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I., Background: Human induced pluripotent stem cell (hiPSC) derived cardiomyocytes (CMs) have been utilized in drug toxicity evaluation, drug discovery, and treating heart failure patients, showing substantial effects. Ensuring the quality, purity, and maturation of hiPSC-CMs during large-scale production is crucial. There is a growing demand for a novel method to characterize cell molecular profiles without labels and without causing damage. II., Methods: In this study, we employed label-free Raman microscopy to evaluate hiPSC-derived CMs. The study involved the characterization of cell molecular profiles without labels and without causing damage. The correlation between Raman spectroscopy of specific components, such as cytochrome c and myoglobin, and CM purity and maturation following hiPSC differentiation was investigated. Additionally, the validation of this correlation was performed by assessing mixtures of commercially available CMs (iCell cardiomyocytes2) and fibroblasts at various ratios as well as hiPSC-derived CMs with different efficiencies. Furthermore, CMs were matured using rapid pacing of traveling waves, and the Raman profiles of matured CMs were compared to those of immature ones. III., Results: Raman spectroscopy indicated that the cytochrome c and myoglobin showed correlation with the purity and maturation of CMs following differentiation of hiPSCs. This correlation was validated through experiments involving different CM-fibroblast mixtures and hiPSC-derived CMs with varying efficiencies. Moreover, matured CMs exhibited markedly different Raman profiles compared to immature ones, indicating the potential of Raman imaging as a tool for assessing CM maturation. IV., Conclusions: We discovered that Raman spectroscopy of certain components, such as cytochrome c and myoglobin, correlates with the CM purity and maturation following hiPSC differentiation. The findings of this study highlight the potential of label-free Raman microscopy as a nondestructive, high-content, and time-efficient method for quality control of hiPSC-derived CMs. This approach could significantly contribute to ensuring the quality and maturity of hiPSC-CMs for various applications in drug discovery and regenerative medicine.

In the realm of disease diagnostics, particularly for conditions such as proteinuria and hemoglobinuria, the quest for a method that combines accurate, label-free detection of protein compositions and their conformational changes remains a formidable challenge. In this study, we introduce an innovative Ag/Au plasmonic hybrid coupling nanoarray (Ag/Au PHCN) architecture marked by sub-10 nm interparticle gaps. These nanoarrays, leveraging plasmonic hybrid coupling and synergistic enhancement mechanisms, create a plethora of uniform surface-enhanced Raman spectroscopy (SERS) hotspots. The Ag/Au PHCN substrates demonstrated unparalleled sensitivity in the unmarked detection of hemoglobin (HGB), bovine serum albumin (BSA), and cytochrome C (Cyt.C) in bodily fluids, incorporating the advantages of high sensitivity, high reproducibility, durability, recyclability, and biocompatibility. Notably, the detection limits for BSA and HGB are unprecedented at 0.5 and 5 ng/mL, respectively. This achievement sets a new benchmark for label-free protein detection using two-dimensional nanostructures. Crucially, the Ag/Au PHCNs possess the novel capability to discern protein conformational changes post denaturation, underscoring their potential in probing protein functionalities. Most importantly, these nanoarrays can differentiate between normal and proteinuria-affected urine samples and monitor protein content variations over time, heralding a new era in clinical diagnostics with particular relevance to proteinuria and hemoglobinuria detection.

Mass spectrometry (MS) using an electron multiplier for intact protein analysis remains limited. Because of the massive size and complex structure of proteins, the slow flight speed of their ions results in few secondary electrons and thus low detection sensitivity and poor spectral resolution. Thus, we present a compact ion trap-mass spectrometry approach to directly detect ion packets and obtain the high-resolution molecular signature of proteins. The disturbances causing deviations of ion motion and mass conversion have been clarified in advance. The radio frequency waveform used to manipulate ions is proposed to be a sequence of constant-frequency steps, interconnected by short time-outs, resulting in least dispersive distortion. Furthermore, more such constant-phase conjunctions are arranged in each step to compensate for fluctuations resulting from defects in the system and operation. In addition, two auxiliary pulses are generated in the right phase of each step to select ions of a specific secular state to detect one clean and sharp spectral line.This study demonstrates a top-down approach for the MS measurement of cytochrome C molecules, resulting in a spectral profile of the protein in its natural state at a resolution of 20 Da. Additionally, quick MS scans of other proteins were performed.

Cytochrome c (cyt c) has been found to play a function in apoptosis in cell-free models. This work presents the creation of molecularly imprinted conducting poly(3, 4-ethylenedioxythiopene) (MIPEDOT) on the surface of a screen printed carbon electrode (SPCE) for cyt c. Cyt c was imprinted by electropolymerization due to the presence of an EDOT monomer hydrophobic functional group on SPCE, using CV to obtain highly selective materials with excellent molecular recognition ability. MIPEDOT was characterized by CV, EIS, and DPV using ferricyanide/ferrocyanide as a redox probe. Further, the characterization of the sensor was accomplished using SEM for surface morphological confirmation. Using CV, the peak current measured at the potential of +1 to -1 V (vs. Ag/AgCl) is linear in the cyt c concentration range from 1 to 1200 pM, showing a remarkably low detection limit of 0.5 pM (sensitivity:0.080 μA pM). Moreover, the applicability of the approach was successfully confirmed with the detection of cyt c in biological samples (human plasma). Similarly, our research has proven a low-cost, simple, and efficient sensing platform for cyt c detection, rendering it a viable tool for the future improvement of reliable and exact non-encroaching cell death detection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The design of synthetic analogs of cytochrome c oxidase (CcO) is a formidable task due to its intricate structure encompassing multiple metal prosthetic sites and protein subunits. In recent years, artificial enzymes based on alloy nanomaterials have garnered significant attention due to the alloy design approach holds promise for the effective tuning of the properties of metal catalysts. In this study, we present copper-cerium alloy nanozymes (Cu-Ce-a NEs), where Cu mimics the active site of CcO, while Ce endows the alloy phase and enhances the capacity to catalyze the oxidation to cytochrome c (Cyt c). Cu-Ce-a NEs functionally mimics CcO, the terminal enzyme in the respiratory electron transport chain (ETC), by catalyzing the four-electron reduction of dioxygen to water. Utilizing the CcO-like properties of Cu-Ce-a NEs, we successfully implemented the electrochemical detection of Cyt c. The Cu-Ce-a NEs based electrochemical sensor revealed a favorable linear range spanning from 2 to 20 μM Cyt c, with a detection limit (LOD) of 2 μM. This method demonstrates high accuracy in Cyt c quantitation in pharmaceuticals, with results closely aligning with the actual concentrations. This finding not only offers new perspectives in the design of enzyme analogs, but also underscores the potential of this method for clinical Cyt c detection, highlighting its significance in biomedical research and diagnostics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background and Objectives : Most patients who are successfully resuscitated from cardiac arrest remain comatose, and only half regain consciousness 72 h after the arrest. Neuroprognostication methods can be complex and even inconclusive. As mitochondrial components have been identified as markers of post-cardiac-arrest injury and associated with survival, we aimed to investigate cytochrome c and mtDNA in comatose patients after cardiac arrest to compare neurological outcomes and to evaluate the markers' neuroprognostic value. Materials and Methods : This prospective observational study included 86 comatose post-cardiac-arrest patients and 10 healthy controls. Cytochrome c and mtDNA were determined at admission. Neuron-specific enolase (NSE) was measured after 72 h. Additional neuroprognostication methods were performed when patients remained unconscious. Cerebral performance category (CPC) was determined. Results : Cytochrome c was elevated in patients compared to healthy controls (2.029 [0.85-4.97] ng/mL vs. 0 [0.0-0.16], p < 0.001) but not mtDNA (95,228 [52,566-194,060] vs. 41,466 [28,199-104,708] copies/μL, p = 0.074). Compared to patients with CPC 1-2, patients with CPC 3-5 had higher cytochrome c (1.735 [0.717-3.40] vs. 4.109 [1.149-8.457] ng/mL, p = 0.011), with no differences in mtDNA (87,855 [47,598-172,464] vs. 126,452 [69,447-260,334] copies/μL, p = 0.208). Patients with CPC 1-2 and CPC 3-5 differed in all neuroprognostication methods. In patients with good vs. poor neurological outcome, ROC AUC was 0.664 ( p = 0.011) for cytochrome c, 0.582 ( p = 0.208) for mtDNA, and 0.860 ( p < 0.001) for NSE. The correlation between NSE and cytochrome c was moderate, with a coefficient of 0.576 ( p < 0.001). Conclusions : Cytochrome c was higher in comatose patients after cardiac arrest compared to healthy controls and higher in post-cardiac-arrest patients with poor neurological outcomes. Although cytochrome c correlated with NSE, its neuroprognostic value was poor. We found no differences in mtDNA.

Cytochrome c (Cyt-c) was commonly an intrinsic biomarker for a variety of cellular characteristics, such as respiration, energy levels, and apoptosis. Herein, a simple fluorescence sensor was constructed for the detection of Cyt-c in buffer and real serum samples. The carbon dots doped with Tb 3+ on the premise of 1-(2-pyridylazo)-2-naphthol (PAN) were fabricated and used as a dual-emission ratiometric fluorescent probe for detecting Cyt-c based on the internal filtering effect (IFE). As a fluorescent probe for ultra-sensitive detection, Cyt-c was quantitatively detected at different concentrations from 1 to 1000 nM. The fluorescent detection method for Cyt-c showed a good linear relationship from 1 to 50 nM, and the limit of detection (LOD) was 0.35 nM. In the recovery range of 101.27-103.39 % in human serum samples, the relative standard deviation (RSD) was less than 3.27 % (n = 3). In the end, the possible structures of CDs were predicted by DFT theoretical simulation calculations. All the results proved the ability of carbon dots as fluorescent probes to detect biomarkers and the application prospects in bioanalysis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: The concentration of cytochrome C is demonstrated to be an effective indicator of the microbial corrosion strength of metals. Traditional cytochrome C sensor can detect cytochrome C with a low detection limit, but their use is limited by their high cost, cumbersome operation, and susceptibility to malignant environments. In addition, studies on the monitoring of cytochrome C in the field of microbial corrosion has still not been carried out. Therefore, there is a need for a highly sensitive, selective, low-cost, anti-interference, and stable cytochrome C sensor with online monitoring and remote sensing capabilities for in-situ measurement of microbial corrosion strength., Results: This paper proposed a highly sensitive label-free fiber-optic sensor based on Mach-Zehnder interferometer (MZI) for in-situ measurement of the microbial corrosion marker cytochrome C. Two-dimensional Ti 2 C-MXene material is uniformly immobilized onto the surface of the sensing area to improve the sensitivity, hydrophilicity, and specific surface area of the sensing area, as well as to facilitate the immobilization of specific sensitive materials. The cytochrome C antibody is modified on the surface of Ti 2 C-MXene to specifically recognize cytochrome C, whose concentration variation can be measured by monitoring the spectral shift of MZI sensor. Results demonstrate a measurement sensitivity of 1.428 nm/μM for cytochrome C concentrations ranging from 0 to 7.04 μM. The detection limit of the sensor is calculated to be 0.392 μM with remarkable performance, including selectivity, stability, and reliability. Besides, the measurement result of the proposed sensor in real microbial corrosive environment is consistent with that of the ideal environment., Significance and Novelty: This is the first instance of achieving in-situ and label-free measurement of cytochrome C by using a fiber-optic MZI sensor, which undoubtedly provides a feasible solution for the effective monitoring of microbial metal corrosion in the environment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

A recently developed proteolytic reactor, designed for protein structural investigation, was coupled to ion mobility mass spectrometry to monitor collisional cross section (CCS) evolution of model proteins undergoing trypsin-mediated mono enzymatic digestion. As peptides are released during digestion, the CCS of the remaining protein structure may deviate from the classical 2/3 power of the CCS-mass relationship for spherical structures. The classical relationship between CCS and mass (CCS = A × M 2/3 ) for spherical structures, assuming a globular shape in the gas phase, may deviate as stabilizing elements are lost during digestion. In addition, collision-induced unfolding (CIU) experiments on partially digested proteins provided insights into the CCS resilience in the gas phase to ion activation, potentially due to the presence of stabilizing elements. The study initially investigated a model peptide ModBea (3 kDa), assessing the impact of disulfide bridges on CCS resilience in both reduced and oxidized forms. Subsequently, β-lactoglobulin (2 disulfide bridges), calmodulin (Ca 2+ coordination cation), and cytochrome c (heme) were selected to investigate the influence of common structuring elements on CCS resilience. CIU experiments probed the unfolding process, evaluating the effect of losing specific peptides on the energy landscapes of partially digested proteins. Comparisons of the TW CCS N2→He to trend curves describing the CCS/mass relationship revealed that proteins with structure-stabilizing elements consistently exhibit TW CCS N2→He and greater resilience toward CIU compared to proteins lacking these elements. The integration of online digestion, ion mobility, and CIU provides a valuable tool for identifying structuring elements in biopolymers in the gas phase.

This study explores the innovative field of pulsed direct current arc-induced nanoelectrospray ionization mass spectrometry (DCAI-nano-ESI-MS), which utilizes a low-temperature direct current (DC) arc to induce ESI during MS analyses. By employing a 15 kV output voltage, the DCAI-nano-ESI source effectively identifies various biological molecules, including angiotensin II, bradykinin, cytochrome C, and soybean lecithin, showcasing impressive analyte signals and facilitating multicharge MS in positive- and negative-ion modes. Notably, results show that the oxidation of fatty acids using a DC arc produces [M + O - H] - ions, which aid in identifying the location of C═C bonds in unsaturated fatty acids and distinguishing between isomers based on diagnostic ions observed during collision-induced dissociation tandem MS. This study presents an approach for identifying the sn -1 and sn -2 positions in phosphatidylcholine using phosphatidylcholine and nitrate adduct ions, accurately determining phosphatidylcholine molecular configurations via the Paternò-Büchi reaction. With all the advantages above, DCAI-nano-ESI holds significant promise for future analytical and bioanalytical applications.

This study investigated changes in mitochondrial lipid molecules and their potential associations with Longissimus lumborum (LL) and Psoas major (PM) quality during storage. A total of 1610 lipid species that matched with 36 lipid classes were identified from isolated mitochondria. PM had more key lipid molecules at storage d-1 including diacylglycerol and triacylglycerol (may play roles in membrane stability), phosphatidylethanolamine, acyl carnitine and cardiolipin (involved in energy metabolism), and cardiolipin and phosphatidylserine (important factors in apoptosis). Correspondingly, with extended storage time, mitochondrial structure, cytochrome c and reactive oxygen species (ROS) were changed, and muscle oxidation intensified. These changes have close associations with shear force and water holding capacity (WHC). Compared with LL, PM had higher content of lipid classes, more mitochondrial ROS, greater muscle oxidation, and lower shear force and WHC. These findings provided new insights into the effects of lipidome on mitochondrial structure, ROS and cytochrome c, and their potential associations with beef quality., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Candida albicans  is a widespread disease-causing yeast affecting humankind, which leads to urinary tract, cutaneous and various lethal systemic infections. As this infection rate steadily increases, it is becoming a significant public health problem. Recently, Caesalpinia bonduc  has received much attention from researchers due to its diverse pharmacological properties, including antimicrobial effects. Accordingly, we first planned to explore the in-vitro anticandidal potential of three extracts obtained from  C. bonduc  seeds against four  Candida  species. Initially, the anticandidal activity of the seed extracts was checked by the microdilution technique. Out of three seed extracts tested, ethanolic extract of  C. bonduc  seed (EECS) recorded the best activity against  C. albicans . Hence, we next aimed to find out the anticandidal mechanism of EECS in  C. albicans . The liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) analysis showed that the major compounds present in the EECS were tocopherols, fucosterol, linoleic acid, β-amyrin, β-sitosterol, campesterol, cassane furanoditerpene, Norcassane furanoditerpene and other diterpenes. To evaluate the cell death mechanism in  C. albicans , a series of parameters related to apoptosis, viz., reactive oxygen species (ROS) production, membrane permeability, mitochondrial membrane potential, release of cytochrome c , DNA fragmentation, nuclear condensation, increased Ca 2+ level in cytosolic and mitochondrial and activation of metacaspase, were analyzed. The results showed that EECS treatment resulted in the elevation of ROS, which leads to plasma membrane permeability   in C. albicans . Annexin V staining further confirms the early stage of apoptosis through phosphatidylserine (PS) externalization. We further inspected the late apoptotic stage using DAPI and TUNEL staining assays. From the results, it can be concluded that EECS triggered mitochondrial dysfunction by releasing high levels of ROS, cytochrome c and Ca 2+ resulting in the activation of metacaspase mediated apoptosis, which is the central mechanism behind the cell death of  C. albicans . Finally, a  Galleria mellonella - C. albicans  infection system was employed to assess the  in-vivo  potential of EECS. The outcomes displayed that the EECS considerably enhanced the recovery rate of  G. mellonella  larvae from infection after the treatment. Additionally, EECS also recorded low hemolytic activity. This study thus spotlights the anticandidal potential and mechanism of action of EECS against  C. albicans  and thus delivers a promising treatment approach to manage  C. albicans  infection in the future., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Sasidharan, Nishanth and Nair.)

The implementation of a reliable, rapid, inexpensive, and simple method for whole-proteome identification would greatly benefit cell biology research and clinical medicine. Proteins are currently identified by cleaving them with proteases, detecting the polypeptide fragments with mass spectrometry, and mapping the latter to sequences in genomic/proteomic databases. Here, we demonstrate that the polypeptide fragments can instead be detected and classified at the single-molecule limit using a nanometer-scale pore formed by the protein aerolysin. Specifically, three different water-soluble proteins treated with the same protease, trypsin, produce different polypeptide fragments defined by the degree by which the latter reduce the nanopore's ionic current. The fragments identified with the aerolysin nanopore are consistent with the predicted fragments that trypsin could produce.

Nanowire-based optical biosensors with high sensitivity are highly desired for the detection of biological microenvironments and analysis of cellular processes. However, the current nanowire biosensors are mostly fabricated with metal and semiconductor materials, which are not suitable for long-term use in biological environments due to their incompatible and nondegradable properties. Biosensors based on biofriendly materials (e.g., spider silk) often do not have high enough sensitivity due to high losses or micron sizes. Here, polylactic acid (PLA), a polymer with high optical transparency, good biocompatibility, biodegradability, and flexibility, is used to fabricate nanowires using a directly drawing method for the first time. Because of the strong evanescent wave and abundant carboxyl groups on the surface of nanowires, an ultralow concentration sensing of cytochrome c is achieved with a limit of detection of 1.38 × 10 -17 M, which is much lower than other detection results using semiconductor/metal-based nanosensors (10 -6 to 10 -12 M). On this basis, a label-free and real-time monitoring of cell apoptosis is realized. In addition, by doping quantum dots, the functionalized PLA nanowires can also sense a change in pH. These results are suggestive of the potential for PLA nanowires applied in multifunctional biosensing and biodetection, pushing forward the photomedicine field.

To investigate the effects of Hericium erinaceus polysaccharide (HEP) on immunity in Muscovy duck reovirus (MDRV)-infected ducklings and explore its mechanism of action, an MDRV contact-infection model was established. Then, we investigated the influence of HEP on morphology of main immune organs in MDRV-infected ducklings by HE staining, while antioxidant capacity (T-AOC, MDA), serum protein levels (TP, ALB, GLO), complement levels (C 3 , C 4 ) and antibody levels (IgA, IgM, IgG) were detected. Apoptotic indexes (apoptosisi rate and FAS-L) were also quantified by TUNEL method and immunohistochemical staining. Meanwhile, FADD and CytC (apoptosis-related genes), were tested by quantitative RT-PCR. Results showed that HEP could reduce the injuries of immune organs caused by MDRV. Additionally, HEP markedly diminished MDA (p < 0.01), while significantly increased T-AOC, TP, ALB, GLO, C 3 , C 4 , IgA, IgM and IgG (p < 0.01 or p < 0.05). Then, HEP shifted apoptosis time to an early MDRV-infected stage and reduced apoptosis at later MDRV-infected stage. This was associated with changes of FADD and CytC. Collectively, our data suggested that HEP could reduce the immunesuppression by many ways, such as decreasing organs' injuries, improving antioxidant capacity, serum proteins levels, antibody levels and complement levels, while diminish the apoptosis by lowering the FADD and CytC., Competing Interests: Declaration of competing interest None., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Vibrational circular dichroism (VCD) spectroscopy has emerged as a powerful platform to quantify chirality, a vital biological property that performs a pivotal role in the metabolism of life organisms. With a photoelastic modulator (PEM) integrated into an infrared spectrometer, the differential response of a sample to the direction of circularly polarized light can be used to infer conformation handedness. However, these optical components inherently exhibit chromatic behavior and are typically optimized at discrete spectral frequencies. Advancements of discrete frequency infrared (DFIR) spectroscopic microscopes in spectral image quality and data throughput are promising for use toward analytical VCD measurements. Utilizing the PEM advantages incorporated into a custom-built QCL microscope, we demonstrate a point scanning VCD instrument capable of acquiring spectra rapidly across all fingerprint region wavelengths in transmission configuration. Moreover, for the first time, we also demonstrate the VCD imaging performance of our instrument for site-specific chirality mapping of biological tissue samples. This study offers some insight into future possibilities of examining small, localized changes in tissue that have major implications for systemic diseases and their progression, while also laying the groundwork for additional modeling and validation in advancing the capability of VCD spectroscopy and imaging.

Sample flow rate is one of the parameters that influence the sensitivity of electrospray ionization (ESI) mass spectrometry. By varying the sample flow rate, initial droplets of different sizes can be generated. Protein molecules in small droplets may form gas-phase ions earlier than the ones in large droplets. Here, we have systematically studied the influence of sample flow rate on the ESI charge state distributions (CSDs) of model proteins. A dedicated programmable sample flow rate scanner was used to infuse protein samples at different flow rates into a mass spectrometer. The synergistic influence of sample flow rate and various electrolytes (ammonium acetate, ammonium bicarbonate, ammonium formate, and piperidine) was studied. Significant alterations to the CSDs with increasing flow rate were observed. For example, in the presence of ammonium acetate, at low flow rates, lower charge states of proteins showed high intensities, while at high flow rates, ions related to higher charge states of proteins dominated the spectra. On the other hand, in the presence of piperidine, a significant reduction in the ion currents of all charge states was observed during the flow rate scan. Our observations suggest that at low flow rates the protein molecules follow a charged residue model of ionization mechanism, and at high flow rates-due to structural changes in protein molecules in large ESI droplets-the charged residue and chain ejection models can possibly coexist. We propose the use of sample flow rate scan as a way to reveal the influence of flow rate on the CSDs of the studied proteins.

Rationale: Ginsenosides are considered to be the main functional components in ginseng and possess various important pharmacological activities. The study of the interactions between ginsenosides and proteins is indispensable for understanding the pharmacological activities of ginsenosides. In this work, the interactions of ginsenosides with cytochrome c (cyt c) were investigated by native mass spectrometry and molecular docking simulations., Methods: The interactions of four ginsenosides (Rb 1 , Rb 3 , Rf, Rg 1 ) and cyt c in NH 4 OAc solution were investigated by electrospray ionization linear ion trap mass spectrometry (ESI-LTQ-MS). Molecular docking simulations of cyt c complexes were carried out by AutoDock., Results: The native mass spectrometry results showed that the four ginsenosides were directly bound to cyt c, with stoichiometric ratios of 1:1 and 2:1 in NH 4 OAc. The order of relative binding abilities of ginsenosides to cyt c obtained by ESI-MS was Rb 1  > Rb 3  > Rf > Rg 1 , which was consistent with the docking results. Moreover, molecular docking simulations also indicated potential binding sites of cyt c and ginsenosides. Hydrogen-bond interaction played a very important role in cyt c binding with ginsenosides., Conclusions: It has been demonstrated that native MS is a useful tool to investigate the interactions of ginsenosides with cyt c. Molecular docking is a good complement to ESI analysis, and can provide information on potential binding sites of cyt c-ginsenoside complexess. This strategy will be helpful to further understand the interactions of proteins and small molecules., (© 2020 John Wiley & Sons, Ltd.)

Single-cell DNA analysis technology has provided unprecedented insights into many physiological and pathological processes. In contrast, technologies that allow protein analysis in single cells have lagged behind. Herein, a method called single-cell Plasmonic ImmunoSandwich Assay (scPISA) that is capable of measuring signaling proteins and protein complexes in single living cells is described. scPISA is straightforward, comprising specific in-cell extraction and ultrasensitive plasmonic detection. It is applied to evaluate the efficacy and kinetics of cytotoxic drugs. It reveals that different drugs exhibit distinct proapoptotic properties at the single-cell level. A set of new parameters is thus proposed for comprehensive and quantitative evaluation of the efficacy of anticancer drugs. It discloses that metformin can dramatically enhance the overall anticancer efficacy when combined with actinomycin D, although it itself is significantly less effective. Furthermore, scPISA reveals that survivin interacts with cytochrome C and caspase-3 in a dynamic fashion in single cells during continuous drug treatment. As compared with conventional assays, scPISA exhibits several significant advantages, such as ultrahigh sensitivity, single-cell resolution, fast speed, and so on. Therefore, this approach may provide a powerful tool for wide, important applications from basic research to clinical applications, particularly precision medicine.

Designing a stable and sensitive luminol-based electrochemiluminescence (ECL) analytical platform in the neutral condition has attracted a lot of attention. Here, gold-nanoparticle-functionalized cobalt-nickel phosphate three-dimensional nanoice creams (Au@Co 3 Ni 3 (PO 4 ) 4 NICs) are successfully prepared via electrostatic interaction. Generally, cobalt-nickel phosphate nanoice creams (Co 3 Ni 3 (PO 4 ) 4 NICs) are synthesized via a mild hydrothermal method and functionalized via polyethylenimine (PEI). Then, Au NPs are adsorbed on the surface of Co 3 Ni 3 (PO 4 ) 4 NICs via Au-N weak interaction to fabricate Au@Co 3 Ni 3 (PO 4 ) 4 NICs. Owing to the important roles of Au@Co 3 Ni 3 (PO 4 ) 4 in exhibiting excellent electrocatalytic activity, as well as preventing the deposition of negatively charged oxidation product induced electrode passivation, luminol in the nanohybrids (LH-Au@Co 3 Ni 3 (PO 4 ) 4 ) gives strong and stable ECL intensity in the neutral conditions. Moreover, the ECL emission of luminol is obviously quenched based on the resonance energy transfer (RET) between luminol as donor and cytochrome c (Cyt c) as acceptor. Hence, a sensitive ECL biosensor is successfully fabricated for the quantitative determination of Cyt c in cell lysates and exhibits wide linear ranges of 1.0 × 10 -4 -0.5 × 10 -5 and 0.5 × 10 -5 -1.0 × 10 -8 M as well as a low detection limit of 2.48 nM. This novel sensing strategy will broaden the application of transition metal (Co, Ni) phosphates in bioassays.

The aim of this study was to investigate the combination effect of exercise training and eugenol supplementation on the hippocampus apoptosis induced by CPF. 64 adult male albino rats were randomly selected and devided into eight groups of eight including: control, exercise (EXE), chlorpyrifos (CPF), Control + Oil (Co + Oil), Control + DMSO (Co + DMSO), chlorpyrifos + eugenol (CPF + Sup), chlorpyrifos + exercise (CPF + Exe) and, chlorpyrifos + exercise + eugenol (CPF + Exe + Eu). Four experimental groups received intraperitoneal injection (5 days a week) of 3.0 mg/kg body weight CPF in DMSO for 6 consecutive weeks. The exercise groups performed aerobic 5 days per week over 4 weeks. Eugenol were administered by gavage. Finally, the animals were sacrificed using CO 2 gas (a half of the rats were anesthetized with ketamine and xylazine and then perfused) to evaluate hippocampus histology and parameters. The results of this study showed that CPF injection significantly decreased BDNF, AChE and ATP in CA1 area of the hippocampus (p ˂ 0.05). Also, CA1 apoptosis by tunnel assay, it was found that CPF receiving groups with different dosage, showed a significant increase compared to other groups, which was confirmed by increasing cytochrome C and procaspase-3 in CPF groups (p ˂ 0.05). The result of this study show that 4 weeks of exercise training and eugenol supplementation does not improve the destructive effects of CPF in CA1 area of the hippocampus. As a result, it is recommended that future studies longer periods for treatment with exercise and eugenol supplementation.

Traumatic brain injury (TBI), due to its high mortality and morbidity, is an important research topic. Apoptosis plays a pathogenic role in a series of neurological disorders, from neurodegenerative diseases to acute neurological lesions.In this study, we analyzed the association between apoptosis and the Glasgow Outcome Scale (GOS), to examine the potential of apoptosis as a biomarker for a TBI outcome. Patients with severe TBI were recruited at the Department of Neurosurgery, Wujin Hospital Affiliated with Jiangsu University, between January 2018 and December 2019. As a control group, healthy subjects were recruited. The concentrations of caspase-3, cytochrome c, sFas, and caspase-9 in the cerebrospinal fluid (CSF) were analyzed by enzyme-linked immunosorbent assay (ELISA). The association between the GOS and the clinical variables age, sex, initial Glasgow Coma Scale (GCS) score, intracranial pressure (ICP), cerebral perfusion pressure (CPP), initial computed tomography (CT) findings, and apoptotic factors was determined using logistic regression. The area under the receiver operator characteristic (ROC) curve (AUC), and thus the sensitivity and specificity of each risk factor, were obtained.The levels of caspase-3, cytochrome c, sFas, and caspase-9 in the TBI group were significantly higher than those in the control group (P < .05). The logistic regression results showed that ICP and caspase-3 were significant predictors of outcome at 6 months post-TBI (P < .05). The AUC was 0.925 and 0.888 for ICP and caspase-3, respectively. However, the AUC for their combined prediction was 0.978, with a specificity and sensitivity of 96.0% and 95.2%, respectively, showing that the combined prediction was more reliable than that of the 2 separate factors.We demonstrated that caspase-3, cytochrome C, sFas, and caspase-9 were significantly increased in the CSF of patients following severe TBI. Furthermore, we found that ICP and caspase-3 were more reliable for outcome prediction in combination, rather than separately.

A new amide-imine conjugate, 2-hydroxybenzoic acid-(2-hydroxybenzylidene)-hydrazide (L 1 ), is employed to prepare a single crystal X-ray structurally characterized poly-nuclear Cu(ii) complex (M1). M1 selectively and spatially interacts with cytochrome C (Cyt C) to allow fluorescence imaging of intracellular translocation events in living cells. Thus, direct visualization of a Cyt C translocation event during an apoptotic process is achieved for the first time. The binding constant and LOD are 7.52 × 10 4 M -1 and 34.0 nM, respectively.

Oxidative stress may result in different modes of cell death, such as necrosis, apoptosis and necroptosis. Currently, researchers are still striving to develop efficient tools/methods to distinguish the cell death modes in direct and label-free ways. In this study, we attempted to employ Raman micro-spectroscopy to observe the molecular changes in Candida utilis cells under oxidative stress induced by low-temperature plasma (LTP) and explore the spectroscopic biomarkers for the modes of cell death under oxidative stress. In this research, we confirmed that LTP could impose oxidative stress on the yeast cells, and recorded the changes of Raman signals of cytochrome c in the cells under LTP oxidative stress. Subsequently, we identified the biochemical and morphological characteristic features corresponding to different modes of cell death. Interestingly, we found that LTP under certain conditions could induce oxidative stress which caused the yeast cell death mainly by means of necroptosis, which was verified by Annexin V/PI, HMGB1 location assay and immunoprecipitation assay of the RIP1/RIP3 necrosome. Correspondingly, we also showed that the LTP induced necroptosis, associated with the increase of cytoplasmic Ca 2+ and mitochondrial ROS, the decrease of mitochondrial membrane potential, the release of oxidized cytochrome c from the mitochondrion to the cytoplasm, and the destruction of mitochondria in yeast cells. This work has therefore demonstrated that monitoring the redox state of cytochrome c using Raman micro-spectroscopy is very useful for distinguishing the modes of cell death and particularly may unveil the unique necroptosis process of cells under extrinsic oxidative stress.

Valorisation of industrial low-value waste residues was preconized. Hence, carbon dots (C-dots) were synthesized from wastewaters of the cork industry-an abundant and affordable, but environmentally-problematic industrial effluent. The carbon nanomaterials were structurally and morphologically characterised, and their photophysical properties were analysed by an ensemble of spectroscopy techniques. Afterwards, they were successfully applied as highly-sensitive fluorescence probes for the direct detection of haemproteins. Haemoglobin, cytochrome c and myoglobin were selected as specific targets owing to their relevant roles in living organisms, wherein their deficiencies or surpluses are associated with several medical conditions. For all of them, remarkable responses were achieved, allowing their detection at nanomolar levels. Steady-state and time-resolved fluorescence, ground-state UV-Vis absorption and electronic circular dichroism techniques were used to investigate the probable mechanisms behind the fluorescence turn-off of C-dots. Extensive experimental evidence points to a static quenching mechanism. Likewise, resonance energy transfer and collisional quenching have been discarded as excited-state deactivating mechanisms. It was additionally found that an oxidative, photoinduced electron transfer occurs for cytochrome c , the most electron-deficient protein. Besides, C-dots prepared from citric acid/ethylenediamine were comparatively assayed for protein detection and the differences between the two types of nanomaterials highlighted.

We report on a direct comparison of the separation performance in capillary nano-LC between commercial packed bed columns and the small-domain silica monoliths in applications. Octadecylsilylated monolithic silica capillary columns with a 50 and 100 µm inner diameter (i.d.) were prepared with a procedure providing domain sizes in the sub-2 µm range. The fabricated monolith columns could provide plate heights (H) of 4.0‒4.2 µm for hexylbenzene (retention factor (k) = 3.6) at an optimal linear velocity range under an isocratic condition, while showing a column permeability (K v0  = 1.6‒1.8 × 10 -14 m 2 ) comparable to that of a column packed with 3‒3.5 µm particles. When the peak capacity (n p ) for a cytochrome C digest was compared for variable gradient times (t G  = 15, 30, 60, and 120 min) and constant gradient steepness (b'), the present monolith columns could show a 30‒40% higher n p -value than the packed capillary column with 2 µm particles (e.g. n p  = 180 versus n p  = 259 at t G  = 30 min). The produced monolith columns showed a high chromatographic repeatability for both isocratic and gradient elution (e.g. relative standard deviation (n = 3, RSD (%)) = 0.5% for H, 2,6% for k, and 5.6% for K v0 in the isocratic mode using the 100 µm i.d.-columns). The present results show that the domain sizes which can now be achieved for capillary silica monoliths are sufficiently small to result in separation efficiencies that can successfully compete with the commercial packed bed columns available for use in nano-LC applications., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019. Published by Elsevier B.V.)

Protein analysis by desorption electrospray ionization mass spectrometry (DESI-MS) is limited and often accompanied by a mass-dependent loss in sensitivity as protein molecular weight increases. Previously, incomplete dissolution was identified as a potential contributing factor to this limitation for larger proteins. Here, we developed a unique two-step configuration in which a prewetting solvent is applied to the sample surface proximal to DESI analysis by a wetting quill to increase dissolution time and the detection of larger proteins. After optimizing the system with a mixture of proteins containing cytochrome c, myoglobin, and chymotripsinogen, we demonstrate the ability of delayed desorption to improve the analysis of larger proteins such as bovine serum albumin. Albumin and other serum proteins, including even larger ones, were also detected directly from diluted goat serum. An additional feature of this technique is the ability to deliver multiple solvents with potential synergistic or cooperative effects. For example, when using acetonitrile solutions of formic acid and ammonium bicarbonate as the prewetting and DESI spray solvent, respectively, the intensity of chymotrypsinogen improved dramatically compared to controls but less so for smaller proteins such as myoglobin and cytochrome c. Adduct removal was also observed for all proteins. These early results demonstrate the ability of this two-step technique for the use of multiple additives and increased dissolution times compared to standard DESI-MS experiments.

A luminescent metal organic framework was prepared by encapsulating Zn-Ag-In-S quantum dots into "French fries"-like MIL-68(In) metal organic frameworks (ZAISQDs@MIL-68(In)). The ZAISQDs@MIL-68(In) had a maximum excitation wavelength at 370 nm and maximum emission wavelength at 620 nm. It was found that the ZAISQDs@MIL-68(In) was efficiently quenched by cytochrome c (Cyt c), which is an important biomarker of early cell apoptosis. The quenching mechanism was ascribed to be an inner filter effect and dynamic quenching of Cyt c towards the ZAISQDs@MIL-68(In), and the enrichment effect of MIL-68(In). Benefiting from the multiple advantages, ZAISQDs@MIL-68(In) was developed as an assay strategy of Cyt c with logarithmic relation between signal quenching and concentration in the range 0.02 to 3.5 μM. The linear equation was (F 0 -F)/F 0  = 0.5043 + 0.2678 × logc Cyt c with a detection limit of 8 nM. Cyt c released by drug induced apoptotic cells was determined by ZAISQDs@MIL-68(In), and this strategy has been utilized for the screening of anticancer drug activity. Graphical abstract Schematic representation of the synthesis of ZAISQDs@MIL-68(In) and its application for Cyt c and screening anticancer drug activity.

A new adsorbent based on pH-responsive polymer assisted aptamer functionalized magnetic nanoparticles was developed for specific recognition and efficient adsorption of proteins. Arising from the synergistic effect of specific affinity of apatamer on protein and tunable hydrophobic/hydrophilic property of pH-responsive polymer, the adsorbent exhibited excellent adsorption capacity for target protein. Notably, because of the pH-responsive property of the polymer, the adsorption and desorption process could be regulated through varying environmental pH. The resultant adsorbent that immobilized with lysozyme binding aptamer was successfully applied in specific recognition and efficient adsorption of lysozyme in egg white samples and good recovery results in the range of 95.2-103.2% were obtained. Moreover, the adsorbent immobilized with cytochrome C binding aptamer also exhibited satisfactory adsorption to cytochrome C. The synergistic effect of pH-responsive polymer and aptamer promoted the recognition selectivity and adsorption capacity to target protein, illustrating a facile way for construction of more specific protein adsorbents., (Copyright © 2019 Elsevier B.V. All rights reserved.)

To extend the ion structure analysis capability of Fourier transform mass spectrometry (FT-MS), both time-domain and frequency-domain methods have been developed to extract ion collision cross sections (CCS) from high resolution mass spectra in Fourier transform ion cyclotron resonance (FT-ICR) cells. In this study, a new frequency-domain method, namely the line shape fitting method, was proposed to calculate ion CCSs from FT-ICR mass spectra line shape. Besides experimental data, simulated data with precisely controlled signal to noise levels and decay factors were also applied to characterize this method. Compared with the linewidth correction method previously proposed by our group, this line shape fitting method is more tolerant to noise, data length, and sampling rate, thus providing more consistent results. More importantly, CCS measurements of angiotensin I, bradykinin, ubiquitin and cytochrome c show that the resolving power is improved with the new method., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Exposure of electrospray droplets to organic vapors was shown to dramatically reduce alkali-metal adduction on protein ions and shift protein charge states. Since DESI-MS is affected by similar adduct species as ESI-MS and shares similar ionization mechanisms, polar organic vapor additives should likewise also improve the DESI-MS analysis of proteins. Here the DESI spray was exposed to a variety of polar organic vapor additives. Head space vapors of polar organic solvents were entrained in nitrogen gas and delivered to the atmosphere inside a semi-enclosed plastic enclosure surrounding the spray plume. The vapors of acetone, acetonitrile, ethyl acetate, methanol, and water were investigated. Vapor dependent effects were observed with respect to changes in protein charge state distributions and signal intensities. With ethyl acetate vapor addition, the signal intensities of all proteins investigated were significantly increased, including proteins larger than 25 kDa such as carbonic anhydrase II and bovine serum albumin.

An enzyme-free aptameric nanosensor is presented for apoptosis assay. The method exploits the peroxidase-mimicking property of silver/platinum alloy nanoclusters (Ag/Pt NCs) and uses a Cyt c binding ssDNA aptamer. An extra-strand polycytosine (C 14 ) aptamer was designed as a template for synthesis of the Ag/Pt NCs. If cell lysate or purified Cyt c is placed in a polystyrene microplate, Cyt c will bind to the surface of the wells of a microtiterplate. On addition of Apt@Ag/PtNCs, it will associate with Cyt c and then catalytically oxidize colorless tetramethylbenzidine (TMB) in the presence of H 2 O 2 to give a blue colored oxidation product (TMB ox ) due to the peroxidase-mimicking property of the Ag/Pt NCs. Under optimal conditions, the absorbance of TMB at 660 nm is linearly enhanced as the concentration of Cyt c increases from 50.0 fM to 500 nM, and the detection limit is ~10 pM. The assay is simple, sensitive and cost effective in that it is enzyme-free, antibody-free and label-free. Graphical abstractSchematic diagram of the apoptosis assay on the basis of microplate well-coated mitochondrial cytochrome c releasing by using Aptamer@Ag/Pt NCs.

Titanium carbide quantum dots functionalized with ε-poly-L-lysine (PLL) were synthesized by sonication cutting and hydrothermal synthesis. The deprotonated Ti 3 C 2 MXene quantum dots (Ti 3 C 2 MQDs) exhibit excitation wavelength-dependent blue photoluminescence with typical excitation/emission peaks at 330/415 nm and a quantum yield of 22% due to strong quantum confinement. The fluorescence of ε-poly-L-lysine protected Ti 3 C 2 MQDs (PLL-protected Ti 3 C 2 MQDs) is reduced via an inner filter effect after the addition of cytochrome c (cyt-c). Response to cyt-c is linear in the 0.2 to 40 μM concentration range and the detection limit is 20.5 nM. In the presence of trypsin, cyt-c is hydrolyzed to small peptides, and the Fe 3+ ion in cyt-c probably is reduced to Fe 2+ with the aid of the digestive enzyme. This results in the restoration of the blue fluorescence of the modified MQDs. Fluorescence increases linearly in the 0.5 to 80 μg mL -1 trypsin concentration range with the detection limit of 0.1 μg mL -1 . The method was successfully applied to the determination of cyt-c and trypsin in spiked serum samples. Graphical abstractSchematic of a method for the fluorometric "turn-off-on" determination of cytochrome c and trypsin based on ε-poly-L-lysine (PLL) protect MXene quantum dots (Ti 3 C 2 MQDs).

We report on facile synthesis and characterization of phosphate-functionalized polymer dots (PDs) by doping tributyl phosphate (TBP) in a semiconducting polymer poly[9,9-dioctylfluorenyl-2,7-diyl)-co-1,4-benzo-{2,10-3}-thiadiazole)] (PFBT). Then, the prepared TBP@PFBT PDs were used to develop a very high sensitive probe for detection Fe 3+ , Cu 2+ ions and Cytochrome c based on aggregation induced fluorescence off mechanism. The PDs exhibited a linear dynamic range for Fe 3+ from 0.1 to 2 nM with a detection limit of 30 pM and for Cu 2+ from 2.0 to 50.0 nM with a detection limit of 0.35 nM. Meanwhile, this probe showed a linear dynamic range for Cyt c from 175 to 1750 pM with a detection limit of 32.7 pM. The TBP@PFBT PDs is a simple, one-step, fast, non-invasive, label-free, and inexpensive probe that is capable of online apoptosis monitoring response to drugs with an ever-present opportunity to contribute in a variety of in-vitro and in-vivo biological applications. We also obtained sharp, specific 2D and 3D imaging results for early stage apoptosis in breast cancer cells. Moreover, this technique possesses the advantage of rapid determination of Fe 3+ ion in biological or environmental samples. Importantly, this label-free assay provides short determination time of only a few min, easy operation and very low LOD allowing 100-4000 times increased in sensitivity over previously reported probes, together with high selectivity without need to using biorecognition elements like enzymes, antibodys and/or aptamers. Such excellent features make the TBP@PFBT PDs an excellent probe for successful apoptosis imaging in live cells., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A chemical tag enhances peptide detection by multiple orders in mass spectrometry. The substantial improvement in the peptide mapping along with simplified and enhanced fragmentation pattern enables the unambiguous sequencing of a protein and antibody. The chemoselective sensitivity booster provides a tool for remarkably improved analysis of protein bioconjugates.

Protein structures respond to changes in their chemical and physical environment. However, studying such conformational changes is notoriously difficult, as many structural biology techniques are also affected by these parameters. Here, the use of photo-crosslinking, coupled with quantitative crosslinking mass spectrometry (QCLMS), offers an opportunity, since the reactivity of photo-crosslinkers is unaffected by changes in environmental parameters. In this study, we introduce a workflow combining photo-crosslinking using sulfosuccinimidyl 4,4'-azipentanoate (sulfo-SDA) with our recently developed data-independent acquisition (DIA)-QCLMS. This novel photo-DIA-QCLMS approach is then used to quantify pH-dependent conformational changes in human serum albumin (HSA) and cytochrome C by monitoring crosslink abundances as a function of pH. Both proteins show pH-dependent conformational changes resulting in acidic and alkaline transitions. 93% and 95% of unique residue pairs (URP) were quantifiable across triplicates for HSA and cytochrome C, respectively. Abundance changes of URPs and hence conformational changes of both proteins were visualized using hierarchical clustering. For HSA we distinguished the N-F and the N-B form from the native conformation. In addition, we observed for cytochrome C acidic and basic conformations. In conclusion, our photo-DIA-QCLMS approach distinguished pH-dependent conformers of both proteins.

Heteroatom doping can endow graphene quantum dots (GQDs) with various new or improved structural, optical and physicochemical properties. In contrast to the widely reported oxygen, nitrogen or sulfur doping in GQDs, simple and scalable synthesis of boron-doped GQDs (B-GQDs) with high yield and quantum yields remains challenge. In this work, B-GQDs are one-step synthesized and serve as the fluorescence probes for the fabrication of sensors towards Fe 3+ ion or phosphate (Pi) as well as biosensor towards cytochrome C (Cyt C). The B-GQDs are facile synthesized using one-step bottom-up molecular fusion between 1,3,6-trinitropyrene and borax in sodium hydroxide under hydrothermal process. The synthesis can be performed using large volume autoclave (500 ml) with a high yield of 71%, indicating possibility for gram-scale production of B-GQDs. The as-prepared B-GQDs exhibit single or bilayer graphene structure, high crystallinity, uniform size, bright (absolute photoluminescence quantum yield of 16.8%) and excitation-independent green fluorescence (maximum excitation wavelength and emission wavelength of 480 nm and 520 nm, respectively). Successful doping of B atoms in the lattice of GQDs enables high selectivity towards Fe 3+ . Based on quenching of fluorescence of B-GQDs by Fe 3+ (turn-off model), detection of Fe 3+ (with limit of detection-LOD of 31.2 nM) and Fe 3+ -rich Cyt C (with LOD of 5.9 μg/ml) are demonstrated. As Pi can recover Fe 3+ -quenched fluorescence of B-GQDs (turn-off-on model), indirect fluorescent detection of Pi is also achieved with LOD of 340 nM. In addition, detection of Fe 3+ , Cyt C and Pi in real samples is achieved., (Copyright © 2019 Elsevier B.V. All rights reserved.)

During apoptosis process, the release of cytochrome c (Cyt c) is considered to be a key factor in the intrinsic pathway and is often defined as no regression point. Quantitative detection of intracellular Cyt c remains a challenge. Herein, we have developed surface-enhanced Raman scattering (SERS)-fluorescence dual-mode nanosensors for the quantitative assay of Cyt c in living cells. Dual signal detection was achieved by constructing gold nanotriangles (AuNTs) nanosensors capable of specifically recognizing Cyt c. The nanosensors were prepared by modifying the aptamer of Cyt c on AuNTs and connecting the complementary strands modified with Cy5. The AuNTs provided both enhanced SERS signals and fluorescence quenching effects. Once cells were induced by external stimulus (such as toxins) to release Cyt c, Cyt c would specifically bind to its aptamer, and the complementary strands modified with Cy5 would detach which would result in weakened SERS signal and recovery of fluorescence signal. The experimental results showed that the nanosensors not only had excellent selectivity and sensitivity but also realized real-time monitoring of Cyt c translocation event from mitochondria to cytoplasm. The SERS and fluorescence intensity showed good linear relationship with Cyt c concentration ranging from 0.044 to 9.95 μM and achieved a minimum limit of detection (LOD) of 0.02 μM in living cells. The accuracy of intracellular Cyt c quantitative results was more than 90% compared with the ELISA results.

A stable sandwiched electrochemiluminescence (ECL) aptasensor was originally constructed established upon Ru(bpy) 3 2+ -doped silica nanoparticles (RuSiO 2 NPs) with ferrocene carboxylic acid-aptamer (Fc-aptamer) to quantitatively detect cytochrome c (Cyt C). Herein, RuSiO 2 NPs and Fc-aptamer were respectively prepared through the microemulsion method and amide reaction to fabricate the ECL aptasensor. Furthermore, Fc-aptamer was used as quenching probe for quenching the ECL emission of RuSiO 2 NPs. In detail, RuSiO 2 NPs were primarily immobilized onto the electrodes by the film-forming function of chitosan. Subsequently, the aptamer was incubated onto the decorated GCE via crosslinking with glutaraldehyde (GA). After Cyt C was connected to the GCE via immunoreaction, Fc-aptamer was immobilized onto the modified electrodes owing to the specific recognition between antigens and aptamer. Ultimately, ECL signals markedly descended owing to the poor electricity conductivity of proteins and superior quenching effect of Fc-aptamer. Under optimum conditions, the designed ECL aptasensor indicated an accurate analysis for Cyt C in a rang of 0.001-100 nM with a detection limit of 0.48 pM (S/N = 3)., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The release of cytochrome C (Cyt C) plays an important role in apoptosis. In this study, selective and sensitive detection of Cyt C based on an aptamer strategy coupled with MCE was developed. Following the binding of a specific aptamer to Cyt C, the aptamer exhibited an irregular state, reducing the binding affinity of a fluorescent probe, and thus preventing the aptamer-Cyt C complexes from detection within the MCE. The height of the detection peak of the residual aptamer linearly decreased, and therefore, the difference in peak height of residual aptamer compared to that of the initial aptamer was used to quantify the captured protein concentration. Experimental conditions such as incubation time, pH, temperature, and ionic strength were optimized. A measurement of Cyt C concentration by MCE was achieved within 135 s, with a limit of detection as low as 0.4 nM. The proposed method has high selectivity and good stability for the detection of Cyt C. The experimental results demonstrate that this method is quick, consumes only a small quantity of sample, is highly selectivity and exhibits high sensitivity., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Quantitative detection of protein biomarkers is crucial to medical diagnosis. Fluorescent probes have been frequently used for protein detection, but they suffered from various weaknesses such as lack of versatility. In particular, most of the reported probes were not capable of simultaneous qualitative and quantitative detection for various proteins. In this paper, we developed novel nanoparticle array-based near-infrared (NIR) ratiometric probes for potent protein analysis, in which the specific protein was able to be distinguished and quantitated within a group of 11 common proteins. The activity of β-galactosidases (β-gal) was temporarily inhibited by the adsorption to magnetic nanoparticles and restored to certain content by replacement with detected proteins, leading to distinctive readout of the enzyme-activatable NIR probe (DCM-β-gal). The readout of the sensor array against 11 proteins, as verified by isothermal titration calorimetry, was processed and transformed into canonical factors with the help of linear discrimination analysis. Moreover, the ratiometric signals of DCM-β-gal were translated to quantitatively detect proteins within the concentration range of 0-100 μg/mL. Based on clear differentiation within both two-dimensional and three-dimensional plots, different proteins could be detected with 100% accuracy with their concentration simultaneously determined, which endowed the sensing system with great potential in clinical diagnosis.

Previous studies have suggested that the loss in sensitivity of DESI-MS for large molecules such as proteins is due to the poor dissolution during the short time scale of desorption and ionization. An investigation into the effect of serine as a solvent additive leads to the interesting observation that there is a concentration-dependent improvement in protein signal intensity when micromolar to low millimolar concentrations of serine is combined with a suitable co-additive in DESI spray. This effect, however, was not observed during similar ESI-MS experiments, where the same solvents and proteins were sprayed directly into the MS inlet. This suggests that the mechanism of signal improvement in DESI is associated with the desorption step of proteins, possibly by facilitating dissolution or improving solubility of proteins on the surface in the solvent micro-layer formed during DESI. Other than poor dissolution, cation adduction such as by sodium ions is also a major contributing factor to the mass-dependent loss in sensitivity in both ESI and DESI, leading to an increase in limits of detection for larger proteins. The adduction becomes a more pressing issue in native-state studies of proteins, as lower charge states are more susceptible to adduction. Previous studies have shown that addition of amino acids to the working spray solution during ESI-MS reduces sodium adduction and can help in stabilization of native-state proteins. Similar to the observed reduction in sodium adducts during native-state ESI-MS, when serine is added to the desorbing spray in DESI-MS, the removal of up to 10 mM NaCl is shown. A selection of proteins with high and low pI and molecular weights was analyzed to investigate the effects of serine on signal intensity by improvements in protein solubility and adduct removal. Graphical Abstract.

A pepsin/FPXIII (protease from Aspergillus saitoi, type XIII) mixed bed column significantly improved the resolution of bottom-up hydrogen/deuterium exchange mass spectrometry (HDX-MS) data compared with a pepsin-only column. The HDX-MS method using the mixed bed column determined 65 amide hydrogen exchange rates out of one hundred cytochrome c backbone amide hydrogens. Different cleavage specificities of the two enzymes generated 138 unique high-quality peptic fragments, which allows fine sub-localization of deuterium. The exchange rates determined in this method are consistent within the current study as well as with the previous HDX-NMR study. High-resolution HDX-MS data can determine the exchange rate of each residue not the deuterium buildup curve of a peptic fragment. The exchange rates provide more precise and quantitative measurements of protein dynamics in a more reproducible manner. Graphical Abstract ᅟ.

Determination of collisional cross sections (CCS) by travelling wave ion mobility mass spectrometry (TWIM-MS) requires calibration against standards for which the CCS has been measured previously by drift tube ion mobility mass spectrometry (DTIM-MS). The different extents of collisional activation in TWIM-MS and DTIM-MS can give rise to discrepancies in the CCS of calibrants across the two platforms. Furthermore, the conditions required to ionize and transmit large, folded proteins and assemblies may variably affect the structure of the calibrants and analytes. Stable hetero-oligomeric phospholipase A 2 (PDx) and its subunits were characterized as calibrants for TWIM-MS. Conditions for acquisition of native-like TWIM (Synapt G1 HDMS) and DTIM (Agilent 6560 IM-Q-TOF) mass spectra were optimized to ensure the spectra exhibited similar charge state distributions. CCS measurements (DTIM-MS) for ubiquitin, cytochrome c, holo-myoglobin, serum albumin and glutamate dehydrogenase were in good agreement with other recent results determined using this and other DTIM-MS instruments. PDx and its β and γ subunits were stable across a wide range of cone and trap voltages in TWIM-MS and were stable in the presence of organic solvents. The CCS of PDx and its subunits were determined by DTIM-MS and were used as calibrants in determination of CCS of native-like cytochrome c, holo-myoglobin, carbonic anhydrase, serum albumin and haemoglobin in TWIM-MS. The CCS values were in good agreement with those measured by DTIM-MS where available. These experiments demonstrate conditions for analysis of native-like proteins using a commercially available DTIM-MS instrument, characterize robust calibrants for TWIM-MS, and present CCS values determined by DTIM-MS and TWIM-MS for native proteins to add to the current literature database. Graphical Abstract ᅟ.

Aim: The subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria in skeletal muscle appear to have distinct biochemical properties affecting metabolism in health and disease. The isolation of mitochondrial subpopulations has been a long-time challenge while the presence of a continuous mitochondrial reticulum challenges the view of distinctive SSM and IFM bioenergetics. Here, a comprehensive approach is developed to identify the best conditions to separate mitochondrial fractions., Methods: The main modifications to the protocol to isolate SSM and IFM from rat skeletal muscle were: (a) decreased dispase content and homogenization speed; (b) trypsin treatment of SSM fractions; (c) recentrifugation of mitochondrial fractions at low speed to remove subcellular components. To identify the conditions preserving mitochondrial function, integrity, and maximizing their recovery, microscopy (light and electron) were used to monitor effectiveness and efficiency in separating mitochondrial subpopulations while respiratory and enzyme activities were employed to evaluate function, recovery, and integrity., Results: With the modifications described, the total mitochondrial yield increased with a recovery of 80% of mitochondria contained in the original skeletal muscle sample. The difference between SSM and IFM oxidative capacity (10%) with complex-I substrate was significant only with a saturated ADP concentration. The inner and outer membrane damage for both subpopulations was <1% and 8%, respectively, while the respiratory control ratio was 16., Conclusion: Using a multidisciplinary approach, conditions were identified to maximize SSM and IFM recovery while preserving mitochondrial integrity, biochemistry, and morphology. High quality and recovery of mitochondrial subpopulations allow to study the relationship between these organelles and disease., (© 2018 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

Intrinsic properties of nickel have enabled its wide applications as an effective catalyst. In this study, nickel nanowires (Ni NWs) as electron donors for oxidized cytochrome c (Cyt c) are investigated, which are NW diameter, temperature, and pH value-dependent. The reductive and magnetic properties facilitate the Ni NWs to rapidly and conveniently reduce Cyt c in complicated biological samples. Moreover, we find that the Ni NWs combined with resonance Raman spectroscopy have specificity toward Cyt c detection in real biological samples, which is successfully used to distinguish the redox state of the released Cyt c from isolated mitochondria in apoptotic Hela cells. Moreover, rapid label-free Cyt c quantification can be achieved by surface-enhanced Raman spectroscopy with a limit of detection of 1 nM and long concentration linear range (1 nM-1 μM). The proposed Ni NWs-based reduction approach will significantly simplify the traditional biological methods and has great potential in the application of Cyt c-related apoptotic studies.

Coumarin (1,2-benzopyrone), an aromatic oxygen-containing heterocyclic compound, has various biological functions. Previous studies have demonstrated that coumarin and its derivatives exhibit antifungal activity against Candida albicans . In this study, we investigated the exact mechanism by which coumarin works against this fungus using Annexin V-FITC/PI double staining, TUNEL assay, and DAPI staining, and found that it induced a series of apoptotic features, including phosphatidylserine (PS) externalization, DNA fragmentation, and nuclear condensation. Moreover, it also induced cytochrome c release from the mitochondria to the cytoplasm and metacaspase activation. Further study revealed that intracellular reactive oxygen species (ROS) levels were increased and mitochondrial functions, such as mitochondrial membrane potential and mitochondrial morphology, were altered after treatment with coumarin. Cytosolic and mitochondrial Ca 2+ levels were also found to be elevated. However, pretreatment with ruthenium red (RR), a known mitochondrial Ca 2+ channel inhibitor, attenuated coumarin-mediated DNA fragmentation and metacaspase activity, indicating that the coumarin-induced C. albicans apoptosis is associated with mitochondrial Ca 2+ influx. Finally, coumarin was found to be low-toxic and effective in prolonging the survival of C. albicans -infected mice. This study highlights the antifungal activity and mechanism of coumarin against C. albicans and provides a potential treatment strategy for C. albicans infection.

Gas-phase hydrogen/deuterium exchange measured by mass spectrometry (gas-phase HDX-MS) is a fast method to probe the conformation of protein ions. The use of gas-phase HDX-MS to investigate the structure and interactions of protein complexes is however mostly unharnessed. Ionizing proteins under conditions that maximize preservation of their native structure (native MS) enables the study of solution-like conformation for milliseconds after electrospray ionization (ESI), which enables the use of ND 3 -gas inside the mass spectrometer to rapidly deuterate heteroatom-bound non-amide hydrogens. Here, we explored the utility of gas-phase HDX-MS to examine protein-protein complexes and inform on their binding surface and the structural consequences of gas-phase dissociation. Protein complexes ranging from 24 kDa dimers to 395 kDa 24mers were analyzed by gas-phase HDX-MS with subsequent collision-induced dissociation (CID). The number of exchangeable sites involved in complex formation could, therefore, be estimated. For instance, dimers of cytochrome c or α-lactalbumin incorporated less deuterium/subunit than their unbound monomer counterparts, providing a measure of the number of heteroatom-bound side-chain hydrogens involved in complex formation. We furthermore studied if asymmetric charge-partitioning upon dissociation of protein complexes caused intermolecular H/D migration. In larger multimeric protein complexes, the dissociated monomer showed a significant increase in deuterium. This indicates that intermolecular H/D migration occurs as part of the asymmetric partitioning of charge during CID. We discuss several models that may explain this increase deuterium content and find that a model where only deuterium involved in migrating charge can account for most of the deuterium enrichment observed on the ejected monomer. In summary, the deuterium content of the ejected subunit can be used to estimate that of the intact complex with deviations observed for large complexes accounted for by charge migration. Graphical abstract ᅟ.

A quartz crystal microbalance (QCM) sensor for detecting cytochrome c based on an oriented surface epitope imprinted polymer was fabricated in this paper. By using the palmitic acid-modified epitope of cytochrome c as the template and the 3-aminopropyltriethoxysilane as the monomer, we prepared a new oriented surface epitope imprinted polymer by the reverse microemulsion polymerization. The prepared oriented imprinted polymer had better imprinting effect than the non-oriented imprinted polymer. And compared to previous studies, this polymerization method is simple and could be carried out at room temperature in the presence of oxygen, under regular atmospheric conditions. Then, by combining the advantages of molecularly imprinted polymers and QCM sensors, we used the prepared polymer to establish a QCM sensor. The described sensor showed good sensitivity and selectivity towards cytochrome c. The linear range was from 0.005 μg mL -1 to 0.050 μg mL -1 and the detection limit was 3.6 ng mL -1 which is lower than most of previous works. Besides, it could be used for real sample analysis and had satisfactory reproducibility and accuracy. This work proposed a new way of fabricating oriented surface epitope imprinted polymers-based QCM sensors for selectively detecting proteins at very low concentrations., (Copyright © 2018 Elsevier B.V. All rights reserved.)

We have performed a systematic study to analyze the effect of ssDNA length, nucleobase composition, and the type of two-dimensional nanoparticles (2D-nps) on the desorption response of 36 two-dimensional nanoassemblies (2D-NAs) against several proteins. The studies were performed using fluorescently labeled polyA, polyC, and polyT with 23, 18, 12, and 7 nucleotide-long sequences. The results suggest that the ssDNAs with polyC and longer sequences are more resistant to desorption, compared to their counterparts. In addition, 2D-NAs assembled using WS 2 were least susceptible to desorption by the proteins tested, whereas nGO 2D-NAs were the most susceptible nanoassemblies. Later, the results of these systematic studies were used to construct a sensor array for discrimination of seven model proteins (BSA, lipase, alkaline phosphatase, acid phosphatase, protease, β-galactosidase, and Cytochrome c). Neither the ssDNAs nor the 2D-nps have any specific interaction with the proteins tested. Only the displacement of the ssDNAs from the 2D-np surface was measured upon the disruption of the existing forces within 2D-NAs. A customized sensor array with five 2D-NAs was developed as a result of a careful screening/filtering process. The sensor array was tested against 200 nM of protein targets, and each protein was discriminated successfully. The results suggest that the systematic studies performed using various ssDNAs and 2D-nps enabled the construction of a sensor array without a bind-and-release sensing mechanism. The studies also demonstrate the significance of systematic investigations in the construction of two-dimensional DNA nanoassemblies for functional studies.