Nucleic Acid-based Biosensors Research Articles

Polydopamine functionalized dendritic fibrous silica nanoparticles as a generic platform for nucleic acid-based biosensing

Accurate and rapid detection of nucleic acid sequences is of utmost importance in various fields, including disease monitoring, clinical treatment, gene analysis and drug discovery. In this study, we developed a "turn-on" fluorescence biosensor that enables simple and highly efficient detection of nucleic acid biomarkers. Our approach involves the utilization of 6-carboxyfluorescein modified single-stranded DNA (FAM-ssDNA) as molecular recognition element, along with polydopamine-functionalized dendritic fibrous nanosilica (DFNS). FAM-ssDNA serves as both specific molecular recognition element for the target analyte and reporter capable of transducing a detectable signal through Watson–Crick base pairing. The polydopamine-functionalized DFNS (DFNS@DA) exhibits strong binding to FAM-ssDNA via polyvalent metal mediated coordination leading to effective quenching by fluorescence resonance energy transfer. In the presence of a complementary target sequence, FAM-ssDNA forms hybridized structure and detaches from DFNS@DA, which causes an increased fluorescence emission. The analytical system based on FAM-ssDNA and DFNS@DA demonstrates exceptional sensitivity, selectivity, and rapid response for the detection of nucleic acid sequences, leveraging the high adsorption and quenching properties of DFNS@DA. For the first proof of concept, we demonstrated the successful detection of microRNA (miR-21) in cancer cells using the FAM-ssDNA/DFNS@DA system. Our results highlight the promising capabilities of DFNS@DA and nucleic acid-based biosensors, offering a generic and cost-effective solution for the detection of nucleic acid-related biomarkers.Graphical abstract

Mn2+-induced structural flexibility enhances the entire catalytic cycle and the cleavage of mismatches in prokaryotic argonaute proteins.

Prokaryotic Argonaute (pAgo) proteins, a class of DNA/RNA-guided programmable endonucleases, have been extensively utilized in nucleic acid-based biosensors. The specific binding and cleavage of nucleic acids by pAgo proteins, which are crucial processes for their applications, are dependent on the presence of Mn2+ bound in the pockets, as verified through X-ray crystallography. However, a comprehensive understanding of how dissociated Mn2+ in the solvent affects the catalytic cycle, and its underlying regulatory role in this structure-function relationship, remains underdetermined. By combining experimental and computational methods, this study reveals that unbound Mn2+ in solution enhances the flexibility of diverse pAgo proteins. This increase in flexibility through decreasing the number of hydrogen bonds, induced by Mn2+, leads to higher affinity for substrates, thus facilitating cleavage. More importantly, Mn2+-induced structural flexibility increases the mismatch tolerance between guide-target pairs by increasing the conformational states, thereby enhancing the cleavage of mismatches. Further simulations indicate that the enhanced flexibility in linkers triggers conformational changes in the PAZ domain for recognizing various lengths of nucleic acids. Additionally, Mn2+-induced dynamic alterations of the protein cause a conformational shift in the N domain and catalytic sites towards their functional form, resulting in a decreased energy penalty for target release and cleavage. These findings demonstrate that the dynamic conformations of pAgo proteins, resulting from the presence of the unbound Mn2+ in solution, significantly promote the catalytic cycle of endonucleases and the tolerance of cleavage to mismatches. This flexibility enhancement mechanism serves as a general strategy employed by Ago proteins from diverse prokaryotes to accomplish their catalytic functions and provide useful information for Ago-based precise molecular diagnostics.

Survey of Conductive Polymers for the Fabrication of Conformation Switching Nucleic Acid-Based Electrochemical Biosensors

Survey of Conductive Polymers for the Fabrication of Conformation Switching Nucleic Acid-Based Electrochemical Biosensors

Studies on Biosensors- Recent Developments and Future Perspectives on Biotechnological Applications: An Overview

The current review compiles the recent trends and future applications of biosensors in many fields of Biotechnology to detect various biological and biochemical entities in the fields of environmental biotechnology, food biotechnology, medical science and agriculture biotechnology. A biosensor is a an analytical or bioreceptor-integrated device that determines the concentration of analyte in a medium and provides quantitative information. The biosensors that monitors the air, soil and water are whole cell-based biosensors that use microbial cell to detect polycyclic aromatic hydrocarbons (PAH), heavy metals, waterborne bacterial pathogens, measure indoor air quality (IAQ), etc. In this review, plant diseases caused by various pathogens are detected by nucleic acid-based affinity biosensors. In this article, authors also focus on medical-based biosensors that monitor the concentration of various biomolecules such as human chorionic gonadotropin hormone (hCG), glucose, etc. to detect pregnancy and other diseases. Apart from this, it also includes food technology and processing-based biosensors to determine and verify the food quality by detecting microbial contaminants, nutrient contents and product freshness by detecting biogenic amines, lactic acid, etc. This review provides new insights into developing certain fascinating and economically interesting models likely to be eco-friendly. GRAPHICAL ABSTRACT

Nucleic acid-based electrochemical biosensor for detection of influenza B by gold nanoparticles.

The influenza virus is a pervasive pathogen that exhibits increased prevalence during colder seasons, resulting in a significant annual occurrence of infections. Notably, pharmaceutical interventions effective against influenza A strains often exhibit limited efficacy against influenza B variants. Against this backdrop, the need for innovative approaches to accurately and swiftly differentiate and detect influenza B becomes evident. Biosensors play a pivotal role in this detection process, offering rapid, specific, and sensitive identification of the virus, facilitating timely intervention and containment efforts. Oligonucleotide sequences targeting the conserved B/Victoria/2/87 influenza virus NP region were designed. Nasopharyngeal swabs were collected from patients suspected of influenza virus infection, and viral RNA was extracted. RNA quality was assessed through one-step PCR. cDNA synthesis was performed using random hexamers, and real-time PCR quantified the influenza genome. Gold nanoparticles were immobilized on a surface to immobilize the specific DNA probe, and electrochemical hybridization was electrochemically followed. The biosensor exhibited high selectivity and effective distinction of complementary sequences from mismatches and influenza virus cDNA genome. The biosensor successfully detected the influenza B virus genome in real samples. Non-influenza samples yielded no significant hybridization signals. The comparison between the results obtained from the biosensor and real-time PCR revealed full agreement of these methods. The biosensor utilized electrochemical detection of hybridization and proved effective in detecting the influenza B virus genome with high specificity, sensitivity, and selectivity. Comparative analysis with real-time PCR underscored the accuracy and potential applicability of the biosensor in rapid and specific virus detection. This innovative approach holds promise for future diagnostic and epidemiological applications in detecting influenza B virus and other pathogens.

Functional nucleic acid-based biosensors for virus detection

A growing number of viruses pose significant threats to human health, impact agricultural production and compromise food safety. Minimizing the impact of viral infections demands for effective technologies that can deliver sensitive viral detection under various settings. Functional nucleic acids (FNAs) – natural or synthetic nucleic acid molecules with catalytic and/or binding properties – have been increasingly explored as key components for setting up diagnostic tests for viral detection. FNA-based viral biosensors offer distinct advantages, such as the ability to achieve sensitive viral detection in complex biological samples, ease of use, scalability, and cost-effectiveness. This mini-review will be dedicated to appraising progress made in recent years in FNA-based biosensors for diagnosing diverse human and animal viruses. We will first describe virus structures and potential biomarkers that can be utilized as the targets for FNA probe engineering. This is followed by reviewing the latest advances in two classes of FNAs - DNAzyme and DNA aptamers, with focuses on DNAzyme- and DNA aptamer-based biosensors that employ diverse signal transduction mechanisms to achieve sensitive viral detections. Finally, we will discuss current challenges in the field and suggest future research directions that can address these challenges.

Nanotechnology-Assisted Biosensors for the Detection of Viral Nucleic Acids: An Overview.

The accurate and rapid diagnosis of viral diseases has garnered increasing attention in the field of biosensors. The development of highly sensitive, selective, and accessible biosensors is crucial for early disease detection and preventing mortality. However, developing biosensors optimized for viral disease diagnosis has several limitations, including the accurate detection of mutations. For decades, nanotechnology has been applied in numerous biological fields such as biosensors, bioelectronics, and regenerative medicine. Nanotechnology offers a promising strategy to address the current limitations of conventional viral nucleic acid-based biosensors. The implementation of nanotechnologies, such as functional nanomaterials, nanoplatform-fabrication techniques, and surface nanoengineering, to biosensors has not only improved the performance of biosensors but has also expanded the range of sensing targets. Therefore, a deep understanding of the combination of nanotechnologies and biosensors is required to prepare for sanitary emergencies such as the recent COVID-19 pandemic. In this review, we provide interdisciplinary information on nanotechnology-assisted biosensors. First, representative nanotechnologies for biosensors are discussed, after which this review summarizes various nanotechnology-assisted viral nucleic acid biosensors. Therefore, we expect that this review will provide a valuable basis for the development of novel viral nucleic acid biosensors.

Recent Progress on Optical Biosensors Developed for Nucleic Acid Detection Related to Infectious Viral Diseases.

Optical biosensors have many advantages over traditional analytical methods. They enable the identification of several biological and chemical compounds directly, instantly, and without the need of labels. Their benefits include excellent specificity, sensitivity, compact size, and low cost. In this review, the main focus is placed on the nucleic acid-based optical biosensor technologies, including colorimetric, fluorescence, surface plasmon resonance (SPR), Evanescent-Wave Optical, Fiber optic and bioluminescent optical fibre. The fundamentals of each type of biosensor are briefly explained, and particular emphasis has been placed on the achievements which have been gained in the last decade on the field of diagnosis of infectious viral diseases. Concluding remarks concerning the perspectives of further developments are discussed.

Microfluidic Chip for the Electrochemical Detection of MicroRNAs: Methylene Blue Increasing the Specificity of the Biosensor.

MicroRNAs (miRNAs) are biomarkers involved in biological processes that are released by cells and found in biological fluids such as blood. The development of nucleic acid-based biosensors has significantly increased in the past 10 years because the detection of such nucleic acids can easily be applied in the field of early diagnosis. These biosensors need to be sensitive, specific, and fast in order to be effective. This work introduces a newly-built electrochemical biosensor that enables a fast detection in 30 min and, as a result of its integration in microfluidics, presents a limit of detection as low as 1 aM. The litterature concerning the specificity of electrochemical biosensors includes several studies that report one base-mismatch, with the base-mismatch located in the middle of the strand. We report an electrochemical nucleic acid biosensor integrated into a microfluidic chip, allowing for a one-base-mismatch specificity independently from the location of the mismatch in the strand. This specificity was improved using a solution of methylene blue, making it possible to discriminate a partial hybridization from a complete and complementary hybridization.

Directional migration propensity of calf thymus DNA in a gradient of metal ions.

Herein we report the migrational behaviour and spatial distribution of calf thymus DNA in a gradient of different physiologically relevant monovalent and divalent cations under two different conditions - (i) microfluidic and (ii) evaporating droplets. Amplified phoresis toward high concentrations of Mg2+ and Ca2+ compared to other ions and non-uniform DNA coating in a gradient of ions were observed. This process, which was governed by the effective charge and diffusion coefficient of the DNA-metal ion complex, can have potential applications in nucleic acid-based spatiotemporal surface patterning, biosensors, and dynamic biocolloidal assembly and transport.

Nucleic acid-based biosensors: analytical devices for prevention, diagnosis and treatment of diseases

BACKGROUND : Biosensing techniques have been the subject of exponentially increasing interest due to their performance advantages such as high selectivity and sensitivity, easy operation, low cost, short analysis time, simple sample preparation, and real-time detection. Biosensors have been developed by integrating the unique specificity of biological reactions and the high sensitivity of physical sensors. Therefore, there has been a broad scope of applications for biosensing techniques, and nowadays, they are ubiquitous in different areas of environmental, healthcare, and food safety. Biosensors have been used for environmental studies, detecting and quantifying pollutants in water, air, and soil. Biosensors also showed great potential for developing analytical tools with countless applications in diagnosing, preventing, and treating diseases, mainly by detecting biomarkers. Biosensors as a medical device can identify nucleic acids, proteins, peptides, metabolites, etc.; these analytes may be biomarkers associated with the disease status. Bacterial food contamination is considered a worldwide public health issue; biosensor-based analytical techniques can identify the presence or absence of pathogenic agents in food. OBJECTIVES: The present review aims to establish state-of-the-art, comprising the recent advances in the use of nucleic acid-based biosensors and their novel application for the detection of nucleic acids. Emphasis will be given to the performance characteristics, advantages, and challenges. Additionally, food safety applications of nucleic acid-based biosensors will be discussed. METHODS: Recent research articles related to nucleic acid-based biosensors, biosensors for detecting nucleic acids, biosensors and food safety, and biosensors in environmental monitoring were reviewed. Also, biosensing platforms associated with the clinical diagnosis and food industry were included. RESULTS: It is possible to appreciate that multiple applications of nucleic acid-based biosensors have been reported in the diagnosis, prevention, and treatment of diseases, as well as to identify foodborne pathogenic bacteria. The use of PNA and aptamers opens the possibility of developing new biometric tools with better analytical properties. CONCLUSIONS: Biosensors could be considered the most important tool for preventing, treating, and monitoring diseases that significantly impact human health. The aptamers have advantages as biorecognition elements due to the structural conformation, hybridization capacity, robustness, stability, and lower costs. It is necessary to implement biosensors in situ to identify analytes with high selectivity and lower detection limits.

Oxidative Cleavage-Based Three-Dimensional DNA Biosensor for Ratiometric Detection of Hypochlorous Acid and Myeloperoxidase.

Methods to detect and quantify disease biomarkers with high specificity and sensitivity in biological fluids play a key role in enabling clinical diagnosis, including point-of-care testing. Myeloperoxidase (MPO) is an emerging biomarker for the detection of inflammation, neurodegenerative diseases, and cardiovascular disease, where excess MPO can lead to oxidative damage to biomolecules in homeostatic systems. While numerous methods have been developed for MPO analysis, most techniques are challenging in clinical applications due to the lack of amplification methods, high cost, or other practical drawbacks. Enzyme-linked immunosorbent assays are currently used for the quantification of MPO in clinical practice, which is often limited by the availability of antibodies with high affinity and specificity and the significant nonspecific binding of antibodies to the analytical surface. In contrast, nucleic acid-based biosensors are of interest because of their simplicity, fast response time, low cost, high sensitivity, and low background signal, but detection targets are limited to nucleic acids and non-nucleic acid biomarkers are rare. Recent studies reveal that the modification of a genome in the form of phosphorothioate is specifically sensitive to the oxidative effects of the MPO/H2O2/Cl- system. We developed an oxidative cleavage-based three-dimensional DNA biosensor for rapid, ratiometric detection of HOCl and MPO in a "one-pot" method, which is simple, stable, sensitive, specific, and time-saving and does not require a complex reaction process, such as PCR and enzyme involvement. The constructed biosensor has also been successfully used for MPO detection in complex samples. This strategy is therefore of great value in disease diagnosis and biomedical research.

An appraisal of various pathogen detection methods in eggs and poultry

Abstract: To ensure safety in egg and poultry products, timely detection of pathogenic microbes is of paramount importance. This review offers an appraisal of different routinely used and novel emerging pathogen detection methods in egg, poultry and their products. Timely detection of pathogens is decisive to curtail outbreak risks, reduce hospitalisation, and provide product assurance. It will also reduce the cost of holding food products in cold storage and reduces product recalls. Some crucial issues need to be taken care of in choosing or developing a foodborne pathogen detection method. They are requirement of costly or sophisticated equipment, portability, trained personnel, viable but non-culturable bacteria (may give false-negative results), dead microbes (may give false-positive results), stressed or sub-lethally damaged bacteria and slow-growing microbes (require enrichment). In this review, the focus has been given on culture-based methods, nucleic acid-based methods, immunological methods and biosensor based methods. Keywords: Egg; poultry; detection methods; product assurance; safety.

Immunomagnetic separation: An effective pretreatment technology for isolation and enrichment in food microorganisms detection.

The high efficiency and accurate detection of foodborne pathogens and spoilage microorganisms in food are a task of great social, economic, and public health importance. However, the contamination levels of target bacteria in food samples are very low. Owing to the background interference of food ingredients and negative impact of nontarget flora, the establishment of efficient pretreatment techniques is very crucial for the detection of food microorganisms. With the significant advantages of high specificity and great separation efficiency, immunomagnetic separation (IMS) assay based on immunomagnetic particles (IMPs) has been considered as a powerful system for the separation and enrichment of target bacteria. This paper mainly focuses on the development of IMS as well as their application in food microorganisms detection. First, the basic principle of IMS in the concentration of food bacteria is presented. Second, the effect of different factors, including the sizes of magnetic particles (MPs), immobilization of antibody and operation parameters (the molar ratio of antibody to MPs, the amount of IMPs, incubation time, and bacteria concentration) on the immunocapture efficiency of IMPs are discussed. The performance of IMPs in different food samples is also evaluated. Finally, the combination of IMS and various kinds of detection methods (immunology-based methods, nucleic acid-based methods, fluorescence methods, and biosensors) to detect pathogenic and spoilage organisms is summarized. The challenges and future trends of IMS are also proposed. As an effective pretreatment technique, IMS can improve the detection sensitivity and shorten their testing time, thus exhibiting broad prospect in the field of food bacteria detection.

A smart deoxyribozyme-based fluorescent sensor for in vitro detection of androgen receptor mRNA.

Nowadays a variety of biosensors are widely used in different fields, including biomedical diagnostics and self-testing. Nucleic acid-based biosensors are typically applied to detect another nucleic acid, proteins, ions, and several other types of compounds. It is most promising to develop simple and effective biosensors for the use in situations where traditional methods are not available due to their complexity and laboriousness. In this project, a novel smart deoxyribozyme-based fluorescent sensor for the detection of androgen receptor mRNA was developed. It consists of several functional modules including two deoxyribozymes 10–23, an RNA-dependent split malachite green aptamer, and an oligonucleotide platform. Deoxyribozymes specifically release a 27-nucleotide RNA fragment that is readily available for the interaction with the aptamer module. This solves a problem of secondary structure in hybridization with the target sequence of full-length mRNA. It was shown that within 24 hours the proposed sensor specifically recognized both a synthetic 60-nucleotide RNA fragment (LOD is 1.4 nM of RNA fragment at 37 °C) and a full-sized mRNA molecule of the androgen receptor. The constructed sensor is easy to use, has high efficiency and selectivity for the RNA target, and can be reconstructed for the detection of various nucleic acid sequences due to its modular structure. Thus, similar biosensors may be useful for the differential diagnosis.

A carbon nanoparticle and DNase I-Assisted amplified fluorescent biosensor for miRNA analysis

Nucleic acid-based biosensors have become powerful tools in biomedical applications. But the stability issue seriously limits their wide applications. Fortunately, the emergence of carbon nanoparticles (CNPs), which can effectively protect DNA probes from enzymatic digestion and unspecific protein binding, provides a good solution. In this work, a DNase I-aided cyclic enzymatic amplification method (CEAM) for microRNA analysis has been developed based on the coupling use of nucleic acid probes with specific molecular recognition ability as well as CNPs with excellent biostability. The method is simple and sensitive, with a detection limit down to 3.2 pM. Furthermore, satisfactory results are achieved for miRNA analysis in breast cancer cell lysate, demonstrating the applicability in disease diagnosis. The ingenious combination of CNPs and nucleic acid probes can open a new chapter in the development of versatile analytical strategies that holds great potentials for clinical diagnosis, food safety, and environmental monitoring.

Review—Chemical and Biological Sensors for Viral Detection

Infectious diseases commonly occur in contaminated water, food, and bodily fluids and spread rapidly, resulting in death of humans and animals worldwide. Among infectious agents, viruses pose a serious threat to public health and global economy because they are often difficult to detect and their infections are hard to treat. Since it is crucial to develop rapid, accurate, cost-effective, and in-situ methods for early detection viruses, a variety of sensors have been reported so far. This review provides an overview of the recent developments in electrochemical sensors and biosensors for detecting viruses and use of these sensors on environmental, clinical and food monitoring. Electrochemical biosensors for determining viruses are divided into four main groups including nucleic acid-based, antibody-based, aptamer-based and antigen-based electrochemical biosensors. Finally, the drawbacks and advantages of each type of sensors are identified and discussed.

Peptide nucleic acid-based electrochemical biosensor for simultaneous detection of multiple microRNAs from cancer cells with catalytic hairpin assembly amplification

Herein we demonstrated a facile electrochemical method for simultaneous detection of miRNA21 and miRNA155 using a peptide nucleic acids (PNAs)-modified gold electrode coupled with the target-catalyzed hairpin assembly (CHA) strategy. In the presence of the target miRNA, the CHA was triggered selectively between two hairpins with one ferrocene (Fc) or methylene blue (MB) labelled. The resulting redox-active group modified CHA products (Fc-CHA21 or MB-CHA155) were then specifically captured by the PNA probes (PNA21 or PNA155) attached on the surface of a gold electrode, which bring the Fc and MB labels into close proximity to generate apparently enhanced electrochemical signals for sensitive and simultaneous detecting of low amount miRNA21 and miRNA155 in cancer cells. This assay was highly selective for discriminating miRNAs with similar sequences and has detection limits of 2.49 fM and 11.63 fM for miRNA21 and miRNA155, respectively. The feasibility of the method for sensitive determination of miRNA21 and miRNA155 from human cancer cells was also demonstrated. This method thus has great potential to be applied for simultaneous detecting of a variety of miRNA biomarkers for clinic applications due to its simple, sensitive and accurate features.

Rationally Engineered Nucleic Acid Architectures for Biosensing Applications.

Development of biosensing platforms plays a key role in research settings for identification of biomarkers and in clinical applications for diagnostics. Biosensors based on nucleic acids have taken many forms, from simple duplex-based constructs to stimuli-responsive nucleic acid nanostructures. In this review, we look at various nucleic acid-based biosensors, the different read-out strategies employed, and their use in chemical and biological sensing. We also look at current developments in DNA nanotechnology-based biosensors and how rational design of such constructs leads to more efficient biosensing platforms.

Anomalous Trends in Nucleic Acid-Based Electrochemical Biosensors with Nanoporous Gold Electrodes.

Molecular diagnostics have significantly advanced the early detection of diseases, where electrochemical sensing of biomarkers has shown considerable promise. For a nucleic acid-based electrochemical sensor with signal-off behavior, the performance is evaluated by percent signal suppression (% ss), which indicates the change in current after hybridization. The % ss is generally due to more redox molecules (e.g., methylene blue) associating with the probe DNA bases in the single-strand form than the double-strand form upon hybridization with the target nucleic acid. Nanostructured electrodes generally enhance electrochemical sensor performance via several mechanisms, including increased number of capture probes per electrode volume and unique nanoscale transport phenomena. Here, we employ nanoporous gold (np-Au) as a model electrode material to study the influence of probe immobilization solution concentration on sensor performance and the underlying mechanisms. Unlike planar gold (pl-Au) electrodes, where % ss reaches a steady state with increasing concentration of the grafting solution, the % ss displays peak performance at certain grafting solution concentrations followed by rapid deterioration and reversal of the % ss polarity, suggesting an unexpected case of increased charge transfer upon hybridization. Fluorometric assessments of electrochemically desorbed nucleic acids for different electrode morphologies reveal that a significant amount of DNA molecules (unhybridized and hybridized) remain within the nanopores posthybridization. Analysis of electrochemical signals (e.g., square wave voltammogram shape) suggests that the large unbound nucleic acid concentration may be altering the modes of methylene blue interaction with the nucleic acids and charge transfer to the electrode surfaces.