Continuous Production of a Shelf‐Stable Living Material as a Biosensor Platform

Abstract

analyze biomarkers in human diseases, such as nitrogen oxides involved in inflammation response. [11] This organismbased type of sensor systems is referred to as whole-cell biosensors, since the sensing functionality and signal transduction require intact, living cells. The basic working principle of a whole-cell biosensor relies on an input, which activates a certain pathway in the cell leading to an output signal such as color, fluorescence, or luminescence that can be measured by the user. [12,13] Cells genetically engineered to recognize a specific target can be produced at low cost compared to other biological diagnostic systems such as enzyme-or antibody-based assays. Nevertheless, the readout of whole-cell biosensors and the quantification of the measured signal require high-priced laboratory equipment such as fluorometers, spectrophotometers, or luminometers and trained personnel to operate these devices.An alternative approach for the quantification of whole-cell biosensors systems has been presented by Mora et al. by embedding genetically modified Escherichia coli cells into an agarose hydrogel. [14] The concentration of different sugars in solution, which were applied to the surface of the hydrogel, was quantified by the direct correlation between the diffusion in the material and the fluorescence induction by the same sugars in the E. coli cells. This living material-based analytical sensor showed quantification precision comparable to those of HPLC-MS and a fluorometric lactose/galactose enzymatic assay. As a drawback, the system requires a cold chain to maintain the viability of the embedded organisms, resulting in limited shelf life of the sensor. Additionally, the material was produced in a batch process and had, due to the fragile nature of the agarose gel, limited mechanical stability. Both factors limit the production to a laboratory scale and the use to a controlled environment, where continuous cooling and short periods of time between production and application are ensured. These restrictions forbid the application of the sensors in areas where an undeveloped infrastructure would compromise the functionality of the material.To facilitate the use of whole-cell biosensors in the field, a platform is required that is versatile in its application. Additionally, it has to be cheap in production, robust toward storage and changing environmental conditions, and can be The advancements made in biological engineering have led to the development of whole-cell biosensors and their successful application in diagnostics. However, due to the sensitivity of the systems, as well as the need for advanced readout devices, the use of such sensors is limited to a laboratory environment. Here, a biosensor platform for the diffusion-based quantification of a small molecule analyte is presented. The platform consists of Bacillus subtilis endospores carrying a genetic reporter construct. The spores are embedded in a poly(vinyl alcohol) matrix casted to a poly(ethylene terephthalate) support material. It is shown th...

Stimuli-responsive engineered living materials

“…Additional techniques used for the fabrication of ELMs include wet spinning and roll-to-roll processes. 104,105 Both wet-spinning and roll-to-roll techniques are highly scalable. For wet spinning methods used in ELMs, polyvinyl alcohol (PVA) microfibers containing dispersed Micrococcus luteus or Nitrobacter winogradskyi bacteria were used for gold sequestration and nitrate bioremediation, respectively.…”

“…It was observed that cells retained functionality during this process as they were capable of generating a fluorescence signal after IPTG sensing. 105 In summary, the combination of synthetic biology and materials science offers new methods to develop stimuli-responsive ELMs with well-defined functions. High specificity to a variety of molecules can be programmed into cells to produce composites that sense, respond, and modify the physical or chemical properties of the material itself or the surrounding environment.…”

Regulating bacterial behavior within hydrogels of tunable viscoelasticity

“…[1][2][3] Examples of ELMs include biofilters to sequester metals [4] or viruses, [5] bacterial hydrogels for biosensing, [6] shape-morphing composites, [7] self-healing adhesives, [8] photosynthetic biogarments [9] or self-regulated drug delivery devices. [10] A common feature in these constructs is the encapsulation of the organisms within matrices including natural polymers like agarose, [11,12] alginate, [13] and dextran, [14] synthetic polymers like polyvinyl alcohol [15] and Pluronic, [16,17] or inorganic matrices like porous silica. [18] Alternatively, proteinaceous [19] or cellulose [6] matrices produced by the organisms themselves, as in a biofilm, can serve as encapsulating networks.…”

Applications, challenges, and needs for employing synthetic biology beyond the lab

“…As an example, a portable system for spore-based whole-cell biosensing of arsenite and zinc in a centrifugal compact disk microfluidic platform was functionally stable for 1 year under elevated temperature and a variety of humidity/drought conditions 85 . A living material polymer device containing B. subtilis spores encapsulated in a PVA matrix for continuous sensing of small-molecule analytes could be stored for extended time periods at room temperature without compromising sensor function 86 . Incorporating hydrogel encapsulation with a paper-based device, Weaver et al 87 first developed a whole-cell yeast paper-based biosensor device to yield a biologically based paper analytical device (BioPAD), which could be stored at 4 °C and 37 °C and remain viable for at least 6 months and 56 days respectively.…”