Haixin Zhu

Characterization of deep wet etching of fused silica glass for single cell and optical sensor deposition

The development of a high-throughput single-cell metabolic rate monitoring system relies on the use of transparent substrate material for a single cell-trapping platform. The high optical transparency, high chemical resistance, improved surface quality and compatibility with the silicon micromachining process of fused silica make it very attractive and desirable for this application. In this paper, we report the results from the development and characterization of a hydrofluoric acid (HF) based deep wet-etch process on fused silica. The pin holes and notching defects of various single-coated masking layers during the etching are characterized and the most suitable masking materials are identified for different etch depths. The dependence of the average etch rate and surface roughness on the etch depth, impurity concentration and HF composition are also examined. The resulting undercut from the deep HF etch using various masking materials is also investigated. The developed and characterized process techniques have been successfully implemented in the fabrication of micro-well arrays for single cell trapping and sensor deposition. Up to 60 µm deep micro-wells have been etched in a fused silica substrate with over 90% process yield and repeatability. To our knowledge, such etch depth has never been achieved in a fused silica substrate by using a non-diluted HF etchant and a single-coated masking layer at room temperature.

Deep reactive ion etching of fused silica using a single-coated soft mask layer for bio-analytical applications

In this note, we present our results from process development and characterization of reactive ion etching (RIE) of fused silica using a single-coated soft masking layer (KMPR® 1025, Microchem Corporation, Newton, MA). The effects of a number of fluorine-radical-based gaseous chemistries, the gas flow rate, RF power and chamber pressure on the etch rate and etching selectivity of fused silica were studied using factorial experimental designs. RF power and pressure were found to be the most important factors in determining the etch rate. The highest fused silica etch rate obtained was about 933 A min−1 by using SF6-based gas chemistry, and the highest etching selectivity between the fused silica and KMPR® 1025 was up to 1.2 using a combination of CF4, CHF3 and Ar. Up to 30 µm deep microstructures have been successfully fabricated using the developed processes. The average area roughness (Ra) of the etched surface was measured and results showed it is comparable to the roughness obtained using a wet etching technique. Additionally, near-vertical sidewalls (with a taper angle up to 85°) have been obtained for the etched microstructures. The processes developed here can be applied to any application requiring fabrication of deep microstructures in fused silica with near-vertical sidewalls. To our knowledge, this is the first note on deep RIE of fused silica using a single-coated KMPR® 1025 masking layer and a non-ICP-based reactive ion etcher.

High Throughput Micropatterning of Optical Oxygen Sensor for Single Cell Analysis

In this paper, we present our results from process development and characterization of optical oxygen sensors that are patterned by traditional UV lithography. An oxygen sensitive luminescent probe, platinum octaethylporphyrin, was encapsulated in commercially purchased photoresist (AZ5214) to form uniform thin sensor films on fused silica substrates. Plasticizer ethoxylated trimethylolpropane triacrylate (SR454) was added to the dye-photoresist sensor mixtures to improve the oxygen sensitivity. The optimum sensor mixture composition that can be patterned with maximum sensitivity was identified. The microfabrication process conditions, cell adherence and oxygen sensitivity results from patterned structures were characterized in detail. Down to 3 μm features have been fabricated on fused silica substrates using the developed techniques. The result implies that the developed methods can provide a feasible way to miniaturize the optical sensor system for single cell analysis with precise control of sensor volume and response.

Fabrication of implantable polyimide based neural implants with flexible regions to accommodate micromovement

A unique structure for chronically implantable cortical electrodes using polyimide polymer was devised, which provides both flexibility between brain tissues and skull and stiffness for easy insertion. The fabricated implants are tri-shanks with 5 recording sites (20/spl times/20 /spl mu/m) and 2 vias per electrode of 40/spl times/40 /spl mu/m. Each recording site was connected to the external circuitry via a 15-channel connector, which is especially designed to facilitate processing of neural signals to the external circuitry. Measured impedance values are in /spl sim/2 Mohm at 1 KHz. For a 5 /spl mu/m thick silicon backbone electrode, the stiffness was improved 10 times larger than that of the electrode without silicon backbone layer. Stiff electrodes with 5 /spl mu/m and 10 /spl mu/m thick backbone silicon penetrated pia of rat without buckling.

Characterization of KMPR ® 1025 as a masking layer for deep reactive ion etching of fused silica

In this paper we report our results from the process development and characterization of KMPR®1025 as a complimentary metal-oxide semiconductor (CMOS) process compatible masking layer for the deep reactive ion etching (RIE) of fused silica. The processing conditions and the etch resistivity of KMPR®1025 as a function of different parameters like pressure, gaseous composition, gas flow rate and hard-bake conditions are examined in details in this study.

Automated platform for multiparameter stimulus response studies of metabolic activity at the single-cell level

We have developed a fully automated platform for multiparameter characterization of physiological response of individual and small numbers of interacting cells. The platform allows for minimally invasive monitoring of cell phenotypes while administering a variety of physiological insults and stimuli by means of precisely controlled microfluidic subsystems. It features the capability to integrate a variety of sensitive intra- and extra-cellular fluorescent probes for monitoring minute intra- and extra-cellular physiological changes. The platform allows for performance of other, post- measurement analyses of individual cells such as transcriptomics. Our method is based on the measurement of extracellular metabolite concentrations in hermetically sealed ~200-pL microchambers, each containing a single cell or a small number of cells. The major components of the system are a) a confocal laser scan head to excite and detect with single photon sensitivity the emitted photons from sensors; b) a microfluidic cassette to confine and incubate individual cells, providing for dynamic application of external stimuli, and c) an integration module consisting of software and hardware for automated cassette manipulation, environmental control and data collection. The custom-built confocal scan head allows for fluorescence intensity detection with high sensitivity and spatial confinement of the excitation light to individual pixels of the sensor area, thus minimizing any phototoxic effects. The platform is designed to permit incorporation of multiple optical sensors for simultaneous detection of various metabolites of interest. The modular detector structure allows for several imaging modalities, including high resolution intracellular probe imaging and extracellular sensor readout. The integrated system allows for simulation of physiologically relevant microenvironmental stimuli and simultaneous measurement of the elicited phenotypes. We present details of system design, system characterization and metabolic response analysis of individual eukaryotic cells.

High throughput micropatterning of optical oxygen sensor

In this paper, we present our results from process development and characterization of optical oxygen sensors that are patterned by traditional UV lithography. An oxygen sensitive luminescent probe, platinum octaethylporphyrin (PtOEP), was encapsulated in commercially purchased photoresist (AZ5214) to form uniform thin sensor films on fused silica substrates. Plasticizer ethoxylated trimethylolpropane triacrylate (SR454) was added to the dye-photoresist sensor mixtures to improve the oxygen sensitivity. The optimum sensor mixture composition that can be patterned with maximum sensitivity was identified. The microfabrication process conditions, cell adherence and oxygen sensitivity results from patterned structures were characterized in detail. Down to 3 µm features have been fabricated on fused silica substrates using the developed techniques. The result implies the developed methods can provide a feasible way to miniaturize the optical sensor system for single cell analysis with precise control of sensor volume and response

Simultaneous multiparameter cellular energy metabolism profiling of small populations of cells

Functional and genomic heterogeneity of individual cells are central players in a broad spectrum of normal and disease states. Our knowledge about the role of cellular heterogeneity in tissue and organism function remains limited due to analytical challenges one encounters when performing single cell studies in the context of cell-cell interactions. Information based on bulk samples represents ensemble averages over populations of cells, while data generated from isolated single cells do not account for intercellular interactions. We describe a new technology and demonstrate two important advantages over existing technologies: first, it enables multiparameter energy metabolism profiling of small cell populations (<100 cells)—a sample size that is at least an order of magnitude smaller than other, commercially available technologies; second, it can perform simultaneous real-time measurements of oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and mitochondrial membrane potential (MMP)—a capability not offered by any other commercially available technology. Our results revealed substantial diversity in response kinetics of the three analytes in dysplastic human epithelial esophageal cells and suggest the existence of varying cellular energy metabolism profiles and their kinetics among small populations of cells. The technology represents a powerful analytical tool for multiparameter studies of cellular function.

Multiple sensor arrays for single cell metabolic analysis

We present the design, fabrication and characterization of multiple micro-pocket lid arrays used in live single cell metabolic analysis. In previous work we reported a platform for quantifying single cell oxygen consumption rates realized using a fused silica deep wet etching process. Here we extend that work to a dual-depth wet etching process for microfabrication of multiple sensor trapping (MST) lid arrays. Each lid comprises multiple micro-pockets. Oxygen, pH, other extra-cellular sensors, and reference dye were deposited in the pockets. In order to achieve simultaneous monitoring of multiple metabolic parameters, the lid array serves to hermetically seal arrays of microwells, each containing a single cell. The dual-depth etching process we developed can be easily applied to other glass-based microfabrication purposes requiring dual- or multiple-depth microstructures.