AGE modifications of three corresponding troponin-I residues  and two corresponding troponin-T residues  were confirmed in cardiac tissue extracts from an experimental rodent diabetic model. Additionally, novel human troponin-I phosphorylation sites were detected . Accelerated AGE modification of troponin-C was evident in vitro with hexose sugar exposure. This study provides the first demonstration of the occurrence of cardiac troponin complex AGE-modifications. These irreversible AGE modifications are situated in regions of the troponin complex known to be important in myofilament relaxation, and may be of particular pathological importance in the pro-glycation environment of diabetic cardiomyopathy.Dynamic movements of the cardiac troponin complex are an important component of the cardiac cycle. Whether cardiac troponins are subjected to irreversible advanced glycation end-product (AGE) modification is unknown. This study interrogated human and rat cardiac troponin-C, troponin-I and troponin-T to identify endogenous AGE modifications using mass spectrometry (LC-MS/MS). AGE modifications were detected on two amino acid residues of human troponin-C (Lys The trimeric complex comprises troponin-C, which binds Ca2+ in low affinity binding sites during systolic Ca2+ influx; troponin-I, which inhibits the actin-myosin interaction and is tightly regulated by phosphorylation; and troponin-T, which anchors the troponin complex into the thin filament structure1. Post-translational modification of the troponin subunits is an important mechanism for mediating modulation of cardiomyocyte contractility in health and disease2, and novel reports of citrullination3, O-GlcNAcylation5, S-nitrosylation7, and phosphorylation11 sites on troponins have emerged in recent years. Post-translational modification of cardiac proteins for biomarker evaluation is also gaining interest14. To date, only dynamic post-translational modifications have been investigated, as a mode of reversible signaling regulation of the troponin complex. Susceptibility of troponins to irreversible post-translational modifications such as advanced glycation end-product (AGE) formation, has not been previously evaluated.The cardiac troponin complex facilitates cardiomyocyte contraction via dynamic interaction between the troponin subunits and the thin myofilaments, actin and tropomyosin. Troponins control the positioning of tropomyosin on the actin filament to regulate myosin-actin cross-bridge formation in response to Ca15, providing evidence that small changes in the troponin protein sequence may have major implications for cardiac function. Both Ca2+ sensitizing and Ca2+ desensitizing troponin mutations have been identified, associated with either a hypertrophic/restrictive cardiomyopathy or a dilated cardiomyopathy phenotype respectively15. Similarly, modified troponin 3-dimensional structure due to the presence of glycated amino acid residues would be expected to contribute to alterations in Ca2+ sensitivity and could underlie acquired cardiopathologies such as heart failure and/or diabetic cardiomyopathy. Surprisingly, no studies to date have investigated this proposition - perhaps reflecting a presumption that the relatively short half-life of troponins (3–5 days)16 would preclude a functional impact of a reputedly slow ‘permanent’ glycation modification . Yet emerging evidence suggests that intracellular cardiac proteins with normally high turnover rates are indeed modified by glycation. Mass spectrometry studies have shown that sarcoplasmic reticulum Ca2+ ATPase and ryanodine receptor proteins are present in AGE modified states in vivo in diabetic rat hearts19, despite half-lives of 5–8 days20. The mechanism and time-course of intracellular AGE-modification is not well established, and presence of glycated proteins considered to be relatively high turnover may reflect AGE-induced delayed protein degradation and/or an environment of accelerated AGE formation.Troponin mutations are detected in many inherited cardiomyopathies21. A physiological role for AGEs is unknown. Conflicting hypotheses have been posed that AGEs may act as either signals for protein degradation22 or inhibitors of protein degradation23. A growing body of evidence suggests that AGEs are primarily a form of environment-induced pathological protein damage with no known enzymatic processes for addition or removal. Although extracellular AGE-crosslinking of myocardial collagen is well-described24, few studies have investigated the role of AGE formation on intracellular proteins.AGEs are formed by non-enzymatic free sugar attachment to proteins and these adducts are converted through oxidation into fixed moieties which have potential to confer protein deformation and rigidity26, and high myocardial fructose levels (up to 60-fold elevated) in diabetic rat hearts has been reported, likely driven by upregulation of the sorbitol pathway27. In type 2 diabetic rats, myocardial levels of Nε-carboxymethyllysine (CML), a common AGE modification, are correlated with impaired left ventricular relaxation28. In particular AGE-modification of troponin proteins has potential to impair the trimeric complex operation and contribute to cardiomyocyte dysfunction in pathological settings.The rationale for investigation of troponin-AGE involvement in diabetic cardiomyopathy etiology is compelling. Not only do diabetic cardiomyocytes exhibit the pro-glycation environment of increased AGE precursors and oxidative stress, but increased exposure to fructose sugar, a potent glycation agent, may also play a role. An understanding of cardiomyocyte fructose actions is emerging119 and Thr123) on human cardiac troponin-I have been identified. Additionally, susceptibility of human cardiac troponin-C to accelerated AGE formation under in vitro conditions of high glucose and fructose exposure was established.The goal of this study was to investigate human cardiac troponins , seeking evidence of endogenous AGE modifications using mass spectrometry screening techniques. As an outcome of AGE discovery our aim was to link the amino acid residue modification sites to functional domains in the protein sequence, and thus obtain biological insight relating to potential cardiac functional impact of these irreversible post-translational modification types. Our studies provide the first evidence that all three troponin subunits exhibit endogenous AGE-modification and demonstrate that human cardiac troponin-I is particularly susceptible to glycation events, occurring at amino acid sites of functional importance. Two novel phosphorylation sites , and Arg79, Arg148, Arg162 and Arg204 were modified by methylglyoxal-derived hydroimidazolone (MG-H). Exemplar spectra for two of the troponin-I peptides are shown in Fig. 193  of trypsin-digested purified human cardiac troponin-C, troponin-I and troponin-T was performed. All AGE modifications detected above identity threshold are detailed in Table 193 Fig.  exhibite120 Fig.  exhibite120 Fig.  to allow120 in both unglycated and glycated form . AGEs were detected on Lys59, Lys121, Lys194 of troponin-I derived peptides from diabetic but not control samples . For these troponin-I AGE modifications, which could only be detected in the diabetic myocardium, selected exemplar spectra of unglycated and glycated troponin-I (Lys121) are provided in Fig. 107, Lys125 and Lys227. Figure 227 and Lys125 in both unglycated and glycated form are shown in Fig. 109, Lys202, Lys228) confirming two of the corresponding AGE-modification sites identified on human troponin-T . In addition, one new AGE-modification site was detected at Lys202  were confirmed in the purified human cardiac troponin-I samples in this study. The previously-reported dual Ser42/44 troponin-I phosphorylation events were not detected in these samples, likely due to the small size of the tryptic peptide containing these residues. Novel unreported troponin-I phosphorylation sites (Thr119 and Thr123) were also identified. Spectra of these troponin-I peptides in the presence and absence of phosphorylation at Thr119 and Thr123 are shown in Fig. 120 shown in Table 120 has the potential to interfere with regulatory phosphorylation sites. All spectra where novel phosphorylation occurrence was detected on troponin-I (Thr119 and Thr123) are provided in the Supplementary Information.LC-MS/MS analysis of trypsin-digested human cardiac troponin-I peptides identified 7 phosphorylation sites  may have important implications for movement of this domain in response to Ca2+ and for phosphorylation of Ser166 and Ser199. Figure 107, Lys125 and Lys227 are located within the central- and C-domain of troponin-T. Thus, glycated troponin residues may modify interaction of the troponin subunits and influence phosphorylation-mediated signaling regulation of the troponin complex.As depicted in Fig. in vitro, and to evaluate site-specific ‘glycation-vulnerability’, human purified cardiac troponin-C was incubated with 2 M glucose or 2 M fructose for 7 days at 37 C. Of the three purified troponin preparations, only troponin-C is soluble in the absence of urea. Thus troponin-C is suitable for in vitro investigations as urea-induced carbamylation of potential AGE sites (Lys and Arg) is avoided. Control , glucose- and fructose-incubated troponin-C samples were trypsin-digested and processed by LC-MS/MS. Given that the conventional view is that the formation of AGEs can take weeks to months, we hypothesized that hexose attachments (AGE initiation) or Amadori products (AGE-precursors) might be evident. We anticipated that it would be unlikely that AGE formation (e.g. CML) would be observed within this time period. Surprisingly, CML-modification of troponin-C was observed after only 7-days of incubation with glucose or fructose , although not all CML-modified residues reached the identity threshold of detection  were counted and presented as the number of modified peptides as a percentage of the total number of peptides (modified and unmodified). Comparisons were made between incubation groups for any single modification site. No differences in hexose modification were observed between PBS control, glucose or fructose incubated troponin-C for Lys17 and Lys92 sites . These findings also confirm that AGE formation on troponin-C can occur within 7 days, a finding consistent with the observation that intracellular proteins of relatively short half-life exhibit AGE modification 119 and Thr123). In vitro experiments with human cardiac troponin-C have demonstrated that AGE formation is promoted in a hexose-enhanced incubation setting, and that fructose in particular is an AGE accelerant. Collectively these findings provide new evidence that the human cardiac troponin complex is a potential target for glycation events at numerous sites, including locations of key importance for cardiomyocyte mechano-operation. Such molecular events may be of particular relevance in pathological settings, including diabetic cardiomyopathy, where hexose intracellular milieu is disturbed and reactive oxygen species levels are elevated.This study provides the first evidence that all subunits of the human cardiac troponin complex are modified by endogenous AGE formation. Our data demonstrate that human cardiac troponin-I is particularly susceptible to AGE modification, with 13 glycation sites detected, many of which are located adjacent to phosphorylation sites or within protein-protein interaction regions. The findings show that some, but not all, troponin lysine and arginine residues exhibit AGE modification indicating that particular residues are more vulnerable to glycation than others. Analysis of troponins isolated from rodent hearts revealed troponin-I derived from diabetic but not control rat myocardium exhibited AGE modification. These studies have also revealed preliminary evidence for two novel phosphorylation sites on human cardiac troponin-I , this study provides preliminary evidence for two novel phosphorylation sites . Interestingly, these sites span the identified robust CML and CEL modification of Lys120. Given that these residues are located within the troponin-I/troponin-T α-helical coiled coil1, phosphorylation and AGE-modification at these sites may be important for troponin complex structural backbone stability and tropomyosin anchoring. Further analysis of the Thr119 and Thr123 phosphorylation sites is now warranted, including identification of the relevant kinase(s) and characterization of functional implications.Several troponin-I domains are intrinsically disordered6 and Lys39) and 3 troponin-T amino acid residues . Spectral counting methods revealed that the overall percentage of troponin-C peptides with CML modification was very low (<2%) but increased after 1 week of in vitro hexose exposure (~70% for Lys106 exposed to fructose). Glucose incubation appeared to promote the initiation of AGE modification of troponin-C evidenced by increased hexose attachments on Lys106, while fructose accelerated AGE formation beyond the hexose initiation step, evidenced by lower hexose attachments but higher CML modification coincident with higher oxidation of methionine residues. Small changes in AGE modification of cardiac troponins would be expected to influence protein structure and function - a recent study demonstrated that a 6% change in Ser199 pseudo-phosphorylation status of troponin-I is sufficient to alter myofilament Ca2+ sensitivity8. These findings now prompt further investigation. As noted above, due to differing ionization efficiencies between peptides comparison could not be made between AGE modification sites. Development of site-specific quantitation methodology is required for further comparative analysis of in vitro and in vivo AGE accumulation. Further exploration of troponin-C AGE vulnerability is required, including more extensive proteolytic studies involving AspN. Additionally, systematically assessing the role of ambient hexose and oxidative stress conditions on AGE formation will yield important pathophysiologic insight. The in vitro hexose conditions utilized in this study are clearly artificial, contrived to achieve strongest evidence of AGE-site susceptibility. Similarly, while the atmospheric incubation environment utilized provides a starting point, a range of conditions should be investigated and the influence of subcellular compartmentalization, concentrating hexose and reactive oxygen species considered.Troponin-C and troponin-T exhibited fewer endogenous AGE-modification sites than troponin-I. CML-modification was detected on 2 troponin-C amino acid residues 44, further corroborated by the extensive observation of AGE modifications of troponin proteins considered to have short life times. AGE formation may delay protein turnover - in diabetic states where pronounced and sustained intracellular metabolic stress is evident, proteins and any accompanying AGE precursors  may persist longer in cytosolic locations due to dysfunction of intracellular degradative processes such as the glyoxalase45 and ubiquitin-proteasome system46 or autophagy pathways47. It could be expected that AGE modification of intracellular cardiac proteins may be promoted in the pro-glycation environment of altered hexose flux and oxidative stress in diabetic cardiomyocytes. Based on the ‘proof of concept’ data generated in this investigation, detailed evaluation of site-specific AGE modification accumulation and associated cardiomyocyte functional deficits in human and rodent diabetic heart tissues is warranted.The 7 day While robust, the findings reported in this investigation involve aspects of inherent bias – both sampling and technical. As a ‘proof of concept’ study, the evaluation of purified proteins involved samples derived from a limited number of human subjects. More extensive studies are required to evaluate a range of tissue samples obtained from diabetic and non-diabetic patients. To maximize identification of AGE modification sites present in the samples evaluated in this study, technical replicates were utilized, and this may confer false positive risk. Conversely, a relatively conservative algorithm was employed to establish detection events of AGE-modified proteins, with a high threshold ‘identity’ requirement. This approach may produce an AGE ‘under-detection’ bias, the extent of which can only be determined by further investigation. These considerations highlight the imperative for further exploration of the role of troponin AGE-modification in myocardial pathophysiology.2+-sensitive troponin complex conformational change with each cardiac cycle, it is likely that obstructive irreversible glycation modifications would elicit effects on cardiomyocyte function. Troponin-I AGE modifications were detected in regions of protein-protein interaction, adjacent to phosphorylation sites and in key regions of conformational change. The findings presented in this study generate new biological insight relating to potential cardiac functional impact of irreversible post-translational modification of cardiac troponins and provide the platform for new in vivo investigations to advance understanding of the functional outcomes of cardiac troponin AGE modification. In particular, investigations into the relative AGE abundance on cardiac troponins in a diabetic setting, where hexose flux disturbance and oxidative stress coincide to generate a pro-glycation environment, will be an important priority. Glycation of cardiac troponins may represent a novel mechanism underlying cardiomyocyte relaxation defects in diabetic cardiomyopathy, and offer an important new target for therapeutic intervention. Further studies of AGE occurrence in cardiac disease states are now warranted.This is the first study to demonstrate that all three human cardiac troponin subunits can exhibit AGE modification. Human cardiac troponin-I appears most susceptible to glycation, with thirteen modified amino acid residues detected. Given the importance of troponin-I in mediating the Cain vivo rat TnI glycation data. All animals were cared for in accordance with the Australian Code of practice for the Care and Use of Animals for Scientific Purposes . All procedures conducted as part of this study were approved by the Animal Ethics Committee of The University of Melbourne .Sprague Dawley rats purchased from the Animal Resource Centre  were housed in polypropylene cages in a temperature-controlled room at a 12:12 hour light and dark cycle at the Biomedical Sciences Animal Facility at the University of Melbourne and provided access to water and standard chow ad libitum for the duration of the experiment. Type 1 diabetes was induced in male rats at 8 weeks of age via a single 55 mg/kg tail vein injection of streptozotocin  with weight and blood glucose monitored for 8 weeks. Hyperglycemia was present in STZ-treated animals from 2 days post injection and at 8 weeks post-injection, all STZ rats exhibited blood glucose >30 mmol/L vs. control animals 6.45 ± 0.16 mmol/L. At 8 weeks after diabetes induction, rats were euthanized by cervical dislocation (isoflurane anaesthesia), hearts were excised, and left ventricles were dissected and frozen at 80 °C. Frozen tissue was homogenized in 100 mM Tris·HCl, 5 mM EGTA, and 5 mM EDTA (Sigma-Aldrich) buffer containing protease and phosphatase inhibitors . STZ and Control hearts  were used to generate 3 cubes, destained for 2 hours with 50% acetonitrile in 50 mM triethylammonium bicarbonate buffer (TEAB), dehydrated with 100% acetonitrile for 30 minutes, reduced with 10 mM tris(2-carboxyethyl)phosphine  for 45 minutes at 55 °C and alkylated with 50 mM iodoacetamide  for 30 minutes, shielded from light. After alkylation, gel pieces were washed 3 times in 50 mM TEAB for 10 minutes, dehydrated with 100% acetonitrile for 30 minutes and digested overnight in sequencing-grade trypsin  or sequencing-grade AspN  in 25 mM TEAB at 37 °C with shaking. Samples were then acidified with 1% formic acid and centrifuged at 16,000 g for 10 minutes. The supernatant (20 μL) containing digested peptides was then transferred to Exigen vials, and stored at 4 oC pending LC-MS/MS acquisition49.Troponin-C, troponin-I and troponin-T were purified from human cardiac tissue harvested from non-myocardial infarction human donors by Life Diagnostics . Human troponin-I, C and T samples were each typically purified from a single donor heart. Each aliquot acquired from a troponin I (n = 10), C (n = 5) or T (n = 5) sample was considered a technical replicate for these studies. Troponin-I and C were purified by urea extraction of cardiac muscle, ion-exchange chromatography followed by hydrophobic affinity chromatography (TnC) or troponin-C affinity chromatography (TnI). Troponin-T was purified by salt extraction and fractionation of cardiac muscle followed by ion-exchange chromatography. The purity was confirmed by the manufacturer to be >98% for troponin-I and troponin-T, and >95% for troponin-C. For analysis of endogenous AGE- and phosphorylation-modifications, coomassie-stained troponin bands were excised from SDS-PAGE gels, diced into 1 mmoC sealed under ambient atmospheric conditions for 7 days. Short incubation pilot experiments showed that hexose attachments formed within 2 days in 2 M glucose or 2 M fructose conditions so a longer 7 day incubation was selected to assess the formation of AGE-modifications. Following the incubation period, troponin-C (4 μg) was diluted with 50 mM TEAB, and reduced with 5 mM TCEP  at 60 oC for 10 minutes. Samples were digested in sequencing-grade trypsin  at 37 oC overnight in TEAB with shaking followed by acidification in 1% v/v formic acid. Following centrifugation , the supernatant (20 µL) containing digested peptides was transferred to Exigen vials, and stored at 4 oC pending LC-MS/MS acquisition48.To determine whether particular lysine or arginine residues are more ‘glycation-vulnerable’ purified human cardiac troponin-C  was incubated in PBS alone or containing 2 M glucose or 2 M fructose at 37 Digested peptides were separated by liquid chromatography using a nanoflow-reversed-phase-HPLC . The digested peptides were loaded onto an Acclaim Pepmap nano-trap column  with 3% acetonitrile/0.1% formic acid  and switched in-line to an Acclaim Pepmap RSLC analytical column  with solvent A, 0.1% formic acid and solvent B, 100% acetylnitrile/0.1% formic acid . All spectra were acquired in positive mode with full scan MS with spectra range m/z 375–1400 at 70,000 resolution, automatic gain control target of 3e6 ions, maximum accumulation time of 50 ms and a lock mass of 445.120024 m/z. The 15 most intense peptide ions with charge states ≥2–5 were isolated (1.2 m/z window) and fragmented with normalized collision energy of 30 and spectra acquired in positive mode MS with 17,500 resolution, automatic gain control target of 1e5 ions, and maximum accumulation time of 100 ms. Mass spectra of peptide ion fragments were acquired using a Thermo Scientific OrbiTRAP Elite MS/MS, or a Thermo Scientific Q-Exactive plus mass spectrometer.50. Peptide spectra were compared against theoretical in silico peptide signatures in the Mass Spectrometry protein sequence Database  to identify sequence coverage of troponin-C, troponin-I and troponin-T. MSDB database trawling was restricted to i) Homo sapien , and ii) peptides were produced via trypsin proteolysis (C-term lysine and arginine cleavage only) or AspN proteolysis  with up to 2 missed cleavages per peptide permitted. Peptide scores were assigned based on the number of ions detected and matched with theoretical ions, and signal intensity relative to background noise. Peak match tolerance was 0.2 Da. An identity threshold score was assigned to represent a peptide score with 95% confidence. Criteria used to determine a protein match included: combined score >1000, 5 or more unique peptides detected, peptide scores greater than identity threshold scores and false discovery rate <5%. Peptide identity scores were determined using a ‘probability based scoring’ algorithm to evaluate the observed match between the experimental data and the database sequence50. The corresponding identity threshold calculation was based on the number of peptides that fell within the precursor mass tolerance window and the significance threshold that was selected (0.05). Probing for post-translational modification sites were limited to hexose, CML, CEL, MG-H, and phosphorylation (MASCOT bioinformatic searching platform). Methionine oxidation was selected as a variable modification. Carbamidomethyl cysteine was selected as a fixed modification as it is a result of iodoacetamide incubation during sample preparation (capping of cysteine residues to inhibit re-formation of disulphide bonds following TCEP reduction). Modifications were confirmed by manually matching theoretical ion masses to spectra with known mass shifts relating to hexose +162 Da, CML + 58 Da, CEL + 72 Da, and MG-H + 54 Da. Phosphorylation sites were identified as mass shifts of +80 Da, and/or by neutral loss scanning of phosphoric acid (−98 Da). Doubly charged peptides will present with half the mass shift e.g.(+29 for CML) and triply charged peptide ions will present a third of the expected mass shift e.g. (+19.3 for CML). Quantification of hexose, AGE- and oxidation-modification of troponin-C was performed by counting modified peptide spectra normalized to total (modified and unmodified) peptide counts (above homology threshold) for a particular peptide of interest. Amino acid residues were numbered as presented in the Uniprot database (UniProtKB - P63316 (TNNC1_HUMAN), P19429 (TNNI3_HUMAN), P45379 (TNNT2_HUMAN)) where full sequence data may be obtained.Mass spectra were collected and compiled using the MSILE2 platform  and bioinformatics analysis performed using the MASCOT Pipeline Data are presented as mean ± sem. Statistical analyses were performed using GraphPad Prism V7.0 . Data were analyzed by one-way ANOVA with Bonferroni post-hoc tests where appropriate. A p-value of < 0.05 was considered statistically significant.Supplementary information


With the naturally separated frontier molecular orbitals, exciplexes are capable of thermally activated delayed fluorescence emitters for organic light‐emitting diodes (OLEDs). And, the current key issue for exciplex emitters is improving their exciton utilization. In this work, a strategy of building exciplex emitters with three components is proposed to realize multiple reverse intersystem crossing (RISC) channels, improving their exciton utilization by enhancing upconversion of nonradiative triplet excitons. Accordingly, a tricomponent exciplex DBT‐SADF:PO‐T2T:CDBP is constructed with three RISC channels respectively on DBT‐SADF, DBT‐SADF:PO‐T2T, and CDBP:PO‐T2T. Furthermore, its photoluminescence quantum yield and rate constant of the RISC process are successfully improved. In the OLED, DBT‐SADF:PO‐T2T:CDBP exhibits a remarkably high maximum external quantum efficiency (EQE) of 20.5%, which is the first report with an EQE over 20% for the OLEDs based on exciplex emitters to the best of our knowledge. This work not only demonstrates that introducing multiple RISC channels can effectively improve the exciton utilization of exciplex emitters, but also proves the superiority of the tricomponent exciplex strategy for further development of exciplex emitters. The traditional fluorescent emitters, phosphorescent emitters, and TADF emitters all realized superior performance in exciplex hosts than in conventional host materials, exhibiting lower driving voltages and improved efficiency roll‐off in the devices.ΦRISC) and fluorescence quantum yield of singlet excitons (Φf).Φfs are hard to be evidently improved due to such natural separation between HOMO and LUMO. As a result, further enhancing ΦRISCs of exciplex emitters should be the most possible way to improve their excitons utilization. As shown in FigureIt is generally understood that the exciton utilization of TADF emitter is decided by its reverse intersystem crossing efficiency of triplet excitons ΦRISC and ΦRISCs. As shown in Figure b,d]thiophene 5,5‐dioxide):PO‐T2T:CDBP, were constructed. Among them, 13AB, PO‐T2T, and CDBP are conventional constituting materials, while DBT‐SADF is a TADF constituting material; and 13AB, CDBP, and DBT‐SADF all can act as the D molecule to form TADF exciplexes with PO‐T2T. Particularly, CDBP:PO‐T2T has the highest energy gap than 13AB:PO‐T2T and DBT‐SADF:PO‐T2T. Therefore, tricomponent exciplex 13AB:PO‐T2T:CDBP will emit the emission of 13AB:PO‐T2T and possess two RISC channels on 13AB:PO‐T2T and CDBP:PO‐T2T; while DBT‐SADF:PO‐T2T:CDBP will emit the emission of DBT‐SADF:PO‐T2T and even have three RISC channels respectively on DBT‐SADF, DBT‐SADF:PO‐T2T, and CDBP:PO‐T2T. With the additional RISC channel, tricomponent exciplexes successfully exhibit much higher photoluminescence quantum yields (ΦPLs) and rate constants of RISC process (kRISCs) than their corresponding bicomponent exciplexes . In the devices, 13AB:PO‐T2T:CDBP exhibits a maximum EQE of 15.5%, while 13AB:PO‐T2T exhibits a maximum EQE of 12.4%. Moreover, with the three RISC channels, DBT‐SADF:PO‐T2T:CDBP exhibits a remarkably high maximum EQE of 20.5% in the device, which is much higher than DBT‐SADF:PO‐T2T with a maximum EQE of 16.9%. To the best of our knowledge, this is the first report with the EQE achieving over 20% at room temperature for the OLEDs based on exciplex emitters.To address this issue, in this work, we proposed a novel strategy of building exciplex emitters with three components to realize multiple RISC channels in the exciplex systems, improving their excitons utilization with further enhanced 2Figure1H, 13C nuclear magnetic resonance spectroscopies and mass spectroscopy, and purified by sublimation before further characterizations. As shown in Figure S1a in the Supporting Information, the fluorescence and phosphorescence of DBT‐SADF doped in mCP  film were investigated at 77 K, and its ΔEST is determined to be 0.030 eV from the difference between the peaks of the fluorescence and phosphorescence spectra, which is basically required for TADF emitters. To further demonstrate the TADF characteristic of DBT‐SADF, the temperature‐dependent transient photoluminescence (PL) decays of DBT‐SADF doped in mCP film were shown in Figure S1b in the Supporting Information. From 100 to 300 K, the delayed parts are remarkably increased due to the enhanced RISC process with the increased temperature, which is the direct evidence for TADF behavior of DBT‐SADF.EST is determined to be 0.56 eV. The temperature‐dependent transient photoluminescence decays of 13AB were also measured, and the trend of delayed parts is consistent with blank reference at 300 K, which is the direct evidence for non‐TADF behavior of 13AB. Besides, we also investigated the carrier transport characteristics of 13AB and DBT‐SADF, and both the 13AB and DBT‐SADF are hole‐dominated materials.Molecular structures of the constituting materials are shown in 1 energy levels of 13AB, CDBP, DBT‐SADF, and PO‐T2T are 3.01, 3.02, 2.53, and 2.95 eV, respectively. These values are evidently higher than the energy values of their corresponding exciplexes, which can ensure the exciplexes harvest both singlet and triplet excitons.ESTs and temperature‐dependent transient PL decays for 13AB:PO‐T2T and DBT‐SADF:PO‐T2T to prove their TADF behaviors. As shown in Figure S5 in the Supporting Information, 13AB:PO‐T2T and DBT‐SADF:PO‐T2T respectively exhibit extremely small ΔESTs of 0.047 and 0.032 eV from the differences between the peaks of their fluorescence and phosphorescence spectra at 77 K. And from 100 to 300 K, more significant decays in the microsecond range were observed with increased temperatures, confirming the existence of TADF characteristic. As shown in Figure Figure Based on three bicomponent TADF exciplexes, we constructed two tricomponent exciplexes, 13AB:PO‐T2T:CDBP and DBT‐SADF:PO‐T2T:CDBP, according to our strategy. As shown in Figure ΦPLs of 13AB:PO‐T2T, 13AB:PO‐T2T:CDBP, DBT‐SADF:PO‐T2T, and DBT‐SADF:PO‐T2T:CDBP by using an integrating sphere under oxygen‐free condition. And the absolute ΦPLs are measured to be 15.0% for 13AB:PO‐T2T, 40.0% for 13AB:PO‐T2T:CDBP, 38.0% for DBT‐SADF:PO‐T2T, and 61.0% for DBT‐SADF:PO‐T2T:CDBP. With the increasing RISC channels, the ΦPLs of the exciplexes are evidently enhanced, and the highest ΦPL of 61.0% is realized with three RISC channels. These results indicate the multiple RISC channels in tricomponent exciplexes will indeed enhance the utilization of excitons. To better understand the superiority of multiple RISC channels in tricomponent exciplexes, we further measured the transient PL decays of four exciplex emitters at room temperature and calculated their key kinetic parameters. For blue and green fluorescent emitters, the internal conversion process of singlet excitons could be ignored compared with fluorescence decay and intersystem crossing process of singlet excitons,ΦF and ΦTADF are the quantum efficiencies of prompt and delayed fluorescence; ΦISC is the efficiency of ISC process; kp, kF, kISC,kTADF, and kRISC are rate constants of prompt fluorescence, fluorescence decay, ISC process from S1 to T1 state, delayed fluorescence decay, and RISC process, respectively. ΦF and ΦTADF of these exciplex emitters are estimated from the ΦPL with a relative ratio which was calculated from the transient PL results.TablekFs and relatively large kISCs, resulting in all four ΦFs with small values. However, for the RISC process, kRISCs are evidently increased from 0.05 × 105 for 13AB:PO‐T2T with only one RISC channel to 0.64 × 105 and 1.17 × 105 for 13AB:PO‐T2T:CDBP and DBT‐SADF:PO‐T2T with two RISC channels, and finally to 14.2 × 105 for DBT‐SADF:PO‐T2T:CDBP with three RISC channels. As a result, ΦTADFs are also increased from 6.7% for 13AB:PO‐T2T to 28.0% for 13AB:PO‐T2T:CDBP and 33.0% for DBT‐SADF:PO‐T2T, and to 58.0% for DBT‐SADF:PO‐T2T:CDBP. Therefore, the evidently improved ΦPL of DBT‐SADF:PO‐T2T:CDBP is actually ascribed to the better upconversion of triplet excitons with three RISC channels and prove the feasibility of our strategy to improve the exciton utilization of exciplex emitters.We then measured the N,N‐di(p‐tolyl)amino]phenyl]‐cyclohexane) and PO‐T2T were used as hole‐transporting layer and electron‐transporting layer, respectively; LiF was acted as electron injection layer; 13AB, DBT‐SADF, and CDBP were used as electron‐blocking layer; exciplexes with the optimal mixing weigh ratios were used as the emitting layer. As listed in TableFigure−1, 43.8 lm W−1, and 12.4% for current efficiency (CE), power efficiency (PE), and EQE, respectively. The EQE value of 12.4% is far lower than the 20–30% theoretical limit of TADF‐based OLEDs, suggesting significant exciton loss. Correspondingly, 13AB:PO‐T2T:CDBP and DBT‐SADF:PO‐T2T respectively realize maximum CEs of 49.9 and 52.4 cd A−1, maximum PEs of 55.9 and 61.0 lm W−1, and maximum EQEs of 15.5% and 16.9% in the devices. One additional RISC channel has already improved the device performance of exciplexes evidently, which is consistent with our previous report.−1, 69.7 lm W−1, and 20.5% in the device. From 13AB:PO‐T2T to 13AB:PO‐T2T:CDBP, DBT‐SADF:PO‐T2T to DBT‐SADF:PO‐T2T:CDBP, the maximum EQE values of the devices are evidently improved from 12.4% to 20.5%, suggesting multiple RISC channels can effectively improve the exciton utilization of exciplex emitters. Moreover, to the best of our knowledge, this is the first report with an EQE over 20% for the OLEDs based on exciplex emitters at room temperature  performance of two tricomponent exciplexes and their contrastive bicomponent exciplexes by using them as the emitters. As the mixing ratios of exciplexes would change their charge balance and affect the device performance,N,N′‐Di(1‐naphthyl)‐N,N′‐diphenyl‐‐4,4′‐diamine (NPB) was chosen to replace the donor CDBP. As NPB has a higher HOMO energy level (−5.3 eV) than DBT‐SADF, the lower‐energy exciplex emitter should be NPB:PO‐T2T. Thus, to well analyze this three‐component exciplex emitter DBT‐SADF:PO‐T2T:NPB, the OLEDs using two exciplexes NPB:PO‐T2T and DBT‐SADF:PO‐T2T:NPB as the emitters were investigated with configurations of ITO/TAPC (35 nm)/NPB (10 nm)/NPB:PO‐T2T /PO‐T2T (45 nm)/LiF (1 nm)/Al (100 nm) and ITO/TAPC (35 nm)/DBT‐SADF (10 nm)/DBT‐SADF:PO‐T2T:NPB /PO‐T2T (45 nm)/LiF (1 nm)/Al (100 nm), respectively. Two devices exhibit quite low turn‐on voltages around 2.3 and 2.5 V. As shown in Figure−1, 13.7 lm W−1, and 4.1% for CE, PE, and EQE, respectively; while the maximum CE, PE, and EQE of NPB:PO‐T2T‐based device are respectively 2.0 cd A−1, 1.84 lm W−1, and 0.89%. The maximum efficiencies of the tricomponent device based on DBT‐SADF:POT2T:NPB are nearly five times higher than that of the bicomponent device based on NPB:PO‐T2T. These results further prove both the complete energy transfer from higher‐energy exciplex to lower‐energy exciplex in tricomponent exciplex system and the enhanced excitons utilization of multiple reverse intersystem crossing channels. Meanwhile, we also chose a classical electron‐transporting host material bis[2‐(diphenylphosphino)phenyl]ether oxide (DPEPO) with a wide gap material to replace the donor CDBP molecule, and constructed a tricomponent exciplex system DBT‐SADF:PO‐T2T:DPEPO. As DPEPO has a higher LUMO energy level and HOMO energy level than DBT‐SADF, only exciplex DBT‐SADF:PO‐T2T can be formed in such tricomponent system. The OLEDs based on DBT‐SADF:PO‐T2T:DPEPO were fabricated with configurations of ITO/TAPC (35 nm)/DBT‐SADF (10 nm)/DBT‐SADF:PO‐T2T:DPEPO (30 nm)/PO‐T2T (45 nm)/LiF (1 nm)/Al (100 nm). As shown in Figure −1, 67.6 lm W−1, and 17.3% for CE, PE, and EQE, respectively, which are also nearly consistent with the DBT‐SADF:PO‐T2T‐based device, suggesting the positive impact of the higher‐energy exciplex in tricomponent exciplex systems.To further confirm the proposed multiple reverse intersystem crossing channels in the tricomponent exciplex emitter system, a typical hole‐transport material with (y level −.3 eV tha3ΦPL (61.0%) and kRISC (14.2 × 105) than other contrasted exciplexes. In the OLED, DBT‐SADF:PO‐T2T:CDBP exhibits a low turn‐on voltage of 2.4 V and high maximum efficiencies of 60.0 cd A−1 CE, 69.7 lm W−1 PE, and 20.5% EQE. To the best of our knowledge, this is the first report for exciplex emitter–based OLEDs with an over 20% EQE at room temperature. Such high performance demonstrates introducing multiple RISC channels can effectively improve the exciton utilization of exciplex emitters, and our novel strategy of tricomponent exciplexes will provide a valuable approach for further development of exciplex emitters.In conclusion, to further improve the exciton utilization, a novel tricomponent exciplex strategy was proposed to realize exciplex emitters with multiple RISC channels in this work. With a newly designed and synthesized single‐molecule TADF emitter DBT‐SADF, a tricomponent exciplex DBT‐SADF:PO‐T2T:CDBP was developed with three RISC channels accordingly. And DBT‐SADF:PO‐T2T:CDBP successfully realizes much higher The authors declare no conflict of interest.SupplementaryClick here for additional data file.


This article emphasizes additional data on the fabrication of metal-free gC3N4 nanofibres (gC3N4NFs) and its associated structural and composition analysis compared with Au/Pd co-doped gC3N4 nanofibres (Au/Pd/gC3N4NFs). The data is including the typical fabrication process of metal-free gC3N4 nanofibers and its SEM, TEM, and element mapping analysis beside Raman, and FTIR spectra relative to Au/Pd/gC3N4NFs. We also investigated the catalytic CO oxidation durability testes on Au/Pd/gC3N4NFs compared to Pd/gC3N4NFs and Au/gC3N4NFs. The presented data are associated with the research article entitled “Rational synthesis of one-dimensional carbon nitride-based nanofibers atomically doped with Au/Pd for efficient carbon monoxide oxidation.” [1].Precise design of graphitic carbon nitride (gC This is in addition to the CO oxidation durability of Au/Pd/gC3N4NFs and its compositional analysis after CO oxidation reaction. Particularly, the data involves the SEM, TEM, and elemental mapping images of gC3N4NFs  are shown in  in an aqueous solution of isopropanol  under stirring at 40 °C. Then, an aqueous solution of nitric acid  was added to the previous solution under stirring at 40 °C. The as-formed white precipitate was filtered and washed with isopropanol solution before being dried at 100 °C for 12 h. Finally, the obtained powder was subsequently annealed under nitrogen at 550 °C for 2 h (5 °C min−1).3  without resolving any other undesired shapes such as spherical and sheets a. The av2.23N4NFs and gC3N4NFs were evaluated using the Fourier transform infrared (FTIR) analysis. Both Au/Pd/gC3N4NFs and gC3N4NFs revealed the peaks attributed to the stretching vibration of triazine at 810 cm−1 and several peaks for C–N heterocycles from 1000 to 1750 cm−1  band, which indicates the high degree of graphitization of the as-obtained materials , [2


VO2), and heart rate (HR) at peak level and ventilatory thresholds during synchronous arm crank ergometry.To examine the effects of stage duration on power output (PO), oxygen uptake  and peak level were compared among the protocols: a repeated measures analysis of variance was performed to test for systematic differences, while intraclass correlation coefficients (ICC) and Bland–Altman plots were calculated to determine relative and absolute agreement.Nineteen healthy participants completed a ramp, 1-min stepwise, and 3-min stepwise graded arm crank exercise test. PO, p ≤ 0.01). No systematic differences for HR or VO2 were found among the protocols. VT1 and VT2 were identified at 52% and 74% of VO2peak, respectively. The relative agreement among protocols varied (ICC 0.02–0.97), while absolute agreement was low with small-to-large systematic error and large random error.Systematic differences among the protocols were found for PO at VT1, VT2, and peak level. At peak level, PO differed significantly among all protocols (ramp: 115 ± 37 W; 1-min stepwise: 108 ± 34 W; 3-min stepwise: 94 ± 31 W, VO2 showed no differences. Therefore, training zones based on PO determined in short-stage protocols might give an overestimation. Moreover, due to large random error in HR at VTs between the protocols, it is recommended that different protocols should not be used interchangeably within individuals.PO at VTs and peak level was significantly higher in short-stage protocols compared with the 3-min stepwise protocol, whereas HR and  Handcycling is a rapidly growing sport for people with disabilities worldwide, especially in persons with spinal cord injury (SCI), muscular disease, or leg amputation , PO and HR, at both peak exercise and VTs during synchronous arm exercise.To become physically fit or to prepare optimally for such an event, a valid and reliable individualised training scheme is necessary. The intensity of the aerobic endurance training in this training scheme is based on the cardiorespiratory fitness of the individual, measured during a graded exercise test (GXT). Results of the GXT are used to develop individualised schemes based on percentages of peak power output (POpeak) or peak heart rate (HRpeak), or based on training zones delineated by power output (PO) or heart rate (HR) at ventilatory thresholds (VTs) , whereas Maher and Cowan (N = 38). The protocols were designed in such a way that patterns of work rate increase, external work, and test duration was comparable between protocols. Both studies found no significant differences in VO2peak, HRpeak, and POpeak between the different protocols. In addition, Smith et al.  was not significantly different between protocols   30 ± 10 years with range: 21–58 years, body mass: 71.6 ± 9.9 kg, height: 1.78 ± 0.07 m. All participants were healthy and physically active. They participated recreationally in sports such as fitness, soccer, and running with an average of four hours a week. They were non-specifically arm trained and had no experience with GXT on an arm crank ergometer. They had no restrictions or injuries of the upper extremities, and did not suffer from chronic diseases, such as heart or lung disease, diabetes, or obesity. Before the start of the test, participants were medically screened using the Physical Activity Readiness Questionnaire (PAR-Q) . The ergometer was wall-mounted using a height-adjustable bracket. Participants were seated, so that the axis of rotation of the arm crank was at the same height as the axis of rotation of the shoulder joint, and positioned at a comfortable distance from the ergometer allowing for a slight bend (15°–20°) of the participant’s elbow at the furthest point of the range of movement , minute ventilation (VE), and respiratory exchange ratio (RER), were collected and analysed per 10 s by mixing-chamber technique using the Cortex . The equipment was calibrated before each test. Criteria for a peak test were: RPE ≥ 8 at the end of the test and RERpeak ≥ 1.10 . HRpeak was defined as the highest 10-s average value achieved. In the 1-min stepwise protocol, POpeak was defined as the last completed PO step, plus ½ times the PO increment for each 30-s block in the non-completed PO step. In the 3-min stepwise protocol, POpeak was defined as the last completed PO step, plus 1/6 times the PO increment for each 30-s block in the non-completed PO step  the ventilatory equivalents of oxygen (VE/VO2) and carbon dioxide (VE/VCO2) vs time. VT1 was defined as an increase in slope of more than 1 in the first plot (V-slope method)   VE/VO2 and VE/VCO2 vs time; and (3) VCO2 vs VO2. VT2 was defined as the inflection in the VE vs VCO2 slope in the first plot  in the second plot  VE vs N = 48) and 0.94 (95% CI 0.90–0.97) for VT2 (based on N = 48). On average, there was a 4.1% and 0.6% difference between raters for VT1 and VT2, respectively. When the raters did not have the exact same point in time for a VT (N = 65), they examined the plots together to come to a mutually agreed VT. This value was then used for further analysis.Two trained researchers independently examined the plots visually to determine both VTs. Thereafter, results were compared. The interrater reliability (intraclass correlation coefficient (ICC)) of the determined VTs was 0.93 (95% CI 0.84–0.97) for VT1  × time (s)] at that point in time. The relative values of VO2, HR, PO, and TWD at both VTs were calculated as the absolute value at that VT divided by the peak value of that respective outcome measure .Thereafter, z scores for skewness and kurtosis. The peak physiological responses and test duration, and the VO2, HR, PO and TWD at VT1 and VT2, were compared among protocols. To test for systematic differences, a repeated measures analysis of variance (ANOVA) was performed. Mauchly’s test was used to test the assumption of sphericity. A Bonferroni post-hoc test for multiple comparisons was used for pairwise comparisons. Due to the potential risk of bias, imputation of data was not considered. Cohen’s d effect sizes were calculated and were evaluated according to Hopkins  unless otherwise stated.Data were analysed using IBM SPSS Statistics 24 . The data were tested for normality using the Kolmogorov–Smirnov test with Lilliefors Significance Correction, the Shapiro–Wilk test, and  Hopkins  as trivi Hopkins . The fol Hopkins . Values All participants completed all tests successfully resulting in a total of 57 tests. In total, 101 out of 114 (89%) VTs could be determined. Of the 13 undetermined VTs, 2 were related to the ramp protocol, 5 to the 1-min stepwise protocol, and 6 to the 3-min stepwise protocol. Eight were VT1 and 5 were VT2 of these 13 undetermined VTs. Outcomes were normally distributed. Peak values and threshold characteristics are shown in Table VO2, RER, and HR were not significantly different among protocols. POpeak differed significantly among all three protocols, with the highest value for the ramp protocol (115 ± 37 W), followed by the 1-min stepwise (108 ± 35 W) and 3-min stepwise protocol (94 ± 31 W). Test duration differed significantly among all three protocols, with the shortest test duration for the 1-min stepwise protocol (10.8 ± 2.4 min), followed by the ramp protocol (11.5 ± 2.1 min) and 3-min stepwise protocol (17.8 ± 3.8 min). TWD was significantly lower for the ramp (41 ± 20 kJ) compared with the 3-min stepwise protocol (59 ± 29 kJ) and for the 1-min stepwise (40 ± 19 kJ) compared with the 3-min stepwise protocol.Results of the repeated measures ANOVA are shown in Table VO2 were not significantly different among protocols. The relative VO2 as a percentage of VO2peak at VT2 was significantly higher for the 1-min stepwise protocol compared with the 3-min stepwise protocol. Absolute and relative values of HR at VT1 and VT2 were not significantly different among protocols. At VT1, PO was significantly higher for the ramp protocol (51 ± 22 W) compared with the 3-min stepwise protocol (36 ± 14 W). At VT2, PO was significantly higher for the ramp (80 ± 22 W) compared with the 3-min stepwise protocol (61 ± 18 W) and for the 1-min stepwise (75 ± 23 W) compared with the 3-min stepwise protocol. The relative PO as a percentage of POpeak, at both VT1 and VT2, was not significantly different among protocols. Absolute and relative values of TWD at VT1 and VT2 were not significantly different among protocols.At both VTs, absolute values of The relative agreement varied Table . Twelve VO2, HR, PO, and TWD. For POpeak and TWD, the lower boundaries of the confidence interval were, however, negative for two comparisons. Figure At peak level, the relative agreement was high to very high for VO2, low to high for HR, and low to very high for PO and TWD. The agreement of the relative values at both VTs was in general none to low for VO2, HR, PO, and TWD. Figure At both VTs, the relative agreement was moderate to high for VO2, and HR at both peak physiological responses and VTs during synchronous arm crank ergometry. The results at peak level demonstrate that PO showed the highest value for the ramp protocol, followed by the 1-min stepwise and 3-min stepwise protocol. At VT1, PO was significantly higher for the ramp protocol compared with the 3-min stepwise protocol, but there was no significant difference between the ramp and 1-min stepwise and the 1-min stepwise and 3-min stepwise protocols. At VT2, PO was significantly higher for both short-stage protocols compared with the 3-min stepwise protocol. No systematic differences for HR and VO2 were found among protocols, at both VTs and peak level. The relative agreement among protocols varied with low absolute agreement.The aim of the present study was to examine the effects of stage duration with a ramp protocol, 1-min stepwise protocol, and 3-min stepwise protocol on PO, VO2peak and HRpeak among protocols with varying stage duration. The differences in POpeak between the 3-min stepwise protocol and the protocols with short-stage duration (1-min stepwise and ramp) are in line with the previous cycling literature  PO value at the end of the test, whereas examples exist in which the final 30-s average PO value . Consequently, training zones for PO based on a ramp protocol or 1-min stepwise protocol will have a higher intensity than zones based on a 3-min stepwise protocol  within participants during synchronous arm ergometry. It might be interesting to investigate agreement at VT1, VT2 and peak level with repeated testing of the same exercise protocol in arm exercise, focussing on both trained and untrained individuals and subgroups, such as individuals with paraplegia or tetraplegia. In the light of the present study, it would be interesting to investigate the agreement of PO at both VTs within the 3-min stepwise protocol. It should, however, be considered that protocols with long test duration might be less feasible for individuals with a low physical capacity or limited arm function . Especially, for this population, agreement within short-stage protocols is warranted.VO2 were found among protocols. However, PO differed significantly among all protocols at peak level, with the highest value for the ramp protocol, followed by the 1-min stepwise and 3-min stepwise protocol. At VT1, PO was significantly higher for the ramp protocol compared with the 3-min stepwise protocol. At VT2, PO was significantly higher for both short-stage protocols compared with the 3-min stepwise protocol. The relative agreement between protocols varied with low absolute agreement. Consequently, it is recommended that the ramp, 1-min stepwise, and 3-min stepwise arm crank ergometry protocol should never be used interchangeably within persons to assess cardiorespiratory fitness and/or monitor adaptations to training programmes. Furthermore, training prescription based on PO at VTs assessed in short-stage protocols might give an overestimation with training zones that could result in overreaching. Individual training prescription based on PO at VTs would be most secure based on 3-min stepwise protocols; however, protocols with long test duration might not be feasible for certain patient populations with a very low physical capacity. Future studies should pay attention to the effect of stage duration on both peak physiological responses and VTs during arm crank ergometry in subgroups with different abilities and to the consequences of these differences in training zones on training response and overreaching.This study showed that stage duration affects outcomes at both VTs and peak level during synchronous arm crank ergometry in able-bodied participants. No systematic differences for HR and


Förster resonance energy transfer (FRET) between two molecules in nanoscale distances is utilized in significant number of applications including biological and chemical applications, monitoring cellular activities, sensors, wireless communications and recently in nanoscale microfluidic radar design denoted by the vibrating FRET (VFRET) exploiting hybrid resonating graphene membrane and FRET design. In this article, a low hardware complexity and novel microfluidic viscosity monitoring system architecture is presented by exploiting VFRET in a novel microfluidic system design. The donor molecules in a microfluidic channel are acoustically vibrated resulting in VFRET in the case of nearby acceptor molecules detected with their periodic optical emission signals. VFRET does not require complicated hardware by directly utilizing molecular interactions detected with the conventional photodetectors. The proposed viscosity measurement system design is theoretically modeled and numerically simulated while the experimental challenges are discussed. It promises point-of-care and environmental monitoring applications including viscosity characterization of blood or polluted water. Viscosity is one of the most important characteristics of the fluids with important implications in biomedicine such as early diagnosis of diseases using blood viscosity measurements . ViscosiPure mechanical method to get measurements without complicated electronic hardware.Nanoscale nature with acousto-optical wireless excitation allowing utilization in challenging applications.Energy harvesting properties from optical and acoustic excitations in the neighborhood of the donor-acceptor pairs.In-vivo measurement capability for future system designs by collecting the emitted photons remotely with in-body photodetectors and acoustic actuation.Flexible and robust system design by tuning the lumped size of the donor/acceptor molecules and the acoustic excitation frequency.Scalability for both nanoscale and microscale sensor applications.Remote monitoring and wireless data collection capability with optical emissions.In this article, a novel viscosity measurement system method is proposed based on vibrating Förster resonance energy transfer (VFRET) introduced in ,14,15,16The rest of this paper is organized as follows. In The system includes a microfluidic platform consisting of acoustic actuators and photodetectors with the fluid including vibrating and large size spherical particles as lump of the donor molecules as shown in If the fluid is assumed to vibrate with the velocity  follows :(1)Uf,maparticle .t is modeled as 2), −1·cm−1 and d and The number of photons emitted in the VFRET mechanism for the time duration of  follows ,14:(3)α≡Then, the final equality representing the relation between the viscosity Next, multiple periods of the received photon count is detected to estimate both T with the sample interval of A physical search mechanism is applied for getting measurements from the large donor particle for the unknown position. The simple proof-of-concept acceptor sensor is physically moved back and forth to search donor and take vibrating measurements. It is assumed that the measurements corresponding to the form in model in  18]. Mu. Mud0>UpThen, the final estimation is found with the following by getting average from each measurement:In Monte Carlo simulation studies, MATLAB built-in function nlinfit is utilized as the lgorithm .The proposed theoretical modeling allows utilization of different pairs of donor/acceptor pairs while still utilizing the fundamental Equation  with dift 5.5 nm . Both mat 5.5 nm . PreventThere are various experimental challenges to be solved for realizing the proof-of-concept experimental implementation. Donor molecules are assumed to form lumps with a spherical geometry and a large diameter. The method to form lumps with the desired particle size is an open issue. In addition, the interaction of the lumped donor sphere and acceptor layer with the fluid should be modeled and analyzed in detail. If the donor molecule disintegrates, then the speed of disintegration should be measured and the vibration frequency should be optimized with the appropriate measurement time.The fluid medium is assumed to be water with the simulation parameters shown in The oscillation magnitude The estimation of the viscosity The proposed viscosity measurement system is promising for utilization as a low cost micromechanical system in various POC and environmental monitoring applications. The viscosity measurement of the blood with a simple and low cost architecture allows early detection of various cardiovascular or cancer related diseases . SimilarThere are significant advantages of the proposed system due to the low hardware complexity relying on a completely mechanical set-up without requiring any electronic circuitry inside the medium to be monitored. In addition, the collection of the optical signals remotely allows for wireless applications and remote detection of the viscosity in challenging environments. These environments could include an in-vivo medium or energy scarce applications. Monitoring energy is harvested from the remote acoustic vibration and optical excitation. Besides that, adaptive formation of lumped spherical donor molecules provides robustness to different environments. The challenging future environmental applications include the analysis of deep water with tuned size of donor and acceptor molecules allowing measurable VFRET by scanning a set of acoustic frequencies.The design with respect to the non-Newtonian characteristics of the fluids including blood such as modeling based on shear rate. Flowing effects and unstable movement of the donor particles should be accurately modeled with respect to the fluid mechanics to have the formulation depending on the viscosity.Utilization of the multiple donor lump spheres to improve the optical emission performance.In-vivo system design with the floating acceptor layers and the donor molecules with drastically different vibration properties.Extension to the flow rate measurement with the complex network of the donor and the acceptor pairs distributed to the medium with the corresponding algorithm.The proof of concept design of the microfluidic chip including the photodetection, acoustic excitation and the viscosity calculating circuit as an all-in-one POC unit.There are various open issues and future works for the proposed architecture to be verified experimentally and to be improved for more advanced applications listed as follows:A novel microfluidic viscosity monitoring system architecture is presented with an acousto-optical mechanism by exploiting recently introduced VFRET. It is completely mechanical and adaptable to different environments as a low complexity solution compared with the state-of-the-art. The donor molecules spread to the medium are acoustically vibrated resulting in VFRET with the nearby acceptor molecules. The collection of optical signals remotely provides the advantages of remote and wireless monitoring capability. Theoretical modeling and numerical simulation of the proposed monitoring system are provided with experimentally feasible parameters of the set-up. Finally, open issues and experimental challenges are discussed including the utilization for point-of-care and environmental monitoring applications such as water or blood viscosity monitoring for widespread usage.


Rodents extract information about nearby objects from the movement of their whiskers through dynamic computations that are carried out by a network of forebrain structures that includes the thalamus and the primary sensory (S1BF) and motor (M1wk) whisker cortices. The posterior nucleus (Po), a higher order thalamic nucleus, is a key hub of this network, receiving cortical and brainstem sensory inputs and innervating both motor and sensory whisker-related cortical areas. In a recent study in rats, we showed that Po inputs differently impact sensory processing in S1BF and M1wk. Here, in C57BL/6 mice, we measured Po synaptic bouton layer distribution and size, compared cortical unit response latencies to “in vivo” Po activation, and pharmacologically examined the glutamatergic receptor mechanisms involved. We found that, in S1BF, a large majority (56%) of Po axon varicosities are located in layer (L)5a and only 12% in L2–L4, whereas in M1wk this proportion is inverted to 18% and 55%, respectively. Light and electron microscopic measurements showed that Po synaptic boutons in M1wk layers 3–4 are significantly larger (~ 50%) than those in S1BF L5a. Electrical Po stimulation elicits different area-specific response patterns. In S1BF, responses show weak or no facilitation, and involve both ionotropic and metabotropic glutamate receptors, whereas in M1wk, unit responses exhibit facilitation to repetitive stimulation and involve ionotropic NMDA glutamate receptors. Because of the different laminar distribution of axon terminals, synaptic bouton size and receptor mechanisms, the impact of Po signals on M1wk and S1BF, although simultaneous, is likely to be markedly different.The online version of this article (10.1007/s00429-019-01862-4) contains supplementary material, which is available to authorized users. Rodent brains compute relationships between motor commands to whisker muscles and signals from whisker mechanoreceptors to infer the position, size and texture of objects in their near environment. These computations are carried out by multi-level sensorimotor neuronal loops . In addition, we examined the “in vivo” responses to Po electrical stimulation and pharmacologically investigated the glutamatergic receptor mechanisms involved. Our findings show that Po axon terminals are remarkably different both in structure and in functional impact in M1wk and S1BF.Experiments were performed in adult  male C57BL/6 mice. Five animals were used for axon tracing experiments (3 for light microscopy and 2 for EM) and 11 further animals for electrophysiological recordings. Animals were housed under standard colony conditions with food and water ad libitum under a 12-h light/dark cycle. All procedures involving live animals were conducted at the Autónoma de Madrid University under protocols approved by our University ethics committee and the competent Regional Government agency , in accordance with the European Community Council Directive 2010/63/UE. Efforts were made to minimize the number of animals required.All surgical procedures were conducted under isoflurane anesthesia (0.5–1% in oxygen) following induction with a combination of ketamine  and xylazine  for anatomical experiments or just a higher dose of isoflurane (3%) for electrophysiological recordings. During recordings, the appropriate level of anesthesia was monitored by the presence of delta frequency waves (1–4 Hz) of high amplitude (> 50 µV) in the field potentials, as well as by the absence of both spontaneous whisker movements and pinch withdrawal reflex. Buprenorphine hydrochloride  was administered for post-surgical analgesia in anatomical experiments. At the time of sacrifice, animals were overdosed with sodium pentobarbital .To anterogradely label the axons arising from populations of Po neurons, animals were positioned in a stereotaxic apparatus  and placed on a water-heated pad at 37 °C. The midline of the scalp was sectioned and retracted, and a small craniotomy was opened. Borosilicate glass micropipettes  were loaded with a 3% solution of lysine-fixable biotinylated dextran amine (BDA) of 10,000 MW  in 0.01 M phosphate buffer  and stereotaxically positioned into Po following coordinates according to the Franklin and Paxinos  mouse atThe animal was placed in a rodent stereotaxic frame as described above. After the scalp incision, lidocaine (1%) was applied locally. Three small craniotomies were made. The first over the M1wk which was identified based on the literature as the region where minimal intensity stimulation produces selective whisker movements in mice  according to the Franklin and Paxinos  mouse atWe applied a protocol of 50 single pulses at different frequencies  separated by 20-s intervals without stimulation. Also, three paired-pulse stimulus paradigms were used in which four stimuli with the same current amplitude were delivered into Po with an inter-stimulus interval (ISI) of 50, 100 or 300 ms.Unit recordings were performed through tungsten microelectrodes  lowered stereotaxically into M1wk or S1BF. Units were recorded throughout the entire cortical depth. For analysis, data were pooled and compared between superficial (DV: 100–400 µm) or deep (DV: 500–1100 µm) cortical layers in each area. An unanalyzed gap of 100 µm was thus left between these compartments as a safeguard against potential errors in laminar localization due to small misalignments or drift of the electrode. In each experiment, we obtained two or three superficial layer recordings and two or three further recordings in deep cortical layers. Recordings were filtered at 0.3–3 kHz and amplified using a DAM80 preamplifier (WPI). Data were recorded continuously, sampled at 10 kHz, via an analog-to-digital converter built in to the Power 1401 data acquisition unit  and digitally stored in a computer until further use. Mice were sacrificed with an overdose of anesthetics (see above) immediately after recordings were completed.To examine the various glutamate receptor components involved in cortical responses to Po thalamocortical activation, the NMDA receptor antagonist D-2-amino-5-phosphonopentanoate , the non-selective group I/group II metabotropic glutamate receptor antagonist (+)-alpha-methyl-4-carboxyphenylglycine , and saline (control) were injected into M1wk and S1BF cortices in different animals. These were injected near the location of the tungsten microelectrode in each area . Drugs were slowly delivered through a cannula connected to a Hamilton syringe  over a 1-min period and recordings were performed 10 min after drug application.Animals were first perfused transcardially with 6 ml of saline for 1 min, followed by 50 ml of 4% paraformaldehyde  for 8 min. Brains were then removed from the skull and postfixed overnight at 4 °C in the same fixative. Subsequently, brains were cryoprotected by soaking in 30% sucrose . Two parallel series of coronal sections (60 µm thick) were obtained on a freezing microtome .In the axonal labeling experiments, sections were processed using avidin–biotin–peroxidase  and diaminobenzidine–glucose oxidase, with nickel enhancement  were collected in PB as two parallel series, and subsequently immersed in sucrose (30% in 0.1 M PB) overnight.Both series of sections underwent three consecutive rapid freezing–thawing cycles in liquid nitrogen to increase membrane permeability, and were then processed, in parallel, to develop BDA as described above, with the exception that in the second series, the oxygen–peroxide blocking step and the nickel sulfate enhancement were omitted. The first series was used to localize labeled Po axon arborizations and BDA deposits. The parallel sections from the second series containing the region with the heaviest cortical labeling underwent further processing for EM. Free-floating sections were first incubated for 45 min in 1% osmium tetroxide in 0.1 M PB, washed in PB, rinsed in 50% ethanol, incubated for 40 min in 1% uranyl acetate diluted in 70% ethanol in the dark, and finally dehydrated in an ascending ethanol series to absolute ethanol. The dehydrated sections were transferred to acetonitrile, and then immersed in epoxy resin  overnight. Finally, sections were flat embedded in Durcupan™ and polymerized at 60 °C for 48 h. Under a dissection stereomicroscope, samples containing the appropriate layers in M1wk or S1BF cortices were cut out, and then glued onto pre-polymerized resin blocks.Embedded tissue blocks were cut on an ultramicrotome (Leica Ultracut UCT) into ultrathin sections (~ 60 nm thickness) and collected on pioloform-coated slot copper grids. Thereafter, they were counterstained with lead citrate  equipped with a MT12 microcator , a X–Y–Z high-precision motorized microscope stage , and an Olympus DP-71 digital camera connected to a computer running the NewCAST stereology software package .3 and a step length area of between 10,000 and 22,500 µm2 . We considered and corrected the inhomogeneous shrinkage in the z-axis calculating the number-weighted mean section thickness, as previously described  and “density of varicosities” (number of varicosities per volume) in each of the three cases included in the study. For this purpose, we estimated the volume of each layer across the cortical sector containing the entire Po-labeled axon population. This volume was estimated using the Cavalieri principle (Gundersen and Jensen 2).Under bright-field optics in Nikon Eclipse 600 microscope  the Po axonal arborizations appeared as sharply labeled filaments with frequent varicose swellings. To estimate and compare the size of varicosities (putative synaptic boutons), we measured the maximal projection area from live images using a Nikon DMX1200 camera fitted to the microscope and the NIS-Elements imaging software tools . To this end, the major perimeter of each varicosity was focused at 1000× and delineated over the computer screen using the polyline and polygon software tools. For each cortical area and for each animal, 500 randomly selected varicosities were measured. Three animals were analyzed . We did not include varicosities with cross-sectional areas  near or below the microscope resolution limit  equipped with a bottom-mounted Proscan 2K digital camera . Bouton profiles were analyzed on digital images obtained using the Multi-Image Acquisition software  at a final magnification of 8000×. For each cortical region, the surface areas of 50 boutons were measured with OpenCAR software as described in detail in Rodriguez-Moreno et al. .Single-unit activity was extracted with the aid of Spike2 software (Cambridge Electronic Design) for spike waveform identification and analysis. In some cases, antidromic activity was present, but these records were excluded from the analysis. The sorted spikes were analyzed and quantified using peri-stimulus time histograms , where time 0 ms corresponded to the onset for the first stimulus in the stimulation train. The PSTH was calculated from 50 successive Po electrical stimuli. Response magnitude was defined as the total number of spikes per stimulus occurring between stimulus onset and 50 ms afterwards. To analyze Po-evoked responses during the application of stimulation trains, we calculated the response magnitude variation as the percentage change of the spikes measured in the second (P2), the third (P3) and the fourth (P4) pulses with respect to the first pulse .Paired-pulse ratios (PPR) were defined as the cortical unit response to P2, P3 or P4 in any given train divided by the response to P1. Thus, a PPR of 1 reflects no change between pulses; PPR < 1 indicates depression of the response, and PPR > 1 indicates facilitation.Initial (onset) and final (offset) latencies and response duration were measured in the PSTH. Initial latency was considered the time elapsed between stimulus onset and when the number of spikes doubled those in the spontaneous activity period. Final latency was considered the time when the number of spikes decayed to the same level as during the spontaneous activity period.p < 0.05, indicating this as (*), p < 0.01 as (**) and p < 0.001 as (***).Statistical analysis was computed using GraphPad Prism 5 software  or SPSS Statistics software . The threshold level of significance was set at U test (M–WUT).For the stereological dataset, comparison was performed by one-way ANOVA and either Bonferroni or Games–Howell post hoc pair-wise comparison methods, depending on the homogeneity of variances. Varicosity size distribution and medians were compared between pairs of areas/layers with a two-sample Kolmogorov–Smirnov (K–S) test and the Mann–Whitney t-test or M-WUT, depending whether the data were normally distributed or not. For multiple comparisons, we used a two-way ANOVA, plus Bonferroni’s multiple comparison tests as a post hoc comparison method.For the electrophysiological dataset, first we applied the Shapiro–Wilk normality test. Then, we used the paired Student’s two-tailed To examine possible structural differences between Po axonal terminals in S1BF and M1wk, we selectively labeled Po populations of axons by means of iontophoretic BDA injections in the thalamus. Three experiments in which a tracer deposit was confined to Po were analyzed . Conversely, axonal length was lower by threefold in L2–4 of S1BF (14.9% ± 5.7) than in the equivalent layers of M1wk . Differences in the layer-specific distribution of axonal varicosities were even more striking: 56.2% ± 11.2 were located in L5a of S1BF but only 18.4% ± 7.1 were observed in the same layer of M1wk (p < 0.001). Conversely, while just 12.5% ± 6.4 Po varicosities were located in L2–4 of S1BF, the equivalent layers of M1wk contained 55.5% ± 8.1 (p < 0.001). The sharp inversion observed in the distribution of Po axonal varicosities in L2–4 and L5a of the two areas seems all the more significant in view that distributions in the remaining cortical layers were similar.In M1wk and S1BF, a marked layer-specific distribution of axonal arborizations was observed Fig. , were ob2) than in those in S1BF L5a , and had a significantly different frequency in size distribution . Moreover, Po synaptic boutons had a different frequency of size distribution in the above two areas  and in S1BF .In several glutamatergic projection systems, including thalamocortical axons, differences in synaptic bouton size have been correlated with specific functional properties (reviewed in Sherman and Guillery p = 0.193) or deep layers (p = 0.327). Units in M1wk superficial layers increased their firing at higher stimulation frequencies , while those in deep layers did not change  spaced at different intervals (ISI) of 50, 100 or 300 ms were applied in each protocol . First responses consistently appeared in S1BF deep layers 4.6 ± 0.2 ms after Po stimulation (p = 0.005), then 5.6 ± 0.3 ms in S1BF superficial layers and 5.8 ± 0.3 ms in M1wk superficial layers. Finally, responses occurred with a slightly longer latency in the deep layers of M1wk . No differences were found in response durations between M1wk and S1BF .Besides receiving inputs from Po, M1wk and S1BF are known to be heavily linked by cortico-cortical connections  spikes at P4 increased by nearly threefold when compared with spikes counted after P1 (p < 0.001). A weaker facilitation effect was observed with 100-ms inter-stimuli intervals . No change was observed when pulses were separated by 300 ms . In contrast, using the same Po stimulation protocol (ISI 50 ms), unit responses were significantly decreased after P2 in S1BF superficial layers , and no changes were observed after P3 or P4, nor during the 100 or 300 ms ISI protocols .The above four-pulse Po stimulation trains elicited a marked facilitation of unit responses in the superficial layers of M1wk Fig. c. In eacF = 2.78, p = 0.046). In S1BF, response facilitation was observed with both 50- and 100-ms ISI protocols . Statistical comparison between response changes in deep layers during the 100-ms ISI protocol demonstrated that neurons in each area, as a population, displayed significantly different temporal response profiles (p = 0.026).Although less striking, changes in spike counts during the four successive pulses of Po stimulation were also observed in the deep layers of both areas Fig. d. In M1wp = 0.001; P3 and P4, p < 0.001). Remarkably, in S1BF superficial layers the ratio was 0.8 ± 0.09 in P2, indicative for depression of the response. In deep layers of both areas  and all ratios indicated facilitation.Analysis of average paired-pulse ratios for the ISI 50-ms protocol confirmed the statistical significance of the above differences Fig. e, f. ExaTo investigate whether the different response profiles of S1BF and M1wk cells reflect the involvement of specific glutamate receptor types at Po synaptic boutons in each area, the effect of different glutamate receptor antagonists on unit response profiles was performed. Using the ISI 50-ms train stimulation protocol, responses in each area after a local injection of saline (control), D-AP5 (a competitive NMDA antagonist) or MCPG . However, no spike count changes were observed following the application of MCPG in this area  are close to that of our VPM thalamocortical varicosities in the L4 S1BF barrels  and that the two areas are at least 3 mm apart Supplementary material 2 (TIF 9365 KB)