Naimish P. Sardesai

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.

Electrochemiluminescent immunosensor for detection of protein cancer biomarkers using carbon nanotube forests and [Ru-(bpy)(3)](2+)-doped silica nanoparticles.

We report the first electrochemiluminescent immunosensor combining single-wall carbon nanotube forests with RuBPY-silica-secondary antibody nanoparticles for sensitive detection of cancer biomarker prostate specific antigen.

Automated Multiplexed ECL Immunoarrays for Cancer Biomarker Proteins

Point-of-care diagnostics based on multiplexed protein measurements face challenges of simple, automated, low-cost, and high-throughput operation with high sensitivity. Herein, we describe an automated, microprocessor-controlled microfluidic immunoarray for simultaneous multiplexed detection of small protein panels in complex samples. A microfluidic sample/reagent delivery cassette was coupled to a 30-microwell detection array to achieve sensitive detection of four prostate cancer biomarker proteins in serum. The proteins are prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), platelet factor-4 (PF-4), and interlukin-6 (IL-6). The six channel system is driven by integrated micropumps controlled by an inexpensive programmable microprocessor. The reagent delivery cassette and detection array feature channels made by precision-cut 0.8 mm silicone gaskets. Single-wall carbon nanotube forests were grown in printed microwells on a pyrolytic graphite detection chip and decorated with capture antibodies. The detection chip is housed in a machined microfluidic chamber with a steel metal shim counter electrode and Ag/AgCl reference electrode for electrochemiluminescent (ECL) measurements. The preloaded sample/reagent cassette automatically delivers samples, wash buffers, and ECL RuBPY-silica-antibody detection nanoparticles sequentially. An onboard microcontroller controls micropumps and reagent flow to the detection chamber according to a preset program. Detection employs tripropylamine, a sacrificial reductant, while applying 0.95 V vs Ag/AgCl. Resulting ECL light was measured by a CCD camera. Ultralow detection limits of 10-100 fg mL(-1) were achieved in simultaneous detection of the four protein in 36 min assays. Results for the four proteins in prostate cancer patient serum gave excellent correlation with those from single-protein ELISA.

Electroanalytical evaluation of antioxidant activity of cerium oxide nanoparticles by nanoparticle collisions at microelectrodes.

We describe a simple, cost-effective and rapid electrochemical screening approach to evaluate antioxidant activity of cerium oxide nanoparticles (CeO2 NPs) by single nanoparticle collision at microelectrodes. The method is based on direct monitoring of the interaction between a Pt microelectrode and surface bound superoxo and peroxo anions of CeO2 NPs (Ce-O2(-)/O2(2-)) formed upon exposure to H2O2, selected here as a model reactive oxygen species. We observe an increase in spike current frequency for CeO2 NPs exposed to H2O2, which we attribute to the reduction of surface bound oxygen species when the particles collide with the microelectrode. The results were confirmed with spectroscopic techniques that demonstrate changes in surface reactivity and composition. The spike frequency was found to correlate well with the superoxide dismutase activity of these particles. This approach could enable routine screening of antioxidant NPs using a rapid and inexpensive assay.

Resistive-pulse measurements with nanopipettes: detection of Au nanoparticles and nanoparticle-bound anti-peanut IgY

Solid-state nanopores have been widely employed in sensing applications from Coulter counters to DNA sequencing devices. The analytical signal in such experiments is the change in ionic current flowing through the orifice caused by the large molecule or nanoparticle translocation through the pore. Conceptually similar nanopipette-based sensors can offer several advantages including the ease of fabrication and small physical size essential for local measurements and experiments in small spaces. This paper describes the first evaluation of nanopipettes with well characterized geometry for resistive-pulse sensing of Au nanoparticles (AuNP), nanoparticles coated with an allergen epitope peptide layer, and AuNP-peptide particles with bound antipeanut antibodies (IgY) on the peptide layer. The label-free signal produced by IgY-conjugated particles was strikingly different from those obtained with other analytes, thus suggesting the possibility of selective and sensitive resistive-pulse sensing of antibodies.

Applications and implications of nanoceria reactivity: measurement tools and environmental impact

Cerium oxide nanoparticles or nanoceria have a unique structure and interesting and unusual redox and catalytic properties that vary with the size, shape, charge, surface coating and chemical reactivity. This paper highlights applications and environmental implications of nanoceria, and describes methodologies for the assessment of the reactivity and potential toxicological effects of these particles. The physical and chemical properties in the particle design that are responsible for their reactivity and transformation in environmental and biological conditions are described. Processes such as surface oxidation, formation of surface complexes and potential interaction with redox active components of the environment are discussed. An overview of analytical characterization methods for study of nanoceria properties, reactivity and impact, highlighting methodological challenges and limitations is presented. Examples discussed include strategies to determine physicochemical properties, cytotoxicity and antioxidant or pro-oxidant activity in various exposure environments. Development of new measurement tools to facilitate rapid assessment and accelerate screening of these particles for their reactivity and effects is discussed. Future research needs for environmental assessment of benefits and potential risks associated with the use of nanoceria are also provided.

Platinum-Doped Ceria Based Biosensor for in Vitro and in Vivo Monitoring of Lactate during Hypoxia

Measurements of lactate concentrations in blood and tissues are an important indication of the adequacy of tissue oxygenation and could be useful for monitoring the state and progress of a variety of diseases. This paper describes the fabrication, analytical characterization, and physiological application of an amperometric microbiosensor based on lactate oxidase and oxygen-rich platinum doped ceria (Pt-ceria) nanoparticles for monitoring lactate levels during hypoxic conditions. The Pt-ceria nanoparticles provided electrocatalytic amplification for the detection of the enzymatically produced hydrogen peroxide and acted as an internal oxygen source for the enzyme, enabling lactate monitoring in an oxygen depleted tissue. In vitro evaluation of the biosensor demonstrated high selectivity against physiological levels of ascorbic acid, a storage stability of 3 weeks, a fast response time of 6 s, and good, linear sensitivity over a wide concentration range. In vivo experiments performed by placing the biosensor in the hippocampus of anesthetized rats demonstrated the feasibility of continuous lactate monitoring over 2 h ischemia and reperfusion. The results demonstrate that Pt-ceria is a versatile material for use in implantable enzyme bioelectrodes, which may be used to assess the pathophysiology of tissue hypoxia. In addition to measurements in hypoxic conditions, the detection limit of this biosensor was low, 100 pM, and the materials used to fabricate this biosensor can be particularly useful in ultrasensitive devices for monitoring lactate levels in a variety of conditions.

Assessing DNA damage from enzyme-oxidized single-walled carbon nanotubes

Peroxidase enzyme digests of oxidized single-wall carbon nanotubes (SWCNT) were shown to damage DNA in potentially genotoxic reactions for the first time using an electro-optical array with and without metabolic activation.

Nanoscience-Based Electrochemical Sensors and Arrays for Detection of Cancer Biomarker Proteins

Measurement of panels of biomarker proteins in serum, tissue or saliva holds great promise for future cancer diagnostics. Broad implementation of this approach in the clinic requires new, low cost devices for multiplexed protein detection. Advanced nanomaterials coupled with electrochemical detection have provided new opportunities for development of such devices. This chapter reviews recent research in using nanoparticle labels and multiplexed detection in protein immunosensors. It focuses in part on research in our own laboratories on ultrasensitive protein immunosensors combining nanostructured electrodes with detection particles with up to 500,000 labels that detect as little as 1 fg/mL protein in diluted serum. Our most mature multiple protein detection arrays are multiplexed microfluidic devices with 8-nanostructured sensors utilizing massively labeled magnetic particles or polymers. This approach provides reliable detection for multiple proteins at levels well below 1 pg/mL, and shows excellent correlation with referee methods. The importance of validating panels of biomarkers for reliable cancer diagnostics is also stressed.