An automated optical biosensor system based on fluorescence excitation and detection in the evanescent field of a quartz fiber was used to detect 16-mer oligonucleotides in DNA hybridization assays. A biotinylated capture probe was immobilized on the fiber surface via avidin or streptavidin. The hybridization with fluorescein-labeled complementary strands was monitored in real time by fluorescence detection. The double strands formed by hybridization could be dissociated by chemical or thermal regeneration, allowing one to perform hundreds of assay cycles with the same fiber. The signal loss during longtime measurements, i.e., consecutive hybridization assays, can be described by a single-exponential function. Over more than 200 cycles, the net signal decreased by 50% with a signal variation of 2.4% after correction for this signal loss. By binding the capture probe with the 5'-end to the optical fiber surface, and by using a 50% (w/w) aqueous urea solution for chemical regeneration, the duration of an assay cycle could be reduced to 3 min. By applying longer assay cycles, the detection limit for the hybridization with a complementary fluorescein-labeled oligonucleotide was 2.0 x 10(-13) M (24 fmol). To detect an unlabeled complementary 16-mer oligonucleotide, competitive hybridization assays were performed, resulting in a detection limit of 1.1 x 10(-9) M (132 pmol). Poly-(acrylic acid) 5100 sodium salt and Tween 20 were used in the hybridization buffer to prevent nonspecific binding caused by ionic or hydrophobic interaction. The amount of nonspecific binding of noncomplementary oligonucleotides was in the range of 1-2%, compared with the specific binding in the different hybridization assays.

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DNA microarrays have become a powerful tool for expression profiling and other genomics applications. A critical factor for their sensitivity is the interfacial coating between the chip substrate and the bound DNA. Such a coating has to embrace the divergent requirements of tightly binding the capture probe DNA during the spotting process and of minimizing the nonspecific binding of target DNA during the hybridization assay. To fulfill these conditions, most coatings require a passivation step. Here we demonstrate how the chain density of a graft copolymer with a polycationic backbone, poly(l-lysine)-graft-poly(ethylene glycol), can be tuned such that the binding capacity during capture probe deposition is maximized while the nonspecific binding during hybridization assays is kept to a minimum, thus alleviating the requirement for a separate passivation procedure. Evidence for the superior performance of such coatings in terms of signal-to-noise ratio and spot quality is presented using an evanescent field-based fluorescent sensing technique (the ZeptoREADER). The surface architecture is further characterized using optical waveguide lightmode spectroscopy and time-of-flight secondary ion mass spectrometry. Finally, in a model assay, we demonstrate that expression changes can be detected from 1 microg of total mRNA sample material with a limit of detectable differential expression of +/-1.5.

We have developed a (bio)chemical analysis system based on luminescence generation and detection in the evanescent field associated with light guiding in an optical fiber. Our intention was directed towards optimization of not only the sensor, including the sensor handling and the immobilization of biochemical recognition elements, but also of the assay chemistiy, with special emphasis on methods used for sensor regeneration, of the fluidic system, and of the experimental control software. Goals of this optimization process were not only to achieve high sensitivity, reproducibility and the related precision of the results, but also maximum regenerabiity of the sensors and system flexibility for a variety ofdifferent applications.Four examples of different bioafflnity assays, established on our sensor system in, are presented: a competitive immunoassay for atrazine, a sandwich immunoassay for hirudin, a DNA hybridization assay, and first studies for the development of sensors based on membrane-bound receptors. In the atrazine assay, the sensor could be regenerated more than 300 times. In the hybridization assay, a detection limit of 7.5x i014 M complementary fluorescem-labeled DNA was achieved. The performance ofour system is compared with an established enzyme-linked imniunosorbent assay (ELISA) on the example ofthe hirudin assay. In the concluding section of this paper, advantages and disadvantages of our fiberoptic, luminescence-based system, compared with commercialized systems, based on detection of changes of the effective refractive index, are discussed. SPIE Vol. 2631 / 15Downloaded From: http://proceedings.spiedigitallibrary.org/ on 06/24/2016 Terms of Use: http://spiedigitallibrary.org/ss/TermsOfUse.aspx