Benjamin L. Legendre

Binding Properties of Near-IR Dyes to Proteins and Separation of the Dye/Protein Complexes Using Capillary Electrophoresis with Laser-Induced Fluorescence Detection

The noncovalent binding of the near-infrared (NIR) dyes, DTTCI (cationic) and IR-125 (anionic), to several model proteins was investigated with the use of steady-state and picosecond laser fluorescence measurements. In an aqueous borate buffer (pH = 9.2), minimal fluorescence emission from these NIR dyes was observed. When a protein was added to the solution, enhancements in the fluorescence emission were found for both dyes. Time-resolved fluorescence measurements for IR-125 in the presence of the protein, β-casein, indicated a biexponential decay with lifetimes of 195 and 682 ps (χ2 = 1.94). Our data suggest that these dyes distribute themselves between the hydrophobic core of the protein and the interstitial aqueous solution. The dye molecules residing in the interior of the protein exhibit enhancements in their fluorescence due to a more favorable microenvironment. The binding and enhanced fluorescence properties allowed the use of these dyes as noncovalent stains for the low-level detection of proteins separated via capillary electrophoresis (CE). Detection limits for some model proteins separated by CE and stained with these NIR dyes were found to be superior to those obtained by using UV detection in CE.

Near-infrared, laser-induced fluorescence detection for DNA sequencing applications

Laser-induced fluorescence detection has become the detection strategy of choice in many large-scale DNA sequencing applications due to its ease of Implementation, sensitivity and the ability to identify the constituent bases of DNA in a single separation lane when the probes used have a distinct spectral characteristic. While the common strategy is to use fluorescent dyes which show absorption and emission properties in the visible region (400-600 nm) of the electromagnetic spectrum, our efforts have been directed toward developing near-IR (700-1000 nm) fluorescence as a viable detection strategy for DNA sequencing. In this paper, we discuss our results concerning the use of near-IR fluorescence detection for DNA sequencing carried out in a capillary gel column, where the capillary column has an internal diameter of 75 /spl mu/m, and the loading level of DNA onto this column is in the nL regime, requiring ultra-sensitive detection. In addition, we discuss our efforts toward the development of a highly efficient, single lane, single fluor, base-calling strategy using lifetime discrimination of heavy-atom modified near-IR dyes. The dyes developed for this application contain an intramolecular heavy atom (halogen) on a remote section of the chromophore, resulting in a perturbation in the fluorescence lifetime without altering the absorption or emission maximum of the base chromophore. This will allow the dye series to be excited with a single laser with the fluorescence processed on a single detector and the identity of the terminal base accomplished via lifetime discrimination. In order to effectively carry out lifetime measurements during capillary electrophoretic separation of the oligonucleotides, a simple solid-state time-correlated single photon counting instrument was constructed.

Error Analysis of Simple Algorithms for Determining Fluorescence Lifetimes in Ultradilute Dye Solutions

We have evaluated the use of two simple algorithms for determining the decay parameters describing a single exponential process for dyes with nanosecond and subnanosecond fluorescence lifetimes in the limit of low concentrations and high backgrounds from scattered photons generated by the solvent using experimental and Monte Carlo simulation results. These algorithms, the maximum likelihood estimator (MLE) and the rapid lifetime determination (RLD), are computationally easy to perform, allowing the evaluation of large amounts of data quickly and efficiently. The MLE and RLD methods were used to calculate the fluorescence lifetimes of three near-IR dyes with lifetimes spanning the range of 0.57 ns to 1.12 ns. For low-concentration conditions and high background-to-fluorescence ratios, the MLE method resulted in larger errors when compared to RLD, although both methods yielded comparable standard deviations. However, when the interval over which the lifetime was calculated within the decay profile was shifted to latter times in order to reduce the amount of scattered photons included in the calculation, significant improvements in the accuracy were observed with the use of MLE. Shifting the start channel of the calculation to latter time channels within the decay profile did not affect the lifetime with the use of RLD. Inclusion of large amounts of scattering photons was found to bias the calculated lifetime to lower values, reducing the accuracy of the determination. The relative standard deviations for MLE and RLD were found to be approximately 2–3% at a background-to-fluorescence ratio of 0.5. The absolute relative error in the methods at the 0.50 background-to-fluorescence ratio ranged from 14 to 27% for MLE and 8 to 18% for the RLD method when the calculation was initiated at t = 0. This error was found to decrease to < 1% with the use of MLE when the calculation was initiated at t ≈ 100 ps.

Single-Molecule Detection in the Near-IR Using Continuous-Wave Diode Laser Excitation with an Avalanche Photon Detector

While single-molecule detection in flowing sample streams has been reported by a number of groups, the instrumentation can be somewhat prohibitive for many applications due to the complexity and extensive expertise required to operate such a device. In this paper we report on the construction of a single-molecule detection device that is rugged, compact, inexpensive, and easily operated by individuals not well trained in optics and laser operations. The single-molecule detection apparatus consists of a semiconductor diode laser operating in a continuous-wave (CW) mode and a single photon avalanche diode transducer for converting the detected photons into transistor–transistor logic (TTL) pulses for displaying the data. In addition, the sampling volume is produced by a single-component lens, to create a volume on the order of 1 pL, allowing the sampling of microliter volumes of material on reasonable time scales. The device is targeted for operation in the near-IR region (700–1000 nm), where matrix interferences are minimal. Our data will demonstrate the detection of single molecules for the near-IR dyes IR-132 and IR-125, in methanol solvents in flowing sample streams at sampling rates of 100–250 samples/s. Detection efficiencies for the investigated near-IR dyes were found to be 98% for IR-132 and 50% for IR-125. Previous attempts in our laboratory to detect single molecules of IR-125 using time-gated detection were unsuccessful because of the short upper-state lifetime of this fluorophore (τf = 472 ps).

Micro-DNA sequence analysis using capillary electrophoresis and near-IR fluorescence detection

Our group is developing optical solid-state, near-IR (NIR) fluorescence detection systems for the analyses of DNA in restriction mapping and sequencing applications. Specifically, we are investigating a base-calling scheme using fluorescence lifetime discrimination in the NIR implementing intramolecular heavy-atom modified NIR fluorescence dyes which can be configured in a single lane, single fluor format. Results are presented concerning the ability to perform lifetime measurements on-line during capillary gel electrophoresis. Due to the high sensitivity associated with NIR fluorescence detection and small injection volumes in capillary electrophoresis, micro-reactor systems are also being developed which will potentially reduce the sample size for preparation of DNA sequencing ladders. The Sanger dideoxy-terminated fragments are prepared using solid-phase sequencing strategies in these micro-capillary reaction chambers. Our discussion focuses on the stability of the immobilized DNA template under typical sequencing conditions and the ability to sequence long templates using this strategy. Finally, we discuss our work on the preparation of nuclear staining dyes which show absorption and emission properties in the NIR for the low-level detection of restriction fragments. Fluorescence spectra of these dyes in the presence of dsDNAs is presented.

A rapid biochemical test to assess postharvest deterioration of sugarcane and milled juice

The delivery of consignments of deteriorated sugarcane to factories can detrimentally affect multiple process units, and even lead to a factory shut-down. An enzymatic factory method was used to measure mannitol, a major degradation product of sugarcane Leuconostoc deterioration in the U.S., in press (consignment) and crusher juices collected across the 2004 processing season at a Louisiana factory. Weather conditions varied markedly across the season causing periods of the delivery of deteriorated sugarcane to the factory. A strong polynomial relationship existed between mannitol and haze dextran (R2=0.912) in press and crusher juices. Mannitol concentrations were usually higher than concentrations of monoclonal antibody dextran, which indicates: (i) the usefulness and sensitivity of mannitol to predict sugarcane deterioration from Leuconostoc and other bacteria, and (ii) the underestimation by sugar industry personnel of the relatively large amounts of mannitol present in deteriorated sugarcane that can affect processing. Greater than ∼250–500 ppm/Brix of mannitol in sugarcane juice predicted downstream processing problems. The enzymatic method is quantitative and could be used in a sugarcane payment formula. Approximately > 300 ppm/Brix of haze dextran in raw sugar indicated that the majority of the crystals were elongated. Approximately > 600 ppm/Brix of antibody dextran indicated when elongated crystals were predominant in the raw sugar. The enzymatic mannitol method underestimates mannitol in raw sugars.

All-solid-state TCPC instrument for dynamic lifetime measurements in sensitive DNA analysis

We have constructed a compact, all solid state, time correlated single photon counting device for sensitive measurement of the time resolved behavior of DNA fragments passing through a capillary tube. The instrument consists of a pulsed diode laser PDL, a single photon avalanche diode SPCM-211 and a PC board containing all TCPC electronics. We achieved an overall instrument response function of the system of less than 300 ps. This is fast enough for measurement of fluorescence lifetimes. We demonstrated the utility and the sensitivity of the instrument; dynamic measurements of fluorescence decays for NIR dye-labelled nucleotide bases were measured during capillary electrophoresis. The results indicate that in a two-dye experiment the characteristic lifetime of the probe could be used to identify the terminal nucleotide base. The lifetimes of the tow dyes bound to DNA fragments were determined to be 670 ps and 530 ps.

Heavy-atom modified near-IR fluorescent dyes for DNA sequencing applications: synthesis and photophysical characterization

A series of near-IR fluorescent dyes have been prepared which contain an intramolecular heavy atom for altering the photophysics to produce a set of probes appropriate for single lane DNA sequencing applications. The identification of the terminal nucleotide base will be affected by temporal discrimination using fluorescence lifetime determination. The heavy-atom modification consists of an intramolecular halogen (mono- or disubstituted) situated on a remote section of the chromophore in order to minimize the perturbation on the photophysics. The series of dyes prepared showed similar absorption and emission maxima as well as fluorescence quantum yields that were similar. However, the lifetimes of these dyes were found to vary with the identity of the halogen substitution, yielding an apparent inverse heavy atom effect, with the heavier atom showing the longest fluorescence lifetime. Nanosecond flash photolysis spectroscopy of these dyes indicated that the intersystem crossing rates in the series increased with the heavier atom, consistent with known heavy-atom effects. The apparent inverse heavy atom effect resulted from decreases in the internal conversion rate of the base chromophore, with the heavier atom showing a smaller rate of internal conversion compared to that of the dye with the lighter heavy-atom modification.

New directions in near-IR fluorescence detection for capillary electrophoresis

Because of the small sample sizes that are typically inserted onto the separation column in capillary electrophoresis (1-100 nL), ultrasensitive detection strategies are required. The common detection approach used in CE is laser-induced fluorescence with He-Cd, Ar or Kr ion laser excitation. We are developing a detector system which utilizes solid-state diode lasers and avalanche photodiodes to produce a low-cost, durable and ultrasensitive fluorescence detector for CE applications. Along these lines, we have prepared some labeling dyes which readily conjugate to primary bioamines and show absorption and emission properties in the near-IR allowing low-level analyses of these target analytes in complex sample matrices. Our discussion will focus on the properties of diode lasers and avalanche diodes for fluorescence detection in CE applications. In addition, we discuss the characteristics of these near-IR dyes and tagging dyes synthesized in our laboratory for the covalent labeling of bioamines and their use in CE. The specific bioanalysis examples that we present utilizing near-IR fluorescence detection for CE are amino acids separations. In addition, we also discuss the ability to do time-resolved fluorescence measurements during CE for peak identification purposes.

Picosecond laser studies of the solvent-dependent nonradiative pathways in near-IR fluorescent dyes: implications on their use in ultrasensitive analysis

Photophysical investigations using steady-state and picosecond, time-resolved fluorescence techniques on several NIR polymethine dyes were performed in order to elucidate solvent- dependent nonradiative pathways.