Fluorescent peptide sensor for the selective detection of Cu

A new fluorescent peptidyl chemosensor for Cu²⁺ ions with fluorescence resonance energy transfer (FRET) capabilities has been synthesized via Fmoc solid-phase peptide synthesis. The metal chelating unit, which is flanked by the fluorophores tryptophan (donor) and dansyl chloride (acceptor), consists of the amino acids glycine and aspartic acid (Gly-Gly-Asp-Gly-Gly-Asp-Gly-Gly-Asp-Gly-Gly-Asp-Gly-Gly). Coordination of the Cu²⁺ ions to the metal chelating unit results in fluorescent quenching of both the donor and acceptor fluorophores. Although it was determined that Cu²⁺ binding causes no change in FRET efficiency, emission and Cu²⁺-induced quenching of the acceptor dye can be used to monitor the concentration of the copper ions, with a detection limit of 32 μg L⁻¹. The sensor also demonstrated sensitivity, reversibility and selectivity towards Cu²⁺ in a transition metal matrix at pH 7.0.     

Fluorescence lifetime based distance measurement illustrates conformation changes of PYL10-CL2 upon ABA binding in solution state

The fluorescence lifetime based FRET distance measurements using sitespecific incorporated unnatural amino acid HC and Alexa488 as FRET pair revealed the different conformations of PYL10-CL2 upon ABA binding.     

General and reliable quantitative measurement of fluorescence resonance energy transfer using three fluorescence channels

In this paper, we describe a comprehensive general system adapted for quantitative fluorescence resonance energy transfer (FRET) measurement using signals from three channels of a fluorescence instrument. The general FRET measurement system involves two established methods, as well as two novel approaches. Unlike the previous measurements, which can be taken correctly only when the quantity of the acceptor is greater than or equal to that of the donor, one of our novel methods can overcome this obstacle and take quantitative FRET measurements when the donor is in excess of the acceptor. Hence the general FRET measurement system allowed one to determine the exact distance when the donor and acceptor were present in different quantities, and integrated the methods for quantitative FRET measurements. The uniformity of measured values and utility of each method were validated using molecular standards based on DNA oligonucleotide rulers. We also discussed and validated the use of a novel method for estimating the relative quantities of the donor and acceptor fluorophores when they were not known before an appropriate method of this system can be selected.     

A generic sandwich-type biosensor with nanomolar detection limits

A quantitative and highly sensitive, yet simple and rapid, biosensor system was developed for the detection of nucleic acid sequences that can also be adapted to the detection of antigens. A dipstick-type biosensor with liposome amplification, based on a sandwich assay format with optical detection, was combined with a simple coupling reaction that allows the transformation of the generic biosensor components to target specific ones by a mere incubation step. This biosensor platform system was developed and optimized, and its principle was proven using DNA oligonucleotides that provided a nucleic acid biosensor for the specific detection of RNA and DNA sequences. However, the coupling reaction principle chosen can also be used for the immobilization of antibodies or receptor molecules, and therefore for the development of immunosensors and receptor-based biosensors. The generic biosensor consists of liposomes entrapping sulforhodamine B that are coated with streptavidin on the outside, and polyethersulfone membranes with anti-fluorescein antibodies immobilized in the detection zone. In order to transform the generic biosensor into a specific DNA/RNA biosensor, two oligonucleotides that are able to hybridize to the target sequence were labeled with a biotin and a fluorescein molecule, respectively. By simultaneously incubating the liposomes, both oligonucleotides, and the target sequence in a hybridization buffer for 20–30 min at 42 ^°C, a sandwich complex was formed. The mixture was applied to the polyethersulfone membrane. The complex was captured in the detection zone and quantified using a handheld reflectometer. The system was tested using RNA sequences from B. anthracis, C. parvum and E. coli. Quantitation of concentrations between 10 fmol and 1000 fmol (10–1000 nM) was possible without altering any biosensor assay conditions. In addition, no changes to hybridization conditions were required when using authentic nucleic acid sequence-based amplified RNA sequences, and the generic biosensor compared favorably with those previously developed specifically for the RNA sequences. Therefore, the universal biosensor described is an excellent tool, for use in laboratories or at test sites, for rapidly investigating and quantifying any nucleic acid sequence of interest, as well as potentially any antigen of interest that can be bound by two antibodies simultaneously.     

Detection of DNA based on fluorescence resonance energy transfer of polyelectrolyte-protected CdTe quantum dots as energy donors

The approach for DNA detection was established by using a fluorescence resonance energy transfer (FRET) system, in which the energy donor was poly-diallyldimethylammonium chloride-protected quantum dots and the energy receptor was ethidium bromide (EB) inserting into the double stranded DNA. The concentration of the probe DNA, EB and NaCl was optimized. Under the optimized conditions, the FRET system has a stable signal and good reproducibility. The linear range is 7.7-61.6 nM with the correlation coefficient of 0.998 and the limit of detection is 7.7 nM. This method is simple and sensitive, and makes the label-free DNA detection come true.     

Design and characterization of two-dye and three-dye binary fluorescent probes for mRNA detection

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 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 with 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 slow 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.     

Rapid detection of pathogenic bacteria based on a universal dual-recognition FRET sensing system constructed with aptamer-quantum dots and lectin-gold nanoparticles

The threat to public health from bacterial infections has led to an urgent need to develop simpler, faster and more reliable bacterial detection methods. In this work, we developed a universal dual-recognition based sandwich fluorescence resonance energy transfer (FRET) sensor by using specific aptamer-modified quantum dots (Aptamer-QDs) as energy donor and lectin concanavalin A (Con A) modified gold nanoparticles (Con A-AuNPs) as energy acceptor to achieve rapid and sensitive detection of Escherichia coli (E. coli) within 0.5 h. In the presence of the target E. coli, the energy donor of Aptamer-QDs and acceptor of Con A-AuNPs were close to each other, causing changes of FRET signals. Based on the constructed FRET sensor, a linear detection range of from 10² cfu/mL to 2 × 10⁸ cfu/mL with the detection limit of 45 cfu/mL for E. coli was achieved. Furthermore, the FRET sensor was applied to detect E. coli in the milk and orange juice with the detection limit of 300 cfu/mL and 200 cfu/mL, respectively and recovery rate from 83.1% to 112.5%. The strategy holds great promise in pathogenic bacteria detection due to its rapid and sensitivity.     

Biotin induced fluorescence enhancement in resonance energy transfer and application for bioassay

A novel method to significantly enhance fluorescence resonance energy transfer (FRET) signal which occurred from fluoresceine isothiocyanate (FITC) to Dylight 549 was studied in this paper. Streptavidin was labeled with the donor fluorophore FITC and biotinamide was conjugated to the acceptor Dylight 549. When biotinamide bound to streptavidin, FRET would occur from FITC to Dylight 549 while a remarkable fluorescence enhancement of streptavidin-FITC was observed. The fluorescence enhancement of streptavidin-FITC in the presence of biotin was utilized in the FRET system to obtain higher fluorescence signal. Increase of fluorescence intensity of FITC and decrease of Dylight 549 depended on the concentration of competitive biotin. A homogeneous analysis method was established based on the fluorescence recovery of FITC in the FRET system with fluorescence enhancement. This method is highly sensitive and simple to determine the concentration of biotin. The detection limit for biotin was 0.5 nM and the linear range of the assay was 0.8-9.8 nM. The response time is no more than 15 min during the one-step assay due to the high affinity between streptavidin and biotin.     

Ratiometric sensing of mercury(II) based on a FRET process on silica core-shell nanoparticles acting as vehicles

We report on a fluorescence resonance energy transfer (FRET)-based ratiometric sensor for the detection of Hg(II) ion. First, silica nanoparticles were labeled with a hydrophobic fluorescent nitrobenzoxadiazolyl dye which acts as a FRET donor. A spirolactam rhodamine was then covalently linked to the surface of the silica particles. Exposure of the nanoparticles to Hg(II) in water induced a ring-opening reaction of the spirolactam rhodamine moieties, leading to the formation of a fluorescent derivative that can serve as the FRET acceptor. Ratiometric sensing of Hg(II) was accomplished by ratioing the fluorescence intensities at 520 nm and 578 nm. The average decay time for the donor decreases from 9.09 ns to 7.37 ns upon addition of Hg(II), which proves the occurrence of a FRET process. The detection limit of the assay is 100 nM (ca. 20 ppb). The sensor also exhibits a large Stokes shift (>150 nm) which can eliminate backscattering effects of excitation light.

Fluorescence resonance energy transfer in polydiacetylene liposomes

Conjugated polydiacetylene (PDA) possessing stimuli-responsive properties has been intensively investigated for developing efficient sensors. We report here fluorescence resonance energy transfer (FRET) in liposomes synthesized using different molar ratios of dansyl-tagged diacetylene and diacetylene-carboxylic acid monomers. Photopolymerization of diacetylene resulted in cross-linked PDA liposomes. We used steady-state electronic absorption, emission, and fluorescence anisotropy (FA) analysis to characterize the thermal-induced FRET between dansyl fluorophores (donor) and PDA (acceptor). We found that the monomer ratio of acceptor to donor ( R ad) and length of linkers (functional part that connects dansyl fluorophores to the diacetylene group in the monomer) strongly affected FRET. For R ad = 10 000, the acceptor emission intensity was amplified by more than 18 times when the liposome solution was heated from 298 to 338 K. A decrease in R ad resulted in diminished acceptor emission amplification. This was primarily attributed to lower FRET efficiency between donors and acceptors and a higher background signal. We also found that the FRET amplification of PDA emissions after heating the solution was much higher when dansyl was linked to diacetylene through longer and flexible linkers than through shorter linkers. We attributed this to insertion of dansyl in the bilayer of the liposomes, which led to an increased dansyl quantum yield and a higher interaction of multiple acceptors with limited available donors. This was not the case for shorter and more rigid linkers where PDA amplification was much smaller. The present studies aim at enhancing our understanding of FRET between fluorophores and PDA-based conjugated liposomes. Furthermore, receptor tagged onto PDA liposomes can interact with ligands present on proteins, enzymes, and cells, which will produce emission sensing signal. Therefore, using the present approach, there exist opportunities for designing FRET-based highly sensitive and selective chemical and biochemical sensors.     

A new nanobiosensor for glucose with high sensitivity and selectivity in serum based on fluorescence resonance Energy transfer (FRET) between CdTe quantum dots and Au nanoparticles

A novel assembled nanobiosensor QDs-ConA-beta-CDs-AuNPs was designed for the direct determination of glucose in serum with high sensitivity and selectivity. The sensing approach is based on fluorescence resonance energy transfer (FRET) between CdTe quantum dots (QDs) as an energy donor and gold nanoparticles (AuNPs) as an energy acceptor. The specific combination of concanavalin A (ConA)-conjugated QDs and thiolated beta-cyclodextrins (beta-SH-CDs)-modified AuNPs assembles a hyperefficient FRET nanobiosensor. In the presence of glucose, the AuNPs-beta-CDs segment of the nanobiosensor is displaced by glucose which competes with beta-CDs on the binding sites of ConA, resulting in the fluorescence recovery of the quenched QDs. Experimental results show that the increase in fluorescence intensity is proportional to the concentration of glucose within the range of 0.10-50 muM under the optimized experimental conditions. In addition, the nanobiosensor has high sensitivity with a detection limit as low as 50 nM, and has excellent selectivity for glucose over other sugars and most biological species present in serum. The nanobiosensor was applied directly to determine glucose in normal adult human serum, and the recovery and precision of the method were satisfactory. The unique combination of high sensitivity and good selectivity of this biosensor indicates its potential for the clinical determination of glucose directly and simply in serum, and provides the possibility to detect low levels of glucose in single cells or bacterial cultures. Moreover, the designed nanobiosensor achieves direct detection in biological samples, suggesting the use of nanobiotechnology-based assembled sensors for direct analytical applications in vivo or in vitro.     

Application of PARAFAC to a two-component system exhibiting fluorescence resonance energy transfer: from theoretical prediction to experimental validation

One of the conventionally accepted requirements for parallel factor analysis (PARAFAC) to handle the fluorescence excitation emission matrices (EEMs) is the independence of each component's absorption and emission spectra in simple mixtures of fluorophores. EEMs of samples in which F?rster resonance energy transfer (FRET) occurs between fluorophores seem to fail to meet this requirement. A rigorous theoretical treatment of the steady-state kinetics in the present work indicates that the fluorescence in the presence of FRET, excited by relatively weak excitation light intensity, can be reasonably separated into additive contributions from three parts: donors, acceptors and FRET. This prediction is for the first time verified experimentally in sodium dodecyl sulfate micellar solutions containing biphenyl as the energy donor and 2,5-diphenyloxazole as the energy acceptor. The experimental EEMs were well fitted to three components as predicted. A well accepted diagnostic test called core consistency (CC), specifically designed for modeling simple mixtures of fluorophores with PARAFAC, was found to be negative for the 3-component model in the present study. The simultaneous occurrence of good model fit and significantly negative CC when modeling fluorophore mixtures by conventional PARAFAC would be indicative of the presence of physical/chemical processes (e.g., FRET) that deviate from the conventional working requirements for PARAFAC. The extent of FRET has been independently measured or calculated by three methods: 1) decrease in steady state fluorescence of donor; 2) lifetime measurements with population analysis; and 3) Poisson statistics based on PARAFAC-determined distribution constants. The results of the three methods are consistent. The normalized scores of the three components found by PARAFAC also agree to within a few percent with relative concentrations in aqueous and micelle phases determined from distribution constants for the solutions prepared with nine different combinations of total donor and acceptor concentrations. Our theoretical treatment also for the first time spells out in detail the relationship between the PARAFAC scores and concentrations of components, in terms of photophysical constants of the components and spectral shape factors.     

Multi-walled Carbon Nanotubes and Gold Nanorod Decorated Biosensor for Detection of microRNA-126

In this article, we introduced a novel electrochemical biosensor for the detection of microRNA-126. The biosensor utilizes a hybridization assay combined with multi-walled carbon nanotubes and gold nanorod-decorated screen-printed carbon electrodes. For electrode preparation, gold nanorods were first immobilized onto the surface of bare and multi-walled carbon nanotube-modified screen-printed carbon electrodes, and the thiol tagged-capture probe was immobilized on the electrode surface through gold and thiol group interaction. After the immobilization, thiol tagged-capture probe hybridized with the target sequence. Under optimum conditions, we determined limit of detection (LOD) and limit of quantification (LOQ) as high as 11 nM and 36 nM, respectively.     

A DNA hybridization detection based on fluorescence resonance energy transfer between dye-doped core-shell silica nanoparticles and gold nanoparticles

A novel and efficient method to evaluate the DNA hybridization based on a fluorescence resonance energy transfer (FRET) system, with fluorescein isothiocyanate (FITC)-doped fluorescent silica nanoparticles (SiNPs) as donor and gold nanoparticles (AuNPs) as acceptor, has been reported. The strategy for specific DNA sequence detecting is based on DNA hybridization event, which is detected via excitation of SiNPs-oligonucleotide conjugates and energy transfer to AuNPs-oligonucleotide conjugates. The proximity required for FRET arises when the SiNPs-oligonucleotide conjugates hybridize with partly complementary AuNPs-oligonucleotide conjugates, resulting in the fluorescence quenching of donors, SiNPs-oligonucleotide conjugates, and the formation of a weakly fluorescent complex, SiNPs-dsDNA-AuNPs. Upon the addition of the target DNA sequence to SiNPs-dsDNA-AuNPs complex, the fluorescence restores (turn-on). Based on the restored fluorescence, a homogeneous assay for the target DNA is proposed. Our results have shown that the linear range for target DNA detection is 0-35.0 nM with a detection limit (3σ) of 3.0 picomole. Compared with FITC-dsDNA-AuNPs probe system, the sensitivity of the proposed probe system for target DNA detection is increased by a factor of 3.4-fold.     

A 'turn-on' FRET peptide sensor based on the mercury binding protein MerP

A new fluorescent peptidyl chemosensor based on the mercury binding MerP protein with fluorescence resonance energy transfer (FRET) capabilities has been synthesized via Fmoc solid-phase peptide synthesis. The metal chelating unit, which is flanked by the fluorophores tryptophan (donor) and dansyl (acceptor), contains amino acids from MerP's metal binding loop (sequence: dansyl-Gly-Gly-Thr-Leu-Ala-Val-Pro-Gly-Met-Thr-Cys-Ala-Ala-Cys-Pro-Ile-Thr-Val-Lys-Lys-Gly-Gly-Trp-CONH(2)). A FRET enhancement or 'turn-on' response was observed for Hg(2+) as well as for Zn(2+), Cd(2+) and Ag(+) in a pure aqueous solution at pH 7.0. The emission intensity of the acceptor was used to monitor the concentration of these metals ions with detection limits of 280, 6, 103 and 496 microg L(-1), respectively. No response was observed for the other transition, alkali and alkaline earth metals tested. The fluorescent enhancement observed is unique for Hg(2+) since this metal generally quenches fluorescence. The acceptor fluorescence increase resulting from metal binding-induced FRET suggests a sensor that is inherently more sensitive than one based on quenching by the binding event.     

Study on the fluorescence resonance energy transfer between CdTe QDs and butyl-rhodamine B in the presence of CTMAB and its application on the detection of Hg(II)

The thioglycolic acid-functionalized CdTe quantum dots (QDs) were synthesized in aqueous solution using safe and low-cost inorganic salts as precursors. Fluorescence resonance energy transfer (FRET) system was constructed between CdTe QDs (donor) and butyl-rhodamine B (BRB) (acceptor) in the presence of cetyltrimethylammonium bromide (CTMAB). CTMAB micelles formed in water reduced the distance between the donor and the acceptor significantly and thus improved the FRET efficiency, which resulted in an obvious fluorescence enhancement of the acceptor. Several factors which impacted the fluorescence spectra of the FRET system were studied. The energy transfer efficiency (E) and the distance (r) between CdTe and BRB were obtained. The feasibility of the prepared FRET system as fluorescence probe for detecting Hg(II) in aqueous solution was demonstrated. At pH 6.60, a linear relationship could be established between the quenched fluorescence intensity of BRB and the concentration of Hg(II) in the range of 0.0625-2.5mumolL(-1). The limit of detection was 20.3nmolL(-1). The developed method was proved to be sensitive and repeatable to detect Hg(II) in a wide range in aqueous solutions.     

Fluorescence resonance energy transfer between quantum dot donors and dye-labeled protein acceptors

We used luminescent CdSe-ZnS core-shell quantum dots (QDs) as energy donors in fluorescent resonance energy transfer (FRET) assays. Engineered maltose binding protein (MBP) appended with an oligohistidine tail and labeled with an acceptor dye (Cy3) was immobilized on the nanocrystals via a noncovalent self-assembly scheme. This configuration allowed accurate control of the donor-acceptor separation distance to a range smaller than 100 A and provided a good model system to explore FRET phenomena in QD-protein-dye conjugates. This QD-MBP conjugate presents two advantages: (1) it permits one to tune the degree of spectral overlap between donor and acceptor and (2) provides a unique configuration where a single donor can interact with several acceptors simultaneously. The FRET signal was measured for these complexes as a function of both degree of spectral overlap and fraction of dye-labeled proteins in the QD conjugate. Data showed that substantial acceptor signals were measured upon conjugate formation, indicating efficient nonradiative exciton transfer between QD donors and dye-labeled protein acceptors. FRET efficiency can be controlled either by tuning the QD photoemission or by adjusting the number of dye-labeled proteins immobilized on the QD center. Results showed a clear dependence of the efficiency on the spectral overlap between the QD donor and dye acceptor. Apparent donor-acceptor distances were determined from efficiency measurements and corresponding F?rster distances, and these results agreed with QD bioconjugate dimensions extracted from structural data and core size variations among QD populations.     

Label-free electrochemical impedance spectroscopy biosensor for the determination of human immunoglobulin G

A simple and sensitive biosensor was developed for the determination of human immunoglobulin G (IgG). Protein A was employed as molecular receptor and electrochemical impedance spectroscopy (EIS) was used as detection technique. The biosensor was obtained by self-assembling protein A on a gold electrode. The surface morphology of the self-assembled layer before and after interaction with IgG was studied by atomic force microscopy. Protein A bound specifically to the Fc portion of IgG, and this caused a change in the resistance of the interfacial electron transfer when using a ferrocyanide redox couple as a probe. The increase of the resistance of the electron transfer was linearly related to the concentration of IgG in the range from 10 ng.mL^?1 to 1.0 μg.mL^?1, with a detection limit of 5 ng.mL^?1. The work demonstrates that protein A is a versatile matrix for the immobilization of antibodies.