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FRET – Förster Resonance Energy Transfer Meeting the need for an up-to-date and detailed primer on all aspects of the topic, this ready reference reflects the incredible expansion in the application of FRET and its derivative techniques over the past decade, especially in the biological sciences. This wide diversity is equally mirrored in the range of expert contributors. The book itself is clearly subdivided into four major sections. The first provides some background, theory, and key concepts, while the second section focuses on some common FRET techniques and applications, such as in vitro sensing and diagnostics, the determination of protein, peptide and other biological structures, as well as cellular biosensing with genetically encoded fluorescent indicators. The third section looks at recent developments, beginning with the use of fluorescent proteins, followed by a review of FRET usage with semiconductor quantum dots, along with an overview of multistep FRET. The text concludes with a detailed and greatly updated series of supporting tables on FRET pairs and Förster distances, together with some outlook and perspectives on FRET. Written for both the FRET novice and for the seasoned user, this is a must-have resource for office and laboratory shelves.
This book provides a comprehensive review of established, cutting-edge, and future trends in the exponentially growing field of nanomaterials and their applications in biosensors and bioanalyses. Part I focuses on the key principles and transduction approaches, reviewing the timeline featuring the important historical milestones in the development and application of nanomaterials in biosensors and bioanalyses. Part II reviews various architectures used in nanobiosensing designs focusing on nanowires, one- and two-dimensional nanostructures, and plasmonic nanobiosensors with interferometric reflectance imaging. Commonly used nanomaterials, functionalization of the nanomaterials, and development of nanobioelectronics are discussed in detail in Part III with examples from screen-printed electrodes, nanocarbon films, and semiconductor quantum dots. Part IV reviews the current applications of carbon nanotubes, nanoneedles, plasmonic sensors, electrochemical scanning microscopes, and field-effect transistors with the future outlook for emerging technologies. Attention is also given to potential challenges, in particular, of taking these technologies at the point-of-need. The book concludes by providing a condensed summary of the contents, with emphasis on future directions. Nanomaterials have become an essential part of biosensors and bioanalyses in the detection and monitoring of medical, pharmaceutical, and environmental conditions, from cancer to chemical warfare agents. This book, with its distinguished editors and international team of expert contributors, will be an essential guide for all those involved in the research, design, development, and application of nanomaterials in biosensors and bioanalyses.
This book provides a broad introduction to all major aspects of quantum dot properties including fluorescence, electrochemical, photochemical and electroluminescence. Such properties have been produced for applications in biosensing, cell tracking, in vivo animal imaging and so on. It focuses on their special applications in DNA biosensing and provides readers with detailed information on the preparation and functionalization of quantum dots and the fabrication of DNA biosensors, using examples to show how these properties can be used in DNA biosensor design and the advantages of quantum dots in DNA biosensing. Further new emerging quantum dots such as metal nanoclusters and graphene dots and their applications in DNA biosensing have also been included.
The unique optical properties of quantum dots (QDs) are of interest in the development of nucleic acid diagnostics. The potential for a simultaneous two-colour diagnostic scheme for nucleic acids operating on the basis of fluorescence resonance energy transfer (FRET) has been demonstrated. Upon ultraviolet excitation, two-colours of CdSe/ZnS quantum dots with conjugated oligonucleotide probes acted as energy donors yielding FRET-sensitized acceptor emission upon hybridization with fluorophore labeled target oligonucleotides. The use of an intercalating dye to improve signal-to-noise was also demonstrated. The major limitation of the system was the non-specific adsorption of oligonucleotides, which was characterized extensively. Adsorptive interactions were found to affect the conformation of oligonucleotides conjugated to QDs, the kinetics of hybridization with QD-DNA conjugates, and the thermal stability of those hybrids. In addition, it was found that thiol-alkyl-acid capped QDs exhibited pKa correlated ligand-chromism and radiative decay rate-driven changes in quantum yield.
The unique spectroscopic properties of quantum dots (QDs) are of interest for application in intracellular studies of gene expression. QDs derivatized with single-stranded probe oligonucleotides were used to detect complementary target sequences via hybridization and fluorescence resonance energy transfer (FRET). As nucleic acid targets are not labeled within cells, a displacement assay for nucleic acid detection featuring QDs as FRET donors was developed. QDs conjugated with oligonucleotide probes and then pre-hybridized with labeled target yielded efficient FRET in vitro. Studies in vitro confirmed that displacement kinetics of pre-hybridized target was a function of the stability of the initial hybridized complex. Displacement was observed as reduction in FRET intensity coupled with regeneration of QD fluorescence. By engineering the sequence of the labeled target, faster displacement was possible. The QD-probe+target system was successfully delivered into cells via transfection. Although QDs with their cargo remained sequestered in endosomal vesicles, fluorescent properties were retained.
The potential for an electrokinetically driven, FRET-based microfluidic biosensor for SNP discrimination has been explored. The method functionalized the glass wall of a PDMS/glass microfluidics channel with multidentate thiol ligands to noncovalently immobilize MPA capped CdSe/ZnS QDs. Single stranded probe DNA could then be immobilized to QDs and target material could be delivered electrokinetically to the sensing surface. SNP discrimination could then occur by manipulation of shear, electrical and thermal effects derived from the applied voltage. The stability of immobilized QDs was investigated by EOF experiments that applied 500 V and 100 V voltages for 10 minutes to initiate electrokintetic flow. Fluorescence intensity measurements showed nearly complete removal of QDs from slides when compared with controls at both voltages. Pressure driven flow experiments demonstrated reduced dissociation of immobilized of QDs compared to channels exposed to EOF. A covalent approach is likely necessary to ensure stability of immobilized QDs during EOF.
The research that is presented herein explores the development of a solid-phase DNA hybridization assay in an electrokinetically controlled biochip. Transduction of nucleic acid hybridization is accomplished by fluorescence resonance energy transfer (FRET) from a layer of immobilized quantum dots (QDs) in microfluidic channels. The chip assay platform was assembled as a composite of glass and polydimethylsiloxane (PDMS), where the glass surface was functionalized with immobilization chemistry to support the spontaneous assembly of QDs. Probe oligonucleotides were subsequently conjugated to the immobilized QDs and hybridization served as the selective interaction for target binding. Since the entire microchannel was derivatized with the transduction element, hybridization of dye labeled oligonucleotides along the channel length created well defined spatial profiles of FRET sensitized acceptor emission. The length of the FRET spatial profiles was related to the quantity of nucleic acid delivered and enabled quantitative transduction of tens of fmol amounts of target within minutes. This chip based assay offered multiplexed analysis where the concurrent detection of two targets was possible with a dynamic range spanning more than an order of magnitude. The robustness of the assay was demonstrated by transduction of nucleic acid targets in a variety of complex matrices including sample solutions that contained an excess of genomic DNA and also serum proteins. Furthermore, the assay offered excellent selectivity toward determination of the presence of a single nucleotide polymorphism (SNP), with contrast ratios exceeding 100:1. A non-traditional approach to SNP transduction was explored, where the size of the QD was found to impact the stringency of interfacial hybridization. Detection of unlabeled target oligonucleotide using a sandwich assay approach enabled transduction of oligonucleotides up to 40 nucleotides in length. The research described herein advances the development of a selective interfacial transduction strategy for the detection of nucleic acid markers such as might be characteristic of disease or pathogens. The on-chip assay format is amenable to point-of-care analysis and can be combined with nucleic acid amplification technologies for highly sensitive on demand DNA analysis.
Combining high sensitivity with simultaneous analysis of numerous biomarkers (multiplexing) is an essential requirement for significantly improving the field of biomedical diagnostics. Such progresses would allow earlier diagnosis, which is required for numerous diseases such as cancer or cardiac diseases. FRET-immunoassays are based on biomolecular recognition events that occur between biomarkers and two specific antibodies conjugated with different fluorophores. The spatial proximity of the two fluorophores can lead to Förster resonance energy transfer (FRET), which can be detected for biomarker quantification. To date, such assays are established using lanthanide complexes as FRET donors and fluorescence dyes as FRET acceptors. However, these assays do not provide sufficient multiplexing capability due to spectral overlap, when several acceptor dyes are used. This project aims at exploiting the exceptional photophysical properties of terbium complexes (Tb) and semiconductor quantum dots (QDs) to provide ultrasensitive multiplexed FRETimmunoassays. We also studied the optical and morphological properties of novel core and core/shell upconverting nanoparticles doped with ytterbium (Yb) and erbium (Er) ions as possible FRET-donors for biosensing.