ABSTRACT
Each cell in a multicellular organism permanently adjusts the concentration of its cell surface proteins. In particular, epithelial cells tightly control the number of carriers, transporters and cell adhesion proteins at their plasma membrane. However, sensitively measuring the cell surface concentration of a particular protein of interest in live cells and in real time represents a considerable challenge. Here, we introduce a novel approach based on split luciferases, which uses one luciferase fragment as a tag on the protein of interest and the second fragment as a supplement to the extracellular medium. Once the protein of interest arrives at the cell surface, the luciferase fragments complement and generate luminescence. We compared the performance of split Gaussia luciferase and split Nanoluciferase by using a system to synchronize biosynthetic trafficking with conditional aggregation domains. The best results were achieved with split Nanoluciferase, for which luminescence increased more than 6000-fold upon recombination. Furthermore, we showed that our approach can separately detect and quantify the arrival of membrane proteins at the apical and basolateral plasma membrane in single polarized epithelial cells by detecting the luminescence signals with a microscope, thus opening novel avenues for characterizing the variations in trafficking in individual epithelial cells.
Subject(s)
Epithelial Cells , Membrane Proteins , Membrane Proteins/metabolism , Epithelial Cells/metabolism , Cell Membrane/metabolism , Luciferases/genetics , Luciferases/metabolism , Cell PolarityABSTRACT
Gibberellins (GAs) are major regulators of developmental and growth processes in plants. Using the degradation-based signaling mechanism of GAs, we have built transcriptional regulator (DELLA)-based, genetically encoded ratiometric biosensors as proxies for hormone quantification at high temporal resolution and sensitivity that allow dynamic, rapid and simple analysis in a plant cell system, i.e. Arabidopsis protoplasts. These ratiometric biosensors incorporate a DELLA protein as a degradation target fused to a firefly luciferase connected via a 2A peptide to a renilla luciferase as a co-expressed normalization element. We have implemented these biosensors for all five Arabidopsis DELLA proteins, GA-INSENSITIVE, GAI; REPRESSOR-of-ga1-3, RGA; RGA-like1, RGL1; RGL2 and RGL3, by applying a modular design. The sensors are highly sensitive (in the low pm range), specific and dynamic. As a proof of concept, we have tested the applicability in three domains: the study of substrate specificity and activity of putative GA-oxidases, the characterization of GA transporters, and the use as a discrimination platform coupled to a GA agonists' chemical screening. This work demonstrates the development of a genetically encoded quantitative biosensor complementary to existing tools that allow the visualization of GA in planta.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Biosensing Techniques , Gibberellins , Protoplasts , Signal Transduction , Gibberellins/metabolism , Biosensing Techniques/methods , Arabidopsis/metabolism , Arabidopsis/genetics , Protoplasts/metabolism , Arabidopsis Proteins/metabolism , Arabidopsis Proteins/genetics , Plant Growth Regulators/metabolism , Transcription Factors/metabolism , Transcription Factors/geneticsABSTRACT
Phase separation of biomolecules into condensates is a key mechanism in the spatiotemporal organization of biochemical processes in cells. However, the impact of the material properties of biomolecular condensates on important processes, such as the control of gene expression, remains largely elusive. Here, the material properties of optogenetically induced transcription factor condensates are systematically tuned, and probed for their impact on the activation of target promoters. It is demonstrated that transcription factors in rather liquid condensates correlate with increased gene expression levels, whereas stiffer transcription factor condensates correlate with the opposite effect, reduced activation of gene expression. The broad nature of these findings is demonstrated in mammalian cells and mice, as well as by using different synthetic and natural transcription factors. These effects are observed for both transgenic and cell-endogenous promoters. The findings provide a novel materials-based layer in the control of gene expression, which opens novel opportunities in optogenetic engineering and synthetic biology.
ABSTRACT
Optogenetics is the genetic approach for controlling cellular processes with light. It provides spatiotemporal, quantitative and reversible control over biological signaling and metabolic processes, overcoming limitations of chemically inducible systems. However, optogenetics lags in plant research because ambient light required for growth leads to undesired system activation. We solved this issue by developing plant usable light-switch elements (PULSE), an optogenetic tool for reversibly controlling gene expression in plants under ambient light. PULSE combines a blue-light-regulated repressor with a red-light-inducible switch. Gene expression is only activated under red light and remains inactive under white light or in darkness. Supported by a quantitative mathematical model, we characterized PULSE in protoplasts and achieved high induction rates, and we combined it with CRISPR-Cas9-based technologies to target synthetic signaling and developmental pathways. We applied PULSE to control immune responses in plant leaves and generated Arabidopsis transgenic plants. PULSE opens broad experimental avenues in plant research and biotechnology.
Subject(s)
Gene Expression Regulation, Plant , Light , Optogenetics , Arabidopsis/genetics , Arabidopsis/immunology , CRISPR-Cas Systems/genetics , Models, Theoretical , Plants, Genetically ModifiedABSTRACT
Recent far-reaching advances in synthetic biology have yielded exciting tools for the creation of new materials. Conversely, advances in the fundamental understanding of soft-condensed matter, polymers and biomaterials offer new avenues to extend the reach of synthetic biology. The broad and exciting range of possible applications have substantial implications to address grand challenges in health, biotechnology and sustainability. Despite the potentially transformative impact that lies at the interface of synthetic biology and biomaterials, the two fields have, so far, progressed mostly separately. This Perspective provides a review of recent key advances in these two fields, and a roadmap for collaboration at the interface between the two communities. We highlight the near-term applications of this interface to the development of hierarchically structured biomaterials, from bioinspired building blocks to 'living' materials that sense and respond based on the reciprocal interactions between materials and embedded cells.
Subject(s)
Biocompatible Materials , Synthetic Biology , PolymersABSTRACT
The link between cancer and aberrant glycosylation has recently become evident. Glycans and their altered forms, known as tumour-associated carbohydrate antigens (TACAs), are diverse, complex and difficult to target therapeutically. Lectins are naturally occurring glycan-binding proteins that offer a unique opportunity to recognise TACAs. T cells expressing chimeric antigen receptors (CARs) have proven to be a successful immunotherapy against leukaemias, but so far have shown limited success in solid tumours. We developed a panel of lectin-CARs that recognise the glycosphingolipid globotriaosylceramide (Gb3), which is overexpressed in various cancers, such as Burkitt's lymphoma, colorectal, breast and pancreatic. We have selected the following lectins: Shiga toxin's B-subunit from Shigella dysenteriae, LecA from Pseudomonas aeruginosa, and the engineered lectin Mitsuba from Mytilus galloprovincialis as antigen-binding domains and fused them to a well-known second-generation CAR. The Gb3-binding lectin-CARs have demonstrated target-specific cytotoxicity against Burkitt's lymphoma-derived cell lines as well as solid tumour cells from colorectal and triple-negative breast cancer. Our findings reveal the big potential of lectin-based CARs as therapeutical applications to target Gb3 and other TACAs expressed in haematological malignancies and solid tumours.
Subject(s)
Burkitt Lymphoma , Colorectal Neoplasms , Receptors, Chimeric Antigen , Burkitt Lymphoma/metabolism , Burkitt Lymphoma/therapy , Humans , Lectins/metabolism , Polysaccharides/metabolism , T-LymphocytesABSTRACT
Controlling the time and dose of nanoparticulate drug delivery by administration of small molecule drugs holds promise for efficient and safer therapies. This study describes a versatile approach of exploiting antibody-ligand interactions for the design of small molecule-responsive nanocarrier and nanocomposite systems. For this purpose, antibody fragments (scFvs) specific for two distinct small molecule ligands are designed. Subsequently, the surface of nanoparticles (liposomes or adeno-associated viral vectors, AAVs) is modified with these ligands, serving as anchor points for scFv binding. By modifying the scFvs with polymer tails, they can act as a non-covalently bound shielding layer, which is recruited to the anchor points on the nanoparticle surface and prevents interactions with cultured mammalian cells. Administration of an excess of the respective ligand triggers competitive displacement of the shielding layer from the nanoparticle surface and restores nanoparticle-cell interactions. The same principle is applied for developing hydrogel depots that can release integrated AAVs or liposomes in response to small molecule ligands. The liberated nanoparticles subsequently deliver their cargoes to cells. In summary, the utilization of different antibody-ligand interactions, different nanoparticles, and different release systems validates the versatility of the design concept described herein.
Subject(s)
Liposomes , Nanoparticles , Animals , Genetic Vectors , Ligands , Mammals , Nanoparticles/chemistry , PolymersABSTRACT
In late 2019 SARS-CoV-2 rapidly spread to become a global pandemic, therefore, measures to attenuate chains of infection, such as high-throughput screenings and isolation of carriers were taken. Prerequisite for a reasonable and democratic implementation of such measures, however, is the availability of sufficient testing opportunities (beyond reverse transcription PCR, the current gold standard). We, therefore, propose an electrochemical, microfluidic multiplexed polymer-based biosensor in combination with CRISPR/Cas-powered assays for low-cost and accessible point-of-care nucleic acid testing. In this study, we simultaneously screen for and identify SARS-CoV-2 infections (Omicron-variant) in clinical specimens (Sample-to-result time: â¼30 min), employing LbuCas13a, whilst bypassing reverse transcription as well as target amplification of the viral RNA (LODs of 2,000 and 7,520 copies/µl for the E and RdRP genes, respectively, and 50 copies/ml for combined targets), both of which are necessary for detection via PCR and other isothermal methods. In addition, we demonstrate the feasibility of combining synthetic biology-driven assays based on different classes of biomolecules, in this case protein-based ß-lactam antibiotic detection, on the same device. The programmability of the effector and multiplexing capacity (up to six analytes) of our platform, in combination with a miniaturized measurement setup, including a credit card sized near field communication (NFC) potentiostat and a microperistaltic pump, provide a promising on-site tool for identifying individuals infected with variants of concern and monitoring their disease progression alongside other potential biomarkers or medication clearance.
ABSTRACT
Clinical assessment based on a single biomarker is in many circumstances not sufficient for adequate diagnosis of a disease or for monitoring its therapy. Multiplexing, the measurement of multiple analytes from one sample and/or of the same target from different samples simultaneously, could enhance the accuracy of the diagnosis of diseases and their therapy success. Thus, there is a great and urgent demand for multiplexed biosensors allowing a low-cost, easy-to-use, and rapid on-site testing. In this work, we present a simple, flexible, and highly scalable strategy for implementing microfluidic multiplexed electrochemical biosensors (BiosensorX). Our technology is able to detect 4, 6, or 8 (different) analytes or samples simultaneously using a sequential design concept: multiple immobilization areas, where the assay components are adsorbed, followed by their individual electrochemical cells, where the amperometric signal readout takes place, within a single microfluidic channel. Here, first we compare vertical and horizontal designs of BiosensorX chips using a model assay. Owing to its easier handling and superior fluidic behavior, the vertical format is chosen as the final multiplexed chip design. Consequently, the feasibility of the BiosensorX for multiplexed on-site testing is successfully demonstrated by measuring meropenem antibiotics via an antibody-free ß-lactam assay. The multiplexed biosensor platform introduced can be further extended for the simultaneous detection of other anti-infective agents and/or biomarkers (such as renal or inflammation biomarkers) as well as different (invasive and non-invasive) sample types, which would be a major step towards sepsis management and beyond.
Subject(s)
Biosensing Techniques , Microfluidics , Biomarkers , Oligonucleotide Array Sequence AnalysisABSTRACT
Biological signals are sensed by their respective receptors and are transduced and processed by a sophisticated intracellular signaling network leading to a signal-specific cellular response. Thereby, the response to the signal depends on the strength, the frequency, and the duration of the stimulus as well as on the subcellular signal progression. Optogenetic tools are based on genetically encoded light-sensing proteins facilitating the precise spatiotemporal control of signal transduction pathways and cell fate decisions in the absence of natural ligands. In this review, we provide an overview of optogenetic approaches connecting light-regulated protein-protein interaction or caging/uncaging events with steering the function of signaling proteins. We briefly discuss the most common optogenetic switches and their mode of action. The main part deals with the engineering and application of optogenetic tools for the control of transmembrane receptors including receptor tyrosine kinases, the T cell receptor and integrins, and their effector proteins. We also address the hallmarks of optogenetics, the spatial and temporal control of signaling events.
Subject(s)
Optogenetics/methods , Signal Transduction/genetics , Signal Transduction/physiology , Animals , Cell Communication/physiology , Cell Differentiation/physiology , Humans , Integrins/metabolism , Light , Receptor Protein-Tyrosine Kinases/metabolismABSTRACT
Activation of T cells by agonistic peptide-MHC can be inhibited by antagonistic ones. However, the exact mechanism remains elusive. We used Jurkat cells expressing two different TCRs and tested whether stimulation of the endogenous TCR by agonistic anti-Vß8 antibodies can be modulated by ligand-binding to the second, optogenetic TCR. The latter TCR uses phytochrome B tetramers (PhyBt) as ligand, the binding half-life of which can be altered by light. We show that this half-life determined whether the PhyBt acted as a second agonist (long half-life), an antagonist (short half-life) or did not have any influence (very short half-life) on calcium influx. A mathematical model of this cross-antagonism shows that a mechanism based on an inhibitory signal generated by early recruitment of a phosphatase and an activating signal by later recruitment of a kinase explains the data.
Subject(s)
Optogenetics , Receptors, Antigen, T-Cell/antagonists & inhibitors , Antibodies/metabolism , Cell Membrane/metabolism , Green Fluorescent Proteins/metabolism , HEK293 Cells , Half-Life , Humans , Jurkat Cells , Ligands , Models, Biological , Receptors, Antigen, T-Cell/metabolismABSTRACT
For the first time, a flow-based regenerable chemiluminescence receptor assay is established that is eminently suited as screening method for the detection of widely used tetracyclines (TCs) in environmental and food samples. The complex functionality and high reactivity of TCs complicate the creation of immunogens which is currently the bottleneck for developing sensitive immunoassays. In this case, competitive bioreceptor assays for the analysis of small organic molecules are preferable and, moreover, flow-based regenerable bioassays are optimally suited for automated analysis applications. Therefore, the solution for rapid and sensitive analysis of TCs is the regenerable CL receptor assay with a covalently immobilized DNA oligonucleotide containing the specific operator sequence tetO to which the repressor protein TetR binds only in the absence of TCs. The TC measurements are performed on the CL microarray analysis platform MCR 3 within 30 min per sample. The LoD in spiked tap water was determined to be 0.1 µg L-1, and for 1 µg L-1 TET, recoveries of 77% ± 16% were obtained. Due to the stability of the immobilized DNA oligonucleotide and the resulting regenerability of the assay for various measurements, the new method is highly cost- and resource-efficient and ideally suited for the monitoring of environmental samples in the field. Graphical abstract.
Subject(s)
Anti-Bacterial Agents/analysis , Environmental Monitoring/methods , Immobilized Nucleic Acids/chemistry , Luminescent Measurements/methods , Tetracyclines/analysis , Water Pollutants, Chemical/analysis , Biosensing Techniques/methods , Environmental Monitoring/instrumentation , Equipment Design , Luminescent Agents/chemistry , Luminescent Measurements/instrumentation , Luminol/chemistry , Microfluidic Analytical Techniques/instrumentation , Microfluidic Analytical Techniques/methodsABSTRACT
The engineering of enzymes for the purpose of controlling their activity represents a valuable approach to address challenges in both fundamental and applied research. Here, we describe and compare different design strategies for the generation of a human rhinovirus-14 (HRV14) 3C protease-inducible caspase-3 (CASP3). We exemplify the application potential of the resulting protease by controlling the activity of a synthetic enzyme cascade, which represents an important motif for the design of artificial signal transduction networks. In addition, we use our engineered CASP3 to characterize the effect of aspartate mutations on enzymatic activity. Besides the identification of mutations that render the enzyme inactive, we find the CASP3-D192E mutant (aspartate-to-glutamate exchange at position 192) to be inaccessible for 3C protease-mediated cleavage. This indicates a structural change of CASP3 that goes beyond a slight misalignment of the catalytic triad. This study could inspire the design of additional engineered proteases that could be used to unravel fundamental research questions or to expand the collection of biological parts for the design of synthetic signaling pathways.
Subject(s)
Caspase 3/genetics , Cysteine Endopeptidases/genetics , Protein Engineering , Rhinovirus/enzymology , Viral Proteins/genetics , 3C Viral Proteases , Aspartic Acid/metabolism , Caspase 3/chemistry , Caspase 3/metabolism , Catalytic Domain/genetics , Cysteine Endopeptidases/chemistry , Cysteine Endopeptidases/metabolism , Glutamic Acid/metabolism , Humans , Mutation , Viral Proteins/chemistry , Viral Proteins/metabolismABSTRACT
Cells receive many different environmental clues to which they must adapt accordingly. Therefore, a complex signal transduction network has evolved. Cellular signal transduction is a highly dynamic process, in which the specific outcome is a result of the exact spatial and temporal resolution of single sub-events. While conventional techniques, like chemical inducer systems, have led to a sound understanding of the architecture of signal transduction pathways, the spatiotemporal aspects were often impossible to resolve. Optogenetics, based on genetically encoded light-responsive proteins, has the potential to revolutionize manipulation of signal transduction processes. Light can be easily applied with highest precision and minimal invasiveness. This review focuses on examples of optogenetic systems which were generated and applied to manipulate non-neuronal mammalian signaling processes at various stages of signal transduction, from cell membrane through cytoplasm to nucleus. Further, the future of optogenetic signaling will be discussed.
Subject(s)
Light , Mammals/metabolism , Optogenetics/methods , Signal Transduction , Animals , Humans , Membrane Proteins/metabolismABSTRACT
Scaffold proteins are hubs for the coordination of intracellular signaling networks. The scaffold protein CNK1 promotes several signal transduction pathway. Here we demonstrate that sterile motif alpha (SAM) domain-dependent oligomerization of CNK1 stimulates CNK1-mediated signaling in growth factor-stimulated cells. We identified Ser22 located within the SAM domain as AKT-dependent phosphorylation site triggering CNK1 oligomerization. Oligomeric CNK1 increased the affinity for active AKT indicating a positive AKT feedback mechanism. A CNK1 mutant lacking the SAM domain and the phosphorylation-defective mutant CNK1S22A antagonizes oligomerization and prevents CNK1-driven cell proliferation and matrix metalloproteinase 14 promoter activation. The phosphomimetic mutant CNK1S22D constitutively oligomerizes and stimulates CNK1 downstream signaling. Searching the COSMIC database revealed Ser22 as putative target for oncogenic activation of CNK1. Like the phosphomimetic mutant CNK1S22D, the oncogenic mutant CNK1S22F forms clusters in serum-starved cells comparable to clusters of CNK1 in growth factor-stimulated cells. CNK1 clusters induced by activating Ser22 mutants correlate with enhanced cell invasion and binding to and activation of ADP ribosylation factor 1 associated with tumor formation. Mutational analysis indicate that EGF-triggered phosphorylation of Thr8 within the SAM domain prevents AKT binding and antagonizes CNK1-mediated AKT signaling. Our findings reveal SAM domain-dependent oligomerization by AKT as switch for CNK1 activation.
Subject(s)
Feedback, Physiological , Gene Expression Regulation, Neoplastic , Intracellular Signaling Peptides and Proteins/genetics , Proto-Oncogene Proteins c-akt/genetics , Sterile Alpha Motif , Cell Adhesion , Cell Movement , Cell Proliferation , Databases, Genetic , HEK293 Cells , HeLa Cells , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Matrix Metalloproteinase 14/genetics , Matrix Metalloproteinase 14/metabolism , Molecular Mimicry , Mutation , Phosphorylation , Promoter Regions, Genetic , Protein Multimerization , Proto-Oncogene Proteins c-akt/metabolism , Signal TransductionABSTRACT
Nanobodies, the smallest possible antibody format, have become of considerable interest for biotechnological and immunotherapeutic applications. They show excellent robustness, are non-immunogenic in humans, and can easily be engineered and produced in prokaryotic hosts. Traditionally, nanobodies are selected from camelid immune libraries involving the maintenance and treatment of animals. Recent advances have involved the generation of nanobodies from naïve or synthetic libraries. However, such approaches demand large library sizes and sophisticated selection procedures. Here, we propose an alternative, two-step approach for the design and generation of nanobodies. In a first step, complementarity-determining regions (CDRs) are grafted from conventional antibody formats onto nanobody frameworks, generating weak antigen binders. In a second step, the weak binders serve as templates to design focused synthetic phage libraries for affinity maturation. We validated this approach by grafting toxin- and hapten-specific CDRs onto frameworks derived from variable domains of camelid heavy-chain-only antibodies (VHH). We then affinity matured the hapten binder via panning of a synthetic phage library. We suggest that this strategy can complement existing immune, naïve, and synthetic library based methods, requiring neither animal experiments, nor large libraries, nor sophisticated selection protocols.
Subject(s)
Protein Engineering/methods , Single-Domain Antibodies/biosynthesis , Single-Domain Antibodies/chemistry , Amino Acid Sequence , Animals , Antigens/metabolism , Camelus , Complementarity Determining Regions , Fluorescein/metabolism , Haptens/metabolism , Immunoglobulin Heavy Chains/chemistry , Immunoglobulin Heavy Chains/metabolism , Immunoglobulin Variable Region/chemistry , Peptide Library , Toxins, Biological/metabolismABSTRACT
Synthetic biology aims to create functional devices, systems and organisms with novel and useful functions on the basis of catalogued and standardized biological building blocks. Although they were initially constructed to elucidate the dynamics of simple processes, designed devices now contribute to the understanding of disease mechanisms, provide novel diagnostic tools, enable economic production of therapeutics and allow the design of novel strategies for the treatment of cancer, immune diseases and metabolic disorders, such as diabetes and gout, as well as a range of infectious diseases. In this Review, we cover the impact and potential of synthetic biology for biomedical applications.
Subject(s)
Biomimetics/methods , Biotechnology/methods , Genetic Engineering/methods , Models, Biological , Synthetic Biology , Animals , Aptamers, Nucleotide/therapeutic use , Biomimetic Materials/therapeutic use , Biomimetics/trends , Biotechnology/trends , Cell Line , Cell Line, Tumor , Drug Design , Epigenomics/methods , Gene Regulatory Networks , Genes, Synthetic , Genetic Engineering/trends , Humans , Light Signal Transduction , Mice , Preventive Medicine/methods , Preventive Medicine/trends , Proteomics/methods , Synthetic Biology/methods , Synthetic Biology/trends , Systems Integration , Transcription, Genetic , Vaccines, Synthetic/therapeutic useABSTRACT
Novel tin oxide field-effect-transistors (SnO2 NW-FET) for pH and protein detection applicable in the healthcare sector are reported. With a SnO2 NW-FET the proof-of-concept of a bio-sensing device is demonstrated using the carrier transport control of the FET channel by a (bio-) liquid modulated gate. Ultra-thin Al2O3 fabricated by a low temperature atomic layer deposition (ALD) process represents a sensitive layer to H+ ions safeguarding the nanowire at the same time. Successful pH sensitivity is demonstrated for pH ranging from 3 to 10. For protein detection, the SnO2 NW-FET is functionalized with a receptor molecule which specifically interacts with the protein of interest to be detected. The feasibility of this approach is demonstrated via the detection of a biotinylated protein using a NW-FET functionalized with streptavidin. An immediate label-free electronic read-out of the signal is shown. The well-established Enzyme-Linked Immunosorbent Assay (ELISA) method is used to determine the optimal experimental procedure which would enable molecular binding events to occur while being compatible with a final label-free electronic read-out on a NW-FET. Integration of the bottom-up fabricated SnO2 NW-FET pH- and biosensor into a microfluidic system (lab-on-a-chip) allows the automated analysis of small volumes in the 400 µl range as would be desired in portable on-site point-of-care (POC) devices for medical diagnosis.
Subject(s)
Biosensing Techniques , Lab-On-A-Chip Devices , Nanowires/chemistry , Repressor Proteins/analysis , Tin Compounds/chemistry , Transistors, Electronic , Aluminum Oxide/chemistry , Biotin/chemistry , Enzyme-Linked Immunosorbent Assay , Humans , Hydrogen-Ion Concentration , Limit of Detection , Point-of-Care Systems , Streptavidin/chemistryABSTRACT
Fluorogenic RNAs that are based on the complex formed by 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) derivatives and the RNA aptamer named Spinach were used to engineer a new generation of in vitro and in vivo sensors for bioanalytics. With the resolved crystal structure of the RNA/small molecule complex, the engineering map becomes available, but comprehensive information regarding the thermodynamic profile of the molecule is missing. Here, we reconstructed the full thermodynamic binding and stability landscapes between DFHBI and a truncated sequence of first-generation Spinach. For this purpose, we established a systematic screening procedure for single- and double-point mutations on a microfluidic large-scale integrated chip platform for 87-nt long RNAs. The thermodynamic profile with single base resolution was used to engineer an improved fluorogenic spinach generation via a directed rather than evolutional approach.
Subject(s)
Aptamers, Nucleotide/chemistry , Benzyl Compounds/chemistry , Fluorescent Dyes/chemistry , Imidazolines/chemistry , Aptamers, Nucleotide/genetics , Lab-On-A-Chip Devices , Point Mutation , ThermodynamicsABSTRACT
The excessive use of antibiotics in human and veterinary medicine causes the emergence of multidrug resistant bacteria. In this context, the surveillance of many different antibiotics provokes a worldwide challenge. Hence, fast and versatile multianalyte single-use biosensors are of increasing interest for many fields such as medical analysis or environmental and food control. Here we present a microfluidic platform enabling the electrochemical readout of up to eight enzyme-linked assays (ELAs), simultaneously. To demonstrate the applicability of this platform for the surveillance and monitoring of antibiotics, we used highly sensitive biomolecular sensor systems for the simultaneous detection of two commonly employed antibiotic classes tetracycline and streptogramin. Thus, microfluidic channel networks are designed, comprising distinct numbers of immobilization sections with a very low volume of 680 nL each. These passively metered sections can be actuated separately for an individual assay procedure. The limits of detection (LOD) are determined, with high precision, to 6.33 and 9.22 ng mL-1 for tetracycline and pristinamycin, respectively. The employed channel material, dry film photoresist (DFR), allows an easy storage of preimmobilized assays with a shelf life of at least 3 months. Multianalyte measurements in a complex medium are demonstrated by the simultaneous detection of both antibiotics in spiked human plasma within a sample-to-result time of less than 15 min.