Time-resolved Luminescence Resonance Energy Transfer Research Articles

A persistent luminescence resonance energy transfer-based molecular beacon probe for the highly sensitive detection of microRNA in biological samples

Herein, a time-resolved luminescence resonance energy transfer (TR-LRET) molecular beacon (MB) probe employing persistent luminescence nanoparticles (PLNPs) as the energy donors was first constructed, and further designed for microRNA21 (miR21) sensing. This probe (named as PLNPs-MB) was facilely fabricated by covalent bioconjugation between poly-(acrylic acid) (PAA) modified near-infrared (NIR) emissive PLNPs i.e. ZnGa2O4:Cr3+ and functionalized MB oligonucleotide (5′-NH2 and 3′-BHQ3). Accordingly, PLNPs and BHQ3 were in close proximity to each other, leading to the occurrence of LRET and obvious persistent luminescence (PL) quenching. In the presence of miR21, loop of the PLNP-MB was hybridized, accompanying BHQ3 away from PLNPs and the restraint of LRET process. As a result, PL of the PLNPs was recovered, which built the foundation of miR21 quantification. The probe provided a linear response range from 0.1 to 10 nM for miR21 detection. Quantification limit of this probe was competitive and about 1–2 orders of magnitude lower than that of other reported MB probes for nucleic acid. Moreover, the proposed probe was successfully adopted for miR21 detection in biological fluids (human serum, cell extraction). This work also provided a sensitive detection nanoplatform for other targets through modifying diverse MBs onto the surface of PLNPs.

Single-Chain Lanthanide Luminescence Biosensors for Cell-Based Imaging and Screening of Protein-Protein Interactions.

SummaryLanthanide-based, Förster resonance energy transfer (LRET) biosensors enabled sensitive, time-gated luminescence (TGL) imaging or multiwell plate analysis of protein-protein interactions (PPIs) in living cells. We prepared stable cell lines that expressed polypeptides composed of an alpha helical linker flanked by a Tb(III) complex-binding domain, GFP, and two interacting domains at each terminus. The PPIs examined included those between FKBP12 and the rapamycin-binding domain of m-Tor (FRB) and between p53 (1–92) and HDM2 (1–128). TGL microscopy revealed dramatic differences (>500%) in donor- or acceptor-denominated, Tb(III)-to-GFP LRET ratios between open (unbound) and closed (bound) states of the biosensors. We observed much larger signal changes (>2,500%) and Z′-factors of 0.5 or more when we grew cells in 96- or 384-well plates and analyzed PPI changes using a TGL plate reader. The modular design and exceptional dynamic range of lanthanide-based LRET biosensors will facilitate versatile imaging and cell-based screening of PPIs.

Inorganic-Organic Hybrid Tongue-Mimic for Time-Resolved Luminescent Noninvasive Pattern and Chiral Recognition of Thiols in Biofluids toward Healthcare Monitoring.

In this work, manganese(II)-doped zinc/germanium oxide nanoparticles (Mn@ZGNPs) have been hydrothermally synthesized to equip with appealing time-resolved luminescence (TRL). Interestingly, we reveal that they can be readily quenched ("turn off") via a facile surface coating with bioinspired polydopamine (PDA) polymerized from dopamine (DA), resulting from PDA-triggered TRL resonance energy transfer (TRL-RET). Byintegrated with the thiol-induced inhibition of PDA formation, an ingenious inorganic-organic hybrid tongue-mimic sensor array is thus unveiled for noninvasive pattern recognition of thiols in biofluids in a TRL-RET-reversed "turn on" format toward healthcare monitoring. The sensing principle is based on the new finding that there are differential inhibitions from thiols against the polymerization of DA with various concentrations. Furthermore, density function theory (DFT) studies excellently prove our sensing principle and experimental results, reinforcing the power of the presented system. More importantly, chiral recognition of varied concentrations and mixtures of cysteine enantiomers using our platform are also been demonstrated, promising its practical usage. This is a novel concept of inorganic-organic hybrid-based pattern and chiral recognition platform for TRL background-free sensing and would sprout more novel relevant strategies toward broader applications.

Luminometric Nanoparticle-Based Assay for High Sensitivity Detection of β-Amyloid Aggregation.

A nanoparticle-based assay utilizing time-resolved luminescence resonance energy transfer (TR-LRET) was developed for the detection of β-amyloid aggregation. The assay is based on the competitive adsorption of the sample and the acceptor-labeled protein to donor europium(III) polystyrene nanoparticles. The performance of the assay was demonstrated by following the fibrillization of β-amyloid peptide 1-42 (Aβ42) as a function of time and by comparing to the reference methods atomic force microscopy (AFM) and thioflavin T (ThT) assay. The fibrillization leads to reduced adsorption of Aβ42 to the nanoparticles increasing the TR-LRET signal. The investigated methods detected fibril formation with equal sensitivities. Eight potential fibrillization inhibitor compounds reported in the literature were tested and the results obtained with each method were compared. It was shown with AFM imaging that the inhibition of fibril formation was not complete with any of the compounds. The developed TR-LRET nanoparticle assay gave corresponding results with the AFM imaging. However, the ThT assay led to contradictory results, as low fluorescence signal was measured in the presence of all tested compounds suggesting inhibition of fibrillization. Our results suggest that the developed TR-LRET nanoparticle assay can be exploited for screening of potential β-amyloid aggregation inhibitors, whereas some of the tested compounds may be measured as false positive inhibitors with the much-utilized ThT assay.

Nonspecific luminometric assay for monitoring protein adsorption efficiency and coverage on nanoparticles.

Nonspecific assays utilizing time-resolved luminescence resonance energy transfer (TR-LRET) are developed for two applications: to monitor protein adsorption efficiency and to assess the degree of surface coverage on the solid phase. We successfully measure the adsorption efficiency of non-sedimenting nanoparticles since this has been notoriously difficult to determine. Monitoring of the protein adsorption on nanoparticles does not require the nanoparticles with the adsorbed protein to be washed and it is based on the competitive adsorption between the non-adsorbed analyte protein and the acceptor-labeled protein to donor europium(iii) polystyrene nanoparticles. The application for assessing the degree of surface coverage is performed with the final coated and washed analyte nanoparticles and it requires no fluorescent labeling of the studied protein. This application utilizes the competitive adsorption of the acceptor-labeled protein on analyte nanoparticles partly covered with protein and donor europium(iii) polystyrene nanoparticles. The developed methods detect either non-adsorbed protein or uncovered nanoparticle surface, not the proteins adsorbed on the nanoparticles. This is not achievable with the traditional total protein quantification assays applied for monitoring protein adsorption since both non-adsorbed and adsorbed protein are detected and their separation is required. Thus, the developed application for monitoring protein adsorption is user-friendly, requires no centrifugal instrumentation, and is applicable also for small nanoparticles requiring ultracentrifugation. No special expertise or investment in costly instruments is required compared to the existing methods, such as spectroscopic techniques, isothermal titration calorimetry, and surface plasmon resonance. The application for assessing the degree of surface coverage is compared to a reference literature method that comprised the analysis of adsorbed fluorescently labeled protein. The saturation reached at similar protein concentrations showing the reliability of the assay. Our results suggest that the developed applications could be exploited as rapid tools for protein adsorption studies on nanoparticles and for quality control and characterization of the coating processes.

Quantitative and discriminative analysis of nucleic acid samples using luminometric nonspecific nanoparticle methods.

Homogeneous simple assays utilizing luminescence quenching and time-resolved luminescence resonance energy transfer (TR-LRET) were developed for the quantification of nucleic acids without sequence information. Nucleic acids prevent the adsorption of a protein to europium nanoparticles which is detected as a luminescence quenching of europium nanoparticles with a soluble quencher or as a decrease of TR-LRET from europium nanoparticles to the acceptor dye. Contrary to the existing methods based on fluorescent dye binding to nucleic acids, equal sensitivities for both single- (ssDNA) and double-stranded DNA (dsDNA) were measured and a detection limit of 60 pg was calculated for the quenching assay. The average coefficient of variation was 5% for the quenching assay and 8% for the TR-LRET assay. The TR-LRET assay was also combined with a nucleic acid dye selective to dsDNA in a single tube assay to measure the total concentration of DNA and the ratio of ssDNA and dsDNA in the mixture. To our knowledge, such a multiplexed assay is not accomplished with commercially available assays.

Method for determination of polyethylene glycol molecular weight.

A method utilizing competitive adsorption between polyethylene glycols (PEGs) and labeled protein to nanoparticles was developed for the determination of PEG molecular weight (MW) in a microtiter plate format. Two mix-and-measure systems, time-resolved luminescence resonance energy transfer (TR-LRET) with donor europium(III) polystyrene nanoparticles and acceptor-labeled protein and quenching with quencher gold nanoparticles and fluorescently labeled protein were compared for their performance. MW is estimated from the PEG MW dependent changes in the competitive adsorption properties, which are presented as the luminescence signal vs PEG mass concentration. The curves obtained with the TR-LRET system overlapped for PEGs larger than 400 g/mol providing no information on MW. Distinctly different curves were obtained with the quenching system enabling the assessment of PEG MW within a broad dynamic range. The data was processed with and without prior knowledge of the PEG concentration to measure PEGs over a MW range from 62 to 35,000 g/mol. The demonstration of the measurement independent of the PEG concentration suggests that the estimation of MW is possible with quenching nanoparticle system for neutrally charged and relatively hydrophilic polymeric molecules widening the applicability of the simple and cost-effective nanoparticle-based methods.

Simple Nanoparticle-Based Luminometric Method for Molecular Weight Determination of Polymeric Compounds

A nanoparticle-based method utilizing time-resolved luminescence resonance energy transfer (TR-LRET) was developed for molecular weight determination. This mix-and-measure nanoparticle method is based on the competitive adsorption between the analyte and the acceptor-labeled protein to donor Eu(III) nanoparticles. The size-dependent adsorption of molecules enables the molecular weight determination of differently sized polymeric compounds down to a concentration level of micrograms per liter. The molecular weight determination from 1 to 10 kDa for polyamino acids and from 0.3 to 70 kDa for polyethylene imines is demonstrated. The simple and cost-effective nanoparticle method as microtiter plate assay format shows great potential for the detection of the changes in molecular weight or for quantification of differently sized molecules in biochemical laboratories and in industrial polymeric processes.

Luminescence stability improvement in liposome-based homogeneous luminescence resonance energy transfer

A stable liposome-based time-resolved luminescence resonance energy transfer (TR–LRET) assay was developed based on the interaction of biotinylated lipids and streptavidin. Eu3+ ion chelated to 4,4,4-trifluoro-1-(2-naphthalenyl)-1,3-butanedione and trioctylphosphine oxide was incorporated into liposomes. Acceptor-labeled streptavidin bound to biotinylated lipids of the liposomes enables TR–LRET. A stable assay performance was achieved by optimization. High Eu3+ signal and stability, low variation, and sensitivity below 100pM for free biotin was achieved by incorporating the chelate into liposomes containing cholesterol in a carbonate buffer. Potentially, the stable assay compared with the assay without cholesterol offers an improved platform to liposome-based detection systems.

Protein Quantification Using Resonance Energy Transfer between Donor Nanoparticles and Acceptor Quantum Dots

A homogeneous time-resolved luminescence resonance energy transfer (TR-LRET) assay has been developed to quantify proteins. The competitive assay is based on resonance energy transfer (RET) between two luminescent nanosized particles. Polystyrene nanoparticles loaded with Eu(3+) chelates (EuNPs) act as donors, while protein-coated quantum dots (QDs), either CdSe/ZnS emitting at 655 nm (QD655-strep) or CdSeTe/ZnS with emission wavelength at 705 nm (QD705-strep), are acceptors. In the absence of analyte protein, in our case bovine serum albumin (BSA), the protein-coated QDs bind nonspecifically to the EuNPs, leading to RET. In the presence of analyte proteins, the binding of the QDs to the EuNPs is prevented and the RET signal decreases. RET from the EuNPs to the QDs was confirmed and characterized with steady-state and time-resolved luminescence spectroscopy. In accordance with the Förster theory, the approximate average donor-acceptor distance is around 15 nm at RET efficiencies, equal to 15% for QD655 and 13% for QD705 acceptor, respectively. The limits of detection are below 10 ng of BSA with less than a 10% average coefficient of variation. The assay sensitivity is improved, when compared to the most sensitive commercial methods. The presented mix-and-measure method has potential to be implemented into routine protein quantification in biological laboratories.

Sensitive Method for Determination of Protein and Cell Concentrations Based on Competitive Adsorption to Nanoparticles and Time-Resolved Luminescence Resonance Energy Transfer between Labeled Proteins

A sensitive mix-and-measure method for the determination of protein and cell concentrations was developed. It is based on the competitive adsorption between the analyte and donor- and acceptor-labeled proteins to carboxylate-modified polystyrene nanoparticles. A high time-resolved luminescence resonance energy transfer (TR-LRET) signal is detected in the absence of the analyte due to the close proximity of the nanoparticle-adsorbed labeled proteins. The increased concentration of the analyte decreases the adsorption of the labeled proteins, leading to the loss of proximity and thus a decrease in the TR-LRET. The detection limit of the assay was 2.6 ng of proteins, which is higher than that of the most sensitive commercial methods. The method was also applied to cell counting, and 200 eukaryotic cells were measured in a microtiter well under the optimized conditions. The average coefficient of variation for both developed assays was approximately 10%, and the protein-to-protein variability for 11 different proteins was no more than 20%. The developed method requires no labeled particles, making the concept optimally applicable to varying targets as the material of the particle may be selected according to the application.

High Sensitivity Luminescence Nanoparticle Assay for the Detection of Protein Aggregation

Nanoparticle assay utilizing time-resolved luminescence resonance energy transfer (TR-LRET) was developed for the detection of protein aggregation. This mix-and-measure nanoparticle assay is based on the competitive adsorption of the sample and the acceptor-labeled protein to donor europium(III) polystyrene particles. The protein aggregation was detected with the developed TR-LRET nanoparticle assay, UV240 absorbance and dynamic light scattering (DLS). All methods well equally detected the aggregation and aggregates, whose size ranged from single protein to more than 1000 nm aggregates. The developed method allowed the aggregation detection of the entire size range at more than 10,000 times lower concentration, 30 μg/L, compared to UV240 and DLS. The simple-to-use and sensitive nanoparticle assay with existing microtiter plate luminometric instrumentation can find use as a routine tool for protein aggregation studies in biochemical laboratories and for quality assessment of protein products in industry.

Time-resolved luminescence resonance energy transfer imaging of protein–protein interactions in living cells

Förster resonance energy transfer (FRET) with fluorescent proteins permits high spatial resolution imaging of protein-protein interactions in living cells. However, substantial non-FRET fluorescence background can obscure small FRET signals, making many potential interactions unobservable by conventional FRET techniques. Here we demonstrate time-resolved microscopy of luminescence resonance energy transfer (LRET) for live-cell imaging of protein-protein interactions. A luminescent terbium complex, TMP-Lumi4, was introduced into cultured cells using two methods: (i) osmotic lysis of pinocytic vesicles; and (ii) reversible membrane permeabilization with streptolysin O. Upon intracellular delivery, the complex was observed to bind specifically and stably to transgenically expressed Escherichia coli dihydrofolate reductase (eDHFR) fusion proteins. LRET between the eDHFR-bound terbium complex and green fluorescent protein (GFP) was detected as long-lifetime, sensitized GFP emission. Background signals from cellular autofluorescence and directly excited GFP fluorescence were effectively eliminated by imposing a time delay (10 micros) between excitation and detection. Background elimination made it possible to detect interactions between the first PDZ domain of ZO-1 (fused to eDHFR) and the C-terminal YV motif of claudin-1 (fused to GFP) in single microscope images at subsecond time scales. We observed a highly significant (P<10(-6)), six-fold difference between the mean, donor-normalized LRET signal from cells expressing interacting fusion proteins and from control cells expressing noninteracting mutants. The results show that time-resolved LRET microscopy with a selectively targeted, luminescent terbium protein label affords improved speed and sensitivity over conventional FRET methods for a variety of live-cell imaging and screening applications.

Liposome-based homogeneous luminescence resonance energy transfer

There is an increasing need for developing simple assay formats for biomedical screening purposes. Assays on cell membranes have become important in studies of receptor–ligand interactions and signal pathways. Here luminescence energy transfer was studied on liposomes containing europium ion chelated to 4,4,4-trifluoro-1-(2-naphthalenyl)-1,3-butanedione and trioctylphosphine oxide. Energy transfer efficiency was characterized with biotin–streptavidin interaction, and a model assay concept for a homogeneous time-resolved luminescence resonance energy transfer (LRET) assay was developed. Acceptor-labeled streptavidin was bound to biotinylated lipids on the liposomes, leading to close proximity of the LRET pair. The liposome-based LRET assay was optimized for dye incorporation and concentration, biotinylation degree, liposome size, and kinetics. Sensitivity for a competitive biotin assay was at a picomolar range with a coefficient of variation from 7 to 20%. The developed lipid membrane-based system was feasible in separation free LRET assay concept with high sensitivity, indicating that the assay principle can potentially be used for biologically more relevant target molecules.