A population of identical proteins has the same amino acid sequence, but there may be subtle differences in local folding that lead to variations in activity. Single molecule studies allow us to understand these subtle differences. Single molecule experiments are usually time consuming and difficult because only a few molecules are observed in one experiment. To address this problem, we have developed an assay where we can simultaneously measure the activity of multiple individual molecules of a protease, α-chymotrypsin. The assay utilizes a synthetic chymotrypsin substrate that is non-fluorescent before cleavage by chymotrypsin, but is intensely fluorescent after. To study the activity of individual enzymes, the enzyme and substrate are encapsulated in micron-sized droplets of water surrounded by silicone oil. On average, each micro-droplet contains less than one enzyme. The fluorescence of these droplets is recorded over time using a microscope and a CCD camera system. Software tracks individual droplets over time and records fluorescence. The kinetics of individual chymotrypsin molecules is calculated through the increase of fluorescence intensity of the same individual droplet over time. The activity profiles of the individual enzymes and the bulk sample of the enzyme are very similar. This validates the assay and demonstrates that the average of a few individual molecules can be representative of the behavior of the bulk population.
The particular form of electrochemiluminescence (ECL) used for analytical assays relies upon the discovery that tris(2,2'-bipyridyl)ruthenium(II) [Ru(bpy)32+] emits a 620 nm photon when adjacent to an electrode held at about one volt relative to Ag/AgCl. This reaction occurs within nanometers of the electrode. The enormous economic investment in nanoscale lithography tools is leading to tools capable of routinely producing 32 nm features by 2009. We propose that these two technologies could be combined to produce a nanoscale microscopy system. We constructed a macroscopic test-bed and performed tests on it to explore the feasibility of such a system. We tested an ECL solution containing 1 mM Ru(bpy)32+ 0.2 mM ammonium oxalate monohydrate in a 0.1 M ammonium acetate buffer at pH 5.0. Using this solution, we found that the ECL light was most intense at an applied voltage of 1.6 Volts, that the effect had excellent reproducibility and that the time to reach maximum intensity was several seconds after applying a voltage.
Using gold electrodes lithographically fabricated onto microscope cover slips, DNA and proteins are interrogated both optically (through fluorescence) and electronically (through conductance measurements). Dielectrophoresis is used to position DNA and proteins at well-defined positions on a chip. For the electronic manipulations, quadrupole electrode geometries are used with gaps ranging from 3 to 100 μm; AC field strengths are typically 106 V/m with frequencies between 10 kHz and 30 MHz. Nanoparticles (20 nm latex beads) are also manipulated. A technique of in situ impedance monitoring is tested for the first time to measure the conductance of the electronically manipulated DNA and proteins. The electrical resistance of DNA and proteins is measured to be larger than 40 MΩ under the experimental conditions used.
Monitoring of biologically active agents such as bacteria, viruses, proteins and small molecules in environmental samples poses complex analytical problems. The particulate nature of the analytes and potential interferents is of particular concern for microfluidic systems in which the channels may not be much larger than the particles themselves. For this reason, sample preconditioning upstream of a chemical analytical device will usually be required. However, the small dimensions of microfluidic devices also allow unique methods of sample purification, concentration, and detection. In our laboratory we have developed a series of microfluidic chemical analytical devices for such purposes. These devices rely on the low Reynolds number flow conditions. In such conditions field flow fractionation based on sedimentation, diffusion and electrophoresis perpendicular to the flow direction can be profitably harnessed to precondition samples. The H-filter is one such device in which a simple 4-port device that allows two fluids to be brought into adjacent flow, and then separated downstream into two (or more) flow streams after exchange of material under the influence of one or more fields. It can be fabricated using anodically bonded silicon and Pyrex channels, or using polymeric devices formed using `soft lithography' techniques. We have tested the ability of this device to be used for purification of bacteria and their spores from complex samples containing silica and other interferent particles. We will present results of our tests of this device, as well as initial attempts to integrate the H-filter into a sample preconditioning system that includes on-chip pumps.
Microfluidic devices fabricated in silicon are quickly finding use in many areas of technology. Exploration of new applications of this technology has shown both advantages and disadvantages to extreme miniaturization of chemical assays. While accuracy, efficiency and smaller sample volumes are among the advantages, interactions between the walls of the micro-channels and the fluid or particles it contains are among the disadvantages. Our group is applying this technology to chemical and biological warfare (CBW) agent purification and detection. We present preliminary result towards achieving a long-term antifouling surface in our detection system. A microfluidic device was anisotropically etched in a (100) silicon wafer and attached to a Pyrex glass slip to create an enclosed channel. Poly(ethylene glycol) (PEG) silane was covalently bonded to the hydroxyls of an oxide layer on the silicon device and the Pyrex cover slip. Fluorescently labeled ovalbumin, a CBW simulant, was in contact with an unmodified and PEG-modified channel. The extent of adsorption was determined using fluorescence microscopy.
We will discuss two recent directions of our work: (1) The influence of submicron length scales on polymer dynamics, (2) Ultra-rapid mixing via sub-micron hydrodynamic focusing. (1) Polymer dynamics at sub-micron length scales. We have explored the changes in the dynamics of long polymers as the thickness of the quasi-2 dimensional space is varied from 0.09 microns to 10 microns. We will show how the thickness of this space, scaled with the persistence length of the polymer, changes the dynamics of the polymer. The consequences of this qualitative change in polymer dynamics is quite important, since it controls the elongation of the polymer at a given force field and hence the ability of he array to fractionate the polymer. (2) Mixing at the sub- micron length scale cannot be tubulent but only diffusive in nature. We will show how it is possible using hydrodynamics to produce liquid jets of width under 20 nanometers which can mix fluids in under 1 microsecond times.
We demonstrate a novel hydrodynamic shear activation of leucocyte adhesion, using physiological flow conditions and a microfabricated array of channels with length scales similar to those of human capillaries. Vital chromosome stains and cell specific fluorochrome labeled antibodies reveal that the eventual adhesion of the leukocytes to the silicon array displays a strong dependence on cell type and nuclear morphology, with granulocytes activating more rapidly with distance and penetrating a smaller distance than lymphocytes. Further, the granulocytes interact with the lymphocytes in a self-exclusionary manner under shearing flow with the eventual separation of the two cell types in the array. Such arrays of microfabricated obstacles thus have an interesting potential for sorting white blood cells by type from a 10 microliter drop of whole blood.
Microfabricated fluidic systems allow complex chemical analyses to be performed on sub-nanoliter volumes of sample. Compared to macroscopic systems, these devices offer many advantages, including the promise of performing some analytical functions more rapidly and on smaller samples. However, miniaturization of analytic instruments is not simply a matter of reducing their size. At small scales, different effects become more prominent, rendering some processes inefficient and others useless. The small scales also permit the creation of novel devices, such as the H- filter, which we are using to extract analytes from whole blood. Fluid flow in microfluidic systems is entirely dominated by viscous forces, making diffusion the sole mechanism of mixing. In addition, a larger fraction of molecules are lost to surface adsorption as devices shrink. This paper examines some of the issues involved in device miniaturization, specifically those phenomena that become increasingly dominant.