Multiplexed Short-wave Infrared Imaging Highlights Anatomical Structures in Mice.

While multiplexed fluorescence imaging is frequently used for in vitro microscopy, extending the technique to whole animal imaging in vivo has remained challenging due to the attenuation and scattering of visible and traditional near infrared (NIR-I) wavelengths. Fluorescence imaging using short-wave infrared (SWIR, 1000 - 1700 nm, a.k.a. NIR-II) light enables deeper tissue penetration for preclinical imaging compared to previous methods due to reduced tissue scattering and minimal background autofluorescence in this optical window. Combining NIR-I excitation wavelengths with multiple distinct SWIR emission peaks presents a tremendous opportunity to distinguish multiple fluorophores with high precision for non-invasive, multiplexed anatomical imaging in small animal models. SWIR-emitting semiconductor quantum dots (QDs) with tunable emission peaks and optical stability have emerged as powerful contrast agents, but SWIR imaging demonstrations have yet to move beyond two-color imaging schemes. In this study, we engineered a set of three high quantum yield lead sulfide/cadmium sulfide (PbS/CdS) core/shell QDs with distinct SWIR emissions ranging from 1100 - 1550 nm and utilize these for simultaneous three-color imaging in mice. We first use QDs to non-invasively track lymphatic drainage, highlighting the detailed network of lymphatic vessels with high-resolution with a widefield imaging over a 2 hr period. We then perform multiplexed imaging with all three QDs to distinctly visualize the lymphatic system and spatially overlapping vasculature network. This work establishes optimized SWIR QDs for next-generation multiplexed preclinical imaging, moving beyond the capability of previous dual-labeling techniques. The capacity to discriminate several fluorescent labels through non-invasive NIR-I excitation and SWIR detection unlocks numerous opportunities for studies of disease progression, drug biodistribution, and cell trafficking dynamics in living organisms.

Minimizing near-infrared autofluorescence in preclinical imaging with diet and wavelength selection.

Significance: Preclinical fluorescence imaging with NIR-I (700 to 900 nm) illumination and short-wave infrared or NIR-II (1000 to 1700 nm) emission increases tissue penetration depth and improves resolution through decreased scattering. Background autofluorescence decreases signal-to-background ratios (SBR) in fluorescence imaging; maximizing SBR will further improve the impact of deep tissue imaging. Aim: The impact of rodent diet, illumination wavelength, and emission range on the background fluorescence and contrast agent SBR were determined to assist with the experimental design of future imaging studies. Approach: Following illumination with 670, 760, or 808 nm, autofluorescence in the NIR-I ( < 975 nm ), NIR-II ( > 1000 nm ), and NIR-II LP ( > 1250 nm ) regions was assessed in mice fed chow or a purified diet using an IR VIVO preclinical imager (Photon, Etc.). Comparison of the SBR of liver-localized indocyanine green in the various imaging conditions indicated when gut autofluorescence was a problematic confounder. Results: Mice fed chow exhibit high levels of background autofluorescence in the gastrointestinal tract and, to a lesser extent, skin when illuminated with 670 nm light for NIR-I imaging (700 to 975 nm), interfering with the identification of fluorescently labeled tissue. Background autofluorescence was reduced by more than two orders of magnitude by any of the following changes: (1) purified diet; (2) excitation with 760 or 808 nm illumination; or (3) emission in the NIR-II (1000 to 1600 or 1250 to 1600 nm). Although the SBR was generally sufficient for feature identification except when imaging of chow-fed mice with 670 nm excitation and NIR-I emission, switching to a purified diet, using longer excitation wavelengths, or using longer emission wavelengths improved SBR significantly. Conclusions: Systematic comparison of imaging conditions and diet highlights the reduction in autofluorescence and increase in SBR enabled by intentional choices in the experimental parameters including diet, excitation wavelength, and emission wavelength range.

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.

Wavelength-Dependent Bifunctional Plasmonic Photocatalysis in Au/Chalcopyrite Hybrid Nanostructures.

Excited, or "hot" charge carrier generation and transfer driven by the decay of localized surface plasmon resonances (LSPRs) are key steps in plasmonic photocatalysis. Hybrid structures that contain both metal and semiconductor building blocks facilitate the extraction of reactive charge carriers and their utilization for photoelectrocatalysis. Additional functionality arises from hybrid structures that combine noble metal nanostructures with semiconductor components, such as chalcopyrite (CuFeS2) nanocrystals (NCs), which by themselves support quasistatic resonances. In this work, we use a hybrid membrane to integrate Au nanorods (NRs) with a longitudinal LSPR at 745 nm and CuFeS2 NCs with a resonance peak at 490 nm into water-stable nanocomposites for robust and bifunctional photocatalysis of oxygen and hydrogen evolution reactions in a wavelength-dependent manner. Excitation of NRs or NCs in the nanocomposite correlates with increased hydrogen or oxygen evolution, respectively, consistent with a light-driven electron transfer between the metal and semiconductor building blocks, the direction of which depends on the wavelength. The bifunctional photoreactivity of the nanocomposite is enhanced by Cu(I)/Cu(II)-assisted catalysis on the surface of the NCs.

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.

Effective Mass for Holes in Paramagnetic, Plasmonic Cu5FeS4 Semiconductor Nanocrystals.

The impact of a magneto-structural phase transition on the carrier effective mass in Cu5FeS4 plasmonic semiconductor nanocrystals was examined using Magnetic Circular Dichroism (MCD). Through MCD, the sample was confirmed as p-type from variable temperature studies from 1.8 - 75 K. Magnetic field dependent behavior is observed, showing an asymptotic behavior at high field with an m∗ value 5.98 m∗∕me at 10 T and 2.73 m∗∕me at 2 T. Experimentally obtained results are holistically compared to SQUID magnetization data and DFT results, highlighting a dependency on vacancy driven polaronic coupling, magnetocrystalline anisotropy, and plasmon coupling of the magnetic field all contributing to an overall decrease in the hole mean free path dependent on the magnetic field applied to Cu5FeS4.

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.

Controlled Synthesis and Exploration of CuxFeS4 Bornite Nanocrystals.

Plasmonic semiconductor nanocrystals (NCs) are a new and exciting class of materials that enable higher control of their localized surface plasmon resonance (LSPR) than metallic counterparts. Additionally, earth-abundant and non-toxic materials such as copper iron sulfides are gaining interest as alternatives to heavy metal-based semiconductor materials. Colloidal bornite (Cu5FeS4) is an interesting but underexplored example of a heavy metal-free plasmonic semiconductor. This report details the hot-injection synthesis of bornite yielding NCs ranging from 2.7 to 6.1 nm in diameter with stoichiometric control of the copper and iron content. The absorbance spectra of bornite NCs with different Cu:Fe ratios change at different rates as the particles oxidize and develop LSPR in the near-infrared region. X-ray photoelectron spectroscopy results indicate that oxidation produces sulfates rather than metal oxides as well as a decrease in the iron content within the NCs. Additionally, increasing iron content leads to decreases in carrier density and effective mass of the carrier, as determined by the Drude model. This controlled synthesis, combined with a further understanding of the relationship between the particle structure and optical properties, will enable the continued development and application of these fascinating heavy metal-free plasmonic semiconductor nanoparticles.

Correlating ZnSe Quantum Dot Absorption with Particle Size and Concentration.

The focus on heavy metal-free semiconductor nanocrystals has increased interest in ZnSe semiconductor quantum dots (QDs) over the past decade. Reliable and consistent incorporation of ZnSe cores into core/shell heterostructures or devices requires empirical fit equations correlating the lowest-energy electron transition (1S peak) to their size and molar extinction coefficients (ε). While these equations are known and heavily used for CdSe, CdTe, CdS, PbS, etc., they are not well established for ZnSe and are nonexistent for ZnSe QDs with diameters <3.5 nm. In this study, a series of ZnSe QDs with diameters ranging from 2 to 6 nm were characterized by small-angle X-ray scattering (SAXS), transmission electron microscopy (TEM), UV-vis spectroscopy, and microwave plasma atomic emission spectroscopy (MP-AES). SAXS-based size analysis enabled the practical inclusion of small particles in the evaluation, and elemental analysis with MP-AES elucidates a nonstoichiometric Zn:Se ratio consistent with zinc-terminated spherical ZnSe QDs. Using these combined results, empirical fit equations correlating QD size with its lowest-energy electron transition (i.e., 1S peak position), Zn:Se ratio, and molar extinction coefficients for 1S peak, 1S integral, and high-energy wavelengths are reported. Finally, the equations are used to track the evolution of a ZnSe core reaction. These results will enable the consistent and reliable use of ZnSe core particles in complex heterostructures and devices.

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.

Encapsulating Quantum Dots in Lipid-PEG Micelles and Subsequent Copper-Free Click Chemistry Bioconjugation.

The utility of quantum dots (QDs) for biological applications is predicated on stably dispersing the particles in aqueous media. During transfer from apolar organic solvents to water, the optical properties of the fluorescent nanoparticles must be maintained; additionally, the resulting colloid should be monodisperse and stable against aggregation. Furthermore, the hydrophilic coating should confer functional groups or conjugation handles to the QDs, as biofunctionalization is often critical to biosensing and bioimaging applications. Micelle encapsulation is an excellent technique for conferring hydrophilicity and conjugation handles to QDs. One interesting conjugation handle that can easily be added to the QDs is an azide group, which conjugates to strained alkynes via strain promoted azide-alkyne cycloaddition (SPAAC) reactions. SPAAC, or copper-free click chemistry, utilizes very mild reaction conditions, involves reactive groups that are bio-orthogonal, and is nearly quantitative. Micelle encapsulation is also very mild and preserves the optical properties of the QDs nearly perfectly. The combination of these approaches comprises a mild, effective, and straightforward approach to preparing functionalized QDs for biological applications.

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.

Shell-Free Copper Indium Sulfide Quantum Dots Induce Toxicity in Vitro and in Vivo.

Semiconductor quantum dots (QDs) are attractive fluorescent contrast agents for in vivo imaging due to their superior photophysical properties, but traditional QDs comprise toxic materials such as cadmium or lead. Copper indium sulfide (CuInS2, CIS) QDs have been posited as a nontoxic and potentially clinically translatable alternative; however, previous in vivo studies utilized particles with a passivating zinc sulfide (ZnS) shell, limiting direct evidence of the biocompatibility of the underlying CIS. For the first time, we assess the biodistribution and toxicity of unshelled CIS and partially zinc-alloyed CISZ QDs in a murine model. We show that bare CIS QDs breakdown quickly, inducing significant toxicity as seen in organ weight, blood chemistry, and histology. CISZ demonstrates significant, but lower, toxicity compared to bare CIS, while our measurements of core/shell CIS/ZnS are consistent with literature reports of general biocompatibility. In vitro cytotoxicity is dose-dependent on the amount of metal released due to particle degradation, linking degradation to toxicity. These results challenge the assumption that removing heavy metals necessarily reduces toxicity: indeed, we find comparable in vitro cytotoxicity between CIS and CdSe QDs, while CIS caused severe toxicity in vivo compared to CdSe. In addition to highlighting the complexity of nanotoxicity and the differences between the in vitro and in vivo outcomes, these unexpected results serve as a reminder of the importance of assessing the biocompatibility of core QDs absent the protective ZnS shell when making specific claims of compositional biocompatibility.

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.