Time-gated Förster Resonance Energy Transfer Research Articles

Quantitative and Multiplexed Chopper-Based Time-Gated Imaging for Bioanalysis on a Smartphone.

Smartphones are emerging platforms for point-of-care diagnostics (POCDs), where the on-board camera is, for example, used to image fluorescence. Many laboratory instruments are capable of time-gated (TG) photoluminescence (PL) measurements─an analytical method leveraged by multiple commercial assay kits. When paired with long-lived PL emitters such as luminescent lanthanide complexes (LLCs), time-gating eliminates background from sample autofluorescence and many other sources. This capability is amenable to minimally processed samples and would thus be useful for POCDs on a smartphone-based platform. Here, we report a double-chopper design for TG PL imaging using a portable, 3D-printed, smartphone-based device. The rotation speed, dimensions, and overlap of the chopper blades and gaps set the timing parameters, with delay times on the order of hundreds of microseconds to milliseconds. The device was capable of quantitative TG imaging of PL from terbium(III) and europium(III) LLCs, including rejection of short-lived PL background from serum and tissue phantoms, spectral and temporal multiplexing, a model time-gated Förster resonance energy transfer (TG-FRET) assay, and imaging of cells. As the first smartphone-based demonstrations of these important analytical capabilities, this work is an important foundation for developing POCD methods based on TG PL imaging.

A Dendrimer-Based Time-Gated Concentric FRET Configuration for Multiplexed Sensing

Förster resonance energy transfer (FRET) is widely used for the development of biological probes and sensors. In this context, the norm for multiplexed detection is deployment of multiple probes, each a discrete donor-acceptor pair. Concentric FRET (cFRET) probes enable multiplexed sensing with a single vector but, to date, have only been developed around semiconductor quantum dots, which may limit the scope of biological applications for such probes. Here, we demonstrate that dendrimers labeled with a luminescent terbium complex (Tb) are a viable and advantageous alternative platform for cFRET probes. Polyamidoamine dendrimers were functionalized with Tb, biotin, NeutrAvidin, and three types of dye-labeled oligonucleotide probes to establish a network of competitive and sequential Tb-to-dye and dye-to-dye FRET pathways. These probes were characterized physically and photophysically, and a time-gated multiplexed assay for DNA targets was demonstrated. The time-gating offered by the Tb allowed the rejection of background autofluorescence from serum. More broadly, this dendrimer-based architecture shows that cFRET is a general concept and is an important step toward a new generation of probes for biological sensing.

Rapid and Wash-Free Time-Gated FRET Histamine Assays Using Antibodies and Aptamers.

Histamine (HA) is an indicator of food freshness and quality. However, high concentrations of HA can cause food poisoning. Simple, rapid, sensitive, and specific quantification can enable efficient screening of HA in food and beverages. However, conventional assays are complicated and time-consuming, as they require multiple incubation, washing, and separation steps. Here, we demonstrate that time-gated Förster resonance energy transfer (TG-FRET) between terbium (Tb) complexes and organic dyes can be implemented in both immunosensors and aptasensors for simple HA quantification using a rapid, single-step, mix-and-measure assay format. Both biosensors could quantify HA at concentrations relevant in food poisoning with limits of detection of 0.19 μg/mL and 0.03 μg/mL, respectively. Excellent specificity was documented against the structurally similar food components tryptamine and l-histidine. Direct applicability of the TG-FRET assays was demonstrated by quantifying HA in spiked fish and wine samples with both excellent concentration recovery and agreement with conventional multistep enzyme-linked immunosorbent assays (ELISAs). Our results show that the simplicity and rapidity of TG-FRET assays do not compromise sensitivity, specificity, and reliability, and both immunosensors and aptasensors have a strong potential for their implementation in advanced food safety screening.

In Situ Rolling Circle Amplification Förster Resonance Energy Transfer (RCA-FRET) for Washing-Free Real-Time Single-Protein Imaging.

Fluorescence signal enhancement via isothermal nucleic acid amplification is an important approach for sensitive imaging of intra- or extracellular nucleic acid or protein biomarkers. Rolling circle amplification (RCA) is frequently applied for fluorescence in situ imaging but faces limitations concerning multiplexing, dynamic range, and the required multiple washing steps before imaging. Here, we show that Förster resonance energy transfer (FRET) between fluorescent dyes and between lanthanide (Ln) complexes and dyes that hybridize to β-actin-specific RCA products in HaCaT cells can afford washing-free imaging of single β-actin proteins. Proximity-dependent FRET could be monitored directly after or during (real-time monitoring) dye or Ln DNA probe incubation and could efficiently distinguish between photoluminescence from β-actin-specific RCA and DNA probes freely diffusing in solution or nonspecifically attached to cells. Moreover, time-gated FRET imaging with the Ln-dye FRET pairs efficiently suppressed sample autofluorescence and improved the signal-to-background ratio. Our results present an important proof of concept of RCA-FRET imaging with a strong potential to advance in situ RCA toward easier sample preparation, higher-order multiplexing, autofluorescence-free detection, and increased dynamic range by real-time monitoring of in situ RCA.

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.

Triplexed CEA-NSE-PSA Immunoassay Using Time-Gated Terbium-to-Quantum Dot FRET

Time-gated Förster resonance energy transfer (TG-FRET) between Tb complexes and luminescent semiconductor quantum dots (QDs) provides highly advantageous photophysical properties for multiplexed biosensing. Multiplexed Tb-to-QD FRET immunoassays possess a large potential for in vitro diagnostics, but their performance is often insufficient for their application under clinical conditions. Here, we developed a homogeneous TG-FRET immunoassay for the quantification of carcinoembryonic antigen (CEA), neuron-specific enolase (NSE), and prostate-specific antigen (PSA) from a single serum sample by multiplexed Tb-to-QD FRET. Tb–IgG antibody donor conjugates were combined with compact QD-F(ab’)2 antibody acceptor conjugates with three different QDs emitting at 605, 650, and 705 nm. Upon antibody–antigen–antibody sandwich complex formation, the QD acceptors were sensitized via FRET from Tb, and the FRET ratios of QD and Tb TG luminescence intensities increased specifically with increasing antigen concentrations. Although limits of detection (LoDs: 3.6 ng/mL CEA, 3.5 ng/mL NSE, and 0.3 ng/mL PSA) for the triplexed assay were slightly higher compared to the single-antigen assays, they were still in a clinically relevant concentration range and could be quantified in 50 µL serum samples on a B·R·A·H·M·S KRYPTOR Compact PLUS clinical immunoassay plate reader. The simultaneous quantification of CEA, NSE, and PSA at different concentrations from the same serum sample demonstrated actual multiplexing Tb-to-QD FRET immunoassays and the potential of this technology for translation into clinical diagnostics.

Quantification of Cellular Deoxyribonucleoside Triphosphates by Rolling Circle Amplification and Förster Resonance Energy Transfer.

The quantification of cellular deoxyribonucleoside triphosphate (dNTP) levels is important for studying pathologies, genome integrity, DNA repair, and the efficacy of pharmacological drug treatments. Current standard methods, such as enzymatic assays or high-performance liquid chromatography, are complicated, costly, and labor-intensive, and alternative techniques that simplify dNTP quantification would present very useful complementary approaches. Here, we present a dNTP assay based on isothermal rolling circle amplification (RCA) and rapid time-gated Förster resonance energy transfer (TG-FRET), which used a commercial clinical plate reader system. Despite the relatively simple assay format, limits of detection down to a few picomoles of and excellent specificity for each dNTP against the other dNTPs, rNTPs, and dUTP evidenced the strong performance of the assay. Direct applicability of RCA-FRET to applied nucleic acid research was demonstrated by quantifying all dNTPs in CEM-SS leukemia cells with and without hydroxyurea or auranofin treatment. Both pharmacological agents could reduce the dNTP production in a time- and dose-dependent manner. RCA-FRET provides simple, rapid, sensitive, and specific quantification of intracellular dNTPs and has the potential to become an advanced tool for both fundamental and applied dNTP research.

Discrimination of the V600E Mutation in BRAF by Rolling Circle Amplification and Förster Resonance Energy Transfer.

The quantification of very low concentrations of circulating tumor DNA (ctDNA) biomarkers from liquid biopsies has become an important requirement for clinical diagnostics and personalized medicine. In particular, the simultaneous detection of wild-type (WT) dsDNA and their cancer-related counterparts presenting single-point mutations with simple, sensitive, specific, and reproducible technologies is paramount for ctDNA assays in clinical practice. Here, we present the development and evaluation of an amplified dsDNA assay based on a combination of isothermal rolling circle amplification (RCA) and time-gated Förster resonance energy transfer (TG-FRET) between a Tb donor and two dye (Cy3.5 and Cy5.5) acceptors. The RCA-FRET assay is free of washing and separation steps and can quantify both WT and mutated (MT) (V600E) dsDNA in the BRAF gene from a single sample in the 75 fM to 4.5 pM (4.5 × 105 to 2.7 × 107 copies) concentration range. This assay includes all steps from denaturation of the dsDNA targets to the final duplexed quantification of WT and MT targets. High assay performance at different dsDNA sequence lengths and high target specificity even in the presence of a large excess of nonspecific cell-free DNA from human plasma samples demonstrated the applicability to clinical samples. The RCA-FRET single-point mutation sensor has the potential to become an important complementary technique for analyzing liquid biopsies in advanced cancer diagnostics.

Simple, Amplified, and Multiplexed Detection of MicroRNAs Using Time-Gated FRET and Hybridization Chain Reaction.

The hybridization chain reaction (HCR) is a simple and sensitive method for quantifying nucleic acids. Current approaches cannot combine a washing-free sensing format with multiplexed target quantification at low concentrations, which would be highly desirable for detection both in solution and in situ. Here, we demonstrate the implementation of time-gated Förster resonance energy transfer (TG-FRET) between terbium donors and dye acceptors into HCR for multiplexed quantification of microRNAs (miR-20a and miR-21) and their DNA analogues. HCR-TG-FRET provided washing-free nucleic acid quantification with very low limits of detection down to 240 amol (1.7 pM) of microRNA and 123 amol (0.88 pM) of DNA. Efficient distinction from very homologous microRNAs demonstrated high target specificity. Multiplexing with a single measurement, a single excitation wavelength, and a single FRET pair allowed for a simultaneous quantification of miR-20a and miR-21 at concentrations between 30 and 300 pM from the same sample. HCR-TG-FRET showed similar performance for serum-free and serum-containing samples without the use of RNase inhibitors. Our results present a significant improvement in current HCR approaches regarding simplicity, sensitivity, and multiplexing. The versatile diagnostic performance of HCR-TG-FRET even in challenging biological environments presents an important advantage for advanced nucleic acid biosensing.

Three-Dimensional FRET Multiplexing for DNA Quantification with Attomolar Detection Limits.

Photoluminescence (PL) multiplexing usually relies on spectral or temporal separation. A combination into higher-order multiplexing for biosensing is extremely challenging because the PL intensity is required for target quantification at very low concentrations and the interplay of color, lifetime, and intensity must be carefully adapted. Here, we demonstrate time-gated Förster resonance energy transfer (TG-FRET) from a long-lifetime Tb complex to Cy3.5 and Cy5.5 dyes for spectrotemporal multiplexing of four different DNA targets in the same sample by single-color excitation and two-color detection. We used rolling circle amplification (RCA) for high specificity and sensitivity and for placing Tb donors and dye acceptors at controlled distances within the amplified DNA concatemers. This precise distance tuning led to target-specific PL decays of the FRET pairs and simple, separation-free, and higher-order multiplexed quantification of DNA. The RCA-FRET DNA assay could distinguish very homologous target sequences and provided limits of detection down to 40 zeptomoles (300 aM).

Advanced microRNA-based cancer diagnostics using amplified time-gated FRET.

MicroRNAs (miRNAs) play an important role in cellular functions and in the development and progression of cancer. Precise quantification of endogenous miRNAs from different clinical patient and control samples combined with a one-to-one comparison to standard technologies is a challenging but necessary endeavor that is largely neglected by many emerging fluorescence technologies. Here, we present a simple, precise, sensitive, and specific ratiometric assay for absolute quantification of miRNAs. Isothermally amplified time-gated Förster resonance energy transfer (TG-FRET) between Tb donors and dye acceptors resulted in miRNA assays with single-nucleotide variant specificity and detection limits down to 4.2 ± 0.5 attomoles. Quantification of miR-21 from human tissues and plasma samples revealed the relevance for breast and ovarian cancer diagnostics. Analysis of miR-132 and miR-146a from acute monocytic leukemia cells (THP-1) demonstrated the broad applicability to different miRNAs and other types of clinical samples. Direct comparison to the gold standard RT-qPCR showed advantages of amplified TG-FRET concerning precision and specificity when quantifying low concentrations of miRNAs as required for diagnostic applications. Our results demonstrate that a careful implementation of rolling circle amplification and TG-FRET into one straightforward nucleic acid detection method can significantly advance the possibilities of miRNA-based cancer diagnostics and research.

Bridging Lanthanide to Quantum Dot Energy Transfer with a Short-Lifetime Organic Dye.

Semiconductor nanocrystals or quantum dots (QDs) should act as excellent Förster resonance energy transfer (FRET) acceptors due to their large absorption cross section, tunable emission, and high quantum yields. Engaging this type of FRET can be complicated due to direct excitation of the QD acceptor along with its longer excited-state lifetime. Many cases of QDs acting as energy transfer acceptors are within time-gated FRET from long-lifetime lanthanides, which allow the QDs to decay before observing FRET. Efficient QD sensitization requires the lanthanide to be in close proximity to the QD. To overcome the lifetime mismatch issues and limited transfer range, we utilized a Cy3 dye to bridge the energy transfer from an extremely long lived terbium emitter to the QD. We demonstrated that short-lifetime dyes can be used as energy transfer relays between extended lifetime components and in this way increased the distance of terbium-QD FRET to ∼14 nm.

Terbium-based time-gated Förster resonance energy transfer imaging for evaluating protein-protein interactions on cell membranes.

Fluorescence imaging of cells and subcellular compartments is an essential tool to investigate biological processes and to evaluate the development and progression of diseases. In particular, protein-protein interactions can be monitored by Förster resonance energy transfer (FRET) between two proximal fluorophores that are attached to specific recognition biomolecules such as antibodies. We investigated the membrane expression of E- and N-cadherins in three different cell lines used as model systems to study epithelial to mesenchymal transition (EMT) and a possible detection of circulating tumour cells (CTCs). EMT is a key process in cancer metastasis, during which epithelial markers (such as E-cadherin) are down-regulated in the primary tumour whereas mesenchymal markers (such as N-cadherin) are up-regulated, leading to enhanced cell motility, intravasation, and appearance of CTCs. Various FRET donor-acceptor pairs and protein recognition strategies were utilized, in which Lumi4-Tb terbium complexes (Tb) and different organic dyes were conjugated to several distinct E- and N-cadherin-specific antibodies. Pulsed excitation of Tb at low repetition rates (100 Hz) and time-gated (TG) imaging of both the Tb-donor and the dye-acceptor photoluminescence (PL) allowed efficient detection of the EMT markers as well as FRET in the case of sufficient donor-acceptor proximity. Efficient FRET was observed only between two E-cadherin-specific antibodies and further experiments indicated that these antibodies recognized the same E-cadherin molecule, suggesting a limited accessibility of cadherins when they are clustered at adherens junctions. The investigated Tb-to-dye FRET systems provided reduced photobleaching compared to the AlexaFluor 488-568 donor-acceptor pair. Our results demonstrate the applicability and advantages of Tb-based TG FRET for efficient and stable imaging of antibody-antibody interactions on different cell lines. They also reveal the limitations of interpreting colocalization on cell membranes in the case of lacking FRET signals.

Multiplexed Tracking of Protease Activity Using a Single Color of Quantum Dot Vector and a Time-Gated Förster Resonance Energy Transfer Relay

Semiconductor quantum dots (QDs) are attractive probes for optical sensing and imaging due to their unique photophysical attributes and nanoscale size. In particular, the development of assays and biosensors based on QDs and Förster resonance energy transfer (FRET) continues to be a prominent focus of research. Here, we demonstrate the application of QDs as simultaneous donors and acceptors in a time-gated FRET relay for the multiplexed detection of protease activity. In contrast to the current state-of-the-art, which uses multiple colors of QDs, multiplexing was achieved using only a single color of QD. The other constituents of the FRET relay, a luminescent terbium complex and fluorescent dye, were assembled to QDs via peptides that were selected as substrates for the model proteases trypsin and chymotrypsin. Loss of prompt FRET between the QD and dye signaled the activity of chymotrypsin; loss of time-gated FRET between the terbium and QD signaled the activity of trypsin. We applied the FRET relay in a series of quantitative, real-time kinetic assays of increasing biochemical complexity, including multiplexed sensing, measuring inhibition in a multiplexed format, and tracking the proteolytic activation of an inactive pro-protease to its active form in a coupled, multienzyme system. These capabilities were derived from a ratiometric analysis of the two FRET pathways in the relay and permitted extraction of initial reaction rates, enzyme specificity constants, and apparent inhibition constants. This work adds to the growing body of research on multifunctional nanoparticles and introduces multiplexed sensing as a novel capability for a single nanoparticle vector. Furthermore, the ability to track both enzymes within a coupled biological system using one vector represents a significant advancement for nanoparticle-based biosensing. Prospective applications in biochemical research, applied diagnostics, and drug discovery are discussed.

Quantum Dots as Simultaneous Acceptors and Donors in Time-Gated Förster Resonance Energy Transfer Relays: Characterization and Biosensing

The unique photophysical properties of semiconductor quantum dot (QD) bioconjugates offer many advantages for active sensing, imaging, and optical diagnostics. In particular, QDs have been widely adopted as either donors or acceptors in Förster resonance energy transfer (FRET)-based assays and biosensors. Here, we expand their utility by demonstrating that QDs can function in a simultaneous role as acceptors and donors within time-gated FRET relays. To achieve this configuration, the QD was used as a central nanoplatform and coassembled with peptides or oligonucleotides that were labeled with either a long lifetime luminescent terbium(III) complex (Tb) or a fluorescent dye, Alexa Fluor 647 (A647). Within the FRET relay, the QD served as a critical intermediary where (1) an excited-state Tb donor transferred energy to the ground-state QD following a suitable microsecond delay and (2) the QD subsequently transferred that energy to an A647 acceptor. A detailed photophysical analysis was undertaken for each step of the FRET relay. The assembly of increasing ratios of Tb/QD was found to linearly increase the magnitude of the FRET-sensitized time-gated QD photoluminescence intensity. Importantly, the Tb was found to sensitize the subsequent QD-A647 donor-acceptor FRET pair without significantly affecting the intrinsic energy transfer efficiency within the second step in the relay. The utility of incorporating QDs into this type of time-gated energy transfer configuration was demonstrated in prototypical bioassays for monitoring protease activity and nucleic acid hybridization; the latter included a dual target format where each orthogonal FRET step transduced a separate binding event. Potential benefits of this time-gated FRET approach include: eliminating background fluorescence, accessing two approximately independent FRET mechanisms in a single QD-bioconjugate, and multiplexed biosensing based on spectrotemporal resolution of QD-FRET without requiring multiple colors of QD.