RESUMEN
Genetically encoded fluorescent biosensors are powerful tools for monitoring biochemical activities in live cells, but their multiplexing capacity is limited by the available spectral space. We overcome this problem by developing a set of barcoding proteins that can generate over 100 barcodes and are spectrally separable from commonly used biosensors. Mixtures of barcoded cells expressing different biosensors are simultaneously imaged and analyzed by deep learning models to achieve massively multiplexed tracking of signaling events. Importantly, different biosensors in cell mixtures show highly coordinated activities, thus facilitating the delineation of their temporal relationship. Simultaneous tracking of multiple biosensors in the receptor tyrosine kinase signaling network reveals distinct mechanisms of effector adaptation, cell autonomous and non-autonomous effects of KRAS mutations, as well as complex interactions in the network. Biosensor barcoding presents a scalable method to expand multiplexing capabilities for deciphering the complexity of signaling networks and their interactions between cells.
Asunto(s)
Técnicas Biosensibles/métodos , Células/ultraestructura , Microscopía Fluorescente/métodos , Análisis de la Célula Individual/métodos , Línea Celular Tumoral , HumanosRESUMEN
Heterotrimeric G-proteins (Gαßγ) are the main transducers of signals from GPCRs, mediating the action of countless natural stimuli and therapeutic agents. However, there are currently no robust approaches to directly measure the activity of endogenous G-proteins in cells. Here, we describe a suite of optical biosensors that detect endogenous active G-proteins with sub-second resolution in live cells. Using a modular design principle, we developed genetically encoded, unimolecular biosensors for endogenous Gα-GTP and free Gßγ: the two active species of heterotrimeric G-proteins. This design was leveraged to generate biosensors with specificity for different heterotrimeric G-proteins or for other G-proteins, such as Rho GTPases. Versatility was further validated by implementing the biosensors in multiple contexts, from characterizing cancer-associated G-protein mutants to neurotransmitter signaling in primary neurons. Overall, the versatile biosensor design introduced here enables studying the activity of endogenous G-proteins in live cells with high fidelity, temporal resolution, and convenience.
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Transferencia de Energía por Resonancia de Bioluminiscencia/instrumentación , Transferencia de Energía por Resonancia de Bioluminiscencia/métodos , Técnicas Biosensibles/instrumentación , Técnicas Biosensibles/métodos , Guanosina Trifosfato/metabolismo , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Secuencias de Aminoácidos , Animales , Células Cultivadas , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/química , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Factores de Intercambio de Guanina Nucleótido/antagonistas & inhibidores , Guanosina Trifosfato/química , Células HEK293 , Células HeLa , Humanos , Ratones , Ratones Endogámicos C57BL , Mutación , Neoplasias/genética , Neoplasias/metabolismo , Neuronas/química , Neuronas/metabolismo , Neuronas/fisiología , Transducción de Señal , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/metabolismoRESUMEN
Reactive oxygen species (ROS) encompass a collection of intricately linked chemical entities characterized by individually distinct physicochemical properties and biological reactivities. Although excessive ROS generation is well known to underpin disease development, it has become increasingly evident that ROS also play central roles in redox regulation and normal physiology. A major challenge in uncovering the relevant biological mechanisms and deconvoluting the apparently paradoxical roles of distinct ROS in human health and disease lies in the selective and sensitive detection of these transient species in the complex biological milieu. Small-molecule-based fluorescent sensors enable molecular imaging of ROS with great spatial and temporal resolution and have thus been appreciated as excellent tools for aiding discoveries in modern redox biology. We review a selection of state-of-the-art sensors with demonstrated utility in biological systems. By providing a systematic overview based on underlying chemical sensing mechanisms, we wish to highlight the strengths and weaknesses in prior sensor works and propose some guiding principles for the development of future probes.
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Técnicas Biosensibles/métodos , Especies Reactivas de Oxígeno/análisis , Colorantes Fluorescentes , Imagen Óptica , Oxidación-Reducción , Estrés OxidativoRESUMEN
Riboswitches are common gene regulatory units mostly found in bacteria that are capable of altering gene expression in response to a small molecule. These structured RNA elements consist of two modular subunits: an aptamer domain that binds with high specificity and affinity to a target ligand and an expression platform that transduces ligand binding to a gene expression output. Significant progress has been made in engineering novel aptamer domains for new small molecule inducers of gene expression. Modified expression platforms have also been optimized to function when fused with both natural and synthetic aptamer domains. As this field expands, the use of these privileged scaffolds has permitted the development of tools such as RNA-based fluorescent biosensors. In this review, we summarize the methods that have been developed to engineer new riboswitches and highlight applications of natural and synthetic riboswitches in enzyme and strain engineering, in controlling gene expression and cellular physiology, and in real-time imaging of cellular metabolites and signals.
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Aptámeros de Nucleótidos/metabolismo , Técnicas Biosensibles/métodos , Regulación Bacteriana de la Expresión Génica , Ingeniería Genética/métodos , Riboswitch , Aptámeros de Nucleótidos/síntesis química , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Ligandos , Imagen Molecular/métodos , Rhodocyclaceae/genética , Rhodocyclaceae/metabolismoRESUMEN
Genetically encoded optical tools have revolutionized modern biology by allowing detection and control of biological processes with exceptional spatiotemporal precision and sensitivity. Natural photoreceptors provide researchers with a vast source of molecular templates for engineering of fluorescent proteins, biosensors, and optogenetic tools. Here, we give a brief overview of natural photoreceptors and their mechanisms of action. We then discuss fluorescent proteins and biosensors developed from light-oxygen-voltage-sensing (LOV) domains and phytochromes, as well as their properties and applications. These fluorescent tools possess unique characteristics not achievable with green fluorescent protein-like probes, including near-infrared fluorescence, independence of oxygen, small size, and photosensitizer activity. We next provide an overview of available optogenetic tools of various origins, such as LOV and BLUF (blue-light-utilizing flavin adenine dinucleotide) domains, cryptochromes, and phytochromes, enabling control of versatile cellular processes. We analyze the principles of their function and practical requirements for use. We focus mainly on optical tools with demonstrated use beyond bacteria, with a specific emphasis on their applications in mammalian cells.
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Técnicas Biosensibles/métodos , Proteínas Luminiscentes/química , Optogenética , Arabidopsis/química , Deinococcus/química , Proteínas Luminiscentes/genética , Fitocromo/química , Ingeniería de Proteínas , Rhodopseudomonas/químicaRESUMEN
Increasing evidence has shown that population dynamics are qualitatively different from single-cell behaviors. Reporters to probe dynamic, single-cell behaviors are desirable yet relatively scarce. Here, we describe an easy-to-implement and generalizable technology to generate reporters of kinase activity for individual cells. Our technology converts phosphorylation into a nucleocytoplasmic shuttling event that can be measured by epifluorescence microscopy. Our reporters reproduce kinase activity for multiple types of kinases and allow for calculation of active kinase concentrations via a mathematical model. Using this technology, we made several experimental observations that had previously been technicallyunfeasible, including stimulus-dependent patterns of c-Jun N-terminal kinase (JNK) and nuclear factor kappa B (NF-κB) activation. We also measured JNK, p38, and ERK activities simultaneously, finding that p38 regulates the peak number, but not the intensity, of ERK fluctuations. Our approach opens the possibility of analyzing a wide range of kinase-mediated processes in individual cells.
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Técnicas Biosensibles/métodos , Fosfotransferasas/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Quinasas JNK Activadas por Mitógenos/química , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Ratones , Datos de Secuencia Molecular , Alineación de Secuencia , Análisis de la Célula IndividualRESUMEN
Transient molecules in the gastrointestinal tract such as nitric oxide and hydrogen sulfide are key signals and mediators of inflammation. Owing to their highly reactive nature and extremely short lifetime in the body, these molecules are difficult to detect. Here we develop a miniaturized device that integrates genetically engineered probiotic biosensors with a custom-designed photodetector and readout chip to track these molecules in the gastrointestinal tract. Leveraging the molecular specificity of living sensors1, we genetically encoded bacteria to respond to inflammation-associated molecules by producing luminescence. Low-power electronic readout circuits2 integrated into the device convert the light emitted by the encapsulated bacteria to a wireless signal. We demonstrate in vivo biosensor monitoring in the gastrointestinal tract of small and large animal models and the integration of all components into a sub-1.4 cm3 form factor that is compatible with ingestion and capable of supporting wireless communication. With this device, diseases such as inflammatory bowel disease could be diagnosed earlier than is currently possible, and disease progression could be more accurately tracked. The wireless detection of short-lived, disease-associated molecules with our device could also support timely communication between patients and caregivers, as well as remote personalized care.
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Biomarcadores , Técnicas Biosensibles , Sulfuro de Hidrógeno , Inflamación , Óxido Nítrico , Animales , Biomarcadores/análisis , Biomarcadores/metabolismo , Técnicas Biosensibles/instrumentación , Técnicas Biosensibles/métodos , Enfermedades Inflamatorias del Intestino/diagnóstico , Enfermedades Inflamatorias del Intestino/metabolismo , Modelos Animales , Tracto Gastrointestinal/metabolismo , Tracto Gastrointestinal/microbiología , Cápsulas/administración & dosificación , Probióticos/metabolismo , Bacterias/metabolismo , Luminiscencia , Progresión de la Enfermedad , Inflamación/diagnóstico , Inflamación/metabolismo , Óxido Nítrico/análisis , Óxido Nítrico/metabolismo , Sulfuro de Hidrógeno/análisis , Sulfuro de Hidrógeno/metabolismo , Tecnología Inalámbrica/instrumentación , Administración Oral , Tecnología de Sensores Remotos/instrumentación , Tecnología de Sensores Remotos/métodos , Factores de Tiempo , Humanos , Tamaño CorporalRESUMEN
Real-time chemical sensing is crucial for applications in environmental and health monitoring1. Biosensors can detect a variety of molecules through genetic circuits that use these chemicals to trigger the synthesis of a coloured protein, thereby producing an optical signal2-4. However, the process of protein expression limits the speed of this sensing to approximately half an hour, and optical signals are often difficult to detect in situ5-8. Here we combine synthetic biology and materials engineering to develop biosensors that produce electrical readouts and have detection times of minutes. We programmed Escherichia coli to produce an electrical current in response to specific chemicals using a modular, eight-component, synthetic electron transport chain. As designed, this strain produced current following exposure to thiosulfate, an anion that causes microbial blooms, within 2 min. This amperometric sensor was then modified to detect an endocrine disruptor. The incorporation of a protein switch into the synthetic pathway and encapsulation of the bacteria with conductive nanomaterials enabled the detection of the endocrine disruptor in urban waterway samples within 3 min. Our results provide design rules to sense various chemicals with mass-transport-limited detection times and a new platform for miniature, low-power bioelectronic sensors that safeguard ecological and human health.
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Técnicas Biosensibles , Conductividad Eléctrica , Contaminantes Ambientales , Escherichia coli , Humanos , Técnicas Biosensibles/métodos , Disruptores Endocrinos/análisis , Escherichia coli/química , Escherichia coli/metabolismo , Nanoestructuras/química , Factores de Tiempo , Contaminantes Ambientales/análisis , Biología Sintética , Transporte de Electrón , Tiosulfatos/análisis , Contaminantes del Agua/análisisRESUMEN
Naturally occurring protein switches have been repurposed for the development of biosensors and reporters for cellular and clinical applications1. However, the number of such switches is limited, and reengineering them is challenging. Here we show that a general class of protein-based biosensors can be created by inverting the flow of information through de novo designed protein switches in which the binding of a peptide key triggers biological outputs of interest2. The designed sensors are modular molecular devices with a closed dark state and an open luminescent state; analyte binding drives the switch from the closed to the open state. Because the sensor is based on the thermodynamic coupling of analyte binding to sensor activation, only one target binding domain is required, which simplifies sensor design and allows direct readout in solution. We create biosensors that can sensitively detect the anti-apoptosis protein BCL-2, the IgG1 Fc domain, the HER2 receptor, and Botulinum neurotoxin B, as well as biosensors for cardiac troponin I and an anti-hepatitis B virus antibody with the high sensitivity required to detect these molecules clinically. Given the need for diagnostic tools to track the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)3, we used the approach to design sensors for the SARS-CoV-2 spike protein and antibodies against the membrane and nucleocapsid proteins. The former, which incorporates a de novo designed spike receptor binding domain (RBD) binder4, has a limit of detection of 15 pM and a luminescence signal 50-fold higher than the background level. The modularity and sensitivity of the platform should enable the rapid construction of sensors for a wide range of analytes, and highlights the power of de novo protein design to create multi-state protein systems with new and useful functions.
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Anticuerpos Antivirales/análisis , Técnicas Biosensibles/métodos , Virus de la Hepatitis B/inmunología , SARS-CoV-2/química , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/análisis , Troponina I/análisis , Anticuerpos Antivirales/inmunología , Técnicas Biosensibles/normas , Toxinas Botulínicas/análisis , Proteínas de la Nucleocápside de Coronavirus/inmunología , Inmunoglobulina G/análisis , Inmunoglobulina G/inmunología , Límite de Detección , Luminiscencia , Fosfoproteínas/inmunología , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Receptor ErbB-2/análisis , Sensibilidad y Especificidad , Proteínas de la Matriz Viral/inmunologíaRESUMEN
We developed a significantly improved genetically encoded quantitative adenosine triphosphate (ATP) sensor to provide real-time dynamics of ATP levels in subcellular compartments. iATPSnFR2 is a variant of iATPSnFR1, a previously developed sensor that has circularly permuted superfolder green fluorescent protein (GFP) inserted between the ATP-binding helices of the ε-subunit of a bacterial F0-F1 ATPase. Optimizing the linkers joining the two domains resulted in a ~fivefold to sixfold improvement in the dynamic range compared to the previous-generation sensor, with excellent discrimination against other analytes, and affinity variants varying from 4 µM to 500 µM. A chimeric version of this sensor fused to either the HaloTag protein or a suitable spectrally separated fluorescent protein provides an optional ratiometric readout allowing comparisons of ATP across cellular regions. Subcellular targeting the sensor to nerve terminals reveals previously uncharacterized single-synapse metabolic signatures, while targeting to the mitochondrial matrix allowed direct quantitative probing of oxidative phosphorylation dynamics.
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Adenosina Trifosfato , Proteínas Fluorescentes Verdes , Animales , Humanos , Adenosina Trifosfato/análisis , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Técnicas Biosensibles/métodos , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Fluorescentes Verdes/genética , Fosforilación Oxidativa , ATPasas de Translocación de Protón/metabolismo , ATPasas de Translocación de Protón/genéticaRESUMEN
G protein-coupled receptors (GPCRs) transduce the effects of many neuromodulators including dopamine, serotonin, epinephrine, acetylcholine, and opioids. The localization of synthetic or endogenous GPCR agonists impacts their action on specific neuronal pathways. In this paper, we show a series of single-protein chain integrator sensors that are highly modular and could potentially be used to determine GPCR agonist localization across the brain. We previously engineered integrator sensors for the mu- and kappa-opioid receptor agonists called M- and K-Single-chain Protein-based Opioid Transmission Indicator Tool (SPOTIT), respectively. Here, we engineered red versions of the SPOTIT sensors for multiplexed imaging of GPCR agonists. We also modified SPOTIT to create an integrator sensor design platform called SPOTIT for all GPCRs (SPOTall). We used the SPOTall platform to engineer sensors for the beta 2-adrenergic receptor (B2AR), the dopamine receptor D1, and the cholinergic receptor muscarinic 2 agonists. Finally, we demonstrated the application of M-SPOTIT and B2AR-SPOTall in detecting exogenously administered morphine, isoproterenol, and epinephrine in the mouse brain via locally injected viruses. The SPOTIT and SPOTall sensor design platform has the potential for unbiased agonist detection of many synthetic and endogenous neuromodulators across the brain.
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Receptores Acoplados a Proteínas G , Animales , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Humanos , Ratones , Células HEK293 , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D1/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Receptores Adrenérgicos beta 2/genética , Receptor Muscarínico M2/agonistas , Receptor Muscarínico M2/metabolismo , Isoproterenol/farmacología , Receptores Opioides mu/agonistas , Receptores Opioides mu/metabolismo , Morfina/farmacología , Encéfalo/metabolismo , Encéfalo/efectos de los fármacos , Encéfalo/diagnóstico por imagen , Receptores Opioides kappa/agonistas , Receptores Opioides kappa/metabolismo , Técnicas Biosensibles/métodosRESUMEN
Real-time visualization of a wide range of biochemical processes in living systems is being made possible through the development and application of genetically encoded fluorescent reporters. These versatile biosensors have proven themselves tailor-made to the study of signal transduction, and in this review, we discuss some of the unique insights that they continue to provide regarding the spatial organization and dynamic regulation of intracellular signaling networks. In addition, we explore the more recent push to expand the scope of biological phenomena that can be monitored using these reporters, while also considering the potential to integrate this highly adaptable technology with a number of emerging techniques that may significantly broaden our view of how networks of biochemical processes shape larger biological phenomena.
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Técnicas Biosensibles/métodos , Transferencia Resonante de Energía de Fluorescencia/métodos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Transducción de Señal/fisiología , Animales , Calcio/metabolismo , Colorantes Fluorescentes/química , Colorantes Fluorescentes/metabolismo , Proteínas Fluorescentes Verdes/química , Membranas Intracelulares/química , Modelos MolecularesRESUMEN
A grand challenge in biosensor design is to develop a single-molecule, fluorescent protein-based platform that can be easily adapted to recognize targets of choice. Here, we created a family of adaptable, turn-on maturation (ATOM) biosensors consisting of a monobody (circularly permuted at one of two positions) or a nanobody (circularly permuted at one of three positions) inserted into a fluorescent protein at one of three surface loops. Multiplexed imaging of live human cells coexpressing cyan, yellow and red ATOM sensors detected biosensor targets that were specifically localized to various subcellular compartments. Fluorescence activation involved ligand-dependent chromophore maturation with turn-on ratios of up to 62-fold in cells and 100-fold in vitro. Endoplasmic reticulum- and mitochondria-localized ATOM sensors detected ligands that were targeted to those organelles. The ATOM design was validated with three monobodies and one nanobody inserted into distinct fluorescent proteins, suggesting that customized ATOM sensors can be generated quickly.
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Técnicas Biosensibles , Proteínas , Humanos , Transferencia Resonante de Energía de Fluorescencia/métodos , Colorantes Fluorescentes/metabolismo , Técnicas Biosensibles/métodosRESUMEN
Fluorescent carbon nanomaterials have broadly useful chemical and photophysical attributes that are conducive to applications in biology. In this review, we focus on materials whose photophysics allow for the use of these materials in biomedical and environmental applications, with emphasis on imaging, biosensing, and cargo delivery. The review focuses primarily on graphitic carbon nanomaterials including graphene and its derivatives, carbon nanotubes, as well as carbon dots and carbon nanohoops. Recent advances in and future prospects of these fields are discussed at depth, and where appropriate, references to reviews pertaining to older literature are provided.
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Técnicas Biosensibles , Grafito , Nanoestructuras , Nanotubos de Carbono , Colorantes Fluorescentes , Técnicas Biosensibles/métodos , Diagnóstico por ImagenRESUMEN
In the era of Internet-of-things, many things can stay connected; however, biological systems, including those necessary for human health, remain unable to stay connected to the global Internet due to the lack of soft conformal biosensors. The fundamental challenge lies in the fact that electronics and biology are distinct and incompatible, as they are based on different materials via different functioning principles. In particular, the human body is soft and curvilinear, yet electronics are typically rigid and planar. Recent advances in materials and materials design have generated tremendous opportunities to design soft wearable bioelectronics, which may bridge the gap, enabling the ultimate dream of connected healthcare for anyone, anytime, and anywhere. We begin with a review of the historical development of healthcare, indicating the significant trend of connected healthcare. This is followed by the focal point of discussion about new materials and materials design, particularly low-dimensional nanomaterials. We summarize material types and their attributes for designing soft bioelectronic sensors; we also cover their synthesis and fabrication methods, including top-down, bottom-up, and their combined approaches. Next, we discuss the wearable energy challenges and progress made to date. In addition to front-end wearable devices, we also describe back-end machine learning algorithms, artificial intelligence, telecommunication, and software. Afterward, we describe the integration of soft wearable bioelectronic systems which have been applied in various testbeds in real-world settings, including laboratories that are preclinical and clinical environments. Finally, we narrate the remaining challenges and opportunities in conjunction with our perspectives.
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Técnicas Biosensibles , Dispositivos Electrónicos Vestibles , Humanos , Inteligencia Artificial , Técnicas Biosensibles/métodos , Electrónica , Atención a la SaludRESUMEN
Luminescence imaging is a powerful and versatile technique for investigating cell physiology and pathology in living systems, making significant contributions to life science research and clinical diagnosis. In recent years, luminescent transition metal complexes have gained significant attention for diagnostic and therapeutic applications due to their unique photophysical and photochemical properties. In this Review, we provide a comprehensive overview of the recent development of luminescent transition metal complexes for bioimaging and biosensing applications, with a focus on transition metal centers with a d6, d8, and d10 electronic configuration. We elucidate the structure-property relationships of luminescent transition metal complexes, exploring how their structural characteristics can be manipulated to control their biological behavior such as cellular uptake, localization, biocompatibility, pharmacokinetics, and biodistribution. Furthermore, we introduce the various design strategies that leverage the interesting photophysical properties of luminescent transition metal complexes for a wide variety of biological applications, including autofluorescence-free imaging, multimodal imaging, organelle imaging, biological sensing, microenvironment monitoring, bioorthogonal labeling, bacterial imaging, and cell viability assessment. Finally, we provide insights into the challenges and perspectives of luminescent transition metal complexes for bioimaging and biosensing applications, as well as their use in disease diagnosis and treatment evaluation.
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Técnicas Biosensibles , Complejos de Coordinación , Elementos de Transición , Técnicas Biosensibles/métodos , Elementos de Transición/química , Humanos , Complejos de Coordinación/química , Animales , Imagen Óptica/métodos , Sustancias Luminiscentes/química , LuminiscenciaRESUMEN
The quantum spin properties of nitrogen-vacancy defects in diamond enable diverse applications in quantum computing and communications1. However, fluorescent nanodiamonds also have attractive properties for in vitro biosensing, including brightness2, low cost3 and selective manipulation of their emission4. Nanoparticle-based biosensors are essential for the early detection of disease, but they often lack the required sensitivity. Here we investigate fluorescent nanodiamonds as an ultrasensitive label for in vitro diagnostics, using a microwave field to modulate emission intensity5 and frequency-domain analysis6 to separate the signal from background autofluorescence7, which typically limits sensitivity. Focusing on the widely used, low-cost lateral flow format as an exemplar, we achieve a detection limit of 8.2 × 10-19 molar for a biotin-avidin model, 105 times more sensitive than that obtained using gold nanoparticles. Single-copy detection of HIV-1 RNA can be achieved with the addition of a 10-minute isothermal amplification step, and is further demonstrated using a clinical plasma sample with an extraction step. This ultrasensitive quantum diagnostics platform is applicable to numerous diagnostic test formats and diseases, and has the potential to transform early diagnosis of disease for the benefit of patients and populations.
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Técnicas Biosensibles/métodos , Diagnóstico Precoz , Infecciones por VIH/diagnóstico , Infecciones por VIH/virología , VIH-1/genética , Nanodiamantes/química , ARN Viral/sangre , Avidina/química , Técnicas Biosensibles/instrumentación , Biotina/química , Fluorescencia , Oro/química , VIH-1/aislamiento & purificación , Humanos , Límite de Detección , Nanopartículas del Metal/química , Microfluídica/instrumentación , Microfluídica/métodos , Microondas , Técnicas de Amplificación de Ácido Nucleico , Papel , Plasma/virología , Teoría Cuántica , Sensibilidad y Especificidad , Imagen Individual de Molécula , TemperaturaRESUMEN
CRISPR-Cas systems with dual functions offer precise sequence-based recognition and efficient catalytic cleavage of nucleic acids, making them highly promising in biosensing and diagnostic technologies. However, current methods encounter challenges of complexity, low turnover efficiency, and the necessity for sophisticated probe design. To better integrate the dual functions of Cas proteins, we proposed a novel approach called CRISPR-Cas Autocatalysis Amplification driven by LNA-modified Split Activators (CALSA) for the highly efficient detection of single-stranded DNA (ssDNA) and genomic DNA. By introducing split ssDNA activators and the site-directed trans-cleavage mediated by LNA modifications, an autocatalysis-driven positive feedback loop of nucleic acids based on the LbCas12a system was constructed. Consequently, CALSA enabled one-pot and real-time detection of genomic DNA and cell-free DNA (cfDNA) from different tumor cell lines. Notably, CALSA achieved high sensitivity, single-base specificity, and remarkably short reaction times. Due to the high programmability of nucleic acid circuits, these results highlighted the immense potential of CALSA as a powerful tool for cascade signal amplification. Moreover, the sensitivity and specificity further emphasized the value of CALSA in biosensing and diagnostics, opening avenues for future clinical applications.
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Técnicas Biosensibles , Sistemas CRISPR-Cas , ADN de Cadena Simple , Oligonucleótidos , Humanos , Oligonucleótidos/química , Oligonucleótidos/genética , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , ADN de Cadena Simple/química , Técnicas Biosensibles/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , ADN/química , ADN/genética , Línea Celular Tumoral , CatálisisRESUMEN
Electronic detection of DNA oligomers offers the promise of rapid, miniaturized DNA analysis across various biotechnological applications. However, known all-electrical methods, which solely rely on measuring electrical signals in transducers during probe-target DNA hybridization, are prone to nonspecific electrostatic and electrochemical interactions, subsequently limiting their specificity and detection limit. Here, we demonstrate a nanomechanoelectrical approach that delivers ultra-robust specificity and a 100-fold improvement in detection limit. We drive nanostructural DNA strands tethered to a graphene transistor to oscillate in an alternating electric field and show that the transistor-current spectra are characteristic and indicative of DNA hybridization. We find that the inherent difference in pliability between unpaired and paired DNA strands leads to the spectral characteristics with minimal influence from nonspecific electrostatic and electrochemical interactions, resulting in high selectivity and sensitivity. Our results highlight the potential of high-performance DNA analysis based on miniaturized all-electronic settings.
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Técnicas Biosensibles , Grafito , ADN/química , Hibridación de Ácido Nucleico , Sondas de ADN/química , Grafito/química , Hibridación Genética , Técnicas Biosensibles/métodosRESUMEN
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.