Copper-crosslinked carbon dot hydrogel nanozyme for colorimetric - tert-butylhydroquinone biosensing and smartphone-assisted visual ratiometric assay.

Due to the potential environment and health risks of tert-butylhydroquinone (TBHQ), rapid, portable, selective and sensitive quantification of TBHQ in food and the environment are strictly essential. With this in mind, a selective, sensitive and rapid colorimetric TBHQ biosensor was developed using rationally designed copper-crosslinked carbon dot hydrogel nanozyme (BC-CDs@Cu). The BC-CDs@Cu had a high peroxidase-like activity toward the chromogenic reaction of hydrogen peroxide with dopamine via the generation of hydroxyl radicals and electron transfer process. The Michaelis-Menten constants of BC-CDs@Cu for dopamine and hydrogen peroxide were determined to be 0.86 and 0.91 mM. The added TBHQ markedly inhibited the BC-CDs@Cu-catalyzed dopamine oxidation by hydrogen peroxide, ascribing to the highly effective and rapid scavenging of hydroxyl radicals and the suppression of electron transfer. The inhibitory extent was applied for well quantifying TBHQ in the range of 0.5 - 20.0 µM with a detection limit of 70 nM. The proposed biosensor had a negligible response to various interfering substances. Moreover, a smartphone-assisted visual ratiometric biosensor was fabricated, and used to accomplish portable quantification of TBHQ in edible oils and water samples. This work reveals the enormous potential of hydrogel nanozyme, which will open a new situation for the detection of hazardous substances.

Water-Soluble Photoluminescent Adenosine-Functionalized Gold Nanoclusters as Highly Sensitive and Selective Receptors for Riboflavin Detection in Rat Brain.

Simple, selective, and sensitive detection of cerebral riboflavin is of great significance due to the vital roles of riboflavin in physiological and pathological processes. In the work, water-soluble photoluminescent adenosine-functionalized gold nanoclusters (Ade-AuNCs) are exploited as highly sensitive and selective receptors for cerebral riboflavin detection. The Ade-AuNCs are prepared under aqueous conditions by the one-step "synthesis-functionalization integration" strategy, using chloroauric acid as gold precursors and adenosine as outer-shell ligands. During the Ade-AuNCs synthesis process, adenosine and ascorbic acid are demonstrated to respectively serve as a stabilizer and a reductant, and citrate buffer plays multiple roles including a pH regulator, reductant, and complexing agent. The added riboflavin causes photoluminescence quenching of Ade-AuNCs, and the quenching photoluminescence is applied for well quantifying riboflavin in the range of 0.005-0.1 nM with a detection limit of 0.002 nM. The detailed analytical characterizations reveal that the photoluminescence quenching results from the static photoinduced electron transfer process from the surface functional Ade-AuNCs to riboflavin and the strong affinity between Ade-AuNCs and riboflavin. Moreover, the Ade-AuNC-based sensor exhibits a high selectivity for riboflavin over metal ions, anions, amino acids, and biological substances that possibly exist in the rat brain. Finally, by coupling the microdialysis technique, the proposed sensor is successfully applied to detect riboflavin in living rat brain microdialysates with a basal value of 13.1 ± 2.5 nM (n = 3), and the results are comparable well with those from a reference high-performance liquid chromatography method.

Copper-carbon dot aerogel: a high-performance mimetic peroxidase and its application for versatile colorimetric bioassays.

Three-dimensional hierarchically porous copper-carbon dot aerogels (Cu-CDA) were fabricated on a large scale using copper as metal nodes and carbon dots derived from adenine and sodium citrate as building blocks. Benefiting from the electron transfer process and generation of hydroxyl radicals, Cu-CDA exhibited a high peroxidase-like activity. Combining the observation with multienzyme cascade reactions, versatile colorimetric bioassays of hydrogen peroxide as well as diabetes-associated glucose and α-glucosidase were explored and then extended to screen α-glucosidase inhibitors that are used as antidiabetic and antiviral agents.

Ratiometric electrochemical sensor for bisphenol A detection using a glassy carbon electrode modified with a poly(toluidine blue)/gold nanoparticle composite.

A ratiometric electrochemical sensor for bisphenol A (BPA) detection is developed using a glassy carbon electrode modified with a poly(toluidine blue)/gold nanoparticle composite (PTB/AuNP/GCE). The ratiometric signal, namely, the oxidation peak current ratio of BPA to PTB, increases linearly with BPA concentration in the 0.2-5.0 µM range, with a detection limit of 0.15 µM. The electrochemical mechanism of BPA is studied at the PTB/AuNP/GCE, and the results show that BPA undergoes an electrooxidation process of two electrons and two protons at the PTB/AuNP/GCE. The proposed sensor has high sensitivity, high stability and good selectivity. The application of BPA in water samples is successfully verified using the proposed ratiometric electrochemical sensor.

Electropolymerization of poly(methylene blue) on flower-like nickel-based MOFs used for ratiometric electrochemical sensing of total polyphenolic content in chrysanthemum tea.

A ratiometric electrochemical sensor for caffeic acid (CAE) detection was constructed using a glassy carbon electrode modified with poly(methylene blue) and flower-like nickel-based metal organic frameworks (PMB@Ni-TPA/GCE). The electrochemical behavior of CAE was investigated at the PMB@Ni-TPA/GCE, and was found to follow a two-electron, two-proton electrooxidation process. PMB was used as the internal reference probe, and Ni-TPA can enhance the electrochemical signals of both CAE and PMB. As the CAE concentration increases, the oxidation peak current of CAE is enhanced but that of PMB keeps almost unchanged. The oxidation peak current ratio between CAE and PMB recorded by differential pulse voltammetry changes linearly with CAE concentration over the range of 0.25-15.0 µM, with a detection limit of 0.2 µM. The proposed sensor was successfully employed to evaluate the total polyphenolic content as CAE equivalent in chrysanthemum tea, and the results were comparable with those given by the reference Folin-Ciocalteu spectrophotometry.

A dual-functional nanovehicle with fluorescent tracking and its targeted killing effects on hepatocellular carcinoma cells.

All-in-one drug delivery nanovehicles with low cytotoxicity, high clinical imaging tracking capability, and targeted- and controlled-releasing performances are regarded as promising nanoplatforms for tumor theranostics. Recently, the design of these novel nanovehicles by low molecular weight amphiphilic chitosan (CS) was proposed. Based on fluorescent gold nanoclusters (AuNCs), a tumor-targeting nanovehicle (i.e. AuNCs-CS-AS1411) was prepared via electrostatic attraction between AuNC-conjugated chitosan (i.e. AuNCs-CS) and the anti-nucleolin aptamer, AS1411. After that, the anticancer drug methotrexate (MTX) was encapsulated into the nanovehicles and then the dual-functional nano-drug (i.e. MTX@AuNCs-CS-AS1411) was comparatively supplied to the human hepatocellular carcinoma cell line HepG2 and the human normal liver cell line LO2, to exhibit its "all in one" behavior. Under the conditions of the same concentration of MTX, MTX@AuNCs-CS-AS1411 demonstrates more intensive cytotoxicity and apoptosis-inducing activity against HepG2 cells than those against normal LO2 cells, mainly due to the targeting effect of AS1411 on the nucleolins that were found at high levels on the surface of tumor cells, but are at low levels or absent on normal cells. On the other hand, the MTX release from the MTX@AuNCs-CS-AS1411 was much faster in mildly acidic solution than that in neutral pH. Thus, it may provide a possibility to more significantly release MTX in intracellular lysosome of tumor cells, rather than let loose MTX during transport of the drug from blood vessels to tumor tissue. In conclusion, our dual-functional nanovehicle possesses high fluorescence efficiency and photostability, low cytotoxicity, pH-dependent controlled release, high sensitivity and target-specificity to cancer cells which allowed concurrent targeted imaging and delivery in cancer chemotherapies.

Adenine-stabilized carbon dots for highly sensitive and selective sensing of copper(II) ions and cell imaging.

Adenine-stabilized carbon dots (A-CDs) are shown to be a viable fluorescent probe for highly sensitive detection and imaging of Cu2+. The probe has a linear fluorometric response in the 1-700 nM concentration range and a 0.3 nM detection limit. The probe, with excitation/emission maxima at 380/435 nm, is highly selective for Cu2+ over other metal ions, anions, amino acids, and biomolecules. The fluorescence quenching mechanism of the A-CDs by Cu2+ is investigated using transmission electron microscopy images coupled with elemental mapping, X-ray photoelectron spectroscopy, X-ray-excited Auger electron spectroscopy, fluorescence lifetime, UV-visible spectroscopy, and cyclic voltammetry. The experimental results show that the fluorescence quenching is caused by the combination of Cu2+-coordination-induced aggregation of the A-CDs, the reduction of Cu2+ by the A-CDs, and the nonradiative photoinduced electron transfer process from the A-CDs to Cu2+ or metallic Cu. The high sensitivity and high selectivity of the sensor are ascribed to the chemical interactions between the A-CDs and Cu2+, the photophysical process between the A-CDs and Cu2+, and the high fluorescence quantum yield of the A-CDs (44.6%). The A-CDs have excellent water solubility, good stability to variation of pH values, high photostability, fast response time, and low cytotoxicity. They are successfully employed for intracellular imaging of Cu2+ in HepG2 cells and Cu2+ detection in the tap water samples.

Rapid, one-pot, protein-mediated green synthesis of water-soluble fluorescent nickel nanoclusters for sensitive and selective detection of tartrazine.

In the work, water-soluble bovine serum albumin-protected fluorescent nickel nanoclusters (BSA-NiNCs) are used as fluorescent probes to construct a label-free fluorescence quenching sensor for sensitive and selective detection of tartrazine. The fluorescent BSA-NiNCs are synthesized in one pot using BSA as both the template and reducing agent, and hydrogen peroxide as the additive. The as-prepared NiNCs are characterized by using various analytical techniques like transmission electron microscopy, X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, UV-Vis absorption spectroscopy, circular dichroism spectroscopy, and fluorescence spectroscopy. The synthesized BSA-NiNCs have a quantum yield of ca. 8% by using quinine sulfate as a standard. The sensor for tartrazine detection shows a wide linear range of 0.01-3.5 µM, with a low detection limit of 4 nM. The fluorescence quenching very likely results from the combination of the intermolecular interactions and the secondary inner filter effect between BSA-NiNCs and tartrazine. Then, the proposed sensor is successfully employed for tartrazine detection in drink samples, and the results are comparable with those based on a reference HPLC method.

Green synthesis of luminescent graphitic carbon nitride quantum dots from human urine and its bioimaging application.

A hydrothermal synthetic approach is developed for the preparation of graphitic carbon nitride quantum dots (g-C3N4 QDs) from human urine. The reported synthetic method is green, simple, low-cost, less time-consuming, and can be used for the large-scale production of the g-C3N4 QDs. The as-prepared g-C3N4 QDs possess a high quantum yield of 15.7% by using quinine sulfate as a reference, and display excitation-wavelength dependent fluorescent emission. In addition, the g-C3N4 QDs exhibit high photostability, low cytotoxicity. and are successfully used as fluorescent probes for cell multicolor imaging. It is believed that the valuable nanomaterials, g-C3N4 QDs, which are transformed from the human bodily wastes, are promising in diverse chemical applications.

Electrochemical determination of 2,4,6-trinitrophenol using a hybrid film composed of a copper-based metal organic framework and electroreduced graphene oxide.

A metal organic framework (MOF) of the type copper(II)-1,3,5-benzenetricarboxylic acid (Cu-BTC) was electrodeposited on electroreduced graphene oxide (ERGO) placed on a glassy carbon electrode (GCE). The modified GCE was used for highly sensitive electrochemical determination of 2,4,6-trinitrophenol (TNP). The fabrication process of the modified electrode was characterized by scanning electron microscopy and electrochemical impedance spectroscopy. Differential pulse voltammetry (DPV) demonstrates that the Cu-BTC/ERGO/GCE gives stronger signals for TNP reduction than Cu-BTC/GCE or ERGO/GCE alone. DPV also shows TNP to exhibit three reduction peaks, the first at a potential of -0.42 V (vs. SCE). This potential was selected because the other three similarly-structured compounds (2-nitrophenol, 4-nitrophenol, 2,4-dinitrophenol) do not give a signal at this potential. Response is linear in the 0.2 to 10 µM TNP concentration range, with a 0.1 µM detection limit (at S/N = 3) and a 15.98 µA∙µM-1∙cm-2 sensitivity under optimal conditions. The applicability of the sensor was evaluated by detecting TNP in spiked tap water and lake water samples. Recoveries ranged between 95 and 101%. Graphical abstract Schematic presentation of an electrochemical sensor that was fabricated by electrodeposition of the metal-organic framework (MOF) of copper(II)-1,3,5-benzenetricarboxylic acid (Cu-BTC) onto the surface of electroreduced graphene oxide (ERGO) modified glassy carbon electrode (GCE). It was applied to sensitive and selective detection of 2,4,6-trinitrophenol (TNP).

Synthesis-identification integration: One-pot hydrothermal preparation of fluorescent nitrogen-doped carbon nanodots for differentiating nucleobases with the aid of multivariate chemometrics analysis.

Most of the conventional multidimensional differential sensors currently need at least two-step fabrication, namely synthesis of probe(s) and identification of multiple analytes by mixing of analytes with probe(s), and were conducted using multiple sensing elements or several devices. In the study, we chose five different nucleobases (adenine, cytosine, guanine, thymine, and uracil) as model analytes, and found that under hydrothermal conditions, sodium citrate could react directly with various nucleobases to yield different nitrogen-doped carbon nanodots (CDs). The CDs synthesized from different nucleobases exhibited different fluorescent properties, leading to their respective characteristic fluorescence spectra. Hence, we combined the fluorescence spectra of the CDs with advanced chemometrics like principle component analysis (PCA), hierarchical cluster analysis (HCA), K-nearest neighbor (KNN) and soft independent modeling of class analogy (SIMCA), to present a conceptually novel "synthesis-identification integration" strategy to construct a multidimensional differential sensor for nucleobase discrimination. Single-wavelength excitation fluorescence spectral data, single-wavelength emission fluorescence spectral data, and fluorescence Excitation-Emission Matrices (EEMs) of the CDs were respectively used as input data of the differential sensor. The results showed that the discrimination ability of the multidimensional differential sensor with EEM data set as input data was superior to those with single-wavelength excitation/emission fluorescence data set, suggesting that increasing the number of the data input could improve the discrimination power. Two supervised pattern recognition methods, namely KNN and SIMCA, correctly identified the five nucleobases with a classification accuracy of 100%. The proposed "synthesis-identification integration" strategy together with a multidimensional array of experimental data holds great promise in the construction of differential sensors.

Multi-functionalized chitosan nanoparticles for enhanced chemotherapy in lung cancer.

Chemotherapy-based treatment for cancer has made great progress in the past decades. However, there is still a big challenge for the treatment of lung cancer. Herein, a multifunctional nanocarrier was developed through electrostatic interaction between the fluorescent gold nanocluster-conjugated chitosan and the nucleolin targeting AS1411 aptamer. Then methotrexate was loaded into the multifunctional nanocarrier through hydrophobic interaction to obtain the nanodrug carrier systems. The prepared nanodrug carrier systems have an average nanoparticle size of 200 nm with 13.8% drug loading efficiency. The drug release is pH-dependent. The in vitro results demonstrated that the nanodrug carrier systems were selectively taken up by cancer cells in a time-dependent manner and exhibited significantly enhanced anticancer activity in a model of lung cancer A549 cells. The in vivo results showed that intravenous administration of nanodrug carrier systems into BALB/c mice led to accumulation of methotrexate at the tumor site and significantly inhibited the tumor growth but without overt toxicity. The present study suggests that the prepared multifunctional nanocarrier can be used as an effective drug delivery system for anticancer drugs and exhibits great potential in clinical applications.

Label-free photoluminescence assay for nitrofurantoin detection in lake water samples using adenosine-stabilized copper nanoclusters as nanoprobes.

In this paper, we constructed a novel label-free analytical strategy for highly sensitive and selective detection of nitrofurantoin (NFT) based on adenosine-stabilized copper nanoclusters (CuNCs) as nanoprobes. It was found that NFT caused a rapid decrease in the photoluminescence intensity of CuNCs. The photoluminescence quenching was likely attributed to the inner filter effect between NFT and CuNCs. The CuNCs exhibited a wide linear range of 0.05-4.0µM with the detection limit of 30nM (7.1ngmL-1) for detection of NFT. And it was successfully applied for NFT detection in lake water samples.

One-Pot Aqueous Synthesis of Nucleoside-Templated Fluorescent Copper Nanoclusters and Their Application for Discrimination of Nucleosides.

A facile, one-pot synthetic method has been proposed to prepare water-soluble fluorescent copper nanoclusters (CuNCs) templated by nucleosides. The nucleoside-templated fluorescent CuNCs were further characterized by using various analytical techniques, such as transmission electron microscopy, X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, and fluorescence spectroscopy. The role of various reactants such as ascorbic acid, nucleoside, and citrate buffer in the synthesis process of fluorescent CuNCs was explored. The results showed that nucleoside and ascorbic acid were very likey to respectively act as a stabilizer and a reductant to form nanoclusters, and citrate buffer acted as both pH regulator solution and a reducing agent. The fluorescence spectra of various nucleoside-templated CuNCs were finally combined with multivariate chemometrics analysis for discrimination of different nucleosides.

One-step synthesis of graphitic carbon nitride nanosheets with the help of melamine and its application for fluorescence detection of mercuric ions.

A facile, simple, and relatively environment-friendly hydrothermal approach was developed for one-step synthesis of graphitic carbon nitride nanosheets (GCNNs) using melamine and sodium citrate as the precursors. The prepared GCNNs emit strong fluorescence with a high quantum yield of 48.3%. The GCNNs were then characterized by various techniques including transmission electron microscopy, atomic force microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, and UV-Vis absorption spectroscopy. In addition, the fluorescence quenching behavior of the GCNNS by mercuric ions (Hg2+) was exploited to fabricate a label-free fluorescence quenching sensor for sensitive and selective detection of Hg2+. The results showed that there existed a linear relationship between the fluorescence intensity and the concentration of Hg2+ from 0.001 to 1.0µM with a detection limit of 0.3nM. Finally, the sensor was successfully used to detection of Hg2+ in water and milk samples.

Label-free fluorescent catalytic biosensor for highly sensitive and selective detection of the ferrous ion in water samples using a layered molybdenum disulfide nanozyme coupled with an advanced chemometric model.

In this work, we developed a novel layered molybdenum disulfide (MoS2) nanosheet peroxidase mimetic-based fluorescent catalytic biosensor for the sensitive and selective detection of Fe(2+). It was found that Fe(2+) remarkably enhanced the catalytic activity of the MoS2 nanosheet for oxidation of OPD to form a highly fluorescent substance, 2,3-diaminophenazine (DAPN), and the MoS2/OPD/H2O2 biosensor displayed substantial fluorescence enhancement after addition of Fe(2+) in a concentration-dependent manner. The fluorescence intensity was proportional to the concentration of Fe(2+) over a range of 0.005-0.20 µM with a limit of detection of 3.5 nM (signal/noise = 3). When compared with the OPD/H2O2 biosensor, the MoS2/OPD/H2O2 biosensor provided a higher sensitivity and selectivity for Fe(2+), suggesting the validity of the use of the MoS2 nanosheets. To further demonstrate the feasibility of the MoS2/OPD/H2O2 biosensor for Fe(2+) detection in real water samples, we measured the three-dimensional excitation-emission spectra of the real system, and submitted the excitation-emission matrix (EEM) data to an advanced chemometrics model based on parallel factor analysis (PARAFAC). The results showed that the use of the PARAFAC model could further enhance the selectivity of the biosensor and determine Fe(2+) concentration in the presence of unexpected interferents from real water samples. This work opens up new opportunities for the use of the catalytic properties of the MoS2 nanosheets and advanced chemometrics models in the field of biosensors.

Enhancing sensitivity and selectivity in a label-free colorimetric sensor for detection of iron(II) ions with luminescent molybdenum disulfide nanosheet-based peroxidase mimetics.

In the present study, we demonstrated that the luminescent molybdenum disulfide (MoS2) nanosheets, which were prepared hydrothermally by using sodium molybdate and thiourea as precursors, possessed peroxidase-like activity, and could catalyze the oxidation of peroxidase substrate o-phenylenediamine (OPD) in the presence of hydrogen peroxide (H2O2) to produce a yellow color reaction. Further addition of Fe(2+) into the nanosheets led to peroxidase mimetics with greatly enhanced catalytic activity. The observation was exploited to develop a label-free colorimetric nanozyme sensor for detection of Fe(2+). The fabricated MoS2/OPD/H2O2 sensor showed a wide linear range of 0.01-0.8 µM with a detection limit of 7 nM. Moreover, it was found that the MoS2/OPD/H2O2 sensor displayed enhanced sensitivity and selectivity toward Fe(2+) compared with the OPD/H2O2 sensor, suggesting that the MoS2 nanosheets could improve the performance of the Fe(2+) sensor. An advanced chemometrics algorithm, multivariate curve resolution by alternating least squares (MCR-ALS), was further applied to interpret the origin of enhancing sensitivity and selectivity in the Fe(2+) sensor with the MoS2 nanosheets. The time-dependent UV-vis spectral data of the studied systems were collected, and submitted to the MCR-ALS. The results showed that the increased sensitivity and selectivity of the MoS2/OPD/H2O2 sensor for Fe(2+) detection likely arose from its large reaction rate constant. Finally, the proposed MoS2/OPD/H2O2 sensor was successfully applied for detection of Fe(2+) in water samples.

Solid-phase synthesis of graphene quantum dots from the food additive citric acid under microwave irradiation and their use in live-cell imaging.

The work demonstrated that solid citric acid, one of the most common food additives, can be converted to graphene quantum dots (GQDs) under microwave heating. The as-prepared GQDs were further characterized by various analytical techniques like transmission electron microscopy, atomic force microscopy, X-ray diffraction, X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, fluorescence and UV-visible spectroscopy. Cytotoxicity of the GQDs was evaluated using HeLa cells. The result showed that the GQDs almost did not exhibit cytotoxicity at concentrations as high as 1000 µg mL(-1). In addition, it was found that the GQDs showed good solubility, excellent photostability, and excitation-dependent multicolor photoluminescence. Subsequently, the multicolor GQDs were successfully used as a fluorescence light-up probe for live-cell imaging.

Facile Microwave-Assisted Solid-Phase Synthesis of Highly Fluorescent Nitrogen-Sulfur-Codoped Carbon Quantum Dots for Cellular Imaging Applications.

Carbon quantum dots (CQDs) have recently attracted significant attention for both their fundamental science and technological applications as a new class of fluorescent zero-dimensional nanomaterials with a size below 10 nm. However, the reported methods of synthesis were generally less suitable for the large-scale production of the CQDs with high-fluorescent quantum yield (QY). In the paper, a novel one-pot microwave-assisted drying synthesis approach was presented to prepare CQDs with high QY of 61.3 % for the first time. The production yield of CQDs was 35±3 % in weight. The as-prepared CQDs were characterized by various techniques such as TEM, AFM, XRD, XPS, FTIR spectroscopy, UV/Vis absorption spectroscopy, and fluorescence spectroscopy. The results showed that the high QY of CQDs was largely attributed to the dual doping of nitrogen and sulphur into CQDs. Such CQDs were then used as live-cell imaging reagents due to their high QY, good water dispersibility, fine biocompatibility, high photostability, and low cytotoxicity.

New photostable naphthalimide-based fluorescent probe for mitochondrial imaging and tracking.

Monitoring mitochondria morphological changes temporally and spatially exhibits significant importance for diagnosing, preventing and treating various diseases related to mitochondrial dysfunction. However, the application of commercially available mitochondria trackers is limited due to their poor photostability. To overcome these disadvantages, we designed and synthesized a mitochondria-localized fluorescent probe by conjugating 1,8-naphthalimide with triphenylphosphonium (i.e. NPA-TPP). The structure and characteristic of NPA-TPP was characterized by UV-vis, fluorescence spectroscopy, (1)HNMR, (13)CNMR, FTIR, MS, etc. The photostability and cell imaging were performed on the laser scanning confocal microscopy. Moreover, the cytotoxicity of NPA-TPP on cells was evaluated using (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay. The results showed that NPA-TPP not only has high sensitivity and specificity to mitochondria, but also exhibits super-high photostability, negligible cytotoxicity and good water solubility. In short, NPA-TPP indicates great potential for targeting mitochondria and enables a real-time and long-term tracking mitochondrial dynamics changes.