James F. Rusling

Targeted Killing of Cancer Cells in Vivo and in Vitro with EGF-Directed Carbon Nanotube-Based Drug Delivery

Carbon nanotube-based drug delivery holds great promise for cancer therapy. Herein we report the first targeted, in vivo killing of cancer cells using a drug-single wall carbon nanotube (SWNT) bioconjugate, and demonstrate efficacy superior to nontargeted bioconjugates. First line anticancer agent cisplatin and epidermal growth factor (EGF) were attached to SWNTs to specifically target squamous cancer, and the nontargeted control was SWNT-cisplatin without EGF. Initial in vitro imaging studies with head and neck squamous carcinoma cells (HNSCC) overexpressing EGF receptors (EGFR) using Qdot luminescence and confocal microscopy showed that SWNT-Qdot-EGF bioconjugates internalized rapidly into the cancer cells. Limited uptake occurred for control cells without EGF, and uptake was blocked by siRNA knockdown of EGFR in cancer cells, revealing the importance of EGF-EGFR binding. Three color, two-photon intravital video imaging in vivo showed that SWNT-Qdot-EGF injected into live mice was selectively taken up by HNSCC tumors, but SWNT-Qdot controls with no EGF were cleared from the tumor region in <20 min. HNSCC cells treated with SWNT-cisplatin-EGF were also killed selectively, while control systems that did not feature EGF-EGFR binding did not influence cell proliferation. Most significantly, regression of tumor growth was rapid in mice treated with targeted SWNT-cisplatin-EGF relative to nontargeted SWNT-cisplatin.

Ultrasensitive immunosensor for cancer biomarker proteins using gold nanoparticle film electrodes and multienzyme-particle amplification.

A densely packed gold nanoparticle platform combined with a multiple-enzyme labeled detection antibody-magnetic bead bioconjugate was used as the basis for an ultrasensitive electrochemical immunosensor to detect cancer biomarkers in serum. Sensitivity was greatly amplified by synthesizing magnetic bioconjugates particles containing 7500 horseradish peroxidase (HRP) labels along with detection antibodies (Ab2) attached to activated carboxyl groups on 1 microm diameter magnetic beads. These sensors had sensitivity of 31.5 microA mL ng(-1) and detection limit (DL) of 0.5 pg mL(-1) for prostate specific antigen (PSA) in 10 microL of undiluted serum. This represents an ultralow mass DL of 5 fg PSA, 8-fold better than a previously reported carbon nanotube (CNT) forest immunosensor featuring multiple labels on carbon nanotubes, and near or below the normal serum levels of most cancer biomarkers. Measurements of PSA in cell lysates and human serum of cancer patients gave excellent correlations with standard ELISA assays. These easily fabricated AuNP immunosensors show excellent promise for future fabrication of bioelectronic arrays.

Peroxidase activity of enzymes bound to the ends of single-wall carbon nanotube forest electrodes

This communication reports the first example, to our knowledge, of enzymes covalently attached onto the ends of vertically oriented single-wall carbon nanotube (SWNT) forest arrays used as electrodes. Quasi-reversible FeIII/FeII voltammetry was observed for the iron heme enzymes myoglobin and horseradish peroxidase coupled to carboxylated ends of the nanotube forests by amide linkages. Results suggest that the “trees” in the nanotube forest behaved electrically similar to a metal, conducting electrons from the external circuit to the redox sites of the enzymes. Electrochemically manifested peroxidase activity of myoglobin and horseradish peroxidase attached to the SWNT forests was demonstrated, with detection limits for hydrogen peroxide in buffer solutions of ∼100 nM. These prototype SWNT-forest biosensors are easy to prepare, and enzyme layers were stable for weeks.

Ultrasensitive Electrochemical Immunosensor for Oral Cancer Biomarker IL-6 using Carbon Nanotube Forest Electrodes and Multilabel Amplification

Squamous cell carcinomas of head and neck (HNSCC) are associated with immune, inflammatory, and angiogenic responses involving interleukin-6 (IL-6). This article reports an ultrasensitive electrochemical immunosensor for human IL-6 and proof-of-concept studies of IL-6 detection in HNSCC cells. Single wall carbon nanotube (SWNT) forests with attached capture antibodies (Ab(1)) for IL-6 were used in an electrochemical sandwich immunoassay protocol using enzyme label horseradish peroxidase (HRP) to measure very low (<or=30 pg mL(-1)) and elevated levels of IL-6. Two levels of multienzyme labeling were used to measure a broad concentration range of IL-6 in a representative panel of HNSCC cells. Secondary antibodies (Ab(2)) attached to carboxylated multiwall carbon nanotubes with 106 HRP labels per 100 nm gave the highest sensitivity of 19.3 nA mL (pg IL-6)(-1) cm(-2) and the best detection limit (DL) of 0.5 pg mL(-1) (25 fM) for IL-6 in 10 microL of calf serum. For more concentrated samples, biotinylated Ab(2) bound to streptavidin-HRP to provide 14-16 labels per antigen was used. These immunosensors accurately measured secreted IL-6 in a wide range of HNSCC cells demonstrated by excellent correlations with standard enzyme-linked immunosorbent assays (ELISA), suggesting that SWNT immunosensors combined with multilabel detection have excellent promise for detecting IL-6 in research and clinical applications.

Carbon Nanotube Microwell Array for Sensitive Electrochemiluminescent Detection of Cancer Biomarker Proteins

This paper describes fabrication of a novel electrochemiluminescence (ECL) immunosensor array featuring capture-antibody-decorated single-wall carbon nanotube (SWCNT) forests residing in the bottoms of 10-μL wells with hydrophobic polymer walls. Silica nanoparticles containing [Ru(bpy)(3)](2+) and secondary antibodies (RuBPY-silica-Ab(2)) are employed in this system for highly sensitive two-analyte detection. Antibodies to prostate specific antigen (PSA) and interleukin-6 (IL-6) were attached to the same RuBPY-silica-Ab(2) particle. The array was fabricated by forming the wells on a conductive pyrolytic graphite chip (1 in. × 1 in.) with a single connection to a potentiostat to achieve ECL. The sandwich immunoassay protocol employs antibodies attached to SWCNTs in the wells to capture analyte proteins. Then RuBPY-silica-Ab(2) is added to bind to the captured proteins. ECL is initiated in the microwells by electrochemical oxidation of tripropyl amine (TprA), which generates excited state [Ru(bpy)(3)](2+) in the 100-nm particles, and is measured with a charge-coupled device (CCD) camera. Separation of the analytical spots by the hydrophobic wall barriers enabled simultaneous immunoassays for two proteins in a single sample without cross-contamination. The detection limit (DL) for PSA was 1 pg mL(-1) and for IL-6 was 0.25 pg mL(-1) (IL-6) in serum. Array determinations of PSA and IL-6 in patient serum were well-correlated with single-protein ELISAs. These microwell SWCNT immunoarrays provide a simple, sensitive approach to the detection of two or more proteins.

Microfluidic electrochemical immunoarray for ultrasensitive detection of two cancer biomarker proteins in serum

A microfluidic electrochemical immunoassay system for multiplexed detection of protein cancer biomarkers was fabricated using a molded polydimethylsiloxane channel and routine machined parts interfaced with a pump and sample injector. Using off-line capture of analytes by heavily-enzyme-labeled 1 μm superparamagnetic particle (MP)-antibody bioconjugates and capture antibodies attached to an 8-electrode measuring chip, simultaneous detection of cancer biomarker proteins prostate specific antigen (PSA) and interleukin-6 (IL-6) in serum was achieved at sub-pg mL⁻¹ levels. MPs were conjugated with ∼90,000 antibodies and ∼200,000 horseradish peroxidase (HRP) labels to provide efficient off-line capture and high sensitivity. Measuring electrodes feature a layer of 5 nm glutathione-decorated gold nanoparticles to attach antibodies that capture MP-analyte bioconjugates. Detection limits of 0.23 pg mL⁻¹ for PSA and 0.30 pg mL⁻¹ for IL-6 were obtained in diluted serum mixtures. PSA and IL-6 biomarkers were measured in serum of prostate cancer patients in total assay time 1.15 h and sensor array results gave excellent correlation with standard enzyme-linked immunosorbent assays (ELISA). These microfluidic immunosensors employing nanostructured surfaces and off-line analyte capture with heavily labeled paramagnetic particles hold great promise for accurate, sensitive multiplexed detection of diagnostic cancer biomarkers.

Attomolar Detection of a Cancer Biomarker Protein in Serum by Surface Plasmon Resonance Using Superparamagnetic Particle Labels

Methods to measure protein biomarkers with ultralow detection limit (DL) and high sensitivity promise to provide valuable tools for early diagnosis of diseases such as cancer, and for monitoring therapy and post-surgical recurrence.[i, ii] Surface plasmon resonance (SPR) utilizing nanoparticle-antibody labels for signal amplification in immunoassays is an emerging approach for detecting proteins in biomedical samples.[iii–x] Herein, we show for the first time that clustering of superparamagnetic labels on SPR surfaces leads to unprecedented sensitivity and ultralow DL for protein biomarkers in serum. Specifically, antibody bioconjugates on 1 µm diameter superparamagnetic particles (MP) used for off-line antigen capture enabled SPR detection of cancer biomarker prostate specific antigen (PSA) in serum at an ultralow DL of 10 fg mL−1 (~300 aM). This approach opens doors for accurate diagnostics based on new protein biomarkers with inherently low concentrations.

An amperometric biosensor with human CYP3A4 as a novel drug screening tool.

We developed a biosensor based on the redox properties of human CYP3A4 to directly monitor electron transfer to the heme protein. Enzyme films were assembled on gold electrodes by alternate adsorption of a CYP3A4 layer on top of a polycation layer. Direct, reversible electron transfer between the electrode and CYP3A4 was observed with voltammetry under anaerobic conditions. In the presence of oxygen, the oxidation peak of the hemoprotein disappeared, and the reduction peak increased 2- to 3-fold. Addition of CYP3A4 substrates (verapamil, midazolam, quinidine, and progesterone) to the oxygenated solution caused a concentration-dependent increase in the reduction current in cyclic voltammetric and amperometric experiments. Product analyses after electrolysis with the enzyme film showed catalytic activity of the biosensor depending on substrate concentration, its inhibition by ketoconazole, and a minor contribution of H(2)O(2) to the catalytic cycle. These results suggest that electron exchange between the electrode and the immobilized CYP3A4 occurred, and that metabolic activity of the enzyme was maintained. Thus, important requirements for the application of human CYP biosensors in order to identify drugs or drug candidates as substrates or inhibitors to the attached enzyme are fulfilled.

Direct electron injection from electrodes to cytochromeP450cam in biomembrane-like films

Direct, reversible, electron transfer has been demonstrated between electrodes and native cytochrome P450 cam (cyt P450 cam ) in lipid films. Electron injection directly from electrodes into the haem Fe III of cyt P450 cam has been confirmed by the influence of the known reaction of cyt P450 cam Fe II with CO on the voltammetric midpoint potential. Analysis of square wave voltammograms suggested a distribution of enzyme formal potentials in the films. With oxygen present, more than one electron was injected into the enzyme in films, mimicking in vivo electron acceptance by cyt P450Fe III and cyt P450Fe II –O 2 during catalytic oxidations. Cyt P450 cam films also catalysed electrochemically driven reduction of trichloroacetic acid in anaerobic solutions. These stable enzyme–lipid films may find applications in fundamental biochemical and toxicity studies, biocatalysis and biosensors.

Nanostructured Immunosensor for Attomolar Detection of Cancer Biomarker Interleukin-8 Using Massively Labeled Superparamagnetic Particles

Extremely sensitive and accurate clinical measurements of biomarker proteins for early detection and monitoring of cancer pose a formidable challenge. However, successful inexpensive devices for reliable on-the-spot cancer diagnosis promise to lead to improved therapeutic outcomes with lower cost, decreased patient stress, and new targeted therapies.[1–3] Such devices will also provide tools for a better fundamental understanding of disease progression, and enable biomarker-based monitoring of therapy.[4] Herein, we report an ultra-sensitive immunosensor based on a glutathione-protected gold nanoparticle (GSH-AuNP) sensor surface. When combined with novel massively labeled paramagnetic particles for the electrochemical detection of cancer biomarker interleukin 8 (IL-8), we obtained an unprecedented detection limit (DL) of 1 fgmL−1 (100 aM) for IL-8, the lowest protein level yet detected in serum. Accuracy was demonstrated by determining IL-8 in conditioned media from head and neck squamous cell carcinoma (HNSCC) cells.