Polypeptide- and Protein-Based Conjugate Nanoparticles via Aqueous Ring-Opening Polymerization-Induced Self-Assembly (ROPISA).

Protein-polymer conjugates and polymeric nanomaterials hold great promise in many applications including biomaterials, medicine, or nanoelectronics. In this work, the first polymerization-induced self-assembly (PISA) approach performed in aqueous medium enabling protein-polymer conjugates and nanoparticles entirely composed of amino acids is presented by using ring-opening polymerization (ROP). It is indeed shown that aqueous ring-opening polymerization-induced self-assembly (ROPISA) can be used with protein or peptidic macroinitiators without prior chemical modification and afford the simple preparation of nanomaterials with protein-like property, for example, to implement biomimetic thermoresponsivity in drug delivery.

Tuning the Aggregates of Thiophene-based Trimers by Methyl Side-chain Engineering for Photocatalytic Hydrogen Evolution.

Organic π-conjugated semiconductors (OCSs) have recently emerged as a promising alternative to traditional inorganic materials for photocatalysis. However, the aggregation of OCSs in photocatalytic aqueous solution caused by self-assembly, which closely relates to the photocatalytic activity, has not yet been studied. Here, the relationship between the aggregation of 4,7-Bis(thiophen-2-yl) benzothiadiazole (TBT) and the photocatalytic activity was systematically investigated by introducing and varying the position of methyl side chains on the two peripheral thiophene units. Experimental and theoretical results indicated that the introduction of -CH3 group at the 3-position of TBT resulted in the smallest size and best crystallinity of aggregates compared to that of TBT, 4- and 5-positions. As a result, TBT-3 exhibited an excellent photocatalytic activity towards H2 evolution, ascribed to the shorten charge carrier transport distance and solid long-range order. These results suggest the important role of aggregation behavior of OCSs for efficient photocatalysis.

Transcription-Factor-Induced Aggregation of Biomimetic Oligonucleotide-b-Protein Micelles.

Polymeric micelles and especially those based on natural diblocks are of particular interest due to their advantageous properties in terms of molecular recognition, biocompatibility, and biodegradability. We herein report a facile and straightforward synthesis of thermoresponsive elastin-like polypeptide (ELP) and oligonucleotide (ON) diblock bioconjugates, ON-b-ELP, through copper-catalyzed azide-alkyne cycloaddition. The resulting thermosensitive diblock copolymer self-assembles above its critical micelle temperature (CMT ∼30 °C) to form colloidally stable micelles of ∼50 nm diameter. The ON-b-ELP micelles hybridize with an ON complementary strand and maintain their size and stability. Next, we describe the capacity of these micelles to bind proteins, creating more complex structures using the classic biotin-streptavidin pairing and the specific recognition between a transcription factor protein and the ON strand. In both instances, the micelles are intact, form larger structures, and retain their sensitivity to temperature.

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.

FRET-mediated quenching of BODIPY fluorescent nanoparticles by methylene blue and its application to bacterial imaging.

High resolution and a good signal to noise ratio are a requirement in cell imaging. However, after labelling with fluorescent entities, and after several washing steps, there is often an unwanted fluorescent background that reduces the images resolution. For this purpose, we developed an approach to remove the signal from extra-cellular fluorescent nanoparticles (FNPs) during bacteria imaging, without the need for any washing steps. Our idea is to use methylene blue to quench > 90% of the emission of BODIPY-based fluorescent polymer nanoparticle by a FRET process. This "Hide-and-Seek Game" approach offers a novel strategy to apply fluorescence quenching in bioimaging to improve image accuracy.

An Allosteric Transcription Factor DNA-Binding Electrochemical Biosensor for Progesterone.

We describe an electrochemical strategy to transduce allosteric transcription factor (aTF) binding affinity to sense steroid hormones. Our approach utilizes square wave voltammetry to monitor changes in current output as a progesterone (PRG)-specific aTF (SRTF1) unbinds from the cognate DNA sequence in the presence of PRG. The sensor detects PRG in artificial urine samples with sufficient sensitivity suitable for clinical applications. Our results highlight the capability of using aTFs as the biorecognition elements to develop electrochemical point-of-care biosensors for the detection of small-molecule biomarkers and analytes.

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.

Fluorescent Copolymers for Bacterial Bioimaging and Viability Detection.

Novel fluorescent labels with high photostability and high biocompatibility are required for microbiological imaging and detection. Here, we present a green fluorescent polymer chain (GFPC), designed to be nontoxic and water-soluble, for multicolor bioimaging and real-time bacterial viability determination. The copolymer is synthesized using a straightforward one-pot reversible addition-fragmentation chain-transfer (RAFT) polymerization technique. We show that GFPC does not influence bacterial growth and is stable for several hours in a complex growth medium and in the presence of bacteria. GFPC allows the labeling of the bacterial cytoplasm for multicolor bacterial bioimaging applications. It can be used in combination with propidium iodide (PI) to develop a rapid and reliable protocol to distinguish and quantify, in real time, by flow cytometry, live and dead bacteria.

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.

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 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.

Aqueous Ring-Opening Polymerization-Induced Self-Assembly (ROPISA) of N-Carboxyanhydrides.

Reported here is the first aqueous ring-opening polymerization (ROP) of N-carboxyanhydrides (NCAs) using α-amino-poly(ethylene oxide) as a macroinitiator to protect the NCA monomers from hydrolysis through spontaneous in situ self-assembly (ISA). This ROPISA process affords well-defined amphiphilic diblock copolymers that simultaneously form original needle-like nanoparticles.

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.

Core-shell polymeric nanoparticles comprising BODIPY and fluorescein as ultra-bright ratiometric fluorescent pH sensors.

A new ratiometric fluorescent pH nanosensor is presented. It is based on ultrabright nanoparticles containing two spatially separated fluorophores: BODIPY covalently linked to the polystyrene core and fluorescein grafted to the nanoparticle shell. The nanoparticles comprise a large number (≥2500) of both fluorescent moieties. Their spectroscopic characteristics were studied at different pH and ionic strength. They could successfully be used to determine the solution pH between 5.5 and 7.5 by measuring the fluorescence intensity ratio of the sensor molecule (fluorescein) relative to the reference dye (BODIPY).

Semiconductor Nanoplatelets: A New Class of Ultrabright Fluorescent Probes for Cytometric and Imaging Applications.

Fluorescent semiconductor nanoplatelets (NPLs) are a new generation of fluorescent probes. NPLs are colloidal two-dimensional materials that exhibit several unique optical properties, including high brightness, photostability, and extinction coefficients, as well as broad excitation and narrow emission spectra from the visible to the near-infrared spectrum. All of these exceptional fluorescence properties make NPLs interesting nanomaterials for biological applications. However, NPLs are synthesized in organic solvents and coated with hydrophobic ligands that render them insoluble in water. A current challenge is to stabilize NPLs in aqueous media compatible with biological environments. In this work, we describe a novel method to disperse fluorescent NPLs in water and functionalize them with different biomolecules for biodetection. We demonstrate that ligand exchange enables the dispersion of NPLs in water while maintaining optical properties and long-term colloidal stability in biological environments. Four different colors of NPLs were functionalized with biomolecules by random or oriented conformations. For the first time, we report that our NPLs have a higher brightness than that of standard fluorophores, like phycoerythrin or Brilliant Violet 650 (BV 650), for staining cells in flow cytometry. These results suggest that NPLs are an interesting alternative to common fluorophores for flow cytometry and imaging applications in multiplexed cellular targeting.

A novel type of quantum dot-transferrin conjugate using DNA hybridization mimics intracellular recycling of endogenous transferrin.

Colloidal nanoparticles such as Quantum Dots (QDs) are promising alternatives to organic fluorophores, especially for long duration bioimaging. For specific targeting applications, QDs frequently require functionalization with selected proteins. In this regard, conjugation of proteins to QDs such that the nanobioconjugates retain the endogenous behavior of the coupled protein remains challenging. We have developed a novel method to conjugate a protein, transferrin (Tf), to QDs using DNA hybridization. These conjugates are characterized biochemically, and the trafficking properties in live cells are investigated. Although the internalization kinetics into the cells is much reduced compared to Tf labelled with organic dye, we could show that DNA hybridization-based QD-Tf conjugates are the first for which recycling from endosomes to the plasma membrane can be observed. This recycling occurs with kinetics that is similar to dye labelled Tf. We could image and follow the trajectories of recycling of individual vesicles for several tens of minutes. The conjugation of QDs to proteins mediated by DNA hybridization yields a new generation of ultra-bright and photostable probes that preserves the intracellular properties of the dye labelled protein better than previously reported QD conjugates using other surface chemistries for direct coupling.

Quantum dot-loaded monofunctionalized DNA icosahedra for single-particle tracking of endocytic pathways.

Functionalization of quantum dots (QDs) with a single biomolecular tag using traditional approaches in bulk solution has met with limited success. DNA polyhedra consist of an internal void bounded by a well-defined three-dimensional structured surface. The void can house cargo and the surface can be functionalized with stoichiometric and spatial precision. Here, we show that monofunctionalized QDs can be realized by encapsulating QDs inside DNA icosahedra and functionalizing the DNA shell with an endocytic ligand. We deployed the DNA-encapsulated QDs for real-time imaging of three different endocytic ligands-folic acid, galectin-3 (Gal3) and the Shiga toxin B-subunit (STxB). Single-particle tracking of Gal3- or STxB-functionalized QD-loaded DNA icosahedra allows us to monitor compartmental dynamics along endocytic pathways. These DNA-encapsulated QDs, which bear a unique stoichiometry of endocytic ligands, represent a new class of molecular probes for quantitative imaging of endocytic receptor dynamics.

Rapid and accurate detection of Escherichia coli growth by fluorescent pH-sensitive organic nanoparticles for high-throughput screening applications.

Rapid detection of bacterial growth is an important issue in the food industry and for medical research. Here we present a novel kind of pH-sensitive fluorescent nanoparticles (FANPs) that can be used for the rapid and accurate real-time detection of Escherichia coli growth. These organic particles are designed to be non-toxic and highly water-soluble. Here we show that the coupling of pH sensitive fluoresceinamine to the nanoparticles results in an increased sensitivity to changes in pH within a physiologically relevant range that can be used to monitor the presence of live bacteria. In addition, these FANPs do not influence bacterial growth and are stable over several hours in a complex medium and in the presence of bacteria. The use of these FANPs allows for continuous monitoring of bacterial growth via real-time detection over long time scales in small volumes and can thus be used for the screening of a large number of samples for high-throughput applications such as screening for the presence of antibiotic resistant strains.

Fast, Efficient, and Stable Conjugation of Multiple DNA Strands on Colloidal Quantum Dots.

A novel method for covalent conjugation of DNA to polymer coated quantum dots (QDs) is investigated in detail. This method is fast and efficient: up to 12 DNA strands can be covalently conjugated per QD in optimized reaction conditions. The QD-DNA conjugates can be purified using size exclusion chromatography and the QDs retain high quantum yield and excellent stability after DNA coupling. We explored single-stranded and double-stranded DNA coupling, as well as various lengths. We show that the DNA coupling is most efficient for short (15 mer) single-stranded DNA. The DNA coupling has been performed on QDs emitting at four different wavelengths, as well as on gold nanoparticles, suggesting that this technique can be generalized to a wide range of nanoparticles.

One-pot synthesis of pegylated fluorescent nanoparticles by RAFT miniemulsion polymerization using a phase inversion process.

Water-soluble and fluorescent core-shell nanoparticles (FNP) are synthesized in a miniemulsion reversible addition-fragmentation transfer (RAFT) polymerization and are shown to respond to pH. The particles are obtained from a hydrophilic PEO-b-PAA macromolecular RAFT agent which is block-extended with styrene and a fluorescent BODIPY monomer. A miniemulsion is then formed with the residual hydrophobic monomers. After completion of the polymerization, FNP of ≈ 60 nm in diameter are obtained. The fluorescence of the BODIPY dye in the particles is found to remain (0.2 quantum yield). The particles can be precipitated in acidic pH and redispersed upon addition of base without loss of their integrity or noticeable rearrangement.