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Antibodies and their conjugates of fluorescent labels are widely applied in life sciences research and clinical pathology. Among diverse label types, compact quantum dots (QDs) provide advantages of multispectral multiplexing, bright signals in the deep red and infrared, and low steric hindrance. However, QD-antibody conjugates have random orientation of the antigen-binding domain which may interfere with labeling and are large (20-30 nm) and heterogeneous, which limits penetration into biospecimens. Here, we develop conjugates of compact QDs and Fab' antibody fragments as primary immunolabels. Fab' fragments are conjugated site-specifically through sulfhydryl groups distal to antigen-binding domains, and the multivalent conjugates have small and homogeneous sizes (∼12 nm) near those of full-sized antibodies. Their performance as immunolabels for intracellular antigens is evaluated quantitatively by metrics of microtubule labeling density and connectivity in fixed cells and for cytological identification in fixed brain specimens, comparing results with probes based on spectrally-matched dyes. QD-Fab' conjugates outperformed QD conjugates of full-sized antibodies and could be imaged with bright signals with 1-photon and 2-photon excitation. The results demonstrate a requirement for smaller bioaffinity agents and site-specific orientation for the success of nanomaterial-based labels to enhance penetration in biospecimens and minimize nonspecific staining.

While nanoscale quantum emitters are effective tags for measuring biomolecular interactions, their utilities for applications that demand single-unit observations are limited by the requirements for large numerical aperture (NA) objectives, fluorescence intermittency, and poor photon collection efficiency resulted from omnidirectional emission. Here, we report a nearly 3000-fold signal enhancement achieved through multiplicative effects of enhanced excitation, highly directional extraction, quantum efficiency improvement, and blinking suppression through a photonic crystal (PC) surface. The approach achieves single quantum dot (QD) sensitivity with high signal-to-noise ratio, even when using a low-NA lens and an inexpensive optical setup. The blinking suppression capability of the PC improves the QDs on-time from 15% to 85% ameliorating signal intermittency. We developed an assay for cancer-associated miRNA biomarkers with single-molecule resolution, single-base mutation selectivity, and 10-attomolar detection limit. Additionally, we observed differential surface motion trajectories of QDs when their surface attachment stringency is altered by changing a single base in a cancer-specific miRNA sequence., (© 2022. The Author(s).)

Macrophages are white blood cells with diverse functions contributing to a healthy immune response as well as the pathogenesis of cancer, osteoarthritis, atherosclerosis, and obesity. Due to their pleiotropic and dynamic nature, tools for imaging and tracking these cells at scales spanning the whole body down to microns could help to understand their role in disease states. Here we report fluorescent and radioisotopic quantum dots (QDs) for multimodal imaging of macrophage cells in vivo , ex vivo , and in situ . Macrophage specificity is imparted by click-conjugation to dextran, a biocompatible polysaccharide that natively targets these cell types. The emission spectral band of the crystalline semiconductor core was tuned to the near-infrared for optical imaging deep in tissue, and probes were covalently conjugated to radioactive iodine for nuclear imaging. The performance of these probes was compared with all-organic dextran probe analogues in terms of their capacity to target macrophages in visceral adipose tissue using in vivo positron emission tomography/computed tomography (PET/CT) imaging, in vivo fluorescence imaging, ex vivo fluorescence, post-mortem isotopic analyses, and optical microscopy. All probe classes exhibited equivalent physicochemical characteristics in aqueous solution and similar in vivo targeting specificity. However, dextran-mimetic QDs provided enhanced signal-to-noise ratio for improved optical quantification, long-term photostability, and resistance to chemical fixation. In addition, the vascular circulation time for the QD-based probes was extended 9-fold compared with dextran, likely due to differences in conformational flexibility. The enhanced photophysical and photochemical properties of dextran-mimetic QDs may accelerate applications in macrophage targeting, tracking, and imaging across broad resolution scales, particularly advancing capabilities in single-cell and single-molecule imaging and quantification.

In this chapter we describe the use of fluorescent quantum dots (QDs) as labels for microbial mRNA transcripts using fluorescence in situ hybridization (FISH). Unlike organic dyes, which are the standard labels in modern FISH methods, QDs provide fluorescence signals that are much brighter and resistant to photobleaching, with an expanded spectral range for multiplexing. We describe the preparation of QDs with compact sizes necessary for accurate labeling, their application for analyzing lacZ transcripts in Escherichia coli cells using FISH, and an assessment of signal stability. We further discuss differences between methods for mammalian cells and bacteria, for which individual nucleic acids cannot be discretely counted due to the small cell size and the optical diffraction limit.

Quantum dots (QDs) are nanocrystals with bright fluorescence and long-term photostability, attributes particularly beneficial for single-molecule imaging and molecular counting in the life sciences. The size of a QD nanocrystal determines its physicochemical and photophysical properties, both of which dictate the success of imaging applications. Larger nanocrystals typically have better optical properties, with higher brightness, red-shifted emission, reduced blinking, and greater stability. However, larger nanocrystals introduce molecular-labeling biases due to steric hindrance and nonspecific binding. Here, we systematically analyze the impact of nanocrystal size on receptor labeling in live and fixed cells. We designed three (core)shell QDs with red emission (600-700 nm) and crystalline sizes of 3.2, 5.5, and 8.3 nm. After coating with the same multidentate polymer, hydrodynamic sizes were 9.2 nm (QD 9.2 ), 13.3 nm (QD 13.3 ), and 17.4 nm (QD 17.4 ), respectively. The QDs were conjugated to streptavidin and applied as probes for biotinylated neurotransmitter receptors. QD 9.2 exhibited the highest labeling specificity for receptors in the narrow synaptic cleft (∼20-30 nm) in living neurons. However, for dense receptor labeling for molecular counting in live and fixed HeLa cells, QD 13.3 yielded the highest counts. Nonspecific binding rose sharply for hydrodynamic sizes larger than 13.3 nm, with QD 17.4 exhibiting particularly diminished specificity. Our comparisons further highlight needs to continue engineering the smallest QDs to increase single-molecule intensity, suppress blinking frequency, and inhibit nonspecific labeling in fixed and permeabilized cells. These results lay a foundation for designing QD probes with further reduced sizes to achieve unbiased labeling for quantitative and single-molecule imaging.

Quantum dots (QDs) are a class of fluorescent nanocrystals in development as labels for molecular imaging in cells and tissues. Recently, coatings for quantum dots based on multidentate polymers have improved labeling performance in a range of bioanalytical applications, primarily due to reduced probe hydrodynamic size. Now, an ongoing challenge is to eliminate nonspecific binding between these small probes and cellular components that mask specifically labeled molecules. Here, we describe insights into controlling and minimizing intermolecular interactions governing nonspecific binding using multidentate polymers with tunable hydrophilic functional groups that are cationic, anionic, zwitterionic (ZW), or nonionic (oligoethylene glycol; OEG). By fixing surface-binding groups and polymer length, coated colloids have similar sizes but diverse physicochemical properties. We measure binding to globular proteins, fixed cells, and living cells and observe a substantial improvement in nonspecific binding resistance when surfaces are functionalized with a combination of ZW and OEG. The independent underlying effects of counterion adsorption and flexibility appear to synergistically resist adsorption when combined, particularly for fixed cells enriched in both charged and hydrophobic moieties. We further show that ZW-OEG QDs are stable under diverse conditions and can be self-assembled with antibodies to specifically label surface antigens on living cells and cytoplasmic proteins in fixed cells. This surface engineering strategy can be adopted across the diverse range of colloidal materials currently in use and in development for biomedical applications to optimize their molecular labeling specificity.

Materials with short-wave infrared (SWIR) emission are promising contrast agents for in vivo animal imaging, providing high-contrast and high-resolution images of blood vessels in deep tissues. However, SWIR emitters have not been developed as molecular labels for microscopy applications in the life sciences, which require optimized probes that are bright, stable, and small. Here, we design and synthesize semiconductor quantum dots (QDs) with SWIR emission based on Hg x Cd 1- x Se alloy cores red shifted to the SWIR by epitaxial deposition of thin Hg x Cd 1- x S shells with a small band gap. By tuning alloy composition alone, the emission can be shifted across the visible-to-SWIR (VIR) spectra while maintaining a small and equal size, allowing direct comparisons of molecular labeling performance across a broad range of wavelength. After coating with click-functional multidentate polymers, the VIR-QD spectral series has high quantum yield in the SWIR (14-33%), compact size (13 nm hydrodynamic diameter), and long-term stability in aqueous media during continuous excitation. We show that these properties enable diverse applications of SWIR molecular probes for fluorescence microscopy using conjugates of antibodies, growth factors, and nucleic acids. A broadly useful outcome is a 10-55-fold enhancement of the signal-to-background ratio at both the single-molecule level and the ensemble level in the SWIR relative to visible wavelengths, primarily due to drastically reduced autofluorescence. We anticipate that VIR-QDs with SWIR emission will enable ultrasensitive molecular imaging of low-copy number analytes in biospecimens with high autofluorescence.

In this chapter, we describe the preparation of fluorescent quantum dots for imaging and measuring protein expression in cells. Quantum dots are nanocrystals that have numerous advantages for biomolecular detection compared with organic dyes and fluorescent proteins, but their large size has been a limiting factor. We describe the synthesis of nanoparticles smaller than 10 nm (smaller than an antibody), their attachment to monoclonal antibodies through click chemistry, characterization of the conjugates, and use for labeling of cellular antigens. We further discuss the unique advantages and challenges associated with this approach compared with conventional immunofluorescence techniques.

The distribution of single-cell properties across a population of cells can be measured using diverse tools, but no technology directly quantifies the biochemical stimulation events regulating these properties. Here we report digital counting of growth factors in single cells using fluorescent quantum dots and calibrated three-dimensional deconvolution microscopy (QDC-3DM) to reveal physiologically relevant cell stimulation distributions. We calibrate the fluorescence intensities of individual compact quantum dots labeled with epidermal growth factor (EGF) and demonstrate the necessity of near-infrared emission to overcome intrinsic cellular autofluoresence at the single-molecule level. When applied to human triple-negative breast cancer cells, we observe proportionality between stimulation and both receptor internalization and inhibitor response, reflecting stimulation heterogeneity contributions to intrinsic variability. We anticipate that QDC-3DM can be applied to analyze any peptidic ligand to reveal single-cell correlations between external stimulation and phenotypic variability, cell fate, and drug response.

Fluorescence in situ hybridization (FISH) is the primary technology used to image and count mRNA in single cells, but applications of the technique are limited by photophysical shortcomings of organic dyes. Inorganic quantum dots (QDs) can overcome these problems but years of development have not yielded viable QD-FISH probes. Here we report that macromolecular size thresholds limit mRNA labeling in cells, and that a new generation of compact QDs produces accurate mRNA counts. Compared with dyes, compact QD probes provide exceptional photostability and more robust transcript quantification due to enhanced brightness. New spectrally engineered QDs also allow quantification of multiple distinct mRNA transcripts at the single-molecule level in individual cells. We expect that QD-FISH will particularly benefit high-resolution gene expression studies in three dimensional biological specimens for which quantification and multiplexing are major challenges.

Inefficient delivery of macromolecules and nanoparticles to intracellular targets is a major bottleneck in drug delivery, genetic engineering, and molecular imaging. Here we apply live-cell single-quantum-dot imaging and tracking to analyze and classify nanoparticle states after intracellular delivery. By merging trajectory diffusion parameters with brightness measurements, multidimensional analysis reveals distinct and heterogeneous populations that are indistinguishable using single parameters alone. We derive new quantitative metrics of particle loading, cluster distribution, and vesicular release in single cells, and evaluate intracellular nanoparticles with diverse surfaces following osmotic delivery. Surface properties have a major impact on cell uptake, but little impact on the absolute cytoplasmic numbers. A key outcome is that stable zwitterionic surfaces yield uniform cytosolic behavior, ideal for imaging agents. We anticipate that this combination of quantum dots and single-particle tracking can be widely applied to design and optimize next-generation imaging probes, nanoparticle therapeutics, and biologics.

Nanocrystals composed of mixed chemical domains have diverse properties that are driving their integration in next-generation electronics, light sources, and biosensors. However, the precise spatial distribution of elements within these particles is difficult to measure and control, yet profoundly impacts their quality and performance. Here we synthesized a unique series of 42 different quantum dot nanocrystals, composed of two chemical domains (CdS:CdSe), arranged in 7 alloy and (core)shell structural classes. Chemometric analyses of far-field Raman spectra accurately classified their internal structures from their vibrational signatures. These classifications provide direct insight into the elemental arrangement of the alloy as well as an independent prediction of fluorescence quantum yield. This nondestructive, rapid approach can be broadly applied to greatly enhance our capacity to measure, predict and monitor multicomponent nanomaterials for precise tuning of their structures and properties.

Quantum dots are fluorescent nanoparticles used to detect and image proteins and nucleic acids. Compared with organic dyes and fluorescent proteins, these nanocrystals have enhanced brightness, photostability, and wavelength tunability, but their larger size limits their use. Recently, multidentate polymer coatings have yielded stable quantum dots with small hydrodynamic dimensions (≤10 nm) due to high-affinity, compact wrapping around the nanocrystal. However, this coating technology has not been widely adopted because the resulting particles are frequently heterogeneous and clustered, and conjugation to biological molecules is difficult to control. In this article we develop new polymeric ligands and optimize coating and bioconjugation methodologies for core/shell CdSe/Cd(x)Zn(1-x)S quantum dots to generate homogeneous and compact products. We demonstrate that "ligand stripping" to rapidly displace nonpolar ligands with hydroxide ions allows homogeneous assembly with multidentate polymers at high temperature. The resulting aqueous nanocrystals are 7-12 nm in hydrodynamic diameter, have quantum yields similar to those in organic solvents, and strongly resist nonspecific interactions due to short oligoethylene glycol surfaces. Compared with a host of other methods, this technique is superior for eliminating small aggregates identified through chromatographic and single-molecule analysis. We also demonstrate high-efficiency bioconjugation through azide-alkyne click chemistry and self-assembly with hexa-histidine-tagged proteins that eliminate the need for product purification. The conjugates retain specificity of the attached biomolecules and are exceptional probes for immunofluorescence and single-molecule dynamic imaging. These results are expected to enable broad utilization of compact, biofunctional quantum dots for studying crowded macromolecular environments such as the neuronal synapse and cellular cytoplasm.

As molecular labels for cells and tissues, fluorescent probes have shaped our understanding of biological structures and processes. However, their capacity for quantitative analysis is limited because photon emission rates from multicolour fluorophores are dissimilar, unstable and often unpredictable, which obscures correlations between measured fluorescence and molecular concentration. Here we introduce a new class of light-emitting quantum dots with tunable and equalized fluorescence brightness across a broad range of colours. The key feature is independent tunability of emission wavelength, extinction coefficient and quantum yield through distinct structural domains in the nanocrystal. Precise tuning eliminates a 100-fold red-to-green brightness mismatch of size-tuned quantum dots at the ensemble and single-particle levels, which substantially improves quantitative imaging accuracy in biological tissue. We anticipate that these materials engineering principles will vastly expand the optical engineering landscape of fluorescent probes, facilitate quantitative multicolour imaging in living tissue and improve colour tuning in light-emitting devices.

Immunofluorescence, a powerful technique to detect specific targets using fluorescently labeled antibodies, has been widely used in both scientific research and clinical diagnostics. The probes should be made with small antibodies and high brightness. We conjugated GFP binding protein (GBP) nanobodies, small single-chain antibodies from llamas, with new ∼7 nm quantum dots. These provide simple and versatile immunofluorescence nanoprobes with nanometer accuracy and resolution. Using the new probes we tracked the walking of individual kinesin motors and measured their 8 nm step sizes; we tracked Piezo1 channels, which are eukaryotic mechanosensitive channels; we also tracked AMPA receptors on living neurons. Finally, we used a new super-resolution algorithm based on blinking of (small) quantum dots that allowed ∼2 nm precision.

Quantum dots (QDs) offer distinct advantages over organic dyes and fluorescent proteins for biological imaging applications because of their brightness, photostability, and tunability. However, a major limitation is that single QDs emit fluorescent light in an intermittent on-and-off fashion called "blinking". Here we report the development of blinking-suppressed, relatively compact QDs that are able to maintain their favorable optical properties in aqueous solution. Specifically, we show that a linearly graded alloy shell can be grown on a small CdSe core via a precisely controlled layer-by-layer process, and that this graded shell leads to a dramatic suppression of QD blinking in both organic solvents and water. A substantial portion (>25%) of the resulting QDs does not blink (more than 99% of the time in the bright or "on" state). Theoretical modeling studies indicate that this type of linearly graded shell not only can minimize charge carrier access to surface traps but also can reduce lattice defects, both of which are believed to be responsible for carrier trapping and QD blinking. Further, we have evaluated the biological utility of blinking-suppressed QDs coated with polyethylene glycol (PEG)-based ligands and multidentate ligands. The results demonstrate that their optical properties are largely independent of surface coatings and solvating media, and that the blinking-suppressed QDs can provide continuous trajectories in live-cell receptor tracking studies.

We developed a coating method to produce functionalized small quantum dots (sQDs), about 9 nm in diameter, that were stable for over a month. We made sQDs in four emission wavelengths, from 527 to 655 nm and with different functional groups. AMPA receptors on live neurons were labeled with sQDs and postsynaptic density proteins were visualized with super-resolution microscopy. Their diffusion behavior indicates that sQDs access the synaptic clefts significantly more often than commercial QDs., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Quantum-confined nanostructures are considered 'artificial atoms' because the wavefunctions of their charge carriers resemble those of atomic orbitals. For multiple-domain heterostructures, however, carrier wavefunctions are more complex and still not well understood. We have prepared a unique series of cation-exchanged Hg(x)Cd(1-x)Te quantum dots (QDs) and seven epitaxial core-shell QDs and measured their first and second exciton peak oscillator strengths as a function of size and chemical composition. A major finding is that carrier locations can be quantitatively mapped and visualized during shell growth or cation exchange simply using absorption transition strengths. These results reveal that a broad range of quantum heterostructures with different internal structures and band alignments exhibit distinct carrier localization patterns that can be used to further improve the performance of optoelectronic devices and enhance the brightness of QD probes for bioimaging.

Semiconductor quantum dots (QDs) are light-emitting particles on the nanometer scale that have emerged as a new class of fluorescent labels for chemical analysis, molecular imaging, and biomedical diagnostics. Compared with traditional fluorescent probes, QDs have unique optical and electronic properties such as size-tunable light emission, narrow and symmetric emission spectra, and broad absorption spectra that enable the simultaneous excitation of multiple fluorescence colors. QDs are also considerably brighter and more resistant to photobleaching than are organic dyes and fluorescent proteins. These properties are well suited for dynamic imaging at the single-molecule level and for multiplexed biomedical diagnostics at ultrahigh sensitivity. Here, we discuss the fundamental properties of QDs; the development of next-generation QDs; and their applications in bioanalytical chemistry, dynamic cellular imaging, and medical diagnostics. For in vivo and clinical imaging, the potential toxicity of QDs remains a major concern. However, the toxic nature of cadmium-containing QDs is no longer a factor for in vitro diagnostics, so the use of multicolor QDs for molecular diagnostics and pathology is probably the most important and clinically relevant application for semiconductor QDs in the immediate future.

Single-molecule imaging is an important tool for understanding the mechanisms of biomolecular function and for visualizing the spatial and temporal heterogeneity of molecular behaviors that underlie cellular biology (1-4). To image an individual molecule of interest, it is typically conjugated to a fluorescent tag (dye, protein, bead, or quantum dot) and observed with epifluorescence or total internal reflection fluorescence (TIRF) microscopy. While dyes and fluorescent proteins have been the mainstay of fluorescence imaging for decades, their fluorescence is unstable under high photon fluxes necessary to observe individual molecules, yielding only a few seconds of observation before complete loss of signal. Latex beads and dye-labeled beads provide improved signal stability but at the expense of drastically larger hydrodynamic size, which can deleteriously alter the diffusion and behavior of the molecule under study. Quantum dots (QDs) offer a balance between these two problematic regimes. These nanoparticles are composed of semiconductor materials and can be engineered with a hydrodynamically compact size with exceptional resistance to photodegradation (5). Thus in recent years QDs have been instrumental in enabling long-term observation of complex macromolecular behavior on the single molecule level. However these particles have still been found to exhibit impaired diffusion in crowded molecular environments such as the cellular cytoplasm and the neuronal synaptic cleft, where their sizes are still too large (4,6,7). Recently we have engineered the cores and surface coatings of QDs for minimized hydrodynamic size, while balancing offsets to colloidal stability, photostability, brightness, and nonspecific binding that have hindered the utility of compact QDs in the past (8,9). The goal of this article is to demonstrate the synthesis, modification, and characterization of these optimized nanocrystals, composed of an alloyed HgxCd1-xSe core coated with an insulating CdyZn1-yS shell, further coated with a multidentate polymer ligand modified with short polyethylene glycol (PEG) chains (Figure 1). Compared with conventional CdSe nanocrystals, HgxCd1-xSe alloys offer greater quantum yields of fluorescence, fluorescence at red and near-infrared wavelengths for enhanced signal-to-noise in cells, and excitation at non-cytotoxic visible wavelengths. Multidentate polymer coatings bind to the nanocrystal surface in a closed and flat conformation to minimize hydrodynamic size, and PEG neutralizes the surface charge to minimize nonspecific binding to cells and biomolecules. The end result is a brightly fluorescent nanocrystal with emission between 550-800 nm and a total hydrodynamic size near 12 nm. This is in the same size range as many soluble globular proteins in cells, and substantially smaller than conventional PEGylated QDs (25-35 nm).

We report a new strategy based on mercury cation exchange in nonpolar solvents to prepare bright and compact alloyed quantum dots (QDs) (Hg(x)Cd(1-x)E, where E = Te, Se, or S) with equalized particle size and broadly tunable absorption and fluorescence emission in the near-infrared. The main rationale is that cubic CdE and HgE have nearly identical lattice constants but very different band gap energies and electron/hole masses. Thus, replacement of Cd(2+) by Hg(2+) in CdTe nanocrystals does not change the particle size, but it greatly alters the band gap energy. After capping with a multilayer shell and solubilization with a multidentate ligand, this class of cation-exchanged QDs are compact (6.5 nm nanocrystal size and 10 nm hydrodynamic diameter) and very bright (60-80% quantum yield), with narrow and symmetric fluorescence spectra tunable across the wavelength range from 700 to 1150 nm.

Semiconductor nanocrystals are tiny light-emitting particles on the nanometer scale. Researchers have studied these particles intensely and have developed them for broad applications in solar energy conversion, optoelectronic devices, molecular and cellular imaging, and ultrasensitive detection. A major feature of semiconductor nanocrystals is the quantum confinement effect, which leads to spatial enclosure of the electronic charge carriers within the nanocrystal. Because of this effect, researchers can use the size and shape of these "artificial atoms" to widely and precisely tune the energy of discrete electronic energy states and optical transitions. As a result, researchers can tune the light emission from these particles throughout the ultraviolet, visible, near-infrared, and mid-infrared spectral ranges. These particles also span the transition between small molecules and bulk crystals, instilling novel optical properties such as carrier multiplication, single-particle blinking, and spectral diffusion. In addition, semiconductor nanocrystals provide a versatile building block for developing complex nanostructures such as superlattices and multimodal agents for molecular imaging and targeted therapy. In this Account, we discuss recent advances in the understanding of the atomic structure and optical properties of semiconductor nanocrystals. We also discuss new strategies for band gap and electronic wave function engineering to control the location of charge carriers. New methodologies such as alloying, doping, strain-tuning, and band-edge warping will likely play key roles in the further development of these particles for optoelectronic and biomedical applications.

Strain can have a large influence on the properties of materials at the nanoscale. The effect of lattice strain on semiconductor devices has been widely studied, but its influence on colloidal semiconductor nanocrystals is still poorly understood. Here we show that the epitaxial deposition of a compressive shell (ZnS, ZnSe, ZnTe, CdS or CdSe) onto a soft nanocrystalline core (CdTe) to form a lattice-mismatched quantum dot can dramatically change the conduction and valence band energies of both the core and the shell. In particular, standard type-I quantum-dot behaviour is replaced by type-II behaviour, which is characterized by spatial separation of electrons and holes, extended excited-state lifetimes and giant spectral shifts. Moreover, the strain induced by the lattice mismatch can be used to tune the light emission--which displays narrow linewidths and high quantum yields--across the visible and near-infrared part of the spectrum (500-1,050 nm). Lattice-mismatched core-shell quantum dots are expected to have applications in solar energy conversion, multicolour biomedical imaging and super-resolution optical microscopy.

We report one-pot synthesis, encapsulation, and solubilization of high-quality quantum dots (QDs) based on the use of amphiphilic and multidentate polymer ligands. In this "all-in-one" procedure, the resulting QDs are first capped by the multidentate ligand and are then spontaneously encapsulated and solubilized by a second layer of the same multidentate polymer upon exposure to water. In addition to providing better control of nanocrystal nucleation and growth kinetics (including resistance to Ostwald ripening), this procedure allows for in situ growth of an inorganic passivating shell on the nanocrystal core, enabling one-pot synthesis of both type-I and type-II core-shell QDs with tunable light emission from visible to near-infrared wavelengths.

We report a new strategy to minimize the hydrodynamic size of quantum dots (QDs) and to overcome their colloidal stability and photobleaching problems based on the use of multifunctional and multidentate polymer ligands. A novel finding is that a balanced composition of thiol (-SH) and amine (-NH 2) coordinating groups grafted to a linear polymer chain leads to highly compact nanocrystals with exceptional colloidal stability, a strong resistance to photobleaching, and high fluorescence quantum yields. In contrast to the standing brushlike conformation of PEGylated dihydrolipoic acid molecules, mutlidentate polymer ligands can wrap around the QDs in a closed "loops-and-trains" conformation. This structure is highly stable thermodynamically and is responsible for the excellent colloidal and optical properties. We have optimized this process for the preparation of ultrastable CdTe nanocrystals and have found the strategy to be broadly applicable to a wide range of nanocrystalline materials and heterostructures. This work has led to a new generation of bright and stable QDs with small hydrodynamic diameters between 5.6 and 9.7 nm with tunable fluorescence emission from the visible (515 nm) to the near-infrared (720 nm). These QDs are well suited for molecular and cellular imaging applications in which the nanoparticle hydrodynamic size must be minimized.

We report quenching and chemical degradation of polymer-coated quantum dots by reactive oxygen species (ROS), a group of oxygen-containing molecules that are produced by cellular metabolism and are involved in both normal physiological and disease processes such as oxidative signaling, cancer, and atherosclerosis. A major new finding is that hypochlorous acid (HOCl) in its neutral form is especially potent in degrading encapsulated QDs, due to its small size, neutral charge, long half-life, and fast reaction kinetics under physiologic conditions. Thus, small and neutral molecules such as HOCl and hydrogen peroxide (H2O2) are believed to diffuse across the polymer coating layer, leading to chemical oxidation of sulfur or selenium atoms on the QD surface. This "etching" process first generates lattice structural defects (which cause fluorescence quenching) and then produces soluble metal (e.g., cadmium and zinc) and chalcogenide (e.g., sulfur and selenium) species. We also find that significant fluorescence quenching occurs before QD dissolution and that localized surface defects can be repaired or "annealed" by UV light illumination. These results have important implications regarding the long-term fate and potential toxicity of semiconductor nanocrystals in vivo.

Semiconductor quantum dots (QDs) are tiny light-emitting particles on the nanometer scale, and are emerging as a new class of fluorescent labels for biology and medicine. In comparison with organic dyes and fluorescent proteins, they have unique optical and electronic properties, with size-tunable light emission, superior signal brightness, resistance to photobleaching, and broad absorption spectra for simultaneous excitation of multiple fluorescence colors. QDs also provide a versatile nanoscale scaffold for designing multifunctional nanoparticles with both imaging and therapeutic functions. When linked with targeting ligands such as antibodies, peptides or small molecules, QDs can be used to target tumor biomarkers as well as tumor vasculatures with high affinity and specificity. Here we discuss the synthesis and development of state-of-the-art QD probes and their use for molecular and cellular imaging. We also examine key issues for in vivo imaging and therapy, such as nanoparticle biodistribution, pharmacokinetics, and toxicology.

Quantum-dot (QD) nanocrystals are promising fluorescent probes for multiplexed staining assays in biological applications. However, nonspecific QD binding to cellular membranes and proteins remains a limiting factor in detection sensitivity and specificity. Here we report a new class of hydroxyl (-OH)-coated QDs for minimizing nonspecific cellular binding and for overcoming the bulky size problems encountered with previous surface coatings. The hydroxylated QDs are prepared from carboxylated (-COOH) dots via a hydroxylation and cross-linking process. With a compact hydrodynamic size of 13-14 nm (diameter), they are highly fluorescent (>60% quantum yields) and stable under both basic and acidic conditions. By using human cancer cells, we have evaluated their superior nonspecific binding properties against that of carboxylated, protein-coated, and poly(ethylene glycol) (PEG)-coated QDs. Quantitative cellular staining data indicate that the hydroxylated QDs result in a dramatic 140-fold reduction in nonspecific binding relative to that of carboxylated dots and a still significant 10-20-fold reduction relative to that of PEG- and protein-coated dots.

A number of procedures are currently available to encapsulate and solubilize hydrophobic semiconductor Quantum Dots (QDs) for biological applications. Most of these procedures are based on the use of small-molecule coordinating ligands, amphiphilic polymers, or amphiphilic lipids. However, it is still not clear how these different surface coating molecules affect the optical, colloidal, and chemical properties of the solubilized QDs. Here we report a systematic study to examine the effects of surface coating chemistry on the hydrodynamic size, fluorescence quantum yield, photostability, chemical stability, and biocompatibility of water-soluble QDs. The results indicate that quantum dots with the smallest hydrodynamic sizes are best prepared by direct ligand exchange with hydrophilic molecules, but the resulting particles are less stable than those encapsulated in amphiphilic polymers. For stability against chemical oxidation, QDs should be protected with a hydrophobic bilayer. For high stability under acidic conditions, the best QDs are prepared by using hyperbranched polyethylenimine. For stability in high salt buffers, it is preferable to have uncharged, sterically-stabilized QDs, like those coated with polyethylene glycol (PEG). These insights are expected to benefit the development of quantum dots and related nanoparticle probes for molecular and cellular imaging applications.

Nanometer-sized particles, such as semiconductor quantum dots and iron oxide nanocrystals, have novel optical, electronic, magnetic or structural properties that are not available from either molecules or bulk solids. When linked with tumor-targeting ligands, such as monoclonal antibodies, peptide fragments of tumor-specific proteins or small molecules, these nanoparticles can be used to target tumor antigens (biomarkers) and tumor vasculatures with high affinity and specificity. In the mesoscopic size range of 5-100 nm diameter, quantum dots and related nanoparticles have large surface areas and functional groups that can be linked to multiple diagnostic (e.g., optical, radioisotopic or magnetic) and therapeutic (e.g., anticancer) agents. In this review, recent advances in the development and applications of bioconjugated quantum dots and multifunctional nanoparticles for in vivo tumor imaging and targeting are discussed.

In the pursuit of sensitive and quantitative methods to detect and diagnose cancer, nanotechnology has been identified as a field of great promise. Semiconductor quantum dots are nanoparticles with intense, stable fluorescence, and could enable the detection of tens to hundreds of cancer biomarkers in blood assays, on cancer tissue biopsies, or as contrast agents for medical imaging. With the emergence of gene and protein profiling and microarray technology, high-throughput screening of biomarkers has generated databases of genomic and expression data for certain cancer types, and has identified new cancer-specific markers. Quantum dots have the potential to expand this in vitro analysis, and extend it to cellular, tissue and whole-body multiplexed cancer biomarker imaging.

Semiconductor quantum dots (QDs) are a new class of fluorescent labels with broad applications in biomedical imaging, disease diagnostics, and molecular and cell biology. In comparison with organic dyes and fluorescent proteins, quantum dots have unique optical and electronic properties such as size-tunable light emission, improved signal brightness, resistance against photobleaching, and simultaneous excitation of multiple fluorescence colors. Recent advances have led to multifunctional nanoparticle probes that are highly bright and stable under complex in vitro and in vivo conditions. New designs involve encapsulating luminescent QDs with amphiphilic block copolymers, and linking the polymer coating to tumor-targeting ligands and drug-delivery functionalities. These improved QDs have opened new possibilities for real-time imaging and tracking of molecular targets in living cells, for multiplexed analysis of biomolecular markers in clinical tissue specimens, and for ultrasensitive imaging of malignant tumors in living animal models. In this article, we briefly discuss recent developments in bioaffinity QD probes and their applications in molecular profiling of individual cancer cells and clinical tissue specimens.

Semiconductor quantum dots are luminescent nanoparticles that are under intensive development for use as a new class of optical imaging contrast agents. Their novel properties such as optical tunability, improved photostability, and multicolor light emission have opened new opportunities for imaging living cells and in vivo animal models at unprecedented sensitivity and spatial resolution. Combined with biomolecular engineering strategies for tailoring the particle surfaces at the molecular level, bio-conjugated quantum dot probes are well suited for imaging single-molecule dynamics in living cells, for monitoring protein-protein interactions within specific intracellular locations, and for detecting diseased sites and organs in deep tissue. In this article, we describe the engineering principles for preparing high-quality quantum dots and for conjugating the dots to biomolecular ligands. We also discuss recent advances in using quantum dots for in vivo molecular and cellular imaging.

Semiconductor quantum dots (QD) are nanometer-sized crystals with unique photochemical and photophysical properties that are not available from either isolated molecules or bulk solids. In comparison with organic dyes and fluorescent proteins, QD are emerging as a new class of fluorescent labels with improved brightness, resistance against photobleaching and multicolor fluorescence emission. These properties could improve the sensitivity of biological detection and imaging by at least 10- to 100-fold. Further development in high-quality near-infrared-emitting QD should allow ultrasensitive and multicolor imaging of molecular targets in deep tissue and living animals. Here, we discuss recent developments in QD synthesis and bioconjugation, applications in molecular and cellular imaging as well as promising directions for future research.

Quantum dots are tiny light-emitting particles on the nanometer scale. They are emerging as a new class of biological labels with properties and applications that are not available with traditional organic dyes and fluorescent proteins. Their novel properties such as improved brightness, resistance against photobleaching, and multicolor light emission, have opened new possibilities for ultrasensitive chemical analysis and cellular imaging. In this Research Highlight article , we discuss the unique optical properties of semiconductor quantum dots, surface chemistry and bioconjugation, current applications in bioanalytical chemistry and cell biology, and future research directions.