Biomedical Engineering Reference
In-Depth Information
quantitative analyses is that there are different statistical models that are applied to ensem-
ble averages and to individual events (stochastic processes). In the extreme, a bioanalytical
or biosensing system based on single molecule detection must invoke an element of time
and the concept of “on” and “off” switching. Theoretically, detection of a single molecule of
target in a sample by a single molecular receptor in a measurement system only requires that
the two species eventually collide in an associative interaction. Practically, this can take time
periods of years if sample volumes are large and processes are diffusion controlled. This
implies the need for rethinking of the common definitions in analytical chemistry of limit-
of-detection and sensitivity so as to accommodate stochastic processes.
2.4.1
DNA Sequencing and DNA Fragment Sizing
DNA sequencing methods often involve incorporation of fluorescently tagged nucleotides
by the use of a polymerase chain reaction (PCR). In subsequent steps, the labeled DNA
strand is anchored to a surface in a flowing stream, exonuclease enzymes are used to
release the tagged nucleotides from the free end of the DNA, and then the DNA sequence
is determined after detection and identification of the cleaved nucleotides by their fluo-
rescent tags (11). Single molecule detection has the potential for sequencing about 10
4
bases with high speed, and this is attractive in comparison to gel-based sequencing tech-
nologies where detection is often limited to lengths of less than 1,000 bases and can be sen-
sitive to molecular conformation. Keller and coworkers (59) were the first to use this novel
method for rapid DNA sequencing, and several other research groups have also used such
single molecule detection methods for DNA sequencing (60-65).
It has been established that the number of fluorescent dye molecules that are incorpo-
rated by the DNA fragment of interest is related to its physical properties, such as size, sur-
face area, volume, and reactivity (15). In the case of intercalating fluorescent dyes that have
no sequence preference and are uniformly distributed along the DNA, the fluorescence
intensity is linearly related to the fragment length of DNA. There are two single molecule
detection methods that are used for rapid DNA fragment size analysis by measurement of
fluorescent intensity from intercalators. One method introduces a solution that contains
DNA fragments into a sheath flow cuvette, and DNA fragments can then be detected by a
downstream laser beam. The smallest detectable fragment size is approximately 1,000 base
pairs (66). The second approach is to stretch DNA molecules on a glass surface and then
cut them with restriction enzymes. The relaxation of strands at cleaved sites form microm-
eter-wide gaps, and these can be seen using an optical microscope. The integrated fluo-
rescence signals of individual fragments reflect the initial fragment size of the DNA
sequence. Note that a single molecule detection scheme based on intercalating dyes can-
not achieve single-base pair resolution because DNA intercalates at a density of about 3-5
base pairs per dye molecule (16).
2.4.2
Single-Pair Fluorescence Resonance Energy Transfer
Fluorescence resonance energy transfer has been widely used in structural biology, bio-
chemistry, and polymer science for measuring separation distances (15). In FRET, energy of
a donor fluorescence molecule is transferred to an acceptor by a through-space dipole inter-
action. Because FRET is strongly dependent on distance, the process can be used as a tool to
evaluate distances, as well as to signal changes of distances between acceptor-donor species.
If the acceptor and donor are in close proximity to one another, the acceptor can absorb
the emission energy of the donor in a nonradiative mechanism as long as the pair is within
the Förster distance for that particular set of donor and acceptor species. The donor and
acceptor can both be fluorescent dyes where the wavelength of excitation for the acceptor is
