Mark R. Holl

A series of naphthalimide derivatives as intra and extracellular pH sensors

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.

Differential blood cell counts obtained using a microchannel based flow cytometer

This paper reports results demonstrating the ability to use single microfabricated silicon flow channels for the differential counting of granulocytes, lymphocytes, monocytes, red blood cells (RBCs), and platelets, in a sample of blood by means of laser light scattering. The microfabrication-based flow cytometer described does not rely on sheath flow in order to align the blood cells.

Automated Selection and Placement of Single Cells Using Vision-Based Feedback Control

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.

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.

Compact optical diagnostic device for isothermal nucleic acids amplification

Abstract We recently reported the successful use of the loop-mediated isothermal amplification (LAMP) reaction for hepatitis B virus (HBV) DNA amplification and its optimal primer design method. In this study, we report the development of an integrated isothermal device for both amplification and detection of targeted HBV DNA. It has two major components, a disposable polymethyl methacrylate (PMMA) micro-reactor and a temperature-regulated optical detection unit (base apparatus) for real-time monitoring of the turbidity changes due to the precipitation of DNA amplification by-product, magnesium pyrophosphate. We have established a correlation curve (R 2 =0.99) between the concentration of pyrophosphate ions and the level of turbidity by using a simulated chemical reaction to evaluate the characteristics of our device. For the applications of rapid pathogens detection, we also have established a standard curve (R 2 =0.96) by using LAMP reaction with a standard template in our device. Moreover, we also have successfully used the device on seven clinical serum specimens where HBV DNA levels have been confirmed by real-time PCR. The result indicates that different amounts of HBV DNA can be successfully detected by using this device within 1h.

Results Obtained using A Prototype Microfluidics-Based Hematology Analyzer

Microfluidic laminate-based structures incorporating hydrodynamic focusing and flow channels with dimensions much less than 1 mm were fabricated and used to transport and analyze blood samples. Optically transparent windows integral to the flow channels were used to intercept the sample streams with a tightly focused diode laser probe beam. The size and structure of the blood cells passing through the laser beam determined the intensity and directional distribution of the scattered light generated. Forward and small angle light scattering channels were used to count and differentiate platelets, red blood cells, and various populations of white blood cells. All the blood samples used were characterized using a commercial hematology analyzer for comparison and validation purposes.

A New Approach for Measuring Single-Cell Oxygen Consumption Rates

A novel system that has enabled the measurement of single-cell oxygen consumption rates is presented. The experimental apparatus includes a temperature controlled environmental chamber, an array of microwells etched in glass, and a lid actuator used to seal cells in the microwells. Each microwell contains an oxygen sensitive platinum phosphor sensor used to monitor the cellular metabolic rates. Custom automation software controls the digital image data collection for oxygen sensor measurements, which are analyzed using an image-processing program to yield the oxygen concentration within each microwell versus time. Two proof-of-concept experiments produced oxygen consumption rate measurements for A549 human epithelial lung cancer cells of 5.39 and 5.27 fmol/min/cell, closely matching published oxygen consumption rates for bulk A549 populations.

Simultaneous Self-Referencing Analyte Determination in Complex Sample Solutions Using Microfabricated Flow Structures (T-Sensors™)

In microfluidic channels, fluids with viscosities similar to or higher than water and flowing at low velocities show laminar behavior. This allows the movement of different layers of fluid and particles next to each other in a channel without mixing other than by diffusion. A sample solution (e.g., whole blood), and a receptor solution (e. g., an indicator solution), and a reference solution (a known analyte standard) are introduced in a common channel (T-Sensor™), and flow next to each other until they exit the structure. Smaller particles such as ions or small proteins diffuse rapidly across the fluid boundaries, whereas larger molecules diffuse more slowly. Large particles (e. g., blood cells) show no significant diffusion within the time the two flow streams are in contact. Two interface zones are formed between the fluid layers. The ratio of a property (e. g., fluorescence intensity) of the two interface zones is a function of the concentration of the analyte, and is largely free of cross-sensitivities to other sample components and instrument parameters. This device allows, for example, one-time or continuous monitoring of the concentration of analytes in microliters of whole blood without the use of membranes or prior removal of blood cells.

New ratiometric optical oxygen and pH dual sensors with three emission colors for measuring photosynthetic activity in Cyanobacteria

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.

A cellular isolation system for real-time single-cell oxygen consumption monitoring

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.