Infrared Spectroscopy of Synovial Fluid Shows Accuracy as an Early Biomarker in an Equine Model of Traumatic Osteoarthritis.

Osteoarthritis is a leading cause of lameness and joint disease in horses. A simple, economical, and accurate diagnostic test is required for routine screening for OA. This study aimed to evaluate infrared (IR)-based synovial fluid biomarker profiling to detect early changes associated with a traumatically induced model of equine carpal osteoarthritis (OA). Unilateral carpal OA was induced arthroscopically in 9 of 17 healthy thoroughbred fillies; the remainder served as Sham-operated controls. The median age of both groups was 2 years. Synovial fluid (SF) was obtained before surgical induction of OA (Day 0) and weekly until Day 63. IR absorbance spectra were acquired from dried SF films. Following spectral pre-processing, predictive models using random forests were used to differentiate OA, Sham, and Control samples. The accuracy for distinguishing between OA and any other joint group was 80%. The classification accuracy by sampling day was 87%. For paired classification tasks, the accuracies by joint were 75% for OA vs. OA Control and 70% for OA vs. Sham. The accuracy for separating horses by group (OA vs. Sham) was 68%. In conclusion, SF IR spectroscopy accurately discriminates traumatically induced OA joints from controls.

Plasma and Synovial Fluid Cell-Free DNA Concentrations Following Induction of Osteoarthritis in Horses.

Biomarkers for osteoarthritis (OA) in horses have been extensively investigated, but translation into clinical use has been limited due to cost, limited sensitivity, and practicality. Identifying novel biomarkers that overcome these limitations could facilitate early diagnosis and therapy. This study aimed to compare the concentrations of synovial fluid (SF) and plasma cell-free DNA (cfDNA) over time in control horses with those with induced carpal OA. Following an established model, unilateral carpal OA was induced in 9 of 17 healthy Thoroughbred fillies, while the remainder were sham-operated controls. Synovial fluid and plasma samples were obtained before induction of OA (Day 0) and weekly thereafter until Day 63, and cfDNA concentrations were determined using fluorometry. The SF cfDNA concentrations were significantly higher for OA joints than for sham-operated joints on Days 28 (median 1430 µg/L and 631 µg/L, respectively, p = 0.017) and 63 (median 1537 µg/L and 606 µg/L, respectively, p = 0.021). There were no significant differences in plasma cfDNA between the OA and the sham groups after induction of carpal OA. Plasma cfDNA measurement is not sufficiently sensitive for diagnostic purposes in this induced model of OA. Synovial fluid cfDNA measurement may be used as a biomarker to monitor early disease progression in horses with OA.

Bone quality changes as measured by Raman and FTIR spectroscopy in primiparous cows with humeral fracture from New Zealand.

The occurrence of spontaneous humeral fractures in primiparous dairy cows from New Zealand prompted the study of bone material from affected cows to further characterize this condition and to outline a likely pathogenesis. Previous studies indicate that these cows developed osteoporosis due to periods of suboptimal bone formation followed by increased bone resorption during the period of lactation complicated by copper deficiency. We hypothesized that there are significant differences in the chemical composition/bone quality in bones from cows with spontaneous humeral fracture compared to cows without humeral fractures. In this study, Raman and Fourier transform infrared spectroscopy band ratios were, for the first time, measured, calculated, and compared in bone samples from 67 primiparous dairy cows that suffered a spontaneous fracture of the humerus and 14 age-matched post-calving cows without humeral fractures. Affected bone showed a significantly reduced mineral/matrix ratio, increased bone remodeling, newer bone tissue with lower mineralization and, lower carbonate substitution, and reduced crystallinity. As such, is likely that these have detrimentally impacted bone quality and strength in affected cows.

Complexation of Anthocyanin-Bound Blackcurrant Pectin and Whey Protein: Effect of pH and Heat Treatment.

A complexation study between blackcurrant pectin (BCP) and whey protein (WP) was carried out to investigate the impact of bound anthocyanins on pectin−protein interactions. The effects of pH (3.5 and 4.5), heating (85 °C, 15 min), and heating sequence (mixed-heated or heated-mixed) were studied. The pH influenced the color, turbidity, particle size, and zeta-potential of the mixtures, but its impact was mainly significant when heating was introduced. Heating increased the amount of BCP in the complexes­especially at pH 3.5, where 88% w/w of the initial pectin was found in the sedimented (insoluble) fraction. Based on phase-separation measurements, the mixed-heated system at pH 4.5 displayed greater stability than at pH 3.5. Heating sequence was essential in preventing destabilization of the systems; mixing of components before heating produced a more stable system with small complexes (<300 nm) and relatively low polydispersity. However, heating WP before mixing with BCP prompted protein aggregation­producing large complexes (>400 nm) and worsening the destabilization. Peak shifts and emergence (800−1200 cm−1) in infrared spectra confirmed that BCP and WP functional groups were altered after mixing and heating via electrostatic, hydrophobic, and hydrogen bonding interactions. This study demonstrated that appropriate processing conditions can positively impact anthocyanin-bound pectin−protein interactions.

Optical Detection of CoV-SARS-2 Viral Proteins to Sub-Picomolar Concentrations.

The emergence of a new strain of coronavirus in late 2019, SARS-CoV-2, led to a global pandemic in 2020. This may have been preventable if large scale, rapid diagnosis of active cases had been possible, and this has highlighted the need for more effective and efficient ways of detecting and managing viral infections. In this work, we investigate three different optical techniques for quantifying the binding of recombinant SARS-CoV-2 spike protein to surface-immobilized oligonucleotide aptamers. Biolayer interferometry is a relatively cheap, robust, and rapid method that only requires very small sample volumes. However, its detection limit of 250 nM means that it is not sensitive enough to detect antigen proteins at physiologically relevant levels (sub-pM). Surface plasmon resonance is a more sensitive technique but requires larger sample volumes, takes longer, requires more expensive instrumentation, and only reduces the detection limit to 5 nM. Surface-enhanced Raman spectroscopy is far more sensitive, enabling detection of spike protein to sub-picomolar concentrations. Control experiments performed using scrambled aptamers and using bovine serum albumin as an analyte show that this apta-sensing approach is both sensitive and selective, with no appreciable response observed for any controls. Overall, these proof-of-principle results demonstrate that SERS-based aptasensors hold great promise for development into rapid, point-of-use antigen detection systems, enabling mass testing without any need for reagents or laboratory expertise and equipment.

Rational Synthesis, Structures and Properties of the Ionic Liquid Binary Iodine-Bromine Octahalide Series [In Br8-n ]2- (n=0, 2, 3, 4).

A series of octanuclear iodine-bromine interhalides [In Br8-n ]2- (n=0, 2, 3, 4) were prepared systematically in two steps. Firstly, addition of a dihalogen (Br2 or IBr) to the triaminocyclopropenium bromide salt [C3 (NEt2 )3 ]Br forms the corresponding trihalide salt with Br3 - or IBr2 - anions, respectively. Secondly, addition to Br3 - of half an equivalent of Br2 gives the octabromine polyhalide [Br8 ]2- , whereas addition to IBr2 - of half an equivalent of Br2 , IBr or I2 gives the corresponding interhalides: [I2 Br6 ]2- , [I3 Br5 ]2- , and [I4 Br4 ]2- , respectively. The four octahalides were characterized by X-ray crystallography, computational studies, Raman and Far-IR spectroscopies, as well as by TGA and melting point. All of the salts were found to be ionic liquids.

The Binary Iodine-Chlorine Octahalide Series [In Cl8-n ]2- (n=3, 3.6, 4).

The octanuclear iodine-chlorine interhalides [I4 Cl4 ]2- and [I3 Cl5 ]2- were prepared in two steps. Firstly, addition of ICl to the triaminocyclopropenium chloride salt [C3 (NEt2 )3 ]Cl forms the trihalide ICl2 - salt, secondly, addition of half an equivalent of I2 or ICl, respectively, gave the desired products upon crystallization at low temperature. The non-stoichiometric octahalide [I3.6 Cl4.4 ]2- was obtained after heating a CH2 Cl2 solution of the ICl2 - salt to reflux for 2 hours followed crystallization. [I4 Cl4 ]2- is best described as two ICl2 - anions bridged by I2 , whereas [I3 Cl5 ]2- is best described as an [I2 Cl3 ]- pentahalide with a weak halogen bond to an ICl2 - trihalide. The octahalides were characterized by X-ray crystallography, computational studies, Raman and Far-IR spectroscopies, as well as by TGA and melting point.

Diels-Alder Reaction of Anthranilic Acids: A Versatile Route to Dense Monolayers on Flat Edge and Basal Plane Graphitic Carbon Substrates.

Methods that reliably yield monolayers of covalently anchored modifiers on graphene and other planar graphitic materials are in demand. Covalently bonded groups can add functionality to graphitic carbon for applications ranging from sensing to supercapacitors and can tune the electronic and optical properties of graphene. Limiting modification to a monolayer gives a layer with well-defined concentration and thickness providing a minimum barrier to charge transfer. Here we investigate the use of anthranilic acid derivatives for grafting aryl groups to few layer graphene and pyrolyzed photoresist film (PPF). Under mild conditions, anthranilic acids generate arynes, which undergo Diels-Alder cycloadditions. Using spectroscopy, electrochemistry, and atomic force microscopy, we demonstrate that the reaction yields monolayers of aryl groups on graphene and PPF with maximum surface coverages consistent with densely packed layers. Our study confirms that anthranilic acids offer a convenient route to covalent modification of planar graphitic carbons (both basal and edge plane materials).

Toward an iron(II) spin-crossover grafted phosphazene polymer.

Two new cyclotriphosphazene ligands with pendant 2,2':6',2″-terpyridine (Terpy) moieties, namely, (pentaphenoxy){4-[2,6-bis(2-pyridyl)]pyridoxy}cyclotriphosphazene (L(1)), (pentaphenoxy){4-[2,6-terpyridin-4-yl]phenoxy}cyclotriphosphazene (L(2)), and their respective polymeric analogues, L(1P) and L(2P), were synthesized. These ligands were used to form iron(II) complexes with an Fe(II)Terpy(2) core. Variable-temperature resonance Raman, UV-visible, and Mössbauer spectroscopies with magnetic measurements aided by density functional theory calculations were used to understand the physical characteristics of the complexes. By a comparison of measurements, the polymers were shown to behave in the same way as the cyclotriphosphazene analogues. The results showed that spin crossover (SCO) can be induced to start at high temperatures by extending the spacer length of the ligand to that in L(2) and L(2P); this combination provides a route to forming a malleable SCO material.

Innovative approach to investigating the microstructure of calcified tissues using specular reflectance Fourier transform-infrared microspectroscopy and discriminant analysis.

Although bone fracture has become a serious global health issue, current clinical assessments of fracture risk based on bone mineral density are unable to accurately predict whether an individual is likely to suffer a fracture. There is increasing recognition that the chemical structure and composition, or microstructure, of mineralized tissues has an important role to play in determining the fracture resistance of bone. The objective of this preliminary study was to evaluate the use of specular reflectance Fourier transform infrared (SR FT-IR) microspectroscopy in conjunction with discriminant analysis as an innovative technique for providing future insights into the origins of orthopedic abnormalities. The impetus for this approach was that SR FT-IR microspectroscopy would offer several advantages over conventional transmission methods. Bone samples were obtained from young racehorses at known fracture predilection sites and spectra were successfully obtained from calcified cartilage and subchondral bone for the first time. By applying discriminant analysis to the spectral data set in biologically relevant regions, microstructural differences between groups of individuals were found to be related to features associated with both the mineral and organic components of the bone. The preliminary findings also suggest that differences in bone microstructure may exist between healthy individuals of the same age, raising important questions around the normal limits of individual variation and whether individuals may be predisposed to later fracture as a result of detrimental microstructural changes during early growth and development.

Luminescent rhenium(I)-dipyrrinato complexes.

The first Re(I)-dipyrrinato complexes are reported. Complexes with the general formulas fac-[ReL(CO)(3)Cl](-), fac-[ReL(CO)(3)PR(3)], and [ReL(CO)(2)(PR(3))(PR'(3))] have been prepared, where L is one of a series of meso-aryl dipyrrinato ligands. Access to these complexes proceeds via the reaction of [Re(CO)(5)Cl] with the dipyrrin (LH) to produce fac-[ReL(CO)(3)Cl](-). A subsequent reaction with PR(3) (R = phenyl, butyl) leads to displacement of the chloride ligand to generate fac-[ReL(CO)(3)PR(3)], and further reaction with PR'(3) leads to the displacement of the CO ligand trans to the first PR(3) ligand to give trans(P), cis(C)-[ReL(CO)(2)(PR(3))(PR'(3))]. The structures of the complexes were determined in the solid state by X-ray crystallography and in solution by (1)H NMR spectroscopy. Electronic absorption spectroscopy reveals a prominent band in the visible region at relatively low energy (472-491 nm) for all complexes, which is assigned as a π-π* transition of the dipyrrin chromophore. Weak emission (λ(ex) = 485 nm, quantum yields <0.01) was observed for [ReL(CO)(3)Cl](-) and [ReL(CO)(3)PR(3)] complexes, but no emission was generally evident from the [ReL(CO)(2)(PR(3))(PR'(3))] complexes. On the basis of the large Stokes shift (~6000 cm(-1)), the emission is ascribed to phosphorescence from a triplet excited state. The emission intensity is sensitive to dissolved oxygen and methyl viologen; a Stern-Volmer plot in the latter case gave a straight line. Photochemical ligand substitution reactions of [ReL(CO)(3)PR(3)] were induced by excitation with a 355 nm laser in acetonitrile. [ReL(CO)(2)(PR(3))(CH(3)CN)] is formed as a putative intermediate, which reacts thermally with added PR'(3) to produce [ReL(CO)(2)(PR(3))(PR'(3))] complexes.

Mechanically interlocked gold and silver nanoparticles using metallosupramolecular catenane chemistry.

We have employed the toolbox of metallosupramolecular chemistry to mechanically interlock gold and silver nanoparticles. A specifically designed PEGthiol-functionalized bis(phenanthroline)copper(I) complex acts to 'catenate' the nanoparticles. The interlocked assemblies were characterised by three complementary techniques: DLS, SERS and TEM.

Exciton coupling in coordination compounds.

This Perspective reviews the impact of exciton coupling on the spectroscopic properties of coordination compounds. Exciton coupling features arise in electronic absorption and circular dichroism spectra when chromophores are brought into close spatial proximity, for example by coordination to a metal centre. The analysis of these features can reveal much information such as the geometry of a complex and its absolute configuration. The extension of the exciton coupling model to polynuclear metallosupramolecular arrays is discussed.

Strongly absorbing pi-pi* states in heteroleptic dipyrrin/2,2'-bipyridine ruthenium complexes: excited-state dynamics from resonance raman spectroscopy.

A recently reported new class of ruthenium complexes containing 2,2'-bipyridine and a dipyrrin ligand in the coordination sphere exhibit both strong metal-to-ligand charge-transfer (MLCT) and pi-pi* transitions. Quantitative analysis of the resonance Raman scattering intensities and absorption spectra reveals only weak electronic interactions between these states despite direct coordination of the bipyridyl and dipyrrin ligands to the central ruthenium atom. On the basis of DFT calculations and time-dependent DFT (TD-DFT), we propose that the electronic excited states closely resemble "pure" MLCT and pi-pi* states. Resonance Raman intensity analysis demonstrates that a large amplitude transannular torsional motion provides a mechanism for relaxation on the pi-pi* excited-state surface. We assert that this result is generally applicable to a range of dipyrrin complexes such as boron-dipyrrin and metallodipyrrin systems. Despite the large torsional distortion between the phenyl ring and the dipyrromethene plane, pi-pi* excitation extends out onto the phenyl ring which may have important consequences in solar-energy-conversion applications of ruthenium-dipyrrin complexes.

Chromophoric dipyrrin complexes capable of binding to TiO2: synthesis, structure and spectroscopy.

The synthesis, characterisation, and TiO2 binding studies of a series of chromophoric complexes of 5-(4-carboxyphenyl)-4,6-dipyrrin (L(b)) are presented. The synthesis of [Ru(bipy)(L(b))2] (bipy = 2,2-bipyridine), [Rh(L(b))3], and [Pd(L(b))2] was achieved by initial coordination of 5-(4-methoxycarbonylphenyl)-4,6-dipyrrin (L(a)) followed by hydrolysis of the ester group. The carboxyl groups that are located on the peripheries of these complexes are able to engage in intermolecular hydrogen bonding interactions in the solid state, as revealed by X-ray crystallography. These groups also allow the complexes to anchor to the surface of TiO2 nanoparticles, as evidenced by colouration of the TiO2 and FT-IR spectroscopy. The ability of these complexes to capture a significant fraction of sunlight and to adhere to TiO2 surfaces renders them viable dyes for photochemical devices such as dye sensitised solar cells.

Heteroleptic dipyrrin/bipyridine complexes of ruthenium(II).

The synthesis and characterization of a series of heteroleptic dipyrrinato/2,2'-bipyridine complexes of ruthenium(II) are reported. Spectroscopic analysis, including resonance Raman, indicates that the complexes are only weakly emissive and that the dipyrrin and Ru --> bipyridine (metal-to-ligand charge transfer) chromophores are uncoupled.

Metal-metal communication in copper(II) complexes of cyclotetraphosphazene ligands.

Copper(II) chloride and bromide react with the pyridyloxy-substituted cyclotetraphosphazene ligands, octakis(2-pyridyloxy)cyclotetraphosphazene (L(1)), and octakis(4-methyl-2-pyridyloxy)cyclotetraphosphazene (L(2)), to form the dimetallic complexes, [L(CuX2)2] (L = L(1), X = Br; L = L(2), X = Cl or Br), and [{L(1)(CuCl2)2}n]. Single crystal X-ray crystallography shows the complex [{L(1)(CuCl2)2}n] to be a coordination polymer propagated by interligand "Cu(mu-Cl)2Cu" bridges whereas [L(2)(CuCl2)2] forms discrete dimetallic cyclotetraphosphazene-based moieties. The variable temperature magnetic susceptibility data for [{L(1)(CuCl2)2}n] are consistent with a weak antiferromagnetic exchange interaction between the copper(II) centers occurring via the bridging chloride ions. [L(2)(CuCl2)2] and [L(CuBr2)2] (L = L(1) and L(2)) exhibit normal Curie-like susceptibilities. The abstraction of a chloride ion, using [Ag(MeCN)4](PF6), from each copper site in [L(2)(CuCl2)2], affords the new complex, [L(2)(CuCl)2](PF6)2, in which the two copper(II) ions are separated by "N-P=N-P=N" phosphazene bridges. Electron spin resonance and variable temperature magnetic measurements indicate the occurrence of weak antiferromagnetic coupling between the unpaired electrons on the copper(II) centers. Density Functional Theory (DFT) calculations on the [L(2)(CuCl)2](2+) dication and the related cyclotriphosphazene complex, [L(4)(CuCl2)2] (L(4) = hexakis(4-methyl-2-pyridyloxy)cyclotriphosphazene), have identified "electron-density-bridge" molecular orbitals which involve Cu 3d orbitals overlapping with the non-bonding N-based molecular orbitals on the phosphazene rings as the pathway for this interaction.

Using internal coordinates to describe photoinduced geometry changes in MLCT excited states.

A resonance Raman intensity analysis of the metal-to-ligand charge-transfer (MLCT) transition for the rhenium compound Re(2-(2'-pyridyl)quinoxaline)(CO)(3)Cl (RePQX) is presented. Photoinduced geometry changes are calculated, and the results are presented using the vibrational normal modes and the redundant internal coordinates. A density functional theory calculation is used to determine the ground-state nonresonant Raman spectrum and a transformation matrix that transforms the redundant internal coordinates into the normal modes. The normal modes nu(37) (rhenium coordination sphere distortion) and nu(75) (ligand skeletal stretch) show the largest photoinduced geometry change (Delta = 1.0 and 0.7, respectively). A single carbonyl mode is enhanced in the resonance Raman spectra. Time-dependent density functional theory is used to calculate excited-state geometry changes, which are subsequently used to determine the signs of the photoinduced normal mode displacements. Transforming to internal coordinates reveals that all the CO bond lengths are displaced in the excited state. The Re-C and C-C ligand bond lengths are also displaced in the excited state. The results are discussed in terms of a simple one-electron picture for the electronic transition. Many bond angles and torsional coordinates are also displaced by the metal-to-ligand charge transfer, and most of these are associated with the rhenium coordination sphere. It is demonstrated that using internal coordinates presents a clear picture of the geometry changes associated with photoinduced electron transfer in metal polypyridyl systems.

Resonance raman excitation profile of a ruthenium(II) complex of dipyrido[2,3-a:3',2'-c]phenazine.

The lowest energy transition of [Ru(CN)(4)(ppb)](2-) (ppb = dipyrido[2,3-a:3',2'-c]phenazine), a metal-to-ligand charge transfer, has been probed using resonance Raman spectroscopy with excitation wavelengths (488, 514, 530, and 568 nm) spanning the lowest energy absorption band centered at 522 nm. Wave packet modeling was used to simultaneously model this lowest energy absorption band and the cross sections of the resonance Raman bands at the series of excitation wavelengths across this absorption band. A fit to within +/-20% was obtained for the Raman cross sections, close to the experimental uncertainty which is typically 10-20%. Delta values of 0.1-0.4 were obtained for modes which were either localized on the ppb ligand (345-1599 cm(-1)) or the CN modes (2063 and 2097 cm(-1)). DFT calculations reveal that the resonance Raman bands observed are due to modes delocalized over the entire ppb ligand.

Structural changes upon photoexcitation into the metal-to-ligand charge-transfer state of [Cu(pqx)(PPh3)2]+ probed by resonance Raman spectroscopy and density functional theory.

The structural changes that occur when [Cu(pqx)(PPh(3))(2)](+) (pqx is 2-(2'-pyridyl)quinoxaline) undergoes excitation through a metal-to-ligand charge-transfer (MLCT) transition are investigated using resonance Raman excitation profiles coupled with density functional theory (DFT). The DFT calculations predict bond lengths to within 3 pm and absolute deviations of 7 cm(-1) for the vibrational frequencies of [Cu(pqx)(PPh(3))(2)](+). TD-DFT calculations of oscillator strengths (f = 0.089) and band positions (419 nm) showed close agreement with experiment (f = 0.07, 431 nm). Resonance Raman spectra show the 527 cm(-1) (nu(29)) and 1476 cm(-1) (nu(75)) modes undergo the largest dimensionless displacement (Delta = 1.5 and 1.1, respectively) following photoexcitation into the MLCT Franck-Condon region. The solvent couples strongly to the MLCT transition and resonance Raman intensity analysis (RRIA) gives a solvent reorganization energy of 3400 cm(-1) for dichloromethane and 2800 cm(-1) for chloroform solutions. A large inner-sphere reorganization of 3430 cm(-1) in dichloromethane solution (3520 cm(-1) in chloroform solution) was found for [Cu(pqx)(PPh(3))(2)](+), indicating that the molecule as a whole undergoes significant distortion following MLCT excitation.