Highly sensitive electrochemical detection of proteins using aptamer-coated gold nanoparticles and surface enzyme reactions.
ABSTRACT 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.
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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 01/2014; 9 Suppl 1:2619-26. · 4.20 Impact Factor
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ABSTRACT: In this paper, we propose a sensitive electrochemical immunosensor synthesized using a surface-initiated atom transfer radical polymerization process for the detection of prostate-specific antigen (PSA). Electrochemical immunosensors based on polymer brush [oligo(ethylene glycol)methacrylate-co-glycidyl methacrylate] (OEGMA-co-GMA) were grown on plane Au and nanostructured (NS) Au electrodes, characterized and compared for their sensitivity to detect PSA. Due to a large capacity for antibody loading and high resistance to nonspecific antibody adsorption of POEGMA-co-GMA brush, the Au-NS immunosensor exhibited detection in a wide dynamic range of five orders of magnitude with an improved lower limit of detection of 2pgml(-1), which was better than the synthesized immunosensor with the polymer brush grown on plane Au electrode. The Au-NS electrode showed improved detection sensitivity of 4.9μAng(-1)ml for PSA detection, which was almost 2 times better than the plane Au electrode. Finally, the use of silica nanoparticles (Si-NPs) conjugated with polyclonal antibody enhanced the response of the immunosensor. The proposed electrochemical immunosensor would be an exciting addition in medical diagnostics for the early detection of cancer biomarkers, e.g., PSA due to improved limit of detection (LOD); eventually helpful in circumventing cancer metastasis.Bioelectrochemistry. 01/2015; 101:75-83.
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ABSTRACT: The development of simple and low-cost approaches to the detection of immunoglobulin E (IgE) would provide a method for the early diagnosis and prevention of atopic diseases. The current methods of detection are generally tedious, multi-step processes and are limited by the high cost of the labeled proteins. We describe here a label-free structure-switching colorimetric method for the simple measurement of IgE using DNA pseudoknot probes and gold nanoparticles. In the absence of a target the IgE aptamer probe adopts a pseudoknot conformation that dissociates a capture probe from the unmodified gold nanoparticles. However, when IgE binds to the aptamer probe, the pseudoknot is resolved, leading to a favorable hybridization between the 3' terminal loop of the aptamer probe and the capture probe; this induces the aggregation of the gold nanoparticles. As a result, the colorimetric IgE sensor using this structure-switching mechanism is sensitive, specific and convenient, and the assay works even when challenged with complicated biological matrixes such as vaginal fluids. The proposed method is expected to be of great clinical value for IgE detection and could be used, after appropriate design, for sensing applications of other structured aptamers.The Analyst 05/2014; · 3.91 Impact Factor