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.

Diverse high-affinity DNA aptamers for wild-type and B.1.1.7 SARS-CoV-2 spike proteins from a pre-structured DNA library.

We performed in vitro selection experiments to identify DNA aptamers for the S1 subunit of the SARS-CoV-2 spike protein (S1 protein). Using a pool of pre-structured random DNA sequences, we obtained over 100 candidate aptamers after 13 cycles of enrichment under progressively more stringent selection pressure. The top 10 sequences all exhibited strong binding to the S1 protein. Two aptamers, named MSA1 (Kd = 1.8 nM) and MSA5 (Kd = 2.7 nM), were assessed for binding to the heat-treated S1 protein, untreated S1 protein spiked into 50% human saliva and the trimeric spike protein of both the wildtype and the B.1.1.7 variant, demonstrating comparable affinities in all cases. MSA1 and MSA5 also recognized the pseudotyped lentivirus of SARS-CoV-2 with respective Kd values of 22.7 pM and 11.8 pM. Secondary structure prediction and sequence truncation experiments revealed that both MSA1 and MSA5 adopted a hairpin structure, which was the motif pre-designed into the original library. A colorimetric sandwich assay was developed using MSA1 as both the recognition element and detection element, which was capable of detecting the pseudotyped lentivirus in 50% saliva with a limit of detection of 400 fM, confirming the potential of these aptamers as diagnostic tools for COVID-19 detection.

A Simple Colorimetric Au-on-Au Tip Sensor with a New Functional Nucleic Acid Probe for Food-borne Pathogen Salmonella typhimurium.

An Au-on-Au tip sensor is developed for the detection of Salmonella typhimurium (Salmonella), using a new synthetic nucleic acid probe (NAP) as a linker for the immobilization of a DNA-conjugated Au nanoparticle (AuNP) onto a DNA-attached thin Au layer inside a pipette tip. In the presence of Salmonella, RNase H2 from Salmonella (STH2) cleaves the NAP and the freed DNA-conjugated AuNP can be visually detected by a paper strip. This portable biosensor does not require any electronic, electrochemical or optical equipment. It delivers a detection limit of 3.2×103  CFU mL-1 for Salmonella in 1 h without cell-culturing or signal amplification and does not show cross-reactivity with several control bacteria. Further, the sensor reliably detects Salmonella spiked in food samples, such as ground beef and chicken, milk, and eggs. The sensor can be reused and is stable at ambient temperature, showing its potential as a point-of-need device for the prevention of food poisoning by Salmonella.

Three on Three: Universal and High-Affinity Molecular Recognition of the Symmetric Homotrimeric Spike Protein of SARS-CoV-2 with a Symmetric Homotrimeric Aptamer.

Our previously discovered monomeric aptamer for SARS-CoV-2 (MSA52) possesses a universal affinity for COVID-19 spike protein variants but is ultimately limited by its ability to bind only one subunit of the spike protein. The symmetrical shape of the homotrimeric SARS-CoV-2 spike protein presents the opportunity to create a matching homotrimeric molecular recognition element that is perfectly complementary to its structural scaffold, causing enhanced binding affinity. Here, we describe a branched homotrimeric aptamer with three-fold rotational symmetry, named TMSA52, that not only possesses excellent binding affinity but is also capable of binding several SARS-CoV-2 spike protein variants with picomolar affinity, as well as pseudotyped lentiviruses expressing SARS-CoV-2 spike protein variants with femtomolar affinity. Using Pd-Ir nanocubes as nanozymes in an enzyme-linked aptamer binding assay (ELABA), TMSA52 was capable of sensitively detecting diverse pseudotyped lentiviruses in pooled human saliva with a limit of detection as low as 6.3 × 103 copies/mL. The ELABA was also used to test 50 SARS-CoV-2-positive and 60 SARS-CoV-2-negative patient saliva samples, providing sensitivity and specificity values of 84.0 and 98.3%, respectively, thus highlighting the potential of TMSA52 for the development of future rapid tests.

Functional Nucleic Acids for Pathogenic Bacteria Detection.

Pathogens have long presented a significant threat to human lives, and hence the rapid detection of infectious pathogens is vital for improving human health. Current detection methods lack the means to detect infectious pathogens in a simple, rapid, and reliable manner at the time and point of need. Functional nucleic acids (FNAs) have the potential to overcome these limitations by acting as key components for point-of-care (POC) biosensors due to their distinctive advantages that include high binding affinities and specificities, excellent chemical stability, ease of synthesis and modification, and compatibility with a variety of signal-amplification and signal-transduction mechanisms.This Account summarizes the work completed in our groups toward developing FNA-based biosensors for detecting bacteria. In vitro selection has led to the isolation of many RNA-cleaving fluorogenic DNAzymes (RFDs) and DNA aptamers that can recognize infectious pathogens, including Escherichia coli, Clostridium difficile, Helicobacter pylori, and Legionella pneumophila. In most cases, a "many-against-many" approach was employed using a DNA library against a crude cellular mixture of an infectious pathogen containing diverse biomarkers as the target to isolate RFDs, with combined counter and positive selections ensuring high specificity toward the desired target. This procedure allows for the isolation of pathogen-specific FNAs without first identifying a suitable biomarker. Multiple target-specific DNA aptamers, including anti-glutamate dehydrogenase (GDH) circular aptamers, anti-degraded toxin B aptamers, and anti-RNase HII aptamers, have also been isolated for the detection of bacteria such as Clostridium difficile. The isolated FNAs have been integrated into fluorescent, colorimetric, and electrochemical biosensors using various signal transduction mechanisms. Both simple-to-use paper-based analytical devices and hand-held electrical devices with integrated FNAs have been developed for POC applications. In addition, signal-amplification strategies, including DNA catenane enabled rolling circle amplification (RCA), DNAzyme feedback RCA, and an all-DNA amplification system using a four-way junction and catalytic hairpin assembly (CHA), have been designed and applied to these systems to further increase their detection sensitivity. The use of these FNA-based biosensors to detect pathogens directly in clinical samples, such as urine, blood, and stool, has now been demonstrated with an outstanding sensitivity of as low as 10 cells per milliliter, highlighting the tremendous potential of using FNA-based sensors in clinical applications. We further describe strategies to overcome the challenges of using FNA-based biosensors in clinical applications, including strategies to improve the stability of FNAs in biological samples and prevent their nonspecific degradation from nucleases and strategies to deal with issues such as signal loss caused by nonspecific binding and biofouling. Finally, the remaining roadblocks for employing FNA-based biosensors in clinical applications are discussed.

A Universal DNA Aptamer that Recognizes Spike Proteins of Diverse SARS-CoV-2 Variants of Concern.

Invited for the cover of this issue are John Brennan, Yingfu Li, and co-workers at McMaster University. The image depicts MSA52 as a universal DNA aptamer that recognizes spike proteins of diverse SARS-CoV-2 variants of concern. Read the full text of the article at 10.1002/chem.202200078.

A Universal DNA Aptamer that Recognizes Spike Proteins of Diverse SARS-CoV-2 Variants of Concern.

We report on a unique DNA aptamer, denoted MSA52, that displays universally high affinity for the spike proteins of wildtype SARS-CoV-2 as well as the Alpha, Beta, Gamma, Epsilon, Kappa, Delta and Omicron variants. Using an aptamer pool produced from round 13 of selection against the S1 domain of the wildtype spike protein, we carried out one-round SELEX experiments using five different trimeric spike proteins from variants, followed by high-throughput sequencing and sequence alignment analysis of aptamers that formed complexes with all proteins. A previously unidentified aptamer, MSA52, showed Kd values ranging from 2 to 10 nM for all variant spike proteins, and also bound similarly to variants not present in the reselection experiments. This aptamer also recognized pseudotyped lentiviruses (PL) expressing eight different spike proteins of SARS-CoV-2 with Kd values between 20 and 50 pM, and was integrated into a simple colorimetric assay for detection of multiple PL variants. This discovery provides evidence that aptamers can be generated with high affinity to multiple variants of a single protein, including emerging variants, making it well-suited for molecular recognition of rapidly evolving targets such as those found in SARS-CoV-2.

Selection and Characterization of an RNA-Cleaving DNAzyme Activated by Legionella pneumophila.

Legionella pneumophila is a deadly bacterial pathogen that has caused numerous Legionnaires' disease outbreaks, where cooling towers were the most common source of exposure. Bacterial culturing is used for L. pneumophila detection, but this method takes approximately 10 days to complete. In this work, an RNA-cleaving fluorogenic DNAzyme, named LP1, was isolated. Extensive characterization revealed that LP1 is reactive with multiple infectious isolates of L. pneumophila but inactive with 25 other common bacterial species. LP1 is likely activated by a protein target, capable of generating a detectable signal in the presence of as few as 10 colony-forming units of L. pneumophila, and able to maintain its activity in cooling tower water from diverse sources. Given that similar DNAzymes have been incorporated into many sensitive assays for bacterial detection, LP1 holds the potential for the development of biosensors for monitoring the contamination of L. pneumophila in exposure sources.

High-Affinity Dimeric Aptamers Enable the Rapid Electrochemical Detection of Wild-Type and B.1.1.7 SARS-CoV-2 in Unprocessed Saliva.

We report a simple and rapid saliva-based SARS-CoV-2 antigen test that utilizes a newly developed dimeric DNA aptamer, denoted as DSA1N5, that specifically recognizes the spike proteins of the wildtype virus and its Alpha and Delta variants with dissociation constants of 120, 290 and 480 pM, respectively, and binds pseudotyped lentiviruses expressing the wildtype and alpha trimeric spike proteins with affinity constants of 2.1 pM and 2.3 pM, respectively. To develop a highly sensitive test, DSA1N5 was immobilized onto gold electrodes to produce an electrochemical impedance sensor, which was capable of detecting 1000 viral particles per mL in 1:1 diluted saliva in under 10 min without any further sample processing. Evaluation of 36 positive and 37 negative patient saliva samples produced a clinical sensitivity of 80.5 % and specificity of 100 % and the sensor could detect the wildtype virus as well as the Alpha and Delta variants in the patient samples, which is the first reported rapid test that can detect any emerging variant of SARS-CoV-2.

Highly Sensitive RNA-Cleaving DNAzyme Sensors from Surface-to-Surface Product Enrichment.

The engineering of easy-to-use biosensors with ultra-low detection sensitivity remains a major challenge. Herein, we report a simple approach for creating such sensors through the use of an RNA-cleaving DNAzyme (RcD) and a strategy designed to concentrate its cleavage product significantly. The assay uses micron-sized beads loaded with a target-responsive RcD and a paper strip containing a microzone covered with a DNA oligonucleotide capable of capturing the cleavage product of the RcD through Watson-Crick hybridization. Placing the beads and the paper strip in a target-containing test sample allows the bead-bound RcD molecules to undergo target-induced RNA cleavage, releasing a DNA fragment that is captured by the paper strip. This strategy, though simple, is very effective in achieving high levels of detection sensitivity, being able to enrich the concentration of the cleavage product by three orders of magnitude. It is also compatible with both fluorescence-based and colorimetric reporting mechanisms. This work provides a simple platform for developing ultrasensitive biosensors that take advantage of the widely available RcDs as molecular recognition elements.

A Rapid Assay to Assess Nitrification Inhibition Using a Panel of Bacterial Strains and Partial Least Squares Modeling.

As a proof of concept, a rapid assay consisting of a cell-based biosensor (CBB) panel of pure bacterial strains, a fluorescent dye, and partial least squares (PLS) modeling was developed to assess the nitrification inhibition potential of industrial wastewater (WW) samples. The current standard method used to assess the nitrification inhibition potential is the specific nitrification rate (SNR) batch test, which requires approximately 4 h to complete under the watch of an experienced operator. In this study, we exposed the CBB panel of seven bacterial strains (nitrifying and non-nitrifying) to 28 different industrial WW samples and then probed both the membrane integrity and cellular activity using a commercially available "live/dead" fluorescent dye. The CBB panel response acts as a surrogate measurement for the performance of nitrification. Of the seven strains, four (Nitrospira, Escherichia coli, Bacillus subtilis, Bacillus cereus) were identified via the modeling technique to be the most significant contributors for predicting the nitrification inhibition potential. The key outcome from this work is that the CBB panel fluorescence data (collected in approximately 10 min) can accurately predict the outcome of an SNR batch test (that takes 4 h) when performed with the same WW samples and has a strong potential to approximate the chemical composition of these WW samples using PLS modeling. Overall, this is a powerful technique that can be used for point-of-use detection of nitrification inhibition.

Deposited Nanoparticles Can Promote Air Clogging of Piezoelectric Inkjet Printhead Nozzles.

Piezoelectric inkjet printing is susceptible to printhead clogging when printing with inks that contain dispersed particles. This paper investigates the mechanisms by which 28-530 nm nanoparticle dispersions induce printhead clogging without forming large aggregates or thick deposited layers on printhead surfaces. Printing experiments were combined with nanoparticle deposition studies and with experiments where inks were pumped through printheads at a constant flow rate with a syringe pump. Submonolayer coverages of hydrophobic cationic polystyrene nanoparticles adhering to printhead surfaces promote rapid clogging by trapped air that enters from the nozzle opening. We propose that the deposited particles distort the shape of the ink/air meniscus, possibly causing air entrainment, and promote air bubble adhesion to the interior printhead surfaces. The printer's purge-blot cleaning procedure removes air clogs, but the clogs quickly reform when printing is resumed because the adsorbed nanoparticles are not removed by the cleaning procedure. Nondepositing anionic hydrophobic nanoparticles cause much less clogging, possibly because of filtration of trace large aggregates. Colloidal stability is a necessary but not sufficient criterion for ink dispersions; the ink particles must not adsorb onto the printhead surfaces. Thus, alternate surface chemistries for the printhead and ink particle surfaces may be required to print hydrophobic ink materials.

A DNAzyme-Based Colorimetric Paper Sensor for Helicobacter pylori.

The reliable detection of pathogenic bacteria in complex biological samples using simple assays or devices remains a major challenge. Herein, we report a simple colorimetric paper device capable of providing specific and sensitive detection of Helicobacter pylori (H. pylori), a pathogen strongly linked to gastric carcinoma, gastric ulcers, and duodenal ulcers, in stool samples. The sensor molecule, an RNA-cleaving DNAzyme obtained through in vitro selection, is activated by a protein biomarker from H. pylori. The colorimetric paper sensor, designed on the basis of the RNA-cleaving property of the DNAzyme, is capable of sensitive detection of H. pylori in human stool samples with minimal sample processing and provides results in minutes. It remains fully functional under storage at ambient temperature for at least 130 days. This work lays a foundation for developing DNAzyme-enabled paper-based point-of-care diagnostic devices for monitoring pathogens in complex samples.

Autonomously Self-Adhesive Hydrogels as Building Blocks for Additive Manufacturing.

We report a simple method of preparing autonomous and rapid self-adhesive hydrogels and their use as building blocks for additive manufacturing of functional tissue scaffolds. Dynamic cross-linking between 2-aminophenylboronic acid-functionalized hyaluronic acid and poly(vinyl alcohol) yields hydrogels that recover their mechanical integrity within 1 min after cutting or shear under both neutral and acidic pH conditions. Incorporation of this hydrogel in an interpenetrating calcium-alginate network results in an interfacially stiffer but still rapidly self-adhesive hydrogel that can be assembled into hollow perfusion channels by simple contact additive manufacturing within minutes. Such channels withstand fluid perfusion while retaining their dimensions and support endothelial cell growth and proliferation, providing a simple and modular route to produce customized cell scaffolds.

Print to detect: a rapid and ultrasensitive phage-based dipstick assay for foodborne pathogens.

Foodborne pathogens are a burden to the economy and a constant threat to public health. The ability to rapidly detect the presence of foodborne pathogens is a vital component of any strategy towards establishing a safe and secure food supply chain. Bacteriophages (phages) are viruses capable of infecting and replicating within bacteria in a strain-specific manner. The ubiquitous and selective nature of phages makes them ideal for the detection and biocontrol of bacteria. Therefore, the objective of this research was to develop and test a phage-based paper dipstick biosensor for the detection of various foodborne pathogens in food matrices. The first step was to identify the best method for immobilizing phages on paper such that their biological activity (infectivity) was preserved. It was found that piezoelectric inkjet printing resulted in lower loss of phage infectivity when compared with other printing methods (namely gravure and blade coating) and that ColorLok paper was ideally suited to create functional sensors. The phage-based bioactive papers developed with use of piezoelectric inkjet printing actively lysed their target bacteria and retained this antibacterial activity for up to 1 week when stored at room temperature and 80% relative humidity. These bioactive paper strips in combination with quantitative real-time PCR were used for quantitative determination of target bacteria in broth and food matrices. A phage dipstick was used to capture and infect Escherichia coli O157:H7, E. coli O45:H2, and Salmonella Newport in spinach, ground beef and chicken homogenates, respectively, and quantitative real-time PCR was used to detect the progeny phages. A detection limit of 10-50 colony-forming units per millilitre was demonstrated with a total assay time of 8 h, which was the duration of a typical work shift in an industrial setting. This detection method is rapid and cost-effective, and may potentially be applied to a broad range of bacterial foodborne pathogens. Graphical abstract ᅟ.

RNA Protection is Effectively Achieved by Pullulan Film Formation.

RNA is a functionally versatile polymer but suffers from susceptibility to spontaneous and RNase-catalyzed degradation. This vulnerability makes it difficult to preserve RNA for extended periods of time, thus limiting its use in various contexts, including practical applications as functional nucleic acids. Here we present a simple method to preserve RNA by pullulan (a complex sugar produced by Aureobasidium pullulans fungus) film formation. This strategy can markedly suppress both spontaneous and RNase degradation. Importantly, the pullulan film readily dissolves in aqueous solution, thus allowing retrieval of fully functional RNA species. In order to illustrate the advantage of this protective method in a practical application, we engineered a simple paper sensor containing a bacteria-detecting RNA-cleaving DNAzyme. This detection capability of the device was unchanged after storage at room temperature for six months.

Target-Induced and Equipment-Free DNA Amplification with a Simple Paper Device.

We report on a paper device capable of carrying out target-induced rolling circle amplification (RCA) to produce massive DNA amplicons that can be easily visualized. Interestingly, we observed that RCA was more proficient on paper than in solution, which we attribute to a significantly higher localized concentration of immobilized DNA. Furthermore, we have successfully engineered a fully functional paper device for sensitive DNA or microRNA detection via printing of all RCA-enabling molecules within a polymeric sugar film formed from pullulan, which was integrated with the paper device. This encapsulation not only stabilizes the entrapped reagents at room temperature but also enables colorimetric bioassays with minimal steps.

Printed paper sensors for serum lactate dehydrogenase using pullulan-based inks to immobilize reagents.

In this study, a paper-based point-of-care (POC) colorimetric biosensor was developed for the detection of lactate dehydrogenase in serum using a nonporous, oxygen impermeable reversibly gelling polysaccharide material based on pullulan. The pullulan could be printed onto paper surfaces along with all required assay reagents, providing a means for high-stability immobilization of all reagents on paper. Serum containing lactate dehydrogenase (LDH) was directly spotted on to the pullulan-coated bioactive paper and provided quantitative colorimetric data that was comparable to that obtained with a conventional plate-reader method. The paper strip was found to be highly stable and could be stored at 4 °C for at least 10 weeks with no loss in performance, as compared to a complete loss in performance within 1 day when the reagents were printed without the stabilizing polysaccharide. The ease of fabrication coupled with the high stability of the printed reagents provides a facile platform for easily manufactured POC sensors.

Poly(oligoethylene glycol methacrylate) dip-coating: turning cellulose paper into a protein-repellent platform for biosensors.

The passivation of nonspecific protein adsorption to paper is a major barrier to the use of paper as a platform for microfluidic bioassays. Herein we describe a simple, scalable protocol based on adsorption and cross-linking of poly(oligoethylene glycol methacrylate) (POEGMA) derivatives that reduces nonspecific adsorption of a range of proteins to filter paper by at least 1 order of magnitude without significantly changing the fiber morphology or paper macroporosity. A lateral-flow test strip coated with POEGMA facilitates effective protein transport while also confining the colorimetric reporting signal for easier detection, giving improved performance relative to bovine serum albumin (BSA)-blocked paper. Enzyme-linked immunosorbent assays based on POEGMA-coated paper also achieve lower blank values, higher sensitivities, and lower detection limits relative to ones based on paper blocked with BSA or skim milk. We anticipate that POEGMA-coated paper can function as a platform for the design of portable, disposable, and low-cost paper-based biosensors.

An inkjet-printed bioactive paper sensor that reports ATP through odour generation.

We describe an inkjet printed paper-based sensor that reports ATP by the enzyme catalysed hydrolysis of S-methyl-L-cysteine generating an odour (methyl mercaptan) that is easily detectable by the human nose.