Xu-dong Wang

Ultra-Small, Highly Stable, and Sensitive Dual Nanosensors for Imaging Intracellular Oxygen and pH in Cytosol

We report on the first dual nanosensors for imaging of pH values and oxygen partial pressure in cells. The sensors have a unique nanostructure in that a soft core structure is rigidized with a silane reagent, while poly(ethylene glycol) chains form an outer shell. Lipophilic oxygen-sensitive probes and reference dyes are encapsulated inside the hydrophobic core, while a pH-sensitive probe is covalently attached to the poly(ethylene glycol) end-group on the shell. The core/shell structure renders the nanosensors well dispersed and highly stable in various kinds of aqueous media. Their average size is 12 nm, and they respond to both pH and oxygen in the physiological range. They do not pass cell membranes, but can be internalized into the cellular cytosol by electroporation, upon which they enable sensing and imaging of pH values and oxygen with high spatial resolution. The nanosensor strategy shown here is expected to be applicable to the development of various other kinds of multiple nanosensors for in vivo studies.

Self-referenced RGB colour imaging of intracellular oxygen

Intracellular oxygen is an important indicator for cell metabolism and respiration. We have designed self-referenced RGB PEBBLEs that enable a simple readout of the intracellular oxygen distribution with conventional wide-field microscopy and a standard RGB digital camera. The RGB PEBBLEs consist of a hydrophobic matrix covered with amino groups on the surface to confer water-dispersibility. Two luminophores are incorporated in a hydrophobic polystyrene matrix that is highly permeable to oxygen. In polystyrene, the dyes are largely protected from quenching or aggregation by cellular components. The dyes have been selected to match the green and the red channel of digital cameras. While the red emission of the oxygen probe is highly sensitive to oxygen with a quenching response of 74%, the green emission of the reference dye is stable under varying oxygen concentrations. Ratiometric images of intracellular oxygen have been acquired that are inherently resistant to fluctuations in absolute signal intensities. As RGB PEBBLEs respond within seconds to changing oxygen concentrations, they are amenable to monitoring fast cellular dynamics.

Photographing Oxygen Distribution

O2, where are you? The spatial distribution of oxygen can be imaged with a conventional digital camera by making use of a specially designed fluorescent sensor film containing a quenchable red-emitting probe for oxygen along with a green-emitting reference fluorophore. The technique exploits the RGB channels involved in digital photography (see picture) to deliver a simple method for quantitative sensing and imaging of this important species.

High-Resolution Colorimetric Assay for Rapid Visual Readout of Phosphatase Activity Based on Gold/Silver Core/Shell Nanorod

Nanostructure-based visual assay has been developed for determination of enzymatic activity, but most involve in poor visible color resolution and are not suitable for routine utilization. Herein, we designed a high-resolution colorimetric protocol based on gold/silver core/shell nanorod for visual readout of alkaline phosphatase (ALP) activity by using bare-eyes. The method relied on enzymatic reaction-assisted silver deposition on gold nanorod to generate significant color change, which was strongly dependent on ALP activity. Upon target ALP introduction into the substrate, the ascorbic acid 2-phosphate was hydrolyzed to form ascorbic acid, and then, the generated ascorbic acid reduced silver ion to metal silver and coated on the gold nanorod, thereby resulting in the blue shift of longitudinal localized surface plasmon resonance peak of gold nanorod accompanying a perceptible color change from red to orange to yellow to green to cyan to blue and to violet. Under optimal conditions, the designed method exhibited the wide linear range 5-100 mU mL(-1) ALP with a detection limit of 3.3 mU mL(-1). Moreover, it could be used for the semiquantitative detection of ALP from 20 to 500 mU mL(-1) by using the bare-eyes. The coefficients of variation for intra- and interassay were below 3.5% and 6.2%, respectively. Finally, this method was validated for the analysis of real-life serum samples, giving results matched well with those from the 4-nitrophenyl phosphate disodium salt hexahydrate (pNPP)-based standard method. In addition, the system could even be utilized in the enzyme-linked immunosorbent assay (ELISA) to detect IgG at picomol concentration. With the merits of simplification, low cost, user-friendliness, and sensitive readout, the gold nanorod-based colorimetric assay has the potential to be utilized by the public and opens a new horizon for bioassays.

Fluorescent pH‐Sensitive Nanoparticles in an Agarose Matrix for Imaging of Bacterial Growth and Metabolism

Living color: fluorescent pH-sensitive nanoparticles 12 nm in diameter were prepared and incorporated into agarose gel in a Petri dish to image pH changes during bacterial growth and metabolism.

Fabrication of a Colorimetric Electrochemiluminescence Sensor

A colorimetric electrochemiluminescence (ECL) sensor was fabricated for the first time, based on a dual-color system including a strong red Ru(bpy)(3)(2+) ECL and a green reference light from a light emitting diode. Traditional ECL intensity information can be easily transformed into a color variation with this sensor, and the color variation can be directly monitored using the naked eye or a commercial CCD camera. The sensor has been successfully used to determine the concentration of tripropylamine, proline (enhancing system), and dopamine (quenching system). The results indicated that the color variation obtained corresponded to the concentration of target analytes. This sensor has potential application in rapid and semiquantitative ECL analysis.

Preparation of Reversible Colorimetric Temperature Nanosensors and Their Application in Quantitative Two-Dimensional Thermo-Imaging

Reversible colorimetric temperature nanosensors were prepared using a very simple precipitation method to encapsulate two color luminescent dyes. These nanosensors presented obvious reversible temperature response and enabled both rapid colorimetric temperature estimation using the eyes and quantitative two-dimensional thermo-imaging. Heat-exchange induced fluid motion was, for the first time, rapidly, precisely, and quantitatively imaged by just taking color pictures, and this presented good temporal and spatial resolution for studying heat-driven hydrodynamics. These nanosensors should have great application in micro/nanoscale research and also fabrication into films for macroscopic study.

Ratiometric luminescence 2D in vivo imaging and monitoring of mouse skin oxygenation

Tissue oxygenation plays a critical role in the pathogenesis of various diseases, but non-invasive, robust and user-friendly methods for its measurement in vivo still need to be established. Here, we are presenting an in vivo oxygen-detection system that uses ratiometric luminescence imaging (RLI) as a readout scheme to determine the skin oxygen tension of mouse hind footpads via side-by-side comparison with more established techniques including luminescence-lifetime imaging using planar sensor films and the polarographic electrode as the gold standard. We also demonstrate that this technology allows the detection of changes in mouse skin tissue oxygenation induced by subjecting mice to systemic hypoxia. The data demonstrate oxygen imaging based on RLI to be a most useful tool for reliably and easily analyzing and monitoring skin tissue oxygenation in vivo. This technology will advance our understanding of local regulation of skin tissue oxygenation in various disease conditions.

Multifunctional Silica Nanoparticles for Covalent Immobilization of Highly Sensitive Proteins.

A convenient reverse micellar one-pot reaction yields multifunctional silica nanoparticles, which can be tailored to effectively suppress non-specific adsorption and, at the same time, enable efficient specific covalent immobilization of proteins. Using two highly sensitive proteins, it is demonstrated that the new particles provide a suitable microenvironment to maintain the proteins activity.

An optical biosensing film for biochemical oxygen demand determination in seawater with an automatic flow sampling system

An on-line roboticized apparatus, including an optical biosensing film with an automatic flow sampling system, has been developed for biochemical oxygen demand (BOD) determination of seawater. The sensing film employed in the apparatus consisted of an organically modified silicate (ORMOSIL) film embedded with tri(4,7-diphenyl-1,10-phenanthroline) ruthenium(II) perchlorate. Three species of microorganism cultivated from seawater were immobilized in an ORMOSIL-polyvinyl alcohol matrix. Possible factors affecting BOD determination were studied, including sampling frequency, temperature, pH and sodium chloride concentration. Based on measurements of the linear fluctuant coefficients and the reproducibility of its response to seawater, the BOD apparatus showed the advantages of high veracity and short response time. Generally, the linear fluctuant coefficient (R2) in the BOD range 0.2–40 mg l−1 was 0.9945 when using a glucose/glutamate (GGA) BOD standard solution. A reproducible response for the BOD sensing film of within ±2.8% could be obtained in the 2 mg l−1 GGA solution. The BOD apparatus was applied to the BOD determination of seawater, and the values estimated by this biosensing apparatus correlated well with those determined by the conventional 5 day BOD (BOD5) test.