In vivo O2 measurement inside single photosynthetic cells.

The oxygen evolution of single cells was investigated using a nano-probe with an ultra-micro electrode (UME) in a submicron sized system in combination with a micro-fluidic system. A single cell was immobilized in the micro-fluidic system and a nano-probe was inserted into the cytosolic space of the single cell. Then, the UME was used for an in vivo amperometric experiment at a fixed potential and electrochemical impedance spectroscopy to detect oxygen evolution of the single cell under various light intensities.

Direct extraction of photosynthetic electrons from single algal cells by nanoprobing system.

There are numerous sources of bioenergy that are generated by photosynthetic processes, for example, lipids, alcohols, hydrogen, and polysaccharides. However, generally only a small fraction of solar energy absorbed by photosynthetic organisms is converted to a form of energy that can be readily exploited. To more efficiently use the solar energy harvested by photosynthetic organisms, we evaluated the feasibility of generating bioelectricity by directly extracting electrons from the photosynthetic electron transport chain before they are used to fix CO(2) into sugars and polysaccharides. From a living algal cell, Chlamydomonas reinhardtii, photosynthetic electrons (1.2 pA at 6000 mA/m(2)) were directly extracted without a mediator electron carrier by inserting a nanoelectrode into the algal chloroplast and applying an overvoltage. This result may represent an initial step in generating "high efficiency" bioelectricity by directly harvesting high energy photosynthetic electrons.

Open micro-fluidic system for atomic force microscopy-guided in situ electrochemical probing of a single cell.

Ultra-sharp nano-probes and customized atomic force microscopy (AFM) have previously been developed in our laboratory for in situ sub-cellular probing of electrochemical phenomena in living plant cells during their photosynthesis. However, this AFM-based electrochemical probing still has numerous engineering challenges such as immobilization of the live cells, compatibility of the immobilization procedure with AFM manipulation of the probe, maintenance of biological activity of the cells for an extended time while performing the measurements, and minimization of electrochemical noise. Thus, we have developed an open micro-fluidic channel system (OMFC) in which individual cells can be immobilized in micro-traps by capillary flow. This system affords easy AFM access and allows for maintenance of the cells in a well-defined chemical environment, which sustains their biological activity. The use of micro-channels for making the electrochemical measurements significantly reduces parasitic electrical capacitances and allows for current detection in the sub-pico-ampere range at high signal bandwidths. The OMFC was further studied using simulation packages for optimal design conditions. This system was successfully used to measure light-dependent oxidation currents of a few pico-amperes from the green alga Chlamydomonas reinhardtii.

Fabrication of multi-layered biodegradable drug delivery device based on micro-structuring of PLGA polymers.

A programmable and biodegradable drug delivery device is desirable when a drug needs to be administered locally. While most local drug delivery devices made of biodegradable polymers relied on the degradation of the polymers, the degradation-based release control is often limited by the property of the polymers. Thus, we propose micro-geometry as an alternative measure of controlling drug release. The proposed devices consist of three functional layers: diffusion control layer via micro-orifices, diffusion layer, and drug reservoir layers. A micro-fabrication technology was used to shape an array of micro-orifices and micro-cavities in 85/15PLGA layers. A thin layer of fast degrading 50/50PLGA was placed as the diffusion layer between the 85/15PLGA layers to prevent any burst-type release. To modulate the release of the devices, the dimension and location of the micro-orifices were varied and the responding in vitro release response of tetracycline was monitored over 2 weeks. The release response to the different micro-geometry was prominent and further analyzed by FEM simulation. Comparison of the experiments to the simulated results identified that the variation of micro-geometry influenced also the volume-dependent degradation rate and induced the osmotic pressure.

The construction of three-dimensional micro-fluidic scaffolds of biodegradable polymers by solvent vapor based bonding of micro-molded layers.

It is increasingly important to control cell growth into and within artificial scaffolds. Tissues such as skin, blood vessels, and cartilage have multi-layer structures with different cells in each layer. With the aid of micro-fabrication technology, a novel scaffolding method for biodegradable polymers such as polylactic acid (PLA), polyglycolic acid (PGA), and the copolymers poly(lactide-co-glycolide)(PLGA), was developed to construct three-dimensional multi-layer micro-fluidic tissue scaffolds. The method emphasizes micro-fluidic interconnections between layers within the scaffolds and maintenance of high-resolution geometries during the bonding process for the creation of multi-layered scaffolds. Micro-holes (10-100 microm), micro-channels, and micro-cavities were all created by micro-molding. Solvent-vapor based bonding of micro-molded layers preserved 20 microm sized structures. Sample scaffolds were constructed for purposes such as channel-directed cell growth and size-based cell sorting. Further extension of these techniques to create a micro-vascular network within or between layers is possible. Culturing of human coronary artery endothelial cells (HCAECs) on the sample scaffolds demonstrated the biocompatibility of the developed process and the strong influence of high-resolution micro-geometries on HCAEC growth.