We describe herein the combination of electrochemical immunosensors using single-wall carbon nanotube (SWNT) forest platforms with multi-label secondary antibody-nanotube bioconjugates for highly sensitive detection of a cancer biomarker in serum and tissue lysates. Greatly amplified sensitivity was attained by using bioconjugates featuring horseradish peroxidase (HRP) labels and secondary antibodies (Ab 2 ) linked to carbon nanotubes (CNT) at high HRP/Ab 2 ratio. This approach provided a detection limit of 4 pg mL −1 (100 amol mL −1 ), for prostate specific antigen (PSA) in 10 μL of undiluted calf serum, a mass detection limit of 40 fg. Accurate detection of PSA in human serum samples was demonstrated by comparison to standard ELISA assays. PSA was quantitatively measured in prostate tissue samples for which PSA could not be differentiated by the gold standard immunohistochemical staining method. These easily fabricated SWNT immunosensors show excellent promise for clinical screening of cancer biomarkers and point-of-care diagnostics.
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 non-targeted bioconjugates. First line anti-cancer agent cisplatin and epidermal growth factor (EGF) were attached to SWNTs to specifically target squamous cancer, and the non-targeted control was SWNT-cisplatin without EGF. Initialin 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 EGFEGFR binding. Three color, two-photon intra-vital video imagingin 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 non-targeted SWNT-cisplatin.
Alternate layer-by-layer polyion adsorption onto gold electrodes coated with chemisorbed mercaptopropanesulfonic acid gave stable, electroactive multilayer films containing the proteins myoglobin and cytochrome P450cam. Direct, reversible, electron exchange between gold electrodes and proteins involved heme FeIII/FeII redox couples. With oxygen in solution, electrons were also transferred to the FeII−O2 complexes of these proteins, a key step for oxidative enzyme catalysis. Film assembly for Mb was done by sequential adsorption with poly(styrenesulfonate) (PSS), DNA, or poly(dimethyl diallyl) ammonium chloride (PDDA). Cyt P450cam was assembled with layers of PSS or PDDA. Quartz crystal microbalance and voltammetric studies on the same films allowed quantitation of electroactive and nonelectroactive protein. At pH 5.5, the first protein monolayer in all films was fully electroactive. A second monolayer added 30−40% redox activity, but additional protein layers did not communicate with the electrode. Using various film construction strategies, Mb monolayers were also placed at distances from the electrodes of 0.5, 1.8, and 3.8 nm. Full electroactivity was found at 0.5 nm, and about 70−80% electroactivity at 1.8 and 3.8 nm. Results suggest the possibility of enhanced electron transport by partial intermixing of protein and nonprotein layers. Polyion films containing Mb and cyt P450cam were active for enzyme-like catalysis of styrene epoxidation in aerobic solutions.
This critical review evaluates progress toward viable point-of-care protein biomarker measurements for cancer detection and diagnostics. The ability to measure panels of specific, selective cancer biomarker proteins in physicians' surgeries and clinics has the potential to revolutionize cancer detection, monitoring, and therapy. The dream envisions reliable, cheap, automated, technically undemanding devices that can analyze a patient's serum or saliva in a clinical setting, allowing on-the-spot diagnosis. Existing commercial products for protein assays are reliable in laboratory settings, but have limitations for point-of-care applications. A number of ultrasensitive immunosensors and some arrays have been developed, many based on nanotechnology. Multilabel detection coupled with high capture molecule density in immunosensors and arrays seems to be capable of detecting a wide range of protein concentrations with sensitivity ranging into the sub pg mL −1 level. Multilabel arrays can be designed to detect both high and ultralow abundance proteins in the same sample. However, only a few of the newer ultrasensitive methods have been evaluated with real patient samples, which is key to establishing clinical sensitivity and selectivity.
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 µm diameter magnetic beads. These sensors had sensitivity of 31.5 µA mL ng−1 and detection limit (DL) of 0.5 pg mL−1 for prostate specific antigen (PSA) in 10 µL of undiluted serum. This represents an ultralow mass DL of 5 fg PSA, eight 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.
He also joined the faculty of the University of Connecticut in 1979, where he is now Professor of Chemistry. His research interests include bioelectrochemistry, electroenzymology, electroorganic synthesis in microemulsions, electrochemical catalysis, surface spectroelectrochemistry, and computer data analysis. He is also a musician, and plays in Irish and Country-Bluegrass bands.
The unique electronic and optical properties of carbon nanotubes, in conjunction with their size and mechanically robust nature, make these nanomaterials crucial to the development of next-generation biosensing platforms. In this Review, we present recent innovations in carbon nanotube-assisted biosensing technologies, such as DNA-hybridization, protein-binding, antibody-antigen and aptamers. Following a brief introduction on the diameter-and chirality-derived electronic characteristics of single-walled carbon nanotubes, the discussion is focused on the two major schemes for electronic biodetection, namely biotransistor-and electrochemistry-based sensors. Key fabrication methodologies are contrasted in light of device operation and performance, along with strategies for amplifying the signal while minimizing nonspecific binding. This Review is concluded with a perspective on future optimization based on array integration as well as exercising a better control in nanotube structure and biomolecular integration.
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