Figure 3. Protein-quantum dot bioconjugate. Copyright National
Academy of Sciences of the United States of America 2004.
It has been discussed that the structure and function of the protein can
be influenced by many factors such as the size of the nanoparticle, the
nanoparticle ligand, the material of the nanoparticle, the stoichiometry
of the conjugate, the labeling site of the protein and the nature of the
linkage.20 Folded proteins have an average size of a
few nanometers (typically a diameter of 5-10 nm). Thus, ideally, the
nanoparticle should be much smaller than that to avoid physical
interference over the morphological dynamics of the protein. Up to now,
the smallest reported synthesized nanoparticle has a size of a few nm.
Another characteristic that should be avoided is fluorescence resonance
energy transfer (FRET), in which energy migrates from the excited
luminescent temperature probe to the conjugated protein. FRET must be
avoided as (a) it may decrease the intensity of the luminescence from
the nanoparticle, which is used for thermometry, and (b) it may induce
change of the protein energy state. Additionally, direct photon
excitation of the protein must be avoided. As the nanoparticle-protein
conjugates are much smaller than the spatial resolution of the optical
system, typically larger than a few hundred nanometers (the Rayleigh
diffraction limit), photons of the excitation source strike both the
nanoparticle and protein. Therefore, excitation sources have to operate
at wavelengths outside the absorption bands of electronic transitions of
the organic structure, the so-called biological windows. Another
important point that should be taken into consideration is the
excitation power density. If the excitation power density is too high,
the reading of temperature may be compromised by optically induced
heating caused by strong absorption of light by the nanoparticle. A
possible solution is the use of very small excitation powder densities,
which besides avoiding optical heating also prevents nonlinear optical
processes such as multi-photon absorption in both the nanoparticle and
the protein. As the luminescence intensity is proportional to the
excitation power density and the amount of luminophores, the intensity
of the luminescence is expected to be very low for a single
nanoparticle. A mechanism called photon avalanche have been proposed as
a way to improve imaging resolution and brightness at a single
nanoparticle level.18 For thermometry application,
fundamental parameters are the absolute temperature sensitivity S,
defined as the ratio in which the thermometric parameter changes with
temperature, and the uncertainty in temperature δT.21To detect small thermal fluctuations, as should be the case found at a
molecular level, S has to be very high while δT has to be extremally
small. Engineering nanomaterials with tailored structural and bonding
characteristics helps improve S. For example, luminescence thermometry
has been studied using the luminescence intensity ratio (LIR)
technique.21 Assuming a Boltzmann population
distribution among two thermally coupled electronic states (TCES) of
lanthanide ions in solids, it is observed that S is a function of the
barycenter of the energy bandgap between the TCES. In this case, the
choice of the host material is a key factor because the crystal field
influences the spectral peak position of emission bands. The parameter
δT, on the other hand, is related to different factors such as, for
example, the signal-to-noise ratio and integration time of the reading
of the thermometric parameter. Thermal fluctuations related to
nonequilibrium thermodynamic states are expected to be fast compared
with other time scales of events happening during the protein activity
and dynamics (microseconds to seconds). As a result, integration time of
the reading of the thermometric parameter during the thermal fluctuation
event has to be relatively short. Thus, the need of advanced instrument
technology with superior spectral and temporal resolutions associated
with high electronic noise reduction is fundamental to minimize δT.
Conclusion
Proteins have a fundamental role in biological processes. The dynamics
of such biochemical activities are complex and a complete understanding
of nonequilibrium states is currently lacking. In this scenario, there
must be a tremendous effort to overcome all the current limitations that
restrains measurements of thermal fluctuations involved in
nonequilibrium thermodynamic states present on the complex dynamics of
proteins. However, the insight to be gained with those measurements will
help better understand the mechanisms involved in the energy balance of
important biochemical reactions and processes involved in the
correct/incorrect operation of those key biomolecules. Luminescence
thermometry is an attractivity option to probe protein activity. Despite
our current experimental limitations, it is matter of time for the
progress in technological tools and methods to achieve a level in which
it will be feasible to unveil the details of the fascinating world of
proteins at work, especially the thermodynamics of nonequilibrium
states.