Microfluidic Sensors with Impregnated Fluorophores for Simultaneous

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Microfluidic Sensors with Impregnated Fluorophores for Simultaneous Imaging of Spatial Structure and Chemical Oxygen Gradients Jay W Grate, Bingwen Liu, Ryan T. Kelly, Norman C. Anheier, and Thomas Schmidt ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.8b00924 • Publication Date (Web): 04 Jan 2019 Downloaded from http://pubs.acs.org on January 6, 2019

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Microfluidic Sensors with Impregnated Fluorophores for Simultaneous Imaging of Spatial Structure and Chemical Oxygen Gradients Jay W. Grate,*1 Bingwen Liu,1† Ryan T. Kelly,1†† Norman C. Anheier,1 and Thomas M. Schmidt2 1Pacific

Northwest National Laboratory, P.O. Box 999, Richland, WA of Michigan, Ann Arbor, MI KEYWORDS Oxygen, microfluidic, sensor, chemical imaging, pore network, polystyrene, fluorophore, impregnation 2University

ABSTRACT: Interior surfaces of polystyrene microfluidic structures were impregnated with the oxygen sensing dye Pt(II) tetra(pentafluorophenyl)porphyrin (PtTFPP) using a solvent-induced fluorophore impregnation (SIFI) method. Using this technique, microfluidic oxygen sensors are obtained that enable simultaneous imaging of both chemical oxygen gradients and the physical structure of the microfluidic interior. A gentle method of fluorophore impregnation using acetonitrile solutions of PtTFPP at 50oC was developed leading to a 10-µm-deep region containing fluorophore. This region is localized at the surface to sense oxygen in the interior fluid during use. Regions of the device that do not contact the interior fluid pathways lack fluorophores and are dark in fluorescent imaging. The technique was demonstrated on straight microchannel and pore network devices, the latter having pillars of 300 µm diameter spaced center to center at 340 µm providing pore throats of 40 µm. Sensing within channels or pores, and imaging across the pore network devices were performed using a Lambert LIFA-P frequency domain fluorescence lifetime imaging system on a Leica microscope platform. Calibrations of different devices prepared by the SIFI method were indistinguishable. Gradient imaging showed fluorescent regions corresponding to the fluid pore network, dark pillars, and fluorescent lifetime varying across the gradient, thus providing both physical and chemical imaging. More generally, the SIFI technique can impregnate the interior surfaces of other polystyrene containers, such as cuvettes or cell and tissue culture containers, to enable sensing of interior conditions.

The measurement of dissolved oxygen is of increasing interest in field and laboratory environments. Oxygen is among the most important electron acceptors in biology, and plays a critical role in cellular function, tissue culture, cancerous biomass, and in the structure and function of microbial communities in diverse settings.1-3 Oxic-anoxic transition zones (OATZ), where gradients in oxygen occur, are important at a variety of scales in the natural world in biological and biogeochemical processes. In aquatic environments, the watersediment interface, or benthic region, is normally the site of an OATZ and high biological activity.4 Some bacteria have mechanisms to navigate to the OATZ.5 The mammalian gut offers an example of an OATZ within a higher organism; in the lower gut, the OATZ consists of a gradient from the oxic host tissue of the gut wall into the anoxic lumen. This gradient occurs over a range of the order 100 µm,6 and is the location of distinctive microbial communities in the gut mucosa.7-11 The development of fluorescent optodes for oxygen measurements3 , 12-15 enables more robust point sensors that require less frequent calibrations than traditional electrochemical sensors. In addition, these planar optodes enable dissolved oxygen imaging across two dimensional areas. Optode measurement capabilities have been applied to environmental and biological studies involving microbial or mammalian cell cultures, single cells, biofilms, microbial

communities in sediments, and in oxygen-controlling microfluidic structures.16-17, 18 , 19-29 The typical optode consists of a fluorescent dye such as a platinum porphyrin distributed in a polymer matrix as a thin film. Collisional interactions between oxygen molecules and excited state dye molecules in the film lead to measurable reductions in both the emission intensity and the fluorescent lifetime. Here we will describe a new method for creating dispersed fluorophore molecules in a thin surface-localized region of a bulk polystyrene matrix, and the application of this approach to create microfluidic structures with internal oxygen sensing and imaging surfaces. We are particularly interested in using microfluidic approaches to create spatial structure by design and fabrication, and to create dissolved oxygen gradients by fluidic inputs and routing. Pore network devices represent one type of spatial structure of interest in physical and biological processes.30-38 3132, 38-41 Pore networks may be fabricated with pillars of solid material within a wide fluidic pathway, creating pores and pore throats. Processes within the pore network devices can be followed by microscopic imaging, often with fluorescence microscopy. Our own research has focused on pore networks with fluorescence imaging to show immiscible fluid displacement processes39-40, 42-45 or biological processes related to oxic microbial respiration of cellulose.46-47

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Recently a new solvent-based method has been developed that enables impregnation of dyes in a near-surface region of a solid plastic polymer matrix , as shown in Figure 1.48 Using appropriate solvents, polymers and temperatures, the exposure of a glassy plastic surface to a solvent leads to the creation of a gel layer. Diffusion of solvent molecules is very rapid within this gel layer, while diffusion within the bulk glassy polymer remains very slow. This process represents an example of Case II sorption.49 A distinct interface develops between the nearsurface gel region and the bulk glassy material; the depth of the interface increases with time.

Figure 1. Cross sectional imaging of polystyrene impregnated with NPO dye in acetonitrile solution at 50oC for 30 sec duration. After drying, the sheet material was fractured. Images are shown with three illumination conditions: white light (left), white light plus UV light to excite the fluorophore (center), and UV light only with false blue color (right). In each image the solid is on the left while the air is on the right.

When dye molecules are present in the bulk solution, they can diffuse into this gel layer with the solvent. Upon removing the surface from contact with solution, dye and solvent molecules remain in this surface localized region. However, the solvent molecules leave by diffusion and evaporation, and the polymer returns to a glassy solid state. The dye molecules remain and are thus immobilized in the near surface region of a bulk piece of solid plastic. The near-surface region is ideal as a location for sensing reporters that puts the dyes in close proximity to the medium being sensed. The creation of surface localized region containing a fluorescent dye is shown in Figure 1, using a solution of the dye 2-(1-naphthyl)-5-phenyloxazole (NPO) in acetonitrile solution at 50oC for 30 sec. The surface localization of the dye in polystyrene is clearly apparent, with dye penetration limited by the interface between the bulk glassy polymer and the gel layer. Polystyrene, a glassy polymer, is very well suited for this approach. Polystyrene is already known as a good medium for fluorescent oxygen sensing films. Polystyrene is also a material that has been preferred for cell and tissue culture containers and devices,50-54 an observation that has stimulated efforts in polystyrene microfluidic device methodology.51, 53 Our approach is distinguished from conventional sensor layers by not having a separate sensing film on a surface of another material, where such films may be susceptible to failure by delamination or crazing. Instead, in our approach, the sensing region is part of a much thicker volume of the same material. Because the gel layer is soft, it is possible to also imprint three dimensional structures for microfluidics, in which case the process has been described as Solvent Induced Imprint Lithography or SIIL.46, 48, 55-56 Here, where we are only

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impregnating dye to create a near-surface chemically sensitive region, and not imprinting features, we simply refer to it as solvent induced fluorophore impregnation, or SIFI. In this paper, we describe the use of SIFI to create oxygensensitive internal surfaces in polystyrene-based microfluidic devices, using the well-known Pt(II) tetra(pentafluorophenyl)porphyrin (PtTFPP) dye.17-18, 57-58 Oxygen molecules diffuse from the contacting fluid into the polymer and interact with PtTFPP molecules by a collisional quenching mechanism. The contacting fluid can be a gas sample containing oxygen, or an aqueous medium containing oxygen (i.e., dissolved oxygen). In the past, we have described the incorporation of optode features in microfluidic devices with emphasis on methods that enable visualization of spatial structure, i.e., fluid channels versus solid regions, in the same fluorescent images that report dissolved oxygen.47 The present technique also creates fluorescent images that coincide with the microfluidic structure, because only those surfaces that contact fluids are impregnated with the fluorescent dye. More generally, this method can impregnate the internal surfaces of a variety of polystyrene containers, polystyrene being the traditionally preferred material for cell and tissue culture devices.50 One can envision using SIFI to convert commercial polystyrene microfluidic chips, cuvettes, or culture containers into dissolved oxygen reporting containers.

EXPERIMENTAL Materials, Devices, and Impregnation Procedures. Polystyrene sheet was obtained from Goodfellow as 1.2 mm transparent crystal sheet material. Fluorescent dye 2-(1naphthyl)-5-phenyloxazole (NPO) (Sigma Aldrich) was used for visualization during method development, while the dye Pt(II) meso-tetra(pentafluorophenyl) porphine (PtTFPP) (Frontier Scientific, Logan, UT) was used as the dye for creating oxygen sensitive surfaces for sensing and imaging. The polystyrene sheet material was used for two purposes. 1) To investigate impregnation conditions, small pieces of ca. 2 cm by 2 cm were cut. Individual pieces were submerged in solutions containing dyes for specific periods of time and at specific temperatures. In some cases, solvent mixtures were used. After drying, pieces were fractured to expose a cross section for imaging. Once a good fracture surface was obtained, the sheet material was cut approximately 2 mm away from the fractured surface. Final pieces were typically the 1.2 mm sheet thickness, 1–2 cm long, and 2 mm high (with the fracture surface facing down on an inverted microscope. 2) Microfluidic devices of polystyrene were fabricated as described in the Supporting Information and shown in Figure SI_1. Both the microfluidic structure and the cover plate were prepared from the polystyrene sheet material. Polystyrene bonding was done in an AML wafer bonder. The structured polystyrene and another piece of blank polystyrene sheet as the cover were sandwiched between two glass slides, and placed in the bonder. When the temperature was increased to and stabilized at 100°C, a 2000 N bonding force was applied for 20 min. The pore network device in polystyrene was prepared by hot embossing polystyrene against a glass-backed PDMS structure using the AML wafer bonder. The straight microchannel was 20 mm long, 100 µm deep, and 300 µm wide, within a total device dimension of approximately 25 mm x 40 mm. The pore network device dimensions were ca. 35 mm x


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60 mm, with an area of the pore network of 10 mm x 20 mm. The depth was 100 µm. The diameter of pillars was 300 µm, the spacing of pillars center to center was 340 µm, the pore throats (narrowest region between pillars) were 40 µm, and the pore size (largest distance between pillars) was 181 µm. The device has two input regions on one end and a single outlet on the other. The single outlet was used to inject dye solutions during impregnation. Inside surfaces were impregnated by injecting a solution of the dye in the solvent, with the device and solution preheated to the desired impregnation temperature of 50oC. The dye solution concentration was 5 mg/mL PtTFPP in acetonitrile. The solution readily flowed into the pore network pulled by capillary forces. After impregnating for the desired length of time, the solution was displaced out the exit port with air and the device was allowed to dry. Imaging. Cross sectional bright field and fluorescence images were taken with an Etaluma Lumascope 600, which utilizes a Semrock Brightline Pinkel filter set optimized for the 405, 488, and 594 LED light sources. This is an inverted microscope, and cross-sectional fractured surfaces were placed facing down to the lens. When the sample still has residual solvent in the former gel layer volume, this volume has optical differences with the unaffected glassy material, such as refractive index. The layer can be visualized even without a dye, and it may guide light. Care was taken to image each sample repeatedly, vary the intensity of the excitation light, and limit the thickness of the sample from fractured surface to top surface. Fluorescence lifetime images were obtained with a Lambert LIFA-P system (LI2CAM-P, Lambert Instruments, Netherlands) mounted on a Leica inverted microscope (Leica DMI6000) platform. The Lambert system uses LEDs for excitation and an intensified camera for imaging, and determines lifetime by a frequency domain technique. For FLIM with PtTFPP, the LED was 399 nm, a long pass filter at 600 nm was used for the emission, and the modulation frequency was 5 kHz. Lifetime values were derived from the phase measurement. Images of overall structures were also obtained using a conventional chemical laboratory UV light (Mineralight Lamp Model UVGL-58, Upland, CA) and a CCD camera (Sony DSCTX20) Oxygen concentrations. All calibrations were performed with continuously flowing gas or liquid samples. Slight diffusion of oxygen through the polystyrene device material is negligible compared to the oxygen concentration supplied by the flowing sample. Gaseous samples for calibration were prepared from compressed air and compressed nitrogen bottles, with gas flows controlled by electronic mass flow controllers. All tubing upstream from the MFCs were metal, and tubing downstream was PEEK. The two gas streams were mixed with a tee junction for a total flow of 200 mL/min at a known gas mixture. For dissolved oxygen, water flow was pumped from an air-saturated reservoir using a milliGAT pump (Global FIA, Inc. Fox Island, WA) regulated with controller and software from GlobalFIA. This flow was routed through the fluid path of a Micromodule G591 membrane device (www.Liquid-cel.com) which has a cross flow of gas through hydrophobic polypropylene hollow fiber membranes.59 An image of this setup is provided in the Supporting Information, as Figure SI_2. The liquid fluid output of this device is equilibrated with

the gas concentration of the gas flow, which was prepared with the MFCs as described. The Micromodule was fitted with tube fitting and the path from the micromodule to NanoPort (IDEX) input of the microfluidic device was metal. This provides a simple way to regulate dissolved oxygen concentrations immediately upstream from the microfluidic device while avoiding bubble formation, which can be problematic in microfluidic devices.

RESULTS AND DISCUSSION Dye impregnation conditions. To impregnate the inside surfaces of a microfluidic device with fine spatial structure, it was necessary to find mild impregnation conditions. Aggressive solvents such as acetone or 2-butanone produce a sufficiently fluid gel layer that fine structure can degrade, and the layer can distort under flow conditions during injection and subsequent displacement of the dye-containing solution within the device. At the same time, by using the solvent impregnation method, the goal is to obtain a dye-loaded near-surface region whose thickness is limited by the interface between the gel layer and the bulk glassy polymer. Figure 1 shows images of the near surface region containing NPO dye. This dye is convenient for development purposes because it is bright under normal atmospheric conditions in the presence of oxygen. The depth of the gel layer interface depends on the solvent, solvent mixture, polymer, and temperature. We investigated acetonitrile at room temperature and 50oC as a solvent that would be less aggressive than acetone or 2-butanone, preserving fine microstructure and enabling reasonable control over the process for confining the dye in thin regions on the order of 10 µm thick at the fluid surface. Depending on solvent and temperature, the dye penetrates no further than the gel layer thickness; dye molecules larger than the solvent molecules may diffuse more slowly and hence not completely fill the gel layer region. The thickness of the dye-containing gel layer in experiments using PtTFPP as the dye is shown in Figure 2 as a function of impregnation time and temperature.

Figure 2. Impregnation depth as a function of time at room temperature and 50oC using solutions of PtTFPP in acetonitrile.

As expected, the depth after drying is greater at 50oC than at room temperature. In practice, we found that at room temperature, this large dye molecule did not effectively


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penetrate the gel layer. At 50oC, the dye had diffused into the gel layer and surface-localized dye in polystyrene was achieved. Our standard conditions for thin sensing regions in the surface were using acetonitrile solution at 50oC for 30 sec. Images for PtTFPP impregnation under these conditions are shown in Figure 3, yielding dye-containing material to ca. 10 microns depth at most. This thickness is small enough to retain the dye near the surface for a rapid response to oxygen diffusing into the material, while being thick enough to contain sufficient dye for measuring the luminescent signal. Dye penetration and speed can also be increased over that of pure acetonitrile by creating a solvent mixture with acetone. Results for solvent mixtures are shown in the Supporting Information in Figure SI_3. We found that 60:40 acetonitrile:acetone at room temperature gets to a similar gel layer depth as pure acetonitrile at 50°C in about twice the time. (i.e., to ca. 100 microns in 10 min in 50oC acetonitrile, and 20 minutes at room temperature in acetonitrile with 40% acetone.) We found it practical to work with dye solutions in pure acetonitrile at 50oC for controlled impregnation processes. Figure 3 shows the impregnation of PtTFPP in polystyrene under these conditions for 30 seconds, yielding a region approximately 10 microns thick at the surface containing the dye. The Supporting Information also shows images in Figure SI_4 for impregnation using 1:1 acetone:acetonitrile at room temperature with NPO dye for visualization.

Figure 3. Images showing impregnation with oxygen sensitive dye PtTFPP in acetonitrile solution at 50oC for 30 sec, imaged in sulfite solution in water to eliminate oxygen so that the luminescence is not quenched during imaging. Imaging was carried out with the Etaluma Lumascope 600, excitation by LED with a band of 370-410 nm, with a window for transmission available at 612-680 for the emission.

Sensing and imaging in microfluidic devices. Straight channel and pore network devices were fabricated in polystyrene, with thermally-bonded polystyrene cover plates, using the methods described in the Supporting Information. Polystyrene is favorable for two reasons. 1) Polystyrene is known as a good polymer matrix for optodes, and 2) polystyrene is a material often used for cell and tissue culture. Interior surfaces were impregnated with Pt using acetonitrile at 50oC for 30 sec as described in the Experimental. NanoPorts were used to make fluidic connections to the ports. Examples of these devices are shown in Figure 4. These results show successful impregnation into simple and complex microfluidic

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structures. Because the dye is only impregnated into the surfaces in contact with the interior flow channels, the luminescence of the dye produces images that show the spatial structure of the device. Regions with no fluid, such as the solid pillars forming the pore network, remain dark in fluorescence images.

Figure 4. Images of the microchannel device (A) and the twoinput region of the pore network device(B) Each shows the RGB image and the red channel only, from the CCD camera (inset on the pore network device image.

Figure 5. Images and profiles for the pore network device taken with the Lambert LIFA-P system, showing clockwise from the upper left, an intensity images, an intensity profile across the pillars and pores, a lifetime profile plot across the pores and pillars, and a lifetime image. These images were taken under gaseous nitrogen at 200 mL/min flow rate.

Intensity and lifetime images of a portion of the pore network structure under nitrogen are shown in Figure 5, along with profile plots along selected long axes. The pillar regions are dark. The fluid-contacting surfaces have intensity from impregnated dye, with pillar surfaces appearing brighter because dye is present along the vertical surface of the pillar. The FLIM images however, normalize out variations in intensity; pore regions are a relatively uniform gray, with lifetimes approaching 60 microseconds under gaseous nitrogen (no data are present in pillar areas), as expected for this dye under anoxic conditions. Using continuously flowing sample gases, the sensing microfluidic structures responded to changes in gas


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concentration in seconds, i.e. in the time from adjusting MFC flows to taking the lifetime imaging data. The fluorescence lifetime as a function of gaseous oxygen concentration is shown in Figure 6. The expected nonlinear decline in lifetime with increasing oxygen is seen. Error bars represent the standard deviation of the pixel to pixel variation as reported by the LIFAP system. Data for both the microchannel device and the pore network are shown. It is striking that these two devices, of different structure and fabricated on different days, have identical overlapping calibration curves. These results speak to the reproducibility of the device fabrication method, using SIFI, and the use of FLIM as a read out.

Figure 6. Calibration curves for microchannel (MC, squares) and pore network (PN, diamonds) microfluidic sensors to gaseous mixtures of air and nitrogen, showing fluorescence lifetime (left axis) and Stern-Volmer representation (right axis, solid circle markers).

Figure 7. Phasor plots in gaseous nitrogen and air using data from a single pore area in the pore network device.

The microchannel data are converted to a Stern Volmer representation58, 60-62 in the same plot. For an ideal system of collisional quenching involving a single-lifetime luminescent species, i.e., the dye molecules each in their polymer matrix microenvironments, it is expected that the Stern Volmer representation should linearize the data. Instead, our data for PtTFPP in polystyrene is curved. Such curved Stern-Volmer plots are known for Pt porphyrin based oxygen sensors, and double quenching site Stern–Volmer calibration curves have been applied in the past.20, 55, 57, 63A detailed description of the

calibration using a two-site quenching model, as it applies to PtTFPP in polystyrene, has been presented.57 The interpretation is that luminescence decay is not a single monoexponential decay process for all molecules of this dye in the polymer matrix. Support for this conclusion can be found in the phasor plot in Figure 7. The phasor plot, a polar plot representation 64-67 easily generated by the Lambert LIFA-P software from frequency domain lifetime measurements, is a two-dimensional histogram where each pixel in the image contributes a point. The data for the pixels under nitrogen center are largely centered on the semicircle shown, which is known as the universal semicircle; these results are consistent with primarily a monoexponential luminescence decay process from a single species. However, under air, the center of the pixel data clearly falls inside the semicircle rather than directly on the semicircle. These are not consistent with a monoexponential decay process and are taken as indicating a multiexponential decay.55 Thus, the luminescence decay under air is complex and is different from luminescence under nitrogen; variations with different oxygen concentrations have been observed previously.55 Dissolved oxygen gradients. Solutions of varying dissolved oxygen concentration were generated using a Liqui-Cel Micromodule membrane device, equilibrating a flowing water solution to a known generated gaseous oxygen concentration, and delivering this continuously flowing solution to the device input through a short metal tubing. (See experimental and Supporting Information.) The calibration curve generated to dissolved oxygen is shown in Figure SI_5. This representation is directly comparable to the calibration to gaseous oxygen conditions in Figure 6. However, the range of the response is slightly compressed under aqueous conditions. The sensing material condition is different in these two cases. Under gas flow, the polymer at the fluid surface is rigorously dry, but when sensing dissolved oxygen, the material is hydrated with water. The two-input pore network device was set up with air saturated water on one input, and nitrogen purged water on the other input. These experiments were conducted to generate a gradient within the pore network with continuously flowing oxic and anoxic input solutions. A region near the junction of the two inputs is shown in Figure 8 depicting fluorescence lifetime on a color scale from 10 microseconds (blue) to 65 microseconds(red). There is a clear gradient in dissolved oxygen indicated by the color gradient in the image, while the dark circles representing pillars show the spatial structure within which the chemical gradient is occurring. The gradient can be made steeper by increasing the input flow rates. The gradient becomes broader as you move downstream from the position where the two flows merge. Transverse diffusional mixing in two input pore network micromodels has been described previously,35-37, 68, and lattice Boltzman methods were used to develop steady-state advection-diffusion-reaction equations with boundary conditions defined by the microfluidic conditions.36-37 Two dimensional imaging, as represented in Figure 8, provides quantitative measurements of the fluorescence lifetime at each pixel. By applying a calibration model to the fluorescence lifetime data, (e.g. from calibrations such as those presented in Figure SI_5.) the pixel data of lifetimes can be transformed to a chemical image of pixels representing dissolved oxygen concentrations.


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culture. Images showing polystyrene cuvettes with fluorescent PtTFPP containing interior surfaces produced by SIFI are shown in the supplementary material (Figure SI_6) as proof of principle. Such interior surfaces can readily reveal whether interior conditions are oxic or anoxic, providing a turn-on signal if they are anoxic. Interior concentrations may be measured from the outside using fiber optic frequency domain techniques (e.g. the PreSens FiBox, with appropriate calibration for this fluorophore film).

ASSOCIATED CONTENT SUPPORTING INFORMATION

Figure 8. Fluorescence lifetime imaging across the gradient in a two-input pore network device. Red is anoxic and blue is oxic. Liquid flow rates to each input were 1 microliter per second. The left pore is where the two input flows meet, with flow from left to right

Discussion. The final figure of the pore network device demonstrates simultaneous imaging of spatial structure and chemical oxygen gradient in a pore network structure. This result is possible because the SIFI method only deposits fluorophore within the interior surfaces of fluid channels, and the method was developed as a mild technique that retains fine spatial structure in the device. This approach is distinguished from microfluidic devices in which a fluorescent film is coated over the entire surface of a cover plate prior to device assembly, and fluorescent signal is observable over this entire surface whether it contacts interior fluid channels or not. The SIFI technique is well controlled and produces sensors that, when read out using a fluorescent lifetime imaging technique, have reproducible sensitivity in various microfluidic channel designs. If, for some pragmatic reason, one wanted to observe spatial structure and oxygen using separate fluorophores and hence separate images, there is nothing to prevent one from impregnating two fluorophores at the same time, one for chemical imaging and the other for spatial structure. The approach is simple and may be generalizable in two important ways. First, in microfluidic or biomedical microdevices, so long as there is a polystyrene surface in contact with the interior fluid channel, sensing interior surfaces may be produced that match the interior spatial structure. The entire microfluidic device need not be polystyrene 69-71 like the examples in this paper; hybrid devices can be designed. Quantitatively, or qualitatively, sensing interiors may serve as turn-on sensors to signal anoxic conditions, as cellular behavior can be sensitive to such environmental changes.54, 69 For quantitative work where flow inputs or respiratory processes are slow, and internal measurements might be biased by even limited diffusion of external oxygen molecules through the material of a thin all-polystyrene device, two approaches may be envisioned; polystyrene devices might be used within an anoxic chamber, i.e. so that there is no external oxygen, or hybrid devices with oxygen impermeable layers of materials such as glass may be used to prevent external oxygen diffusion into the fluidic interior. The second generalizable concept is that the interior surfaces of other polystyrene labware can be impregnated to obtain labware with oxygen sensing interior surfaces for cell and tissue

The Supporting Information is available free of charge on the ACS Publications website. A single pdf file contains microfabrication details and Supplemental Figures S1-S6, showing the microfabrication process, gas exchange device set up, impregnation rates by solvent mixtures, impregnation layer, dissolved oxygen calibration, and impregnation inside a polystyrene container.

AUTHOR INFORMATION Corresponding Author * Email: [email protected]

Present Addresses † Bingwen Liu, Chemistry and Biochemistry, The University of Arizona, Tucson AZ 85721. Email: [email protected] †† Ryan Kelly, Chemistry and Biochemistry, Brigham Young University, Provo, UT 84602. Email: [email protected]

ORCID

Jay W. Grate: 0000-0002-2163-8383 Bingwen Liu: 0000-0002-0092-6685 Ryan T. Kelly: 0000-0002-3339-4443 Thomas M. Schmidt: 0000-0002-8209-6055 Notes

The authors declare no competing financial interests.

ACKNOWLEDGMENT The Pacific Northwest National Laboratory (PNNL) is a multiprogram national laboratory operated for the U.S. Department of Energy (DOE) by Battelle Memorial Institute under contract number DE-AC05-76RL01830. Thomas M. Schmidt at the University of Michigan received support from the National Institutes of Health contract number 5R01GM099549. Concepts for this research collaboration were originally developed under the DOE PNNL Laboratory Directed Research and Development (LDRD) Program under PNNL’s Microbial Communities Initiative. Research in solvent impregnation for the creation of oxygen sensing surfaces for microfluidics and biology has been supported by the DOE Office of Biological and Environmental Research, DOE Laboratory Directed Research and Development, and the National Institutes of Health. A portion of this research was performed using the William R. Wiley Environmental Molecular Sciences Laboratory (EMSL), a national scientific user facility sponsored by the DOE’s Office of Biological and Environmental Research and located at PNNL.

REFERENCES


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1. Fenchel, T.; Finlay, B., Oxygen and the Spatial Structure of Microbial Communities. Biological Reviews 2008, 83 (4), 553-569. 2. Klimant, I.; Kuehl, M.; Glud, R. N.; Holst, G., Optical measurement of oxygen and temperature in microscale: strategies and biological applications. Sens. Actuators, B 1997, B38 (1-3), 29-37. 3. Wang, X.-d.; Wolfbeis, O. S., Optical methods for sensing and imaging oxygen: materials, spectroscopies and applications. Chem. Soc. Rev. 2014, 43 (10), 3666-3761. 4. Kuhl, M., Biogeochemical microsensors for boundary layer studies. In The Benthic Boundary Layer, Boubreau, B. P.; Jørgensen, B. B., Eds. Oxford University Press: New York, 2001; pp 180-210. 5. He, G.; Shankar, R. A.; Chzhan, M.; Samouilov, A.; Kuppusamy, P.; Zweier, J. L., Noninvasive measurement of anatomic structure and intraluminal oxygenation in the gastrointestinal tract of living mice with spatial and spectral EPR imaging. Proceedings of the National Academy of Sciences of the United States of America 1999, 96 (8), 4586-4591. 6. Marteyn, B.; West, N. P.; Browning, D. F.; Cole, J. A.; Shaw, J. G.; Palm, F.; Mounier, J.; Prevost, M.-C.; Sansonetti, P.; Tang, C. M., Modulation of Shigella virulence in response to available oxygen in vivo. Nature 2010, 465 (7296), 355-358. 7. Morris, R. L.; Schmidt, T. M., Shallow breathing: bacterial life at low O-2. Nature Reviews Microbiology 2013, 11 (3), 205-212. 8. Schmidt, T. M.; Kao, J. Y., A Little O-2 May Go a Long Way in Structuring the GI Microbiome. Gastroenterology 2014, 147 (5), 956-959. 9. Stevenson, B. S.; Eichorst, S. A.; Wertz, J. T.; Schmidt, T. M.; Breznak, J. A., New strategies for cultivation and detection of previously uncultured microbes. Appl. Environ. Microbiol. 2004, 70 (8), 4748-4755. 10. Vital, M.; Penton, C. R.; Wang, Q.; Young, V. B.; Antonopoulos, D. A.; Sogin, M. L.; Morrison, H. G.; Raffals, L.; Chang, E. B.; Huffnagle, G. B.; Schmidt, T. M.; Cole, J. R.; Tiedje, J. M., A gene-targeted approach to investigate the intestinal butyrateproducing bacterial community. Microbiome 2013, 1 (8), DOI: 10.1186/2049-2618-1-8. 11. Young, V. B.; Schmidt, T. M., Overview of the gastrointestinal microbiota. In Gi Microbiota and Regulation of the Immune System, Huffnagle, G. B.; Noverr, M. C., Eds. 2008; Vol. 635, pp 29-40. 12. Klimant, I.; Meyer, V.; Kuhl, M., Fiberoptic Oxygen Microsensors, a New Tool in Aquatic Biology. Limnol. Oceanogr. 1995, 40 (6), 1159-1165. 13. Chu, C.-S.; Lo, Y.-L.; Sung, T.-W., Review on recent developments of fluorescent oxygen and carbon dioxide optical fiber sensors. Photonic Sensors 2011, 1 (3), 234-250. 14. Quaranta, M.; Borisov, S. M.; Klimant, I., Indicators for optical oxygen sensors. Bioanalytical Reviews 2012, 4 (2-4), 115-157. 15. Wolfbeis, O. S., Luminescent sensing and imaging of oxygen: Fierce competition to the Clark electrode. Bioessays 2015, 37 (8), 921-928. 16. Oguri, K.; Kitazato, H.; Glud, R. N., Platinum octaetylporphyrin based planar optodes combined with an UV-LED excitation light source: An ideal tool for high-resolution O-2 imaging in O-2 depleted environments. Mar. Chem. 2006, 100 (1-2), 95-107. 17. Kuhl, M.; Polerecky, L., Functional and structural imaging of phototrophic microbial communities and symbioses. Aquat. Microb. Ecol. 2008, 53 (1), 99-118. 18. Thomas, P. C.; Halter, M.; Tona, A.; Raghavan, S. R.; Plant, A. L.; Forry, S. P., A Noninvasive Thin Film Sensor for Monitoring Oxygen Tension during in Vitro Cell Culture. Anal. Chem. 2009, 81 (22), 9239-9246. 19. Dragavon, J.; Molter, T.; Young, C.; Strovas, T.; McQuaide, S.; Holl, M.; Zhang, M.; Cookson, B.; Jen, A.; Lidstrom, M.; Meldrum, D.; Burgess, L., A cellular isolation system for real-time single-cell oxygen consumption monitoring. Journal of the Royal Society Interface 2008, 5, S151-S159. 20. Molter, T. W.; McQuaide, S. C.; Suchorolski, M. T.; Strovas, T. J.; Burgess, L. W.; Meldrum, D. R.; Lidstrom, M. E., A microwell array device capable of measuring single-cell oxygen consumption rates. Sens. Actuator B-Chem. 2009, 135 (2), 678-686.

21. Strovas, T. J.; Dragavon, J. M.; Hankins, T. J.; Callis, J. B.; Burgess, L. W.; Lidstrom, M. E., Measurement of respiration rates of Methylobacterium extorquens AM1 cultures by use of a phosphorescence-based sensor. Appl. Environ. Microbiol. 2006, 72 (2), 1692-1695. 22. Vollmer, A. P.; Probstein, R. F.; Gilbert, R.; Thorsen, T., Development of an integrated microfluidic platform for dynamic oxygen sensing and delivery in a flowing medium. Lab Chip 2005, 5 (10), 1059-1066. 23. Lo, J. F.; Sinkala, E.; Eddington, D. T., Oxygen gradients for open well cellular cultures via microfluidic substrates. Lab Chip 2010, 10 (18), 2394-2401. 24. Thomas, P. C.; Raghavan, S. R.; Forry, S. P., Regulating Oxygen Levels in a Microfluidic Device. Anal. Chem. 2011, 83 (22), 8821-8824. 25. Park, J.; Bansal, T.; Pinelis, M.; Maharbiz, M. M., A microsystem for sensing and patterning oxidative microgradients during cell culture. Lab Chip 2006, 6 (5), 611-622. 26. Lam, R. H. W.; Kim, M.-C.; Thorsen, T., Culturing Aerobic and Anaerobic Bacteria and Mammalian Cells with a Microfluidic Differential Oxygenator. Anal. Chem. 2009, 81 (14), 5918-5924. 27. Kühl, M.; Rickelt, L. F.; Thar, R., Combined Imaging of Bacteria and Oxygen in Biofilms. Appl. Environ. Microbiol. 2007, 73 (19), 6289-6295; DOI: 10.1128/AEM.01574-07 28. Mosshammer, M.; Strobl, M.; Kuhl, M.; Klimant, I.; Borisov, S. M.; Koren, K., Design and Application of an Optical Sensor for Simultaneous Imaging of pH and Dissolved O-2 with Low CrossTalk. ACS Sens. 2016, 1 (6), 681-687. 29. Prest, E. I.; Staal, M.; Kuhl, M.; van Loosdrecht, M. C. M.; Vrouwenvelder, J. S., Quantitative measurement and visualization of biofilm O-2 consumption rates in membrane filtration systems. J. Membr. Sci. 2012, 392, 66-75. 30. Levache, B.; Azioune, A.; Bourrel, M.; Studer, V.; Bartolo, D., Engineering the surface properties of microfluidic stickers. Lab Chip 2012, 12, 3028–3031. 31. Gunda, N. S. K.; Bera, B.; Karadimitriou, N. K.; Mitra, S. K.; Hassanizadeh, S. M., Reservoir-on-a-Chip (ROC): A new paradigm in reservoir engineering. Lab Chip 2011, 11 (22), 3785-3792. 32. Wu, M.; Xiao, F.; Johnson-Paben, R. M.; Retterer, S. T.; Yin, X.; Neeves, K. B., Single- and two-phase flow in microfluidic porous media analogs based on Voronoi tessellation. Lab Chip 2012, 12 (2), 253-261. 33. Berejnov, V.; Djilali, N.; Sinton, D., Lab-on-chip methodologies for the study of transport in porous media: energy applications. Lab Chip 2008, 8 (5), 689-693. 34. Schneider, M. H.; Tabeling, P., Lab-on-Chip Methodology in the Energy Industry: Wettability Patterns and Their Impact on Fluid Displacement in Oil Reservoir Models. American Journal of Applied Sciences 2011, 8 (10), 927-932. 35. Zhang, C. Y.; Dehoff, K.; Hess, N.; Oostrom, M.; Wietsma, T. W.; Valocchi, A. J.; Fouke, B. W.; Werth, C. J., Pore-Scale Study of Transverse Mixing Induced CaCO3 Precipitation and Permeability Reduction in a Model Subsurface Sedimentary System. Environ Sci. Technol. 2010, 44 (20), 7833-7838. 36. Zhang, C. Y.; Kang, Q. J.; Wang, X.; Zilles, J. L.; Muller, R. H.; Werth, C. J., Effects of Pore-Scale Heterogeneity and Transverse Mixing on Bacterial Growth in Porous Media. Environ Sci. Technol. 2010, 44 (8), 3085-3092. 37. Willingham, T. W.; Werth, C. J.; Valocchi, A. J., Evaluation of the effects of porous media structure on mixing-controlled reactions using pore-scale modeling and micromodel experiments. Environ Sci. Technol. 2008, 42 (9), 3185-3193. 38. Lenormand, R., Visualization of Flow Patterns in 2D Model Networks. In Experimental Methods in the Physical Sciences, Celotta, R.; Lucatorto, T., Eds. Academic Press: San Diego, 1999; Vol. 35, pp 43-67. 39. Zhang, C. Y.; Oostrom, M.; Grate, J. W.; Wietsma, T. W.; Warner, M. G., Liquid CO2 Displacement of Water in a DualPermeability Pore Network Micromodel. Environ. Sci. Technol. 2011, 45 (7), 7581-7588.


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40. Zhang, C. Y.; Oostrom, M.; Wietsma, T. W.; Grate, J. W.; Warner, M. G., Influence of Viscous and Capillary Forces on Immiscible Fluid Displacement: Pore-Scale Experimental Study in a Water-Wet Micromodel Demonstrating Viscous and Capillary Fingering. Energy Fuels 2011, 25 (8), 3493-3505. 41. Keller, A. A.; Blunt, M. J.; Roberts, P. V., Micromodel observation of the role of oil layers in three-phase flow. Transp. Porous Media 1997, 26 (3), 277-297. 42. Grate, J. W.; Warner, M. G.; Pittman, J. W.; Dehoff, K. J.; Wietsma, T. W.; Zhang, C. Y.; Oostrom, M., Silane modification of glass and silica surfaces to obtain equally oil-wet surfaces in glasscovered silicon micromodel applications. Water Resour. Res. 2013, 49 (8), 4724-4729. 43. Grate, J. W.; Dehoff, K. J.; Warner, M. G.; Pittman, J. W.; Wietsma, T. W.; Zhang, C.; Oostrom, M., Correlation of Oil–Water and Air–Water Contact Angles of Diverse Silanized Surfaces and Relationship to Fluid Interfacial Tensions. Langmuir 2012, 28 (18), 7182-7188. 44. DeHoff, K. J.; Oostrom, M.; Zhang, C.; Grate, J. W., Evaluation of Two-Phase Relative Permeability and Capillary Pressure Relations for Unstable Displacements in a Pore Network. Vadose Zone Journal 2012, 11 (4), vzj2012.0024. doi: https://doi.org/10.2136/vzj2012.0024. 45. Grate, J. W.; Zhang, C. Y.; Wietsma, T. W.; Warner, M. G.; Anheier, N. C.; Bernacki, B. E.; Orr, G.; Oostrom, M., A note on the visualization of wetting film structures and a nonwetting immiscible fluid in a pore network micromodel using a solvatochromic dye. Water Resour. Res. 2010, 46 (11), W11602. doi:10.1029/2010WR009419. 46. Grate, J. W.; Mo, K. F.; Shin, Y.; Vasdekis, A.; Warner, M. G.; Kelly, R. T.; Orr, G.; Hu, D. H.; Dehoff, K. J.; Brockman, F. J.; Wilkins, M. J., Alexa Fluor-Labeled Fluorescent Cellulose Nanocrystals for Bioimaging Solid Cellulose in Spatially Structured Microenvironments. Bioconjugate Chem. 2015, 26 (3), 593-601. 47. Grate, J. W.; Kelly, R. T.; Suter, J.; Anheier, N. C., Siliconon-glass pore network micromodels with oxygen-sensing fluorophore films for chemical imaging and defined spatial structure. Lab Chip 2012, 12 (22), 4796-4801. 48. Vasdekis, A. E.; Wilkins, M. J.; Grate, J. W.; Kelly, R. T.; Konopka, A. E.; Xantheas, S. S.; Chang, T. M., Solvent immersion imprint lithography. Lab Chip 2014, 14 (12), 2072-2080. 49. Windle, A. H., Case II Sorption. In Polymer Permeability, Comyn, J., Ed. Springer: Netherlands, 1986; pp 75-118. 50. Berthier, E.; Young, E. W. K.; Beebe, D., Engineers are from PDMS-land, Biologists are from Polystyrenia. Lab Chip 2012, 12 (7), 1224-1237. 51. Young, E. W. K.; Berthier, E.; Guckenberger, D. J.; Sackmann, E.; Lamers, C.; Meyvantsson, I.; Huttenlocher, A.; Beebe, D. J., Rapid prototyping of arrayed microfluidic systems in polystyrene for cell-based assays. Anal. Chem. 2011, 83 (4), 1408-1417. 52. Nge, P. N.; Rogers, C. I.; Woolley, A. T., Advances in Microfluidic Materials, Functions, Integration, and Applications. Chem. Rev. 2013, 113 (4), 2550-2583. 53. Nargang, T. M.; Brockmann, L.; Nikolov, P. M.; Schild, D.; Helmer, D.; Keller, N.; Sachsenheimer, K.; Wilhelm, E.; Pires, L.; Dirschka, M.; Kolew, A.; Schneider, M.; Worgull, M.; Giselbrecht, S.; Neumann, C.; Rapp, B. E., Liquid polystyrene: a room-temperature photocurable soft lithography compatible pour-and-cure-type polystyrene. Lab Chip 2014, 14 (15), 2698-2708. 54. Halldorsson, S.; Lucumi, E.; Gómez-Sjöberg, R.; Fleming, R. M. T., Advantages and challenges of microfluidic cell culture in polydimethylsiloxane devices. Biosens. Bioelectron. 2015, 63, 218231. 55. Moore, J. S.; Xantheas, S. S.; Grate, J. W.; Wietsma, T. W.; Gratton, E.; Vasdekis, A. E., Modular Polymer Biosensors by Solvent Immersion Imprint Lithography. J. Polym. Sci. Pt. B-Polym. Phys. 2016, 54 (1), 98-103.

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56. Denis, L.; H., N. S.; E., V. A., Solvent-assisted prototyping of microfluidic and optofluidic microsystems in polymers. J. Polym. Sci., Part B: Polym. Phys. 2016, 54 (17), 1681-1686. 57. Vieweg, M.; Trauth, N.; Fleckenstein, J. H.; Schmidt, C., Robust Optode-Based Method for Measuring in Situ Oxygen Profiles in Gravelly Streambeds. Environ Sci. Technol. 2013, 47 (17), 98589865. 58. Lee, S. K.; Okura, I., Photostable optical oxygen sensing material: Platinum tetrakis(pentafluorophenyl)porphyrin immobilized in polystyrene. Anal. Commun. 1997, 34 (6), 185-188. 59. Gospodinova, K.; McNichol, A. P.; Gagnon, A.; Shah Walter, S. R., Rapid extraction of dissolved inorganic carbon from seawater and groundwater samples for radiocarbon dating. Limnol. Oceanogr. Methods 2016, 14 (1), 24-30. 60. John, G. T.; Klimant, I.; Wittmann, C.; Heinzle, E., Integrated optical sensing of dissolved oxygen in microtiter plates: A novel tool for microbial cultivation. Biotechnol. Bioeng. 2003, 81 (7), 829-836. 61. Mills, A.; Lepre, A., Controlling the Response Characteristics of Luminescent Porphyrin Plastic Film Sensors for Oxygen. Anal. Chem. 1997, 69 (22), 4653-4659. 62. Tengberg, A.; Hovdenes, J.; Andersson, H. J.; Brocandel, O.; Diaz, R.; Hebert, D.; Arnerich, T.; Huber, C.; Kortzinger, A.; Khripounoff, A.; Rey, F.; Ronning, C.; Schimanski, J.; Sommer, S.; Stangelmayer, A., Evaluation of a lifetime-based optode to measure oxygen in aquatic systems. Limnology and Oceanography-Methods 2006, 4, 7-17. 63. Molter, T. W.; Holl, M. R.; Dragavon, J. M.; McQuaide, S. C.; Anderson, J. B.; Young, A. C.; Burgess, L. W.; Lidstrom, M. E.; Meldrum, D. R., A New Approach for Measuring Single-Cell Oxygen Consumption Rates. IEEE Transactions on Automation Science and Engineering 2008, 5 (1), 32-42. 64. Digman, M. A.; Caiolfa, V. R.; Zamai, M.; Gratton, E., The Phasor Approach to Fluorescence Lifetime Imaging Analysis. Biophys. J. 2008, 94 (2), L14-L16. 65. Clayton, A. H. A.; Hanley, Q. S.; Verveer, P. J., Graphical representation and multicomponent analysis of single-frequency fluorescence lifetime imaging microscopy data. Journal of Microscopy 2004, 213 (1), 1-5. 66. Gadella, T. W. J.; Clegg, R. M.; Jovin, T. M., Fluorescence lifetime imaging microscopy: Pixel-by-pixel analysis of phasemodulation data. Bioimaging 1994, 2 (3), 139-159. 67. Redford, G. I.; Clegg, R. M., Polar Plot Representation for Frequency-Domain Analysis of Fluorescence Lifetimes. Journal of Fluorescence 2005, 15 (5), 805-815. 68. Nambi, I. M.; Werth, C. J.; Sanford, R. A.; Valocchi, A. J., Pore-scale analysis of anaerobic halorespiring bacterial growth along the transverse mixing zone of an etched silicon pore network. Environ Sci. Technol. 2003, 37 (24), 5617-5624. 69. Park, J.; Koito, H.; Li, J.; Han, A., Microfluidic compartmentalized co-culture platform for CNS axon myelination research. Biomed. Microdevices 2009, 11 (6), 1145-1153. 70. Han, A.; Wang, O.; Graff, M.; Mohanty, S. K.; Edwards, T. L.; Han, K.-H.; Bruno Frazier, A., Multi-layer plastic/glass microfluidic systems containing electrical and mechanical functionality. Lab Chip 2003, 3 (3), 150-157. 71. “Rapid technique for UV-curable adhesive bonding of glass coverslips to polystyrene microdevices”, David J. Guckenberger, Jake Kanack, Loren Stallcop, David J. Beebe, Chips and Tips, http://blogs.rsc.org/chipsandtips/2015/07/31/rapid-technique-for-uvcurable-adhesive-bonding-of-glass-coverslips-to-polystyrenemicrodevices/?doing_wp_cron=1533595711.64095902442932128906 25, sampled 8/6/2018. .


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Table of contents graphic:

9 ACS Paragon Plus Environment

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Figure 1. Cross sectional imaging of polystyrene impregnated with NPO dye in acetonitrile solution at 50oC for 30 sec duration. After drying, the sheet material was fractured. Images are shown with three illumination conditions: white light (left), white light plus UV light to excite the fluorophore (center), and UV light only with false blue color (right). In each image the solid is on the left while the air is on the right.


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Figure 2. Impregnation depth as a function of time at room temperature and 50oC using solutions of PtTFPP in acetonitrile.


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Figure 3. Images showing impregnation with oxygen sensitive dye PtTFPP in acetonitrile solution at 50oC for 30 sec, imaged in sulfite solution in water to eliminate oxygen so that the luminescence is not quenched during imaging. Imaging was carried out with the Etaluma Lumascope 600, excitation by LED with a band of 370-410 nm, with a window for transmission available at 612-680 for the emission.


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Figure 4. Images of the microchannel device (A) and the two-input region of the pore network device(B) Each shows the RGB image and the red channel only, from the CCD camera (inset on the pore network device image. 254x292mm (300 x 300 DPI)


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Figure 5. Images and profiles for the pore network device taken with the Lambert LIFA-P system, showing clockwise from the upper left, an intensity images, an intensity profile across the pillars and pores, a lifetime profile plot across the pores and pillars, and a lifetime image. These images were taken under gaseous nitrogen at 200 mL/min flow rate.


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Figure 6. Calibration curves for microchannel (MC, squares) and pore network (PN, diamonds) microfluidic sensors to gas-eous mixtures of air and nitrogen, showing fluorescence life-time (left axis) and SternVolmer representation (right axis, solid circle markers).


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Figure 7.

Phasor plots in gaseous nitrogen and air using data from a single pore area in the pore network device.


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Figure 8. Fluorescence lifetime imaging across the gradient in a two-input pore network device. Red is anoxic and blue is oxic. Liquid flow rates to each input were 1 microliter per second. The left pore is where the two input flows meet, with flow from left to right. 61x45mm (300 x 300 DPI)


Microfluidic Sensors with Impregnated Fluorophores for Simultaneous

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