Upconversion fluorescence resonance energy transfer in a homogeneous immunoassay for estradiol

We recently described a novel homogeneous assay principle based on upconversion fluorescence resonance energy transfer (UC-FRET), where an upconverting phosphor (UCP) is utilized as a donor. The UC-FRET has now been applied to a competitive homogeneous immunoassay for 17beta-estradiol (E2) in serum, using a small-molecular dye as an acceptor. The assay was constructed by employing an UCP coated with an E2-specific recombinant antibody Fab fragment as a donor and an E2-conjugated small-molecular dye, Oyster-556, as an acceptor. Standard curves for the assay were produced both in buffer and in male serum. Sensitized acceptor emission was measured at 600 nm under continuous laser diode excitation at 980 nm. In buffer, the IC50 value of the assay was 1 nM and in serum 3 nM. The lower limits of detection (mean of zero calibrators, 3 SD) were 0.4 and 0.9 nM, respectively. The measurable concentration range extended up to 3 nM in buffer and 9 nM in serum. Equilibrium in the assay was reached in 30 min. The novel principle of UC-FRET has unique advantages compared to present homogeneous luminescence-based methods and can enable an attractive assay system platform for clinical diagnostics and for high-throughput screening approaches.     

Homogeneous immunoassay based on two-photon excitation fluorescence resonance energy transfer

A two-photon excitable small organic molecule (abbreviated as TP-NH 2) with large two-photon absorption cross section and competitive fluorescence quantum yield was prepared, which emitted fluorescence in the visible region upon excitation at 800 nm. Using the TP-NH 2 molecule as an energy donor, a two-photon excitation fluorescence resonance energy-transfer (TPE-FRET) based homogeneous immunoassay method was proposed. The donor and the acceptor (DABS-Cl, a dark quencher) were labeled to bovine serum albumin (BSA) separately, and anti-BSA protein was determined by employing an antibody bridging assay scheme. Rabbit anti-BSA serum containing other biomolecules was intentionally used as the sample to introduce interference. A parallel assay was performed using the traditional one-photon excitation FRET model, which failed to carry out quantitative determination due to the serious background luminescence arising from those biomolecules in the sample. The TPE-FRET model showed its strong ability to overcome the problem of autofluorescence and provided satisfying analytical performance. Quite good sensitivity and wide linear range (0.05-2.5 nM) for anti-BSA protein was obtained. The results of this work suggest that TPE-FRET could be a promising technique for homogeneous assays excluding separation steps, especially in complicated biological sample matrixes.     

A homogeneous and noncompetitive immunoassay based on the enhanced fluorescence resonance energy transfer by leucine zipper interaction

Fluorescence resonance energy transfer (FRET) between two GFP variants is a powerful technique to describe protein-protein interaction in a biological system. However, it has a limitation that the two variants tethered to the respective proteins have to be in sufficient proximity upon binding, which is often difficult to attain by simple N- or C-terminal fusions. Here we describe a novel method to significantly enhance FRET between GFP variant-tagged proteins with the use of leucine zippers. For the homogeneous sandwich immunoassay of a high molecular weight antigen human serum albumin (HSA), two separate single-chain Fvs recognizing distant epitopes of HSA were respectively fused with fluorescence donor ECFP or acceptor EYFP, and FRET between the two was analyzed by fluorescence spectrometry. Because these two proteins did not give any detectable FRET uponantigen addition, we tethered each protein with a leucine zipper motif (c-Jun or FosB) at the C-terminus to help the neighborhood of the GFP variants. Upon antigen addition, the new pairs showed significant antigen-dependent FRET. By exchanging the binding domains, the method will find a range of applications for the assay of other proteins and their interactions in vitro or in vivo.     

Homogeneous noncompetitive immunoassay for 17beta-estradiol based on fluorescence resonance energy transfer

We previously presented a homogeneous noncompetitive assay principle based on quenching of the fluorescence of europium(III) chelate. This assay principle has now been applied to the measurement of 17beta-estradiol (E2) using europium(III) chelate labeled estradiol specific antibody Fab fragment (Eu(III)-Fab) as a donor and E2 conjugated with nonfluorescent QSY21 dye as an acceptor. Fluorescence could be measured only from those Eu(III)-Fab that were bound to E2 of the sample, while the fluorescence of the Eu(III)-Fab that were not occupied by E2 were quenched by E2-QSY21 conjugates. The performance of the assay was tested both in buffer and in the presence of serum. Because approximately half of the Fabs were incapable of binding to E2, the maximum quenching efficiency was only 55%. The lowest limits of detection were 18 and 64 pM in buffer and serum-based calibrators, respectively. The highest measurable concentrations were 0.4 nM in buffer and 1 nM in serum. The presented noncompetitive assay principle requires only one binder, and it can be applied to other haptens as well, providing that a suitable antibody is available.     

Detection of pathogenic bacteria using a homogeneous immunoassay based on shear alignment of virus particles and linear dichroism

Biomolecular detection has for a long time depended on a relatively small number of established methodologies. Many of these depend on the detection of a ligand-antibody complex using some kind of optical technique, e.g., chemiluminescence. Before this measurement can be made, the ligand-antibody complex generally has to be separated from bulk contaminants. This process involves the implementation of a heterogeneous assay format involving immobilization of the antibody onto a solid support to enable washing of the unbound ligand. This approach has a number of inherent issues including being slow and complex and requiring the use of expensive reagents. In this paper, we demonstrate how we have harnessed a biologically inspired nanoparticle to provide the basis for a homogeneous assay which requires no immobilization. The method relies on using fluid shear flow to align a fiber-like nanoparticle formed from a filamentous virus, M13, combined with a ligand-specific antibody. This renders the particle visible to linear dichroic spectroscopy, which provides an easily interpretable signal. The addition of the target ligand (in this case Escherichia coli O157) leads to the formation of a nanoparticle-ligand particle that is unable to align, leading to the perturbation of the linear dichroism signal.     

Highly adaptable and sensitive protease assay based on fluorescence resonance energy transfer

Proteases are widely used in analytical sciences and play a central role in several widespread diseases. Thus, there is an immense need for highly adaptable and sensitive assays for the detection and monitoring of various proteolytic enzymes. We established a simple protease fluorescence resonance energy transfer (pro-FRET) assay for the determination of protease activities, which could in principle be adapted for the detection of all proteases. As proof of principle, we demonstrated the potential of our method using trypsin and enteropeptidase in complex biological mixtures. Briefly, the assay is based on the cleavage of a FRET peptide substrate, which results in a dramatic increase of the donor fluorescence. The assay was highly sensitive and fast for both proteases. The detection limits for trypsin and enteropeptidase in Escherichia coli lysate were 100 and 10 amol, respectively. The improved sensitivity for enteropeptidase was due to the application of an enzyme cascade, which leads to signal amplification. The pro-FRET assay is highly specific as even high concentrations of other proteases did not result in significant background signals. In conclusion, this sensitive and simple assay can be performed in complex biological mixtures and can be easily adapted to act as a versatile tool for the sensitive detection of proteases.     

Determination of the magneto-optical relaxation of magnetic nanoparticles as a homogeneous immunoassay

The interaction between human eotaxin (hEotaxin) and its polyclonal antibody anti-human eotaxin (anti-hEotaxin) was investigated by means of a novel liquid-phase immunoassay using the magneto-optical relaxation of ferrofluids. The binding quality as well as kinetic properties of the binding partners was determined using specifically binding magnetic probes. For this purpose, magnetic nanoparticles (MNP; DDM128N, Meito Sangyo, Japan) were initially functionalized with streptavidin. The biotin-nylated antibody was conjugated with streptavidin-MNP applying the streptavidin-biotin binding system. Binding reactions were detected by measuring the relaxation of the optical birefringence signal occurring when a pulsed magnetic field is applied to the ferrofluid. The addition of hEotaxin to anti-hEotaxin conjugated MNP in different amounts yielded an enlargement of the mean relaxation time due to the formation of MNP aggregates. In order to express the observed increase of the particles' effective diameter in terms of elementary kinetic processes between antigen and antibody, a kinetic model was introduced. Here, the binding reactions are described by a process of stepwise polymerization. The obtained results were compared with data received from surface plasmon resonance biosensor analysis, a standard tool for biomolecular interaction analysis.     

Assay for glucosamine 6-phosphate using a ligand-activated ribozyme with fluorescence resonance energy transfer or CE-laser-induced fluorescence detection

A naturally occurring aptazyme, the glmS ribozyme, is adapted to an assay for glucosamine 6-phosphate, an effector molecule for the aptazyme. In the assay, binding of analyte allosterically activates aptazyme to cleave a fluorescently labeled oligonucleotide substrate. The extent of reaction, and hence analyte concentration, is detected by either fluorescence resonance energy transfer (FRET) or capillary electrophoresis with laser-induced fluorescence (CE-LIF). With FRET, assay signal is the rate of increase in FRET in presence of analyte. With CE-LIF, the assay signal is the peak height of cleavage product formed after a fixed incubation time. The assay has a linear response up to 100 (CE-LIF) or 500 microM (FRET) and detection limit of approximately 500 nM for glucosamine 6-phosphate under single-turnover conditions. When substrate is present in excess of the aptazyme, it is possible to amplify the signal by multiple turnovers to achieve a 13-fold improvement in sensitivity and detection limit of 50 nM. Successful signal amplification requires a temperature cycle to alternately dissociate cleaved substrate and allow fresh substrate to bind aptazyme. The results show that aptazymes have potential utility as analytical reagents for quantification of effector molecules.     

An efficient and biocompatible fluorescence resonance energy transfer system based on lanthanide-doped nanoparticles

This work demonstrates an efficient and bio-friendly fluorescence resonance energy transfer (FRET) system based on lanthanide-doped inorganic nanoparticles. A facile aqueous route was used to synthesize the CePO(4):Tb nanorods with homogeneous colloidal dispersion, which emits a bright green light with a high quantum yield (～0.36) and a long fluorescence lifetime (～3.50 ms) upon UV excitation. Upon treatment of CePO(4):Tb with aqueous Rhodamine B (RhB), an efficient FRET occurs from the Tb(3+) to the RhB molecules, giving rise to well resolved and ratiometric emissions of donors and acceptors, respectively, with an energy transfer efficiency of up to 0.85. When incubated with HeLa cells at 37?°C, the CePO(4):Tb treated with RhB shows bright intracellular luminescence, indicating that it can be successfully internalized inside the cells and the FRET remains in the living cells. Moreover, the cytotoxic measurements demonstrate good biocompatibility and low cytotoxicity of our present FRET system. The advantages presented above including high quantum yield of donors, high energy transfer efficiency, ratiometric fluorescent emission and good biocompatibility, indicate the high potential of the CePO(4):Tb/RhB FRET system for monitoring biological events.     

Microcapsule biosensors using competitive binding resonance energy transfer assays based on apoenzymes

This paper reports the first demonstration of a fluorescence resonance energy transfer based glucose sensor, wherein a competitive binding (CB) assay is encapsulated into polyelectrolyte microcapsules. The work supports the concept that microcapsules are superior to hydrogel systems or other matrixes for competitive-binding-based system, as they provide free movement of the sensing elements within the capsule interior while constant total sensing assay concentration is maintained. The transduction approach employed in these preliminary experiments is also a novel CB system based on a model apoenzyme, apo-glucose oxidase (AG), which is highly specific to beta-d-glucose, as the model target-binding protein. The glucose sensitivity of the fluorescein isothiocyanate (FITC)-dextran and tetramethylrhodamine isothiocyanate-AG encapsulated in microcapsules showed 5 times greater specificity for beta-D-glucose over other sugars, with sensitivity (change in intensity ratio) in the range of 2-6%/mM. It was observed that the sensitivity and range of the response can be tailored by controlling the assay concentration using different FITC-dextran molecular weight and total capsule concentration. The findings support the concepts of using microcapsules to encapsulate CB assays for reversible and stable sensors and the use of apoenzymes as specific molecular recognition elements in CB assays. Further, characterization results for microcapsule glucose sensors demonstrate their suitability for monitoring physiological glucose levels.     

Microchip device with 64-site electrode array for multiplexed immunoassay of cell surface antigens based on electrochemiluminescence resonance energy transfer

This paper describes a novel on-chip microarray platform based on an electrochemiluminescence resonance energy transfer (ECL-RET) strategy for rapid assay of cancer cell surface biomarkers. This platform consists of 64 antigen-decorated CdS nanorod spots with the diameter of 1.0 cm uniformly distributed on 16 indium tin oxide (ITO) strips, which is coated with a multichannel decorated polydimethylsiloxane (PDMS) slice to realize multiplexed determination of antigens. To shorten the immune reaction time in the microchannels and simplify the device, magnetic stirring and four-channel universal serial bus (USB) ports for plug-and-play were used. When Ru(bpy)(3)(2+) labeled antibodies were selectively captured by the corresponding antigens on the CdS nanorod spot array, ECL-RET from the CdS nanorod (donor) by cathodic emission in the presence of K(2)S(2)O(8) to Ru(bpy)(3)(2+) (acceptor) occurred. With signal amplification of Ru(bpy)(3)(2+) and competitive immunoassay, carcinoembryonic antigen (CEA), α-fetoprotein (AFP), and prostate specific antigen (PSA) as models were detected on this microfluidic device via recording the increased ECL-RET signals on electrode surfaces. Furthermore, this multiplexed competitive immunoassay was successfully used for detecting cancer cell surface antigens via the specific antibody-cell interactions and cell counting via cell surface receptors and antigens on the CdS nanorod surface. This platform provides a rapid and simple but sensitive approach with microliter-level sample volume and holds great promise for multiplexed detection of antigens and antigen-specific cells.     

Effect of a homogeneous chemical reaction on heat transfer

Heat and mass transfer processes are investigated in the presence of homogeneous physico-chemical conversions of a substance.Institutes of Mathematics and Mechanics, Academy of Sciences of Turkmenistan, Ashkhabad. Translated from Inzhenerno-Fizicheskii Zhurnal, Vol. 64, No. 6, pp. 734–739, June, 1993.     

Concentration gradient immunoassay. 1. An immunoassay based on interdiffusion and surface binding in a microchannel

We describe a novel microfluidic immunoassay method based on the diffusion of a small-molecule analyte into a parallel-flowing stream containing a cognate antibody. This interdiffusion results in a steady-state gradient of antibody binding site occupancy transverse to convective flow. In contrast to the diffusion immunoassay (Hatch, A.; Kamholz, A. E.; Hawkins, K. R.; Munson, M. S.; Schilling, E. A.; Weigl, B. H.; Yager, P. Nat. Biotechnol. 2001, 19, 461-465.), this antibody occupancy gradient is interrogated by a sensor surface coated with a functional analogue of the analyte. Antibodies with at least one unoccupied binding site may specifically bind to this functionalized surface, leading to a quantifiable change in surface coverage by the antibody. SPR imaging is used to probe the spatial distribution of antibody binding to the surface and, therefore, the outcome of the assay. We show that the pattern of antibody binding to the SPR sensing surface correlates with the concentration of a model analyte (phenytoin) in the sample stream. Using an inexpensive disposable microfluidic device, we demonstrate assays for phenytoin ranging in concentration from 75 to 1000 nM in phosphate buffer. At a total volumetric flow rate of 90 nL/s, the assays are complete within 10 min. Inclusion of an additional flow stream on the side of the antibody stream opposite to that of the sample enables simultaneous calibration of the assay. This assay method is suitable for rapid quantitative detection of low molecular weight analytes for point-of-care diagnostic instrumentation.     

Upconversion fluorescence resonance energy transfer based biosensor for ultrasensitive detection of matrix metalloproteinase-2 in blood

Matrix metalloproteinase-2 (MMP-2) is a very important biomarker in blood. Presently, sensitive and selective determination of MMP-2 directly in blood samples is still a challenging job because of the high complexity of the sample matrix. In this work, we reported a new homogeneous biosensor for MMP-2 based on fluorescence resonance energy transfer (FRET) from upconversion phosphors (UCPs) to carbon nanoparticles (CNPs). A polypeptide chain (NH(2)-GHHYYGPLGVRGC-COOH) comprising both the specific MMP-2 substrate domain (PLGVR) and a π-rich motif (HHYY) was designed and linked to the surface of UCPs at the C terminus. The FRET process was initiated by the π-π interaction between the peptide and CNPs, which thus quenched the fluorescence of the donor. Upon the cleavage of the substrate by the protease at the amide bond between Gly and Val, the donor was separated from the acceptor while the π-rich motif stayed on the acceptor. As a result, the fluorescence of the donor was restored. The fluorescence recovery was found to be proportional to the concentration of MMP-2 within the range from 10-500 pg/mL in an aqueous solution. The quantification limit of this sensor was at least 1 order of magnitude lower than that of other reported assays for MMP-2. The sensor was used to determine the MMP-2 level directly in human plasma and whole blood samples with satisfactory results obtained. Owing to the hypersensitivity of the method, clinical samples of only less than 1 μL were needed for accurate quantification, which can be meaningful in MMP-2-related clinical and bioanalytical applications.     

Efficient single-molecule fluorescence resonance energy transfer analysis by site-specific dual-labeling of protein using an unnatural amino acid

Single-molecule fluorescence resonance energy transfer (smFRET) measurement provides a unique and powerful approach to understand complex biological processes including conformational and structural dynamics of individual biomolecules. For effective smFRET analysis of protein, site-specific dual-labeling with two fluorophores as an energy donor and an acceptor is crucial. Here we demonstrate that site-specific dual-labeling of protein via incorporation of unnatural amino acid provides a clearer picture for the folded and unfolded states of the protein in smFRET analysis than conventional labeling using double cysteines. As a model study, maltose-binding protein (MBP) was dually labeled via incorporation of ρ-azido-l-phenylalanine and cysteine at specific positions, immobilized on a surface, and subjected to smFRET analysis under native and denaturing conditions. The resulting histograms show that site-specific dual-labeling results in a more homogeneous distribution in protein populations, enabling a precise smFRET analysis of protein.     

Assessing phytoplankton using a two-rank database based on excitation-emission fluorescence spectra

The feasibility of using a two-rank database of reference spectra based on in vivo fluorescence excitation-emission matrix (EEMs) spectra to assess dominant groups of phytoplankton was explored. Twenty-six species belonging to 20 genera of seven divisions were studied. First, fluorescent characteristics of these EEMs were extracted using Daubechies-7 wavelet analysis. Second, the optimal characteristic spectra of scale vectors (SOCS) and time-series vectors (TOCS) were selected; phytoplankton of different genera were classified using Fisher linear discriminant analysis. Third, SOCS and TOCS reference spectra databases were obtained by hierarchical cluster analysis. Using non-negative least squares as the method to assess the phytoplankton, a two-rank reference spectra database was established according to their respective ability to identify the 2818 single-species samples. Using this fluorimetric technique, the correct identification rates (CIRs) for single-species samples were 88.8-100% at the genus level and 98.8-100% at the division level. Dominant species in the 465 laboratory mixtures had corresponding CIRs of 85.6% and 96.1%. Moreover, 15 of the 19 species used as dominants were correctly identified at the genus level. In 27 natural seawater samples, Prorocentrum donghaiense, Thalassiosira nordenskioldi, and Chaetoceros socialis (bloom-forming species with a density of about 10(7) cell L(-1)), and Alexandrium sp. (dominant species with abundance of about 10(6) cell L(-1)) were qualitatively identified at the genus level; other dominant species, with densities of 10(5) to 10(6) cell L(-1), were identified at the division level. The quantitative identification was relatively poor in the natural water samples, and several potential resolutions, including trying both new measuring methods and calculating methods, for future study are given.     

Enhancement of the sensitivity of a capillary electrophoresis immunoassay for estradiol with laser-induced fluorescence based on a fluorescein-labeled secondary antibody

A competitive immunoassay for estradiol (E2) based on capillary electrophoresis was established. This method was based on the competitive reaction of complete antigen and E2 with a limited amount of monoclonal antibody, with fluorescein isothiocyanate (FITC)-labeled secondary antibody as a fluorescence probe. The addition of the thermally reversible hydrogel, poly-N-isopropylacrylamide (pNIPA) in the buffer as a replaceable packing material improved reproducibility and resolution of the method. This capillary electrophoresis immunoassay with laser-induced fluorescence can be applied to determine E2 with good precision at concentrations as low as 9 pg/mL. Details of typical separations of immunological complex and free reactants are presented.     

On-chip transduction of nucleic acid hybridization using spatial profiles of immobilized quantum dots and fluorescence resonance energy transfer

The glass surface of a glass-polydimethylsiloxane (PDMS) microfluidic channel was modified to develop a solid-phase assay for quantitative determination of nucleic acids. Electroosmotic flow (EOF) within channels was used to deliver and immobilize semiconductor quantum dots (QDs), and electrophoresis was used to decorate the QDs with oligonucleotide probe sequences. These processes took only minutes to complete. The QDs served as energy donors in fluorescence resonance energy transfer (FRET) for transduction of nucleic acid hybridization. Electrokinetic injection of fluorescent dye (Cy3) labeled oligonucleotide target into a microfluidic channel and subsequent hybridization (within minutes) provided the proximity for FRET, with emission from Cy3 being the analytical signal. The quantification of target concentration was achieved by measurement of the spatial length of coverage by target along a channel. Detection of femtomole quantities of target was possible with a dynamic range spanning an order of magnitude. The assay provided excellent resistance to nonspecific interactions of DNA. Further selectivity of the assay was achieved using 20% formamide, which allowed discrimination between a fully complementary target and a 3 base pair mismatch target at a contrast ratio of 4:1.     

Detection of C-reactive protein based on immunoassay using antibody-conjugated magnetic nanoparticles

We report a detection method for C-reactive protein (CRP) based on competitive immunoassay using magnetic nanoparticles under magnetic fields. Functional magnetic nanoparticles were prepared and conjugated with anti-CRP for immunoassay. Magnetic nanoparticles labeled with anti-CRP were flowed through a separation channel to form depositions for selective capture of CRP under magnetic fields. Free CRP and a fixed number of CRP-labeled particles were used to compete for a limited number of anti-CRP binding sites on the magnetic nanoparticles. The deposited percentages of CRP-labeled particles at various concentrations of free CRP were determined and used as a reference plot. The determination of CRP in the unknown sample was deduced from the reference plot using the deposited percentages. The running time was less than 10 min. The CRP concentration of serum sample was linearly over the range of 1.2-310 microg/mL for deposited percentages of CRP-labeled particles. The detection limit of this method was 0.12 microg/mL which was approximately 8-fold lower than the typical clinical cutoff concentration (1 microug/mL). This method can provide a fast, simple, and sensitive way for protein detection based on competitive immunoassay using magnetic nanoparticles under magnetic fields.     

Development of a time-resolved fluorometric detection system using diffusion-enhanced energy transfer

A novel detection system using both emission energy transfer and time-resolved fluorometry (TRF) was developed, with a europium chelate as the energy donor and a novel fluorophore SNR1, excitable with long-wavelength light corresponding to europium emission, as the energy acceptor. When the donor and acceptor molecules were mixed in solution, energy transfer was observed without direct attachment of the donor and the acceptor, via a diffusion-enhanced energy-transfer mechanism. Thus, the acceptor emission can be detected as a long-lifetime fluorescence in TRF. When the fluorescence properties of the acceptor molecule are changed by interaction with an enzyme or other bioactive molecule, the change can be detected as a long-lived sensitized emission. If we develop or select suitable acceptor molecules, this simple and convenient system should be applicable to a wide variety of bioactive molecules. Since it is based on TRF, it can be used for high-resolution assay.