Mediated amperometric immunosensing using single walled carbon nanotube forests

A prototype amperometric immunosensor was evaluated based on the adsorption of antibodies onto perpendicularly oriented assemblies of single wall carbon nanotubes called SWNT forests. The forests were self-assembled from oxidatively shortened SWNTs onto Nafion/iron oxide coated pyrolytic graphite electrodes. The nanotube forests were characterized using atomic force microscopy and resonance Raman spectroscopy. Anti-biotin antibody strongly adsorbed to the SWNT forests. In the presence of a soluble mediator, the detection limit for horseradish peroxidase (HRP) labeled biotin was 2.5 pmol ml[-1] (2.5 nM). Unlabelled biotin was detected in a competitive approach with a detection limit of 16 nmol ml[-1] (16 μM) and a relative standard deviation of 12%. The immunosensor showed low non-specific adsorption of biotin-HRP (approx. 0.1%) when blocked with bovine serum albumin. This immunosensing approach using high surface area, patternable, conductive SWNT assemblies may eventually prove useful for nano-biosensing arrays

DSG3 As a Biomarker for the Ultrasensitive Detection of Cccult Lymph Node Metastasis in Oral Cancer Using Nanostructured Immunoarrays

OBJECTIVES: The diagnosis of cervical lymph node metastasis in head and neck squamous cell carcinoma (HNSCC) patients constitutes an essential requirement for clinical staging and treatment selection. However, clinical assessment by physical examination and different imaging modalities, as well as by histological examination of routine lymph node cryosections can miss micrometastases, while false positives may lead to unnecessary elective lymph node neck resections. Here, we explored the feasibility of developing a sensitive assay system for desmoglein 3 (DSG3) as a predictive biomarker for lymph node metastasis in HNSCC. MATERIALS AND METHODS: DSG3 expression was determined in multiple general cancer- and HNSCC-tissue microarrays (TMAs), in negative and positive HNSCC metastatic cervical lymph nodes, and in a variety of HNSCC and control cell lines. A nanostructured immunoarray system was developed for the ultrasensitive detection of DSG3 in lymph node tissue lysates. RESULTS: We demonstrate that DSG3 is highly expressed in all HNSCC lesions and their metastatic cervical lymph nodes, but absent in non-invaded lymph nodes. We show that DSG3 can be rapidly detected with high sensitivity using a simple microfluidic immunoarray platform, even in human tissue sections including very few HNSCC invading cells, hence distinguishing between positive and negative lymph nodes. CONCLUSION: We provide a proof of principle supporting that ultrasensitive nanostructured assay systems for DSG3 can be exploited to detect micrometastatic HNSCC lesions in lymph nodes, which can improve the diagnosis and guide in the selection of appropriate therapeutic intervention modalities for HNSCC patients

Oxidised guanidinohydantoin (Ghox) and spiroiminodihydantoin (Sp) are major products of iron- and copper-mediated 8-oxo-7,8-dihydroguanine and 8-oxo-7,8-dihydro-2-deoxyguanosine oxidation

8-Oxo-7,8-dihydroguanine (8-oxoGua), an important biomarker of DNA damage in oxidatively generated stress, is highly reactive towards further oxidation. Much work has been carried out to investigate the oxidation products of 8-oxoGua by one-electron oxidants, singlet oxygen, and peroxynitrite. This report details for the first time, the iron- and copper-mediated Fenton oxidation of 8-oxoGua and 8-oxo-7,8-dihydro-29-deoxyguanosine (8-oxodGuo). Oxidised guanidinohydantoin (Ghox) was detected as the major product of oxidation of 8-oxoGua with iron or copper and hydrogen peroxide, both at pH 7 and pH 11. Oxaluric acid was identified as a final product of 8-oxoGua oxidation. 8-oxodGuo was subjected to oxidation under the same conditions as 8-oxoGua. However, dGhox was not generated. Instead, spiroiminodihydantoin (Sp) was detected as the major product for both iron and copper mediated oxidation at pH 7. It was proposed that the oxidation of 8-oxoGua was initiated by its one-electron oxidation by the metal species, which leads to the reactive intermediate 8-oxoGua?+, which readily undergoes further oxidation. The product of 8-oxoGua and 8-oxodGuo oxidation was determined by the 29-deoxyribose moiety of the 8-oxodGuo, not whether copper or iron was the metal involved in the oxidation

Multiplexed electrochemical protein detection and translation to personalized cancer diagnostics

Measuring diagnostic panels of multiple proteins promises a new, personalized approach to early detection and therapy of diseases like cancer. Levels of biomarker proteins in patient serum can provide a continually updated record of disease status. Research in electrochemical detection of proteins has produced exquisitely sensitive, approaches. Most utilize ELISA-like sandwich immunoassays incorporating various aspects of nanotechnology. Several of these ultrasensitive methodologies have been extended to microfluidic multiplexed protein detection, but engineered, solutions are needed to measure more proteins in a single device from a small patient sample such as a drop of blood or tissue lysate. To achieve. clinical or point-of-care (POC) use simplicity and low cost are essential. In multiplexed microfluidic immunoassays, required reagent additions and washing steps pose a significant problem calling for creative engineering. A grand challenge is to develop a general cancer screening device to accurately measure 50-100 proteins in a simple, cost-effective fashion. This will require creative solutions to simplified reagent addition and multiplexing

High Sensitivity Carbon Nanotube Based Electrochemiluminescence Sensor Array

Ink jet printed carbon nanotube forest arrays capable of detecting picomolar concentrations of immunoglobulin G (IgG) using electrochemiluminescence (ECL) are described. Patterned arrays of vertically aligned single walled carbon nanotube (SWCNT) forests were printed on indium tin oxide (ITO) electrodes. Capture anti-IgG antibodies were then coupled through peptide bond formation to acidic functional groups on the vertical nanotubes. IgG immunoassays were performed using silica nano particles (Si NP) functionalized with the ECL luminophore [Ru(bpy)2 PICH2]2+], and IgG labelled G1.5 acid terminated PAMAM dendrimers. PAMAM is poly(amido amine), bpy is 2,2′-bipyridyl and PICH2 is (2-(4-carboxyphenyl)imidazo[4,5-f][1,10]phenanthroline). The carboxyl terminal of [Ru(bpy)2 PICH2]2+ (fluorescence lifetime ≈682 ± 5 ns) dye was covalently coupled to amine groups on the 800 nm diameter silica spheres in order to produce significant ECL enhancement in the presence of sodium oxalate as co-reactant in PBS at pH 7.2). Significantly, this SWCNT-based sensor array shows a wide linear dynamic range for IgG coated spheres (106 to 1012 spheres) corresponding to IgG concentrations between 20 pM and 300 nM. A detection limit of 1.1 ± 0.1 pM IgG is obtained under optimal conditions

Multiplexed electrochemical protein detection and translation to personalized cancer diagnostics

Measuring diagnostic panels of multiple proteins promises a new, personalized approach to early detection and therapy of diseases like cancer. Levels of biomarker proteins in patient serum can provide a continually updated record of disease status. Research in electrochemical detection of proteins has produced exquisitely sensitive, approaches. Most utilize ELISA-like sandwich immunoassays incorporating various aspects of nanotechnology. Several of these ultrasensitive methodologies have been extended to microfluidic multiplexed protein detection, but engineered, solutions are needed to measure more proteins in a single device from a small patient sample such as a drop of blood or tissue lysate. To achieve. clinical or point-of-care (POC) use simplicity and low cost are essential. In multiplexed microfluidic immunoassays, required reagent additions and washing steps pose a significant problem calling for creative engineering. A grand challenge is to develop a general cancer screening device to accurately measure 50-100 proteins in a simple, cost-effective fashion. This will require creative solutions to simplified reagent addition and multiplexing