A versatile pumpless multi-channel fluidics system for maintenance and real-time functional assessment of tissue and cells.

To address the needs of the life sciences community and the pharmaceutical industry in pre-clinical drug development to both maintain and continuously assess tissue metabolism and function with simple and rapid systems, we improved on the initial BaroFuse to develop it into a fully functional, pumpless, scalable multi-channel fluidics instrument that continuously measures changes in oxygen consumption and other endpoints in response to test compounds. We and several other laboratories assessed it with a wide range of tissue types including retina, pancreatic islets, liver, and hypothalamus with both aqueous and gaseous test compounds. The setup time was less than an hour for all collaborating groups, and there was close agreement between data obtained from the different laboratories. This easy-to-use system reliably generates real-time metabolic and functional data from tissue and cells in response to test compounds that will address a critical need in basic and applied research.

Bacterio- and Isobacteriodilactones by Stepwise or Direct Oxidations of meso-Tetrakis(pentafluorophenyl)porphyrin.

Porpholactones are porphyrinoids in which one or more ß,ß'-bonds of the parent chromophore were replaced by lactone moieties. Accessible to varying degrees by direct and nonselective oxidations of porphyrins, the rational syntheses of all five dilactone isomers along stepwise, controlled, and high-yielding routes via porphyrin → tetrahydroxyisobacteriochlorin metal complexes → isobacteriochlorindilactone metal complexes or porphyrin → tetrahydroxybacteriochlorin → bacteriochlorindilactone (and related) pathways, respectively, are described. A major benefit of these complementary routes over established methods is the simplicity of the isolation of the dilactones because of the reduced number of side products formed. In an alternative approach we report the direct and selective conversion of free base meso-tetrakis(pentafluorophenyl)porphyrin to all isomers of free base isobacteriodilactones using the oxidant cetyltrimethylN+MnO4-. The solid-state structures of some of the isomers and their precursors are reported, providing data on the conformational modulation induced by the derivatizations. We also rationalize computationally their differing thermodynamic stability and electronic properties. In making new efficient routes toward these dilactone isomers available, we enable the further study of this diverse class of porphyrinoids.

BaroFuse, a novel pressure-driven, adjustable-throughput perfusion system for tissue maintenance and assessment.

OBJECTIVES: Microfluidic perfusion systems are used for assessing cell and tissue function while assuring cellular viability. Low perfusate flow rates, desired both for conserving reagents and for extending the number of channels and duration of experiments, conventionally depend on peristaltic pumps to maintain flow yet such pumps are unwieldy and scale poorly for high-throughput applications requiring 16 or more channels. The goal of the study was to develop a scalable multichannel microfluidics system capable of maintaining and assessing kinetic responses of small amounts of tissue to drugs or changes in test conditions. METHODS: Here we describe the BaroFuse, a novel, multichannel microfluidics device fabricated using 3D-printing technology that uses gas pressure to drive large numbers of parallel perfusion experiments. The system is versatile with respect to endpoints due to the translucence of the walls of the perifusion chambers, enabling optical methods for interrogating the tissue status. The system was validated by the incorporation of an oxygen detection system that enabled continuous measurement of oxygen consumption rate (OCR). RESULTS: Stable and low flow rates (1-20 µL/min/channel) were finely controlled by a single pressure regulator (0.5-2 psi). Control of flow in 0.2 µL/min increments was achieved. Low flow rates allowed for changes in OCR in response to glucose to be well resolved with very small numbers of islets (1-10 islets/channel). Effects of acetaminophen on OCR by precision-cut liver slices of were dose dependent and similar to previously published values that used more tissue and peristaltic-pump driven flow. CONCLUSIONS: The very low flow rates and simplicity of design and operation of the BaroFuse device allow for the efficient generation of large number of kinetic profiles in OCR and other endpoints lasting from hours to days. The use of flow enhances the ability to make measurements on primary tissue where some elements of native three-dimensional structure are preserved. We offer the BaroFuse as a powerful tool for physiological studies and for pharmaceutical assessment of drug effects as well as personalized medicine.

Quantification of Low-Level Drug Effects Using Real-Time, in vitro Measurement of Oxygen Consumption Rate.

There is a general need to detect toxic effects of drugs during preclinical screening. We propose that increased sensitivity of xenobiotics toxicity combined with improved in vitro physiological recapitulation will more accurately assess potentially toxic perturbations of cellular biochemistry that are near in vivo pharmacological exposure levels. Importantly, measurement of such cytopathologies avoids activating mechanisms mediating toxicity at suprapharmacologic levels not relevant to in vivo effects. We present a sensitive method to measure changes in oxygen consumption rate (OCR), a well-established parameter reflecting a potential hazard, in response to exposure to pharmacologic levels of drugs using a flow culture system and state of the art oxygen sensing system. We tested metformin and acetaminophen on rat liver slices to illustrate the method. The features of the method include continuous and very stable measurement of OCR over the course of 48 h in liver slices in a continuous flow chamber with the ability to resolve changes as small as 0.3%/h. Kinetic modeling of metformin inhibition of OCR over a wide range of concentrations revealed both a slow and fast mechanism, where the fast mechanism activated only at concentrations above 0.6 mM. For both drugs, small amounts of inhibition were reversible, but higher decrements were irreversible. Overall the study highlights the advantages of measuring low-level toxicity so as to avoid the common extrapolations made about drug toxicity based on effects of drugs tested at suprapharmacologic levels.

High pH sensing with water-soluble porpholactone derivatives and their incorporation into a Nafion® optode membrane.

The known optical high pH sensing chromophores, free base and metal complexes (M = 2H, Zn(ii), Pt(ii)) of meso-tetrakis(pentafluorophenyl)porpholactone, and the as yet untested Ga(iii) complex, were made freely water-soluble by derivatization at the aryl group with PEG chains. Their halochromic response profiles were determined and found to be surprisingly shifted toward greater base sensitivity when compared to the parent sensors in aqueous solution in the presence of a surfactant. Select PEG-derivatized chromophores were also incorporated into Nafion®-based membranes. The immobilized sensor was shown to be suitable for a moderately rapid (response time in minutes) sensing of high concentrations of hydroxides (pH 11 and above, up to 5 M NaOH concentrations). The lesser sensitivity of the indicators in the membrane is rationalized by the anionic nature of the membrane material. The membrane shows a perfectly reversible response and remains transparent and stable even under extended times of exposure to very caustic environments, and no leaching of the chromophore is observed. The membrane might find use in fiber optics-based optodes suitable for the monitoring of high hydroxide environments inside chemical reactors or fuel cells.

Characterization of multi-dye pressure-sensitive microbeads.

The response times of pressure-sensitive particles to passing shockwaves were measured to investigate their ability to accurately determine pressure changes in unsteady flows. The particles tested were loaded with novel pressure-sensitive dyes such as Pt (II) meso-tetra(pentafluorophenyl)porphine, Pt(II) octaethylporphine, bis(3,5-difluoro-2-(2-pyridyl)phenyl-(2-carboxypyridyl))iridium III, and iridium(III) bis(4-phenylthieno[3,2-c] pyridinato-N,C2')acetylacetonate. For this work, porous silicon dioxide pressure-sensitive beads (PSBeads) were used. Two synthetic procedures were used to fabricate the particles. In the first, a one-step method loaded dyes during the synthesis of microbeads, in the second a two-step method synthesized the microbeads first, then loaded the dyes. The shock tube facility was used to measure the response times of microbeads to fast pressure jumps. The study involved testing multiple luminophors loaded in microbeads with various size distributions. Response times for the silica-based microbeads ranged between 26 µs and 462 µs (at 90% of the amplitude response), which are much faster than previously reported polystyrene-based microbead response times, which range from 507 µs to 1582 µs (at 90% of the amplitude response) [F. Kimura, M. Rodriguez, J. McCann, B. Carlson, D. Dabiri, G. Khalil, J. B. Callis, Y. Xia, and M. Gouterman, "Development and characterization of fast responding pressure sensitive microspheres," Rev. Sci. Instrum. 79, 074102 (2008)].

Microencapsulated 3-dimensional sensor for the measurement of oxygen in single isolated pancreatic islets.

BACKGROUND: Oxygen consumption reflects multiple processes in pancreatic islets including mechanisms contributing to insulin secretion, oxidative stress and viability, providing an important readout in studies of islet function, islet viability and drug testing. Due to the scarcity, heterogeneity, and intrinsic kinetic properties of individual islets, it would be of great benefit to detect oxygen consumption by single islets. We present a novel method we have developed to image oxygen in single islets. METHODOLOGY/PRINCIPAL FINDINGS: Using a microfluidics system, individual islets and a fluorescent oxygen-sensitive dye were encased within a thin alginate polymer layer. Insulin secretion by the encapsulated islets was normal. Fluorescent signal from the encased dye, detected using a standard inverted fluorescence microscope and digital camera, was stable and proportional to the amount of oxygen in the media. When integrated into a perifusion system, the sensing system detected changes in response to metabolic substrates, mitochondrial poisons, and induced-oscillations. Glucose responses averaged 30.1±7.1% of the response to a metabolic inhibitor (cyanide), increases were observed in all cases (n = 6), and the system was able to resolve changes in oxygen consumption that had a period greater than 0.5 minutes. The sensing system operated similarly from 2-48 hours following encapsulation, and viability and function of the islets were not significantly affected by the encapsulation process. CONCLUSIONS/SIGNIFICANCE: An oxygen-dependent dye situated around and within a pancreatic islet encapsulated by a thin layer of alginate was sensitive to changes in oxygen consumption, and was not harmful to the function or viability of islets over the course of two days. The microcapsule-based sensing method is particularly suited to assessing the effects of compounds (dose responses and time courses) and chronic changes occurring over the course of days. The approach should be applicable to other cell types and dyes sensitive to other biologically important molecules.

Simultaneous velocity and pressure measurements using luminescent microspheres.

Using the technique of modified rapid lifetime determination, pressure-sensitive microspheres, known as PrSBeads, were used to make quantitative oxygen measurements over two-dimensional areas within gaseous flows. Aerosolized PrSBeads in carrier gases of varying oxygen concentrations demonstrated point measurement precisions on the order of 0.1%-1%. A charge-coupled device featuring a double image frame (DIF) feature was used to make spatially resolved pressure measurements within gas phase flows. Errors on the order of 0.5 atm for one standard deviation were demonstrated when 2 x 2 pixel binning (162 x 128 pixel overall resolution) was used, but improved to 0.003-0.005 atm with the use of 32 x 32 pixel binning (10 x 8 pixel overall resolution). Experiments demonstrate the ability to resolve the oxygen concentration differences between a N(2) jet and the surrounding ambient air environment and the ability to measure instantaneous air pressure changes within a square syringe as the plunger is moved in and out. In addition, instantaneous velocity measurements of the airborne PrSBeads in a square syringe were achieved using digital particle image velocimetry at frame rates of 6.4 Hz, thus validating PrSBeads as a tool to simultaneously measure the velocity and pressure within an aerodynamic flow.

meso-Tetraarylporpholactones as high pH sensors.

The ability of meso-tetra(pentafluorophenyl)porpholactone (T(F)PL) and its Pt(II) complex [meso-tetra(pentafluorophenyl)porpholactonato]Pt(II) (T(F)PLPt) to function as optical high pH sensors is described. Under strongly alkaline or high methoxide conditions, their UV-vis spectra undergo dramatic and reversible red-shifts. The dynamic range for the sensor T(F)PLPt in solution is from pH 11.5 to 13.2. Using (1)H, (19)F, and (13)C NMR, UV-vis and IR spectroscopy, mass spectrometry, and the use of model compounds, the molecular origin of this optical shift is deduced to be a nucleophilic attack of OH(-)/MeO(-) on the lactone carbonyl of the chromophore, representing a novel mechanism for porphyrin-based sensors. The sensing compound was solubilized with Cremophor EL for use in aqueous solutions and embedded in polymer matrixes for testing as optical fiber-based optodes and planar sheet optode materials.

Synthesis, structure, and optical properties of the platinum(II) complexes of indaphyrin and thiaindaphyrin.

The novel free base meso-di(5'-methylthien-2'-yl)thiaindaphyrin, 10, was prepared from the corresponding meso-tetra(thien-2-yl)porphyrin using a methodology analogous to that for the preparation of known meso-diphenylindaphyrin, 5: beta,beta'-Dihydroxylation of the porphyrin is followed by oxidative diol cleavage. The resulting aldehyde moieties undergo an acid-catalyzed intramolecular Friedel-Crafts alkylation of the adjacent meso-thienyl groups with concomitant oxidation. Insertion of Pt(II) into either of the chromophores is facile, producing 5Pt and 10Pt. The crystal structure of 5Pt, the first for any indaphyrin, shows that the conformation of the indaphyrinato ligand is strongly ruffled, while the N(4) donor set that coordinates the central Pt(II) maintains a near-perfect square-planar coordination geometry around the central metal ion (crystal data for C(44)H(24)N(4)O(2)Pt: triclinic space group P1 with a = 8.8735(4) A, b = 12.9285(6) A, c = 14.3297(6) A, alpha = 88.785(1) degrees, beta = 82.248(1) degrees, gamma = 72.422(1) degrees; Z = 2). The UV-vis and emission spectra, triplet yields, and lifetimes of the Pt(II) complexes 5Pt and 10Pt were determined. Both complexes luminesce (in EtOH at 77 K) in the NIR (5Pt: lambda(max-emission) = 864, 974 nm, lifetime 2 micros; 10Pt: lambda(max-emission) = 990, 1112, 1276 nm) with modest to low quantum yields (Phi(p) approximately 1% and approximately 6 x 10(-3) %, respectively).

Development and characterization of fast responding pressure sensitive microspheres.

The response times of pressure sensitive paint (PSP) and pressure sensitive microspheres to passing shockwaves were measured to investigate their ability to accurately determine pressure changes in unsteady flows. The PSPs tested used platinum tetra(pentafluorophenyl)porphine (PtTFPP), platinum octaethylporphine (PtOEP), and a novel set of osmium-based organometallic complexes as pressure sensitive luminophors incorporated into polymer matrices of dimethylsiloxane bisphenol A-polycarbonate block copolymer or polystyrene. Two types of pressure sensitive microspheres were used, the first being PtOEP-doped polystyrene microspheres (PSBeads) and the second being porous silicon dioxide microspheres containing the novel, pressure sensitive osmium complexes. Response times for the platinum-based PSPs ranged from 47.2 to 53.0 micros, while the osmium-based PSPs ranged between 37.6 and 58.9 micros. For the microspheres, 2.5 microm diameter PSBeads showed a response time of 3.15 ms, while the osmium-based silicon dioxide microspheres showed a response time ranging between 13.6 and 18.9 micros.

Singlet molecular oxygen by direct excitation.

Direct excitation at 1064 nm and detection of singlet molecular oxygen at 1270 nm is made possible by the availability of powerful YAG-lasers and sensitive NIR photomultipliers. Singlet oxygen was generated in condensed phase at 77 K by direct excitation at 1064 nm (without the use of sensitizers). Several luminescing species were observed by time resolved luminescence spectroscopy and luminescence lifetime measurements, including the single molecule (1)Delta(g)and (1)Sigma(g)(+)states as well as luminescence from the [(1)Delta(g)](2) simultaneous transition. As an application we propose a novel method for obtaining quantitative non-intrusive mapping of the 2-D oxygen concentrations and pressure at cryogenic temperatures, which is of importance in aircraft design for high altitudes.

Observation of phenyl-fused porphyrinoids during the ESI mass spectrometric analysis of meso-pentafluorophenyl-substituted porphyrin and corrole.

The results of ESI(+) and ESI(-) mass spectrometry/mass spectrometry investigations of meso-tetrakisphenylporphyrin (TPP) and meso-trisphenylcorrole (TPC) in comparison with their meso-tetrakis(pentafluorophenyl)-substituted analogs T(F)PP and T(F)PC are reported. The fragmentation patterns of TPP and TPC show the expected loss of meso-aryl radicals. Analyzing the meso-pentafluorophenyl substituted analogs T(F)PP and T(F)PC, we found ESI(-) to be an excellent ionization mode. Rich and well-defined HF-elimination fragmentation patterns unique to the presence of meso-tetrafluorophenyl groups and evocative for the formation of fragments with direct covalent o-phenyl-to-beta-linkages are observed. A computation of the feasibility and relative energies of the resulting species corroborates the interpretation of the experimental findings. The computations indicate the presence of cooperative interactions between the linkages that direct the linkage formations to occur in a unidirectional fashion. MS/MS/MS experiments also provide indications for the regioselectivity of the fusions. Our observations further detail earlier reports of similar HF-eliminations and allow a generalization of the findings. The results presented may also point to strategies towards the bulk synthesis of novel porphyrinoid structures.

Electrospray ionization (ESI) tandem mass spectrometric analysis of meso-tetrakis(Heptafluoropropyl)porphyrin.

The results of an electrospray ionization (ESI(+) and ESI(-)) collision-induced fragmentation investigation of meso-tetrakis(heptafluoropropyl)porphyrin (T(F)HP) are reported. The fragmentation patterns of T(F)HP show, in both ionization modes, the formation of fragments with direct covalent meso-alkyl-to-beta-linkages on elimination of HF. In addition, in the ESI(+) mode, the losses of perfluoroalkyl radicals are observed. A detailed analysis of the fragmentation patterns allows conclusions to be drawn on the identification of the fragment species. Comparable cyclized products were previously produced using radical-initiated solution-phase syntheses. The results presented will help to analyze meso-perfluoroalkyl-derivatized porphyrinic macrocycles that are becoming increasingly popular.

Synthesis of polystyrene beads loaded with dual luminophors for self-referenced oxygen sensing.

Dispersion polymerization has been successfully applied to synthesize monodisperse polystyrene beads loaded with SiOEP and PtOEP for self-referenced oxygen sensing. The polystyrene beads became larger in size as the concentration of initiator was increased due to the reduction of primary particles precipitated from the polymerization medium. The dual luminophors showed similar absorption spectra but two distinctive emission spectra with peaks at 580 and 650nm for SiOEP and PtOEP, respectively. While the emission of SiOEP exhibited no response to oxygen, the luminescence intensity of PtOEP was monotonically dependent on the concentration of oxygen. From the Stern-Volmer plot, we observed a linear correlation between the intensity ratio of SiOEP at 580nm to PtOEP at 650nm and the concentration of oxygen, which could be used to reliably monitor the partial pressure of oxygen in a system.

Regulation of ATP/ADP in pancreatic islets.

ATP and ADP levels are critical regulators of glucose-stimulated insulin secretion. In many aerobic cell types, the phosphorylation potential (ATP/ADP/P(i)) is controlled by sensing mechanisms inherent in mitochondrial metabolism that feed back and induce compensatory changes in electron transport. To determine whether such regulation may contribute to stimulus-secretion coupling in islet cells, we used a recently developed flow culture system to continuously and noninvasively measure cytochrome c redox state and oxygen consumption as indexes of electron transport in perifused isolated rat islets. Increasing substrate availability by increasing glucose increased cytochrome c reduction and oxygen consumption, whereas increasing metabolic demand with glibenclamide increased oxygen consumption but not cytochrome c reduction. The data were analyzed using a kinetic model of the dual control of electron transport and oxygen consumption by substrate availability and energy demand, and ATP/ADP/P(i) was estimated as a function of time. ATP/ADP/P(i) increased in response to glucose and decreased in response to glibenclamide, consistent with what is known about the effects of these agents on energy state. Therefore, a simple model representing the hypothesized role of mitochondrial coupling in governing phosphorylation potential correctly predicted the directional changes in ATP/ADP/P(i). Thus, the data support the notion that mitochondrial-coupling mechanisms, by virtue of their role in establishing ATP and ADP levels, may play a role in mediating nutrient-stimulated insulin secretion. Our results also offer a new method for continuous noninvasive measures of islet cell phosphorylation potential, a critical metabolic variable that controls insulin secretion by ATP-sensitive K(+)-dependent and -independent mechanisms.

Continuous measurement of oxygen consumption by pancreatic islets.

The rate of oxygen consumption is an important measure of mitochondrial function in all aerobic cells. In pancreatic beta cells, it is linked to the transduction mechanism that mediates glucose-stimulated insulin secretion. However, measurement of oxygen consumption over long periods of time is technically difficult owing to the error resulting from baseline drift and the challenge of measuring small changes in oxygen tension. We have adapted an ultrastable oxygen sensor based on the detection of the decay of the phosphorescent emission from an oxygen-sensitive dye to a previously developed islet flow culture system. The drift of the sensor is approximately 0.3%/24 h, allowing for the continuous measurement of oxygen consumption by 300 islets (or about 6 x 10(5) cells) for hours or days. Rat islets placed in the perifusion chamber for 24 h were well maintained as reflected by membrane integrity, insulin secretion, and oxygen consumption. Both acute changes in oxygen consumption as induced by glucose and chronic changes as induced by sequential pulses of azide were resolved. The features of the flow culture system--aseptic conditions, fine temporal control of the composition of the media, and the collection of outflow fractions for measurement of insulin, and other products--facilitate a systematic approach to assessing metabolic and functional viability in responses to a variety of stimuli. Applications to the measurement of effects of hypoxia on insulin secretion, membrane integrity, and the redox state of cytochromes are demonstrated. The system has particular application to the field of human islet transplantation, where assessment and the study of islet viability have been hampered by a lack of experimental methods.