Fighting Mutations with Mutations: Evolutionarily Adapting a DNA Aptamer for High-Affinity Recognition of Mutated Spike Proteins of SARS-CoV-2.

An on-going challenge with COVID-19, which has huge implications for future pandemics, is the rapid emergence of viral variants that makes diagnostic tools less accurate, calling for rapid identification of recognition elements for detecting new variants caused by mutations. We hypothesize that we can fight mutations of the viruses with mutations of existing recognition elements. We demonstrate this concept via rapidly evolving an existing DNA aptamer originally selected for the spike protein (S-protein) of wildtype SARS-CoV-2 to enhance the interaction with the same protein of the Omicron variants. The new aptamer, MBA5SA1, has acquired 22 mutations within its 40-nucleotide core sequence and improved its binding affinity for the S-proteins of diverse Omicron subvariants by > 100-fold compared to its parental aptamer (improved from nanomolar to picomolar affinity). Deep sequencing analysis reveals dynamic competitions among several MBA5SA1 variants in response to increasing selection pressure imposed during in vitro selection, with MBA5SA1 being the final winner of the competition. Additionally, MBA5SA1 was implemented into an enzyme-linked aptamer binding assay (ELABA), which was applied for detecting Omicron variants in the saliva of infected patients. The assay produced a sensitivity of 86.5% and a specificity of 100%, which was established with 83 clinical samples.

High-Precision Viral Detection Using Electrochemical Kinetic Profiling of Aptamer-Antigen Recognition in Clinical Samples and Machine Learning.

High-precision viral detection at point of need with clinical samples plays a pivotal role in the diagnosis of infectious diseases and the control of a global pandemic. However, the complexity of clinical samples that often contain very low viral concentrations makes it a huge challenge to develop simple diagnostic devices that do not require any sample processing and yet are capable of meeting performance metrics such as very high sensitivity and specificity. Herein we describe a new single-pot and single-step electrochemical method that uses real-time kinetic profiling of the interaction between a high-affinity aptamer and an antigen on a viral surface. This method generates many data points per sample, which when combined with machine learning, can deliver highly accurate test results in a short testing time. We demonstrate this concept using both SARS-CoV-2 and Influenza A viruses as model viruses with specifically engineered high-affinity aptamers. Utilizing this technique to diagnose COVID-19 with 37 real human saliva samples results in a sensitivity and specificity of both 100 % (27 true negatives and 10 true positives, with 0 false negative and 0 false positive), which showcases the superb diagnostic precision of this method.

An Omniphobic Spray Coating Created from Hierarchical Structures Prevents the Contamination of High-Touch Surfaces with Pathogens.

Engineered surfaces that repel pathogens are of great interest due to their role in mitigating the spread of infectious diseases. A robust, universal, and scalable omniphobic spray coating with excellent repellency against water, oil, and pathogens is presented. The coating is substrate-independent and relies on hierarchically structured polydimethylsiloxane (PDMS) microparticles, decorated with gold nanoparticles (AuNPs). Wettability studies reveal the relationship between surface texturing of micro- and/or nano-hierarchical structures and the omniphobicity of the coating. Studies of pathogen transfer with bacteria and viruses reveal that an uncoated contaminated glove transfers pathogens to >50 subsequent surfaces, while a coated glove picks up 104 (over 99.99%) less pathogens upon first contact and transfers zero pathogens after the second touch. The developed coating also provides excellent stability under harsh conditions. The remarkable anti-pathogen properties of this surface combined with its ease of implementation, substantiate its use for the prevention of surface-mediated transmission of pathogens.

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.

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.

Transparent and Highly Flexible Hierarchically Structured Polydimethylsiloxane Surfaces Suppress Bacterial Attachment and Thrombosis Under Static and Dynamic Conditions.

The surface fouling of biomedical devices has been an ongoing issue in healthcare. Bacterial and blood adhesion in particular, severely impede the performance of such tools, leading to poor patient outcomes. Various structural and chemical modifications have been shown to reduce fouling, but all existing strategies lack the combination of physical, chemical, and economic traits necessary for widespread use. Herein, a lubricant infused, hierarchically micro- and nanostructured polydimethylsiloxane surface is presented. The surface is easy to produce and exhibits the high flexibility and optical transparency necessary for incorporation into various biomedical tools. Tests involving two clinically relevant, priority pathogens show up to a 98.5% reduction in the biofilm formation of methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa. With blood, the surface reduces staining by 95% and suppresses thrombin generation to background levels. Furthermore, the surface shows applicability within applications such as catheters, extracorporeal circuits, and microfluidic devices, through its effectiveness in dynamic conditions. The perfusion of bacterial media shows up to 96.5% reduction in bacterial adhesion. Similarly, a 95.8% reduction in fibrin networks is observed following whole blood perfusion. This substrate stands to hold high applicability within biomedical systems as a means to prevent fouling, thus improving performance.

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.

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.

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.

Development of Nucleic-Acid-Based Electrochemical Biosensors for Clinical Applications.

Nucleic acids are remarkable molecules. In addition to Watson-Crick base pairing, the different structural motifs of these molecules can bind non-nucleic acid targets or catalyze chemical reactions. Additionally, nucleic acids are easily modified with different molecules or functional groups. These properties make nucleic acids, particularly DNA, ideally suited for use in electrochemical biosensors, both as biorecognition elements and redox reporter probes. In this Minireview, we will review the historical evolution of nucleic acids as probes in electrochemical biosensors. We will then review the specific examples of nucleic-acid-based biosensors that have been evaluated for clinical use in the areas of infectious disease, cancer, or cardiovascular health.

A DNA Barcode-Based Aptasensor Enables Rapid Testing of Porcine Epidemic Diarrhea Viruses in Swine Saliva Using Electrochemical Readout.

Pen-side testing of farm animals for infectious diseases is critical for preventing transmission in herds and providing timely intervention. However, most existing pathogen tests have to be conducted in centralized labs with sample-to-result times of 2-4 days. Herein we introduce a test that uses a dual-electrode electrochemical chip (DEE-Chip) and a barcode-releasing electroactive aptamer for rapid on-farm detection of porcine epidemic diarrhea viruses (PEDv). The sensor exploits inter-electrode spacing reduction and active field mediated transport to accelerate barcode movement from electroactive aptamers to the detection electrode, thus expediting assay operation. The test yielded a clinically relevant limit-of-detection of 6 nM (0.37 µg mL-1 ) in saliva-spiked PEDv samples. Clinical evaluation of this biosensor with 12 porcine saliva samples demonstrated a diagnostic sensitivity of 83 % and specificity of 100 % with a concordance value of 92 % at an analysis time of one hour.

Two-Step Competitive Hybridization Assay: A Method for Analyzing Cancer-Related microRNA Embedded in Extracellular Vesicles.

With an increased understanding of the role of microRNAs (miRNAs) in cancer evolution, there is a growing interest in the use of these non-coding nucleic acids in cancer diagnosis, prognosis, and treatment monitoring. miRNAs embedded in extracellular vesicles (EVs) are of particular interest given that circulating EVs carry cargo that are strongly correlated to their cells of origin such as tumor cells while protecting them from degradation. As such, there is a tremendous interest in new simple-to-operate vesicular microRNA analysis tools for widespread use in performing liquid biopsies. Herein, we present a two-step competitive hybridization assay that is rationally designed to translate low microRNA concentrations to large electrochemical signals as the measured signal is inversely proportional to the microRNA concentration. Using this assay, with a limit-of-detection of 122 aM, we successfully analyzed vesicular miRNA 200b from prostate cancer cell lines and human urine samples, demonstrating the expected lower expression levels of miRNA 200b in the EVs from prostate cancer cells and in the prostate cancer patient's urine samples compared to healthy patients and non-tumorigenic cell lines, validating the suitability of our approach for clinical analysis.

Electron Microscopy Imaging Applications of Room Temperature Ionic Liquids in the Biological Field: A Review.

For biological imaging using electron microscopy (EM), the use of room-temperature ionic liquids (RTILs) has been proposed as an alternative to traditional lengthy preparation methods. With their low vapor pressures and conductivity, RTILs can be applied onto hard-to-image soft and/or wet samples without dehydration - allowing for a more representative, hydrated state of material and opening the possibility for visualization of in situ physiological processes using conventional EM systems. However, RTILs have yet to be utilized to their full potential by microscopists and microbiologists alike. To this end, this review aims to provide a comprehensive summary of biological applications of RTILs for EM to bridge the RTIL, in situ microscopy, and biological communities. We outline future research avenues for the use of RTILs for the EM observation of biological samples, notably i) RTIL selection and optimization, ii) applications for live cell processes and iii) electron beam and ionic liquid interaction studies.

Enhancing the Sensitivity of Photoelectrochemical DNA Biosensing Using Plasmonic DNA Barcodes and Differential Signal Readout.

Photoelectrochemical biosensors hold great promise for sensitive bioanalysis; however, similar to their electrochemical analogues, they are highly affected by the variable backgrounds caused by biological matrices. We developed a new PEC biosensing strategy that uses differential signal generation, combining signals from two separate but correlated binding events on the biosensor, for improving the limit-of-detection, sensitivity, and specificity of PEC DNA biosensors in biological samples. In this assay, the binding of unlabeled target DNA is followed by the capture of a signal amplification barcode featuring a plasmonic nanoparticle. The interaction of the plasmonic barcode with the semiconductive building blocks of the biosensor results in significant signal amplification, and together with differential signal processing enhances the limit of detection and sensitivity of the assay by up to 15- and three-fold, respectively, compared to the previously-used PEC assays with a single binding event, demonstrating a limit of detection of 3 fM.

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.

Hierarchical Structures, with Submillimeter Patterns, Micrometer Wrinkles, and Nanoscale Decorations, Suppress Biofouling and Enable Rapid Droplet Digitization.

Liquid repellant surfaces have been shown to play a vital role for eliminating thrombosis on medical devices, minimizing blood contamination on common surfaces as well as preventing non-specific adhesion. Herein, an all solution-based, easily scalable method for producing liquid repellant flexible films, fabricated through nanoparticle deposition and heat-induced thin film wrinkling that suppress blood adhesion, and clot formation is reported. Furthermore, superhydrophobic and hydrophilic surfaces are combined onto the same substrate using a facile streamlined process. The patterned superhydrophobic/hydrophilic surfaces show selective digitization of droplets from various solutions with a single solution dipping step, which provides a route for rapid compartmentalization of solutions into virtual wells needed for high-throughput assays. This rapid solution digitization approach is demonstrated for detection of Interleukin 6. The developed liquid repellant surfaces are expected to find a wide range of applications in high-throughput assays and blood contacting medical devices.

Electron energy-loss spectroscopy of surface plasmon activity in wrinkled gold structures.

The surface plasmon response of a cross-sectional segment of a wrinkled gold film is studied using electron energy loss spectroscopy (EELS). EELS data demonstrate that wrinkled gold structures act as a suitable substrate for surface plasmons to propagate. The intense surface variations in these structures facilitate the resonance of a wide range of surface plasmons, leading to the broadband surface plasmon response of these geometries from the near-infrared to visible wavelengths. The metallic nanoparticle boundary element method toolbox is used to simulate plasmon eigenmodes in these structures. Eigenmode simulations show how the diverse morphology of the wrinkled structure leads to its high spectral complexity. Micron-sized structural features that do not provide interactions between segments of the wrinkle have only a small effect on the surface plasmon resonance response, whereas nanofeatures strongly affect the resonant modes of the geometry. According to eigenmode calculations, different eigenenergy shifts around the sharp folds contribute to the broadband response and infrared activity of these structures; these geometrical features also support higher energy (shorter wavelength) symmetric and anti-symmetric plasmon coupling across the two sides of the folds. It is also shown that additional plasmon eigenstates are introduced from hybridization of modes across nanogaps between structural features in close proximity to each other. All of these factors contribute to the broadband response of the wrinkled gold structures.

Dynamic Bio-Barcode Assay Enables Electrochemical Detection of a Cancer Biomarker in Undiluted Human Plasma: A Sample-In-Answer-Out Approach.

There is a need for biosensing systems that can be operated at the point-of-care (POC) for disease screening and diagnostics and health monitoring. In spite of this, simple to operate systems with the required analytical sensitivity and specificity in clinical samples, using a sample-in-answer-out approach, remain elusive. Reported here is an electrochemical bio-barcode assay (e-biobarcode assay) that integrates biorecognition with signal transduction using molecular (DNA/protein) machines and signal readout using nanostructured electrodes. The e-biobarcode assay eliminates multistep processing and uses a single step for analysis following sample collection into the reagent tube. A clinically relevant performance for the analysis of prostate specific antigen (PSA) in undiluted and unprocessed human plasma: a log-linear range of 1 ng mL-1 -200 ng mL-1 and a LOD of 0.4 ng mL-1 , was achieved. The e-biobarcode assay offers a realistic solution for biomarker analysis at the POC.

Liquid Cell Transmission Electron Microscopy Sheds Light on The Mechanism of Palladium Electrodeposition.

Electrodeposition is widely used to fabricate tunable nanostructured materials in applications ranging from biosensing to energy conversion. A model based on 3D island growth is widely accepted in the explanation of the initial stages of nucleation and growth in electrodeposition. However, there are regions in the electrodeposition parameter space where this model becomes inapplicable. We use liquid cell transmission electron microscopy along with post situ scanning electron microscopy to investigate electrodeposition in this parameter space, focusing on the effect of the supporting electrolyte, and to shed light on the nucleation and growth of palladium. Using a collection of electron microscopy images and current time transients recorded during electrodeposition, we discover that electrochemical aggregative growth, rather than 3D island growth, best describes the electrodeposition process. We then use this model to explain the change in the morphology of palladium electrodeposits from spherical to open clusters with nonspherical morphology when HCl is added to the electrolyte solution. The enhanced understanding of the early stages of palladium nucleation and growth and the role of electrolyte in this process provides a systematic route toward the electrochemical fabrication of nanostructured materials.