Reusable split-aptamer-based biosensor for rapid detection of cocaine in serum by using an all-fiber evanescent wave optical biosensing platform

A rapid, facile, and sensitive assay of cocaine in biological fluids is important to prevent illegal abuse of drugs. A two-step structure-switching aptasensor has been developed for cocaine detection based on evanescent wave optical biosensing platform. In the proposed biosensing platform, two tailored aptamer probes were used to construct the molecular structure switching. In the existence of cocaine, two fragments of cocaine aptamer formed a three-way junction quickly, and the fluorophore group of one fragment was effectively quenched by the quencher group of the other one. The tail of the three-way junction hybridized with the cDNA sequences immobilized on the optical fiber biosensor. Fluorescence was excited by evanescent wave, and the fluorescence signal was proportional to cocaine concentration. Cocaine was detected in 450 s (300 s for incubation and 150 s for detection and regeneration) with a limit of detection (LOD) of 165.2 nM. The proposed aptasensor was evaluated in human serum samples, and it exhibited good recovery, precision, and accuracy without complicated sample pretreatments.     

A rational approach to minimal high-resolution cross-reactive arrays

We report a rational approach to the construction of cross-reactive arrays for steroids consisting of five to seven sensors incorporating modified oligonucleotides. The sensors for our arrays were selected to maximize their differential responses to the two steroids most different in an arbitrarily chosen parameter named "shape-length". The arrays incorporated three previously unreported types of sensors identified through a massive screening effort: (1) three-way junction sensors with neutralized charges within junction; (2) "self-aggregating sensors"; and (3) sensors incorporating fluorophore directly in a three-way junction as a spacer. The arrays were tested on seven steroids and an alkaloid (cocaine) over a range of concentrations, and achieved 92-96% accuracy in class assignments, despite the close structural similarities between steroids.     

Capillary electrophoresis and fluorescence studies on molecular beacon-based variable length oligonucleotide target discrimination

Molecular beacons (MBs) are oligonucleotide probes having a compact hairpin structure, with a fluorophore attached to one end and a quencher molecule attached to the other end. In its native state, the fluorophore is quenched by virtue of its proximity to the quencher molecule. Upon hybridization with its complementary oligonucleotide target, fluorescence is elicited due to a conformational change that results in separation of the fluorophore and quencher molecule. The present study describes the hybridization interaction of an MB to various complementary target sequences. The effects of temperature and length of complementary target sequences on hybridization were investigated using capillary electrophoresis and solution-based fluorescence techniques. Hybridization efficiency was dependent on the ability of the target sequences to destabilize the stem region by binding directly to the stem region. Optimal hybridization occurred between 40 and 50 degrees C for all targets tested, with the true target forming a more stable hybrid complex.     

Principal factors that determine the extension of detection range in molecular beacon aptamer/conjugated polyelectrolyte bioassays

A strategy to extend the detection range of weakly-binding targets is reported that takes advantage of fluorescence resonance energy transfer (FRET)-based bioassays based on molecular beacon aptamers (MBAs) and cationic conjugated polyelectrolytes (CPEs). In comparison to other aptamer-target pairs, the aptamer-based adenosine triphosphate (ATP) detection assays are limited by the relatively weak binding between the two partners. In response, a series of MBAs were designed that have different stem stabilities while keeping the constant ATP-specific aptamer sequence in the loop part. The MBAs are labeled with a fluorophore and a quencher at both termini. In the absence of ATP, the hairpin MBAs can be opened by CPEs via a combination of electrostatic and hydrophobic interactions, showing a FRET-sensitized fluorophore signal. In the presence of ATP, the aptamer forms a G-quadruplex and the FRET signal decreases due to tighter contact between the fluorophore and quencher in the ATP/MBA/CPE triplex structure. The FRET-sensitized signal is inversely proportional to [ATP]. The extension of the detection range is determined by the competition between opening of the ATP/MBA G-quadruplex by CPEs and the composite influence by ATP/aptamer binding and the stem interactions. With increasing stem stability, the weak binding of ATP and its aptamer is successfully compensated to show the resistance to disruption by CPEs, resulting in a substantially broadened detection range (from millimolar up to nanomolar concentrations) and a remarkably improved limit of detection. From a general perspective, this strategy has the potential to be extended to other chemical- and biological-assays with low target binding affinity.     

Intramolecular dimers: a new strategy to fluorescence quenching in dual-labeled oligonucleotide probes

Many genomics assays use profluorescent oligonucleotide probes that are covalently labeled at the 5' end with a fluorophore and at the 3' end with a quencher. It is generally accepted that quenching in such probes without a stem structure occurs through F?rster resonance energy transfer (FRET or FET) and that the fluorophore and quencher should be chosen to maximize their spectral overlap. We have studied two dual-labeled probes with two different fluorophores, the same sequence and quencher, and with no stem structure: 5'Cy3.5-beta-actin-3'BHQ1 and 5'FAM-beta-actin-3'BHQ1. Analysis of their absorption spectra, relative fluorescence quantum yields, and fluorescence lifetimes shows that static quenching occurs in both of these dual-labeled probes and that it is the dominant quenching mechanism in the Cy3.5-BHQ1 probe. Absorption spectra are consistent with the formation of an excitonic dimer, an intramolecular heterodimer between the Cy3.5 fluorophore and the BHQ1 quencher.     

Fluorescence resonance energy transfer by quencher adsorption into hydrogels containing fluorophores

We synthesized anionic hydrogels containing fluorophores and investigated the adsorption of a cationic quencher having an amino group into hydrogels by fluorescence resonance energy transfer (FRET). FRET from the fluorophore to the quencher in hydrogels was examined by fluorescence intensity and fluorescence decay using a fluorescence spectrophotometer and femtosecond laser spectroscopy. The fluorescence intensity of the fluorophore‐containing hydrogels decreased rapidly with increasing amounts of adsorbed cationic quencher. The fluorescence emission of the fluorophore in the quencher‐adsorbed hydrogels containing fluorophores decayed more rapidly than that of the original hydrogels. The aforementioned result indicates that the fluorescence of the fluorophore‐containing hydrogels is quenched due to FRET from the fluorophore to the quencher as the cationic quenchers can approach the fluorophores in hydrogels by electrostatic interactions. © 2006 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 44: 3245–3252, 2006     

Aptamer switch probe based on intramolecular displacement

A novel aptamer-based molecular probe design employing intramolecular signal transduction is demonstrated. The probe is composed of three elements: an aptamer, a short, partially cDNA sequence, and a PEG linker conjugating the aptamer with the short DNA strand. We have termed this aptamer probe an "aptamer switch probe", or ASP. The ASP design utilizes both a fluorophore and a quencher which are respectively modified at the two termini of the ASP. In the absence of the target molecule, the short DNA will hybridize with the aptamer, keeping the fluorophore and quencher in close proximity, thus switching off the fluorescence. However, when the ASP meets its target, the binding between the aptamer and the target molecule will disturb the intramolecular DNA hybridization, move the quencher away from the fluorophore, and, in effect, switch on the fluorescence. Both ATP and human alpha-thrombin aptamers were engineered to demonstrate this design, and both showed that fluorescence enhancement could be quantitatively mediated by the addition of various amounts of target molecules. Both of these ASPs presented excellent selectivity and prompt response toward their targets. With intrinsic advantages resulting from its intramolecular signal transduction architecture, the ASP design holds promising potential for future applications, such as biochip and in situ imaging, which require reusability, excellent stability, prompt response, and high sensitivity.     

Modulation of intramolecular heterodimer-induced fluorescence quenching of tricarbocyanine dye for the development of fluorescent sensor

Various approaches have been used to modulate the fluorescence changes of sensors in the presence of target analytes, including intramolecular interaction between fluorophores or between fluorophore and other molecular species, like resonance energy transfer (RET). Here, we focus on fluorescence quenching by intramolecular heterodimer complex formation, which can be modulated over a shorter distance range than RET. We synthesized several conjugates of tricarbocyanine, which is a near-infrared fluorophore, with several quencher candidates via flexible short linker structure, and examined their fluorescence properties. Of our synthesized compounds, the dabcyl group proved to be the best quencher via heterodimer complex formation. The fluorescence of tricarbocyanine-dabcyl conjugates in aqueous media was almost completely quenched, and there was a dramatic fluorescence enhancement when heterodimer formation was blocked. These results suggested a design approach to develop fluorescence sensors for probing proximity relationships and structural transitions.     

A double-stranded molecular probe for homogeneous nucleic acid analysis

This paper reports the design and optimization of a double-stranded molecular probe for homogeneous detection of specific nucleotide sequences. The probes are labeled with either a fluorophore or a quencher such that the probe hybridization brings the two labels into close proximity, and this diminishes the fluorescence signal in the absence of a target. In the presence of a target, the fluorophore probe is thermodynamically driven to unzip from its hybridized form and bind with the target. An equilibrium analysis, which successfully describes all the major features of the assay without any fitting parameter, is performed to generalize the design of the probe. Several key parameters affecting the performance of the assay are examined. We show that the dynamic range and the signal-to-noise ratio of the assay can be optimized by the probe concentration, the quencher-to-fluorophore ratio, and the probe strand sequence. By proper design of the sequence, the probe discriminates single nucleotide mismatches in a single step without any separation step or measurement of melting profile.     

Inhibited aptazyme-based catalytic molecular beacon for amplified detection of adenosine

Combining the inhibited aptazyme and molecular beacon(MB),we developed a versatile sensing strategy for amplified detection of adenosine.In this strategy,the adenosine aptamer links to the 8-17 DNAzyme to form an aptazyme.A short sequence,denoted as inhibitor,is designed to form a duplex spanning the aptamer–DNAzyme junction,which blocks the catalytic function of the DNAzyme.Only in the presence of target adenosine,the aptamer binds to adenosine,thus the inhibitor dissociates from the aptamer portion of the aptazyme and can no longer form the stable duplex required to inhibit the catalytic activity of the aptazyme.The released DNAzyme domain will hybridize to the MB and catalyze the cleavage in the presence of Zn2+,making the fluorophore separate from the quencher and resulting in fluorescence signal.The results showed that the detection method has a dynamic range from 10 nmol/L to 1 nmol/L,with a detection limit of 10 nmol/L.     

Molecular beacons for bioanalytical applications

Molecular beacons (MBs) are hairpin-shaped oligonucleotides that contain both fluorophore and quencher moieties. They act like switches and are normally in a closed state, when the fluorophore and the quencher are brought together to turn "off" the fluorescence. When prompted to undergo conformational changes that open the hairpin structure, the fluorophore and the quencher are separated, and fluorescence is turned "on." This Education will outline the principles of MBs and discuss recent bioanalytical applications of these probes for in vitro RNA and DNA monitoring, biosensors and biochips, real-time monitoring of genes and gene expression in living systems, as well as the next generation of MBs for studies on proteins, the MB aptamers. These important applications have shown that MBs hold great potential in genomics and proteomics where real-time molecular recognition with high sensitivity and excellent specificity is critical.     

Application of Single—labelled Probe—primer in PCR Amplification to the Detection of Hepatitis B Virus DNA

A new method based on the incorporation of a single-lablled probe-primer into polymerase chain reaction(PCR) for the detection of PCR-amplified DNA in a closed system is reported.The probeprimerc consists of a specific probe sequence on the 5‘‘‘‘‘‘‘‘-end and a primer sequence on the 3‘‘‘‘‘‘‘‘-end.A flurophore is located at the 5‘‘‘‘‘‘‘‘end.The primeR-quencher is an oligonucleotide,which is complementary to the probe sequence of probe-primer and labelled with a quencher at the 3‘‘‘‘‘‘‘‘-end.In the duplex formed by probe-primer and primer-quencher.the fluorophore and quencher are kept in close proximity to each other.Therefore the fluorescence is quenched.During PCR amplificatio,the specific probe sequence of probeprimer binds to its complement within the same strand of DNA,and is cleaved by Taq DNA polymerase,resulting in the restoration of fluorescence.This system has the same energy transfer mechanism as molecular beacons,and a good quenching effciency can be ensured.Following optimization of PCR conditions,this method was used to detect hepatitis b virus(HBV) dna in patient sera.This technology eliminates the risk of carry-over contamination,simplifies the amplification assay and opens up new possibilities for the real-time detection of the amplified DNA.     

All-trans-N-retinylidenetryptamine Schiff base in surfactant solubilized water pools in heptane—a fluorescence study

All-trns-N-retinylidenetryptamine Schiff base was incorporated into aerosol-OT (AOT, sodium bis(2-ethylhexyl)sulphosuccinate)/heptane reverse micelles. This micellar system was used as a model to study the retinal-tryptophan interactions in retinal proteins. The retinylidene Schiff base remains stable in the presence of reverse micelle-solubilized water pools. Partition coefficient and microviscosity measurements show that the Schiff base is located in the micellar interphase. The results are discussed in terms of the interaction between the retinylidene chromophore and the active site environment of rhodopsin and bacteriorhodopsin. In the present model, the quencher and emitting units are covalently attached, and are separated by two carbon spacer units. The fluorescence emission data obtained for the micelle-intercalated Schiff base chromophore are compared with the fluorescence of the native protein and intermediates in the photochemical cycle of bacteriofhodopsin. A comparison of the data obtained for tryptamine and the Schiff base with the results available for bacteriorhodopsin and bacterioopsin reveals that there is a large degree of quenching on intercalation of the retinylidene chromophore in the vicinity of the fluorophore. Evidence provided by this model suggests that energy transfer to retinal can occur from tryptophan residues located in the retinal pocket in the native protein. Thus the retinylidene unit can act as a quencher of the energy of tryptophan, the nature and extent of which may depend on the conformation and relative orientation of the protein-bound fluorophore.     

Bulge‐like Asymmetric Heterodye Clustering in DNA Duplex Results in Efficient Quenching of Background Emission Based on the Maximized Excitonic Interaction

Asymmetric dye clusters with a single fluorophore (Cy3) and multiple quenchers (4′‐methylthioazobenzene‐4‐carboxylate, methyl red, and 4′‐dimethylamino‐2‐nitroazobenzene‐4‐carboxylate) were prepared. The dye and one‐to‐five quenchers were tethered through D ‐threoninol to opposite strands of a DNA duplex. NMR analysis revealed that the clusters with a single fluorophore and two quenchers formed a sandwich‐like structure (antiparallel H‐aggregates). The melting temperatures of all the heteroclusters were almost the same, although structural distortion should become larger, as the number of quenchers increased. An asymmetric heterocluster of a single fluorophore and two quenchers showed larger excitonic interaction (i.e., hypochromicity of Cy3), than did a single Cy3 and a single quencher. Due to the larger exciton coupling between the dyes, the 1:2 heterocluster suppressed the background emission more efficiently than the 1:1 cluster. However, more quenchers did not enhance quenching efficiency due to the saturation of exciton coupling with two quenchers. Finally, this asymmetric 1:2 heterocluster was introduced into the stem region of a molecular beacon (MB; also known as an in‐stem MB) targeting the fusion site in the L6 BCR‐ABL fusion gene. With this MB design, the signal/background ratio was as high as 68 due to efficient suppression of background emission resulting from the maximized excitonic interaction.     

双链荧光核酸适体探针用于蛋白质的简便快速检测

发展了一种基于双链荧光核酸适体(F-Aptamer)探针的简单快速检测蛋白质的分析方法.该双链荧光Aptamer探针由一条带荧光标记的Aptamer探针和带猝灭标记的互补DNA组成,当靶蛋白存在时,能形成比双链荧光Aptamer探针更稳定的F-Aptamer/蛋白质复合物,并发出荧光,从而实现对蛋白质的简便快速检测,检测线性范围为6~100 nmol/L,检出限为6 nmol/L.该方法设计简单,对核酸适体分子的大小和空间结构没有要求,可作为一种通用的基于F-Aptamer识别机理的蛋白质检测方法.     

The mechanism of pyrene fluorescence quenching by selective and nonselective quenchers during sol–gel transition

The process of pyrene fluorescence quenching by potassium iodide, acrylamide, and gaseous oxygen in silane sol during sol–gel transition was investigated. Pyrene fluorescence emission spectra were recorded vs concentrations of the added quencher on consecutive days of the gelling process. When using 0, 20, and 100% oxygen in the gas mixture as a quencher, time-resolved measurements were also made. On this basis, Stern–Volmer constants were determined on consecutive days of the gelling process, availability of fluorophore molecules to the quencher, and also rate constants of pyrene quenching by acrylamide, iodide ions, and oxygen were specified. Moreover, diffusion coefficients were determined for acrylamide and potassium iodide in the silane gel formed. The mechanism of fluorophore quenching in sol of growing viscosity and in gel was discussed in view of using this carrier in the construction of sensors and optical biosensors.     

Bulge-like Asymmetric Heterodye Clustering in DNA Duplex Results in Efficient Quenching of Background Emission Based on the Maximized Excitonic Interaction

Asymmetric dye clusters with a single fluorophore (Cy3) and multiple quenchers (4'-methylthioazobenzene-4-carboxylate, methyl red, and 4'-dimethylamino-2-nitroazobenzene-4-carboxylate) were prepared. The dye and one-to-five quenchers were tethered through D-threoninol to opposite strands of a DNA duplex. NMR analysis revealed that the clusters with a single fluorophore and two quenchers formed a sandwich-like structure (antiparallel H-aggregates). The melting temperatures of all the heteroclusters were almost the same, although structural distortion should become larger, as the number of quenchers increased. An asymmetric heterocluster of a single fluorophore and two quenchers showed larger excitonic interaction (i.e., hypochromicity of Cy3), than did a single Cy3 and a single quencher. Due to the larger exciton coupling between the dyes, the 1:2 heterocluster suppressed the background emission more efficiently than the 1:1 cluster. However, more quenchers did not enhance quenching efficiency due to the saturation of exciton coupling with two quenchers. Finally, this asymmetric 1:2 heterocluster was introduced into the stem region of a molecular beacon (MB; also known as an in-stem MB) targeting the fusion site in the L6 BCR-ABL fusion gene. With this MB design, the signal/background ratio was as high as 68 due to efficient suppression of background emission resulting from the maximized excitonic interaction.     

Fluorescence quenching of naphthols by Cu2+ in micelles

Effect of the micelles of anionic, cationic and non-ionic surfactants on the fluorescence quenching of 1- and 2-naphthols has been studied in the presence of copper ion. The excited state lifetime, dynamic and static quenching constants for these systems have been determined. Fluorescence quenching in water and SDS micelle is due to the collision of the fluorophore with the quencher with a small static component. The negatively charged naphtholate ions in the excited state are quenched with significantly higher rates than the neutral naphthol molecules, which are located further inside the mesophase. CTAB micelle is less effective than the SDS micelle for fluorescence quenching. The effect of CTAB on water-assisted excited-state deprotonation has been investigated in the presence of ZnSO₄. For TX-100 micelle there is negligible quenching even at higher concentration of the quencher.