Electrochemical immunosensor for salbutamol detection based on CS-Fe3O4-PAMAM-GNPs nanocomposites and HRP-MWCNTs-Ab bioconjugates for signal amplification

An ultrasensitive electrochemical immunosensor based on chitosan-iron oxide-poly(amino-amine) dendrimers-gold nanoparticles (CS-Fe₃O₄-PAMAM-GNPs) nanocomposites and horseradish peroxidase-multiwall carbon nanotubes-antibody (HRP-MWCNTs-Ab) bioconjugates was developed for the detection of salbutamol (SAL). CS-Fe₃O₄-PAMAM-GNPs nanocomposites as immobilization matrix were used to enhance the electroactivity and stability of the electrode. HRP-MWCNTs-Ab bioconjugates as label were used to improve catalytic activity for hydrogen reduction of the electrode. Under the optimized conditions, a calibration plot for SAL was obtained with a linear range between 0.11 ng/mL and 1061 ng/mL (r = 0.9984). The detection limit was 0.06 ng/mL. The immunosensor was examined in real samples for the analysis of SAL.     

New amperometric immunosensor with response enhanced by PAMAM-dendrimers linked via self assembled monolayers for determination of alpha-fetoprotein in human serum

A new amperometric immunosensor for alpha-fetoprotein (AFP), based on nanobiocomposite substrate and with response enhanced by polyamidoaminic (PAMAM) dendrimers was developed and characterized. The nanostructurated substrate obtained by electrochemical deposition of 100 nm-sized gold nanoparticles on glassy carbon electrodes (GCE) was functionalized by deposition of a SAM of 2-aminoethanethiol (AET), used as linker for the subsequent immobilization of polyamidoaminic dendrimers (PAMAM G.1.5). Two different modes were investigated for the reading of the assay: cyclic voltammetry (CV) or Double Step ChronoAmperometry (DSCA). Satisfying results in terms of response range and precision were reached with both methods. Immunosensors were tested and validated for AFP determination in human serum, showing a limit of detection of 3 ng/mL and a limit of quantitation of 15 ng/mL. The enhanced immunosensor has proved an attractive diagnostic tool able to match the needs of clinical monitoring purposes for AFP quantification in human serum at levels useful both for prognosis of pregnancy progression, and for the identification of the occurrence of neoplastic diseases.     

Sensitive electrochemical immunosensor for the detection of cancer biomarker using quantum dot functionalized graphene sheets as labels

Quantum dot (QD) functionalized graphene sheets (GS) were prepared and used as labels for the preparation of sandwich-type electrochemical immunosensors for the detection of a cancer biomarker (i.e., prostate specific antigen (PSA)). The primary anti-PSA antibody was also immobilized onto the GS. The immunosensor displayed a wide range of linear response (0.005-10 ng/mL), low detection limit (3 pg/mL), and good reproducibility, selectivity and stability. The immunosensor was used to detect PSA in patient serum samples with satisfactory results. Thus, this unique immunosensor may provide many applications in clinical diagnosis.     

可再生使用的磁性纳米修饰C反应蛋白电流型免疫传感器

利用Fe3O4(核)/Au(壳)(简称GMPs)标记C反应蛋白酶标抗体(HRP-anti CRP),构建了一类新型的磁性纳米探针(HRP-anti CRP/GMPs),将其修饰在丝网印刷电极(SPCE)表面构建了可再生使用的CRP安培型酶联免疫传感器.首先将多壁碳纳米管(MCNTs) -硫堇(Thi) -Nafion复...     

Development of sulfhydrylated antibody functionalized microcantilever immunosensor for taxol

An immobilization method using a sulfhydrylated monoclonal antibody (mAb) on the gold surface of a microcantilever immunosensor was developed for label-free detection of taxol. The sulfhydrylated anti-taxol mAb was synthesized. Then it was immobilized on the gold surface of the microcantilever. The deflection of the microcantilever corresponding to different taxol concentrations was real time monitored by optical lever technique. The activity of the anti-taxol mAb before and after sulfhydrylation, and the binding of the sulfhydrylated anti-taxol mAb on the gold surface of the microcantilever were evaluated by non-competitive enzyme-linked immunosorbent assay (ELISA). The limit of detection is 1 ng/mL and the method was used for detection of taxol in human plasma sample.     

Determination of 3-nitrotyrosine in human urine samples by surface plasmon resonance immunoassay

This paper describes the detection of a low-molecular weight molecule, 3-nitrotyrosine (3-NT) (∼226 Da), in human urine by coupling indirect inhibition assay with a surface plasmon resonance (SPR) sensor. 3-NT antibody (anti-3-NT Ab, mouse IgG) was used in this assay. An optimal antibody concentration has been measured at 27.9 μg/mL in order to obtain the best performance of the sensor surface. The lowest detection limit for 3-NT with this method is 4.7 ng/mL (S/N = 3). Sensor reliability was demonstrated by good specificity, intra-assay and inter-assay relative standard deviations <8%, average recovery of 107.68 ± 19.4% and sensor surface (self-assembled monolayer) stability through more than 200 regeneration cycles and 15 days of repeated measurement. This is the first SPR biosensor assay of 3-NT in human urine. The high stability of the SPR sensor surface underlies the potential of the SPR method as a low cost diagnostic tool for clinical detection of 3-NT.     

一种基于纳米金／石墨烯／普鲁士蓝（PB）修饰玻碳电极非标记免疫传感器

采用电聚合的方法将普鲁士蓝（PBl聚合在玻碳电极表面,再将石墨烯修饰在PB上面,然后再采用电沉积的方法将HAuCL直接还原成纳米金粒子,沉积在石墨烯表面,最后将羊抗人IgG抗体直接固定于该修饰的玻碳电极表面,制备了用于人IgG抗原检测的非标记电化学免疫传感器。利用循环伏安法和交流阻抗研究了修饰电极表面的电化学特性,用差分脉冲伏安法对人IgG抗原进行了测定。实验表明,此免疫传感器在含不同浓度人IgG的PBS溶液（pH6．98）中测定,响应电流与人IgG浓度在5．55～455．5ng／mL范围内有良好的线性关系,其相关系数r=0．9926,检测限为0．015ng／mL（S／N=3）。该免疫传感器具有制备简单、响应时间快（5min）、稳定性好等特点。     

Ochratoxin A immunoassay with surface plasmon resonance registration: Lowering limit of detection by the use of colloidal gold immunoconjugates

An immunoanalytical system was developed for the determination of ochratoxin A with the use of a surface plasmon resonance (SPR) sensor amplified by the anti-species antibody-colloidal gold particle (CGP) conjugate. The use of the binding of immune complexes to the CGP-anti-species antibody conjugate leads to the SPR signal amplification by a factor of more than 10 and results in the 60 pg/mL limit of detection of ochratoxin A with an assay time of 30 min. These characteristics are superior to those obtained both in the conventional enzyme immunoassay with the use of the same reagents and the SPR assay with unmodified antibodies and specific antibodies conjugated to colloidal gold.     

Multifunctional mesoporous silica nanoparticles as sensitive labels for immunoassay of human chorionic gonadotropin

A novel method to construct amperometric immunosensor for human serum chorionic gonadotrophin (hCG) has been described. In this study, horseradish peroxidase (HRP), Pt nanoparticles and secondary antibody (Ab₂) modified MSN (Pt@MSN/HRP/Ab₂) was synthesized and the multifunctional MSN was used as label for the preparation of immunosensor. With the hCG primary antibody immobilized onto thionine/graphene modified glassy carbon electrode (GCE) via crosslinking with glutaraldehyde, the electrochemical immunosensor was able to realize a reliable determination of hCG in the range of 0.01-12 ng mL⁻¹ with a detection limit of 7.50 pg mL⁻¹. This immunoassay system has many desirable merits including high sensitivity, accuracy, and little instrumentation requirement. Significantly, the new method may be quite promising, with potentially broad applications for clinical immunoassays.     

Direct-binding quartz crystal microbalance immunosensor to detect carp metallothionein

Metallothionein (MT) is a valuable biomarker against xenobiotic heavy metal contamination in organs and blood of fish. In this study, MT was induced in carp blood by cadmium injection and was purified to homogeneity by DEAE-cellulose ion exchange column chromatography to be used dually as a standard and immunogen. A sensitive detection on carp MT was then followed using a batch-type direct-binding quartz crystal microbalance immunosensor system installed with a well cell. Among antibody immobilization methods tested, two methods that were carried out via a heterobifunctional thiolation cross-linker, and via the cross-linker and protein G showed their respective advantages in simplicity and sensitivity. When analyzed using the immunosensor for carp MT in the concentration range of 50-4000 ng/mL, the limit of detection around 250 ng/mL was obtained. Based on its reasonable sensitivity and real-time presentation for the sensor signal, the immunosensor of this study was presumed as a screening tool to monitor possible heavy metal contamination in a fish farm and imported fish.     

Label-free electrochemical immunosensor for sensitive detection of kanamycin

A novel label-free electrochemical immunosensor for sensitive detection of kanamycin based on water-soluble graphene sheet (WGS)/prussian blue-chitosan (PB-CTS)/nanoporous gold (NPG) composited film has been reported. PB was selected as an electron transfer mediator, and was modified onto the electrode together with WGS through electrostatic adsorption. Then NPG was immobilized onto the as-prepared film for biomolecules anchoring. The electroactivity of PB was greatly enhanced in the presence of WGS and NPG. It could mainly be ascribed to the fact that the good conductivity of WGS and NPG promoted electron transfer and enhanced the sensitivity. kanamycin antibody, as a model, was immobilized onto the composite film for the detection of kanamycin. Under optimum conditions, the amperometric signal of PB decreased linearly with kanamycin concentration (0.02-14 ng mL⁻¹), a linear calibration plot (y = 1.3817 + 4.7544x, r = 0.9993), resulting in a low limit of detection (6.31 pg mL⁻¹). The novel immunosensor for the detection of kanamycin in real sample with satisfactory results has been proved. In addition, this method would be easily adapted for the detection of other residual antibiotics in animal derived foods.     

基于纳米金/硫堇修饰金电极的脱落酸安培免疫传感器的研制

提出了一种基于纳米金/硫堇修饰金电极的ABA安培免疫传感器。该传感器基于H2O2-HRP-硫堇催化波体系构建,其中硫堇为传感介质。当HRP存在时,通过加入H2O2,硫堇的还原电流大幅增加,并且电流的增加依赖于HRP活性。HRP活性又由ABA与HRP酶标抗体结合物调控,产生一个减小的催化波。用BSA封闭硫堇单分子层修饰后可能存在的活性位点以避免非特异性吸附。优化了测定条件,包括酶标抗体和硫堇的最佳比例、培育时间、缓冲液的pH值和H2O2浓度。此传感器的还原电流在ABA浓度0.5~1000ng/mL范围内呈线性下降,回归方程为y=0.0209x 17.071,相关系数为0.9922,检测限为0.2ng/mL。     

基于抗体包被纳米金和铜配合物共固定修饰电极的癌胚抗原安培免疫传感器研究

研制了基于抗体包被化学镀纳米金(AuNPs)和[Cu(bpy)2(ONO)]NO3配合物(CuL)共固定修饰玻碳电极(GCE)的安培免疫传感器,并用于血清中癌胚抗原(CEA)的检测。首先将GCE电极表面氧化形成羧基,进而键合上乙二胺。将此胺化电极浸泡在CuL和化学镀金溶液后,CuL可通过π-π堆积作用吸附到GCE表面,并在电极表面还原成30~50nm的纳米金层(GCE|CuL/AuNPs)。将上述电极浸泡在CEA抗体(anti CEA)溶液中,利用AuNPs固定anti CEA,并通过辣根过氧化物酶(HRP)封闭剩余的AuNPs位点,由此构建了一类快速检测CEA的无试剂安培免疫传感器(GCE|CuL-AuNPs/anti CEA-HRP)。其中CuL作为电子媒介体对过氧化脲(CP)有催化还原作用,而且HRP可增强这种作用。当该传感器在37℃下,含CEA的pH6.5PBS溶液中温育30min后,随着温育液中CEA浓度的增加,电极表面形成的免疫复合物也增加,导致CuL对CP的催化电流下降。电流下降百分比1%与CEA浓度在0.1~80ng/mL之间成线性关系,检测限为0.052ng/mL(3σ)。由于采用化学镀法可...     

ZnO modified gold disc: A new route to efficient glucose sensing

Surface of a gold disc was modified by depositing ZnO film electrochemically. AFM analysis of the film shows c-axis oriented pillar like structures grown normal to the surface. Sensor surface was prepared by immobilizing glucose oxidase (GOD) on the ZnO modified gold disc. Different concentrations of glucose (50-1000 ng/ml) were taken to monitor the sensor response. Sensor was found to be highly sensitive to low concentrations of glucose and sensitivity increases linearly in the range of 50-250 ng/ml. The high sensitivity of the ZnO modified gold disc may be attributed to the SPR induced electron transfer between the two systems (i.e. Au and ZnO). The work indicates promising application of the system as a tool for studying sensitive bio-specific interactions, with further development of highly sensitive and selective bio-molecular and chemical SPR based optical sensors.     

A micro amperometric immunosensor for detection of human immunoglobulin

1 Introduction In recent years, electrochemical immunosensors have gained considerable interests as bioanalytical devices[1―3]. They have many attractive features, such as convenience for manipulate, being easy to achieve high sensitivity and excellent d…     

基于囊状银钯铂合金的无标记型癌胚抗原免疫传感器的研制

该文将所合成的中空囊状银钯铂合金和壳聚糖（CHIT）修饰于玻碳电极（GCE）表面,利用中空囊状银钯铂合金比表面积大、反应活性位点多、导电能力良好、催化性能优异和吸附能力强等优点将癌胚抗原的抗体（anti-CEA）固定到电极表面,从而制得高灵敏的无标记型癌胚抗原（CEA）免疫传感器。当抗体与抗原发生免疫反应形成复合物时,会降低银钯铂合金的电催化活性并增加传质阻力。采用示差脉冲伏安法（DPV）检测电化学探针铁氰化钾的响应电流信号的减小程度,实现对CEA的检测。实验考察了电极表面的电化学行为,并对免疫传感器的性能进行了研究。在最优的实验条件下检测癌胚抗原的线性范围为0.5-80.0 ng/mL,线性相关系数为0.989 0,检测下限为0.17 ng/mL。     

Electrochemical DNA aptamer-based biosensor for OTA detection, using superparamagnetic nanoparticles

A novel electrochemical aptasensor, based on disposable screen-printed electrodes was developed for the sensitive detection of the mycotoxin ochratoxin A (OTA). Two strategies were investigated by using an indirect and a direct competitive assay, based on the use of superparamagnetic nanoparticles. The performance of the optimized aptasensors in terms of reproducibility, stability, sensitivity, and analysis time was studied. The best strategy was found to be the direct competitive format. In this assay, free OTA competed with labeled alkaline phosphatase-OTA for the binding to the DNA aptamer immobilized on magnetic beads. The electrochemical detection was thus achieved through a suitable substrate for the enzyme ALP, by Differential Pulse Voltammetry. The aptasensor obtained using this novel approach allowed detection limit of 0.11 ng/mL, and was also validated for real sample analysis.     

Polypyrrole–neutravidin layer for impedimetric biosensor

Polypyrrole synthesis and neutravidin entrapment inside the polymer were performed simultaneously in one step using a controlled anodic current passing. The incorporation of neutravidin within the electrogenerated film was confirmed by FTIR and impedance spectroscopy. Biotinylated anti-triazine Fab fragment was attached to the PPy/neutravidin modified electrode throughout the well-studied biotin–neutravidin interaction. During impedance measurements, the charge transfer resistance increased gradually with atrazine concentration whereas no significant response was recorded after rabbit IgG injection. The immunosensor was very sensitive to atrazine antigen in the range of 0.1–200 ng/ml and the detection limit attained 0.1 ng/ml.     

Amperometric determination of l-dopa by nickel hexacyanoferrate film modified gold nanoparticle graphite composite electrode

Surface modification of graphite electrode with nickel hexacyanoferrate (NiHCF) film through 2-mercaptoethylamine (MEA) capped gold nanoparticle (GNP) was achieved by a novel fabrication approach. The NiHCF modified electrode exhibits a distinct pair of redox peaks corresponding to anodic and cathodic reactions of hexacyanoferrate (II/III) of NiHCF with a formal potential of 0.33 V versus SCE. l-Dopa, is a potential chemotherapeutic drug used for the treatment of a neurodegenerative disease like Parkinson's disease (PD). l-Dopa was successfully determined by NiHCF modified electrode in the concentration range of 0.82 μM to 2.5 mM with a good sensitivity of 0.363 μA/μM. A reduction in overpotential of 200 mV with a detection limit of 0.53 μM was obtained. Determination of l-dopa in commercial tablets was also investigated by the proposed sensor.     

赤潮毒素大田软海绵酸表面等离子共振免疫检测方法研究

结合表面等离子体共振技术与免疫检测技术,研究和建立了一种响应速度快、免标记、低成本新型海洋赤潮毒素大田软海绵酸检测方法.基于SpreetaTM传感器构建了小型表面等离子共振免疫检测系统,采用共价偶联方法在传感器金膜表面修饰大田软海绵酸-牛血清蛋白抗原作为生物敏感膜;测得该方法相对标准偏差为1.51％ (n=12),定量...