An Aptamer-Based Hydrogel Sensing Platform Enabling Low Micromolar Detection of Small Molecules Using Low Sample Volumes.

Poor fluorescence recovery at low analyte dosages and slow ligand binding kinetics are critical challenges currently limiting the use of aptamer-functionalized hydrogels for sensing small molecules. In this paper, we report an adenosine-responsive hydrogel sensor that integrates FRET-signaling aptamer switches into in situ-gelling thin-film hydrogels. The hydrogel sensor is able to entrap a high proportion of the sensing probes (>70% following vigorous washing), delay nucleolytic degradation, stabilize weak aptamer complexes to improve hybridization affinity and suppress fluorescence background, and provide high sensitivity in biological fluids (i.e., undiluted human serum). Furthermore, the developed hydrogel sensors were able to achieve low limits of detection (5.3 µM in buffer and 8.8 µM in serum) within 4 min of exposure to the sample, with signal generation requiring only 20 µL/well of analyte sample. The physical nature of the aptamer encapsulation allows this approach to accommodate virtually any small-molecule aptamer, avoiding the need for covalent anchoring and the complex modification of nucleic acid sequences typically required for effective aptamer-based molecular recognition.

Comprehensive fluorescence profiles of contamination-prone foods applied to the design of microcontact-printed in situ functional oligonucleotide sensors.

With both foodborne illness and food spoilage detrimentally impacting human health and the economy, there is growing interest in the development of in situ sensors that offer real-time monitoring of food quality within enclosed food packages. While oligonucleotide-based fluorescent sensors have illustrated significant promise, the development of such on-food sensors requires consideration towards sensing-relevant fluorescence properties of target food products-information that has not yet been reported. To address this need, comprehensive fluorescence profiles for various contamination-prone food products are established in this study across several wavelengths and timepoints. The intensity of these food backgrounds is further contextualized to biomolecule-mediated sensing using overlaid fluorescent oligonucleotide arrays, which offer perspective towards the viability of distinct wavelengths and fluorophores for in situ food monitoring. Results show that biosensing in the Cyanine3 range is optimal for all tested foods, with the Cyanine5 range offering comparable performance with meat products specifically. Moreover, recognizing that mass fabrication of on-food sensors requires rapid and simple deposition of sensing agents onto packaging substrates, RNA-cleaving fluorescent nucleic acid probes are successfully deposited via microcontact printing for the first time. Direct incorporation onto food packaging yields cost-effective sensors with performance comparable to ones produced using conventional deposition strategies.

Material Breakthroughs in Smart Food Monitoring: Intelligent Packaging and On-Site Testing Technologies for Spoilage and Contamination Detection.

Despite extensive commercial and regulatory interventions, food spoilage and contamination continue to impose massive ramifications on human health and the global economy. Recognizing that such issues will be significantly eliminated by the accurate and timely monitoring of food quality markers, smart food sensors have garnered significant interest as platforms for both real-time, in-package food monitoring and on-site commercial testing. In both cases, the sensitivity, stability, and efficiency of the developed sensors are largely informed by underlying material design, driving focus toward the creation of advanced materials optimized for such applications. Herein, a comprehensive review of emerging intelligent materials and sensors developed in this space is provided, through the lens of three key food quality markers - biogenic amines, pH, and pathogenic microbes. Each sensing platform is presented with targeted consideration toward the contributions of the underlying metallic or polymeric substrate to the sensing mechanism and detection performance. Further, the real-world applicability of presented works is considered with respect to their capabilities, regulatory adherence, and commercial potential. Finally, a situational assessment of the current state of intelligent food monitoring technologies is provided, discussing material-centric strategies to address their existing limitations, regulatory concerns, and commercial considerations.

Miniaturization of an enclosed electrospinning process to enhance reproducibility in the fabrication of rapidly dissolving cell-based biosensors.

There is broad interest in producing electrospun films embedded with biological materials. It is well known that electrospinning requires careful control of the process conditions, especially the environmental conditions such as relative humidity (RH). Given that commercial electrospinning systems are expensive (> $10,000) and are typically too large to be used in standard biological safety cabinets (BSC), we designed and built a miniaturized electrospinning box (E-Box) that will fit inside a BSC, and the RH can be easily controlled using simple instrumentation (gas cylinder, regulator, needle valve, rotameter). It uses an inexpensive computerized numerical control machine to control the spinneret positioning and collector rotational speed-all the parts for the device (except the syringe pump and voltage supply) can be purchased for approximately $1000. We demonstrate the usefulness of our design in optimizing the production of Escherichia coli-embedded pullulan-trehalose films to be used as rapidly dissolving biosensors for environmental monitoring. At a fixed electrospinning recipe, we showed that decreasing the RH from approximately 48% to 22% resulted in the average fiber diameter increasing from 240 (± 11) nm to 314 (± 8) nm. We also demonstrate the usefulness of our design in performing sequential electrospinning experiments to evaluate process performance reproducibility. For example, from just 1 mL of a polymer solution, we produced 16 electrospun films (approximately 3 cm by 8 cm each)-from those films we hole-punched approximately 80 biosensor discs which were then used in subsequent experiments to determine the amount of two different biocides (Grotan BK and triclosan) in aqueous samples. The technique developed in this study is ideal for creating electrospun materials in high quantities that are highly reproducible through the precise control of RH.

Advancing In Situ Food Monitoring through a Smart Lab-in-a-Package System Demonstrated by the Detection of Salmonella in Whole Chicken.

With food production shifting away from traditional farm-to-table approaches to efficient multistep supply chains, the incidence of food contamination has increased. Consequently, pathogen testing via inefficient culture-based methods has increased, despite its lack of real-time capabilities and need for centralized facilities. While in situ pathogen detection would address these limitations and enable individual product monitoring, accurate detection within unprocessed, packaged food products without user manipulation has proven elusive. Herein, "Lab-in-a-Package" is presented, a platform capable of sampling, concentrating, and detecting target pathogens within closed food packaging, without intervention. This system consists of a newly designed packaging tray and reagent-infused membrane that can be paired universally with diverse pathogen sensors. The inclined food packaging tray maximizes fluid localization onto the sensing interface, while the membrane acts as a reagent-immobilizing matrix and an antifouling barrier for the sensor. The platform is substantiated using a newly discovered Salmonella-responsive nucleic acid probe, which enables hands-free detection of 103 colony forming units (CFU) g-1 target pathogen in a packaged whole chicken. The platform remains effective when contamination is introduced with toolsand surfaces, ensuring widespread efficacy. Its real-world use for in situ detection is simulated using a handheld fluorescence scanner with smartphone connectivity.

Tuning the sensitivity of cell-based biosensors for the detection of biocides.

The presence of biocides in wastewater can negatively impact the efficiency of wastewater treatment processes, particularly the process of nitrification. In this paper, we describe the development of cell-based biosensors (CBBs) with tunable levels of sensitivity for rapidly detecting the presence and predicting the type and concentration of biocides. The CBB assay developed is performed by first exposing a panel of bacterial strains (E. coli, B. subtilis, B. cereus) to the sample being tested and to the control sample without biocide, and then adding a fluorescent dye (LIVE/DEAD BacLight). We then compare the fluorescence signals generated by the two samples, and the differences in the signals indicate the presence of a biocide, as previously reported in the literature. We found that the sensitivity of the CBB assay can be improved by 'tuning' the type/salinity of the buffer used to suspend the cells, and by changing the number of cells used in the assay. These changes improved the level of detection (LOD) of the biocide Cetyltrimethylammonium bromide (CTAB) from 10 ppm to 0.625 ppm and the biocide Grotan® BK from 500 ppm to 7.8 ppm. With the optimized conditions for each strain, we also establish that the combined response from the panel of bacterial strains can be used to predict the type and concentration of biocide sample tested. Additionally, we provide evidence that the CBB assay can be performed using a compact, commercially available fluorometer. Overall, the significance of this work will improve point-of-use testing and enable the discrimination between biocide-containing samples of similar toxicity and detection of lower toxicity samples, thereby improving the accuracy of the CBB assay.

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.

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.

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.

Polyvinylamine with moderate binding affinity as a highly effective vehicle for RNA delivery.

Polymeric carriers for RNA therapy offer potential advantages in terms of low immunogenicity, promoting modifiability and accelerating intracellular transport. However, balancing high transfection efficacy with low toxicity remains challenging with polymer-based vehicles; indeed, polyethyleneimine (PEI) remains the "gold standard" polymer for this purpose despite its significant toxicity limitations. Herein, we demonstrate the potential of polyvinylamine (PVAm), a commodity high-charge cationic polymer used in the papermaking industry and has similar structure with PEI, as an alternative carrier for RNA delivery. High levels of transfection of normal, tumor, and stem cells with a variety of RNA cargoes including small interfering RNA (siRNA), microRNA (miRNA), and recombinant RNA can be achieved in vitro under the proper complex conditions. While, both the anti-tumor effect achieved in a xenograft osteosarcoma model and lipid-lowering activity observed in a hyperlipidemia mice indicate the potential for highly effective in vivo activity. Of note, both the transfection efficiency and the cytotoxicity of PVAm compare more favorably with those of PEI, with PVAm offering the additional advantages of simpler purification and significantly lower cost. In addition, the mechanism for the difference in transfection efficiency between PVAm and PEI is explored by molecular docking as well as analyzing the process of association and dissociation between polymers (PVAm and PEI) and nucleic acids. Our research provides a novel, non-toxic, and cost-effective carrier candidate for next generation RNA therapy, and elucidates the potential mechanism of PVAm for its efficient delivery of RNA.

Investigation of discordant SARS-CoV-2 RT-PCR results using minimally processed saliva.

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 106 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.

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.

LISzyme Biosensors: DNAzymes Embedded in an Anti-biofouling Platform for Hands-free Real-Time Detection of Bacterial Contamination in Milk.

Nonspecific binding is a significant challenge associated with biosensors in complex food textures. To overcome this, we have developed LISzymes, which are DNAzymes incorporated in lubricant-infused surfaces (LISs). Using milk as a complex background matrix, we show that LISzyme biosensors are significantly more effective in preventing nonspecific binding compared to other commonly used "blocking" methods. The use of lubricant infusion to treat sensing surfaces results in a 4-fold increase in the signal-to-noise ratio obtained with the DNAzyme with respect to untreated surfaces, when detecting the presence of specific bacteria in milk. This is a striking improvement upon previous DNAzyme sensors. We also show that the use of LISs does not affect the DNAzyme's ability to effectively and specifically detect its target─a protein specifically produced by Escherichia coli (E. coli), in a complex sample matrix such as milk. LISzymes drastically improve DNAzyme performance, resulting in target detection associated with E. coli at concentrations as low as 250 CFU/mL in milk in less than an hour, which is currently not possible using other optical platforms. LISzymes are promising tools for the real-time monitoring of food contamination and may prove valuable within many other biosensing applications.

Quantitative Point-of-Care Colorimetric Assay Modeling Using a Handheld Colorimeter.

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.

DNAzyme-Based Biosensors: Immobilization Strategies, Applications, and Future Prospective.

Since their discovery almost three decades ago, DNAzymes have been used extensively in biosensing. Depending on the type of DNAzyme being used, these functional oligonucleotides can act as molecular recognition elements within biosensors, offering high specificity to their target analyte, or as reporters capable of transducing a detectable signal. Several parameters need to be considered when designing a DNAzyme-based biosensor. In particular, given that many of these biosensors immobilize DNAzymes onto a sensing surface, selecting an appropriate immobilization strategy is vital. Suboptimal immobilization can result in both DNAzyme detachment and poor accessibility toward the target, leading to low sensing accuracy and sensitivity. Various approaches have been employed for DNAzyme immobilization within biosensors, ranging from amine and thiol-based covalent attachment to non-covalent strategies involving biotin-streptavidin interactions, DNA hybridization, electrostatic interactions, and physical entrapment. While the properties of each strategy inform its applicability within a proposed sensor, the selection of an appropriate strategy is largely dependent on the desired application. This is especially true given the diverse use of DNAzyme-based biosensors for the detection of pathogens, metal ions, and clinical biomarkers. In an effort to make the development of such sensors easier to navigate, this paper provides a comprehensive review of existing immobilization strategies, with a focus on their respective advantages, drawbacks, and optimal conditions for use. Next, common applications of existing DNAzyme-based biosensors are discussed. Last, emerging and future trends in the development of DNAzyme-based biosensors are discussed, and gaps in existing research worthy of exploration are identified.

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.

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.

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.

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.