Incoherent merger network for robust ratiometric gene expression response.

A ratiometric response gives an output that is proportional to the ratio between the magnitudes of two inputs. Ratio computation has been observed in nature and is also needed in the development of smart probiotics and organoids. Here, we achieve ratiometric gene expression response in bacteria Escherichia coli with the incoherent merger network. In this network, one input molecule activates expression of the output protein while the other molecule activates an intermediate protein that enhances the output's degradation. When degradation rate is first order and faster than dilution, the output responds linearly to the ratio between the input molecules' levels over a wide range with R2 close to 1. Response sensitivity can be quantitatively tuned by varying the output's translation rate. Furthermore, ratiometric responses are robust to global perturbations in cellular components that influence gene expression because such perturbations affect the output through an incoherent feedforward loop. This work demonstrates a new molecular signal processing mechanism for multiplexed sense-and-respond circuits that are robust to intra-cellular context.

Characterization of synthetic riboswitch in cell-free protein expression systems.

Riboswitches are RNA-based regulatory elements that utilize ligand-induced structural changes in the 5'-untranslated region of mRNA to regulate the expression of associated genes. The majority of synthetic riboswitches have been selected and tested in cell-based systems. Cell-free protein expression systems (CFPS) have several advantages for the development and testing of synthetic riboswitches, including eliminating interactions with complex cellular networks, and the decoupling of transcription and translation processes. To gain a better understanding of the riboswitch regulatory mechanism, to allow for more efficient riboswitch optimization and use for biosensing applications, we studied the performance of a theophylline-responsive synthetic riboswitch coupled with the superfolder green fluorescent protein (sfGFP) reporter gene in E. coli cellular extract and PURE cell-free systems. To monitor the mRNA dynamics, a malachite green aptamer sequence was added to the 3'-untranslated region of sfGFP mRNA. Performance of the theophylline riboswitch was compared with a constitutively expressed sfGFP (control). Transcription dynamics of the riboswitch mRNA was very similar to the transcription of the control mRNA for all theophylline concentrations tested in both E. coli extract and PURE CFPS. However, sfGFP expression in the riboswitch construct was one order of magnitude lower, even at the highest concentration of theophylline. A mathematical model of riboswitch activation governed by the kinetic trapping mechanism was developed. Two factors - a reduced fraction of mRNA in the 'ON' state and a considerably lower translation initiation rate in the riboswitch - contribute to the much lower level of protein expression in the theophylline riboswitch compared to the control construct.

Multiplexed assessment of engineered bacterial constructs for intracellular ß-galactosidase expression by redox amplification on catechol-chitosan modified nanoporous gold.

Synthetic biology approaches for rewiring of bacterial constructs to express particular intracellular factors upon induction with the target analyte are emerging as sensing paradigms for applications in environmental and in vivo monitoring. To aid in the design and optimization of bacterial constructs for sensing analytes, there is a need for lysis-free intracellular detection modalities that monitor the signal level and kinetics of expressed factors within different modified bacteria in a multiplexed manner, without requiring cumbersome surface immobilization. Herein, an electrochemical detection system on nanoporous gold that is electrofabricated with a biomaterial redox capacitor is presented for quantifying ß-galactosidase expressed inside modified Escherichia coli constructs upon induction with dopamine. This nanostructure-mediated redox amplification approach on a microfluidic platform allows for multiplexed assessment of the expressed intracellular factors from different bacterial constructs suspended in distinct microchannels, with no need for cell lysis or immobilization. Since redox mediators present over the entire depth of the microchannel can interact with the electrode and with the E. coli construct in each channel, the platform exhibits high sensitivity and enables multiplexing. We envision its application in assessing synthetic biology-based approaches for comparing specificity, sensitivity, and signal response time upon induction with target analytes of interest.

Screening and selection of artificial riboswitches.

Synthetic riboswitches are engineered to regulate gene expression in response to a variety of non-endogenous small molecules, and a challenge to select this engineered response requires robust screening tools. A new synthetic riboswitch can be created by linking an in vitro-selected aptamer library with a randomized expression platform followed by in vivo selection and screening. In order to determine response to analyte, we developed a dual-color reporter comprising elements of the E. coli fimbriae phase variation system: recombinase FimE controlled by a synthetic riboswitch and an invertible DNA segment (fimS) containing a constitutively active promoter placed between two fluorescent protein genes. Without an analyte, the fluorescent reporter constitutively expressed green fluorescent protein (GFPa1). Addition of the analyte initiated translation of fimE causing unidirectional inversion of the fimS segment and constitutive expression of red fluorescent protein (mKate2). The dual color reporter system can be used to select and to optimize artificial riboswitches in E. coli cells. In this work, the enriched library of aptamers incorporated into the riboswitch architecture reduces the sequence search space by offering a higher percentage of potential ligand binders. The study was designed to produce structure switching aptamers, a necessary feature for riboswitch function and efficiently quantify this function using the dual color reporter system.

Highly multiplexed design of an allosteric transcription factor to sense novel ligands.

Allosteric transcription factors (aTF), widely used as biosensors, have proven challenging to design for detecting novel molecules because mutation of ligand-binding residues often disrupts allostery. We developed Sensor-seq, a high-throughput platform to design and identify aTF biosensors that bind to non-native ligands. We screened a library of 17,737 variants of the aTF TtgR, a regulator of a multidrug exporter, against six non-native ligands of diverse chemical structures - four derivatives of the cancer therapeutic tamoxifen, the antimalarial drug quinine, and the opiate analog naltrexone - as well as two native flavonoid ligands, naringenin and phloretin. Sensor-seq identified novel biosensors for each of these ligands with high dynamic range and diverse specificity profiles. The structure of a naltrexone-bound design showed shape-complementary methionine-aromatic interactions driving ligand specificity. To demonstrate practical utility, we developed cell-free detection systems for naltrexone and quinine. Sensor-seq enables rapid, scalable design of new biosensors, overcoming constraints of natural biosensors.

Cell surface engineering with edible protein nanoshells.

Natural protein (silk fibroin) nanoshells are assembled on the surface of Saccharomyces cerevisiae yeast cells without compromising their viability. The nanoshells facilitate initial protection of the cells and allow them to function in encapsulated state for some time period, afterwards being completely biodegraded and consumed by the cells. In contrast to a traditional methanol treatment, the gentle ionic treatment suggested here stabilizes the shell silk fibroin structure but does not compromise the viability of the cells, as indicated by the fast response of the encapsulated cells, with an immediate activation by the inducer molecules. Extremely high viability rates (up to 97%) and preserved activity of encapsulated cells are facilitated by cytocompatibility of the natural proteins and the formation of highly porous shells in contrast to traditional polyelectrolyte-based materials. Moreover, in a high contrast to traditional synthetic shells, the silk proteins are biodegradable and can be consumed by cells at a later stage of growth, thus releasing the cells from their temporary protective capsules. These on-demand encapsulated cells can be considered a valuable platform for biocompatible and biodegradable cell encapsulation, controlled cell protection in a synthetic environment, transfer to a device environment, and cell implantation followed by biodegradation and consumption of protective protein shells.

Orthogonal cell-based biosensing: fluorescent, electrochemical, and colorimetric detection with silica-immobilized cellular communities integrated with an ITO-glass/plastic laminate cartridge.

This is the first report of a living cell-based environmental sensing device capable of generating orthogonal fluorescent, electrochemical, and colorimetric signals in response to a single target analyte in complex media. Orthogonality is enabled by use of cellular communities that are engineered to provide distinct signals in response to the model analyte. Coupling these three signal transduction methods provides additional and/or complementary data regarding the sample which may reduce the impact of interferants and increase confidence in the sensor's output. Long-term stability of the cells was addressed via 3D entrapment within a nanostructured matrix derived from glycerated silicate, which allows the device to be sealed and stored under dry, ambient conditions for months with significant retention in cellular activity and viability (40% viability after 60 days). Furthermore, the first co-entrapment of eukaryotic and bacterial cells in a silica matrix is reported, demonstrating multianalyte biodetection by mixing disparate cell lines at intimate proximities which remain viable and responsive. These advances in cell-based biosensing open intriguing opportunities for integrating living cells with nanomaterials and macroscale systems.

Bacterial sunscreen: layer-by-layer deposition of UV-absorbing polymers on whole-cell biosensors.

UV-protective coatings on live bacterial cells were created from the assembly of cationic and UV-absorbing anionic polyelectrolytes using layer-by-layer (LbL) methodology. A cationic polymer (polyallylamine) and three different anionic polymers with varying absorbance in the UV range (poly(vinyl sulfate), poly(4-styrenesulfonic acid), and humic acid) were used to encapsulate Escherichia coli cells with two different green fluorescent protein (GFP) expression systems: constitutive expression of a UV-excitable GFP (GFPuv) and regulated expression of the intensely fluorescent GFP from amphioxus (GFPa1) through a theophylline-inducible riboswitch. Riboswitches activate protein expression after specific ligand-RNA binding events. Hence, they operate as a cellular biosensor that will activate reporter protein synthesis after exposure to a ligand target. E. coli cells coated with UV-absorbing polymers demonstrated enhanced protection of GFP stability, metabolic activity, and viability after prolonged exposure to radiation from a germicidal lamp. The results show the effectiveness of LbL coatings to provide UV protection to living cells for biotechnological applications.

Engineering a Synthetic Dopamine-Responsive Riboswitch for In Vitro Biosensing.

The detection of chemicals using natural allosteric transcription factors is a powerful strategy for point-of-use molecular sensing, particularly using fieldable cell-free gene expression (CFE) systems. However, the reliance of detection schemes on characterized protein-based sensors limits the number of measurable analytes. One alternative solution to this issue is to develop new sensors by generating RNA aptamers against the target analyte and then incorporating them directly into a riboswitch scaffold for ligand-inducible genetic control of a reporter protein. However, this strategy has not generated more than a handful of successful portable cell-free molecular sensors. To address this gap, here we convert dopamine-binding aptamers into functional dopamine-sensing riboswitches that regulate gene expression in a freeze-dried CFE reaction. We then develop an assay for direct detection and semi-quantification of dopamine in human urine. We anticipate that this work will be broadly applicable for converting many in vitro-generated RNA aptamers into fieldable molecular diagnostics.

Preparation of Multifunctional Silk-Based Microcapsules loaded with DNA Plasmids Encoding RNA Aptamers and Riboswitches.

We introduce a protocol for the preparation of DNA-laden silk fibroin microcapsules via the Layer-by-Layer (LbL) assembly method on sacrificial spherical cores. Following adsorption of a prime layer and DNA plasmids, the formation of robust microcapsules was facilitated by inducing ß-sheets in silk secondary structure during acute dehydration of a single silk layer. Hence, the layering occurred via multiple hydrogen bonding and hydrophobic interactions. Upon adsorption of multilayered shells, the core-shell structures can be further functionalized with gold nanoparticles (AuNPs) and/or antibodies (IgG) to be used for remote sensing and/or targeted delivery. Adjusting several key parameters during sequential deposition of key macromolecules on silica cores such as the presence of a polymer primer, the concentration of DNA and silk protein, as well as a number of adsorbed layers resulted in biocompatible, DNA-laden microcapsules with variable permeability and DNA loadings. Upon dissolution of silica cores, the protocol demonstrated the formation of hollow and robust microcapsules with DNA plasmids immobilized to the inner surface of the capsule membrane. Creating a selectively permeable biocompatible membrane between the DNA plasmids and the external environment preserved the DNA during long-term storage and played an important role in the improved output response from spatially confined plasmids. The activity of DNA templates and their accessibility were tested during in vitro transcription and translation reactions (cell-free systems). DNA plasmids encoding RNA light-up aptamers and riboswitches were successfully activated with corresponding analytes, as was visualized during localization of fluorescently labeled RNA transcripts or GFPa1 protein in the shell membranes.

A high-throughput pipeline for design and selection of peptides targeting the SARS-Cov-2 Spike protein.

Rapid design, screening, and characterization of biorecognition elements (BREs) is essential for the development of diagnostic tests and antiviral therapeutics needed to combat the spread of viruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To address this need, we developed a high-throughput pipeline combining in silico design of a peptide library specific for SARS-CoV-2 spike (S) protein and microarray screening to identify binding sequences. Our optimized microarray platform allowed the simultaneous screening of ~ 2.5 k peptides and rapid identification of binding sequences resulting in selection of four peptides with nanomolar affinity to the SARS-CoV-2 S protein. Finally, we demonstrated the successful integration of one of the top peptides into an electrochemical sensor with a clinically relevant limit of detection for S protein in spiked saliva. Our results demonstrate the utility of this novel pipeline for the selection of peptide BREs in response to the SARS-CoV-2 pandemic, and the broader application of such a platform in response to future viral threats.

Impact of Porous Matrices and Concentration by Lyophilization on Cell-Free Expression.

Cell-free expression systems have drawn increasing attention as a tool to achieve complex biological functions outside of the cell. Several applications of the technology involve the delivery of functionality to challenging environments, such as field-forward diagnostics or point-of-need manufacturing of pharmaceuticals. To achieve these goals, cell-free reaction components are preserved using encapsulation or lyophilization methods, both of which often involve an embedding of components in porous matrices like paper or hydrogels. Previous work has shown a range of impacts of porous materials on cell-free expression reactions. Here, we explored a panel of 32 paperlike materials and 5 hydrogel materials for the impact on reaction performance. The screen included a tolerance to lyophilization for reaction systems based on both cell lysates and purified expression components. For paperlike materials, we found that (1) materials based on synthetic polymers were mostly incompatible with cell-free expression, (2) lysate-based reactions were largely insensitive to the matrix for cellulosic and microfiber materials, and (3) purified systems had an improved performance when lyophilized in cellulosic but not microfiber matrices. The impact of hydrogel materials ranged from completely inhibitory to a slight enhancement. The exploration of modulating the rehydration volume of lyophilized reactions yielded reaction speed increases using an enzymatic colorimetric reporter of up to twofold with an optimal ratio of 2:1 lyophilized reaction to rehydration volume for the lysate system and 1.5:1 for the purified system. The effect was independent of the matrices assessed. Testing with a fluorescent nonenzymatic reporter and no matrix showed similar improvements in both yields and reaction speeds for the lysate system and yields but not reaction speeds for the purified system. We finally used these observations to show an improved performance of two sensors that span reaction types, matrix, and reporters. In total, these results should enhance efforts to develop field-forward applications of cell-free expression systems.

Improving the Activity of DNA-Encoded Sensing Elements through Confinement in Silk Microcapsules.

Assembling synthetic bioparts into simplified artificial cells holds tremendous promise for advancing studies into the synthesis, biosensing, and delivery of biomolecules. Currently, the most successful techniques for encapsulation of the transcription-translation machinery exploit compartmentalization in liposomal vesicles. However, improvements to these methods may increase permeability to polar molecules, functionalization of the membrane with biologically active elements, and encapsulation efficiency. Microcapsules prepared via templated layer-by-layer (LbL) assembly using natural polymers have the potential to resolve some of the hurdles associated with liposomes. Here, we introduce a design for immobilizing DNA templates encoding translationally activated riboswitches and RNA aptamers into microcapsules prepared from regenerated silk fibroin protein. Adjusting several key parameters such as the presence of a polymer primer, concentration of silk protein, and DNA loadings during LbL assembly resulted in biocompatible, semipermeable, DNA-laden microcapsules. To preserve bioactivity, DNA was immobilized inside of the capsule membrane, which not only promoted stability during long-term storage at ambient conditions but also improved output response from spatially confined DNA-encoded sensing elements (SEs). Multiple copies of mRNA and GFPa1 protein were synthesized upon activation with specific analytes during in vitro transcription/translation reactions, demonstrating that selective permeability of silk microcapsules was essential for the diffusion of components of the cell-free system inside of the capsules. Further functionalization of capsule shells with gold nanoparticles (AuNPs) and antibodies (IgG) demonstrated the applicability of microcompartmentalized colloidal objects carrying SEs for remote sensing and/or targeted delivery. In the future, multifunctional, biocompatible silk-based microcapsules loaded with different RNA sensors can help advance the design of multiplexed biosensors tracking multiple biomarkers in complex media.

FRET-based optical assay for monitoring riboswitch activation.

Riboswitches are regulatory RNAs located in the 5'-untranslated region of mRNA sequences that recognize and bind to small molecules and regulate the expression of downstream genes. Creation of synthetic riboswitches to novel ligands depends on the ability to monitor riboswitch activation in the presence of analyte. In our work, we have coupled a synthetic riboswitch to an optical reporter assay based on fluorescence resonance energy transfer (FRET) between two genetically encoded fluorescent proteins. The theophylline-sensitive riboswitch was placed upstream of the Tobacco Etch Virus (TEV) protease coding sequence. Our FRET construct was composed of eGFP and a nonfluorescent yellow fluorescent protein mutant called REACh (for resonance energy-accepting chromoprotein) connected with a peptide linker containing a TEV protease cleavage site. Addition of theophylline to the E. coli cells activates the riboswitch and initiates the translation of mRNA. Synthesized protease cleaves the linker in the FRET-based fusion protein causing a change in the fluorescence signal. By this method, we observed an 11-fold increase in cellular extract fluorescence in the presence of theophylline. The advantage of using an eGFP-REACh pair is the elimination of acceptor fluorescence. This leads to an improved detection of FRET via better signal-to-noise ratio, allowing us to monitor riboswitch activation in a wide range of analyte concentrations from 0.01 to 2.5 mM.

Quantification of Interlaboratory Cell-Free Protein Synthesis Variability.

Cell-free protein synthesis (CFPS) platforms, once primarily a research tool to produce difficult to express proteins, are increasingly being pursued by the synthetic biology community for applications including biomanufacturing, rapid screening systems, and field-ready sensors. While consistency within individual studies is apparent in the literature, challenges with reproducing results between laboratories, or even between individuals within a laboratory, are discussed openly by practitioners. As the field continues to grow and move toward applications, a quantitative understanding of expected variability for CFPS and the relative contribution of underlying sources will become increasingly important. Here we offer the first quantitative assessment of interlaboratory variability in CFPS. Three laboratories implemented a single CFPS protocol and performed a series of exchanges, both of material and personnel, designed to quantify relative contributions to variability associated with the site, operator, cell extract preparation, and supplemental reagent preparation. We found that materials prepared at each laboratory, exchanged pairwise, and tested at each site resulted in 40.3% coefficient of variation compared to 7.64% for a single operator across days using a single set of materials. Reagent preparations contributed significantly to observed variability; extract preparations, however, surprisingly did not explain any of the observed variability, even when prepared in different laboratories by different operators. Subsequent exchanges showed that both the site and the operator each contributed to observed interlaboratory variability. In addition to providing the first quantitative assessment of interlaboratory variability in CFPS, these results establish a baseline for individual operator variability across days that can be used as an initial benchmark for community-driven standardization efforts. We anticipate that our results will narrow future avenues of investigation to develop best practices that will ultimately drive down interlaboratory variability, accelerating research progress and informing the suitability of CFPS for real-world applications.

Amplifying Riboswitch Signal Output Using Cellular Wiring.

If fieldable riboswitch-based biological sensors are to fulfill their potential, it is necessary to increase their signal output. Here we report a novel modular amplification system using a riboswitch to initiate signaling between a sensing strain and a reporter strain of E. coli. A quorum sensing signaling molecule biologically wires the sensing and reporter strains together. The amplification circuit increased the amount of fluorescence generated on ligand binding compared to when the riboswitch controlled fluorescence expression directly. This had the corollary effect of increasing the sensitivity of the system, and allowed riboswitch-based reporting in E. coli strains that did not produce a detectable output when the riboswitch directly controlled reporter expression. The amplification circuit also reduced the time required to detect a signal output. The modularity of this amplification system coupled with the achievable increases in output can advance the development of riboswitches and biological sensors.

Riboswitch-Based Reversible Dual Color Sensor.

Riboswitches are RNA-based "sensors" that utilize chemically induced structural changes in the 5'-untranslated region of mRNA to regulate expression of downstream genes. Coupling a specific riboswitch with a reporter gene system translates chemical detection by the cell into a quantifiable reporter protein signal. For the majority of reporter gene systems, the readout signal is only expressed in the presence of the target analyte. This makes it difficult to determine the viability and localization of the uninduced biosensor when it is used for "real-word" applications. To address this problem, we developed a dual-color reporter comprising elements of the E. coli fimbriae phase variation system: recombinase FimE controlled by a synthetic riboswitch and an invertible DNA segment (fimS) containing a constitutively active promoter placed between two fluorescent protein genes. Without an analyte, the fluorescent reporter constitutively expressed green fluorescent protein (GFPa1). Addition of the analyte initiated translation of fimE causing unidirectional inversion of the fimS segment and constitutive expression of red fluorescent protein (mKate2). Thus, the sensor is always fluorescent, but its color is determined by detection of a specific analyte. We demonstrate that the recombinase-based dual-color reporter can be successfully applied to monitor the activation of a theophylline synthetic riboswitch that was used as our model system. To show the feasibility of the FimE recombinase-based system to serve as a reporter for monitoring activation of multiple synthetic riboswitches and, therefore, expand the applicability of the system, we tested a number of previously developed synthetic riboswitches responsive to different analytes. We show that the dual-color reporter system can be successfully used to monitor activation of M6 and M6″ riboswitches responsive to ammeline and pyrimido[4,5-d]pyrimidine-2,4-diamine, respectively, and a 2,4,6-trinitrotoluene-responsive riboswitch developed in this study. We also demonstrate that the system can be reversed by HbiF recombinase-mediated fimS inversion to the initial state of the fluorescent reporter, creating a resettable and reusable cell-based sensor.

Immobilization of Recombinant E. coli Cells in a Bacterial Cellulose-Silk Composite Matrix To Preserve Biological Function.

Strategies for the encapsulation of cells for the design of cell-based sensors require efficient immobilization procedures while preserving biological activity of the reporter cells. Here, we introduce an immobilization technique that relies upon the symbiotic relationship between two bacterial strains: cellulose-producing Gluconacetobacter xylinus cells; and recombinant Escherichia coli cells harboring recombinase-based dual-color synthetic riboswitch (RS), as a model for cell-based sensor. Following sequential coculturing of recombinant cells in the cellulose matrix, final immobilization of E. coli cells was completed after reconstituted silk fibroin (SF) protein was added to a "living membrane" generating the composite bacterial cellulose-silk fibroin (BC-SF) scaffold. By controlling incubation parameters for both types of cells, as well as the conformations in SF secondary structure, a variety of robust composite scaffolds were prepared ranging from opaque to transparent. The properties of the scaffolds were compared in terms of porosity, water capacity, distribution of recombinant cells within the scaffolds matrix, onset of cells activation, and ability to protect recombinant function of cells against UV irradiation. The closer-fitted microstructure of transparent BC-SF scaffolds resulted in leakage-free encapsulation of recombinant cells with preserved RS function because of a combination of several parameters that closely matched properties of a biofilm environment. Along with proper elasticity, fine porosity, capacity to retain the water, and ability of SF to absorb UV light, the composite hydrogel material provided necessary conditions to form confined cell colonies that modified cell metabolism and enhanced cell resilience to the stresses induced by encapsulation.

Integrating and amplifying signal from riboswitch biosensors.

Biosensors offer a built-in energy supply and inherent sensing machinery that when exploited correctly may surpass traditional sensors. However, biosensor systems have been hindered by a narrow range of ligand detection capabilities, a relatively low signal output, and their inability to integrate multiple signals. Integration of signals could increase the specificity of the sensor and enable detection of a combination of ligands that may indicate environmental or developmental processes when detected together. Amplifying biosensor signal output will increase detector sensitivity and detection range. Riboswitches offer the potential to widen the diversity of ligands that may be detected, and advances in synthetic biology are illuminating myriad possibilities in signal processing using an orthogonal parts-based engineering approach. In this chapter, we describe the design, building, and testing of a riboswitch-based Boolean logic AND gate in bacteria, where an output requires the activation of two riboswitches, and the biological circuitry required to amplify the output of the AND gate using natural extracellular bacterial communication signals to "wire" cells together.

Printed Dual Cell Arrays for Multiplexed Sensing.

We demonstrated inkjet printing of large-scale dual-type encapsulated bacterial cell arrays for prospective multiplexing sensing. The dual cell arrays were constructed on the basis of two types of bioengineered E. coli cells hosting fluorescent reporters (green-GFPa1 and red-turboRFP) capable of detecting different target chemicals. The versatility of inkjet printing allows for the fabrication of uniform multilayered confined structures composed of silk ionomers that served as nests for in-printing different cells. Furthermore, sequential encapsulation of "red" and "green" cells in microscopic silk nest arrays with the preservation of their function allowed for facile confinement of cells into microscopic silk nests, where cells retained dual red-green response to mixed analyte environment. Whole-cell dual arrays immobilized in microscopic biocompatible silk matrices were readily activated after prolonged storage (up to 3 months, ambient conditions), showing red-green pattern and demonstrating an effective prototype of robust and long-living multiplexed biosensors for field applications.