In the solid state, the resorcinarene podand 1 forms an intramolecular self‐inclusion complex with the pendant p‐toluene group of a podand arm, whereas the resorcinarene podand 2 does not show self‐inclusion. The conformations of the flexible resorcinarene podands in solution were investigated by variable‐temperature experiments using 1D and 2D NMR spectroscopic techniques as well as by computational methods, including a conformational search and subsequent DFT optimisation of representative structures. The 1H NMR spectra of 1 and 2 at room temperature show a single set of proton signals that are in agreement with Cv4 symmetry. At low temperatures, the molecules exist as a mixture of boat conformations featuring slow exchange on the NMR timescale. Energy barriers (ΔG≠298) of 55.5 and 52.0 kJ mol−1 were calculated for the boat‐to‐boat exchange of 1 and 2, respectively. The results of the ROESY experiments performed at 193 K and computational modelling suggest that in solution the resorcinarene podand 1 adopts a similar conformation to that present in its crystal structure, whereas podand 2 populates a more versatile range of conformations in solution.The synthesis of tetramethoxyresorcinarene podands bearing  Self‐inclusion: An intramolecular self‐inclusion complex of a flexible resorcinarene podand stabilised by weak C−H⋅⋅⋅O interactions has been observed in both the crystal structure and in solution (see figure). The solid‐state conformation was determined by XRD and the conformation in solution was investigated by NMR and computational methods, with good agreement observed between the different techniques. Inclusion complexes, along with the control of self‐inclusion behaviour, play an essential role in the preparation and function of self‐assembled nanomaterials.N‐alkyl‐resorcinarene halides.Intermolecular self‐inclusion is most often detected in crystal structures. Self‐included dimers have been observed in several crystal structures of calixarene macrocycles, such as deep cavitands,The control of intermolecular self‐inclusion is important for the preparation of supramolecular materials and self‐assembled structures. A few examples of pillararenes show a concentration‐dependent transition from intramolecular to intermolecular self‐inclusion in solution, which is utilised for the reversible self‐assembly of dimers or supramolecular polymers.Self‐inclusion can also be undesirable for the designed function of a molecular receptor or the self‐assembly of supramolecular aggregates. In the case of a bambusuril anion receptor, solid‐state intramolecular self‐inclusion was observed, and it was expected that this would lower its affinity for guest binding even though self‐inclusion could not be detected in solution.Cv4‐symmetrical crown conformation, resorcinarene podands typically exhibit a Cv2‐symmetrical boat conformation in the solid state  and all but one were in the boat conformation). The boat conformation is defined by two resorcinol rings being almost vertically oriented and the two remaining ones horizontally aligned. Previously, self‐inclusion dimers of resorcinarene tetrapodands have been observed by usResorcinarene podands are macrocycles in which the upper‐rim hydroxy groups of the parent resorcinarene have been functionalised, resulting in conformationally flexible octopus‐like structures. In contrast to resorcinarene cavitands with a covalently bridged p‐toluene podand arms connected by sulfonyloxy or benzyloxy linkers to provide similar length but different hydrogen‐bond donor/acceptor properties. The single‐crystal X‐ray structure of the sulfonyl‐linked podand 1  included in the podand arms of resorcinarenes 1 and 2, respectively 6) according to a literature procedure  connects the tolyl ring with the 1,2‐dioxyethane chain. To prepare an analogous compound to the resorcinarene podand 1 without the sulfonyl group, resorcinarene podand 2 was synthesised by alkylation of 3 with 2‐[(4‐methylbenzyl)oxy]ethyl tosylate (5) in a yield of 44 %. In the podand arm of 2, the ‐CH2O‐ group provides a similar length but lacks the hydrogen‐bond acceptor properties of 1. The tosylate 5 was prepared in two steps from 4‐methylbenzyl chloride and ethylene glycol. The resorcinarene podands were fully characterised by high‐resolution NMR spectroscopy, ESI‐MS and single‐crystal X‐ray diffraction.Resorcinarene podand 1 was isolated after column chromatographic purification and single crystals suitable for X‐ray diffraction analysis were obtained from ethyl acetate/hexane solution. The crystal structure shows that the resorcinarene macrocycle 1 adopts a boat conformation with two opposite resorcinol rings being almost vertically oriented and the other two horizontally aligned . In addition, the oxygen atoms of the sulfonyloxy group in the included arm reside within a distance of 2.73–2.79 Å from the methoxy and ethoxy protons of one of the two horizontally oriented resorcinol rings.The self‐inclusion complex is supported by two intramolecular C−H⋅⋅⋅O bonds formed between the The two podand arms attached to the horizontal resorcinol rings are wrapped around the resorcinarene cavity. The arms have, however, different O‐C‐C‐O dihedral angles. In one arm, an O‐C‐C‐O torsion angle of +70° induces an intramolecular 2.62 Å contact between a methoxy proton and the sulfonyloxy oxygen on one side Figure  a,b and 1, single crystals of resorcinarene podand 2 were grown from an ethanol solution. The crystal structure of 2 contains two resorcinarene molecules per asymmetric unit, both adopting a boat conformation. In contrast to resorcinarene 1, the vertically aligned resorcinol rings lean towards each other, thereby reducing the size of the aromatic cavity of resorcinarene 2  and methoxy oxygen atoms of the vertical resorcinol rings (dH⋅⋅⋅O=2.65–2.82 Å). The podand arms connected to the horizontal resorcinol rings extend away from the cavity and form edge‐to‐face π⋅⋅⋅π interactions with tolyl arms of adjacent molecules. It should be noted that the second molecule in the asymmetric unit forms a weak intermolecular hydrogen bond between the p‐tolyl group attached to the vertical resorcinol ring and a podand arm oxygen of a neighbouring molecule (see the Supporting Information).To compare the role of the sulfonyl group in the crystal structure of the podand 1. In all of these, one vertical arm was folded inside the cavity, similarly to the conformation observed in the crystal structure. The conformations were divided into five categories depending on the folding of the horizontal podand arms and, in particular, the O‐C‐C‐O dihedral angles  at a higher level of theory. The order of increase of the relative energies remained the same as determined in the force‐field energy minimisation  without self‐inclusion and seven examples of a horizontal podand arm folding into the cavity (class 2‐V), which indicates a more versatile range of potential conformations than those found for 1. Interestingly, the benzylic protons of the vertical aryl rings are directed towards the horizontal resorcinol rings in both the open (2‐IV) and self‐inclusion conformations (2‐I–2‐III). This results in a notable difference in the position and angle of the self‐included tolyl group in 2 compared with in 1. In 2, the tolyl group is positioned higher relative to the aromatic cavity and the p‐methyl group points upwards, whereas in 1 it points down.The conformational search for podand 2‐I–2‐V changed after DFT optimisation with the PBE0‐D3 functional, however, structure 2‐I with self‐inclusion of a vertical podand arm remained as the lowest‐energy conformation. To confirm this result, optimisation was repeated with another popular DFT functional (B3LYP‐D3), which found the same trend in energies but a significant discrepancy in the absolute values along with some structural changes in the optimised geometries (see the Supporting Information). This suggests that the potential energy landscape of podand 2 is more versatile than that of podand 1 containing many local minima.The order of increase of the relative energies of structures 1 and 2 in solution was carried out by using NMR spectroscopy. Our aim was to assess how the crystal structures of these conformationally flexible molecules reflect the conformations adopted in solution. The 1H NMR spectrum of the tetratolyl‐resorcinarene 2 at 303 K in CD2Cl2 , a unique singlet for the methoxy groups  and two separate singlets for the aromatic protons of the resorcinol rings . The number and multiplicity of the proton signals are in agreement with Cv4 symmetry, which probably results from the chemical exchange process between two boat conformations being fast on the 1H NMR timescale. The two multiplets observed at δ=3.7 and 4.0 ppm, respectively, were assigned to the protons of the methylene group α to the phenol oxygen atom, which resonate as diastereotopic signals , and the AB quartet at 4.5 ppm was assigned to the ‐CH2O‐ methylene protons α to the toluene ring . In addition to the two singlets of the aromatic resorcinol protons, two sharp doublets corresponding to the protons of the tolyl rings  appear in the aromatic region of the spectrum.The conformational characterisation of the resorcinarene podands 1) exhibited a similar 1H NMR spectrum. The most significant difference is the observation of the methylene protons α to the sulfonyloxy group in 1 appearing as a well‐defined triplet at δ=4.18 ppm. The analogous signal for the methylene protons α to the benzyloxy substituent in 2 resonated slightly upfield (δ=3.65 ppm) owing to a reduction in the inductive effect of the substituent.At room temperature, the tetratosylate resorcinarene (1H NMR spectra of 1 and 2 at different temperatures (303–193 K) to investigate the structural and dynamic properties of the resorcinarene podands at low temperatures, and to determine the energy barriers for the conformational exchange . As the temperature was further decreased to 263 K, the singlet from the lower‐rim protons  broadened significantly due to the slowing of the conformational exchange of the two boat conformations, to that close to the NMR timescale. At 243 K, coalescence of the upper rim protons  and the methoxy groups was observed. Within the temperature range of 223–193 K, new peaks in the aromatic region and at 3.3–4.7 ppm appeared and sharpened, giving rise to three singlets for resorcinol rings , one signal for methine bridges  and two singlets for methoxy groups. In addition, a broad doublet with an integral for four protons at 7.26 ppm and a multiplet arising from 12 protons at 7.12 ppm were observed for the tolyl protons Hc and Hb/Hn/Ho, respectively. The number and multiplicity of the proton signals at 193 K indicate that the podand adopts a Cv2‐symmetrical boat conformation.In the case of 1H NMR experiments of resorcinarene 1 were very similar to those obtained with 2. Coalescence was observed at 243 K, which suggests a similar energy barrier to the boat‐to‐boat exchange to that determined for 2. However, below the coalescence temperature, the lower‐rim methyl signal was split into two broad triplets (Hx and Hl) and the methine bridges appeared as two separate signals (Hv and Hj). Interestingly, half of the tolyl ring protons appeared as sharp doublets at 193 K (Hc and Hb), originating from two magnetically equivalent podand arms, whereas two broad signals were assigned to the tolyl protons (Hn and Ho) of the two remaining podand arms. These broad aromatic signals resonated upfield‐shifted, at 6.60 and 7.10 ppm, relative to their sharply resolved counterparts. Accordingly, the singlet of Ar‐CH3 (Ha) had an integral equivalent of six protons, and the remaining Ar‐CH3 protons (Hm) were most likely located together with the lower‐rim CH2 signals Hw and Hk based on the integral values of these broad resonances.The results of the variable‐temperature 3 protons (Hl and Hx) and upper‐rim resorcinol protons (Hh and Ht) at different temperatures were modelled by line‐shape analysis to obtain the exchange rate constants (k). The exchange rate constants were analysed by means of Eyring plots (see the Supporting Information), which provided Gibbs free energies of activation (ΔG≠298 ) of 55.5 kJ mol−1 for 1 and 52.0 kJ mol−1 for 2 (Table G≠298=54.7 kJ mol−1).The line shapes of the NMR signals arising from the lower‐rim CH1H–1H COSY and ROESY NMR spectroscopy were used to investigate the conformations of the resorcinarene podands 1 and 2 in solution, as well as to confirm the assignment of the protons at low temperature. The ROESY measurements of 2 at 193 K showed strong ROE correlations between aryl protons and methoxy protons, ArHh to Hg and ArHt to Hs, as well as between aryl and ethoxy protons, ArHh to Hf and ArHt to Hr  is significantly shorter than the distance between Hg and Hf , and therefore a more likely source of this correlation. This assignment was made assuming that the similar chemical shifts of the methoxy groups in 1 and 2 allow the assignment of Hg and Hs to horizontally and vertically oriented resorcinol rings also in 1.The ROESY spectrum of K Figure  revealedc and Hb were observed to the resorcinol lower‐rim groups. Proton Hb showed weak correlations with methine Hj and methyl Hl, and proton Hc showed cross‐peaks with the same lower‐rim protons and, interestingly, to the ethoxy He/Hf and methoxy Hs. This combination of ROEs is best explained if the tolyl group bearing Hc and Hb is attached to the horizontal resorcinol ring and wraps around the aromatic cavity thereby approaching the vertical methoxy group Hs and the lower rim of the podand. This interpretation is in agreement with our tentative assignment of the vertically and horizontally aligned resorcinol rings as well as with the crystal structure of 1.ROE correlations from the sharp tolyl group signals H1‐I–1‐V are in excellent agreement with the observed ROEs. In particular, structure 1‐I, which also has the lowest DFT energy, shows all the expected distances from the horizontal podand arm protons Hb and Hc to the lower‐rim protons and the vertical methoxy protons Hs. In the structures 1‐IV and 1‐V, one of the horizontal arms folds under the horizontally aligned resorcinol ring. In this conformation, the distances between the tolyl group protons and lower‐rim protons are too long for the observed ROEs, which suggests that these conformations are less populated in solution, in accordance with their higher DFT energies.The computational structures m, Hn and Ho signals at 193 K and appear upfield‐shifted relative to the corresponding Ha, Hb and Hc signals in the horizontal arm. This indicates that the tolyl groups are engaged in a dynamic process that is intermediate on the NMR chemical shift timescale. Based on the most likely conformation of molecule 1 in solution (ROEs and DFT structures), especially the proximity of the vertical podand arm ethoxy protons Hq to the horizontal ring protons Hg, it can be deduced that the conformation of the vertical podand arm is favourable for tolyl group inclusion into the aromatic resorcinol cavity. The upfield‐shifted and broadened tolyl group signals are indicative of a second exchange process with a low energy barrier involving dynamic inclusion of the tolyl groups in the magnetically shielded environment of the resorcinarene cavity.It is important to note that the tolyl groups of the two podand arms attached to the vertical resorcinol rings show broad Hp‐toluene podand arms, with the sulfonyloxy linker in 1 promoting self‐inclusion complexation of the p‐toluene group and the benzyloxy linker in 2 pinching of the resorcinarene cavity. Variable‐temperature NMR experiments revealed an energetic barrier in the range of 52–55 kJ mol−1 for the boat‐to‐boat exchange for both podands, which indicates that the different podand arm linkers do not influence significantly the exchange rate.We have presented the synthesis of two conformationally flexible resorcinarene podands without permanent cavities that were characterised by a complete set of high‐resolution spectroscopic techniques and X‐ray crystallography. In the solid state, the resorcinarenes exhibit different conformations of the 1 exhibits a similar wrapped conformation of the horizontal podand arms in solution as observed in the solid state. Furthermore, the magnetic shielding of the broad aromatic tolyl group signals assigned to the vertical podand arms and the computational modelling results suggest that the self‐inclusion of the p‐toluene group is a highly favourable conformation also in solution.The conformations and intramolecular interactions of the macrocycles were also investigated by ROESY experiments at low temperature. Furthermore, a conformational search and DFT optimisations were carried out to explore the conformation space of the podands. The observed ROE correlations between the podand arms and resorcinol protons suggest that the tetratosylate resorcinarene podand 2, the 1H NMR and ROESY data suggest that the benzylic protons of the vertical podand arms are involved in C−H⋅⋅⋅π interactions with the horizontally aligned resorcinol rings in solution, which was also observed in all the computational structures. Computational modelling revealed a more versatile conformation space for resorcinarene 2 than for 1, for which self‐inclusion of a vertical or horizontal podand arm was identified as the lowest‐energy structure. However, the conformational search also found an open conformation without self‐inclusion, which deviates from the crystal structure most significantly by a more compact orientation of the horizontal podand arms. Therefore, it is expected that both types of conformations are populated in solution, whereas the extended conformation of the horizontal podand arms is an effect induced by the favourable π⋅⋅⋅π interactions in crystal packing.For the tetratolyl‐resorcinarene 1 is two‐fold. On the one hand, the sulfonyloxy oxygen atoms are suitable hydrogen‐bond acceptors for establishing weak hydrogen bonds that support the self‐inclusion complex, and on the other hand, the oxygen lone pairs direct the podand arms away from the horizontally aligned resorcinol rings. Comparison with resorcinarene 2 clearly indicates that the presence of the sulfonyl group restricts the range of conformations the podand is likely to adopt. Ultimately, we can conclude that the conformation of podand 1 in solution correlates well with its solid‐state structure, whereas the conformation of podand 2 changes between self‐inclusion complexes and open conformations.The role of the sulfonyloxy linker in the self‐inclusion complex of resorcinarene Solvents and reagents were purchased from the commercial suppliers Sigma–Aldrich and VWR. Solvents were dried by using an MBraun solvent purification system. The syntheses were performed with dried glassware (at 120 °C overnight) under a balloon of nitrogen. Flash chromatography was performed with a Combiflash Companion (Teledyne Isco) and Redisep Gold silica columns. Mass spectrometry was performed with a Micromass LCT ESI‐TOF or an ABSciex QSTAR Elite ESI‐Q‐TOF mass spectrometer. Melting points were measured with a Stuart Scientific SMP30 instrument and are uncorrected.1H and 126 MHz for 13C). Chemical shifts (δ) are given in ppm and coupling constants (J) are given in Hz. Variable‐temperature NMR experiments of the podands were performed with a BBI probe. The 1H NMR spectra were recorded in CD2Cl2 at different temperatures  at a concentration of 9 mm for 1 and 11 mm for 2. ROESY and COSY 1H–1H NMR experiments were performed at 193 K. Complete line‐shape analysis was performed with Bruker TopSpin software .NMR spectra were recorded with a Bruker Avance III 500 spectrometer : δH=7.67 , 7.18 , 6.76 , 6.11 , 4.32 , 4.17 , 4.01–3.93 , 3.73–3.62 , 3.51 , 2.27 , 1.87–1.73 , 0.82 ppm ; 13C NMR : δC=156.15, 155.29, 145.65, 133.63, 130.41, 128.29, 127.35, 126.67, 126.45, 98.26, 69.42, 67.54, 55.77, 36.62, 28.82, 21.69, 12.78 ppm.Resorcinarene 2: Tetramethoxyresorcinarene 3 , Cs2CO3  and dibenzo‐18‐crown‐6  were mixed with dry acetonitrile (30 mL) under a nitrogen atmosphere at 100 °C with stirring. A white suspension formed. The mixture was heated at reflux for 15 min before the addition of 5  in dry acetonitrile (20 mL). The resulting suspension was then heated at reflux for 24 h. The solution was filtered while hot through a pad of Celite, and the solid residue was washed with hot acetonitrile. The solvent was evaporated on a rotary evaporator, and the residue was dissolved in dichloromethane (50 mL) and washed with water (20 mL). The organic solution was dried with MgSO4 and filtered, evaporated and purified by flash chromatography on a SiO2 column with a hexane/ethyl acetate gradient (from 8:2 to 0:1). The product was recovered as a colourless oil and recrystallised from ethanol as a white crystalline solid (yield: 44 %). M.p. 92–93 °C; 1H NMR : δH=7.24 , 7.14 , 6.68 , 6.33 , 4.51 , 4.44 , 4.05–3.93 , 3.81–3.71 , 3.71–3.59 , 3.55 , 2.32 , 1.93–1.75 , 0.89 ppm ; 13C NMR : δC=156.12, 155.67, 137.50, 135.90, 129.17, 128.00, 126.35, 126.28, 98.24, 73.49, 69.54, 69.40, 55.79, 37.22, 28.12, 21.10, 12.85 ppm; HRMS (ESI‐TOF): m/z calcd for C80H96O12Na+: 1271.6794 [M+Na+]; found: 1271.6785.Compound 4: KOH  was dissolved in anhydrous ethylene glycol by heating to 90–100 °C under a nitrogen atmosphere. 4‐Methylbenzyl chloride  was added dropwise to the yellow solution and heating was continued at 90 °C for 24 h. The yellow mixture was diluted with Millipore water (30 mL) and extracted with dichloromethane (3×20 mL). The combined extracts were washed once with water and dried with MgSO4 and filtered. The organic solvent was evaporated and the residue was purified by flash chromatography using a SiO2 column and a petroleum ether/ethyl acetate gradient (from 65:35 to 0:1). The product was isolated as a colourless liquid  and used as such in the next step. 1H NMR : δH=7.24 , 7.17 , 4.52 , 3.77–3.72 , 3.60–3.56 , 2.35 ppm ; 13C NMR : δC=137.5, 134.9, 129.1, 127.9, 73.1, 71.1, 61.9, 21.1 ppm.Compound 5: Tosyl chloride  was dissolved in dry dichloromethane (30 mL) under a nitrogen atmosphere, and the solution was cooled in an ice bath. Triethylamine  was added, followed by 2‐O‐(4‐methylbenzyl)ethyl alcohol  in dichloromethane (15 mL). The mixture was then stirred for 1 h in the ice bath and for 24 h at room temperature. The resulting brown solution was washed with water (2×40 mL). The resulting yellowish organic solution was dried with MgSO4 and filtered, and evaporated to dryness. The product was purified by flash chromatography using a SiO2 column and a hexane/ethyl acetate gradient (from 8:2 to 0:1). The product was recovered as a colourless liquid  with traces of ethyl acetate and used as such in the next step. 1H NMR : δH=7.79 , 7.31 , 7.14 , 4.44 , 4.18 , 3.63 , 2.43 , 2.34 ppm ; 13C NMR : δC=144.9, 137.7, 134.6, 133.2, 129.9, 129.2, 128.1, 127.9, 73.3, 69.4, 67.4, 21.8, 21.3 ppm; MS (ESI‐TOF): m/z=343.6 (100) [M+Na]+.X‐ray crystallography: Single‐crystal X‐ray diffraction data were recorded on an Agilent SuperNova, single source at offset, Eos diffractometer using an Agilent Atlas CCD detector with mirror monochromatised MoKα irradiation (λ=0.71073 Å). The data were processed and empirical absorption correction was made by using CrysAlisPro . The structures were solved with the Superflip  structure solution program using Charge Flipping  or a SHELXS  structure solution program using direct methods. Structure refinement was carried out with Olex2  using the SHELXL  refinement package with least‐squares minimisation. Hydrogen atoms were calculated in their idealised positions with isotropic temperature factors (1.2 or 1.5 times the C temperature factor) and refined as riding atoms. EADP and EXYZ constraints, and SADI, DFIX, DELU and SIMU restraints were used in the refinement of the disordered fragments in 2. The crystallographic parameters are shown in the Supporting Information.1) and 1963709 (2)1963708  should be addressed to the authors.SupplementaryClick here for additional data file.


X-ray crystallography was used to elucidate the effect of a single-site mutation on the activity of a native metalloenzyme. The subtle structural modifications around the active site of the enzyme were correlated with the retarded catalytic efficiency in terms of the mechanistic steps and their kinetics. 2) and product (HCO3−) are altered in the V143I variant and the water network in the water-replenishment pathway is restructured, while the proton-transfer pathway remains mostly unaffected. This structural information is then used to estimate the modifications of the reaction rate constants and the corresponding free-energy profiles of CA II catalysis. Finally, the obtained results are used to reveal the effect of the V143I mutation on the measured kinetic parameters (kcat and kcat/Km) at the atomic level. It is believed that the systematic approach outlined in this study may be used as a template to unravel the structure–function relationships of many other biologically important enzymes.Enzymes are catalysts of biological processes. Significant insight into their catalytic mechanisms has been obtained by relating site-directed mutagenesis studies to kinetic activity assays. However, revealing the detailed relationship between structural modifications and functional changes remains challenging owing to the lack of information on reaction intermediates and of a systematic way of connecting them to the measured kinetic parameters. Here, a systematic approach to investigate the effect of an active-site-residue mutation on a model enzyme, human carbonic anhydrase II (CA II), is described. Firstly, structural analysis is performed on the crystallographic intermediate states of native CA II and its V143I variant. The structural comparison shows that the binding affinities and configurations of the substrate (CO WDW occupies the pre-catalytic association site for substrate and is displaced by one of the O atoms of CO2 during binding  is located adjacent to the zinc-bound hydroxide and is responsible for substrate binding ]  waters . The intermediate states are henceforth referred to as native-0atm (PDB entry 6km3), native-7atm (PDB entry 6km4), native-13atm (PDB entry 6km5) and native-15atm (PDB entry 6km6) and as V143I-0atm (PDB entry 6klz), V143I-7atm (PDB entry 6km0), V143I-13atm (PDB entry 6km1) and V143I-15atm (PDB entry 6km2), respectively. Based upon the structural modifications, we successfully estimated the alterations in the reaction rate constants and the corresponding free-energy profiles in the CA II enzymatic mechanism. This study systematically reveals how a single point mutation influences an enzyme’s catalytic pathway at the atomic level, leading to an estimation of the kinetics governing its individual mechanistic steps.In this study, we investigate one of the most challenging cases and describe the subtle effects brought on by a Val143 to Ile (V143I) mutation. As shown in Table 12.supporting information) revealed that the overall protein backbones (tertiary structures) of the native and V143I CA II structures were very similar, with Cα–Cα r.m.s.d. values of less than 0.14 Å (Supplementary Tables S1 and S2). However, careful structural analysis successfully established subtle but clear changes in the active site . The key bound water molecules in native and V143I CA II are listed in Supplementary Tables S3 and S4.Our X-ray studies  in the vicinity of the zinc ion. In native CA II [Figs. 2a)–2d)], the water molecules (WZn/WDW/W1) around the zinc ion are initially well ordered at 0 atm CO2 pressure. At higher CO2 pressures of 7–15 atm, electron density for the CO2 molecule becomes apparent, displacing the deep water (WDW). Concurrently, two intermediate waters WI and WI′ emerge near Thr200, while W1 disappears at higher CO2 pressures. The distance between WI and W2 is ∼4.7 Å, suggesting that the hydrogen-bonded water network that facilitates proton transfer is disrupted when the CO2-binding site is fully occupied.Fig. 23− is stabilized and is observable at 0 atm CO2 pressure with an estimated occupancy of ∼20% [Fig. 2e)]. In the absence of experimentally introduced CO2, it is likely that the captured HCO3− is converted from CO2 absorbed into the crystal from ambient air. As the CO2 pressure increases, the HCO3− occupancy increases to 54% at 13 atm and subsequently decreases slightly to 48% at 15 atm, with increased CO2 occupancy [Figs. 2f)–2h)]. It is likely that the observed decrease in the HCO3− occupancy at 15 atm is owing to steric hindrance from the bound CO2 molecule. When superimposed with the previously reported coordinates of HCO3− bound to native CA II . In addition, the central C atoms of the two superimposed HCO3− molecules in native and V143I CA II are separated by 0.5 Å.On the other hand, the active site of V143I CA II shows noteworthy modifications. The most striking difference is that HCO3− molecule, the CO2 molecule is less stable in V143I CA II. For example, CO2 shows almost full occupancy at 7 atm in native CA II, while no CO2 is visible at 7 atm in V143I CA II, which instead shows the appearance of WDW [Figs. 2b) and 2f)]. The CO2 molecule appears at higher pressures (13 and 15 atm) with a decreased occupancy of ∼50% [Figs. 2g) and 2h)]. The bound CO2 is tilted by ∼6° and is situated closer to the zinc ion by 0.34 Å compared with that in native CA II . The distance between the end carbon (Cδ1) of Ile143 and the CO2 molecule is only 3.0–3.2 Å, and this steric disruption seems to affect the critical interactions between the CO2 molecule and the hydrophobic pocket, thereby destabilizing it in the active site. It is worth noting that in contrast to native CA II, the bound CO2 molecule in V143I CA II is distorted from the plane defined by the bound HCO3− molecule, and this seems to be detrimental to the efficient conversion of CO2 to HCO3−.Unlike the HCOI and WI′ show different behaviour in V143I CA II. The intermediate water WI is visible even in 0 atm V143I CA II but is not present in 0 atm native CA II [Figs. 2a) and 2e)]. However, it is observed that the electron densities of the two intermediate waters were less defined than in native CA II [Figs. 2f)–2h)]. These ‘weaker’ intermediate waters were accompanied by the presence of W1 at all pressures (0–15 atm). It is likely that these weak intermediate waters are related to the perturbed structures and dynamical motions of the EC waters, as explained later.Finally, the two intermediate waters W2.2.2 pressure [Fig. 3a)]. As the CO2 pressure increases, W1 disappears and intermediate waters (WI and WI′) emerge instead [Figs. 3b)–3d)]. The W2 water, which transfers a proton from W1 to His64, shows an alternative position denoted W2′. Considering the steric hindrance between His64in and W2′, it seems that the presence of W2′ pushes His64 towards the ‘out’ conformation (away from the water network). Indeed, His64 shows a net movement from the ‘in’ to the ‘out’ conformation as W2′ becomes prominent at higher CO2 pressures. W3a shows little change, but W3b shows an alternative water position, W3b′, and is found to interact with one of the entrance waters WEC1 .Fig. 32 pressure [Figs. 3e)–3h)]. W3b and WEC1 also show the same alternative positions, although their electron densities are slightly weaker. As the distance between W2 and the N atom (Nδ1) of His64in is relatively long (3.3 Å), efficient proton transfer seems to depend on the dynamical motions of W2/W2′ and His64in/His64out. These motions are quite similar in native and V143I CA II and therefore proton transfer is not significantly impacted in the variant.Similarly, in V143I CA II W2 shows the same alternative position W2′, and His64 shows the same ‘in’ to ‘out’ flip, with similar occupancies as observed in native CA II with increasing CO2.3.EC1/WEC2/WEC3/WEC4/WEC5). In native CA II, the five WEC waters are well ordered at 0 atm CO2 pressure [Fig. 4a)]. As the CO2 pressure increases, WEC1 shows an alternative position W′EC1, and WEC2 shifts to an alternative position W′EC2 [Figs. 4b)–4d)]. These dynamical motions of WEC1 and WEC2 are accompanied by the emergence of the intermediate waters WI and WI′. The two intermediate waters are located deep within the entrance conduit near the active site and are transiently stabilized via hydrogen bonding to several WEC waters . The short distance (2.2 Å) between WI and WI′ suggests that WI′ can rapidly shift to the WI position as WI refills the vacant water positions (W1/WZn/WDW) during catalysis. Previous studies suggest that the intermediate waters (WI and WI′) play a critical role in the rapid replenishment of the active-site water network during catalysis and therefore could influence the overall catalytic rate  [Fig. 4e)]. As the CO2 pressure increases, these alternative positions disappear and both the WEC2 and W′EC2 waters are observed instead [Figs. 4f)–4h)]. Compared with native CA II, W′′EC2, W′′′EC2 and W′′′′EC2 are new alternative positions that are observed only at 0 atm CO2 pressure in V143I CA II. Along with the perturbed dynamical motions of the WEC2 water, much weakened electron densities of the intermediate waters WI and WI′ are observed. It should be noted that among the five EC waters, WEC2 is unique in that it interacts with all three of the key waters, W1, WI and WI′. Therefore, the fact that the structures and dynamical motions of the WEC2 waters are significantly perturbed in V143I CA II is likely to account for the stabilization of W1 but the destabilization of the two intermediate waters.Among the five WEC2 is that its alternative position W′′′′EC2 is situated close to the bound HCO3−. The distances between W′′′′EC2 and the closest O atom and the C atom of HCO3− are 1.4 and 2.5 Å, respectively (Supplementary Table S5). Since W′′′′EC2 is too close to the bound HCO3−, W′′′′EC2 and HCO3− cannot coexist in tandem. Indeed, W′′′′EC2 is only visible in V143I CA II at 0 atm, when the HCO3− occupancy is low, and disappears as the HCO3− occupancy increases at higher CO2 pressures [Figs. 4e)–4h)]. It seems that the relationship between W′′′′EC2 and HCO3− is analogous to the relationship between WDW and CO2. In native CA II, the hydrophobic cavity produces an electrostatic environment in which the deep water (WDW) can be locally stabilized around the zinc ion and then replaced with one of the O atoms of CO2 upon CO2 binding. In V143I CA II, the altered hydrophobic cavity owing to the V143I mutation produces a slightly different electrostatic environment in which an additional water position (W′′′′EC2) can be locally stabilized around the zinc ion and subsequently replaced with one of the O atoms in HCO3− during CA II catalysis. The release of the W′′′′EC2 molecule upon HCO3− binding seems to reduce the entropic cost of the process, analogous to the release of WDW upon CO2 binding in native CA II. Thus, it is likely that the altered multiple conformations of WEC2 allow HCO3− to bind more easily and firmly in a tilted configuration at the active site.Another interesting aspect of W3.2 are slightly restricted and the binding affinity of HCO3− is increased with a distorted configuration and (ii) the EC water network in the water-replenishment pathway is restructured, while (iii) the proton-transfer dynamics are mostly unaffected. These detailed structural insights can now be used to assess the modifications in the reaction rate constants  and the corresponding free-energy profiles during the CO2-hydration reaction of CA II.In this study, we have successfully identified the ‘fine’ structural changes in the CA II intermediates induced by a single-residue mutation at the active site. The V143I mutation in CA II produces steric hindrance and induces subtle changes in the electrostatic environment of the active site. The resulting effects on the CA II intermediates can be summarized as follows: (i) the dynamical motions and the allowed configurations of CO2 molecule within the hydrophobic pocket of CA II. Previous mutational studies on hydrophobic pocket residues  suggest that the hydrophobic pocket of native CA II is involved in ‘ushering’ the CO2 molecule to the zinc hydroxide in the active site, but does not directly interact with the CO2 molecule to hold it in a specific orientation. Therefore, it is most likely that the CO2 molecule in aqueous solution is guided to the hydrophobic pocket of CA II but retains some degrees of freedom with regard to the acceptable configurations (positions and orientations) around the zinc hydroxide that eventually facilitate its rapid conversion into HCO3−. In the V143I variant, although the hydrophobicity of the active-site pocket is increased, the added methyl group appears to sterically restrict the number of favourable configurations and the dynamical motions accessible to the CO2 molecule within the cavity. This estimation is supported by the crystallographic observation that the CO2 molecule is distorted towards the zinc ion, tilted by ∼6° and is destabilized with lower occupancies in the V143I variant. Consequently, interconversion from CO2 to HCO3− becomes less efficient, leading to a reduced k1 (k1V143I < k1native). The lower k1 value also implies an enhanced activation-energy barrier for the step   increases the binding affinity of the HCO3− molecule in the active site. This stronger binding of HCO3− suggests that the free energy of the enzyme–product (EZnHCO3−) complex is lowered in the V143I variant . On the other hand, the alterations of the WEC2 water in the V143I variant make the intermediate waters (WI and WI′) slightly less stable, therefore possibly slowing down the replenishment of the active-site water network and HCO3− dissociation. This results in a reduced k2 value (k2V143I < k2native).Secondly, the V143I mutation induces a slightly different electrostatic environment in the hydrophobic cavity, thereby altering the location and dynamics of the Wnt Fig. 5. In conjZn→W1→W2→His64. This implies that if a mutation induces the destabilization of W1, proton transfer could be significantly perturbed. However, stabilization of W1 as in the V143I variant does not necessarily suggest a faster proton-transfer process. Rather, it is the dynamical motions of W2 and His64 that have a more critical influence on the proton-transfer rate. Our results suggest that both W2 and His64 show very similar dynamical motions. Taken together, it seems that the overall proton-transfer rate k3 is not significantly altered in the V143I mutant (k3V143I ≃ k3native).Thirdly, the V143I mutation seems to have little effect on the kinetics of the proton-transfer reaction. It is observed that W1 is more stabilized in the V143I variant intermediates. It should be noted that the intermolecular proton transfer can occur via the fully established water network Wkcat and kcat/Km; Nair et al., 1991et al., 19932-hydration reaction are listed in Table 1kcat value shows little change, but the second-order rate constant kcat/Km shows an approximately tenfold decrease in the V143I variant. The parameter kcat contains rate constants from the initial enzyme–substrate complex through the remaining steps, including proton transfer. Therefore, for the proposed mechanistic scheme (equations 1 and 2), kcat can effectively be represented as kcat = k2k3/(k2 + k3). On the other hand, the ratio kcat/Km contains rate constants for the initial association of the substrate CO2 through the dissociation of the product HCO3−. Hence, kcat/Km only contains rate constants from equation 1 (and not equation 2) and is represented as kcat/Km = k1k2/(k−1 + k2).The interplay between the modified reaction rate constants, as discussed above, now allows us to determine the effect of the V143I mutation on the measured kinetic parameters (3−-dissociation process (k2) is much faster than the reverse interconversion from HCO3− to CO2 (k−1) and the proton-transfer rate (k3), i.e.k2native ≫ k−1native, k2native ≫ k3native, with k2native ≃ 10k−1native, k2native ≃ 15k3native and k−1native ≃ 1.5k3native , we can estimate the effect of the V143I point mutation on the observed kinetic parameter kcat in the following way. In the native state [kcat]native = k2nativek3native/(k2native + k3native) ≃ k3native (using k2native ≫ k3native), indicating that the turnover rate in the native is almost the same as the proton-transfer rate and that the proton-transfer process is the rate-limiting step. In the V143I variant [kcat]V143I = k2V143Ik3V143I/(k2V143I + k3V143I) ≃ k3native[k2V143I/(k2V143I + k3V143I)] (using k3V143I ≃ k3native), and the experimental measurement ] ≃ 1, or k2V143I ≫ k3V143I. This result indicates that HCO3− dissociation (k2) remains faster than proton transfer (k3) in the V143I variant and that the proton-transfer process is still the rate-limiting step in the V143I variant. The negligible reduction in k2V143I suggests that its activation energy is not significantly increased  ≃ k1native (using k2native ≫ k−1native). This indicates that [kcat/Km]native mostly reflects the CO2-binding step and its interconversion to HCO3−. On the other hand, in the V143I variant both the dissociation of HCO3− (k2) and the reverse interconversion (k−1) from HCO3− to CO2 are retarded (k2V143I < k2native and k−1V143I < k−1native), leaving the relation k2V143I ≫ k−1V143I unresolved. However, the relation k2V143I ≫ k3V143I ≃ k3native obtained from the kcat analysis above suggests that k2V143I is still an order of magnitude larger than k3native, while k−1V143I < k−1native ≃ 1.5k3native suggests that k−1V143I is comparable to or less than k3native, thereby ensuring that the relation k2V143I ≫ k−1V143I remains valid. Thus, we can estimate the kcat/Km in the V143I variant in the following manner: [kcat/Km]V143I = k1V143Ik2V143I/(k−1V143I + k2V143I) ≃ k1V143I (using k2V143I ≫ k−1V143I). This estimation indicates that the reduced [kcat/Km]V143I is mostly owing to the decrease in k1, reflecting the slower CO2 binding and interconversion to HCO3− in the V143I variant. It should be noted that the reduction in the k−1 value has little effect on [kcat/Km]V143I in the direction of CO2 hydration, as long as the reduction in k2 is small enough to keep the relation k2V143I ≫ k−1V143I valid. However, it is expected that the reduction in the k−1 value would have significant consequences for the HCO3−-dehydration direction.The second-order rate constant kcat ≃ k3 and [kcat/Km] ≃ k1 in both native and V143I CA II, the Michaelis constant Km is expressed in the following way: Kmnative = k3native/k1native and KmV143I = k3V143I/k1V143I. Consequently, KmV143I > Kmnative can be obtained using the estimated relationships k1V143I < k1native and k3V143I ≃ k3native. The relationship shows that the substrate concentration needed to reach half of the maximum reaction velocity is larger in V143I CA II mainly owing to the slower CO2 binding and interconversion to HCO3−.Finally, considering that 2-hydration direction, the mutation can first perturb substrate funnelling into the hydrophobic pocket via steric hindrance, thereby limiting the configurations that allow its efficient conversion into product. This influence is directly reflected in kcat/Km, but not in kcat. Secondly, the mutation can structurally distort the proton-transfer pathway by perturbing the water network or its associated stabilizing residues, and this effect is directly reflected in kcat but not in kcat/Km. Thirdly, the mutation can alter the product-dissociation process via direct steric hindrance or perturbations in the water-replenishment pathway. This influence can be intricate and is reflected both in kcat and kcat/Km. On the other hand, the mutation can affect the reverse reaction, the interconversion from product to substrate and substrate dissociation, but this has little influence on either kcat or kcat/Km in the hydration direction, as long as the reverse interconversion process is much slower than the product-dissociation process.Although our study was performed for a single point mutation within the hydrophobic pocket (V143I), our approach and interpretations can be extended to arbitrary mutations in CA II. Considering the forward CO4.We systematically studied the effect of a single-residue mutation on the CA II catalytic pathway at atomic resolution. We have successfully captured the high-resolution intermediate states of the V143I variant and shown clearly that the single point mutation induces noticeable changes in substrate and product binding at the active site and in the water-replenishment pathway, but has little effect on the proton-transfer pathway. The structural information was then utilized to estimate the reaction rate constants and the free-energy profiles during the catalytic cycle, unravelling the effect of the point mutation on the altered kinetic parameters. We believe that the detailed and systematic approach in our CA II study can be extended to identify the specific roles of target amino-acid residues in many other biologically important enzymes. We also anticipate that our detailed descriptions could serve as a reference point for future theoretical and computational studies that may lead to an advanced understanding of enzyme mechanisms at the quantum-chemistry level.5.supporting information for this article: Emsley et al. (2010et al. (1988et al. (1977et al. (2005et al. (2011et al. (2011The following references are cited in the  al. 2010, Forsman al. 1988, Henders al. 1988, Khalifa al. 1977, Kim et  al. 2005, 2016 ▸,2, 6km3human carbonic anhydrase II, native, 0 atm COPDB reference: 2, 6km47 atm COPDB reference: 2, 6km513 atm COPDB reference: 2, 6km615 atm COPDB reference: 2, 6klzV143I variant, 0 atm COPDB reference: 2, 6km07 atm COPDB reference: 2, 6km113 atm COPDB reference: 2, 6km215 atm COPDB reference: 10.1107/S2052252520011008/jt5048sup1.pdfSupplementary Methods, Tables and Figures. DOI:


Usual clinical practice for arterial blood gas analysis (BGA) in conscious patients involves a one-time arterial puncture to be performed after a resting period of 20–30 min. The aim of this study was to evaluate the use of transcutaneous BGA for estimating this gold standard arterial BGA.n = 295). Transcutaneous PO2 (PtcO2) and PCO2 (PtcCO2) were monitored using a transcutaneous monitor  with sensors placed on the chest, forearm, earlobe or forehead. Transcutaneous BGA at 1-min intervals was compared with arterial BGA at 30 min. Reasonable steps to find severe hypercapnia with PaCO2 > 50 mmHg were evaluated.Spontaneously breathing Asian adults  were enrolled (n = 272). The average PCO2 bias was close to 0 mmHg at 4 min, and was almost constant (4–5 mmHg) with PtcCO2 being higher than PaCO2 at ≥8 min. The limit of agreement for PCO2 narrowed over time: ± 13.6 mmHg at 4 min, ± 7.5 mmHg at 12–13 min, and ± 6.3 mmHg at 30 min. The limit of agreement for PO2 also narrowed over time (± 23.1 mmHg at 30 min). Subgroup analyses showed that the PaCO2 and PaO2 levels, gender, and younger age significantly affected the biases. All hypercapnia subjects with PaCO2 > 50 mmHg (n = 13) showed PtcCO2 ≥ 50 mmHg for until 12 min.Sensors on the chest and forearm were equally preferred and used because of small biases (2 is useful, it cannot completely replace PaCO2 because PCO2 occasionally showed large bias. On the other hand, the prediction of PaO2 using PtcO2 was unrealistic in Asian adults. PtcCO2 ≥ 50 mmHg for until 12 min can be used as a screening tool for severe hypercapnia with PaCO2 > 50 mmHg.Although PtcCO Measiffusion , 4. In ciffusion –7. It maiffusion –10.2 and PaCO2 over time, the time courses of transcutaneous data for the estimation of arterial blood gas analysis (BGA) are not well characterized [Although previous studies have investigated the relationship between PtcCOcterized –13. Varicterized . This inA previous study suggested that the correlation between arterial BGA and transcutaneous BGA data via sensors on the chest is stronger than that observed via sensors on the arm; however, this report involved only anesthetized adult patients . The mosIO2 is reached between 10 and 30 min depending on the physiological and pathophysiological conditions of the patient [Arterial blood samples are drawn with the patient being in a steady state . The usu patient . Therefo2 > 50 mmHg) and recommend a reasonable time-saving step for the accurate diagnosis of these patients.We evaluated the transcutaneous BGA data at 1-min intervals comparing the final goal of arterial BGA at 30 min. This novel approach will answer the following questions: “From which time point are the transcutaneous BGA data meaningful?” and “How accurately are the current transcutaneous BGA data predicting arterial BGA?” In addition, the results of the subgroup analyses which may help to understand transcutaneous BGA, are shown. Finally, we discuss the most important subgroup  with a pulse oximeter  was performed to confirm that SpO2 data from each subject were constant during the study .The study was approved by the Ethics Committee of the International University of Health and Welfare . All subjects provided written consent prior to participating in this study. All subjects were adults, aged ≥20 years. Both healthy volunteers and patients who visited the Department of Respiratory Medicine, IUHW Shioya Hospital were invited to participate in the study. Measurements were performed in the supine position at room temperature (24–25 °C). Transcutaneous BGA data from 1 to 30 min and arterial BGA data at 30 min were obtained, and compared through Bland–Altman analysis [2 and PtcCO2 were measured with a transcutaneous gas monitoring system , using the principles of the Clark and Severinghaus electrodes [2 and PtcCO2 were recorded at 1-min intervals. Prior to sensor fixation, the sensor detected the atmospheric PO2 and PCO2. The electromechanical response of the TCM4 device, as shown by breathing on the sensor measuring atmospheric PO2 and PCO2, elicited a decrease in PO2 within 5 s and an increase in PCO2 within 15 s.PtcOectrodes . Calibraectrodes , 8, 9. MArterial BGA was performed at 30 min after sensor fixation in the supine position. Femoral arterial blood (0.5–1.0 ml) was drawn with a 22-gauge needle attached to a heparinized syringe. Samples were immediately analyzed with a blood gas analyzer .2 level and PaO2 level on the agreement data were evaluated. The transcutaneous data obtained via the sensors on the chest or forearm were used. The subjects with a PaCO2 level within the normal range (35–45 mmHg) were compared with those having different levels of hypocapnia or hypercapnia [2 level with the normal range (80–100 mmHg) were compared with those having hypoxemia or hyperoxemia [The effects of gender, age, PaCOercapnia . The suberoxemia .P < 0.05 denoted a statistically significant difference. Data were accumulated from the usual clinical practice at IUHW Shioya Hospital. When the number of subjects using chest or forearm sensor was 163 , PtcCO2 at 30 min was markedly larger than PaCO2 at 30 min (P = 3.8 × 10− 42). In the subgroup analysis, the absolute values of PO2 bias were larger in males than in females with P = 0.050. We calculated the necessary sample size to evaluate this interesting effect of gender. The expected effect size was 4.0 mmHg (SD: 12.1 mmHg); therefore, the standardized effect size was 0.33 [P-value decreased to < 0.01 and the effect of gender was confirmed (data are shown later).Data are expressed as means ± standard deviation (SD), unless otherwise indicated. Concordance of arterial (at 30 min) and transcutaneous (between 1 and 30 min) blood gas data were investigated by Bland–Altman analysis. Therefore, 30 Bland–Altman analyses were performed. Analysis of variance with Tukey’s correction or unpaired t-test (two-tailed) was used for the comparison at 30 min. The Excel Statistics software, 2010 version  was used. was 0.33 . It was The tolerance of local heating for electrode attachment was good. There were no signs of skin irritation or erythema at the end of the monitoring.2. Initially, four sensor positions were randomly used. However, shortly after the study was initiated, the use of the forehead as a sensor location was discontinued because of prominently large biases .A total of 295 spontaneously breathing Asian adults , comprising 10 healthy volunteers and 285 patients with various lung diseases, were enrolled from August 2015 to August 2019. The breakdown of lung diseases is shown in Supplementary Table S1 . The bias of PCO2 was 4.7 mmHg, with PtcCO2 being higher than PaCO2, and the 95% limits of agreement were ± 6.3 mmHg . The bias of PCO2 was 0.2 mmHg at 4 min . At 8 min or later, the bias was 4.1–4.8 mmHg, with PtcCO2 being higher than PaCO2. In contrast, the absolute value of bias of PO2 was lowest  at 1–2 min  are shown in Fig. min Fig. a, and thmin Fig. b. This 12 and PO2 , female , 40–59 years (n = 12), 60–79 years (n = 138), and ≥ 80 years (n = 111)  were significantly lower than those observed in the 40–59 year-old group and ≥ 80 year-old group  and 31 ≤ PaCO2 < 35 mmHg group  for both PCO2 (P < 0.01) and PO2  and > 50 mmHg group  were lower than those measured in the normal PaO2 group (n = 102) for both PCO2 (P < 0.05) and PO2  were larger than those recorded in the normal PaO2 group  are shown in Table 2 therapy. At 4 min, PtcCO2 ≥ 50 mmHg was observed in 69% of these subjects. This ratio increased over time; at 12 min, all these subjects showed PtcCO2 ≥ 50 mmHg. At 13 min, all these subjects showed PtcCO2 ≥ 51 mmHg. Subjects with severe hypercapnia appeared to have more diseases  than those with mild hypercapnia.Data and profiles of subjects with hypercapnia  between PaCO2 and venous PCO2. By waiting longer, we can obtain more accurate PtcCO2 data for the estimation of PaCO2. Several studies have indicated that PtcCO2 is more accurate than end-tidal PCO2 as a surrogate measure of PaCO2 [2 and PaCO2 ranged from 6.9 to 14.4 mmHg, 1.96SD data between PtcCO2 and PaCO2 ranged from 4.6 to 10.4 mmHg. The PtcCO2 data at 12–13 min or later were within the acceptable clinical range of agreement for PtcCO2 (± 7.5 mmHg) recommended in the guideline of the American Association for Respiratory Care [Previously, it was reported that the 1.96SD between venous PCO5.0 mmHg . Venous of PaCO2 –28. Whil2 is possible. It involves initial measurement of PtcCO2 at 4 min without bias, and observation of PtcCO2 at 8 min or later considering a bias of 4–5 mmHg. In a steady state, PtcCO2 is higher than PaCO2 because the former is an epidermal parameter which does not exclusively reflect arterial blood, and CO2 is produced by living epidermal cells [As a whole, the prediction of PaCOal cells , 29, 30.2 and PaO2 displayed a continual decline without an obvious plateau at 30 min. Even the minimal limit of agreement of ±24.0 mmHg at 30 min is not negligible in clinical practice. Therefore, PtcO2 is not an appropriate substitute for PaO2. Kesten et al. reported that the 90% response speed of PtcCO2 in this system was approximately three times faster than that of PtcO2 [2 and CO2 in water and aqueous tissues may be important to understand the difference between these gases [2 has been reported to be 20–25 times higher than that for O2.The 1.96SD between PtcOof PtcO2 . The Krose gases . In wateA change in the baseline level with time is termed “drift” . The cal2 and PaCO2 over time [2 via an arterial catheter and PtcCO2 over time (n = 6), yielding evaluations of individual agreements [2 via an arterial catheter and PtcCO2 over time , demonstrating parallel changes without any description of concordance over time. The present noninvasive study without arterial catheterization is in line with the actual clinical practice for spontaneously breathing patients, and provides data from a larger sample of subjects compared with previous studies [2 and PO2) were larger in the PaCO2 < 31 mmHg group than in the normal group. Arterial vasoconstriction by hyperventilation may be involved in this phenomenon [2 is low, the PCO2 bias may increase and underestimation of hyperventilation might occur. However, Bendjelid et al. reported that the PaCO2 level did not affect the PCO2 bias  [2 and PO2) were smaller in the hypoxemia group than in the normal group. Hypoxic vasodilation may be involved in this phenomenon [A few previous studies have investigated the relationship between PtcCOver time –13. Fukereements . Excellereements  and Stor studies –13. The enomenon . If PaCOans 85%) . The absenomenon .2 data were almost constant in each subject from the sensor fixation to the arterial blood sampling procedure. The effect of changing body position  on PaCO2 has been reported to be smaller than that exerted on PaO2 [Another limitation of the study is that arterial BGA was performed only at 30 min. However, it is worth performing Bland–Altman analysis for the comparison of the single time point arterial BGA data with the minutely obtained transcutaneous data. All gas data were collected during a short period (30–40 min) in the steady state, which was validated by observations that SpO on PaO2 , 35. Bla on PaO2 .2 > 50 mmHg is important to avoid CO2 narcosis. This technology of TCM4 with a Severinghaus electrode is useful in identifying subjects with PaCO2 > 50 mmHg. By performing arterial BGA after detecting PtcCO2 ≥ 50 mmHg during an observation for 12 min, PaCO2 > 50 mmHg can be accurately measured (without exceptions at least in our 13 subjects). We recommend this reasonable step for the efficient use of PtcCO2 data.Correct diagnosis of severe hypercapnia with PaCO2 is useful and can be used as a screening tool for severe hypercapnia, it cannot completely replace PaCO2. On the other hand, the prediction of PaO2 by PtcO2 was unrealistic in Asian adults. Consideration of gender, age, PaCO2 levels, and PaO2 levels may assist in improving the accuracy of estimation. Further investigations are needed to clarify the mechanisms of these factors that influence the biases. This approach may be of potential use to better understand transcutaneous BGA.We compared the agreement between minutely obtained transcutaneous BGA data and the final answer data of arterial BGA at 30 min. The use of sensors on the chest and forearm is equally recommended. Although PtcCOAdditional file 1: Supplementary Table S1. Comparison of subjects among the four groups of different sensor locations. Supplementary Table S2. Comparison of blood gas data among the four groups of different sensor locations.Additional file 2: Supplementary Fig. S1. Comparison of the time course data among the four locations of sensors. Average data are shown. Blue line: forearm (n = 143), red line: chest (n = 129), green line: earlobe (n = 17), and purple line: forehead (n = 6). Trajectories of (a) bias and (b) 1.96SD on PCO2. Trajectories of (c) bias and (d) 1.96SD on PO2. (a): Compared with the forearm or chest sensors, the earlobe or forehead sensors yielded larger bias for PCO2 at 4 min or later. (b): 1.96SD on PCO2 was similar among the four locations. (c): The forearm and chest sensors showed almost the same time course of bias for PO2. The earlobe sensor yielded larger absolute values of bias, whereas the forehead sensor yielded much larger absolute values of bias. (d): 1.96SD of the forehead sensor was larger than that of the forearm or chest sensors. Supplementary Fig. S2. Comparison of the time course data . Transcutaneous data obtained via the chest or forearm sensors were used. Average data are shown. Blue line: males (n = 168), red line: females (n = 104). Trajectories of (a) bias and (b) 1.96SD on PCO2. Trajectories of (c) bias and (d) 1.96SD on PO2. PCO2 bias was similar between the two groups (a). The 1.96SD of females on PCO2 was slightly lower than male (b). The absolute values of female PO2 bias was lower than that of males (c). 1.96SD on PO2 was not affected by gender (d). Supplementary Fig. S3. Comparison of the time course data . Transcutaneous data obtained via the chest or forearm sensors were used. Average data are shown. Blue line: 20–39 years (n = 11), red line: 40–59 years (n = 12), green line: 60–79 years (n = 138), purple line: ≥ 80 (n = 111). Trajectories of (a) bias and (b) 1.96SD on PCO2. Trajectories of (c) bias and (d) 1.96SD on PO2. Crossing the 0 line at approximately 5 min (later than in the other three groups), PCO2 biases in young adults (20–39 years) was slightly lower than those of the other three groups (a). The 1.96SD of young adults (20–39 years) on PCO2 was slightly higher than that of the other three groups at 21 min or later (b). The absolute values of PO2 bias in 40–59 years group seemed a little larger than those of the other three groups at 13 min or later. (c). The 1.96SD of young adults (20–39 years) on PO2 was slightly higher than that of the other three groups at 11 min or later (d). Supplementary Fig. S4. Comparison of the time course data to evaluate effects of hypocapnia. Transcutaneous data obtained via the chest or forearm sensors were used. Average data are shown. Blue line: severe hypocapnia , red line: mild hypocapnia , green line: normal range . Trajectories of (a) bias and (b) 1.96SD on PCO2. Trajectories of (c) bias and (d) 1.96SD on PO2. Crossing the 0 line at 2–3 min (earlier than in the other two groups), PCO2 biases in severe hypocapnia group was larger than those of the other two groups (a). This effect of hypocapnia seemed to be severity-dependent at 9 min or later. The 1.96SD of severe hypocapnia group was higher than that of the other two groups . The absolute values of PO2 bias in severe hypocapnia group were larger than those of the other two groups at 6 min or later. (c). Supplementary Fig. S5. Comparison of the time course data to evaluate effects of hypercapnia. Transcutaneous data obtained via the chest or forearm sensors were used. Average data are shown. Blue line: normal range , red line: mild hypercapnia , green line: severe hypercapnia . Trajectories of (a) bias and (b) 1.96SD on PCO2. Trajectories of (c) bias and (d) 1.96SD on PO2. Crossing the 0 line at about 6 min , PCO2 biases in mild hypocapnia group were smaller than those of normal range group (a). This delay of crossing the 0 line seemed to be severity-dependent. The 1.96SD of severe hypercapnia group on PCO2 was higher than that of the other two groups (b). The absolute values of PO2 bias in mild hypercapnia group were smaller than those of normal range group at 2 min or later (c). 1.96SD on PO2 seemed to be about the same among the three groups (d). Supplementary Fig. S6. Comparison of the time course data to evaluate effects of PaO2 levels. Transcutaneous data obtained via the chest or forearm sensors were used. Average data are shown. Blue line: hypoxemia , red line: normal range , green line: hyperoxemia . Trajectories of (a) bias and (b) 1.96SD on PCO2. Trajectories of (c) bias and (d) 1.96SD on PO2. PCO2 biases in hypoxemia group were smaller than those of the other two groups (a). The 1.96SD on PCO2 seemed to be about the same among the three groups (b). The absolute values of PO2 bias in hypoxemia group were smaller than those of normal range group (c). The absolute values of PO2 bias in hyperoxemia group were larger than those of normal range group (c). The 1.96SD of hyperoxemia group on PO2 was higher than that of the other two groups (d).


A simple dual transformation of T(λ) is proposed and used for increasing the accuracy of computation of its envelopes T+(λ) and T−(λ) accounting for the significant glass substrate absorption especially for λ > 2500 nm. The refractive index n(λ) of As40Te60 and As98Te2 films is determined with a relative error <0.30%. As far as we know, the As80Te20 film is the only one with anomalous dispersion and the thickest, with estimatedk(λ) of any of the three AsxTe100−x films is computed more accurately from the quantity Ti(λ) = [T+(λ)T−(λ)]0.5 compared to its commonly employed computation from T+(λ). The obtained results strengthen our conviction that the AOEM has a capacity for providing most accurate optical characterization of almost every dielectric or semiconductor film with T(λ).Three As Thin films have widespread applications in optics, optoelectronics and magneto-optics ,2, as thT(λ) of a specimen consisting of the studied film deposited on a light transmitting substrate, such as glass , as smaller FOM corresponds to more accurate film characterization.After a given thin film characterization is completed, its quality can be assessed by computation of a reconstructed transmittance spectrum FOM from Equation (7), it was shown in  of the film for every λ. In film characterization based on the AOEM, since different k(λ) are computed e.g., from Tsm(λ) and Equations (1) and (5), T+(λ) and Equation (2), and Ti(λ) and Equation (8), the extinction coefficient of the film is selected to be k(λ) corresponding to the smallest FOM from Equation (7) 0.5 are computed based on the algorithm from [λt(i) with Tsm(λ)’ are determined, completing the forward transformation of transmittance spectra. Thereafter, the first stage of the AOEM is executed by using the points T+(λt)’ and T−(λt)’. However, excluding the several longest λt for which xs(λt) << 1 since xs(λ) is present not only in the numerator of Equations (2) and (9), and substituting the film absorbance by xs(λt)’ = xs(λt)/xs(λt) = 1. At the end of such first stage of the AOEM, m1 are computed as in [In regard to the above, thm from , and alled as in ,29.Tsm(λ) = Tsm(λ)’xs(λ) are calculated, its envelopes T+(λ) = T+(λ)’xs(λ) and T−(λ) = T−(λ)’xs(λ), and Ti(λ) = Ti(λ)’xs(λ). Importantly, the tangency wavelengths of Tsm(λ) are the same as the already determined λt(i) since Tsm, T+ and T− are represented by multiplying their respective Tsm’, T+’ ≥ Tsm’ and T−’ ≤ Tsm’ by the same xs > 0, for every λ. After Tsm(λ), T+(λ), T−(λ), Ti(λ) and all λt(i) are computed, the second stage of the AOEM can be executed as in [xs(λ) ≤ 1.In the reverse transformation, ed as in  taking iTu+(λ) and Tu−(λ) are the upper and lower envelopes of the smoothed transmittance spectrum of the respective specimen with uniform film.An expression about the refractive index of a uniform film on a non-transparent substrate was presented in :(10)n(λ)T+(λ) and T−(λ) are the upper and lower envelopes of the smoothed transmittance spectrum Tsm(λ) of this specimen with non-uniform film [Furthermore, the following relations are valid for a specimen with the same characteristics except that the film, with the same orm film ,39. Morenl(λ) represents a lower limit of n(λ).It is deduced from Equations (10)–(12) that:40Te60, As80Te20, and As98Te2 in [The chemical composition and thickness of the films have been measured by energy-dispersive X-ray microanalysis (EDS) and scanning electron microscope (SEM) using JSM IT-300LV SEM of JEOL with an energy-dispersive attachment X-MaxN 20 of Oxford Instruments at high vacuum and an accelerating voltage of 20 kV. The compositions of the three studied films have been determined as As98Te2 in .40Te60 film on a glass substrate from 3/2 trigonal pyramidal structural units, as confirmed by Raman spectroscopy data in [80Te20 and As98Te2 films also have amorphous structures revealed by similar XRD images.X-ray diffraction (XRD) of film on glass substrate specimens are performed on a Shimadzu XRD-7000 X-ray diffractometer at a fixed time in the range of 10–80° with a step of 0.02°, using CuKα radiation with a wavelength of 1.5406 nm and scanning speed of 2° per minute. The XRD image for the As data in . Further98Te2 film are shown in Rq is determined from AFM image by Gwyddion open software [Rq is 7.7 ± 0.7 nm for the As40Te60 film [80Te20 film [98Te2 film from 40Te60 and As80Te20 films have been destroyed without independent measurement of their average film thickness The surface morphology of the films was monitored at atmospheric conditions using Smena atomic force microscopy (AFM) head, based on the NTEGRA Spectra using HA_NC and HA_C probes NT-MDT , in contact and semi-contact mode. Two-dimensional image of the surface and cross-section of the Assoftware . Rq is 7e60 film , 6.6 ± 0e20 film , and 8.140Te60 film has been prepared by five minutes deposition onto 0.45 mm thick sapphire plate with double-sided polishing manufactured by Monocrystal [80Te20 film or the As98Te2 film have been formed by deposition onto 1 mm thick standard microscope slide glass substrate of Levenhuk [T(λ) and Ts(λ) of these three samples and their substrates, as well as reflectance spectra Rs(λ) of the substrates have been measured by a Cary 5000 double-beam spectrophotometer of Agilent [A sample containing the Asocrystal ,42. SampLevenhuk ,43. Norm Agilent . These t Agilent .ns(λ) and ks(λ) are computed by solving the system of two equations about Ts(λ) and Rs(λ), for every λ, as in [Tsm(λ) of a studied sample is computed by “external smoothing” of its T(λ), as described in the fifth paragraph of the Introduction and in [xTe100–x films based on the AOEM are presented in the next three figures, whereas the results referring to λt(i) are exhibited by open circles, and the most important computed results are in red color. The optimized curve fittings for deriving n(λ) and k0(λ) are performed as described in Equation (6).The substrate characteristics λ, as in . Every sn and in . Data anλ [xs(λ) ≅ 1, Ts(λ)~xs(λ) ≅ constant [T+(λ) and T−(λ) for substrate absorption. Data and characterization results for the As40Te60 film are shown in m1 computed at the end of the first stage of the AOEM are typed in Since sapphire is quasi-transparent over the used interval of λ , its xs of the film decreases with increasing λ, i.e., it has a normal dispersion in the whole studied spectral range, and n(λ) is larger than its lower limit nl(λ) in accordance with Equation (13). Furthermore, k(λ).It is seen from 98Te2 film has been deposited for fifteen minutes on a glass substrate described in λ > 2500 nm as revealed by the significantly lower values of Ts(λ > 2500 nm) from Tsm(λ), described in λt(i) since only it is within the region of significantly lower values of Ts(λ). Data and results from the characterization of the As98Te2 film by the AOEM are exhibited in The AsTsm(λ)’, T+(λ)’ and T−(λ)’, obtained by the forward transformation described in λ > 2500 nm of strong absorption in the substrate, unlike the significantly lower Ts(λ) and T(λ) there. This allows accurate computation of Tsm(λ)’, T+(λ)’, T−(λ)’, Ti(λ)’ and λt(i); followed by accurate computation of Tsm(λ), T+(λ), T−(λ) and Ti(λ) by the reverse transformation described in n(λ) has a normal dispersion in the whole studied spectral range and n(λ) > nl(λ). Besides, the film also has a wide spectral region of quasi-transparency-It is seen from k(λ) ≈ 2 × 10−4 for the As40Te60 film, seen from T(λ) [k(λ) for the As98Te2 film from T(λ).The positive partially coherent light correction Δrom T(λ) . The pos80Te20 film has been deposited for thirty minutes on a glass substrate described in Tsm(λ) is implemented, as the first stage of the AOEM is executed excluding the four longest λt(i) since they are within the region of significantly lower values of Ts(λ) from 80Te20 film by the AOEM are presented in λt(i) participating in the first stage of the characterization is n = 26. The dependence ne(λ) from T+(λ), T−(λ), m1 in Equations (2) and (9) of the envelopes. Solving these two equations with respect to the two unknown ne and ke components of the complex refractive index of the film, for every λ. The longest wavelength of n(λ) crossing ne(λ) is denoted by λb in The AsT(λ) from 80Te20 film has quite different optical characteristics with respect to the As40Te60 and As98Te2 films. For example, the much narrower interference pattern of T(λ) from n(λ) of the As80Te20 film is significantly smaller than that of the As40Te60 and As98Te2 films, as explained in [e of extrema of Tsm(λ) from n(λ)80Te20 film is at least twice larger than that for the As40Te60 and As98Te2 films, as implied by Equation (3). The above data show that the average thickness of the As80Te20 film is significantly larger than 240Te60 film.A comparison of ained in . Also, tTs(λ) − T+(λ) is much larger in the region of the interference pattern from 40Te60 and As98Te2 films, indicating significantly stronger absorption and larger k(λ) of the As80Te20 film. Moreover, Ti(λ) from λ above 2300 nm implying increasing absorption there, which can be attributed to anomalous dispersion in this region. Besides, the difference T+(λ) − T−(λ) from T+(λ) − T−(λ) regarding λ above its apparent minimum at λ ≈ 2100 nm, indicating increasing n(λ) for λ > 2100 nm, thus confirming the presence of anomalous dispersion within the studied spectral region of the As80Te20 film.Also, the difference 80Te20 film by the AOEM does not recognize the commented above presence of anomalous dispersion. It is also apparent from nl(max(λ)) − n(max(λ)) is too large, as n(λ > λb) < ne(λ > λb) ≈ nl(λ > λb) although nl(λ) should be a lower limit of n(λ) according to Equation (13). Moreover, the difference |T(λ > 2600 nm) − Tr(λ > 2600 nm)| reaches too large values in 80Te20 film by the AOEM is inaccurate in the region of anomalous dispersion of the film. A main reason for this is the exclusion of the four longest λt(i) from the execution of the first stage of the AOEM characterization corresponding to λt(i) also belong to the region of anomalous dispersion where T+(λ) − T−(λ) rises significantly with increasing λ.Furthermore, it is seen from m1, which has integer or half-integer value, since it imposes a narrow interval of possible values of the average film thickness 80Te20 film by the AOEM represented by m1.Importantly, accurate film characterization by any EM requires determination of the correct first interference order m1, a simplified AOEM is employed by fixing both m1 to every possible value from Equation (3) and the number of used extrema of Tsm(λ) to Ne, for each one of the three films. At the second stage of the respective simplified characterizations of the film, k(Ti) computed from Equation (8) is used as described in Aiming at selection of the correct n and FOM correspond to the same m1 for each one of the As40Te60 and As98Te2 films, thus confirming the accuracy of their characterizations represented by m1 corresponding to the smallest δd/n is required for accurate computation of n(λ) and k(λ) of the characterized thin dielectric or semiconductor film with It is seen from N and the smallest FOM correspond to different values of m1 for the significantly thicker As80Te20 film. Since the film characterization should provide reconstructed transmittance spectrum Tr(λ) as close as possible to T(λ), another characterization of the As80Te20 film is performed based on using the AOEM with the values of m1 listed in red in However, the smallest δd/80Te20 film, as the difference nl(max(λ)) − n(max(λ)) is significantly smaller than for the characterization from m1 based on determination of min{FOM )}, exemplified by the use of 80Te20 film.Notably, it is seen from 40Te60 and As98Te2 films refer to the characterizations represented by 80Te20 film and designated as “m1 = 11.5, ND” and “m1 = 17.5, AD” refer to the characterizations represented by n(λ) from nl(max(λ)) − n(max(λ)) is quite large, one more characterization of the As80Te20 film is performed using the film data typed in n(λ ≤ λb) from n(λ > λb) = ne(λ > λb). Taking into account that this characterization recognizes the presence of anomalous dispersion, it is denoted as “m1 = 11.5, AD”. However, the FOM data from 80Te20 film.Other data regarding characterizations of the three studied films based on the AOEM are presented in Ti(λ) and Equation (8) rather than from T+(λ) and Equation (2), for all five film characterizations based on the AOEM. This is mainly attributed to the independence of Ti(λ) from possible partial coherence of the light passing through the film and light scattering from the film, as discussed in Importantly, a comparison of the data from the fourth and fifth columns of E0 is the oscillator energy, Ed is the dispersion energy, and E(eV) = 1.2398/λ (µm) is the photon energy [n(E(λt))2−1}−1 versus E(λt)2 [40Te60 and As98Te2 films. The parameters E0 and Ed are determined by a low-energy linear regression to the WD plot, and the static refractive index is The Wemple–DiDomenico (WD) single-effective-oscillator approximation n energy . Regardis E(λt)2  for the β = βc = 0.37 ± 0.04 eV in ‘covalent’ materials, β = βio = 0.26 ± 0.03 eV in ‘ionic’ materials, Nc is the coordination number of the cation nearest neighbor to the anion, Za is the formal chemical valence of the anion, and Nv is the total number of valence electrons (cores excluded) per anion. Using Ed typed in β = βc is estimated the coordination number of the cation (As) to be Nc ≈ 3.5 for the As40Te60 film, and Nc ≈ 3.56 for the As98Te2 film.The dispersion energy, Ed, measures the average strength of the interband optical transitions, and has been found to obey the empirical relationship :(14)Ed is also significantly smaller in accordance with E) is too close to E0 for the As80Te20 film, anomalous dispersion occurs and low-energy linear regression to the WD plot cannot be used for accurate determination of E0, Ed, and n0.Furthermore, T(λ) of the specimen, by optimization of the three parameters of the IEM from [T(λ) offsetting the influence of light scattering, enhanced computation of the envelopes using both iteration and extra points, and computation of n(λ) and k(λ) by optimized curve fitting.The OEM  was deveIEM from , accountIEM from  that theIEM from  providesIEM from , TLUM [3IEM from , and CLUIEM from  selectedIEM from  based onm1 of the film are computed, by minimization of the error metric, usually δd/N > 0. This error metric can be used for estimation of the relative error δd/N.  and T−(λ) as a result of the implementation of the proposed here dual transformation regarding Tsm(λ).The record low value of δd/n/n = δd/λt/λt, according to Equation (3), where δλt/λt is the relative error in computation of λt. Justifiably assuming δλt/λt ≤ 0.15% [n/n < 0.3% for the characterizations of the As40Te60 and As98Te2 films by the AOEM represented by n(λ) and larger k(λ) of the As80Te20 film are attributed to some porosity, oxidation in the film, and its non-stoichiometry in comparison with the As40Te60 and As98Te2 films. In this regard, anomalous dispersion has already been observed for other chalcogenide thin films [Furthermore, the relative error in the computation of the refractive index of the film is δ ≤ 0.15% ,29,30 lein films .xTe100−x films with different compositions and dissimilar T(λ ≤ 3000 nm) of a specimen consisting of the film on a substrate. To cope with the strong light absorption in commonly used glass substrates for λ > 2500 nm, a dual transformation of T(λ) is proposed and implemented for accurate computation of its envelopes T+(λ) and T−(λ). This dual transformation is simpler and should provide more accurate envelopes of T(λ) in comparison with the computation of the envelopes from [n(λ) of the As40Te60 and As98Te2 films by the AOEM.The AOEM is employed for optical characterization of three Aspes from , in case80Te20 film exhibits a region of anomalous dispersion and is significantly thicker than 6600 nm, which makes it very difficult for characterization by envelope method. In this respect, a procedure is proposed for accurate determination of the first interference order m1 for such a thick film, and this procedure is used for characterization of the As80Te20 film based on the AOEM. As far as we are aware of, the As80Te20 film is the only one with anomalous dispersion and the thickest, with estimated It is justified that the AsTi(λ) does not depend on the scattering of light from the film, unlike T+(λ). Consequently, it is shown that computation of k(λ) from Ti(λ) and Equation (8) is more accurate than the commonly employed computation of k(λ) from T+(λ) and Equation (2), for all five characterizations of AsxTe100−x films based on the AOEM and represented in It is also pointed out that T(λ).The obtained results strengthen our conviction that the AOEM has a capacity for providing most accurate optical characterization of almost every dielectric or semiconductor film with


We recently read, with great interest, the article entitled “Comparison of Methods Study between a Photonic Crystal Biosensor and Certified ELISA to Measure Biomarkers of Iron Deficiency in Chronic Kidney Disease Patients” . After r2T is the variance of the test method  and σ2R is the variance of the reference method  for each biomarker, and σ2(δ) is the total inherent imprecision of the test and reference methods together [T + σR. Therefore, when the two methods are identical, it is expected that 68% of differences are distributed around 0 ± 1σ(δ), and 95% of differences are distributed between 0 ± 2σ(δ), as illustrated in the different plots [The authors reported the total analytical error of a photonic crystal (PC) biosensor in the determination of ferritin and soluble transferrin receptor (sTfR) as biomarkers of iron deficiency anemia in chronic kidney disease (CKD) patients against certified ELISAs . They caIn their concluding remarks, by using different chronic kidney disease patients’ data, the authors claim that the PC assay’s inherent analytical imprecision was different than that of the ELISA in the case of the ferritin biomarker. However, in the case of the sTfR biomarker, there was no statistical difference between the methods. Figure 2a for ferritin and Figure 2b for sTfR indicate the difference plots comparing serum ferritin and sTfR concentrations from hemodialysis patients using the PC biosensor against the certified ELISAs. Sensors journal. Figure 2 indicates clearly that “the actual distribution of mean differences was 43% at 0 ± 1σ(δ) and 68% at 0 ± 2σ(δ)”.The problem is, Figure 2a,b indicates that for both serum ferritin and sTfR concentration cases, there is no statistical difference between the PC biosensor against the certified ELISAs. It shows a contradictory statement, as the authors claim in their published article  that theIn this comment, Figure 2a of the published article  is re-nu