A Novel Screening Method for Competitive FRET-Aptamers Applied to <Emphasis Type="Italic">E. coli</Emphasis> Assay Development

A novel high-throughput screening method is described in which a family of DNA aptamers selected against E. coli outer membrane proteins (OMPs) is subjected to PCR in the presence of fluorophore-dUTP conjugates using Deep Vent® exo- polymerase. The fluorophore-doped aptamers and their complementary strands are then heated to render them single-stranded and screened in filter well microtiter plates for fluorescence resonance energy transfer (FRET) assay potential. Using this system, a superior competitive FRET-aptamer designated EcO 4R was identified and the location of its putative binding pocket was determined by individually testing FRET potential in each of the secondary loop structures. By labeling the binding pocket with Alexa Fluor (AF) 647 and binding the aptamer to heavily Black Hole Quencher-3 (BHQ-3)-labeled E. coli bacteria, detection of as few as 30 live unlabeled E. coli per ml was achieved in a competitive displacement FRET assay format. The far red fluorescence emission enables detection in largely blue-green autofluorescent matrices. In addition, the competitive transfer of AF 647-EcO-4R aptamer to unlabeled E. coli cells after a 15 min equilibration period was verified by fluorescence microscopy. The present study also demonstrated that high aptamer affinity is not well correlated with competitive FRET potential.     

A ratiometric fluorescent aptamer homogeneous biosensor based on hairpin structure aptamer for AFB1 detection

On the basis of aptamer (Apt) with hairpin structure and fluorescence resonance energy transfer (FRET), a ratio fluorescent aptamer homogeneous sensor was prepared for the determination of Aflatoxin B₁ (AFB1). Initially, the Apt labeled simultaneously with Cy5, BHQ2, and cDNA labeled with Cy3 were formed a double-stranded DNA through complementary base pairing. The fluorescence signal of Cy3 and Cy5 were restored and quenched respectively. Thus, the ratio change of F_Cy3 to F_Cy5 was used to realized the detection of AFB1 with wider detection range and lower limit of detection (LOD). The response of the optimized protocol for AFB1 detection was wider linear range from 0.05 ng/mL to 100 ng/mL and the LOD was 12.6 pg/mL. The sensor designed in this strategy has the advantages of simple preparation and fast signal response. It has been used for the detection of AFB1 in labeled corn and wine, and has good potential for application in real samples.

     

A Novel Reconfigurable Optical Biosensor Based on DNA Aptamers and a DNA Molecular Beacon

In order to alter a typical molecular aptamer beacon (MAB) to detect a different analyte there is currently a need to change the whole sensor unit including the expensive labeling fluorophores. In this work a DNA-based reconfigurable molecular aptamer beacon was developed. It is composed of two parts: a variable part and a constant part. The variable part comprises an aptamer strand and its complementary strand while the constant part is an oligonucleotide doubly labeled with a Förster Resonance Energy Transfer (FRET) pair and the two parts become joined via DNA hybridization. The sensor exists in two conformations: a folded (high FRET) and an unfolded (low FRET) in the absence and presence of the aptamer-target binding respectively. This sensor can be reconfigured by washing away the aptamer and the complementary strand using proper complementary strands, called washers. As a proof of the principle, a sensor that bound the enzyme thrombin, an analyte with a strong binding, was first constructed and then reconfigured to bind adenosine, selected as an analyte with a weak binding. We believe that the design is of universal use applicable to many types of aptamers.     

Single-Pair Fluorescence Resonance Energy Transfer (spFRET) for the High Sensitivity Analysis of Low-Abundance Proteins Using Aptamers as Molecular Recognition Elements

We have developed a strategy for the detection of single protein molecules, which uses single-pair fluorescence resonance energy transfer (spFRET) as the readout modality and provides exquisite analytical sensitivity and reduced assay turn-around-time by eliminating various sample pre-processing steps. The single-protein detection assay uses two independent aptamer recognition events to form an assembly conducive to intramolecular hybridization of oligonucleotide complements that are tethered to the aptamers. This hybridization brings a donor-acceptor pair within the Förster distance to create a fluorescence signature indicative of the presence of the protein-aptamer(s) association complex. As an example of spFRET, we demonstrate the technique for the analysis of serum thrombin. The assay requires co-association of two distinct epitope-binding aptamers, each of which is labeled with a donor or acceptor fluorescent dye (Cy3 or Cy5, respectively) to produce a FRET response. The FRET response between Cy3 and Cy5 was monitored by single-molecule photon-burst detection, which provides high analytical sensitivity when the number of single-molecule events is plotted versus the target concentration. We are able to identify thrombin with high efficiency based on photon burst events transduced in the Cy5 detection channel. We also demonstrate that the technique can discriminate thrombin molecules from its analogue prothrombin. The analytical sensitivity was >200-fold better than an ensemble measurement.     

Two-Photon Excited Fluorescence Energy Transfer: A Study Based on Oligonucleotide Rulers

The use of two-photon excitation of fluorescence for detection of fluorescence resonance energy transfer (FRET) was studied for a selected fluorescent donor–acceptor pair. A method based on labeled DNA was developed for controlling the distance between the donor and the acceptor molecules. The method consists of hybridization of fluorescent oligonucleotides to a complementary single-stranded target DNA. As the efficiency of FRET is strongly distance dependent, energy transfer does not occur unless the fluorescent oligonucleotides and the target DNA are hybridized. A high degree of DNA hybridization and an excellent FRET efficiency were verified with one-photon excited fluorescence studies. Excitation spectra of fluorophores are usually wider in case of two-photon excitation than in the case of one-photon excitation [1]. This makes the selective excitation of donor difficult and might cause errors in detection of FRET with two-photon excited fluorescence. Different techniques to analyze the FRET efficiency from two-photon excited fluorescence data are discussed. The quenching of the donor fluorescence intensity turned to be the most consistent way to detect the FRET efficiency. The two-photon excited FRET is shown to give a good response to the distance between the donor and the acceptor molecules.     

Highly Sensitive Optical Biosensor for Thrombin Based on Structure Switching Aptamer-Luminescent Silica Nanoparticles

We describe here the construction of a sensitive and selective optical sensor system for the detection of human α-thrombin. The surface functionalized luminescent [Ru(dpsphen)₃]^4? (dpsphen-4,7-diphenyl-1,10-phenanthroline disulfonate) ion doped silica nanoparticles (SiNPs) with a size ~70 nm have been prepared. The DABCYL (2-(4-dimethylaminophenyl)diazenyl-benzoic acid) quencher labeled thrombin binding aptamer is conjugated to the surface of SiNPs using BS³ (bis(sulfosuccinimidyl) suberate) as a cross-linker, resulting in the conformational change of aptamer to form G-quadruplex structure upon the addition of thrombin. The binding event is translated into a change in the luminescence intensity of Ru(II) complex via FRET mechanism, due to the close proximity of DABCYL quencher with SiNPs. The selective detection of thrombin using the SiNPs-aptamer system up to 4 nM is confirmed by comparing its sensitivity towards other proteins. This work demonstrates the application of simple aptamer-SiNPs conjugate as a highly sensitive system for the detection of thrombin and also it is highly sensitive towards thrombin in the presence of other proteins and complex medium such as BSA.     

Development of Aptamer Beacons for Rapid Presumptive Detection of <Emphasis Type="Italic">Bacillus</Emphasis> Spores

A library of 92 DNA aptamer sequences was developed against Bacillus anthracis (nonpathogenic Sterne strain) spores and anthrose sugar immobilized on magnetic beads. The selected DNA sequences were studied for similarities and potential binding pockets between the B. anthracis spore and anthrose aptamers. Several recurring loop structures were identified and tested for their potential to act as aptamer beacons when labeled with TYE 665 dye on their 5′ ends and Iowa Black quencher on their 3′ ends. Of these candidate sequences, two beacons designated BAS-6F and BAS-6R emerged which gave strong fluorescence responses at high spore concentrations (greater than 30,000 spores/ml). These aptamer beacons also detect B. cereus and B. thuringiensis spores with greater fluorescence intensity, but do not strongly detect vegetative cells from an array of other bacterial species. BAS-6F and 6R are also not capable of detecting pure anthrose, thereby probably ruling that epitope out as a spore surface target for these particular beacons. While not extremely sensitive, the BAS-6F and 6R aptamer beacons are potentially valuable for rapid presumptive detection of anthrax or Bacillus spores in suspect powders or bioterrorist activity where spore concentrations are anticipated to be high. The sequence similarities of these beacons to other published Bacillus spore aptamers are also discussed.     

Detection of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> Carrying the Gene for Toxic Shock Syndrome Toxin 1 by Quantum-Dot-Probe Complexes

In this study, a high-sensitive and high-specific method to detect the toxic shock syndrome toxin-1 (TSST-1)-producing Staphylococcus aureus was developed based on quantum dot (QD) and oligonucleotide probe complexes. S. aureus carrying tst gene which is responsible for the production of TSST-1 were detected based on fluorescence resonance energy transfer (FRET) occurring between CdSe/ZnS QD donors and black hole quencher (BHQ) acceptors. QD-DNA probe was prepared by conjugating the carboxyl-modified QD and the amino-modified DNA with the EDC. Photoluminescence (PL) quenching was achieved through FRET after the addition of BHQ-DNA which was attached to tst gene probe by match sequence hybridization. The PL recovery was detected in the presence of target DNA by BHQ-DNA detached from QD-DNA probe because of the different affinities. In contrast, mismatch oligonucleotides and DNAs of other bacteria did not contribute to fluorescence intensity recovery, which exhibits the higher selectivity of the biosensor. The experimental results showed clearly that the intensity of recovered QD PL is linear to the concentration of target DNA within the range of 0.2–1.2 μM and the detection limit was 0.2 μM.     

DNA aptamer beacon assay for C-telopeptide and handheld fluorometer to monitor bone resorption

A novel DNA aptamer beacon is described for quantification of a 26-amino acid C-telopeptide (CTx) of human type I bone collagen. One aptamer sequence and its reverse complement dominated the aptamer pool (31.6% of sequenced clones). Secondary structures of these aptamers were examined for potential binding pockets. Three-dimensional computer models which analyzed docking topologies and binding energies were in agreement with empirical fluorescence experiments used to select one candidate loop for beacon assay development. All loop structures from the aptamer finalists were end-labeled with TYE 665 and Iowa Black quencher for comparison of beacon fluorescence levels as a function of CTx concentration. The optimal beacon, designated CTx 2R-2h yielded a low ng/ml limit of detection using a commercially available handheld fluorometer. The CTx aptamer beacon bound full-length 26-amino acid CTx peptide, but not a shorter 8-amino acid segment of CTx peptide which is a common target for commercial CTx ELISA kits. The prototype assay was shown to detect CTx peptide from human urine after creatinine and urea were removed by size-exclusion chromatography to prevent nonspecific denaturing of the aptamer beacon. This work demonstrates the potential of aptamer beacons to be utilized for rapid and sensitive bone health monitoring in a handheld or point-of-care format.     

Fluorescence Assay Based on Aptamer-Quantum Dot Binding to <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Spores

A novel assay was developed for the detection of Bacillus thuringiensis (BT) spores. The assay is based on the fluorescence observed after binding an aptamer-quantum dot conjugate to BT spores. The in vitro selection and amplification technique called SELEX (Systematic Evolution of Ligands by EXponential enrichment) was used in order to identify the DNA aptamer sequence specific for BT. The 60 base aptamer was then coupled to fluorescent zinc sulfide-capped, cadmium selenide quantum dots (QD). The assay is semi-quantitative, specific and can detect BT at concentrations of about 1,000 colony forming units/ml.     

FRET lasing from self-assembled DNA tetrahedral nanostructures suspended in optofluidic droplet resonators

We demonstrate Förster resonance energy transfer (FRET) lasing from self-assembled tetrahedral DNA complexes labeled with Cy3 and Cy5 dyes and suspended as a gain medium in aqueous microdroplet cavities deposited on a superhydrophobic surface. Threshold fluence and differential efficiency are characterized for DNA complexes containing 1Cy3-3Cy5 and 3Cy3-1Cy5. We demonstrate that at a constant Cy5 concentration, average threshold fluence is reduced 3 to 8 times and average differential efficiency is enhanced 6 to 30 times for 3Cy3-1Cy5 as compared to 1Cy3-3Cy5. Using 3Cy3-1Cy5 nanostructures, FRET lasing is observed at very low concentrations down to ～ 1 μM. This work shows that optofluidic microlasers based on droplet resonators can be combined with DNA nanotechnology to explore applications in bio/chemical sensing and novel photonic devices.     

A Significant Fluorescent Aptamer Sensor Based on Carbon Dots and Graphene Oxide for Highly Selective Detection of Progesterone

In this paper, a fluorescent aptamer sensor was constructed based on the carbon dots (CDs) and graphene oxide (GO). This sensor combines the excellent fluorescence performance of CDs with the high specificity of aptamer, which can detect progesterone (P₄) with high sensitivity and selectivity. In the absence of P₄, the CDs-aptamer system and GO form a fluorescence resonance energy transfer process (FRET), which quenches the fluorescence of the CDs. When P₄ is added, the aptamer specifically binds to it, resulting the fluorescence of the CDs is recovered. At optimal conditions, the fluorescence intensity recovered by the CDs has a linear relationship with the concentration of P₄ in the range of 0.1–120 nM and the detection limit is 3.3?×?10^–11 M. Besides, the sensor has satisfactory detection results of P₄ in milk, indicating that constructed method has enormous potential for application in food safety.

     

Detection of PCR products amplified from DNA of epizootic pathogens using magnetic nanoparticles and SERS

Here we present a novel approach using surface‐enhanced Raman scattering (SERS) spectroscopy for the sequence‐specific detection of DNA utilizing magnetic nanoparticles (MNPs) for the enrichment of the target molecules. To achieve fast and efficient binding of longer DNA strands, e.g. PCR products, the hybridization procedure is performed in solution. To further purify and enrich the DNA strands of interest, MNPs are used for their separation. Following the binding of the target DNA, a dye‐modified, short synthetic ssDNA is hybridized, which serves as label for the SERS detection. The SERS spectra are used to identify the bound molecules. The applicability of this approach was first tested with short synthetic oligonucleotides to evaluate its specificity. Afterward, the system was applied to detect PCR products amplified from DNA of specific agents of epizootic diseases. Sequences of the bacterium Mycoplasma mycoides subspecies mycoides small colony type (MmmSC), causing contagious bovine pleuropneumonia (CBPP) were used as PCR targets. To demonstrate the multiplexing capability of SERS, the simultaneous detection of three different PCR products labeled with three dyes was performed. Copyright © 2010 John Wiley & Sons, Ltd.     

Rapid Fluorescent Detection of Escherichia coli K88 Based on DNA Aptamer Library as Direct and Specific Reporter Combined With Immuno-Magnetic Separation

Nucleic acid aptamers have long demonstrated the capacity to bind cells with high affinity so that they have been utilized to diagnose various important pathogens. In this study, a DNA aptamer library was on initial efforts developed to act as a specific reporter for rapid detection of enter toxigenic Escherichia coli (ETEC) K88 combined with immuno-magnetic separation (IMS). During a Whole-cell Systematic Evolution of Ligands by Exponential Enrichment (CELL-SELEX) procedure, the last selection pool against ETEC K88, which is named “DNA aptamer library” here, was selected and subsequently identified by flow cytometric analysis and confocal imaging. A K88 monoclonal antibody (mAb) with high affinity (K_aff: 1.616?±?0.033?×?10⁸ M^?1) against K88 fimbrial protein was prepared, biotinylated and conjugated to streptavidin-coated magnetic beads (MBs). After the bacteria were effectively captured and enriched from the complex sample by immuno-magnetic beads (IMBs), 5′-FITC modified aptamer library was directly bound to target cells as a specific reporter for its detection. The detection system showed clearly high specificity and sensitivity with the detection limit of 1.1?×?10³ CFU/ml in pure culture and 2.2?×?10³ CFU/g in artificially contaminated fecal sample. The results also indicated that fluorophore-lablled DNA aptamer library as specific reporter could generate more reliable signals than individual aptamer with best affinity against target cells and implied it would have great applied potential in directly reporting bacteria from complex samples combined with IMS technology.     

Improvements in the Sensitivity of Time Resolved Fluorescence Energy Transfer Assays

The effect on fluorescence resonance energy transfer (FRET) of multiple labelling of DNA oligonucleotides with donor lanthanide chelate and acceptor CyDye fluors has been investigated. It is shown that using a multiple donor lanthanide chelate with a single acceptor Cy or Cy5 can increase sensitivity and fluorescence output. The enhanced FRET observed in the multiple donor label system has been utilised in two different DNA based assay formats to demonstrate the advantages over a steady state fluorescence assay and a radiometric assay.     

Exploring antibody polyspecificity using synthetic combinatorial libraries

Summary Extensive mapping studies for seven antigen-antibody interactions have been carried out using both individual analogs and peptide libraries. With competitive ELISA, these studies have revealed that monoclonal antibodies exhibit a broad range of specificities, from antibodies that recognize only conservative substitutions for 1–2 positions of the antigenic determinant, to antibodies that recognize sequences that are completely unrelated to the parent antigen with comparable affinities. Synthetic combinatorial libraries, containing millions of peptide sequences, permit a more systematic and rapid evaluation of the extent of multiple-binding specificities of monoclonal antibodies than individual analogs. The peptide libraries used here comprise mixtures of compounds having specifically defined positions and mixture positions. The same diversity of sequences in different formats, which differ by the numbers of positions singularly defined and different locations defined within the sequence, can be examined. Comparison of the screening results, selection criteria of the most active mixtures, and different approaches used for the deconvolution of active individual compounds are discussed. Synthetic combinatorial libraries greatly facilitate the understanding of antigen-antibody interactions at the amino acid level and will assist in the development of improved immunodiagnostics.     

Detection of <Emphasis Type="Italic">Bifidobacterium</Emphasis> Species-specific 16S rDNA Based on QD FRET Bioprobe

Fluorescence resonance energy transfer (FRET) that consists of quantum dot as donors and organic fluorophore dyes as acceptors has been a very important method to detect biomolecules such as nucleic acids. In this work, we established a new FRET detection system of Bifidobacterium species-specific 16S rDNA using QD—ROX FRET bioprobe, in which 525 nm QD-DNA conjugation consisted of the carboxyl-modified QD and the amino-modified DNA in the presence of EDC. Both ROX-DNA and the conjugation above could hybridize with the target DNA after forming the QD—ROX bioprobe. When the hybridization made the distance between the QD and ROX to meet FRET effect needed, 525 nm QD fluorescence intensity decreased and ROX fluorescence intensity increased. In the control, there was no notable change of fluorescence intensities without target DNA. It is very clear that the change of the QD and ROX fluorescence intensities provide the good base and guaranty for this rapid and simple detection system.     

Comparison of FÖrster-Resonance-Energy-Transfer Acceptors for Tryptophan and Tyrosine Residues in Native Proteins as Donors

Homogenous bioaffinity analysis with tryptophan/tyrosine residues in native proteins as FÖrster-resonance-energy-transfer (FRET) donors is feasible when suitable fluorophors can act as FRET acceptors in ligands (FRET probes) and FRET efficiency in complexes of proteins and FRET probes is high enough. In complexes of proteins and FRET probes, suitable acceptors should have excitation peaks around 335 nm and high rotation freedom, are preferred to have sufficient quantum yields and excitation valleys around 280 nm. In protein binding sites mimicked with mixtures of neutral phosphate buffer and organic solvents, quantum yields of candidate acceptors are altered inconsistently but their excitation peaks show tiny changes. Fluorophores as acceptors in such FRET probes are buried inside glutathione-S-transferase and have low rotation freedom, but are localized on streptavidin surface and display high rotation freedom; FRET efficiency in complexes of streptavidin and its FRET probes is much stronger than that in complexes of glutathione-S-transferase and its FRET probes. Specially, the quantum yield is about 0.70 for free 1-naphthylamine probe in neutral phosphate buffer, about 0.50 for 1-naphthylamine probe bound by streptavidin, and about 0.15 for that bound by glutathione-S-transferase. The quantum yield is about 0.06 for free dansylamide probe, about 0.11 for dansylamide probe bound by streptavidin and about 0.27 for that bound by glutathione-S-transferase. Therefore, 1-naphthylamine and dansylamide are effective acceptors when they localize on surfaces of complexes of proteins and FRET probes.     

Separate quantification of doubly and singly 13C-labeled metabolites by HSQC-filtered J spectroscopy

NMR detection of multiply labeled compounds in biological samples is often used to follow metabolic pathways. Detection of protons bound to 13C atoms offers a more sensitive approach than direct 13C detection, but generally results in the loss of carbon-carbon coupling information. We have modified an HSQC sequence to refocus the carbon chemical shifts in order to obtain a proton-correlated 13C homonuclear J spectrum, which allows us to measure singly and doubly labeled compounds in the same spectrum.     

Single-Pair FRET Microscopy Reveals Mononucleosome Dynamics

We applied spFRET microscopy for direct observation of intranucleosomal DNA dynamics. Mononucleosomes, reconstituted with DNA containing a FRET pair at the dyad axis and exit of the nucleosome core particle, were immobilized through a 30 bp DNA tether on a polyethyleneglycol functionalized slide and visualized using Total Internal Reflection Fluorescence microscopy. FRET efficiency time-traces revealed two types of dynamics: acceptor blinking and intramolecular rearrangements. Both Cy5 and ATTO647N acceptor dyes showed severe blinking in a deoxygenated buffer in the presence of 2% βME. Replacing the triplet quencher βME with 1 mM Trolox eliminated most blinking effects. After suppression of blinking three subpopulations were observed: 90% appeared as dissociated complexes; the remaining 10% featured an average FRET efficiency in agreement with intact nucleosomes. In 97% of these intact nucleosomes no significant changes in FRET efficiency were observed in the experimentally accessible time window ranging from 10 ms to 10’s of seconds. However, 3% of the intact nucleosomes showed intervals with reduced FRET efficiency, clearly distinct from blinking, with a lifetime of 120 ms. These fluctuations can unambiguously be attributed to DNA breathing. Our findings illustrate not only the merits but also typical caveats encountered in single-molecule FRET studies on complex biological systems.