Single molecule detection

FIGURE 1.12 (a) Electrochemical detection of a single molecule, A, trapped between the 

Interestingly, an SECM-based nanogap can be also used to detect the emission of electrogenerated chemiluminescence (ECL) from a single molecule [71], 

With time-resolved detection the temporal behavior of a single molecule can be followed. One example is the measurement of the relaxation time constant of intersystem crossing from the excited singlet state into the triplet state of a dye molecule during the diffusion time through the detection volume [15.15]. 

Molecular impurities in solids can now be detected down to the singlemolecule level [15.16]. These molecules and their interaction with their surroundings can be probed by high-resolution laser spectroscopy. The relevance of such techniques in biology is obvious [15.17]. The development of single-molecule detection for the in vitro and in vivo quantification of biomolecular dynamics is essential for the understanding of biomolecular reactions and for the realization of evolutionary biotechnology [15.18]. 

More examples of lasers in analytical chemistry can be found in [15.19-15.21]. 

The combination of illuminating the sample with a strongly focused laser beam and observing the laser-induced fluorescence or the non-resonant scattered laser light with confocal microscopy allows the detection of single molecules and their diffusion through a liquid medium. Each molecule can emit N = T/2r photons (see Fig. 10.2) where T is the diffusion time through the laser beam and r the upper state lifetime. 

The experimental setup is illustrated in Fig. 10.5. The light scattered by molecules in a diluted solution with concentrations in the nanomole range, which 

---

Kneipp K, Wang Y, Kneipp H, Itzkan I, Dasari R R and Feld M S 1996 Approach to single molecule detection using surface-enhanced Raman scattering ICORS 98 XVth Int. Conf on Raman Spectroscopy ed S A Asher and P B Stein (New York Wley) pp 636-7... 

Two-photon excited fluorescence detection at the single-molecule level has been demonstrated for cliromophores in cryogenic solids [60], room-temperature surfaces [61], membranes [62] and liquids [63, 64 and 65]. Altliough multiphoton excited fluorescence has been embraced witli great entluisiasm as a teclmique for botli ordinary confocal microscopy and single-molecule detection, it is not a panacea in particular, photochemical degradation in multiphoton excitation may be more severe tlian witli ordinary linear excitation, probably due to absorjDtion of more tlian tire desired number of photons from tire intense laser pulse (e.g. triplet excited state absorjDtion) [61],... 

Weston K D, Carson P J, DeAro J A and Buratto S K 1999 Single-molecule detection fluorescence of surface-bound species in vacuum Chem. Phys. Lett. 308 58-64... 

Goodwin P M, Ambrose W P and Keller R A 1996 Single-molecule detection in liquids by laser-induced fluorescence Acc. Chem. Res. 29 607-13... 

Soper S A and Legendre B L Jr 1998 Single-molecule detection in the near-IR using continuous-wave diode laser... 

Mertz J, Xu C and Webb W W 1995 Single-molecule detection by two-photon-excited fluorescence Opt Lett. 20 2532-4... 

Williams J G K 1997 Single-molecule detection of specific nucleic acid sequences in unamplified genomic DNA Anal. Chem. 69 3915-20... 

Haab B B and Mathies R A 1999 Single-molecule detection of DNA separations in microfabricated capillary electrophoresis chips employing focused molecular streams Ana/. Chem. 71 5137-45... 

FIGURE 2-19 Single-molecule detection with SECM. Molecule A is trapped between the tip and the surface. (Reproduced with permission from reference 58.)... 

Single molecule detection, 52, 128 Size exclusion films, 123 Sodium, 155... 

Zander Ch, Enderlein J, Keller RA (2002) (eds) Single molecule detection in solution. Methods and applications. Wiley-VCH, Weinheim, Germany... 

DNA fragment sizing by single molecule detection in submicrometer-sized closed fluidic channels. Anal. Chem. 74 (2002) 1415-1422. 

Kneipp, K., Wang, Y., Kneipp, H., Perelman, L. T., Itzkan, I., Dasari, R. R. and Eeld, M. S. (1997) Single molecule detection using surface-enhanced Raman scattering (SERS). Phys. Rev. Lett., 78, 1667-1670. 

Oswald B, Gruber M, Bohmer M, Lehmann F, Probst M, Wolfbeis OS (2001) Novel diode laser-compatible fluorophores and their application to single molecule detection, protein labeling and fluorescence resonance energy transfer immunoassay. Photochem Photobiol 74 237-245... 

Buehler, C., Stoeckli, K. and Auer, M. (2001). The integration of single molecule detection technologies into miniturized drug screening Current status and future perspectives. In New Trends in Fluorescence Spectroscopy (Valeur, B. and Brochon, J. C., eds.). Springer, Berlin, pp. 331-79. 

Widengren, J., Kudryavtsev, V., Antonik, M., Berger, S., Gerken, M. and Seidel, C. A. (2006). Single-molecule detection and identification of multiple species by multiparameter fluorescence detection. Anal. Chem. 78,2039-50. 

Kneipp K., Kneipp H., Deinum G., Itzkan I., Dasari R.R., Feld M. S., Single-molecule detection of a cyanine dye in silver colloidal solution using near-infrared surface-enhanced Raman scattering, Appl. Spectrosc. 1998 52 175-178. 

Novel techniques as single molecule detection will focus on molecular localization and dynamics and may open insights into the micro-compartmen-talization of cellular systems. 

Armani, A. M. Kulkami, R. P. Fraser, S. E. Flagan, R. C. Vahala, K. J., Label free, single molecule detection with optical microcavities, Science 2007, 317, 783 787... 

---