Guillermo A. Casay

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.

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.

Spectroscopic studies of a near-infrared absorbing pH sensitive aminodienone—carbocyanine dye system

Synthetic red and near-infrared absorbing dyes may be used as probe molecules in a large number of applications. Dyes exhibiting spectral changes with hydrogen ion concentration are useful as pH probes. Those dyes which have their absorption and fluorescence maxima in the long wavelength region of the visible spectral region are specially valuable because of decreased interference and semiconductor laser applications. In this paper we have evaluated an aminodienone dyes 1 which demostrates pH dependent absorption and fluorescence spectra as well as solvent polarity dependence. In organic solvents the long wavelength absorption band of the dye is in the reduced interference region. The absorption maximum is at 535 nm in neutral or alkaline solutions in methanol. The absorption spectra undergo a strong bathochromic shift in the presence of acids (lambda(max) = 709 nm) with a concomitant change in the fluorescence spectra. This pH sensitive dye was found to be specially especially useful for organic solvents. The analytical utility of this and similar near-infrared absorbing dyes is discussed.

Near-Infrared Fluorescence Probes

Optical fiber detectors (OFD) are devices that measure electromagnetic radiation transmitted through optical fibers to produce a quantitative signal in response to the chemical or biochemical recognition of a specific analyte. Ideally, an OFD should produce a specific and accurate measurement, continuously and reversibly, of the presence of a particular molecular species in a given sample medium. Additionally, OFD should provide maximum sensitivity and minimal interferences from superfluous ions or molecules to obtain low detection limits. Other attractive features include the miniaturization of the fiber’s tip to accommodate single-cell analysis and portable instrumentation to allow in situ analysis. OFDs can be divided into two subclasses: (1) optical fiber chemical detectors (OFCD) which detect the presence of chemical species in samples, and (2) optical fiber biomolecular detectors (OFBD) which detect biomolecules in samples. Each subclass can be divided further into probes and sensors, and bioprobes and biosensors, respectively. As a result of the rapid expansion of optical research, these terms have not been clearly defined and to date, the terms “probe” and “sensor” have been used synonymously in the literature. As the number of publications increases, the terminology should be clarified. Although both probes and sensors serve to detect chemicals in samples, they are not identical. The same situation exists with “bioprobes” and “biosensors.” Simply, probes and bioprobes are irreversible to the analyte’s presence, whereas sensors and biosensors monitor compounds reversibly and continuously. Progress in the development of OFD has been parallel to advances in the production of optical fibers. Fiber production and reliability have increased and fiber costs have decreased; therefore, routine use of OFD has become more practical. The increased availability of OFD has produced a surge of publications on its applications including quantitative metal detection, pH determination, immunoassays, and the

Fiber Optic Probe Applications Using Near-Infrared Compounds: Determination of NaOH

Abstract The development of a near-infrared fiber optic probe (FOPP) for the determination of caustic soda will be reported. The utility of the FOPP is demonstrated using an NIR non-commercially available tetrasubstituted chloroaluminum naphthalocyanine NIR dye which is susceptible to pH changes in solutions. The naphthalocyanine derivatives have been synthesized in our laboratories. The spectral characteristics of these NIR dyes, are above 700 nm and the absorbance maximum is a perfect match for the use of semiconductor laser diodes as the excitation source. The response of the Q-band against solution pHs and caustic soda will be reported. The FOPP consists of a semiconductor laser diode (λmax=780 nm), an NIR dye and a detector. The probe made of poly(methyl methacrylate) serves as a support for a permeable polymer that is used for the entrapment of the NIR dye (about < 2mm in diameter). The permeable polymers used were Nafion and Gelatin. As hydrogen ions diffuse through the permeable polymer, a complex...

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 ...

Investigation of a near-infrared fiber optic immunosensor

The designs of NIR optical fiber immunosensors for the detection of biomolecules are discussed. The use of fiber optics combined with laser-excited fluorescence detection ((lambda) max equals 780 nm) and immunochemical techniques has provided the essential components for developing simpler and more practical, sensitive and selective immunosensors. The antibody is immobilized on the distal end of a surface-activated polymethyl methylacrylate sensor. As the probe is placed in different concentrations of antigenic substance, the antibody reacts with its corresponding antigen through sandwich and/or competitive immunoassays. The concentrations varied from 10 - 100 ng/ml. The near-infrared dye labeled antigen - antibody complex is excited and the emitted fluorescence is collected with a silicon photodiode detector equipped with an 820-nm bandpass filter. In order to determine various factors influencing the immunosensors performance, the fluorescence intensity responses are obtained under a variety of conditions. The sensor response depending on the type of surface-activating reagent, surface activation period, incubation time, and other measurement conditions also are discussed.

Design and Development of a Fiber-Optic Immunosensor Utilizing Near-Infrared Fluorophores

The design and application of a fluorescent fiber-optic immunosensor (FFOI) are reported. The FFOI is utilized for the detection of antibody/antigen binding within the near-infrared (NIR) spectral region. The technique is developed through the combined use of fiber-optic, semiconductor laser-excitation, fluorescence detection, NIR dye, and immunochemical techniques. The antibody is immobilized on the FFOI and utilized as a recognition component for trace amounts of specific antigen. The FFOI is constructed to utilize an antibody sandwich technique. The assay involves the immobilization of the capture antibody on the sensing tip of the FFOI followed by the exposure of the immobilized sensing tip to the antigen. The antigen-coated FFOI is then introduced to a second antibody previously labeled with the NIR dye. Typical measurements are performed in about 15 min. A semiconductor laser provides the excitation (780 nm) of the immune complex. The resulting emission is detected by a silicon photodiode detector (820 nm). The intensity of the resulting fluorescence is directly proportional to the concentration of the antigen. The sensitivity of the analysis reaches 10 ng/ml and the response time is 10–15 min.