RESUMEN
This study investigates the controlled release of α-chymotrypsin from an alginate hydrogel matrix. When protein molecules entrapped in the hydrogel matrix have a size smaller than the hydrogel pores, their hold/release from the polymer matrix are controlled by the electrostatic interaction between the guest molecules and host polymer. α-Chymotrypsin, as a model protein, was chemically modified with negatively charged species to change its pI and to convert its attractive interaction with a negatively charged alginate hydrogel matrix to a repulsion interaction allowing its release by pH-triggered signal. Then, bulk pH changes and electrochemically controlled local pH changes resulting from oxygen reduction were used for the controlled release of the enzyme from the alginate hydrogel. Three batches of modified α-chymotrypsin with different linker/enzyme ratios were synthesized, and their release profiles were investigated. The activity of both unmodified and modified α-chymotrypsin was evaluated using a UV-visible spectrophotometer following the standard procedure for the enzymatic assay of α-chymotrypsin (EC 3.4.21.1) and compared across all batches. Direct infusion electrospray ionization mass spectrometry (DI ESI-MS) was used to analyze the protein modifications and their impact on the isoelectric point values.
Asunto(s)
Alginatos , Quimotripsina , Hidrogeles , Quimotripsina/química , Concentración de Iones de Hidrógeno , Alginatos/química , Hidrogeles/química , Técnicas ElectroquímicasRESUMEN
ß-Glucosidase (EC 3.2.1.21) from sweet almond was encapsulated into pH-responsive alginate-polyethylenimine (alginate-PEI) hydrogel. Then, electrochemically controlled cyclic local pH changes resulting from ascorbate oxidation (acidification) and oxygen reduction (basification) were used for the pulsatile release of the enzyme from the composite hydrogel. Activation of the enzyme was controlled by the very same pH changes used for ß-glucosidase release, separating these two processes in time. Importantly, the activity of the enzyme, which had not been released yet, was inhibited due to the buffering effect of PEI present in the gel. Thus, only a portion of the released enzyme was activated. Both enzymatic activity and release were monitored by confocal fluorescence microscopy and regular fluorescent spectroscopy. Namely, commercially available very little or nonfluorescent substrate 4-methylumbelliferyl-ß-d-glucopyranoside was hydrolyzed by ß-glucosidase to produce a highly fluorescent product 4-methylumbelliferone during the activation phase. At the same time, labeling of the enzyme with rhodamine B isothiocyanate was used for release observation. The proposed work represents an interesting smart release-activation system with potential applications in biomedical field.
Asunto(s)
Alginatos , Hidrogeles , Polietileneimina , beta-Glucosidasa , Alginatos/química , Hidrogeles/química , Polietileneimina/química , Concentración de Iones de Hidrógeno , beta-Glucosidasa/metabolismo , beta-Glucosidasa/química , Rodaminas/química , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/metabolismo , Himecromona/química , Activación Enzimática/efectos de los fármacos , Prunus/enzimología , Prunus/química , Ácido Glucurónico/química , Técnicas ElectroquímicasRESUMEN
A metal-organic framework (MOF), ZIF-8, which is stable at neutral and slightly basic pH values in aqueous solutions and destabilized/dissolved under acidic conditions, is loaded with a pH-insensitive fluorescent dye, rhodamine-B isothiocyanate, as a model payload species. Then, the MOF species are immobilized at an electrode surface. The local (interfacial) pH value is rapidly decreased by means of an electrochemically stimulated ascorbate oxidation at +0.4 V (Ag/AgCl/KCl). Oxygen reduction upon switching the applied potential to -0.8 V allows to return the local pH to the neutral/basic pH, then stopping rapidly the release process. The developed method allows electrochemical control over stimulated or inhibited payload release processes from the MOF. The pH variation proceeds in a thin film of the solution near the electrode surface. The switchable release process is realized in a buffer solution and undiluted human serum. As the second option, the pH decrease stimulating the release process is achieved upon an enzymatic reaction using esterase and ester substrate. This approach potentially allows the release activation controlled by numerous enzymes assembled in complex biocatalytic cascades. It is expected that related electrochemical or biocatalytic systems can represent novel signal-responding materials with switchable features for delivering (bio)molecules within biomedical applications.
Asunto(s)
Estructuras Metalorgánicas , Humanos , Estructuras Metalorgánicas/química , Biocatálisis , Esterasas , Agua/química , Colorantes Fluorescentes , ElectrodosRESUMEN
The construction and assembly of artificial allosteric protein switches into information and energy processing networks connected to both biological and non-biological systems is a central goal of synthetic biology and bionanotechnology. However, designing protein switches with the desired input, output and performance parameters is challenging. Here we use a range of reporter proteins to demonstrate that their chimeras with duplicated receptor domains produce YES gate protein switches with large (up to 9,000-fold) dynamic ranges and fast (minutes) response rates. In such switches, the epistatic interactions between largely independent synthetic allosteric sites result in an OFF state with minimal background noise. We used YES gate protein switches based on ß-lactamase to develop quantitative biosensors of therapeutic drugs and protein biomarkers. Furthermore, we demonstrated the reconfiguration of YES gate switches into AND gate switches controlled by two different inputs, and their assembly into signalling networks regulated at multiple nodes.
RESUMEN
Protein biosensors hold a promise to transform the way we collect physiological data by enabling quantification of biomarkers outside of specialized laboratory environment. However, achieving high specificity and sensitivity in homogeneous assay format remains challenging. Here we report construction of fluorescent biosensor arrays based on artificial allosteric α-amylase-activated PQQ-dependent glucose dehydrogenase (Amy-GDH). Amy-GDH was covalently immobilized on silica nanoparticles that were then arrayed on fiberglass sheets. The activity of the biosensor was monitored using a smartphone camera via emergence of bright fluorescence (λex 365 nm) originating from reduced phenazine methosulfate upon glucose oxidation by Amy-GDH. We show that such biosensor arrays demonstrate an apparent Kd of 115 pM for α-amylase with a detection limit of 2 pM. Using the developed biosensor arrays, we were able to specifically and accurately quantify the concentration of α-amylase in biological fluids such as serum and saliva. We propose that the presented approach can enable construction of ultrasensitive point-of-care diagnostic arrays.
Asunto(s)
Técnicas Biosensibles , alfa-Amilasas , Glucosa , Saliva , Oxidación-ReducciónRESUMEN
A micro/nanoshaped system composed of alginate microspheres (microgels) decorated with silica oxide nanoparticles functionalized with nitroavidin was used for on-demand biomolecule release stimulated by different input signals. Enzymes preloaded in the microgels processed the applied signals producing either basic pH locally near the microspheres or generating H2O2 inside the hydrogel, or both simultaneously. The pH increase resulted in cleavage of the affinity bonds between nitroavidin and biotin, then releasing the latter. The H2O2 produced resulted in oxidative cleavage of cross-linking bonds in the alginate matrix, then opening pores and releasing a loaded model protein (bovine serum albumin).
Asunto(s)
Microgeles , Nanogeles , Peróxido de Hidrógeno , Alginatos/química , BiocatálisisRESUMEN
The electrochemically controlled release of proteins was studied in a Ca2+-cross-linked alginate hydrogel deposited on an electrode surface. The electrochemical oxidation of ascorbate or reduction of O2 was achieved upon applying electrical potentials +0.6 or -0.8 V (vs Ag/AgCl/KCl 3 M), respectively, resulting in decreasing or increasing pH locally near an electrode surface. The obtained local acidic solution resulted in the protonation of carboxylic groups in the alginate hydrogel and, as a result, the formation of a hydrophobic shrunken hydrogel film. Conversely, the produced alkaline local environment resulted in a hydrophilic swollen hydrogel film. The release of the proteins was effectively inhibited from the shrunk hydrogel and activated from the swollen hydrogel film. Overall, the electrochemically produced local pH changes allowed control over the biomolecule release process. While the release inhibition by applying +0.6 V was always effective and could be maintained as long as the positive potential was applied, the release activation was different depending on the protein molecular size, being more effective for smaller species, and molecule charge, being more effective for negatively charged species. The repetitive change from the inhibited to stimulated state of the biomolecule release process was obtained upon cyclic application of oxidative and reductive potentials (+0.6 V â -0.8 V). The alginate hydrogel film shrinking-swelling as well as the protein release process were studied and visualized using a confocal fluorescent microscope. In order to be observed, an external surface of the alginate film and the loaded protein molecules were labeled with different fluorescent dyes, which then produced colored fluorescent images under a confocal microscope.
Asunto(s)
Alginatos , Hidrogeles , Hidrogeles/química , Alginatos/química , Proteínas , Oxidación-Reducción , Concentración de Iones de HidrógenoRESUMEN
Precision analysis of the key biological metabolites such as L-lactate has great practical importance for many technological processes in food technology, including beverage production. Here we describe a new, highly selective, and sensitive biosensor for accurate L-lactate assay based on a combination of peroxidase-mimetic nanozymes with microbial lactate oxidase (LOx) immobilized onto the surface of a graphite-rod electrode (GE). The peroxidase-like nanozymes were synthesized using the debris of carbon microfibers (CFs) functionalized with hemin (H) and modified with gold nanoparticles (AuNPs) or platinum microparticles (PtMPs). The nanozyme formed with PtMPs as well as corresponding bioelectrodes based on it (LOx-CF-H-PtMPs/GE) is characterized by preferable catalytic and operational characteristics, so it was selected for the analysis of L-lactate content in real samples of grape must and red wine. The results of the L-lactate analysis obtained by the developed biosensors are highly correlated with a very selective spectrophotometric approach used as a reference. The developed biosensor, due to its high selectivity and sensitivity, is very prospective not only for the beverage industry and food technology, but also for clinical diagnostics and medicine, as well as in other applications where the accurate analysis of L-lactate is highly important.
Asunto(s)
Técnicas Biosensibles , Grafito , Nanopartículas del Metal , Ácido Láctico/análisis , Peroxidasa , Oro/análisis , Estudios Prospectivos , Enzimas Inmovilizadas/metabolismo , Técnicas Biosensibles/métodos , Platino (Metal) , Bebidas/análisisRESUMEN
A new nano-structured platform for fluorescent analysis using PQQ-dependent glucose dehydrogenase (PQQ-GDH) was developed, particularly using a smartphone for transduction and quantification of optical signals. The PQQ-GDH enzyme was immobilized on SiO2 nanoparticles deposited on glass microfiber filter paper, providing a high load of the biocatalytic enzyme. The platform was tested and optimized for glucose determination using a wild type of the PQQ-GDH enzyme. The analysis was based on the fluorescence generated by the reduced form of phenazine methosulfate produced stoichiometrically to the glucose concentration. The fluorescent signals were generated at separate analytical spots on the paper support under wavelength (365 nm) UV excitation. The images of the analytical spots, dependent on the glucose concentration, were obtained using a photo camera of a standard smartphone. Then, the images were processed and quantified using software installed in a smartphone. The developed biocatalytic platform is the first step to assembling a large variety of biosensors using the same platform functionalized with artificial allosteric chimeric PQQ-dependent glucose dehydrogenase activated with different analytes. The future combination of the artificial enzymes, the presently developed analytical platform, and signal processing with a smartphone will lead to novel point-of-care and end-user biosensors applicable to virtually all possible analytes.
Asunto(s)
Glucosa 1-Deshidrogenasa , Cofactor PQQ , Glucosa/análisis , Dióxido de Silicio , Teléfono InteligenteRESUMEN
Local pH changes were produced upon electrochemical reactions. Cyclic application of reductive and oxidative potentials resulted in the formation of pH waves in the form of distinct solution layers. Multiple layers with basic and acidic pH values were visualized with a fluorescence confocal microscope following fluorescence of pH-dependent dyes.
Asunto(s)
Colorantes , Electrodos , Fluorescencia , Concentración de Iones de Hidrógeno , Oxidación-ReducciónRESUMEN
The search for new nanoscale materials with predictable properties to target the timely and fast detection of toxic components in wastewater is one of the most promising directions of modern biosensorics. We have shown that TiO2 nanoparticles modified with sulfur significantly improve the main operational parameters of laccase-based electrodes when compared with controls. The nanoparticle samples were labeled as TiO2S(0.75), TiO2S(1.5), and TiO2S(3.0), in which the numbers in parentheses refer to the quantity of H2SO4 (mL) used in the synthesis. The nanoparticles and enzyme were immobilized by means of Nafion film formed on a carbon rod electrode. It was shown that the modification of Nafion film by TiO2 or TiO2S(1.5) nanoparticles does not affect the size of the nanocavities and defect structure of the main polymer matrix as revealed by positron annihilation spectroscopy. It testifies that the structural-morphological difference between the film samples is rather small, and the improving of the sensor operational parameters for TiO2S(1.5)-based laccase electrodes is connected only with the impact of sulfur doping, but not the difference in membrane properties. The developed bioelectrodes were tested for phenol analysis in real communal wastewater samples spiked with these analytes, demonstrating the high accuracy of the assay.
Asunto(s)
Técnicas Biosensibles , Nanopartículas , Técnicas Biosensibles/métodos , Electrodos , Enzimas Inmovilizadas/química , Lacasa/química , Azufre , Titanio , Aguas ResidualesRESUMEN
Bovine serum albumin (BSA), used as a model protein, was immobilized on a buckypaper electrode by formation of covalent bonds with avidin/iminobiotin or nitroavidin/biotin complexes. pH-sensitive affinity interactions between avidin and iminobiotin or between nitroavidin and biotin allowed splitting of the affinity bonds upon pH variation, thus resulting in BSA release. Local (interfacial) pH was changed electrochemically. The pH was decreased upon electrochemical oxidation of ascorbate or increased upon electrochemical reduction of O2. The local pH change resulted in the weakening of the affinity complexes, resulting in BSA release from the avidin/iminobiotin or nitroavidin/biotin systems when the pH was decreased or increased, respectively. Importantly, protein release was only observed when the number of chemical bonds with the affinity systems was decreased by blocking a part (ca. 50%) of the binding sites in avidin/nitroavidin with iminobiotin/biotin molecules missing the possibility of attaching the protein. Without this blocking effect, multiple bond formation with the protein preserved BSA at the electrode surface, by not allowing its release upon electrochemical pH change.
Asunto(s)
Avidina , Biotina , Avidina/química , Biotina/análogos & derivados , Biotina/química , Electrodos , Concentración de Iones de HidrógenoRESUMEN
Concatenated enzyme-based Boolean logic gates activated with 5 chemical input signals were analyzed with a smartphone photo camera. Simultaneous detection of 32 input combinations was conveniently performed using enzyme-modified fiberglass sensing spots generating fluorescence with different intensities for the 0 and 1 binary outputs. The developed technology offers an easy readout method for multi-channel logic systems.
Asunto(s)
Computadores Moleculares , LógicaRESUMEN
Advances in protein engineering resulted in increased efforts to create protein biosensors that can replace instrumentation-heavy analytical and diagnostic methods. Sensitivity, amenability to multiplexing, and manufacturability remain to be among the key issues preventing broad utilization of protein biosensors. Here, we attempt to address these by constructing arrays utilizing protein biosensors based on the artificial allosteric variant of PQQ-glucose dehydrogenase (GDH). We demonstrated that the silica nanoparticle-immobilized GDH protein could be deposited on fiberglass sheets without loss of activity. The particle-associated GDH activity could be monitored using changes in the fluorescence of the commonly used electron mediator phenazine methosulfate. The constructed biosensor arrays of macrocyclic immunosuppressant drugs cyclosporine A and FK-506 displayed very low background and a remarkable dynamic range exceeding 300-fold that resulted in a limit of detection of 2 pM for both analytes. This enabled us to quantify both drugs in human blood, serum, urine, and saliva. The arrays could be stored in dry form and quantitatively imaged using a smartphone camera, demonstrating the method's suitability for field and point-of-care applications. The developed approach provides a generalizable platform for biosensor array development that is compatible with inexpensive and potentially scalable manufacturing.
Asunto(s)
Técnicas Biosensibles , Tacrolimus , Técnicas Biosensibles/métodos , Ciclosporina , Glucosa 1-Deshidrogenasa/metabolismo , Humanos , Proteínas Recombinantes de FusiónRESUMEN
The biocatalytic activity of electrode-immobilized luciferase followed by bioluminescence emitted from the electrode surface was reversibly tuned and switched by applying electrochemical signals. When a reductive potential (-0.9 V vs. Ag/AgCl) was applied, O2 was consumed at the electrode resulting in its depletion in a thin film near the electrode surface. This resulted in the inhibition of the immobilized luciferase which needs O2 for the biocatalytic reaction. Releasing the potential resulted in diffusional equilibration of the O2 local concentration with the bulk solution, then reactivating luciferase. Reversible inhibition-activation of luciferase was obtained upon cyclic application and releasing of the potential, respectively.
Asunto(s)
Electroquímica , Biocatálisis , Electroquímica/métodos , Electrodos , LuciferasasRESUMEN
A nanostructured surface composed of SiO2 nanoparticles (200 nm) deposited on fiberglass was used as a platform for immobilization of NAD+-dependent enzymes (alcohol and glucose dehydrogenases). The surface functionalized with the enzymes was further loaded with NAD(P)+cofactor and 1-methoxy-5-methylphenazinium methyl sulfate (MPMS) redox species. The designed biocatalytic system was used for fluorescent analysis of substrates (ethanol and glucose used as example analyte substances). Importantly, the output analytical signal was obtained by processing fluorescent images of the sensing spots using a smartphone for handling and digitizing the images. The designed sensing interface is suggested as a universal analytical platform which can be used with many different NAD+-dependent enzymes. The present biosensing/bioanalytic system was studied as a proof-of-the-concept, rather than as a practically important ethanol or glucose biosensor. Many more practically important biosensors based on the same universal platform can be designed with different enzymes for various biomedical, forensic, and homeland security applications as well as environmental monitoring.
Asunto(s)
Técnicas Biosensibles , Teléfono Inteligente , Técnicas Biosensibles/métodos , NAD , Lectura , Dióxido de SilicioRESUMEN
Immobilized avidin-biotin complexes were used to release biotinylated (bio)molecules upon producing local pH changes near an electrode surface by electrochemical reactions. The nitro-avidin complex with biotin was dissociated by increasing local pH with electrochemical O2 reduction. The avidin complex with iminobiotin was split by decreasing local pH with electrochemical oxidation of ascorbate. Both studied systems were releasing molecule cargo species in response to small electrical potentials (-0.4 V or 0.2 V for the O2 reduction or ascorbate oxidation, respectively) applied on the modified electrodes.
Asunto(s)
Avidina , Biotina , Avidina/química , Biotina/química , Electrodos , Concentración de Iones de Hidrógeno , Oxidación-ReducciónRESUMEN
A biocatalytic cascade based on concerted operation of pyruvate kinase and luciferase with a bioluminescent output was switched reversibly between low and high activity by applying an external magnetic field at different positions or removing it. The enzymes participating in the reaction cascade were bound to magnetic nanoparticles to allow their translocation or aggregation/dispersion to be controlled by the magnetic field. The reaction intensity, measured as the bioluminescent output, was dependent on the effective distances between the enzymes transported on the magnetic nanoparticles controlled by the magnets.
Asunto(s)
Fluorescencia , Luciferasas/metabolismo , Nanopartículas de Magnetita/química , Piruvato Quinasa/metabolismo , Aliivibrio fischeri/enzimología , Animales , Biocatálisis , Luciferasas/química , Mediciones Luminiscentes , Campos Magnéticos , Piruvato Quinasa/química , ConejosRESUMEN
This review provides a general overview of different biosensors, mostly concentrating on electrochemical analytical devices, while briefly explaining general approaches to various kinds of biosensors, their construction and performance. A discussion on how all required components of biosensors are brought together to perform analytical work is offered. Different signal-transducing mechanisms are discussed, particularly addressing the immobilization of biomolecular components in the vicinity of a transducer interface and their functional integration with electronic devices. The review is mostly addressing general concepts of the biosensing processes rather than specific modern achievements in the area.
Asunto(s)
Técnicas Biosensibles , Transductores , Técnicas ElectroquímicasRESUMEN
Protein biosensors play an increasingly important role as reporters for research and clinical applications. Here we present an approach for the construction of fully integrated but modular electrochemical biosensors based on the principal component of glucose monitors PQQ-glucose dehydrogenase (PQQ-GDH). We designed allosterically regulated circular permutated variants of PQQ-GDH that show large (>10-fold) changes in enzymatic activity following intramolecular scaffolding of the newly generated N- and C termini by ligand binding domain/ligand complexes. The developed biosensors demonstrated sub-nanomolar affinities for small molecules and proteins in colorimetric and electrochemical assays. For instance, the concentration of Cyclosporineâ A could be measured in 1â µL of undiluted blood with the same accuracy as the leading diagnostic technique that uses 50 times more sample. We further used this biosensor to construct highly porous gold bioelectrodes capable of robustly detecting concentrations of Cyclosporineâ A as low as 20â pM and retained functionality in samples containing at least 60 % human serum.