Targeted Deposition of Antibodies on a Multiplex CMOS Microarray and Optimization of a Sensitive Immunoassay Using Electrochemical Detection

The CombiMatrix ElectraSense microarray is a highly multiplex, complementary metal oxide semiconductor with 12,544 electrodes that are individually addressable. This platform is commercially available as a custom DNA microarray; and, in this configuration, it has also been used to tether antibodies (Abs) specifically on electrodes using complementary DNA sequences conjugated to the Abs.An empirical method is described for developing and optimizing immunoassays on the CombiMatrix ElectraSense microarray based upon targeted deposition of polypyrrole (Ppy) and capture Ab. This process was automated using instrumentation that can selectively apply a potential or current to individual electrodes and also measure current generated at the electrodes by an enzyme-enhanced electrochemical (ECD) reaction. By designating groups of electrodes on the array for different Ppy deposition conditions, we determined that the sensitivity and specificity of a sandwich immunoassay for staphylococcal enterotoxin B (SEB) is influenced by the application of different voltages or currents and the application time. The sandwich immunoassay used a capture Ab adsorbed to the Ppy and a reporter Ab labeled for fluorescence detection or ECD, and results from these methods of detection were different.Using Ppy deposition conditions for optimum results, the lower limit of detection for SEB using the ECD assay was between 0.003 and 0.01 pg/ml, which represents an order of magnitude improvement over a conventional enzyme-linked immunosorbant assay. In the absence of understanding the variables and complexities that affect assay performance, this highly multiplexed electrode array provided a rapid, high throughput, and empirical approach for developing a sensitive immunoassay

Measurements of elliptic and triangular flow in high-multiplicity 3^{3}He++Au collisions at sNN=200\sqrt{s_{_{NN}}}=200 GeV

We present the first measurement of elliptic ($v_2$) and triangular ($v_3$) flow in high-multiplicity $^{3}$He$+$Au collisions at $\sqrt{s_{_{NN}}}=200$ GeV. Two-particle correlations, where the particles have a large separation in pseudorapidity, are compared in $^{3}$He$+$Au and in $p$$+$$p$ collisions and indicate that collective effects dominate the second and third Fourier components for the correlations observed in the $^{3}$He$+$Au system. The collective behavior is quantified in terms of elliptic $v_2$ and triangular $v_3$ anisotropy coefficients measured with respect to their corresponding event planes. The $v_2$ values are comparable to those previously measured in $d$$+$Au collisions at the same nucleon-nucleon center-of-mass energy. Comparison with various theoretical predictions are made, including to models where the hot spots created by the impact of the three $^{3}$He nucleons on the Au nucleus expand hydrodynamically to generate the triangular flow. The agreement of these models with data may indicate the formation of low-viscosity quark-gluon plasma even in these small collision systems.Comment: 630 authors, 9 pages, 4 figures, 2 tables. v2 is the version accepted for publication by Physical Review Letters. Plain text data tables for the points plotted in figures for this and previous PHENIX publications are (or will be) publicly available at http://www.phenix.bnl.gov/papers.htm

Electrochemical detection of SEB binding on an array with Ppy deposited using constant current.

<p>Polypyrrole was deposited using currents from 10 to 260 nA for 0.1, 0.5, 1.0, or 2.0 s. Different concentrations (0, 0.1, 1.0 or 10.0 pg/ml) of SEB were incubated in individual chambers of a four-chamber hyb cap, and binding was detected using biotinylated rabbit anti-SEB with HRP-SA.</p

Fluorescence detection of SEB binding on an array with Ppy deposited using constant current.

<p>Polypyrrole was deposited using constant current from 0 to 980 nA for 1.0 s. Different concentrations (0, 0.1, 1.0, and 10.0 pg/ml) of SEB were incubated in individual chambers of a four-chamber hyb cap, and binding was detected using biotinylated rabbit anti-SEB with Cy5-SA.</p

Detection of ricin binding to murine anti-ricin MAb adsorbed on Ppy.

<p>Polypyrrole was deposited at different voltages followed by adsorption of anti-ricin MAb. Three concentrations of ricin were incubated for 1 h in different chambers of a four-chamber hyb cap. Binding was detected using biotin-labeled goat anti-ricin Ab. A) Results using Cy5-SA showing a scanned fluorescence image of the array and a graph illustrating fluorescence intensities for different groups of electrodes. B) Results using HRP-SA showing a pseudo image of the array and a graph illustrating the ECD signals for different groups of electrodes.</p

Photomicrograph of a single electrode.

<p>The image shows relationships among the different components and their electrical connections.</p

Determination of the LOD for SEB detection.

<p><b>A</b>) Three concentrations (0.003, 0.01, and 0.03 pg/ml) were incubated on a microarray with either anti-SEB MAb (•-•) or casein (▪-▪) adsorbed onto Ppy that was deposited at 40 nA for 1 s. SEB binding was detected using biotinylated rabbit anti-SEB Ab and SA-HRP. Background (0 pg/ml SEB) was 1147±283. <b>B</b>) Different concentrations of SEB were incubated on a microtiter plate with anti-SEB MAb adsorbed onto the surface of each well. SEB binding was detected using biotinylated rabbit anti-SEB Ab and SA-HRP. Background (0 pg/ml SEB) was OD_450–570 2.939±.026.</p

Electrochemical detection of SEB binding on an array with Ppy deposited using constant voltage.

<p>Polypyrrole was deposited using potentials from 0.0 to 2 V for 0.5, 1.0, 2.0, or 5.0 s followed by adsorption of anti-SEB MAb. Different concentrations (0.0, 0.1, 1.0 or 10.0 pg/ml) of SEB were incubated in individual chambers of a four-chamber hyb cap, and binding was detected using biotinylated rabbit anti-SEB with HRP-SA.</p