Electrophoresis with Polarized Fluorescence Detection. Application to Capillary Fluorescence Rejection

In this paper, we integrate the apparatus of electrophoresis with polarized fluorescence detection to suppress the background fluorescence arising from illuminating the coating layer of channel containers with a laser excitation source. It has demonstrated moderate background suppression efficiency, close to the ideal 3‐fold reduction on the surface doped with randomly distributed static fluorophores. In addition, when the fluorescence coverage is coated along the same orientation more uniformly, the background reduction is 10‐fold in our experiments. This detection scheme is applicable to acquire the electrophoregrams of separating small organic molecules and biopolymers, such as nucleic acids, when the loading of staining dyes is not heavy. This apparatus is simple. Only adding a pair of polarizer optics is needed. This detection scheme should work equally well with an incoherent light source.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/112242/1/200500106_ftp.pd

Intra-Arterial Chemotherapy with Doxorubicin and Cisplatin Is Effective for Advanced Hepatocellular Cell Carcinoma

Advanced hepatocellular carcinoma (HCC) remains a fatal disease even in the era of targeted therapies. Intra-arterial chemotherapy (IACT) can provide therapeutic benefits for patients with locally advanced HCC who are not eligible for local therapies or are refractory to targeted therapies. The aim of this retrospective study was to analyze the effect of IACT with cisplatin and doxorubicin on advanced HCC. Methods. Patients with advanced HCC who were not eligible for local therapies or were refractory to sorafenib received doxorubicin (50 mg/m2) and cisplatin (50 mg/m2) infusions into the liver via the transhepatic artery. Between January 2005 and December 2011, a total of 50 patients with advanced HCC received this treatment regimen. The overall response rate (ORR) was 22% in all treated patients. In patients who received at least 2 cycles of IACT, the ORR was 36.7%, and the disease control rate was 70%. Survival rate differed significantly between patients who received only one cycle of IACT (group I) and those who received several cycles (group II). The median progression-free survival was 1.3 months and 5.8 months in groups I and II, respectively (P<0.0001). The median overall survival was 8.3 months for all patients and was 3.1 months and 12.0 months in groups I and II, respectively (P<0.0001). The most common toxicity was alopecia. Four patients developed grade 3 or 4 leukopenia. Worsening of liver function, nausea, and vomiting were uncommon side effects. This study demonstrated clinical efficacy and tolerable side effects of repeated IACT with doxorubicin and cisplatin in advanced HCC. Our regimen can be an alternative choice for patients with adequate liver function who do not want to receive continuous infusion of IACT

Electroseparations in thermoplastic microfabricated channels. Fluorescence detection strategies and effects of electroosmosis.

In this dissertation, several techniques are described that address solutions of problems in plastic micro devices for electroseparations, (1) background fluorescence arising from polymeric substrate, and (2) effects of electroosmotic flow in the plastic micro channels. Additionally, we explore polymer rigidity effects on the DNA electrophoresis separations in dilute polymer solutions, an emerging application area for these devices. Conventionally, microchip background has been reduced with confocal optics or circumvented with specialized long wavelength fluorophores. We show that microchip background can be rejected with analyte velocity modulation. In this scheme the driving voltage is modulated at low frequency (7--20 Hz). Consequently migration velocities and analyte signals are modulated at the same frequency. Microchip fluorescence is unmodulated, so that lock-in detection easily separates the analyte signal from background. The technique does not require a laser source. In our implementation a blue (485nm) LED is the light source. Fluorescence rejection provided by our current system lowers detection limits by approximately one order of magnitude compared to DC measurements with the same optical train. Detection by polarized fluorescence is also used to discriminate against emission arising from fluorescent substrates. Emission is excited with linearly polarized light and detected through a polarizer oriented perpendicular to the excitation polarizer. In this way, only fluorescence of freely rotating species is observed. The technique is shown to provide approximately 3X with background suppression, in bare capillaries, and 10X in capillaries coated with an oriented thin film. Protocols are described for control of electroosmotic flow in microfabricated channels in VivakTM co-polyester. Alkaline hydrolysis of surface ionizable groups alone or in combination with dynamic coating with cetyltrimethylammonium bromide (CTAB) is shown to provide reproducible electroosmotic flow. Video microscopic visualization of DNA electrophoretic dynamics in polymer solutions of different rigidity shows that there is more of DNA extension during entanglement in a solution of a rigid polymer such as hydroxyl ethyl cellulose (EEC), than in a solution of more flexible one, dextran. These visualization results are used to explain different electrophoretic behavior in these two polymer solutions.Ph.D.Analytical chemistryPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/132492/2/9963915.pd

MNHMT2009-18207 ENTRAINMENT OF DNA AND CHARGED NANO-PARTICLES USING AC ELECTROSPRAY

ABSTRACT We examine our earlier finding that AC electrospray cone can preferentially entrain and concentrate low-mobility ions generated at the cone tip [Chetwani, Maheshwari and Chang, Phys Rev Lett., 101, 204501 (2008)]. Instead of tip ionization, we scrutinize entrainment effects on dispersed ionic species in the spray solution. Fluorescence images of ejected ionic species, including anionic oxide ions, organic dyes, charged nano-particles, and stained DNA from AC electrospray cone generated with a 4-5 kV source. Negatively charged species such as anionic dyes concentrate at the tip of AC spray cone before ejection. Cationic dyes are more homogeneously distributed in the cone during ejection. Larger particles with negative charges, including fluorescence dye-tagged nanoparticles (about 50 nm) and dye-stained DNA, have similar spatio-temporal evolutions of anioic dyes within the cone before ejection. These observations extend our previous entrainment and concentration effect to AC spray without ionization. The images will be correlated with tandem Mass Spectrometry and will be used to selectively gate either cationic (protein) or anionic polymers (DNA)

ac electroosmotic pumping induced by noncontact external electrodes

Electroosmotic (EO) pumps based on dc electroosmosis is plagued by bubble generation and other electrochemical reactions at the electrodes at voltages beyond 1 V for electrolytes. These disadvantages limit their throughput and offset their portability advantage over mechanical syringe or pneumatic pumps. ac electroosmotic pumps at high frequency (>100 kHz) circumvent the bubble problem by inducing polarization and slip velocity on embedded electrodes,1 but they require complex electrode designs to produce a net flow. We report a new high-throughput ac EO pump design based on induced-polarization on the entire channel surface instead of just on the electrodes. Like dc EO pumps, our pump electrodes are outside of the load section and form a cm-long pump unit consisting of three circular reservoirs (3 mm in diameter) connected by a 1×1 mm channel. The field-induced polarization can produce an effective Zeta potential exceeding 1 V and an ac slip velocity estimated as 1 mm∕sec or higher, both one order of magnitude higher than earlier dc and ac pumps, giving rise to a maximum throughput of 1 μl∕sec. Polarization over the entire channel surface, quadratic scaling with respect to the field and high voltage at high frequency without electrode bubble generation are the reasons why the current pump is superior to earlier dc and ac EO pumps

Inositol hexaphosphate modulates the behavior of macrophages through alteration of gene expression involved in pathways of pro‐ and anti‐inflammatory responses, and resolution of inflammation pathways

Abstract Inositol hexaphosphate (IP6) is a dietary compound commonly obtained from corn, rice, etc. Although we may consume significant amount of IP6 daily, it is unclear whether this diet will impact macrophages’ fate and function. Therefore, we characterized the underlying relationship between IP6 and macrophage polarization in this study. We specifically examined the signature gene expression profiles associated with pro‐ and anti‐inflammatory responses, and resolution of inflammation pathways in macrophages under the influence of IP6. Interestingly, our data suggested that IP6 polarizes bone marrow‐derived macrophages (BMDM) into an M2a‐like subtype. Our results also demonstrated that IP6 reduces lipopolysaccharide‐induced apoptosis and pro‐inflammatory responses in macrophages. In contrast, the expression levels of genes related to anti‐inflammatory responses and resolution of inflammation pathways are upregulated. Our findings collectively demonstrated that IP6 has profound modulation effects on macrophages, which warrant further research on the therapeutic benefits of IP6 for inflammatory diseases

Enhancement of photoheterotrophic biohydrogen production at elevated temperatures by the expression of a thermophilic clostridial hydrogenase

The working temperature of a photobioreactor under sunlight can be elevated above the optimal growth temperature of a microorganism. To improve the biohydrogen productivity of photosynthetic bacteria at higher temperatures, a [FeFe]-hydrogenase gene from the thermophile Clostridium thermocellum was expressed in the mesophile Rhodopseudomonas palustris CGA009 (strain CGACThydA) using a log-phase expression promoter PpckA to drive the expression of heterogeneous hydrogenase gene. In contrast, a mesophilic Clostridium acetobutylicum [FeFe]- hydrogenase gene was also constructed and expressed in R. palustris (strain CGA-CAhydA). Both transgenic strains were tested for cell growth, in vivo hydrogen production rate, and in vitro hydrogenase activity at elevated temperatures. Although both CGA-CThydA and CGA-CAhydA strains demonstrated enhanced growth over the vector control at temperatures above 38 �C, CGA-CThydA produced more hydrogen than the other strains. The in vitro hydrogenase activity assay, measured at 40 �C, confirmed that the activity of the CGA-CThydA hydrogenase was higher than the CGA-CAhydA hydrogenase. These results showed that the expression of a thermophilic [FeFe]-hydrogenase in R. palustris increased the growth rate and biohydrogen production at elevated temperatures. This transgenic strategy can be applied to a broad range of purple photosynthetic bacteria used to produce biohydrogen under sunlight