scholarly journals Design of a High-Sensitivity Dimeric G-Quadruplex/Hemin DNAzyme Biosensor for Norovirus Detection

Molecules ◽  
2021 ◽  
Vol 26 (23) ◽  
pp. 7352
Author(s):  
Yun Zhang ◽  
Xinao Ma ◽  
Jingtian Zhang ◽  
Feixian Luo ◽  
Wenshu Wang ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Operating Cost ◽  
High Sensitivity ◽  
Cost Effective ◽  
Sequence Detection ◽  
Conserved Sequence ◽  
Synergistic Enhancement ◽  
Target Dna ◽  
G Quadruplex ◽  
Dna Strand

G-quadruplexes can bind with hemin to form peroxidase-like DNAzymes that are widely used in the design of biosensors. However, the catalytic activity of G-quadruplex/hemin DNAzyme is relatively low compared with natural peroxidase, which hampers its sensitivity and, thus, its application in the detection of nucleic acids. In this study, we developed a high-sensitivity biosensor targeting norovirus nucleic acids through rationally introducing a dimeric G-quadruplex structure into the DNAzyme. In this strategy, two separate molecular beacons each having a G-quadruplex-forming sequence embedded in the stem structure are brought together through hybridization with a target DNA strand, and thus forms a three-way junction architecture and allows a dimeric G-quadruplex to form, which, upon binding with hemin, has a synergistic enhancement of catalytic activities. This provides a high-sensitivity colorimetric readout by the catalyzing H2O2-mediated oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline -6-sulfonic acid) diammonium salt (ABTS). Up to 10 nM of target DNA can be detected through colorimetric observation with the naked eye using our strategy. Hence, our approach provides a non-amplifying, non-labeling, simple-operating, cost-effective colorimetric biosensing method for target nucleic acids, such as norovirus-conserved sequence detection, and highlights the further implication of higher-order multimerized G-quadruplex structures in the design of high-sensitivity biosensors.

scholarly journals Simple, sensitive and cost-effective detection of wAlbB Wolbachia in Aedes mosquitoes, using loop mediated isothermal amplification combined with the electrochemical biosensing method

2021 ◽  
Author(s):  
Pattamaporn Kittayapong ◽  
Parinda Thayanukul ◽  
Benchaporn Lertanantawong ◽  
Worachart Sirawaraporn ◽  
Surat Charasmongkolcharoen ◽  
...  
Keyword(s):  
Low Cost ◽  
High Sensitivity ◽  
Cost Effective ◽  
Isothermal Amplification ◽  
Loop Mediated Isothermal Amplification ◽  
Control Programs ◽  
Electrochemical Biosensing ◽  
Dna Strand Displacement ◽  
Displacement Based ◽  
Dna Strand

Background Wolbachia is an endosymbiont bacterium generally found in about 40% of insects, including mosquitoes, but it is absent in Aedes aegypti which is an important vector of several arboviral diseasesdengue, chikungunya, Zika, and yellow fever. The evidence that Wolbachia trans-infected Ae. aegypti mosquitoes lost their vectorial competence and became less capable of transmitting arboviruses to human hosts highlights the potential of using Wolbachia - based approaches for prevention and control of arboviral diseases. Recently, release of Wolbachia trans-infected Ae. aegypti has been deployed widely in many countries for the control of mosquito-borne viral diseases. Field surveillance and monitoring of Wolbachia presence in released mosquitoes is important for the success of these control programs. So far, a number of studies have reported the development of loop mediated isothermal amplification (LAMP) assays to detect Wolbachia in mosquitoes, but the methods still have some specificity issues. Methodology/Principal Findings We describe here the development of a LAMP combined with the DNA strand displacement-based electrochemical sensor (BIOSENSOR) method to detect wAlbB Wolbachia in trans-infected Ae . aegypti . Our developed LAMP primers were more specific to wAlbB detection than those of the previous published ones if  the assays were conducted with low-cost and non-specific detecting dyes. The detection capacity of our LAMP technique was 3.8 nM and the detection limit reduced to 2.16 fM when combined with the BIOSENSOR. Our study demonstrates that the BIOSENSOR can also be applied as a stand-alone method for detecting Wolbachia ; and it showed high sensitivity when used with the crude DNA extracts of macerated mosquito samples without DNA purification. Conclusions/Significance Our results suggest that both LAMP and BIOSENSOR, either used in combination or stand-alone, are robust and sensitive. The methods have good potential for routine detection of Wolbachia in mosquitoes during field surveillance and monitoring of Wolbachia -based release programs, especially in countries with limited resources.

scholarly journals An Enzyme- and Label-Free Fluorescence Aptasensor for Detection of Thrombin Based on Graphene Oxide and G-Quadruplex

Sensors ◽  
2019 ◽  
Vol 19 (20) ◽  
pp. 4424 ◽  
Author(s):  
Yani Wei ◽  
Luhui Wang ◽  
Yingying Zhang ◽  
Yafei Dong
Keyword(s):  
Graphene Oxide ◽  
Cost Effective ◽  
Target Pair ◽  
Label Free ◽  
Detection Scheme ◽  
G Quadruplex ◽  
Universal Detection ◽  
Dna Strand ◽  
Aptamer Sequence

An enzyme- and label-free aptamer-based assay is described for the determination of thrombin. A DNA strand (S) consisting of two parts was designed, where the first (Sa) is the thrombin-binding aptamer and the second (Se) is a G-quadruplex. In the absence of thrombin, Sa is readily adsorbed by graphene oxide (GO), which has a preference for ss-DNA rather than for ds-DNA. Upon the addition of the N-methyl-mesoporphyrin IX (NMM), its fluorescence (with excitation/emission at 399/610 nm) is quenched by GO. In contrast, in the presence of thrombin, the aptamer will bind thrombin, and thus, be separated from GO. As a result, fluorescence will be enhanced. The increase is linear in the 0.37 µM to 50 µM thrombin concentration range, and the detection limit is 0.37 nM. The method is highly selective over other proteins, cost-effective, and simple. In our perception, it represents a universal detection scheme that may be applied to other targets according to the proper choice of the aptamer sequence and formation of a suitable aptamer-target pair.

scholarly journals Molecular mechanism of off-target effects in CRISPR-Cas9

2018 ◽  
Author(s):  
Clarisse G. Ricci ◽  
Janice S. Chen ◽  
Yinglong Miao ◽  
Martin Jinek ◽  
Jennifer A. Doudna ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Md Simulations ◽  
Target Sequence ◽  
Protospacer Adjacent Motif ◽  
Target Dna ◽  
Dna Mismatches ◽  
Open Issue ◽  
Conformational Freedom ◽  
Target Binding ◽  
Dna Strand

AbstractCRISPR-Cas9 is the state-of-the-art technology for editing and manipulating nucleic acids. However, the occurrence of off-target mutations can limit its applicability. Here, all-atom enhanced molecular dynamics (MD) simulations – using Gaussian accelerated MD (GaMD) – are used to decipher the mechanism of off-target binding at the molecular level. GaMD reveals that base pair mismatches in the target DNA at specific distal sites with respect to the Protospacer Adjacent Motif (PAM) induce an extended opening of the RNA:DNA heteroduplex, which leads to newly discovered interactions between the unwound nucleic acids and the protein counterpart. The conserved interactions between the target DNA strand and the L2 loop of the catalytic HNH domain constitute a “lock” effectively decreasing the conformational freedom of the HNH domain and its activation for cleavage. Remarkably, depending on their position at PAM distal sites, DNA mismatches leading to off-target cleavages are unable to “lock” the HNH domain, thereby identifying the ability to “lock” HNH as a key determinant. Consistently, off-target sequences hampering the catalysis have been shown to “trap” somehow the HNH domain in an inactive “conformational checkpoint” state (Dagdas et al. Sci Adv, 2017). As such, this mechanism identifies the molecular basis underlying off-target cleavages and contributes in clarifying a long-lasting open issue of the CRISPR-Cas9 function. It also poses the foundation for designing novel and more specific Cas9 variants, which could be obtained by magnifying the “locking” interactions between HNH and the target DNA in the presence of any incorrect off-target sequence, thus preventing undesired cleavages.

scholarly journals Kinetics of DNA Strand Displacement Systems with Locked Nucleic Acids

2017 ◽  
Vol 121 (12) ◽  
pp. 2594-2602 ◽  
Author(s):  
Xiaoping Olson ◽  
Shohei Kotani ◽  
Bernard Yurke ◽  
Elton Graugnard ◽  
William L. Hughes
Keyword(s):  
Nucleic Acids ◽  
Strand Displacement ◽  
Dna Strand Displacement ◽  
Locked Nucleic Acids ◽  
Dna Strand ◽  
Kinetics Of

scholarly journals Multiplexed detection of SARS-CoV-2 and other respiratory infections in high throughput by SARSeq

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ramesh Yelagandula ◽  
◽  
Aleksandr Bykov ◽  
Alexander Vogt ◽  
Robert Heinen ◽  
...  
Keyword(s):  
Influenza A ◽  
Respiratory Infections ◽  
High Sensitivity ◽  
Cost Effective ◽  
Massively Parallel ◽  
Rt Pcr ◽  
Viral Spread ◽  
Saliva Analysis ◽  
Double Blinded ◽  
Generation Sequencing

AbstractThe COVID-19 pandemic has demonstrated the need for massively-parallel, cost-effective tests monitoring viral spread. Here we present SARSeq, saliva analysis by RNA sequencing, a method to detect SARS-CoV-2 and other respiratory viruses on tens of thousands of samples in parallel. SARSeq relies on next generation sequencing of multiple amplicons generated in a multiplexed RT-PCR reaction. Two-dimensional, unique dual indexing, using four indices per sample, enables unambiguous and scalable assignment of reads to individual samples. We calibrate SARSeq on SARS-CoV-2 synthetic RNA, virions, and hundreds of human samples of various types. Robustness and sensitivity were virtually identical to quantitative RT-PCR. Double-blinded benchmarking to gold standard quantitative-RT-PCR performed by human diagnostics laboratories confirms this high sensitivity. SARSeq can be used to detect Influenza A and B viruses and human rhinovirus in parallel, and can be expanded for detection of other pathogens. Thus, SARSeq is ideally suited for differential diagnostic of infections during a pandemic.

scholarly journals Mechanistic insights into the R-loop formation and cleavage in CRISPR-Cas12i1

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Bo Zhang ◽  
Diyin Luo ◽  
Yu Li ◽  
Vanja Perčulija ◽  
Jing Chen ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dna Cleavage ◽  
Binary Complex ◽  
Loop Formation ◽  
Dna Duplex ◽  
Target Dna ◽  
Before And After ◽  
Dna Strand ◽  
Type V ◽  
Functionally Diverse

AbstractCas12i is a newly identified member of the functionally diverse type V CRISPR-Cas effectors. Although Cas12i has the potential to serve as genome-editing tool, its structural and functional characteristics need to be investigated in more detail before effective application. Here we report the crystal structures of the Cas12i1 R-loop complexes before and after target DNA cleavage to elucidate the mechanisms underlying target DNA duplex unwinding, R-loop formation and cis cleavage. The structure of the R-loop complex after target DNA cleavage also provides information regarding trans cleavage. Besides, we report a crystal structure of the Cas12i1 binary complex interacting with a pseudo target oligonucleotide, which mimics target interrogation. Upon target DNA duplex binding, the Cas12i1 PAM-interacting cleft undergoes a remarkable open-to-closed adjustment. Notably, a zipper motif in the Helical-I domain facilitates unzipping of the target DNA duplex. Formation of the 19-bp crRNA-target DNA strand heteroduplex in the R-loop complexes triggers a conformational rearrangement and unleashes the DNase activity. This study provides valuable insights for developing Cas12i1 into a reliable genome-editing tool.

scholarly journals A two-stage amplified PZT sensor for monitoring lung and heart sounds in discharged pneumonia patients

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Hongbin Chen ◽  
Shuai Yu ◽  
Haiyang Liu ◽  
Jie Liu ◽  
Yongguang Xiao ◽  
...  
Keyword(s):  
High Sensitivity ◽  
Cost Effective ◽  
High Ratio ◽  
Heart Sounds ◽  
Two Stage ◽  
Diagnosis And Prognosis ◽  
Sensor Device ◽  
Cost Effective Alternative ◽  
A Charge ◽  
Highly Sensitive Detection

AbstractAssessment of lung and heart states is of critical importance for patients with pneumonia. In this study, we present a small-sized and ultrasensitive accelerometer for continuous monitoring of lung and heart sounds to evaluate the lung and heart states of patients. Based on two-stage amplification, which consists of an asymmetric gapped cantilever and a charge amplifier, our accelerometer exhibited an extremely high ratio of sensitivity to noise compared with conventional structures. Our sensor achieves a high sensitivity of 9.2 V/g at frequencies less than 1000 Hz, making it suitable to use to monitor weak physiological signals, including heart and lung sounds. For the first time, lung injury, heart injury, and both lung and heart injuries in discharged pneumonia patients were revealed by our sensor device. Our sound sensor also successfully tracked the recovery course of the discharged pneumonia patients. Over time, the lung and heart states of the patients gradually improved after discharge. Our observations were in good agreement with clinical reports. Compared with conventional medical instruments, our sensor device provides rapid and highly sensitive detection of lung and heart sounds, which greatly helps in the evaluation of lung and heart states of pneumonia patients. This sensor provides a cost-effective alternative approach to the diagnosis and prognosis of pneumonia and has the potential for clinical and home-use health monitoring.

scholarly journals Transforming biorefinery designs with ‘Plug-In Processes of Lignin’ to enable economic waste valorization

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Zhi-Hua Liu ◽  
Naijia Hao ◽  
Yun-Yan Wang ◽  
Chang Dou ◽  
Furong Lin ◽  
...  
Keyword(s):  
Cellulosic Ethanol ◽  
Cost Effective ◽  
Substantial Improvement ◽  
Selling Price ◽  
Waste Valorization ◽  
Lignin Valorization ◽  
Capital Costs ◽  
Carbon Efficiency ◽  
Synergistic Enhancement ◽  
Total Capital

AbstractBiological lignin valorization has emerged as a major solution for sustainable and cost-effective biorefineries. However, current biorefineries yield lignin with inadequate fractionation for bioconversion, yet substantial changes of these biorefinery designs to focus on lignin could jeopardize carbohydrate efficiency and increase capital costs. We resolve the dilemma by designing ‘plug-in processes of lignin’ with the integration of leading pretreatment technologies. Substantial improvement of lignin bioconversion and synergistic enhancement of carbohydrate processing are achieved by solubilizing lignin via lowering molecular weight and increasing hydrophilic groups, addressing the dilemma of lignin- or carbohydrate-first scenarios. The plug-in processes of lignin could enable minimum polyhydroxyalkanoate selling price at as low as $6.18/kg. The results highlight the potential to achieve commercial production of polyhydroxyalkanoates as a co-product of cellulosic ethanol. Here, we show that the plug-in processes of lignin could transform biorefinery design toward sustainability by promoting carbon efficiency and optimizing the total capital cost.

scholarly journals Nanopore Technology for the Application of Protein Detection

Nanomaterials ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1942
Author(s):  
Xiaoqing Zeng ◽  
Yang Xiang ◽  
Qianshan Liu ◽  
Liang Wang ◽  
Qianyun Ma ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Detection ◽  
High Sensitivity ◽  
Single Molecule Detection ◽  
Sequence Detection ◽  
Fast Detection ◽  
Material Basis ◽  
Sensing Technology ◽  
Detection Technology ◽  
Structure Recognition

Protein is an important component of all the cells and tissues of the human body and is the material basis of life. Its content, sequence, and spatial structure have a great impact on proteomics and human biology. It can reflect the important information of normal or pathophysiological processes and promote the development of new diagnoses and treatment methods. However, the current techniques of proteomics for protein analysis are limited by chemical modifications, large sample sizes, or cumbersome operations. Solving this problem requires overcoming huge challenges. Nanopore single molecule detection technology overcomes this shortcoming. As a new sensing technology, it has the advantages of no labeling, high sensitivity, fast detection speed, real-time monitoring, and simple operation. It is widely used in gene sequencing, detection of peptides and proteins, markers and microorganisms, and other biomolecules and metal ions. Therefore, based on the advantages of novel nanopore single-molecule detection technology, its application to protein sequence detection and structure recognition has also been proposed and developed. In this paper, the application of nanopore single-molecule detection technology in protein detection in recent years is reviewed, and its development prospect is investigated.

A high sensitivity wireless mass-loading surface acoustic wave DNA biosensor

2014 ◽  
Vol 28 (07) ◽  
pp. 1450056 ◽  
Author(s):  
Hua-Lin Cai ◽  
Yi Yang ◽  
Yi-Han Zhang ◽  
Chang-Jian Zhou ◽  
Cang-Ran Guo ◽  
...  
Keyword(s):  
Acoustic Wave ◽  
Surface Acoustic Wave ◽  
Dna Sequences ◽  
Biological Treatment ◽  
High Performance ◽  
Processing System ◽  
High Sensitivity ◽  
Treatment Method ◽  
Saw Sensor ◽  
Target Dna

In this paper, a surface acoustic wave (SAW) biosensor with gold delay area on LiNbO 3 substrate detecting DNA sequences is proposed. By well-designed device parameters of the SAW sensor, it achieves a high performance for highly sensitive detection of target DNA. In addition, an effective biological treatment method for DNA immobilization and abundant experimental verification of the sensing effect have made it a reliable device in DNA detection. The loading mass of the probe and target DNA sequences is obtained from the frequency shifts, which are big enough in this work due to an effective biological treatment. The experimental results show that the biosensor has a high sensitivity of 1.2 pg/ml/Hz and high selectivity characteristic is also verified by the few responses of other substances. In combination with wireless transceiver, we develop a wireless receiving and processing system that can directly display the detection results.

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