Conformational change-based fluorometric aptasensor for sensitive cadmium(II) detection in fruits and vegetables.

Cadmium (Cd2+) is a highly toxic heavy metal that can accumulate in the human body through contaminated food and water, posing great health risks. In this study, a label-free fluorescent aptasensor based on SYBR Green I (SGI) for the rapid and sensitive detection of Cd2+ in food samples was designed. The aptasensor utilizes a Cd2+-specific aptamer (Cd-(21)) and its complementary strand (CSCd-(21)) to form a double-stranded DNA (dsDNA) structure in the absence of Cd2+. SGI intercalates into the dsDNA, resulting in a strong fluorescence signal. In the presence of Cd2+, the aptamer undergoes a conformational change, preventing the formation of dsDNA and leading to a decrease in fluorescence intensity. Under optimized conditions, the aptasensor exhibited a linear response to Cd2+ concentrations ranging from 0.11 to 157.37 ng mL-1, with a limit of detection (LOD) of 0.07 ng mL-1. The aptasensor demonstrated high specificity and was successfully applied to detect Cd2+ in fruits and vegetables, with satisfactory recovery rates (95-111%). The proposed aptasensor provides a promising tool for the rapid and sensitive detection of Cd2+ in food.

Catalyst and base-free, direct oxidation of chitin to lactic acid with hydrogen peroxide.

In recent years, the research on the conversion of chitin to high value-added chemicals has attracted more and more attention. At present, the method of preparing lactic acid from chitin mostly uses strong base or catalyst. The reaction system under alkaline condition not only corrodes the container but also easily harms the human body. Herein, a simple and effective method to convert chitin to organic acids in catalyst and base-free conditions is developed. The use of H2O2 only can efficiently convert chitin to organic acids in the absence of bases and catalysts. Under the optimal conditions of 30 mg chitin, 2.1 mL water, 0.9 mL H2O2 at 230 °C for 1.5 h, the lactic acid yield of chitin can reach 58.2 % and the total organic acid yield can reach 84.0 %. This work provides an efficient method for the resource utilization of chitin biomass.

Tag-free fluorometric aptasensor for detection of chromium(VI) in foods via SYBR Green I signal amplification and aptamer structure transition.

BACKGROUND: In response to growing concerns regarding heavy metal contamination in food, particularly chromium (Cr)(VI) contamination, this study presented a simple, sensitive and practical method for Cr(VI) detection. RESULTS: A magnetic separation-based capture-exponential enrichment ligand system evolution (SELEX) method was used to identify and characterize DNA aptamers with a high affinity for Cr(VI). An aptamer, Cr-15, with a dissociation constant (Kd) of 4.42 ± 0.44 µmol L-1 was obtained after only eight rounds of selection. Further innovative methods combining molecular docking, dynamic simulation and thermodynamic analysis revealed that CrO4 2- could bind to the 19th and 20th guanine bases of Cr-15 via hydrogen bonds. Crucially, a label-free fluorometric aptasensor based on SYBR Green I was successfully constructed to detect CrO4 2-, achieving a linear detection range of 60-300 nmol L-1 with a lower limit of detection of 44.31 nmol L-1. Additionally, this aptasensor was able to quantitatively detect CrO4 2- in grapes and broccoli within 40 min, with spike recovery rates ranging from 89.22% to 108.05%. The designed fluorometric aptasensor exhibited high selectivity and could detect CrO4 2- in real samples without sample processing or target pre-enrichment. CONCLUSION: The aptasensor demonstrated its potential as a reliable tool for monitoring Cr(VI) contamination in fruit and vegetable products. © 2024 Society of Chemical Industry.

Advances in colorimetric aptasensors for heavy metal ion detection utilizing nanomaterials: a comprehensive review.

Heavy metal ion contamination poses significant environmental and health risks, necessitating rapid and efficient detection methods. In the last decade, colorimetric aptasensors have emerged as powerful tools for heavy metal ion detection, owing to their notable attributes such as high specificity, facile synthesis, adaptability to modifications, long-term stability, and heightened sensitivity. This comprehensive overview summarizes the key developments in this field over the past ten years. It discusses the principles, design strategies, and innovative techniques employed in colorimetric aptasensors using nanomaterials. Recent advancements in enhancing sensitivity, selectivity, and on-site applicability are highlighted. The review also presents application studies of successful heavy metal ion detection using colorimetric aptasensors, underlining their potential for environmental monitoring and health protection. Finally, future directions and challenges in the continued evolution of these aptasensors are outlined.

Improving aptamer performance: key factors and strategies.

Aptamers are functional single-stranded oligonucleotide fragments isolated from randomized libraries by Systematic Evolution of Ligands by Exponential Enrichment (SELEX), exhibiting excellent affinity and specificity toward targets. Compared with traditional antibody reagents, aptamers display many desirable properties, such as low variation and high flexibility, and they are suitable for artificial and large-scale synthesis. These advantages make aptamers have a broad application potential ranging from biosensors, bioimaging to therapeutics and other areas of application. However, the overall performance of aptamer pre-selected by SELEX screening is far from being satisfactory. To improve aptamer performance and applicability, various post-SELEX optimization methods have been developed in the last decade. In this review, we first discuss the key factors that influence the performance or properties of aptamers, and then we summarize the key strategies of post-SELEX optimization which have been successfully used to improve aptamer performance, such as truncation, extension, mutagenesis and modification, splitting, and multivalent integration. This review shall provide a comprehensive summary and discussion of post-SELEX optimization methods developed in recent years. Moreover, by discussing the mechanism of each approach, we highlight the importance of choosing the proper method to perform post-SELEX optimization.

Regulation of Parkin in Cr (VI)-induced mitophagy in chicken hepatocytes.

The large amount of heavy metal chromium emissions from industrial production, ore smelting and sewage treatment plants have made chromium one of the most widespread heavy metal pollutants, with Cr (VI) being the most toxic. In recent years, people have gradually recognized the great harm of heavy metal chromium pollution, but the research on its pathogenic mechanism is still not deep enough. In this study, we treated the Primary cells of chicken liver with Cr (VI) to establish a model of toxicity. The optimal treatment time and Cr (VI) concentration were screened using the CCK-8 test. The intracellular mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) were measured qualitatively and quantitatively by laser confocal and flow cytometry, respectively. This result was confirmed by the fact that Cr (VI) could cause mitophagy by causing damage to mitochondria. Subsequently, this study used LMH cells to construct a Parkin silencing model to further investigate that Parkin exerts the function on the Cr (VI)-induced mitophagy in chicken hepatocytes. The results showed that the knockdown of Parkin effectively blocked p62 degradation and LC3 lipidation and that PINK1 expression was significantly inhibited in LMH cells, further suggesting that the knockdown of Parkin effectively inhibited mitophagy. Mitochondrial morphology, MMP, and ROS were observed using laser confocal. The results showed that Parkin knockdown resulted in mitochondrial fission and increased levels of reactive oxygen species, together with increased depolarization of the mitochondrial membrane potential. These changes led to increased mitochondrial damage. In conclusion, this study showed that Cr (VI) could cause the occurrence of mitophagy by damaging mitochondria, and Parkin played a crucial role in Cr (VI)-induced mitophagy in chicken hepatocytes.

Physiological and transcriptomic analyses revealed gene networks involved in heightened resistance against tomato yellow leaf curl virus infection in salicylic acid and jasmonic acid treated tomato plants.

Tomato yellow leaf curl virus (TYLCV), a member of the genus Begomovirus of the Geminiviridae family, causes leaf curl disease of tomato that significantly affects tomato production worldwide. SA (salicylic acid), JA (jasmonic acid) or the JA mimetic, COR (coronatine) applied exogenously resulted in improved tomato resistance against TYLCV infection. When compared to mock treated tomato leaves, pretreatment with the three compounds followed by TYCLV stem infiltration also caused a greater accumulation of H2O2. We employed RNA-Seq (RNA sequencing) to identify DEGs (differentially expressed genes) induced by SA, JA, COR pre-treatments after Agro-inoculation of TYLCV in tomato. To obtain functional information on these DEGs, we annotated genes using gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) databases. Based on our comparative analysis, differentially expressed genes related to cell wall metabolism, hormone signaling and secondary metabolism pathways were analyzed in compound treated samples. We also found that TYLCV levels were affected in SlNPR1 and SlCOI1 silenced plants. Interestingly, compared to the mock treated samples, SA signaling was hyper-activated in SlCOI1 silenced plants which resulted in a significant reduction in viral titer, whereas in SINPR1 silencing tomato plants, there was a 19-fold increase in viral load. Our results indicated that SA, JA, and COR had multiple impacts on defense modulation at the early stage of TYLCV infection. These results will help us better understand SA and JA induced defenses against viral invasion and provide a theoretical basis for breeding viral resistance into commercial tomato accessions.

One-step synthesis of highly fluorescent carbon dots as fluorescence sensors for the parallel detection of cadmium and mercury ions.

Cadmium (Cd2+) and mercury ions (Hg2+) are essential for the quality control of food samples because of their serious toxicity to human health, but the effective and simple strategy for their parallel detection remains challenging. In this paper, a rapid and simple parallel detection method for Cd2+ and Hg2+ was developed using carbon dots (CDs) as fluorescent sensors. A one-step hydrothermal method with a single precursor l-arginine as both the carbon and nitrogen sources was employed to prepare nitrogen-doped CDs (N-CDs). N-CDs exhibited a uniform particle size and excitation-independent fluorescence emission. The maximum emission wavelength of N-CDs was observed at 354 nm with the excitation wavelength at 295 nm. The quantum yield of N-CDs reached as high as 71.6% in water. By using sodium diphosphate and phytic acid as masking agents, the fluorescent sensor can be quenched by Cd2+ and Hg2+ in the linear range of 0-26.8 µM and 0-49.9 µM within 5 min. Other common ions in farm products showed no significant effect on the fluorescence intensity of the sensing system. The results demonstrated that the sensing system had good selectivity and sensitivity for Cd2+ and Hg2+. The detection limits for Cd2+ and Hg2+ were 0.20 and 0.188 µM, respectively. In addition, the fluorescent sensor had been successfully applied for the detection of Cd2+ and Hg2+ in fruits and vegetables, and the recoveries were 86.44-109.40% and 86.62-115.32%, respectively. The proposed fluorescent sensor provides a rapid, simple, and sensitive detection method for Cd2+ and Hg2+ in food samples and thus a novel quantitative detection method for heavy metal ions in foods.

Selection and colorimetric application of ssDNA aptamers against metamitron based on magnetic bead-SELEX.

Metamitron (MTM) is a typical and widely used triazine herbicide in agricultural production. Its moderate toxicity and high residue in the environment have deleterious impacts on human health. The establishment of a rapid and efficient MTM detection method is of great significance. In this study, a magnetic-bead SELEX (systematic evolution of ligands by exponential enrichment) system was developed to select the MTM aptamers with high affinity and specificity. Through 10 rounds of screening, six candidate aptamers with the highest abundance were obtained by high-throughput sequencing. The homology, secondary structure, and affinity analyses were performed. The aptamer named MTM-6 was selected as the optimal aptamer with the dissociation constant (Kd) value of 16 nM. Then, a colorimetric detection method for MTM based on aptamer MTM-6 and the aggregation of gold nanoparticles (AuNPs) induced by NaCl was established with a linear range from 20 to 1000 nM (R = 0.9966) and a limit of detection (LOD) of 4.58 nM. The average recovery rate of MTM in the application of actual aqueous samples ranged from 95.40 to 107.83% with a relative standard deviation (RSD) from 1.11 to 3.48%. With considerable sensitivity and specificity, this colorimetric aptasensor is convenient and efficient, and shows bright application potential in MTM detection in aqueous samples.

Dual-mode colorimetric determination of As(III) based on negatively-charged aptamer-mediated aggregation of positively-charged AuNPs.

The aggregation and redispersion of positively-charged AuNPs ((+)AuNPs) modified with cysteamine (CS) could be regulated by the negatively charged As(III)-specific aptamer (As(III)-apt). In general, (+)AuNPs aggregated with increasing inducer concentration. However, in the present study, it was found that (+)AuNPs re-dispersed after a certain high concentration of As(III)-apt was reached. By optimizing the concentration of As(III)-apt that resulted in the aggregation and redispersion behavior of (+)AuNPs, a dual-mode colorimetric aptasensor for As(III) determination was established. It was not only able to quantify As(III) sensitively over a ranges of As(III) concentrations, but also to selectively differentiate As(III) and eliminate false results from other control ions by dual-mode.

Fluorescent carbon dots from water hyacinth as detection sensors for ferric ions: the preparation and optimisation using response surface methodology.

The search for alternatives to chemicals from natural products as precursors for the preparation of highly doped carbon dots (CDs) remains challenging. Novel CDs (W-CDs) were synthesised using a one-step pyrolysis method with wastewater hyacinth as the sole carbon and nitrogen source at a mild temperature without using any surface-activating reagents or salt. The obtained W-CDs emitted strong blue fluorescence under 365 nm UV light excitation, with a quantum yield of 15.12%. The Box-Behnken design of the response surface methodology was applied to optimize the W-CD preparation conditions, including the reaction temperature, reaction time and weight of water hyacinths. The temperature was found to be the most important factor affecting the fluorescence intensity of the W-CDs. Additionally, the fluorescence sensor based on W-CDs demonstrated excellent selectivity towards ferric (Fe) ions, with a limit of detection of 2.35 µM. The fluorescent sensor was successfully applied for detecting Fe3+ in real water samples with a recovery of 97.80-103.10%. Hence, the pyrolysis of water hyacinth is proven to be a rapid, effective and green approach for CDs and provides a novel method for recycling water hyacinth.

Selection and identification of a DNA aptamer for fluorescent detection of netilmicin.

Netilmicin (NET) is an antibiotic widely used in healthcare and agriculture, but it can accumulate in the environment to threat human health. Netilmicin (NET) is an antibiotic used for veterinary purposes, for human therapy and for agricultural purposes. Therefore, there is a need to develop high-sensitive measuring methods to detect NET. Aptamer-based detecting methods are highly sensitive, inexpensive, and portable. In this study, we developed an aptamer-based fluorescence method to detect and quantify NET. NET was first conjugated to magnetic beads by amidation reaction and then NET-coated beads were used as the stationary phase to isolate aptamers by systematic evolution of ligands by exponential enrichment (SELEX) screening method. After ten rounds of SELEX screening, 32 aptamers with NET-binding affinity were obtained and the candidate aptamer APT-21 was finally chosen by comprehensively comparing their secondary structure characters and NET-binding affinity. APT-21 bound to NET with high affinity (Kd = 194.1 nmol/L) and high specificity that it displayed low cross-binding activities on 7 different structural analogs. We also developed a fluorometric assay using SYBR Green I (SG-I) and the APT-21. Key experimental parameters were optimized, including buffer system, SG-I and APT-21 reaction time, SG-I concentration, and aptamer concentration, to improve the detecting sensitivity. Our results suggest that the low limit of detection (LOD) of this method reached a low level of 1.95 nM and it also exhibited a good linear range up to 200 nM. Moreover, we successfully applied our method to detect the NET spiked in tap water and river water with good recoveries in the range from 97% to 111%. In conclusion, our current study isolated a NET-specific aptamer and developed an aptamer-based quantification method, which is promising to apply to detect NET in environmental samples.

Protective effects of agrimonolide on hypoxia-induced H9c2 cell injury by maintaining mitochondrial homeostasis.

Cardiomyocyte death caused by hypoxia is one of the main causes of myocardial infarction or heart failure, and mitochondria play an important role in this process. Agrimonolide (AM) is a monomeric component extracted from Agrimonia pilosa L. and has antioxidant, antitumor, and anti-inflammatory effects. This study aimed to investigate the role and mechanism of AM in improving hypoxia-induced H9c2 cell damage. The results showed that low AM concentrations promote H9c2 cell proliferation and increase cellular ATP content. Transcriptome sequencing showed that AM induces differential expression of genes in H9c2 cells. Gene ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses revealed that these genes were concentrated in mitochondrial function. Subsequent experiments confirmed that AM regulates hypoxia-induced cell cycle arrest. AM inhibited the rate of apoptosis by regulating the expression of apoptosis-related proteins, reducing the level of cleaved Caspase 3 and Bax, and increasing the level of Bcl2, thereby protecting H9c2 cells from hypoxia-induced apoptosis. AM restored the mitochondrial membrane potential, inhibited the generation of ROS, maintained the normal shape of the mitochondria, improved the level of the mitochondrial functional proteins OPA1, MFN1, MFN2, Tom20, and increased the level of ATP. In conclusion, AM protects H9c2 cells from hypoxia-induced cell damage.

Inflammatory Injury and Mitophagy in the Cock Heart Induced by the Oral Administration of Hexavalent Chromium.

As a highly toxic heavy metal, chromium has caused a certain threat to public health and livestock breeding in recent years. In poultry, as one of our most commonly consumed meat product, its health issues will seriously threaten the safety of human life. As previous studies have confirmed, when cells are stimulated by the external environment, mitochondria, as an organelle that provides energy to the cells, can cause damage and autophagy. The purpose of this study is to confirm whether Cr(VI) can cause mitophagy in cock heart. We first randomly divided 32 cocks into four groups to explore the mechanism of this effect. The cocks were then separately exposed to four different dose levels, namely, the control level and 10, 30, and 50 mg/kg levels, via daily oral intake into the body through mixed feeding for 45 days. After 45 days, we sampled and detected pathological changes and the levels of inflammatory factors (IL-6, TNF-α, and IFN-γ), mitochondrial membrane potential (MMP), adenosine triphosphatases (ATPases), and mitophagy-related proteins (LC3, p62/SQTM1, TOMM20, and Parkin). We found that IL-6, TNF-α, IFN-γ, and LC3II contents increased with the increase in Cr(VI) concentration. However, MMP, ATPases, p62/SQTM1, and TOMM20 levels decreased with the increase in Cr(VI) concentration. At the same time, Cr(VI) exposure caused heart tissue damages and Parkin translocation. In conclusion, our results proved that inflammatory damage, mitochondrial function damage, and mitophagy in cock heart tissues were dependent on Cr(VI) concentration.

Platycodon grandiflorus polysaccharides inhibit Pseudorabies virus replication via downregulating virus-induced autophagy.

Pseudorabies virus (PRV) is one of the common pathogens in farms. Platycodon grandiflorus polysaccharide (PGPS) has been reported with a variety of biological activities. Autophagy is one of the vital mechanisms for cells to cope with virus infection, and it may also inhibit or promote virus replication. This study was conducted to investigate the antiviral activity of total PGPS(PGPSt) against PRV and the role of virus-induced autophagy in the anti-PRV effect of PGPSt in PK-15 cells. First, we established an infection model and detected the autophagy induced by PRV in PK-15 cells. Then, the protective effect of PGPSt against PRV was evaluated, and the effect of PGPSt on PRV replication and virus-induced autophagy were analysed by quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, Western blot and confocal immunofluorescence. Results showed that PGPSt can reduce the PRV replication. PRV infection resulted in the accumulation of autophagosomes, which were inhibited by PGPSt. Moreover, PGPSt upregulated the Akt/mammalian target of rapamycin (mTOR) signalling pathway repressed by PRV infection, whereas rapamycin attenuated the anti-PRV effect of PGPSt. These findings suggest that PGPSt possess a protective effect against PRV infection and can inhibit PRV replication through relieving PRV-induced autophagy. This article can provide ideas for the development of antiviral drugs.

Intracellular Staphylococcus aureus inhibits autophagy of bovine mammary epithelial cells through activating p38α.

Staphylococcus aureus is a common pathogen of bovine mastitis which can induce autophagy and inhibit autophagy flux, resulting in intracellular survival and persistent infection. The aim of the current study was to investigate the role of p38α in the autophagy induced by intracellular S. aureus in bovine mammary epithelial cells. An intracellular infection model of MAC-T cells was constructed, and activation of p38α was examined after S. aureus invasion. Through activating/inhibiting p38α by anisomycin/SB203580, the autophagosomes, LC3 and p62 level were analyzed by immunofluorescence and western blot. To further study the detailed mechanism of p38α, phosphorylation of ULK1ser757 was also detected. The results showed that intracellular S. aureus activated p38α, and the activation developed in a time-dependent manner. Inhibition of p38α promoted intracellular S. aureus-induced autophagy flow, up-regulated the ratio of LC3 II/I, reduced the level of p62 and inhibited the phosphorylation of ULK1ser757, whereas the above results were reversed after activation of p38α. The current study indicated that intracellular S. aureus can inhibit autophagy flow by activating p38α in bovine mammary epithelial cells.

Inflammatory injury and mitophagy of the brain in chicken exposed to Cr(VI).

The aim of this study is to determine whether Cr(VI) can induce inflammatory injury in chicken brain and influence mitophagy and related mechanisms. A total of 120 hyline brown chickens (1 day old, 20±3g) were selected and randomly divided into four groups and given different doses of Cr(VI) (0, 10, 30, and 50 mg/kg) every day at 45 days. Results showed that excessive intake of Cr(VI) led to increased tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) levels and decreased interferon-gamma (IF-γ) level. Cr(VI) increased the production of mitochondrial reactive oxygen species (ROS) in chicken brain cells, causing the decline of mitochondrial membrane potential (MMP) and formation of autophagosomes for mitophagy. In addition, Cr(VI) promoted the translocation of Parkin to the mitochondrial outer membrane, increased LC3-II protein level, and inhibited p62 and TOM20 protein expression. In conclusion, excessive Cr(VI) intake can induce inflammatory injury and mitophagy in chicken brain.

Cr(VI) promotes tight joint and oxidative damage by activating the Nrf2/ROS/Notch1 axis.

This study aimed to investigate whether Cr(VI) induced tight joint and oxidative damage in the small intestine, as mediated by the nuclear factor erythroid 2-related factor 2 (Nrf2)/reactive oxygen species (ROS)/Notch1 axis crosstalk. Thirty-two ICR mice were obtained and subjected to Cr(VI) via intragastric administration daily for 5 days. Western blot (WB) analysis, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC) staining, and immunofluorescence (IF) staining were applied to detect small intestinal damage, Nrf2, Notch1, and respective downstream targets in this research. Results showed that Cr(VI) led to the tight joint and oxidative damage in the small intestine of mice. Nrf2 was stimulated, and Notch1 (Notch intracellular domain, NICD1) was activated to translocate into the nucleus and activate an antioxidant action. These findings were validated by WB analysis and IF staining. ROS levels increased as the Cr(VI) concentration increased. The colocalization analysis of Nrf2 and NICD1 implied that a crosstalk between Nrf2 and Notch1 existed. Therefore, this study indicated that the Nrf2/ROS/Notch1 axis crosstalk could aggravate the tight joint and oxidative damage in the small intestine after Cr(VI) treatment.

Mitophagy Induced by Mitochondrial Function Damage in Chicken Kidney Exposed to Cr(VI).

Cr(VI) is a heavy metal environmental pollutant and carcinogen. Excessive Cr(VI) exposure injures kidneys. This study aimed to investigate mitophagy induced by mitochondrial function damage in chicken kidney exposed to Cr(VI). To explore the mechanism involved, we randomly divided 40 one-day-old Hy-line Brown cockerels into four groups, with each group exposed to different concentrations of Cr(VI), i.e., 0, 10, 30 and 50 mg kg-1, which were orally administered daily for 45 days. Excessive Cr(VI) increased tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and chemokine (C-X-C motif) ligand 1(CXCL1) expression and decreased Ca2+-adenosine triphosphatase (Ca2+-ATPase), Mg2+-ATPase and Na+/k+-ATPase activities in chicken kidney. Furthermore, Cr(VI) significantly increased reactive oxygen species (ROS) production and induced mitochondrial membrane potential (MMP) collapse and typical autophagosome formation. With the increase of Cr(VI) concentration, the Parkin translocation, value of LC3-II increased and decreased the content of p62/SQSTM1 and the translocase of outer mitochondrial membrane 20 (TOMM20). In summary, our findings explicated that mitochondrial function damage and mitophagy-related indicators were related to Cr(VI) concentration in chicken kidney.