Programmable polymer materials empowered by DNA nanotechnology

Over the past century, synthetic polymers have had a transformative impact on human life, replacing nature-derived materials in many areas. Yet, despite their many advantages, the structure and function of synthetic polymers still appear rudimentary compared to biological matter: cells use dynamic self-assembly to construct complex materials and operate sophisticated macromolecular devices. The field of DNA nanotechnology has demonstrated that synthetic DNA molecules can be programmed to undergo predictable self-assembly, offering unparalleled control over the formation and dynamic properties of artificial nanostructures. Intriguingly, the principles of DNA nanotechnology can be applied to the engineering of soft programmable materials, bringing the abilities of synthetic polymers closer to their biological counterparts. In this perspective, we discuss the unique features of DNA-functionalized polymer materials. We describe design principles that allow researchers to build complex supramolecular architectures with predictable and dynamically adjustable material properties. Finally, we highlight two key application areas where this biologically inspired material class offers particularly promising opportunities: (1) as dynamic matrices for 3D cell and organoid culture and (2) as smart materials for nucleic acid sequencing and pathogen detection.

Antibody- and aptamer-free SERS substrate for ultrasensitive and anti-interference detection of SARS-CoV-2 spike protein in untreated saliva.

Sensitive and anti-interference detection of targeted signal(s) in body fluids is one of the paramount tasks in biosensing. Overcoming the complication and high cost of antibody/aptamer-modification, surface-enhanced Raman spectroscopy (SERS) based on antibody/aptamer-free (AAF) substrates has shown great promise, yet with rather limited detection sensitivity. Herein, we report ultrasensitive and anti-interference detection of SARS-CoV-2 spike protein in untreated saliva by an AAF SERS substrate, applying the evanescent field induced by the high-order waveguide modes of well-defined nanorods for SERS for the first time. A detection limit of 3.6 × 10-17 M and 1.6 × 10-16 M are obtained in phosphate buffered saline and untreated saliva, respectively; the detection limits are three orders of magnitude improved than the best records from AAF substrates. This work unlocks an exciting path to design AAF SERS substrates for ultrasensitive biosensing, not limited to detection of viral antigens.

Molecular Crowding Modulates SARS-CoV-2 Aptamer Affinity

SARS-CoV-2 aptamer is a favorable candidate for the recognition and detection of SARS-CoV-2, owing to its small size and easy synthesis. However, the issue of compromised binding affinities in real samples and targeting mutant SARS-CoV-2 hinder wide applications of the aptamer. In this study, it is discovered that molecular crowding could increase binding affinity of CoV2-6C3 aptamer against RBD (Receptor Binding Domain) of SARS-CoV-2 via increasing the absolute value of the enthalpy change. The values of the equilibrium dissociation constant in molecular crowding decrease by 70% and 150%, respectively, against wild-type and mutant RBD compared with those in buffer without crowding. Moreover, the detection limit of SARS-CoV-2 pseudovirus is up to 5 times lower under molecular crowding compared to dilute conditions. The discovery deepens the understanding of aptamer-target interaction mechanisms in crowding conditions and provides an effective way to apply SARS-CoV-2 aptamer for virus recognition and detection.

Nucleocapsid protein binding DNA aptamers for detection of SARS-COV-2.

The severe acute respiratory syndrome coronavirus (SARS-CoV-2) has infected millions of individuals and continues to be a major health concern worldwide. While reverse transcription-polymerase chain reaction remains a reliable method for detecting infections, limitations of this technology, particularly cost and the requirement of a dedicated laboratory, prevent rapid viral monitoring. Antigen tests filled this need to some extent but with limitations including sensitivity and specificity, particularly against emerging variants of concern. Here, we developed aptamers against the SARS-CoV-2 Nucleocapsid protein to complement or replace antibodies in antigen detection assays. As detection reagents in ELISA-like assays, our DNA aptamers were able to detect as low as 150 pg/mL of the protein and under 150 k copies of inactivated SARS-CoV-2 Wuhan Alpha strain in viral transport medium with little cross-reactivity to other human coronaviruses (HCoVs). Further, our aptamers were reselected against the SARS-CoV-2 Omicron variant of concern, and we found two sequences that had a more than two-fold increase in signal compared to our original aptamers when used as detection reagents against protein from the Omicron strain. These findings illustrate the use of aptamers as promising alternative detection reagents that may translate for use in current tests and our findings validate the method for the reselection of aptamers against emerging viral strains.

Aptamer-based diagnosis of various SARS-CoV2 strains isolated from clinical specimens.

The emergence of the SARS-CoV-2 virus, an unknown strain of coronavirus, has resulted in severe acute respiratory syndrome with high mortality rates worldwide. Due to the possibility of asymptomatic carriers, late diagnosis of infected individuals can lead to uncontrollable transmission of the disease, making early and accurate detection crucial in controlling the spread of the virus. In this study we identified high-binding-affinity aptamers targeting various strains of the SARS-CoV2 (COVID-19) virus, using the GO-Cell-SELEX (Graphene Oxide- Systematic Evolution of Ligands by Exponential Enrichment) strategy. A total of 96 aptamers were developed through 11 rounds of GO-Cell-SELEX from a random 40 nucleotide single-strand DNA (ssDNA) aptamer library. Using the surface plasmon resonance (SPR) method, the dissociation constant (Kd) values of all aptamers were calculated and two aptamers 52 and 91 with Kd 50 and 61 were selected for enzyme-linked apta-sorbent assay (ELASA). Aptamer 91 could detect various strains of the virus in above 97% of clinical samples obtained from nasopharyngeal swaps (NPS) specimens kept in viral transport media (VTM), confirmed by real-time PCR assay at COVID-19 Reference Diagnostic Laboratory of Iran, Pasture Institute. Aptamer 52 could detect the SARS-CoV2 virus in a competitive lateral flow assay (LFA) to be considered for a future designed kit. These two simple, specific, and sensitive tests can be used in combination for rapid and early diagnosis of various strains of the COVID-19 virus. Our results suggest that these two discovered aptamers present an opportunity for developing a new rapid aptamer-based coronavirus diagnostic kit.

Development of Aptasensor for the Diagnosis of Covid-19

Objectives: The objective is to develop a low-cost, practical, portable aptasensor platform for the diagnosis of COVID-19. Materials -Methods: Amino-terminated aptamers to be used for the design of an aptasensor were synthesized by SELEX method, and interaction of aptamers with SARS-CoV-2 S1 protein was investigated by isothermal titration calorimetry (ITC). Gold electrodes were used to design the biosensor platform. After the electrode surface was functionalized with cysteamine, the amino-terminated aptamer was conjugated to the surface via glutaraldehyde crosslinker. Then, the surface characterization and analytical parameters of the designed sensing platform were determined by adding commercial S1 proteins on the surface using differential pulse voltammetry (DPV), cyclic voltammetry (CV) and impedance spectroscopy (EIS). To evaluate the working performance of the system, S1 proteins were added to the synthetic serum samples using the standard addition method and the measurements were repeated. Result(s): Surface characterization of the platform designed with EIS and CV measurements was performed and it was found that the modification was successfully performed. In addition, DPV results and analytical parameters of the platform (calibration plot, limit of detection(LOD) , repeatability, coefficient of variation) were determined and the working performance of system was evaluated. Moreover, working performance of the biosensor in real samples and its specificity for COVID -19 were determined by experiments with synthetic serum and influenza A and B proteins. Conclusion(s): According the results, the system has potential to be used for the detection of COVID -19, and also it can be rapidly adapted in different pandemic situations that may occur in the future.

Recent Progress in Development and Application of DNA, Protein, Peptide, Glycan, Antibody, and Aptamer Microarrays.

Microarrays are one of the trailblazing technologies of the last two decades and have displayed their importance in all the associated fields of biology. They are widely explored to screen, identify, and gain insights on the characteristics traits of biomolecules (individually or in complex solutions). A wide variety of biomolecule-based microarrays (DNA microarrays, protein microarrays, glycan microarrays, antibody microarrays, peptide microarrays, and aptamer microarrays) are either commercially available or fabricated in-house by researchers to explore diverse substrates, surface coating, immobilization techniques, and detection strategies. The aim of this review is to explore the development of biomolecule-based microarray applications since 2018 onwards. Here, we have covered a different array of printing strategies, substrate surface modification, biomolecule immobilization strategies, detection techniques, and biomolecule-based microarray applications. The period of 2018-2022 focused on using biomolecule-based microarrays for the identification of biomarkers, detection of viruses, differentiation of multiple pathogens, etc. A few potential future applications of microarrays could be for personalized medicine, vaccine candidate screening, toxin screening, pathogen identification, and posttranslational modifications.

Multiplex Lithographic SERS Aptasensor for Detection of Several Respiratory Viruses in One Pot.

Rapid and reliable techniques for virus identification are required in light of recurring epidemics and pandemics throughout the world. Several techniques have been distributed for testing the flow of patients. Polymerase chain reaction with reverse transcription is a reliable and sensitive, though not rapid, tool. The antibody-based strip is a rapid, though not reliable, and sensitive tool. A set of alternative tools is being developed to meet all the needs of the customer. Surface-enhanced Raman spectroscopy (SERS) provides the possibility of single molecule detection taking several minutes. Here, a multiplex lithographic SERS aptasensor was developed aiming at the detection of several respiratory viruses in one pot within 17 min. The four labeled aptamers were anchored onto the metal surface of four SERS zones; the caught viruses affect the SERS signals of the labels, providing changes in the analytical signals. The sensor was able to decode mixes of SARS-CoV-2 (severe acute respiratory syndrome coronavirus two), influenza A virus, respiratory syncytial virus, and adenovirus within a single experiment through a one-stage recognition process.

CRISPR/Cas-based Aptasensor as an Innovative Sensing Approaches for Food Safety Analysis: Recent Progresses and New Horizons.

Food safety is one of the greatest public problems occurring around the world. Chemical, physical, and microbiological hazards could lead to food safety problems, which might occur at all stages of the supply chain. To tackle food safety problems and protect consumer health, specific, accurate, and rapid diagnosis techniques meeting various requirements are the imperative measures to ensure food safety. CRISPR-Cas system, a novel emerging technology, is effectively repurposed in (bio)sensing and has shown a tremendous capability to develop on-site and portable diagnostic methods with high specificity and sensitivity. Among numerous existing CRISPR/Cas systems, CRISPR/Cas13a and CRISPR/Cas12a are extensively employed in the design of biosensors, owing to their ability to cleave both non-target and target sequences. However, the specificity limitation in CRISPR/Cas has hindered its progress. Nowadays, nucleic acid aptamers recognized for their specificity and high-affinity characteristics for their analytes are incorporated into CRISPR/Cas systems. With the benefits of reproducibility, high durability, portability, facile operation, and cost-effectiveness, CRISPR/Cas-based aptasensing approaches are an ideal choice for fabricating highly specific point-of-need analytical tools with enhanced response signals. In the current study, we explore some of the most recent progress in the CRISPR/Cas-mediated aptasensors for detecting food risk factors including veterinary drugs, pesticide residues, pathogens, mycotoxins, heavy metals, illegal additives, food additives, and other contaminants. The nanomaterial engineering support with CRISPR/Cas aptasensors is also signified to achieve a hopeful perspective to provide new straightforward test kits toward trace amounts of different contaminants encountered in food samples.

Aptamer-based CRISPR-Cas powered diagnostics of diverse biomarkers and small molecule targets.

CRISPR-Cas systems have been widely used in genome editing and transcriptional regulation. Recently, CRISPR-Cas effectors are adopted for biosensor construction due to its adjustable properties, such as simplicity of design, easy operation, collateral cleavage activity, and high biocompatibility. Aptamers' excellent sensitivity, specificity, in vitro synthesis, base-pairing, labeling, modification, and programmability has made them an attractive molecular recognition element for inclusion in CRISPR-Cas systems. Here, we review current advances in aptamer-based CRISPR-Cas sensors. We briefly discuss aptamers and the knowledge of Cas effector proteins, crRNA, reporter probes, analytes, and applications of target-specific aptamers. Next, we provide fabrication strategies, molecular binding, and detection using fluorescence, electrochemical, colorimetric, nanomaterials, Rayleigh, and Raman scattering. The application of CRISPR-Cas systems in aptamer-based sensing of a wide range of biomarkers (disease and pathogens) and toxic contaminants is growing. This review provides an update and offers novel insights into developing CRISPR-Cas-based sensors using ssDNA aptamers with high efficiency and specificity for point-of-care setting diagnostics.

Harnessing the CRISPR-Cas13d System for Protein Detection via Dual-aptamer-based Transcription Amplification.

CRISPR-based biosensing technology has been emerging as a revolutionary diagnostics for many diseases-related biomarkers. In particular, RspCas13d, a newly identified RNA-guided Cas13d ribonuclease derived from Ruminococcus sp., has shown great promise for accurate and sensitive detection of RNA due to its RNA sequence-specific recognition and robust collateral trans-cleavage activity. However, its diagnostic utility is limited to detect nucleic-acid-related biomarkers. To address this limitation, we herein present a proof-of-concept demonstration of a target-responsive CRISPR-Cas13d sensing system for protein biomarkers. Such a system is rationally designed by integrating a Dual-Aptamer-based Transcription Amplification Strategy with CRISPR-Cas13d (DATAS-Cas13d), in which the protein binding initiates the in vitro RNA transcription followed by the activation of RspCas13d. Using a short fluorescent ssRNA as the signal reporter and cardiac troponin I (cTnI) as the model analyte, the DATAS-Cas13d system showed a wide linear range, low detection limit and high specificity for the detection of cTnI in buffer and human serum. Thanks to the facile integration of various bioreceptors into the DATAS-Cas13d system, the method could be adapted to detecting a broad range of clinically relevant protein biomarkers, and thus broaden the medical applications of Cas13d-based diagnostics.

Nanopore single-molecule analysis of biomarkers: Providing possible clues to disease diagnosis

Biomarker detection has attracted increasing interest in recent years due to the minimally or non-invasive sampling process. Single entity analysis of biomarkers is expected to provide real-time and accurate biological information for early disease diagnosis and prognosis, which is critical to the effective disease treatment and is also important in personalized medicine. As an innovative single entity analysis method, nanopore sensing is a pioneering single-molecule detection technique that is widely used in analytical bioanalytical fields. In this review, we overview the recent progress of nanopore biomarker detection as new approaches to disease diagnosis. In highlighted studies, nanopore was focusing on detecting biomarkers of different categories of communicable and noncommunicable diseases, such as pandemic COVID-19, AIDS, cancers, neurologic diseases, etc. Various sensitive and selective nanopore detecting strategies for different types of biomarkers are summarized. In addition, the challenges, opportunities, and direction for future development of nanopore-based biomarker sensors are also discussed.Copyright © 2023 Elsevier B.V.

Design and Development of Nanoscale Aptasensors for Viral Diagnostics

Viral infection may be a serious threat for human beings. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a highly transmissible virus causing coronavirus disease 2019 (COVID-19) in humans and creating a universal pandemic outbreak. The current methods for detection of SARS-CoV-2 include real-time reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and loop-mediated isothermal amplification (LAMP). Though the methods are widely used for the diagnosis of COVID-19, they too have their limitations such as time-consuming process, sophisticated instrumental setup, which requires highly skilled personnel for operation, and prevalence of false positive/negative reports. Therefore, there is a pressing need to develop alternative tools such as point-of-care testing (POCT) devices to detect SARS-CoV-2 rapidly, accurately, and user-friendly. Here, the authors propose a one-step diagnostic method using aptamer-based sensing technology. The intended design of aptamer-based biosensors (also known as aptasensors) utilizes the optical properties of gold nanoparticles (AuNP) conjugated with angiotensin-converting enzyme-2 (ACE-2) aptamers targeting SARS-CoV-2 using lateral flow assay (LFA). This study leads to the development of portable nanoscale aptasensors for viral diagnostics. © 2022 by the authors.

Stimulus-Responsive DNA Hydrogel Biosensors for Food Safety Detection.

Food safety has always been a major global challenge to human health and the effective detection of harmful substances in food can reduce the risk to human health. However, the food industry has been plagued by a lack of effective and sensitive safety monitoring methods due to the tension between the cost and effectiveness of monitoring. DNA-based hydrogels combine the advantages of biocompatibility, programmability, the molecular recognition of DNA molecules, and the hydrophilicity of hydrogels, making them a hotspot in the research field of new nanomaterials. The stimulus response property greatly broadens the function and application range of DNA hydrogel. In recent years, DNA hydrogels based on stimulus-responsive mechanisms have been widely applied in the field of biosensing for the detection of a variety of target substances, including various food contaminants. In this review, we describe the recent advances in the preparation of stimuli-responsive DNA hydrogels, highlighting the progress of its application in food safety detection. Finally, we also discuss the challenges and future application of stimulus-responsive DNA hydrogels.

ß-Cyclodextrin Polymer-Based Fluorescence Enhancement Strategy via Host-Guest Interaction for Sensitive Assay of SARS-CoV-2.

Nucleocapsid protein (N protein) is an appropriate target for early determination of viral antigen-based severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We have found that ß-cyclodextrin polymer (ß-CDP) has shown a significant fluorescence enhancement effect for fluorophore pyrene via host-guest interaction. Herein, we developed a sensitive and selective N protein-sensing method that combined the host-guest interaction fluorescence enhancement strategy with high recognition of aptamer. The DNA aptamer of N protein modified with pyrene at its 3' terminal was designed as the sensing probe. The added exonuclease I (Exo I) could digest the probe, and the obtained free pyrene as a guest could easily enter into the hydrophobic cavity of host ß-CDP, thus inducing outstanding luminescent enhancement. While in the presence of N protein, the probe could combine with it to form a complex owing to the high affinity between the aptamer and the target, which prevented the digestion of Exo I. The steric hindrance of the complex prevented pyrene from entering the cavity of ß-CDP, resulting in a tiny fluorescence change. N protein has been selectively analyzed with a low detection limit (11.27 nM) through the detection of the fluorescence intensity. Moreover, the sensing of spiked N protein from human serum and throat swabs samples of three volunteers has been achieved. These results indicated that our proposed method has broad application prospects for early diagnosis of coronavirus disease 2019.

Aptamer-based nanointerferometer enables amplification-free ultrasensitive detection and differentiation of SARS-CoV-2 variants.

The state-of-the-art SARS-CoV-2 detection methods include qRT-PCR and antibody-based lateral flow assay (LFA) point-of-care tests. Despite the high sensitivity and selectivity, qRT-PCR is slow, expensive and needs well-trained operators. On the other extreme, LFA suffers from low sensitivity albeit its fast detection speed, low detection cost and ease of use. Therefore, the continuing COVID-19 pandemic calls for a SARS-CoV-2 detection method that is rapid, convenient and cost-effective without compromise in sensitivity. Here we provide a proof-of-principle demonstration of an optimized aptamer-based nanointerferometer that enables rapid and amplification-free detection of SARS-CoV-2 spike protein-coated pseudovirus directly from human saliva with the limit of detection (LOD) of about 400 copies per mL. This LOD is on par with that of qRT-PCR, making it 1000 to 100,000-fold more sensitive than commercial LFA tests. Using various combinations of negative selections during the screens for the aptamer targeting the receptor binding domain of the spike protein of SARS-CoV-2, we isolated two aptamers that can distinguish the Omicron and Delta variants. Integrating these two aptamers with LFA strips or the nanointerferometer sensors allows both detection and differentiation of the Omicron and Delta variants which has the potential to realize rapid triage of patients infected different SARS-CoV-2 variants.

bioTCIs: Middle-to-Macro Biomolecular Targeted Covalent Inhibitors Possessing Both Semi-Permanent Drug Action and Stringent Target Specificity as Potential Antibody Replacements.

Monoclonal antibody therapies targeting immuno-modulatory targets such as checkpoint proteins, chemokines, and cytokines have made significant impact in several areas, including cancer, inflammatory disease, and infection. However, antibodies are complex biologics with well-known limitations, including high cost for development and production, immunogenicity, a limited shelf-life because of aggregation, denaturation, and fragmentation of the large protein. Drug modalities such as peptides and nucleic acid aptamers showing high-affinity and highly selective interaction with the target protein have been proposed alternatives to therapeutic antibodies. The fundamental limitation of short in vivo half-life has prevented the wide acceptance of these alternatives. Covalent drugs, also known as targeted covalent inhibitors (TCIs), form permanent bonds to target proteins and, in theory, eternally exert the drug action, circumventing the pharmacokinetic limitation of other antibody alternatives. The TCI drug platform, too, has been slow in gaining acceptance because of its potential prolonged side-effect from off-target covalent binding. To avoid the potential risks of irreversible adverse drug effects from off-target conjugation, the TCI modality is broadening from the conventional small molecules to larger biomolecules possessing desirable properties (e.g., hydrolysis resistance, drug-action reversal, unique pharmacokinetics, stringent target specificity, and inhibition of protein-protein interactions). Here, we review the historical development of the TCI made of bio-oligomers/polymers (i.e., peptide-, protein-, or nucleic-acid-type) obtained by rational design and combinatorial screening. The structural optimization of the reactive warheads and incorporation into the targeted biomolecules enabling a highly selective covalent interaction between the TCI and the target protein is discussed. Through this review, we hope to highlight the middle to macro-molecular TCI platform as a realistic replacement for the antibody.

Aptamers as Novel Binding Molecules on an Antimicrobial Peptide-Armored Composite Hydrogel Wound Dressing for Specific Removal and Efficient Eradication of Pseudomonas aeruginosa.

Here we present for the first time a potential wound dressing material implementing aptamers as binding entities to remove pathogenic cells from newly contaminated surfaces of wound matrix-mimicking collagen gels. The model pathogen in this study was the Gram-negative opportunistic bacterium Pseudomonas aeruginosa, which represents a considerable health threat in hospital environments as a cause of severe infections of burn or post-surgery wounds. A two-layered hydrogel composite material was constructed based on an established eight-membered focused anti-P. aeruginosa polyclonal aptamer library, which was chemically crosslinked to the material surface to form a trapping zone for efficient binding of the pathogen. A drug-loaded zone of the composite released the C14R antimicrobial peptide to deliver it directly to the bound pathogenic cells. We demonstrate that this material combining aptamer-mediated affinity and peptide-dependent pathogen eradication can quantitatively remove bacterial cells from the "wound" surface, and we show that the surface-trapped bacteria are completely killed. The drug delivery function of the composite thus represents an extra safeguarding property and thus probably one of the most important additional advances of a next-generation or smart wound dressing ensuring the complete removal and/or eradication of the pathogen of a freshly infected wound.

Ultra-sensitive label-free SERS biosensor with high-throughput screened DNA aptamer for universal detection of SARS-CoV-2 variants from clinical samples.

COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused an ongoing global pandemic with economic and social disruption. Moreover, the virus has persistently and rapidly evolved into novel lineages with mutations. The most effective strategy to control the pandemic is suppressing virus spread through early detection of infections. Therefore, developing a rapid, accurate, easy-to-use diagnostic platform against SARS-CoV-2 variants of concern remains necessary. Here, we developed an ultra-sensitive label-free surface-enhanced Raman scattering-based aptasensor as a countermeasure for the universal detection of SARS-CoV-2 variants of concern. In this aptasensor platform, we discovered two DNA aptamers that enable binding to SARS-CoV-2 spike protein via the Particle Display, a high-throughput screening approach. These showed high affinity that exhibited dissociation constants of 1.47 ± 0.30 nM and 1.81 ± 0.39 nM. We designed a combination with the aptamers and silver nanoforest for developing an ultra-sensitive SERS platform and achieved an attomolar (10-18 M) level detection limit with a recombinant trimeric spike protein. Furthermore, using the intrinsic properties of the aptamer signal, we demonstrated a label-free aptasensor approach, enabling use without the Raman tag. Finally, our label-free SERS-combined aptasensor succeeded in detecting SARS-CoV-2 with excellent accuracy, even in clinical samples with variants of concern, including the wild-type, delta, and omicron variants.

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.