Modular Protein Engineering Approach to the Functionalization of Gold Nanoparticles for Use in Clinical Diagnostics

Functional protein–gold nanoparticle (AuNP) conjugates have a wide variety of applications including biosensing and drug delivery. Correct protein orientation, which is important to maintain functionality on the nanoparticle surface, can be difficult to achieve in practice, and dedicated protein scaffolds have been used on planar gold surfaces to drive the self-assembly of oriented protein arrays. Here we use the transmembrane domain of <i>Escherichia coli</i> outer membrane protein A (OmpA_TM) to create protein–AuNP conjugates. The addition of a single cysteine residue into a periplasmic loop, to create cysOmpA_TM, drives oriented assembly and increased equilibrium binding. As the protein surface concentration increases, the sulfur–gold bond in cysOmpA_TM creates a more densely populated AuNP surface than the poorly organized wtOmpA_TM layer. The functionalization of AuNP improved both their stability and homogeneity. This was further exploited using multidomain protein chimeras, based on cysOmpA_TM, which were shown to form ordered protein arrays with their functional domains displayed away from the AuNP surface. A fusion with protein G was shown to specifically bind antibodies via their Fc region. Next, an in vitro selected single chain antibody (scFv)-cysOmpA_TM fusion protein, bound to AuNP, detected influenza A nucleoprotein, a widely used antigen in diagnostic assays. Finally, using the same scFv-cysOmpA_TM–AuNP conjugates, a prototype lateral flow assay for influenza demonstrated the utility of fully recombinant self-assembling sensor layers. By simultaneously removing the need for both animal antibodies and a separate immobilization procedure, this technology could greatly simplify the development of a range of in vitro diagnostics

Analysis of the rotor temperature.

<p>(A) Temperature values obtained in different instruments of the spinning rotor, as measured in the iButton at 1,000 rpm after temperature equilibration, while the set point for the console temperature is 20°C (indicated as dotted vertical line). The box-and-whisker plot indicates the central 50% of the data as solid line, with the median displayed as vertical line, and individual circles for data in the upper and lower 25% percentiles. The mean and standard deviation is 19.62°C ± 0.41°C. (B) Correlation between iButton temperature and measured BSA monomer <i>s</i>-values corrected for radial magnification, scan time, scan velocity, but not viscosity (symbols). In addition to the data from the present study as shown in (A) (circles), also shown are measurements from the pilot study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126420#pone.0126420.ref027" target="_blank">27</a>] where the same experiments were carried out on instruments not included in the present study (stars). The dotted line describes the theoretically expected temperature-dependence considering solvent viscosity.</p

Examples for the determination of radial magnification errors.

<p>(A) Radial intensity profile measured in scans of the precision mask. Blue lines are experimental scans, and shaded areas indicate the regions expected to be illuminated on the basis of the known mask geometry. In this example, the increasing difference between the edges corresponds to a calculated radial magnification error of -3.1%. (B—D) Examples for differences between the experimentally measured positions of the light/dark transitions (blue circles, arbitrarily aligned for absolute mask position) and the known edge distances of the mask. The solid lines indicate the linear or polynomial fit. (B) Approximately linear magnification error with a slope corresponding to an error of -0.04%. Also indicated as thin lines are the confidence intervals of the linear regression. (C) A bimodal shift pattern of left and right edges, likely resulting from out-of-focus location of the mask, with radial magnification error of -1.7%. (D) A non-linear distortion leading to a radial magnification error of -0.53% in the <i>s</i>-values from the analysis of back-transformed data. The thin grey lines in C and D indicate the best linear fit through all data points.</p

Absence of a long-term trend in <i>s</i>_{<i>20T</i>,<i>t</i>,<i>r</i>,<i>v</i>}-values of the BSA monomer with time of experiment for the three kits (blue, green, and magenta).

<p>Highlighted as bold solid line is the overall average, and the grey area indicates one standard deviation.</p

Corrected best-fit apparent monomer molecular mass from integration of the <i>c</i>(<i>s</i>) peak when scanned with the absorbance system (green) and the interference system (magenta).

<p>Only data with rmsd less than 0.01 OD or 0.01 fringes were included. The box-and-whisker plot indicates the central 50% of the data as solid line and draws the smaller and larger 25% percentiles as individual circles. The median is displayed as a vertical line.</p

Observed fraction of dimer (as a ratio of dimer peak area to the sum of monomer plus dimer peak areas).

<p>The box-and-whisker plot indicates the central 50% of the data as solid line and draws the smaller and larger 25% percentiles as individual circles. The median displayed as vertical line. The mean and standard deviations are 18.5% ± 1.1% for the absorbance system, and 19.0% ± 2.1% for the interference system.</p

Examples of transient changes in the console temperature reading during the SV experiment, as saved in the scan file data.

<p>For comparison, the maximum adiabatic cooling of -0.3°C would be expected after approximately 300 sec, recovering to the equilibrium temperature after approximately 1,200 s (see Fig 3 in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126420#pone.0126420.ref033" target="_blank">33</a>]).</p

Histogram and box-and-whisker plot of <i>s</i>-values of the BSA monomer after different corrections: Raw experimental <i>s</i>-values (black, with grey histogram), scan time corrected <i>s</i>_<i>t</i>-values (blue), rotor temperature corrected <i>s</i>_<i>20T</i>-values (green), or radial magnification corrected <i>s</i>_<i>r</i>-values (cyan), and fully corrected <i>s</i>_{<i>20T</i>,<i>t</i>,<i>r</i>,<i>v</i>}-values (red with red histogram).

<p>The box-and-whisker plots indicate the central 50% of the data as solid line and draw the smaller and larger 25% percentiles as individual circles. The median for each group is displayed as a vertical line.</p

Distributions of calculated BSA monomer signals for the different kits and the different optical systems.

<p>The box-and-whisker plots indicate the central 50% of the data as solid line and draw the smaller and larger 25% percentiles as individual circles. The median for each group is displayed as vertical line.</p

Correlations of the <i>s</i>_{<i>20T</i>,<i>t</i>,<i>r</i>,<i>v</i>}-values of the BSA monomer with the difference of the best-fit meniscus from the mean meniscus value, separately for absorbance data sets (A) and interference data sets (B).

<p>The difference of the best-fit meniscus to the mean was calculated separately for each kit, to eliminate offsets due to different sample volumes in each kit, and then merged into groups for the optical systems. Data are shown as a histogram with frequency values indicated in the colorbar. The dotted lines show the theoretically expected dependence of the apparent <i>s</i>-value on errors in the absolute radial position.</p