Wearable Aptalyzer Integrates Microneedle and Electrochemical Sensing for in Vivo Monitoring of Glucose and Lactate in live Animals.

Continuous monitoring of clinically relevant biomarkers within the interstitial fluid (ISF) using microneedle (MN)-based assays, has the potential to transform healthcare. This study introduces the Wearable Aptalyzer, an integrated system fabricated by combining biocompatible hydrogel microneedle (HMN) arrays for ISF extraction with an electrochemical aptamer-based biosensor for in situ monitoring of blood analytes. The use of aptamers enables continuous monitoring of a wide range of analytes, beyond what is possible with enzymatic monitoring. The Wearable Aptalyzer is used for real-time and multiplexed monitoring of glucose and lactate in ISF. Validation experiments using live mice and rat models of Type 1 Diabetes demonstrate strong correlation between the measurements collected from the Wearable Aptalyzer in ISF and those obtained from gold-standard techniques for blood glucose and lactate, for each analyte alone and in combination. The Wearable Aptalyzer effectively addresses the limitations inherent in enzymatic detection methods as well as solid MN biosensors and addresses the need for reliable and multiplexed bioanalytical monitoring in vivo. This article is protected by copyright. All rights reserved.

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.

Impact of palladium/palladium hydride conversion on electrochemical CO2 reduction via in-situ transmission electron microscopy and diffraction.

Electrochemical conversion of CO2 offers a sustainable route for producing fuels and chemicals. Pd-based catalysts are effective for converting CO2 into formate at low overpotentials and CO/H2 at high overpotentials, while undergoing poorly understood morphology and phase structure transformations under reaction conditions that impact performance. Herein, in-situ liquid-phase transmission electron microscopy and select area diffraction measurements are applied to track the morphology and Pd/PdHx phase interconversion under reaction conditions as a function of electrode potential. These studies identify the degradation mechanisms, including poisoning and physical structure changes, occurring in PdHx/Pd electrodes. Constant potential density functional theory calculations are used to probe the reaction mechanisms occurring on the PdHx structures observed under reaction conditions. Microkinetic modeling reveals that the intercalation of *H into Pd is essential for formate production. However, the change in electrochemical CO2 conversion selectivity away from formate and towards CO/H2 at increasing overpotentials is due to electrode potential dependent changes in the reaction energetics and not a consequence of morphology or phase structure changes.

Superomniphobic and Photoactive Surface Presents Antimicrobial Properties by Repelling and Killing Pathogens.

Healthcare-acquired infections place a significant burden on the cost and quality of patient care in hospitals. Reducing contamination on surfaces within healthcare environments is critical for halting the spread of these infections. Herein, we report a bifunctional─repel and kill─surface developed using photoactive TiO2 nanoparticles integrated into a hierarchical scaffold (OmniKill). To quantify the repellency of OmniKill, we developed a touch-based assay, capable of simulating the transfer of individual pathogens, multiple pathogens, or pathogen-latent fecal matter from hands to surfaces. OmniKill repels bacterial pathogens by at least 2.77-log (99.8%). The photoactive material within OmniKill further reduces the viability of transferred pathogens on the surface by an additional 2.43-log (99.6%) after 1 h of light exposure. The antipathogenic effects─repel and kill─remain robust under complex biological contaminates such as feces. These findings show the potential use of OmniKill in reducing the physical transmission of bacterial pathogens in healthcare settings.

A universal bacterial sensor created by integrating a light modulating aptamer complex with photoelectrochemical signal readout.

Photoelectrochemical (PEC) signal transduction is of great interest for ultrasensitive biosensing; however, signal-on PEC assays that do not require target labeling remain elusive. In this work, we developed a signal-on biosensor that uses nucleic acids to modulate PEC currents upon target capture. Target presence removes a biorecognition probe from a DNA duplex carrying a gold nanoparticle, bringing the gold nanoparticle in direct contact to the photoelectrode and increasing the PEC current. This assay was used to develop a universal bacterial detector by targeting peptidoglycan using an aptamer, demonstrating a limit-of-detection of 82 pg/mL (13 pM) in buffer and 239 pg/mL (37 pM) in urine for peptidoglycan and 1913 CFU/mL forEscherichia coliin urine. When challenged with a panel of unknown targets, the sensor identified samples with bacterial contamination versus fungi. The versatility of the assay was further demonstrated by analyzing DNA targets, which yielded a limit-of-detection of 372 fM.

Liquid NanoBiosensors Enable One-Pot Electrochemical Detection of Bacteria in Complex Matrices.

There is a need for point-of-care bacterial sensing and identification technologies that are rapid and simple to operate. Technologies that do not rely on growth cultures, nucleic acid amplification, step-wise reagent addition, and complex sample processing are the key for meeting this need. Herein, multiple materials technologies are integrated for overcoming the obstacles in creating rapid and one-pot bacterial sensing platforms. Liquid-infused nanoelectrodes are developed for reducing nonspecific binding on the transducer surface; bacterium-specific RNA-cleaving DNAzymes are used for bacterial identification; and redox DNA barcodes embedded into DNAzymes are used for binding-induced electrochemical signal transduction. The resultant single-step and one-pot assay demonstrates a limit-of-detection of 102 CFU mL-1 , with high specificity in identifying Escherichia coli amongst other Gram positive and negative bacteria including Klebsiella pneumoniae, Staphylococcus aureus, and Bacillus subtilis. Additionally, this assay is evaluated for analyzing 31 clinically obtained urine samples, demonstrating a clinical sensitivity of 100% and specify of 100%. When challenging this assay with nine clinical blood cultures, E. coli-positive and E. coli-negative samples can be distinguished with a probability of p < 0.001.

A Bifunctional Spray Coating Reduces Contamination on Surfaces by Repelling and Killing Pathogens.

Surface-mediated transmission of pathogens is a major concern with regard to the spread of infectious diseases. Current pathogen prevention methods on surfaces rely on the use of biocides, which aggravate the emergence of antimicrobial resistance and pose harmful health effects. In response, a bifunctional and substrate-independent spray coating is presented herein. The bifunctional coating relies on wrinkled polydimethylsiloxane microparticles, decorated with biocidal gold nanoparticles to induce a "repel and kill" effect against pathogens. Pathogen repellency is provided by the structural hierarchy of the microparticles and their surface chemistry, whereas the kill mechanism is achieved using functionalized gold nanoparticles embedded on the microparticles. Bacterial tests with methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa reveal a 99.9% reduction in bacterial load on spray-coated surfaces, while antiviral tests with Phi6─a bacterial virus often used as a surrogate to SARS-CoV-2─demonstrate a 98% reduction in virus load on coated surfaces. The newly developed spray coating is versatile, easily applicable to various surfaces, and effective against various pathogens, making it suitable for reducing surface contamination in frequently touched, heavy traffic, and high-risk surfaces.

Integrating Water Purification with Electrochemical Aptamer Sensing for Detecting SARS-CoV-2 in Wastewater.

Wastewater analysis of pathogens, particularly SARS-CoV-2, is instrumental in tracking and monitoring infectious diseases in a population. This method can be used to generate early warnings regarding the onset of an infectious disease and predict the associated infection trends. Currently, wastewater analysis of SARS-CoV-2 is almost exclusively performed using polymerase chain reaction for the amplification-based detection of viral RNA at centralized laboratories. Despite the development of several biosensing technologies offering point-of-care solutions for analyzing SARS-CoV-2 in clinical samples, these remain elusive for wastewater analysis due to the low levels of the virus and the interference caused by the wastewater matrix. Herein, we integrate an aptamer-based electrochemical chip with a filtration, purification, and extraction (FPE) system for developing an alternate in-field solution for wastewater analysis. The sensing chip employs a dimeric aptamer, which is universally applicable to the wild-type, alpha, delta, and omicron variants of SARS-CoV-2. We demonstrate that the aptamer is stable in the wastewater matrix (diluted to 50%) and its binding affinity is not significantly impacted. The sensing chip demonstrates a limit of detection of 1000 copies/L (1 copy/mL), enabled by the amplification provided by the FPE system. This allows the integrated system to detect trace amounts of the virus in native wastewater and categorize the amount of contamination into trace (<10 copies/mL), medium (10-1000 copies/mL), or high (>1000 copies/mL) levels, providing a viable wastewater analysis solution for in-field use.

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.

Electrochemical DNAzyme-based biosensors for disease diagnosis.

DNAzyme-based electrochemical biosensors provide exceptional analytical sensitivity and high target recognition specificity for disease diagnosis. This review provides a critical perspective on the fundamental and applied impact of incorporating DNAzymes in the field of electrochemical biosensing. Specifically, we highlight recent advances in creating DNAzyme-based electrochemical biosensors for diagnosing infectious diseases, cancer and regulatory diseases. We also develop an understanding of challenges around translating the research in the field of DNAzyme-based electrochemical biosensors from labs to clinics, followed by a discussion on different strategies that can be applied to enhance the performance of the currently existing technologies to create truly point-of-care electrochemical DNAzyme biosensors.

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.

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.

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.

DNAzyme-Immobilizing Microgel Magnetic Beads Enable Rapid, Specific, Culture-Free, and Wash-Free Electrochemical Quantification of Bacteria in Untreated Urine.

Rapid, ultrasensitive, and specific detection and identification of bacteria in unprocessed clinical specimens is critically needed to enable point-of-care diagnosis of infectious diseases. However, existing systems require sample processing and/or analyte enrichment for direct bacterial analysis in clinical samples, which significantly adds to the assay time and complexity. Herein, we integrate RNA-cleaving DNAzymes specific to Escherichia coli (E. coli) and programmed for electrochemical signal transduction, multifunctional microgel magnetic beads for immobilizing the DNAzyme into a hydrated and three-dimensional scaffold, and hierarchical electrodes for ultrasensitive electrochemical readout to achieve rapid bacterial analysis in undiluted and unprocessed urine collected from symptomatic patients suspected of having urinary tract infections (UTIs). The microgel magnetic bead assay enables highly efficient conjugation and hydration of the immobilized DNAzymes, resulting in low limits-of-detection of 6 CFU/mL in buffer and 138 CFU/mL in unprocessed urine with high specificity against multiple urinary pathogens within a 1 hour assay time. The assay successfully identifies which patients are infected with E. coli as the causative organism for their UTI symptoms, indicating the clinical relevance of this assay.

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.

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.

Pathogen-Repellent Plastic Warp with Built-In Hierarchical Structuring Prevents the Contamination of Surfaces with Coronaviruses.

Amidst the COVID-19 pandemic, it is evident that viral spread is mediated through several different transmission pathways. Reduction of these transmission pathways is urgently needed to control the spread of viruses between infected and susceptible individuals. Herein, we report the use of pathogen-repellent plastic wraps (RepelWrap) with engineered surface structures at multiple length scales (nanoscale to microscale) as a means of reducing the indirect contact transmission of viruses through fomites. To quantify viral repellency, we developed a touch-based viral quantification assay to mimic the interaction of a contaminated human touch with a surface through the modification of traditional viral quantification methods (viral plaque and TCID50 assays). These studies demonstrate that RepelWrap reduced contamination with an enveloped DNA virus as well as the human coronavirus 229E (HuCoV-229E) by more than 4 log 10 (>99.99%) compared to a standard commercially available polyethylene plastic wrap. In addition, RepelWrap maintained its repellent properties after repeated 300 touches and did not show an accumulation in viral titer after multiple contacts with contaminated surfaces, while increases were seen on other commonly used surfaces. These findings show the potential use of repellent surfaces in reducing viral contamination on surfaces, which could, in turn, reduce the surface-based spread and transmission.

Producing Fluorine- and Lubricant-Free Flexible Pathogen- and Blood-Repellent Surfaces Using Polysiloxane-Based Hierarchical Structures.

High-touch surfaces are known to be a major route for the spread of pathogens in healthcare and public settings. Antimicrobial coatings have, therefore, garnered significant attention to help mitigate the transmission of infectious diseases via the surface route. Among antimicrobial coatings, pathogen-repellent surfaces provide unique advantages in terms of safety in public settings such as instant repellency, affordability, biocompatibility, and long-term stability. While there have been many advances in the fabrication of biorepellent surfaces in the past two decades, this area of research continues to suffer challenges in scalability, cost, compatibility with high-touch applications, and performance for pathogen repellency. These features are critical for high-touch surfaces to be used in public settings. Additionally, the environmental impact of manufacturing repellent surfaces remains a challenge, mainly due to the use of fluorinated coatings. Here, we present a flexible hierarchical coating with straightforward and cost-effective manufacturing without the use of fluorine or a lubricant. Hierarchical surfaces were prepared through the growth of polysiloxane nanostructures using n-propyltrichlorosilane (n-PTCS) on activated polyolefin (PO), followed by heat shrinking to induce microscale wrinkles. The developed coatings demonstrated repellency, with contact angles over 153° and sliding angles <1°. In assays mimicking touch, these hierarchical surfaces demonstrated a 97.5% reduction in transmission of Escherichia coli (E.coli), demonstrating their potential as antimicrobial coatings to mitigate the spread of infectious diseases. Additionally, the developed surfaces displayed a 93% reduction in blood staining after incubation with human whole blood, confirming repellent properties that reduce bacterial deposition.