Single-Molecule Detection in Capillary Electrophoresis:  Molecular Shot Noise as a Fundamental Limit to Chemical Analysis

Abstract

Capillary electrophoresis is coupled with a single molecule detector based on laser-induced fluorescence. Individual molecules migrating from the capillary are detected and counted with 50% efficiency. Injection of 30 000 analyte molecules generates a reproducible peak consisting of at least five components. However, injection of 3000 or fewer molecules leads to a noisy and irreproducible peak. Monte Carlo simulation demonstrates that this irreproducibility results from molecular shot noise or stochastic fluctuations in the number of injected molecules. The model predicts that the relative standard deviations of peak area, peak center, and peak width are inversely proportional to the square root of the number of injected molecules. At least 104 analyte molecules (17 zmol) are required to define peak area and width with 1% relative precision. Fluctuation in the number of molecules taken for chemical analysis is a fundamental and irreducible source of uncertainty.

Statistics of single‐molecule measurements: Applications in flow‐cytometry sizing of DNA fragments

“…The DNA fingerprint in Figure 6a shows only seven peaks due to our inability to resolve the 2,322-bp and 2,027-bp fragments. The total number of events comprising the fingerprint shown in Figure 6a is 8,991. This corresponds to roughly 1,100 digested genomes.…”

“…Determining analyte concentration, by SMD or any other technique, is a counting operation and is governed by Poisson statistics. Chen and Dovichi (8) present a thorough summary of Poisson statistics, relative to SMD concentration measurements in capillary electrophoresis. Briefly, the authors show that the uncertainties in the number of detected molecules, as well as peak shape and position, are determined by counting statistics and limited by molecular shot noise for small samples.…”

Peer Reviewed: Analytical Applications of Single-Molecule Detection

“…Approximately 2% of the injected molecules were detected. For ultradilute samples and femtoliter detection volumes, the relative precision of the peak area, peak position, and peak standard deviation are dominated by the diffusion of the molecules into and out of the detection volume (14) and the inverse square root of the number of detected molecules (34). Concentration detection limits (>99% confidence) were reported to be 1.7 pM for R6G and 8.5 pM for RB.…”

Automation for Genomics, Part Two: Sequencers, Microarrays, and Future Trends

“…A variety of novel approaches based on single molecule analysis are being investigated to provide revolutionary approaches for DNA sequencing and analysis (Davis et al 1991;Chen and Dovichi 1996;Fernandes 1998;Haab and Mathies 1998;Nie and Zare 1998;Rich 1998;Akeson et al 1999;Herrick and Bensimon 1999;Ishikawa 1999;Moore et al 1999;Yeung 1999;Bustamante et al 2000;Deamer and Akeson 2000;Jett et al 2000;Krylov and Dovichi 2000;Van Craenenbroeck and Engelborghs 2000). One example is the research by Dr. Deamer at the University of California, Santa Cruz and Dr. Branton at Harvard University to develop single molecule electrophoresis (Akeson et al 1999;Akeson and Deamer 1999;Deamer and Akeson 2000;Service 2000).…”