Fred P. Milanovich

High-Throughput Droplet Digital PCR System for Absolute Quantitation of DNA Copy Number

Digital PCR enables the absolute quantitation of nucleic acids in a sample. The lack of scalable and practical technologies for digital PCR implementation has hampered the widespread adoption of this inherently powerful technique. Here we describe a high-throughput droplet digital PCR (ddPCR) system that enables processing of ∼2 million PCR reactions using conventional TaqMan assays with a 96-well plate workflow. Three applications demonstrate that the massive partitioning afforded by our ddPCR system provides orders of magnitude more precision and sensitivity than real-time PCR. First, we show the accurate measurement of germline copy number variation. Second, for rare alleles, we show sensitive detection of mutant DNA in a 100 000-fold excess of wildtype background. Third, we demonstrate absolute quantitation of circulating fetal and maternal DNA from cell-free plasma. We anticipate this ddPCR system will allow researchers to explore complex genetic landscapes, discover and validate new disease associations, and define a new era of molecular diagnostics.

A fiber-optic sensor for CO2 measurement

A fiber-optic sensor has been prepared which responds to carbon dioxide at physiologically significant concentrations. It is based on pH modulation by dissolved carbon dioxide in a sensing layer of fluorescent dye. By use of a previously developed methodology by which the sensing chemistry is bonded directly to the glass fiber tip, the miniature size of the sensor is preserved. This method involves consecutive applications of solution polymers to the fiber tip rather than mechanical attachment of sensor reagents. Preparations of polymer-immobilized dyes and polymer membranes are described.

Laser-fiber-optic "optrode" for real time in vivo blood carbon dioxide level monitoring

A fiber-optic sensor has been developed to measure and monitor the partial pressure of carbon dioxide in blood via fluorescence or phosphorescence changes in sensing dyes. The pCO 2 optrode uses a nanoliter size droplet of a concentrated fluorescein derivative in a buffered sodium ascorbate solution as a pH indicator. The pH of the droplet equilibrates with the volatile constituents of blood across an air bubble trapped at the tip of the capillary surrounding the fiber. Ascorbic acid is used to reduce photobleaching, which is further minimized by working on the right portion of the concentration quenching curve. Signal levels of 300 000 counts per second are obtained at 0.5 μW using the 488-nm line of an argon ion laser. A response range of 25 percent was obtained when the sensor was evaluated from 1.97 to 7.40 percent CO 2 in air at controlled humidity and temperature.

Raman spectroscopy of Kevlar 49 fibre

Abstract The Raman spectrum of poly( p -phenylene terephthalate) fibre, Kevlar 49, is reported. Fibre which had been exposed to heat, stress and radiation indicated that within experimental error, no general change takes place in the chemical structure of the material as a whole. However the spectrum of a filament under stress shows that all bands in the spectrum become increasingly polarized with increased stress. The spectral characteristics of the stress fibre lead to a possible explanation of the polymers response to deformation — opening of the angles in the amide linkage of the polymeric chain and improved alignment of crystallites along the fibre axis.

Raman spectroscopic investigations of sarcoplasmic reticulum membranes.

Raman spectra are presented for sarcoplasmic reticulum membranes. Interpretation of the 1000-1130 cm-1 region of the spectrum indicates that the sarcoplasmic reticulum membrane may be more fluid than erythrocyte membranes that have been examined by the I portion of the membrane spectrum with a strong 1658 cm-1 band characteristic of C=C stretching in hydrocarbon side chains exhibiting cis conformation. This band is unaltered in intensity and position in H2O and in 2H2O thus obscuring amide I protein conformation. Of particular interest is the appearance of strong, resonantly enhanced bands at 1160 and 1527 cm-1 attributable to membrane-associated carotenoids.

Remote Detection of Organochlorides With a Fiber Optic Based Sensor

ABSTRACT Optical fibers, analytical chemistry, and fluorescence spectroscopy have been integrated to form the new technology of remote fiber fluorimetry. Among the many potential applications of this technology is the measurement of volatile organochlorides in ground water and in the vadose zone. The key to this application was the development of a fiber optic chemical sensor (FOCS) with sub-ppm sensitivity for, at least, chloroform and trichlorethylene (TCE). We present here the concept and instrumentation of remote fiber fluorimetry, the design and laboratory evaluation of an organochloride FOCS, and preliminary field results of the application of the FOCS to contaminated well water.

High-laser-damage-threshold potassium dihydrogen phosphate crystals

Development of solid‐state laser drivers of higher operating fluence is dependent upon the damage resistance of the frequency conversion crystals. We have demonstrated the correlation between the purity of the crystal growth solution and the laser damage threshold of single‐crystal potassium dihydrogen phosphate (KDP). Impurities introduced in the growth process can be atomic species, inorganic or organic compounds (dissolved or particulate), or bacteria. We have developed a purification process for KDP that minimizes contamination: initial recrystallization of bulk KDP followed by ozonation to remove oxidizable material, ultrafiltration to remove nonoxidized particles, and UV‐light exposure to suppress bacterial growth. We have also developed a crystal growth method that excludes load‐bearing surfaces, which are a potential source of particulate contamination, from the growth environment. The method, which incorporates continuous filtration and continuous flow of the growth solution has yielded crystals with damage thresholds of 28.6 J/cm2 at 355 nm and greater than 64 J/cm2 at 1064 nm (10‐ns pulse length).Development of solid‐state laser drivers of higher operating fluence is dependent upon the damage resistance of the frequency conversion crystals. We have demonstrated the correlation between the purity of the crystal growth solution and the laser damage threshold of single‐crystal potassium dihydrogen phosphate (KDP). Impurities introduced in the growth process can be atomic species, inorganic or organic compounds (dissolved or particulate), or bacteria. We have developed a purification process for KDP that minimizes contamination: initial recrystallization of bulk KDP followed by ozonation to remove oxidizable material, ultrafiltration to remove nonoxidized particles, and UV‐light exposure to suppress bacterial growth. We have also developed a crystal growth method that excludes load‐bearing surfaces, which are a potential source of particulate contamination, from the growth environment. The method, which incorporates continuous filtration and continuous flow of the growth solution has yielded crystals ...

High sensitivity atmospheric transmission measurements using a cavity ringdown technique

The design and operation of an absorption spectrometer is described. The spectrometer isused to measure water vapor absorption in the ambient laboratory atmosphere.(AIP)

Raman spectroscopic analysis of Dutch Belt rabbit erythrocyte ghosts

Abstract Dutch Belt rabbit erythrocyte ghosts have been examined by Raman spectroscopy. An unusually high signal-to-noise spectrum was obtained which enabled assessment of vibrations within 300 cm −1 of the exciting radiation. Assignment of the observed bands to specific vibrations yielded information concerning membrane fluidity, the conformations of the peptide backbones and disulfide bonds of membrane proteins, and the configurations of lipid unsaturated hydrocarbon side chains.

Clinical measurements using fiber optics and optrodes

Fiber optics, optrodes, and fluorescence spectroscopy have been combined to form the new technology of remote fiber fluorimetry (RFF). Both in-vivo and in-vitro clinical measurements can be made by using this technique. The optrode, a fiber termination with preselected chemical or physical properties, is attached to the distal end of the optical fiber so that specific, in-situ measurements can be made. RFF systems for pH, blood pressure, oxygen, and carbon dioxide are being completed, and other optrodes are in the development stages.