By creating a paper-based cell-free gold biosensing mechanism, we aim to provide an accessible, environmentally-friendly and portable tool for the detection and measurement of gold levels from mining samples. Current methods for gold detection rely heavily on expensive immobile facilities that are almost exclusively available to large-scale mining companies. Our proposed device hopes to drastically improve the cost and environmental impact of gold sensing while still providing adequate specificity and reproducibility. Using GolS, a gold-binding transcriptional activator coupled with LacZ, a well-studied reporter, our biosensor aims to produce a colorimetric display sensitive to the concentration of gold ions present in a mining sample. GolS is a goldbinding transcriptional regulator, natively found in the gramnegative bacteria Salmonella enterica. In the presence of gold, GolS induces the activation of the gol gene cluster by binding to the pGolB promoter. It has been shown that by fusing a promoterless LacZ construct to pGolB, E. coli is able to express the LacZ reporter in the presence of goldassociated GolS in a concentrationdependent manner. LacZ cleaves the yellow chlorophenol redβD-galactopyranoside substrate within the biosensor into a purple chlorophenol red product. The proteins are anchored onto a matrix of paper wells that can be individually analyzed. Using a smartphone camera application, the colour change in each well can be used as an indirect indicator of gold concentration. Given specific input information, the app performs image processing, colour analysis, and estimation of reporter gene expression. A complete explanation of the functioning of the camera app can be found in the dry-lab section.
A major problem with using GolS in a biosensing circuit is its crossreactivity with other metal ions – in particular, Cu(I). GolS belongs to the MerR family of transcriptional activators, which also include CueR. These sensors coordinate metal ions using conserved C112 and C120 residues located on their metalbinding sites. As such, they share similar affinities for Cu(I). However, transcriptional activity of these regulators does not rely on their metal binding affinity, but rather on the ability of the metal ions to properly activate them through inducing conformational change favorable to pGolB binding. Using visualization softwares to identify the interacting residues between the metal ion and GolS as well as insights from the work Ibanez et al on metal ion detection, we designed GolS mutants with a binding pocket less “sensitive” to Cu(I). It was predicted that inserting A118 in place of L118 would make GolS almost unresponsive to copper, while only slightly decreasing its selectivity for gold. Reproducibility While the paper-based cell-free detection system will require further optimization before reproducible data can be obtained from a variety of mining samples, the camera app has been designed to produce a reliable output on a consistent basis. For instance, in order to ensure accuracy in colorimetric analysis, any color distortions from the true values due to ambient light must be adjusted for. To account for these issues, our app will apply the Robust Auto White Balance API developed by Huo et al. Moreover, we have engineered an Image Color Summarizer API which allows us to negate the effects of small variations in lightness and saturation for either yellow or purple pigment. It also takes into account the additive values of the two colours (i.e. yellow and purple) and approximates how much of each colour is present in the mix. Adjustments to account for extraneous variability in the image input as well as effective measurement of pigmentation in the paper wells allow the application to provide a more reproducible output.
Cost and impact on society
Practically speaking, the cost and portability of our design remain its key strengths. Our policy and practices team estimates that the cost of the biosensing paper could be as low as $0.02 once production starts. This greatly improves upon current methods which rely on expensive high-maintenance equipment. Using the smartphone app as an analytical tool also reduces the overall cost of the process. Our product is also much more portable and allows for rapid on-site results with the prototype only taking a few minutes to achieve the optimal visual display for analysis. The lower cost of the product is expected to improve the accessibility of gold detection technologies to artisanal miners, a group that has largely become marginalized by the monopoly of large corporations in mining communities. A thorough examination of the social impact of our prototype can be found in the policy and practices section.
Our paper-based cell-free gold detection system aims at producing a portable and accessible solution to the measurement of gold levels in mining samples. By developing a genetic circuit consisting of a highly specific gold-binding transcriptional regulator and a standardized reporter gene, our prototype couples a sensitive cell-free expression system with a highly optimized camera app to provide rapid on-site analysis of samples that do not require expensive high-maintenance equipment. The modular design for paper-based metal ion detection and camera-based colorimetric analysis also opens up interesting avenues for the development of cheap and portable biosensing devices.