Screening Metabolic Biomarkers in KRAS Mutated Mouse Acinar and Human Pancreatic Cancer Cells via Single-Cell Mass Spectrometry.

Pancreatic cancer is a highly aggressive and rapidly progressing disease, often diagnosed in advanced stages due to the absence of early noticeable symptoms. The KRAS mutation is a hallmark of pancreatic cancer, yet the underlying mechanisms driving pancreatic carcinogenesis remain elusive. Cancer cells display significant metabolic heterogeneity, which is relevant to the pathogenesis of cancer. Population measurements may obscure information about the metabolic heterogeneity among cancer cells. Therefore, it is crucial to analyze metabolites at the single-cell level to gain a more comprehensive understanding of metabolic heterogeneity. In this study, we employed a 3D-printed ionization source for metabolite analysis in both mice and human pancreatic cancer cells at the single-cell level. Using advanced machine learning algorithms and mass spectral feature selection, we successfully identified 23 distinct metabolites that are statistically significantly different in KRAS mutant human pancreatic cancer cells and mouse acinar cells bearing the oncogenic KRAS mutation. These metabolites encompass a variety of chemical classes, including organic nitrogen compounds, organic acids and derivatives, organoheterocyclic compounds, benzenoids, and lipids. These findings shed light on the metabolic remodeling associated with KRAS-driven pancreatic cancer initiation and indicate that the identified metabolites hold promise as potential diagnostic markers for early detection in pancreatic cancer patients.

Cooperative transcriptional regulation by ATAF1 and HY5 promotes light-induced cotyledon opening in Arabidopsis thaliana.

The opening of the embryonic leaves (cotyledons) as seedlings emerge from the dark soil into the light is crucial to ensure the survival of the plant. Seedlings that sprout in the dark elongate rapidly to reach light but keep their cotyledons closed. During de-etiolation, the transition from dark to light growth, elongation slows and the cotyledons open. Here, we report that the transcription factor ACTIVATING FACTOR1 (ATAF1) participates in de-etiolation and facilitates light-induced cotyledon opening. The transition from dark to light rapidly induced ATAF1 expression and ATAF1 accumulation in cotyledons. Seedlings lacking or overexpressing ATAF1 exhibited reduced or enhanced cotyledon opening, respectively, and transcriptomic analysis indicated that ATAF1 repressed the expression of genes associated with growth and cotyledon closure. The activation of the photoreceptor phytochrome A (phyA) by far-red light induced its association with the ATAF1 promoter and stimulation of ATAF1 expression. The transcription factor ELONGATED HYPOCOTYL5 (HY5), which is also activated in response far-red light, cooperated with phyA to induce ATAF1 expression. ATAF1 and HY5 interacted with one another and cooperatively repressed the expression of growth-promoting and cotyledon closure genes. Together, our study reveals a mechanism through which far-red light promotes cotyledon opening.

Non-invasive monitoring of microbiota and host metabolism using secondary electrospray ionization-mass spectrometry.

The metabolic "handshake" between the microbiota and its mammalian host is a complex, dynamic process with major influences on health. Dissecting the interaction between microbial species and metabolites found in host tissues has been a challenge due to the requirement for invasive sampling. Here, we demonstrate that secondary electrospray ionization-mass spectrometry (SESI-MS) can be used to non-invasively monitor metabolic activity of the intestinal microbiome of a live, awake mouse. By comparing the headspace metabolome of individual gut bacterial culture with the "volatilome" (metabolites released to the atmosphere) of gnotobiotic mice, we demonstrate that the volatilome is characteristic of the dominant colonizing bacteria. Combining SESI-MS with feeding heavy-isotope-labeled microbiota-accessible sugars reveals the presence of microbial cross-feeding within the animal intestine. The microbiota is, therefore, a major contributor to the volatilome of a living animal, and it is possible to capture inter-species interaction within the gut microbiota using volatilome monitoring.

Mitochondrial alternative oxidase enhanced ABA-mediated drought tolerance in Solanum lycopersicum.

The phytohormone abscisic acid (ABA) plays essential roles in modulating drought stress responses. Mitochondrial alternative oxidase (AOX) is critical for reactive oxygen species (ROS) scavenging in drought stress responses. However, whether ABA signal in concert with AOX to moderate drought stress response remains largely unclear. In our study, we uncover the positive role of AOX in ABA-mediated drought tolerance in tomato (Solanum lycopersicum). Here, we report that ABA participates in the regulation of alternative respiration, and the increased AOX was found to improve drought tolerance by reducing total ROS accumulation. We also found that transcription factor ABA response element-binding factor 1 (SlAREB1) can directly bind to the promoter of AOX1a to activate its transcription. Virus-induced gene silencing (VIGS) of SlAREB1 compromised the ABA-induced alternative respiratory pathway, disrupted redox homeostasis and decreased plant resistance to drought stress, while overexpression of AOX1a in TRV2-SlAREB1 plants partially rescued the severe drought phenotype. Taken together, our results indicated that AOX1a plays an essential role in ABA-mediated drought tolerance partially in a SlAREB1-dependent manner, providing new insights into how ABA modulates ROS levels to cope with drought stress by AOX.

Genome-wide identification and functional analysis of Cellulose synthase gene superfamily in Fragaria vesca.

The Cellulose synthase (CesA) and Cellulose synthase-like (Csl) gene superfamilies encode key enzymes involved in the synthesis of cellulose and hemicellulose, which are major components of plant cell walls, and play important roles in the regulation of fruit ripening. However, genome-wide identification and functional analysis of the CesA and Csl gene families in strawberry remain limited. In this study, eight CesA genes and 25 Csl genes were identified in the genome of diploid woodland strawberry (Fragaria vesca). The protein structures, evolutionary relationships, and cis-acting elements of the promoter for each gene were investigated. Transcriptome analysis and quantitative real-time PCR (qRT-PCR) results showed that the transcript levels of many FveCesA and FveCsl genes were significantly decreased during fruit ripening. Moreover, based on the transcriptome analysis, we found that the expression levels of many FveCesA/Csl genes were changed after nordihydroguaiaretic acid (NDGA) treatment. Transient overexpression of FveCesA4 in immature strawberry fruit increased fruit firmness and reduced fresh fruit weight, thereby delaying ripening. In contrast, transient expression of FveCesA4-RNAi resulted in the opposite phenotypes. These findings provide fundamental information on strawberry CesA and Csl genes and may contribute to the elucidation of the molecular mechanism by which FveCesA/Csl-mediated cell wall synthesis regulates fruit ripening. In addition, these results may be useful in strawberry breeding programs focused on the development of new cultivars with increased fruit shelf-life.

Metabolic reconstitution of germ-free mice by a gnotobiotic microbiota varies over the circadian cycle.

The capacity of the intestinal microbiota to degrade otherwise indigestible diet components is known to greatly improve the recovery of energy from food. This has led to the hypothesis that increased digestive efficiency may underlie the contribution of the microbiota to obesity. OligoMM12-colonized gnotobiotic mice have a consistently higher fat mass than germ-free (GF) or fully colonized counterparts. We therefore investigated their food intake, digestion efficiency, energy expenditure, and respiratory quotient using a novel isolator-housed metabolic cage system, which allows long-term measurements without contamination risk. This demonstrated that microbiota-released calories are perfectly balanced by decreased food intake in fully colonized versus gnotobiotic OligoMM12 and GF mice fed a standard chow diet, i.e., microbiota-released calories can in fact be well integrated into appetite control. We also observed no significant difference in energy expenditure after normalization by lean mass between the different microbiota groups, suggesting that cumulative small differences in energy balance, or altered energy storage, must underlie fat accumulation in OligoMM12 mice. Consistent with altered energy storage, major differences were observed in the type of respiratory substrates used in metabolism over the circadian cycle: In GF mice, the respiratory exchange ratio (RER) was consistently lower than that of fully colonized mice at all times of day, indicative of more reliance on fat and less on glucose metabolism. Intriguingly, the RER of OligoMM12-colonized gnotobiotic mice phenocopied fully colonized mice during the dark (active/eating) phase but phenocopied GF mice during the light (fasting/resting) phase. Further, OligoMM12-colonized mice showed a GF-like drop in liver glycogen storage during the light phase and both liver and plasma metabolomes of OligoMM12 mice clustered closely with GF mice. This implies the existence of microbiota functions that are required to maintain normal host metabolism during the resting/fasting phase of circadian cycle and which are absent in the OligoMM12 consortium.

Hybrid Ionization Source Combining Nanoelectrospray and Dielectric Barrier Discharge Ionization for the Simultaneous Detection of Polar and Nonpolar Compounds in Single Cells.

Single-cell metabolomics is expected to deliver fast and dynamic information on cell function; therefore, it requires rapid analysis of a wide variety of very small quantities of metabolites in living cells. In this work, a hybrid ionization source that combines nanoelectrospray ionization (nanoESI) and dielectric barrier discharge ionization (DBDI) is proposed for single-cell analysis. A capillary with a 1 µm i.d. tip was inserted into cells for sampling and then directly used as the nanoESI source for ionization of polar metabolites. In addition, a DBDI source was employed as a post-ionization source to improve the ionization of apolar metabolites in cells that are not easily ionized by ESI. By increasing the voltage of the DBDI source from 0 to 3.2 kV, the classes of detected metabolites can be shifted from mostly polar to both polar and apolar to mainly apolar. Plant cells (onion) and human cells (PANC-1) were investigated in this study. After optimization, 50 compounds in onion cells and 40 compounds in PANC-1 cells were observed in ESI mode (3.5 kV) and an additional 49 compounds in onion cells and 73 compounds in PANC-1 cells were detected in ESI (3.5 kV)-DBDI (2.6 kV) hybrid mode. This hybrid ionization source improves the coverage, ionization efficiency, and limit of detection of metabolites with different polarities and could potentially contribute to the fast-growing field of single-cell metabolomics.

High-Throughput Single-Cell Mass Spectrometry Reveals Abnormal Lipid Metabolism in Pancreatic Ductal Adenocarcinoma.

Even populations of clonal cells are heterogeneous, which requires high-throughput analysis methods with single-cell sensitivity. Here, we propose a rapid, label-free single-cell analytical method based on active capillary dielectric barrier discharge ionization mass spectrometry, which can analyze multiple metabolites in single cells at a rate of 38 cells/minute. Multiple cell types (HEK-293T, PANC-1, CFPAC-1, H6c7, HeLa and iBAs) were discriminated successfully. We found evidence for abnormal lipid metabolism in pancreatic cancer cells. We also analyzed gene expression in a cancer genome atlas dataset and found that the mRNA level of a critical enzyme of lipid synthesis (ATP citrate lyase, ACLY) was upregulated in human pancreatic ductal adenocarcinoma (PDAC). Moreover, both an ACLY chemical inhibitor and a siRNA approach targeting ACLY could suppress the viability of PDAC cells. A significant reduction in lipid content in treated cells indicates that ACLY could be a potential target for treating pancreatic cancer.

Risk factors and prediction model of urosepsis in patients with diabetes after percutaneous nephrolithotomy.

OBJECTIVE: To analyze the risk factors of patients with diabetes mellitus (DM) and urosepsis after percutaneous nephrolithotomy (PCNL) for upper urinary tract stones and to develop a nomogram to predict postoperative urosepsis according to the risk factors. METHODS: The data of patients with type 2 diabetes who underwent one-stage PCNL due to upper urinary tract stones were retrospectively analyzed. The risk factors of patients with postoperative urosepsis were evaluated by univariate and multivariate logistic regression analysis, and the nomogram prediction model was developed according to the regression coefficient. RESULTS: One-stage PCNL was successfully completed in 241 patients with DM, and urosepsis occurred in 41 (17.0%) patients after PCNL. Based on multivariate logistic regression analysis, the independent risk factors associated with postoperative urosepsis included preoperative leukocyte elevation (OR = 3.973, P = 0.005), positive urine nitrite (OR = 3.697, P = 0.010), and positive urine culture (OR = 3.562, P = 0.002). According to the results of the logistic regression analysis model, staghorn stones (OR = 2.049, P < 0.1) and complete intraoperative stone clearance (OR = 0.431, P < 0.1), were used to develop the nomogram. Internal validation of the nomogram showed that the concordance index (C-index) was 0.725. Additionally, the Hosmer-Lemeshow test was performed, P = 0.938 > 0.05. CONCLUSION: Preoperative leukocyte elevation, positive urine nitrite, and positive urine culture are independent risk factors for urosepsis after one-stage PCNL for patients with DM with upper urinary tract stones. The nomogram, which is based on independent risk factors that combine stone morphology and intraoperative stone clearance, can help predict the risk of postoperative urosepsis.

Minimizing ion competition boosts volatile metabolome coverage by secondary electrospray ionization orbitrap mass spectrometry.

Secondary electrospray ionization high-resolution mass spectrometry (SESI-HRMS) is an emerging technique for the detection of volatile metabolites. However, sensitivity and reproducibility of SESI-HRMS have limited its applications in untargeted metabolomics profiling. Ion suppression in the SESI source has been considered to be the main cause. Here, we show that besides ion suppression, ion competition in the C-trap of Orbitrap instruments is another important factor that influences sensitivity and reproducibility of SESI-MS. Instead of acquiring the full mass-to-charge ratio (m/z) range, acquisition of consecutive m/z windows to minimize the ion competition effect allows the detection of more features. m/z window ranges are optimized to fill the C-trap either with an equal number of features or an equal cumulative intensity per window. Considering a balance between maximizing scanning speed and minimizing ion competition, splitting the m/z = 50-500 range into 4 windows is selected for measuring human breath and bacterial culture samples on SESI-Orbitrap MS, corresponding to a duty cycle of 2.3 s at a resolution of 140'000. In a small cohort of human subjects, the proposed splitting into 4 windows allows three times more features to be detected compared to the classical full m/z range method.

Monitoring peppermint washout in the breath metabolome by secondary electrospray ionization-high resolution mass spectrometry.

In this study, a secondary electrospray ionization-high resolution mass spectrometer (SESI-HRMS) system was employed to profile the real-time exhaled metabolome of ten subjects who had ingested a peppermint oil capsule. In total, six time points were sampled during the experiment. Using an untargeted way of profiling breath metabolome, 2333m/zunique metabolite features were determined in positive mode, and 1322 in negative mode. To benchmark the performance of the SESI-HRMS setup, several additional checks were done, including determination of the technical variation, the biological variation of one subject within three days, the variation within a time point, and the variation across all samples, taking allm/zfeatures into account. Reproducibility was good, with the median technical variation being ∼ 18% and the median variation within biological replicates being ∼ 34%. Both variations were lower than the variation across individuals. Washout profiles of compounds from the peppermint oil, including menthone, limonene, pulegone, menthol and menthofuran were determined in all subjects. Metabolites of the peppermint oil were also determined in breath, for example, cis/trans-carveol, perillic acid and menthol glucuronide. Butyric acid was found to be the major metabolite that reduce the uptake rate of limonene. Pathways related to limonene metabolism were examined, and meaningful pathways were identified from breath metabolomics data acquired by SESI using an untargeted analysis.

Real-time breath analysis of exhaled compounds upon peppermint oil ingestion by secondary electrospray ionization-high resolution mass spectrometry: technical aspects.

Breath analysis by secondary electrospray ionization high-resolution mass spectrometry (SESI-HRMS) has potential for clinical diagnosis and drug monitoring. However, there is still a lack of benchmarking data that shows the capability of this technique and allows comparability with other breath analysis techniques. In this regard, the goal of this study was the identification of volatile compounds upon ingestion of a specific peppermint oil capsule to get benchmark data for real-time breath analysis with SESI-HRMS. This was done in the framework of a consortium set up by the International Association of Breath Research (IABR), aimed at comparing several analytical instruments for breath analysis. Breath temporal profiles of two subjects were analyzed with SESI-HRMS before and after ingestion of a peppermint oil capsule. The measurements were performed at two different locations using identical SESI-HRMS platforms to allow for comparability and benchmarking. Remarkably, along with the four major compounds (monoterpenes/cineole, menthone, menthofuran and menthol) reported by other members of the consortium, we detected 57 additional features significantly associated (ρ > 0.8) with the peppermint oil capsule, suggesting that this relatively simple intervention might trigger a more complex metabolic cascade than initially expected. This observation was made on both sites. Additional replicate experiments for one of the subjects suggested that a core of 35-40 unique molecules are consistently detected in exhaled breath upon ingestion of the capsule. In addition, we illustrate the analytical capabilities of real-time SESI-HRMS/MS to assist in the identification of unknown compounds. The results outlined herein showcase the performance of SESI-HRMS and enable comparison with other breath analysis techniques. Along with that, they strengthen the potential of this analytical technique for non-invasive drug monitoring and clinical diagnostic purposes.

Mitochondrial translation and dynamics synergistically extend lifespan in C. elegans through HLH-30.

Mitochondrial form and function are closely interlinked in homeostasis and aging. Inhibiting mitochondrial translation is known to increase lifespan in C. elegans, and is accompanied by a fragmented mitochondrial network. However, whether this link between mitochondrial translation and morphology is causal in longevity remains uncharacterized. Here, we show in C. elegans that disrupting mitochondrial network homeostasis by blocking fission or fusion synergizes with reduced mitochondrial translation to prolong lifespan and stimulate stress response such as the mitochondrial unfolded protein response, UPRMT. Conversely, immobilizing the mitochondrial network through a simultaneous disruption of fission and fusion abrogates the lifespan increase induced by mitochondrial translation inhibition. Furthermore, we find that the synergistic effect of inhibiting both mitochondrial translation and dynamics on lifespan, despite stimulating UPRMT, does not require it. Instead, this lifespan-extending synergy is exclusively dependent on the lysosome biogenesis and autophagy transcription factor HLH-30/TFEB. Altogether, our study reveals the mechanistic crosstalk between mitochondrial translation, mitochondrial dynamics, and lysosomal signaling in regulating longevity.

A Conserved Mito-Cytosolic Translational Balance Links Two Longevity Pathways.

Slowing down translation in either the cytosol or the mitochondria is a conserved longevity mechanism. Here, we found a non-interventional natural correlation of mitochondrial and cytosolic ribosomal proteins (RPs) in mouse population genetics, suggesting a translational balance. Inhibiting mitochondrial translation in C. elegans through mrps-5 RNAi repressed cytosolic translation. Transcriptomics integrated with proteomics revealed that this inhibition specifically reduced translational efficiency of mRNAs required in growth pathways while increasing stress response mRNAs. The repression of cytosolic translation and extension of lifespan from mrps-5 RNAi were dependent on atf-5/ATF4 and independent from metabolic phenotypes. We found the translational balance to be conserved in mammalian cells upon inhibiting mitochondrial translation pharmacologically with doxycycline. Lastly, extending this in vivo, doxycycline repressed cytosolic translation in the livers of germ-free mice. These data demonstrate that inhibiting mitochondrial translation initiates an atf-5/ATF4-dependent cascade leading to coordinated repression of cytosolic translation, which could be targeted to promote longevity.

Characterizing the iron loading pattern of ferritin using high-mass matrix-assisted laser desorption ionization mass spectrometry.

RATIONALE: Ferritin is an iron storage protein assembly, usually formed by a 24-subunit protein shell and an iron core. The ferritin shell has been well studied using various structural biology tools such as X-ray diffraction and cryo-electron microscopy, whereas the iron status of ferritin is less studied and no well-established method exists for characterizing the distribution of the iron loading of ferritin. Recent advances in mass spectrometry (MS) have expanded the observable m/z range, making the measurement of ferritin possible with MS. In this study, matrix-assisted laser desorption ionization (MALDI)-MS was employed to quantify the iron content of ferritin. METHODS: The iron content of ferritin was quantified using a MALDI-MS system coupled with a commercially available ion conversions dynode high-mass detector. IgG1 antibody and its aggregates were used as external mass calibrants. The stability of HoloFt and ApoFt was also assessed in this study under different conditions, including various buffer pH, crosslinking agents and MALDI laser intensities. RESULTS: The differences in peak width of HoloFt, ApoFt and IgG1 indicate the existence of mineral adducts in both HoloFt and ApoFt, and the mineral loading is heterogeneous among the HoloFt and ApoFt population. An average of 2773 ± 1584 iron atoms were determined for a commercial HoloFt sample. The iron core inside the ferritin complex is shown to stabilize and maintain the intact globular complex structure of ferritin. CONCLUSIONS: This work introduces a MALDI-MS-based workflow for characterizing the ferritin iron loading pattern, which is meaningful for clinical analysis of iron deficiency/overload. In addition, the stability of ferritin is examined under various conditions, providing a guideline for further method development related to ferritin complex.

Quantifying and Localizing the Mitochondrial Proteome Across Five Tissues in A Mouse Population.

We have used SWATH mass spectrometry to quantify 3648 proteins across 76 proteomes collected from genetically diverse BXD mouse strains in two fractions (mitochondria and total cell) from five tissues: liver, quadriceps, heart, brain, and brown adipose (BAT). Across tissues, expression covariation between genes' proteins and transcripts-measured in the same individuals-broadly aligned. Covariation was however far stronger in certain subsets than others: only 8% of transcripts in the lowest expression and variance quintile covaried with their protein, in contrast to 65% of transcripts in the highest quintiles. Key functional differences among the 3648 genes were also observed across tissues, with electron transport chain (ETC) genes particularly investigated. ETC complex proteins covary and form strong gene networks according to tissue, but their equivalent transcripts do not. Certain physiological consequences, such as the depletion of ATP synthase in BAT, are thus obscured in transcript data. Lastly, we compared the quantitative proteomic measurements between the total cell and mitochondrial fractions for the five tissues. The resulting enrichment score highlighted several hundred proteins which were strongly enriched in mitochondria, which included several dozen proteins were not reported in literature to be mitochondrially localized. Four of these candidates were selected for biochemical validation, where we found MTAP, SOAT2, and IMPDH2 to be localized inside the mitochondria, whereas ABCC6 was in the mitochondria-associated membrane. These findings demonstrate the synergies of a multi-omics approach to study complex metabolic processes, and this provides a resource for further discovery and analysis of proteoforms, modified proteins, and protein localization.

Systematic Evaluation of the Use of Human Plasma and Serum for Mass-Spectrometry-Based Shotgun Proteomics.

Over the last two decades, EDTA-plasma has been used as the preferred sample matrix for human blood proteomic profiling. Serum has also been employed widely. Only a few studies have assessed the difference and relevance of the proteome profiles obtained from plasma samples, such as EDTA-plasma or lithium-heparin-plasma, and serum. A more complete evaluation of the use of EDTA-plasma, heparin-plasma, and serum would greatly expand the comprehensiveness of shotgun proteomics of blood samples. In this study, we evaluated the use of heparin-plasma with respect to EDTA-plasma and serum to profile blood proteomes using a scalable automated proteomic pipeline (ASAP2). The use of plasma and serum for mass-spectrometry-based shotgun proteomics was first tested with commercial pooled samples. The proteome coverage consistency and the quantitative performance were compared. Furthermore, protein measurements in EDTA-plasma and heparin-plasma samples were comparatively studied using matched sample pairs from 20 individuals from the Australian Imaging, Biomarkers and Lifestyle (AIBL) Study. We identified 442 proteins in common between EDTA-plasma and heparin-plasma samples. Overall agreement of the relative protein quantification between the sample pairs demonstrated that shotgun proteomics using workflows such as the ASAP2 is suitable in analyzing heparin-plasma and that such sample type may be considered in large-scale clinical research studies. Moreover, the partial proteome coverage overlaps (e.g., ∼70%) showed that measures from heparin-plasma could be complementary to those obtained from EDTA-plasma.

Interactions between carbon nanodots with human serum albumin and γ-globulins: The effects on the transportation function.

Carbon nanodots (C-dots) have attracted great attention as a new class of luminescent nanomaterials due to their superior physical and chemical properties. In order to better understand the basic behavior of C-dots in biological systems, a series of photophysical measurements were applied to study the interactions of C-dots with human serum albumin (HSA) and γ-globulins. The fluorescence of proteins was quenched by the dynamic mechanism rather than the formation of a protein/C-dots complex. The apparent dissociation constants of the C-dots bound to HSA and γ-globulins were of the same order of magnitude. Furthermore, it is proven that C-dots showed little influence on the conformation of HSA and γ-globulins. In addition, Fourier transform infrared and fluorescence spectroscopic studies demonstrated that the interaction between C-dots and two kinds of serum proteins was driven by hydrophobic and van der waals forces. Since the bioavailability of drugs can be modulated by their interactions with proteins, the variations of binding constants of three drugs with HSA and γ-globulins in the presence of different concentrations of C-dots (0-84 µmol L(-1)) have also been analyzed in this work, to reflect the effect of C-dots on the transportation function of HSA and γ-globulins.

Highly Photoluminescent Nitrogen-Doped Carbon Nanodots and Their Protective Effects against Oxidative Stress on Cells.

Highly photoluminescent (PL) (quantum yield = 54%) nitrogen doped carbon nanodots (C-dots) have been prepared through one-step carbonizing citric acid and tris(hydroxymethyl)aminomethane and using oleic acid as solvent. The synthesized C-dots are monodisperse with narrow size distribution (average 1.7 nm). The PL properties of C-dots are pH dependent, and hence, using C-dots as sophisticated pH sensor to detect pH values between 7 and 9 can be expected. In addition, the PL intensity of C-dots remains stable under high ionic strength. The C-dots can protect cells from oxidative stress, which shows potential to expand the biological application of C-dots, especially in medical treatment. The protective mechanism is associated with intracellular reactive oxygen species elimination and the intracellular superoxide dismutase production.

Mechanistic studies on the reversible photophysical properties of carbon nanodots at different pH.

The pH-dependent photoluminescence (PL) behavior of carbon nanodots (C-dots) and its mechanism has been exhaustively studied in this work. The PL and UV-vis absorption spectra are reversible in the pH between 3 and 13. We speculate that two kinds of reactions (fast and slow) occurring at the surface of C-dots may contribute to this pH-dependent PL behavior. When C-dots solutions are switched to acidic conditions, they will quickly self-assembled aggregate into larger particles and surface oxygen-related groups of C-dots would be slowly oxidized at room temperature. Moreover, it should be noted that this is the first direct observation of self-assembled aggregation of C-dots under acidic conditions. In addition, the optimal PL spectra of C-dots blue-shift while their sizes increase, so-called 'inverse PL shift' phenomenon is also observed. Meanwhile, as the solution is adjusted to alkaline conditions, a structural tautomerization of C-dots rapidly takes place and hydrogenation/deoxygenation reaction proceeds in a much slower rate. Furthermore, through distinct decay dynamics as well as the characterizations of C-dots at different pHs, the PL properties are proposed to be mainly related to the surface states of C-dots.