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Traditionally, visible fluorophores have been used as labels in DNA sequencing. They absorb and fluoresce in a region of the spectrum that is susceptible to biological interferences in sequencing samples. The increased noise level due to autofluorescence of glass, solvent, or impurities can greatly reduce the potential sensitivity of the analysis. In an attempt to increase the sensitivity, we have investigated the use of near-infrared (near-IR) fluorophores as labels in DNA sequencing. Near-IR fluorophores possess spectral properties which are observed between 700 and 1200 nm, a region with characteristically little interference by biomolecules. Therefore, the detection of near-IR dyes is not limited by noise levels, but rather by detector capabilities. Near-IR dyes are also suitable for selective excitation with commercially available laser diodes which can further enhance the observed fluorescence signal. We have covalently linked functionalized near-IR dyes to modified DNA oligomers for use in DNA sequencing. We report the synthesis and chromatographic purification of near-IR-labeled DNA oligomers. We further discuss the inherent properties of the conjugates and their use in DNA sequencing procedures.Fluorescent labels are being used increasingly in bioanalytical applications.1-6 They have been attached successfully to a variety of biomolecules such as enzymes, antibodies, and nucleic acids for use in immunoassay, flow cytometry, and sequencing

The construction of a near-infrared (near-IR) fluorescence detector for measuring picomolar levels of near-IR laser dyes is described. The detector is designed for use in an immunoassay technique that employs antibodies labeled with near-IR polymethine cyanine dyes. These dyes possess spectral properties that are exclusive to the near-IR region (650-1100 nm). The instrumentation is characterized, including its hardware and data acquisition software components. The detector is capable of measuring fluorescence in both solution and solid-phase environments. Data on the detector's performance is presented.

No abstract

Physical measurement of tumor volume reduction is the most commonly used approach to assess tumor progression and treatment efficacy in mouse tumor models. However, it is relatively insensitive, and often requires long treatment courses to achieve gross physical tumor destruction. As alternatives, several non-invasive imaging methods such as bioluminescence imaging (BLI), fluorescence imaging (FLI) and positron emission tomography (PET) have been developed for more accurate measurement. As tumors have elevated glucose metabolism, 18F-fludeoxyglucose (18F-FDG) has become a sensitive PET imaging tracer for cancer detection, diagnosis, and efficacy assessment by measuring alterations in glucose metabolism. In particular, the ability of 18F-FDG imaging to detect drug-induced effects on tumor metabolism at a very early phase has dramatically improved the speed of decision-making regarding treatment efficacy. Here we demonstrated an approach with FLI that offers not only comparable performance to PET imaging, but also provides additional benefits, including ease of use, imaging throughput, probe stability, and the potential for multiplex imaging. In this report, we used sorafenib, a tyrosine kinase inhibitor clinically approved for cancer therapy, for treatment of a mouse tumor xenograft model. The drug is known to block several key signaling pathways involved in tumor metabolism. We first identified an appropriate sorafenib dose, 40 mg/kg (daily on days 0–4 and 7–10), that retained ultimate therapeutic efficacy yet provided a 2–3 day window post-treatment for imaging early, subtle metabolic changes prior to gross tumor regression. We then used 18F-FDG PET as the gold standard for assessing the effects of sorafenib treatment on tumor metabolism and compared this to results obtained by measurement of tumor size, tumor BLI, and tumor FLI changes. PET imaging showed ~55–60% inhibition of tumor uptake of 18F-FDG as early as days 2 and 3 post-treatment, without noticeable changes in tumor size. For comparison, two FLI probes, BombesinRSense™ 680 (BRS-680) and Transferrin-Vivo™ 750 (TfV-750), were assessed for their potential in metabolic imaging. Metabolically active cancer cells are known to have elevated bombesin and transferrin receptor levels on the surface. In excellent agreement with PET imaging, the BRS-680 imaging showed 40% and 79% inhibition on days 2 and 3, respectively, and the TfV-750 imaging showed 65% inhibition on day 3. In both cases, no significant reduction in tumor volume or BLI signal was observed during the first 3 days of treatment. These results suggest that metabolic FLI has potential preclinical application as an additional method for detecting drug-induced metabolic changes in tumors.