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.

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.

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.

Innenrücktitelbild: A DNA Barcode‐Based Aptasensor Enables Rapid Testing of Porcine Epidemic Diarrhea Viruses in Swine Saliva Using Electrochemical Readout (Angew. Chem. 31/2022)

Animal Testing, Aptamers, Coronavirus, Electrochemical Biosensors, Porcine Epidemic Diarrhea Viruses Keywords: Animal Testing;Aptamers;Coronavirus;Electrochemical Biosensors;Porcine Epidemic Diarrhea Viruses EN Animal Testing Aptamers Coronavirus Electrochemical Biosensors Porcine Epidemic Diarrhea Viruses 1 1 1 07/27/22 20220801 NES 220801 B Schnelle und reagenzienfreie Erregertests b werden dringend benötigt. Innenrücktitelbild: A DNA Barcode-Based Aptasensor Enables Rapid Testing of Porcine Epidemic Diarrhea Viruses in Swine Saliva Using Electrochemical Readout (Angew. [Extracted from the article] Copyright of Angewandte Chemie is the property of John Wiley & Sons, Inc. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full . (Copyright applies to all s.)

Inside Back Cover: A DNA Barcode‐Based Aptasensor Enables Rapid Testing of Porcine Epidemic Diarrhea Viruses in Swine Saliva Using Electrochemical Readout (Angew. Chem. Int. Ed. 31/2022)

Keywords: Animal Testing;Aptamers;Coronavirus;Electrochemical Biosensors;Porcine Epidemic Diarrhea Viruses EN Animal Testing Aptamers Coronavirus Electrochemical Biosensors Porcine Epidemic Diarrhea Viruses 1 1 1 07/27/22 20220801 NES 220801 B Rapid and reagent-free pathogen tests b are urgently needed. Inside Back Cover: A DNA Barcode-Based Aptasensor Enables Rapid Testing of Porcine Epidemic Diarrhea Viruses in Swine Saliva Using Electrochemical Readout (Angew. Animal Testing, Aptamers, Coronavirus, Electrochemical Biosensors, Porcine Epidemic Diarrhea Viruses. [Extracted from the article] Copyright of Angewandte Chemie International Edition is the property of John Wiley & Sons, Inc. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full . (Copyright applies to all s.)

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.

Front Cover: A Universal DNA Aptamer that Recognizes Spike Proteins of Diverse SARS‐CoV‐2 Variants of Concern (Chem. Eur. J. 15/2022)

A unique DNA aptamer, denoted MSA52, displays universally high affinity for the spike proteins of the wild‐type SARS‐CoV‐2 as well as its Alpha, Beta, Gamma, Epsilon, Kappa, Delta and Omicron variants. This aptamer also recognizes pseudotyped lentiviruses expressing eight different spike proteins of SARS‐CoV‐2 with very high affinity, exhibiting dissociation constants (Kd) of 20–50 pM for these viruses. More information can be found in the Research Article by J. D. Brennan, Y. Li et al. (DOI: 10.1002/chem.202200078).

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.

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.

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.

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.

Roadmap to the Bioanalytical Testing of COVID-19: From Sample Collection to Disease Surveillance.

The disease caused by SARS-CoV-2, coronavirus disease 2019 (COVID-19), has led to a global pandemic with tremendous mortality, morbidity, and economic loss. The current lack of effective vaccines and treatments places tremendous value on widespread screening, early detection, and contact tracing of COVID-19 for controlling its spread and minimizing the resultant health and societal impact. Bioanalytical diagnostic technologies have played a critical role in the mitigation of the COVID-19 pandemic and will continue to be foundational in the prevention of the subsequent waves of this pandemic along with future infectious disease outbreaks. In this Review, we aim at presenting a roadmap to the bioanalytical testing of COVID-19, with a focus on the performance metrics as well as the limitations of various techniques. The state-of-the-art technologies, mostly limited to centralized laboratories, set the clinical metrics against which the emerging technologies are measured. Technologies for point-of-care and do-it-yourself testing are rapidly emerging, which open the route for testing in the community, at home, and at points-of-entry to widely screen and monitor individuals for enabling normal life despite of an infectious disease pandemic. The combination of different classes of diagnostic technologies (centralized and point-of-care and relying on multiple biomarkers) are needed for effective diagnosis, treatment selection, prognosis, patient monitoring, and epidemiological surveillance in the event of major pandemics such as COVID-19.

Antimicrobial Nanomaterials and Coatings: Current Mechanisms and Future Perspectives to Control the Spread of Viruses Including SARS-CoV-2.

The global COVID-19 pandemic has attracted considerable attention toward innovative methods and technologies for suppressing the spread of viruses. Transmission via contaminated surfaces has been recognized as an important route for spreading SARS-CoV-2. Although significant efforts have been made to develop antibacterial surface coatings, the literature remains scarce for a systematic study on broad-range antiviral coatings. Here, we aim to provide a comprehensive overview of the antiviral materials and coatings that could be implemented for suppressing the spread of SARS-CoV-2 via contaminated surfaces. We discuss the mechanism of operation and effectivity of several types of inorganic and organic materials, in the bulk and nanomaterial form, and assess the possibility of implementing these as antiviral coatings. Toxicity and environmental concerns are also discussed for the presented approaches. Finally, we present future perspectives with regards to emerging antimicrobial technologies such as omniphobic surfaces and assess their potential in suppressing surface-mediated virus transfer. Although some of these emerging technologies have not yet been tested directly as antiviral coatings, they hold great potential for designing the next generation of antiviral surfaces.