J. M. Ramsey

Integrated microdevice for DNA restriction fragment analysis.

An integrated monolithic device (8 mm × 10 mm) that performs an automated biochemical procedure is demonstrated. The device mixes a DNA sample with a restriction enzyme in a 0.7-nL reaction chamber and after a digestion period injects the fragments onto a 67-mm-long capillary electrophoresis channel for sizing. Materials are precisely manipulated under computer control within the channel structure using electrokinetic transport. Digestion of the plasmid pBR322 by the enzyme HinfI and fragment analysis are completed in 5 min using 30 amol of DNA and 2.8 × 10(-3) unit of enzyme per run.

Low temperature bonding for microfabrication of chemical analysis devices

Abstract A low temperature bonding process was developed for the fabrication of microchip devices for liquid and heterogeneous phase chemical analysis. Photolithographically etched microchannels on glass substrates were closed by bonding a glass cover plate using a spin-on sodium silicate layer as an adhesive. Good channel sealing was achieved by curing at 90°C for 1 h or room temperature overnight. The fluidic performance of the device was evaluated by monitoring the electroosmotic flow on the chip. The results compared well with those obtained from devices made by high temperature direct bonding of the substrate and cover plate. The dielectric and mechanical strength for bonds, created using the low and high temperature methods, were compared. A dielectric strength of 400 kV cm−1 was obtained for the sodium silicate bonding and 1100 kV cm−1 for the high temperature bonding. Mechanical strength measurements gave a surface energy value of ≈2.7 J m−2 for sodium silicate bonding, compared to 6.5 J m−2 for direct bonding. The mechanical strength of glass bonds obtained with sodium silicate at low temperature was comparable to that reported for the sodium silicate bonding of silicon wafers at >200°C or by conventional direct bonding of oxidized silicon at 1400°C. The low temperature bonding performance is adequate for microfabricated fluidic devices that employ electrokinetic transport phenomena. The reduced temperature of the bonding process will allow chemical surface modification prior to bonding.

Minimizing the number of voltage sources and fluid reservoirs for electrokinetic valving in microfluidic devices.

A microchip gated valve is demonstrated that uses a single voltage source and three fluid reservoirs. The fluidic valve is a cross intersection, and the channels are dimensioned to perform the appropriate voltage division, simplifying the voltage control hardware. A single voltage source is applied directly to the sample reservoir and through a high-voltage relay to the buffer reservoir, and the waste reservoir is grounded. The volume of sample dispensed is determined by the duration that the high-voltage relay is open. Volumetric reproducibility is demonstrated to be <0.5% relative standard deviation for volumes of ≥20 pL. The valve is tested for the minimum applied voltage necessary for leakage-free operation, i.e., sample diffusing from the cross intersection into the analysis channel. Moreover, appropriate channel dimensions are used to minimize the number of fluid reservoirs allowing effluent from the analysis and waste channels to be combined into a single reservoir.

The Elucidation of Charge-Transfer-Induced Matrix Effects in Environmental Aerosols Via Real-Time Aerosol Mass Spectral Analysis of Individual Airborne Particles

Matrix effects in real-time aerosol mass spectrometry (RTAMS) were investigated using Standard Reference Materials (SRMs) obtained from the National Institute of Standards and Technology (NIST). Suppression of major component ions by much less concentrated species was observed. Attempts were made to mimic the ion suppression using binary systems but were unsuccessful. Data are presented that suggest the origin of the matrix effect is charge transfer induced neutralization in the ablation plume.

Homogeneous linewidths of Rhodamine 6G at room temperature from cavity-enhanced spontaneous emission rates

Fluorescence lifetimes of Rhodamine 6G in levitated micron‐sized droplets have been measured using a time‐correlated photon counting technique. The coupling of emission into spherical cavity modes of the droplet results in significant emission rate enhancements which allow estimation of the homogeneous linewidth at room temperature.

Direct Observation of the Evolution of the Soot Carbonization Process in an Acetylene Diffusion Flame via Real-Time Aerosol Mass Spectrometry

Abstract Real-time aerosol mass spectrometry was used to measure the size and composition of individual soot particles in an acetylene diffusion flame. This on-line data analysis technique permits for the first time direct observation of the evolution of the carbonization process, determination of the degree of carbonization of each measured particle, and measurement of the size distribution of both polycyclic aromatic hydrocarbon (PAH)-containing and mature soot particles. The carbonization process is characterized by rapid exchange of hydrogen between the PAH and pyrolytic addition of small hydrocarbons to form larger PAH molecules. The hydrogen exchange rate builds until carbon–carbon bond rearrangement becomes facile. The structural rearrangement/dehydrogenation process is very rapid once started. Rapid carbonization permits unambiguous size measurement of both PAH-containing and mature soot particles. Rapid hydrogen exchange yields a low activation energy path for making radicals in the particle phase and permits the PAH-containing media to act as a hydrogen sink. The presence of substantial amounts of labile hydrogen in the PAH-containing particle is demonstrated by the presence of preferentially hydrogenated PAHs. The absence of ethynylated PAHs and the presence of the hydrogenated PAHs are the result of flame pyrolysis of the PAH-containing particles. Optical images of flame particles collected by an independent sampling method conclusively confirm the presence of the micron sized PAH-containing particles in the flame and strongly suggest that mature soot aggregates are formed directly from the micron-sized PAH-containing particles. The optical images and the size distribution data cast doubt on the currently accepted mechanisms for mature soot aggregate formation. A new mature soot aggregate formation mechanism found in the aerosol literature is presented. This mechanism readily explains our results and many of the universal characteristics of soot. The implications of these measurements are discussed.

Real-time detection of individual airborne bacteria

Airborne bacteria and bacterial spores were directly sampled by an ion trap mass spectrometer with an atmospheric pressure inlet system. Samples were aerosolized from suspensions of single species. The organisms were individually characterized in real time by laser ablation mass spectrometry. Either positive or negative ions could be studied. Ions of a particular value of m/z (mass to charge ratio) could be further characterized by tandem mass spectrometry in the ion trap.

Confinement and manipulation of individual molecules in attoliter volumes.

We report observation of fluorescence from individual rhodamine 6G molecules in streams of charged 1-μm-diameter water droplets. With this approach, illumination volumes comparable to diffraction-limited fluorescence microscopy techniques (≤500 aL) are achieved, resulting in similarly high contrast between single-molecule fluorescence signals and nonfluorescent background. However, since the fluorescent molecules are confined to electrically charged droplets, in situ electrodynamic manipulation (e.g., focusing, switching, or merging) can be accomplished in a straightforward manner, allowing experimental control over both the delivery of molecules of interest to the observation region and the laser-molecule interaction time. As illustrated by photocount statistics that are independent of molecular diffusion and spatial characteristics of the excitation field, individual rhodamine 6G molecules in 1-μm droplets are reproducibly delivered to a target a few micrometers in diameter at a rate of between 10 and 100 Hz, with laser beam transit times more than 1 order of magnitude longer than diffusion-limited laser-molecule interaction times in equivalent volumes of free solution.

A CCD based approach to high-precision size and refractive index determination of levitated microdroplets using Fraunhofer diffraction

We describe a fast and convenient method of high precision size and refractive index determination of electrodynamically levitated microdroplets using Fraunhofer diffraction. The diffraction data were obtained with a 16-bit, unintensified charge coupled device (CCD) camera, and converted into angle-resolved elastic scattering intensity patterns by means of a carefully determined set of transformation parameters. The angular scattering patterns were analyzed without any a priori estimate of the droplet size and only a nominal estimate (≈2%) of the refractive index. Experimental angular scattering patterns were fit to calculated patterns from Mie theory using a graded step-size and scaling algorithm and optimized with respect to both droplet diameter and refractive index (real part only) with a precision of ⩽3 parts in 104 and 1 part in 103, respectively. Potential application to quantitative fluorescence and Raman spectroscopy, as well as mixture analysis in microdroplets is discussed.

Real-time observation of single-molecule fluorescence in microdroplet streams

We report real-time observation of fluorescence bursts from individual Rhodamine 6G molecules in streams of microdroplets (peak signal-to-noise ratios, approximately 30) whose trajectories are constrained with a linear electric quadrupole. This approach offers a reasonable dynamic range in droplet size (3- 12-microm diameter) with <1% shot-to-shot size fluctuations and sensitivity comparable with that of droplet levitation techniques with at least 10(3) higher analysis rates. Applications to the study of single-molecule microcavity effects and stimulated emission are discussed.