Xue Qiu

Rapid and Multiplexed MicroRNA Diagnostic Assay Using Quantum Dot-Based Förster Resonance Energy Transfer

The detection of next generation microRNA (miRNA) biomarkers has become a highly important aspect for clinical diagnostics. We use multiplexed Förster resonance energy transfer (FRET) between a luminescent Tb complex and three different semiconductor quantum dots (QDs) to sensitively detect three different miRNAs from a single 150 μL sample with ca. 1 nM (subpicomol) detection limits. The rapid and amplification-free mix-and-measure assay format is based on careful design of miRNA base pairing and stacking to selectively detect different miRNAs with very strong sequence homologies. Clinical applicability is demonstrated by sensitive multiplexed quantification of three miRNAs at low (2 to 10 nM) and varying concentrations in samples that contained up to 10% serum.

Time-gated FRET nanoassemblies for rapid and sensitive intra- and extracellular fluorescence imaging

The time-gated FRET technique is used for rapid, sensitive intra- and extracellular imaging. Time-gated Förster resonance energy transfer (FRET) using the unique material combination of long-lifetime terbium complexes (Tb) and semiconductor quantum dots (QDs) provides many advantages for highly sensitive and multiplexed biosensing. Although time-gated detection can efficiently suppress sample autofluorescence and background fluorescence from directly excited FRET acceptors, Tb-to-QD FRET has rarely been exploited for biomolecular imaging. We demonstrate Tb-to-QD time-gated FRET nanoassemblies that can be applied for intra- and extracellular imaging. Immunostaining of different epitopes of the epidermal growth factor receptor (EGFR) with Tb- and QD-conjugated antibodies and nanobodies allowed for efficient Tb-to-QD FRET on A431 cell membranes. The broad usability of Tb-to-QD FRET was further demonstrated by intracellular Tb-to-QD FRET and Tb-to-QD-to-dye FRET using microinjection as well as cell-penetrating peptide–mediated endocytosis with HeLa cells. Effective brightness enhancement by FRET from several Tb to the same QD, the use of low nanomolar concentrations, and the quick and sensitive detection void of FRET acceptor background fluorescence are important advantages for advanced intra- and extracellular imaging of biomolecular interactions.

Multiplexed Nucleic Acid Hybridization Assays Using Single‐FRET‐Pair Distance‐Tuning

Multiplexed photoluminescence (PL) detection plays an important role in chemical and biological sensing. Here, it is shown that time-gated (TG) detection of a single terbium-donor-based Förster resonance energy transfer (FRET) pair can be used to selectively quantify low nanomolar concentrations of multiple DNAs or microRNAs in a single sample. This study demonstrates the applicability of single-TG-FRET-pair multiplexing for molecular (Tb-to-dye) and nanoparticle (Tb-to-quantum-dot) biosensing. Both systems use acceptor-sensitization and donor-quenching for quantifying biomolecular recognition and modification of the donor-acceptor distance for tuning the PL decays. TG intensity detection provides extremely low background noise and a quick and simple one-step assay format. Single-TG-FRET-pair multiplexing can be combined with spectral and spatial resolution, paving the way for biosensing with unprecedented high-order multiplexing capabilities.

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.

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).

Terbium complex to quantum dot Förster resonance energy transfer for homogeneous and multiplexed microRNA assay (Conference Presentation)

The importance of microRNA (miRNA) dysregulation in the development and progression of diseases has made these short-length nucleic acids to next generation biomarkers. Tb-to-QD Förster resonance energy transfer (FRET) has several unique advantages over organic dye-based FRET systems for biomolecular sensing. Large Förster distances (6-11 nm) offer much high FRET efficiencies, exceptionally long Tb excited-state lifetimes (ms) enable time-gated detection void of autofluorecence background, and the narrow, symmetric, and tunable emission bands of QDs provide unrivaled potential for multiplexing. Here we report a rapid and homogeneous method to sensitively detect three different miRNAs (hsa-miR-20a-5p, hsa-miR-20b-5p, and hsa-miR-21-5p) from a single 150 µL sample based on multiplexed FRET between a luminescent Lumi4-Tb complex and three different QDs. The biosensing approach exploits both base pairing and stacking. Careful design and optimization of sequence lengths and orientations of the QD and Tb-DNA conjugates was performed to provide maximum selectivity and sensitivity for all three miRNA biomarkers. The assays work at room temperature and were designed for their application on a KRYPTOR diagnostic plate reader system.Only 30 min of sample incubation and 7.5 s of measurement are required to obtain ca. 1 nM (subpicomol) detection limits. We also demonstrate precise multiplexed measurements of these miRNAs at different and varying concentrations and the feasibility of adapting the technology to point-of-care testing (POCT) in buffer containing 10% serum. Our assay does not only demonstrate an important milestone for the integration of quantum dots to multiplexed clinical diagnostics but also a unique rapid miRNA detection technology that is complimentary to the rather complicated high-throughput and high-sensitivity approaches that are established today.