Wieslaw Stryjewski

Single molecule detection of double-stranded DNA in poly(methylmethacrylate) and polycarbonate microfluidic devices

Single photon burst techniques were used to detect double‐stranded DNA molecules in poly(methylmethacrylate) (PMMA) and polycarbonate (PC) microfluidic devices. A confocal epi‐illumination detection system was constructed to monitor the fluorescence signature from single DNA molecules that were multiply labeled with the mono‐intercalating dye, TOPRO‐5, which possessed an absorption maximum at 765 nm allowing excitation with a solid‐state diode laser and fluorescence monitoring in the near‐infrared (IR). Near‐IR excitation minimized autofluorescence produced from the polymer substrate, which was found to be significantly greater when excitation was provided in the visible range (488 nm). A solution containing ‐DNA (48.5 kbp) was electrokinetically transported through the microfluidic devices at different applied voltages and solution pH values to investigate the effects of polymer substrate on the transport rate and detection efficiency of single molecular events. By applying an autocorrelation analysis to the data, we were able to obtain the molecular transit time of the individual molecules as they passed through the 7 νm laser beam. It was observed that the applied voltage for both devices affected the transport rate. However, solution pH did not alter the transit time for PMMA‐based devices since the electroosmotic flow of PMMA was independent of solution pH. In addition, efforts were directed toward optimizing the sampling efficiency (number of molecules passing through the probe volume) by using either hydrodynamically focused flows from a sheath generated by electrokinetic pumping from side channels or reducing the channel width of the microfluidic device. Due to the low electroosmotic flows generated by both PMMA and PC, tight focusing of the sample stream was not possible. However, in PMMA devices, flow gating was observed by applying field strengths >–120 V/cm to the sheath flow channels. By narrowing the microchannel width, the number of molecular events detected per unit time was found to be four times higher in channels with 10 νm widths compared to those of 50 νm, indicating improved sampling efficiency for the narrower channels without significantly deteriorating detection efficiency. Attempts were made to do single molecule sizing of ‐DNA, M13 (7.2 kbp) and pUC19 (2.7 kbp) using photon burst detection. While the average number of photons for each DNA type were different, the standard deviations were large due to the Gaussian intensity profile of the excitation beam. To demonstrate the sensitivity of single molecule analysis in the near‐IR using polymer microfluidic devices, the near‐IR chromophore, NN382, was analyzed using our confocal imager. A detection efficiency of 94% for single NN382 molecules was observed in the PC devices.

Time-resolved fluorescence of the single tryptophan of Bacillus stearothermophilus phosphofructokinase

The fluorescence of the single tryptophan in Bacillus stearothermophilus phosphofructokinase was characterized by steady-state and time-resolved techniques. The enzyme is a tetramer of identical subunits, which undergo a concerted allosteric transition. Time-resolved emission spectral data were fitted to discrete and distributed lifetime models. The fluorescence decay is a double exponential with lifetimes of 1.6 and 4.4 ns and relative amplitudes of 40 and 60%. The emission spectra of both components are identical with maxima at 327 nm. The quantum yield is 0.31 +/- 0.01. The shorter lifetime is independent of temperature; the longer lifetime has weak temperature dependence with activation energy of 1 kcal/mol. The fluorescence intensity and decay are the same in H2O and D2O solutions, indicating that the indole ring is not accessible to bulk aqueous solution. The fluorescence is not quenched significantly by iodide, but it is quenched by acrylamide with bimolecular rate constant of 5 x 10(8) M-1 s-1. Static and dynamic light scattering measurements show that the enzyme is a tetramer in solution with hydrodynamic radius of 40 A. Steady-state and time-resolved fluorescence anisotropies indicate that the tryptophan is immobile. The allosteric transition has little effect on the fluorescence properties. The fluorescence results are related to the x-ray structure.

Optimization of sequencing conditions using near-infrared lifetime identification methods in capillary gel electrophoresis

We have investigated the sample preparation and electrophoresis conditions necessary to prepare DNA sequencing samples appropriate for use with near‐infrared (IR) fluorescent labels with dye identification accomplished via lifetime techniques. It was found that several sample preparation protocols required attention to maximize the fluorescence yields of the labeling dyes, such as thermal cycling conditions, choice of counter ion used for the ethanol precipitation step and also, dye‐primer versus dye‐terminator chemistries. In addition, several different sieving matrices were investigated for their effects on both the fluorescence properties of the labeling dyes and electrophoretic resolution. Extended times used for the high temperature denaturing of duplexed DNA fragments during cycle sequencing produced cleavage products, in which the covalently attached dye to the sequencing primer was released through attack by dithiothreitol (DTT). Even under optimized thermal cycling conditions, free dye was generated that masked readable data from the sequencing traces. Ethanol precipitation was necessary to remove this free dye with the proper choice of counter ion (sodium). The results using different sieving matrices indicated that linear polyacrylamides (LPAs) were appropriate for any fluorescence measurement, since they could readily be replaced between runs minimizing deleterious memory effects associated with cross‐linked polyacrylamide gels. After investigation of several different sieving LPAs, the commercially available POP6 was found to be particularly attractive, since it produced good electrophoretic resolution, single exponential behavior for the near‐IR dye series investigated herein, and also, discernible lifetime differences within the dye set. Finally, dye‐terminator chemistry was also found to minimize bleeding in the gel matrix produced by large amounts of unextended dye‐primer within the gel lane.

Macintosh/LabVIEW based control and data acquisition system for a single photon counting fluorometer

A flexible software system has been developed for controlling fluorescence decay measurements using the virtual instrument approach offered by LabVIEW. The time‐correlated single photon counting instrument operates under computer control in both manual and automatic mode. Implementation time was short and the equipment is now easier to use, reducing the training time required for new investigators. It is not difficult to customize the front panel or adapt the program to a different instrument. We found LabVIEW much more convenient to use for this application than traditional, textual computer languages.

Self-diffusion of a semiflexible polymer measured across the lyotropic liquid-crystalline-phase boundary

The self-diffusion of fluorescently tagged poly(γ-benzyl-α,L-glutamate), a helical, semiflexible synthetic homopolypeptide, has been measured in isotropic and cholesteric liquid-crystalline solutions by pattern fluorescence photobleaching recovery. On the isotropic side of the sharp isotropic–liquid-crystalline (ISO–LC) phase boundary, the rodlike polymers assume all possible orientations in a three-dimensional space, becoming enmeshed. In liquid-crystalline solutions, as first shown by Robinson [Trans. Faraday Soc. 52, 571 (1956)], spontaneous alignment of the cholesteric screw axis parallel to the optical (z) axis of the instrument produces small monodomains in which parallel rodlike polymers are organized into planes. Each horizontal plane is twisted slightly compared to its neighbors. Over the thickness of the sample, the rodlike polymers assume all possible orientations in this two-dimensional space. Despite the small size of the monodomains, it was possible to determine the self-diffusion coefficien...

A fiber-optic-based multichannel time-correlated single-photon-counting device with subnanosecond time resolution

A fiber-optic-based multichannel time-correlated single-photon-counting device with subnanosecond time resolution was developed. A passively mode-locked Ti:sapphire laser provided the excitation source for 12 separate time-correlated single-photon counting channels in which three single-photon avalanche diode detectors were used to analyze the fluorescence from the channels. Single-mode optical fibers provided the conduit for the excitation light as well as the fluorescence emission. We determined that the excitation pulses undergo a temporal spread of 50 fs, a spectral spread of 0.2 nm, and remained nearly transform limited. The average instrument response function of each channel was determined to be 181 ps (full width at half maximum). The fluorescence lifetime of a representative near-infrared dye, aluminum tetrasulfonated naphthalocyanine, was determined to be 3.08 ns using this fiber-optic-based multichannel time-correlated single-photon-counting device.

Detection of low abundant mutations in DNA using single molecule FRET and ligase detection reactions

New strategies for analyzing molecular signatures of disease states in real time using single pair fluorescence resonance energy transfer (spFRET) were developed to rapidly detect point mutations in unamplified genomic DNA (DNA diagnostics). The assay was carried out using allele-specific primers, which flanked the point mutation in the target gene fragment and were ligated using a thremostable ligase enzyme only when the genomic DNA carried this mutation (ligase detection reaction, LDR). We coupled LDR with spFRET to identify a single base mutation in codon 12 of a K-ras oncogene that has high diagnostic value for colorectal cancers. A simple diode laser-based fluorescence system capable of interrogating single fluorescent molecules undergoing FRET was used to detect photon bursts generated from the MB probes formed upon ligation. We demonstrated the ability to rapidly discriminate single base differences in heterogeneous populations having as little as 600 copies of human genomic DNA without PCR amplification. Single base difference in the K-ras gene was discriminated in less than 5 min at a frequency of 1 mutant DNA per 10 normals using only a single LDR thermal cycle of genomic DNA. Real time analyses of point mutations were also performed in PMMA microfluidic device.

Multiplexed analysis using time-resolved near-IR fluorescence for the detection of genomic material

While fluorescence continues to be an important tool in genomics, new challenges are being encountered due to increased efforts toward miniaturization reducing detection volumes and the need for screening multiple targets simultaneously. We have initiated work on developing time- resolved near-IR fluorescence as an additional tool for the multiplexed analyses of DNA, either for sequencing or mutation detection. We have fabricated simple and compact time-resolved fluorescence microscopes for reading fluorescence from electrophoresis or DNA microarrays. These microscopes consist of solid-state diode lasers and diode detectors and due to their compact size, the optical components and laser head can be mounted on high-speed micro-translational stages to read fluorescence from either multi-channel capillary electrophoresis instruments or micro fabricated DNA sorting devices. The detector is configured in a time-correlated single photon counting format to allow acquisition of fluorescence lifetimes on-the-fly during data acquisition in the limit of low counting statistics. In multiplexed analyses, lifetime discrimination serves as a method for dye-reporter identification using only a single readout channel. Also, coupled to multi-color systems, lifetime identification can significantly increase the number of probes monitored in a single instrument. In this work, near-IR fluorescence, including dye-labels and hardware, will be discussed as well as the implementation of near-IR fluorescence in DNA sequencing using slab gel electrophoresis and DNA microarrays.

Multichannel Capillary Electrochromatography PMMA Microdevice with Integrated Pulsed Conductivity Detector

The development of a 16-channel C18 modified poly(methyl methacrylate) (PMMA) microanalytical device utilizing contact conductivity detection for the purification/ separation of PCR products is described. The separation is carried out using reverse-phase ion-pair capillary electrochromatography (RP-IPCEC) in the modified PMMA device.

Solid-state fluorescence lifetime measurements

The intrinsic fluorescence properties of molecules in condensed phases are complicated by intermolecular interactions. In crystalline solids the fluorescence depends on the photophysics of individual molecules as well as the interactions between molecules in the solid state Even a chemically pure crystal has some lattice disorder, which alters the local environment and changes the intermolecular interactions. Sample preparation and experimental technique may influence the fluorescence results.