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Caffeic acid (CA) is a natural polyphenol that can have various positive effects on human health. However, its extraction and processing can cause significant ecological issues. Therefore, it is crucial to detect and degrade CA effectively in the environment. In this study, we have developed a multifunctional magnetic luminescent nanozyme, Fe3O4@CeO2/Tb-MOF, which combines peroxidase activity to detect and degrade CA. The fluorescence of the nanozyme was significantly attenuated due to the specific nucleophilic reaction between its boronic acid moiety and the o-diphenol hydroxyl group of CA, energy competition absorption and photo-induced electron transfer (PET) effect. This nanozyme demonstrates a linear detection range from 50 nM to 500 µM and an exceptionally low detection limit of 18.9 nM, along with remarkable selectivity and stability. Moreover, the synergistic catalysis of Fe3O4 and CeO2 within Fe3O4@CeO2/Tb-MOF fosters peroxidase activity, leading to the generation of substantial free radicals catalyzed by H2O2, which ensures the efficient degradation of CA (â¼95%). The superparamagnetic property of Fe3O4 further enables the efficient reuse and recycling of the nanozyme. This research provides a novel approach for the concurrent detection and remediation of environmental contaminants.
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Adeno-associated virus (AAV) requires co-infection with helper virus for efficient replication. We previously reported that Human Bocavirus 1 (HBoV1) genes, including NP1, NS2, and BocaSR, were critical for AAV2 replication. Here, we first demonstrate the essential roles of the NP1 protein in AAV2 DNA replication and protein expression. We show that NP1 binds to single-strand DNA (ssDNA) at least 30 nucleotides (nt) in length in a sequence-independent manner. Furthermore, NP1 colocalized with the BrdU-labeled AAV2 DNA replication center, and the loss of the ssDNA-binding ability of NP1 by site-directed mutation completely abolished AAV2 DNA replication. We used affinity-tagged NP1 protein to identify host cellular proteins associated with NP1 in cells cotransfected with the HBoV1 helper genes and AAV2 duplex genome. Of the identified proteins, we demonstrate that NP1 directly binds to the DBD-F domain of the RPA70 subunit with a high affinity through the residues 101-121. By reconstituting the heterotrimer protein RPA in vitro using gel filtration, we demonstrate that NP1 physically associates with RPA to form a heterologous complex characterized by typical fast-on/fast-off kinetics. Following a dominant-negative strategy, we found that NP1-RPA complex mainly plays a role in expressing AAV2 capsid protein by enhancing the transcriptional activity of the p40 promoter. Our study revealed a novel mechanism by which HBoV1 NP1 protein supports AAV2 DNA replication and capsid protein expression through its ssDNA-binding ability and direct interaction with RPA, respectively.IMPORTANCERecombinant adeno-associated virus (rAAV) vectors have been extensively used in clinical gene therapy strategies. However, a limitation of these gene therapy strategies is the efficient production of the required vectors, as AAV alone is replication-deficient in the host cells. HBoV1 provides the simplest AAV2 helper genes consisting of NP1, NS2, and BocaSR. An important question regarding the helper function of HBoV1 is whether it provides any direct function that supports AAV2 DNA replication and protein expression. Also of interest is how HBoV1 interplays with potential host factors to constitute a permissive environment for AAV2 replication. Our studies revealed that the multifunctional protein NP1 plays important roles in AAV2 DNA replication via its sequence-independent ssDNA-binding ability and in regulating AAV2 capsid protein expression by physically interacting with host protein RPA. Our findings present theoretical guidance for the future application of the HBoV1 helper genes in the rAAV vector production.
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Proteínas do Capsídeo , Capsídeo , DNA de Cadeia Simples , DNA Viral , Proteínas de Ligação a DNA , Dependovirus , Bocavirus Humano , Proteínas Virais , Humanos , Capsídeo/metabolismo , Proteínas do Capsídeo/biossíntese , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Dependovirus/genética , Dependovirus/crescimento & desenvolvimento , Dependovirus/metabolismo , DNA de Cadeia Simples/biossíntese , DNA de Cadeia Simples/metabolismo , DNA Viral/biossíntese , DNA Viral/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação Viral da Expressão Gênica , Bocavirus Humano/genética , Bocavirus Humano/metabolismo , Cinética , Mutagênese Sítio-Dirigida , Mutação , Regiões Promotoras Genéticas , Ligação Proteica , Domínios Proteicos , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação ViralRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Cornus officinalis var. koreana Kitam (Cornus officinalis) is a commonly used Chinese herbal medicine and has a good clinical efficacy in kidney and liver diseases. Recent years, a number of studies reported the significant effects of Cornus officinalis on renal fibrosis. However, it is still unclear about the underlying specific mechanism, the bioactive ingredients, and the target gene regulatory network. AIM OF THE STUDY: We investigated the impact of Cornus officinalis extract on cadmium-induced renal fibrosis, screened the bioactive ingredients of Cornus officinalis using a pharmacological sub-network analysis, and explored the regulatory effects of Cornus officinalis extracts on target gene matrix metallopeptidase 9 (MMP9). METHODS: Male C57BL/6N mice were treated with single or combinatorial agents such as saline, cadmium chloride, Cornus officinalis, Isoginkgetin and FSL-1. Isoginkgetin is a compound with anti-MMP9 activity. FSL-1 can induce MMP9 expression. Masson staining and Western blot and immunohistochemistry analyses were used for assessing renal fibrosis. In addition, wound healing model was established using BUMPT (Boston university mouse proximal tubular) cells to investigate how Cornus officinalis affected cadmium-induced cell migration. The main Cornus officinalis bioactive compounds were identified by UHPLC-MS (Ultra-high-performance liquid chromatography - mass spectrometry). The MMP9 target for Cornus officinalis active ingredients were confirmed through a pharmacological sub-network analysis. RESULTS: Aqueous extracts of Cornus officinalis protected from renal dysfunction and kidney fibrosis induced by cadmium chloride in mice. In vitro experiments validated that Cornus officinalis extracts inhibited cell migration ability especially in cadmium chloride condition. The sub-network analysis and chemical components profiling technique revealed the active compounds of Cornus officinalis. Cellular thermal shift assay verified the binding abilities of three active components Daidzein, N-Acetyl-L-tyrosine or Swertisin with matrix metalloproteinase-9. Gelatin zymography assay revealed that the activity of MMP9 was inhibited by the three active components. We further confirmed that MMP9 was involved in the process of Cornus officinalis extracts reducing renal fibrosis. Cornus officinalis attenuated the cadmium-induced renal fibrosis was correlated with decreased expression of MMP9, collagen I, α-SMA (alpha-smooth muscle actin) and vimentin. CONCLUSIONS: This study demonstrated that Cornus officinalis extracts could alleviate the cadmium chloride-induced renal fibrosis by targeting MMP9, and might provide new insights into the mechanism of treating renal fibrosis by Cornus officinalis.
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Cornus , Nefropatias , Humanos , Masculino , Camundongos , Animais , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Extratos Vegetais/química , Cornus/química , Cádmio/toxicidade , Metaloproteinase 9 da Matriz , Cloreto de Cádmio , Camundongos Endogâmicos C57BL , Nefropatias/induzido quimicamente , Nefropatias/tratamento farmacológico , Nefropatias/prevenção & controle , FibroseRESUMO
Carcinoembryonic antigen (CEA), an acidic protein, is a characteristic antigen produced by the tumor of various cancers (eg, breast, cervical, rectal, lung, etc.). Therefore, the detection of cancer antigens is very important for the early diagnosis and treatment of cancer. In this study, a novel of "signal off" strategy for electrochemical immunosensor was developed to detect CEA. To this end, Prussian blue nanoparticles (PB NPs), an electroactive substance, were used as the immunological platform. In addition, CuO2@SiO2 nanocomposites, which release Cu2+ and H2O2 under acidic conditions, were synthesized. The generated Cu2+ can replace the high spin iron (FeIII) in PB NPs, which in turn reduces the oxidation peak current of PB NPs. Due to the peroxidase-like nature of PB NPs, they can react with self-generated H2O2 to generate hydroxyl radicals (·OH), which can further convert 4-chloro-1 naphthol (4-CN) into a non-conductive polymer that accumulates on the electrode surface, this leads to a further reduction in the electrical signal of the PB NPs. Moreover, the self-generated Cu2+ and H2O2 can reduce the introduction of exogenous substances and improve the detection accuracy. Square wave voltammetry (SWV) revealed that the electrical signal of PB NPs gradually decreased with increasing CEA concentration. In addition, the electrical signal of PB NPs exhibited a good linearity in the range from 0.01 pg mL-1 to 80 ng mL-1, where in the logarithm of CEA concentration and the detection limit was as low as 0.0032 pg mL-1.
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Técnicas Biossensoriais , Nanopartículas Metálicas , Antígeno Carcinoembrionário/química , Naftóis , Técnicas Eletroquímicas , Ouro/química , Nanopartículas Metálicas/química , Peróxido de Hidrogênio/química , Limite de Detecção , Compostos Férricos , Dióxido de Silício , Imunoensaio , Polímeros/química , Peroxidases , FerroRESUMO
The effective amplification of the signal is the prerequisite for the ultrasensitive detection of electrochemical immunosensors. To quantitatively detect alpha-fetoprotein (AFP), we prepared a sandwich-type electrochemical immunosensor. Using a gold nanoparticles (Au NPs) modified glassy carbon electrode (GCE) as the sensing platform and Cu/Cu2O-rGO@Au as the signal label, differential pulse voltammetry (DPV) was used to achieve sensitive detection of AFP. We found that the nanomaterials can undergo electro-oxidation and electro-reduction reactions between Cu(I) and Cu(II) in a buffer solution of pH = 6.0. It is worth mentioning that the incorporation of metals into metal oxide substrates is a new strategy to combine the catalytic activity of metal oxides with the electrical conductivity of metals. Reduced graphene oxide (rGO), which is rich in oxygen-containing groups, can load more Cu/Cu2O and Au NPs and increase the conductivity. The modification of Au NPs makes them have better biocompatibility and conductivity. Under the best detection conditions, the prepared immunosensor realizes the specific and ultrasensitive detection of AFP. The detection range is 0.01-50 ng mL-1 and the limit of detection (LOD) was as low as 0.589 pg mL-1 (S/N = 3); meanwhile it also has good practical application ability. Therefore, this immunosensor provides an important means for the early screening and detection of AFP.
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Técnicas Biossensoriais , Nanopartículas Metálicas , Técnicas Eletroquímicas , Ouro , Grafite , Imunoensaio , Óxidos , alfa-FetoproteínasRESUMO
Carcinoembryonic antigen levels in the human body reflect the conditions associated with a variety of tumors and can be used for the identification, development, monitoring, and prognosis of lung cancer, colorectal cancer, and breast cancer. In this study, an amperometric immunosensor with CuCo/carbon nanocubes (CuCo/CNC) as the signal label is constructed. The bimetal-doped carbon skeleton structure has a high specific surface area and exhibits good electrocatalytic activity. In addition, Au/g-C3N4 nanosheets (Au/g-C3N4 NSs) are used to modify the substrate platform, facilitating the loading of more capture antibodies. The reaction mechanism was explored through electrochemical methods, X-ray powder diffraction, X-ray photoelectron spectroscopy, and other methods. Kinetic studies have shown that CuCo/CNC have good peroxidase-like activity. In addition, the electrocatalytic reduction ability of CuCo/CNC on hydrogen peroxide can be monitored using amperometric i-t curve (- 0.2 V, vs. SCE), and the response current value is positively correlated with the CEA antigen concentration. The prepared electrochemical immunosensor has good selectivity, precision, and stability. The dynamic range of the sensor was 0.0001-80 ng/mL, and the detection limit was 0.031 pg/mL. In addition, the recovery and relative standard deviation in real serum samples were 97.7-103 % and 3.25-4.13 %, respectively. The results show that the sensor has good analytical capabilities and can provide a new method for the clinical monitoring of CEA.
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Cobalto/química , Cobre/química , Técnicas Eletroquímicas/métodos , Nanopartículas Metálicas/química , Técnicas Biossensoriais , Antígeno Carcinoembrionário , Eletrodos , Ouro , Nitrilas/químicaRESUMO
Electrochemical immunoassays are commonly used to detect biomarkers and Ti3C2 MXene anchored CuAu-LDH two-dimensional hydroxide heterojunctions for dual-mode electrochemical immunosensors were fabricated in this work. Layered double hydroxides have a large surface area, high chemical stability, tunable metal composition and interchangeable anions, however, the insulating nature of LDH further limits its catalytic performance. For this reason, Ti3C2 Mxenes were introduced to improve this problem. 2D layers of Ti3C2 Mxenes with large specific surface area and excellent conductivity have been well proven and widely used. And the surface of Ti3C2 Mxenes (due to the presence of abundant surface functional groups), will facilitate the anchoring of metal ions and the nucleation of LDH. In addition, its excellent electrical conductivity will facilitate the electron transfer between Cu2+ and Cu+. The immunosensor not only showed a heavy square wave voltammetry (SWV) signal. It also exhibited high electrocatalytic activity for H2O2 redox reactions and improves the sensitivity of the Ampere Current (i-t) detection. The CEA immunosensor developed in this study showed a wide linear response (0.0001-80 ng/mL) and the lowest detection limits (SWV: 33.6 fg/mL and i-t: 45.4 fg/mL S/N = 3). The results confirmed the excellent analytical capability of the immunosensor.
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Técnicas Biossensoriais/métodos , Antígeno Carcinoembrionário/análise , Técnicas Eletroquímicas/métodos , Imunoensaio/métodos , Neoplasias/diagnóstico , Humanos , Nanopartículas Metálicas/químicaRESUMO
Effective signal amplification is a prerequisite for electrochemical immunosensors to achieve ultra-sensitive detection. In this work, we prepared a sandwich-type electrochemical immunosensor for the quantitative detection of carcinoembryonic antigen (CEA). As a base platform, Ag NPs modified aminated two-dimensional nitrogen carbide nanosheets (Ag/g-C3N4) have good biocompatibility and conductivity. In addition, with the layered structure of Au@SiO2/Cu2O as the signal label, the response current value of H2O2 was monitored by the Amperometric i-t Curve (i-t), so as to realize the accurate measurement of CEA. The presence of SiO2 nanoframes not only reduces the agglomeration of Au NPs and Cu2O but also provides good biocompatibility to facilitate the connection of secondary antibodies. Finally, we also verified the signal amplification mechanism of the immunosensor through XPS and other means, and calculated the kinetic parameters of the signal tag, which proved the good peroxidase-like activity of Au@SiO2/Cu2O. Under the best test conditions, the prepared immunosensor has a detection range from 0.01 pg/mL to 80 ng/mL, and the detection limit is as low as 0.0038 pg/mL. The results show that the immunosensor has good analytical performance and it can provide a new method for the clinical diagnosis of CEA.
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Anticorpos Imobilizados/química , Técnicas Biossensoriais/métodos , Antígeno Carcinoembrionário/sangue , Técnicas Eletroquímicas/métodos , Nanopartículas Metálicas/química , Humanos , Imunoensaio/métodosRESUMO
A new type of ultrasensitive electrochemical immunosensor with "signal on" strategy was designed for quantitative detection of CEA. The sensing strategy design is based on the following principles: We use HMSNs-Cu2+@HA as the signal probe, the structure of HA is destroyed under acidic conditions, and the released Cu2+ activates the substrate material MMoO4 (M = Co, Ni) Peroxidase activity initiates the reaction of catalytic H2O2 and realizes the "signal on" condition of electrical signals. This strategy has the following advantages: (1) HA coating of HMSNs-Cu2+ can prevent Cu2+ leakage, has good biocompatibility and can be connected with more antibodies. (2) The prepared sensor has the characteristics of high sensitivity and a low detection limit. When the electrode substrate was CoMoO4, the detection range of the immunosensor was 0.01 pg/mL-40 ng/mL, and the detection limit was 0.0035 pg/mL (S/N = 3). This work innovatively applies the catalytic activity of metal ion-activated nanozymes in the detection of CEA, providing a new perspective for the monitoring and analysis of cancer markers.
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Técnicas Biossensoriais , Nanopartículas Metálicas , Antígeno Carcinoembrionário , Técnicas Eletroquímicas , Ouro , Humanos , Peróxido de Hidrogênio , Imunoensaio , Limite de Detecção , PeroxidaseRESUMO
Signal amplification is very important for electrochemical immunoassays. We report an enzyme-free electrochemical immunosensor based on an electrochemical-chemical-chemical (ECC) redox cycle advanced (RCA) signal amplification strategy for the ultrasensitive detection of Carcinoembryonic antigen (CEA). In this scheme, CeO2/Au NPs@SiO2 is connected with ferrocene as the redox indicator, and the detection buffer is composed of the reducing agent hydroquinone (HQ) and tris(2-carboxyethyl) phosphine (TCEP). First, ferrocenecarboxylic acid (FcA) is oxidized to FcA+ on the electrode. Then, HQ reduces FcA+ to FcA to trigger the cyclic reaction of the inner ring. Second, the oxidation product of HQ is catalyzed by TCEP. The product is reduced to HQ again to complete the cyclic reaction of the outer ring, so the entire cyclic reaction forms a closed loop. The system realizes the high-efficiency regeneration of the electroactive material Fc, thereby ensuring the full amplification of the electrical signal. The results show that the immunosensor exhibits good analytical performance, the detection range is 0.01 pg/mL-80 ng/mL, the detection limit is 0.0037 pg/mL, and the immunosensor has excellent selectivity and stability and performs well in the detection of actual samples. This strategy provides a new method for the early screening of CEA.