The quantum dot vs. organic dye conundrum for ratiometric FRET-based biosensors: which one would you chose?

Förster resonance energy transfer (FRET) is a widely used and ideal transduction modality for fluorescent based biosensors as it offers high signal to noise with a visibly detectable signal. While intense efforts are ongoing to improve the limit of detection and dynamic range of biosensors based on biomolecule optimization, the selection of and relative location of the dye remains understudied. Herein, we describe a combined experimental and computational study to systematically compare the nature of the dye, i.e., organic fluorophore (Cy5 or Texas Red) vs. inorganic nanoparticle (QD), and the position of the FRET donor or acceptor on the biomolecular components. Using a recently discovered transcription factor (TF)-deoxyribonucleic acid (DNA) biosensor for progesterone, we examine four different biosensor configurations and report the quantum yield, lifetime, FRET efficiency, IC50, and limit of detection. Fitting the computational models to the empirical data identifies key molecular parameters driving sensor performance in each biosensor configuration. Finally, we provide a set of design parameters to enable one to select the fluorophore system for future intermolecular biosensors using FRET-based conformational regulation in in vitro assays and new diagnostic devices.

Electric field induced macroscopic cellular phase of nanoparticles.

A suspension of nanoparticles with very low volume fraction is found to assemble into a macroscopic cellular phase that is composed of particle-rich walls and particle-free voids under the collective influence of AC and DC voltages. Systematic study of this phase transition shows that it was the result of electrophoretic assembly into a two-dimensional configuration followed by spinodal decomposition into particle-rich walls and particle-poor cells mediated principally by electrohydrodynamic flow. This mechanistic understanding reveals two characteristics needed for a cellular phase to form, namely (1) a system that is considered two dimensional and (2) short-range attractive, long-range repulsive interparticle interactions. In addition to determining the mechanism underpinning the formation of the cellular phase, this work presents a method to reversibly assemble microscale continuous structures out of nanoscale particles in a manner that may enable the creation of materials that impact diverse fields including energy storage and filtration.

Engineering Brightness Matched Indium Phosphide Quantum Dots.

The size-dependent optoelectronic properties of semiconductor nanocrystals quantum dots (QDs) are hugely beneficial for color tunability but induce an inherent relative PL brightness mismatch in QDs emitting different colors, as larger emitters absorb more incident photons than smaller particles. Here, we examine the effect of core composition, shell composition, and shell thickness on optical properties including high energy absorption, quantum yield (QY), and the relative brightness of InP/ZnS and InP/ZnSe core/shell and InP/ZnSe/ZnS core/shell/shell QDs at different excitation wavelengths. Our analysis reveals that the presence of an intermediate ZnSe shell changes the wavelength of enhanced absorption onset and leads to highly excitation wavelength dependent QYs. Switching from commercial CdSe/ZnS to InP/ZnS reduces the brightness-mismatch between green and red emitters from 33- to 5-fold. Incorporating a 4-monolayer thick optically absorbing ZnSe shell into the QD heterostructure and heating the QDs in a solution of zinc oleate and trioctylphosphine produces InP/ZnSe/ZnS QDs that are ~10-fold brighter than their InP/ZnS counterparts. In contrast to CdSe/CdS/ZnS core/shell/shell QDs, which only photoluminesce at red wavelengths with thicker CdS shells due to their Quasi-Type II bandstructure, Type I InP/ZnSe/ZnS QDs are uniquely suited to creating a rainbow of visible-emitting, brightness matched emitters. By tailoring the thickness of the intermediate ZnSe shell, heavy metal-free, brightness-matched green and red emitters are produced. This study highlights the ability to overcome the inherent brightness mismatch seen in QDs through concerted materials design of heterostructured core/shell InP-based QDs.

Extending the Near-Infrared Emission Range of Indium Phosphide Quantum Dots for Multiplexed In Vivo Imaging.

This report of the reddest emitting indium phosphide quantum dots (InP QDs) to date demonstrates tunable, near-infrared (NIR) photoluminescence (PL) as well as PL multiplexing in the first optical tissue window while avoiding toxic constituents. This synthesis overcomes the InP "growth bottleneck" and extends the emission peak of InP QDs deeper into the first optical tissue window using an inverted QD heterostructure, specifically ZnSe/InP/ZnS core/shell/shell nanoparticles. The QDs exhibit InP shell thickness-dependent tunable emission with peaks ranging from 515-845 nm. The high absorptivity of InP yields effective photoexcitation of the QDs with UV, visible, and NIR wavelengths. These nanoparticles extend the range of tunable direct-bandgap emission from InP-based nanostructures, effectively overcoming a synthetic barrier that has prevented InP-based QDs from reaching their full potential as NIR imaging agents. Multiplexed lymph node imaging in a mouse model demonstrates the potential of the NIR-emitting InP particles for in vivo imaging.

Transcription Factor Based Small-Molecule Sensing with a Rapid Cell Phone Enabled Fluorescent Bead Assay.

Recently, allosteric transcription factors (TFs) were identified as a novel class of biorecognition elements for in vitro sensing, whereby an indicator of the differential binding affinity between a TF and its cognate DNA exhibits dose-dependent responsivity to an analyte. Described is a modular bead-based biosensor design that can be applied to such TF-DNA-analyte systems. DNA-functionalized beads enable efficient mixing and spatial separation, while TF-labeled semiconductor quantum dots serve as bright fluorescent indicators of the TF-DNA bound (on bead) and unbound states. The prototype sensor for derivatives of the antibiotic tetracycline exhibits nanomolar sensitivity with visual detection of bead fluorescence. Facile changes to the sensor enable sensor response tuning without necessitating changes to the biomolecular affinities. Assay components self-assemble, and readout by eye or digital camera is possible within 5 minutes of analyte addition, making sensor use facile, rapid, and instrument-free.

Hydrogel-Embedded Quantum Dot-Transcription Factor Sensors for Quantitative Progesterone Detection.

Immobilization of biosensors in or on a functional material is critical for subsequent device development and translation to wearable technology. Here, we present the development and assessment of an immobilized quantum dot-transcription factor-nucleic acid complex for progesterone detection as a first step toward such device integration. The sensor, composed of a polyhistidine-tagged transcription factor linked to a quantum dot and a fluorophore-modified cognate DNA, is embedded within a hydrogel as an immobilization matrix. The hydrogel is optically transparent, soft, and flexible as well as traps the quantum dot-transcription factor DNA assembly but allows free passage of the analyte, progesterone. Upon progesterone exposure, DNA dissociates from the quantum dot-transcription factor DNA assembly resulting in an attenuated ratiometric fluorescence output via Förster resonance energy transfer. The sensor performs in a dose-dependent manner with a limit of detection of 55 nM. Repeated analyte measurements are similarly successful. Our approach combines a systematically characterized hydrogel as an immobilization matrix and a transcription factor-DNA assembly as a recognition/transduction element, offering a promising framework for future biosensor devices.

Surface Immobilized Nucleic Acid-Transcription Factor Quantum Dots for Biosensing.

Immobilization of biosensors on surfaces is a key step toward development of devices for real-world applications. Here the preparation, characterization, and evaluation of a surface-bound transcription factor-nucleic acid complex for analyte detection as an alternative to conventional systems employing aptamers or antibodies are described. The sensor consists of a gold surface modified with thiolated Cy5 fluorophore-labeled DNA and an allosteric transcription factor (TetR) linked to a quantum dot (QD). Upon addition of anhydrotetracycline (aTc)-the analyte-the TetR-QDs release from the surface-bound DNA, resulting in loss of the Förster resonance energy transfer signal. The sensor responds in a dose-dependent manner over the relevant range of 0-200 µm aTc with a limit of detection of 80 nm. The fabrication of the sensor and the subsequent real-time quantitative measurements establish a framework for the design of future surface-bound, affinity-based biosensors using allosteric transcription factors for molecular recognition.

Quantum dot to quantum dot Förster resonance energy transfer: engineering materials for visual color change sensing.

In this work, quantum dots (QDs) of various heterostructured compositions and shell thicknesses are used as Förster or fluorescence resonance energy transfer (FRET) donors and acceptors to optimize QD-QD FRET sensing through materials design. While several reports have highlighted the advantages of using QD-dye, rather than dye-dye, FRET in sensing applications, QD-QD FRET has lagged behind in development as a result of high background signal from direct acceptor excitation. However, in designing sensors for longitudinal studies, QD-dye sensors are limited by the photostability of the fluorescent dye. While fluorescence generally affords higher sensitivity than absorbance-based readouts, the instrumentation needed for detecting fluorescence can be expensive, motivating the development of sensors bright enough to be seen by eye or imaged with cheap consumer electronics. Harnessing the exceptional brightness of QDs, our study focuses on the development of QD-QD FRET pairs where color change is achieved for visual readout and instrument-free sensing. We demonstrate that bulk semiconductor material characteristics can be used to a priori predict and tailor the behavior of QD-QD FRET systems, and our findings show that it is possible to create QD donors that are brighter than their acceptors through concerted compositional and morphological choices in heterostructured QDs. This is significant for developing visual sensors, as we show that the most profound color change occurs when the direct acceptor emission is lower than that of the donor. Finally, the use of an optimal cadmium-free QD-QD FRET pair is presented in a pH sensor that shows a large range of pH-dependent color change with bright, instrument-free readout.

Phase Transfer and DNA Functionalization of Quantum Dots Using an Easy-to-Prepare, Low-Cost Zwitterionic Polymer.

Small, stable, and bright quantum dots (QDs) are of interest in many biosensing and biomedical imaging applications, but current methodologies for obtaining these characteristics can be highly specialized or expensive. We describe a straightforward, low-cost protocol for functionalizing poly(isobutylene-alt-maleic anhydride) (PIMA) with moieties that anchor to the QD surface (histamine), impart hydrophilicity [(2-aminoethyl)trimethylammonium chloride (Me3N+-NH2)], and provide a platform for biofunctionalization via click chemistry (dibenzocyclooctyne (DBCO)). Guidelines to successfully use this polymer for QD ligand exchange are presented, and an example of biofunctionalization with DNA is shown. Stable QD-DNA conjugates are obtained with high yield and without requiring additional purification steps.

A Förster Resonance Energy Transfer-Based Ratiometric Sensor with the Allosteric Transcription Factor TetR.

A recent description of an antibody-free assay is significantly extended for small molecule analytes using allosteric transcription factors (aTFs) and Förster resonance energy transfer (FRET). The FRET signal indicates the differential binding of an aTF-DNA pair with a dose-dependent response to its effector molecule, i.e., the analyte. The new sensors described here, based on the well-characterized aTF TetR, demonstrate several new features of the assay approach: 1) the generalizability of the sensors to additional aTF-DNA-analyte systems, 2) sensitivity modulation through the choice of donor fluorophore (quantum dots or fluorescent proteins, FPs), and 3) sensor tuning using aTF variants with differing aTF-DNA binding affinities. While all of these modular sensors self-assemble, the design reported here based on a recombinant aTF-FP chimera with commercially available dye-labeled DNA uses readily accessible sensor components to facilitate easy adoption of the sensing approach by the broader community.

A progesterone biosensor derived from microbial screening.

Bacteria are an enormous and largely untapped reservoir of biosensing proteins. We describe an approach to identify and isolate bacterial allosteric transcription factors (aTFs) that recognize a target analyte and to develop these TFs into biosensor devices. Our approach utilizes a combination of genomic screens and functional assays to identify and isolate biosensing TFs, and a quantum-dot Förster Resonance Energy Transfer (FRET) strategy for transducing analyte recognition into real-time quantitative measurements. We use this approach to identify a progesterone-sensing bacterial aTF and to develop this TF into an optical sensor for progesterone. The sensor detects progesterone in artificial urine with sufficient sensitivity and specificity for clinical use, while being compatible with an inexpensive and portable electronic reader for point-of-care applications. Our results provide proof-of-concept for a paradigm of microbially-derived biosensors adaptable to inexpensive, real-time sensor devices.

Ligands and media impact interactions between engineered nanomaterials and clay minerals.

The exponential growth in technologies incorporating engineered nanomaterials (ENMs) requires plans to handle waste ENM disposal and accidental environmental release throughout the material life cycle. These scenarios motivate efforts to quantify and model ENM interactions with diverse background particles and solubilized chemical species in a variety of environmental systems. In this study, quantum dot (QD) nanoparticles and clay minerals were mixed in a range of water chemistries in order to develop simple assays to predict aggregation trends. CdSe QDs were used as a model ENM functionalized with either negatively charged or zwitterionic small molecule ligand coatings, while clays were chosen as an environmentally relevant sorbent given their potential as an economical water treatment technology and ubiquitous presence in nature. In our unbuffered experimental systems, clay type impacted pH, which resulted in a change in zwitterionic ligand speciation that favored aggregation with kaolinite more than with montmorillonite. With kaolinite, the zwitterionic ligand-coated QD exhibited greater than ten times the relative attachment efficiency for QD-clay heteroaggregation compared to the negatively charged ligand coated QD. Under some conditions, particle oxidative dissolution and dynamic sorption of ions and QDs to surfaces complicated the interpretation of the removal kinetics. This work demonstrates that QDs stabilized by small molecule ligands and electrostatic surface charges are highly sensitive to changes in water chemistry in complex media. Natural environments enable rapid dynamic physicochemical changes that will influence the fate and mobility of ENMs, as seen by the differential adsorption of water-soluble QDs to our clay media.

A versatile and accessible polymer coating for functionalizable zwitterionic quantum dots with high DNA grafting efficiency.

Efficient and versatile functionalization of poly(anhydride maleic-alt-isobutylene) (PIMA), with economical commercial reagents, results in the one-step/one-day production of a copper-free click chemistry-ready carboxybetaine-like coating for quantum dots (QDs). The QDs are bright and stable in aqueous media and easily grafted with DNA with >95% efficiency.

Measuring Nanoparticle Polarizability Using Fluorescence Microscopy.

Using a novel method developed to quantify the polarizability of photoluminescent nanoparticles in water, we present experimental observations of the extraordinary polarizability exhibited by nanoparticles of commensurate size with the Debye screening length, confirming previously reported theory. Semiconductor quantum dots (QDs) are ideal model nanoparticles to demonstrate this assay, due to their tunable size and bright photoluminescence. This assay is based upon microfluidic chambers with microelectrodes that generate trapping potentials that are weaker than thermal energy. By comparing the local electric field strength and variations in QD concentration, their polarizability was computed and found to agree with estimates based upon the hydrodynamic diameter found using light scattering. Strikingly, the polarizability of the nanoparticles increased 30-fold in low salt conditions compared to high salt conditions due to the increased thickness of the Debye layer relative to the particle radius. In addition to providing evidence that corroborates theoretical work studying direct solutions to the Poisson-Nernst-Planck equations, these observations provide an explanation for the previously observed conductivity dependence of biomolecule polarizability. As the polarizability of nanoparticles is of high importance to the electrically directed assembly of particles, as well as their interactions with other materials in complex environments, we anticipate that these results will be highly relevant to ongoing efforts in materials by design and nanomedicine.

Sensing with photoluminescent semiconductor quantum dots.

Fluorescent sensors benefit from high signal-to-noise and multiple measurement modalities, enabling a multitude of applications and flexibility of design. Semiconductor nanocrystal quantum dots (QDs) are excellent fluorophores for sensors because of their extraordinary optical properties. They have high thermal and photochemical stability compared to organic dyes or fluorescent proteins and are extremely bright due to their large molar cross-sections. In contrast to organic dyes, QD emission profiles are symmetric, with relatively narrow bandwidths. In addition, the size tunability of their emission color, which is a result of quantum confinement, make QDs exceptional emitters with high color purity from the ultra-violet to near infrared wavelength range. The role of QDs in sensors ranges from simple fluorescent tags, as used in immunoassays, to intrinsic sensors that utilize the inherent photophysical response of QDs to fluctuations in temperature, electric field, or ion concentration. In more complex configurations, QDs and biomolecular recognition moieties like antibodies are combined with a third component to modulate the optical signal via energy transfer. QDs can act as donors, acceptors, or both in energy transfer-based sensors using Förster resonance energy transfer (FRET), nanometal surface energy transfer (NSET), or charge or electron transfer. The changes in both spectral response and photoluminescent lifetimes have been successfully harnessed to produce sensitive sensors and multiplexed devices. While technical challenges related to biofunctionalization and the high cost of laboratory-grade fluorimeters have thus far prevented broad implementation of QD-based sensing in clinical or commercial settings, improvements in bioconjugation methods and detection schemes, including using simple consumer devices like cell phone cameras, are lowering the barrier to broad use of more sensitive QD-based devices.

Shell thickness effects on quantum dot brightness and energy transfer.

Heterostructured core/shell quantum dots (QDs) are prized in biomedical imaging and biosensing applications because of their bright, photostable emission and effectiveness as Förster resonance energy transfer (FRET) donors. However, as nanomaterials chemistry has progressed beyond traditional QDs to incorporate new compositions, ultra-thick shells, and alloyed structures, few of these materials have had their optical properties systematically characterized for effective application. For example, thick-shelled QDs, also known as 'giant' QDs (gQDs) are useful in single-particle tracking microscopy because of their reduced blinking, but we know only that CdSe/CdS gQDs are qualitatively brighter than thin-shelled CdSe/CdS in aqueous media. In this study, we quantify the impact of shell thickness on the nanoparticle molar extinction coefficient, quantum yield, brightness, and effectiveness as a FRET donor for CdSe/xCdS core/shell and CdSe/xCdS/ZnS core/shell/shell QDs, with variable thicknesses of the CdS shell (x). Molar extinction coefficients up to three orders of magnitude higher than conventional dyes and forty-fold greater than traditional QDs are reported. When thick CdS shells are combined with ZnS capping, quantum yields following thiol ligand exchange reach nearly 40%-5-10× higher than either the commercially available QDs or gQDs without ZnS caps treated the same way. These results clearly show that thick CdS shells and ZnS capping shells work in concert to provide the brightest possible CdSe-based QDs for bioimaging applications. We demonstrate that thicker shelled gQDs are over 50-fold brighter than their thin-shelled counterparts because of significant increases in their absorption cross-sections and higher quantum yield in aqueous milieu. Consistent with the point-dipole approximation commonly used for QD-FRET, these data show that thick shells contribute to the donor-acceptor distance, reducing FRET efficiency. Despite the reduction in FRET efficiency, even the thickest-shell gQDs exhibited energy transfer. Through this systematic study, we elucidate the tradeoffs between signal output, which is much higher for the gQDs, and FRET efficiency, which decreases with shell thickness. This study serves as a guide to nanobiotechnologists striving to use gQDs in imaging and sensing devices.