Mansoor Nasir

Multi-wavelength microflow cytometer using groove-generated sheath flow

A microflow cytometer was developed that ensheathed the sample (core) fluid on all sides and interrogated each particle in the sample stream at four different wavelengths. Sheathing was achieved by first sandwiching the core fluid with the sheath fluid laterally via fluid focusing. Chevron-shaped groove features fabricated in the top and bottom of the channel directed sheath fluid from the sides to the top and bottom of the channel, completely surrounding the sample stream. Optical fibers inserted into guide channels provided excitation light from diode lasers at 532 and 635 nm and collected the emission wavelengths. Two emission collection fibers were connected to PMTs through a multimode fiber splitter and optical filters for detection at 635 nm (scatter), 665 nm and 700 nm (microsphere identification) and 565 nm (phycoerythrin tracer). The cytometer was capable of discriminating microspheres with different amounts of the fluorophores used for coding and detecting the presence of a phycoerythrin antibody complex on the surface of the microspheres. Assays for Escherichia coli were compared with a commercial Luminex flow cytometer.

Multiplexed detection of bacteria and toxins using a microflow cytometer.

A microfabricated flow cytometer was used to demonstrate multiplexed detection of bacteria and toxins using fluorescent coded microspheres. Antibody-coated microspheres bound biothreat targets in a sandwich immunoassay format. The microfluidic cytometer focused the microspheres in three dimensions within the laser interrogation region using passive groove structures to surround the sample stream with sheath fluid. Optical analysis at four different wavelengths identified the coded microspheres and quantified target bound by the presence of phycoerythrin tracer. The multiplexed assays in the microflow cytometer had performance approaching that of a commercial benchtop flow cytometer. The respective limits of detection for bacteria (Escherichia coli, Listeria, and Salmonella) were found to be 10(3), 10(5), and 10(4) cfu/mL for the microflow cytometer and 10(3), 10(6), and 10(5) cfu/mL for the commercial system. Limits of detection for the toxins (cholera toxin, staphylococcal enterotoxin B, and ricin) were 1.6, 0.064, and 1.6 ng/mL for the microflow cytometer and 1.6, 0.064, and 8.0 ng/mL for the commercial system.

Hydrodynamic focusing – a versatile tool

The control of hydrodynamic focusing in a microchannel has inspired new approaches for microfluidic mixing, separations, sensors, cell analysis, and microfabrication. Achieving a flat interface between the focusing and focused fluids is dependent on Reynolds number and device geometry, and many hydrodynamic focusing systems can benefit from this understanding. For applications where a specific cross-sectional shape is desired for the focused flow, advection generated by grooved structures in the channel walls can be used to define the shape of the focused flow. Relative flow rates of the focused flow and focusing streams can be manipulated to control the cross-sectional area of the focused flows. This paper discusses the principles for defining the shape of the interface between the focused and focusing fluids and provides examples from our lab that use hydrodynamic focusing for impedance-based sensors, flow cytometry, and microfabrication to illustrate the breadth of opportunities for introducing new capabilities into microfluidic systems. We evaluate each example for the advantages and limitations integral to utilization of hydrodynamic focusing for that particular application.

Porphyrin-Embedded Silicate Materials for Detection of Hydrocarbon Solvents

The development of porphyrin-embedded mesoporous organosilicate materials for application to the detection of volatile hydrocarbon solvents is described. Design of the receptor and optical indicator construct begins with parallel selection of the porphyrin indicator and design of the mesoporous sorbent. For the porphyrin indicator, high binding affinity and strong changes in spectrophotometric character upon target interaction are desired. The sorbent should provide high target binding capacity and rapid binding kinetics. A number of porphyrin/metalloporphyrin variants and organosilicate sorbents were evaluated to determine the characteristics of their interaction with the targets, benzene, toluene, and hexane. The selected porphyrin candidates were covalently immobilized within a benzene-bridged sorbent. This construct was applied to the detection of targets using both fluorescence- and reflectance-based protocols. The use of red, green, and blue (RGB) color values from the constructs in a highly simplified detection scheme is described.

Hydrodynamic focusing of conducting fluids for conductivity-based biosensors.

Hydrodynamic focusing of a conducting fluid by a non-conducting fluid to form a constricted current path between two sensing electrodes is implemented in order to enhance the sensitivity of a 4-electrode conductance-based biosensor. The sensor has a simple two-inlet T-junction design and performs four-point conductivity measurements to detect particles immobilized between the sensing electrode pair. Computational simulations conducted in conjunction with experimental flow studies using confocal microscopy show that a flat profile for the focused layer is dependent on the Reynolds number for the chosen flow parameters. The results also indicate that a flat focused layer is desirable for both increased sensitivity as well as surface-binding efficiency. Proof of concept for conductance measurements in a hydrodynamically focused conducting fluid was demonstrated with entrapped magnetic beads.

Hydrodynamic and electrical considerations in the design of a four-electrode impedance-based microfluidic device

AbstractA four-electrode impedance-based microfluidic device has been designed with tunable sensitivity for future applications to the detection of pathogens and functionalized microparticles specifically bound to molecular recognition molecules on the surface of a microfluidic channel. In order to achieve tunable sensitivity, hydrodynamic focusing was employed to confine the electric current by simultaneous introduction of two fluids (high- and low-conductivity solutions) into a microchannel at variable flow-rate ratios. By increasing the volumetric flow rate of the low-conductivity solution (sheath fluid) relative to the high-conductivity solution (sample fluid), increased focusing of the high-conductivity solution over four coplanar electrodes was achieved, thereby confining the current during impedance interrogation. The hydrodynamic and electrical properties of the device were analyzed for optimization and to resolve issues that would impact sensitivity and reproducibility in subsequent biosensor applications. These include variability in the relative flow rates of the sheath and sample fluids, changes in microchannel dimensions, and ionic concentration of the sample fluid. A comparative analysis of impedance measurements using four-electrode versus two-electrode configurations for impedance measurements also highlighted the advantages of using four electrodes for portable sensor applications. A four-electrode sensor with hydrodynamic focusing to confine that the current was characterized for tunable sensitivity

Toward In Situ Monitoring of Water Contamination by Nitroenergetic Compounds

We have previously described the application of novel porous organosilicate materials to the preconcentration of nitroenergetic targets from aqueous solution prior to HPLC analysis. The performance of the sorbents and the advantages of these types of materials over commercially available solid phase extraction sorbents have been demonstrated. Here, the development of systems for application of those sorbents to in situ monitoring is described. Considerations such as column pressure, particulate filtration, and component durability are discussed. The diameter of selected column housings, the sorbent bed depth, and the frits utilized significantly impact the utility of the sorbent columns in the prototype system. The impact of and necessity for improvements in the morphological characteristics of the sorbents as they relate to reduction in column pressure are detailed. The results of experiments utilizing a prototype system are presented. Data demonstrating feasibility for use of the sorbents in preconcentration prior to ion mobility spectrometry is also presented.