We measured light scattered from gold nanoparticles with darkfield microscopy in order to perform single molecule detection based on tethered particle motion (TPM). This combination results in a signal to noise ratio of about 40 dB, which allowed us to use 80 nm diameter gold particles as reporters instead of the typically used polystyrene particles whose sizes are up to 1 µm. The particle size is crucial in TPM experiments as it can induce a volume-exclusion effect, which results in a stretching force acting on the DNA tether. This affects both the biophysical and statistical properties of the anchored DNA and hence the interpretation of the experimental data. We demonstrated that the gold nanoparticles and darkfield microscopy can be used to characterize the confined Brownian motion of dsDNA-tethered gold particles with a spatial precision of 3 nm. Physical parameters such as the spring constant of the tethered DNA fragment and the persistence length can be derived from the two dimensional (2D) (x, y) projected image data. We have applied this method to various MgCl2 and glycerol concentrations as a proof of principle.
Single-molecule techniques continue to gain in popularity in research disciplines such as the study of intermolecular
interactions. These techniques provide information that otherwise would be lost by using bulk measurements that deal
with a large number of molecules. We describe in this report the motion of tethered DNA molecules that have been
tagged with gold nanobeads and observed under dark field microscopy to study single molecular interactions (SMI). We
further report on the derivation and use of several physical parameters and how these parameters change under differing
experimental conditions.
The study of DNA-protein interactions is gaining increased attention due to their importance in cellular processes. Only a well-functioning interaction guaranties that such a process can take place without errors. So far, only a small percentage of these interactions have been unraveled, partially due to their complexity but also due to the fact that there are only a few techniques that permit the study of these interactions. In this report we describe the development of a research tool based on tethered bead motion and Resonance Light Scattering (RLS) from gold beads. This method permits the study of DNA-protein interactions and the screening of proteins binding to a specific DNA sequence.
In previous publications we have shown that we can perform enzymatic reactions in nanoarrays by means of a microarray-reader based on a conventional microscope. In this publication we report on a modification of this system in order to monitor the aggregation kinetics of the natively unfolded protein α-synuclein. We describe the motivation for this development, the problems associated with the miniaturization of the aggregation assay, and the validation of our modifications.
The intense research in proteomics is demanding for fast, reliable and easy-to-use methods in order to study the proteome. In this proceeding we report the development of such a novel research tool based on spectral imaging and Resonance Light Scattering gold particles. This method will allow the study of DNA-protein interactions. We suggest a broad range of applications: the screening of proteins binding to a specific DNA sequence, the analysis of binding affinities between protein and DNA, and the investigation of the influence of environmental conditions on the binding. We will explain the principle, first experiments and first results based on Brownian motion.
Traditional light sources for fluorescence microscopy have been mercury lamps, xenon lamps, and lasers. These sources have been essential in the development of fluorescence microscopy but each can have serious disadvantages: lack of near monochromaticity, heat generation, cost, lifetime of the light source, and possible distortions due to coherence effects.
We are examining the possibility of using the new high-power LED light sources as alternatives to the above mentioned sources. LED sources are near monochromatic, are inexpensive, produce little heat, have no coherence problems, have extended lifetimes, are small, and can easily be modulated.
In this presentation we will describe experiments comparing various LEDs to other light sources. We will compare, for example, a 530 nm LED to the 546 nm line from a mercury lamp on a fluorophore whose absorption maximum is broad and in the middle between these two wavelengths.
In previous publications and presentations we have described our construction of a laboratory-on-a-chip based on nanoliter capacity wells etched in silicon. We have described methods for dispensing reagents as well as samples, for preventing evaporation, for embedding electronics in each well to measure fluid volume per well in real-time, and for monitoring the production or consumption of NADH in enzyme-catalyzed reactions such as those found in the glycolytic pathway of yeast. In this paper we describe the use of light sensors (photodiodes) in each well to measure both fluorescence (such as that evidenced in NADH) as well as bioluminescence (such as evidenced in ATP assays). We show that our detection limit for NADH fluorescence in 100 μM and for ATP/luciferase bioluminescence is 2.4 μM.
This paper explores the use of photo patternable polymers for integrated high-speed screening arrays, where enzyme reactions are monitored in nano liter volume reactors using fluorescence of NADH and photodiode detection. Implementing the array of nano liter volume wells using a low-temperature CMOS-compatible process allows wells to be patterned after the photodiode array and electronics fabrication is completed. We demonstrate filling of 400 X 400 micron square, 25 micron deep photoresist-on-silicon wells with liquid samples by electro spray and wetting. We also demonstrate usability of the wells on NADH samples by measuring the fluorescence of 0.1, 0.5 and 1 millimolar NADH solutions using external optics.
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