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We report a study on chemiluminescence-based chemical analyses using luminol molecules covalently attached to 10 nm diameter gold nanoparticles (GNPs). Chemiluminescence (CL) has been systematically studied under two schemes by varying the concentrations of luminol-labeled GNPs and [Fe(CN)6](3-) catalyst, respectively. The CL signal of luminol-labeled GNPs is enhanced by 5 to 10 times compared to the bulk luminol solutions of the same concentration. The log-log plot of the CL signal versus the number of luminol-labeled GNPs suspended in a standard 96-well plate shows two characteristic linear curves with distinct slopes across eight orders of magnitude variation in the GNP quantity (from 1.82 × 10(2) to 1.82 × 10(10) GNPs per well). The detection limit represented by the cross-point of these two curves can reach down to ~6.1 × 10(5) GNPs per well (corresponding to 1.0 × 10(-14) M GNP and 2.4 × 10(-11) M equivalent luminol concentration). The attachment of luminol molecules to GNP nano-carriers allows a large amount of luminol to be placed in a greatly reduced volume (or area) toward developing miniaturized CL sensors. We have demonstrated this by preloading dried luminol-labeled GNPs in homemade microwell arrays (with a volume of ~12 µL per well). A linear log-log curve can be obtained across the full range from 1 × 10(3) to 1 × 10(10) GNPs per microwell. The CL signal was detectable with as few as ~1000 GNPs. We have further applied this microwell method to the detection of highly diluted blood samples, in both intact and lysed forms, which releases Fe(3+)-containing hemoglobin to catalyze luminol CL. The lysed blood sample can be detected even after a 10(8) fold dilution (corresponding to ~0.18 cells per well). This ultrasensitive CL detection method may be readily adapted for developing various miniaturized multiplex biosensors for rapid chemical/biochemical analyses.

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We report an electrochemical method for measuring the activity of proteases using nanoelectrode arrays (NEAs) fabricated with vertically aligned carbon nanofibers (VACNFs). The VACNFs of ~150 nm in diameter and 3 to 5 µm in length were grown on conductive substrates and encapsulated in SiO2 matrix. After polishing and plasma etching, controlled VACNF tips are exposed to form an embedded VACNF NEA. Two types of tetrapeptides specific to cancer-mediated proteases legumain and cathepsin B are covalently attached to the exposed VACNF tip, with a ferrocene (Fc) moiety linked at the distal end. The redox signal of Fc can be measured with AC voltammetry (ACV) at ~1 kHz frequency on VACNF NEAs, showing distinct properties from macroscopic glassy carbon electrodes due to VACNF's unique interior structure. The enhanced ACV properties enable the kinetic measurements of proteolytic cleavage of the surface-attached tetrapeptides by proteases, further validated with a fluorescence assay. The data can be analyzed with a heterogeneous Michaelis-Menten model, giving "specificity constant" kcat /Km as (4.3 ± 0.8) × 104 M-1s-1 for cathepsin B and (1.13 ± 0.38) × 104 M-1s-1 for legumain. This method could be developed as portable multiplex electronic techniques for rapid cancer diagnosis and treatment monitoring.

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This work describes efficient manipulation of bacteriophage virus particles using a nanostructured DEP device. The nonuniform electric field for DEP is created by utilizing a nanoelectrode array (NEA) made of vertically aligned carbon nanofibers versus a macroscopic indium tin oxide electrode in a "points-and-lid" configuration integrated in a microfluidic channel. The capture of the virus particles has been systematically investigated versus the flow velocity, sinusoidal AC frequency, peak-to-peak voltage, and virus concentration. The DEP capture at all conditions is reversible and the captured virus particles are released immediately when the voltage is turned off. At the low virus concentration (8.9 × 10(4) pfu/mL), the DEP capture efficiency up to 60% can be obtained. The virus particles are individually captured at isolated nanoelectrode tips and accumulate linearly with time. Due to the comparable size, it is more effective to capture virus particles than larger bacterial cells with such NEA-based DEP devices. This technique can be potentially utilized as a fast sample preparation module in a microfluidic chip to capture, separate, and concentrate viruses and other biological particles in small volumes of dilute solutions in a portable detection system for field applications.

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Laccases are copper-containing oxidases that are involved in sclerotization of the cuticle of mosquitoes and other insects. Oxidation of exogenous compounds by insect laccases may have the potential to produce reactive species toxic to insects. We investigated two classes of substituted phenolic compounds, halogenated di- and trihydroxybenzenes and substituted di-tert-butylphenols, on redox potential, oxidation by laccase and effects on mosquito larval growth. An inverse correlation between the oxidation potentials and laccase activity of halogenated hydroxybenzenes was found. Substituted di-tert-butylphenols however were found to impact mosquito larval growth and survival. In particular, 2,4-di-tert-butyl-6-(3-methyl-2-butenyl)phenol (15) caused greater than 98% mortality of Anophelesgambiae larvae in a concentration of 180nM, whereas 2-(3,5-di-tert-butyl-4-hydroxyphenyl)-2-methylpropanal oxime (13) and 6,8-di-tert-butyl-2,2-dimethyl-3,4-dihydro-2H-chromene (33) caused 93% and 92% mortalities in concentrations of 3.4 and 3.7µM, respectively. Larvae treated with di-tert-butylphenolic compounds died just before pupation.

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Laccase-2 is a highly conserved multicopper oxidase that functions in insect cuticle pigmentation and tanning. In many species, alternative splicing gives rise to two laccase-2 isoforms. A comparison of laccase-2 sequences from three orders of insects revealed eleven positions at which there are conserved differences between the A and B isoforms. Homology modeling suggested that these eleven residues are not part of the substrate binding pocket. To determine whether the isoforms have different kinetic properties, we compared the activity of laccase-2 isoforms from Tribolium castaneum and Anopheles gambiae. We partially purified the four laccases as recombinant enzymes and analyzed their ability to oxidize a range of laccase substrates. The predicted endogenous substrates tested were dopamine, N-acetyldopamine (NADA), N-ß-alanyldopamine (NBAD) and dopa, which were detected in T. castaneum previously and in A. gambiae as part of this study. Two additional diphenols (catechol and hydroquinone) and one non-phenolic substrate (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)) were also tested. We observed no major differences in substrate specificity between the A and B isoforms. Dopamine, NADA and NBAD were oxidized with catalytic efficiencies ranging from 51 to 550 min⁻¹ mM⁻¹. These results support the hypothesis that dopamine, NADA and NBAD are endogenous substrates for both isoforms of laccase-2. Catalytic efficiencies associated with dopa oxidation were low, ranging from 8 to 30 min⁻¹ mM⁻¹; in comparison, insect tyrosinase oxidized dopa with a catalytic efficiency of 201 min⁻¹ mM⁻¹. We found that dopa had the highest redox potential of the four endogenous substrates, and this property of dopa may explain its poor oxidation by laccase-2. We conclude that laccase-2 splice isoforms are likely to oxidize the same substrates in vivo, and additional experiments will be required to discover any isoform-specific functions.

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This paper reports capture and detection of pathogenic bacteria based on AC dielectrophoresis (DEP) and electrochemical impedance spectroscopy (EIS) employing an embedded vertically aligned carbon nanofiber (VACNF) nanoelectrode array (NEA) versus a macroscopic indium-tin-oxide (ITO) transparent electrode in "points-and-lid" configuration. The nano-DEP device was fabricated using photolithography processes to define an exposed active region on a randomly distributed NEA and a microfluidic channel on ITO to guide the flow of labeled Escherichia coli cells, respectively, and then bond them into a fluidic chip. A high-frequency (100 kHz) AC field was applied to generate positive DEP at the tips of exposed CNFs. Enhanced electric field gradient was achieved due to reduction in electrode size down to nanometer scale which helped to overcome the large hydrodynamic drag force experienced by E. coli cells at high flow velocities (up to 1.6 mm/s). This DEP device was able to effectively capture a significant number of E. coli cells. Significant decrease in the absolute impedance at the NEA was observed in EIS experiments. The results obtained in this study suggest the possibility of integration of a fully functional electronic device for rapid, reversible and label-free capture and detection of pathogenic bacteria.

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The effect of the interior structure of carbon nanomaterials on their electrochemical properties is not well understood. We report here the electron transfer rate (ETR) of ferrocene (Fc) molecules covalently attached to the exposed end of carbon nanofibers (CNFs) in an embedded nanoelectrode array. The ETR in normal DC voltammetry was found to be limited by the conical graphitic stacking structure interior of CNFs. AC voltammetry, however, can cope with this intrinsic materials property and provide over 100 times higher ETR, likely by a new capacitive pathway. This provides a new method for high-performance electroanalysis using CNF nanoelectrodes.