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1.
Anal Chem ; 91(5): 3239-3245, 2019 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-30704241

RESUMO

Reversing the switching of DNA scissors with precisely control remains a compelling goal. Herein, based on strand displacement reaction within single step, the DNA scissor realized reversible switching and further controlled the distance of end strands along the movement of DNA scissor, which has been applied for the development of a regenerated sensing platform for the ultrasensitive detection of microRNA-21 (miRNA-21) with the electrochemiluminescence (ECL) complex (PEI-Ru(II)) as luminophores and diethylenetriamine (DETA) as the coreactant. In the presence of ferrocene-labeled DNA (Fc-DNA), the DETA-labeled DNA scissor clockwise switched to "off" state based on strand displacement reaction, resulting in the significant ECL quenching of Ru(II) system. Next, by using miRNA-21 as the motive fuel, the configuration of DNA scissor could be anticlockwise switched, which significantly enhanced the ECL intensity of Ru(II) complex due to the releasing of Fc-DNA and the proximity between DETA and Ru(II) complex. The reversible switching of DNA scissor led to the remarkably enhancing of ECL signal, realizing ultrasensitive detection of miRNA-21 with an excellent detection limit of 0.17 fM, which was also applied in miRNA detection successfully from different cancer cells. Impressively, the reversible switching of DNA scissor biosensor was able to realize the regeneration of the biosensing platform by adding an additional single stranded DNA (ssDNA) based on strand displacement reaction within a single step, providing a novel concept for constructing simple and sensitive regenerated biosensor.


Assuntos
Técnicas Biossensoriais/métodos , MicroRNAs/análise , Linhagem Celular Tumoral , Complexos de Coordenação/química , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Técnicas Eletroquímicas , Compostos Ferrosos/química , Humanos , Limite de Detecção , Medições Luminescentes , Metalocenos/química , MicroRNAs/química , MicroRNAs/metabolismo , Poliaminas/química , Rutênio/química
2.
Biotechnol Appl Biochem ; 63(6): 757-764, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27992074

RESUMO

A simple and high sensitive cholesterol amperometric biosensor, which is based on in situ electropolymerization of multi-walled carbon nanotube-polyaniline (MWCNT-PANI) nanocomposite and electrodeposition of platinum nanoparticle (nano-Pt) films onto the glassy carbon electrode surface for cholesterol oxidase immobilization, was constructed in this study. The preparation process of the modified electrode was characterized by cyclic voltammetry, electrochemical impedance spectroscopy, scanning electron microscopy, and chronoamperometry. Because of the synergistic electrocatalytic activity between MWCNT-PANI nanocomposites and nano-Pt, the cholesterol biosensor exhibited an excellent performance with a linear range of 2.0-510.0 µM, a detection limit of 0.8 µM (signal-to-noise ratio = 3), a high sensitivity of 109.9 µA mM-1 , and a short response time within 5 Sec. Moreover, the reproducibility, stability, and selectivity of the biosensor were also investigated.


Assuntos
Compostos de Anilina/química , Técnicas Biossensoriais/métodos , Colesterol/análise , Nanocompostos/química , Nanotubos de Carbono/química , Platina/química , Carbono/química , Colesterol/sangue , Colesterol/química , Eletroquímica , Eletrodos , Humanos , Peróxido de Hidrogênio/análise , Limite de Detecção , Nanopartículas Metálicas/química , Polimerização
3.
Biosens Bioelectron ; 81: 382-387, 2016 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-26994365

RESUMO

A simple electrochemical proximity immunoassay (ECPA) system for the total protein of Nosema bombycis (TP N.b) detection has been developed on the basis of a new amplification strategy combined with target-induced proximity hybridization. The desirable ECPA system was achieved through following process: firstly, the methylene blue (MB) labeled hairpin DNA (MB-DNA) were immobilized on electrode through Au-S bonding. Then, the antibody labeled complementary single-stranded DNA probe (Ab1-S1) hybridized with MB-DNA to open its hairpin structure, which led to the labeled MB far away from electrode surface. After that, the presence of target biomarker (TP N.b) and antibody labeled single-stranded DNA (Ab2-S2) triggered the typical sandwich reaction and proximity hybridization, which resulted in the dissociation of Ab1-S1 from electrode and the transformation of the MB-DNA into a hairpin structure with MB approaching to electrode surface. In consequence, the hairpin-closed MB was electrocatalyzed by the modified magnetic nanoparticles (Fe3O4NPs), leading to an increased and amplified electrochemical signal for the quantitative detection of TP N.b. In the present work, Fe3O4NPs were acted as catalyst to electrocatalyze the reduction of electron mediator MB for signal amplification, which could not only overcome the drawbacks of protein enzyme in electrocatalytic signal amplification but also shorten the interaction distance between catalyst and substance. Under optimal condition, the proposed ECPA system exhibited a wide linear range from 0.001ngmL(-1) to 100ngmL(-)(1) with a detection limit (LOD) of 0.54pgmL(-1). Considering the desirable sensitivity and specificity, as well as the novel and simple features, this signal amplified ECPA system opened an opportunity for quantitative analysis of many other kinds of protein biomarker.


Assuntos
Técnicas Eletroquímicas/métodos , Proteínas Fúngicas/análise , Nanopartículas de Magnetita/química , Azul de Metileno/química , Nosema/química , Anticorpos Imobilizados/química , Técnicas Biossensoriais , Catálise , DNA de Cadeia Simples/química , Ácidos Nucleicos Imobilizados/química , Imunoensaio/métodos , Hibridização de Ácido Nucleico/métodos
4.
Biosens Bioelectron ; 55: 313-7, 2014 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-24412428

RESUMO

Here, an ultrasensitive electrochemiluminescence (ECL) aptasensor using in situ generated proline and polyamidoamine (PAMAM) dendrimers as coreactant for bis(2,2'-bipyridyl)(5-amino-1,10-phenanthroline) ruthenium(II) (Ru) was successfully constructed for detection of thrombin (TB). Firstly, PAMAM combined with PtNPs was used as platform to assemble substantial Ru, prolidase (GPDA) and avidin labeled thrombin aptamer (avidin-TBA) to form PAMAM-PtNPs-Ru-GDPA-TBA bioconjugate. With the double aptamer-based sandwich assay, the bioconjugate was successfully modified on the electrode surface. The proposed aptasensor possessed three attractive advantages: PAMAM, as a tertiary amine substance, not only served as a platform to immobilize the PtNPs for further assembling prolidase (GPDA) and avidin-TBA, but also used as a coreactant of Ru to amplify the ECL signal. Furthermore, putting the coreactant and luminescence reagent together onto the electrode surface could effectively shorten the reaction time, improve the efficiency of electron transfer, and enhance the ECL signal. Lastly, the in situ generated coreactant proline for Ru catalyzed by GPDA could rapidly increase the concentration of proline around the electrode surface, which could also greatly amplify the ECL signal. With the several amplification factors mentioned above, the proposed ECL aptasensor showed a wide linear range from 0.01 pmol L(-1) to 10 nmol L(-1) with the detection limit of 5.0 fmol L(-1). The experimental results also indicated that the aptasensor exhibited excellent selectivity, stability and reproducibility.


Assuntos
Aptâmeros de Peptídeos/química , Técnicas Biossensoriais/instrumentação , Dendrímeros/química , Medições Luminescentes/instrumentação , Prolina/química , Trombina/análise , Desenho de Equipamento , Análise de Falha de Equipamento , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Trombina/química , Trombina/genética
5.
Chem Commun (Camb) ; 49(66): 7328-30, 2013 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-23851915

RESUMO

We for the first time fabricate a pseudo triple-enzyme cascade electrocatalytic electrochemical aptasensor by using the alcohol dehydrogenase (ADH) as well as the autonomously assembled hemin-G-quadruplex that simultaneously acted as an NADH oxidase and HRP-mimicking DNAzyme.


Assuntos
Aptâmeros de Nucleotídeos/química , DNA Catalítico/química , Eletroquímica , Quadruplex G , Hemina/química , Nanofios/química , Álcool Desidrogenase/química , Catálise , Limite de Detecção
6.
Anal Chim Acta ; 783: 49-55, 2013 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-23726099

RESUMO

In the present study, a novel and ultrasensitive electrochemiluminescence (ECL) immunosensor based on luminol cathodic ECL was fabricated by using Au nanoparticles and Pt nanoparticles (nano-AuPt) electrodeposited on graphene-carbon nanotubes nanocomposite as platform for the detection of carcinoembryonic antigen (CEA). For this introduced immunosensor, graphene (GR) and single wall carbon nanotubes (CNTs) dispersed in chitosan (Chi-GR-CNTs) were firstly decorated on the bare gold electrode (GE) surface. Then nano-AuPt were electrodeposited (DpAu-Pt) on the Chi-GR-CNTs modified electrode. Subsequently, glucose oxidase (GOD) was employed to block the non-specific sites of electrode surface. When glucose was present in the working buffer solution, GOD immediately catalyzed the oxidation of glucose to in situ generate hydrogen peroxide (H2O2), which could subsequently promote the oxidation of luminol with an amplified cathodic ECL signal. The proposed immunosensor was performed at low potential (-0.1 to 0.4V) and low concentration of luminol. The CEA was determined in the range of 0.1 pg mL(-1) to 40 ng mL(-1) with a limit of detection down to 0.03 pg mL(-1) (SN(-1)=3). Moreover, with excellent sensitivity, selectivity, stability and simplicity, the as-proposed luminol-based ECL immunosensor provided great potential in clinical applications.


Assuntos
Ligas/química , Glucose Oxidase/química , Ouro/química , Grafite/química , Medições Luminescentes/métodos , Luminol/química , Nanotubos de Carbono/química , Platina/química , Antígeno Carcinoembrionário/análise , Eletrodos , Humanos , Limite de Detecção , Microscopia Eletrônica de Varredura
7.
Biosens Bioelectron ; 43: 63-8, 2013 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-23277341

RESUMO

A novel signal amplification strategy of mimicking bi-enzyme synergetic catalysis to generate coreactant in situ was designed to fabricate an ultrasensitive peroxydisulfate electrochemiluminescence (ECL) immunosensor for detection of Streptococcus suis serotype 2 (SS2). It was the first time to detect SS2 by using ECL. Through the interaction between l-cysteine (l-cys) and hollow PtPd bimetal alloy nanoparticles (HPtPd) to form ((l-cys-HPtPd)n) nanocomposites, the loading amount of l-cys and HPtPd was greatly increased, which could greatly enhance the ECL signal of peroxydisulfate. At the same time, Glucose Oxidase (GOD), used to block nonspecific binding sites of (l-cys-HPtPd)n nanocomposites, could rapidly oxidize d-glucose in the detection solution into gluconic acid accompanying with the generation of H2O2, which was further catalyzed by HPtPd to generate O2. And O2, acted as the coreactant of peroxydisulfate, could greatly amplify the ECL signal. In the process, HPtPd could be regarded as mimicking enzyme, the effect of which was similar to horseradish peroxidase (HRP) in generating O2. With the several amplification factors of a sandwich-type structure we designed, a wide linear ranged from 0.0001 to 100ngmL(-1) was acquired with a relatively low detection limit of 33fgmL(-1) for SS2. The present work demonstrated that the novel strategy had the great advantages in sensitivity, selectivity and reproducibility which might hold a new promise for highly sensitive bioassays applied in clinical detection.


Assuntos
Técnicas Biossensoriais/instrumentação , Condutometria/instrumentação , Cisteína/química , Eletrodos , Imunoensaio/instrumentação , Medições Luminescentes/instrumentação , Streptococcus suis/isolamento & purificação , Materiais Biomiméticos/química , Catálise , Desenho de Equipamento , Análise de Falha de Equipamento
8.
Biosens Bioelectron ; 42: 474-80, 2013 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-23238321

RESUMO

A novel sensitive sandwich-type pseudobienzyme aptasensor was developed by dendrimer functionalized reduced graphene oxide (PAMMA-rGO) as nanocarrier and hemin/G-quadruplex as NADH oxidase and HRP-mimicking DNAzyme. Greatly enhanced sensitivity for the target thrombin was achieved by using a dual signal amplification strategy: first, the PAMMA-rGO not only constructed an effective platform for anchoring larger amounts of electron mediator thionine (TH) and hemin/G-quadruplex bioelectrocatalytic complex with high stability and bioactivity but also accelerated the electron transfer process assisted by the conductive rGO with amplified electrochemical signal output. Second, the hemin/G-quadruplex simultaneously acting as an NADH oxidase and HRP-mimicking DNAzyme for constructing pseudobienzyme amplifying system could in situ biocatalyze formation of H2O2 with high local concentrations and low transfer loss that lead to obvious signal enhancements. On the basis of the dual signal amplification strategy of PAMMA-rGO and the pseudobienzyme amplifying, the developed aptasensor could respond to 0.1 pM thrombin with a linear calibration range from 0.0002 to 30.0 nM. Compared with protein enzymes assisted bienzyme aptasensor, this new aptasensor avoided the fussy labeling process and the spatial distribution of each sequentially acting enzyme, which provided ideal candidate for the development of sensitive and simple bioanalytical platform.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Grafite/química , Nanopartículas/química , DNA Catalítico/química , Dendrímeros/química , Quadruplex G , Ouro/química , Hemina/química , Peróxido de Hidrogênio/química , Limite de Detecção , Nanopartículas Metálicas/química , Óxidos/química , Poliaminas/química
9.
Chemistry ; 18(44): 14186-91, 2012 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-23001991

RESUMO

A simple wet-chemical strategy for the synthesis of 3,4,9,10-perylenetetracarboxylic acid (PTCA)/hemin nanocomposites through π-π interactions is demonstrated. Significantly, the hemin successfully conciliates PTCA redox activity with a pair of well-defined redox peaks and intrinsic peroxidase-like activity, which provides potential application of the PTCA self-derived redox activity as redox probes. Additionally, PTCA/hemin nanocomposites exhibit a good membrane-forming property, which not only avoids the conventional fussy process for redox probe immobilization, but also reduces the participation of the membrane materials that act as a barrier of electron transfer. On the basis of these unique properties, a pseudobienzyme-channeling amplified electrochemical aptasensor is developed that is coupled with glucose oxidase (GOx) for thrombin detection by using PTCA/hemin nanocomposites as redox probes and electrocatalysts. With the addition of glucose to the electrolytic cell, the GOx on the aptasensor surface bioelectrocatalyzed the reduction of glucose to produce H(2)O(2), which in turn was electrocatalyzed by the PTCA/hemin nanocomposites. Cascade schemes, in which an enzyme is catalytically linked to another enzyme, can produce signal amplification and therefore increase the biosensor sensitivity. As a result, a linear relationship for thrombin from 0.005 to 20 nM and a detection limit of 0.001 nM were obtained.


Assuntos
Aptâmeros de Nucleotídeos/química , Hemina/química , Nanocompostos/química , Perileno/análogos & derivados , Técnicas Biossensoriais , Catálise , Técnicas Eletroquímicas , Glucose Oxidase/química , Glucose Oxidase/metabolismo , Oxirredução , Perileno/química
10.
Anal Chim Acta ; 726: 67-72, 2012 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-22541015

RESUMO

A novel tracer, 3,4,9,10-perylenetetracarboxylic dianhydride (PTCDA) functionalized graphene sheet (GS) composite (GS-TCDA), is employed to label the secondary anti-thrombin aptamer (TBA) to construct an ultrasensitive electrochemiluminescent sandwich-type aptasensor. The GS provided large surface area for loading abundant PTCDA and TBA with good stability and biocompatibility. Because of the excellent electroconductivity of GS and the desirable optical properties of PTCDA, the as-formed Apt II bioconjugate considerably amplified the electrochmiluminescence (ECL) signal of peroxydisulfate (S(2)O(8)(2-)) and worked as the desirable label for Apt II. On the basis of the considerably amplified ECL signal and sandwich format, an extremely wide range from 1 fM to 1 nM with an ultralow detection limit of 0.33 fM for thrombin was obtained. Additionally, the selectivity and stability of the proposed aptasensor were also excellent. Thus, this procedure has great promise for detection of thrombin present at ultra-trace levels during early stage of diseases.


Assuntos
Anidridos/química , Grafite/química , Medições Luminescentes , Perileno/análogos & derivados , Trombina/análise , Aptâmeros de Nucleotídeos/química , Técnicas Eletroquímicas , Perileno/química
11.
Chem Commun (Camb) ; 48(38): 4621-3, 2012 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-22466956

RESUMO

For the first time, hemin/G-quadruplex was employed to simultaneously serve as NADH oxidase and an HRP-mimicking DNAzyme for constructing a simple and sensitive pseudobienzyme-amplifying electrochemical aptasensor for thrombin detection.


Assuntos
DNA Catalítico/metabolismo , Técnicas Eletroquímicas , Quadruplex G , Hemina/química , Trombina/análise , Biocatálise , Materiais Biomiméticos/metabolismo , Peroxidase do Rábano Silvestre/metabolismo , Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/metabolismo
12.
Biosens Bioelectron ; 34(1): 25-9, 2012 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-22387036

RESUMO

A solid-state electrochemiluminescence (ECL) aptasensor based on target-induced aptamer displacement for highly sensitive detection of thrombin was developed successfully using 4-(dimethylamino)butyric acid (DMBA)@PtNPs labeling as enhancer. Such a special aptasensor included three main parts: ECL substrate, ECL intensity amplification and target-induced aptamer displacement. The ECL substrate was made by modifying the complex of Pt nanoparticles (PtNPs) and tris(2,2-bipyridyl) ruthenium (II) (Ru(bpy)(3)(2+)) (Ru-PtNPs) onto nafion@multi-walled carbon nanotubes (nafion@MWCNTs) modified electrode surface. A complementary thrombin aptamer labeled by DMBA@PtNPs (Aptamer II) acted as the ECL intensity amplification. The thrombin aptamer (TBA) was applied to hybridize with the labeled complementary thrombin aptamer, yielding a duplex complex of TBA-Aptamer II on the electrode surface. The introduction of thrombin triggered the displacement of Aptamer II from the self-assembled duplex into the solution and the association of inert protein thrombin on the electrode surface, decreasing the amount of DMBA@PtNPs and increasing the electron transfer resistance of the aptasensor and thus resulting large decrease in ECL signal. With the synergistic amplification of DMBA and PtNPs to Ru(bpy)(3)(2+) ECL, the aptasensor showed an enlarged ECL intensity change before and after the detection of thrombin. As a result, the change of ECL intensity has a direct relationship with the logarithm of thrombin concentration in the range of 0.001-30 nM. The detection limit of the proposed aptasensor is 0.4 pM. Thus, the approach is expected to open new opportunities for protein diagnostics in clinical as well as bioanalysis in general.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos , Luminescência , Trombina/análise , 9,10-Dimetil-1,2-benzantraceno/química , Técnicas Biossensoriais/instrumentação , Humanos , Nanopartículas Metálicas/química , Nanotubos de Carbono/química , Platina/química
13.
Biosens Bioelectron ; 30(1): 123-7, 2011 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-21944184

RESUMO

Graphene/3,4,9,10-perylenetetracarboxylic acid (GPD) with three-dimensional porous structure has been successfully synthesized and served as redox probe to construct ultrasensitive electrochemical aptasensor. The GPD nanocomposite shows promoted electrochemical redox-activity of 3,4,9,10-perylenetetracarboxylic acid (PTCA) with an obvious well-defined cathodic peak from -0.7 to 0 V that never been seen from graphene or PTCA, which avoids miscellaneous redox peaks of PTCA in electrochemical characterization, offering a novel redox probe for electrochemical sensors with highly electrochemical active area and conductivity. To the best of our knowledge, this is the first study that utilizes PTCA self-derived redox-activity as redox probe in electrochemical sensors. Moreover, the interesting GPD possesses the advantages of membrane-forming property, providing a direct immobilization of redox probes on electrode surface. This simple process not only diminishes the conventional fussy immobilization of redox probes on the electrode surface, but also reduces the participation of the membrane materials that acted as a barrier of the electron propagation in redox probe immobilization. With thrombin as a model target, the redox probe-GPD based label-free electrochemical aptasensor shows a much higher sensitivity (a detection range from 0.001 nM to 40 nM with a detection limit of 200 fM) to that of analogous aptasensors produced from other redox probes.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/instrumentação , Condutometria/instrumentação , Grafite/química , Nanopartículas/química , Perileno/análogos & derivados , Trombina/análise , Desenho de Equipamento , Análise de Falha de Equipamento , Nanotecnologia/instrumentação , Perileno/química , Coloração e Rotulagem , Trombina/química
14.
Biotechnol Appl Biochem ; 58(4): 256-60, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21838800

RESUMO

A sensitive label-free electrochemical aptasensor was successfully fabricated for thrombin detection with platinum nanoparticles (Pt) and blocking reagent horseradish peroxidase (HRP) as enhancers. A Nafion®-graphene-coated electrode was first modified with an electrochemical probe of methylene blue (MB) through electrostatic interaction. Then Pt was electrodeposited onto an electrode for immobilization of the thrombin aptamer (TBA). Subsequently, HRP served as blocking reagent instead of bovine serum albumin (BSA). With the synergistic effect between Pt and HRP, the prepared aptasensor showed a superior catalytic efficiency toward H(2) O(2) in the presence of MB. After the combination of target thrombin on electrode surface, the TBA-thrombin complex made a barrier for electrocatalysis of Pt and HRP and inhibited the electrotransfer, resulting in a greater decrease in MB signals. As a result, the proposed approach showed a high sensitivity and a much wider linearity to thrombin in the range from 0.005 to 50 nM with a detection limit of 1 pM.


Assuntos
Técnicas Eletroquímicas , Nanopartículas Metálicas/química , Platina/química , Trombina/análise , Aptâmeros de Peptídeos/química , Grafite/química , Peroxidase do Rábano Silvestre/química , Humanos , Peróxido de Hidrogênio/química , Limite de Detecção , Azul de Metileno/química
15.
Bioprocess Biosyst Eng ; 34(9): 1081-5, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21643688

RESUMO

A sensitive label-free electrochemical aptasensor was successfully fabricated for thrombin detection with nafion@graphene as platform. With electrostatic interaction between nafion and methylene blue (MB), positive charged MB was successfully assembled on nafion@graphene modified electrode surface, which provided amounts of redox probes for electrochemical aptasensor. In the presence of thrombin, the thrombin aptamer (TBA) on the electrode surface would catch the target on the electrode interface, which made a barrier for electrons and inhibits the electro-transfer, resulting in the decreased differential pulse voltammetry signals of MB. As a result, the proposed approach showed a high sensitivity and a wider linearity to thrombin in the range 0.01-50 nM with a detection limit of 6 pM.


Assuntos
Eletroquímica/métodos , Polímeros de Fluorcarboneto/química , Grafite/química , Trombina/análise , Técnicas Biossensoriais , Eletrodos , Desenho de Equipamento , Ouro/química , Humanos , Imunoglobulina G/química , Azul de Metileno/análise , Azul de Metileno/química , Oxirredução , Sensibilidade e Especificidade , Eletricidade Estática , Trombina/química , Fatores de Tempo
16.
Biosens Bioelectron ; 26(10): 4236-40, 2011 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-21536422

RESUMO

A simple electrochemical aptasensor for sensitive detection of thrombin was fabricated with G-quadruplex horseradish peroxidase-mimicking DNAzyme (hemin/G-quadruplex system) and blocking reagent-horseradish peroxidase as dual signal-amplification scheme. Gold nanoparticles (nano-Au) were firstly electrodeposited onto single wall nanotube (SWNT)-graphene modified electrode surface for the immobilization of electrochemical probe of nickel hexacyanoferrates nanoparticles (NiHCFNPs). Subsequently, another nano-Au layer was electrodeposited for further immobilization of thrombin aptamer (TBA), which later formed hemin/G-quadruplex system with hemin. Horseradish peroxidases (HRP) then served as blocking reagent to block possible remaining active sites and avoided the non-specific adsorption. In the presence of thrombin, the TBA binded to thrombin and the hemin released from the hemin/G-quadruplex electrocatalytic structure, increasing steric hindrance of the aptasensor and decomposing hemin/G-quadruplex electrocatalytic structure, which finally decreased the electrocatalytic efficiency of aptasensor toward H(2)O(2) in the presence of NiHCFNPs with a decreased electrochemical signal. On the basis of the synergistic amplifying action, a detection limit as low as 2 pM for thrombin was obtained.


Assuntos
Técnicas Biossensoriais/métodos , Trombina/análise , Aptâmeros de Nucleotídeos , Técnicas Biossensoriais/estatística & dados numéricos , DNA Catalítico , Técnicas Eletroquímicas , Quadruplex G , Ouro , Hemina , Peroxidase do Rábano Silvestre , Nanopartículas Metálicas , Reprodutibilidade dos Testes
17.
Bioprocess Biosyst Eng ; 32(1): 109-16, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18465149

RESUMO

A flow-through quartz crystal microbalance (QCM) immunoassay method has been developed based on aflatoxin B(1) antibody (anti-AFB(1))-functionalized magnetic core-shell Fe(3)O(4)/SiO(2) composite nanoparticles (bionanoparticles) in this study. To construct such an assay protocol, anti-AFB(1), as a model protein, was initially covalently immobilized onto the Fe(3)O(4)/SiO(2) surface, and then the functionalized nanoparticles were attached to the surface of the QCM probe with an external magnet. The binding of target molecules onto the immobilized antibodies decreased the sensor's resonant frequency, and the frequency shift was proportional to the AFB(1) concentration in the range of 0.3-7.0 ng/ml. The regeneration of the developed immunosensor was carried out via attaching or detaching the external magnet from the detection cell. In addition, the selectivity, reproducibility, and stability of the proposed immunoassay system were acceptable. Compared with the conventional ELISAs, the proposed immunoassay system was simple and rapid without multiple labeling and separation steps. Importantly, the proposed immunoassay method could be further developed for the immobilization of other antigens or biocompounds.


Assuntos
Aflatoxina B1/análise , Óxido Ferroso-Férrico/química , Imunoensaio/métodos , Nanopartículas Metálicas/química , Nanotecnologia/métodos , Aflatoxina B1/química , Materiais Biocompatíveis , Ensaio de Imunoadsorção Enzimática , Concentração de Íons de Hidrogênio , Imunoensaio/instrumentação , Ferro/química , Magnetismo , Microscopia de Força Atômica , Quartzo , Silício/química , Dióxido de Silício/química , Espectroscopia de Infravermelho com Transformada de Fourier
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