Direct monitoring of pulmonary disease treatment biomarkers using plasmonic gold nanorods with diffusion-sensitive OCT.

The solid concentration of pulmonary mucus (wt%) is critical to respiratory health. In patients with respiratory disease, such as Cystic Fibrosis (CF) and Chronic Obstructive Pulmonary Disorder (COPD), mucus hydration is impaired, resulting in high wt%. Mucus with high wt% is a hallmark of pulmonary disease that leads to obstructed airways, inflammation, and infection. Methods to measure mucus hydration in situ and in real-time are needed for drug development and personalized therapy. We employed plasmonic gold nanorod (GNR) biosensors that intermittently collide with macromolecules comprising the mucus mesh as they self-diffuse, such that GNR translational diffusion (DT) is sensitive to wt%. GNRs are attractive candidates for bioprobes due to their anisotropic optical scattering that makes them easily distinguishable from native tissue using polarization-sensitive OCT. Using principles of heterodyne dynamic light scattering, we developed diffusion-sensitive optical coherence tomography (DS-OCT) to spatially-resolve changing DT in real-time. DS-OCT enables, for the first time, direct monitoring of changes in nanoparticle diffusion rates that are sensitive to nanoporosity with spatial and temporal resolutions of 4.7 µm and 0.2 s. DS-OCT therefore enables us to measure spatially-resolved changes in mucus wt% over time. In this study, we demonstrate the applicability of DS-OCT on well-differentiated primary human bronchial epithelial cells during a clinical mucus-hydrating therapy, hypertonic saline treatment (HST), to reveal, for the first time, mucus mixing, cellular secretions, and mucus hydration on the micrometer scale that translate to long-term therapeutic effects.

Natural electrophoresis of norepinephrine and ascorbic acid.

The electric field produced by cell membranes, extending only a few nanometers, is 1000 times stronger than the electric fields required to produce dissociation of molecular complexes. Using the complex formed by norepinephrine (NE) and ascorbic acid (AA), we have demonstrated the quantitative binding of AA to NE, the use of capillary electrophoresis to measure quantitative binding of nonelectrolyte complexes, the determination of a dissociation constant (Kd) from electric field-dissociation constants (Ke), and a model for natural dissociation of the NE-AA complex due to the electric field generated by a cell membrane. NE-AA dissociation constants show little effect of NE concentration or pH changes. NE-related compounds also bind AA: epinephrine > norepinephrine > tyrosine > histamine > phenylalanine. Serotonin does not bind AA. Phosphorylated AA and glucose also bind NE at 0.05 and 0.08 of the AA binding, respectively. Natural electrophoresis of molecular complexes allows compounds to travel through the body in a protected state and still be available for physiological activity upon reaching a membrane.

Differential coupling of smooth and skeletal muscle pyruvate kinase to creatine kinase.

The interaction of pyruvate kinase from skeletal (SKPK) and smooth (SMPK) muscle with MM-creatine kinase (MMCK) and BB-creatine kinase (BBCK) was assessed using temporal absorbance changes, variations in absorbance at different wavelengths, concentration dependence, association in an electric field, and PK kinetic activity. SKPK exhibits a time course of absorbance increase in the presence of MMCK with a time constant of 29.5 min. This increase occurs at all wavelength from 240 to 1000 nm. At 195 nm, the combination of SKPK and MMCK produces a decrease in absorption with electric fields of both 0 and 204 V/cm. The change in SKPK-MMCK is saturable. SKPK activity is significantly increased by the presence of MMCK in solutions of 0-32% ethanol. These results indicate specific SKPK-MMCK interaction. SMPK and BBCK did not exhibit similar coupling when the BBCK concentration dependence of absorbance or SMPK activity in solutions of 0-32% ethanol was determined. Both MMCK and BBCK increased SKPK activity; neither MMCK nor BBCK increased SMPK activity. The ability to form diazymatic complexes with creatine kinase appears to reside in SKPK. This coupling may account for the increased flux through PK without significant substrate changes seen during skeletal muscle activation. This coupling will not occur in smooth muscle.

Capillary electrophoretic measurement of tissue metabolites.

A method for the measurement of tissue metabolites from rabbit urinary bladder using capillary electrophoresis (CE) has been developed. The method generates a reproducible electropherogram containing > 20 peaks, including NAD, NADH, lactate, UDP-glucose, phosphocreatine, creatine, ATP, ADP, GTP, and UTP, from < 20 nl of extract solution generated from 1.1 nl (or approximately 1.2 micrograms) of tissue in < 40 min. Multiple samples from the same bladder produce SE comparable with enzymatic or nuclear magnetic resonance (NMR) measurements of metabolites: phosphorus-NMR measurement requires 10(6) more tissue than CE; individual enzymatic measurements using 100 microliters/sample require 2,000 microliters, a 10(5) greater volume than required by CE for the same number of metabolites. CE detects about three times more peaks than phosphorus-NMR on a similar time scale. Comparable measurements using enzymatic analysis would require approximately 10 times longer. The combination of minimal tissue volume requirements, rapid measurement, and reproducibility makes CE a valuable tool in the investigation of simultaneous changes in multiple metabolites from minute tissue samples.