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The 10-23 DNAzyme is one of the most active DNA-based enzymes, and in theory, can be designed to target any purine-pyrimidine junction within an RNA sequence for cleavage. However, purine-pyrimidine junctions within a large, structured RNA (lsRNA) molecule of biological origin are not always accessible to 10-23, negating its general utility as an RNA-cutting molecular scissor. Herein, we report a generalizable strategy that allows 10-23 to access any purine-pyrimidine junction within an lsRNA. Using three large SARS-CoV-2 mRNA sequences of 566, 584 and 831 nucleotides in length as model systems, we show that the use of antisense DNA oligonucleotides (ASOs) that target the upstream and downstream regions flanking the cleavage site can restore the activity (kobs) of previously poorly active 10-23 DNAzyme systems by up to 2000-fold. We corroborated these findings mechanistically using in-line probing to demonstrate that ASOs reduced 10-23 DNAzyme target site structure within the lsRNA substrates. This approach represents a simple, efficient, cost-effective, and generalizable way to improve the accessibility of 10-23 to a chosen target site within an lsRNA molecule, especially where direct access to the genomic RNA target is necessary., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Over the last three decades, significant advancements have been made in the development of biosensors and bioassays that use RNA-cleaving DNAzymes (RCDs) as molecular recognition elements. While early examples of RCDs were primarily responsive to metal ions, the past decade has seen numerous RCDs reported for more clinically relevant targets such as bacteria, cancer cells, small metabolites, and protein biomarkers. Over the past 5 years several RCD-based biosensors have also been evaluated using either spiked biological matrixes or patient samples, including blood, serum, saliva, nasal mucus, sputum, urine, and faeces, which is a critical step toward regulatory approval and commercialization of such sensors. In this review, an overview of the methods used to generate RCDs and the properties of key RCDs that have been utilized for in vitro testing is first provided. Examples of RCD-based assays and sensors that have been used to test either spiked biological samples or patient samples are then presented, highlighting assay performance in different biological matrixes. A summary of current prospects and challenges for development of in vitro diagnostic tests incorporating RCDs and an overview of future directions of the field is also provided., (© 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems are increasingly used in biosensor development. However, directly translating recognition events for non-nucleic acid targets by CRISPR into effective measurable signals represents an important ongoing challenge. Herein, it is hypothesized and confirmed that CRISPR RNAs (crRNAs) in a circular topology efficiently render Cas12a incapable of both site-specific double-stranded DNA cutting and nonspecific single-stranded DNA trans cleavage. Importantly, it is shown that nucleic acid enzymes (NAzymes) with RNA-cleaving activity can linearize the circular crRNAs, activating CRISPR-Cas12a functions. Using ligand-responsive ribozymes and DNAzymes as molecular recognition elements, it is demonstrated that target-triggered linearization of circular crRNAs offers great versatility for biosensing. This strategy is termed as "NAzyme-Activated CRISPR-Cas12a with Circular CRISPR RNA (NA3C)." Use of NA3C for clinical evaluation of urinary tract infections using an Escherichia coli-responsive RNA-cleaving DNAzyme to test 40 patient urine samples, providing a diagnostic sensitivity of 100% and specificity of 90%, is further demonstrated., (© 2023 Wiley-VCH GmbH.)

The first protein-binding allosteric RNA-cleaving DNAzyme (RCD) obtained by direct in vitro selection against eosinophil peroxidase (EPX), a validated marker for airway eosinophilia, is described. The RCD has nanomolar affinity for EPX, shows high selectivity against related peroxidases and other eosinophil proteins, and is resistant to degradation by mammalian nucleases. An optimized RCD was used to develop both fluorescence and lateral flow assays, which were evaluated using 38 minimally processed patient sputum samples (23 non-eosinophilic, 15 eosinophilic), producing a clinical sensitivity of 100 % and specificity of 96 %. This RCD-based lateral flow assay should allow for rapid evaluation of airway eosinophilia as an aid for guiding asthma therapy., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

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., (© 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Eosinophils are granulocytes that play a significant role in the pathogenesis of asthma and other airway diseases. Directing patient treatment based on the level of eosinophilia has been shown to be extremely effective in reducing exacerbations and therefore has tremendous potential as a routine clinical test. Herein, we describe the in vitro selection and optimization of DNA aptamers that bind to eosinophil peroxidase (EPX), a protein biomarker unique to eosinophils. Fifteen rounds of magnetic bead aptamer selection were performed prior to high throughput DNA sequencing. The top 10 aptamer candidates were assessed for EPX binding using a mobility shift assay. This process identified a lead aptamer candidate termed EAP1-05 with low nanomolar affinity and high specificity for EPX over other common sputum proteins. This aptamer sequence was further optimized through truncation and used to develop an easy-to-use colourimetric pull-down assay that can detect EPX over a concentration range from 1 - 100 nM in processed sputum. Forty-six clinical samples were processed using a new sputum dispersal method, appropriate for a rapid assessment assay, that avoids centrifugation and lengthy processing times. The assay showed 89% sensitivity and 96% specificity to detect eosinophilia (compared to gold standard sputum cytometry), with results being produced in under an hour. This assay could allow for an easy assessment of eosinophil activity in the airway to guide anti-inflammatory therapy for several airway diseases., (© 2022. The Author(s).)

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 × 10 3 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 (FNAs), including DNA aptamers and DNAzymes, are finding increasing use as molecular recognition elements for point-of-care (POC) assays and sensors. An ongoing challenge in the development of FNA-based POC sensors is the ability to achieve detection of low levels of analyte without compromising assay time and ease of use. Rolling circle amplification (RCA) is a leading nucleic acid (NA) isothermal amplification method which can be coupled with FNAs for the ultrasensitive detection of non-NA targets. Herein we examine the key considerations required when designing FNA-coupled biosensors utilizing RCA. Specifically, we describe methods for using FNAs as inputs to regulate RCA, various modes of RCA amplification, and methods to detect the output of the RCA reaction, along with how these can be combined to allow detection of non-NA targets. Recent progress on development of portable optical and electrochemical POC devices that incorporate RCA is then described, followed by a summary of key challenges and opportunities in the field.

The SARS-CoV-2 virus and COVID-19 pandemic continue to demand effective diagnostic and therapeutic solutions. Finding these solutions requires highly functional molecular recognition elements. Nucleic acid aptamers represent a possible solution. Characterized by their high affinity and specificity, aptamers can be rapidly identified from random-sequence nucleic acid libraries. Over the past two years, many labs around the world have rushed to create diverse aptamers that target two important structural proteins of SARS-CoV-2: the spike (S) protein and nucleocapsid (N) protein. These have led to the identification of many aptamers that show real promise for the development of diagnostic tests and therapeutic agents for SARS-CoV-2. Herein we review all these developments, with a special focus on the development of diverse aptasensors for detecting SARS-CoV-2. These include electrochemical and optical sensors, lateral flow devices, and aptamer-linked immobilized sorbent assays., Competing Interests: The authors declare no conflict of interest., (© 2022 Wiley‐VCH GmbH.)

The cover feature image shows nucleic acid aptamers armed and ready for our battle against the monstrous SARS-CoV-2 virus. Often thought of as "chemical antibodies", these molecular recognition elements are equipped with several unique benefits and have thus been a popular research subject worldwide. Many aptamers for recognizing the spike and nucleocapsid proteins of SARS-CoV-2 have been developed and examined as diagnostic and therapeutic weaponry for the war against COVID-19 and future pandemics. More information can be found in the Review by J. D. Brennan, Y. Li, and co-workers., (© 2022 Wiley‐VCH GmbH.)

We report on a paper-based sensor capable of performing template-independent DNA synthesis by terminal deoxynucleotidyl transferase (TdT). Importantly, we observed that TdT efficiently incorporates fluorescently labeled dUTP on to 3'-OH ends of DNA strands in a strictly controllable manner on cellulose paper, in comparison to its distributive mode of DNA synthesis in solution. Due to the high roughness and porous nature of cellulose paper, we attribute this controllable DNA polymerization to the pore confinement effect on the catalytic behaviour of TdT. Taking advantage of this finding, we proposed a paper-assisted TdT (PAT) assay for absolute quantification of alkylated DNA lesions (N7-methylguanine), DNA deamination (cytosine-to-uracil) and DNA oxidation (8-oxo-7,8-dihydroguanine) by combining various DNA glycosylases. This PAT assay provides a low-cost, high throughput and easy to use method for quantifying the absolute levels of various types of DNA lesions, thus making it well-suited for drug development, genotoxicity testing, and environmental toxicology., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

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., (© 2022 Wiley-VCH GmbH.)

Saliva is an attractive sample for coronavirus disease 2019 testing due its ease of collection and amenability to detect viral RNA with minimal processing. Using a direct-to-RT-PCR method with saliva self-collected from confirmed COVID-19 positive volunteers, we observed 32% false negative results. Confirmed negative and healthy volunteer samples spiked with 10 6 genome copies/mL of heat-inactivated severe acute respiratory syndrome coronavirus 2 showed false negative results of 10% and 13%, respectively. Additional sample heating or dilution of the false negative samples conferred only modest improvements. These results highlight the potential to significantly underdiagnose COVID-19 infections when testing directly from minimally processed heterogeneous saliva samples., (© 2022. The Author(s).)

Detection of pathogenic bacteria in complex biological matrices remains a major challenge. Herein, we report the selection and optimization of a new DNAzyme for Staphylococcus aureus (SA) and the use of the DNAzyme to develop a simple lateral flow device (LFD) for detection of SA in nasal mucus. The DNAzyme was generated by in vitro selection using a crude extra/intracellular mixture derived from SA, which could be used directly for simple solution or paper-based fluorescence assays for SA. The DNAzyme was further modified to produce a DNA cleavage fragment that acted as a bridging element to bind DNA-modified gold nanoparticles to the test line of a LFD, producing a simple colorimetric dipstick test. The LFD was evaluated with nasal mucus samples spiked with SA, and demonstrated that SA detection was possible in minutes with minimal sample processing., (© 2021 Wiley-VCH GmbH.)

We report a new method for biosensing based on the target-mediated resistance of DNA aptamers against 5'-exonuclease digestion, allowing them to act as primers for rolling circle amplification (RCA). A target-bound DNA strand containing an aptamer region on the 5'-end and a primer region on the 3'-end is protected from 5'-exonuclease digestion by RecJ exonuclease in a target-dependent manner. As the protected aptamer is at the 5'-end, the exposed primer on the 3'-end can participate in RCA in the presence of a circular template to generate a turn-on sensor. Without target, RecJ digests the primer and prevents RCA from occurring, allowing quantitative fluorescence detection of both thrombin, a protein, and ochratoxin A (OTA), a small molecule, at picomolar concentrations., (© 2021 Wiley-VCH GmbH.)

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., (© 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

We report a generalizable strategy for biosensing that takes advantage of the resistance of DNA aptamers against nuclease digestion when bound with their targets, coupled with toehold mediated strand displacement (TMSD) and rolling circle amplification (RCA). A DNA aptamer containing a toehold extension at its 5'-end protects it from 3'-exonuclease digestion by phi29 DNA polymerase (phi29 DP) in a concentration-dependent manner. The protected aptamer can participate in RCA in the presence of a circular template that is designed to free the aptamer from its target via TMSD. The absence of the target leads to aptamer digestion, and thus no RCA product is produced, resulting in a turn-on sensor. Using two different DNA aptamers, we demonstrate rapid and quantitative real-time fluorescence detection of two human proteins: platelet-derived growth factor (PDGF) and thrombin. Sensitive detection of PDGF was also achieved in human serum and human plasma, demonstrating the selectivity of the assay., (© 2021 Wiley-VCH GmbH.)

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.

This article provides a comprehensive review of biosensing with DNAzymes, providing an overview of different sensing applications while highlighting major progress and seminal contributions to the field of portable biosensor devices and point-of-care diagnostics. Specifically, the field of functional nucleic acids is introduced, with a specific focus on DNAzymes. The incorporation of DNAzymes into bioassays is then described, followed by a detailed overview of recent advances in the development of in vivo sensing platforms and portable sensors incorporating DNAzymes for molecular recognition. Finally, a critical perspective on the field, and a summary of where DNAzyme-based devices may make the biggest impact are provided.

Colorimetric assays typically offer a rapid and convenient method to assess analytes that span healthcare monitoring to water quality testing. However, such tests can only provide qualitative results when employed in resource-limited settings or require bulky and expensive equipment such as lab spectrophotometers to allow quantitative measurements. In this paper, we report on the use of a handheld colorimeter to quantitatively determine the concentration of analytes in a manner that is independent of ambient lighting or initial sample color. The method combines the response of the sensor with first-principles modeling that better describes the nature of the assay compared to linear-in-parameters regression modeling that is typically performed in other studies. This method was successfully demonstrated using a number of colorimetric assays: (1) determination of solution pH using a universal indicator, (2) quantification of the DNase presence using a DNA-gold nanoparticle assay, and (3) quantification of the concentration of the antibiotic tetracycline using a cell-based assay., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

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., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

DNAzymes are synthetic functional nucleic acids that have found widespread use in biosensing applications both for molecular recognition and signal generation. Two classes of DNAzymes have proved particularly effective for use in proof-of-concept biosensing systems, namely RNA-cleaving DNAzymes (RCDs) and peroxidase mimicking DNAzymes (PMDs). RCDs catalyze the site-specific cleavage reaction of an RNA dinucleotide junction, generating two cleavage fragments. PMDs are capable of catalyzing peroxidation reactions of chromophores, thereby generating a measurable signal. Herein, we review the use of these DNAzymes in biomedical assays and diagnostics, and show that this emerging field should have great promise for biosensor development over the next few decades., (Copyright © 2021 Elsevier B.V. All rights reserved.)

φ29 DNA polymerase (Polφ29) is capable of synthesizing long-chain single-stranded (ss) DNA molecules by copying the sequence of a small ss circular DNA template (ssCDT) in a process known as rolling circle amplification (RCA). The use of a ssCDT in RCA, however, comes with a key drawback: the rate of DNA synthesis is significantly reduced. We hypothesize that this issue can be overcome using a very long linear ssDNA template with a repeating sequence. To test this idea, we engineered a DNA assembly, which we denote "micrometer-sized DNA track" (μDT). This μDT, with an average length of ≈13.5 μm, is made of a long chain DNA with a primer-binding domain at its 3' end and ≈1000 repeating sequence units at its 5' end, each carrying a DNA anchor. We find that Polφ29 copies μDT at a speed ≈5-time faster than it does a related ssCDT. We use this to design a simple all-in-one printed paper device for rapid and sensitive detection of microRNA let-7. This paper sensor is capable of detecting 1 pM let-7a in 10 minutes., (© 2020 Wiley-VCH GmbH.)

We report on a programmable all-DNA biosensing system that centers on the use of a 4-way junction (4WJ) to transduce a DNAzyme reaction into an amplified signal output. A target acts as a primary input to activate an RNA-cleaving DNAzyme, which then cleaves an RNA-containing DNA substrate that is designed to be a component of a 4WJ. The formation of the 4WJ controls the release of a DNA output that becomes an input to initiate catalytic hairpin assembly (CHA), which produces a second DNA output that controls assembly of a split G-quadruplex as a fluorescence signal generator. The 4WJ can be configured to produce either a turn-off or turn-on switch to control the degree of CHA, allowing target concentration to be determined in a quantitative manner. We demonstrate this approach by creating a sensor for E. coli that could detect as low as 50 E. coli cells mL -1 within 85 min and offers an amplified bacterial detection method that does not require a protein enzyme., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Protein biomarkers often exist as degradation fragments in biological samples, and affinity agents derived using a purified protein may not recognize them, limiting their value for clinical diagnosis. Herein, we present a method to overcome this issue, by selecting aptamers against a degraded form of the toxin B protein, which is a marker for diagnosing toxigenic Clostridium difficile infections. This approach has led to isolation of a DNA aptamer that recognizes degraded toxin B, fresh toxin B, and toxin B spiked into human stool samples. DNA aptamers selected using intact recombinant toxin B failed to recognize degraded toxin B, which is the form present in stored stool samples. Using this new aptamer, we produced a simple paper-based analytical device for colorimetric detection of toxin B in stool samples, or in the NAP1 strain of Clostridium difficile. The combined aptamer-selection and paper-sensing strategy can expand the practical utility of DNA aptamers in clinical diagnosis., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We report a method to detect proteins via suppression of rolling circle amplification (RCA) by using an appropriate aptamer as the linear primer (denoted as an aptaprimer) to initiate RCA. In the absence of a protein target, the aptaprimer is free to initiate RCA, which can produce long DNA products that are detected via binding of a fluorescent intercalating dye. Introduction of a target causes the primer region within the aptamer to become unavailable for binding to the circular template, inhibiting RCA. Using SYBR Gold or QuantiFluor dyes as fluorescent probes to bind to the RCA reaction product, it is possible to produce a generic protein-modulated RCA assay system that does not require fluorophore- or biotin-modified DNA species, substantially reducing complexity and cost of reagents. Based on this modulation of RCA, we demonstrate the ability to produce both solution and paper-based assays for rapid and quantitative detection of proteins including platelet derived growth factor and thrombin., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Screening for a deficiency of glucose-6-phosphate dehydrogenase (G6PD) in red blood cells is vital for determining the potentially life-threatening presence of congenital, hereditary or induced hemolytic anemias. In this study, a "sample-to-readout" paper-based point-of-care (POC) colorimetric biosensor was developed for direct detection of G6PD in whole blood by simple visual comparison to a color card. The G6PD paper sensor was highly stable with no observable loss in performance after room temperature storage for at least 6 weeks, and worked equally well at room temperature and 37 °C. The simple printed paper format and the stability of the colorimetric reagents facilitates scalable manufacturing. The ability to utilize well established sample collection and preparation protocols along with a colorimetric visual readout should facilitate future transfer of this proof-of-concept POC biosensor to remote or resource-poor locations.

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., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We report on the first effort to select DNA aptamers from a circular DNA library, which resulted in the discovery of two high-affinity circular DNA aptamers that recognize the glutamate dehydrogenase (GDH) from Clostridium difficile, an established antigen for diagnosing Clostridium difficile infection (CDI). One aptamer binds effectively in both the circular and linear forms, the other is functional only in the circular configuration. Interestingly, these two aptamers recognize different epitopes on GDH, demonstrating the advantage of selecting aptamers from circular DNA libraries. A sensitive diagnostic test was developed to take advantage of the high stability of circular DNA aptamers in biological samples and their compatibility with rolling circle amplification. This test is capable of identifying patients with active CDI using stool samples. This work represents a significant step forward towards demonstrating the practical utility of DNA aptamers in clinical diagnosis., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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 paper based litmus test has been developed using modulation of urease enzyme activity for detection of C-C mismatch single nucleotide polymorphisms (SNPs) by the naked eye. Urease is first inactivated with silver ions and printed onto paper microzones. Addition of DNA containing C-C mismatches reactivates urease via binding of Ag(I), allowing restoration of urease activity, hydrolysis of urea to produce ammonia, and an increase in pH, which is monitored colorimetrically using a pH indicator with a limit of detection of 11 nM DNA in 40 min. The assay system is easy to use, portable, and stable for at least 30 days at ambient temperature. To assess the versatility and practical application of the paper sensor, we used it to identify a G > C transversion present in human genomic DNA from a ductal carcinoma cell line, a mutation commonly found in breast cancer. We believe this new assay system has the potential to be a low-cost method for rapidly identifying DNA with the C-C mismatch SNP as a means of cancer screening in resource-limited areas.

Micrometer-sized functional nucleic acid (FNA) superstructures (denoted as 3D DNA) were examined as a unique class of biorecognition elements to produce highly functional bioactive paper surfaces. 3D DNA containing repeating sequences of either a DNA aptamer or DNAzyme was created from long-chain products of rolling circle amplification followed by salt aging. The resulting 3D DNA retained its original spherical shape upon inkjet printing and adhered strongly to the paper surface via physisorption. 3D DNA paper sensors showed resistance to degradation by nucleases, suppressed nonspecific protein adsorption, and provided a much higher surface density of functional DNA relative to monomeric FNAs, making such species ideally suited for development of paper-based biosensors., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We report a paper-based aptasensor platform that uses two reaction zones and a connecting bridge along with printed multifunctional bio/nano materials to achieve molecular recognition and signal amplification. Upon addition of analyte to the first zone, a fluorescently labelled DNA or RNA aptamer is desorbed from printed graphene oxide, rapidly producing an initial fluorescence signal. The released aptamer then flows to the second zone where it reacts with printed reagents to initiate rolling circle amplification, generating DNA amplicons containing a peroxidase-mimicking DNAzyme, which produces a colorimetric readout that can be read in an equipment-free manner or with a smartphone. The sensor was demonstrated using an RNA aptamer for adenosine triphosphate (a bacterial marker) and a DNA aptamer for glutamate dehydrogenase (Clostridium difficile marker) with excellent sensitivity and specificity. These targets could be detected in spiked serum or feacal samples, demonstrating the potential for testing clinical samples., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Technologies capable of linking DNA amplification to molecular recognition are very desirable for ultrasensitive biosensing applications. We have developed a simple but powerful isothermal DNA amplification method, termed DNAzyme feedback amplification (DFA), that is capable of relaying molecular recognition to exponential DNA amplification. The method incorporates both an RNA-cleaving DNAzyme (RCD) and rolling circle amplification (RCA) carried out by a special DNA polymerase using a circular DNA template. DFA begins with a stimulus-dependent RCA reaction, producing tandemly linked RCDs in long-chain DNA products. These RCDs cleave an RNA-containing DNA sequence to form additional primers that hybridize to the circular DNA molecule, giving rise to DNA assemblies that act as the new inputs for RCA. The RCA reaction and the cleavage event keep on feeding each other autonomously, resulting in exponential growth of repetitive DNA sequences that can be easily detected. This method can be used for the detection of both nucleic acid based targets and non-nucleic acid analytes. In this article, we discuss the conceptual framework of the feedback amplification approach, the essential features of this method as well as remaining challenges and possible solutions., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Rapid and accurate diagnosis of Clostridium difficile infections (CDI) is crucial for patient treatment, infection control and epidemiological monitoring. As an important antigen, glutamate dehydrogenase (GDH) has been proposed as a preliminary screening test target for CDI. However, current assays based on GDH activity or GDH immunoassays have suboptimal sensitivity and specificity. Herein, we describe the selection and characterization of single-stranded DNA aptamers that specifically target GDH. After 10 rounds of selection, high-throughput sequencing was used to identify enriched aptamer candidates. Of 10 candidates, three aptamers for GDH were identified. Gel shift assays showed that these aptamers exhibited low nanomolar affinities. One aptamer was optimized based on structural analysis and further engineered into a structure-switching fluorescence signaling aptamer, wherein desorption from reduced graphene oxide (RGO) upon binding of GDH led to an increase in fluorescence emission. This method allowed for quantitative detection of GDH with a detection limit of 1 nM, providing great potential for its further application in CDI diagnosis., (Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

We present a simple all-in-one paper-based sensor for E. coli detection using a composite ink made of a fluorogenic DNAzyme probe for bacterial recognition and signal generation, lysozyme that lyses whole bacterial cells, and pullulan/trehalose sugars that stabilize printed bioactive molecules. The paper sensor is capable of producing a fluorescence signal as a readout within 5 minutes upon contacting E. coli, can achieve a limit of detection of 100 cells/mL, in a variety of sample matrixes, without sample enrichment, and remains stable for at least 6 months when stored at ambient temperature. Therefore, this simple paper sensor provides rapid bacterial testing on site, and can be shipped and stored under ambient conditions to benefit users living in resource-limited regions.

Sol-gel-derived bio/inorganic hybrid materials have been examined for diverse applications, including biosensing, affinity chromatography and drug discovery. However, such materials have mostly been restricted to the interaction between entrapped biorecognition elements and small molecules, owing to the requirement for nanometer-scale mesopores in the matrix to retain entrapped biorecognition elements. Herein, we report on a new class of macroporous bio/inorganic hybrids, engineered through a high-throughput materials screening approach, that entrap micron-sized concatemeric DNA aptamers. We demonstrate that the entrapment of these long-chain DNA aptamers allows their retention within the macropores of the silica material, so that aptamers can interact with high molecular weight targets such as proteins. Our approach overcomes the major limitation of previous sol-gel-derived biohybrid materials by enabling molecular recognition for targets beyond small molecules., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We report a signal amplification strategy termed DNAzyme feedback amplification (DFA) that takes advantage of rolling-circle amplification (RCA) and an RNA-cleaving DNAzyme (RCD). DFA employs two specially programmed DNA complexes, one composed of a primer and a circular template containing the antisense sequence of an RCD, and the other composed of the same circular template and an RNA-containing substrate for the RCD. RCA is initiated at the first complex to produce RCD elements that go on to cleave the substrate in the second complex. This cleavage event triggers the production of more input complexes for RCA. This reaction circuit continues autonomously, resulting in exponential DNA amplification. We demonstrate the versatility of this approach for biosensing through the design of DFA systems capable of detecting a microRNA sequence and a bacterium, with sensitivity improvements of 3-6 orders of magnitude over conventional methods., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We describe a versatile and simple method to perform sequential reactions on paper analytical devices by stacking dry pullulan films on paper, where each film contains one or more reagents or acts as a delay layer. Exposing the films to an aqueous solution of the analyte leads to sequential dissolution of the films in a temporally controlled manner followed by diffusive mixing of the reagents, so that sequential reactions can be performed. The films can be easily arranged for lateral flow assays or for spot tests (reactions take place sequentially in the z-direction). We have tested the general feasibility of the approach using three different model systems to demonstrate different capabilities: 1) pH ramping from low to high and high to low to demonstrate timing control; 2) rapid ready-to-use two-step Simon's assays on paper for detection of drugs of abuse utilizing a 2-layer stack containing two different reagents to demonstrate the ability to perform assays in the z-direction; and 3) sequential cell lysing and colorimetric detection of an intracellular bacterial enzyme, to demonstrate the ability of the method to perform sample preparation and analysis in the form of a spot assay. Overall, these studies demonstrate the potential of stacked pullulan films as useful components to enable multi-step assays on simple paper-based devices.

With over 4.8 million Canadians suffering from chronic airway diseases, respiratory exacerbations are currently the leading cause of hospitalization in Canada. In cases of bacterial infection, neutrophil cell density increases from ∼10 million cells per gram to over 15 million cells per gram. As sputum is a direct discharge from the primarily affected areas of respiratory diseases, quantification of granulocytes (including neutrophils) can be used to effectively determine a course of patient treatment. Unfortunately this quantification is currently limited to labour-intensive and time-consuming cell counts. In the present study, we describe a simple one-step lateral flow test (LFT) that can semi-quantitatively determine myeloperoxidase (MPO), a biomarker found in neutrophils, in minimally-processed sputum samples. This point-of-need (PON) diagnostic device provides positive results observable to the naked eye after 15 minutes. 37 human sputum samples were quantified for MPO using the developed LFT and compared to neutrophil levels quantified through traditional cell counting. A trend between sputum MPO concentration and total neutrophils was observed, suggesting that the LFT has the potential to replace cell counting for neutrophil approximation to aid in directing therapies quickly at the point of need.

Many rationally engineered DNA nanostructures use mechanically interlocked topologies to connect individual DNA components, and their physical connectivity is achieved through the formation of a strong linking duplex. The existence of such a structural element also poses a significant topological constraint on functions of component rings. Herein, we hypothesize and confirm that DNA catenanes with a strong linking duplex prevent component rings from acting as the template for rolling circle amplification (RCA). However, by using an RNA-containing DNA [2] catenane with a strong linking duplex, we show that a stimuli-responsive RNA-cleaving DNAzyme can linearize one component ring, and thus enable RCA, producing an ultra-sensitive biosensing system. As an example, a DNA catenane biosensor is engineered to detect the model bacterial pathogen Escherichia coli through binding of a secreted protein, with a detection limit of 10 cells ml(-1), thus establishing a new platform for further applications of mechanically interlocked DNA nanostructures.

Many biodetection systems employ labile enzymes and substrates that need special care, making it hard to routinely use them for point-of-care or field applications. In this work we provide a simple solution to this challenging problem through the creation of all-inclusive pullulan assay tablets. The proposed tablet system not only enhances the long-term stability of both enzymes and organic substrates, but also simplifies the assay procedure. The enhanced stability is attributed to two factors: the restriction of the molecular motion of proteins and impermeability to molecular oxygen afforded by the tables. These tablets dissolve rapidly upon addition to testing samples, making the test very easy to perform. Using the ATP-detecting luciferase-luciferin system as an example, we show that the tablet-based assay can achieve highly sensitive detection of ATP in biological samples and that the activity of the assay tablets remains unchanged for over a month at room temperature.

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., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The utility of hydrophobic wax barriers in paper-based lateral flow and multiwell devices for containment of aqueous solvents was extended to organic solvents and challenging aqueous surfactant solutions by preparation of a three layer barrier, consisting of internal pullulan impregnated paper barriers surrounded by external wax barriers. When paper impregnated with pullulan solution dries, the polymer forms solvent blocking lenses in the paper structure. Lens formation was illustrated by forming pullulan lenses in glass capillaries. The lens shapes were less curved compared to the predictions of a model based upon minimizing surface area. For barriers on Whatman # 1 filter paper, the pullulan molecular weight must be greater than ∼70 kDa, the mass fraction of pullulan in the barrier zone must be at least 32%, and there are restrictions on the minimum width of the pullulan impregnated zone.

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.

ϕ29 DNA polymerase (ϕ29DP) is able to carry out repetitive rounds of DNA synthesis using a circular DNA template by rolling circle amplification (RCA). It also has the ability to execute 3'-5' digestion of single-stranded but not double-stranded DNA. A biosensor engineering strategy is presented that takes advantage of these two properties of ϕ29DP coupled with structure-switching DNA aptamers. The design employs a DNA assembly made of a circular DNA template, a DNA aptamer, and a pre-primer. The DNA assembly is unable to undergo RCA in the absence of cognate target owing to the formation of duplex structures. The presence of the target, however, triggers a structure-switching event that causes nucleolytic conversion of the pre-primer by ϕ29DP into a mature primer to facilitate RCA. This method relays target detection by the aptamer to the production of massive DNA amplicons, giving rise to dramatically enhanced detection sensitivity., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

There is growing interest in developing printable paper sensors to enable rapid testing of analytes for environmental, food safety, and clinical applications. A major challenge is to find suitable bioinks that are amenable to high-speed printing and remain functional after printing. We report on a simple and effective approach wherein an aqueous ink composed of megadalton-sized tandem repeating structure-switching DNA aptamers (concatemeric aptamers) is used to rapidly create patterned paper sensors on filter paper by inkjet printing. These concatemeric aptamer reporters remain immobilized at the point of printing through strong adsorption but retain sufficient segmental mobility to undergo structure switching and fluorescence signaling to provide both qualitative and quantitative detection of small molecules and protein targets. The convenience of inkjet printing allows for the patterning of internally referenced sensors with multiplexed detection, and provides a generic platform for on-demand printing of sensors even in remote locations., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)