Paper-based colorimetric detection of COVID-19 using aptasenor based on biomimetic peroxidase like activity of ChF/ZnO/CNT nano-hybrid.

Corona Virus Disease 2019 (COVID-19) as the infectious disease caused the pandemic disease around the world through infection by SARS-CoV-2 virus. The common diagnosis approach is Quantitative RT-PCR (qRT-PCR) which is time consuming and labor intensive. In the present study a novel colorimetric aptasensor was developed based on intrinsic catalytic activity of chitosan film embedded with ZnO/CNT (ChF/ZnO/CNT) on 3,3',5,5'-tetramethylbenzidine (TMB) substrate. The main nanocomposite platform was constructed and functionalized with specific COVID-19 aptamer. The construction subjected with TMB substrate and H2O2 in the presence of different concentration of COVID-19 virus. Separation of aptamer after binding with virus particles declined the nanozyme activity. Upon addition of virus concentration, the peroxidase like activity of developed platform and colorimetric signals of oxidized TMB decreased gradually. Under optimal conditions the nanozyme could detect the virus in the linear range of 1-500 pg mL and LOD of 0.05 pg mL. Also, a paper-based platform was used for set up the strategy on applicable device. The paper-based strategy showed a linear range between 50 and 500 pg mL with LOD of 8 pg mL. The applied paper based colorimetric strategy showed reliable results for sensitive and selective detection of COVID-19 virus with the cost-effective approach.

Aptamers Targeting Membrane Proteins for Sensor and Diagnostic Applications.

Many biological processes (physiological or pathological) are relevant to membrane proteins (MPs), which account for almost 30% of the total of human proteins. As such, MPs can serve as predictive molecular biomarkers for disease diagnosis and prognosis. Indeed, cell surface MPs are an important class of attractive targets of the currently prescribed therapeutic drugs and diagnostic molecules used in disease detection. The oligonucleotides known as aptamers can be selected against a particular target with high affinity and selectivity by iterative rounds of in vitro library evolution, known as Systematic Evolution of Ligands by EXponential Enrichment (SELEX). As an alternative to antibodies, aptamers offer unique features like thermal stability, low-cost, reuse, ease of chemical modification, and compatibility with various detection techniques. Particularly, immobilized-aptamer sensing platforms have been under investigation for diagnostics and have demonstrated significant value compared to other analytical techniques. These "aptasensors" can be classified into several types based on their working principle, which are commonly electrochemical, optical, or mass-sensitive. In this review, we review the studies on aptamer-based MP-sensing technologies for diagnostic applications and have included new methodological variations undertaken in recent years.

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.

Investigating the competition between ACE2 natural molecular interactors and SARS-CoV-2 candidate inhibitors.

The SARS-CoV-2 pandemic still poses a threat to the global health as the virus continues spreading in most countries. Therefore, the identification of molecules capable of inhibiting the binding between the ACE2 receptor and the SARS-CoV-2 spike protein is of paramount importance. Recently, two DNA aptamers were designed with the aim to inhibit the interaction between the ACE2 receptor and the spike protein of SARS-CoV-2. Indeed, the two molecules interact with the ACE2 receptor in the region around the K353 residue, preventing its binding of the spike protein. If on the one hand this inhibition process hinders the entry of the virus into the host cell, it could lead to a series of side effects, both in physiological and pathological conditions, preventing the correct functioning of the ACE2 receptor. Here, we discuss through a computational study the possible effect of these two very promising DNA aptamers, investigating all possible interactions between ACE2 and its experimentally known molecular partners. Our in silico predictions show that some of the 10 known molecular partners of ACE2 could interact, physiologically or pathologically, in a region adjacent to the K353 residue. Thus, the curative action of the proposed DNA aptamers could recruit ACE2 from its biological functions.

Ultrasensitive detection of SARS-CoV-2 S protein with aptamers biosensor based on surface-enhanced Raman scattering.

A rapid and accurate diagnostic modality is essential to prevent the spread of SARS-CoV-2. In this study, we proposed a SARS-CoV-2 detection sensor based on surface-enhanced Raman scattering (SERS) to achieve rapid and ultrasensitive detection. The sensor utilized spike protein deoxyribonucleic acid aptamers with strong affinity as the recognition entity to achieve high specificity. The spherical cocktail aptamers-gold nanoparticles (SCAP) SERS substrate was used as the base and Au nanoparticles modified with the Raman reporter molecule that resonates with the excitation light and spike protein aptamers were used as the SERS nanoprobe. The SCAP substrate and SERS nanoprobes were used to target and capture the SARS-CoV-2 S protein to form a sandwich structure on the Au film substrate, which can generate ultra-strong "hot spots" to achieve ultrasensitive detection. Analysis of SARS-CoV-2 S protein was performed by monitoring changes in SERS peak intensity on a SCAP SERS substrate-based detection platform. This assay detects S protein with a LOD of less than 0.7 fg mL-1 and pseudovirus as low as 0.8 TU mL-1 in about 12 min. The results of the simulated oropharyngeal swab system in this study indicated the possibility of it being used for clinical detection, providing a potential option for rapid and accurate diagnosis and more effective control of SARS-CoV-2 transmission.

In vitro Selection of Aptamers to Differentiate Infectious from Non-Infectious Viruses.

Virus infections have a major impact on society; most methods of detection have difficulties in determining whether a detected virus is infectious, causing delays in treatment and further spread of the virus. Developing new sensors that can inform on the infectability of clinical or environmental samples will meet this unmet challenge. However, very few methods can obtain sensing molecules that can recognize an intact infectious virus and differentiate it from the same virus that has been rendered non-infectious by disinfection methods. Here, we describe a protocol to select aptamers that can distinguish infectious viruses vs non-infectious viruses using systematic evolution of ligands by exponential enrichment (SELEX). We take advantage of two features of SELEX. First, SELEX can be tailor-made to remove competing targets, such as non-infectious viruses or other similar viruses, using counter selection. Additionally, the whole virus can be used as the target for SELEX, instead of, for example, a viral surface protein. Whole virus SELEX allows for the selection of aptamers that bind specifically to the native state of the virus, without the need to disrupt of the virus. This method thus allows recognition agents to be obtained based on functional differences in the surface of pathogens, which do not need to be known in advance.

Aptamer-Linked Photonic Crystal Assay for High-Throughput Screening of HIV and SARS-CoV-2.

We present a microplate assay for the detection of HIV and SARS-CoV-2 which involves the preadsorption of carboxy-modified polystyrene microspheres to the microplate wells and their self-assembly leading to the formation of a photonic crystal colloidal array (PCCA). PCCA is then cross-linked with amino-modified aptamers selected for viral cell surface glycoproteins, i.e., S1-protein of SARS-CoV-2 and gp120 of the human immunodeficiency virus (HIV), to develop an aptamer-linked photonic crystal assay (ALPA). ALPA is then utilized as a proof-of-concept method for the detection of S1-protein, gp120, and two whole viruses, i.e., SARS-CoV-2 and HIV, as well. The aptamers are stable at room temperature and can bind with the viruses' proteins via hydrogen bonding. This binding leads to color generation from PCCA, and the signal can easily be measured and quantified by a UV/vis spectrometer. The assay carries the advantage of a two-step detection process by the addition of the virus sample directly to a 96-well microplate and incubation of 5 min followed by convenient detection through a UV/vis-spectrometer. The assay does not require any additional reagents and can be customized for similar viruses utilizing specific aptamers targeting their cell surface receptors.

Highly efficacious and safe neutralizing DNA aptamer of SARS-CoV-2 as an emerging therapy for COVID-19 disease.

BACKGROUND: The paucity of SARS-CoV-2-specific virulence factors has greatly hampered the therapeutic management of patients with COVID-19 disease. Although available vaccines and approved therapies have shown tremendous benefits, the continuous emergence of new variants of SARS-CoV-2 and side effects of existing treatments continue to challenge therapy, necessitating the development of a novel effective therapy. We have previously shown that our developed novel single-stranded DNA aptamers not only target the trimer S protein of SARS-CoV-2, but also block the interaction between ACE2 receptors and trimer S protein of Wuhan origin, Delta, Delta plus, Alpha, Lambda, Mu, and Omicron variants of SARS-CoV-2. We herein performed in vivo experiments that administer the aptamer to the lungs by intubation as well as in vitro studies utilizing PBMCs to prove the efficacy and safety of our most effective aptamer, AYA2012004_L. METHODS: In vivo studies were conducted in transgenic mice expressing human ACE2 (K18hACE2), C57BL/6J, and Balb/cJ. Flow cytometry was used to check S-protein expressing pseudo-virus-like particles (VLP) uptake by the lung cells and test the immuogenicity of AYA2012004_L. Ames test was used to assess mutagenicity of AYA2012004_L. RT-PCR and histopathology were used to determine the biodistribution and toxicity of AYA2012004_L in vital organs of mice. RESULTS: We measured the in vivo uptake of VLPs by lung cells by detecting GFP signal using flow cytometry. AYA2012004_L specifically neutralized VLP uptake and also showed no inflammatory response in mice lungs. In addition, AYA2012004_L did not induce inflammatory response in the lungs of Th1 and Th2 mouse models as well as human PBMCs. AYA2012004_L was detectable in mice lungs and noticeable in insignificant amounts in other vital organs. Accumulation of AYA2012004_L in organs decreased over time. AYA2012004_L did not induce degenerative signs in tissues as seen by histopathology and did not cause changes in the body weight of mice. Ames test also certified that AYA2012004_L is non-mutagenic and proved it to be safe for in vivo studies. CONCLUSIONS: Our aptamer is safe, effective, and can neutralize the uptake of VLPs by lung cells when administered locally suggesting that it can be used as a potential therapeutic agent for COVID-19 management.

An ultrasensitive fluorescence aptasensor for SARS-CoV-2 antigen based on hyperbranched rolling circle amplification.

Sensitive and accurate diagnosis of SARS-CoV-2 infection at early stages can help to attenuate the effects of the COVID-19. Compared to RNA and antibodies detection, direct detection of viral antigens could reflect infectivity more appropriately. However, it is still a great challenge to construct a convenient, accurate and sensitive biosensor with a suitable molecular recognition element for SARS-CoV-2 antigens. Herein, we report a HRCA-based aptasensor for simple, ultrasensitive and quantitative detection of SARS-CoV-2 S1 protein and pseudovirus. The aptamer sequence used here is selected from several published aptamers by enzyme-linked oligonucleotide assay and molecular docking simulation. The sensor forms an antibody-target-aptamer sandwich complex on the surface of microplates and elicits HRCA for fluorescent detection. Without complicated operations or special instruments and reagents, the aptasensor can detect S1 protein with a LOD of 89.7 fg/mL in the linear range of 100 fg/mL to 1 µg/mL. And it can also detect SARS-CoV-2 spike pseudovirus in artificial saliva with a LOD of 51 TU/µL. Therefore, this simple and ultrasensitive aptasensor has the potential to detect SARS-CoV-2 infection at early stages. It may improve the timeliness and accuracy of SARS-CoV-2 diagnosis and demonstrate a strategy to conduct aptasensors for other targets.

Sandwich-like electrochemical aptasensing of heat shock protein 70 kDa (HSP70): Application in diagnosis/prognosis of coronavirus disease 2019 (COVID-19).

In this research, by using aptamer-conjugated gold nanoparticles (aptamer-AuNPs) and a modified glassy carbon electrode (GCE) with reduced graphene oxide (rGO) and Acropora-like gold (ALG) nanostructure, a sandwich-like system provided for sensitive detection of heat shock protein 70 kDa (HSP70), which applied as a functional biomarker in diagnosis/prognosis of COVID-19. Initially, the surface of the GCE was improved with rGO and ALG nanostructures, respectively. Then, an aptamer sequence as the first part of the bioreceptor was covalently bound on the surface of the GCE/rGO/ALG nanostructures. After adding the analyte, the second part of the bioreceptor (aptamer-AuNPs) was immobilized on the electrode surface to improve the diagnostic performance. The designed aptasensor detected HSP70 in a wide linear range, from 5 pg mL-1 to 75 ng mL-1, with a limit of detection (LOD) of ∼2 pg mL-1. The aptasensor was stable for 3 weeks and applicable in detecting 40 real plasma samples of COVID-19 patients. The diagnostic sensitivity and specificity were 90% and 85%, respectively, compared with the reverse transcription-polymerase chain reaction (RT-PCR) method.

Ultrasensitive FRET-based aptasensor for interleukin-6 as a biomarker for COVID-19 progression using nitrogen-doped carbon quantum dots and gold nanoparticles.

A label-free and specific FRET-based interleukin-6 (IL-6) aptasensor was developed using a DNA aptamer modified with nitrogen-doped carbon quantum dots (NCDs) and gold nanoparticles (AuNPs) as a donor-quencher pair. The assayed target was capable of disrupting the donor-acceptor assemblies yielding a concentration-related fluorescence recovery of NCDs (λem = 445 nm and λex = 350 nm). By designing two different probes, the interaction of DNA aptamers with IL-6 protein was studied using FRET efficiency. It appeared that the sensing probes showed slightly different sensing profiles. One of the aptasensors showed a linear response of 1.5-5.9 pg/mL for IL-6 with a coefficient of determination of R2 ≥ 0.99 and the a detection limit of 0.82 pg/mL (at S/N = 3). The experimental results indicated that the biosensor can be applied to determine IL-6 in human serum (with recovery of 95.7-102.9%). Due to the high sensitivity, excellent selectivity, and simplicity of the procedure, this strategy represents a promising alternative for IL-6 sensing in clinical applications.

Aptasensors for the detection of infectious pathogens: design strategies and point-of-care testing.

The epidemic of infectious diseases caused by contagious pathogens is a life-threatening hazard to the entire human population worldwide. A timely and accurate diagnosis is the critical link in the fight against infectious diseases. Aptamer-based biosensors, the so-called aptasensors, employ nucleic acid aptamers as bio-receptors for the recognition of target pathogens of interest. This review focuses on the design strategies as well as state-of-the-art technologies of aptasensor-based diagnostics for infectious pathogens (mainly bacteria and viruses), covering the utilization of three major signal transducers, the employment of aptamers as recognition moieties, the construction of versatile biosensing platforms (mostly micro and nanomaterial-based), innovated reporting mechanisms, and signal enhancement approaches. Advanced point-of-care testing (POCT) for infectious disease diagnostics are also discussed highlighting some representative ready-to-use devices to address the urgent needs of currently prevalent coronavirus disease 2019 (COVID-19). Pressing issues in aptamer-based technology and some future perspectives of aptasensors are provided for the implementation of aptasensor-based diagnostics into practical application.

Aptamer-Gated Mesoporous Silica Nanoparticles for N Protein Triggered Release of Remdesivir and Treatment of Novel Coronavirus (2019-nCoV).

Since the 2019-nCoV outbreak was first reported, hundreds of millions of people all over the world have been infected. There is no doubt that improving the cure rate of 2019-nCoV is one of the most effective means to deal with the current serious epidemic. At present, Remdesivir (RDV) has been clinically proven to be effective in the treatment of SARS-CoV-2. However, the uncertain side effects make it important to reduce the use of drugs while ensuring the self-healing effect. We report an approach here with targeted therapy for the treatment of SARS-CoV-2 and other coronaviruses illness. In this study, mesoporous silica was used as the carrier of RDV, the nucleocapsid protein (N protein) aptamer was hybridized with the complementary chain, and the double-stranded DNA was combined with gold nanoparticles as the gates of mesoporous silica pores. When the RDV-loaded mesoporous silica is incubated with the N protein, aptamer with gold nanoparticles dissociate from the complementary DNA oligonucleotide on the mesoporous silica surface and bind to the N protein. The releasing of RDV was determined by detecting the UV-vis absorption peak of RDV in the solution. These results show that the RDV delivery system designed in this work has potential clinical application for the treatment of 2019-nCoV.

Aptamer-Based Antibacterial and Antiviral Therapy against Infectious Diseases.

Nucleic acid aptamers are single-stranded DNA or RNA molecules selected in vitro that can bind to a broad range of targets with high affinity and specificity. As promising alternatives to conventional anti-infective agents, aptamers have gradually revealed their potential in the combat against infectious diseases. This article provides an overview on the state-of-art of aptamer-based antibacterial and antiviral therapeutic strategies. Diverse aptamers targeting pathogen-related components or whole pathogenic cells are summarized according to the species of microorganisms. These aptamers exhibited remarkable in vitro and/or in vivo inhibitory effect for pathogenic invasion, enzymatic activities, or viral replication, even for some highly drug-resistant strains and biofilms. Aptamer-mediated drug delivery and controlled drug release strategies are also included herein. Critical technical barriers of therapeutic aptamers are briefly discussed, followed by some future perspectives for their implementation into clinical utility.

Electrochemical Aptasensing of SARS-CoV-2 Based on Triangular Prism DNA Nanostructures and Dumbbell Hybridization Chain Reaction.

Development of convenient, accurate, and sensitive methods for rapid screening of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) infection is highly desired. In this study, we have developed a facile electrochemical aptasensor for the detection of the SARS-CoV-2 S1 protein amplified by dumbbell hybridization chain reaction (DHCR). A triangular prism DNA (TPDNA) nanostructure is first assembled and modified at the electrode interface. Due to the multiple thiol anchors, the immobilization is quite stable. The TPDNA nanostructure also provides an excellent scaffold for better molecular recognition efficiency on the top single-strand region (DHP0). The aptamer sequence toward the SARS-CoV-2 S1 protein is previously localized by partial hybridization with DHP0. In the presence of the target protein, the aptamer sequence is displaced and DHP0 is exposed. After further introduction of the fuel stands of DHCR, compressed DNA linear assembly occurs, and the product can be stacked on the TPDNA nanostructure for the enrichment of electrochemical species. This electrochemical method successfully detects the target protein in clinical samples, which provides a simple, robust, and accurate platform with great potential utility.

Aptamers: A prospective tool for infectious diseases diagnosis.

It is well known that people's health is seriously threatened by various pathogens (such as Mycobacterium tuberculosis, Treponema pallidum, Novel coronavirus, HIV, Mucor, etc.), which leads to heavy socioeconomic burdens. Therefore, early and accurate pathogen diagnosis is essential for timely and effective therapies. Up to now, diagnosing human contagious diseases at molecule and nano levels is remarkably difficult owing to insufficient valid probes when it comes to determining the biological markers of pathogens. Aptamers are a set of high-specificity and high-sensitivity plastic oligonucleotides screened in vitro via the selective expansion of ligands by exponential enrichment (SELEX). With the advent of aptamer-based technologies, their merits have aroused mounting academic interest. In recent years, as new detection and treatment tools, nucleic acid aptamers have been extensively utilized in the field of biomedicine, such as pathogen detection, new drug development, clinical diagnosis, nanotechnology, etc. However, the traditional SELEX method is cumbersome and has a long screening cycle, and it takes several months to screen out aptamers with high specificity. With the persistent development of SELEX-based aptamer screening technologies, the application scenarios of aptamers have become more and more extensive. The present research briefly reviews the research progress of nucleic acid aptamers in the field of biomedicine, especially in the diagnosis of contagious diseases.

Model of the SARS-CoV-2 Virus for Development of a DNA-Modified, Surface-Enhanced Raman Spectroscopy Sensor with a Novel Hybrid Plasmonic Platform in Sandwich Mode.

The recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has posed a great challenge for the development of ultra-fast methods for virus identification based on sensor principles. We created a structure modeling surface and size of the SARS-CoV-2 virus and used it in comparison with the standard antigen SARS-CoV-2-the receptor-binding domain (RBD) of the S-protein of the envelope of the SARS-CoV-2 virus from the Wuhan strain-for the development of detection of coronaviruses using a DNA-modified, surface-enhanced Raman scattering (SERS)-based aptasensor in sandwich mode: a primary aptamer attached to the plasmonic surface-RBD-covered Ag nanoparticle-the Cy3-labeled secondary aptamer. Fabricated novel hybrid plasmonic structures based on "Ag mirror-SiO2-nanostructured Ag" demonstrate sensitivity for the detection of investigated analytes due to the combination of localized surface plasmons in nanostructured silver surface and the gap surface plasmons in a thin dielectric layer of SiO2 between silver layers. A specific SERS signal has been obtained from SERS-active compounds with RBD-specific DNA aptamers that selectively bind to the S protein of synthetic virion (dissociation constants of DNA-aptamer complexes with protein in the range of 10 nM). The purpose of the study is to systematically analyze the combination of components in an aptamer-based sandwich system. A developed virus size simulating silver particles adsorbed on an aptamer-coated sensor provided a signal different from free RBD. The data obtained are consistent with the theory of signal amplification depending on the distance of the active compound from the amplifying surface and the nature of such a compound. The ability to detect the target virus due to specific interaction with such DNA is quantitatively controlled by the degree of the quenching SERS signal from the labeled compound. Developed indicator sandwich-type systems demonstrate high stability. Such a platform does not require special permissions to work with viruses. Therefore, our approach creates the promising basis for fostering the practical application of ultra-fast, amplification-free methods for detecting coronaviruses based on SARS-CoV-2.

A highly selective and sensitive competitive aptasensor capable of quantifying thrombin elevated with side effects in severe COVID-19.

To protect critical injury from blood clots with side effects in severe COVID-19, a highly selective and sensitive biosensor was developed for the quantification of trace levels of thrombin using the combination of a DNA aptamer (TBA) of thrombin and a complementary strand of TBA. TBA rapidly binds with thrombin, whereas it slowly binds with the complementary strand to form a double stranded DNA (dsDNA). SFC green intercalated into dsDNA cannot emit light in 1,1'-oxalyldiimidazole chemiluminescence (ODI-CL) reaction because high-energy intermediates formed from ODI-CL reaction cannot transfer energy to SFC trapped in dsDNA. However, SFC freely existing with the formation of G-quadruplex from the reaction of thrombin and TBA emits bright chemiluminescence because the high-energy intermediates can transfer energy to SFC (or camel) in solution. Thus, the brightness of light emitted in ODI-CL reaction was proportionally enhanced with the increase of thrombin in a sample due to the increase of G-quadruplex and reduction of dsDNA. The limit of detection (LOD) of the label free aptasensor operated with good linear calibration curve (10-320 mU/ml) was as low as 3 mU/ml (or 43 pM). Also, the biosensor was quantified trace levels of thrombin with good accuracy, precision, and reliability.

Ultrasensitive aptamer-functionalized Cu-MOF fluorescent nanozyme as an optical biosensor for detection of C-reactive protein.

In the present work, an aptasensing method based on integration of RNA on Cu-MOF was developed for detection of C-Reactive Protein (CRP). Cu-MOF showed stimulated fluorescence and mimetic peroxidase enzymatic activity at the time and can be used as dual-signal transduction. CRP binding RNA was used as a highly selective recognition element and immobilized on the Cu-MOF. The immobilized RNA can block the peroxidase activity and fluorescence of the signal traducer probe. Adding CRP to the RNA/Cu-MOF will release RNA from the surface of Cu-MOF and recover both the stimulated fluorescence and peroxidase activity. A biosensor was built for detection of CRP using the two modes of transduction, either colorimetry or fluorometry. A dynamic linear range was obtained from 0.1 to 50 ng mL -1with a limit of detection (LOD) as small as 40 pg mL -1was calculated in fluorescence mode and 240 pg mL -1 as LOD in colorimetry mode. The LODs are lower than the LOD of nephelometric techniques used in clinical practice and is comparable to the normal clinical cutoff value in high-sensitivity CRP assays (1 µg/mL). The aptasensor was successfully applied for detection of CRP in Covid-19 patients with spike recoveries between 84 and 102% and RSD from 0.94% to 2.05%.

Smart Approach for the Design of Highly Selective Aptamer-Based Biosensors.

Aptamers are chemically synthesized single-stranded DNA or RNA oligonucleotides widely used nowadays in sensors and nanoscale devices as highly sensitive biorecognition elements. With proper design, aptamers are able to bind to a specific target molecule with high selectivity. To date, the systematic evolution of ligands by exponential enrichment (SELEX) process is employed to isolate aptamers. Nevertheless, this method requires complex and time-consuming procedures. In silico methods comprising machine learning models have been recently proposed to reduce the time and cost of aptamer design. In this work, we present a new in silico approach allowing the generation of highly sensitive and selective RNA aptamers towards a specific target, here represented by ammonium dissolved in water. By using machine learning and bioinformatics tools, a rational design of aptamers is demonstrated. This "smart" SELEX method is experimentally proved by choosing the best five aptamer candidates obtained from the design process and applying them as functional elements in an electrochemical sensor to detect, as the target molecule, ammonium at different concentrations. We observed that the use of five different aptamers leads to a significant difference in the sensor's response. This can be explained by considering the aptamers' conformational change due to their interaction with the target molecule. We studied these conformational changes using a molecular dynamics simulation and suggested a possible explanation of the experimental observations. Finally, electrochemical measurements exposing the same sensors to different molecules were used to confirm the high selectivity of the designed aptamers. The proposed in silico SELEX approach can potentially reduce the cost and the time needed to identify the aptamers and potentially be applied to any target molecule.