Optothermal properties of plasmonic inorganic nanoparticles for photoacoustic applications.

Plasmonic systems are becoming a favourable alternative to dye molecules in the generation of photoacoustic signals for spectroscopy and imaging. In particular, inorganic nanoparticles are appealing because of their versatility. In fact, as the shape, size and chemical composition of nanoparticles are directly correlated with their plasmonic properties, the excitation wavelength can be tuned to their plasmon resonance by adjusting such traits. This feature enables an extensive spectral range to be covered. In addition, surface chemical modifications can be performed to provide the nanoparticles with designed functionalities, e.g., selective affinity for specific macromolecules. The efficiency of the conversion of absorbed photon energy into heat, which is the physical basis of the photoacoustic signal, can be accurately determined by photoacoustic methods. This review contrasts studies that evaluate photoconversion in various kinds of nanomaterials by different methods, with the objective of facilitating the researchers' choice of suitable plasmonic nanoparticles for photoacoustic applications.

Surface Enhanced Raman Spectroscopy for In-Field Detection of Pesticides: A Test on Dimethoate Residues in Water and on Olive Leaves.

Dimethoate (DMT) is an organophosphate insecticide commonly used to protect fruit trees and in particular olive trees. Since it is highly water-soluble, its use on olive trees is considered quite safe, because it flows away in the residual water during the oil extraction process. However, its use is strictly regulated, specially on organic cultures. The organic production chain certification is not trivial, since DMT rapidly degrades to omethoate (OMT) and both disappear in about two months. Therefore, simple, sensitive, cost-effective and accurate methods for the determination of dimethoate, possibly suitable for in-field application, can be of great interest. In this work, a quick screening method, possibly useful for organic cultures certification will be presented. DMT and OMT in water and on olive leaves have been detected by surface enhanced Raman spectroscopy (SERS) using portable instrumentations. On leaves, the SERS signals were measured with a reasonably good S/N ratio, allowing us to detect DMT at a concentration up to two orders of magnitude lower than the one usually recommended for in-field treatments. Moreover, detailed information on the DMT distribution on the leaves has been obtained by Raman line- (or area-) scanning experiments.

A straightforward synthesis of phenyl boronic acid (PBA) containing BODIPY dyes: new functional and modular fluorescent tools for the tethering of the glycan domain of antibodies.

We report here on the efficient and straightforward synthesis of a series of modular and functional PBA-BODIPY dyes 1-4. They are an outstanding example of the efficient merge of the versatility of the 3,5-dichloro-BODIPY derivatives and the receptor-like ability of the PBA moiety. The potential bioanalytical applicability of these tools was assessed by measuring the binding to glycan chains of antibodies by a Quartz Crystal Microbalance (QCM).

Silver nanowires as infrared-active materials for surface-enhanced Raman scattering.

Surface-enhanced Raman scattering (SERS) is increasing in significance as a bioanalytical tool. Novel nanostructured metal substrates are required to improve performances and versatility of SERS spectroscopy. In particular, as biological tissues are relatively transparent in the infrared wavelength range, SERS-active materials suitable for infrared laser excitation are needed. Nanowires appear interesting in this respect as they show a very broad localized surface plasmon resonance band, ranging from near UV to near infrared wavelengths. The SERS activity of silver nanowires has been tested at three wavelengths and a fair enhancement at 1064 and 514 nm has been observed, whereas a very weak enhancement was present when exciting close to the nanowire extinction maximum. These experimentally measured optical properties have been contrasted with finite element method simulations. Furthermore, laser-induced optoacoustic spectroscopy measurements have shown that the extinction at 1064 nm is completely due to scattering. This result has an important implication that no heating occurs when silver nanowires are utilized as SERS-active substrates, thereby preventing possible thermal damage.

Structural determinants of ligand binding in truncated hemoglobins: Resonance Raman spectroscopy of the native states and their carbon monoxide and hydroxide complexes.

The ligand binding characteristics of heme-containing proteins are determined by a number of factors, including the nature and conformation of the distal residues and their capability to stabilize the heme-bound ligand via hydrogen-bonding and electrostatic interactions. In this regard, the heme pockets of truncated hemoglobins (TrHbs) constitute an interesting case study as they share many common features, including a number of polar cavity residues. In this review, we will focus on three proteins of group II TrHbs, from Thermobifida fusca (Tf-HbO) and Pseudoalteromonas haloplanktis TAC125 (Ph-HbO). Although the residues in positions G8 (Trp) and B10 (Tyr) are conserved in all three proteins, the CD1 residue is a Tyr in T. fusca and a His in P. haloplanktis. Comparison of the ligand binding characteristics of these proteins, in particular the hydroxo and CO ligands by means of resonance Raman spectroscopy, reveals that this single difference in the key heme cavity residues markedly affects their ligand binding capability and conformation. Furthermore, although the two Ph-HbOs (Ph-HbO-2217 and Ph-HbO-0030) have identical key cavity residues, they display distinct ligand binding properties.

Synthesis, characterization and DNA interactions of [Pt3(TPymT)Cl3], the trinuclear platinum(II) complex of the TPymT ligand.

The triplatinum complex of the 2,4,6-Tris(2-pyrimidyl)-1,3,5-triazine ligand, Pt3TPymT hereafter, has been prepared and characterized for the first time. NMR studies point out that the three platinum(II) centers possess an identical coordination environment. The interactions of Pt3TPymT with DNA were explored in comparison to the free ligand. Specifically, fluorescence, mass spectrometry, viscometry and melting measurements were carried out. In contrast to expectations, the obtained data reveal that no intercalative binding takes place; we propose that binding of Pt3TPymT to DNA mainly occurs through external/groove binding.

Site-Specific SERS Assay for Survivin Protein Dimer: From Ensemble Experiments to Correlative Single-Particle Imaging.

An assay for Survivin, a small dimeric protein which functions as modulator of apoptosis and cell division and serves as a promising diagnostic biomarker for different types of cancer, is presented. The assay is based on switching on surface-enhanced Raman scattering (SERS) upon incubation of the Survivin protein dimer with Raman reporter-labeled gold nanoparticles (AuNP). Site-specificity is achieved by complexation of nickel-chelated N-nitrilo-triacetic acid (Ni-NTA) anchors on the particle surface by multiple histidines (His6 -tag) attached to each C-terminus of the centrosymmetric protein dimer. Correlative single-particle analysis using light sheet laser microscopy enables the simultaneous observation of both elastic and inelastic light scattering from the same sample volume. Thereby, the SERS-inactive AuNP-protein monomers can be directly discriminated from the SERS-active AuNP-protein dimers/oligomers. This information, i.e. the percentage of SERS-active AuNP in colloidal suspension, is not accessible from conventional SERS experiments due to ensemble averaging. The presented correlative single-particle approach paves the way for quantitative site-specific SERS assays in which site-specific protein recognition by small chemical and in particular supramolecular ligands can be tested.

Tuning the interactions of PEG-coated gold nanorods with BSA and model proteins through insertion of amino or carboxylate groups.

Gold nanorods (GNRs) are important platforms for biosensing and drug delivery. As for most nanomaterials, appropriate coatings such as polyethylene glycol (PEG) are needed to stabilize GNRs within biological fluids. We show here that the interactions of GNRs with proteins can be finely modulated through surface modification using PEG-containing chains bearing charged headgroups. Interestingly, introduction of amino or carboxylate groups produces relevant and differential changes in GNR interactions with three representative proteins: lysozyme, cytochrome c, and bovine serum albumin. These effects were explored through the direct monitoring of plasmonic bands of the GNRs and are supported by independent dynamic light scattering (DLS) and circular dichroism (CD) determinations. Notably, GNR-protein interactions observed for these charged GNRs can be almost completely reversed by salt addition. These observations demonstrate the importance of electrostatic effects in governing GNR-protein interactions, and provide a basis for new sensing and delivery platforms.

Reactivity of inorganic sulfide species toward a heme protein model.

The reactivity of inorganic sulfide species toward heme peptides was explored under biorelevant conditions in order to unravel the molecular details of the reactivity of the endogenous hydrogen sulfide toward heme proteins. Unlike ferric porphyrinates, which are reduced by inorganic sulfide, some heme proteins can form stable Fe(III)-sulfide adducts. To isolate the protein factors ruling the redox chemistry, we used as a system model, the undecapeptide microperoxidase (MP11), a heme peptide derived from cytochrome c proteolysis that retains the proximal histidine bound to the Fe(III) atom. Upon addition of gaseous hydrogen sulfide (H2S) at pH 6.8, the UV-vis spectra of MP11 closely resembled those of the low-spin ferric hydroxo complex (only attained at an alkaline pH) and cysteine or alkylthiol derivatives, suggesting that the Fe(III) reduction was prevented. The low-frequency region of the resonance Raman spectrum revealed the presence of an Fe(III)-S band at 366 cm(-1) and the general features of a low-spin hexacoordinated heme. Anhydrous sodium sulfide (Na2S) was the source of sulfide of choice for the kinetic evaluation of the process. Theoretical calculations showed no distal stabilization mechanisms for bound sulfide species in MP11, highlighting a key role of the proximal histidine for the stabilization of the Fe(III)-S adducts of heme compounds devoid of distal counterparts, which is significant with regard to the biochemical reactivity of endogenous hydrogen sulfide.

Interplay of the H-bond donor-acceptor role of the distal residues in hydroxyl ligand stabilization of Thermobifida fusca truncated hemoglobin.

The unique architecture of the active site of Thermobifida fusca truncated hemoglobin (Tf-trHb) and other globins belonging to the same family has stimulated extensive studies aimed at understanding the interplay between iron-bound ligands and distal amino acids. The behavior of the heme-bound hydroxyl, in particular, has generated much interest in view of the relationships between the spin-state equilibrium of the ferric iron atom and hydrogen-bonding capabilities (as either acceptor or donor) of the OH(-) group itself. The present investigation offers a detailed molecular dynamics and spectroscopic picture of the hydroxyl complexes of the WT protein and a combinatorial set of mutants, in which the distal polar residues, TrpG8, TyrCD1, and TyrB10, have been singly, doubly, or triply replaced by a Phe residue. Each mutant is characterized by a complex interplay of interactions in which the hydroxyl ligand may act both as a H-bond donor or acceptor. The resonance Raman stretching frequencies of the Fe-OH moiety, together with electron paramagnetic resonance spectra and MD simulations on each mutant, have enabled the identification of specific contributions to the unique ligand-inclusive H-bond network typical of this globin family.

Photoacoustic excitation profiles of gold nanoparticles.

The wavelength dependence of the laser-induced photoacoustic signal amplitude has been measured for water dispersions of 10, 61, and 93 nm diameter gold nanospheres. The whole region of the localized surface plasmon resonance has been covered. This "photoacoustic excitation profile" can be overlayed with the extinction spectrum between 450 nm and 600 nm in the case of the smallest nanoparticles. At variance, the larger-sized nanoparticles display a progressive deviation from the extinction spectrum at longer wavelength, where the photoacoustic signal becomes relatively smaller. Considering that photoacoustics is intrinsically insensitive to light scattering, at least for optically thin samples, the results are in agreement with previous theoretical work predicting (i) an increasing contribution of scattering to extinction when the nanoparticle size increases and (ii) a larger scattering component at longer wavelengths. Therefore, the method has a general validity and can be applied to selectively determine light absorption by plasmonic systems.

Concentration-controlled formation of myoglobin/gold nanosphere aggregates.

Gold nanoparticles are being increasingly proposed as biotechnological tools for medical diagnosis and therapy purposes. Their safety for human beings and the environment is therefore becoming an emerging issue, which calls for basic research on the interactions between nanostructured gold particles and biological materials, including physicochemical studies of model systems. In this Article, we focus on the "reaction products" of a widely known nanoparticle type, citrate-capped 30 nm gold nanospheres, with a model protein, horse myoglobin. Protein adsorption and partial denaturation were accompanied by the formation of nanoparticle aggregates with strongly distinct optical spectroscopy properties and shapes, as observed by transmission electron microscopy. We singled out the concentration of myoglobin as the determinant of these differences, and verified on this basis that surface-enhanced Raman scattering (SERS) spectra can only be obtained by aggregates with strong interparticle optical coupling, which are obtained at low protein concentration. The results can be useful both in improving the spectroscopy of biomolecules and in understanding the formation of the protein corona in biomedical applications.

Ligand uptake modulation by internal water molecules and hydrophobic cavities in hemoglobins.

Internal water molecules play an active role in ligand uptake regulation, since displacement of retained water molecules from protein surfaces or cavities by incoming ligands can promote favorable or disfavorable effects over the global binding process. Detection of these water molecules by X-ray crystallography is difficult given their positional disorder and low occupancy. In this work, we employ a combination of molecular dynamics simulations and ligand rebinding over a broad time range to shed light into the role of water molecules in ligand migration and binding. Computational studies on the unliganded structure of the thermostable truncated hemoglobin from Thermobifida fusca (Tf-trHbO) show that a water molecule is in the vicinity of the iron heme, stabilized by WG8 with the assistance of YCD1, exerting a steric hindrance for binding of an exogenous ligand. Mutation of WG8 to F results in a significantly lower stabilization of this water molecule and in subtle dynamical structural changes that favor ligand binding, as observed experimentally. Water is absent from the fully hydrophobic distal cavity of the triple mutant YB10F-YCD1F-WG8F (3F), due to the lack of residues capable of stabilizing it nearby the heme. In agreement with these effects on the barriers for ligand rebinding, over 97% of the photodissociated ligands are rebound within a few nanoseconds in the 3F mutant case. Our results demonstrate the specific involvement of water molecules in shaping the energetic barriers for ligand migration and binding.

H-bonding networks of the distal residues and water molecules in the active site of Thermobifida fusca hemoglobin.

The ferric form of truncated hemoglobin II from Thermobifida fusca (Tf-trHb) and its triple mutant WG8F-YB10F-YCD1F at neutral and alkaline pH, and in the presence of CN(-) have been characterized by resonance Raman spectroscopy, electron paramagnetic resonance spectroscopy, and molecular dynamics simulations. Tf-trHb contains three polar residues in the distal site, namely TrpG8, TyrCD1 and TyrB10. Whereas TrpG8 can act as a potential hydrogen-bond donor, the tyrosines can act as donors or acceptors. Ligand binding in heme-containing proteins is determined by a number of factors, including the nature and conformation of the distal residues and their capability to stabilize the heme-bound ligand via hydrogen-bonding and electrostatic interactions. Since both the RR Fe-OH(-) and Fe-CN(-) frequencies are very sensitive to the distal environment, detailed information on structural variations has been obtained. The hydroxyl ligand binds only the WT protein giving rise to two different conformers. In form 1 the anion is stabilized by H-bonds with TrpG8, TyrCD1 and a water molecule, in turn H-bonded to TyrB10. In form 2, H-bonding with TyrCD1 is mediated by a water molecule. Unlike the OH(-) ligand, CN(-) binds both WT and the triple mutant giving rise to two forms with similar spectroscopic characteristics. The overall results clearly indicate that H-bonding interactions both with distal residues and water molecules are important structural determinants in the active site of Tf-trHb. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.

Following ligand migration pathways from picoseconds to milliseconds in type II truncated hemoglobin from Thermobifida fusca.

CO recombination kinetics has been investigated in the type II truncated hemoglobin from Thermobifida fusca (Tf-trHb) over more than 10 time decades (from 1 ps to ∼100 ms) by combining femtosecond transient absorption, nanosecond laser flash photolysis and optoacoustic spectroscopy. Photolysis is followed by a rapid geminate recombination with a time constant of ∼2 ns representing almost 60% of the overall reaction. An additional, small amplitude geminate recombination was identified at ∼100 ns. Finally, CO pressure dependent measurements brought out the presence of two transient species in the second order rebinding phase, with time constants ranging from ∼3 to ∼100 ms. The available experimental evidence suggests that the two transients are due to the presence of two conformations which do not interconvert within the time frame of the experiment. Computational studies revealed that the plasticity of protein structure is able to define a branched pathway connecting the ligand binding site and the solvent. This allowed to build a kinetic model capable of describing the complete time course of the CO rebinding kinetics to Tf-trHb.

Fluoride as a probe for H-bonding interactions in the active site of heme proteins: the case of Thermobifida fusca hemoglobin.

The structural and functional properties of the active site of the bacterial hemoglobin from Thermobifida fusca are largely determined by three polar amino acids: TrpG8, TyrCD1, and TyrB10. We have exploited the availability of a combinatorial set of mutants, in each of which these three amino acids have been singly, doubly, or triply replaced by a Phe residue, to perform a detailed study on H-bonding interactions between the protein and heme-bound fluoride. By appropriate choice of the excitation conditions, ν(Fe-F) stretching bands have been detected in the resonance Raman spectra. In the wild-type protein and one of the mutants, two ν(Fe-F) bands have been observed and assigned to the presence of two protein conformers where fluoride is singly or doubly H-bonded. Furthermore, by plotting the CT1 charge-transfer transition energy vs the ν(Fe-F) wavenumbers, an empirical correlation has been found. The data are well fitted by a straight line with a positive slope. The position along the correlation line can be considered as a novel, general spectroscopic indicator of the extent of H-bonding in the active site of heme proteins. In agreement with the spectroscopic results, we have observed that the rate of ligand dissociation in stopped-flow kinetic measurements progressively increases upon substitution of the H-bonding amino acids. Molecular dynamics simulations have been performed on the fluoride complexes of native and mutated forms, indicating the prevalent interactions at the active site. All the techniques yield evidence that TrpG8 and TyrCD1 can form strong H bonds with fluoride, whereas TyrB10 plays only a minor role in the stabilization of the ligand.

The optical spectra of fluoride complexes can effectively probe H-bonding interactions in the distal cavity of heme proteins.

Fluoride complexes of heme proteins are characterized by unique spectroscopic properties, that provide a simple and direct means to monitor the interactions of the distal heme pocket environment with the iron-bound ligand. In particular, a strong correlation has been demonstrated between the wavelength of the iron-porphyrin charge transfer band at 600-620nm (CT1) and the strength of H-bonding donation from the distal amino acid side chains to the fluoride ion. In parallel, resonance Raman spectra with excitation within either the CT1 band or the charge transfer band at 450-460nm (CT2) have revealed that the iron-fluoride stretching frequency is directly affected by H-bonding to the fluoride ion. On this basis, globins and peroxidases display distinct spectroscopic features, which are strongly dependent on the capability of their distal residues (i.e. histidine, arginine and tryptophan) to be involved in H-bonding with the ligand. In particular, in peroxidases strong H-bonding corresponds to a low iron-fluoride stretching frequency and to a red-shifted CT1 band. The reverse is observed in myoglobin. Interestingly, a truncated hemoglobin of microbial origin (Thermobifida fusca) investigated in the present work, displays the specific spectroscopic signature of a peroxidase, in agreement with the presence of strong H-bonding residues, i.e., tyrosine and tryptophan, within the distal pocket.

Histidine E7 dynamics modulates ligand exchange between distal pocket and solvent in AHb1 from Arabidopsis thaliana.

The distal His residue in type 1 nonsymbiotic hemoglobin AHb1 from Arabidopsis thaliana plays a fundamental role in stabilizing the bound ligand. This residue might also be important in regulating the accessibility to the distal cavity. The feasibility of this functional role has been examined using a combination of experimental and computational methods. We show that the exchange of CO between the solvent and the reaction site is modulated by a swinging motion of the distal His, which opens a channel that connects directly the distal heme pocket with the solvent. The nearby PheB10 aids the distal His in the stabilization of the bound ligand by providing additional protection against solvation. Overall, these findings provide evidence supporting the functional implications of the conformational rearrangement found for the distal His in AHb1, which mimics the gating role proposed for the same residue in myoglobin.

Heme pocket structural properties of a bacterial truncated hemoglobin from Thermobifida fusca.

An acidic surface variant (ASV) of the "truncated" hemoglobin from Thermobifida fusca was designed with the aim of creating a versatile globin scaffold endowed with thermostability and a high level of recombinant expression in its soluble form while keeping the active site unmodified. This engineered protein was obtained by mutating the surface-exposed residues Phe107 and Arg91 to Glu. Molecular dynamics simulations showed that the mutated residues remain solvent-exposed, not affecting the overall protein structure. Thus, the ASV was used in a combinatorial mutagenesis of the distal heme pocket residues in which one, two, or three of the conserved polar residues [TyrB10(54), TyrCD1(67), and TrpG8(119)] were substituted with Phe. Mutants were characterized by infrared and resonance Raman spectroscopy and compared with the wild-type protein. Similar Fe-proximal His stretching frequencies suggest that none of the mutations alters the proximal side of the heme cavity. Two conformers were observed in the spectra of the CO complexes of both wild-type and ASV protein: form 1 with ν(FeC) and ν(CO) at 509 and 1938 cm(-1) and form 2 with ν(FeC) and ν(CO) at 518 and 1920 cm(-1), respectively. Molecular dynamics simulations were performed for the wild-type and ASV forms, as well as for the TyrB10 mutant. The spectroscopic and computational results demonstrate that CO interacts with TrpG8 in form 1 and interacts with both TrpG8 and TyrCD1 in form 2. TyrB10 does not directly interact with the bound CO.

Sulfide binding properties of truncated hemoglobins.

The truncated hemoglobins from Bacillus subtilis (Bs-trHb) and Thermobifida fusca (Tf-trHb) have been shown to form high-affinity complexes with hydrogen sulfide in their ferric state. The recombinant proteins, as extracted from Escherichia coli cells after overexpression, are indeed partially saturated with sulfide, and even highly purified samples still contain a small but significant amount of iron-bound sulfide. Thus, a complete thermodynamic and kinetic study has been undertaken by means of equilibrium and kinetic displacement experiments to assess the relevant sulfide binding parameters. The body of experimental data indicates that both proteins possess a high affinity for hydrogen sulfide (K = 5.0 x 10(6) and 2.8 x 10(6) M(-1) for Bs-trHb and Tf-trHb, respectively, at pH 7.0), though lower with respect to that reported previously for the sulfide avid Lucina pectinata I hemoglobins (2.9 x 10(8) M(-1)). From the kinetic point of view, the overall high affinity resides in the slow rate of sulfide release, attributed to hydrogen bonding stabilization of the bound ligand by distal residue WG8. A set of point mutants in which these residues have been replaced with Phe indicates that the WG8 residue represents the major kinetic barrier to the escape of the bound sulfide species. Accordingly, classical molecular dynamics simulations of SH(-)-bound ferric Tf-trHb show that WG8 plays a key role in the stabilization of coordinated SH(-) whereas the YCD1 and YB10 contributions are negligible. Interestingly, the triple Tf-trHb mutant bearing only Phe residues in the relevant B10, G8, and CD1 positions is endowed with a higher overall affinity for sulfide characterized by a very fast second-order rate constant and 2 order of magnitude faster kinetics of sulfide release with respect to the wild-type protein. Resonance Raman spectroscopy data indicate that the sulfide adducts are typical of a ferric iron low-spin derivative. In analogy with other low-spin ferric sulfide adducts, the strong band at 375 cm(-1) is tentatively assigned to a Fe-S stretching band. The high affinity for hydrogen sulfide is thought to have a possible physiological significance as H(2)S is produced in bacteria at metabolic steps involved in cysteine biosynthesis and hence in thiol redox homeostasis.