Photon counting optical fiber biosensor for the characterization of nucleic acid hybridization

摘要

Optical fiber biosensors offer great promise for rapid and sensitive detection of a variety of chemical and biological analyte in this paper, we report our progress in developing an optical fiber biosensor for the characterization of solid phase nucleic acid hybridization. The principle of the measurement is based on the combination of photon counting and laser induced fluoresence in the evanescent field on the optical fiber surface. We used long, fused silica fibers that had several centimeters of cladding removed along the distal end for the fabrication of sensor probe. 5'-amino-terminal oligonucleotide probes were covalently immobilized via glutaraldehyde cross-linking to an aminosilane on the exposed fiber core to form a thin layer of oligonucleotide probes. The evanescent excitation of fluorescence was achieved through the evanescent wave penetrating into the oligonucleotide probes layer when an Ar ion laser beam was guided in the optical fiber. In situ hybridization of 5'-fluorescein isothiocyanate (FITC)-labeled complementary oligonucleotides to their immobilized probes was monitored at the proximal end of the sensing fiber through the fluorescent signal which generated in the evanescent wave region of the uncladded portion as they coupled back to the fiber core. Hybridization specificity was tested by addition of fluorescein isothiocyanate (FITC)-labeled noncomplementary oligonucleotides to the immobilized probes. Regeneration of the surface-immobilized probe was possible, allowing reuse without a significant loss of hybridization activity. Dilution experiments showed an apparent lower limit of detection at 1 nM oligonucleotides. This performance was comparable to other previously reported DNA biosensors.