of the ocular surface. Tears hydrate and lubricate the mucous membranes constituting the ocular surface, supply nourishment to the avascular corneal epithelium, and provide a smooth optical surface essential for visual acuity. The drainage of tears via the lacrimal puncta fl ushes contaminants and irritants out of the eye, thereby functioning as a fi rst line of defense for the anterior eye against invading pathogens ( 1, 2 ). The typical volume of tears in normal eyes ranges from 3.4 to 10.7 l per eye ( 3 ). Despite its small volume, tears represent a biological fl uid of immense complexities with a wide array of proteins/peptides, electrolytes, lipids, and small molecule metabolites contributed by distinct sources ( 1 ). The precise balance of these various metabolites is crucial in ensuring proper physiological function and maintaining biophysical integrity of the precorneal tear fi lm. Perturbations in this delicate equilibrium may be manifested in various ocular conditions such as dry eye syndrome (DES) and blepharitis ( 1,4,5 ).Recent decades have witnessed tremendous progress in the systematic profi ling of proteins as well as small molecule metabolites present in tears ( 1, 6-9 ). Furthermore, with technological advancements in MS and nuclear magnetic Abstract The tear fi lm covers the anterior eye and the precise balance of its various constituting components is critical for maintaining ocular health. The composition of the tear fi lm amphiphilic lipid sublayer, in particular, has largely remained a matter of contention due to the limiting concentrations of these lipid amphiphiles in tears that render their detection and accurate quantitation tedious. Using systematic and sensitive lipidomic approaches, we validated dif ferent tear collection techniques and report the most comprehensive human tear lipidome to date; comprising more than 600 lipid species from 17 major lipid classes. The tear fl uid covers the anterior surface of the cornea and serves critical functions in maintaining the homeostasis

We combine nanofluidics and nanoplasmonics for surface-plasmon resonance (SPR) sensing using flow-through nanohole arrays. The role of surface plasmons on resonant transmission motivates the application of nanohole arrays as surface-based biosensors. Research to date, however, has focused on dead-ended holes, and therefore failed to harness the benefits of nanoconfined transport combined with SPR sensing. The flow-through format enables rapid transport of reactants to the active surface inside the nanoholes, with potential for significantly improved time of analysis and biomarker yield through nanohole sieving. We apply the flow-through method to monitor the formation of a monolayer and the immobilization of an ovarian cancer biomarker specific antibody on the sensing surface in real-time. The flow-through method resulted in a 6-fold improvement in response time as compared to the established flow-over method.

Simple, rapid, and sensitive technologies to detect nucleic acid modifications have important applications in genetic analysis, clinical diagnosis, and molecular biology. Because genetic modifications such as single nucleotide polymorphisms (SNP), DNA methylation, and other lesions can serve as hallmarks of human disease, interest in such methods has increased in recent years. This Account describes a new strategy for the optical detection of these DNA targets using cationic conjugated polymers (CCPs). Because of their unique signal amplification properties, researchers have extensively investigated conjugated polymers as optical transducers in highly sensitive biosensors. Recently, we have shown that cationic polyfluorene can detect SNPs within the DNA of clinical samples. When we incorporated deoxyguanosine triphosphate (dGTP-Fl) into the DNA chain at an SNP site where the target/probe pair is complementary, we observed higher fluorescence resonance energy transfer (FRET) efficiency between cationic polyfluorene and fluorescein label on the dGTP. By monitoring the change in emission intensity of cationic polyfluorene or fluorescein, we identified the homozygous or heterozygous SNP. The high sensitivity of this assay results from the 10-fold enhancement of fluorescein emission intensity by the FRET from polyfluorene. This method can detect allele frequencies (the proportion of all copies of a gene that is made up of a particular gene variant) as low as 2%. Using this novel method, we clearly discriminated among the SNP genotypes of 76 individuals of Chinese ancestry. Improving on this initial system, we designed a method for multicolor and one-tube SNP genotyping assays based on cationic polyfluorene using fluorescein-labeled deoxyuridine triphosphate (dUTP-Fl) and Cy3-labeled deoxycytidine triphosphate (dCTP-Cy3) in extension reactions. We also developed a one-step method for direct detection of SNP genotypes from genomic DNA by combining allele-specific PCR with CCPs. In 2008, we developed a new method for DNA methylation detection based on single base extension reaction and CCPs. Treatment of DNA with bisulfite followed by PCR amplification converts unmethylated DNA into a C/T polymorphism, which allows us to characterize the methylation status of the target DNA. Furthermore, we used CCPs to detect DNA lesions caused by ultraviolet light irradiation for the first time. By monitoring the color change of cationic polythiophene before and after DNA cleavage, we also detected oxidative damage to DNA by hydroxyl radical. These CCP-based new assays avoid primer labeling, cumbersome workups, and sophisticated instruments, leading to simpler procedures and improved sensitivity. We expect that these features could lead to major advances in human disease diagnostics and genomic study in the near future.