Application of a biotin functionalized QD assay for determining available binding sites on electrospun nanofiber membrane.

BACKGROUND: The quantification of surface groups attached to non-woven fibers is an important step in developing nanofiber biosensing detection technologies. A method utilizing biotin functionalized quantum dots (QDs) 655 for quantitative analysis of available biotin binding sites within avidin immobilized on electrospun nanofiber membranes was developed. RESULTS: A method for quantifying nanofiber bound avidin using biotin functionalized QDs is presented. Avidin was covalently bound to electrospun fibrous polyvinyl chloride (PVC 1.8% COOH w/w containing 10% w/w carbon black) membranes using primary amine reactive EDC-Sulfo NHS linkage chemistry. After a 12 h exposure of the avidin coated membranes to the biotin-QD complex, fluorescence intensity was measured and the total amount of attached QDs was determined from a standard curve of QD in solution (total fluorescence vs. femtomole of QD 655). Additionally, fluorescence confocal microscopy verified the labeling of avidin coated nanofibers with QDs. The developed method was tested against 2.4, 5.2, 7.3 and 13.7 mg spray weights of electrospun nanofiber mats. Of the spray weight samples tested, maximum fluorescence was measured for a weight of 7.3 mg, not at the highest weight of 13.7 mg. The data of total fluorescence from QDs bound to immobilized avidin on increasing weights of nanofiber membrane was best fit with a second order polynomial equation (R(2) = .9973) while the standard curve of total fluorescence vs. femtomole QDs in solution had a linear response (R(2) = .999). CONCLUSION: A QD assay was developed in this study that provides a direct method for quantifying ligand attachment sites of avidin covalently bound to surfaces. The strong fluorescence signal that is a fundamental characteristic of QDs allows for the measurement of small changes in the amount of these particles in solution or attached to surfaces.

Molecular optical imaging of therapeutic targets of cancer.

Recent progress in discerning the molecular events that accompany carcinogenesis has led to development of new cancer therapies directly targeted against the molecular changes of neoplasia. Molecular-targeted therapeutics have shown significant improvements in response rates and decreased toxicity as compared to conventional cytotoxic therapies which lack specificity for tumor cells. In order to fully explore the potential of molecular-targeted therapy, a new set of tools is required to dynamically and quantitatively image and monitor the heterogeneous molecular profiles of tumors in vivo. Currently, molecular markers can only be visualized in vitro using complex immunohistochemical staining protocols. In this chapter, we discuss emerging optical tools to image in vivo a molecular profile of risk-based hallmarks of cancer for selecting and monitoring therapy. We present the combination of optically active, targeted nanoparticles for molecular imaging with advances in minimally invasive optical imaging systems, which can be used to dynamically image both a molecular and phenotypic profile of risk and to monitor changes in this profile during therapy.

Subcellular-resolution molecular imaging within living tissue by fiber microendoscopy.

Conventional histopathology involves sampling, sectioning and staining of tissue specimens prior to microscopic evaluation, and provides diagnostic information at a single location and point in time. In vivo microscopy and molecular-targeted optical labeling are two rapidly developing fields, which together have the potential to provide anatomical and functional indications of disease by staining and imaging tissue in situ. To address the need for high-resolution imaging instrumentation, we have developed a compact, robust, and inexpensive fiber-optic microendoscopy system based around wide-field LED illumination, a flexible 1 mm diameter fiber-optic bundle, and a color CCD camera. We demonstrate the sub-cellular resolution imaging capabilities of the system through a series of experiments, beginning with simultaneous imaging of three different cancer cell lines in culture, each targeted with a distinct fluorescent label. We used the narrow diameter probe to access subcutaneous tumors in an in vivo murine model, allowing direct comparison of microendoscopy images with macroscopic images and histopathology. A surgically resected tissue specimen from the human oral cavity was imaged across the clinical margin, demonstrating qualitative and quantitative distinction between normal and cancerous tissue based on sub-cellular image features. Finally, the fiber-optic microendoscope was used on topically-stained normal human oral mucosa in vivo, resolving epithelial cell nuclei and membranes in real-time fluorescence images. Our results demonstrate that this imaging system can potentially complement conventional diagnostic techniques, and support efforts to translate emerging molecular-diagnostic and therapeutic agents into clinical use.

Fluorescent nanocrystals for use in early cervical cancer detection.

BACKGROUND: Quantum dots (qdots) are a promising alternative to organic fluorophores for biological imaging. Advantages of quantum dots over organic fluorophores include broad excitation coupled with narrow, tunable emission, high resistance to chemical and metabolic degradation, a higher photobleaching threshold and finally the ability to be modified with a targeting ligand. These many properties allow quantum dots to be used in conjunction with optical detection methods for imaging. METHODS: We are investigating the use of quantum dots to detect precancerous biomarkers. We have directly targeted epidermal growth factor receptors with quantum dots conjugated to anti-EGFR antibodies. RESULTS: Compared to appropriate controls, we do see specific labeling of EGF receptors. CONCLUSIONS: Quantum dots provide a promising alternative to conventional organic dyes for biological imaging. Combined with optical imaging technologies, quantum dots can help visualize changes in cervical cancer at the molecular level. This ability may alert health care providers to the need for intervention before a cancer can metastasize.