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.     

A time-resolved near-infrared fluorescence assay for glucose: opportunities for trans-dermal sensing

We report a time-resolved near-infrared fluorescence assay for glucose detection that incorporates pulsed diode laser excitation. Reduction in fluorescence resonance energy transfer to a malachite green-Dextran complex from allophycocyanin bound to concanavalin A (ConA) due to displacement of the complex by glucose from ConA provides the basis of the assay. The fluorescence quenching kinetics are analysed and discussed in detail. The change in fluorescence decay kinetics in the presence of glucose is found from dimensionality studies to be brought about by a change in the distribution of malachite green-Dextran acceptors. Glucose concentrations are measured in solution to within +/- 10% over the range 0-30 mM.     

Reusable optical bioassay platform with permeability-controlled hydrogel pads for selective saccharide detection

A reusable optical bioassay platform using permeability-controlled hydrogel pads for selective saccharide detection has been developed. An optical glucose detection assay based on fluorescence resonance energy transfer (FRET) between dye-labeled dextran and Concanavalin A (ConA) was incorporated into hydrogel pads by entrapment. The hydrogel pads are constructed from hemispherical hydrogel attached onto hydrophobic surfaces of a microtiter plate. The resulted hemispherical hydrogel pads entrapping the sensing biological materials were further surface coated with polyelectrolyte multilayers through a Layer-by-Layer (LbL) self-assembly process to create a permeability-controlled membrane with nanometer thickness. The selective permeable LbL film deposited on the hydrogel surface allows small molecular weight analytes to diffuse into the hydrogel pads while the large molecular weight sensing biological molecules are immobilized. An encapsulation efficiency of 75% for the ConA/Dextran complex within the coated hydrogel pads was achieved and no significant leakage of the complex was observed. Glucose calibration curve with linear range from 0 to 10 mM glucose was obtained. Selective permeability of the hydrogel pads has been demonstrated by measurement of saccharides with various molecular weights. The LbL hydrogel pads could selectively detect monosaccharides (glucose, MW = 180) and disaccharides (sucrose, MW = 342) while polysaccharides (dextran, MW ∼ 70 kDa) cannot diffuse through the LbL layer and are excluded. LbL hydrogel pads allow regeneration of the FRET system with good signal reproducibility of more than 90% to construct a reusable and reagentless optical bioassay platform.     

Fluorescence energy transfer-sensitized photobleaching of a fluorescent label as a tool to study donor-acceptor distance distributions and dynamics in protein assemblies: studies of a complex of biotinylated IgM with streptavidin and aggregates of concanavalin A

A photokinetic method of detection of fluorescence resonance energy transfer (FRET) between special fluorescent labels is applied to study time-averaged spatial distribution of labeled proteins in protein assemblies. Prolonged irradiation of a sample at the absorption maximum of the energy donor initiates FRET-sensitized fluorescence photobleaching of the energy acceptor label, which was monitored by steady-state fluorimetric measurements. Kinetics of the acceptor photobleaching and kinetics of decreasing the efficiency of FRET from donors to unbleached acceptors were determined. The FRET efficiency was found from measuring sensitization of acceptor fluorescence. Analysis of the photokinetic data permits to estimate the time-averaged distribution of acceptors on donor-acceptor distances in the range of characteristic distances of FRET. Dynamic processes influencing donor-acceptor distances can be also investigated by the method. Application of the method is demonstrated by the studies of a complex of biotinylated IgM with streptavidin and aggregates composed of concanavalin A and sodium dodecyl sulphate. A new thiadicarbocyanine dye was used as the acceptor label. R-phycoerythrin and tetramethylrhodamine isothiocyanate were the donor labels. In the IgM-streptavidin complex, 16% of acceptors most contributed to FRET provided 90% of FRET efficiency, whereas acceptors made about the same time-averaged contribution to FRET in the concanavalin A aggregates.     

Measurements of FRET in a glucose-sensitive affinity system with frequency-domain lifetime spectroscopy

We report measurements of fluorescence resonance energy transfer (FRET) for glucose sensing in an established concanavalin A–dextran affinity system using frequency‐domain lifetime spectroscopy. A dextran (MW 2000000) labeled with a small fluorescent donor molecule, Alexa Fluor 568, was used to competitively bind to a sugar‐binding protein, concanavalin A, labeled with acceptor molecule, Alexa Fluor 647, in the presence of glucose. The FRET‐quenching kinetics of the donor were analyzed from frequency‐domain measurements as a function of both glucose and acceptor‐protein concentrations using a Förster‐type decay kinetics model. The results show that the frequency‐domain measurements and donor decay kinetics can quantitatively indicate changes in the competitive binding of 0.09 μM dextran to labeled concanavalin A at a solution concentration of 10.67 μM in the presence of glucose at concentrations ranging from 0 to 224 mg/dL.     

A New Robust and Highly Sensitive FRET Donor–Acceptor Pair: Synthesis,Characterization, and Application in a Thrombin Assay

The synthesis of a new, robust fluorescence‐resonance‐energy‐transfer (FRET) system is described. Its donor chromophore is derived from an N‐allyl‐substituted quinolinone attached to 4‐bromophenylalanine via Heck cross‐coupling. The resulting Fmoc‐protected derivative 11 was used as building block in solid‐phase peptide synthesis (SPPS). As FRET acceptor, a sulfonylated ruthenium(II)–bathophenanthroline complex with a peripheral COOH function was prepared for covalent attachment to target molecules. The UV/VIS absorption and emission spectra of peptides bearing only the donor (D) or acceptor (A) dye showed a good overlap of the emission band of the donor with the absorption band of the acceptor. The fluorescence spectra of a peptide bearing both dyes revealed an additional emission after excitation of the donor, which is due to indirect excitation of the acceptor via FRET. The long fluorescence lifetime of the Ru^II complex (0.53 μs) makes it well‐suited for time‐resolved measurements. As a first application of this new FRET system, the peptide 18 , with the recognition sequence for the protease thrombin, flanked by the two dyes, was synthesized and successfully cleaved by the enzyme. The change in the ratio of the fluorescence intensities could be determined.     

Fluorescence resonance energy transfer from tryptophan in human serum albumin to a bioactive indoloquinolizine system

The interaction between a bioactive molecule, 3-acetyl-4-oxo-6,7-dihydro-12H indolo-[2,3-a] quinolizine (AODIQ), with human serum albumin (HSA) has been studied using steady-state absorption and fluorescence techniques. A 1:1 complex formation has been established and the binding constant (K) and free energy change for the process have been reported. The AODIQ-HSA complex results in fluorescence resonance energy transfer (FRET) from the tryptophan moiety of HSA to the probe. The critical energy-transfer distance (R ₀) for FRET and the Stern-Volmer constant (K _sv) for the fluorescence quenching of the donor in the presence of the acceptor have been determined. Importantly, K _SV has been shown to be equal to the binding constant itself, implying that the fluorescence quenching arises only from the FRET process. The study suggests that the donor and the acceptor are bound to the same protein at different locations but within the quenching distance.     

Highly sensitive FRET-based fluorescence immunoassay for aflatoxin B1 using cadmium telluride quantum dots

We report on a competitive immunoassay for the determination of aflatoxin B1 using fluorescence resonance energy transfer (FRET) from anti-aflatoxin B1 antibody (immobilized on the shell of CdTe quantum dots) to Rhodamine 123 (Rho 123-labeled aflatoxin B1 bound to albumin). The highly specific immunoreaction between the antibody against aflatoxin B1 on the QDs and the labeled-aflatoxin B1 brings the Rho 123 fluorophore (acting as the acceptor) and the QDs (acting as the donor) in close spatial proximity and causes FRET to occur upon photoexcitation of the QDs. In the absence of unlabeled aflatoxin B1, the antigen-antibody complex is stable, and strong emission resulting from the FRET from QDs to labeled aflatoxin B1 is observed. In the presence of aflatoxin B1, it will compete with the labeled aflatoxin B1-albumin complex for binding to the antibody-QDs conjugate so that FRET will be increasingly suppressed. The reduction in the fluorescence intensity of the acceptor correlates well with the concentration of aflatoxin B1. The feasibility of the method was established by the detection of aflatoxin B1 in spiked human serum. There is a linear relationship between the increased fluorescence intensity of Rho 123 with increasing concentration of aflatoxin B1 in spike human serum, over the range of 0.1–0.6 μmol·mL^?1. The limit of detection is 2?×?10^?11 M. This homogeneous competitive detection scheme is simple, rapid and efficient, and does not require excessive washing and separation steps.

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.     

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.     

Application of a Highly Robust and Efficient Fluorescence‐Resonance‐Energy‐Transfer (FRET) System in DNA

We report a feasibility study for the application of our newly developed highly efficient and robust fluorescence‐resonance‐energy‐transfer (FRET) system to DNA. A 2′‐oligodeoxynucleotide, 12 , equipped with a quinolinone derivative as donor and a (bathophenanthroline)ruthenium(II) complex as acceptor and having a single uridine as potential cleavage site under basic conditions revealed an intensive FRET, which almost vanished after cleavage of the oligonucleotide under basic conditions (Fig. 7). Furthermore, in the arrangement of a molecular beacon (MB) DNA (see 13 ), a significant decrease of the FRET was observed after hybridization to a target sequence (Fig. 9). Due to the long decay times of the fluorescence of the Ru‐complex, the system allows for highly sensitive time‐gated measurements.     

Manipulating FRET with polymeric vesicles: development of a "mix-and-detect" type fluorescence sensor for bacterial toxin

A simple "mix-and-detect" type of fluorescence sensor for cholera toxin (CT) is reported. The sensor consists of a BODIPY lipid dye and polydiacetylene (PDA) vesicles and utilizes the lipid insertion and FRET mechanism to offer a direct and fluorescence "turn-on" detection of the analyte. BODIPY conjugated GM1, dissolved in a Tris buffer through aggregate formation, demonstrated substantial fluorescence quenching with addition of PDA vesicle solution. The close proximity of the dye molecules to the conjugated chains as a result of lipid insertion enables energy transfer from dye to the polymer backbone, yielding the observed phenomenon. When CT is present, the binding of BO-GM1 to CT results in formation of a complex that prohibits it from membrane insertion, leading to the blocking of the quenching process. The fluorescence signal was found to be proportional to the CT concentration. The method is very simple and allows specific and sensitive detection of the protein toxin with just a few mixing steps. It can be further developed into a general sensing strategy for detection of other proteins with amplified FRET mechanism.     

A novel assembly of Au NPs-beta-CDs-FL for the fluorescent probing of cholesterol and its application in blood serum

A novel assembly of Au NPs-beta-CDs-FL for the fluorescent probing of cholesterol (Cho) is provided. Gold nanoparticles (Au NPs) possessing a high extinction coefficient function can be used as excellent fluorescent quenchers in Au NP-fluorophore composites. Inclusion of fluorescein (FL) into beta-cyclodextrin (beta-CD) makes fluorescence resonance energy transfer (FRET) occur through the donor and quencher nearby. FRET switches off because of the cholesterol-induced release of FL from beta-CD cavity, which results in the fluorescence recovery of the quenched dye. Spectral analysis supported the idea that the signal enhancement was attributed to the formation of an inclusion complex of the cholesterol moiety in beta-CD, resulting in separation of FL from the Au NPs. This phenomenon is explained by the guest-induced location change of the FL from inside to outside the cavity, suggesting that the assembly of Au NPs-beta-CDs-FL is effective as a fluorescent probe for cholesterol recognition. The fluorescence increase is proportional to the concentration of cholesterol in the range of approx. 30 nM to 15 muM. A concentration of cholesterol as low as 9 nM would be readily detected. The precision of the method applied to the determination of quantities of cholesterol present in human blood serum were satisfactory.     

Fluorescent carbon dots for sensitive determination and intracellular imaging of zinc(II) ion

We describe the preparation of carbon dots (CDs) from glucose that possess high stability, a quantum yield of 0.32, and low toxicity (according to an MTT assay). They were used, in combination with the fluorogenic zinc(II) probe quercetin to establish a fluorescence resonance energy transfer (FRET) system for the determination of Zn(II). The CDs are acting as the donor, and the quercetin-Zn(II) complex as the acceptor. This is possible because of the strong overlap between the fluorescence spectrum of CDs and the absorption spectrum of the complex. The method enables Zn(II) to be determined in the 2 to 100 μM concentration range, with a 2 μM detection limit. The method was applied to image the distribution of Zn(II) ions in HeLa cells.

Based on the fluorescence resonance energy transfer (FRET) between carbon dots and quercetin (QCT)-Zn²⁺, the fluorescence indicator was established, which displays high sensitivity and selectivity in the detection of Zn²⁺. The method was also applied to image the distribution of Zn(II) ions in HeLa cells.

     

Gauging the flexibility of fluorescent markers for the interpretation of fluorescence resonance energy transfer

Intramolecular distances in proteins and other biomolecules can be studied in living cells by means of fluorescence resonance energy transfer (FRET) in steady-state or pulsed-excitation experiments. The major uncertainty originates from the unknown orientation between the optical dipole moments of the fluorescent markers, especially when the molecule undergoes thermal fluctuations in physiological conditions. We introduce a statistical method based on the von Mises-Fisher distribution for the interpretation of fluorescence decay dynamics in donor-acceptor FRET pairs that allows us to retrieve both the orientation and the extent of directional fluctuations of the involved dipole moments. We verify the method by applying it to donor-acceptor pairs controllably attached to DNA helices and find that common assumptions such as complete rotational freedom or fully hindered rotation of the dipoles fail a physical interpretation of the fluorescence decay dynamics. This methodology is applicable in single-molecule and ensemble measurements of FRET to derive more accurate distance estimates from optical experiments, without the need for more complex and expensive NMR studies.     

Steady-state fluorescence quenching applications for studying protein structure and dynamics

Fluorescence quenching methods are useful to obtain information about the conformational and/or dynamic changes of proteins in complex macromolecular systems. In this review steady-state methods are described and the data interpretation is thoroughly discussed. As a special case of fluorescence quenching mechanism, fluorescence resonance energy transfer (FRET) phenomenon is also presented. Application of a FRET based method to characterize the temperature dependence of the flexibility of protein matrix is clearly demonstrated.     

An enzyme-free quartz crystal microbalance biosensor for sensitive glucose detection in biological fluids based on glucose/dextran displacement approach

A sensitive and facile quartz crystal microbalance (QCM) biosensor for glucose detection in biological fluids was developed by means of a displacement-type assay mode between glucose and its analogy dextran for concanavalin A (ConA) binding sites on a graphene-based sensing platform. To construct such a displacement-based sensor, phenoxy-derived dextran (DexP) molecules were initially assembled onto the surface of graphene-coated QCM probe via π-π stacking interaction, and ConA molecules were then immobilized on the dextran through the dextran-ConA interaction. Upon addition of glucose, the analyte competed with the dextran for the ConA, and displaced it from the QCM probe, leading to a change in the frequency. Under optimal conditions, the frequency change relative to the basic resonant frequency was proportional to glucose concentration, and exhibited a dynamic range from 0.01 to 7.5 mM with a low detection limit (LOD) of 5.0 μM glucose (at 3σ). The relative standard deviations (RSDs) were below 6.2% and 9.0% for the reproducibility and selectivity of the QCM glucose sensors, respectively. In addition, the assay system was evaluated with glucose spiking samples into the distilled water and blank cattle serum, receiving in excellent correlation with the referenced values.     

Selective determination of tryptophan-containing peptides through precolumn derivatization and liquid chromatography using intramolecular fluorescence resonance energy transfer detection.

A selective and sensitive fluorometric determination method for native fluorescent peptides has been developed. This method is based on intramolecular fluorescence resonance energy transfer (FRET) detection in a liquid chromatography (LC) system following precolumn derivatization of the amino groups of tryptophan (Trp)-containing peptides. In this detection process, we monitored the FRET from the native fluorescent Trp moieties (donor) to the derivatized fluorophore (acceptor). From a screening study involving 10 fluorescent reagents, we found that o-phthalaldehyde (OPA) generated FRET most effectively. The OPA derivatives of the native fluorescent peptides emitted OPA fluorescence (445 nm) through an intramolecular FRET process when they were excited at the excitation maximum wavelength of the Trp-containing peptides (280 nm). The generation of FRET was confirmed through comparison with the analysis of a non-fluorescent peptide (C-reactive protein fragment (77 - 82)) performed using LC and a three-dimensional fluorescence detection system. We were able to separate the OPA derivatives of the Trp-containing peptides when performing LC on a reversed-phase column. The detection limits (signal-to-noise ratio = 3) for the Trp-containing peptides, at a 20-microL injection volume, were 41 - 180 fmol. The sensitivity of the intramolecular FRET-forming derivatization method is higher than that of the system that takes advantage of the conventional detection of OPA derivatives. Moreover, native non-fluorescent amines and peptides in the sample monitored at FRET detection are weaker than those of conventional fluorescence detection.     

A new FRET nanoprobe for trypsin using a bridged β-cyclodextrin dimer-dye complex and its biological imaging applications

A new self-assembly nanoprobe, mercaptoethylamine-modified-gold nanoparticles-Lysine-bridged-bis(β-cyclodextrins)-fluorescein (MGNPs-Lys-bis(β-CDs)-FL), based on fluorescence resonance energy transfer (FRET) was developed for determination of trypsin firstly in biological systems. With the Lys-bis(β-CDs)-FL complex as an energy donor and mercaptoethylamine (MEA)-modified gold nanoparticles (MGNPs) as an energy acceptor, the two parts assemble an efficient FRET nanoprobe through an amide bond. Trypsin is specific for the hydrolysis of amide linkages of lysine. Therefore, in the presence of trypsin, the nanoprobe is cleaved by trypsin on the binding sites of amide with good specificity and sensitivity, resulting in the fluorescence recovery of the quenched FL. The nanoprobe has good biological applicability and provides a potential assay for further clinical research of trypsin in biosystems.