High altitude hypoxia and oxidative stress: The new hope brought by free radical scavengers.

Various strategies can be employed to prevent and manage altitude illnesses, including habituation, oxygenation, nutritional support, and medication. Nevertheless, the utilization of drugs for the prevention and treatment of hypoxia is accompanied by certain adverse effects. Consequently, the quest for medications that exhibit minimal side effects while demonstrating high efficacy remains a prominent area of research. In this context, it is noteworthy that free radical scavengers exhibit remarkable anti-hypoxia activity. These scavengers effectively eliminate excessive free radicals and mitigate the production of reactive oxygen species (ROS), thereby safeguarding the body against oxidative damage induced by plateau hypoxia. In this review, we aim to elucidate the pathogenesis of plateau diseases that are triggered by hypoxia-induced oxidative stress at high altitudes. Additionally, we present a range of free radical scavengers as potential therapeutic and preventive approaches to mitigate the occurrence of common diseases associated with hypoxia at high altitudes.

Characteristics and Functions of MYB (v-Myb avivan myoblastsis virus oncogene homolog)-Related Genes in Arabidopsis thaliana.

The MYB (v-Myb avivan myoblastsis virus oncogene homolog) transcription factor family is one of the largest families of plant transcription factors which plays a vital role in many aspects of plant growth and development. MYB-related is a subclass of the MYB family. Fifty-nine Arabidopsis thaliana MYB-related (AtMYB-related) genes have been identified. In order to understand the functions of these genes, in this review, the promoters of AtMYB-related genes were analyzed by means of bioinformatics, and the progress of research into the functions of these genes has been described. The main functions of these AtMYB-related genes are light response and circadian rhythm regulation, root hair and trichome development, telomere DNA binding, and hormone response. From an analysis of cis-acting elements, it was found that the promoters of these genes contained light-responsive elements and plant hormone response elements. Most genes contained elements related to drought, low temperature, and defense and stress responses. These analyses suggest that AtMYB-related genes may be involved in A. thaliana growth and development, and environmental adaptation through plant hormone pathways. However, the functions of many genes do not occur independently but instead interact with each other through different pathways. In the future, the study of the role of the gene in different pathways will be conducive to a comprehensive understanding of the function of the gene. Therefore, gene cloning and protein functional analyses can be subsequently used to understand the regulatory mechanisms of AtMYB-related genes in the interaction of multiple signal pathways. This review provides theoretical guidance for the follow-up study of plant MYB-related genes.

Comprehensive Investigation of the Influence of High-Altitude Hypoxia on Clopidogrel Metabolism and Gut Microbiota.

BACKGROUND: The amount of metabolites converted into active metabolites is correspondingly reduced since only more than 50% of clopidogrel is absorbed. OBJECTIVE: Exploring the effect of gut microbiota altered by altitude hypoxia on the pre-absorption metabolism of clopidogrel. METHODS: In vitro and in vivo experiments were conducted to analyze the metabolism of clopidogrel through LCMS/ MS, while 16S rRNA analysis was used to investigate the changes in the gut microbiota of high-altitude animals. RESULTS: We demonstrated that the intestinal flora is involved in the metabolism of clopidogrel through in vivo and in vitro experiments. In addition, the plateau environment caused changes in the number and composition of intestinal microbes. Intriguingly, alterations in the microbial population could lead to an increase in the pre-absorption metabolism of clopidogrel after rapid entry into the plateau, the amount of absorbed blood is thus reduced, which may affect the bioavailability and therapeutic effect of clopidogrel. CONCLUSION: Our results not only as a first clinical reference for dose adjustment of clopidogrel in high-altitude environments but also would be helpful to provide a statement on the broader significance within the field of pharmacokinetics or personalized medicine.

Identification of Brassica rapa BrEBF1 homologs and their characterization in cold signaling.

EIN3-binding F-box 1 (EBF1) is involved in cold tolerance in Arabidopsis; however, its exact roles in cold signaling in Brassica rapa remain uncertain. Herein, we demonstrated that EBF1 homologs are highly conserved in Brassica species, but their copy numbers are diverse, with some motifs being species specific. Cold treatment activated the expression of EBF1 homologs BrEBF1 and BrEBF2 in B. rapa; however, their expression schemas were diverse in different cold-resistant varieties of the plant. Subcellular localization analysis revealed that BrEBF1 is a nuclear-localized F-box protein, and cold treatment did not alter its localization but induced its degradation. BrEBF1 overexpression enhanced cold tolerance, reduced cold-induced ROS accumulation, and enhanced MPK3 and MPK6 kinase activity in Arabidopsis. Our study revealed that BrEBF1 positively regulates cold tolerance in B. rapa and that BrEBF1-regulated cold tolerance is associated with ROS scavenging and MPK3 and MPK6 kinase activity through the C-repeat binding factor pathway.

Pharmacokinetics, absorption and transport mechanism for ginseng polysaccharides.

BACKGROUND: Ginseng polysaccharide (GP) is one of the most abundant components in Panax ginseng. However, the absorption pathways and mechanisms of GPs have not been investigated systematically due to the challenges of their detection. METHODS: The fluorescein isothiocyanate derivative (FITC) was employed to label GP and ginseng acidic polysaccharide (GAP) to obtain target samples. HPLC-MS/MS assay was used to determine the pharmacokinetics of GP and GAP in rats. The Caco-2 cell model was used to investigate the uptake and transport mechanisms of GP and GAP in rats. RESULTS: Our results demonstrated that the absorption of GAP was more than that of GP in rats after gavage administration, while there was no significant difference between both after intravenous administration. In addition, we found that GAP and GP were more distributed in the kidney, liver and genitalia, suggesting that GAP and GP are highly targeted to the liver, kidney and genitalia. Importantly, we explored the uptake mechanism of GAP and GP. GAP and GP are endocytosed into the cell via lattice proteins or niche proteins. Both are transported lysosomally mediated to the endoplasmic reticulum (ER) and then enter the nucleus through the ER, thus completing the process of intracellular uptake and transportation. CONCLUSION: Our results confirm that the uptake of GPs by small intestinal epithelial cells is primarily mediated via lattice proteins and the cytosolic cellar. The discovery of important pharmacokinetic properties and the uncovering of the absorption mechanism provide a research rationale for the research of GP formulation and clinical promotion.

Characteristics analysis of Early Responsive to Dehydration genes in Arabidopsis thaliana (AtERD).

Early Responsive to Dehydration (ERD) genes are rapidly induced in response to various biotic and abiotic stresses, such as bacteria, drought, light, temperature and high salt in Arabidopsis thaliana. Sixteen ERD of Arabidopsis thaliana (AtERD) genes have been previously identified. The lengths of the coding region of the genes are 504-2838 bp. They encode 137-745 amino acids. In this study, the AtERD genes structure and promoter are analyzed through bioinformatics, and a overall function is summarized and a systematic signal pathway involving AtERD genes is mapped. AtERD9, AtERD11 and AtERD13 have the GST domain. AtERD10 and AtERD14 have the Dehyd domain. The promoters regions contain 32 light responsive elements, 23 ABA responsive elements, 5 drought responsive elements, 5 meristem expression related elements and 132 core promoter elements. The study provides a theoretical guidance for subsequent studies of AtERD genes.

The protein phosphatase 2C clade A TaPP2CA interact with calcium-dependent protein kinases, TaCDPK5/TaCDPK9-1, that phosphorylate TabZIP60 transcription factor from wheat (Triticum aestivum L.).

Previously we have found that TabZIP60 from the ABF/AREB (ABRE-binding factor/ABA-responsive element-binding protein) subfamily of bZIP transcription factor (TF) was involved in salt stress response. However, the regulatory mechanism of TabZIP60 is unknown. In the present study, we identified two calcium-dependent protein kinase (CDPK) genes, TaCDPK5/TaCDPK9-1, which were clustered into group Ⅰ and were induced by salt, abscisic acid (ABA), and polyethylene glycol (PEG) treatments. RT-qPCR results showed that the expression level of salt-induced TabZIP60 was drastically inhibited by Ca2+ channel blocker LaCl3. TaCDPK5/TaCDPK9-1 were involved in interaction with TabZIP60 protein in vivo and in vitro. And TaCDPK5/TaCDPK9-1 could autophosphorylate and phosphorylate TabZIP60 protein in a Ca2+-dependent way. Mutational analysis indicated that Serine-110 of TabZIP60 was essential for TaCDPK5/TaCDPK9-1-TabZIP60 interaction and was the phosphorylation site of TaCDPK5/TaCDPK9-1 kinases. Yeast two-hybrid assay results showed the interactions between TaCDPK5/TaCDPK9-1 and wheat protein phosphatase 2 C clade A TaPP2CA116/ TaPP2CA121 separately. These findings demonstrate that the phosphorylation status of TabZIP60 controlled by TaPP2CA116/ TaPP2CA121 and TaCDPK5/TaCDPK9-1 might play a crucial role in wheat during salt stress.

Comparative Transcriptome Analysis Revealed the Freezing Tolerance Signaling Events in Winter Rapeseed (Brassica rapa L.).

Winter rapeseed (Brassica rapa L.) is an important oilseed crop in northwest China. Freezing stress severely limits its production and geographical distribution, and frequent extreme freezing events caused by climate change are increasing the chances of winter freeze-injury. However, the underlying mechanism of B. rapa response to freezing stress remains elusive. Here, B. rapa genome (v3.0) was used as a reference for the comparative transcriptomic analysis of Longyou 6 and Tianyou 2 (strong and weak cold tolerance, respectively) under different freezing stress. Before and after freezing stress, 5,982 and 11,630 unique differentially expressed genes (DEGs) between two cultivars were identified, respectively. After freezing stress, the GO terms in Tianyou 2 were mainly involved in "macromolecule biosynthetic process", and those in Longyou 6 were involved in "response to stimulus" and "oxidoreductase activity". Morphological and physiological results indicated that Longyou 6 retained a higher basal freezing resistance than Tinayou 2, and that cold acclimation could strengthen the basal freezing resistance. Freezing stress could activate the MAPK signal cascades, and the phosphorylation level of Longyou 6 showed a higher increase in response to freezing treatment than Tianyou 2. Based on our findings, it was speculated that the cell membrane of B. rapa perceives external signals under freezing stress, which are then transmitted to the nucleus through the cold-activated MAPK cascades and Ca2+-related protein kinase pathway, thus leading to activation of downstream target genes to enhance the freezing resistance of B. rapa.

Exogenous sucrose influences KEA1 and KEA2 to regulate abscisic acid-mediated primary root growth in Arabidopsis.

Arabidopsis K+-efflux antiporter (KEA)1 and KEA2 are chloroplast inner envelope membrane K+/H+ antiporters that play an important role in plastid development and seedling growth. However, the function of KEA1 and KEA2 during early seedling development is poorly understood. In this work, we found that in Arabidopsis, KEA1 and KEA2 mediated primary root growth by regulating photosynthesis and the ABA signaling pathway. Phenotypic analyses revealed that in the absence of sucrose, the primary root length of the kea1kea2 mutant was significantly shorter than that of the wild-type Columbia-0 (Col-0) plant. However, this phenotype could be remedied by the external application of sucrose. Meanwhile, HPLC-MS/MS results showed that in sucrose-free medium, ABA accumulation in the kea1kea2 mutant was considerably lower than that in Col-0. Transcriptome analysis revealed that many key genes involved in ABA signals were repressed in the kea1kea2 mutant. We concluded that KEA1 and KEA2 deficiency not only affected photosynthesis but was also involved in primary root growth likely through an ABA-dependent manner. This study confirmed the new function of KEA1 and KEA2 in affecting primary root growth.

Phospholipase Dδ and H2S increase the production of NADPH oxidase-dependent H2O2 to respond to osmotic stress-induced stomatal closure in Arabidopsis thaliana.

Osmotic stress is one of the main stresses that seriously affects the survival of plants, destroying normal cell activities, and potentially leading to plant death. Phospholipase D (PLD), a major lipid hydrolase, hydrolyzes membrane phospholipids to produce phosphatidic acid (PA) and responds to many abiotic stresses. Hydrogen sulfide (H2S) emerges as the third gaseous signaling molecule involved in the complex network of signaling events. Hydrogen peroxide (H2O2) plays a crucial role as a signaling molecule in plant development and growth, and responds to various abiotic and biotic stresses. In this study, the functions and the relationship of PLDδ, H2S, and H2O2 in osmotic stress-induced stomatal closure were explored. By using the seedlings of ecotype (WT), PLDδ-deficient mutant (pldδ), l-cysteine desulfhydrase (LCD)-deficient mutant (lcd), and pldδlcd double mutant, atrbohD, and atrbohF mutant as materials, and the stomatal aperture were analyzed. The relative water loss of pldδ, lcd, and pldδlcd was higher than that of WT. Exogenous PA and NaHS could partially alleviate the leaf wilting and yellowing phenotypes of pldδ, lcd, and pldδlcd under osmotic stress, but the mutants could not be restored to the same phenotype as WT. The fluorescence intensity of H2O2 in guard cells of pldδ, lcd, and pldδlcd was lower than that of WT, indicating that PLDδ and LCD were involved in the production of H2O2 in guard cells. Exogenous application of H2O2 to WT, pldδ, lcd, and pldδlcd significantly induced stomatal closure under osmotic stress. Exogenous NaHS induced stomatal closure of WT, but could not induce stomatal closure of atrbohD and atrbohF under osmotic stress. These results suggest that the accumulation of H2O2 was essential to induce stomatal closure under osmotic stress, and PLDδ and LCD acted upstream of H2O2.

Cloning and functional identification of setaria italica somatic embryogenesis receptor-like kinase1 gene (SiSERK1).

Plant somatic embryogenesis receptor-like kinases (SERK), members of leucine-rich repeat receptor-like kinases (LRR-RLKs) subfamily, are widely involved in plant growth, development and innate immunity. In this study, the setaria italica somatic embryogenesis receptor-like kinase1 gene (SiSERK1) was cloned by gateway technology, and transferred into a brasssinosteroid (BR) receptor mutant of Arabidopsis thaliana WS2 (bri1-5). After BL treatment, the transgenic plants could partially restore the phenotype of bri1-5. After Pst DC3000 treatment, the CFU value of SiSERK1 overexpression plant pathogen was between WS2 and bri1-5. Stomatal opening and plant height were also between them. Therefore, it is speculated that SiSERK1 gene is involved in BR signaling pathway and can improve the resistance of bri1-5 to Pst DC3000 through SA and NHP mediated systemic acquired resistance (SAR).

Triggered peroxidase-like activity of Au decorated carbon dots for colorimetric monitoring of Hg2+ enrichment in Chlorella vulgaris.

Developing a rapid, low-cost, and multimode detection method for heavy metal ions remains a compelling goal for many applications, including food safety, environmental and biological analysis. This study investigated the influence of Hg2+ on the peroxidase-like activity of gold nanoparticles (GNPs) decorated on carbon dots (CDs) from lysine (denoted as GNP@CDs). A new type of Hg2+-triggered peroxidase-like activity of GNP@CDs was discovered, which could catalyze the oxidation of the colorless 3,3',5,5'-tetramethylbenzidine (TMB) into blue TMB. Based on the regulation of the catalytically triggered activity, a sensitive colorimetric method for the detection of Hg2+ was developed, with a linear range of 7-150 nM, providing a limit of detection as low as 3.7 nM. The sensor is simple and rapid, and was successfully applied to the detection of Hg2+ enrichment in chlorella, suggesting a promising application in biological analysis.

Alleviation of osmotic stress by H2S is related to regulated PLDα1 and suppressed ROS in Arabidopsis thaliana.

Hydrogen sulfide (H2S) is an important gaseous molecule responding to osmotic stress in plant. Phospholipase Dα1 (PLDα1) and reactive oxygen species (ROS) are involved in many biotic or abiotic stress responses. Using the seedlings of Arabidopsis thaliana ecotype (WT), PLDα1 deficient mutant (pldα1) and the L-cysteine desulfhydrase (L-DEs) deficient mutant (lcd) as materials, the effect of H2S responding to osmotic stress and the functions of PLDα1 and ROS in this response were investigated. The results showed that H2S, PLDα1 and ROS were involved in osmotic stress resistance. Exogenous sodium hydrosulfide (NaHS) promoted the endogenous H2S content and up-regulated the expression of LCD in WT, lcd and plda1. Exogenous phosphatidic acid (PA) enhanced the H2S content and up-regulated the expressions of LCD in WT and plda1 but had no significant effect on the H2S content and LCD expression in lcd under osmotic stress. This suggested that H2S was located downstream of PLDα1 to participate in the osmotic stress signal response. Exogenous NaHS treatment regulated the antioxidant enzymes (SOD, POD, and CAT). The activities and the gene relative expressions of antioxidant enzymes in pldα1 and lcd were higher than those in WT under osmotic stress. This indicated that H2S and PLD regulated the antioxidant enzyme system under osmotic stress. The ROS level, electrolyte leakage (EL), malondialdehyde (MDA) were decreased by NaHS under osmotic stress, demonstrating H2S maintained the membrane integrity. All of these results revealed that H2S alleviated the osmotic stress by elevating PLD and suppressing ROS in A. thaliana.

Enhanced His@AuNCs oxidase-like activity by reduced graphene oxide and its application for colorimetric and electrochemical detection of nitrite.

Enzyme-mimicking (nanozyme)-based biosensors are attractive owing to their unique catalytic efficiency, multifunctionality, and tunable activity, but examples of oxidase-like nanozymes are quite rare. Herein, we demonstrated that histidine-capped gold nanoclusters (His@AuNCs) possessed intrinsic oxidase-like activity, which could directly oxidize 3,3',5,5'-tetramethylbenzidine (TMB) to blue colored ox-TMB without H2O2. The assembly of reduced graphene oxide (RGO) with His@AuNCs could further improve its oxidase-like activity and the His@AuNCs/RGO nanocomposites had a lower Michaelis constant (Km) and higher catalytic constant (Kcat) for TMB oxidation. Furthermore, compared to other nanomaterials, the as-prepared His@AuNCs/RGO also exhibited enhanced electrocatalytic activity toward TMB. Interestingly, nitrite inhibited the catalytic (chromogenic) and electrocatalytic processes of His@AuNCs/RGO in the oxidation of TMB. The oxidase-like and electrocatalytic activity of His@AuNCs/RGO was evaluated with nitrite and TMB as substrates, and the results indicated that TMB and nitrite might share the same catalytic active sites. On the basis of these findings, a colorimetric and electrochemical sensor was developed with the His@AuNCs/RGO composite as an oxidase mimic for determination of nitrite with linear ranges of 10-500 µM and 2.5-5700 µM, respectively. The developed method was successfully applied to the detection of nitrites in real samples. The present work suggests that the oxidase-like nanozyme is not only suitable for colorimetric assay but also for development of electrochemical sensors in bioanalysis. Graphical abstract The colorimetric and electrochemical detection of nitrite using His@AuNCs/RGO.

A novel "dual-potential" ratiometric electrochemiluminescence DNA sensor based on enhancing and quenching effect by G-quadruplex / hemin and Au-Luminol bifunctional nanoparticles.

An ultrasensitive and stable "dual-potential" ratiometric electrochemiluminescence (ECL) sensor is reported for specific DNA, the femtomolar detection limit (0.12 fM, S/N = 3) and high selectivity insure its potential applications in cancer biomarkers searching or monitoring. The excellent performance of the sensor comes from simultaneously fabricated layer by layer structure "target DNA + Hemin / Au-Luminol NPs / DNA* / sl DNA / TGA / QDs / MWNTs / GCE" mode which was based on the enhancing effect of luminol by G-quadruplex / hemin and Au nanoparticles and the quenching effect of CdSe/ZnS by G-quadruplex / hemin. (i) DNA-SH could combine with Au-Luminol NPs via S-Au bond to solve the problem of poor solubility and weak ECL intensity of luminol in neutral medium. (ii) Target DNA and Hemin formed the G-quadruplex / hemin peroxidase mimicking DNAzyme could enhance the ECL of luminol and quench the ECL of CdSe/ZnS simultaneously. (iii) DNA* was employed to increase a certain distance between CdSe/ZnS and Au-Luminol for enhancing the CdSe/ZnS QDs initial ECL intensity. The dual-potential ratiometric mode lower the influence of background and side reaction of the ECL sensor which were the most important factors in trace sensing.

Constructing a novel 8-hydroxy-2'-deoxyguanosine electrochemical sensor and application in evaluating the oxidative damages of DNA and guanine.

8-Hydroxy-2'-deoxyguanosine (8-OHdG) is commonly identified as a biomarker of oxidative DNA damage. In this work, a novel and facile 8-OHdG sensor was developed based on the multi-walled carbon nanotubes (MWCNTs) modified glassy carbon electrode (GCE). It exhibited good electrochemical responses toward the oxidation of 8-OHdG, and the linear ranges were 5.63×10(-8)-6.08×10(-6)M and 6.08×10(-6)-1.64×10(-5)M, with the detection limit of 1.88×10(-8)M (S/N=3). Moreover, the fabricated sensor was applied for the determination of 8-OHdG generated from damaged DNA and guanine, respectively, and the oxidation currents of 8-OHdG increased along with the damaged DNA and guanine within certain concentrations. These results could be used to evaluate the DNA damage, and provide useful information on diagnosing diseases caused by mutation and deficiency of the immunity system.

Bioassay Guided Fractionation Identified Hederagenin as a Major Cytotoxic Agent from Cyclocarya paliurus Leaves.

An ethanol extract prepared from the leaves of Cyclocarya paliurus, also known as sweet tea, which is one of the most popular teas utilized in traditional Chinese medicine, exhibited significant cytotoxicity against human lung and breast cancer cells. Using a bioassay-guided fractionation, we purified a pentacyclic triterpenoid, hederagenin, which exhibited superior and selective cytotoxicity against human breast and lung cancer cells. Evaluation of the structure-activity relationship between hederagenin and seven other pentacyclic triterpenoids revealed that the C3 hydroxyl group, the C17 carboxyl group and the Δ (12,13) double bond could be important active groups for the bioactivity of pentacyclic triterpenoids, whereas introduction of a hydroxyl group at C2 or C23 might reduce their bioactivity. We also investigated the cytotoxic activity of hedeargenin and demonstrated that it induces apoptosis, increases the cell membrane permeability, reduces the mitochondria potential, and suppresses NF-κB activation.

Determination of malachite green in fish based on magnetic molecularly imprinted polymer extraction followed by electrochemiluminescence.

A novel procedure for selective extraction of malachite green (MG) from fish samples was set up by using magnetic molecularly imprinted polymers (MMIP) as the solid phase extraction material followed by electrochemiluminescence (ECL) determination. MMIP was prepared by using Fe3O4 magnetite as magnetic component, MG as template molecule, methacrylic acid (MAA) as functional monomer and ethylene glycol dimethacrylate (EGDMA) as crosslinking agent. MMIP was characterized by SEM, TEM, FT-IR, VSM and XRD. Leucomalachite green (LMG) was oxidized in situ to MG by 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). And then MMIP was successfully used to selectively enrich MG from fish samples. Adsorbed MG was desorbed and determined by ECL. Under the optimal conditions, calibration curve was good linear in the range of 0.29-290 µg/kg and the limit of detection (LOD) was 7.3 ng/kg (S/N=3). The recoveries of MMIP extraction were 77.1-101.2%. In addition, MMIP could be regenerated. To the best of our knowledge, MMIP coupling with ECL quenching of Ru(bpy)3(2+)/TPA for the determination of MG has not yet been developed.

Ultrasensitive determination of DNA sequences by flow injection chemiluminescence using silver ions as labels.

We presented a new strategy for ultrasensitive detection of DNA sequences based on the novel detection probe which was labeled with Ag(+) using metallothionein (MT) as a bridge. The assay relied on a sandwich-type DNA hybridization in which the DNA targets were first hybridized to the captured oligonucleotide probes immobilized on Fe3O4@Au composite magnetic nanoparticles (MNPs), and then the Ag(+)-modified detection probes were used to monitor the presence of the specific DNA targets. After being anchored on the hybrids, Ag(+) was released down through acidic treatment and sensitively determined by a coupling flow injection-chemiluminescent reaction system (Ag(+)-Mn(2+)-K2S2O8-H3PO4-luminol) (FI-CL). The experiment results showed that the CL intensities increased linearly with the concentrations of DNA targets in the range from 10 to 500 pmol L(-1) with a detection limit of 3.3 pmol L(-1). The high sensitivity in this work may be ascribed to the high molar ratio of Ag(+)-MT, the sensitive determination of Ag(+) by the coupling FI-CL reaction system and the perfect magnetic separation based on Fe3O4@Au composite MNPs. Moreover, the proposed strategy exhibited excellent selectivity against the mismatched DNA sequences and could be applied to real samples analysis.

Simultaneous detection of multiple DNA targets based on encoding metal ions.

We present a novel strategy for simultaneous electrochemical detection of multiple DNA targets based on the use of different encoding metal ions as tags. The principle of this scheme is that metal ions bound to metallothionein (MT) molecules can be released down after hybridization with DNA targets and then be detected by stripping voltammetry. The novel detection probes, ssDNA/MT conjugates, covered with different metal ions were synthesized for the first time, then three encoding metal ions (Zn(2+), Cd(2+), and Pb(2+)) were used to differentiate the signals of three virus DNA due to their well-defined anodic stripping peaks at -1.13 V (Zn), -0.78 V (Cd), and -0.52 V (Pb) at BiFE, respectively. The anodic peak currents increased linearly with the concentrations of DNA targets in the range from 0.1 nM to 10nM with a detection limit of 33 pM. In addition, the one-base mismatched target was effectively discriminated from the complementary target. The described results demonstrated that this method possesses high sensitivity and selectivity for multi-target DNA assay and has great potential in applications for detection of even more targets in biological assays, particularly immunoassays.