In this work, we utilized a unique polyhistidine peptide-DNA to conjugate with DHLA-capped QD625 (QD625) and different lengths of ssDNAs which were complementary to different parts of polyhistidine peptide-DNA to conjugate with Tb, and therefore, Tb are located away from the surface of QD with the length of polyhistidine peptide in addition of the length of ssDNA of 0, 2, 4, 6, 10 and 14 bases, or the lengths of dsDNA of 10, 14, 18, 22 and 26 base pairs, respectively. The lifetime of QD became longer and longer as Tb was moving away from QD. The distances calculated from Tb and QD channels by fitting were in an excellent agreement with the model that demonstrated temporal multiplexing FRET using a single Tb-QD FRET pair is successfully developed and can be used as biosensor.
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.
Nanobioconjugates have been synthesized using cadmium selenide quantum dots (QDs), europium complexes (EuCs), and biotin. In those conjugates, long-lived photoluminescence (PL) is provided by the europium complexes, which efficiently transfer energy via Förster resonance energy transfer (FRET) to the QDs in close spatial proximity. As a result, the conjugates have a PL emission spectrum characteristic for QDs combined with the long PL decay time characteristic for EuCs. The nanobioconjugates synthesis strategy and photophysical properties are described as well as their performance in a time-resolved streptavidin-biotin PL assay. In order to prepare the QD-EuC-biotin conjugates, first an amphiphilic polymer has been functionalized with the EuC and biotin. Then, the polymer has been brought onto the surface of the QDs (either QD655 or QD705) to provide functionality and to make the QDs water dispersible. Due to a short distance between EuC and QD, an efficient FRET can be observed. Additionally, the QD-EuC-biotin conjugates’ functionality has been demonstrated in a PL assay yielding good signal discrimination, both from autofluorescence and directly excited QDs. These newly designed QD-EuC-biotin conjugates expand the class of highly sensitive tools for bioanalytical optical detection methods for diagnostic and imaging applications.
Luminescent semiconductor nanocrystals or quantum dots (QDs) contain favorable photonic properties (e.g., resistance to photobleaching, size-tunable PL, and large effective Stokes shifts) that make them well-suited for fluorescence (Förster) resonance energy transfer (FRET) based applications including monitoring proteolytic activity, elucidating the effects of nanoparticles-mediated drug delivery, and analyzing the spatial and temporal dynamics of cellular biochemical processes. Herein, we demonstrate how unique considerations of temporal and spatial constraints can be used in conjunction with QD-FRET systems to open up new avenues of scientific discovery in information processing and molecular logic circuitry. For example, by conjugating both long lifetime luminescent terbium(III) complexes (Tb) and fluorescent dyes (A647) to a single QD, we can create multiple FRET lanes that change temporally as the QD acts as both an acceptor and donor at distinct time intervals. Such temporal FRET modulation creates multi-step FRET cascades that produce a wealth of unique photoluminescence (PL) spectra that are well-suited for the construction of a photonic alphabet and photonic logic circuits. These research advances in bio-based molecular logic open the door to future applications including multiplexed biosensing and drug delivery for disease diagnostics and treatment.
Semiconductor quantum dots (QDs) are highly interesting fluorophores for a large variety of spectroscopic applications.
Although their fluorescence properties are well investigated, accurate size determination of QDs is still a problem. TEM
techniques can image the inorganic core/shell system of QDs, but size determination of polymer coated QDs is difficult.
SEC (size exclusion chromatography) compares the QD size only with standard polymers and their sizes, and is therefore
not easy to use on nanoparticles. As QDs are fluorescent, single molecule spectroscopy methods such as fluorescence
correlation spectroscopy (FCS) can be used to determine QDs diffusion coefficients and hence their hydrodynamic radii.
Moreover, this method for size determination requires only very low QD concentrations, which is a mayor advantage
compared to other techniques such as dynamic light scattering.
Within our contribution we present the size determination of commercially available and self-modified QDs with FCS.
The commercial QDs (QD525, QD565, QD605, QD655 and QD705 - purchased from Invitrogen Inc.) have a rather
thick polymer shell and are functionalized with streptavidin, biotin or carboxylic groups. The self-modified QDs consist
of the same commercial core/shell QDs and are modified with a polymer shell and several bio-functionalization groups.
For all nanoparticles the diffusion coefficients were measured by FCS and the hydrodynamic radii were calculated according
to the Stokes-Einstein equation. The obtained results are in good agreement with the size information provided
by Invitrogen Inc., which demonstrates that FCS is an important technique for QD size determination at very low concentrations.
Semiconductor nanocrystals (quantum dots - QDs) possess unique photophysical properties that make them highly interesting
for many biochemical applications. Besides their common use as fluorophores in conventional spectroscopy and
microscopy, QDs are well-suited for studying Förster resonance energy transfer (FRET). Size-dependent broadband
absorption and narrow emission bands offer several advantages for the use of QDs both as FRET donors and acceptors.
QD-based FRET pairs can be efficiently used as biological and chemical sensors for highly sensitive multiplexed detection.
In this contribution we present the use of several commercially available QDs (Qdot® Nanocrystals - Invitrogen) as
FRET donors in combination with commercial organic dyes as FRET acceptors. In order to investigate the FRET process
within our donor-acceptor pairs, we used biotinylated QDs and streptavidin-labeled dyes. The well-known biotinstreptavidin
molecular recognition enables effective FRET from QDs to dye molecules and provides defined distances
between donor and acceptor. Steady-state and time-resolved fluorescence measurements were performed in order to
investigate QD-to-dye FRET. Despite a thick polymer shell around the QDs, our results demonstrate the potential of
these QDs as efficient donors both for steady-state and time-resolved FRET applications in nano-biotechnology.
Holographic surface relief gratings written in azobenzene containing films were studied for the use as masters for
polymeric thin film distributed feedback (DFB) lasers. Light induced mass transport driven by E-Z isomerization in
azobenzene containing materials have shown to be attractive for all optical and one-step fabrication of periodic surface
structures with varying parameters for different optical applications. Based on new azobenzene materials and their
holographic processing deep surface relief gratings were generated with grating pitches in the range of 400 nm as
resonant structures for second order DFB lasers emitting in the VIS range. Nanoimprint techniques enabled multiple
duplications of azobenzene master gratings in UV adhesives. The replicas were coated via spin casting with thin films of
red light emitting polymer layers to form DFB thin film lasers. These active layers are guest-host-systems consisting of
an UV-light absorbing conjugated polymer as host transferring its excitation via Förster resonant energy transfer to a red
emitting conjugated polymer. Simple adjustment of grating depth via controlling of illumination time allowed it to
investigate the influence of the corrugation depth and thereby the coupling of laser light and grating on the lasing
behavior of second order DFB lasers in the red region. For this purpose multiple surface structures with different
corrugation depths of up to 130 nm were generated holographically, duplicated and coated.
An innovative approach for voltage-tunable optical gratings based on dielectric elastomer actuators (DEAs) using electro active polymers is presented. Sinusoidal surface gratings, holographically written into azobenzene containing films, are transferred via nanoimprinting to DEAs of different carrier materials. We demonstrate that the surface relief deformation depends on the mechanical and geometrical properties of the actuators. The tested DEAs were made using commercially available elastomers, including a tri-block copolymer poly-styrene-ethylene-butadiene-styrene (SEBS), a silicone polydimethylsiloxane rubber (PDMS) and commonly used polyacrylic glue. The polyacrylic glue is ready to use, whereas the SEBS and the PDMS precursors have to be processed into thin films via different casting methods. The DEA material was pre-stretched, fixed to a stiff frame and coated with stretchable electrodes in appropriate designs. Since the actuation strain of the DEA depends strongly upon the conditions such as material properties, pre-stretch and geometry, the desired voltage-controllable deformations can be optimized during manufacturing of the DEA and also in the choice of materials in the grating transfer process. A full characterization of the grating deformation includes measurements of the grating pitch and depth modulation, plus the change of the diffraction angle and efficiency. The structural surface distortion was characterized by measuring the shape of the transmitted and diffracted laser beam with a beam profiling system while applying an electro-mechanical stress to the grating. Such surface distortions may lead to decreasing diffraction efficiency and lower beam quality. With properly chosen manufacturing parameters, we found a period shift of up to 9 % in a grating with 1 μm pitch. To describe the optical behavior, a model based on independently measured material parameters is presented.
An optical multiplexed homogeneous (liquid phase) immunoassay based on FRET from a terbium complex to eight
different fluorescent dyes is presented. We achieved highly sensitive parallel detection of four different lung cancer
specific tumor markers (CEA, NSE, SCC and CYFRA21-1) within a single assay and show a proof-of-principle for 5-
fold multiplexing. The method is well suited for fast and low-cost miniaturized point-of-care testing as well as for highthroughput
screening in a broad range of in-vitro diagnostic applications.
KEYWORDS: Fluorescence resonance energy transfer, Quantum dots, Luminescence, Terbium, Technetium, Multiplexing, Energy transfer, Semiconductors, Ions, Resonance energy transfer
The efficient use of luminescent semiconductor quantum dots (QDs) as Förster Resonance Energy Transfer
(FRET) acceptors can be accomplished with terbium complexes (TCs) as donors. TCs exhibit long excited state
lifetimes (in the millisecond range) up to 105 times longer than typical QD lifetimes. When FRET occurs from TCs
to QDs the measured TC luminescence decay times decrease (FRET quenching), whereas the QD decay times
increase (FRET sensitization). Due to the large difference between the TC and QD excited state lifetimes the FRET
formalism can be applied to both the TC donors as well as the QD acceptors. This is a big advantage because the
FRET information from one experiment can be received from both sides of the FRET pair allowing for the use of
different detection channels and wavelengths for donor and acceptor. Thus, a multiplexing format becomes possible
with one single donor (TC) and several different acceptors (different QDs). In this contribution we show the
theoretical background for simultaneously applying FRET to donor and acceptor and give an example with a
commercially available TC-QD donor-acceptor pair.
We present the use of luminescent terbium complexes (LTCs) as FRET donors and luminescent semiconductor quantum
dots (QDs) as FRET acceptors for spectroscopic ruler measurements. The LTCs were labeled to polyhistidine-appended
peptides which self-assembled onto three different QDs allowing optical multiplexed measurements. Forster distances
were in the range of 60-75 Å and FRET efficiencies of up to 97 % were realized. Time-resolved analysis allowed the
determination of donor-acceptor separation distances. The results suggests the efficient use of our LTC-to-QD FRET
systems for multiplexed optical size determination, molecular ruler measurements and multi-parameter diagnostics.
In this contribution we present the application of five different commercially available semiconductor core/shell quantum
dots (Qdot® Nanocrystals - Invitrogen Corp.) as multiplexing FRET acceptors together with a commercial
supramolecular terbium complex (Lumi4®-Tb - Lumiphore Inc.) as donor in a homogeneous immunoassay format. To
realize the molecular recognition necessary for a FRET assay, the terbium complex was labeled to streptavidin (sAv-
Lumi4-Tb) and the QDs were surface functionalized with biotin (Biot-QD). The biotin-streptavidin binding serves as a
proof-of-principle representative for many biological interactions taking place on the nanometer scale (e.g.
immunoassays). The presented FRET system can be efficiently used for the detection of inter- and intramolecular
processes for clinical diagnostics and biomedical spectroscopy as well as molecular ruler applications and microscopy.
Homogeneous immunoassays have the benefit that they do not require any time-consuming separation steps. FRET is
one of the most sensitive homogeneous methods used for immunoassays. Due to their extremely strong absorption over a
broad wavelength range the use of quantum dots as FRET acceptors allows for large Foerster radii, an important
advantage for assays in the 5 to 10 nm distance range. Moreover, because of their size-tunable emission, quantum dots of
different sizes can be used with a single donor for the detection of different analytes (multiplexing). As the use of
organic dyes with short fluorescence decay times as donors is known to be inefficient with quantum dot acceptors,
lanthanide complexes with long luminescence decays are very efficient alternatives.
In this contribution we present the application of commercially available biocompatible CdSe/ZnS core/shell quantum
dots as multiplexing FRET acceptors together with a single terbium complex as donor in a homogeneous immunoassay
system. Foerster radii of 10 nm and FRET efficiencies of 75 % are demonstrated. The high sensitivity of the terbium-toquantum
dot FRET assay is shown by sub-100-femtomolar detection limits for two different quantum dots (emitting at
605 and 655 nm) within the same biotin-streptavidin assay. Direct comparison to the FRET immunoassay "gold standard" (FRET from Eu-TBP to APC) yields a three orders of magnitude sensitivity improvement, demonstrating the
big advantages of quantum dots not only for multiplexing but also for highly sensitive nanoscale analysis.
Homogeneous FRET (Forster Resonance Energy Transfer) fluoroimmunoassays (FIA) allow fast, inexpensive
and highly sensitive monitoring of biochemical processes occurring on the nanometer scale. The technique
is widely applied in high throughput screening (HTS) for in vitro diagnostics (IVD). Quantum dots (qdots) are usually applied as energy donors for FRET experiments in solution. In this contribution we show that commercially available biotinylated CdSe/ZnS core/shell qdots (Qdot 665 Biotin conjugate, Invitrogen Corp., USA) are excellent FRET acceptors in a time-resolved FIA based on interaction with lanthanide complex-labeled streptavidin as energy donor. The energy transfer experiments were performed on a modified commercial FIA reader system (KRYPTOR, Cezanne SA, France) using three di.erent lanthanide chelates (1 of terbium and 2 of europium). All three FRET donors showed efficient energy transfer to the qdots, evidenced both by nanocrystals emission sensitization and by a thousand fold increase of the qdot luminescence decay time, reaching some
hundreds of microseconds. In a control experiment the unlabeled donors were used to rule out dynamic energy
transfer between lanthanides and qdots. Due to the very high qdot absorption extremely large Forster radii of
104 A for terbium and 96 A for europium were achieved. FRET efficiency was up to 67 % and sub-picomolar detection limits were obtained for qdots in this type of homogeneous FIA. The use of qdots as energy acceptors potentially offers a broad scope of scientific and commercial applications such as ultra-sensitive FIA, the study of interactions within very large molecules, biomedical HTS and multiplexed analysis.
We demonstrate the improvement of fluorescence immunoassay (FIA) diagnostics in deploying a newly developed compact diode-pumped solid state (DPSS) laser with emission at 315 nm. The laser is based on the quasi-three-level transition in Nd:YAG at 946 nm. The pulsed operation is either realized by an active Q-switch using an electro-optical device or by introduction of a Cr4+:YAG saturable absorber as passive Q-switch element. By extra-cavity second harmonic generation in different nonlinear crystal media we obtained blue light at 473 nm. Subsequent mixing of the fundamental and the second harmonic in a β-barium-borate crystal provided pulsed emission at 315 nm with up to 20 μJ maximum pulse energy and 17 ns pulse duration. Substitution of a nitrogen laser in a FIA diagnostics system by the DPSS laser succeeded in considerable improvement of the detection limit. Despite significantly lower pulse energies (7 μJ DPSS laser versus 150 μJ nitrogen laser), in preliminary investigations the limit of detection was reduced by a factor of three for a typical FIA.
KEYWORDS: Fluorescence resonance energy transfer, Luminescence, Quantum dots, Terbium, Lanthanides, Quantum efficiency, Energy transfer, Ions, Absorption, Resonance energy transfer
Due to their extraordinary photophysical properties CdSe/ZnS core/shell nanocrystals (quantum dots) are excellent luminescence dyes for fluorescence resonance energy transfer (FRET) systems. By using a supramolecular lanthanide complex with central terbium cation as energy donor, we show that commercially available biocompatible biotinilated quantum dots are excellent energy acceptors in a time-resolved FRET fluoroimmunoassay (FRET-FIA) using streptavidin-biotin binding as biological recognition process. The efficient energy transfer is demonstrated by quantum dot emission sensitization and a thousandfold increase of the nanocrystal luminescence decay time. A Foerster Radius of 90 Å and a picomolar detection limit were achieved in quantum dot borate buffer. Regarding biological applications the influence of bovine serum albumin (BSA) and sodium azide (a frequently used preservative) to the luminescence behaviour of our FRET-system is reported.
KEYWORDS: Fluorescence resonance energy transfer, Luminescence, Europium, Energy transfer, Quantum dots, Molecules, Absorption, Resonance energy transfer, Optical properties, Energy efficiency
Quantum dots (QDs) are common as luminescing markers for imaging in biological applications because their optical properties seem to be inert against their surrounding solvent. This, together with broad and strong absorption bands and intense, sharp tuneable luminescence bands, makes them interesting candidates for methods utilizing Forster Resonance Energy Transfer (FRET), e. g. for sensitive homogeneous fluoroimmunoassays (FIA). In this work we demonstrate energy transfer from Eu3+-trisbipyridin (Eu-TBP) donors to CdSe-ZnS-QD acceptors in solutions with and without serum. The QDs are commercially available CdSe-ZnS core-shell particles emitting at 655 nm (QD655). The FRET system was achieved by the binding of the streptavidin conjugated donors with the biotin conjugated acceptors. After excitation of Eu-TBP and as result of the energy transfer, the luminescence of the QD655 acceptors also showed lengthened decay times like the donors. The energy transfer efficiency, as calculated from the decay times of the bound and the unbound components, amounted to 37%. The Forster-radius, estimated from the absorption and emission bands, was ca. 77Å. The effective binding ratio, which not only depends on the ratio of binding pairs but also on unspecific binding, was obtained from the donor emission dependent on the concentration. As serum promotes unspecific binding, the overall FRET efficiency of the assay was reduced. We conclude that QDs are good substitutes for acceptors in FRET if combined with slow decay donors like Europium. The investigation of the influence of the serum provides guidance towards improving binding properties of QD assays.
Two examples of our biophotonic research utilizing nanoparticles are presented, namely laser-based fluoroimmuno analysis and in-vivo optical oxygen monitoring. Results of the work include significantly enhanced sensitivity of a homogeneous fluorescence immunoassay and markedly improved spatial resolution of oxygen gradients in root nodules of a legume species.
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