Biomedical subjects

Hanbin Mao

Publications and source records attributed to Hanbin Mao.

6 recordsLinked to original sources

PubMed2003

Investigations of bivalent antibody binding on fluid-supported phospholipid membranes: the effect of hapten density.

Investigations of ligand-receptor binding between bivalent antibodies and membrane-bound ligands are presented. The purpose of these studies was to explore binding as a function of hapten density in a two-dimensionally fluid environment. A novel microfluidic strategy in conjunction with total internal reflection fluorescence microscopy was designed to achieve this. The method allowed binding curves to be acquired with excellent signal-to-noise ratios while using only minute quantities of protein solution. The specific system investigated was the interaction between anti-DNP antibodies and phospholipid membranes containing DNP-conjugated lipids. Binding curves for ligand densities ranging from 0.1 to 5.0 mol % were obtained. Two individual dissociation constants could be extracted from the data corresponding to the two sequential binding events. The first dissociation constant, K(D1), was 2.46 x 10(-)(5) M, while the second was K(D2) = 1.37 x 10(-)(8) mol/m(2). This corresponded to a positively cooperative binding effect with an entropic difference between the two events of 62.3 +/- 2.7 J/(mol.K). Furthermore, the percentage of monovalently and bivalently bound protein was determined at each ligand density.

2,4-Dinitrophenol↗

PubMed2003

A sensitive, versatile microfluidic assay for bacterial chemotaxis.

We have developed a microfluidic assay for bacterial chemotaxis in which a gradient of chemoeffectors is established inside a microchannel via diffusion between parallel streams of liquid in laminar flow. The random motility and chemotactic responses to L-aspartate, L-serine, L-leucine, and Ni(2+) of WT and chemotactic-mutant strains of Escherichia coli were measured. Migration of the cells was quantified by counting the cells accumulating in each of 22 outlet ports. The sensitivity of the assay is attested to by the significant response of WT cells to 3.2 nM L-aspartate, a concentration three orders of magnitude lower than the detection limit in the standard capillary assay. The response to repellents was as robust and easily recorded as the attractant response. A surprising discovery was that L-leucine is sensed by Tar as an attractant at low concentrations and by Tsr as a repellent at higher concentrations. This assay offers superior performance and convenience relative to the existing assays to measure bacterial tactic responses, and it is flexible enough to be used in a wide range of different applications.

Chemotaxis↗

PubMed2003

Measuring LCSTs by novel temperature gradient methods: evidence for intermolecular interactions in mixed polymer solutions.

Herein we describe studies of molecular interactions in thermoresponsive polymers as they go through phase transitions in aqueous solutions. By using our recently reported linear temperature gradient setup for studying the effects of temperature on chemical processes, we demonstrate the ability to probe lower critical solution temperature (LCST) behavior with excellent precision. This method also provides a simple and convenient way to assay the LCST of solutions containing more than one polymer and follow the clouding kinetics of polymer mixtures in real time.

Journal Article↗

PubMed2002

Reusable platforms for high-throughput on-chip temperature gradient assays.

This paper describes a reusable platform that can apply a linear temperature gradient to a lab-on-a-chip device. When a planar microfluidic device with a series of microchannels is placed on top of the platform with the channels perpendicular to the gradient, each channel is held at a discrete temperature. This allows temperature-dependent data for chemical or biochemical species flowed into the device to be obtained in a concurrent fashion. As a demonstration, a melting curve for dsDNA is performed by collecting all the data simultaneously. The gradient is stable enough to easily distinguish between 30-mers where the complement strand contains a single C-A mismatch or a single T-G mismatch or is a perfect match. On the other hand, a temperature gradient can be formed parallel to the direction of flow of the microchannels. This allows the temperature in each channel to vary continuously as the liquid flows downstream. If each microchannel in the array contains a distinct pH value, ionic strength, species concentration, or chemical composition, then a high-throughput two-variable experiment can be performed. We demonstrate this mode of data collection by measuring the fluorescence yield of fluorescein dye molecules in aqueous solution simultaneously as a function of concentration and temperature.

Journal Article↗

PubMed2002

A microfluidic device with a linear temperature gradient for parallel and combinatorial measurements.

Methods for obtaining combinatorial and array-based data as a function of temperature are needed in the chemical and biological sciences. It is presently quite difficult to employ temperature as a variable using standard wellplate formats simply because it is very inconvenient to keep each well at a distinct temperature. In microfluidics, however, the situation is very different due to the short length scales involved. In this article, it is shown how a simple linear temperature gradient can be generated across dozens of parallel microfluidic channels simultaneously. This result is exploited to rapidly obtain activation energies from catalytic reactions, melting point transitions from lipid membranes, and fluorescence quantum yield curves from semiconductor nanocrystal probes as a function of temperature. The methods developed here could quite easily be extended to protein crystallization, phase diagram measurements, chemical reaction optimization, or multivariable experiments.

Alkaline Phosphatase↗

PubMed2002

Design and characterization of immobilized enzymes in microfluidic systems.

Herein we report the fabrication, characterization, and use of total analytical microsystems containing surface-immobilized enzymes. Streptavidin-conjugated alkaline phosphatase was linked to biotinylated phospholipid bilayers coated inside poly(dimethylsiloxane) microchannels and borosilicate microcapillary tubes. Rapid determination of enzyme kinetics at many different substrate concentrations was made possible by carrying out laminar flow-controlled dilution on-chip. This allowed Lineweaver-Burk analysis to be performed from a single experiment with all the data collected simultaneously. The results revealed an enzyme turnover number of 51.1 +/- 3.2 s(-1) for this heterogeneous system. Furthermore, the same enzyme immobilization strategy was extended to demonstrate that multiple chemical reactions could be performed in sequence by immobilizing various enzymes in series. Specifically, the presence of glucose was detected by two coupled steps employing immobilized avidinD-conjugated glucose oxidase and streptavidin-conjugated horseradish peroxidase.

Bacterial Proteins↗