RESUMEN
BACKGROUND: Ex vivo and in vivo detection and imaging of adenosine triphosphate (ATP) is critically important for the diagnosis and treatment of diseases, which still remains challenges up to present. RESULTS: We herein demonstrate that ATP could be fluorescently detected and imaged ex vivo and in vivo. In particular, we fabricate a kind of fluorescent ATP probes, which are made of titanium carbide (TC) nanosheets modified with the ROX-tagged ATP-aptamer (TC/Apt). In the constructed TC/Apt, TC shows superior quenching efficiency against ROX (e.g., ~ 97%). While in the presence of ATP, ROX-tagged aptamer is released from TC surface, leading to the recovery of fluorescence of ROX under the 545-nm excitation. Consequently, a wide dynamic range from 1 µM to 1.5 mM ATP and a high sensitivity with a limit of detection (LOD) down to 0.2 µM ATP can be readily achieved by the prepared TC/Apt. We further demonstrate that the as-prepared TC/Apt probe is feasible for accurate discrimination of ATP in different samples including living cells, body fluids (e.g., mouse serum, mouse urine and human serum) and mouse tumor models. CONCLUSIONS: Fluorescence detection and imaging of ATP could be readily achieved in living cells, body fluids (e.g., urine and serum), as well as mouse tumor model through a new kind of fluorescent ATP nanoprobes, offering new powerful tools for the treatment of diseases related to abnormal fluctuation of ATP concentration.
Asunto(s)
Adenosina Trifosfato/química , Adenosina Trifosfato/aislamiento & purificación , Colorantes Fluorescentes , Imagen Óptica/métodos , Animales , Técnicas Biosensibles/métodos , Líquidos Corporales , Femenino , Fluorescencia , Células HeLa , Humanos , Límite de Detección , Células MCF-7 , RatonesRESUMEN
OBJECTIVES: To develop a colorimetric assay for ATP based on the blue-pigment synthesising non-ribosomal peptide synthetase (NRPS) BpsA, and to demonstrate its utility in defining the substrate specificity of other NRPS enzymes. RESULTS: BpsA is able to convert two molecules of L-glutamine into the readily-detected blue pigment indigoidine, consuming two molecules of ATP in the process. We showed that the stoichiometry of this reaction is robust and that it can be performed in a microplate format to accurately quantify ATP concentrations to low micromolar levels in a variety of media, using a spectrophotometric plate-reader. We also demonstrated that the assay can be adapted to evaluate the amino acid substrate preferences of NRPS adenylation domains, by adding pyrophosphatase enzyme to drive consumption of ATP in the presence of the preferred substrate. CONCLUSIONS: The robust nature and simplicity of the reaction protocol offers advantages over existing methods for ATP quantification and NRPS substrate analysis.
Asunto(s)
Adenosina Trifosfato/aislamiento & purificación , Técnicas Biosensibles , Colorimetría , Péptido Sintasas/química , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Glutamina/química , Piperidonas/químicaRESUMEN
Surface-enhanced Raman spectroscopy (SERS) as one of the effective tools for sensitive and selective detection of biomolecules has attracted tremendous attention. Here, we construct a versatile biomolecular detection platform based on photo-induced enhanced Raman spectroscopy (PIERS) effect for ultrasensitive detection of multiple analytes. In our PIERS sensor, we exploit the molecular recognition capacity of aptamers and the high affinity of aptamers with analyte to trigger TiO2@AgNP substrates binding with Raman tag-labeled gold nanoparticles probes via analyte, thus forming sandwich complexes. Additionally, combining plasmonic nanoparticles with photo-activated substrates allows PIERS sensor to achieve increased sensitivity beyond the normal SERS effect upon ultraviolet irradiation. Accordingly, the PIERS can be implemented for analysis of multiple analytes by designing different analyte aptamers, and we further demonstrate that the constructed PIERS sensor can serve as a versatile detection platform for sensitively analyzing various biomolecules including small molecules (adenosine triphosphate (ATP), limit of detection (LOD) of 0.1â¯nM), a biomarker (thrombin, LOD of 50 pM), and a drug (cocaine, LOD of 5â¯nM). Therefore, this versatile biomolecular detection platform based on PIERS effect for ultrasensitive detection of multiple analytes holds great promise to be a practical tool.
Asunto(s)
Adenosina Trifosfato/aislamiento & purificación , Técnicas Biosensibles , Nanopartículas del Metal/química , Trombina/aislamiento & purificación , Adenosina Trifosfato/química , Aptámeros de Nucleótidos/química , Oro/química , Límite de Detección , Espectrometría Raman , Trombina/químicaRESUMEN
Four fluorescent DNA-stabilized fluorescent silver nanoclusters (DNA-AgNCs) were designed and synthesized with differences in lengths of cytosine-rich DNA strand (as the stabilizing agent) and target-specific strand DNA aptamers for adenosine triphosphate (ATP) and cytochrome c (Cyt c). After their nanohybrid formation with graphene oxide (GO), it was unexpectedly found that, depending on the composition of the base and length of the strand DNA aptamer, the fluorescence intensity of three of the nanohybrids significantly enhanced. Our experimental observations and quantum mechanical calculations provided an insight into the mechanisms underlying the behavior of DNA-AgNCs/GO nanohybrids. The enhanced fluorescence was found to be attributed to the aggregation-induced emission enhancement (AIE) characteristic of the DNA-AgNCs adsorbed on the GO surface, as confirmed evidently by both fluorescence and transmission electron microscopies. The AIE is a result of hardness and oxidation properties of GO, which lead to enhanced argenophilic interaction and thus to increased Ag(I)-DNA complex shell aggregation. Consequently, two of the DNA-AgNCs/GO nanohybrids were successfully extended to construct highly selective, sensitive, label-free, and simple aptasensors for biosensing of ATP (LOD = 0.42 nM) and Cyt c (LOD = 2.3 nM) in lysed Escherichia coli DH5 α cells and mouse embryonic stem cells, respectively. These fundamental findings are expected to significantly influence the designing and engineering of new AgNCs/GO-based AIE biosensors.
Asunto(s)
Adenosina Trifosfato/aislamiento & purificación , Aptámeros de Nucleótidos/química , Técnicas Biosensibles , Citocromos c/aislamiento & purificación , Adenosina Trifosfato/química , Animales , Citocromos c/química , Escherichia coli/química , Grafito/química , Nanopartículas del Metal/química , Ratones , Células Madre Embrionarias de Ratones/químicaRESUMEN
In this study, we have developed a method to assess adenosine 5'-triphosphate by adsorptive extraction using surface adenosine 5'-triphosphate-imprinted polymer over polystyrene nanoparticles (412 ± 16 nm) for selective recognition/separation from urine. Molecularly imprinted polymer was synthesized by emulsion copolymerization reaction using adenosine 5'-triphosphate as a template, functional monomers (methacrylic acid, N-isopropyl acrylamide, and dimethylamino ethylmethacrylate) and a crosslinker, methylenebisacrylamide. The binding capacities of imprinted and non-imprinted polymers were measured using high-performance liquid chromatography with UV detection with a detection limit of 1.6 ± 0.02 µM of adenosine 5'-triphosphate in the urine. High binding affinity (QMIP , 42.65 µmol/g), and high selectivity and specificity to adenosine 5'-triphosphate compared to other competitive nucleotides including adenosine 5'-diphosphate, adenosine 5'-monophosphate, and analogs such as adenosine, adenine, uridine, uric acid, and creatinine were observed. The imprinting efficiency of imprinted polymer is 2.11 for urine (QMIP , 100.3 µmol/g) and 2.51 for synthetic urine (QMIP , 48.5 µmol/g). The extraction protocol was successfully applied to the direct extraction of adenosine 5'-triphosphate from spiked human urine indicating that this synthesized molecularly imprinted polymer allowed adenosine 5'-triphosphate to be preconcentrated while simultaneously interfering compounds were removed from the matrix. These submicron imprinted polymers over nano polystyrene spheres have a potential in the pharmaceutical industries and clinical analysis applications.
Asunto(s)
Adenosina Trifosfato/química , Adenosina Trifosfato/aislamiento & purificación , Impresión Molecular , Nanosferas/química , Polímeros/química , Poliestirenos/química , Adsorción , Biomarcadores/orina , Industria Farmacéutica , Estructura Molecular , Polímeros/síntesis química , Propiedades de SuperficieRESUMEN
Electrochemical aptamer receptor/transducer systems are key elements of emerging E-AB sensors (aptasensor) used for the detection of various kinds of targets. However, the performance of these amperometric sensors is often limited by the low density of receptors attached to the sensor surface and high background signals. In the present work, interdigitated organic electrochemical transistors (iOECT) were used as a transducer to enhance the sensitivity and dynamic detection range of aptasensors. Therefore, the electrode of an amperometric sensor was utilized as gate electrode to operate the iOECT. This device was used to detect the low weight target molecule adenosine triphosphate (ATP), a common biomarker, which plays an important role for cardiovascular, neurodegenerative, and immune deficiency diseases. The novel aptasensor can selectively detect ATP with ultrahigh sensitivity down to the concentration of 10 pM, which is four orders of magnitude lower than the detection limit of the same aptasensor using an amperometric transducer principle (limit-of-detection of 106â¯nM) and most other previously reported electrochemical sensors. Furthermore, sensor regeneration was demonstrated, which facilitates reusability of OECT aptasensors. The small device size in combination with high transconductances paves the way for the development of highly sensitive integrated micro-biosensors for point-of-care applications.
Asunto(s)
Adenosina Trifosfato/aislamiento & purificación , Aptámeros de Nucleótidos/química , Técnicas Biosensibles , Técnicas Electroquímicas , Adenosina Trifosfato/química , Electrodos , Oro , Humanos , Límite de DetecciónRESUMEN
This work illustrates the highly selective fluorescence detection of ATP in the presence of other competing anions, such as AMP, ADP, PPi and other phosphates by using a set of hydroxide-bridged dizinc(ii) complexes offering a cavity lined with hydrogen bonds and other interactive forces. ATP, as a whole, was recognized by the synergic combination of Zn-phosphate bonding, ππ stacking between the adenine ring of ATP and the pyridine ring of the dizinc complex and hydrogen bonding interactions that modulate the cavity structure of the dizinc complexes.
Asunto(s)
Adenosina Difosfato/química , Adenosina Monofosfato/química , Adenosina Trifosfato/química , Complejos de Coordinación/química , Difosfatos/química , Hidróxidos/química , Zinc/química , Adenosina Difosfato/aislamiento & purificación , Adenosina Monofosfato/aislamiento & purificación , Adenosina Trifosfato/aislamiento & purificación , Cristalografía por Rayos X , Difosfatos/aislamiento & purificación , Fluorescencia , Enlace de Hidrógeno , Modelos Moleculares , Estructura MolecularRESUMEN
Purinergic signals, such as extracellular adenosine triphosphate (ATP) and adenosine diphosphate (ADP), mediate intercellular communication and stress responses throughout mammalian tissues, but the dynamics of their release and clearance are still not well understood. Although physiochemical methods provide important insight into physiology, genetically encoded optical sensors have proven particularly powerful in the quantification of signaling in live specimens. Indeed, genetically encoded luminescent and fluorescent sensors provide new insights into ATP-mediated purinergic signaling. However, new tools to detect extracellular ADP are still required. To this end, in this study, we use protein engineering to generate a new genetically encoded sensor that employs a high-affinity bacterial ADP-binding protein and reports a change in occupancy with a change in the Förster-type resonance energy transfer (FRET) between cyan and yellow fluorescent proteins. We characterize the sensor in both protein solution studies, as well as live-cell microscopy. This new sensor responds to nanomolar and micromolar concentrations of ADP and ATP in solution, respectively, and in principle it is the first fully-genetically encoded sensor with sufficiently high affinity for ADP to detect low levels of extracellular ADP. Furthermore, we demonstrate that tethering the sensor to the cell surface enables the detection of physiologically relevant nucleotide release induced by hypoosmotic shock as a model of tissue edema. Thus, we provide a new tool to study purinergic signaling that can be used across genetically tractable model systems.
Asunto(s)
Adenosina Difosfato/aislamiento & purificación , Adenosina Trifosfato/aislamiento & purificación , Técnicas Biosensibles , Edema/diagnóstico , Adenosina Difosfato/química , Adenosina Trifosfato/química , Comunicación Celular/genética , Edema/genética , Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes/química , Humanos , Proteínas Luminiscentes/química , Presión Osmótica , Unión Proteica/genéticaRESUMEN
Many chemical reactions in the cell are thermodynamically unfavorable. To overcome this barrier, the energy released from the hydrolysis of adenosine triphosphate (ATP) is coupled to these reactions via ATP hydrolyzing enzymes known as ATPases. These enzymes are ubiquitous in nature and frequently act as molecular motors in processes ranging from DNA replication to protein degradation. Assays that characterize ATPase activity in vitro are important tools to gain insight into their functions in vivo. Here, we describe a direct and flexible thin-layer chromatography method for detecting ATPase activity using radiolabeled ATP. Additionally, we describe a high-throughput coupled reaction assay pairing ATP hydrolysis with nicotinamide adenine dinucleotide (NADH) oxidation to monitor ATP hydrolysis in real time.
Asunto(s)
Adenosina Trifosfato/aislamiento & purificación , Ensayos Analíticos de Alto Rendimiento/métodos , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Cromatografía en Capa Delgada/métodos , Hidrólisis , NAD/metabolismo , Oxidación-Reducción , TermodinámicaRESUMEN
The need for technologies to monitor cell health is increasing with advancements in the field of cell therapy and regenerative medicine. In this study, we demonstrated unlabeled optical metabolic imaging of cultured living cells. This imaging technique is based on motion vector analysis with a block-matching algorithm to compare sequential time-lapse images. Motion vector analysis evaluates the movement of intracellular granules observed with a phase-contrast microscope. We demonstrated that the motion speed of intracellular movement reflects adenosine triphosphate (ATP)-dependent intracellular trafficking in cells. We also confirmed that intracellular motion speed is correlated with the ATP concentrations of the cells. This assay can measure cellular viability at a single-cell level without requiring any reagents.
Asunto(s)
Rastreo Celular/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Microscopía de Contraste de Fase/métodos , Análisis de la Célula Individual/métodos , Adenosina Trifosfato/química , Adenosina Trifosfato/aislamiento & purificación , Algoritmos , Línea Celular , Movimiento Celular/genética , HumanosRESUMEN
Inspired by the design of folding greeting cards and tissue drawing covers, a photoelectrochemical (PEC) lab-on-paper device with a controllable fluid separator, producing both reaction zone and detection zone, was explored for ultrasensitive detection of adenosine 5'-triphosphate (ATP) via mimic peroxidase-transfer enhancement of photocurrent response. To realize it, the DNA1, aptamer, and DNA2 as well as the mimic peroxidase of G-quadruplex/hemin modified Au nanocubes were linked on the graphene oxide-functionalized reaction zone via the DNA hybridization. Meanwhile, three-dimensional CuO nanoflowers (CuO NFs) as a photoactive material with outstanding electron transfer ability and absorption of light were grown in situ on the detection zone, providing a PEC active interface. Besides, an innovative fluid separator was elaborately designed by assembling a strip of paper with a hydrophilic channel, providing an effective way to bridge the gap between the two zones with a controllable drawing way, which could successfully avoid the signal interference caused by modifying biomolecules layer by layer on photosensitive materials. In the presence of ATP, the G-quadruplex/hemin modified in the reaction zone was dissociated due to the specific recognition of ATP with aptamer and released into the detection zone with the assistance of controllable fluid separator. The free G-quadruplex/hemin could catalyze hydrogen peroxide to generate oxygen for the consumption of photo-induced electrons from CuO NFs, which could further promote the electron-hole carriers separation efficiency, and eventually resulting in the enhancement of PEC signal. The proposed PEC lab-on-paper device could be employed for specific detection of ATP in the range from 5.0 to 3.0â¯×â¯103 nM with a detection limit of 2.1â¯nM.
Asunto(s)
Adenosina Trifosfato/aislamiento & purificación , Técnicas Biosensibles , Técnicas Electroquímicas , Adenosina Trifosfato/química , G-Cuádruplex , Grafito/química , Hemina/química , Peróxido de Hidrógeno/química , Límite de Detección , Nanoestructuras/química , Hibridación de Ácido Nucleico , Peroxidasas/química , Procesos Fotoquímicos , Puntos Cuánticos/químicaRESUMEN
Biomarkers detection at an ultra-low concentration in biofluids (blood, serum, saliva, etc.) is a key point for the early diagnosis success and the development of personalized therapies. However, it remains a challenge due to limiting factors like (i) the complexity of analyzed media, and (ii) the aspecificity detection and the poor sensitivity of the conventional methods. In addition, several applications require the integration of the primary sensors with other devices (microfluidic devices, capillaries, flasks, vials, etc.) where transducing the signal might be difficult, reducing performances and applicability. In the present work, we demonstrate a new class of optical biosensor we have developed integrating an optical waveguide (OWG) with specific plasmonic surfaces. Exploiting the plasmonic resonance, the devices give consistent results in surface enhanced Raman spectroscopy (SERS) for continuous and label-free detection of biological compounds. The OWG allows driving optical signals in the proximity of SERS surfaces (detection area) overcoming spatial constraints, in order to reach places previously optically inaccessible. A rutile prism couples the remote laser source to the OWG, while a Raman spectrometer collects the SERS far field scattering. The present biosensors were implemented by a simple fabrication process, which includes photolithography and nanofabrication. By using such devices, it was possible to detect cell metabolites like Phenylalanine (Phe), Adenosine 5-triphosphate sodium hydrate (ATP), Sodium Lactate, Human Interleukin 6 (IL6), and relate them to possible metabolic pathway variation.
Asunto(s)
Técnicas Biosensibles/métodos , Óptica y Fotónica/métodos , Espectrometría Raman/métodos , Adenosina/química , Adenosina/aislamiento & purificación , Adenosina Trifosfato/química , Adenosina Trifosfato/aislamiento & purificación , Humanos , Interleucina-6/química , Interleucina-6/aislamiento & purificación , Dispositivos Laboratorio en un Chip , Límite de Detección , Fenilalanina/química , Fenilalanina/aislamiento & purificación , Lactato de Sodio/química , Lactato de Sodio/aislamiento & purificación , Resonancia por Plasmón de Superficie , Propiedades de SuperficieRESUMEN
A fluorescence-based enzymatic microplate intracellular glucose assay was designed and fully validated. The method was tested in a hepatocellular cancer cell line (HepG2). Our novel one-step extraction reagent gave stable cell lysates for glucose, adenosine triphosphate (ATP), and total protein determination from the same sample. Limit of detection for glucose was 0.13 µM (26 pmol/well), which is superior to commercially available glucose assays. Both intra- and interday assay imprecision in HepG2 cultures were less than 12% coefficient of variance (CV). In cell lysates spiked with glucose, recovery at two levels varied between 83.70% and 91.81%, and both linearity and stability were acceptable. HepG2 cells treated with agents affecting glucose uptake/metabolism (phloretin, quercetin, quercetin-3'-sulfate, NaF, 3-bromopyruvate, NaN3, oligomycin A, ochratoxin A, cytochalasin B, and anti-GLUT1 antibody) showed dose-dependent changes in glucose and ATP levels without total protein (cell) loss. Finally, we performed flow cytometric glucose uptake measurement in the treated cells using 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose fluorescent glucose analog. Glucose uptake did not always mirror the intracellular glucose levels, which most likely reflects the differences between the two methodologies. However, interpreting data obtained by both methods and taking ATP/protein levels at the same time, one can get information on the mode of action of the compounds.
Asunto(s)
Adenosina Trifosfato/análisis , Glucosa/análisis , Hepatocitos/química , Espectrometría de Fluorescencia/métodos , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/aislamiento & purificación , Adenosina Trifosfato/metabolismo , Transporte Biológico , Citometría de Flujo , Colorantes Fluorescentes/química , Colorantes Fluorescentes/metabolismo , Glucosa/aislamiento & purificación , Glucosa/metabolismo , Células Hep G2 , Hepatocitos/metabolismo , Humanos , Indicadores y Reactivos , Límite de Detección , Proteínas/análisis , Proteínas/aislamiento & purificaciónRESUMEN
We report a series of Cu(II) and Zn(II) complexes with different ligands containing a dipicolyl unit functionalized with urea groups that may contain or not a phenylboronic acid function. These complexes were designed for the recognition of phosphorylated anions through coordination to the metal ion reinforced by hydrogen bonds involving the anion and NH groups of urea. The complexes were isolated and several adducts with pyrophosphate were characterized using Xray diffraction measurements. Coordination of one of the urea nitrogen atoms to the metal ion promoted the hydrolysis of the ligands containing 1,3-diphenylurea units, while ligands bearing 1-ethyl-3-phenylurea groups did not hydrolyze significantly at room temperature. Spectrophotometric titrations, combined with ¹H and 31P NMR studies, were used in investigating the binding of phosphate, pyrophosphate (PPi), and nucleoside 5'-polyphosphates (AMP, ADP, ATP, CMP, and UMP). The association constants determined in aqueous solution (pH 7.0, 0.1 M MOPS) point to a stronger association with PPi, ADP, and ATP as compared with the anions containing a single phosphate unit. The [CuL4]2+ complex shows important selectivity for pyrophosphate (PPi) over ADP and ATP.
Asunto(s)
Ácidos Borónicos/química , Complejos de Coordinación/química , Urea/química , Adenosina Difosfato/química , Adenosina Difosfato/aislamiento & purificación , Adenosina Monofosfato/química , Adenosina Monofosfato/aislamiento & purificación , Adenosina Trifosfato/química , Adenosina Trifosfato/aislamiento & purificación , Aniones/química , Cobre/química , Difosfatos/química , Ligandos , Espectroscopía de Resonancia Magnética , Estructura Molecular , Fosfatos , Ácidos Picolínicos/química , Agua/química , Zinc/químicaRESUMEN
In this work, single Au nanowire electrodes (AuNWEs) were fabricated by laser-assisted pulling/hydrofluoric acid (HF) etching process, which then were characterized by transmission electron microscopy (TEM), electrochemical method and finite-element simulation. The as-prepared single AuNWEs were used to construct electrochemical aptamer-based nanosensors (E-AB nanosensors) based on the formation of Au-S bond that duplex DNA tagged with methylene blue (MB) was modified on the surface of electrode. In the presence of adenosine triphosphate (ATP), the MB-labeled aptamer dissociated from the duplex DNA due to the strong specific affinity between aptamer and target, which lead to the reduction of MB electrochemical signals. Moreover, BSA was employed to further passivate electrode surface bonding sites for the stable of the sensor. The as-prepared E-AB nanosensor has been used for ATP assay with excellent sensitivity and selectivity, even in a complex system like cerebrospinal fluid of rat brain. Considering the unique properties of good stability, larger surface area and smaller overall dimensions, this E-AB nanosensor should be an ideal platform for widely sensing applications in living bio-system.
Asunto(s)
Adenosina Trifosfato/aislamiento & purificación , Técnicas Biosensibles , Técnicas Electroquímicas , Adenosina Trifosfato/química , Aptámeros de Nucleótidos/química , Oro/química , Nanocables/químicaRESUMEN
Stimuli-responsive carriers have extensively attracted attention in recent years. However, long-term and real-time tracking ability with stimuli-responsive carrier is still in its infant stage due to the limitations such as, low efficacy, instability and cytotoxicity in a bio-environment. In this work, we developed a reduction-sensitive carrier composed of lipoic acid-modified low molecular weight polyethyleneimine (LA-PEI) and large surface ratio MoS2 nanosheet integrated via disulfide bond to mimic a high molecular weight PEI. The positively charged carriers loading negatively charged aptamer enter the cells for a real time long-term tracking of adenosine triphosphate (ATP) metabolism in glioma stem cells (GSCs) when stimulated by TGFß factor secreted from HUVECs. We envision that MoS2-LA-PEI carrier has a promising potential for delivery and monitoring the changes in live cells with low cytotoxicity and high efficiency.
Asunto(s)
Adenosina Trifosfato/metabolismo , Técnicas Biosensibles , Células Endoteliales/metabolismo , Glioma/metabolismo , Adenosina Trifosfato/aislamiento & purificación , Técnicas de Cocultivo , Células Endoteliales/patología , Glioma/patología , Células HeLa , Células Endoteliales de la Vena Umbilical Humana , Humanos , Dispositivos Laboratorio en un Chip , Nanocompuestos/química , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Polietileneimina/química , Factor de Crecimiento Transformador beta1/química , Factor de Crecimiento Transformador beta1/genéticaRESUMEN
Bioluminescence has been widely used for important biosensing applications such as the measurement of adenosine triphosphate (ATP), the energy unit in biological systems and an indicator of vital processes. The current technology for detection is mainly based on large equipment such as readers and imaging systems, which require intensive and time-consuming procedures. A miniaturised bioluminescence sensing system, which would allow sensitive and continuous monitoring of ATP, with an integrated and low-cost disposable microfluidic chamber for handling of biological samples, is highly desirable. Here, we report the design, fabrication and testing of 3D printed microfluidics chips coupled with silicon photomultipliers (SiPMs) for high sensitive real-time ATP detection. The 3D microfluidic chip reduces reactant consumption and facilitates solution delivery close to the SiPM to increase the detection efficiency. Our system detects ATP with a limit of detection (LoD) of 8nM and an analytical dynamic range between 15nM and 1µM, showing a stability error of 3%, and a reproducibility error below of 20%. We demonstrate the dynamic monitoring of ATP in a continuous-flow system exhibiting a fast response time, ~4s, and a full recovery to the baseline level within 17s. Moreover, the SiPM-based bioluminescence sensing system shows a similar analytical dynamic range for ATP detection to that of a full-size PerkinElmer laboratory luminescence reader.
Asunto(s)
Adenosina Trifosfato/aislamiento & purificación , Técnicas Biosensibles , Técnicas Analíticas Microfluídicas/métodos , Adenosina Trifosfato/química , Dispositivos Laboratorio en un Chip , Mediciones Luminiscentes , Impresión , Silicio/químicaRESUMEN
The construction of reliable sensors for adenosine triphosphate (ATP) detection gains increasing interest because of its important roles in various enzymatic activities and biological processes. Based on a cascaded, significant signal amplification approach by the integration of the aptazymes and catalytic hairpin assembly (CHA), we have developed a sensitive electrochemical sensor for the detection of ATP. The target ATP leads to the conformational change of the aptazyme sequences and their association with the hairpin substrates to form active aptazymes, in which the hairpin substrates are cyclically cleaved by the metal ion cofactors in buffer to release the enzymatic sequences that can also bind the hairpin substrates to generate active DNAzymes. The catalytic cleavage of the hairpin substrates in the aptazymes/DNAzymes thus results in the generation of a large number of intermediate sequences. Subsequently, these intermediate sequences trigger catalytic capture of many methylene blue-tagged signal sequences on the electrode surface through CHA, producing significantly amplified current response for sensitive detection of ATP at 0.6nM. Besides, the developed sensor can discriminate ATP from analogous interference molecules and be applied to human serum samples, making the sensor a useful addition to the arena for sensitive detection of small molecules.
Asunto(s)
Adenosina Trifosfato/aislamiento & purificación , Técnicas Biosensibles , ADN Catalítico/química , Técnicas Electroquímicas , Adenosina Trifosfato/química , Aptámeros de Nucleótidos/química , ADN Catalítico/genética , Humanos , Secuencias Invertidas Repetidas/genética , Límite de Detección , Conformación de Ácido NucleicoRESUMEN
Electrochemical aptamer (EA) sensors based on aptamer-cDNA duplex probes (cDNA: complementary DNA) and target induced strand displacement (TISD) recognition are sensitive, selective and capable of detecting a wide variety of target analytes. While substantial research efforts have focused on engineering of new signaling mechanisms for the improvement of sensor sensitivity, little attention was paid to the enhancement of sensor response rate. Typically, the previous TISD based EA sensors exhibited relatively long response times larger than 30min, which mainly resulted from the suboptimal aptamer-cDNA probe structure in which most of aptamer bases were paired to the cDNA bases. In an effort to improve the response rate of this type of sensors, we report here the rational engineering of a quickly responsive and sensitive aptamer-cDNA probe by employing the conception of bivalent interaction in supramolecular chemistry. We design a bivalent cDNA strand through linking two short monovalent cDNA sequences, and it is simultaneously hybridized to two electrode-immobilized aptamer probes to form a bivalent binding (BB) aptamer-cDNA probe. This class of BB probe possesses the advantages of less aptamer bases paired to the cDNA bases for quick response rate and good structural stability for high sensor sensitivity. By use of the rationally designed BB aptamer-cDNA probe, a TISD based EA sensor against ATP with significantly enhanced response rate (with a displacement equilibrium time of 4min) and high sensitivity was successfully constructed. We believe that our BB probe conception will help guide future designs and applications of TISD based EA sensors.
Asunto(s)
Adenosina Trifosfato/aislamiento & purificación , Aptámeros de Nucleótidos/química , Técnicas Biosensibles , Técnicas Electroquímicas , Adenosina Trifosfato/química , ADN Complementario/químicaRESUMEN
Mitochondria are essential for the production and maintenance of ATP in the eukaryotic cell. To image and monitor intracellular ATP level without cell breakage, biological and chemical sensors were developed in the last years. Here, we have internalized a rhodamine-based sensor RSL+ into living cells and monitored the mitochondrial ATP levels in cultured mouse embryonic fibroblasts. To evaluate the robustness of the sensor we imaged the changes of the mitochondrial ATP levels under non-physiological conditions upon incubation with FCCP, oligomycin, azide, deoxyglucose or phosphoenolpyruvate; all compounds that interfere with ATP homeostasis of the cell. The ATP sensor allowed us to determine the mitochondrial ATP levels in human skin fibroblasts where we observe a similar amount of ATP compared to mouse embryonic fibroblasts. We propose the RSL+ to be a valuable tool for the assessment of mitochondrial dysfunction in human cells derived from mitochondrial OXPHOS patients and for basic studies on bioenergetics metabolism.