[show abstract][hide abstract] ABSTRACT: We report a novel method for wafer level, high throughput optical chemical sensor patterning, with precise
control of the sensor volume and capability of producing arbitrary microscale patterns. Monomeric
oxygen (O2) and pH optical probes were polymerized with 2-hydroxyethyl methacrylate (HEMA) and
acrylamide (AM) to form spin-coatable and further crosslinkable polymers. A micro-patterning method
based on micro-fabrication techniques (photolithography, wet chemical process and reactive ion etch)
was developed to miniaturize the sensor film onto glass substrates in arbitrary sizes and shapes. The
sensitivity of fabricated micro-patterns was characterized under various oxygen concentrations and pH
values. The process for spatially integration of two sensors (oxygen and pH) on the same substrate surface
was also developed, and preliminary fabrication and characterization results were presented. To the
best of our knowledge, it is the first time that poly(2-hydroxylethyl methacrylate)-co-poly(acrylamide)
(PHEMA-co-PAM)-based sensors had been patterned and integrated at the wafer level with micron
scale precision control using microfabrication techniques. The developed methods can provide a feasible
way to miniaturize and integrate the optical chemical sensor system and can be applied to any
lab-on-a-chip system, especially the biological micro-systems requiring optical sensing of single or
Sensors and Actuators B Chemical 08/2012; · 3.54 Impact Factor
[show abstract][hide abstract] ABSTRACT: Intercellular heterogeneity is a key factor in a variety of core cellular processes including proliferation, stimulus response, carcinogenesis, and drug resistance. However, cell-to-cell variability studies at the single-cell level have been hampered by the lack of enabling experimental techniques. We present a measurement platform that features the capability to quantify oxygen consumption rates of individual, non-interacting and interacting cells under normoxic and hypoxic conditions. It is based on real-time concentration measurements of metabolites of interest by means of extracellular optical sensors in cell-isolating microwells of subnanoliter volume. We present the results of a series of measurements of oxygen consumption rates (OCRs) of individual non-interacting and interacting human epithelial cells. We measured the effects of cell-to-cell interactions by using the system's capability to isolate two and three cells in a single well. The major advantages of the approach are: 1. ratiometric, intensity-based characterization of the metabolic phenotype at the single-cell level, 2. minimal invasiveness due to the distant positioning of sensors, and 3. ability to study the effects of cell-cell interactions on cellular respiration rates.
Journal of Biomedical Optics 03/2012; 17(3):037008. · 2.88 Impact Factor
[show abstract][hide abstract] ABSTRACT: A pico-liter pump is developed and integrated into a robotic manipulation system that automatically selects and transfers individual living cells of interest to analysis locations. The pump is a displacement type pump comprising one cylindrical chamber connected to a capillary micropipette. The top of the chamber is a thin diaphragm which, when deflected, causes the volume of the fluid-filled cylindrical chamber to change thereby causing fluid in the chamber to flow in and out of the micropipette. This enables aspirating and dispensing individual living cells. The diaphragm is deflected by a piezoelectric actuator that pushes against its center. The pump aspirates and dispenses volumes of fluid between 500 pL and 250 nL at flow rates up to 250 nL/s. The piezo-driven diaphragm arrangement provides exquisite control of the flow rate in and out of the capillary orifice. This feature, in turn, allows reduced perturbation of live cells by controlling and minimizing the applied shear stresses.
[show abstract][hide abstract] ABSTRACT: Photosynthetic algae and cyanobacteria have been proposed for producing biofuels through a direct photoconversion process. To accelerate the efforts of discovering and screening microbes for biofuel production, sensitive and high throughput methods to measure photosynthetic activity need to be developed. Here we report the development of new ratiometric optical oxygen and pH dual sensors with three emission colors for measuring photosynthetic activities directly. The dual sensor system can measure oxygen (O(2)) generation and pH increase resulted from carbon dioxide (CO(2)) consumption simultaneously. The sensor was prepared by a copolymerization of three monomeric probes, an intra-reference probe (IRP) which does not respond to pH or O(2), a probe for pH sensing (pHS), and an O(2) probe for O(2) sensing (OS) with 2-hydroxyethyl methacrylate (HEMA) and acrylamide (AM). After polymerization, the three probes were chemically immobilized in an ion and O(2) permeable poly(2-hydroxyethyl methacrylate)-co-polyacrylamide (PHEMA-co-PAM) matrix. The resulted sensing films (membranes) exhibited three emission colors with well separated emission spectra, covering blue, green, and red emission windows, under 380 nm light excitation. Responses of the sensors to pH and dissolved O(2) were investigated in buffers and cyanobacterial cell cultures (Synechocystis sp. PCC 6803). In spite of the strong autofluorescence from cyanobacteria, the sensors were able to determine the pH values and dissolved O(2) concentrations accurately and reproducibly. The measured results using the optical sensors were well in accordance with measurements using electrodes with minimal experimental variations. The sensors were further applied for evaluation of photosynthetic activities of Synechocystis sp. PCC 6803 at the exponential and stationary phases. The results were consistent with biological observation that the photosynthetic activity in the exponential phase was higher than that in the stationary phase.
Journal of Materials Chemistry 01/2011; 2011(48):19293-192301. · 5.97 Impact Factor
[show abstract][hide abstract] ABSTRACT: Oxygen consumption is a fundamental component of metabolic networks, mitochondrial function, and global carbon cycling. To date there is no method available that allows for replicate measurements on attached and unattached biological samples without compensation for extraneous oxygen leaking into the system. Here we present the Respiratory Detection System, which is compatible with virtually any biological sample. The RDS can be used to measure oxygen uptake in microliter-scale volumes with a reversibly sealed sample chamber, which contains a porphyrin-based oxygen sensor. With the RDS, one can maintain a diffusional seal for up to three hours, allowing for the direct measurement of respiratory function of samples with fast or slow metabolic rates. The ability to easily measure oxygen uptake in small volumes with small populations or dilute samples has implications in cell biology, environmental biology, and clinical diagnostics.
Advances in bioscience and biotechnology (Print). 12/2010; 5(5):398-408.
[show abstract][hide abstract] ABSTRACT: Oxygen sensing films were synthesized by a chemical conjugation of functional platinum porphyrin probes in silica gel, polystyrene (PS), and poly(2-hydroxyethyl methacrylate) (PHEMA) matrices. Responses of the sensing films to gaseous oxygen and dissolved oxygen were studied and the influence of the matrices on the sensing behaviors was investigated. Silica gel films had the highest fluorescence intensity ratio from deoxygenated to oxygenated environments and the fastest response time to oxygen. PHEMA films had no response to gaseous oxygen, but had greater sensitivity and a faster response time for dissolved oxygen than those of PS films. The influence of matrices on oxygen response, sensitivity and response time was discussed. The influence is most likely attributed to the oxygen diffusion abilities of the matrices. Since the probes were chemically immobilized in the matrices, no leaching of the probes was observed from the sensing films when applied in aqueous environment. One sensing film made from the PHEMA matrix was used to preliminarily monitor the oxygen consumption of Escherichia coli (E. coli) bacteria. E. coli cell density and antibiotics ampicillin concentration dependent oxygen consumption was observed, indicating the potential application of the oxygen sensing film for biological application.
Sensors and Actuators B Chemical 10/2010; 150(2):579-587. · 3.54 Impact Factor
[show abstract][hide abstract] ABSTRACT: A series of new naphthalimide derivatives were synthesized and studied. Three of the materials (SM1, SM2, and SM3) possess methacrylate(s) moieties as pH sensor monomers, enabling these compounds to be polymerized with other monomers for thin film preparation for extracellular pH sensing. Herein, poly(2-hydroxyethyl methacrylate)-co-poly(acrylamide) (PHEMA-co-PAM) was chosen as the polymer matrix. Structure influences on pH responses and pK(a) values were studied. The film P3 composed of the sensing moiety SM3 has a pK(a) close to the usual biological environmental pH of approximately 7. It was used as an extracellular pH sensor to monitor pH change during the metabolism of prokaryotic Escherichia coli (E. coil). On the other hand, the three sensor monomers are new intracellular biomarkers to sense lysosomes of eukaryotic cells since (1) their pK(a) values are in a range of 5.9-6.8; (2) their emission intensities at acidic conditions (such as at pH 5) are much stronger than those at a neutral condition of pH 7; (3) lysosomes range in size from 0.1 to 1.2 mum in diameter with pH ranging from 4.5 to 5.0, which is much more acidic than the pH value of the cytoplasm (usually with a pH value of approximately 7.2); and (4) the acidity of lysosomes enables a protonation of the amino groups of the pH probes making the sensors emit brightly in acidic organelles by inhibiting the photo-induced electron transfer from the amino groups to the fluorophores. Lysosome sensing was demonstrated using live human brain glioblastoma U87MG cell line, human cervical cancer HeLa cell line, and human esophagus premalignant CP-A and CP-D cell lines by observations of small acidic spherical organelles (lysosomes) and significant colocalizations (82-95%) of the sensors with a commercially available lysosome-selective staining probe LysoTracker Red under confocal fluorescence microscopy.
[show abstract][hide abstract] ABSTRACT: We present a robotic manipulation system for automated selection and transfer of individual living cells to analysis locations. We begin with a commonly used cell transfer technique using glass capillary micropipettes to aspirate and release living cells suspended in liquid growth media. Using vision-based feedback and closed-loop process control, two individual three-axis robotic stages position the micropipette tip in proximity to the cell of interest. The cell is aspirated and the tip is moved to a target location where the cell is dispensed. Computer vision is used to monitor and inspect the success of the dispensing process. In our initial application, the target cell destination is a microwell etched in a fused silica substrate. The system offers a robust and flexible technology for cell selection and manipulation. Applications for this technology include embryonic stem cells transfer, blastomere biopsy, cell patterning, and cell surgery.
IEEE Transactions on Automation Science and Engineering 08/2010; · 1.67 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Journal of Micromechanics and Microengineering 01/2009; 80(19). · 1.79 Impact Factor
[show abstract][hide abstract] ABSTRACT: Haploid Saccharomyces cerevisiae yeast cells use a prototypic cell signalling system to transmit information about the extracellular concentration of mating pheromone secreted by potential mating partners. The ability of cells to respond distinguishably to different pheromone concentrations depends on how much information about pheromone concentration the system can transmit. Here we show that the mitogen-activated protein kinase Fus3 mediates fast-acting negative feedback that adjusts the dose response of the downstream system response to match the dose response of receptor-ligand binding. This 'dose-response alignment', defined by a linear relationship between receptor occupancy and downstream response, can improve the fidelity of information transmission by making downstream responses corresponding to different receptor occupancies more distinguishable and reducing amplification of stochastic noise during signal transmission. We also show that one target of the feedback is a previously uncharacterized signal-promoting function of the regulator of G-protein signalling protein Sst2. Our work suggests that negative feedback is a general mechanism used in signalling systems to align dose responses and thereby increase the fidelity of information transmission.
[show abstract][hide abstract] ABSTRACT: Extended abstract Eukaryotic cells use signal transduction pathways to sense and transmit information about external conditions. Studies of these systems generally focus on time scales that overlook rapid system behaviors potentially important for information encoding and transmission. By measuring multiple system activities at much shorter timescales, we identified a new, rapid feedback mechanism that helps maximize the amount of information transmitted through a prototypic multi-step GPCR and MAPK signal transduction pathway, the yeast pheromone response system . This negative feedback aligns the dose response of downstream system points with the receptor-pheromone binding response. By doing so, the feedback minimizes degradation of information about external pheromone dose transmitted through the cell into the nucleus.
[show abstract][hide abstract] ABSTRACT: The development of a cellular isolation system (CIS) that enables the monitoring of single-cell oxygen consumption rates in real time is presented. The CIS was developed through a multidisciplinary effort within the Microscale Life Sciences Center (MLSC) at the University of Washington. The system comprises arrays of microwells containing Pt-porphyrin-embedded polystyrene microspheres as the reporter chemistry, a lid actuator system and a gated intensified imaging camera, all mounted on a temperature-stabilized confocal microscope platform. Oxygen consumption determination experiments were performed on RAW264.7 mouse macrophage cells as proof of principle. Repeatable and consistent measurements indicate that the oxygen measurements did not adversely affect the physiological state of the cells measured. The observation of physiological rates in real time allows studies of cell-to-cell heterogeneity in oxygen consumption rate to be performed. Such studies have implications in understanding the role of mitochondrial function in the progression of inflammatory-based diseases, and in diagnosing and treating such diseases.
Journal of The Royal Society Interface 10/2008; 5 Suppl 2:S151-9. · 4.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: One goal of systems biology is to understand how genome-encoded parts interact to produce quantitative phenotypes. The Alpha Project is a medium-scale, interdisciplinary systems biology effort that aims to achieve this goal by understanding fundamental quantitative behaviours of a prototypic signal transduction pathway, the yeast pheromone response system from Saccharomyces cerevisiae. The Alpha Project distinguishes itself from many other systems biology projects by studying a tightly bounded and well-characterised system that is easily modified by genetic means, and by focusing on deep understanding of a discrete number of important and accessible quantitative behaviours. During the project, the authors have developed tools to measure the appropriate data and develop models at appropriate levels of detail to study a number of these quantitative behaviours. The authors have also developed transportable experimental tools and conceptual frameworks for understanding other signalling systems. In particular, the authors have begun to interpret system behaviours and their underlying molecular mechanisms through the lens of information transmission, a principal function of signalling systems. The Alpha Project demonstrates that interdisciplinary studies that identify key quantitative behaviours and measure important quantities, in the context of well-articulated abstractions of system function and appropriate analytical frameworks, can lead to deeper biological understanding. The authors' experience may provide a productive template for systems biology investigations of other cellular systems.
IET Systems Biology 10/2008; 2(5):222-33. · 1.54 Impact Factor
[show abstract][hide abstract] ABSTRACT: A robotic manipulation system for automated selection and transfer of individual living cells of interest to analysis locations is presented. The system is the first in a series of operational components comprising an integrated system for the analysis of cell expression and metabolism at the single cell level. In our approach, we begin with a commonly used cell transfer technique, which uses glass capillary micropipettes to aspirate and release cells. Using a vision-based feedback closed-loop control process, two individual three-axis robotic stages are used to position the micropipette tip in proximity to the cell of interest. The cell is aspirated and the tip is moved to a target location, where the cell is then dispensed. For our initial application, the target location for living cells of interest is a microwell containing sensors that measure metabolic parameters. The system, when complete with fully integrated functionality, will offer a robust and flexible technology for cell selection and manipulation. Applications for this technology include precise tools for multi-parameter analysis of single cells, cell patterning, tissue engineering, and cell surgery.
Automation Science and Engineering, 2008. CASE 2008. IEEE International Conference on; 09/2008
[show abstract][hide abstract] ABSTRACT: In general, a formal specification for hardware-software integration is imperative for systematic design of automated systems. This specification requires a critical understanding of hardware and software systems at various levels of abstraction. The objective of the research we present is to develop a framework that serves as a specification for the implementation of an integrated system for the analysis of cell function - a classic application for laboratory automation. The proposed framework addresses a specific set of integrated system requirements comprising the placement of single cells in analysis locations, the analyses of these cells throughout application of external stimuli, and post stimuli end-point analyses of these same cells. The same framework can be directly extended to automate cell-to-cell and tissue analyses. It has been developed using principles from unified modeling language (UML) to maximize potential for integration with other standard laboratory automation systems. The research presented provides the foundation for efficient use of UML with the proposed framework, and serves as an exemplar for others pursuing similar laboratory automation challenges.
Automation Science and Engineering, 2008. CASE 2008. IEEE International Conference on; 09/2008
[show abstract][hide abstract] ABSTRACT: Major advances in the understanding in the biological sciences have been achieved by the precision automation of repetitive analytical procedures. The requirements for precision are pushed to the extreme when considering the analysis of cell function at the single cell level. Such analyses may include examination of gene expression, protein synthesis, and metabolic activity. The repetitive nature of performing measurement of these parameters requires not only effective signal transduction and amplification, significant attention to system integration and automation is paramount to instrument development objectives. Here we present the systems integration and software development for a multi-spectral imaging and phosphorescence lifetime measurement tool. This tool represents and early engineering prototype of one subsystem of an integrated automation pipeline comprising robotics for microwell-array movement, microwell-array sealing for metabolic rate measurements, control of experimental protocol sequencing and real-time imaging of dynamic experiments. A graphical user interface and underlying control software allows for flexible construction of experimental procedures. The engineering prototype has demonstrated experimental operation times up to 10 times faster than previous manual methods.
Automation Science and Engineering, 2008. CASE 2008. IEEE International Conference on; 09/2008