53 research outputs found
Design Strategies for Aptamer-Based Biosensors
Aptamers have been widely used as recognition elements for biosensor construction, especially in the detection of proteins or small molecule targets, and regarded as promising alternatives for antibodies in bioassay areas. In this review, we present an overview of reported design strategies for the fabrication of biosensors and classify them into four basic modes: target-induced structure switching mode, sandwich or sandwich-like mode, target-induced dissociation/displacement mode and competitive replacement mode. In view of the unprecedented advantages brought about by aptamers and smart design strategies, aptamer-based biosensors are expected to be one of the most promising devices in bioassay related applications
Fluorescent sensors using DNA-functionalized graphene oxide
The final publication is available at Springer via http://dx.doi.org/10.1007/s00216-014-7888-3In the past few years, graphene oxide (GO) has emerged as a unique platform for developing DNA-based biosensors, given the DNA adsorption and fluorescence-quenching properties of GO. Adsorbed DNA probes can be desorbed from the GO surface in the presence of target analytes, producing a fluorescence signal. In addition to this initial design, many other strategies have been reported, including the use of aptamers, molecular beacons, and DNAzymes as probes, label-free detection, utilization of the intrinsic fluorescence of GO, and the application of covalently linked DNA probes. The potential applications of DNA-functionalized GO range from environmental monitoring and cell imaging to biomedical diagnosis. In this review, we first summarize the fundamental surface interactions between DNA and GO and the related fluorescence-quenching mechanism. Following that, the various sensor design strategies are critically compared. Problems that must be overcome before this technology can reach its full potential are described, and a few future directions are also discussed.University of Waterloo ||
Natural Sciences and Engineering Research Council ||
Ontario Ministry of Research and Innovation ||
Foundation for Shenghua Scholar ||
National Natural Science Foundation of China || Grant No. 81301258, 21301195
Postdoctoral Science Foundation of Central South University and Hunan province ||Grant No. 124896
China Postdoctoral Science Foundation || Grant No. 2013M540644
Hunan Provincial Natural Science Foundation of China || Grant No. 13JJ4029
Specialized Research Fund for the Doctoral Program of Higher Education of China || Grant No. 2013016212007
Nanoscaled aptasensors for multi-analyte sensing
Introduction:
Nanoscaled aptamers (Aps), as short single-stranded DNA or RNA
oligonucleotides, are able to bind to their specific targets with high
affinity, upon which they are considered as powerful diagnostic and
analytical sensing tools (the so-called "aptasensors"). Aptamers are
selected from a random pool of oligonucleotides through a procedure
known as "systematic evolution of ligands by exponential enrichment".
Methods:
In this work, the most recent studies in the field of aptasensors are
reviewed and discussed with a main focus on the potential of aptasensors
for the multi-analyte detection(s).
Results:
Due to the specific folding capability of aptamers in the presence of
analyte, aptasensors have substantially successfully been exploited for
the detection of a wide range of small and large molecules (e.g., drugs
and their metabolites, toxins, and associated biomarkers in various
diseases) at very low concentrations in the biological fluids/samples
even in presence of interfering species.
Conclusion:
Biological samples are generally considered as complexes in the real
biological media. Hence, the development of aptasensors with capability
to determine various targets simultaneously within a biological matrix
seems to be our main challenge. To this end, integration of various key
scientific dominions such as bioengineering and systems biology with
biomedical researches are inevitable
Développement de nouveaux outils analytiques à base d'acides nucléiques aptamÚres pour la détection de petites molécules
La dĂ©tection de petites molĂ©cules est d'un grand intĂ©rĂȘt dans les domaines pharmaceutique, environnemental, alimentaire et de la biologie clinique. Les aptamĂšres, sĂ©lectionnĂ©s par la mĂ©thode SELEX (pour Systematic Evolution of Ligands by Exponential Enrichment), sont des oligonuclĂ©otides qui se lient Ă une cible donnĂ©e avec une affinitĂ© et une spĂ©cificitĂ© importantes. L'objectif de ce travail est d'Ă©tablir de nouvelles mĂ©thodologies analytiques basĂ©es sur l'utilisation des aptamĂšres pour la dĂ©tection de petites molĂ©cules. Dans un premier temps, une mĂ©thodologie par Ă©lectrophorĂšse capillaire, dĂ©rivĂ©e du concept de dĂ©placement du brin complĂ©mentaire de l'aptamĂšre, est dĂ©crite pour la dĂ©tection simultanĂ©e de plusieurs analytes dans un seul capillaire. La deuxiĂšme Ă©tude se focalise sur le dĂ©veloppement d'un aptacapteur colorimĂ©trique simple, rapide et peu coĂ»teux, qui utilise le concept gĂ©nĂ©ral de protection enzymatique de l'aptamĂšre et les nanoparticules d'or en tant que systĂšme de transduction. Enfin, deux mĂ©thodes par polarisation de fluorescence, basĂ©es sur le concept de dĂ©placement (du brin complĂ©mentaire ou de l'aptamĂšre lui-mĂȘme), sont prĂ©sentĂ©es afin d'accroitre les potentialitĂ©s des aptacapteurs dĂ©diĂ©s Ă la dĂ©tection des petites molĂ©cules.Small biomolecule detection is of great interest and importance in the pharmaceutical, environmental, food and clinical fields. Aptamers, selected by SELEX (Systematic Evolution of Ligands by Exponential Enrichment), are oligonucleotides that bind to a target with high affinity and specificity. The objective of the work is to establish novel methodologies of aptamer-based assays for the small biomolecule detection. In the first work, a rationalized capillary electrophoresis strategy, derived from the structure-switching aptamer concept, is described for the design of simultaneous detection of multiple analytes. The second work based on a gold nanoparticle colorimetric sensing strategy allows a rapid, label-free, homogeneous assay for small molecule using an aptamer enzymatic cleavage protection strategy. In the third work, two aptamer-based fluorescence polarization approaches, using the displacement concept, are described to improve the potentialities of the small molecule-dedicated aptasensors.SAVOIE-SCD - Bib.Ă©lectronique (730659901) / SudocGRENOBLE1/INP-Bib.Ă©lectronique (384210012) / SudocGRENOBLE2/3-Bib.Ă©lectronique (384219901) / SudocSudocFranceF
Native and Synthetic G-quartet-based DNAzyme Systems â Artificial Enzymes for Biotechnological Applications
Catalysis of chemical reactions is crucial for both chemical industry and research. However, scientists are not the first ones to use catalysts in their laboratory. In fact, they are also essential for nature which designs plenty of biocatalysts, playing a pivotal role in living systems. For a long time, it was thought that only enzymes had this property. However, since the beginning of the 1980s, it is known that ribonucleic acids (also termed RNA) can acquire this ability, making them compulsory for key reactions (e.g., for the translation of messenger RNA in the ribosome). Based on that, chemists designed several synthetic DNA catalysts (termed DNAzymes) for a large variety of reactions and applications. Among the DNA structures used, G-quadruplexes are guanine-rich noncanonical DNA structures (i.e., differing from duplex DNA) composed of native G-quartets and particularly interesting for their ability to catalyze reactions of peroxidation. This peroxidase-mimicking system found plenty of applications detailed in this chapter. Moreover, optimizations of experimental conditions are also discussed and highlight the versatility and easy-to-use characteristics of G-quadruplexes DNA. Also, synthetic G-quartets, mainly TASQ (for template-assembled synthetic G-quartets), developed by chemists showed their ability to mimic G-quadruplexes, thanks to the presence of a G-quartet. Thus, synthetic G-quartets proved their capability to catalyze peroxidase-mimicking reactions, and these new exciting nature-mimicking catalytic systems are presented in detail in this chapter
Aptasensors versus immunosensorsâWhich will prevail?
Since the invention of the first biosensors 70 years ago, they have turned into valuable and versatile tools for various applications, ranging from disease diagnosis to environmental monitoring. Traditionally, antibodies have been employed as the capture probes in most biosensors, owing to their innate ability to bind their target with high affinity and specificity, and are still considered as the gold standard. Yet, the resulting immunosensors often suffer from considerable limitations, which are mainly ascribed to the antibody size, conjugation chemistry, stability, and costs. Over the past decade, aptamers have emerged as promising alternative capture probes presenting some advantages over existing constraints of immunosensors, as well as new biosensing concepts. Herein, we review the employment of antibodies and aptamers as capture probes in biosensing platforms, addressing the main aspects of biosensor design and mechanism. We also aim to compare both capture probe classes from theoretical and experimental perspectives. Yet, we highlight that such comparisons are not straightforward, and these two families of capture probes should not be necessarily perceived as competing but rather as complementary. We, thus, elaborate on their combined use in hybrid biosensing schemes benefiting from the advantages of each biorecognition element
Development Of Molecular Biosensors And Multifunctional Graphene-Based Nanomaterials
In the first project, a simple, rapid, and reversible fluorescent DNA INHIBIT logic gate has been developed for sensing mercury (Hg2+) and iodide (I-) ions based on a molecular beacon (MB). In this logic gate, a mercury ion was introduced as the first input into the MB logic gate system to assist in the hybridization of the MB with an assistant DNA probe through the thymineâHg2+âthymine interaction, which eventually restored the fluorescence of MB as the output. With this signal-on process, mercury ions can be detected with a limit of detection as low as 7.9 nM. Furthermore, when iodide ions were added to the Hg2+/MB system as the second input, the fluorescence intensity decreased because Hg2+ in the thymineâHg2+âthymine complex was grabbed by I- due to a stronger binding force. Iodide ions can be detected with a limit of detection of 42 nM. Meanwhile, we studied the feasibility and basic performance of the DNA INHIBIT logic gate, optimized the logic gate conditions, and investigated its sensitivity and selectivity. The results showed that the MB based logic gate is highly selective and sensitive for the detection of Hg2+ and I- over other interfering cations and anions.
In the second project, an ultrasensitive and rapid turn-on fluorescence assay has been developed for the detection of 3â-5â exonuclease activity of exonuclease III (Exo III) using molecular beacons (MBs). This method has a linear detection range from 0.04 to 8.00 U mL-1 with a limit of detection of 0.01 U mL-1. In order to improve the selectivity of the method, a dual-MB system has been developed to distinguish between different exonucleases. With the introduction of two differently designed MBs which respond to different exonucleases, the T5 exonuclease, Exo III and RecJf exonucleases can be easily distinguished from each other. Furthermore, fetal bovine serum and fresh mouse serum were used as complex samples to investigate the feasibility of the dual-MB system for the detection of the enzymatic activity of Exo III. As a result, the dual-MB system showed a similar calibration curve for the detection of Exo III as in the ideal buffer solution. The designed MB probe could be a potential sensor for the detection of Exo III in biological samples.
In the third project, A sensitive label-free fluorescence assay for monitoring T4 polynucleotide kinase (T4 PNK) activity and inhibition was developed based on a coupled λ exonuclease cleavage reaction and SYBR Green I. In this assay, a double-stranded DNA (dsDNA) was stained with SYBR Green I and used as a substrate for T4 PNK. After the 5ĂÂŽ-hydroxyl termini of the dsDNA was phosphorylated by the T4 PNK, the coupled λ exonuclease began to digest the dsDNA to form mononucletides and single-stranded DNA (ssDNA). At this moment, the fluorescence intensity of the SYBR Green I decreased because less affinity with ssDNA than dsDNA. The decrease extent was proportional to the concentration of the T4 PNK. After optimization of the detection conditions, including the concentration of ATP, amount of λ exonuclease and reaction time, the activity of T4 PNK was monitored by the fluorescence measurement, with the limit of detection of 0.11 U/mL and good linear correlation between 0.25-1.00 U/mL (R2=0.9896). In this assay, no label was needed for the fluorescence detection. Moreover, the inhibition behaviours of the T4 PNKâs inhibitors were evaluated by this assay. The result indicated a potential of using this assay for monitoring of phosphorylation-related process.
In the fourth project, a facile bottom-up method for the synthesis of highly fluorescent graphene quantum dots (GQDs) has been developed using a one-step pyrolysis of a natural amino acid, L-glutamic acid, with the assistance of a simple heating mantle device. The developed GQDs showed strong blue, green and red luminescence under irradiation with ultra-violet, blue and green light, respectively. Moreover, the GQDs emitted near-infrared (NIR) fluorescence in the range 800â850 nm with an excitation-dependent manner. This NIR fluorescence has a large Stokes shift of 455 nm, providing a significant advantage for the sensitive determination and imaging of biological targets. The fluorescence properties of the GQDs, such as the quantum yields, fluorescence life times, and photostability, were measured and the fluorescence quantum yield was as high as 54.5%. The morphology and composites of the GQDs were characterized using TEM, SEM, EDS, and FT-IR. The feasibility of using the GQDs as a fluorescent biomarker was investigated through in vitro and in vivo fluorescence imaging. The results showed that the GQDs could be a promising candidate for bioimaging. Most importantly, compared to the traditional quantum dots (QDs), the GQDs are chemically inert. Thus, the potential toxicity of the intrinsic heavy metal in the traditional QDs would not be a concern for GQDs. In addition, the GQDs possessed an intrinsic peroxidase-like catalytic activity that was similar to graphene sheets and carbon nanotubes. Coupled with 2,20-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), the GQDs can be used for the sensitive detection of hydrogen peroxide with a limit of detection of 20 mM.
In the fifth project, a general, environmental-friendly, one-pot method for the fabrication of reduced graphene oxide (RGO)/metal (oxide) (e.g. RGO/Au, RGO/Cu2O, and RGO/Ag) nanocomposties was developed using glucose as the reducing agent and stabilizer. The RGO/metal (oxide) nanocomposites were characterized using STEM, FE-SEM, EDS, UV-vis absorption spectroscopy, XRD, FT-IR and Raman spectroscopy. The reducing agent, glucose, not only reduced GO effectively to RGO, but it also reduced the metal precursors to form metal (oxide) nanoparticles on the surface of RGO. Moreover, the RGO/metal (oxide) nanocomposites were stabilized by gluconic acid on the surface of RGO. Finally, the developed nanomaterials were successfully applied to simultaneous electrochemical analysis of L-ascorbic acid (L-AA), dopamine (DA) and uric acid sing RGO/Au nanocomposite as an electrode catalyst.
In the sixth project, a reduced graphene oxide/silver nanoparticle (RGO/Ag) nanocomposite using glucose as the environmental-friendly reducing agent was developed. The antibacterial activity of RGO/Ag nanocomposite was carefully investigated using Escherichia coli (E. coli) and Klebsiella pneumoniae (Kp) as bacterial models. We found that, compared with AgNPs, graphene oxide (GO) and RGO, RGO/Ag nanocomposite had higher antibacterial efficiency. Furthermore, under the near-infrared (NIR) irradiation, RGO/Ag nanocomposite demonstrated enhanced synergetic antibacterial activity through the photothermal effect. Almost 100 % of E. coli and Kp were killed by the treatment of 15 Ôg/mL and 20 Ôg/mL, respectively, with NIR irradiation. Moreover, the membrane integrity assay and ROS species assay demonstrated that RGO/Ag nanocomposite under NIR irradiation caused the cell membranes disruption and generation of ROS species, providing other possible mechanisms for their high antibacterial activity besides photothermal effect.
In the seventh project, a rigid distance spacer, silica shell, was used between GO and dyes in this work to elucidate the quenching ability of GO. First, an organic dye was doped in silica nanoparticles, followed by the modification of another layer of silica shell with a different thickness. Due to the electrostatic interaction between GO and positively charged silica nanoparticles, GO wrapped the silica nanoparticles when they were mixed together. Therefore, the distance between GO and organic dyes was adjusted by the thickness of the silica shell. The quenching efficiency of GO to two different organic dyes, including Tetramethylrhodamine (TAMRA) and Tris(bipyridine)ruthenium(II) chloride (Rubpy), was measured at various distances. This quenching ability investigation of GO to dyes with distance-dependent manner would provide a guideline for the design of the fluorescent functional composite using GO in the future.
In the eighth project, we characterized the antibacterial activity of GO in both cell culture and animal models. Klebsiella pneumoniae (Kp) is one of the most common multidrug resistant (MDR) pathogens in causing persistent nosocomial infections and is very difficult to eradicate once established in the host. First, we demonstrated that GO exerted direct killing of Kp in agar dishes and afforded the protection of alveolar macrophages (AM) from Kp infection in culture. We then evaluated the mortality, tissue damage, polymorphonuclear neutrophil (PMN) penetration, and bacterial dissemination in Kp-infected mice. Our results revealed that GO can counteract the invasive ability of Kp in vivo, resulting in lessened tissue injury, significant but subdued inflammatory response, and prolonged mouse survival. These findings indicate that GO may be an alternative agent for controlling MDR pathogens in clinics
Molecular beacon strategies for sensing purpose
The improvement of nucleic acid probes as vital molecular engineering devices will cause a noteworthy contribution to developments in bioimaging, biosensing, and disorders diagnosis. The molecular beacon (MB) which was designed by Tyagi and Kramer in 1996, are loop-stem hairpin-designed oligonucleotides armed with a quencher and a dye (also named reporter groups) at the 30 or 50 ends. This construction allows that MBs in the absence of their target complementary molecules do not fluoresce. Through hybridization with their specific targets a spontaneous configuration change on MBs occur and the dye and quencher separate from each other, resulting in emitting the fluorescence. MBs are effective probes for biosensing because of their extraordinary target-specificity, unique structure, inherent fluorescent signal transduction mechanism, low background fluorescence emission, recognition without separation, and favorable thermodynamic properties. In comparison to other probes (such as linear DNA sequences), MBs with the same number of complementary nucleotides matching their target, are multitasking probes. They have advantages of thermodynamic and photostability, flexible ability for conjugation, higher efficient intrinsic signal switching, and ultra-sensitivity. MBs not only are useful for identifying a nucleic acid target but can also be employed for recognition of various non-nucleic acid goals, including heavy metals and cations, enzymes, cells, ATP, etc. Hence, this review highlights the potential of MBs in the improvement of biosensors and their usage in detection of different analytes such as miRNA, mRNA, cocaine, methamphetamine, actin, thrombin, heavy metal and cations and so on. (C) 2020 Elsevier B.V. All rights reserved.Peer reviewe
DESIGN AND APPLICATIONS OF DNA-BASED DEVICES FOR SELF-ASSEMBLY, MOLECULAR CIRCUITS, AND SOFT MATERIALS
Biologically inspired synthetic materials have led to novel technologies due of their ability to sense, influence, or adapt to their environment. One way to build these materials and devices is to utilize the high sequence specificity and innate biocompatibility of DNA. While once considered as a material useful for only storing genetic information, DNA-based devices are now being realized as molecular tools in fields such as therapeutics, diagnostics, regenerative medicine, and soft robotics. In this dissertation, we investigate the use of DNA to build programmable tools to control self-assembly, implement molecular computation, and direct material change processes.
DNA origami nanostructures are useful tools for controlling the spatial patterns of proteins, nanoparticles, and fluorophores because they contain hundreds of independently functionalizable locations that can be engineered with nanoscale precision. However, the addressable surface area is currently limited by the size of single origami structures, and efficient, high-yield self-assembly of multiple origami into higher-order assemblies continues to be a challenge. To investigate the factors important for heterogeneous self-assembly of multiple origami, we experimentally measure the equilibrium distribution of four origami tiles in the monomer, intermediate, and final tetramer states as a function of temperature. We find that the thermodynamics of the self-assembly process is determined by the binding interface between origami. Simulations of the assembly kinetics suggest assembly occurs primarily via hierarchical pathways.
Next, we engineer a DNA-based timer circuit that can be used in computational devices for molecular release or material control. The circuit releases target DNA sequences into solution at a programmable time with a tunable, constant rate. Multiple timer circuits can operate simultaneously, each releasing their target sequences at independent rates and times.
We further develop the utility of the timer and similar DNA-based circuits as a means to control molecular events in biological environments, such as serum-supplemented cell media, where DNA-degrading nucleases can reduce the functional stability and lifetime of DNA-based devices. By implementing DNA circuit-protective design principles and by adding screening molecules to reduce nuclease activity, the functional lifetime of simple DNA circuits can be significantly increased. We develop a model by fitting parameters for reactions between nucleases and simple DNA circuits. Using the model, we can qualitatively predict the behavior of more complex circuits: multiple circuits in series and circuits containing competitive reactions.
Finally, we investigate how DNA-based circuits can be used to trigger the high-degree swelling response of DNA-crosslinked metamorphic hydrogels. By coupling signal amplification to the triggering process, we demonstrate modular control over the timescale and degree of swelling. Further, we show control over the identity of the trigger molecule using molecular translators and computational controllers capable of converting complex chemical inputs into mechanical actuation
G-Quadruplex Reporters: Structural Studies and Application for Visual and Fluorescent Detection of Point Mutations in Nucleic Acid Sequences
DNA-based diagnostics traditionally utilize hybridization probes: strands of DNA complimentary to a target sequence that, upon binding, generate a signal to indicate the presence of the target. The classic hybridization probes are the molecular beacon (MB) and TaqMan probes, both single DNA strands with a fluorophore and quencher at opposing ends. Despite their widespread use in applications such as qPCR due to their ability to multiplex with a variety of bound fluorophores, these probes have several shortcomings: temperature limitations for selective target recognition and differentiating single-nucleotide polymorphisms, intrinsic design limitations to interrogate some target sequences, and a high relative cost for synthesis and purification. A split probe design in combination with label-free reporters overcomes these shortcomings. Split probes break the target-recognition sequence into two shorter pieces, each equipped with a portion of a signal reporting unit. The shorter length both allows for the probes to efficiently function at ambient temperatures, opposed to the elevated temperatures generally required for monomer probes, and provides greater ability to discriminate single-nucleotide polymorphisms. These structures will only generate a signal if complete, which may only occur when both halves of the probe are properly bound to the target. By utilizing label-free reporters such as light-up aptamers and/or (deoxy)ribozymes, split probes offer cost-efficiency advantages over other fluorescent probes. In this dissertation, advances in the usage of split probes with G-quadruplex-based signal transducing units are detailed. An alphanumeric display comprised of tandem probes utilizing the peroxidase-like deoxyribozyme for colorimetric output demonstrates the instrument-free usage of these systems. Additionally, the promiscuous activity of the dapoxyl light-up aptamer with a variety of arylmethane, offers a label-free fluorescent split probe that is capable of discerning single-nucleotide polymorphisms without expensive chemical modifications
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