Selective DNA-mediated assembly of gold nanoparticles on electroded substrates.

Motivated by the technological possibilities of electronics and sensors based on gold nanoparticles (Au NPs), we investigate the selective assembly of such NPs on electrodes via DNA hybridization. Protocols are demonstrated for maximizing selectivity and coverage using 15mers as the active binding agents. Detailed studies of the dependences on time, ionic strength, and temperature are used to understand the underlying mechanisms and their limits. Under optimized conditions, coverage of Au NPs on Au electrodes patterned on silicon dioxide (SiO2) substrates was found to be approximately 25-35%. In all cases, Au NPs functionalized with non-complementary DNA show no attachment and essentially no nonspecific adsorption is observed by any Au NPs on the SiO2 surfaces of the patterned substrates. DNA-guided assembly of multilayers of NPs was also demonstrated and, as expected, found to further increase the coverage, with three deposition cycles resulting in a surface coverage of approximately 60%.

An engineered virus as a bright fluorescent tag and scaffold for cargo proteins--capture and transport by gliding microtubules.

We have demonstrated substantial capture and transport of fluorescently-labeled engineered cowpea mosaic virus (CPMV) using Drosophila kinesin-driven microtubules (MTs). The capture occurred through both NeutrAvidin (NA)-biotin and antibody (IgG)-antigen interactions. The MTs were derivatized with rabbit anti-chicken IgG or biotin, and the virus was conjugated with chicken IgG or NA. The CPMV conjugate was introduced into standard MT motility assays via convective flow at concentrations as high as 1.36 nM, and became bound to the MTs in densities as high as one virus per microm of MT length. When the CPMV conjugate was present at 17 pM, the average speed of the MTs bearing the NA-virus was 0.59 +/- 0.08 microm/sec, and that of those bearing IgG-virus was 0.52 +/- 0.15 microm/sec. These speeds are comparable to those of the unladen MTs (0.61 +/- 0.09 microm/sec), the presence of the virus on the MT causing only a small decrease in MT gliding speeds. The fluorescent CPMV appears to be superior to fluorescent polystyrene spheres of the same size, as both a reporter tag and a scaffold for MT-transported cargo proteins, because of its negligible non-specific adsorption and superior brightness. This work is important for the development of sensors based on nanolocomotion and biological recognition, or new strategies for the nanoassembly of biological structures.

A portable array biosensor for detecting multiple analytes in complex samples.

The Multi-Analyte Array Biosensor (MAAB) has been developed at the Naval Research Laboratory (NRL) with the goal of simultaneously detecting and identifying multiple target agents in complex samples with minimal user manipulation. This paper will focus on recent improvements in the biochemical and engineering aspects of this instrument. These improvements have enabled the expansion of the repertoire of analytes detected to include Salmonella typhimurium and Listeria monocytogenes, and also expanded the different sample matrices tested. Furthermore, all components of the biochemical assays could be prepared well in advance of sample testing, resulting in a "plug-and-play" methodology. Simultaneous detection of three toxins (ricin, staphylococcal enterotoxin B, and cholera toxin) was demonstrated using a novel fluidics cube module that limits the number of manipulations to only the initial sample loading. This work demonstrates the utility of the MAAB for rapid analysis of complex samples with multianalyte capability, with a minimum of operator manipulations required for either sample preparation or final analysis.

Kinetics of antigen binding to arrays of antibodies in different sized spots.

A fluorescence-based array biosensor has been developed which can measure the binding kinetics of an antigen to an immobilized antibody in real time. A patterned array of antibodies immobilized on the surface of a planar waveguide was used to capture a Cy5-labeled antigen present in a solution that was continuously flowed over the surface. The CCD image of the waveguide was monitored continuously for 25 min. The resulting exponential rise in fluorescence signal was determined by image analysis software and fitted to a reaction-limited kinetics model, giving a kf of 3.6 x 10(5) M(-1) s(-1). Different spot sizes were then patterned on the surface of the waveguide using either a PDMS flow cell or laser exposure, producing width sizes ranging from 80 to 1145 microm. It was demonstrated that under flow conditions, the reduction of spot size did not alter the association rate of the antigen with immobilized antibody; however, as the spot width decreased to < 200 nm, the signal intensity also decreased.

Optical biosensing of nitrite ions using cytochrome cd1 nitrite reductase encapsulated in a sol-gel matrix.

Nitrite is an important human health and environmental analyte. As such, the European Union (EU) has imposed a limit for nitrite in potable water of 0.1 mg l-1 (2.18 microM). In order to develop an optical biosensing system for the determination of nitrite ions in environmental waters, cytochrome cd1 nitrite reductase has been extracted and purified from the bacterium Paracoccus pantotrophus. The protein has been spectroscopically characterised in solution and important kinetic parameters of nitrite reduction of the cytochrome cd1 enzyme, i.e., Km, Vmax and kcat have been determined. The influence of pH on the activity of the cytochrome cd1 has been investigated and the results suggest that this enzyme can be used for the determination of nitrite in the pH range 6-9. Biosensing experiments with the cytochrome cd1 in solution suggested that the decrease in intensity of the absorption band associated with the d1 haem (which is the nitrite binding site), at 460 nm, with increasing nitrite concentrations would enable the measurement of this analyte with the optimum limit of detection. The cytochrome cd1 has been encapsulated in a bulk sol-gel monolith with no structural changes observed and retention of enzymatic activity. The detection of nitrite ions in the range 0.075-1.250 microM was achieved, with a limit of detection of 0.075 microM. In order to increase the speed of response, a sol-gel sandwich thin film structure was formulated with the cytochrome cd1. This structure enabled the determination of nitrite concentrations within ca. 5 min. The sol-gel sandwich entrapped cytochrome cd1 enzyme was found to be stable for several months when the films were stored at 4 degrees C.