Cross-Platform DNA Encoding for Single-Cell Imaging of Gene Expression.

Integration of imaging data across different molecular target types can provide in-depth insight into cell physiology and pathology, but remains challenging owing to poor compatibility between target-type-specific labeling methods. We show that cross-platform imaging analysis can be readily achieved through DNA encoding of molecular targets, which translates the molecular identity of various target types into a uniform in situ array of ssDNA tags for subsequent labeling with complementary imaging probes. The concept was demonstrated through multiplexed imaging of mRNAs and their corresponding proteins with multicolor quantum dots. The results reveal heterogeneity of cell transfection with siRNA and outline disparity in RNA interference (RNAi) kinetics at the level of both the mRNA and the encoded protein.

Eliminating Size-Associated Diffusion Constraints for Rapid On-Surface Bioassays with Nanoparticle Probes.

Nanoparticle probes enable implementation of advanced on-surface assay formats, but impose often underappreciated size-associated constraints, in particular on assay kinetics and sensitivity. The present study highlights substantially slower diffusion-limited assay kinetics due to the rapid development of a nanoprobe depletion layer next to the surface, which static incubation and mixing of bulk solution employed in conventional assay setups often fail to disrupt. In contrast, cyclic solution draining and replenishing yields reaction-limited assay kinetics irrespective of the probe size. Using common surface bioassays, enzyme-linked immunosorbent assays and immunofluorescence, this study shows that this conceptually distinct approach effectively "erases" size-dependent diffusion constraints, providing a straightforward route to rapid on-surface bioassays employing bulky probes and procedures involving multiple labeling cycles, such as multicycle single-cell molecular profiling. For proof-of-concept, the study demonstrates that the assay time can be shortened from hours to minutes with the same probe concentration and, at a typical incubation time, comparable target labeling can be achieved with up to eight times lower nanoprobe concentration. The findings are expected to enable realization of novel assay formats and stimulate development of rapid on-surface bioassays with nanoparticle probes.

Multiplexed In-cell Immunoassay for Same-sample Protein Expression Profiling.

In-cell immunoassays have become a valuable tool for protein expression analysis complementary to established assay formats. However, comprehensive molecular characterization of individual specimens has proven challenging and impractical due to, in part, a singleplex nature of reporter enzymes and technical complexity of alternative assay formats. Herein, we describe a simple and robust methodology for multiplexed protein expression profiling on the same intact specimen, employing a well-characterized enzyme alkaline phosphatase for accurate quantification of all targets of interest, while overcoming fundamental limitations of enzyme-based techniques by implementing the DNA-programmed release mechanism for segregation of sub-sets of target-bound reporters. In essence, this methodology converts same-sample multi-target labeling into a set of isolated singleplex measurements performed in a parallel self-consistent fashion. For a proof-of-principle, multiplexed detection of three model proteins was demonstrated on cultured HeLa cells, and two clinically-relevant markers of dementia, ß-amyloid and PHF-tau, were profiled in formalin-fixed paraffin embedded brain tissue sections, uncovering correlated increase in abundance of both markers in the "Alzheimer's disease" cohort. Featuring an analytically powerful yet technically simple and robust methodology, multiplexed in-cell immunoassay is expected to enable insightful same-sample protein profiling studies and become broadly adopted in biomedical research and clinical diagnostics.

An aggregation-induced-emission platform for direct visualization of interfacial dynamic self-assembly.

An in-depth understanding of dynamic interfacial self-assembly processes is essential for a wide range of topics in theoretical physics, materials design, and biomedical research. However, direct monitoring of such processes is hampered by the poor imaging contrast of a thin interfacial layer. We report in situ imaging technology capable of selectively highlighting self-assembly at the phase boundary in real time by employing the unique photophysical properties of aggregation-induced emission. Its application to the study of breath-figure formation, an immensely useful yet poorly understood phenomenon, provided a mechanistic model supported by direct visualization of all main steps and fully corroborated by simulation and theoretical analysis. This platform is expected to advance the understanding of the dynamic phase-transition phenomena, offer insights into interfacial biological processes, and guide development of novel self-assembly technologies.

Solid-phase bioconjugation of heterobifunctional adaptors for versatile assembly of bispecific targeting ligands.

High-throughput generation of bispecific molecules promises to expedite the discovery of new molecular therapeutics and guide engineering of novel multifunctional constructs. However, high synthesis complexity and cost have hampered the discovery of bispecific molecules in drug development and biomedical research. Herein we describe a simple solid-phase bioconjugation procedure for preparation of Protein A(G,L)-PEG-Streptavidin heterobifunctional adaptors (with 1:1:1 stoichiometry), which enable self-assembly of unmodified antibodies and biotinylated molecules into bispecific targeting ligands in a versatile mix-and-use manner. Utility of such adaptors is demonstrated by assembly of anti-CD3 and anti-Her2 antibodies into bispecific CD3xHer2 targeting ligands, which efficiently drive T-cell-mediated lysis of Her2-positive cancer cells. In comparison to bioconjugation in solution, the solid-phase procedure described here offers precise stoichiometry control, ease of purification, and high yield of functional conjugates. Simplicity and versatility should prove this methodology instrumental for preparation of bispecific ligands, as well as for high-throughput screening of bispecific combinations, before proceeding to synthesis of lead candidates via recombinant engineering or chemical cross-linking.

Addressing Key Technical Aspects of Quantum Dot Probe Preparation for Bioassays.

Fluorescent semiconductor nanoparticles, or quantum dots, have become a promising platform for the engineering of biofunctional probes for a variety of biomedical applications, ranging from multicolor imaging to single-molecule tracking to traceable drug delivery. Advances in organometallic synthesis have enabled preparation of hydrophobic quantum dots with high quantum yields and narrow size distribution, offering bright optical materials with narrow size-tunable emission profiles. At the same time, polymer encapsulation procedures provided a simple and versatile methodology for transferring hydrophobic nanoparticles into physiologically-relevant aqueous buffers. Taken together, hydrophobic nanoparticle platforms and polymer encapsulation should offer great flexibility for implementation of novel probe designs. However, the success of the encapsulation and purification depends on many factors often overlooked in the scientific literature, such as close match between nanoparticle and polymer physicochemical properties and dimensions, slow dynamics of polymer arrangement on the nanoparticle surface, and the size and charge similarity of resultant polymer-coated quantum dots and empty byproduct polymer micelles. To make this general hydrophobic nanoparticle modification strategy accessible by a broad range of biomedical research groups, we focus on the important technical aspects of nanoparticle polymer encapsulation, purification, bioconjugation, and characterization.

Multicolor multicycle molecular profiling with quantum dots for single-cell analysis.

Here we present a detailed protocol for molecular profiling of individual cultured mammalian cells using multicolor multicycle immunofluorescence with quantum dot probes. It includes instructions for cell culture growth and processing (2 h + 48-72 h for cell growth), preparation and characterization of universal quantum dot probes (4.5 h + overnight incubation), cyclic cell staining (∼4.5 h per cycle) and image analysis (varies by application). The use of quantum dot fluorescent probes enables highly multiplexed, robust quantitative molecular imaging with a conventional fluorescence microscopy setup, whereas the probe preparation methodology, using a self-assembly between protein A-decorated universal quantum dots and intact primary antibodies, offers a fast, simple and purification-free route for an on-demand preparation of antibody-functionalized quantum dot libraries. As a result, this protocol can be used by biomedical researchers for a variety of cell staining applications, and, with further optimization, for staining of other biological specimens (e.g., clinical tissue sections).

Quantum dot imaging platform for single-cell molecular profiling.

Study of normal cell physiology and disease pathogenesis heavily relies on untangling the complexity of intracellular molecular mechanisms and pathways. To achieve this goal, comprehensive molecular profiling of individual cells within the context of microenvironment is required. Here we report the development of a multicolour multicycle in situ imaging technology capable of creating detailed quantitative molecular profiles for individual cells at the resolution of optical imaging. A library of stoichiometric fluorescent probes is prepared by linking target-specific antibodies to a universal quantum dot-based platform via protein A in a quick and simple procedure. Surprisingly, despite the potential for multivalent binding between protein A and antibody and the intermediate affinity of this non-covalent bond, fully assembled probes do not aggregate or exchange antibodies, facilitating highly multiplexed parallel staining. This single-cell molecular profiling technology is expected to open new opportunities in systems biology, gene expression studies, signalling pathway analysis and molecular diagnostics.

Quantum dots as a platform for nanoparticle drug delivery vehicle design.

Nanoparticle-based drug delivery (NDD) has emerged as a promising approach to improving upon the efficacy of existing drugs and enabling the development of new therapies. Proof-of-concept studies have demonstrated the potential for NDD systems to simultaneously achieve reduced drug toxicity, improved bio-availability, increased circulation times, controlled drug release, and targeting. However, clinical translation of NDD vehicles with the goal of treating particularly challenging diseases, such as cancer, will require a thorough understanding of how nanoparticle properties influence their fate in biological systems, especially in vivo. Consequently, a model system for systematic evaluation of all stages of NDD with high sensitivity, high resolution, and low cost is highly desirable. In theory, this system should maintain the properties and behavior of the original NDD vehicle, while providing mechanisms for monitoring intracellular and systemic nanocarrier distribution, degradation, drug release, and clearance. For such a model system, quantum dots (QDots) offer great potential. QDots feature small size and versatile surface chemistry, allowing their incorporation within virtually any NDD vehicle with minimal effect on overall characteristics, and offer superb optical properties for real-time monitoring of NDD vehicle transport and drug release at both cellular and systemic levels. Though the direct use of QDots for drug delivery remains questionable due to their potential long-term toxicity, the QDot core can be easily replaced with other organic drug carriers or more biocompatible inorganic contrast agents (such as gold and magnetic nanoparticles) by their similar size and surface properties, facilitating translation of well characterized NDD vehicles to the clinic, maintaining NDD imaging capabilities, and potentially providing additional therapeutic functionalities such as photothermal therapy and magneto-transfection. In this review we outline unique features that make QDots an ideal platform for nanocarrier design and discuss how this model has been applied to study NDD vehicle behavior for diverse drug delivery applications.

Rapid multitarget immunomagnetic separation through programmable DNA linker displacement.

Immunomagnetic separation has become an essential tool for high-throughput and low-cost isolation of biomolecules and cells from heterogeneous samples. However, as magnetic selection is essentially a "black-and-white" assay, its application has been largely restricted to single-target and single-parameter studies. To address this issue, we have developed an immunomagnetic separation technology that can quickly sort multiple targets in high yield and purity using selectively displaceable DNA linkers. We envision that this technology will be readily adopted for experiments requiring high-throughput selection of multiple targets or further adapted for selection of a single target based on multiple surface epitopes.

Designing multifunctional quantum dots for bioimaging, detection, and drug delivery.

The emerging field of bionanotechnology aims at revolutionizing biomedical research and clinical practice via introduction of nanoparticle-based tools, expanding capabilities of existing investigative, diagnostic, and therapeutic techniques as well as creating novel instruments and approaches for addressing challenges faced by medicine. Quantum dots (QDs), semiconductor nanoparticles with unique photo-physical properties, have become one of the dominant classes of imaging probes as well as universal platforms for engineering of multifunctional nanodevices. Possessing versatile surface chemistry and superior optical features, QDs have found initial use in a variety of in vitro and in vivo applications. However, careful engineering of QD probes guided by application-specific design criteria is becoming increasingly important for successful transition of this technology from proof-of-concept studies towards real-life clinical applications. This review outlines the major design principles and criteria, from general ones to application-specific, governing the engineering of novel QD probes satisfying the increasing demands and requirements of nanomedicine and discusses the future directions of QD-focused bionanotechnology research (critical review, 201 references).

Encapsulation of single quantum dots with mesoporous silica.

A new generation of silica encapsulated single quantum dots (QDs) was synthesized based on recent breakthroughs made in coating magnetic nanoparticles and their clusters. In comparison with the traditional Stöber sol-gel method, this new approach is significantly simpler, resulting in QDs with excellent luminescence, stability, size monodispersity, and tunable silica shell thickness. An important finding was that unlike previous reported magnetic and metallic nanoparticles, the QDs coated with only a layer of surfactant molecules were highly unstable and sensitive to the environment. As a consequence, the surfactant stabilized QDs must be prepared fresh and stored in dark before silica coating. The QDs became stable once silica shell formed on their surface and excess surfactants were removed. Further development of this technology particularly by incorporating drugs into the mesosized silica pores will open exciting opportunities in traceable delivery and controlled release of therapeutic agents.

Multifunctional Quantum Dots for Personalized Medicine.

Successes in biomedical research and state-of-the-art medicine have undoubtedly improved the quality of life. However, a number of diseases, such as cancer, immunodeficiencies, and neurological disorders, still evade conventional diagnostic and therapeutic approaches. A transformation towards personalized medicine may help to combat these diseases. For this, identification of disease molecular fingerprints and their association with prognosis and targeted therapy must become available. Quantum dots (QDs), semiconductor nanocrystals with unique photo-physical properties, represent a novel class of fluorescence probes to address many of the needs of personalized medicine. This review outlines the properties of QDs that make them a suitable platform for advancing personalized medicine, examines several proof-of-concept studies showing utility of QDs for clinically relevant applications, and discusses current challenges in introducing QDs into clinical practice.