A combination tapered fiber-optic biosensor (CTFOB) dip probe for rapid and cost-effective quantification of proteins in serum samples has been developed. This device relies on diode laser excitation and a charged-coupled device spectrometer and functions on a technique of sandwich immunoassay. As a proof of principle, this technique was applied in a quantitative estimation of interleukin IL-6. The probes detected IL-6 at picomolar levels in serum samples obtained from a patient with lupus, an autoimmune disease, and a patient with lymphoma. The estimated concentration of IL-6 in the lupus sample was 5.9 ± 0.6 pM, and in the lymphoma sample, it was below the detection limit. These concentrations were verified by a procedure involving bead-based xMAP technology. A similar trend in the concentrations was observed. The specificity of the CTFOB dip probes was assessed by analysis with receiver operating characteristics. This analysis suggests that the dip probes can detect 5-pM or higher concentration of IL-6 in these samples with specificities of 100%. The results provide information for guiding further studies in the utilization of these probes to quantify other analytes in body fluids with high specificity and sensitivity.
This document reports a novel method of measuring association rate constant (ka) for antibody-antigen interaction using
evanescent wave-based combination tapered fiber-optic biosensor (CTFOB) dip-probes. The method was demonstrated
by measuring association rate constant for bovine serum albumin (BSA) and anti-BSA antibody interaction. "Direct
method" was used for detection; goat anti-BSA "capture" antibodies were immobilized on the probe surfaces while the
antigen (BSA) was directly labeled with Alexa 488 dye. The probes were subsequently submerged in 3nM Labeled BSA
in egg albumin (1 mg/ml). The fluorescence signal recorded was proportional to BSA anti-BSA conjugates and
continuous signal was acquired suing a fiber optic spectrometer (Ocean Optics, Inc.). A 476 nm diode laser was use as
an excitation source. Association constant was estimated from a plot of signal as a function of time. Measured
association rate constant ka for the binding of BSA with anti-BSA at room temperature is (8.33 ± 0.01) x 104M-1s-1.
A rapid and cost-effective combination tapered fiber-optic biosensor (CTFOB) dip-probe was used for quantitative
estimation of interleukin (IL)-6 in serum/plasma samples. Sandwich immunoassay was used as the detection technique.
Probes could successfully detect presence of IL-6 in two serum samples, non-neoplastic autoimmune patient (lupus)
sample and lymphoma patient sample. The estimated amount of IL-6 in lupus patient sample was 4.8 ± 0.9 pM and in
lymphoma patient sample was 2 ± 1 pM. It is demonstrated that the developed CTFOB dip-probe is capable of
quantitative estimation of proteins in serum/plasma samples with high specificity.
The document reports a novel method of measuring dissociation constant (kD) of antibody-antigen interaction using
evanescent wave based combination tapered fiber-optic biosensor (CTFOB) dip-probes. The method was demonstrated by
measuring the dissociation constant of human Interleukin-6 (IL-6) and anti-IL-6 interaction. Sandwich immunoassay was
used to generate fluorescence signal proportional to antigen-antibody conjugate pairs. A portable CCD based spectrometer
setup was used to record spectral profile of the fluorescence signal. The measured value of dissociation constant kD for IL-6 and capture anti-IL-6 (clone MQ2-13A5) antibodies at room temperature is588 ±19 pM .
We are reporting detection of IL-8 in a mixed protein solution, using combination tapered fiber-optic biosensor
(CTFOB) dip-probe. Sandwich immunoassay was used as the detection technique. The specificity of the sensor was
established by using two types of negative control probes. It is demonstrated that with the help of these CTFOB dipprobe
we could successfully detect IL-8 with high specificity in protein mixture. The lowest detected concentration of
IL-8 was 150 pM.
A theoretical model using ray tracing method is developed. The results predicted by the model were confirmed by experimental results. The model could explain the experimentally observed fact that the maximum signal for a given realistic tapered length is at a probe radius smaller than that expected from V-number matching condition. It is shown that for obtaining maximum fluorescence signal from an evanescent wave fiber optic biosensor a realistic optimum taper length needs to be chosen. We found that different detection environments require different taper lengths at a given taper angle. These facts were confirmed experimentally.
In this paper we are reporting the development of a highly sensitive evanescent wave combination tapered fiber optic
fluorosensor. We have demonstrated detection of 5 pM Bovine Serum Albumin (BSA) protein using these fiber optic
sensors. The sensor can be easily adopted for detection of other proteins. Six identical probes were prepared and affinity
pure Goat anti-BSA antibodies were immobilized on the probe surface. We could detect signal from all the probes kept
in 5 pM to 1 nM BSA solution while no signal was detected from the probes kept in 20 nM labeled ESA solution.
Imaging in biological systems has become one of the most relied upon tools in the study of human disease. Two-photon excitation methodology in laser scanning microscopy has resulted in 3D-imaging capability not easily achieved in one- photon systems. Our Institute, in conjunction with Andrew Schally (Noble Laureate, Tulane University), has used two- photon laser scanning microscopy (TPLSM) to understand the real time cellular transport of the chemotherapeutic agent, Luteinizing Hormone-Releasing Hormone-Doxorubicin (AN152) covalently coupled to a novel two-photon fluorophore (C625). At the Institute, new and highly efficient two-photon fluorophores that fluoresce at different wavelengths have been developed. The coupling of LH-RH and AN152 with two-photon fluorophores having different spectroscopic profiles allows for the simultaneous determination of their cellular compartmentalization. Coupled with the two-photon microspectrofluorometer, we acquired localized fluorescence spectra from the inside of living cells to differentiate the cytoplasmic and nuclear localization of the LH-RH and AN152 respectively. The ability of these new dyes to fluoresce at different wavelengths using the same excitation wavelength provides a major advantage over single photon dyes. This technology has great promise in imaging the dynamic changes or events occurring in living cells over short periods of time. Another approach to bioimaging at the Institute is the integration of two-photon and nanosized technologies. Nanoclinics (20 - 30 nm silica bubbles) can be fabricated to contain these two photon fluorophores and the surface functionalized with biological agents which can target specific cells. These highly fluorescent nanoclinics are sufficiently small in size to allow for tissue penetration, allowing for the multiple probing for different cellular functions in normal and cancerous tissues.
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