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Inorganic−Organic Hybrid Luminescent Binary Probe for DNA Detection Based on Spin-Forbidden Resonance Energy Transfer
Abstract
We describe the design of new fluorescent binary probe sensors for DNA detection based on spin-forbidden resonance energy transfer (SF-RET). Binary probes consist of a donor and acceptor fluorophores that are attached to two different oligonucleotides and serve as a resonance energy transfer (RET) donor-acceptor pair when hybridized to adjacent sites of a target sequence. In the absence of target, excitation of the donor results in fluorescence only from the donor, but when the probes hybridize to the target, the fluorophores are brought into close proximity favoring RET, yielding fluorescence mainly from the acceptor fluorophore. These new binary probes use the metal complex Ru(bpy')(DIP)(2)(2+) as the energy donor and an organic fluorophore (Cy5) as the energy acceptor. Energy transfer from the MLCT state of the Ru complex to singlet Cy5 is spin forbidden and produces a delayed fluorescence of Cy5. This paper demonstrates that fluorescence delay of Cy5 can be used to time resolve the emission of the probe from the intense fluorescence background of a model system for cellular background; this provides the reported system to overcome intense autofluorescence, an important and general advantage over "classical" spin-allowed steady-state probes.
... Among the various examples of these novel BPs, spin-forbidden resonance energy transfer (SF-RET) BPs have been successfully applied to monitor nucleic acids in a highly fluorescent cell medium. In this approach, a ruthenium (Ru) complex acting as the energy donor and an organic fluorophore (F) acting as the energy acceptor are attached to the ends of two oligonucleotide strands (Fig. 6a) [43]. The strongest emission from the organic fluorophore and the Ru complex is expected to be observed in the presence and absence of the target, respectively. ...
... 7 Fluorescence decay traces of strongly fluorescent cell medium (green) and SF-RET BPs (red). Long-lived SF-RET BPs allow timegated detection and increase the signal-to-background ratio (modified from [43]) [43]. b Pyrene excimer BPs with a pyrene moiety attached to each probe [44]. ...
... 7 Fluorescence decay traces of strongly fluorescent cell medium (green) and SF-RET BPs (red). Long-lived SF-RET BPs allow timegated detection and increase the signal-to-background ratio (modified from [43]) [43]. b Pyrene excimer BPs with a pyrene moiety attached to each probe [44]. ...
Due to their high sensitivity and selectivity, minimum interference with living biological systems, and ease of design and synthesis, fluorescent hybridization probes have been widely used to detect nucleic acids both in vivo and in vitro. Molecular beacons (MBs) and binary probes (BPs) are two very important hybridization probes that are designed based on well-established photophysical principles. These probes have shown particular applicability in a variety of studies, such as mRNA tracking, single nucleotide polymorphism (SNP) detection, polymerase chain reaction (PCR) monitoring, and microorganism identification. Molecular beacons are hairpin oligonucleotide probes that present distinctive fluorescent signatures in the presence and absence of their target. Binary probes consist of two fluorescently labeled oligonucleotide strands that can hybridize to adjacent regions of their target and generate distinctive fluorescence signals. These probes have been extensively studied and modified for different applications by modulating their structures or using various combinations of fluorophores, excimer-forming molecules, and metal complexes. This review describes the applicability and advantages of various hybridization probes that utilize novel and creative design to enhance their target detection sensitivity and specificity.
... Work to date has included a [Ru(bpy) 3 ] 2+ -based enzyme-cleavable sensor 25 and DNA sequences covalently labeled with RPCs to examine DNA− DNA assembly. 26,27 Studies employing a DNA-binding RPC as a FRET donor are even rarer, yet the potential for this was explored by Lakowicz et al., who demonstrated successful RET between [Ru(bpy) 2 (dppz)] 2+ and BO-PRO 3 when both molecules were intercalated to DNA. 28,29 A disadvantage of this was that two reversibly binding DNA molecules were employed, thereby introducing an additional variable that makes assay development problematic. ...
Ruthenium(II) polypyridyl complexes (RPCs) that emit from metal-to-ligand charge transfer (MLCT) states have been developed as DNA probes and are being examined as potential anticancer agents. Here, we report that MLCT-emissive RPCs that bind DNA undergo Förster resonance energy transfer (FRET) with Cy5.5-labeled DNA, forming mega-Stokes shift FRET pairs. Based on this discovery, we developed a simple and rapid FRET binding assay to examine DNA-binding interactions of RPCs with diverse photophysical properties, including non-"light switch" complexes [Ru(dppz)2(5,5'dmb)]2+ and [Ru(PIP)2(5,5'dmb)]2+ (dppz = dipyridophenazine, 5,5'dmb = 5,5'-dimethyl-2,2'-bipyridine, PIP = 2-phenyl-imidazo[4,5-f][1,10]phenanthroline). Binding affinities toward duplex, G-quadruplex, three-way junction, and mismatch DNA were determined, and derived FRET donor-acceptor proximities provide information on potential binding sites. Molecules characterized by this method demonstrate encouraging anticancer properties, including synergy with the PARP inhibitor Olaparib, and mechanistic studies indicate that [Ru(PIP)2(5,5'dmb)]2+ acts to block DNA replication fork progression.
... The presence of heavy metal atoms introduces attractive chemical and photophysical properties, leading-with the choice of proper ligands and metal ions-to luminescent compounds with high emission quantum yields, tunable emission color in the visible region, long excited state lifetimes and large Stokes shifts, properties very useful in biosensing and bioimaging [9][10][11][12][13]. Because of that, ruthenium [14][15][16][17][18][19][20], iridium [21][22][23][24][25][26][27][28][29] and platinum [30][31][32][33][34][35] complexes have been successfully employed as in-vitro and in-vivo luminescent biolabels. Moreover, as a consequence of the strong spin-orbit coupling due to the presence of the heavy atom, the intersystem crossing processes lead to an efficient population of energetically low-lying triplet excited states, often very close to the excited states of molecular oxygen, with consequent energy transfer processes and generation of singlet oxygen. ...
In this mini review, we highlight advances in the last five years in light-activated cancer theranostics by using hybrid systems consisting of transition metal complexes (TMCs) and plasmonic gold nanostructures (AuNPs). TMCs are molecules with attractive properties and high potential in biomedical application. Due to their antiproliferative abilities, platinum-based compounds are currently first-choice drugs for the treatment of several solid tumors. Moreover, ruthenium, iridium and platinum complexes are well-known for their ability to photogenerate singlet oxygen, a highly cytotoxic reactive species with a key role in photodynamic therapy. Their potential is further extended by the unique photophysical properties, which make TMCs particularly suitable for bioimaging. Recently, gold nanoparticles (AuNPs) have been widely investigated as one of the leading nanomaterials in cancer theranostics. AuNPs—being an inert and highly biocompatible material—represent excellent drug delivery systems, overcoming most of the side effects associated with the systemic administration of anticancer drugs. Furthermore, due to the thermoplasmonic properties, AuNPs proved to be efficient nano-sources of heat for photothermal therapy application. Therefore, the hybrid combination TMC/AuNPs could represent a synergistic merger of multiple functionalities for combinatorial cancer therapy strategies. Herein, we report the most recent examples of TMC/AuNPs systems in in-vitro in-vivo cancer tharanostics application whose effects are triggered by light-exposure in the Vis–NIR region, leading to a spatial and temporal control of the TMC/AuNPs activation for light-mediated precision therapeutics.
... With the advent of fast-switching electric circuits, shorter lifetimes in the range of 100 ns to several microseconds are nowadays also accessible for TRF detection, and ruthenium complexes have been suggested as suitable probes, which are, however, similarly problematic (Bannwarth et al., 1988;Hennig and Zeckert, 2000;Yun et al., 2003;Kainmüller et al., 2005;Kainmüller and Bannwarth, 2006;Clima et al., 2007;Martí et al., 2007a,b;Kramer et al., 2008;Kolpashchikov, 2010;Guo et al., 2011). Further, pyrene has been explored for its utility in TRF assays with nanosecond lifetimes (Yang et al., 2005;Martí et al., 2006Martí et al., , 2007aConlon et al., 2008;Guo et al., 2011), but large hydrophobic, aromatic surfaces, such as that of pyrene, are wellknown to interfere with biomolecular recognition (Daugherty and Gellman, 1999;Sahoo et al., 2007a). As an appealing alternative, we have introduced 2,3-diazabicyclo[2.2.2]oct-2ene labeled asparagine (Dbo), which exhibits an unquenched lifetime of ca. ...
We report the use of the macrocyclic host cucurbit[7]uril (CB7) as a supramolecular additive in nanosecond time-resolved fluorescence (Nano-TRF) assays for proteases to enhance the discrimination of substrates and products and, thereby, the sensitivity. A peptide substrate was labeled with 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO) as a long-lived (>300 ns) fluorescent probe and 3-nitrotyrosine was established as a non-fluorescent fluorescence resonance energy transfer (FRET) acceptor that acts as a “dark quencher.” The substrate was cleaved by the model proteases trypsin and chymotrypsin and the effects of the addition of CB7 to the enzyme assay mixture were investigated in detail using UV/VIS absorption as well as steady-state and time-resolved fluorescence spectroscopy. This also allowed us to identify the DBO and nitrotyrosine residues as preferential binding sites for CB7 and suggested a hairpin conformation of the peptide, in which the guanidinium side chain of an arginine residue is additionally bound to a vacant ureido rim of one of the CB7 hosts.
... There are a great number of other examples available in the literature. From straight forward binary probes carrying a ruthenium complex and organic fluorophore suited for FRET, [46] to more convoluted approaches, such as a system where each binary probe carries one part of the sequence complementary of a molecular beacon whose hybridization results in an increase on fluorescence. [47] Ribozymes, DNA/RNA sequences with an enzymatic activity, are also common in the field. ...
In den letzten Jahren gab es rasche Weiterentwicklungen auf dem Gebiet der Nukleinsäure-Erkennung. Von microRNA-Quantifizierung zur Untersuchung von Zelltods, --Teilung und -Regulation bis zur Bewertung genetischer Variabilität in Hinblick auf Krankheitsentstehung und -Behandlung: Die Analyse von Nukleinsäuren wird in der zukünftigen Medizin eine zentrale Rolle zukommen. Vor allem die Erkennung von SNPs als Hauptquelle der genetischen Vielfalt, aber aus Analysesicht auch eine der herausforderndsten Mutationen, stellt in dieser Hinsicht einen wesentlichen Aspekt dar. Methoden zur SNP-Erkennung müssen nicht nur sensibel, selektiv und stabil, sondern auch vielfältig sein und eine der wachsenden Analyseanzahl gerecht werdende hohe Verarbeitungsmenge bieten. Im Rahmendieser Arbeit wurde ein chemisches Prüfverfahren zur Erkennung von Nukleinsäuren und Einzelnukleotid-Polymorphismen (SNPs) entwickelt. Das Reaktionssystem zur Nukleinsäuren- Erkennung beruht hierbeiauf der Interaktion zweier modifizierter Peptid-Nukleinsäure (PNS) Oligonukleotiden. Das Erste beinhaltet einen C-terminalen Thioester (Donor-Sonde), die zweite einen N-terminalen Cysteinyl-Rest (Akzeptor-Sonde). Zusätzlich ist die Donor-Sonde durch einenmakrocyclischen Metall Chelatkomplex aus 1,4,7,10-tetraazacyclododecan-1,4,7,10-tetraessigsäure(DOTA) mit einem gebundenen lanthanoid-tag funktionalisiert. In die Akzeptor-Sonde wurde, zurReinigung mit magnetischen Streptavidin Partikeln, Biotin integriert. Der Ziel-DNA-Strang bringt beideSonden in räumliche Nähe zueinander und ermöglicht so eine chemische Reaktion. Das so gewonneneLigationsprodukt beinhaltet den Lanthanoid-Tag und Biotin, über welches das Produkt gereinigt wird,bevor die Detektion mittels ICP-MS erfolgt. Die Lanthanoid Konzentration dient als Indikator desLigationsprodukts welches wiederum den Reporter des Ziel-DNS-Strangs darstellt. Die, mithilfe diesesSystems erreichte, methodische Nachweisgrenze lag bei 29 pM mit einem RSD von 6,8% bei 50 pM(n=5). Zur Erkennung von SNPs wurde das Experiment mit einer Kombination zweier-Sets PNS Sonden mit unterschiedlichen Lanthanoid Tags durchgeführt. Das erste Set zielte auf die SNP beinhaltende Sequenz (Reportersystem) ab, während das zweite an eine benachbarte Sequenz (Kontrollsystem) binden sollte. Zur Erkennung der SNP wurden die Signale bei der Lanthanoide wurden ins Verhältnis gesetzt. Mithilfe dieses Verfahrens konnte durch Messung von sechs Lanthaniden bei einer Konzentration von 5 nM erfolgreich simultan zwischen den Allelen dreier SNPs unterschieden werden.
... 10,11 It has well-dened spectroscopic, photophysical, photochemical and electrochemical properties which are sensitive to variations of external inputs, thus making it useful in the construction of supramolecular systems in sensor applications. 12,13 The results obtained will further enhance the understanding of the complexation behaviour and ability of such systems to selectively bind and recover so toxic metals that have potentially adverse effects on health. ...
A series of complexes with oxathiacrown ethers appended to a [Ru(bpy)2]²⁺ moiety have been synthesized and characterised using ¹H NMR, ¹³C NMR, IR, electronic absorption and emission spectroscopies, mass spectrometry and elemental analyses. The complexes exhibit strong MLCT luminescence bands in the range 608–611 nm and one reversible metal centred oxidation potential in the range 1.00–1.02 V. Their selectivity and sensitivity towards Hg²⁺, Cd²⁺ and Pb²⁺ metal ions have been investigated using electronic absorption, luminescence, cyclic and differential pulse voltammetry titrations. Their responses towards selected cations and anions have also been investigated using electronic absorption and luminescence. While the complexes are selective towards Hg²⁺ and Cd²⁺ ions, none of them is selective towards Pb²⁺ ions. In particular, complex 2 gives a selective change in the UV/Vis absorbance with Hg²⁺ making it possible to detect mercury down to a detection limit of 68 ppm. The binding constants and limits of detection of the complexes have been calculated, with values ranging from 4.37 to 5.38 and 1.4 × 10⁻³ to 6.8 × 10⁻⁵ for log Ks and LOD respectively.
... The repertoire of organic molecules is practically infinite, and the possibilities for developing novel DNA−organic molecular hybrid structures are endless. The judicious selection of organic molecules can result in unique DNA-based nanostructures for application in molecular and cellular biophysics, as biomimetic systems, in energy transfer and photonics, and in diagnostics and therapeutics [18][19][20][21]. Moreover, as a bottom-up technique, such a methodology can contribute to molecular electronics where tunable electronic properties and templated metallization are frequently warranted [22][23][24]. ...
Single-stranded DNA-melamine hybrid molecular building blocks were synthesized using a phosphoramidation cross-coupling reaction with a zero linker approach. The self-assembly of the DNA-organic hybrid molecules was achieved by DNA hybridization. Following self-assembly, two distinct types of nanostructures in the form of linear chains and network arrays were observed. The morphology of the self-assembled nanostructures was found to depend on the number of DNA strands that were attached to a single melamine molecule.
... Measuring the time resolved decay will also reduce problems with autofluorescence background from biological samples. 38 Most importantly, the non-toxic nature of ZnSe/ZnMnS/ZnS:dye sensing NCs will pave inroads for semiconductor nanotechnology in matters of direct concern for human health. Our demonstration of pH sensing with fluorescein NC conjugates show that we may perhaps create highly multiplexed sensing schemes where the core emission from a NC undergoes chemically sensitive energy transfer to one surface bound dye while the dopant emission is coupled to a second, red shifted sensing dye. ...
We have studied phosphor doping of core / shell nanocrystals (NCs) where the impurity emitter resides in the shell. We have found that a two step synthesis can be used to create these non-toxic materials that efficiently transfer energy from the core to the doped shell. These core / shell NCs retain ample brightness when solubilized in water. We explored the functionalization of these materials in water as well to create ratiometric chemical sensing agents. First, we used a method of controlled polymerization to synthesize amphiphilic polymers to solubilize the intrinsically hydrophobic NCs into water. The polymer has a build in "chemical handle" which we use to functionalize the polymers closely bound to the NC with a fluorescent dye in aqueous solution. We have found that there exists efficient Förster resonant energy transfer from the shell doped phosphors to the surface bound dyes. Conjugation of the NC to an environmentally sensitive dye such as fluorescein has also demonstrated that non-toxic doped NCs can be used to develop ratiometric sensing / biological imaging agents. Last, we have found that the same technique can be applied to functionalize non-emissive magnetic nanocrystals as well.
Fluorescence lifetime imaging has been a powerful tool for biomedical research. Recently, fluorescence lifetime‐based multiplexing imaging has expanded imaging channels by using probes that harbor the same spectral channels and distinct excited state lifetime. While it is desirable to control the excited state lifetime of any given fluorescent probes, the rational control of fluorescence lifetimes remains a challenge. Herein, we chose boron dipyrromethene (BODIPY) as a model system and provided chemical strategies to regulate the fluorescence lifetime of its derivatives with varying spectral features. We find electronegativity of structural substituents at the 8’ and 5’ positions are important to control the lifetime for the green‐emitting and red‐emitting BODIPY scaffolds. Mechanistically, such influences are exerted via the photo‐induced electron transfer and the intramolecular charge transfer processes for the 8’ and 5’ positions of BODIPY, respectively. Based on these principles, we have generated a group of BODIPY probes that enable imaging experiments to separate multiple targets using fluorescence lifetime as a signal. In addition to BODIPY, we envision modulation of electronegativity of chemical substituents could serve as a feasible strategy to achieve rational control of fluorescence lifetime for a variety of small molecule fluorophores.
Fluorescence lifetime imaging has been a powerful tool for biomedical research. Recently, fluorescence lifetime‐based multiplexing imaging has expanded imaging channels by using probes that harbor the same spectral channels and distinct excited state lifetime. While it is desirable to control the excited state lifetime of any given fluorescent probes, the rational control of fluorescence lifetimes remains a challenge. Herein, we chose boron dipyrromethene (BODIPY) as a model system and provided chemical strategies to regulate the fluorescence lifetime of its derivatives with varying spectral features. We find electronegativity of structural substituents at the 8′ and 5′ positions is important to control the lifetime for the green‐emitting and red‐emitting BODIPY scaffolds. Mechanistically, such influences are exerted via the photo‐induced electron transfer and the intramolecular charge transfer processes for the 8′ and 5′ positions of BODIPY, respectively. Based on these principles, we have generated a group of BODIPY probes that enable imaging experiments to separate multiple targets using fluorescence lifetime as a signal. In addition to BODIPY, we envision modulation of electronegativity of chemical substituents could serve as a feasible strategy to achieve rational control of fluorescence lifetime for a variety of small molecule fluorophores.
Fluorescence-based assays are widely used in clinical diagnosis, drug development, environmental pollution detection, and chemical biology research due to their high sensitivity and rapid read-out times. Undesirable background fluorescence from complex matrices such as biological samples can be easily suppressed by time-resolved fluorescence (TRF) detection.Therein, long-lived probes are used and a delay time, after which all short-live background fluorescence has decayed, is applied to selectively detect the long-lived emission. In addition, to the established lanthanide-based probes with lifetimes in the millisecond time range, probes with much shorter lifetimes (>10 ns) can be used as well, for which the name Nano-TRF assays has been coined. This book chapter reviews the current state-of-the-art of Nano-TRF assays including probes based on Ru(II) complexes, pyrenes, and fluorazophores, as well as their applications.
One of the driving forces behind the development of sensitive detection methods for trace amounts of samples, is biomolecular detection. These methods are very important for Biology and environmental analysis. Photothermal lens spectroscopy is a powerful optical detection method that can be used to detect biomolecules in very small sample volumes, over a short period of time without destroying the sample. Nowadays, investigations about lab-on-chip and microfluidic devices have attracted the attention of many scientists, especially biologists. Photothermal lens microscopy is one of the best optical detection methods that can be used for detection in microfluidic devices.
The aim of this thesis was the development of photothermal lens technique for the analysis of biomolecules. At first, sensitive determination of DNA based on phosphate-dye interaction using the photothermal lens technique was investigated. For this purpose, to evaluate the performance of the presented method, herring sperm DNA, Escherichia coli bacteria DNA, and partial 16S rRNA gene were examined. After optimization of the measurement conditions, the linear response of the method was obtained for all three types of DNAs at nanomolar concentration range and also the detection limits were at picomolar concentrations.
The second goal of this thesis was the determination of bovine serum albumin by photo thermal lens spectroscopy using dedimerization of methylene blue dye in the methylene blue-sodium dodecyl sulfate system due to the presence of a protein such as bovine serum albumin. Under optimum conditions, the photothermal lens signal was linearly dependent on the concentration of bovine serum albumin in the range of 5×10-7–7.5×10-5 mgL-1 and the detection limit was 3.5×10-7 gmL−1. Eventually, the investigation of microfluidic chip-photothermal lens microscopy for DNA hybridization assay using gold nanoparticles was considered as the final goal. For this purpose, a glass microchip (MC) with a Y-shaped channel was fabricated and also two types of target DNAs were considered. Under the optimized conditions, the results showed that the variation of photothermal lens signal in the focal volume of 105 fL (10−15 L) was linearly proportional to the target DNA concentration over the range of 50–500 nM with detection limits of 30.7 nM and 27.3 nM for target DNA I and II, respectively. The lowest amount of target DNA that was measured using gold nanoparticles was 2.6 zepto (10−21) mole. The obtained results, from this method, were confirmed by two different common methods including gel electrophoresis and fluorescent in situ hybridization monitoring. It is hoped that the efforts made during this thesis will help to develop further advancements in the photothermal lens technique as a sensitive detection method for biomolecular analysis.
Some Ru complexes have extremely promising anticancer or antibacterial properties, but the poor H 2 O solubility and/or low stability of many Ru complexes in aqueous solution under physiological conditions and/or metabolic or biodistribution profiles prevent their therapeutic use. To overcome these drawbacks, various strategies have been developed to improve the delivery of these compounds to their target tissues. The first strategy is based on physical encapsulation of Ru complexes in carriers, such as polymeric micelles, microparticles, nanoparticles and polymer–lipid hybrids, which enables the delivery and controlled release of the active Ru drug candidate. The second strategy involves covalent conjugation of the Ru complex to a polymer to give a prodrug that can be converted into the active drug at a more controllable rate. In this Review, we provide an overview of recent developments in polymer encapsulation of Ru complexes for biological and medicinal applications. We place particular emphasis on how polymer structure affects Ru delivery.
In this Review article, we systematically summarize the design and applications of various kinds of long-lived emissive probes for bioimaging and biosensing via time-resolved photoluminescence techniques. The probes reviewed, including lanthanides, transition-metal complexes, organic dyes, carbon and silicon nanoparticles, metal clusters, and persistent phosphores, exhibit longer luminescence lifetimes than that of autofluorescence from biological tissue and organs. When these probes are internalized into living cells or animals, time-gated photoluminescence imaging selectively collects long-lived signals for intensity analysis, while photoluminescence lifetime imaging reports the decay details of each pixel. Since the long-lived signals are differentiated from autofluorescence in the time domain, the imaging contrast and sensing sensitivity are remarkably improved. The future prospects and challenges in this rapidly growing field are addressed.
Herein, we reported a new fluorescence regulation mechanism of DNA-templated AgNC caused by coil DNA. Based on these phenomena, a novel dual fluorescent AgNC-MB with exponential signal enhancement and remarkable...
A new bis-heteroleptic Ru(II) complex (1) of benzimidazole-substituted 1,2,3-triazole pyridine ligand has been designed and constructed for the photoluminescent detection of cationic and anionic analytes, Ag+and phosphate ions. Compound, 1[PF6]2was fully characterized by various spectroscopic techniques and the solid-state structure was determined via single-crystal X-ray diffraction. The cation and anion sensing properties in 50% aqueous buffer (pH 9.2) and pure acetonitrile were carefully examined in photoluminescence (PL) spectroscopy. The 1[PF6]2was found to be highly selective to pyrophosphate; PPi/HP2O73-and H2PO4-ions in CH3CN. It showed ∼10-fold PL intensity enhancement at 583 nm in the presence of only 1 and 2 equiv of PPi and H2PO4-ions, respectively. The PL titrations of 1[PF6]2with PPi and H2PO4-in CH3CN furnished the association constant (Ka= 3.3 × 103M-1and 6.8 × 103M-1) and the detection limit was as low as 5.73 and 5.19 ppb, respectively. The 1[PF6]2also selectively detected Ag+over other competitive cations through the luminescence light up in 50% aqueous buffer (pH 9.2) media. The PL titration of 1[PF6]2with Ag+showed ∼8-fold luminescence enhancement at 591 nm and yielded association constant, Ka= 3.5 × 104M-1and the detection limit was determined to be 5.05 ppb. A new cation sensing mechanism has been established where the Ag+ion is detected in photoluminescence spectroscopy through the unique cyclometalated Ag+-triazolide complex formation. The high selectivity of 1[PF6]2for phosphates and Ag+was established by PL in the presence of various competing ions. Finally, for biological application, the cytotoxicity study was performed. The probe showed low cytotoxicity and was suitable for intracellular Ag+imaging. The cell imaging and in vitro photoluminescence study revealed that the probe stained the cell nucleoli and specifically bind with ribosomal RNA (rRNA) and, therefore, it can also serve as a luminescent probe for rRNA in the presence of Ag+.
Herein we report a colorimetric biosensing strategy to discriminate single-nucleotide mutation in DNA with high selectivity using unmodified gold nanoparticles (AuNPs) as indicators. In the AuNPs-based colorimetric strategy, binary DNA probes were produced by splitting a long DNA probe in the middle for sensitive differentiation of single-base mismatch. The detection limit of this method toward target DNA was 5 nM. The developed system has superior advantages of utilization of inexpensive materials, simplicity and visualization. Moreover, binary DNA probes not only can distinguish single-base mutation in the target DNA very well, as compared to long DNA probe, but also can construct “AND” logic gate using two distinct target DNAs as inputs, which holds great potential for increasing the accuracy of disease diagnosis in clinical applications.
Live-cell imaging has provided the life sciences with insights into the cell biology and dynamics. Fluorescent labeling of target molecules proves to be indispensable in this regard. In this review, we focus on the current fluorescent labeling strategies for nucleic acids, and in particular messenger RNA (mRNA) and plasmid DNA (pDNA), which are of interest to a broad range of scientific fields. By giving a background of the available techniques and an evaluation of the pros and cons, we try to supply scientists with all the information needed to come to an informed choice of nucleic acid labeling strategy aimed at their particular needs.
A wide variety of probes have been designed and synthesized for detecting oligonucleotides and polynucleotides in vivo and in vitro. Of these, molecular beacons (MBs) and binary probes (BPs) have shown particular applicability to specific problems such as mRNA tracking, single nucleotide polymorphism, and polymerase chain reaction quantization. MBs are hairpin oligonucleotide probes, containing a fluorophore and a quencher, that change their fluorescent properties upon binding to a given target. BPs, on the other hand, consist of two fluorophore-containing oligonucleotide strands that hybridize to adjacent regions of a target sequence, thus favoring energy transfer between the neighboring fluorophores. These probes have been extensively studied and modified to enhance their detection characteristics using different dye combinations, three-dye arrays, excimer-forming molecules and metal complexes. The design, applicability and advantages of these probes for the detection and tracing of oligonuclotides in different media will be discussed.
The main objective of this thesis is the design of chromophores exhibiting optical limiting
properties at telecommunication wavelengths. Two families of near infrared dyes have been
synthesized and studied for this purpose: cyanines dyes and boron dipyrromethene. Their
linear and nonlinear optical properties (second and third order) have been studied. These
compounds display strong Two-Photon Absorption (TPA) around 1500 nm. Their good
solubility allows carrying out optical limiting experiments at these wavelengths. The
phenomenon has been interpreted on the basis of a TPA induced excited state absorption
model. Finally, the synthesis of functionalized chromophores for solid state optical limiting
has been considered.
Introduction Mathematical background Prediction error algorithm for fractional order system identification Fractional order modeling of electrochemical processes Identification of a real electrochemical biochip Conclusion Bibliography
This feature article will cover our efforts to sense biologically relevant molecules using photoluminescent metal complexes. Photoluminescent metal complexes possess large Stokes shifts, long lifetimes and their photoluminescence maxima can be tuned. We have developed probes for DNA and RNA detection containing metal complexes of ruthenium and iridium as photoluminescent reporters. Iridium complexes have also been modified to serve as probes for amino acids such as cysteine, homocysteine, and histidine. Also, probes sensible to the aggregation state of proteins such as amyloid-β and alpha-synuclein using photoluminescence (ruthenium dipyridophenazine complexes) or birefringence (ruthenium red) were developed. Finally, we will present the detection of solvent vapors using a photoluminescent rhenium complex within a zeolite matrix. The long photoluminescence lifetime of the aforementioned complexes has been synergistically combined with advanced time-resolved methods to enhance the detection scope of these probes. For example techniques such as time-gating have been used to detect oligonucleotides, amino acids and protein aggregation even in highly autofluorescent media. Time-gating allows selecting a time-window in a time-resolved emission spectrum where the long-lived photoluminescence of the probes can be preferentially detected from the short-lived autofluorescence of the medium. In addition, we have used time-resolved photoluminescence spectroscopy to extract the photoluminescence of free histidine from the photoluminescence of histidine-containing proteins. Also, a combination of photoluminescence intensity, maximum and lifetime was used to detect solvent vapors. These examples testify on the advantages of time-resolved photoluminescence spectroscopy for enhancing the detection of analytes using probes with long-lived photoluminescence.
DNA-assisted Förster resonance energy transfer (FRET) between an anthracene-based cyclophane () and mono- and bis-intercalators such as propidium iodide () and ethidium homodimer-1 (), respectively, has been studied using various photophysical and biophysical techniques. The cyclophane and exhibited simultaneous binding to DNA at all concentrations studied and showed DNA-assisted FRET from the excimer of cyclophane with a FRET efficiency of ca. 71%. On the other hand, the bis-intercalator , only at lower concentrations (<3 μM), can act as an acceptor for the energy transfer process with a lower efficiency of ca. 44%. At higher concentrations (>15 μM), on account of its higher binding affinity, displaces cyclophane from the DNA scaffold. Employing the ternary system comprising of the cyclophane, DNA and and fine-tuning the concentrations of the components in a molar ratio of 1 : 0.75 : 0.05 ( : DNA : ) we have demonstrated white light emission with CIE coordinates (0.35, 0.37).
Three tripodal ligands H3L1–3 containing imidazole rings were synthesized by the reaction of 1,10-phenanthroline-5,6-dione with 1,3,5-tris[(3-formylphenoxy)methyl]benzene, 1,3,5-tris[(3-formylphenoxy)methyl]-2,4,6-trimethylbenzene, and 2,2′,2"-tris[(3-formylphenoxy)ethyl]amine, respectively. Trinuclear RuII polypyridyl complexes [(bpy)6Ru3H3L1–3](PF6)6 were prepared by the condensation of Ru(bpy)2Cl2·2H2O with ligands H3L1–3. The pH effects on the UV/Vis absorption and fluorescence spectra of the three complexes were studied, and ground- and excited-state ionization constants of the three complexes were derived. The three complexes act as “off-on-off” fluorescence pH switch through protonation and deprotonation of imidazole ring with a maximum on-off ratio of 5 in buffer solution at room temperature.
We studied 15-crown-5 ether mono- and bis(styryl) derivatives of 2,2'-bipyridine ( and ) as a scaffold to construct photoresponsive complexes possessing metallodendrimer structure. The synthesis and optical properties of one dimensional (1D) Ca(2+)-containing and octahedral 3D Zn(2+)-containing complexes with crowned styryl derivatives of 2,2'-bipyridine are described. Bimetallic Ca(2+), Zn(2+)-containing complexes of and possess well-defined structures and particular optical characteristics. The effective size of the complexes was estimated by diffusion-ordered NMR spectroscopy (DOSY) by measuring translation diffusion coefficients. The NMR data on the compositions of the formed complexes are in full agreement with those obtained by an optical method. The properties of the zinc polypyridyl complexes are modified by intermolecular interactions between the crown ether center and the metal ions, so they are potentially useful in the preparation of chemical sensors.
Two polypyridyl ligands, 5-(4′-ethynylbenzo-15-crown-5)-2,2′-bipyridine (L1) and 3-bromo-8-(4′-ethynylbenzo-15-crown-5)-1,10-phenanthroline (L2), and their Ru(II) complexes [(bpy)2RuL](PF6)2 have been prepared and characterized. Both complexes exhibit metal-to-ligand charge transfer absorption at around 452 nm and emission at around 640 nm in MeCN solution. Electrochemical studies of the complexes reveal a Ru(II)-centered oxidation at around 1.31 V and three ligand-centered reductions. The binding ability of the complexes with Na+ has been investigated by UV/Vis absorption, emission, and electrochemical titrations. Addition of Na+ to MeCN solutions of both complexes results in a progressive enhancement of the emission, a red-shift of the UV/Vis absorption, and a progressive cathodic shift of the Ru(II)-centered E
1/2 couple. The stability constants for the 1:1 stoichiometry adducts of the complexes with Na+ have been obtained from the UV/Vis absorption titrations.
The design of novel small molecules for studying the interaction with DNA is one of the most important goals in modern medicinal chemistry. In this paper, we developed two BODIPY-imidazolium salts, 1 and 2, as sensitive and selective fluorescent intercalators toward DNA. The nature and the strength of the stacking interaction between these BODIPYs and DNA has been addressed through a detailed study of the photophysical properties of the bound form and unbound form of the dyes. Strong hypochromism and red-shifted absorption spectra, together with the marked decrease in the positive CD band of ct-DNA, are consistent with strong intercalation of the chromophores with DNA. More important, these dyes give substantial increases in fluorescence on DNA binding and may prove useful as sensitive fluorescent turn-on probes for DNA. The time-resolved fluorescence shows single exponential decay with longer lived excited-state lifetime observed upon intercalative interaction. The intercalation binding model is also supported by steady-state emission quenching experiments using KI as a quencher.
More than one thousand photochemistry papers were published in the most cited twenty-four scientific journals in 2007. With nanosecond, picosecond, and femtosecond laser techniques widely applied, detection of reactive intermediates, products of chemical bond dissociation, isomerizations, rearrangements, as well as the initial results of intramolecular energy or electron transfer, become facile. Complex photoluminescence, DNA sequence-directed structure and dynamics studies, photopolymerization, and photocatalysis remain important topics. Because optical single-molecule detection, photoprobes, and photosensors have been increasingly applied in the chemical and biological sciences, these have received specific attention. The photochemistry of nanoparticles and nanostructures has also attracted much attention. Optical devices, photoswitches, and two-photon/multiphoton chemistry have been widely investigated. In this review, we briefly summarize major progress in the photochemical sciences and selected applications are discussed.
Tetrapodal ligands tetrakis{4-((1,10-phenanthroline-[5,6-d]imidazol-2-yl)phenoxy)methyl}methane (L1), tetrakis{3-((1,10-phenanthroline-[5,6-d]imidazol-2-yl)phenoxy)methyl}methane (L2), and corresponding Ru(II) complexes [(bpy)8L1–2(RuII)4](PF6)8, shortly called Ru–L1–2, have been synthesized and characterized. The pH effects on UV–vis absorption and fluorescence spectra of the two complexes are studied, and ground- and excited-state ionization constants of the two complexes are derived. The two complexes act as pH-induced “off–on–off” fluorescence switches through protonation and deprotonation in buffer solution at room temperature.
A terbium chelate formed with diethylenetriaminepentaacetic acid and p-aminosalicylic acid (Tb–DTPA–pAS) was labeled on a 23-mer DNA strand and used as donor in the fluorescence resonance energy transfer (FRET) study of DNA hybridization. The change in donor fluorescence lifetime with and without DNA-bind acceptor carboxytetramethylrhodamine (TAMRA) showed high energy transfer efficiency (87%). The distance between the two ends of the 23-mer double-stranded DNA was estimated. This study provides a possibility of using the Tb-chelate as a probe in the studies of the geometry-dependent interaction between DNA strands or proteins.
The binding of the complex [Co(phen)2(DPQ)]Cl3 to the decanucleotide d(CCGAATGAGG)2 containing two pairs of sheared G:A mispairs was studied by 2D-NMR. There appear many 1H NOE cross-peaks from the complex to the oligonucleotide. The results indicate that the complex, with DPQ, intercalates into the oligonucleotide via its terminal base pairs from the minor groove, which further proved our previous conclusion.
Three bis(bidentate) ligands cis-di(4,5-diazafluoren-9-ylimino)dibenzo-14-crown-4 (BL1), trans-di(4,5-diazafluoren-9-ylimino)dibenzo-14-crown-4 (BL2), and 1,4,8,11-tetraoxacyclotetradecyl[2,3:9,10]bis-dipyrido[3,2-a:2′3′-c]phenazine (BL3), and corresponding bimetallic Ru(II) complexes [(bpy)2RuBL1−3Ru(bpy)2](PF6)4 (Ru–BL1−3) have been synthesized. Cyclic voltammetry of these complexes are consistent with one Ru(II)-centered reversible oxidation and three ligand-centered reductions. These complexes show metal-to-ligand charge transfer absorption at 445–454nm and emission at 575–592nm.
Four polypyridyl bridging ligands BL1−4 containing open-chain crown ether, where BL1−3 formed by the condensation of 4,5-diazafluoren-9-oxime with diethylene glycol di-p-tosylate, triethylene glycol di-p-tosylate, and tetraethylene glycol di-p-tosylate, respectively. BL4 formed by the reaction of 4-(1,10-phenanthrolin-5-ylimino)methylphenol with triethylene glycol di-p-tosylate, have been synthesized. Reaction of Ru(bpy)2Cl2·2H2O with BL, respectively, afforded four bimetallic complexes [(bpy)2RuBL1−4Ru(bpy)2]4+ as [PF6]− salts. Electrochemistry of these complexes is consistent with one RuII-based oxidation and several ligand-based reductions. These complexes show metal-to-ligand charge transfer absorption at 439-452 nm and emission at 570-597 nm.
Hexapodal ligand H6L containing imidazole groups has been prepared by the reaction of 1,2,3,4,5,6-hexakis[(4-formylphenoxy)methyl]benzene with 1,10-phenanthroline-5,6-dione. Hexanuclear complex [RuH6L](PF6)12 has been obtained by the condensation of Ru(bpy)2Cl2·2H2O and H6L, and isolated as PF6− salt. The deprotonated complex [RuL]6+ was achieved by reaction of sodium methoxide with complex [RuH6L]12+ in methanol. The pH effects on the UV–vis absorption and fluorescence spectra of complex [RuH6L](PF6)12 are studied, and ground and excited state ionization constants of the complex are derived. The absorption and fluorescence spectra of the complex are both strongly dependent on the solution pH. Complex [RuH6L](PF6)12 acts as “on–off” fluorescence pH sensor through protonation and deprotonation of imidazole groups with a maximum on–off ratio of 5 in buffer solution at room temperature.
Tripodal ligands 1,3,5-tris{4-((1,10-phenanthroline-[5,6-d]imidazol-2-yl)phenoxy)methyl}-2,4,6-trimethylbenzene (L1), 1,1,1-tris{4-((1,10-phenanthroline-[5,6-d]imidazol-2-yl)phenoxy)methyl}propane (L2), 2,2′,2′′-tris{4-((1,10-phenanthroline-[5,6-d]imidazol-2-yl)phenoxy)ethyl}amine (L3), and corresponding Ru(II) complexes [(bpy)6L1–3(RuII)3](PF6)6, shortly called (Ru–L1–3), have been synthesized. UV–vis absorption and fluorescence spectra of these complexes are both strongly dependent on the pH of the buffer solution. These complexes act as pH-induced off–on–off fluorescence switch through protonation and deprotonation of the imidazole-containing ligands.
Despite the promising photofunctionalities, phosphorescent probes have been examined only to a limited extent, and the molecular features that provide convenient handles for controlling the phosphorescence response have yet to be identified. We synthesized a series of phosphorescence zinc sensors based on a cyclometalated heteroleptic Ir(III) complex. The sensor construct includes two anionic cyclometalating ligands and a neutral diimine ligand that tethers a di(2-picolyl)amine (DPA) zinc receptor. A series of cyclometalating ligands with a range of electron densities and band gap energies were used to create phosphorescence sensors. The sensor series was characterized by variable-temperature steady-state and transient photoluminescence spectroscopy studies, electrochemical measurements, and quantum chemical calculations based on time-dependent density functional theory. The studies demonstrated that the suppression of non-radiative PeT from DPA to the photoexcited IrIV species provided the underlying mechanism that governed the phosphorescent response to zinc ions. Importantly, the Coulombic barrier, which was located on either the cyclometalating ligand or the diimine ligand, negligibly influenced the PeT process. Phosphorescence modulation by PeT strictly obeyed the Rehm-Weller principle, and the process occurred in the Marcus-normal region. These findings provide important guidelines for improving sensing performance; an efficient phosphorescence sensor should include a cyclometalating ligand with a wide band gap energy and a deep oxidation potential. Finally, the actions of the sensor were demonstrated by visualizing the intracellular zinc ion distribution in HeLa cells using a confocal laser scanning microscope and a photoluminescence lifetime imaging microscope.
Nucleic acids are unique molecular recognition elements in biosensors having targets that range from ions, small molecules, peptides, proteins and DNA/RNA to virus and whole cells. Pyrene is a polycyclic aromatic compound with very special photophysical characteristics, including long fluorescence lifetime, high quantum yield, and the capability of forming excited state dimers with large Stokes shift. In recent years, pyrene has been used extensively as a novel signaling element in nucleic acid sensors. In this review, we will discuss the optical properties of pyrene and summarize recent progress in the development of pyrene DNA probes for the sensing of nucleic acids, proteins, and small molecules.
Improving probes so that they can perform more sensitive and accurate detections is at the heart of much fundamental and applied research. Within the past few years a considerable amount of effort has been devoted to the study of photoluminescent probes in combination with time-resolved photoluminescence spectroscopy (TRPS). Although TRPS is a powerful and important technique for improving the sensitivity of long-lived probes, there is a lack of a general methodology that would allow one to unambiguously optimize the parameter affecting this technique. In this manuscript it will be shown how parameters that are probe- and technique-specific can affect the effectiveness of TRPS in improving sensitivity. Furthermore, it will be demonstrated that, when TRPS is used, the sensitivity of the probe is strongly dependent on the time window used to generate the time-resolved emission spectra (TRES). A method will be described that will allow one to remove the uncertainty in the selection of the time window that would yield the optimum improvement in probe performance, as well as the experimental parameters that need to be considered. Molecular beacon probes (MBs) were used to demonstrate these points. These probes show signal-to-background ratios (S/B) of less than 9 when SSPS is used, which can be easily enhanced to 17 using TRPS. The detection limits were also improved when TRPS is used allowing detecting target DNA with concentrations as low as 13.6 nM.
Time-resolved emission data (fluorescence decay and fluorescence depolarization) of two three-color Förster resonance energy transfer (tc-FRET) systems consisting of a carbostyril donor (D), a ruthenium complex (Ru) as relay dye, and a Cy5 derivative (Cy) or, optionally, an anthraquinone quencher (Q) were carefully analyzed using advanced distribution analysis models. Thereby, it is possible to get information on the flexibility and mobility of the chromophores which are bound to double stranded (ds) DNA. Especially the distance distribution based on the analysis of the fluorescence depolarization is an attractive approach to complement data of fluorescence decay time analysis. The distance distributions extracted from the experimental data were in excellent agreement with those determined from accessible volume (AV) simulations. Moreover, the study showed that for tc-FRET systems the combination of dyes emitting on different time scales (e.g., nanoseconds vs microseconds) is highly beneficial in the distribution analysis of time-resolved luminescence data in cases where macromolecules such as DNA are involved. Here, the short lifetimes can yield information on the rotation of the dye molecule itself and the long lifetime can give insight in the overall dynamics of the macromolecule.
Three complexes with 2-[bis(2-pyridylmethyl)amino] propanic acid (Adpa) were synthesized and characterized. The crystal structure
of [(Adpa)CoCl] (1) shows that the cobalt(II) atom is coordinated by three N atoms, one oxygen atom from the Adpa ligand and
one chloride, forming a distorted trigonal bipyramidal geometry. The fluorescence titration data indicate the interactions
of ct-DNA with complexes [(Adpa)FeCl2] (2) and [(Adpa)Fe(H2O)2] (3) are exothermic, but binding of complex (1) with ct-DNA is endothermic. The inhibiting activities of the three complexes on the cancer cells (Mcf-7, Eca-109, A549 and Hela) follow the order: (3)>(2)≫(1),
which is in correlation with their DNA-binding properties.
The title compound, [Cu(NCS)(2)(C(20)H(21)N(3))]·0.5CH(2)Cl(2), crystallized with two independent complex mol-ecules (A and B) in the asymmetric unit, accompanied by one dichloro-methane solvent mol-ecule. Each Cu(II) atom has a square-pyramidal geometry, being coordinated by five N atoms, three from the (4-methyl-benz-yl)bis-(pyridin-2-ylmeth-yl)amine ligand and two from the thio-cyanate ligands. In the crystal, the B mol-ecules are linked via C-H⋯S inter-actions, forming chains propagating along [100].
By intelligently utilizing the different interacting strengths between different moieties according to the displacement method, general biosensors with aggregation-induced emission (AIE) characteristics for biomacromolecules without selectivity were converted to excellent, highly selective probes for one specific biomacromolecule with the aid of graphene oxide (GO) in an aqueous medium. Importantly, thanks to the different interactions between the AIE molecule and biomacromolecules, just by simply changing the AIE molecule the sensing system could detect different types of biomacromolecules, thereby providing a new approach to the development of AIE-based sensors with high selectivity and sensitivity. More specifically, the complex of A(2)HPS⋅HCl-a derivative of hexaphenylsilone (HPS) functionalized by two amino (A(2)) groups (N(CH(2)CH(3))(3))-and GO only gives an "off-on" response to DNA, with a detection limit of 2.3 μg mL(-1) toward DNA-CT (calf thymus); interestingly, the complex of TPE-N(2)C(4) (1,2-bis{4-[4-(N,N,N-triethylammonium)butoxy]phenyl}-1,2-diphenylethene dibromide) and GO could only detect the presence of bovine serum albumin (BSA), whereas other biomacromolecules, including DNA, RNA, and even other proteins have very little influence.
Conservation of angular momentum is a familiar tenet in science but has seldom been invoked to understand (or predict) chemical processes. We have developed a general formalism based on Wigner's original ideas concerning angular momentum conservation to interpret the photo-induced reactivity of two molecular donor-acceptor assemblies with physical properties synthetically tailored to facilitate intramolecular energy transfer. Steady-state and time-resolved spectroscopic data establishing excited-state energy transfer from a rhenium(I)-based charge-transfer state to a chromium(III) acceptor can be fully accounted for by Förster theory, whereas the corresponding cobalt(III) adduct does not undergo an analogous reaction despite having a larger cross-section for dipolar coupling. Because this pronounced difference in reactivity is easily explained within the context of the angular momentum conservation model, this relatively simple construct may provide a means for systematizing a broad range of chemical reactions.
We report the design, synthesis, and characterization of a binary oligonucleotideprobe for selective DNA or RNA detection. The probe is based on fluorescence resonance energy transfer (FRET) from quantum dot (CdSe/ZnS core shell) DNA conjugates to organic dye (cyanine-5) DNA conjugates. Selective hybridization of the donor/acceptor DNA conjugates to target DNA enhances FRET and a change in fluorescence signature was observed.
We report on a new three-color FRET system which we were able to verify in peptides as well as in synthetic DNA. All three chromophores could be introduced by a building block approach avoiding postsynthetic labeling. Additional features are robustness, matching spectroscopic properties, high-energy transfer, and sensitivity. The system was investigated in detail on a set of peptides as well as an array of tailored oligonucleotides. The detailed analysis of the experimental data and comparison with theoretical considerations were in excellent agreement. It is shown that in the case of polypeptides specific interaction with the fluorescence probes has to be considered. In contrast with DNA, the fluorescence probes did not show any indications of such interactions. The novel three-color FRET toolbox revealed the potential for applications studying fundamental processes of three interacting molecules in life science applications.
The aggregation of amyloid-β (Aβ) peptides has been associated with the onset of Alzheimer's disease. Here, we report the use of a luminescent dipyridophenazine ruthenium(II) complex to monitor Aβ fibrillization. This complex is not photoluminescent in aqueous solution nor in the presence of monomeric Aβ, but it presents a strong photoluminescence in the presence of Aβ fibril aggregates. One of the advantages of this metal complex is its large Stokes shift (180 nm). Furthermore, the long-lived photoluminescence lifetime of this ruthenium complex allows its use for the detection of fibrillar proteins in the presence of short-lived fluorescent backgrounds, using time-gating technology. We will present evidence of the advantages of dipyridophenazine ruthenium(II) complexes for monitoring protein fibrillization in highly fluorescent media.
The concept of DNA biosensors is sustained by the need for rapid and highly sensitive analytical tools for genetic detection. Their implementation is based on three key steps: (i) immobilization of single-stranded oligonucleotide probes onto a substrate; (ii) hybridization and (iii) reading. These steps involve complementary knowledge in various disciplinary fields such as surface physics and chemistry, molecular electrochemistry, micro-technologies, optics, electronics and biochemistry. We present here, in a non-exhaustive way, the recent advances in the two steps of immobilization and detection that rely upon increasing integration of the number of reading points or/and of the reading strategy.
We have carried out a detailed photophysical study of the FRET D/A pair consisting of a carbostyril donor and a Ru(II)bathophenanthroline complex acceptor in double-stranded synthetic DNA. Altogether 13 different double-stranded 30 base pair DNAs showing small incremental differences in the distances between donor and acceptor were synthesized. Using the fluorescence of the donor as well as of the acceptor, D/A separations were determined and compared to those derived from a well-established model for DNA distance calculations. The model calculations and anisotropy studies revealed that the donor can nearly be seen as a free rotator allowing the application of the established FRET data evaluation.
The advances in the development of binary probe (BPs) and their improved selectivity in comparison with other hybridization-based techniques were studied. The first BP, which used Förster resonance energy transfer (FRET), was suggested in 1988. A commonly adopted BP architecture employs the different affinity mode BPs. In this design one strand with a longer analyte binding arm binds tightly to the position abutting to the single-nucleotide polymorphisms (SNP) site. A second shorter analyte binding arm interrogates the SNP site by forming stable hybrid only with the perfectly matched sequence. The design of BPs employs self-assembly of more than two nucleic acid components. The same principle is adopted by DNA nanotechnology, which deals with constructing objects and functionally active assemblies from DNA molecules. Newly designed constructs based on aptamers, DNA junctions, and DNA enzymes offer an opportunity to utilize DNA probes that avoid direct covalent attachment with organic dyes.
Three approaches were used to study hybridization of complementary oligodeoxynucleotides by nonradiative fluorescence resonance energy transfer. (i) Fluorescein (donor) and rhodamine (acceptor) were covalently attached to the 5' ends of complementary oligodeoxynucleotides of various lengths. Upon hybridization of the complementary oligodeoxynucleotides, energy transfer was detected by both a decrease in fluorescein emission intensity and an enhancement in rhodamine emission intensity. In all cases, fluorescein emission intensity was quenched by about 26% in the presence of unlabeled complement. Transfer efficiency at 5 degrees C decreased from 0.50 to 0.22 to 0.04 as the distance between donor and acceptor fluorophores in the hybrid increased from 8 to 12 to 16 nucleotides. Modeling of these hybrids as double helices showed that transfer efficiency decreased as the reciprocal of the sixth power of the donor-acceptor separation R, as predicted by theory with a corresponding R0 of 49 A. (ii) Fluorescence resonance energy transfer was used to study hybridization of two fluorophore-labeled oligonucleotides to a longer, unlabeled oligodeoxynucleotide. Two 12-mers were prepared that were complementary to two adjacent sequences separated by four bases on a 29-mer. The adjacent 5' and 3' ends of the two 12-mers labeled with fluorescein and rhodamine exhibited a transfer efficiency of approximately 0.60 at 5 degrees C when they both hybridized to the unlabeled 29-mer. (iii) An intercalating dye, acridine orange, was used as the donor fluorophore to a single rhodamine covalently attached to the 5' end of one oligodeoxynucleotide in a 12-base-pair hybrid. Under these conditions, the transfer efficiency was approximately 0.47 at 5 degrees C. These results establish that fluorescence modulation and nonradiative fluorescence resonance energy transfer can detect nucleic acid hybridization in solution. These techniques, with further development, may also prove useful for detecting and quantifying nucleic acid hybridization in living cells.
The primary or secondary structure of single-stranded nucleic acids has been investigated with fluorescent oligonucleotides,
i.e., oligonucleotides covalently linked to a fluorescent dye. Five different chromophores were used: 2-methoxy-6-chloro-9-amino-acridine,
coumarin 500, fluorescein, rhodamine and ethldlum. The chemical synthesis of derivatized oligonucleotides is described. Hybridization
of two fluorescent oligonucleotides to adjacent nucleic acid sequences led to fluorescence excitation energy transfer between
the donor and the acceptor dyes. This phenomenom was used to probe primary and secondary structures of DNA fragments and the
orientation of ollgodeoxynucleotides synthesized with the alpha-anomers of nucleoside units. Fluorescence energy transfer
can be used to reveal the formation of hairpin structures and the translocation of genes between two chromosomes.
Quantitative protein bioanalysis in complex biological fluids presents considerable challenges in biological studies and disease diagnosis. The major obstacles are the background signals from both the probe and the biological fluids where the proteins reside. We have molecularly engineered light-switching excimer aptamer probes for rapid and sensitive detection of a biomarker protein, platelet-derived growth factor (PDGF). Labeled with one pyrene at each end, the aptamer switches its fluorescence emission from ≈400 nm (pyrene monomer) to 485 nm (pyrene excimer) upon PDGF binding. This fluorescence wavelength change from monomer to excimer emission is a result of aptamer conformation rearrangement induced by target binding. The excimer probe is able to effectively detect picomolar PDGF in homogeneous solutions. Because the excimer has a much longer fluorescence lifetime (≈40 ns) than that of the background (≈5 ns), time-resolved measurements were used to eliminate the biological background. We thus were able to detect PDGF in a cell sample quantitatively without any sample pretreatment. This molecular engineering strategy can be used to develop other aptamer probes for protein monitoring. Combined with lifetime-based measurements and molecular engineering, light-switching excimer aptamer probes hold great potential in protein analysis for biomedical studies.
• aptamer
• biomarker
• platelet-derived growth factor
• pyrene
• time-resolved fluorescence
We report here the design, synthesis and application of pyrene binary oligonucleotide probes for selective detection of cellular
mRNA. The detection strategy is based on the formation of a fluorescent excimer when two pyrene groups are brought into close
proximity upon hybridization of the probes with the target mRNA. The pyrene excimer has a long fluorescence lifetime (>40
ns) compared with that of cellular extracts (∼7 ns), allowing selective detection of the excimer using time-resolved emission
spectra (TRES). Optimized probes were used to target a specific region of sensorin mRNA yielding a strong excimer emission
peak at 485 nm in the presence of the target and no excimer emission in the absence of the target in buffer solution. While
direct fluorescence measurement of neuronal extracts showed a strong fluorescent background, obscuring the detection of the
excimer signal, time-resolved emission measurements indicated that the emission decay of the cellular extracts is ∼8 times
faster than that of the pyrene excimer probes. Thus, using TRES of the pyrene probes, we are able to selectively detect mRNA
in the presence of cellular extracts, demonstrating the potential for application of pyrene excimer probes for imaging mRNAs
in cellular environments that have background fluorescence.
We report the design, synthesis and characterization of binary oligonucleotide probes for mRNA detection. The probes were designed to avoid common problems found in standard binary probes such as direct excitation of the acceptor fluorophore and overlap between the donor and acceptor emission spectra. Two different probes were constructed that contained an array of either two or three dyes and that were characterized using steady-state fluorescence spectroscopy, time-resolved fluorescence spectroscopy and fluorescence depolarization measurements. The three-dye binary probe (BP-3d) consists of a Fam fluorophore which acts as a donor, collecting light and transferring it as energy to Tamra, which subsequently transfers energy to Cy5 when the two probes are hybridized to mRNA. This design allows the use of 488 nm excitation, which avoids the direct excitation of Cy5 and at the same time provides a good fluorescence resonance energy transfer (FRET) efficiency. The two-dye binary probe system (BP-2d) was constructed of Alexa488 and Cy5 fluorophores. Although the overlap between the fluorescence of Alexa488 and the absorption of Cy5 is relatively low, FRET still occurs due to their close physical proximity when the probes are hybridized to mRNA. This framework also decreases the direct excitation of Cy5 and reduces the fluorescence overlap between the donor and the acceptor. Picosecond time-resolved spectroscopy showed a reduction in the fluorescence lifetime of donor fluorophores after the formation of the hybrid between the probes and target mRNA. Interestingly, BP-2d in the presence of mRNA shows a slow rise in the fluorescence decay of Cy5 due to a relatively low FRET rate, which together with the reduction in the Alexa488 lifetime provides a way to improve the signal to background ratio using time-resolved fluorescence spectra (TRES). In addition, fluorescence depolarization measurements showed complete depolarization of the acceptor dyes (Cy5) for both BP-3d (due to sequential FRET steps) and BP-2d (due to the relatively low FRET rate) in the presence of the mRNA target.
Cellular autofluorescence affects the sensitivity of flow cytometric assays by interfering with detection of low level specific fluorescence. These detection limits increase with use of protocols, such as thermocycling and fluorescent in-situ hybridization (FISH), that can increase intrinsic cellular fluorescence to 5,000-20,000 fluorescein isothiocyanate (FITC) equivalents. In order to improve signal to noise ratios when using FITC labeled probes in these procedures, we employed a method using the polyanionic azo dye, trypan blue, to reduce intracellular autofluorescence. Dyes such as these are commonly used in immunofluorescent microscopy to reduce background fluorescence. By using this method, we realized an approximately 5-fold increase in signal to noise ratio (S/N) in the direct detection of RNA target probes using flow cytometry. Trypan blue aided in the resolution of dim surface antibodies, internal markers and probes, and functions to reduce background autofluorescence after thermocycling and hybridization. This technique is rapid and easily applicable for reducing intracellular autofluorescence, and can be used in single and dual color applications.
Metallointercalator-DNA conjugates were prepared by amide bond formation between active esters on the nonintercalating ligands of transition metal complexes and primary amines presented at the 5' or the 3' termini of oligonucleotides attached to solid supports. The conjugates were liberated from the support by aminolysis and purified by HPLC on C18 or C4 stationary phases, which separates the two diastereomeric forms of the conjugates containing either the Lambda or the Delta enantiomer of the octahedral metal complex. The coupling reaction proceeds with approximately 75% conversion of the amino-terminated oligonucleotide into the conjugate; the isolated yield is approximately 200 nmol for syntheses initiated on DNA-synthesis columns with a loading of 2 micromol. The conjugates were characterized by ultraviolet-visible and circular dichorism absorption spectroscopy, electrospray ionization mass spectrometry, enzymatic digestion, and polyacrylamide gel electrophoresis (PAGE). Oligonucleotides bearing [Rh(phi)(2)(bpy')](3+) (phi = 9, 10-phenanthrene quinone diimine; bpy' = 4-butyric acid-4'-methyl bipyridyl) form 1:1 duplexes with the complementary strand, and the electrophoretic mobility under nondenaturating PAGE of duplexes containing Delta-Rh is notably different from duplexes containing Lambda-Rh. High-resolution PAGE of DNA photocleavage reactions initiated by irradiation of the tethered Rh complexes reveal intercalation of the complex only near the tethered end of the duplex. Analogous DNA-binding properties were observed with [Rh(phi)(2)(bpy')](3+) tethered to the 3' terminus. By combining the 3' and 5' modification strategies, a mixed-metal DNA conjugate containing both [Os(phen)(bpy')(Me(2)-dppz)](2+) (Me(2)-dppz = 7, 8-dimethyldipyridophenazine) on the 3' terminus and [Rh(phi)(2)(bpy')](3+) on the 5' terminus was prepared and isolated. Taken together, these strategies for preparing metallointercalator-DNA conjugates offer a useful approach to generate chemical assemblies to probe long-range DNA-mediated charge transfer where the redox initiator is confined to and intercalated in a well-defined binding site.
We observed the expression of human c-fos mRNA in a living transfected Cos7 cell under a fluorescence microscope by detecting hybrid formed with two fluorescently labeled oligodeoxynucleotides (oligoDNAs) and c-fos mRNA in the cytoplasm. Two fluorescent oligoDNAs were prepared, each labeled with a fluorescence molecule different from the other. When two oligoDNAs hybridized to an adjacent sequence on the target mRNA, the distance between the two fluorophores became very close and fluorescence resonance energy transfer (FRET) occurred, resulting in changes in fluorescence spectra. To find sequences of high accessibility of c-fos RNA to oligoDNAs, several sites that included loop structures on the simulated secondary structure were selected. Each site was divided into two halves, and the pair of fluorescent oligoDNAs complementary to the sequence was synthesized. Each site was examined for the efficiency of hybridization to c-fos RNA by measuring changes in fluorescence spectra when c-fos RNA was added to the pair of oligoDNAs in solution. A 40 mer specific site was found, and the pair of oligoDNAs for the site were microinjected into Cos7 cells that expressed c-fos mRNA. To block oligoDNAs from accumulating in the nucleus, oligoDNA was bound to a macromolecule (streptavidin) to prevent passage of nuclear pores. Hybridization of the pair of oligoDNAs to c-fos mRNA in the cytoplasm was detected in fluorescence images indicating FRET.
A number of formats for nucleic acid hybridization have been developed to identify DNA and RNA sequences that are involved in cellular processes and that aid in the diagnosis of genetic and infectious diseases.
The introduction of hybridization probes with interactive fluorophore pairs has enabled the development of homogeneous hybridization assays for the direct identification of nucleic acids. A change in the fluorescence of these probes indicates the presence of a target nucleic acid, and there is no need to separate unbound probes from hybridized probes.
The advantages of homogeneous hybridization assays are their speed and simplicity. In addition, homogeneous assays can be combined with nucleic acid amplification, enabling the detection of rare target nucleic acids. These assays can be followed in real time, providing quantitative determination of target nucleic acids over a broad range of concentrations.
We outline the different approaches taken by our group in the design of fluorescent hybridization sensors. Molecular beacons (MBs) and binary probes (BPs) using two dyes (2d-MB and 2d-BP, respectively) have been synthesized; these sensors serve as switches in emission upon binding to target biomolecules, such as DNA. These sensors allow for ratiometric fluorescence detection of polynucleotides (PNs) by visualization of the probes when bound to a target PN. Additionally, three-dye MBs (3d-MB) and BPs (3d-BP) have been developed, where an energy-transfer cascade is employed to decrease the overlap between the fluorophore emission spectra, resulting in a low direct excitation of the acceptor fluorophore. Pyrene-based MB (Py-MB) and BP (Py-BP), which possess the advantage of long fluorescence lifetimes, have also been synthesized. Time-resolved fluorescence spectra (TRES) can be used to discriminate between short-lived background fluorescence and long-lived fluorescence of the pyrene probes. This technique was demonstrated by time-resolving the signal of a Py-BP from the background fluorescence in Aplysia californica cell extracts.
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University Science Books
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- Mosiman V. L.