Abstract:Herein, we have developed a rapid and enzyme-free nucleic acid amplification detection method that combined the exponential self-assembly of four DNA hairpins and the FRET pair Cy3 and Cy5. This strategy was very ingenious and rapid, and could detect nucleic acids at concentrations as low as 10 pM in 15 min in biological fluids.
“…While the sensing of biomolecules, and of nucleic acids in particular, via isothermal signal amplification has been of intense recent interest, − few examples exist for the sensing of proteins via purely DNA-based enzyme-free methodologies. Many sensing schemes require polymerase, nicking enzymes, or the incorporation of nanoparticle or bead-based approaches.…”
The sensitive detection of proteins is a key objective in many areas of biomolecular science, ranging from biophysics to diagnostics. However, sensing in complex biological fluids is hindered by nonspecific interactions with off-target species. Here, we describe and demonstrate an assay that utilizes both the chemical and physical properties of the target species to achieve high selectivity in a manner not possible by chemical complementarity alone, in complex media. We achieve this objective through a combinatorial strategy, by simultaneously exploiting free-flow electrophoresis for target selection, on the basis of electrophoretic mobility, and conventional affinity-based selection. In addition, we demonstrate amplification of the resultant signal by a catalytic DNA nanocircuit. This approach brings together the inherent solution-phase advantages of microfluidic sample handling with isothermal, enzyme-free signal amplification. With this method, no surface immobilization or washing steps are required, and our assay is well suited to monoepitopic targets, presenting advantages over conventional ELISA techniques.
“…While the sensing of biomolecules, and of nucleic acids in particular, via isothermal signal amplification has been of intense recent interest, − few examples exist for the sensing of proteins via purely DNA-based enzyme-free methodologies. Many sensing schemes require polymerase, nicking enzymes, or the incorporation of nanoparticle or bead-based approaches.…”
The sensitive detection of proteins is a key objective in many areas of biomolecular science, ranging from biophysics to diagnostics. However, sensing in complex biological fluids is hindered by nonspecific interactions with off-target species. Here, we describe and demonstrate an assay that utilizes both the chemical and physical properties of the target species to achieve high selectivity in a manner not possible by chemical complementarity alone, in complex media. We achieve this objective through a combinatorial strategy, by simultaneously exploiting free-flow electrophoresis for target selection, on the basis of electrophoretic mobility, and conventional affinity-based selection. In addition, we demonstrate amplification of the resultant signal by a catalytic DNA nanocircuit. This approach brings together the inherent solution-phase advantages of microfluidic sample handling with isothermal, enzyme-free signal amplification. With this method, no surface immobilization or washing steps are required, and our assay is well suited to monoepitopic targets, presenting advantages over conventional ELISA techniques.
“…When target DNA was present, Cy3 conjugated to hairpin H3 was brought close to Cy5 on the neighboring hairpin H4, and the energy was transferred from Cy3 to Cy5. Thus, the EHA reaction could be detected by observation of the lasing emission from Cy5 at approximate 660 nm 16 .Figure 4( a ) The principle of exponential hairpin assembly (EHA). H1, H2, H3 and H4 were four stable species of DNA hairpins for EHA reaction, and T was target DNA.…”
Section: Resultsmentioning
confidence: 99%
“…The EHA reaction system contained four hairpins H1, H2, H3, and H4 16, 17 . Here, the final concentrations of H1, H2, H3 and H4 were all 5.0 × 10 −7 M (10 mM PBS, 5 mM MgCl 2 , pH 7.4).…”
Nucleic acids have been shown to be versatile molecules and engineered to produce various nanostructures. However, the poor rate of these uncatalyzed nucleic acid reactions has restricted the development and applications. Herein, we reported a novel finding that DNA self-assembly could be nonenzymatically catalyzed by artificial agents with an increasing dissociation rate constant K2. The catalytic role of several artificial agents in DNA self-assembly was verified by real-time fluorescent detection or agarose gel electrophoresis. We found that 20% PEG 200 could significantly catalyze DNA self-assembly and increase the reaction efficiency, such as linear hybridization chain reaction (HCR) and exponential hairpin assembly (EHA). Therefore, we foresee that a fast and efficient DNA self-assembly in structural DNA nanotechnology will be desirable.
“…Moreover, nucleic acidbased biosensors for nucleic acid detection can be classified according to the biorecognition element, the structure of the bioreceptor, or the type of transducer (Figure 6). Accurate, rapid, and sensitive detection of nucleic acids in complex matrices plays an important role but remains challenging (13,104). One of the most investigated strategies for improving sensitivity is signal amplification, which refers to optimizing the biomolecules immobilized in the bioreceptor to enhance signal output and reliability (9).…”
Section: Biosensors For Nucleic Acid Detectionmentioning
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.
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