Fluorescence resonance energy transfer between donor-acceptor pair on two oligonucleotides hybridized adjacently to DNA template

We use fluorescein as the energy donor and rhodamine as the acceptor to measure the efficiency of fluorescence resonance energy transfer (FRET) in a set of hybridized DNA constructs. The two fluorophores are covalently attached via linkers to two separate oligonucleotides with fluorescein at the 3' end of one oligonucleotide and rhodamine at the 5' end or in the middle of another nucleotide. For the FRET analysis both fluorophore-labeled oligonucleotides are hybridized to adjacent sections of the same DNA template to form a three-component duplex with a one base gap between the two labeled oligonucleotides. A similar configuration is implemented for a quantitative real-time polymerase chain reaction (PCR) with LightCycler technology, where a 1-5 base separation between donor and acceptor is recommended to optimize energy transfer efficiencies. Our constructs cover donor-acceptor separations from 2 to 17 base pairs (approximately 10-70 A). The results show that, when the two fluorophores are located at close distances (less than 8 base separation), FRET efficiencies are above 80%, although there may be ground-state interactions between fluorophores when the separation is under about 6 bases. Modeling calculations are used to predict the structure of these three-component constructs. The duplex mostly retains a normal double helical structure, although slight bending may occur near the unpaired base in the DNA template. Stable and reproducible energy transfer is also observed over the distance range investigated here in real-time thermal cycling. The study identifies important parameters that determine FRET response in applications such as real-time PCR.     

Conformational changes of the Ca(2+) regulatory site of the Na(+)-Ca(2+) exchanger detected by FRET

The Na(+)-Ca(2+) exchanger is a plasma membrane protein expressed at high levels in cardiomyocytes. It extrudes 1 Ca(2+) for 3 Na(+) ions entering the cell, regulating intracellular Ca(2+) levels and thereby contractility. Na(+)-Ca(2+) exchanger activity is regulated by intracellular Ca(2+), which binds to a region (amino acids 371-508) within the large cytoplasmic loop between transmembrane segments 5 and 6. Regulatory Ca(2+) activates the exchanger and removes Na(+)-dependent inactivation. The physiological role of intracellular Ca(2+) regulation of the exchanger is not yet established. Yellow (YFP) and cyan (CFP) fluorescent proteins were linked to the NH(2)- and CO(2)H-termini of the exchanger Ca(2+) binding domain (CBD) to generate a construct (YFP-CBD-CFP) capable of responding to changes in intracellular Ca(2+) concentrations by FRET efficiency measurements. The two fluorophores linked to the CBD are sufficiently close to generate FRET. FRET efficiency was reduced with increasing Ca(2+) concentrations. Titrations of Ca(2+) concentration versus FRET efficiency indicate a K(D) for Ca(2+) of approximately 140 nM, which increased to approximately 400 nM in the presence of 1 mM Mg(2+). Expression of YFP-CBD-CFP in myocytes, generated changes in FRET associated with contraction, suggesting that NCX is regulated by Ca(2+) on a beat-to-beat basis during excitation-contraction coupling.     

Synaptic complex formation of two retrovirus DNA attachment sites by integrase: a fluorescence energy transfer study

The integration of retroviral DNA by the viral integrase (IN) into the host genome occurs via assembled preintegration complexes (PIC). We investigated this assembly process using purified IN and viral DNA oligodeoxynucleotide (ODN) substrates (93 bp in length) that were labeled with donor (Cy3) and acceptor fluorophores (Cy5). The fluorophores were attached to the 5' 2 bp overhangs of the terminal attachment (att) sites recognized by IN. Addition of IN to the assay mixture containing the fluorophore-labeled ODN resulted in synaptic complex formation at 14 degrees C with significant fluorescence resonance energy transfer (FRET) occurring between the fluorophores in close juxtaposition (from approximately 15 to 100 A). Subsequent integration assays at 37 degrees C with the same ODN (32P-labeled) demonstrated a direct association of a significant FRET signal with concerted insertion of the two ODNs into the circular DNA target, here termed full-site integration. FRET measurements (deltaF) show that IN binds to a particular set of 3' OH recessed substrates (type I) generating synaptic complexes capable of full-site integration that, as shown previously, exhibit IN mediated protection from DNaseI digestion up to approximately 20 bp from the ODN att ends. In contrast, IN also formed complexes with nonspecific DNA ends and loss-of-function att end substrates (type II) that had significantly lower deltaF values and were not capable of full-site integration, and lacked the DNaseI protection properties. The type II category may exemplify what is commonly understood as "nonspecific" binding by IN to DNA ends. Two IN mutants that exhibited little or no integration activity gave rise to the lower deltaF signals. Our FRET analysis provided the first direct physical evidence that IN forms synaptic complexes with two DNA att sites in vitro, yielding a complex that exhibits properties comparable to that of the PIC.     

The structure of sulfoindocarbocyanine 3 terminally attached to dsDNA via a long, flexible tether

Fluorescence resonance energy transfer (FRET) is an important source of long-range distance information in macromolecules. However, extracting maximum information requires knowledge of fluorophore, donor and acceptor, positions on the macromolecule. We previously determined the structure of the indocarbocyanine fluorophores Cy3 and Cy5 attached to DNA via three-carbon atom tethers, showing that they stacked onto the end of the helix in a manner similar to an additional basepair. Our recent FRET study has suggested that when they are attached via a longer 13-atom tether, these fluorophores are repositioned relative to the terminal basepair by a rotation of ～30°, while remaining stacked. In this study, we have used NMR to extend our structural understanding to the commonly used fluorophore sulfoindocarbocyanine-3 (sCy3) attached to the 5'-terminus of the double-helical DNA via a 13-atom flexible tether (L13). We find that L13-sCy3 remains predominantly stacked onto the end of the duplex, but adopts a significantly different conformation, from that of either Cy3 or Cy5 attached by 3-atom tethers, with the long axes of the fluorophore and the terminal basepair approximately parallel. This result is in close agreement with our FRET data, supporting the contention that FRET data can be used to provide orientational information.     

Orientation of cyanine fluorophores terminally attached to DNA via long, flexible tethers

Cyanine fluorophores are commonly used in single-molecule FRET experiments with nucleic acids. We have previously shown that indocarbocyanine fluorophores attached to the 5′-termini of DNA and RNA via three-carbon atom linkers stack on the ends of the helix, orienting their transition moments. We now investigate the orientation of sulfoindocarbocyanine fluorophores tethered to the 5′-termini of DNA via 13-atom linkers. Fluorescence lifetime measurements of sulfoindocarbocyanine 3 attached to double-stranded DNA indicate that the fluorophore is extensively stacked onto the terminal basepair at 15°C, with properties that depend on the terminal sequence. In single molecules of duplex DNA, FRET efficiency between sulfoindocarbocyanine 3 and 5 attached in this manner is modulated with helix length, indicative of fluorophore orientation and consistent with stacked fluorophores that can undergo lateral motion. We conclude that terminal stacking is an intrinsic property of the cyanine fluorophores irrespective of the length of the tether and the presence or absence of sulfonyl groups. However, compared to short-tether indocarbocyanine, the mean rotational relationship between the two fluorophores is changed by ∼60° for the long-tether sulfoindocarbocyanine fluorophores. This is consistent with the transition moments becoming approximately aligned with the long axis of the terminal basepair for the long-linker species.     

Bidirectional fluorescence resonance energy transfer (FRET) in mutated and chemically modified yellow fluorescent protein (YFP)

Fluorescence resonance energy transfer (FRET) using fluorescent protein variants are used for studying the associations and biomolecular motions of macromolecules inside the cell. Intramolecular FRET utilizing fluorescent chemical labels has been applied in nucleic acid chemistry for detection of specific sequence. However, the biotechnological applications of intramolecular FRET in fluorescent proteins have not been exploited. This study demonstrates the intramolecular FRET between fluorescent protein and conjugated chemical label whereby FRET occurs from inside to outside and vice versa for fluorescent protein. The fluorescent protein is modified for the attachment of chemical fluorophores and the novel FRET pairs created by conjugation are MDCC (435/475)-Citrine (516/529) and Citrine-Alexa fluor (568/603). These protein-label pairs exhibited strong intramolecular FRET and the energy transfer efficiency was determined based on the time evolution of the ratio of emission intensities of labeled and unlabeled proteins. The efficiency was found to be 0.79 and 0.89 for MDCC-Citrine and 0.24 and 0.65 for Citrine-Alexa Fluor pairs when the label is conjugated at different sites in the protein. Fo?rster distance and the average distance between the fluorophores were also determined. The bidirectional approach described here can provide new insights into designing FRET-based sensors.     

Intramolecular fluorescence resonance energy transfer between fused autofluorescent proteins reveals rearrangements of the N- and C-terminal segments of the plasma membrane Ca2+ pump involved in the activation

The blue and green fluorescent proteins (BFP and GFP) have been fused at the N- and C-terminal ends, respectively, of the plasma membrane Ca(2+) pump (PMCA) isoform 4xb (hPMCA4xb). The fusion protein was successfully expressed in yeast and purified by calmodulin affinity chromatography. Despite the presence of the fused autofluorescent proteins BFP-PMCA-GFP performed similarly to the wild-type enzyme with respect to Ca(2+)-ATPase activity and sensitivity to calmodulin activation. In the autoinhibited state BFP-PMCA-GFP exhibited a significant intramolecular fluorescence resonance energy transfer (FRET) consistent with the location of the fluorophores at an average distance of 45A. The FRET intensity in BFP-PMCA-GFP decreased when the enzyme was activated either by Ca(2+)-calmodulin, partial proteolysis, or acidic lipids. Moreover, FRET decreased and became insensitive to calmodulin when hPMCA4xb was activated by mutation D170N in BFP-PMCA(D170N)-GFP. The results suggest that the ends of the PMCA are in close proximity in the autoinhibited conformation, and they separate or reorient when the PMCA achieves its final activated conformation.     

Sphingomyelin composition and physical asymmetries in native acetylcholine receptor-rich membranes

Selective enzymatic hydrolysis, lipid compositional analyses, and fluorescence studies have been carried out on acetylcholine receptor (AChR)-rich membranes from Torpedinidae to investigate the topology of sphingomyelin (SM) in the native membrane and its relationship with the AChR protein. Controlled sphingomyelinase hydrolysis of native membranes showed that SM is predominantly (approximately 60%) localized in the outer half of the lipid bilayer. Differences were also observed in the distribution of SM fatty acid molecular species in the two bilayer leaflets. A fluorescent SM derivative ( N-[10-(1-pyrenyl)decanoyl]sphingomyelin; Py-SM) was used to study protein-lipid interactions in the AChR-rich membrane and in affinity-purified Torpedo AChR reconstituted in liposomes made from Torpedo electrocyte lipid extracts. The efficiency of F?rster resonance energy transfer (FRET) from the protein to the pyrenyl-labeled lipid as a function of acceptor surface density was used to estimate distances and topography of the SM derivative relative to the protein. The dynamics of the lipid acyl chains were explored by measuring the thermal dependence of Py-SM excimer formation, sensitive to the fluidity of the membrane. Differences were observed in the concentration dependence of excimer/monomer pyrenyl fluorescence when measured by direct excitation of the probe as against under FRET conditions, indicating differences in the intermolecular collisional frequency of the fluorophores between bulk and protein-vicinal lipid environments, respectively. Py-SM exhibited a moderate selectivity for the protein-vicinal lipid domain, with a calculated relative affinity K(r) approximately 0.55. Upon sphingomyelinase digestion of the membrane, FRET efficiency increased by about 50%, indicating that the resulting pyrenyl-ceramide species have higher affinity for the protein than the parental SM derivative.     

Fluorescence resonance energy transfer-based intracellular assay for the conformation of hepatitis C virus drug target NS5A

Nonstructural protein 5A (NS5A) is essential for hepatitis C virus (HCV) replication and assembly and is a critical drug target. Biochemical data suggest large parts of NS5A are unfolded as an isolated protein, but little is known about its folded state in the cell. We used fluorescence resonance energy transfer (FRET) to probe whether or not different regions of NS5A are in close proximity within the cell. Twenty-three separate reporter constructs were created by inserting one or more fluorophores into different positions throughout the three domains of NS5A. FRET efficiency was maximal when donor and acceptor fluorophores were positioned next to each other but also could be observed when the two fluorophores flanked NS5A domain 1 or domain 3. Informatic and biochemical analysis suggests that large portions of the carboxy terminus of NS5A are in an unfolded and disordered state. Quercetin, a natural product known to disrupt NS5A function in cells, specifically disrupted a conformationally specific domain 3 FRET signal. Intermolecular FRET indicated that the NS5A amino termini, but not other regions, are in close proximity in multimeric complexes. Overall, this assay provides a new window on the intracellular conformation(s) of NS5A and how the conformation changes in response to cellular and viral components of the replication and assembly complex as well as antiviral drugs.     

Modest Influence of FRET Chromophores on the Properties of Unfolded Proteins

Single-molecule Förster resonance energy transfer (FRET) experiments are often used to study the properties of unfolded and intrinsically disordered proteins. Because of their large extinction coefficients and quantum yields, synthetic heteroaromatic chromophores covalently linked to the protein are often used as donor and acceptor fluorophores. A key issue in the interpretation of such experiments is the extent to which the properties of the unfolded chain may be affected by the presence of these chromophores. In this article, we investigate this question using all-atom explicit solvent replica exchange molecular dynamics simulations of three different unfolded or intrinsically disordered proteins. We find that the secondary structure and long-range contacts are largely the same in the presence or absence of the fluorophores, and that the dimensions of the chain with and without chromophores are similar. This suggests that, at least in the cases studied, extrinsic fluorophores have little effect on the structural properties of unfolded or disordered proteins. We also find that the critical FRET orientational factor κ², has an average value and equilibrium distribution very close to that expected for isotropic orientations, which supports one of the assumptions frequently made when interpreting FRET efficiency in terms of distances.     

An ion-insensitive cAMP biosensor for long term quantitative ratiometric fluorescence resonance energy transfer (FRET) measurements under variable physiological conditions

Ratiometric measurements with FRET-based biosensors in living cells using a single fluorescence excitation wavelength are often affected by a significant ion sensitivity and the aggregation behavior of the FRET pair. This is an important problem for quantitative approaches. Here we report on the influence of physiological ion concentration changes on quantitative ratiometric measurements by comparing different FRET pairs for a cAMP-detecting biosensor. We exchanged the enhanced CFP/enhanced YFP FRET pair of an established Epac1-based biosensor by the fluorophores mCerulean/mCitrine. In the case of enhanced CFP/enhanced YFP, we showed that changes in proton, and (to a lesser extent) chloride ion concentrations result in incorrect ratiometric FRET signals, which may exceed the dynamic range of the biosensor. Calcium ions have no direct, but an indirect pH-driven effect by mobilizing protons. These ion dependences were greatly eliminated when mCerulean/mCitrine fluorophores were used. For such advanced FRET pairs the biosensor is less sensitive to changes in ion concentration and allows consistent cAMP concentration measurements under different physiological conditions, as occur in metabolically active cells. In addition, we verified that the described FRET pair exchange increased the dynamic range of the FRET efficiency response. The time window for stable experimental conditions was also prolonged by a faster biosensor expression rate in transfected cells and a greatly reduced tendency to aggregate, which reduces cytotoxicity. These properties were verified in functional tests in single cells co-expressing the biosensor and the 5-HT(1A) receptor.     

生物大分子相互作用检测技术新进展———三色荧光级联荧光共振能量转移技术

荧光共振能量转移(fluorescenceresonanceenergytransfer,FRET),是指能量从一种受激发的荧光基团(fluorophore)以非辐射的方式转移到另一种荧光基团的物理现象.FRET的能量转移效率是两个荧光基团间距离的函数,并对此距离十分敏感,它的有效响应距离一般在1～10nm之间,因而可被用于测定原子间及分子间的距离.这一特点使FRET技术在大分子构象变化、大分子之间相互作用、细胞信号通路等研究中发挥重要作用,成为生物医学研究中的重要方法.但细胞内的生物学过程常常涉及多于两个的大分子间相互作用,二色荧光基团的FRET技术不能满足这种生物学研究的需求.最近,两个研究小组在这方面取得突破,建立了分别基于共聚焦显微镜和流式细胞仪的三色荧光级联FRET技术.这一技术的出现将会极大地促进生物学及相关研究领域的发展.     

Electroporation and Microinjection Successfully Deliver Single-Stranded and Duplex DNA into Live Cells as Detected by FRET Measurements

Förster resonance energy transfer (FRET) technology relies on the close proximity of two compatible fluorophores for energy transfer. Tagged (Cy3 and Cy5) complementary DNA strands forming a stable duplex and a doubly-tagged single strand were shown to demonstrate FRET outside of a cellular environment. FRET was also observed after transfecting these DNA strands into fixed and live cells using methods such as microinjection and electroporation, but not when using lipid based transfection reagents, unless in the presence of the endosomal acidification inhibitor bafilomycin. Avoiding the endocytosis pathway is essential for efficient delivery of intact DNA probes into cells.     

VenusA206 Dimers Behave Coherently at Room Temperature

Fluorescent proteins (FPs) have revolutionized cell biology by allowing genetic tagging of specific proteins inside living cells. In conjunction with Förster’s resonance energy transfer (FRET) measurements, FP-tagged proteins can be used to study protein-protein interactions and estimate distances between tagged proteins. FRET is mediated by weak Coulombic dipole-dipole coupling of donor and acceptor fluorophores that behave independently, with energy hopping discretely and incoherently between fluorophores. Stronger dipole-dipole coupling can mediate excitonic coupling in which excitation energy is distributed near instantaneously between coherently interacting excited states that behave as a single quantum entity. The interpretation of FP energy transfer measurements to estimate separation often assumes that donors and acceptors are very weakly coupled and therefore use a FRET mechanism. This assumption is considered reasonable as close fluorophore proximity, typically associated with strong excitonic coupling, is limited by the FP β-barrel structure. Furthermore, physiological temperatures promote rapid vibrational dephasing associated with a rapid decoherence of fluorophore-excited states. Recently, FP dephasing times that are 50 times slower than traditional organic fluorophores have been measured, raising the possibility that evolution has shaped FPs to allow stronger than expected coupling under physiological conditions. In this study, we test if excitonic coupling between FPs is possible at physiological temperatures. FRET and excitonic coupling can be distinguished by monitoring spectral changes associated with fluorophore dimerization. The weak coupling mediating FRET should not cause a change in fluorophore absorption, whereas strong excitonic coupling causes Davydov splitting. Circular dichroism spectroscopy revealed Davydov splitting when the yellow FP Venus_A206 dimerizes, and a novel approach combining photon antibunching and fluorescence correlation spectroscopy was used to confirm that the two fluorophores in a Venus_A206 homodimer behave as a single-photon emitter. We conclude that excitonic coupling between Venus_A206 fluorophores is possible at physiological temperatures.     

Quantitative time domain analysis of lifetime‐based Förster resonant energy transfer measurements with fluorescent proteins: Static random isotropic fluorophore orientation distributions

Förster resonant energy transfer (FRET) measurements are widely used to obtain information about molecular interactions and conformations through the dependence of FRET efficiency on the proximity of donor and acceptor fluorophores. Fluorescence lifetime measurements can provide quantitative analysis of FRET efficiency and interacting population fraction. Many FRET experiments exploit the highly specific labelling of genetically expressed fluorescent proteins, applicable in live cells and organisms. Unfortunately, the typical assumption of fast randomization of fluorophore orientations in the analysis of fluorescence lifetime‐based FRET readouts is not valid for fluorescent proteins due to their slow rotational mobility compared to their upper state lifetime. Here, previous analysis of effectively static isotropic distributions of fluorophore dipoles on FRET measurements is incorporated into new software for fitting donor emission decay profiles. Calculated FRET parameters, including molar population fractions, are compared for the analysis of simulated and experimental FRET data under the assumption of static and dynamic fluorophores and the intermediate regimes between fully dynamic and static fluorophores, and mixtures within FRET pairs, is explored. Finally, a method to correct the artefact resulting from fitting the emission from static FRET pairs with isotropic angular distributions to the (incorrect) typically assumed dynamic FRET decay model is presented.      

Development of dual receptor biosensors: an analysis of FRET pairs

The development of a dual receptor detection method for enhanced biosensor monitoring was investigated by analyzing potential fluorescent resonance energy transfer (FRET) pairs. The dual receptor scheme requires the integration of a chemical transducer system with two unique protein receptors that bind to a single biological agent. The two receptors are tagged with special molecular groups (donors and acceptors fluorophores) while the chemical transduction system relies on the well-known mechanisms of FRET. During the binding event, the two FRET labeled receptors dock at the binding sites on the surface of the biological agent. The resulting close proximity of the two fluorophores upon binding will initiate the energy transfer resulting in fluorescence. The paper focuses on the analysis and optimization of the chemical transduction system. A variety of FRET fluorophore pairs were tested in a spectrofluorimeter and promising FRET pairs were then tagged to the protein, avidin and its ligand, biotin. Due to their affinities, the FRET-tagged biomolecules combine in solution, resulting in a stable, fluorescent signal from the acceptor FRET dye with a simultaneous decrease in fluorescent signal from the donor FRET dye. The results indicate that the selected FRET pairs can be utilized in the development of dual receptor sensors.     

Fluorescent nanometer microspheres as a reporter for sensitive detection of simulants of biological threats using multiplexed suspension arrays

We succeeded in using 40 nm FRET (fluorescence resonance energy transfer) microspheres conjugated to antibodies as the fluorescent reporters to perform the multiplexing suspension array measurements on two simulants of biological threats, ricin (A chain) and a crude spore preparation of Bacillus globigii (Bg). The microspheres were impregnated with two types of fluorophores in equal number (approximately 140 fluorophores in total per microsphere) and displayed bright PE-like fluorescence via a fluorescence resonance energy transfer mechanism. Activated microspheres (aldehyde groups) were directly coupled to antibodies and used to form sandwich-type immunoassays in a suspension array. For the crude preparations of Bg, the assay sensitivity using antibody-conjugated microspheres is an order of magnitude higher than that using the conventional fluorescent reporter, R-phycoerythrin (PE). Using the microspheres, Bg at the concentration of 5 ng/mL can be easily detected. For ricin, the assay sensitivity was similar to that obtained using PE as the reporter, but washing the reaction mixtures resulted in the fluorescence signals that were 2-3 times higher compared to those using PE. Ricin at a concentration of 1 ng/mL can be readily identified. Importantly, the two simulants do not interfere with each other in the multiplexing experiments. The 40 nm FRET microspheres are a new sensitive alternative as fluorescent reporters for detection in suspension arrays.     

Modest Influence of FRET Chromophores on the Properties of Unfolded Proteins

Single-molecule Förster resonance energy transfer (FRET) experiments are often used to study the properties of unfolded and intrinsically disordered proteins. Because of their large extinction coefficients and quantum yields, synthetic heteroaromatic chromophores covalently linked to the protein are often used as donor and acceptor fluorophores. A key issue in the interpretation of such experiments is the extent to which the properties of the unfolded chain may be affected by the presence of these chromophores. In this article, we investigate this question using all-atom explicit solvent replica exchange molecular dynamics simulations of three different unfolded or intrinsically disordered proteins. We find that the secondary structure and long-range contacts are largely the same in the presence or absence of the fluorophores, and that the dimensions of the chain with and without chromophores are similar. This suggests that, at least in the cases studied, extrinsic fluorophores have little effect on the structural properties of unfolded or disordered proteins. We also find that the critical FRET orientational factor κ², has an average value and equilibrium distribution very close to that expected for isotropic orientations, which supports one of the assumptions frequently made when interpreting FRET efficiency in terms of distances.     

Development of a novel FRET immunosensor technique

We report on a novel technique to develop an optical immunosensor based on fluorescence resonance energy transfer (FRET). IgG antibodies were labeled with acceptor fluorophores while one of three carrier molecules (protein A, protein G, or F(ab')2 fragment) was labeled with donor fluorophores. The carrier molecule was incubated with the antibody to allow specific binding to the Fc portion. The labeled antibody-protein complex was then exposed to specific and nonspecific antigens, and experiments were designed to determine the 'in solution' response. The paper reports the results of three different donor-acceptor FRET pairs, fluorescein isothiocyanate/tetramethylrhodamine isothiocyanate, Texas Red/Cy5, and Alexa Fluor 546/Alexa Fluor 594. The effects of the fluorophore to protein conjugation ratio (F/P ratio) and acceptor to donor fluorophore ratios between the antibody and protein (A/D ratio) were examined. In the presence of specific antigens, the antibodies underwent a conformational change, resulting in an energy transfer from the donor to the acceptor fluorophore as measured by a change in fluorescence. The non-specific antigens elicited little or no changes. The Alexa Fluor FRET pair demonstrated the largest change in fluorescence, resulting in a 35% change. The F/P and A/D ratio will affect the efficiency of energy transfer, but there exists a suitable range of A/D and F/P ratios for the FRET pairs. The feasibility of the FRET immunosensor technique was established; however, it will be necessary to immobilize the complexes onto optical substrates so that consistent trends can be obtained that would allow calibration plots.