Bhaskara V. Chikkaveeraiah

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.

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.

Ultrasensitive Detection of Cancer Biomarkers in the Clinic by Use of a Nanostructured Microfluidic Array

Multiplexed biomarker protein detection holds unrealized promise for clinical cancer diagnostics due to lack of suitable measurement devices and lack of rigorously validated protein panels. Here we report an ultrasensitive electrochemical microfluidic array optimized to measure a four-protein panel of biomarker proteins, and we validate the protein panel for accurate oral cancer diagnostics. Unprecedented ultralow detection into the 5-50 fg·mL(-1) range was achieved for simultaneous measurement of proteins interleukin 6 (IL-6), IL-8, vascular endothelial growth factor (VEGF), and VEGF-C in diluted serum. The immunoarray achieves high sensitivity in 50 min assays by using off-line protein capture by magnetic beads carrying 400,000 enzyme labels and ~100,000 antibodies. After capture of the proteins and washing to inhibit nonspecific binding, the beads are magnetically separated and injected into the array for selective capture by antibodies on eight nanostructured sensors. Good correlations with enzyme-linked immunosorbent assays (ELISA) for protein determinations in conditioned cancer cell media confirmed the accuracy of this approach. Normalized means of the four protein levels in 78 oral cancer patient serum samples and 49 controls gave clinical sensitivity of 89% and specificity of 98% for oral cancer detection, demonstrating high diagnostic utility. The low-cost, easily fabricated immunoarray provides a rapid serum test for diagnosis and personalized therapy of oral cancer. The device is readily adaptable to clinical diagnostics of other cancers.

Single-Wall Carbon Nanotube Forest Arrays for Immunoelectrochemical Measurement of Four Protein Biomarkers for Prostate Cancer

Protein arrays that measure multiple protein cancer biomarkers in clinical samples hold great promise for reliable early cancer detection. Herein, we report a prototype 4-unit electrochemical immunoarray based on single-wall carbon nanotube forests for the simultaneous detection of multiple protein biomarkers for prostate cancer. Immunoarray procedures were designed to measure prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), platelet factor-4 (PF-4), and interleukin-6 (IL-6) simultaneously in a single serum sample. All of these proteins are elevated in serum of patients with prostate cancer, but they have widely different relative levels of serum concentration. Horseradish peroxidase (HRP) was used as label on detection (secondary) antibodies in a sandwich immunoassay scheme. Biotinylated secondary antibodies (Ab(2)) that bind specifically to streptavidin-HRP conjugates provided 14-16 labels per antibody and gave the necessary higher sensitivity required for PF-4 and IL-6 detection at physiological levels. Conventional singly labeled Ab(2)-HRP conjugates were sufficient for PSA and PSMA detection. Immunoarrays were used to measure four biomarkers in clinical human serum samples of prostate cancer patients and controls with excellent correlation to referee enzyme-linked immunosorbent (ELISA) assays.

On-line protein capture on magnetic beads for ultrasensitive microfluidic immunoassays of cancer biomarkers.

Accurate, sensitive, multiplexed detection of biomarker proteins holds significant promise for personalized cancer diagnostics. Here we describe the incorporation of a novel on-line chamber to capture cancer biomarker proteins on magnetic beads derivatized with 300,000 enzyme labels and 40,000 antibodies into a modular microfluidic immunoarray. Capture and detection chambers are produced from PDMS on machined molds and do not require lithography. Protein analytes are captured from serum or other biological samples in the stirred capture chamber on the beads held in place magnetically. The beads are subsequently washed free of sample components, and wash solutions sent to waste. Removal of the magnet and valve switching sends the magnetic bead-protein bioconjugates into a detection chamber where they are captured on 8 antibody-decorated gold nanoparticle-film sensors and detected amperometrically. Most steps in the immunoassay including protein capture, washing and measurement are incorporated into the device. In simultaneous assays, the microfluidic system gave ultralow detection limits of 5 fg mL(-1) for interleukin-6 (IL-6) and 7 fg mL(-1) for IL-8 in serum. Accuracy was demonstrated by measuring IL-6 and IL-8 in conditioned media from oral cancer cell lines and showing good correlations with standard ELISAs. The on-line capture chamber facilitates rapid, sensitive, repetitive protein separation and measurement in 30 min in a semi-automated system adaptable to multiplexed protein detection.

Magnetic particles in ultrasensitive biomarker protein measurements for cancer detection and monitoring

IMPORTANCE OF THE FIELD: Devices for the reliable detection of panels of biomarker proteins facilitated by magnetic bead-based technologies have the potential to greatly improve future cancer diagnostics. The reason for this review is to highlight promising research on emerging procedures for protein capture, transport and detection featuring magnetic particles. AREAS COVERED IN THIS REVIEW: The review covers applications of magnetic particles in protein immunoassays in emerging research and commercial methods, and stresses multiplexed protein assays for reliable future cancer diagnostics. Research literature over the past dozen years has been surveyed and specific examples are presented in detail. EXPERT OPINION: Magnetic particles are important components of emerging protein detection systems. They need to be integrated into simple inexpensive systems for accurate, sensitive detection of fully validated panels of biomarker proteins to be widely useful in clinical cancer diagnostics.

Targeted Therapeutic Nanotubes Influence the Viscoelasticity of Cancer Cells to Overcome Drug Resistance

Resistance to chemotherapy is the primary cause of treatment failure in over 90% of cancer patients in the clinic. Research in nanotechnology-based therapeutic alternatives has helped provide innovative and promising strategies to overcome multidrug resistance (MDR). By targeting CD44-overexpressing MDR cancer cells, we have developed in a single-step a self-assembled, self-targetable, therapeutic semiconducting single-walled carbon nanotube (sSWCNT) drug delivery system that can deliver chemotherapeutic agents to both drug-sensitive OVCAR8 and resistant OVCAR8/ADR cancer cells. The novel nanoformula with a cholanic acid-derivatized hyaluronic acid (CAHA) biopolymer wrapped around a sSWCNT and loaded with doxorubicin (DOX), CAHA-sSWCNT-DOX, is much more effective in killing drug-resistant cancer cells compared to the free DOX and phospholipid PEG (PL-PEG)-modified sSWCNT formula, PEG-sSWCNT-DOX. The CAHA-sSWCNT-DOX affects the viscoelastic property more than free DOX and PL-PEG-sSWCNT-DOX, which in turn allows more drug molecules to be internalized. Intravenous injection of CAHA-sSWCNT-DOX (12 mg/kg DOX equivalent) followed by 808 nm laser irradiation (1 W/cm2, 90 s) led to complete tumor eradication in a subcutaneous OVCAR8/ADR drug-resistant xenograft model, while free DOX alone failed to delay tumor growth. Our newly developed CAHA-sSWCNT-DOX nanoformula, which delivers therapeutics and acts as a sensitizer to influence drug uptake and induce apoptosis with minimal resistance factor, provides a novel effective means of counteracting the phenomenon of multidrug resistance.

Ultrasensitive nanostructured immunosensor for stem and carcinoma cell pluripotency gatekeeper protein NANOG

AIMS To develop an immunosensor for ultrasensitive detection of the NANOG protein. NANOG regulates pluripotency in stem cells and some cancer cells. This article reports the first electrochemical immunosensor for ultrasensitive detection and absolute quantification of the NANOG protein. The sensor features dense capture antibody-coated gold nanoparticle layers on a pyrolytic graphite underlayer. MATERIALS & METHODS Two separate multilabel detection strategies were used to achieve moderate and ultra-high sensitivity. RESULTS Good sensitivity was achieved for NANOG over the concentration range 0.1-160 pg/ml. The moderate-sensitivity approach gave a detection limit of 25 pg/ml, while the ultrasensitive method achieved a 250-fold lower detection limit of 0.1 pg/ml. Amounts of NANOG detected in human embryonic stem cell lysates correlated well with qualitative western blots and mRNA expression. CONCLUSION The electrochemical gold nanoparticle immunosensor is suitable for measuring NANOG protein expression in stem and carcinoma cell tissue lysates at very low levels.

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.