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.

Electrochemical detection combined with nanostructured sensor surfaces offers potentially low-cost, high-throughput solutions for detection of clinically significant proteins. Inkjet printing offers an inexpensive non-contact fabrication method for microelectronics that is easily adapted for incorporating into protein immunosensor devices. Herein we report the first direct fabrication of inkjet-printed gold nanoparticle arrays, and apply them to electrochemical detection of the cancer biomarker interleukin-6 (IL-6) in serum. The gold nanoparticle ink was printed on a flexible, heat resistant polyimide Kapton substrate and subsequently sintered to create eight-electrode arrays costing <0.2 euro per array. The inkjet-printed working electrodes had reproducible surface areas with RSD <3%. Capture antibodies for IL-6 were linked onto the eight-electrode array, and used in sandwich immunoassays. A biotinylated secondary antibody with 16-18 horseradish peroxidase labels was used, and detection was achieved by hydroquinone-mediated amperometry. The arrays provided a clinically relevant detection limit of 20 pg mL−1 in calf serum, sensitivity of 11.4 nA pg−1 cm−2, and a linear dynamic range of 20–400 pg mL−1.

Silica microbead bioreactors (0.5 µm diameter) coated with DNA and enzymes were fabricated to measure reactive metabolite and DNA-adduct formation rates relevant to genotoxicity screening. Cytochrome (cyt) P450 2E1, cyt P450 cam , and myoglobin (Mb) were incorporated into thin films with DNA using the electrostatic layer-bylayer (LbL) method. The utility of these biocolloids was demonstrated by oxidation of guaiacol, styrene, and (4-methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK). Enzyme turnover rates for formation of reactive metabolites were monitored using gas chromatography/mass spectrometry (GC/MS) and liquid chromatography-mass spectrometry (LC-MS). Capillary LC-MS/MS was employed to determine DNA nucleobase adducts after catalyzing the reactive metabolite formation with DNAenzyme biocolloids and then using neutral thermal hydrolysis on the biocolloids. Dramatic improvements in surface area to volume ratio over similar films on macroscopic surfaces opens new avenues for genotoxicity screening and enabled the first use of pure cyt P450 enzymes in enzyme-DNA films to produce DNA adducts. The method makes possible identification and formation rate measurements of major and minor DNA adducts as well as the metabolites themselves in <5 min of reaction time using relevant human liver enzymes.Toxicity is often not identified sufficiently early in developing new pharmaceutical, agricultural, personal care, and dietary products. Roughly 30% of drug development failures result from toxicity issues, driving drug costs up significantly. 1-4 Conventional in vitro biological tests such as Ames, chromosome aberration, mouse lymphoma, and Comet assays provide qualitative answers to toxicity questions based on bulk DNA damage but do not give chemical structure or site-specific DNA damage information. 5,6 Animal testing may not relate to human exposure because of metabolic and physiological differences between humans and animal models. 2,4,7 While all these toxicity tests are valuable, we believe that rapid, high-throughput toxicity screening methods for new chemicals that provide chemical structure information about reactive intermediates at early stages of commercial development are important goals to complement conventional microbiological and animal tests.Bioactivation of xenobiotic molecules by metabolic enzymes often results in reactive metabolites that damage biomolecules, including DNA, in a major toxicity pathway. [8][9][10] An example is the metabolism of lipophilic compounds by liver cytochrome (cyt) P450 (or CYP) enzymes creating reactive electrophilic metabolites. 4,11 These electrophiles attack nucleophilic DNA sites, primarily guanines, potentially resulting in genotoxicity. Depending on the adduct site, additional metabolism, and DNA repair processes, covalent DNA adducts can lead to mutations, teratogenesis, and carcinogenesis. 12-14 Thus, nucleobase adducts are key biomarkers for predicting cancer risk in humans and for exposure to toxic chemicals, and sensitive liquid chromatography-mass spectromet...

Tear glucose measurements have been suggested as a potential alternative to blood glucose monitoring for diabetic patients. While previous work has reported that there is a correlation between blood and tear glucose levels in humans, this link has not been thoroughly established and additional clinical studies are needed. Herein, we evaluate the potential of using commercial blood glucose test strips to measure glucose in tears. Of several blood glucose strips evaluated, only one brand exhibits the low detection limit required for quantitating glucose in tears. Calibration of these strips in the range of 0-100 μM glucose with an applied potential of 150 mV to the working electrode yields a sensitivity of 0.127 nA/μM and a limit of quantitation (LOQ) of 9 μM. The strips also exhibit ≤13% error (n = 3) for 25, 50, and 75 μM glucose in the presence of 10 μM acetaminophen, 100 μM ascorbic acid, and 100 μM uric acid. Measurements of glucose in tears from nine normal (nondiabetic) fasting human subjects using strips yielded glucose values within the range of 5-148 μM (mean = 47 μM, median = 43 μM), similar to those for human tears reported by others with more complex LC-MS methods. The glucometer strip method could facilitate more clinical studies to determine whether tear glucose and blood glucose levels sufficiently correlate for application to routine measurements in tears to supplement blood glucose testing. This would be especially helpful for children, adolescents, other Type 1 diabetics, and also for Type 2 diabetics who require treatment with insulin and cannot tolerate multiple finger sticks per day.

Characterization studies of a multi-enzyme-antibody-carbon nanotube bioconjugate designed for the amplification of electrochemical immunosensing are described. Secondary antibodies for prostate specific antigen (PSA) were covalently linked to highly carboxylated multi-walled carbon nanotube (CNT) along with multiple horseradish peroxidase (HRP) enzyme labels. These bioconjugates provide ultra-sensitive amperometric detection of PSA on a single-wall carbon nanotube forest sandwich immunosensor platform. A single layer of HRP on the surface of the CNT was suggested by images from atomic force microscopy (AFM) and transmission electron microscopy (TEM). HRP on the bioconjugate surface was visualized by confocal microscopy using in-situ HRP-catalyzed polymerization yielding a fluorescent product, and HRP activity was estimated in a conventional assay. Binding of quantum-dot labeled PSA to antibodies on the bioconjugate was used for visualization by TEM. Combining TEM and enzyme activity results gave estimates of ~82 HRPs and 30 ± 15 secondary antibodies per 100 nm of antibody-HRP-CNT bioconjugate.

We have achieved reversible tunability of local surface plasmon resonance in conjugated polymer functionalized gold nanoparticles. This property was facilitated by the preparation of 3,4-ethylenedioxythiophene (EDOT) containing polynorbornene brushes on gold nanoparticles via surface-initiated ring-opening metathesis polymerization. Reversible tuning of the surface plasmon band was achieved by electrochemically switching the EDOT polymer between its reduced and oxidized states.

An improved planar amperometric nitric oxide (NO) sensor with enhanced selectivity over carbon monoxide (CO), a volatile interfering species for NO sensors that has been largely overlooked until recently, is described. Formation of an oxide film on the inner platinum working electrode via anodic polarization using an inner alkaline electrolyte solution provides the basis for improved selectivity. Cyclic voltammetry reveals that formation of oxidized Pt film inhibits adsorption of CO to the electrode surface, which is a necessary initial step in the electrocatalytic oxidation of CO on Pt. Previous NO gas sensors that employ internal electrolyte solutions have been assembled using acidic internal solutions, that inhibit the formation of a dense platinum oxide film on the working electrode surface. It is demonstrated herein that increasing the internal electrolyte pH promotes oxidized platinum film formation, resulting in improved selectivity over CO. Selectivity coefficients (log KNO,j) for sensors assembled with internal solutions at various pH values range from −0.08 at pH 2.0 to −2.06 at pH 11.7 with average NO sensitivities of 1.24 nA/μM and LOD of <1 nM.

The free energy change (ΔG°) for the unfolding of immobilized yeast iso-1-cytochrome c (Cyt c) at nanoassemblies was measured by surface plasmon resonance (SPR) spectroscopy. Data show that SPR is sensitive to protein conformational changes, and protein solid interface exerts a major influence on bound protein stability. First, Cyt c was self-assembled on the Au film via the single thiol of Cys-102. Then, crystalline sheets of layered α-Zr(O3POH)2 · H2O (α-ZrP) or Zr(O3PCH2CH2COOH)2 · xH2O (α-ZrCEP) were adsorbed to construct α-ZrP/Cyt c/Au or α-ZrCEP/Cyt c/Au nanoassemblies. The construction of each layer was monitored by SPR, in real time, and the assemblies were further characterized by atomic force microscopy and electrochemical studies. Thermodynamic stability of the protein nanoassembly was assessed by urea-induced unfolding. Surprisingly, unfolding is reversible in all cases studied here. Stability of Cyt c in α-ZrP/Cyt c/Au increased by ~4.3 kJ/mol when compared to the unfolding free energy of Cyt c/Au assembly. In contrast, the protein stability decreased by ~1.5 kJ/mol for α-ZrCEP/Cyt c/Au layer. Thus, OH-decorated surfaces stabilized the protein whereas COOH-decorated surfaces destabilized it. These data quantitate the role of specific functional groups of the inorganic layers in controlling bound protein stability.