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.