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.

A microfluidic electrochemical device for high sensitivity biosensing: detection of nanomolar hydrogen peroxide.

We report herein a simple device for rapid biosensing consisting of a single microfluidic channel made from poly(dimethylsiloxane) (PDMS) coupled to an injector, and incorporating a biocatalytic sensing electrode, reference and counter electrodes. The sensing electrode was a gold wire coated with 5 nm glutathione-decorated gold nanoparticles (AuNPs). Sensitive detection of H(2)O(2) based on direct bioelectrocatalysis by horseradish peroxidase (HRP) was used for evaluation. HRP was covalently linked the glutathione-AuNPs. This electrode presented quasi-reversible cyclic voltammetry peaks at -0.01 V vs Ag/AgCl at pH 6.5 for the HRP heme Fe(III)/Fe(II) couple. Direct electrochemical activity of HRP was used to detect H(2)O(2) at high sensitivity with a detection limit of 5 nM in an unmediated system.

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.

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/cm(2), 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.

Electrochemical immunosensors for detection of cancer protein biomarkers.

Bioanalytical methods have experienced unprecedented growth in recent years, driven in large part by the need for faster, more sensitive, more portable ("point of care") systems to detect protein biomarkers for clinical diagnosis. Electrochemical detection strategies, used in conjunction with immunosensors, offer advantages because they are fast, simple, and low cost. Recent developments in electrochemical immunosensors have significantly improved the sensitivity needed to detect low concentrations of biomarkers present in early stages of cancer. Moreover, the coupling of electrochemical devices with nanomaterials, such as gold nanoparticles, carbon nanotubes, magnetic particles, and quantum dots, offers multiplexing capability for simultaneous measurements of multiple cancer biomarkers. This review will discuss recent advances in the development of electrochemical immunosensors for the next generation of cancer diagnostics, with an emphasis on opportunities for further improvement in cancer diagnostics and treatment monitoring. Details will be given for strategies to increase sensitivity through multilabel amplification, coupled with high densities of capture molecules on sensor surfaces. Such sensors are capable of detecting a wide range of protein quantities, from nanogram to femtogram (depending on the protein biomarkers of interest), in a single sample.

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.

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.

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.

Photocatalytic degradation of 2,4-dinitrophenol.

Contamination of the food supply from agricultural waste is an increasing concern worldwide. Numerous hazardous chemicals enter the environment from various industrial sources daily. Many of these pollutants, including 2,4-dinitrophenol (2,4-DNP), are water soluble, toxic, and not easily biodegradable. The solar photocatalytic degradation of 2,4-DNP was investigated in a solution of titanium dioxide (TiO(2)) that was prepared to be an optically clear aqueous solution of nanosized particles of TiO(2). In order to achieve optimal efficiency of the photodegradation, the effects of light intensity and pH were conducted. All experiments were carried out in a batch mode. At a pH of 8, maximum removal of 70% of 2,4-DNP was achieved within 7h of irradiation time. The nearly homogeneous solution of 5.8nm TiO(2) particles, size determined by XDS, were very effective in the photocatalytic degradation of 2,4-DNP.

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.