Ascorbic acid (AA) and alkaline phosphatase (ALP) are important biomarkers for diagnosing scurvy, liver dysfunction, and bone metabolic disorders. In this work, we have synthesized Pt@PCN-224(FeⅢ), a metal-organic framework (MOF) doped with Fe3+ and decorated with Pt nanoparticles for sensitive detection of AA and ALP. By synergistically integrating Fe3+ and Pt into the PCN-224 framework, the material exhibits a 2-fold enhancement in peroxidase-like activity over pristine PCN-224. Using H2O2 as an oxidant, Pt@PCN-224(FeⅢ) efficiently catalyzes the conversion of colorless TMB to generate blue ox-TMB. AA suppresses blue signal generation by reducing ox-TMB to colorless state. This enables AA detection with a good linear response over the range of 5-60 μM and a detection limit of 0.13 μM. For ALP analysis, ALP-mediated hydrolysis of L-ascorbic acid 2-phosphate trisodium (AA2P) generates AA, which reduces ox-TMB. ALP concentration thus inversely correlates with ox-TMB absorbance, showing a linear range of 1-150 U/L with a detection limit of 0.75 U/L. The detection platform demonstrates robust performance in complex biological matrices, with spiked recoveries of 96.5-103.8%. Therefore, this work presents a portable, low-cost point-of-care testing solution that completes the entire detection process within 15 min without sophisticated instrumentation.

Mercury ion (Hg2+) pollution poses severe environmental and health risks, necessitating the development of highly sensitive and reliable detection methods. Although ratiometric and genetically encoded fluorescent probes each offer distinct advantages, their integration for Hg2+ sensing remains largely unexplored. Herein, we report the rational design of a novel ratiometric genetically encoded fluorescent probe through the fusion of a circularly permuted green fluorescent protein (cpEGFP), a mercury-binding domain (MerBD), and a large Stokes shift reference fluorescent protein (LSSmOrange). This probe enables self-calibrated Hg2+ quantification by measuring the fluorescence intensity ratio of the response channel (F512) to the stable reference channel (F572), which effectively minimizes interferences from probe concentration, environmental fluctuations, and instrumental variations. The probe exhibits an ultra-high affinity for Hg2+ (apparent Kd' = 2.71 × 10-13 M) and a detection limit of 4 nM, alongside long-term stability and high selectivity against most common metal ions. Competitive titration, circular dichroism, and fluorescence lifetime analyses reveal that the probe operates through a cooperative conformational-change mechanism, which translates picomolar-level binding into a nanomolar-level fluorescence response. This work not only addresses a critical gap in ratiometric genetically encoded probes for Hg2+ but also provides a robust and versatile platform for accurate Hg2+ monitoring in environmental and biological systems.

Reactions that excel in small-molecule settings typically require metal loadings far exceeding the number of protein reaction sites (often ≥10-fold) once transplanted into proteinaceous media-conditions that are not truly "catalytic." Here, we show that biologically inert metal-ligand complexes based on bathocuproine disulfonic acid disodium salt (BCS) overcome this barrier and enable ligand-accelerated catalysis (LAC) on proteins under substoichiometric conditions. For example, Ni-BCS effects complete deprotection of green fluorescent protein bearing Nε-propargyloxycarbonyl-L-lysine (GFP-ProcLys) at 5 mol% catalyst with an observed turnover number (TON) ≈ 20, surpassing all previously reported metal-catalyzed depropargylation reactions. Mechanistic studies indicate that an in situ Ni-H intermediate mediates multiple transformations on proteins, including reductive deuteration of terminal alkenes/alkynes and efficient decaging across diverse amino acid side chains. Likewise, Cu-BCS enables copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) on proteins at 10 mol% with low residual copper and no protein oxidation, in sharp contrast to the benchmark Cu-BTTAA (tris((1-tert-butyl-1H-1,2,3-triazol-4-yl)methyl)amine) system. These outcomes stem from a screening strategy that prioritized metal-ligand stability, eliminating metal complexes susceptible to protein sequestration and selecting strongly coordinating, physiologically inert pairs. The resulting rational ligand-design framework for protein-level transition-metal catalysis expands the frontier of protein chemistry and paves the way to translate advanced small-molecule LAC strategies onto protein substrates for posttranslational mutagenesis.

Rare earth elements (REEs) are emerging bioaccumulative contaminants threatening health and ecosystems. However, current detection and recovery technologies face three key bottlenecks: poor selectivity between light and heavy REEs, costly multi-step adsorbents, and a lack of visualized adsorption platforms. Here, we develop a bifunctional platform based on REE-responsive Hans-LanM. Inserting Hans-LanM into sfGFP yields a sensitive probe (sfGFP-LanM) with picomolar affinity, 2 nM detection limit for Sm³ ⁺, excellent selectivity for light/medium REEs, reversibility, and long-term stability. Mechanistically, REE-induced conformational ordering of LanM enhances sfGFP fluorescence by stabilizing its chromophore and increasing the radiative rate. Displaying this fusion protein on E. coli creates a whole-cell adsorption system. The engineered strain achieves 81.5% (Nd³⁺) and 72.6% (Sm³⁺) adsorption vs. 32% (Y³⁺) at 500 μM, an equilibrium capacity of 150 mg/g for Sm³ ⁺, and rapid equilibrium within 30 min. Notably, it maintains selective REE adsorption (55%-70%) in a real soil leachate containing a 10⁶-fold excess of competing ions. Its fluorescence signal linearly correlates with adsorbed amount, enabling real-time monitoring and quantification. The system is efficiently regenerated (>85% desorption at pH 2.3) and maintains > 60% efficiency after 5 cycles. Adsorption kinetics follow a quasi-second-order model, indicating monolayer chemical adsorption, consistent with the molecular recognition mechanism of the probe in solution. Multidimensional characterization reveals that Hans-LanM mediates specific adsorption via synergistic coordination of carboxylate, hydroxyl, and amide groups. This study provides a new strategy with both theoretical depth and application potential for the highly sensitive detection and green recovery of rare earth resources.

Phosphate (Pi) and superoxide anion radical ( O_2^· - ) serve as crucial bioactive molecules, with their dysregulated concentrations being strongly implicated in the pathogenesis of multiple disorders, including atherosclerosis and neurodegenerative diseases. This study innovatively developed a dual-functional fluorescent sensor, UiO-66-NH₂@CDs, which achieved high-selective simultaneous detection of Pi and O_2^· - for the first time. A mild room-temperature stirring method was used to construct the amino-functionalized UiO-66-NH₂ carrier, followed by post-synthesis modification to precisely load fluorescent carbon dots (CDs) specific for superoxide anion recognition. Mechanism studies revealed that the redox reaction between CDs and O_2^· - specifically activates the fluorescence signal at 655 nm, while the Zr (IV) clusters in UiO-66-NH2 form Zr-O-P coordination bonds with Pi, leading to fluorescence recovery at 440 nm. The detection limit for Pi was 0.032 μM, and for O2⋅− was 0.21 μM, with no interference between two detection channels. In actual serum sample testing, the recovery was 97.2 O_2^· - in physiological and pathological processes.

This study introduces cpEGFP-MerBD, a novel genetically encoded fluorescent probe for Hg2+ detection, overcoming existing limitations in sensitivity, selectivity, and dynamic monitoring range. Engineered with cpEGFP and a sensing domain MerBD based on the native Tn501 MerR protein, cpEGFP-MerBD offers a broad linear detection range (50-7000 nM), high Hg2+ affinity (Kd ' = 1.91 +/- 0.08 x 10-13 M), and robust anti-interference properties. It enables accurate Hg2+ detection in environmental and biological samples, including real-time tracking of intracellular Hg2+ uptake. This work also elucidates the involvement of Ca2+ ion channels in cellular Hg2+ uptake, offering insights into Hg2+ biological transport. The cpEGFP-MerBD probe represents a significant advancement in mercury research, promising to enhance monitoring and unravel the mechanisms of Hg2+ toxicity.

Coating nanomaterials with natural cell membranes has enabled a wide range of promising nanomedicine platforms. To support the broader translation of this technology, scalable membrane manufacturing is essential. In this study, we present a continuous and scalable process for isolating cell membranes using an affinity chromatography-based approach. This method efficiently separates membranes from cell lysates while preserving their purity and structural integrity. The continuous process yields high-purity membranes across multiple cycles, demonstrating consistent and robust performance. Comparative analyses show that membranes isolated via this method effectively coat synthetic nanoparticle cores. The resulting cell membrane-coated nanoparticles exhibit similar morphology, size, surface charge, colloidal stability, and pharmacokinetic profile to those prepared using membranes obtained by conventional centrifugation method, with no observable acute toxicity. Overall, this work introduces an efficient and scalable membrane isolation method that streamlines cell membrane production and supports the large-scale adoption of cell membrane coating technology for biomedical applications.

Nitrate radical (NO3) and dinitrogen pentoxide (N2O5) play an important role in nocturnal tropospheric chemistry. Here we present the measurements of gas-phase NO3 and N2O5 by a cavity ringdown spectroscopy (CRDS) instrument from June 2 to June 22, 2017.The measurement site was in an urban area of Beijing, China as a part of the Air Pollution and Human Health (APHH) campaign. N2O5 and NO3 showed large day-to-day variations with average (± 1σ) mixing ratios of 88.5 ± 101.7 pptv and 8.3 ± 6.5 pptv, respectively. The heterogeneous N2O5 uptake coefficient (γN2O5) was retrieved from steady-state analysis. The estimated N2O5 uptake coefficient ranged from 0.045 to 0.109, with an average of 0.083 ± 0.027. Further analysis revealed that the average NO3 reactivity by the reaction with volatile organic compounds (VOCs) was 0.024 s− 1. The oxidation of biologic volatile organic compounds (BVOCs) by NO3 was an important pathway for the generations of organic nitrates (ONs) and secondary organic aerosols (SOA) at night. The ON production was 0.057 ppbv/h. The heterogeneous reaction of N2O5 plays an important role in the formation of nitrate. The nitrate formations during the whole night were 24.05 ± 20.56 µg/m3 in Beijing. We highlight the importance of NO3 oxidation of VOCs in the formation of ON and subsequent secondary organic aerosols in summer in Beijing.

Background: The accurate and rapid detection of blood lead concentration is of paramount importance for assessing human lead exposure levels. Fluorescent protein -based probes, known for their high detection capabilities and low toxicity, are extensively used in analytical sciences. However, there is currently a shortage of such probes designed for ultrasensitive detection of Pb 2+ , and no reported probes exist for the quantitative detection of Pb 2+ in blood samples. This study aims to fill this critical void by developing and evaluating a novel fluorescent protein -based probe that promises accurate and rapid lead quantification in blood. Results: A simple and small -molecule fluorescent protein -based probe was successfully constructed herein using a peptide PbrBD designed for Pb 2+ recognition coupled to a single fluorescent protein, sfGFP. The probe retains a three -coordinate configuration to identify Pb 2+ and has a high affinity for it with a K d ' of 1.48 +/- 0.05 x 10 -17 M. It effectively transfers the conformational changes of the peptide to the chromophore upon Pb 2+ binding, leading to fast fluorescence quenching and a sensitive response to Pb 2+ . The probe offers a broad dynamic response range of approximately 37 -fold and a linear detection range from 0.25 nM to 3500 nM. More importantly, the probe can resist interference of metal ions in living organisms, enabling quantitative analysis of Pb 2+ in the picomolar to millimolar range in serum samples with a recovery percentage of 96.64%-108.74 %. Significance: This innovative probe, the first to employ a single fluorescent protein -based probe for ultrasensitive and precise analysis of Pb 2+ in animal and human serum, heralds a significant advancement in environmental monitoring and public health surveillance. Furthermore, as a genetically encoded fluorescent probe, this probe also holds potential for the in vivo localization and concentration monitoring of Pb 2+ .

The calcium/calmodulin-dependent protein kinase II (CaMKII) is a mediator of calcium signals and regulates fatty acid metabolism in mammalian cells. Cmk2p is a yeast homolog of CaMKII and functions as a negative regulator of calcium signaling. However, its substrates remain to be identified. Combination of immunoprecipitation (IP) and mass spectrometry has been proven to be very useful for identification of interacting partner proteins and interactome. In this study, through these approaches, we have identified 65 and 110 potential Cmk2p-interacting proteins in yeast cells in the absence or presence of calcium stress, respectively. In yeast cells expressing both CMK2-HA and FAS1-GFP fusion proteins, in the absence or presence of calcium stress, less amounts of FAS1-GFP proteins are present in cell lysates after IP with anti-HA antibody than cell lysates before IP, while FAS1-GFP proteins are detected on both types of IP beads. However, as an internal control, similar amounts of Pgk1p proteins were detected in both after-IP and before-IP cell lysates but not on the IP beads. Therefore, our biochemical analysis demonstrates that the β subunit Fas1p of fatty acid synthetase interacts with Cmk2p in yeast cells independent of calcium stress. It is also interesting to note that, in addition to the expected 52-kDa CMK2-HA band, a faster-moving 48-kDa CMK2-HA band is present in the calcium-stressed cell lysate but not in the cell lysate without calcium stress. Our data would provide important clues for understanding the functions of CaMKII in the regulation of fatty acid metabolism as well as related diseases such as cancers, diabetes, and obesity.

At present, few genetically encoded fluorescent probes are currently available for the analysis of toxic heavy metal ions, and most have poor performance that cannot meet the requirements of sensitive and dynamic detection in living cells. In this study, we designed a single fluorescent protein-based probe sfGFP-MerBD, which can specifically response to Hg2+ with high binding affinity and wide dynamic range. More importantly, the developing probe can timely and reversibly monitor changes of Hg2+ concentration in living mammalian cells. The excellent performance of this probe is largely due to the recognition element of the probe, MerBD, which adopts an unusual planar trigonometric coordination configuration with Hg2+, and the coordination can cause enough conformational change to influence the fluorescence of skeleton protein sfGFP coupled with it. The small peptide MerBD was delicately designed based on the three-dimensional structure of metalloprotein MerR. This novel design strategy solves the challenging problems that there are few natural functional proteins in the process of constructing fluorescent probes for toxic metal ions and some functional proteins cannot be directly used as recognition elements. Based on the new strategy, more genetically encoded fluorescent probes of toxic heavy metal ions can be efficiently constructed and applied in the future.

Rotavirus is the leading cause of life-threatening diarrhea in infants and young children, posing serious casualties and economic losses every year. Effective vaccines are urgently needed to combat rotavirus and protect the health of young children. In our previous study, a potential vaccine candidate was developed by using the ferritin nanocage as a platform to display the inner capsid protein VP6 of rotavirus on its surface. It could induce highly efficient humoral and mucosal immunogenicity in mice by oral administration. However, the biological macromolecule of recombinant rVP6-Ferritin was expressed as inclusion bodies in prokaryotic expression system, severely limiting its preparations, physical characterizations, and potential applications. In this work, we exploited a soluble, effective, and convenient method for the expression and purification of biological macromolecule rVP6-Ferritin. Similar to ferritin, the recombinant rVP6-Ferritin could self-assembling into spherical nanoparticles in the nanometer range with remarkable uniformity. However, the rVP6-Ferritin nanoparticles exhibited different surface morphology and pH-dependent size behavior from the ferritin nanocages. To our knowledge, this is the first report of expressing the ferritin-based antigen nanoparticles with good solubility in prokaryotic system by cell-surface display technique. Based on this work, more self-assembling biological macromolecule with nanometer scale could be rationally designed and conveniently manufactured for use in vaccines, drug delivery, gene therapy, and materials science.

随着工业的快速发展,汞离子被大量排放到环境中,造成了严重的环境污染.虽然加大了环境治理的力度,但已造成显著的生物毒性,严重的汞污染已对人类健康造成巨大威胁.该研究通过蛋白质工程技术,改变荧光蛋白mCherry发色团周围的环境,成功设计了一种荧光猝灭型的Hg2+荧光生物传感器(mCherry L199C).该荧光生物传感器可以快速、可逆的响应微摩尔级别的Hg2+,并且具有较好的金属离子选择性和较强的抗干扰能力.利用蛋白质固定化技术开发了蛋白琼脂糖凝胶试纸,初步实现了汞污染的现场可视化检测.

Rhizoma paridis is a traditional Chinese medicine that has been reported to have anti-cancer activity. However, the antitumor effect of pennogenin 3-O-β-chacotrioside (Formular: C45H72O17), a steroidal saponin isolated from its extract, in lung cancer have not been well studied, its potential mechanisms remain unclear. In this paper, its effect on the cell viability of non-small cell lung cancer (NSCLC) was examined using the Cell Counting Kit-8 assay, the potential target proteins were screened by antibody microarrays, the potential pharmacological mechanisms were predicted using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and protein–protein interaction (PPI) analyses. Moreover, to further validate the findings, cell apoptosis and cell cycle arrest were measured by flow cytometry, cell invasion and migration were measured by Transwell assays, and apoptosis-related protein expression levels were measured using western blotting. The results showed that, after treatment by pennogenin 3-O-β-chacotrioside, the cell viability was significantly reduced. In addition, thirteen target proteins related to apoptosis and platinum drug resistance were screened out, and their PPIs were constructed. Moreover, it was confirmed to have effects in inducing apoptosis and promoting cell cycle arrest in NSCLC cells. The expression levels of apoptosis-related proteins, such as caspase 8, caspase 9, phospho-ERK1/2, phospho-p38, phospho-Akt, were decreased following the treatment, whereas the expression levels of cleaved caspase 3 and cytochrome C were increased. The findings of the present study highlight the potential of pennogenin 3-O-β-chacotrioside as a novel therapeutic agent.

With increasing industrial activities, mercury has been largely discharged into environment and caused serious environmental problems. The growing level of mercury pollution has become a huge threat to human health due to its significant biotoxicity. Therefore, the simple and fast means for on-site monitoring discharged mercury pollution are highly necessary to protect human beings from its pernicious effects in time. Herein, a “turn off” fluorescent biosensor (mCherry L199C) for sensing Hg2+ was successfully designed based on direct modification of the chromophore environment of fluorescent protein mCherry. For rapid screening and characterization, the designed variant of mCherry (mCherry L199C) was directly expressed on outer-membrane of  Escherichia coli cells by cell surface display technique. The fluorescent biosensor was characterized to have favorable response to Hg2+ at micromole level among other metal ions and over a broad pH range. Further, the cells of the fluorescent biosensor were encapsulated in alginate hydrogel to develop the cells-alginate hydrogel-based paper. The cells-alginate hydrogel-based paper could detect mercury pollution in 5 min with simple operation process and inexpensive equipment, and it could keep fluorescence and activity stable at 4 °C for 24 hr, which would be a high-throughput screening tool in preliminarily reporting the presence of mercury pollution in natural setting.

A signal-amplified mercury sensing biosensor with desired sensitivity was developed through firstly using the GFP mutant with fluorescence increasing response towards Hg2+ as the reporter module. The developed biosensor showed response for Hg2+ in a relatively wide range of 1-10,000 nmol/L, and the detection limit was improved one or two orders of magnitude in comparison with most metal-sensing biosensors in similar constructs. In addition, the biosensor could distinguish Hg2+ easily from multiple metal ions and displayed strong adaptability to extensive pH conditions (pH 4.0-10.0). More importantly, the developed biosensor was able to provide an initial assessment of Hg2+ spiked in the environmental water with the recoveries between 85.70% and 112.50%. The signal-amplified strategy performed by the modified reporter module will be widely applicable to many other whole-cell biosensors, meeting the practical requirements with sufficient sensing performance.

目的 建立一种检测生乳中体细胞含量的快速检测方法(试剂盒法).方法 本试剂盒法通过生物酶溶解奶样中的蛋白质,缓冲液稳定脂肪球和蛋白并改变体细胞通透性,细胞裂解液及荧光染料快速渗透进入体细胞并沾染DNA,培养过程中,染液进入DNA的双螺旋结构,与DNA结合后荧光量子产率明显提高,最后通过荧光信号探测系统捕捉荧光强度来测定生乳中的体细胞数量.结果 该方法与快速仪器法对比,相关系数达94％以上,2种方法在统计学上无显著性差异(P>0.05).与标准方法对比,针对同一样品的检测结果的Log差值小于0.25,2种方法统计学上也无显著性差异(P>0.05).对不同样品进行重复性检测,相对标准偏差均小于15％.结论 该试剂盒法具有较高的准确度和精密度,检测结果与标准方法和市售仪器快速检测法无明显差异,检测时间缩短50％,提升了检测效率.由于无需大型的检测设备及配套试剂,适合牧场和企业实验室的快速定量检测.

A simulated occluded cell was used to investigate pitting corrosion of Grade D Q235 carbon steel in an ammonia (NH3) desulfurization slurry. In the initial stage, the pH drops rapidly, with chloride (Cl–) and fluoride (F–) ions quickly entering the simulated occluded cell. The corrosion rate is high during this period (12.7 mm/y). Later, the presence of F– attenuates the autocatalytic process of acidification, slowing and eventually stabilizing corrosion, pH decline, and the migration of Cl– and F– into the occluded zone. However, the corrosion rate is still large in the later stages (8.5 mm/y), indicating that pitting corrosion is serious.

Mercury pollution has always been a huge threat to human health due to its significant toxicity. Thus, it's the continuing goal to obtain new mercury detection techniques that are cost-effective, operational stable, performance efficient, and applicable to the environmental and biological milieus. In this research, the soluble pigment pyocyanin with anti-bacterial and anti-fungal activities, the biosynthesis pathway of which was engineered under the regulation of Hg2+-dependent transcriptional activator MerR, was firstly used as the visual detection signal in the whole-cell biosensor. The engineered biosensor displayed optical sensing window and a good linearity for Hg2+ in the range of 25-1000 nM, and the detection limit could reach as loW as 10 nM. It permitted on-site detection of bioavailable Hg2+ with extraordinary selectivity and could resist the interferences of extra metal ions. What's more, the developed biosensor performed function well in a wide pH range (pH 4-10) as well as the environmental water. By fully imitating and utilizing the biosystems from nature, the engineered colorimetric biosensor has great economic and performance advantages over most chemosensors as well as whole-cell biosensors in the practical application of detecting Hg2+ in the contaminated aquatic systems. (C) 2019, The Society for Biotechnology, Japan. All rights reserved.

Herein, interaction between graphene oxide (GO) and trypsin was systematically characterized for deep investigations of conformational structure and enzymatic activity of trypsin affected by GO. Results indicated that GO bound with trypsin to form ground state complex with molar ratio of 1 to 1. Intrinsic fluorescence of trypsin was statically quenched by GO through van der Waal interaction, hydrophobic interaction, hydrogen bond, and electrostatic interaction. Both tertiary structure and secondary structure of trypsin were changed obviously after its binding with trypsin, resulting in the structure transformation of trypsin from the β-sheet structure to the α-helix structure. Since GO bound with the allosteric site of trypsin to inhibit its enzymatic activity via non-competitive manner, GO efficiently protected human serum albumin and human cervical carcinoma HeLa cells from the digestion of trypsin. These results explored the exact binding mechanism of GO with protease, which provides more important information for possible biological risk of GO on human beings.