization and functionality of cellular signaling cascades. Through programmable enzyme-DNA nanostructures, SCREEN activates upon interaction with different RNA targets to initiate multi-enzyme catalysis; through system-wide favorable equilibrium shifting, SCREEN directly transduces a single target binding into an amplified electrical signal. To establish collaborative equilibrium coupling in the architecture, a computational model that simulates all reactions to predict overall performance and optimize assay configuration is developed. The developed platform achieves direct and sensitive RNA detection (approaching single-copy detection), fast response (assay reaction is completed within 30 min at room temperature), and robust programmability (across different genetic loci of SARS-CoV-2). When clinically evaluated, the technology demonstrates robust and direct detection in clinical swab lysates to accurately diagnose COVID-19 patients.

A Rolling Circle-Amplified G-Quadruplex/Hemin DNAzyme for Chemiluminescence Immunoassay of the SARS-CoV-2 Protein.

Sensitive detection of the SARS-CoV-2 protein remains a great research interest in clinical screening and diagnosis owing to the coronavirus epidemic. Here, an ultrasensitive chemiluminescence (CL) imaging strategy was developed through proximity hybridization to trigger the formation of a rolling circle-amplified G-quadruplex/hemin DNAzyme for the detection of the SARS-CoV-2 protein. The target protein was first recognized by a pair of DNA-antibody conjugates, Ab-1 and Ab-2, to form a proximity-ligated complex, Ab-1/SARS-CoV-2/Ab-2, which contained a DNA sequence complemental to block DNA and thus induced a strand displacement reaction to release the primer from a block/primer complex. The released primer then triggered a rolling circle amplification to form abundant DNAzyme units in the presence of hemin, which produced a strong chemiluminescent signal for the detection of the target protein by catalyzing the oxidation of luminol by hydrogen peroxide. The proposed assay showed a detectable concentration range over 5 orders of magnitude with the detection limit down to 6.46 fg/mL. The excellent selectivity, simple procedure, acceptable accuracy, and intrinsic high throughput of the imaging technique for analysis of serum samples demonstrated the potential applicability of the proposed detection method in clinical screening and diagnosis.

REVEALR: A Multicomponent XNAzyme-Based Nucleic Acid Detection System for SARS-CoV-2.

Isothermal amplification strategies capable of rapid, inexpensive, and accurate nucleic acid detection provide new options for large-scale pathogen detection, disease diagnosis, and genotyping. Here we report a highly sensitive multicomponent XNA-based nucleic acid detection platform that combines analyte preamplification with X10-23-mediated catalysis to detect the viral pathogen responsible for COVID-19. The platform, termed RNA-Encoded Viral Nucleic Acid Analyte Reporter (REVEALR), functions with a detection limit of ≤20 aM (∼10 copies/µL) using conventional fluorescence and paper-based lateral flow readout modalities. With a total assay time of 1 h, REVEALR provides a convenient nucleic acid alternative to equivalent CRISPR-based approaches, which have become popular methods for SARS-CoV-2 detection. The assay shows no cross-reactivity for other in vitro transcribed respiratory viral RNAs and functions with perfect accuracy against COVID-19 patient-derived clinical samples.

Catalytic hairpin DNA assembly-based chemiluminescent assay for the detection of short SARS-CoV-2 target cDNA.

Colorimetric sensors are recognized as a promising means for target molecule detection as they provide rapid, cost-effective, and facile sensing visible to the naked eye. Challenges remain though in terms of their detection sensitivity and specificity for short-length target genes. Herein, we demonstrate the successful combination of the catalytic hairpin DNA assembly (CHA) approach with enzyme-linked immunosorbent assay (ELISA)-mimicking techniques for a simple, sensitive, and sequence-specific colorimetric assay to detect short SARS-CoV-2 target cDNA. In the developed CHA-based chemiluminescent assay, a low concentration of target cDNA is continuously recycled to amplify dimeric DNA probes from two biotinylated hairpin DNA until the hairpin DNA is completely consumed. The dimeric DNA probes are effectively immobilized in a neutravidin-coated microplate well and then capture neutravidin-conjugated horseradish peroxidase via biotin-neutravidin interactions, resulting in a sensitive and selective colorless-to-blue color change. The developed sensing system exhibits a high sensitivity with a detection limit of ~1 nM for target cDNA as well as the ability to precisely distinguish a single-base mismatched mutant gene within 2 h. As the proposed system does not require complex protocols or expensive equipment to amplify target cDNA, it has the potential to be utilized as a powerful tool to improve the detection sensitivity of target genes for clinical diagnostics with colorimetric detection.

Dual recognition element-controlled logic DNA circuit for COVID-19 detection based on exonuclease III and DNAzyme.

Two fragments of the COVID-19 genome (specific and homologous) were used as two inputs to construct an AND logic gate for COVID-19 detection based on exonuclease III and DNAzyme. The detection sensitivity of the assay can reach fM levels. Satisfactory recovery values were obtained in real sample analysis.