Biomedical Engineering Reference
In-Depth Information
2.5 Conclusions ........................................................................................................................104
References ....................................................................................................................................104
2.1
Introduction
The development of highly sensitive and selective instruments are opening new inroads
for the detection and monitoring of increasingly small amounts of material. The ultimate
in such instrumentation is that which allows investigation of the properties of single mol-
ecules (1-7). The advent of specialized instrumentation techniques, such as the scanning
tunneling microscopy (STM) and atomic force microscopy (AFM), has been a crucial tech-
nical advance that has permitted the area of nanotechnology to move forward. These lat-
ter methods are commonly used for investigations of molecules at solid planar interfaces
(6,7), while optical methods are more commonly used when ensembles of molecules to be
interrogated are located in a three-dimensional medium (1-5). However, various iterations
of optical methods, including laser scanning confocal microscopy (CM), total internal
reflection microscopy (TIRM), and near-field scanning optical microscopy (NSOM), are
also able to provide information with sufficient spatial resolution for investigation of the
location and behavior of individual molecules on surfaces (8-10).
The ability to use optical methods is often based on the fact that one single molecule can
be repetitively cycled between its ground state and excited states many times in 1 s when
using a wavelength that is resonant with this transition (11). The consequence is the abil-
ity to collect a large number of photons for detection and analysis from a single molecule,
resulting in a sufficiently large signal-to-noise ratio for analytical interpretation.
The first papers to suggest practical measurement of single molecules by an optical
method was published by Hirschfeld in 1976 (12), and reported the use of TIRM to detect
a single antibody molecule labeled with nearly 100 fluorophores. Single fluorophore
detection was subsequently reported using a flow cytometry method at room temperature
(2,13). In 1993, using NSOM, the first optically generated image of a single molecule was
obtained at room temperature (14). These were all important steps, culminating in the abil-
ity today to use techniques such as TIRM to produce two- and three-dimensional images
of targets associated with emission by single fluorescent molecules (3-5). The advent of
powerful small laser sources and semiconductor detectors with excellent amplification has
made it possible for biosensor technology to advance and implement optical strategies for
single molecule detection.
A fundamental question to pose at the outset is why detection and study of a single mole-
cule is important in the areas of bioanalytical chemistry and biosensing? Most experiments
typically involve a large number of molecules. The typical static ensemble measurements
only yield average values based on collections of molecules that may actually be in different
states, and such static measurements tend to report about only the final stages of reaction
paths or molecular conformations (15). However, there is much to be learned from the study
of intermediate steps during complicated reaction processes that involve many molecules, as
there is substantial diversity and distribution of states. Dynamic processes would require the
synchronization of all of the individual molecules in a large ensemble, and this is difficult to
achieve and impossible to maintain (16). Therefore, single molecule dynamics and kinetics
are seldom accessible. In contrast, single molecule detection offers observation of individual
states or conformations as correlated with time. Biosensors that are designed to detect and
study selective binding interactions can achieve new levels of confidence in determination of
selective binding interactions by accessing information about individual states rather than
