We have developed a novel microfluidic device constructed from poly(dimethylsiloxane) using multilayer soft lithography technology for the analysis of single cells. The microfluidic network enables the passive and gentle separation of a single cell from the bulk cell suspension, and integrated valves and pumps enable the precise delivery of nanoliter volumes of reagents to that cell. Various applications are demonstrated, including cell viability assays, ionophore-mediated intracellular Ca2+ flux measurements, and multistep receptor-mediated Ca2+ measurements. These assays, and others, are achieved with significant improvements in reagent consumption, analysis time, and temporal resolution over macroscale alternatives.

A novel polarizer based on the dissolution-dynamic nuclear polarization (DNP) method has been designed, built and tested. The polarizer differs from those previously described by being designed with sterile use intent and being compatible with clinical use. The main features are: (1) an integral, disposable fluid path containing all pharmaceuticals constituting a sterile barrier, (2) a closed-cycle cryogenic system designed to eliminate consumption of liquid cryogens and (3) multi-sample polarization to increase throughput. The fluid path consists of a vial with the agent to be polarized, a pair of concentric inlet and outlet tubes connected to a syringe with dissolution medium and a receiver, respectively. The fluid path can operate at up to 400 K and 2.0 MPa and generates volumes as high as 100 mL. An inline filter removes the amount of electron paramagnetic agent in the final product by more than 100-fold in the case of [1-(13)C]pyruvate. The system uses a sorption pump in conjunction with a conventional cryocooler. The system operates through cycles of pumping to low temperature and regeneration of the sorption pump. The magnet accommodates four samples at the same time. A temperature of less than 1 K was achieved for 68 h (no sample heat loads) with a liquid helium volume of 2.4 L. The regeneration of the liquid helium could be achieved in less than 10 h, and the transition to cold (< 1.2 K) was achieved in less than 90 min. A solid state polarization of 36 ± 4% for [1-(13)C]pyruvic acid was obtained with only 10 mW of microwave power. The loading of a sample adds less than 50 J of heat to the helium bath by introducing the sample over 15 min. The heat load imposed on the helium bath during dissolution was less than 70 J. The measured liquid state polarization was 18 ± 2%.

A microfluidic flow injection analysis system has been designed and evaluated. The system incorporates within a single two-layer poly(dimethylsiloxane) monolith multiple pneumatically driven peristaltic pumps, an injection loop, a mixing column, and a transparent window for fluorescence detection. Central to this device is an injection system that mimics the operation of a standard six-port, two-way valve used in conventional liquid chromatography and flow injection experiments. Analyte and carrier solutions continuously flow through this injection system allowing for measurements and sample changes to be performed rapidly and simultaneously. Injection volumes of 1.25 nL generated peak area reproducibility of better than 3% relative standard deviation. The flow injection device was evaluated with fluorescent dyes and demonstrated a detection limit of 400 zmol for fluorescein. A rudimentary sample selection system allowed calibration curves to be rapidly produced, often in less than 10 min. The hydrolysis of fluorescein diphosphate by alkaline phosphatase demonstrates that chemical assays can be carried out with this device in a manner characterized by short analysis times and low sample consumption.

A method has been developed that allows the accurate, standardless measurement of the elemental composition of metal samples from single laser ablation (LA) pulses. This technique provides a fast, low-sample-consumption means for the characterization of samples having a range of matrixes. The method directly compares adjusted elemental signals with the total mass spectrometric signal to produce relative percent composition information. Three mathematical techniques were used to determine the accuracy and precision of single-shot LA measurement. Comparison of the techniques showed that a linear regression calculation, which plots individual elemental signals as a function of the summed signal for all elements in the sample on a point-by-point basis during a laser ablation transient proved superior. The simultaneous extraction capability of time-of-flight mass spectrometry permits the sampling of all analytes from any temporal position within the transient laser ablation pulse, thereby reducing quantitation error. A typical concentration dynamic range of 3 orders of magnitude, from 0.1 to 100%, was achieved. However, by measuring low-abundance isotopes for matrix elements, the dynamic range of the technique was extended to 4 orders of magnitude. The new technique is largely immune to sample matrix effects commonly experienced in laser ablation. By performing a complete elemental analysis from a single ablation pulse, high spatial resolution should be achieved.

Low sample consumption and high achievable spatial resolution combine to make laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) an attractive direct sampling technique for the analysis of solids. These desirable characteristics are best realized when the laser ablation system is operated in a single-shot fashion, in which each laser pulse produces a transient analyte signal. The temporal width of a single-shot LA analyte transient is inversely related to the best achievable signal-to-noise ratio (S/N). Thus, production of fast transient signals is an important consideration in single-shot LA analyses. In this study we explore the effects on transient pulse width of ablation-cell volume, the diameter and length of tubing used to connect the ablation cell to the ICP, and the composition and flow rate of ablation-cell sweep gas. Minimization of ablation-cell volume and the length and diameter of the transfer tube was found to dramatically decrease the peak widths of transient signals. However, use of helium gas to sweep analyte particles from the ablation cell was found to significantly reduce the effect of cell volume on transient width. Use of a cell volume of 0.70 cm3 and optimization of other instrumental parameters produced a transient sample pulse 85 ms in duration and limits of detection in the tens of femtograms range for single-shot laser-ablation events.

Optoelectronic consumer products that are widely employed in the office and home attract attention for optical sensor applications due to (1) their cost advantage over analytical instruments produced only in small quantities, (2) robustness in operation due to the detailed manufacturability improvements, and (3) ease of operation. We demonstrate here a new approach for quantitative chemical/biochemical sensing when analog signals are acquired from conventional optical disk drives, and these signals are used for quantitative detection of optical changes of sensor films deposited on conventional CD and DVD optical disks. Because we do not alter manufacturing process of optical disks, any disk can be employed for deposition and readout of sensor films. The optical disk drives also perform their original function of reading and writing digital content to optical media because no optical modifications are introduced to obtain the analog signal. Such a sensor platform is quite universal and can be applied for chemical and biological quantitative detection, as well as for monitoring of changes of physical properties of regions deposited onto a CD or DVD (e.g., during combinatorial screening of materials). As a model example, we demonstrate the concept using chemical detection of ionic species such as Ca2+ in liquids (e.g., blood, urine, or water). Colorimetric calcium-sensitive sensor films were deposited onto a DVD, exposed to water with different concentrations of Ca2+, and quantified in the optical disk drive. The developed lab-on-DVD system demonstrated a 5 ppm detection limit of Ca2+ determinations, similar or slightly better than that achieved using a conventional fiber-optic portable spectrometer. This detection limit corresponded to a 0.023 absorbance unit resolution, as determined by the measurement of the same colorimetric films with a portable spectrometer. Determinations of Ca2+ unknowns using the lab-on-DVD system demonstrated +/-5 ppm accuracy and 2-5% relative standard deviation precision in predicting 100 ppm Ca2+.

No abstract

The development of hyperpolarized technology utilizing dynamic nuclear polarization (DNP) has enabled the rapid measurement of 13C metabolism in vivo with very high SNR. However, with traditional DNP equipment, consecutive injections of a hyperpolarized compound in an animal have been subject to a practical minimum time between injections governed by the polarization build-up time, which is on the order of an hour for [1-13C]pyruvate. This has precluded the monitoring of metabolic changes occurring on a faster time scale. In this study, we demonstrated the ability to acquire in vivo dynamic magnetic resonance spectroscopy (MRS) and 3D magnetic resonance spectroscopic imaging (MRSI) data in normal rats with a 5 minute interval between injections of hyperpolarized [1-13C]pyruvate using a prototype, sub-Kelvin dynamic nuclear polarizer with the capability to simultaneously polarize up to 4 samples and dissolve them in rapid succession. There were minimal perturbations in the hyperpolarized spectra as a result of the multiple injections, suggesting that such an approach would not confound the investigation of metabolism occurring on this time scale. As an initial demonstration of the application of this technology and approach for monitoring rapid changes in metabolism as a result of a physiological intervention, we investigated the pharmacodynamics of the anti-cancer agent dichloroacetate (DCA), collecting hyperpolarized data before administration of DCA, 1 minute after administration, and 6 minutes after administration. Dramatic increases in 13C-bicarbonate were detected just 1 minute (as well as 6 minutes) after DCA administration.