DNA stabilized fluorescent metal nanoclusters for biosensor development

The final publication is available at Elsevier via https://doi.org/10.1016/j.trac.2013.12.014." © 2014. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/Fluorescent silver, gold and copper nanoclusters (NCs) have emerged for biosensor development. Compared to semiconductor quantum dots, there is less concern about the toxicity of metal NCs, which can be more easily conjugated to biopolymers. These NCs need a stabilizing ligand. Many polymers, proteins and nucleic acids stabilize NCs, and many DNA sequences produce highly-fluorescent NCs. Coupling these DNA stabilizers with other sequences, such as aptamers, has generated a large number of biosensors. We summarize the synthesis of DNA and nucleotide-templated NCs; and, we discuss their chemical interactions. We briefly review properties of NCs, such as fluorescence quantum yield, emission wavelength and lifetime, structure and photostability. We categorize sensor-design strategies using these NCs into: (1) fluorescence de-quenching; (2) generation of templating DNA sequences to produce NCs; (3) change of nearby environment; and, (4) reacting with heavy metal ions or other quenchers. Finally, we discuss future trends.University of Waterloo || Canadian Foundation for Innovation || Ontario Ministry of Research & Innovation || Natural Sciences and Engineering Research Council |

Strand Displacement Amplification for Multiplex Detection of Nucleic Acids

The identification of various targets such as bacteria, viruses, and other cells remains a prerequisite for point-of-care diagnostics and biotechnological applications. Nucleic acids, as encoding information for all forms of life, are excellent biomarkers for detecting pathogens, hereditary diseases, and cancers. To date, many techniques have been developed to detect nucleic acids. However, most of them are based on polymerase chain reaction (PCR) technology. These methods are sensitive and robust, but they require expensive instruments and trained personnel. DNA strand displacement amplification is carried out under isothermal conditions and therefore does not need expensive instruments. It is simple, fast, sensitive, specific, and inexpensive. In this chapter, we introduce the principles, methods, and updated applications of DNA strand displacement technology in the detection of infectious diseases. We also discuss how robust, sensitive, and specific nucleic acid detection could be obtained when combined with the novel CRISPR/Cas system

Towards understanding of poly-guanine activated fluorescent silver nanoclusters

This is an author-created, un-copyedited version of an article accepted for publication in Nanotechnology. The publisher is not responsible for any errors or omissions in this version of the manuscript or any version derived from it. The Version of Record is available online at https://doi.org/10.1088/0957-4484/25/15/155501.It has been recently reported that the fluorescence of some DNA-templated silver nanoclusters (AgNCs) can be significantly enhanced upon by hybridizing with a partially complementary DNA containing a G-rich overhang near the AgNCs. This discovery has found a number of analytical applications but many fundamental questions remain to be answered. In this work, the photostability of these activated AgNCs is reported. After adding the G-rich DNA activator, the fluorescence intensity peaks in ~1 h and then starts to decay, where the decaying rate is much faster with light exposure. The lost fluorescence is recovered by adding NaBH4, suggesting that the bleaching is an oxidative process. Once activated, the G-rich activator can be removed while the AgNCs still maintain most of their fluorescence intensity. UV–vis spectroscopy suggests that new AgNC species are generated upon hybridization with the activator. The base sequence and length of the template DNA have also been varied, leading to different emission colors and color change after hybridization. G-rich aptamers can also serve as activators. Our results indicate that activation of the fluorescence by G-rich DNA could be a convenient method for biosensor development since the unstable NaBH4 is not required for the activation step.University of Waterloo || Canadian Foundation for Innovation || Natural Sciences and Engineering Research Council || Ontario Ministry of Research and Innovation |

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

BIOASSAYS FOR DISEASE-RELATED NUCLEIC ACID DETECTION

Ph.DDOCTOR OF PHILOSOPH

Synthesis and applications in immunoassays of antibody-protected nanoclusters

187 p.Los nanoclusters (NCs) han ganado atención los últimos años debido a sus propiedades ópticas y químicas dependientes del tamaño. En esta tesis se presenta el primer método para la síntesis de NCs empleando anticuerpos como andamios estructurales y sus aplicaciones en inmunoensayos. Las síntesis se realizan empleando condiciones suaves que no alteran la estructura del anticuerpo, manteniendo su estructura secundaria y sus funciones biológicas. Así, los anticuerpos modificados con NCs mantienen la afinidad por su antígeno. Los NCs resultantes según su naturaleza pueden presentar propiedades fluorescentes y fotocatalíticas (CdS NCs) o catalíticas (Ag/Pt NCs y Au/Pt NCs).Estas propiedades se han empleado para dar los primeros pasos hacia el desarrollo de un inmunoensayo homogéneo competitivo basado en FRET empleando los CdS NCs-IgG fluorescentes como donantes de energía. Los NCs bimetálicos se han empleado como anticuerpo de detección en un inmunoensayo colorimétrico tipo sándwich y se ha comparado su rendimiento con el de la comúnmente empleada enzima HRP. Empleando NCs bimetálicos se ha conseguido mejorar el límite de detección 5 veces con los Ag/Pt NCs-IgG y hasta 56 veces empleando los Au/Pt NCs-IgG en comparación con el empleo de un anticuerpo de detección marcado con HRP

Nucleic Acid Architectures for Therapeutics, Diagnostics, Devices and Materials

Nucleic acids (RNA and DNA) and their chemical analogs have been utilized as building materials due to their biocompatibility and programmability. RNA, which naturally possesses a wide range of different functions, is now being widely investigated for its role as a responsive biomaterial which dynamically reacts to changes in the surrounding environment. It is now evident that artificially designed self-assembling RNAs, that can form programmable nanoparticles and supra-assemblies, will play an increasingly important part in a diverse range of applications, such as macromolecular therapies, drug delivery systems, biosensing, tissue engineering, programmable scaffolds for material organization, logic gates, and soft actuators, to name but a few. The current exciting Special Issue comprises research highlights, short communications, research articles, and reviews that all bring together the leading scientists who are exploring a wide range of the fundamental properties of RNA and DNA nanoassemblies suitable for biomedical applications

Fluorescent ZnO−Au Nanocomposite as a Probe for Elucidating Specificity in DNA Interaction

In this work, we report the interaction of a fluorescent ZnO–Au nanocomposite with deoxyribonucleic acid (DNA), leading to AT-specific DNA interaction, which is hitherto not known. For this study, three natural double-stranded (ds) DNAs having different AT:GC compositions were chosen and a ZnO–Au nanocomposite has been synthesized by anchoring a glutathione-protected gold nanocluster on the surface of egg-shell-membrane (ESM)-based ZnO nanoparticles. The ESM-based bare ZnO nanoparticles did not show any selective interaction toward DNA, whereas intrinsic fluorescence of the ZnO–Au nanocomposite shows an appreciable blue shift (Δλmax = 18 nm) in the luminescence wavelength of 520 nm in the presence of ds calf thymus (CT) DNA over other studied DNAs. In addition, the interaction of the nanocomposite through fluorescence studies with single-stranded (ss) CT DNA, synthetic polynucleotides, and nucleobases/nucleotides (adenine, thymine, deoxythymidine monophosphate, deoxyadenosine monophosphate) was also undertaken to delineate the specificity in interaction. A minor blue shift (Δλmax = 5 nm) in the emission wavelength at 520 nm was observed for single-stranded CT DNA, suggesting the proficiency of the nanocomposite for discriminating ss and ds CT DNA. More importantly, fluorescence signals from the nano-bio-interaction could be measured directly without any modification of the target, which is the foremost advantage emanated from this study compared with other previous reports. The AT base-pair-induced enhancement was also found to be highest for the melting temperature of CT DNA (ΔTmCT = 6.7 °C). Furthermore, spectropolarimetric experiments followed by calorimetric analysis provided evidence for specificity in AT-rich DNA interaction. This study would lead to establish the fluorescent ZnO–Au nanocomposite as a probe for nanomaterial-based DNA-binding study, featuring its specific interaction toward AT-rich DNA