Narasimhachari Narayanan

Detection of biomolecules in the near-infrared spectral region via a fiber-optic immunosensor

The design, development, and application of a fluorescent fiber-optic immunosensor (FFOI) procedure for the detection of antibody/antigen binding within the near-infrared (NIR) spectral region is reported. The technique was developed through the combined use of fiber-optics, semiconductor laser excitation, fluorescence detection, NIR dye, and immunochemical techniques. The antibody is immobilized on the FFOIs sensing tip and utilized as a recognition component for trace amounts of specific antigen. The FFOI is constructed to utilize antibody sandwich technique. Three individual immunoassays are reported. The first two assays utilize the FFOI and NN382, a commercial NIR dye, for the detection of human immunoglobulin G (IgG). In these assays, goat anti-human IgG antibody (GAHG) is immobilized on the sensitive terminal of the FFOI followed by the exposure of the antibody-coated terminal to human IgG. The probe is then introduced to GAHG labeled with NN382, generating a signal. The third assay utilizes the FFOI for the detection of trace amounts of Legionella pneumophila serogroup 1 (LPS1). In this assay, rabbit anti-LPS1 antibody is immobilized on the sensitive terminal of the FFOI followed by exposure to LPS1. The antigen-coated probe is then treated with monoclonal anti-LPS1 antibody followed by incubation with GAHG labeled with NN382. The assays are optimized to detect the corresponding antigen via the NIR-FFOI. Typical measurements are performed in 10-15 min. A 780-nm semiconductor laser provides the excitation of the immune complex and the resulting emission is detected by a 820-nm silicon photodiode detector. The intensity of the resulting fluorescence is directly proportional to the concentration of the antigen. Solutions of IgG and LPS1 with concentrations as low as 10(-11) M and 0.5 ng/ml, respectively, have been detected with a minimum interference.

Near-infrared tetra-substituted aluminum 2, 3-naphthalocyanine dyes for optical fiber applications

The synthesis and spectral characterization of several tetra-substituted aluminum 2, 3-naphthalocyanine dyes for the determination of metal ions is reported. The synthesis is done by means of a homogeneous phase reaction, replacing the previously used heterogeneous method. The new scheme allows for improved product yields, higher purity, better product reproducibility and can be monitored at different stages using UV-Vis-near-infrared spectroscopy. The incorporation of electron-donating or -withdrawing groups was found to influence the product yield and to cause a shift in the absorbance maximum. The typical shift in the excitation maximum (of up to 27 nm) enables the dye to match the output of semiconductor laser diodes. In addition the tetra-substituted groups were capable of undergoing an ion-exchange process with the metal ions which produced a change in the fluorescence signal of the dye. Similar results were achieved using an optical fiber metal probe. The detection of metal ions using the near-infrared dyes was accomplished via steady-state fluorescence using both a commerically available instrument and a fiber optic system and also via the fluorescence lifetime technique.

Use of three longer-wavelength fluorophores with the fiber-optic biosensor

Abstract With the availability of fluorescent dyes that are excited above 600 nm and couple easily to proteins, portable biosensors are becoming a reality. The use of diode lasers to excite these days permits systems to be made small and lightweight. An added benefit to switching to the near-infrared (NIR) regime is the reduction in background fluorescence from environmental and clinical samples. Previously, an evanescent wave biosensor was developed at the Naval Research Laboratory (NRL) using tetramethylrhodamine isothiocyanate as the fluorescent tag. Using this biosensor, toxins and other proteins have been detected down to 1 ng ml−1 and bacteria to 3000 cells ml−1. In this study, two new biosensors have been constructed using the same design with only selected components changed for detection of other fluorophores. An antigen (goat IgG) is labelled with one of three fluorophores: tetramethylrhodamine isothiocyanate, Cy5 and a near-infrared dye. The direct binding of each labelled antigen to an antibody-coated fiber-optic probe is measured. Comparisons of signal magnitudes, level of detection achieved, and photo-bleaching have been performed.

Spectral Characterization and Evaluation of Modified Near-Infrared Laser Dyes for DNA Sequencing:

Several derivatives of IR-144, a commercially available near-infrared (near-IR) laser dye, have been synthesized with different moieties, which produce bathochromic shifts of the absorbance maximum without affecting their fluorescence emission spectrum. With this capability, the absorbance maximum of the dye can be adjusted to coincide with the maximum output wavelength of various semiconductor laser diodes. This capability allows compatibility with a multidye fluorescence system that has different excitation sources but a common near-IR detector. The utility of IR-144 derivatives with an absorbance maximum close to the output wavelength of commercially available semiconductor laser diodes was illustrated by coupling oligonucleotide primers that contain an amino linker to a dye derivative and sequencing DNA with the use of an instrument designed specifically for near-IR detection. Before sequencing reactions were performed, the near-IR laser dye-DNA conjugates were purified by reversed-phase high-performance liquid chromatography. We report the synthesis and spectral characterization of IR-144 derivatives. In addition, the conjugation reaction between the near-IR laser dye and amino-modified oligonucleotides and subsequent purification is discussed. Data that validate the subfemptomole detection of the labeled DNA strands are presented.

Near-Infrared Heptamethine Cyanine Dyes: A New Tracer for Solid-Phase Immunoassays:

Near-infrared (near-IR) fluorescence has been used to develop a solid-phase immunoassay that detects trace amounts of human immunoglobulin (HuIgG). Various concentrations of HuIgG bound to a nitrocellulose surface were determined from the fluorescence generated by near-IR labeled goat anti-human antibody (GAHG) bound to the HuIgG. The GAHG was labeled with a heptamethine cyanine fluorophore that has spectral properties in the near-IR region (above 780 nm). These fluorophores are versatile because they can be modified for several bioanalytical applications. Fluorescence was detected with a near-IR fluorescence instrument previously developed in the laboratory. Two cyanine fluorophore labels were evaluated for the ability to selectively bind to GAHG on a nitrocellulose matrix with a minimal amount of background interference. After the most appropriate near-IR fluorophore was selected, the labeling of GAHG was optimized under aqueous conditions. The most effective GAHG–dye conjugates were used to develop an immunoassay to detect various concentrations of HuIgG. The results are presented, here. Solutions of HuIgG with concentrations as low as 10−10 molar have been detected with a minimum of interference.

New near-infrared dyes for applications in bioanalytical methods

The near infrared (NIR) fluorescence has several advantages as a detection system over visible or uv fluorescence counterpart. The spectral properties of cyanine dyes offer advantages for detection in the longer wavelength NIR region. The present work focuses on the synthesis of NIR dyes and their applications in bioanalytical chemistry. Several cyanine dyes have been synthesized. The spectroscopic characteristics such as molar absorptivity, fluorescence quantum yield, excitation and emission maxima have been determined. The potential utilities of these new dyes as probe and reporter molecules in bioanalytical methods have been explored. Dyes bearing reactive functional groups such as isothiocyanate, carboxylic acid or hydroxyl groups can be directly used for covalent labeling of proteins and nucleic acids. Their importance in the synthesis of dye labeled phosphoramidites and dye labeled dideoxynucleotides and use in DNA sequencing have been evaluated.

Spectroscopic Investigations of a Tetrasubstituted Aluminum Naphthalocyanine Near-Infrared Compounds

Abstract The spectroscopic characteristics in methanol of tetra substituted aluminum 2,3-napthalocyanines (2,3-NcAl) derivatives have been investigated. The tetra substitution of an aluminum naphthalocyanine molecule produces a bathochromic shift of the Q and B band. The N band showed almost no effect by the presence of the functional group and remained at a constant frequency; however, multiple overlapping of bands occurred between 25000 cm−1 (400 nm) and 35000 cm−1 (250 nm). The degree of bathochromic shift of the Q band was affected by the electron withdrawing or electron donating property of the substituents. Changes in the absorption of the Q, B and N band will be discussed. The fluorescence, quantum yield and molar absorptivity of the Q-band due to structural modifications are reported. The molar absorptivity (∊) of the dyes was in the order of 3.5 (log ∊); however, higher ∊ values were obtained with sulphonic groups. Solubility of these compounds in various solvents will be discussed. Possible use ...

New NIR Dyes: Synthesis, Spectral Properties and Applications in DNA Analyses

New pentamethine and heptamethine monofunctional asymmetric cyanine dyes have been synthesized. They are suitable for independently exciting at 680nm and 780nm laser diodes respectively. The absorption and fluorescence characteristics such as molar absorptivity and quantum yield have been examined in various solvents. A new spectrofluorometer, an instrument built in house is described. The dyes having a terminal hydroxyl group (1, 3, 4, 5 and 8) have been successfully attached to oligonucleotides on an automated DNA synthesizer through phosphoramidite chemistry. The dyes with carboxyl (2) and isothiocyanate functional groups (7) have been coupled directly to deoxyribonucleotides (dATP). The dye labeled primers and dye labeled dATPs provide excellent sensitivity and high throughput when used for sequencing and genotyping applications on LI-COR’s 4200 automated DNA analyzer which independently detects at two wavelengths.

Near-IR biosensor for evanescent wave immunoassays

Based upon a biosensor design which utilizes standard fluorescent dyes (FITC, TRITC), a new device has been developed which incorporates a laser diode light source to excite novel near infrared (NIR) dyes. The purpose of switching to the NIR regime is to decrease the background fluorescence of biological samples and to decrease the size and power requirements of the biosensor. New dyes which fluoresce in the NIR have been conjugated to protein antigen and immunoassays performed. Assay results using excitation at 780 nm are shown.

Near-infrared fluorescence in fiber optic applications

Fluorescence spectroscopy has long proven to be a valuable tool in fiber optic applications. A large number of publications have addressed different fiber optic applications using fluorescent probe molecules. Since most probe molecules absorb in the UV/Vis part of the electromagnetic spectrum, the majority of these applications address the use of visible fluorophores. However, the utilization of the longer wavelength part of the spectrum may be advantageous due to its relatively low interference. Biological applications of this longer wavelength spectral region may be especially advantageous if semiconductor lasers are used as light sources. Laser diodes have all the properties of other types of lasers with the added benefits of compactness and low price. To utilize these advantages, however, new NIR absorbing probe molecules need to be developed. Certain requirements, e.g., chemical stability, presence of functional groups for binding to the fiber, etc., need to be met for using these NIR chromophores in fiber optic applications. These NIR fluorophores may be incorporated into a fiber optic probe and used for determining analytically important properties. In this paper examples of the use of NIR fluorophores are given.