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Development of orthogonal, restriction-enzyme-based reporters a, Candidate restriction enzymes were evaluated through a four-step screening pipeline designed to find enzymes with high rates of expression and processivity in the cell-free expression system (CFS). Step 1: 37 of 66 commercially available enzymes demonstrated activity in the cell-free (CF) buffer system that replicates the pH, buffer and salt composition found in the complete transcription and translation system. Step 2: 26 of the 37 above restriction enzymes were successfully expressed de novo in the cell-free system (Supplementary Fig. 1). Step 3: 14 of these 26 cell-free expressed enzymes showed high levels of cleavage activity (Supplementary Fig. 2). Step 4: 10 of the 14 enzymes demonstrated high rates of enzyme-mediated cleavage from de novo cell-free expression. b, A summary of the performance of the screened restriction enzymes with the colours matching the categories described in a. c, Representative data of three candidate restriction-enzyme-based reporters in molecular beacon cleavage assays. The data are presented as a percentage of maximum fluorescence for each molecular beacon. Error bars represent the standard error (sample number, N = 3, Supplementary Fig. 3). d, Heat map of the specific enzyme activity. All combinations of restriction enzymes and molecular beacons were tested. The values are the average of three replicates at 180 min.
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The field of synthetic biology has used the engineered assembly of synthetic gene networks to create a wide range of functions in biological systems. To date, gene-circuit-based sensors have primarily used optical proteins (for example, fluorescent, colorimetric) as reporter outputs, which has limited the potential to measure multiple distinct sign...
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Multicellular entities are characterized by intricate spatial patterns, intimately related to the functions they perform. These patterns are often created from isotropic embryonic structures, without external information cues guiding the symmetry breaking process. Mature biological structures also display characteristic scales with repeating distri...
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... To achieve this, analytical devices may employ either multiplexed or hybrid approaches. Multiplexed systems are capable of simultaneously detecting several substances while operating under a single transducing mechanism (electrochemical, optical, etc.) [1][2][3][4][5]. In contrast, hybrid systems combine different techniques to acquire multiple signals acquisition originating from diverse sources, such as physiological, chemical, or biological processes [6][7][8][9][10]. ...
In the field of biosensing and chemical sensing, there is a growing demand for multiplexed detection and quantification of multiple targets within complex matrices. In electrochemical sensing, simultaneous multiplexed analysis is typically performed with multiple electrodes connected to a multichannel potentiostat. An alternative strategy involves using a single electrode capable of discriminating and detecting several analytes in a single measurement, which is, however, unfortunately limited to a selective group of molecules. Herein, we report a novel electrochemical method based on the parallel assembly of a dual-electrochemical cell (PADEC), which enables the simultaneous detection and quantification of solvent-incompatible analytes, prepared separately in two distinct electrochemical cells, using a single-channel potentiostat—thus achieving multichannel-like performance. This approach relies on connecting two electrochemical cells in parallel, allowing the concurrent measurement of distinct electrochemical responses from analytes that otherwise could not be simultaneously determined due to solvent incompatibility. As a proof of concept, the water-soluble vitamin C, and the lipid-soluble vitamin D3 were simultaneously determined, each in its respective optimized medium. The PADEC approach demonstrated performance comparable to individual detection methods, achieving limits of detection of 27 μM for vitamin C and 32 μM for vitamin D3 over a linear range of 20–400 μM. This strategy establishes a new approach for simultaneous, multiplexed electrochemical determination of analytes in different media. Moreover, this innovation may extend applications in electrochemistry beyond (bio)sensing to include areas such as electrocatalysis, energy and corrosion, potentially reducing dependence on multichannel potentiostats.
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... Cell-free gene expression (CFE) offers a promising alternative to cellular manufacturing, creating an opportunity for decentralized production (Borhani et al. 2023;Bundy et al. 2018;Hunt et al. 2024;Lee and Kim 2024;Pardee, Green et al. 2016;Warfel et al. 2023). CFE has offered speed and control for a number of different applications including enzyme prototyping (Ekas et al. 2024a;Karim et al. 2020;Karim and Jewett 2016;Kightlinger et al. 2018Kightlinger et al. , 2019Landwehr et al. 2025;Liew et al. 2022;Vögeli et al. 2022), biosensor development (Ekas et al. 2024b;Nguyen et al. 2021;Pardee et al. 2014, Pardee, Slomovic et al. 2016Sadat Mousavi et al. 2020;Thavarajah et al. 2020), studying protein-protein interactions (Hunt et al. 2023), building artificial cells (Peruzzi et al. 2023), and education (Collias et al. 2019;Collins et al. 2024;Huang et al. 2018;Jung et al. 2023;Rybnicky et al. 2022;Stark et al. 2018Stark et al. , 2019, among others. However, CFE can go beyond prototyping to produce therapeutically relevant products. ...
The SARS‐CoV‐2 pandemic highlighted the urgent need for biomanufacturing paradigms that are robust and fast. Here, we demonstrate the rapid process development and scalable cell‐free production of T7 RNA polymerase, a critical component in mRNA vaccine synthesis. We carry out a 1‐L cell‐free gene expression (CFE) reaction that achieves over 90% purity, low endotoxin levels, and enhanced activity relative to commercial T7 RNA polymerase. To achieve this demonstration, we implement rolling circle amplification to circumvent difficulties in DNA template generation, and tune cell‐free reaction conditions, such as temperature, additives, purification tags, and agitation, to boost yields. We achieve production of a similar quality and titer of T7 RNA polymerase over more than four orders of magnitude in reaction volume. This proof of principle positions CFE as a viable solution for decentralized biotherapeutic manufacturing, enhancing preparedness for future public health crises or emergent threats.
... This technique has gained significant attention due to its high sensitivity, cost-effectiveness, and operational simplicity [1]. It has found diverse applications across multiple domains, including food quality control [2,3], chemical analysis [4][5][6], medical diagnostics [7][8][9], and environmental monitoring [10,11]. ...
The utilization of two-working-electrode mode of interdigitated array (IDA) electrodes and other two-electrode systems has revolutionized electrochemical detection by enabling the simultaneous and independent detection of two species or reactions. In contrast to conventional two-potential electrodes, such as the rotating ring disk electrodes, IDAs demonstrate analogous yet vastly improved performance, characterized by remarkable collection efficiency, sensitivity, and signal amplification resulted from the ‘feedback’ effect. In recent decades, the research surrounding IDAs has garnered escalating interest due to their attractive attributes. This review centers its focus on the recent development on the fabrication of IDA electrodes as well as their applications leveraging the unique electrochemical and structural features. In fabrication, two critical breakthroughs are poised for realization: the achievement of reduced dimensions and the diversification of materials. Established fabrication methods for IDA electrodes encompass photolithography, inkjet printing, and direct laser writing, each affording distinct advantages in terms of size and precision. Photolithography enables the creation with finer structures and higher resolution compared to others. Inkjet printing or laser writing provides a simpler, more cost-effective, and straightforward patterning process, albeit with lower resolution. In terms of applications, IDAs have found utility in diverse fields. This review summarizes recent applications based on their fundamental working principles, encompassing redox cycling, resistance modulation, capacitance variations, and more. This specialized tool shows great promise for further development with enhanced properties. It is also important to note that, micron- or sub-micron-sized IDAs generally cannot be reused, as their small structures cannot be polished. Therefore, controlling the cost of IDA fabrication is crucial for promoting their broader application. Additionally, the distinctive electrochemical properties of ‘feedback’ effect is often underappreciated. The high sensitivity of IDA electrodes, arising from the ‘feedback’ signal amplification mechanism, holds significant potential for the detection of species with short lifetimes or low concentrations.
... biosensors facilitate comprehensive analysis, allowing for the identification of functional correlations between markers that may be overlooked when using traditional single-analyte approaches [2][3][4]. As a result, the development and applications of multiplexed biosensors hold great promise for enhancing our understanding of biological systems and addressing global health challenges. ...
... Some studies have developed nucleic acid probes with terminal thiol groups. For instance, Sadat Mousavi et al. created a sensor array with electrodes functionalized with DNA probes via gold-thiol bonds, enabling the simultaneous measurement of five to ten genes [4]. The thiol group can also be easily conjugated to aptamers for immobilization on gold electrodes, allowing for the detection of a diverse range of analytes, including DNA sequences [27], proteins [28], exosomes [29], small-molecule metabolites [30], and antibiotics [31]. ...
Multiplexed biosensing methods for simultaneously detecting multiple biomolecules are important for investigating biological mechanisms associated with physiological processes, developing applications in life sciences, and conducting medical tests. The development of biosensors, especially those advanced biosensors with multiplexing potentials, strongly depends on advancements in nanotechnologies, including the nano-coating of thin films, micro–nano 3D structures, and nanotags for signal generation. Surface functionalization is a critical process for biosensing applications, one which enables the immobilization of biological probes or other structures that assist in the capturing of biomolecules. During this functionalizing process, nanomaterials can either be the objects of surface modification or the materials used to modify other base surfaces. These surface-functionalizing strategies, involving the coordination of sensor structures and materials, as well as the associated modifying methods, are largely determinative in the performance of biosensing applications. This review introduces the current studies on biosensors with multiplexing potentials and focuses specifically on the roles of nanomaterials in the design and functionalization of these biosensors. A detailed description of the paradigms used for method selection has been set forth to assist understanding and accelerate the application of novel nanotechnologies in the development of biosensors.
... Cell-free gene expression (CFE) offers a promising alternative to cellular manufacturing, creating an opportunity for decentralized production (Borhani et al., 2023;Bundy et al., 2018;Hunt, 2024;Lee & Kim, 2024;Pardee et al., 2016;Silverman, Karim, et al., 2020;Warfel et al., 2023). CFE has offered speed and control for a number of different applications including enzyme prototyping (Ekas, 2024a;Karim et al., 2020;Karim & Jewett, 2016;Kightlinger et al., 2019;Kightlinger et al., 2018;Liew et al., 2022;Vögeli et al., 2022), biosensor development (Ekas, 2024b;Nguyen et al., 2021;Sadat Mousavi et al., 2020;Silverman, Akova, et al., 2020;Thavarajah et al., 2020), studying protein-protein interactions (Hunt et al., 2023), building artificial cells (Peruzzi et al., 2023), and education (Collins et al., 2024;Huang et al., 2018;Stark et al., 2019;Stark et al., 2018), among others. However, CFE can go beyond prototyping to produce therapeutically relevant products. ...
The SARS-CoV-2 pandemic highlighted the urgent need for biomanufacturing paradigms that are robust and fast. Here, we demonstrate the rapid process development and scalable cell-free production of T7 RNA polymerase, a critical component in mRNA vaccine synthesis. We carry out a one-liter cell-free gene expression (CFE) reaction that achieves over 80% purity, low endotoxin levels, and enhanced activity relative to commercial T7 RNA polymerase. To achieve this demonstration, we implement rolling circle amplification to circumvent difficulties in DNA template generation, and tune cell-free reaction conditions, such as temperature, additives, purification tags and agitation to boost yields. We achieve production of a similar quality and titer of T7 RNA polymerase over more than 4 orders of magnitude reaction volume. This proof of principle positions CFE as a viable solution for decentralized biotherapeutic manufacturing, enhancing preparedness for future public health crises or emergent threats.
... For most diagnostic applications, the output from a genetic sensor is an easy to monitor reporter (e.g., a fluorescent protein or luminescence-producing metabolic pathway) [50]. Point-of-care sensors also often use colorimetric outputs that can be read by eye, such as enzymatic reactions and chromoproteins [23,50], or are interfaced with small electronic devices for digital readouts [24,51]. However, it is important to note that genetic sensors are not limited to merely "reporting" on a target; their output can be exchanged for genes that enable more complex responses. ...
... Many THS applications link the sensor output to easily visualized pigment-producing enzymes, or fluorescent proteins for laboratory-based reporting. Recently, real-time monitoring of THS output has been demonstrated by using restriction enzymes as the sensor output and interfacing this with DNA-coated electrodes [51]. In this reporter system, the restriction enzyme cleaves the DNA attached to the electrodes, altering its conductivity. ...
Living cells are exquisitely tuned to sense and respond to changes in their environment. Repurposing these systems to create engineered biosensors has seen growing interest in the field of synthetic biology and provides a foundation for many innovative applications spanning environmental monitoring to improved biobased production. In this review, we present a detailed overview of currently available biosensors and the methods that have supported their development, scale-up, and deployment. We focus on genetic sensors in living cells whose outputs affect gene expression. We find that emerging high-throughput experimental assays and evolutionary approaches combined with advanced bioinformatics and machine learning are establishing pipelines to produce genetic sensors for virtually any small molecule, protein, or nucleic acid. However, more complex sensing tasks based on classifying compositions of many stimuli and the reliable deployment of these systems into real-world settings remain challenges. We suggest that recent advances in our ability to precisely modify nonmodel organisms and the integration of proven control engineering principles (e.g., feedback) into the broader design of genetic sensing systems will be necessary to overcome these hurdles and realize the immense potential of the field.
... A direct gene-circuit/electrode interface for the electrochemical detection of colistin antibiotic resistance genes has been proposed by Mousavi et al. [183]. The colistin resistance genes (i.e., mcr-1, mcr-2, mcr-3 and mcr-4) have recently been identified in livestock globally. ...
... The colistin resistance genes (i.e., mcr-1, mcr-2, mcr-3 and mcr-4) have recently been identified in livestock globally. The interesting strategy described in ref. [183] is based on the production of restriction-enzyme-based reporters to catalyze the release of a reporter DNA (single-stranded DNA, ssDNA, labeled with methylene blue), which hybridizes with a DNA capture probe immobilized onto the electrode surface. The approach begins with the synthesis of toehold switches complementary to the 24 top-ranked binding sites within each mcr gene and the ligation of a unique reporter enzyme gene to each set of switches. ...
... Moreover, the cost-effectiveness and versatility in functionalization makes green-synthetized nanoparticles important tools for contributing to a faster propulsion in genetic science research [48][49][50]. Apart from facilitating the detection of even traces amounts of substances, the science of genetic editing, coupled with the development of nanoparticle-based sensing platforms, also permits simultaneous analysis of multiple substances, achieving multiplexed sensing with exceptional accuracy [51,52]. This convergence of advanced techniques holds significant promise in reshaping the landscape of analytical sciences, fostering innovative solutions in environmental monitoring, healthcare diagnostics, biomedical research, food quality, and safety [53]. ...
Technological progress has led to the development of analytical tools that promise a huge socio-economic impact on our daily lives and an improved quality of life for all. The use of plant extract synthesized nanoparticles in the development and fabrication of optical or electrochemical (bio)sensors presents major advantages. Besides their low-cost fabrication and scalability, these nanoparticles may have a dual role, serving as a transducer component and as a recognition element, the latter requiring their functionalization with specific components. Different approaches, such as surface modification techniques to facilitate precise biomolecule attachment, thereby augmenting recognition capabilities, or fine tuning functional groups on nanoparticle surfaces are preferred for ensuring stable biomolecule conjugation while preserving bioactivity. Size optimization, maximizing surface area, and tailored nanoparticle shapes increase the potential for robust interactions and enhance the transduction. This article specifically aims to illustrate the adaptability and effectiveness of these biosensing platforms in identifying precise biological targets along with their far-reaching implications across various domains, spanning healthcare diagnostics, environmental monitoring, and diverse bioanalytical fields. By exploring these applications, the article highlights the significance of prioritizing the use of natural resources for nanoparticle synthesis. This emphasis aligns with the worldwide goal of envisioning sustainable and customized biosensing solutions, emphasizing heightened sensitivity and selectivity.
... The multiplexed biosensors show great promise in accurately diagnosing/treating of disease, due to their advantages of rapid, sensitive, and cost-effective sensing, and are especially suitable for complex samples with tiny amount [51][52][53]. However, the existing multiplexed biosensors require a slow and complex process resulting in waste of materials [54,55]. ...
Inkjet printing, capable of rapid and template-free fabrication with high resolution and low material waste, is a promising method to construct electrochemical biosensor devices. However, the construction of fully inkjet-printed electrochemical biosensor remains a challenge owing to the lack of appropriate inks, especially the sensing inks of bioactive materials. Herein, we demonstrate a fully inkjet-printed, integrated and multiplexed electrochemical biosensor by combining rationally designed nanoparticle Inks. The stable gold (Au) nanoparticles ink with lower sintering temperature is prepared by using L-cysteine as stabilizer, and it is used to print the interconnects, the counter electrodes, and the working electrodes. The SU-8 ink is used to serve as dielectric layer for the biosensor, whereas the silver electrode is printed on the Au electrode by using commercially silver nanoparticles ink before it is chlorinated to prepare Ag/AgCl reference electrode. Moreover, we synthesize an inkjet-printable and electroactive ink, by the "one-pot method", which is composed of conductive poly 6-aminoindole (PIn-6-NH2) and gold-palladium (Au-Pd) alloy nanoparticle (Au-Pd@PIn-6-NH2) to enhance the sensing performance of gold electrode towards hydrogen peroxide (H2O2). Especially, the amino groups in PIn-6-NH2 canbe further used to immobilizing glucose oxidase (GOx) and lactic acid oxidase (LOx) by glutaraldehyde to prepare printable sensing ink for the detection of glucose and lactate. The fully inkjet-printed electrochemical biosensor enabled by advanced inks can simultaneously detect glucose and lactate with good sensitivity and selectivity, as well as facile and scalable fabrication, showing great promise for metabolic monitoring.
... More recent examples for low-cost diagnostics based on toehold switches comprise the development of electrochemical and bioelectronic sensors. For electrochemical transduction, toehold switches were designed that coded for restriction nucleases rather than GFP [48]. Upon detection of RNA analytes (amplified RNA of a bacterial antibiotic resistance gene), different types of nuclease were expressed, which cleaved off electrochemical labels from DNA transducer molecules that were immobilized on a detector gold electrode. ...
Nucleic acid strand displacement reactions involve the competition of two or more DNA or RNA strands of similar sequence for binding to a complementary strand, and facilitate the isothermal replacement of an incumbent strand by an invader. The process can be biased by augmenting the duplex comprising the incumbent with a single-stranded extension, which can act as a toehold for a complementary invader. The toehold gives the invader a thermodynamic advantage over the incumbent, and can be programmed as a unique label to activate a specific strand displacement process. Toehold-mediated strand displacement processes have been extensively utilized for the operation of DNA-based molecular machines and devices as well as for the design of DNA-based chemical reaction networks. More recently, principles developed initially in the context of DNA nanotechnology have been applied for the de novo design of gene regulatory switches that can operate inside living cells. The article specifically focuses on the design of RNA-based translational regulators termed toehold switches. Toehold switches utilize toehold-mediated strand invasion to either activate or repress translation of an mRNA in response to the binding of a trigger RNA molecule. The basic operation principles of toehold switches will be discussed as well as their applications in sensing and biocomputing. Finally, strategies for their optimization will be described as well as challenges for their operation in vivo.
