Highly Sensitive Ammonia Gas Sensor Based on Single-Crystal Poly(3-hexylthiophene) (P3HT) Organic Field Effect Transistor.

A highly sensitive organic field-effect transistor (OFET)-based sensor for ammonia in the range of 0.01 to 25 ppm was developed. The sensor was fabricated by employing an array of single-crystal poly(3-hexylthiophene) (P3HT) nanowires as the organic semiconductor (OSC) layer of an OFET with a top-contact geometry. The electrical characteristics (field-effect mobility, on/off current ratio) of the single-crystal P3HT nanowire OFET were about 2 orders of magnitude larger than those of the P3HT thin film OFET with the same geometry. The P3HT nanowire OFET showed excellent sensitivity to ammonia, about 3 times higher than that of the P3HT thin film OFET at 25 ppm ammonia. The ammonia response of the OFET was reversible and was not affected by changes in relative humidity from 45 to 100%. The high ammonia sensitivity of the P3HT nanowire OFET is believed to result from the single crystal nature and high surface/volume ratio of the P3HT nanowire used in the OSC layer.

Nanoparticle PEBBLE sensors for quantitative nanomolar imaging of intracellular free calcium ions.

Ca(2+) is a universal second messenger and plays a major role in intracellular signaling, metabolism, and a wide range of cellular processes. To date, one of the most successful approaches for intracellular Ca(2+) measurement involves the introduction of optically sensitive Ca(2+) indicators into living cells, combined with digital imaging microscopy. However, the use of free Ca(2+) indicators for intracellular sensing and imaging has several limitations, such as nonratiometric measurement for the most-sensitive indicators, cytotoxicity of the indicators, interference from nonspecific binding caused by cellular biomacromolecules, challenging calibration, and unwanted sequestration of the indicator molecules. These problems are minimized when the Ca(2+) indicators are encapsulated inside porous and inert polyacrylamide nanoparticles. We present PEBBLE nanosensors encapsulated with rhodamine-based Ca(2+) fluorescence indicators. The rhod-2-containing PEBBLEs presented here show a stable sensing range at near-neutral pH (pH 6-9). Because of the protection of the PEBBLE matrix, the interference of protein-nonspecific binding to the indicator is minimal. The rhod-2 PEBBLEs give a nanomolar dynamic sensing range for both in-solution (K(d) = 478 nM) and intracellular (K(d) = 293 nM) measurements. These nanosensors are useful quantitative tools for the measurement and imaging of the cytosolic nanomolar free Ca(2+) levels.

Two-photon fluorescence imaging super-enhanced by multishell nanophotonic particles, with application to subcellular pH.

A novel nanophotonic method for enhancing the two-photon fluorescence signal of a fluorophore is presented. It utilizes the second harmonic (SH) of the exciting light generated by noble metal nanospheres in whose near-field the dye molecules are placed, to further enhance the dye's fluorescence signal in addition to the usual metal-enhanced fluorescence phenomenon. This method enables demonstration, for the first time, of two-photon fluorescence enhancement inside a biological system, namely live cells. A multishell hydrogel nanoparticle containing a silver core, a protective citrate capping, which serves also as an excitation quenching inhibitor spacer, a pH indicator dye shell, and a polyacrylamide cladding are employed. Utilizing this technique, an enhancement of up to 20 times in the two-photon fluorescence of the indicator dye is observed. Although a significant portion of the enhanced fluorescence signal is due to one-photon processes accompanying the SH generation of the exciting light, this method preserves all the advantages of infrared-excited, two-photon microscopy: enhanced penetration depth, localized excitation, low photobleaching, low autofluorescence, and low cellular damage.

Hydrogel nanoparticles with covalently linked coomassie blue for brain tumor delineation visible to the surgeon.

Delineation of tumor margins is a critical and challenging objective during brain cancer surgery. A tumor-targeting deep-blue nanoparticle-based visible contrast agent is described, which, for the first time, offers in vivo tumor-specific visible color staining. This technology thus enables color-guided tumor resection in real time, with no need for extra equipment or special lighting conditions. The visual contrast agent consists of polyacrylamide nanoparticles covalently linked to Coomassie Blue molecules (for nonleachable blue color contrast), which are surface-conjugated with polyethylene glycol and F3 peptides for efficient in vivo circulation and tumor targeting, respectively.

Methylene blue covalently loaded polyacrylamide nanoparticles for enhanced tumor-targeted photodynamic therapy.

The use of targeted nanoparticles (NPs) as a platform for loading photosensitizers enables selective accumulation of the photosensitizers in the tumor area, while maintaining their photodynamic therapy (PDT) effectiveness. Here two novel kinds of methylene blue (MB)-conjugated polyacrylamide (PAA) nanoparticles, MBI-PAA NPs and MBII-PAA NPs, based on two separate MB derivatives, are developed for PDT. This covalent conjugation with the NPs (i) improves the loading of MB, (ii) prevents any leaching of MB from the NPs and (iii) protects the MB from the effects of enzymes in the biological environment. The loading of MB into these two kinds of NPs was controlled by the input amount, resulting in concentrations with optimal singlet oxygen production. For each of the MB-NPs, the highest singlet oxygen production was found for an MB loading of around 11 nmol mg(-1). After attachment of F3 peptide groups, for targeting, each of these NPs was taken up, selectively, by MDA-MB-435 tumor cells, in vitro. PDT tests demonstrated that both kinds of targeted NPs resulted in effective tumor cell kill, following illumination, while not causing dark toxicity.

Novel methods to incorporate photosensitizers into nanocarriers for cancer treatment by photodynamic therapy.

OBJECTIVE: A hydrophobic photosensitizer, 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a (HPPH), was loaded into nontoxic biodegradable amine functionalized polyacrylamide (AFPAA) nanoparticles using three different methods (encapsulation, conjugation, and post-loading), forming a stable aqueous dispersion. Each formulation was characterized for physicochemical properties as well as for photodynamic performance so as to determine the most effective nanocarrier formulation containing HPPH for photodynamic therapy (PDT). MATERIALS AND METHODS: HPPH or HPPH-linked acrylamide was added into monomer mixture and polymerized in a microemulsion for encapsulation and conjugation, respectively. For post-loading, HPPH was added to an aqueous suspension of pre-formed nanoparticles. Those nanoparticles were tested for optical characteristics, dye loading, dye leaching, particle size, singlet oxygen production, dark toxicity, in vitro photodynamic cell killing, whole body fluorescence imaging and in vivo PDT. RESULTS: HPPH was successfully encapsulated, conjugated or post-loaded into the AFPAA nanoparticles. The resultant nanoparticles were spherical with a mean diameter of 29 ± 3 nm. The HPPH remained intact after entrapment and the HPPH leaching out of nanoparticles was negligible for all three formulations. The highest singlet oxygen production was achieved by the post-loaded formulation, which caused the highest phototoxicity in in vitro assays. No dark toxicity was observed. Post-loaded HPPH AFPAA nanoparticles were localized to tumors in a mouse colon carcinoma model, enabling fluorescence imaging, and producing a similar photodynamic tumor response to that of free HPPH in equivalent dose. CONCLUSIONS: Post-loading is the promising method for loading nanoparticles with hydrophobic photosensitizers to achieve effective in vitro and in vivo PDT.

Nanoencapsulation method for high selectivity sensing of hydrogen peroxide inside live cells.

Reactive oxygen species (ROS) are ubiquitous in life and death processes of cells (Finkel, T.; Holbrook, N. J. Nature 2000, 408 (6809), 239-247), with a major role played by the most stable ROS, hydrogen peroxide (H(2)O(2)). However, the study of H(2)O(2) in live cells has been hampered by the absence of selective probes. Described here is a novel nanoprobe ("nanoPEBBLE") with dramatically improved H(2)O(2) selectivity. The traditional molecular probe, 2',7'-dichlorofluorescin (DCFH), which is also sensitive to most other ROS, was empowered with high selectivity by a nanomatrix that blocks the interference from all other ROS (hydroxyl radical, superoxide, nitric oxide, peroxynitrite, hypochlorous acid, and alkylperoxyl radical), as well as from enzymes such as peroxidases. The blocking is based on the combination of multiple exclusion principles: time barrier, hydrophobic energy barrier, and size barrier. However, H(2)O(2) sensitivity is maintained down to low nanomolar concentrations. The surface of the nanoprobe was engineered to address biological applications, and the power of this new nanoPEBBLE is demonstrated by its use on RAW264.7 murine macrophages. These nanoprobes may provide a powerful chemical detection/imaging tool for investigating biological mechanisms related to H(2)O(2) or other species, with high spatial and temporal resolution.

Near infrared luminescent oxygen nanosensors with nanoparticle matrix tailored sensitivity.

The development of sensors for noninvasive determination of oxygen levels in live cells and tissues is critical for the understanding of cellular functions, as well as for monitoring the status of disease, such as cancer, and for predicting the efficacy of therapy. We describe such nontoxic, targeted, and ratiometric 30 nm oxygen nanosensors made of polyacrylamide hydrogel, near-infrared (NIR) luminescent dyes, and surface-conjugated tumor-specific peptides. They enabled noninvasive real-time monitoring of oxygen levels in live cancer cells under normal and hypoxic conditions. The required sensitivity, brightness, selectivity, and stability were achieved by tailoring the interaction between the nanomatrix and indicator dyes. The developed nanosensors may become useful for in vivo oxygen measurements.

Extremely High Barrier Performance of Organic-Inorganic Nanolaminated Thin Films for Organic Light-Emitting Diodes.

This work presents a novel barrier thin film based on an organic-inorganic nanolaminate, which consists of alternating nanolayers of self-assembled organic layers (SAOLs) and Al2O3. The SAOLs-Al2O3 nanolaminated films were deposited using a combination of molecular layer deposition and atomic layer deposition techniques at 80 °C. Modulation of the relative thickness ratio of the SAOLs and Al2O3 enabled control over the elastic modulus and stress in the films. Furthermore, the SAOLs-Al2O3 thin film achieved a high degree of mechanical flexibility, excellent transmittance (>95%), and an ultralow water-vapor transmission rate (2.99 × 10-7 g m-2 day-1), which represents one of the lowest permeability levels ever achieved by thin film encapsulation. On the basis of its outstanding barrier properties with high flexibility and transparency, the nanolaminated film was applied to a commercial OLEDs panel as a gas-diffusion barrier film. The results showed defect propagation could be significantly inhibited by incorporating the SAOLs layers, which enhanced the durability of the panel.

Inkjet-Assisted Nanotransfer Printing for Large-Scale Integrated Nanopatterns of Various Single-Crystal Organic Materials.

Inkjet-assisted nanotransfer printing (inkjet-NTP) facilitates spatial control of many arrays of various organic functional materials on a single substrate with a high-throughput integration process, enabling monolithic integration of various organic nanopatterns. Inkjet-NTP enables wafer-scale organic electronic circuits composed of field-effect transistors, complementary inverters, and p-n diodes, demonstrating its capability to produce a high-performance, multifunctional organic chip.

Wafer-scale single-domain-like graphene by defect-selective atomic layer deposition of hexagonal ZnO.

Large-area graphene films produced by means of chemical vapor deposition (CVD) are polycrystalline and thus contain numerous grain boundaries that can greatly degrade their performance and produce inhomogeneous properties. A better grain boundary engineering in CVD graphene is essential to realize the full potential of graphene in large-scale applications. Here, we report a defect-selective atomic layer deposition (ALD) for stitching grain boundaries of CVD graphene with ZnO so as to increase the connectivity between grains. In the present ALD process, ZnO with a hexagonal wurtzite structure was selectively grown mainly on the defect-rich grain boundaries to produce ZnO-stitched CVD graphene with well-connected grains. For the CVD graphene film after ZnO stitching, the inter-grain mobility is notably improved with only a little change in the free carrier density. We also demonstrate how ZnO-stitched CVD graphene can be successfully integrated into wafer-scale arrays of top-gated field-effect transistors on 4-inch Si and polymer substrates, revealing remarkable device-to-device uniformity.

Overcoming cancer multidrug resistance by codelivery of doxorubicin and verapamil with hydrogel nanoparticles.

The efficacy of chemotherapy is often inhibited by multidrug resistance (MDR). A highly engineerable hydrogel nanoparticle (NP) serves as a carrier for the optimal codelivery to tumor cells of the chemodrug, doxorubicin (Dox) and the chemosensitizer, verapamil (Vera), aiming at alleviating tumor MDR. The hydrogel NPs are prepared via the copolymerization of acrylamide and 2-carboxyethyl acrylate. Dox and Vera are post-loaded into the respective NPs, with drug loading around 7.7 wt% and 8.0 wt%, respectively. The codelivery of Dox-NPs and Vera-NPs increases the intracellular accumulation of Dox, and significantly enhances the cell killing ability of Dox with respect to NCI/ADR-RES cells in vitro. These findings suggest that such codelivery nanoplatforms provide a promising route for overcoming tumor MDR.

A high-throughput photodynamic therapy screening platform with on-chip control of multiple microenvironmental factors.

We present a novel high-throughput microfluidic platform that enables the evaluation of the anticancer efficacy of photodynamic therapy (PDT) drugs over multiple microenvironmental factors. PDT is uniquely complex, originating from its dependence on three separate but essential elements: drug (also called photosensitizer), oxygen, and light. Thus, obtaining a reliable evaluation of PDT efficacy is highly challenging, requiring considerable effort and time to evaluate all three interdependent parameters. In this paper, we report a high-throughput efficacy screening platform that we implemented by developing microfluidic components that individually control basic PDT elements (photosensitizer concentrations, oxygen levels, and light fluence) and then integrating them into a single triple-layer device. The integrated microfluidic chip consists of an array of small compartments, each corresponding to a specific combination of these three variables. This allows for more than 1000 different conditions being tested in parallel. Cancer cells are cultured within the device, exposed to different PDT conditions, and then monitored for their viability using live/dead fluorescence staining. The entire screening assay takes only 1 hour, and the collected PDT outcomes (cell viability) for combinatorial screening are analysed and reported as traditional dose-response curves or 3D bubble charts using custom software. As a proof of concept, methylene blue is adopted as a photosensitizer and its drug efficacy on C6 glioma cells has been successfully evaluated for a total of 324 PDT conditions using the fabricated chip. This platform can facilitate not only the development of new photosensitizers but also the optimization of current PDT protocols.

Assessing the barriers to image-guided drug delivery.

Imaging has become a cornerstone for medical diagnosis and the guidance of patient management. A new field called image-guided drug delivery (IGDD) now combines the vast potential of the radiological sciences with the delivery of treatment and promises to fulfill the vision of personalized medicine. Whether imaging is used to deliver focused energy to drug-laden particles for enhanced, local drug release around tumors, or it is invoked in the context of nanoparticle-based agents to quantify distinctive biomarkers that could risk stratify patients for improved targeted drug delivery efficiency, the overarching goal of IGDD is to use imaging to maximize effective therapy in diseased tissues and to minimize systemic drug exposure in order to reduce toxicities. Over the last several years, innumerable reports and reviews covering the gamut of IGDD technologies have been published, but inadequate attention has been directed toward identifying and addressing the barriers limiting clinical translation. In this consensus opinion, the opportunities and challenges impacting the clinical realization of IGDD-based personalized medicine were discussed as a panel and recommendations were proffered to accelerate the field forward.

Hydrogen peroxide (H2O2) detection with nanoprobes for biological applications: a mini-review.

Hydrogen peroxide (H2O2) is an important member of the reactive oxygen species, playing various roles in biology and medicine. The conventional detection methods for H2O2 are often restricted by their limited sensitivity, poor selectivity towards H2O2, inappropriate physicochemical properties for detection in biological environments, long response time, etc. We briefly review here some recent nanotechnology--based approaches for H2O2 detection, which present an effective improvement, overcoming some of the limitations of the conventional H2O2 sensing techniques.

Polymer-Protein Hydrogel Nanomatrix for Stabilization of Indocyanine Green towards Targeted Fluorescence and Photoacoustic Bio-imaging.

Indocyanine green (ICG) is an optical contrast agent commonly used for a variety of imaging applications. However, certain limitations of the free dye molecule, concerning its low stability, uncontrolled aggregation and lack of targeting ability, have limited its use. Presented here is a method of embedding ICG in a novel polymer/protein hybrid nanocarrier so as to overcome the above inherent drawbacks of the free molecule. The hybrid nanocarrier consists of a non-toxic and biocompatible polyacrylamide nanoparticle (PAA NP) matrix that incorporates human serum albumin (HSA). This nanocarrier was synthesized through pre-conjugation with HSA and amine functionalized monomer, followed by polymerization using biodegradable cross-linkers, in a water-in-oil emulsion. The ICG dye is loaded into the HSA conjugated PAA nanoparticles (HSA-PAA NPs) through post-loading. Compared to the PAA polymer matrix, the presence of hydrophobic pockets in the HSA-PAA NPs further increases the chemical and physical stability of ICG. This is manifested by lowering the chemical degradation rates under physiological conditions, as well as by improving the thermal- and photo-stability of the dye. A targeting moiety, F3-Cys peptide, was attached to the surface of the NPs, for selective delivery to specific cancer cell lines. The suitability of these NPs for optical imaging applications was demonstrated by performing fluorescence imaging on a rat gliosarcoma cell line (9L). We also present the photoacoustic response of the HSA-PAA NPs, used as imaging contrast agents, in the spectral window of 700 nm to 800 nm.

Nanoparticle PEBBLE sensors in live cells.

Live cell studies are of fundamental importance to the life sciences and their medical applications. Nanoparticle (NP)-based sensor platforms have many advantages as sensors for intracellular measurements, due to their flexible engineerability, noninvasive nature (due to their nano-size and nontoxic matrix), and, for some of the NPs, intrinsic optical properties. NP-based fluorescent sensors for intracellular measurements, so called PEBBLE sensors, have been developed for many important intracellular analytes and functions, including ions, small molecules, reactive oxygen species, physical properties, and enzyme activities, which are involved in many chemical, biochemical, and physical processes taking place inside the cell. PEBBLE sensors can be used with a standard microscope for simultaneous optical imaging of cellular structures and sensing of composition and function, just like investigations performed with molecular probes. However, PEBBLE sensors of any design and matrix can be delivered into cells by several standard methods, unlike dye molecules that need to be cell permeable. Furthermore, new sensing possibilities are enabled by PEBBLE nanosensors, which are not possible with molecular probes. This review summarizes a variety of designs of the PEBBLE sensors, their characteristics, and their applications to cells.

Lifetime-based photoacoustic oxygen sensing in vivo.

The determination of oxygen levels in blood and other tissues in vivo is critical for ensuring proper body functioning, for monitoring the status of many diseases, such as cancer, and for predicting the efficacy of therapy. Here we demonstrate, for the first time, a lifetime-based photoacoustic technique for the measurement of oxygen in vivo, using an oxygen sensitive dye, enabling real time quantification of blood oxygenation. The results from the main artery in the rat tail indicated that the lifetime of the dye, quantified by the photoacoustic technique, showed a linear relationship with the blood oxygenation levels in the targeted artery.

Multifunctional biodegradable polyacrylamide nanocarriers for cancer theranostics--a "see and treat" strategy.

We describe here the development of multifunctional nanocarriers, based on amine-functionalized biodegradable polyacrylamide nanoparticles (NPs), for cancer theranostics, including active tumor targeting, fluorescence imaging, and photodynamic therapy. The structural design involves adding primary amino groups and biodegradable cross-linkers during the NP polymerization, while incorporating photodynamic and fluorescent imaging agents into the NP matrix, and conjugating PEG and tumor-targeting ligands onto the surface of the NPs. The as-synthesized NPs are spherical, with an average diameter of 44 nm. An accelerated biodegradation study, using sodium hydroxide or porcine liver esterase, indicated a hydrogel polymer matrix chain collapse within several days. By using gel permeation chromatography, small molecules were detected, after the degradation. In vitro targeting studies on human breast cancer cells indicate that the targeted NPs can be transported efficiently into tumor cells. Incubating the multifunctional nanocarriers into cancer cells enabled strong fluorescence imaging. Irradiation of the photosensitizing drug, incorporated within the NPs, with light of a suitable wavelength, causes significant but selective damage to the impregnated tumor cells, but only inside the illuminated areas. Overall, the potential of polymeric-based NPs as biodegradable, multifunctional nanocarriers, for cancer theranostics, is demonstrated here.

Polymeric nanoparticles for photodynamic therapy.

Photodynamic therapy is a relatively new clinical therapeutic modality that is based on three key components: photosensitizer, light, and molecular oxygen. Nanoparticles, especially targeted ones, have recently emerged as an efficient carrier of drugs or contrast agents, or multiple kinds of them, with many advantages over molecular drugs or contrast agents, especially for cancer detection and treatment. This paper describes the current status of PDT, including basic mechanisms, applications, and challenging issues in the optimization and adoption of PDT; as well as recent developments of nanoparticle-based PDT agents, their advantages, designs and examples of in vitro and in vivo applications, and demonstrations of their capability of enhancing PDT efficacy over existing molecular drug-based PDT.