Colorimetric detection methods that produce results readable by eye are important for diagnostic tests in resource-limited settings. In this work, we have compared three main types of colorimetric ...methods - enzymatic reactions, silver deposition catalyzed by gold nanoparticles, and polymerization-based amplification - in a paper-based immunoassay for detection of Plasmodium falciparum histidine-rich protein 2, a biomarker of malarial infection. We kept the binding events in the immunoassay constant in order to isolate the effect of the detection method on the outcome of the test. We have highlighted that the optimal readout time in a test can vary significantly - ranging from immediately after addition of a visualization agent to 25 minutes after addition of a visualization agent - depending on the colorimetric method being used, and accurate time keeping is essential to prevent false positives in methods where substantial color develops over time in negative tests. We have also shown that the choice of a colorimetric method impacts the calculated limit-of-detection, the ease of visual perception of the readout, and the total cost of the assay, and therefore directly impacts the feasibility and the ease-of-use of a test in field settings.
Background
Robotic hepatectomy has gained increasing acceptance across the US. Although the robotic approach offers significant technical advantages, it is still bound by the individual surgeon’s ...learning curve. Proficiency in this approach should theoretically lead to improved peri-operative outcomes.
Methods
Between 2017 and 2020, data on 148 consecutive robotic hepatectomies performed by a single surgeon was retrospectively analyzed. Using cumulative sum (CUSUM) method, intraoperative blood loss (EBL) and operative time were used to assess learning curves for robotic major (
n
= 58) and minor (
n
= 90) hepatectomy patients. Perioperative outcomes were compared in regards with proficiency.
Results
Proficiency for robotic major and minor hepatectomy was achieved after 22 cases and 34 cases, respectively. No significant differences were observed in patient demographics or tumor characteristics. For robotic major hepatectomy, when compared to early experience, proficiency was associated with a significant improvement in mean EBL (242 mL vs 118 mL,
p
= 0.0004), operative time (330 min vs 247 min,
p
= 0.0002), decreased overall complication rate (23% vs 3%,
p
= 0.039), and length of hospital stay (5.7 days vs 4.1 days,
p
= 0.004). No difference in conversion rate, mortality or 30 day readmission was seen. For robotic minor hepatectomy, proficiency was associated with significantly decreased mean EBL (115 mL vs 54 mL,
p
= 0.005), operative time (168 vs 125 min,
p
= 0.014), and length of hospital stay (2.8 days vs 2.1 days,
p
= 0.021). No difference was observed in conversion rate, overall complications, mortality or 30 day readmission.
Conclusion
In the modern era, robotic hepatectomy offers a safe approach with excellent perioperative outcomes. Post learning curve proficiency is associated with significant improvements in perioperative outcomes in both major and minor hepatectomy. Results from our study can serve as a guide to surgeons and programs looking to adopt this technique.
Hydrogen peroxide (H2O2) promotes a range of phenotypes depending on its intracellular concentration and dosing kinetics, including cell death. While this qualitative relationship has been well ...established, the quantitative and mechanistic aspects of H2O2 signaling are still being elucidated. Mitochondria, a putative source of intracellular H2O2, have recently been demonstrated to be particularly vulnerable to localized H2O2 perturbations, eliciting a dramatic cell death response in comparison to similar cytosolic perturbations. We sought to improve our dynamic and mechanistic understanding of the mitochondrial H2O2 reaction network in HeLa cells by creating a kinetic model of this system and using it to explore basal and perturbed conditions. The model uses the most current quantitative proteomic and kinetic data available to predict reaction rates and steady-state concentrations of H2O2 and its reaction partners within individual mitochondria. Time scales ranging from milliseconds to one hour were simulated. We predict that basal, steady-state mitochondrial H2O2 will be in the low nM range (2–4 nM) and will be inversely dependent on the total pool of peroxiredoxin-3 (Prx3). Neglecting efflux of H2O2 to the cytosol, the mitochondrial reaction network is expected to control perturbations well up to H2O2 generation rates ~50 μM/s (0.25 nmol/mg-protein/s), above which point the Prx3 system would be expected to collapse. Comparison of these results with redox Western blots of Prx3 and Prx2 oxidation states demonstrated reasonable trend agreement at short times (≤ 15 min) for a range of experimentally perturbed H2O2 generation rates. At longer times, substantial efflux of H2O2 from the mitochondria to the cytosol was evidenced by peroxiredoxin-2 (Prx2) oxidation, and Prx3 collapse was not observed. A refined model using Monte Carlo parameter sampling was used to explore rates of H2O2 efflux that could reconcile model predictions of Prx3 oxidation states with the experimental observations.
Catalytic redox reactions have been employed to enhance colorimetric biodetection signals in point-of-care diagnostic tests, while their time-sensitive visual readouts may increase the risk of false ...results. To address this issue, we developed a dual photocatalyst signal amplification strategy that can be controlled by a fixed light dose, achieving time-independent colorimetric biodetection in paper-based tests. In this method, target-associated methylene blue (MB+) photocatalytically amplifies the concentration of eosin Y by oxidizing deactivated eosin Y (EYH3–) under red light, followed by photopolymerization with eosin Y autocatalysis under green light to generate visible hydrogels. Using the insights from mechanistic studies on MB+-sensitized photo-oxidation of EYH3–, we improved the photocatalytic efficiency of MB+ by suppressing its degradation. Lastly, we characterized 100- to 500-fold enhancement in sensitivity obtained from MB+-specific eosin Y amplification, highlighting the advantages of using dual photocatalyst signal amplification.
Molecular level, mechanistic understanding of the roles of reactive oxygen species (ROS) in a variety of pathological conditions is hindered by the difficulties associated with determining the ...concentration of various ROS species. Here, we present an approach that converts fold-change in the signal from an intracellular sensor of hydrogen peroxide into changes in absolute concentration. The method uses extracellular additions of peroxide and an improved biochemical measurement of the gradient between extracellular and intracellular peroxide concentrations to calibrate the intracellular sensor. By measuring peroxiredoxin activity, we found that this gradient is 650-fold rather than the 7-10-fold that is widely cited. The resulting calibration is important for understanding the mass-action kinetics of complex networks of redox reactions, and it enables meaningful characterization and comparison of outputs from endogenous peroxide generating tools and therapeutics across studies.
Hydrogen peroxide (H2O2) acts as a signaling molecule via its reactions with particular cysteine residues of certain proteins. Determining the roles of direct oxidation by H2O2 versus disulfide ...exchange reactions (i.e. relay reactions) between oxidized and reduced proteins of different identities is a current focus. Here, we use kinetic modeling to estimate the spatial and temporal localization of H2O2 and its most likely oxidation targets during a sudden increase in H2O2 above the basal level in the cytosol. We updated a previous redox kinetic model with recently measured parameters for HeLa cells and used the model to estimate the length and time scales of H2O2 diffusion through the cytosol before it is consumed by reaction. These estimates were on the order of one micron and one millisecond, respectively. We found oxidation of peroxiredoxin by H2O2 to be the dominant reaction in the network and that the overall concentration of reduced peroxiredoxin is not significantly affected by physiological increases in intracellular H2O2 concentration. We used this information to reduce the model from 22 parameters and reactions and 21 species to a single analytical equation with only one dependent variable, i.e. the concentration of H2O2, and reproduced results from the complete model. The reduced kinetic model will facilitate future efforts to progress beyond estimates and precisely quantify how reactions and diffusion jointly influence the distribution of H2O2 within cells.
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•We reduced a kinetic model of 22 parameters and 21 species to a single equation.•We analyzed the fraction of peroxiredoxin oxidized by H2O2.•We estimated length and time scales of H2O2 that is generated within the cytosol.•We used a H2O2 consumption parameter and diffusivity to obtain scale estimates.•Cytosolic H2O2 is consumed within ~1ms and ~1μM of its generation.
Despite the numerous applications of eosin Y as an organic photoredox catalyst, substantial mechanistic aspects of the photoredox process have remained elusive. Through deductive, steady-state ...kinetic studies, we first propose a mechanism for alkaline, aqueous photoredox catalysis using eosin Y, triethanolamine, and oxygen, integrating photo- and nonphotochemical steps. The photoredox cycle begins with a single-electron transfer (SET) induced when eosin Y absorbs green light. This photoinduced SET leads to the formation of a metastable radical trianion that can be fully reduced to inactivated leuco eosin Y via H+/e–/H+ transfer or regenerated to eosin Y via ground-state SET to oxygen. Since the radical trianion absorbs violet light, we tested the effect of radical trianion photoexcitation on catalyst regeneration. We found that excitation of the metastable radical trianion in the presence of a threshold concentration of oxygen enabled ∼100% regeneration of eosin Y. The response to violet light supports the important role of the metastable radical trianion and indicates that the photoredox cycle can be closed via a secondary photoinduced SET event. The idea of photoredox cycles with two consecutive photoinduced electron transfer (PET) steps is not intuitive and is introduced as a tool to increase photocatalyst turnover by selectively favoring regeneration over “death”. This alludes to the Z-scheme in biological photosynthesis, where multiple PET reactions, often triggered by different frequencies, promote highly selective biochemical transformations by precluding unproductive SET events in plants and bacteria. We expect that the simple Z-scheme model introduced here will enable more efficient use of organic photoredox catalysts in organic and materials chemistry.
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