Biomedical Engineering Reference
In-Depth Information
ensemble averages. Furthermore, there continues to be a demand for detection of very small
numbers of targets in complicated biological samples, such as the early detection of
pathogens, or markers associated with genetic mutations that may signal the onset of cancers.
This chapter reviews instrumentation and procedures for single molecule detection by
optical methods, and applications of such methods in bioanalytical chemistry and
biosensor development.
2.2
The Principle of Single Molecule Detection Using Optical Methods
The fluorescence process of a single molecule begins with an electronic excitation caused
by absorption of a photon, followed by internal relaxation to the lowest energy state
within the excited state, and then radiative decay to the ground state. The lifetime of a flu-
orescence cycle is dominated by the radiative and nonradiative relaxation processes, and
tends to fall in a range of approximately 10 ns-10 ps. Multiple excitation and emission
cycles are possible at rates of about 10
7
-10
8
per second when using a 1.0 mW laser beam
focused to the diffraction limit (11). Fluorescence intensity and the time for emission are
related to several deactivation channels. These can involve processes such as collisional
deactivation, photochemical reactions leading to photodestruction of the molecule, energy
transfer, and electron transfer. Photochemical reactions include photooxidation, photoion-
ization, photodissociation, and photoisomerization. Another process that is not commonly
a significant energy loss pathway in bulk solution, but that does influence solids and
immobilized systems, is intersystem crossing from the excited singlet state to the triplet
state, resulting in phosphorescence rather than fluorescence emission (11).
2.2.1
Origins of Fluorescence
One of the fundamental requirements of an optical biosensor is the generation of a photolu-
minescent signal indicative of target analyte binding. Fluorescence emission that is designed
to be indicative of selective reactions can be detected and converted into an analytical signal.
Fluorescence is a type of photoluminescence that is generated when a photon of specific
energy is absorbed by a molecule wherein an electron within the molecule is promoted
from the ground state to an electronically excited state. The excited state can exist as either
a singlet or triplet state (17). The singlet excited state occurs when the electron is paired
with another electron of opposing spin (17). This is a quantum-mechanically allowed tran-
sition, and fluorescence emission occurs as a result of the spin-paired excited electron
returning to the ground state (17). The triplet excited state occurs when the excited elec-
tron is of the same spin as the second electron in the pair, and upon relaxation, the elec-
tron must undergo a “spin-flip” to return to the singlet ground state (17). These processes
are represented in the Jablonski diagram.
Within each of the energy levels of the excited state, there are several nonradiative vibra-
tional energy levels (17). The relaxation of a molecule from an excited vibrational state to
the lowest energy excited vibrational state occurs much faster than the radiative relaxation
of the molecule from the excited electronic state to the ground state; hence, fluorescence
occurs from the thermally equilibrated excited state (17). Because some energy is lost
through nonradiative processes associated with the vibrational levels present within each
of the excited and ground electronic states, there is a shift to longer wavelengths (lower
energy) for the fluorescence emission with respect to wavelength of light required for
excitation. This is referred to as a Stokes' shift. Stokes' shifts are also encountered due to
