A novel electrochemical detection methodology is described for the femtomolar detection of proteins which utilizes both DNA aptamer-functionalized nanoparticles and a surface enzymatic reaction. Immunoglobulin E (IgE) was used as a model protein biomarker, which possesses two distinct epitopes for antibody (anti-IgE) and DNA aptamer binding. A surface sandwich assay format was utilized involving the specific adsorption of IgE onto a gold electrode surface that was pre-modified with a monolayer of aptamer-nanoparticle conjugates followed by the specific interaction of alkaline phosphatase (ALP) conjugated anti-IgE. To clearly demonstrate the signal enhancement associated with nanoparticle use, anodic current measurements of the ALP catalyzed oxidation of the enzyme substrate 4-aminophenylphosphate (APP) were also compared with electrode surfaces upon which the aptamer was directly attached. The detection of an unlabelled protein at concentrations as low as 5 fM is a significant improvement compared to conventional electrochemical-based immunoassay approaches and provides a foundation for the practical use and incorporation of nanoparticle-enhanced detection into electrochemical biosensing technologies.
"The limit of detection was estimated to be 8 fmol L−1, which is at least one order of magnitude lower than those achievable by mass spectrometry and spectroscopy (Table 1). The lower detection limit can be attributed to the multiplex amplification of AuNPs and the high turnover frequency of ALP.19 Moreover, this value is comparable to (or even lower than) those achievable using other electrochemical strategies for detection of glycoproteins including PSA (1.6 pg mL−1),17 sialylated glycoproteins fetuin (0.33 fM) and asialofetuin (0.54 fM),39 lactoferrin (145 pg mL−1),40 α-fetoprotein (4 pg mL−1),41 and ovalbumin (0.83 pg mL−1).42 "
[Show abstract][Hide abstract] ABSTRACT: Ultrasensitive detection of protein biomarkers is essential for early diagnosis and therapy of many diseases. Glycoproteins, differing from other types of proteins, contain carbohydrate moieties in the oligosaccharide chains. Boronic acid can form boronate ester covalent bonds with diol-containing species. Herein, we present a sensitive and cost-effective electrochemical method for glycoprotein detection using 4-mercaptophenylboronic acid (MBA)/biotin-modified gold nanoparticles (AuNPs) (MBA-biotin-AuNPs) as labels. To demonstrate the feasibility and sensitivity of this method, recombinant human erythropoietin (rHuEPO) was tested as a model analyte. Specifically, rHuEPO was captured by the anti-rHuEPO aptamer-covered electrode and then derivatized with MBA-biotin-AuNPs through the boronic acid-carbohydrate interaction. The MBA-biotin-AuNPs facilitated the attachment of streptavidin-conjugated alkaline phosphatase for the production of electroactive p-aminophenol from p-aminophenyl phosphate substrate. A detection limit of 8 fmol L(-1) for rHuEPO detection was achieved. Other glycosylated and non-glycosylated proteins, such as horseradish peroxidase, prostate specific antigen, metallothionein, streptavidin, and thrombin showed no interference in the detection assay.
International Journal of Nanomedicine 05/2014; 9 Suppl 1(1):2619-26. DOI:10.2147/IJN.S62343 · 4.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This work reports the characterization of a modified gold surface as a platform for the development of a label free aptasensor for thrombin detection. The biorecognition platform was obtained by the self-assembly of 4-mercaptobenzoic acid onto a gold surface, covalent attachment of streptavidin and further immobilization of the biotinylated anti-thrombin aptamer. The biosensing platform was characterized by cyclic voltammetry, electrochemical impedance spectroscopy, surface plasmon resonance (SPR) and quartz crystal microbalance with dissipation monitoring. The biorecognition event aptamer-thrombin was detected from changes in the SPR angle produced as a consequence of the molecular interaction between the aptasensor and the target protein. The biosensing platform demonstrated to be highly selective for human thrombin even in the presence of large excess of bovine thrombin, bovine serum albumin, cytochrome C, lysozyme and myoglobin. The relationship between the changes in the SPR angle and thrombin concentration was linear up to 0.19μmolL(-1) (R(2)=0.992) while the detection limit was of 12.0nmolL(-1) (240fmol in the sample). This new sensing approach represents an interesting and promising alternative for the SPR-based quantification of thrombin.
[Show abstract][Hide abstract] ABSTRACT: Progress and development in clinical diagnostics certainly focus upon the advances in the nanomaterials, particularly gold nanoparticles (AuNPs) that offer promise to solve the biocompatible and sensitive detection systems. This paper focuses on the recent application of AuNPs in clinical diagnosis. Various important methods of AuNPs synthesis and their application in clinical detection of various biomolecules using electrochemical detection methods have been described. AuNPs alone and in various composites are also described based on the various biosensors design recently published for the detection of cancer biomarkers, proteins, bacteria, and cancer cells. The effect of AuNPs type and size in clinical detection has also been briefly illustrated.
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