Long-pulsed luminescence for the measurement of dissolved oxygen.

Thin-film luminescent sensors were used to measure dissolved oxygen in picoliter volumes for the purpose of monitoring single-cell oxygen consumption rates, and that work served as the motivation for the development of the method described here. A few different platinum porphyrin sensor materials were examined, with all measurements conducted microscopically. By employing convolution theory to understand observed responses, including an unexpected red luminescent emission from an optic, we developed a new, rapid method for the determination of exponential decay lifetime. This new method of long-pulsed luminescence offers substantially improved signal-to-noise ratios for detected signals as long as self-illumination sources are carefully controlled in the experimental set-up.

Sensitive refractive index detection using a broad-band optical ring resonator.

Broad-band operation is investigated as an alternative means of refractive index detection in an optical ring resonator. In this work, a liquid-core (LCORR, capillary-based) design is used. Optical ring resonators have been recently demonstrated for the detection of a wide variety of analytes, including DNA, viruses, proteins, chemical vapors, and pesticides. In the field of analytical chemistry, much of their value is in the ability to provide enhanced detection of surface analytes in small volumes. Conventional analysis methods that employ a single resonant wavelength and propagation mode are highly dependent on resonator quality. By utilizing the complex and variable response of a multiple-mode resonator and the simultaneous data of more than 40 resonance peaks, impressive results exceeding the single-mode prediction are produced from a very modest quality device. The presented methods also become attractive through the use of inexpensive LED light sources and common UV-vis spectrometers, as well as the ability to also take absorbance measurements without any physical configuration changes. Complex interference spectra are produced from the convolution of multiple wavelengths and modes. Two possible methods for analyzing this type of data are presented-one using Fourier transform deconvolution to extract resonance components from interference spectra, and another using chemometrics by constructing a partial least-squares model. Using isopropyl alcohol and water mixtures, detection limits are the order of 10(-6) RIU (refractive index units), comparable to existing ring resonators devices. To study surface detectability and biomolecule detection feasibility, surface adsorption of bovine serum albumin (BSA) is also analyzed.

Broadband absorbance in a liquid-core optical ring resonator.

We have demonstrated the application of broadband absorption spectroscopy in a liquid-core optical ring resonator. An initial proof of concept of the broadband liquid-core optical ring resonator (BLCORR) was constructed using a thinned-wall, 250-µm-inner-diameter fused silica capillary, tapered multimode optical fibers for input and output coupling, and a light-emitting diode (LED) source. When compared with standard cuvette measurements, an apparent path length as high as 5 cm was observed for methylene blue (MB). MB is a cationic dye that exhibits strong surface interaction with bare silica. Bromothymol blue (BTB), on the other hand, has a similar absorbance spectrum but does not share this same surface activity. On comparing these two dyes, the apparent path length for MB was found to reach more than 50 times that of BTB, confirming the expectation that the sensing region being probed is largely within the evanescent field at the inner surface of the capillary. The BLCORR may also inherit, from attenuated total reflection (ATR) spectroscopy, the ability to analyze highly concentrated chromophores. Concentrations of BTB as high as 10(-2) and 10(-3) M were easily distinguished from each other at the λ(max) in the BLCORR, whereas this was not the case in a 4-mm cuvette cell. Our presented device employs commercially available materials and could incorporate well into microfluidic systems. These benefits, along with the demonstrated ability to take enhanced surface absorbance measurements in a capillary, give the BLCORR potential in a variety of applications.

Direct measurement of oxygen consumption rates from attached and unattached cells in a reversibly sealed, diffusionally isolated sample chamber.

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.

A microwell array device capable of measuring single-cell oxygen consumption rates.

Due to interest in cell population heterogeneity, the development of new technology and methodologies for studying single cells has dramatically increased in recent years. The ideal single cell measurement system would be high throughput for statistical relevance, would measure the most important cellular parameters, and minimize disruption of normal cell function. We have developed a microwell array device capable of measuring single cell oxygen consumption rates (OCR). This OCR device is able to diffusionally isolate single cells and enables the quantitative measurement of oxygen consumed by a single cell with fmol/min resolution in a non-invasive and relatively high throughput manner. A glass microwell array format containing fixed luminescent sensors allows for future incorporation of additional cellular parameter sensing capabilities. To demonstrate the utility of the OCR device, we determined the oxygen consumption rates of a small group of single cells (12 to 18) for three different cells lines: murine macrophage cell line RAW264.7, human epithelial lung cancer cell line A549, and human Barrett's esophagus cell line CP-D.

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.

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.

Coherence loss in light backscattering by random media with nanoscale nonuniformities.

An experimental technique for measuring time-resolved coherence loss and destruction of backscattered wave packets in random media is described. The results of such measurements, performed with a modified Michelson interferometer, contain rich information about the characteristics of media nonuniformities. Experimental data for model nanosuspensions are compared with theoretical expressions developed in the paper which include the effects of Mie-type resonant scattering. We attribute one such observed effect to enhanced ineleastic optical transitions near the surface of nonmetallic nanoparticles. The inverse problem of characterization of multiscattering random media by backscattering is also considered.

Measurement of respiration rates of Methylobacterium extorquens AM1 cultures by use of a phosphorescence-based sensor.

Respiration rates of bacterial cultures can be a powerful tool in gauging the effects of genetic manipulation and environmental changes affecting overall metabolism. We present an optical method for measuring respiration rates using a robust phosphorescence lifetime-based sensor and off-the-shelf technology. This method was tested with the facultative methylotroph Methylobacterium extorquens AM1 to demonstrate subtle mutant phenotypes.

Monitoring of a pharmaceutical nanomilling process using grating light reflection spectroscopy.

An optical diagnostic method, grating light reflection spectroscopy (GLRS), has been demonstrated for the in situ monitoring of properties of heterogeneous matrices in industrial processes. The technique is based on measurements near the critical points of intensity and phase in waves reflected from a transmission diffraction grating in contact with a diagnostic sample. The features contained in the reflection spectrum near these thresholds allow for the simultaneous determination of the real and imaginary parts of the dielectric function of the sample. Using these data, the milling progress of highly concentrated fluid suspensions is observed as the material is milled from approximately 40 mm to 160 nm in diameter. A theoretical model that closely resembles experimentally determined spectra was constructed and applied in combination with principal components analysis (PCA) to demonstrate that GLRS can be used to closely monitor changes in the mean particle size of the nanomilled drug product.

Ultrasonic diffraction grating spectroscopy and characterization of fluids and slurries.

The ultrasonic diffraction grating is formed by machining triangular grooves, 300 microns apart, on a stainless steel surface. The grating surface is in contact with the liquid or slurry. The ultrasonic beam, traveling in the solid, strikes the back of the grating and produces a transmitted m=1 beam in the liquid. The angle of this beam in the liquid increases with decreasing frequency and the critical frequency FCR occurs when the angle is 90 degrees. At frequencies below FCR, this m=1 wave does not exist and its energy is shared with other types of waves. The signal of the reflected m=0 wave is observed and an increase is observed at FCR. This information yields the velocity of sound in the liquid and particle size.

Characterization and use of a Raman liquid-core waveguide sensor using preconcentration principles.

A novel Raman sensor using a liquid-core optical waveguide is reported, implementing a Teflon-AF 2400 tube filled with water. An aqueous analyte mixture of benzene, toluene and p-xylene was introduced using a 1000 microl sample loop to the liquid-core waveguide (LCW) sensor and the analytes were preconcentrated on the inside surface of the waveguide tubing. The analytes were then eluted from the waveguide using an acetonitrile-water solvent mixture injected via a 30 microl eluting solvent loop. The preconcentration factor was experimentally determined to be 14-fold, in reasonable agreement with the theoretical preconcentration factor of 33 based upon the sample volume to elution volume ratio. Raman spectra of benzene, toluene and p-xylene were obtained during elution. It was found that analytically useful Raman signals for benzene, toluene and p-xylene were obtained at 992, 1004 and 1206 cm(-1), respectively. The relative standard deviation of the method was 3% for three replicate measurements. The limit of detection (LOD) was determined to be 730 ppb (parts per billion by volume) for benzene, exceptional for a system that does not resort to surface enhancement or resonance Raman approaches. The Raman spectra of these test analytes were evaluated for qualitative and quantitative analysis utility.

Toward a fully integrated positive-pressure driven microfabricated liquid analyzer.

A versatile integrated analyzer with a flow-programmed injection strategy and multiwavelength detection is described with applications toward sampling, flow injection analysis, and capillary separations. Continuous near-real-time sampling is a major benefit of the flow-programmed injection technique. Injection volumes ranging from 250 pL to several microliters were made without electrophoretic flow. Multiwavelength grating light reflection spectroscopy (GLRS) and transmission absorbance spectroscopy were performed simultaneously in a detection volume of 150 pL. The utility of these detection methods for refractive index (RI) and absorbance detection in capillary channels is demonstrated through analysis of salt, indicator, and dyes. GLRS is a unique, selective, and path-length-independent technique for probing RI, absorbance, and other optical properties. A limit of detection (LOD) of 170 microM was achieved for GLRS interferometric detection of FD&C Red #3, which corresponded to 2.6 fmol of analyte in the 150-pL detection volume. A LOD of 2 mM for phosphate buffer, or 3 fmol in the 150-pL detection volume will also be demonstrated. A siloxane coating on the GLRS grating was employed as a sensing layer to probe interactions between the sample and stationary phase. The combined GLRS interferometric response provided insight into both optical and chromatographic properties of samples. Open tubular capillary liquid chromatography with multidimensional multiwavelength detection is demonstrated for the analysis of three food dyes. Separation efficiency, N, of 16,000 was achieved for an unretained dye peak eluting at 12 min. Integration of novel sampling and detection schemes makes this a broadly applicable liquid analyzer.