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1.
Mikrochim Acta ; 186(4): 247, 2019 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-30879138

RESUMO

The authors describe a fluorometric method for the quantification of tannic acid (TA). MoO3-x quantum dots (QDs) can selectively capture TA via the formation of an organic molybdate complex. This causes an electron transfer effect and an inner filter effect to result in synergistic quenching of the fluorescence of the QDs. TA can be detected via this effect with a linear response in the of 0.1-10 µM concentration range and a lower detection limit of 30 nM within 1 min. The use of such QDs as a quenchable fluorescent probe warrants good selectivity even in the presence of relatively high concentration of potentially interferents and makes the method suitable for real sample analysis. Graphical abstract Tannic acid can be rapidly and selectively detected in food using a MoO3-x quantum dots based fluorometric assay.

2.
Biosens Bioelectron ; 132: 360-367, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30897543

RESUMO

Lateral flow immunoassay (LFIA) is a class and widespread applied point-of-care biosensor in the rapid monitoring field. To address the matched antibodies and antibody labeling dependence in the conventional LFIAs, in this work, an innovative label-free LFIA was proposed for the sensitive detection of Salmonella enteritidis (S. enteritidis) by introducing a new nanoparticles-bacteria-antibody sandwich strategy in the sensor. Surface positively charged nitrogen-rich carbon (pNC) nanoparticles, synthesized via calcination and etching reactions, were used as adsorbent to capture bacteria as well as for generating signals. In the presence of target pathogens, bacterial cells could combine with pNC through electrostatic interaction and hydrogen bonding, then the complex would be captured specifically by the anti-bacteria monoclonal antibody (McAb) coated on the test line (T-line). With the accumulation of nanoparticles-bacteria, the color on T-line would be gradually deepened from nearly colorless to deep black. Importantly, the pNC-based immunoassay could exhibit high sensitivity for target pathogens detection with a linear range of 102-108 cfu mL-1 and a low detection limit of 102 cfu mL-1. Furthermore, this system was validated preliminarily to screen S. enteritidis in different food samples with recoveries ranging from 85% to 110%. Taking advantages of simplicity, label-free, convenience, and sensitivity, the pNC-based LFIA has the application potential for pathogenic microorganisms monitoring in food safety and early clinical diagnosis fields.


Assuntos
Anticorpos Imobilizados/química , Técnicas Biossensoriais/instrumentação , Carbono/química , Análise de Alimentos/instrumentação , Nanopartículas/química , Nitrogênio/química , Salmonella enteritidis/isolamento & purificação , Microbiologia de Alimentos , Humanos , Imunoensaio/instrumentação , Limite de Detecção , Modelos Moleculares , Fitas Reagentes/análise , Infecções por Salmonella/microbiologia
3.
Food Chem ; 268: 242-248, 2018 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-30064753

RESUMO

A deoxynivalenol (DON) epitope clone (D8) was obtained by phage display technology using anti-DON monoclonal antibodies as a target molecule. Subsequently, a DON antigen mimic (D8-maltose-binding protein [MBP]) was synthesized by fusing the mimic epitope peptide with MBP. An enzyme-linked immunosorbent assay (ELISA) and urchin-like gold nanoparticle immunochromatographic assay was developed based on D8-MBP for detection of DON in maize and wheat. The half-maximal inhibitory concentration, lower detection limit, and linear range of the D8-MBP ELISA were 57.98 ±â€¯0.97, 9.83, and 11.32-286.77 ng/mL, respectively. The sensitivity of the D8-MBP ELISA was nearly 2.5 times higher than that of traditional ELISA using DON-bovine serum albumin (BSA). The detection threshold of the colloidal gold immunochromatographic assay for D8-MBP was 25 ng/mL. Thus, D8-MBP could be used to replace the traditional DON-BSA antigen for the immunological detection of DON, permitting low cost, rapid detection of DON.

4.
Biochem Biophys Res Commun ; 499(3): 719-726, 2018 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-29605294

RESUMO

MiR-615 and epidermal growth factor receptor (EGFR) are associated with a number of disease processes and pathogenesis. However, little is known about the mechanisms of miR-615 and EGFR in human glioblastoma multiforme (GBM). Here, we found that down-regulation of miR-615 expression occurred in GBM tissues and cells, and was inversely correlated with overall survival, relapse-free survival, WHO grade as well as EGFR expression. We further determined that miR-615 functions as a tumor suppressor by inhibiting GBM cell proliferation, cell cycle, migration and invasion, and promoting cell apoptosis. In-vivo assay validated the inhibition effect of miR-615 on tumor growth and EGFR expression. Luciferase reporter assays demonstrated that miR-615 targeted the 3'-untranslated region (3'-UTR) of EGFR. Besides, over-expression of EGFR reversed the inhibition effects of miR-615, while silencing of EGFR aggravated these inhibition effects. In conclusions, we identified that miR-615 plays a tumor suppressor role in GBM cell proliferation, migration and invasion by targeting EGFR expression, and miR-615 may act as a novel biomarker for early diagnosis or therapeutic targets of GBM.


Assuntos
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Movimento Celular/genética , Receptores ErbB/metabolismo , Glioblastoma/genética , Glioblastoma/patologia , MicroRNAs/metabolismo , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação para Baixo/genética , Receptores ErbB/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Análise Multivariada , Invasividade Neoplásica , Modelos de Riscos Proporcionais
5.
Biosens Bioelectron ; 102: 9-16, 2018 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-29101785

RESUMO

Here, we for the first time used a thiolated amino-ligand modified multi-branched gold nanoflower as skeleton to encapsulate iron porphyrins (AuNF@FeTPPCl) as alternatives to horseradish peroxidase (HRP). After the FeTPPCl encapsulation, the HRP mimicking activity of FeTPPCl was effectively blocked by the steric hindrance of the hydrophobic layer on the AuNF surface. Upon the addition of ethanol, the loaded FeTPPCl was released into the solution in its free format and exposed the catalytic sites, resulting in the recovery of catalytic activity. The proposed encapsulation method effectively avoided the loss of catalytic activity that originated from the blocking of the catalytic active sites during immobilization. Additionally, the resultant AuNF@FeTPPCl nanocomposite exhibited a high loading level with 2.4 × 106 FeTPPCl molecules per AuNF, and showed considerably high catalytic activity for hydrogen peroxide and 3, 3' 5, 5'-tetramethylbenzidine, which is approximately 40- and 172-folds higher than native HRP. Through using the as-prepared AuNF@FeTPPCl as an alternative of HRP for trace labeling, we successfully developed colorimetric immunosensors for fumonisin B1 (FB1) and hepatitis B surface antigen (HBsAg) with competitive and sandwich-type formats, respectively. The developed AuNF@FeTPPCl-based colorimetric immunosensor exhibited higher detection sensitivity for FB1 and HBsAg than the corresponding HRP-based immunosensors. Thus, the proposed AuNF@FeTPPCl can be used as HRP mimicking analogs for developing highly sensitive colorimetric immunosensor and for loading other hydrophobic iron porphyrins or catalysts.


Assuntos
Técnicas Biossensoriais/métodos , Colorimetria/métodos , Fumonisinas/análise , Ouro/química , Antígenos de Superfície da Hepatite B/análise , Nanopartículas Metálicas/química , Metaloporfirinas/química , Anticorpos Imobilizados/química , Materiais Biomiméticos/química , Hepatite B/sangue , Hepatite B/virologia , Antígenos de Superfície da Hepatite B/sangue , Vírus da Hepatite B/isolamento & purificação , Peroxidase do Rábano Silvestre/química , Humanos , Imunoensaio/métodos
6.
Exp Ther Med ; 14(3): 2511-2516, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28962188

RESUMO

The aim of the study was to investigate dynamic changes in α-melanocyte-stimulating hormone (α-MSH) levels in the serum of patients with craniocerebral trauma. Forty-eight patients with acute craniocerebral injury were selected between January 2015 and October 2016. The patients were divided into three groups: severe (18 cases), moderate (16 cases) and mild (14 cases), according to the Glasgow Coma Scale (GCS) score at the time of admission. At the same time, 10 adults with a similar age distribution to the patients were also selected as a control group. Venous blood was extracted from patients at 1, 3, 5 and 7 days after injury. Serum α-MSH and tumor necrosis factor (TNF)-α levels were measured using an enzyme-linked immunosorbent assay (ELISA). The correlation between α-MSH and TNF-α was analyzed using Pearson's correlation analysis. Serum α-MSH levels in patients with craniocerebral injury were lower than those in the healthy control group (P<0.05). Decreased serum α-MSH levels were usually accompanied with higher degrees of craniocerebral injury. Serum α-MSH levels initially decreased and then later increased, with the lowest α-MSH levels in the mild at 5 days, moderate at 5 days, and severe groups at 3 days after injury (P<0.05). Serum TNF-α levels in all the patient groups were higher than those in the control group at different time points after injury, with higher TNF-α serum levels accompanying higher degrees of brain injury. In all three groups, serum TNF-α levels initially increased and then decreased post-injury, with peak serum TNF-α levels found at 3-day post-injury in all the patient groups (P<0.05). A negative correlation between serum α-MSH content and serum TNF-α levels in patients with craniocerebral trauma at different time points, was noted (P<0.05). Serum α-MSH content in the survival group was higher than that in the death group (P<0.05). Serum α-MSH levels in patients with non-systemic inflammatory response syndrome (SIRS) were higher than in patients with SIRS (P<0.05). Serum α-MSH levels during the early stages after craniocerebral trauma can be used as a factor for the prediction of secondary SIRS, with constant low levels of serum α-MSH suggest poor prognosis.

7.
Anal Bioanal Chem ; 407(24): 7341-8, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26297453

RESUMO

An immunochromatographic strip (ICS) using urchin-like gold nanoparticles (UGNs) for sensitive detection of fumonisin B1 (FB1) was developed to meet the requirement for rapidly monitoring FB1 in grain samples. The sensitivity of the ICS was 5.0 ng/mL, which represents a fourfold increase in sensitivity over conventional strip preparation using colloidal gold as the antibody-labeled probe. Analysis of FB1 in grain samples showed that data obtained from the strip tests were in a good agreement with those obtained from HPLC and enzyme-linked immunosorbent assays (ELISAs). This qualitative test did not require any specialized equipment, and the detection time was less than 5 min, which is suitable for on-site testing of FB1 in grain samples. Overall, to our knowledge, this is the first report of using a UGN as the antibody-labeled probe for sensitive detection of FB1 in grains using an ICS. Graphical Abstract Preparation of ICS using conventional colloidal gold and urchin-like gold nanoparticle, respectively.


Assuntos
Cromatografia de Afinidade/métodos , Grão Comestível/química , Fumonisinas/análise , Ouro/química , Nanopartículas Metálicas/química , Anticorpos Monoclonais/imunologia , Fumonisinas/imunologia , Limite de Detecção
8.
Talanta ; 143: 388-393, 2015 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-26078175

RESUMO

Nanobodies that are small and thermally stable, as well as have high expression level, are leading alternative to produce anti-idiotypic antibodies. These antibodies have the advantage of replacing mycotoxins and their conjugates for immunoassays. In this work, anti-fumonisin B1 (FB1) monoclonal antibody (mAb) was used as the target for biopanning from a naïve alpaca nanobody (Nb) phage display library. After three cycles of panning, one anti-idiotypic nanobody (Ab2ß Nb) was isolated and subjected to a Nb-ELISA for the detection of FB1. Surface plasmon resonance was used to analyze the reaction kinetics between the Ab2ß Nb and anti-FB1 mAb. The developed assay showed a half inhibitory concentration (IC50) of 0.95±0.12 ng/mL, a limit of detection of 0.15 ng/mL, a linear range of 0.27-5.92 ng/mL, and a low cross-reactivity toward FB2 of 4.93%. The sensitivity was enhanced approximately 20-fold compared with that of the chemosynthetic FB1-BSA conjugates. The equilibrium dissociation constant (KD) measured for Ab2ß Nb: anti-FB1 mAb was 164.6 nM. The assay was compared with conventional ELISA (the commercial ELISA kit), and the results indicated the reliability of Ab2ß Nb replacing the antigen-carrier protein conjugates. The use of biotechnology in developing the surrogate is an ideal strategy for replacing conventional synthesized antigens.


Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Fumonisinas/análise , Anticorpos de Domínio Único/imunologia , Fumonisinas/imunologia , Imunoensaio
9.
Talanta ; 142: 206-12, 2015 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-26003713

RESUMO

Immunochromatographic test strips (ICTS) are commonly limited to higher concentrations of analytes. This limitation stems from the relatively low sensitivity of conventional gold nanospheres (AuNSs with a diameter of 20 nm) to emit detectable brightness values. The larger multi-branched gold nanoflowers (AuNFs) with a higher optical brightness as well as good colloidal stability exhibit significant improvements over conventional AuNSs for enhanced sensitivity of ICTS. In this study, blue AuNFs with an average diameter of 75±5 nm were synthetized and employed as a signal amplification probe for ultrasensitive and quantitative detection of aflatoxin B1 (AFB1) in rice. A portable optical strip reader was used to record the optical densities of test and control lines of the strip. Under the optimal conditions, the AuNF based ICTS system accurately detected AFB1 linearly and dynamically over the range of 0.5-25 pg/mL with a half maximal inhibitory concentration at 4.17 pg/mL. The inhibitory concentration was achieved 10 times lower than that of the traditional AuNS based ICTS systems (41.25 pg/mL). The limit of detection for AFB1 in rice extract was achieved at 0.32 pg/mL. In summary, AuNFs are a novel probe that exhibited excellent sensitivity in the ICTS system and could be used for ultrasensitive detection of other analytes in food safety monitoring, and even medical diagnostics.


Assuntos
Aflatoxina B1/análise , Ouro/química , Nanoestruturas/química , Oryza/química , Aflatoxina B1/imunologia , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Cromatografia de Afinidade/instrumentação , Contaminação de Alimentos/análise , Limite de Detecção , Fitas Reagentes
10.
Biosens Bioelectron ; 66: 184-90, 2015 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-25460900

RESUMO

Gold nanoparticle (GNP)-based dynamic light scattering (DLS) assay has been widely used for sensitive detection of small analytes based on analyte binding-induced GNP aggregation. However, the use of this new method to detect large biological objectives, such as pathogenic bacteria, has not been reported. This study is the first to describe a homogeneous GNP-based DLS immunoassay for ultrasensitive detection of Listeria monocytogenes. Compared with small analytes, L. monocytogenes has a larger surface and a higher number of antigen epitopes, which serve as carriers that bind to GNP probes to form "GNP-coated bacteria" complexes. To achieve better analytical performance, various parameters including GNP diameter and concentration, amount of labeled antibodies, and immunoreaction time were systematically investigated and optimized. Under the developed optimum conditions, limit of detection (LOD) for L. monocytogenes reached as low as 3.5×10(1)CFUmL(-1) in 0.01M phosphate-buffered saline. Coupled with a large-volume immunomagnetic separation method, LOD for spiked lettuce samples reached 2.2×10(1)CFUg(-1), which was one order of magnitude lower than the maximum limit imposed in Canada (100CFUg(-1)). The proposed method also exhibited excellent discrimination against 17 common pathogenic bacteria in lettuces. The developed GNP-based DLS immunoassay is highly promising as an approach for detecting large biological objectives.


Assuntos
Técnicas Biossensoriais/métodos , Imunoensaio/métodos , Alface/microbiologia , Listeria monocytogenes/isolamento & purificação , Microbiologia de Alimentos , Ouro/química , Humanos , Nanopartículas Metálicas/química
11.
Chin Med J (Engl) ; 122(11): 1260-6, 2009 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-19567134

RESUMO

BACKGROUND: Glioma is the most common primary brain tumor with poor prognosis. Temozolomide has been used with thalidomide to treat gliomas. We investigated the synergistic mechanism of these two drugs in vitro. METHODS: Human malignant glioma cells U251-MG were cultured and assigned to four groups with different treatments for 3 days: temozolomide group (100 micromol/L), thalidomide group (100 microg/L), temozolomide (100 micromol/L) plus thalidomide group (100 microg/L) and control group. MTT assay was applied to evaluate the cell viability. Cell cycle was analyzed by flow cytometry. The ultra-structural features of autophagosomes were observed with electron microscope. Acridine orange and monodansylcadaverine were adopted to label autophagosomes and flow cytometry was applied for quantification of autophagosomes. The expression of autophagy-associated protein was detected by Western blotting. RESULTS: Proliferation of tumor cell was obviously suppressed by temozolomide with thalidomide treatment than by either drug used alone (P = 0.000 for each day). The combination treatment induced cell cycle arrest at G0/G1 phase. Typical autophagic ultra-structural character was found after the combined treatment. Thalidomide promoted the autophagy induced by temozolomide. The autophagy-associated proteins-microtubule associated protein 1 light chain 3 (MAP1LC3) and Beclin1 were more significantly up-regulated by the combined treatment than temozolomide used alone (MAP1LC3, P = 0.000; Beclin1, P = 0.004). The expression level of phosphatase and tensin homolog deleted on chromosome ten (PTEN), which promoted autophagy by suppressing PI3K/Akt/mTOR signaling pathway, was elevated by thalidomide (thalidomide group: P = 0.000; combined group: P = 0.002). CONCLUSIONS: Thalidomide enhances the cytotoxicity of temozolomide by promoting the autophagy induced by temozolomide. Contributing to the up-regulation of PTEN by thalidomide, the expression of autophagy associated protein-MAP1LC3 and Beclin1 was enhanced, which leads to a reinforced autophagy in the combined treatment of temozolomide and thalidomide in vitro.


Assuntos
Antineoplásicos Alquilantes/farmacologia , Autofagia/efeitos dos fármacos , Dacarbazina/análogos & derivados , Glioma/patologia , Talidomida/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dacarbazina/farmacologia , Humanos , Temozolomida
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