Efficient Extraction and Analysis of Nanoplastics by Ionic Liquid-Assisted Cloud-Point Extraction Coupled with Electromagnetic Heating Pyrolysis Mass Spectrometry.

Nanoplastics have attracted much attention due to their potential hazards. However, analysis of nanoplastics remains challenging. In this study, ionic liquid-assisted cloud-point extraction (IL-assisted CPE) was developed to enrich nanoplastics in the aqueous environment and further coupled with electromagnetic heating pyrolysis mass spectrometry. The use of trace ILs improves the extraction efficiency of CPE for nanoplastics. The effects of ILs (types, contents), nanoplastic properties (type, size), and environmental factors (aging time, humic acid content) were systematically investigated to evaluate the applicability. The limits of detection of poly(methyl methacrylate) (PMMA) and polystyrene (PS) were determined to be 1.78 and 2.67 µg/L, respectively. Real environmental samples including lake water, rainwater, and influent and effluent from wastewater treatment plant were analyzed with good accuracy (79.58-116.87%) and satisfactory precision (RSD ≤ 11.99%). A possible mechanism for ILs being absorbed into the ordered surfactant micellar and generating larger micelles to synergically enclose hydrophobic nanoplastics was proposed. This work provides a simple and efficient approach to the extraction and analysis of nanoplastics in aqueous environments.

One-step online analysis of antibiotics in highly saline seawater by nano-based slug-flow microextraction.

The transition to mass spectrometry (MS) in the analysis of antibiotics in the marine environment is highly desirable, particularly in the enhancement of sensitivity for high-salinity (3.5 wt%) seawater samples. However, the persistence of complex operational procedures poses substantial challenges to this transition. In this study, a rapid method for the online analysis of antibiotics in seawater samples via nano-electrospray ionization (nESI) MS based on slug-flow microextraction (SFME) has been proposed. Comparisons with other methods, complex laboratory setups for sample processing are now seamlessly integrated into a single online step, completing the entire process, including desalination and detection, SFME-nESI-MS provides faster results in less than 2 min while maintaining sensitivity comparable to that of other detection methods. Using SFME-nESI, six antibiotics in high-salinity (3.5 wt%) seawater samples have been determined in both positive and negative ion modes. The proposed method successfully detected clarithromycin, ofloxacin, and sulfadimidine in seawater within a linear range of 1-1000 ng mL-1 and limit of detection (LOD) of 0.23, 0.06, and 0.28 ng mL-1, respectively. The method recovery was from 92.8% to 107.3%, and the relative standard deviation was less than 7.5%. In addition, the response intensity of SFME-nESI-treated high-salinity (3.5 wt%) samples surpassed that of untreated medium-salinity (0.35 wt%) samples by two to five orders of magnitude. This advancement provides an exceptionally simplified protocol for the online rapid, highly sensitive, and quantitative determination of antibiotics in high-salinity (3.5 wt%) seawater.

Determination of amino acid metabolic diseases from dried blood spots with a rapid extraction method coupled with nanoelectrospray ionization mass spectrometry.

In this work, a rapid extraction method of methanol/water (95:5 v/v) with 0.1% formic acid was developed for extraction of amino acids from dried blood spots (DBS) for inherited metabolic diseases (IMDs). The combination of this extraction procedure with nanoelectrospray ionization mass spectrometry (nESI-MS) was used for the rapid analysis of amino acids. This approach with eliminating the chromatographic separation required only 2 min for the extraction of amino acids from DBS, which simplified the configuration and improved the timeliness. Dependence of the sensitivity on the operating parameters was systematically investigated. The LOD of 91.2-262.5 nmol/L and LOQ of 304-875 nmol/L which were lower than the cut-off values were obtained for amino acids within DBS. The accuracy was determined to be 93.82%-103.07% and the precision was determined to be less than 8.30%. The effectiveness of this method was also compared with the gold standard method (e.g., LC-MS/MS). The desalination mechanism was explored with interference mainly originated from the blood. These findings indicated that the rapid extraction procedure coupled with nESI-MS is capable of screening indicators for IMDs in complex biological samples.

Detection of nanoplastics released from consumer plastic food containers by electromagnetic heating pyrolysis mass spectrometry.

Nanoplastics released from consumer plastic food containers are emerging environmental pollutants and directly ingested as part of the diet. However, quantification methods for nanoplastics are still lacking. Herein, a rapid identification and mass quantification approach was developed for nanoplastics analysis by combining electromagnetic heating with pyrolysis mass spectrometry (Eh-Py-MS). The pyrolysis products directly entered into the MS, which omits the gas phase separation process and shortens the detection time. A compact pyrolysis chamber was used and this increased the sample transfer efficiency and lowered power requirement. The operational parameters were systematically examined. The influence of nanoplastic size, additive, humic acid, and aging on detection was investigated, and it was concluded that environmental factors (humic acid, aging) and plastic properties (size, additives) did not influence the detection. The developed chamber showed that the limit of detection of polystyrene (PS) nanoplastics was 15.72 ng. Several typical food packages were demonstrated with satisfactory recovery rates (87.5-110%) and precision (RSD ≤11.36%). These results suggested that the consumer plastic food containers are a significant source of direct exposure to nanoplastics in humans from the environment.

Nicotiana alkaloids-intervened phospholipid ozonolysis at the air-water interface.

Cigarette nicotiana alkaloids associated with lung and cardiovascular diseases attack enormous attention. However, the mechanism at the molecular level between nicotiana alkaloids and phospholipid ozonolysis remains elusive. Herein, we investigated the interfacial ozonolysis of a hung droplet containing 1-palmitoyl-2-oleoyl-sn-phosphatidylglycerol (POPG) intervened by nicotiana alkaloids (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, NNK; rac-N'-nitrosonornicotine, NNN; nicotine; and (R,S)-N-nitrosoanasabine, NAT) and followed by on-line mass spectrometry analysis. NNK and NNN showed an acceleration on the interfacial ozonolysis, while nicotine and NAT inhibited this chemistry. Such acceleration/inhibition on POPG ozonolysis was positively correlated with nicotiana alkaloid concentrations. The reaction rate constants suggested that the ozonolysis of lung phospholipids exposed to cigarette smoke at the air-water interface occurred rapidly. A possible mechanism of the hydrophilic/oleophilic nature of nicotiana alkaloids mediating the packing density of POPG was proposed. NNK and NNN with a hydrophilic nature inserted into the POPG monolayer loosed the packing, but nicotine and NAT with an oleophilic nature let the POPG closely pack and shield the CC double bonds exposed to ozone (O3). These results gain the knowledge of nicotiana alkaloids mediated phospholipid ozonolysis at the molecule level and provide a method for online interfacial reaction studies associated with elevated indoor pollutants on public health.

Recent Advances in Catecholamines Analytical Detection Methods and Their Pretreatment Technologies.

Catecholamines (CAs), including adrenaline, noradrenaline, and dopamine, are neurotransmitters and hormones that play a critical role in regulating the cardiovascular system, metabolism, and stress response in the human body. As promising methods for real-time monitoring of catecholamine neurotransmitters, LC-MS detectors have gained widespread acceptance and shown significant progress over the past few years. Other detection methods such as fluorescence detection, colorimetric assays, surface-enhanced Raman spectroscopy, and surface plasmon resonance spectroscopy have also been developed to varying degrees. In addition, efficient pretreatment technology for CAs is flourishing due to the increasing development of many highly selective and recoverable materials. There are a few articles that provide an overview of electrochemical detection and efficient enrichment, but a comprehensive summary focusing on analytical detection technology is lacking. Thus, this review provides a comprehensive summary of recent analytical detection technology research on CAs published between 2017 and 2022. The advantages and limitations of relevant methods including efficient pretreatment technologies for biological matrices and analytical methods used in combination with pretreatment technology have been discussed. Overall, this review article provides a better understanding of the importance of accurate CAs measurement and offers perspectives on the development of novel methods for disease diagnosis and research in this field.

Aqueous microdroplets promote C-C bond formation and sequences in the reverse tricarboxylic acid cycle.

The reverse tricarboxylic acid cycle (rTCA) is a central anabolic network that uses carbon dioxide (CO2) and may have provided complex carbon substrates for life before the advent of RNA or enzymes. However, non-enzymatic promotion of the rTCA cycle, in particular carbon fixation, remains challenging, even with primordial metal catalysis. Here, we report that the fixation of CO2 by reductive carboxylation of succinate and α-ketoglutarate was achieved in aqueous microdroplets under ambient conditions without the use of catalysts. Under identical conditions, the aqueous microdroplets also facilitated the sequences in the rTCA cycle, including reduction, hydration, dehydration and retro-aldol cleavage and linked with the glyoxylate cycle. These reactions of the rTCA cycle were compatible with the aqueous microdroplets, as demonstrated with two-reaction and four-reaction sequences. A higher selectivity giving higher product yields was also observed. Our results suggest that the microdroplets provide an energetically favourable microenvironment and facilitate a non-enzymatic version of the rTCA cycle in prebiotic carbon anabolism.

Reaction acceleration in microdroplet mass spectrometry: Inlet capillary and solvent composition effects.

RATIONALE: Microdroplet chemistry has attracted tremendous interest in recent years. We have previously reported that microdroplet mass spectrometry (MS) achieves reaction acceleration. Here we systematically investigated the effect of capillary heating of MS inlet and solvent polarity of microdroplets on the conversion ratios of dehydration and phosphorylation reactions. METHODS: The micron-sized droplets generated by high-speed gas encapsulated the compounds. The conversion ratios of dehydration and phosphorylation reactions were investigated at different capillary temperatures of MS inlet between 30°C and 300°C. Subsequently, the effects of solvent polarity of different microdroplets (acetonitrile, acetonitrile/water [v/v: 9:1], and water) on microdroplet reactions were investigated. RESULTS: The microdroplets could be used as reaction vessels for rapid dehydration and phosphorylation reactions. Microdroplet MS is characterized by the completion of the reaction in microseconds. The increase in capillary temperature increased the conversion ratio of dehydration reactions but had little effect on phosphorylation reactions. The stability of compounds supports this phenomenon. In addition, the increase in solvent polarity in microdroplets promoted the dehydration reaction but inhibited the nucleophilic substitution reaction (phosphorylation reaction). CONCLUSIONS: Microdroplet MS achieved an acceleration of the reaction, which was attributed to capillary temperature, microdroplet solvents, and the stability of reaction products. This finding suggested that the inlet capillary and solvent system should be considered in the study and interpretation of microdroplet MS.

Abiotic synthesis with plausible emergence for primitive phospholipid in aqueous microdroplets.

Phospholipids are the protective layer of modern cells, but it is challenging for the formation of phospholipids that require a simple abiotic synthesis before the advent of primitive cells. Here, we reported the abiotic synthesis for lysophosphatidic acids (LPAs) with prebiotically plausible reactants in aqueous microdroplets under ambient conditions. The LPAs formation is carried out by fusing two microdroplets streams: one contains glycerol and pyrophosphate in water and the other one contains fatty acids in acetonitrile. Compared with the bulk solution, LPAs were generated in microdroplets without the addition of catalyst and heating. Conditions of reactant concentrations and microdroplet size varied and suggested that LPAs formation occurred near or at the microdroplet surface. The LPAs formation also showed chemoselective toward on chain-length of fatty acids. Finally, the formation of LPAs underwent two-step reactions with glycerol phosphorylation eliminating one water molecule followed by esterification with fatty acids. These results also implicated that pyrophosphate functioned as both catalysts and precursors in prebiotic LPAs synthesis. The approach using fusion aqueous microdroplets has desirable features in studying the substance exchange and interaction in atmosphere.

Biochemical and Metabolomic Responses of Antarctic Bacterium Planococcus sp. O5 Induced by Copper Ion.

Heavy metal pollution in the Antarctic has gone beyond our imagination. Copper toxicity is a selective pressure on Planococcus sp. O5. We observed relatively broad tolerance in the polar bacterium. The heavy metal resistance pattern is Pb2+ > Cu2+ > Cd2+ > Hg2+ > Zn2+. In the study, we combined biochemical and metabolomics approaches to investigate the Cu2+ adaptation mechanisms of the Antarctic bacterium. Biochemical analysis revealed that copper treatment elevated the activity of antioxidants and enzymes, maintaining the bacterial redox state balance and normal cell division and growth. Metabolomics analysis demonstrated that fatty acids, amino acids, and carbohydrates played dominant roles in copper stress adaptation. The findings suggested that the adaptive mechanisms of strain O5 to copper stress included protein synthesis and repair, accumulation of organic permeable substances, up-regulation of energy metabolism, and the formation of fatty acids.

Adsorption behaviors of triclosan by non-biodegradable and biodegradable microplastics: Kinetics and mechanism.

Microplastics (MPs) pollution has been becoming serious and widespread in the global environment. Although MPs have been identified as vectors for contaminants, adsorption and desorption behaviors of chemicals with non-biodegradable and biodegradable MPs during the aging process is limited. In this work, the adsorption behaviors of triclosan (TCS) by non-biodegradable polyethylene (PE) and polypropylene (PP), and biodegradable polylactic acid (PLA) were investigated. The differences in morphology, chemical structures, crystallization, and hydrophilicity were investigated after the ultraviolet aging process and compared with the virgin MPs. The results show that the water contact angles of the aged MPs were slightly reduced compared with the virgin MPs. The aged MPs exhibited a stronger adsorption capacity for TCS because of the physical and chemical changes in MPs. The virgin biodegradable PLA had a larger adsorption capacity than the non-biodegradable PE and PP. The adsorption capacity presented the opposite trend after aging. The main adsorption mechanism of MPs relied on hydrophobicity interaction, hydrogen bonding, and electrostatic interaction. The work provides new insights into TCS as hazardous environmental contaminants, which will enhance the vector potential of non-biodegradable and biodegradable MPs.

Mass spectrometric observation on free radicals during electrooxidation of dopamine.

It has been challenging to directly observe the o-semiquinone radicals and transient intermediates produced during the oxidation of dopamine (DA). To achieve this goal, we developed an electrochemistry-neutral reionization-mass spectrometry (EC-NR-MS) technique for online studying the electrooxidation process of DA. The EC-NR device mainly composed by a self-designed EC flow cell, a sonic spray ionization source, a heating tube, an ion deflector and an electrospray ionization source. By precisely controlling the oxidation potential at 0.55 V, a series of reaction products include o-quinone (DAQ), Leukodopaminochrome (LDAC), Dopaminochrome (DAC), 5,6-dihydroxyindole (DHI) and DA dimer clearly appeared in the MS spectrum. Based on the ion deflector of EC-NR setup, the neutral o-semiquinone radical DA● and neutral Leukodopaminochrome radical LDAC● were successfully extracted from these ionic products and allowed to be detected by MS. Such finding was further confirmed by spin trapping experiment. The formation of o-semiquinone radical anion (DA●-) has been identified as its metal complex with Zn2+. These results provide conclusive evidence for the DA oxidation mechanism, which consists of proton coupled electron transfer and sequential proton-electron transfer steps.

Aqueous-Microdroplet-Driven Abiotic Synthesis of Ribonucleotides.

Phosphorylation for ribonucleotide formation is a critical step in the origin of life but has had limited success due to the thermodynamic and kinetic constraints in aqueous media. Here, we report that the production of ribonucleotides from ribonucleosides in the presence of monopotassium phosphate (KH2PO4) spontaneously proceeded in aqueous microdroplets under ambient conditions and without using a catalyst. A full set of ribonucleotides including adenosine monophosphate (AMP), guanosine monophosphate (GMP), uridine monophosphate (UMP), and cytidine monophosphate (CMP) were generated on the scale of a few milliseconds. The aqueous microdroplets could transfer the ribonucleotides to oligoribonucleotides and showed mutual compatibility for individual phosphorylation. Conditions established the dependence of the conversion ratio on the droplet size and suggested that the condensation reactions occurred at or near the microdroplets' surface. This aqueous microdroplet approach also provides a route for elucidating phosphorylation chemistry in the prebiotic era.

Metabolomics reveals the mechanism of Antarctic yeast Rhodotorula mucliaginosa AN5 to cope with cadmium stress.

Heavy metal pollution in Antarctica has far exceeded expectations. Antarctic yeast is widely present in polar marine environment. The mechanisms of metabolomics effect of heavy metal on polar yeast have not been reported previously. In this study, gas chromatography-mass spectrometry (GC-MS) wascarried out to performed the metabolite profiling analysis of Antarctic sea-ice yeast Rhodotorula mucilaginosa AN5 exposed to different cadmium (Cd) stresses of 5 mM (HM5), 10 mM (HM10) and 20 mM (HM20), respectively. Metabolic profile analysis showed that the composition and contents of cellular metabolites have been altered by cadmium. 93 different metabolites were identified altogether, among which 23, 58 and 81 different metabolites were found in HM5, HM10 and HM20 group respectively. MetaboAnalyst analysis showed that in HM5, HM10 and HM20 groups, 12, 24 and 31 metabolic pathways were involved in the stress of cadmium to R. mucilaginosa, respectively. By contrasting with Kyoto Encyclopedia of Genes and Genomes database, we discovered that exposure of yeast AN5 to Cd stress resulted in profound biochemical changes including amino acids, organic acids and saccharides. These results will supply a nonnegligible basis of studying the adaptive resistance mechanism of Antarctic yeast Rhodotorula mucilaginosa to heavy metal.

Molecular cloning and sequence analysis of a mitogen-activated protein kinase gene in the Antarctic yeast Rhodotorula mucilaginosa AN5.

BACKGROUND: The mitogen-activated protein kinase (MAPK) cascades play important roles in various signaling transduction networks of biotic and abiotic stress responses. However, MAPK signaling pathways in cold-active yeast Rhodotorula mucilaginosa have not been reported comprehensively. METHODS AND RESULTS: In the present study, MAPK gene (RmMAPK) was first cloned and characterized from Antarctic sea ice yeast R. mucilaginosa AN5. The full length of the RmMAPK gene is 1086 bp and encodes a 361 amino acids protein with a predicted molecular mass of 40.9 kDa and a pI of 5.25. The RmMAPK contains 11 MAPK conserved subdomains and the phosphorylation motif TGY located in the activation loop of the kinase. Quantitative real-time PCR and western blot assay revealed that the expression and phosphorylation level of RmMAPK up-regulated rapidly and significantly when yeast cells were subjected to low temperature (4 °C), high salinity (120‰ NaCl) and heavy metal (2 mmol/L CuCl2). CONCLUSIONS: All data suggested that the MAPK cascades might act as a key function in response to extreme stresses, such as low temperature, high salinity and heavy metal.

Hypochlorous acid initiated lipid chlorination at air-water interface.

There has been a surge of interest in interfacial hypochlorous acid (HOCl) chemistry for indoor air quality and public health. Here we combined nanoelectrospray mass spectrometry (nESI-MS) and acoustic levitation (AL) techniques to study the chlorination chemistry of three model lipids (DPPE, POPG, DOPG) mediated by HOCl at the air-water interface of levitated water droplet. For DPPE with no CC double bonds, HOCl was insensitive to the alkane chains, and showed considerable delay directing to head amino groups compared to that in aqueous environment. Chlorination chemistry, for POPG and DOPG with CC double bonds, preferentially reacted with double bonds of one chain. The mechanism was discussed in light of these observations, and it is concluded that the increased hydrophilicity of the chlorinated chain disturbed the lipid packing and attracted it toward the water phase. In addition, the reaction rate constant and reactive uptake coefficient suggested that the chlorination of lipids exposed to HOCl at the air-water interface is likely to occur rapidly. These results gain the knowledge of HOCl mediated lipid interface reaction at the molecule level, and would better understand the adverse health effects associated with elevated indoor pollutants.

Water Microdroplets Allow Spontaneously Abiotic Production of Peptides.

The chemistry of abiotic synthesis of peptides in the context of their prebiotic origins is a continuing challenge that arises from thermodynamic and kinetic constraints in aqueous media. Here we reported a strategy of microdroplets' mass spectrometry for peptide bonds formed from pure amino acids or a mixture in the presence of phosphoric acids in aqueous microdroplets. In contrast to bulk experiments, the condensation reactions proceed spontaneously under ambient conditions. The microdroplet gave a negative free-energy change (ΔG ∼ -1.1 kcal/mol), and product yields of ∼75% were obtained at the scale of a few milliseconds. Experiments in which nebulization gas pressure and external charge were varied established dependence of peptide production on the droplet size that has a high surface-to-volume ratio. It is concluded that the condensation reactions occurred at or near the air-water interfaces of microdroplets. This aqueous microdroplets approach also provides a route for chemistry synthesis in the prebiotic era.

Cloning, characterization and expression analysis of glutathione S-transferase from the Antarctic yeast Rhodotorula mucilaginosa AN5.

The gene for glutathione S-transferase (GST) in Antarctic sea-ice yeast Rhodotorula mucilaginosa AN5 was cloned and expressed in Escherichia coli and named RmGST. Sequence analysis showed that the RmGST gene contained a 843 bp open reading frame, which encoded 280 amino acid residues with a calculated molecular mass of 30.4 kDa and isoelectric point of 5.40. RmGST has the typical C- and N-terminal double domains of glutathione S-transferase. Recombinant RmGST (rRmGST) was expressed in E. coli to produce heterologous protein that had a high specific activity of 60.2 U/mg after purification. The apparent Km values of rRmGST for glutathione and 1-chloro-2,4-dinitrobenzene were 0.35 mM and 0.40 mM, respectively. Optimum enzyme activity was measured at 35 °C and at pH 7.0 and complete inactivation was observed after incubation at 55 °C for 60 min rRmGST tolerated high salt concentrations (1.0 M NaCl) and was stable at pH 3.0. Additionally, the recombinant protein nearly kept whole activity in Hg2+ and Mn2+, and could tolerate Ca2+, Cu2+, Mg2+, Cd2+, EDTA, thiourea, urea, Tween-80, H2O2 and Triton X-100. Real-time quantitative PCR showed that relative expression of the GST gene was significantly increased under Cu2+ and low temperature stress. These results indicate that rRmGST is a typical low thermostable enzyme, while its other characteristics, heavy metal and low temperature tolerance, might be related to its Antarctic home environment.

Cloning and functional characterization of a novel metallothionein gene in Antarctic sea-ice yeast (Rhodotorula mucilaginosa).

Metallothionein (MT) is a low-molecular-weight protein with a high metal binding capacity and plays a key role in organism adaptation to heavy metals. In this study, a metallothionein gene was successfully cloned and sequenced from Antarctic sea-ice yeast Rhodotorula mucilaginosa AN5. Nucleotide sequencing and analysis revealed that the gene had four exons interrupted by three introns. MTs complementary DNA (named as RmMT) had an open reading frame of 321 bp encoding a 106 amino acid protein with a predicted molecular weight of 10.3 kDa and pI of 8.49. The number of amino acids and distribution of cysteine residues indicated that RmMT was a novel family of fungal MTs. Quantitative real-time polymerase chain reaction analysis showed that RmMT expression was elevated under copper-induced stress. The RmMT gene was transferred into E. coli and the RmMT expressing bacteria showed improved tolerance to copper ion and increased accumulation of heavy metals, such as Cu2+ , Pb2+ , Zn2+ , Cd2+ , and Ag+ . Moreover, in vitro studies, purified recombinant RmMT demonstrated that it could be used as a good scavenger of superoxide anion, hydroxyl, and 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radicals. In summary, these results demonstrate that RmMT plays a key role in the tolerance and bioaccumulation of heavy metals.

Purification and characterization of a novel wild-type α-amylase from Antarctic sea ice bacterium Pseudoalteromonas sp. M175.

A novel wild-type α-amylase named wtAmy175 from Pseudoalteromonas sp. M175 strain was purified through ammonium sulphate precipitation, DEAE cellulose, and Sephadex G-75 sequentially (25.83-fold, 7.67%-yield) for biochemical characterization. SDS-PAGE and zymographic activity staining of purified enzyme showed a single band with a predicted molecular mass of about 61 kDa. The optimum temperature and pH for enzyme activity were 30 °C and 7.5, respectively. Additionally, the enzyme exhibited high activity and remarkable stability in 0-10 mM SDS. The values of Km and Vmax for soluble starch as substrate were 2.47 mg/ml and 0.103 mg/ml/min, respectively. Analysis of hydrolysis products of soluble starch and maltooligosaccharides showed that wtAmy175 cleaved the interior and the terminal α-1,4-glycosidic linkage in starch, and had transglycosylation activity. The result of fluorescence spectroscopy showed that wtAmy175 had strong binding affinity with soluble starch. In brief, this study discovered the first wild-type α-amylase so far with several distinctive properties of cold activity, SDS-resistance, and the mixed activity of α-amylase and α-glucosidase, suggesting that wtAmy175 possess high adaptive capability to endure harsh industrial conditions and would be an excellent candidate in detergent and textile industries.