RESUMO
Human chorionic gonadotropin (HCG), a vital protein for pregnancy determination and a marker for trophoblastic diseases, finds application in monitoring early pregnancy and ectopic pregnancy. This study presents an innovative approach employing electrochemical immunosensors for enhanced HCG detection, utilizing Anti-HCG antibodies and gold nanoparticles (AuNPs) in the sensor platform. Two sensor configurations were optimized: BSA/Anti-HCG/c-AuNPs/MEL/e-AuNPs/SPCE with [Fe(CN)6]3-/4- as a redox probe (1) and BSA/Anti-HCG/PPy/e-AuNPs/SPCE using polypyrrole (PPy) as a redox probe (2). The first sensor offers linear correlation in the 0.10-500.00 pgâmL-1 HCG range, with a limit of detection (LOD) of 0.06 pgâmL-1, sensitivity of 32.25 µAâpg-1âmLâcm-2, RSD <2.47 %, and a recovery rate of 101.03-104.81 %. The second sensor widens the HCG detection range (40.00 fgâmL-1-5.00 pgâmL-1) with a LOD of 16.53 fgâmL-1, ensuring precision (RSD <1.04 %) and a recovery range of 94.61-106.07 % in serum samples. These electrochemical immunosensors have transformative potential in biomarker detection, offering enhanced sensitivity, selectivity, and stability for advanced healthcare diagnostics.
Assuntos
Técnicas Biossensoriais , Gonadotropina Coriônica , Técnicas Eletroquímicas , Ouro , Limite de Detecção , Nanopartículas Metálicas , Polímeros , Pirróis , Gonadotropina Coriônica/sangue , Gonadotropina Coriônica/análise , Gonadotropina Coriônica/imunologia , Ouro/química , Humanos , Nanopartículas Metálicas/química , Técnicas Eletroquímicas/métodos , Técnicas Biossensoriais/métodos , Polímeros/química , Pirróis/química , Imunoensaio/métodos , Imunoensaio/instrumentação , Ferricianetos/química , FemininoRESUMO
It was previously reported that stress induces a cellular production of abscisic acid in plants, but no direct method shows the evidence. Here, an electrochemical microsensor involving an abscisic acid receptor PYL2 modified carbon fiber microelectrode was fabricated by self-assembly method, where the Cu2+ combined with the histidine tag of PYL2 on the surface of microelectrode was used as the detection probe, the mediated reaction between Cu+ and ferricyanide realized the amplification responses and provided the microsensor with a high sensitivity for detection of abscisic acid with a detection limit of 0.8 nM. With use of this microsensor, an increase of extracellular abscisic acid from single rice protoplast induced by sulfate, osmotic and salinity stress was real-time monitored. Direct measurement of free extracellular abscisic acid in single plant cells might offer important new insights into its role in plants challenged by abiotic stresses.
Assuntos
Ácido Abscísico , Microeletrodos , Oryza , Proteínas de Plantas , Protoplastos , Oryza/metabolismo , Oryza/química , Ácido Abscísico/metabolismo , Protoplastos/metabolismo , Proteínas de Plantas/metabolismo , Técnicas Biossensoriais/métodos , Técnicas Biossensoriais/instrumentação , Cobre/metabolismo , Técnicas Eletroquímicas/métodos , Técnicas Eletroquímicas/instrumentação , Ferricianetos/química , Ferricianetos/metabolismoRESUMO
The non-pathogenic ß-proteobacterium Cupriavidus necator has the ability to switch between chemoorganotrophic, chemolithoautotrophic and electrotrophic growth modes, making this microorganism a widely used host for cellular bioprocesses. Oxygen usually acts as the terminal electron acceptor in all growth modes. However, several challenges are associated with aeration, such as foam formation, oxygen supply costs, and the formation of an explosive gas mixture in chemolithoautotrophic cultivation with H2, CO2 and O2. Bioelectrochemical systems in which O2 is replaced by an electrode as a terminal electron acceptor offer a promising solution to these problems. The aim of this study was to establish a mediated electron transfer between the anode and the metabolism of living cells, i.e. anodic respiration, using fructose as electron and carbon source. Since C. necator is not able to transfer electrons directly to an electrode, redox mediators are required for this process. Based on previous observations on the extracellular electron transfer enabled by a polymeric mediator, we tested 11 common biological and non-biological redox mediators for their functionality and inhibitory effect for anodic electron transfer in a C. necator-based bioelectrochemical system. The use of ferricyanide at a concentration of 15 mM resulted in the highest current density of 260.75µAcm-2 and a coulombic efficiency of 64.1 %.
Assuntos
Cupriavidus necator , Oxirredução , Cupriavidus necator/metabolismo , Eletrodos , Transporte de Elétrons , Oxigênio/metabolismo , Fontes de Energia Bioelétrica/microbiologia , Frutose/metabolismo , Técnicas Eletroquímicas/métodos , Ferricianetos/química , Ferricianetos/metabolismoRESUMO
A surface protein-imprinted biosensor was constructed on a screen-printed carbon electrode (SPCE) for the detection of anti-human immunoglobulin G (anti-IgG). The SPCE was successively decorated with aminated graphene (NH2-G) and gold nanobipyramids (AuNBs) for signal amplification. Then 4-mercaptophenylboric acid (4-MPBA) was covalently anchored to the surface of AuNBs for capturing anti-IgG template through boronate affinity binding. The decorated SPCE was then deposited with an imprinting layer generated by the electropolymerization of pyrrole. After removal of the anti-IgG template by the dissociation of the boronate ester in an acidic solution, three-dimensional (3D) cavities complementary to the anti-IgG template were formed in the imprinting layer of polypyrrole (PPy). The molecularly imprinted polymers (MIP)-based biosensor was used for the detection of anti-IgG, exhibiting a wide linear range from 0.05 to 100 ng mL-1 and a low limit of detection of 0.017 ng mL-1 (S/N = 3). In addition, the MIP-based anti-IgG biosensor also shows high selectivity, reproducibility and stability. Finally, the practicability of the fabricated anti-IgG biosensor was demonstrated by accurate determination of anti-IgG in serum sample.
Assuntos
Técnicas Biossensoriais , Boratos/química , Ferricianetos/química , Imunoglobulina G/análise , Proteínas de Membrana/química , Impressão Molecular , Técnicas Eletroquímicas , HumanosRESUMO
We present the in situ formation of a hole-transporting material (bismuth hexacyanoferrate) on the surface of bismuth tungstate aimed at an innovative photoelectrochemical strategy. This approach enabled a competent aptasensing platform for chloramphenicol that was amenable to homogenous, label-free, and split-mode detection.
Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Bismuto/química , Técnicas Eletroquímicas/métodos , Compostos de Tungstênio/química , Animais , Cloranfenicol/análise , Cloranfenicol/química , Ferricianetos/química , Contaminação de Alimentos/análise , Lagos/análise , Limite de Detecção , Leite/química , Processos Fotoquímicos , Poluentes Químicos da Água/análise , Poluentes Químicos da Água/químicaRESUMO
In this study, tin oxidecobalt oxide nanocatalyst was prepared by a simple method, which grew in spherical particles with an average diameter of 30 nm. Tin oxide-cobalt oxide was further wrapped in alginate polymer hydrogel (Alg@tin oxide-cobalt oxide), and both materials were utilized as nanocatalysts for the catalytic transformation of different pollutants. Tin oxide-cobalt oxide and Alg@tin oxide-cobalt oxide nanocatalysts were tested for the catalytic reduction of 4-nitrophenol, congo red, methyl orange, methylene blue (MB) and potassium ferricyanide in which sodium borohydride was used as a reducing agent. Tin oxide-cobalt oxide and Alg@tin oxide-cobalt oxide nanocatalysts synergistically reduced MB in shorter time (2.0 and 4.0 min) compared to other dyes. The reduction conditions were optimized by changing different parameters. The rate constants for MB reduction were calculated and found to be 1.5714 min-1 and 0.6033 min-1 using tin oxide-cobalt oxide and Alg@tin oxide-cobalt oxide nanocatalysts, respectively. Implementing Alg@tin oxide-cobalt oxide nanocatalyst toward MB reduction in real samples proved its efficacy in sea and well water samples. The catalyst could be easily recovered, recycled and revealed a minimal loss of nanoparticles, which offering a competition and replacement with reputable commercial catalysts.
Assuntos
Alginatos/química , Cobalto/química , Nanocompostos , Óxidos/química , Compostos de Estanho/química , Águas Residuárias/química , Poluentes Químicos da Água/química , Purificação da Água , Compostos Azo/química , Boroidretos/química , Catálise , Vermelho Congo/química , Ferricianetos/química , Cinética , Azul de Metileno/química , Nanotecnologia , Nitrofenóis/química , OxirreduçãoRESUMO
Vibrio cholerae cryptochrome-1 (VcCRY-1) is a member of the cryptochrome DASH family. The flavoprotein appears to use blue light both for repair of cyclobutane pyrimidine dimers (CPDs) on DNA and signal transduction. Earlier, we found that it was almost impossible to oxidize the FADH· state upon binding to a CPD, and, in the absence of substrate, the rate of FADH· oxidation was much larger at high pH (Gindt et al. in Biochemistry 54:2802-2805, 2015). Here, we present the pH-dependence of the oxidation of FADH· by ferricyanide, which revealed a switch between slow and fast oxidation with a pKa ≈ 7.0. Stopped-flow mixing was used to measure the oxidation of FADH- to FADH· at pH 6.7 and 7.5. Substrate binding was required to slow down this oxidation such that it could be measured with stopped flow, but there was only a small effect of pH. In addition, resonance Raman measurements of FADH· in VcCRY-1 at pH 6.5 and 7.5 were performed to probe for structural changes near the FAD cofactor related to the observed changes in rate of FADH· oxidation. Only substrate binding seemed to induce a change near the FAD cofactor that may relate to the change in oxidation kinetics. The pH-effect on the FADH· oxidation rate, which is rate-limited by the proton acceptor, does not seem to be due to a protein structural change near the FAD cofactor. Instead, a conserved glutamate in CRY-DASH may control the deprotonation of FADH· and give rise to the pH-effect.
Assuntos
Criptocromos/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Ferricianetos/química , Concentração de Íons de Hidrogênio , Cinética , OxirreduçãoRESUMO
An SU-8 probe with an array of nine, individually addressable gold microband electrodes (100 µm long, 4 µm wide, separated by 4-µm gaps) was photolithographically fabricated and characterized for detection of low concentrations of chemicals in confined spaces and in vivo studies of biological tissues. The probe's shank (6 mm long, 100 µm wide, 100 µm thick) is flexible, but exhibits sufficient sharpness and rigidity to be inserted into soft tissue. Laser micromachining was used to define probe geometry by spatially revealing the underlying sacrificial aluminum layer, which was then etched to free the probes from a silicon wafer. Perfusion with fluorescent nanobeads showed that, like a carbon fiber electrode, the probe produced no noticeable damage when inserted into rat brain, in contrast to damage from an inserted microdialysis probe. The individual addressability of the electrodes allows single and multiple electrode activation. Redox cycling is possible, where adjacent electrodes serve as generators (that oxidize or reduce molecules) and collectors (that do the opposite) to amplify signals of small concentrations without background subtraction. Information about electrochemical mechanisms and kinetics may also be obtained. Detection limits for potassium ferricyanide in potassium chloride electrolyte of 2.19, 1.25, and 2.08 µM and for dopamine in artificial cerebral spinal fluid of 1.94, 1.08, and 5.66 µM for generators alone and for generators and collectors during redox cycling, respectively, were obtained.
Assuntos
Dopamina/líquido cefalorraquidiano , Técnicas Eletroquímicas/instrumentação , Microeletrodos , Animais , Calibragem , Corpo Estriado/cirurgia , Técnicas Eletroquímicas/métodos , Eletrólitos/química , Ferricianetos/análise , Ferricianetos/química , Ouro , Lasers , Masculino , Microeletrodos/efeitos adversos , Microtecnologia , Oxirredução , Polímeros/química , Cloreto de Potássio/química , Ratos Sprague-DawleyRESUMO
Rationale: Acute pancreatitis (AP) is a serious acute condition affecting the abdomen and shows high morbidity and mortality rates. Its global incidence has increased in recent years. Inflammation and oxidative stress are potential therapeutic targets for AP. This study was conducted to investigate the intrinsic anti-oxidative and anti-inflammatory effects of Prussian blue nanozyme (PBzyme) on AP, along with its underlying mechanism. Methods: Prussian blue nanozymes were prepared by polyvinylpyrrolidone modification method. The effect of PBzyme on inhibiting inflammation and scavenging reactive oxygen species was verified at the cellular level. The efficacy and mechanism of PBzyme for prophylactically treating AP were evaluated using the following methods: serum testing in vivo, histological scoring following hematoxylin and eosin staining, terminal deoxynucleotidyl transferase dUTP nick end labeling fluorescence staining, polymerase chain reaction array, Kyoto Encyclopedia of Genes and Genomes analysis and Western blotting analysis. Results: The synthetic PBzyme showed potent anti-oxidative and anti-inflammatory effects in reducing oxidative stress and alleviating inflammation both in vitro and in vivo in the prophylactic treatment of AP. The prophylactic therapeutic efficacy of PBzyme on AP may involve inhibition of the toll-like receptor/nuclear factor-κB signaling pathway and reactive oxygen species scavenging. Conclusion: The single-component, gram-level mass production, stable intrinsic biological activity, biosafety, and good therapeutic efficacy suggest the potential of PBzyme in the preventive treatment of AP. This study provides a foundation for the clinical application of PBzyme.
Assuntos
Terapia Enzimática/métodos , Nanotecnologia/métodos , Pancreatite/terapia , Transdução de Sinais/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , China , Citocinas/metabolismo , Enzimas/metabolismo , Enzimas/farmacologia , Ferricianetos/química , Ferricianetos/uso terapêutico , Ferrocianetos/química , Ferrocianetos/uso terapêutico , Humanos , Inflamação/tratamento farmacológico , Inflamação/patologia , Masculino , Camundongos Endogâmicos BALB C , NF-kappa B/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Pancreatite/metabolismo , Povidona/química , Povidona/uso terapêutico , Reação do Azul da Prússia/métodos , Espécies Reativas de Oxigênio/metabolismo , Receptores Toll-Like/efeitos dos fármacosRESUMO
A non-invasive aptamer-based electrochemical biosensor using disposable screen-printed graphene electrodes (SPGEs) was developed for simple, rapid, and sensitive determination of cortisol levels. Selective detection of cortisol based on a label-free electrochemical assay was achieved by specific recognition of the cortisol DNA aptamer (CApt). The CApt was modified with streptavidin magnetic beads (MBs) before simple immobilization onto the electrode surface using a neodymium magnet. The electrochemical behavior of the aptamer-based biosensor was assessed by using electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) (vs Ag/AgCl). The specific binding between cortisol and CApt resulted in a decrease in charge transfer resistance (Rct) from EIS using [Fe(CN)6]3-/4- with increasing cortisol concentration. Under optimal conditions, a linear range from 0.10 to 100 ng/mL with a low detection limit (3SD/slope) of 2.1 pg/mL was obtained. Furthermore, the proposed biosensing system exhibited a satisfactory recovery in the range 97.4-109.2% with 5.7-6.6% RSD in spiked artificial human sweat. Regarding the applications of this tool, the aptamer-based biosensor has potential to be a versatile and point-of-care (POC) device for simple, sensitive, selective, disposable, and low-cost cortisol detection.
Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos , Hidrocortisona/análise , Técnicas Biossensoriais/instrumentação , Técnicas Eletroquímicas/instrumentação , Eletrodos , Ferricianetos/química , Humanos , Hidrocortisona/química , Ácidos Nucleicos Imobilizados/química , Limite de Detecção , Fenômenos Magnéticos , Reprodutibilidade dos Testes , Suor/químicaRESUMO
Trans-resveratrol (TRA) possesses a variety of pharmacological activities, making important to explore simple, inexpensive, and reliable analytical methods for identification and quantification of it. We report on the synergistic effects originated from layer-by-layer films of graphene (Gr)-gold nanoparticles (Au) and molecularly imprinted polymers (MIPs) modified glassy carbon electrode (GCE) for electrochemical detection of TRA. To construct the TRA electrochemical sensor (GCE|Gr-Au/MIPs), the films of Gr-Au, MIPs were step by step formed onto GCE via in-situ and controllable electrodeposition and polymerization processes. The compositions, morphologies, and electrochemical properties of obtained films were investigated by various methods. Under the optimized experimental conditions, the electrochemical sensor showed superior performance toward selective and sensitive determination of TRA with K3[Fe(CN)6] as electrochemical signal probe. The electrochemical sensor was applied to determine TRA in real samples with good accuracy and recovery, verifying the broad and practical application prospects for foods and medicines analysis.
Assuntos
Técnicas Eletroquímicas/métodos , Resveratrol/análise , Calibragem , Carbono/química , Técnicas Eletroquímicas/normas , Eletrodos , Ferricianetos/química , Análise de Alimentos , Ouro/química , Grafite/química , Isomerismo , Nanopartículas Metálicas/química , Impressão Molecular , Reprodutibilidade dos Testes , Resveratrol/normasRESUMO
A novel and rapid Electrochemical Immunosensing platform was developed for the direct sensing of antibody human immuno globulin gamma (IgG) interaction with virulence factor of S. aureus, staphylococcal protein A (SpA) in the presence of electroactive redox couple ferri/ferro cyanide (K3/K4[Fe(CN)6]). The receptor SpA was attached to BioPE-DOTAP binary lipid bilayer tethered on alkane thiol molecular cushions. Atomic force microscopy (AFM), High-resolution transmission electron microscope (TEM), Fourier-transform infrared spectroscopy (FT-IR), dynamic light scattering (DLS) techniques were used to study the molecular interactions. The AFM images showed array like formation of BioPE-DOTAP on the monolayer surface. The IgG sensor showed a linear range from 10-21 M to 10-16 M.
Assuntos
Técnicas Eletroquímicas/métodos , Imunoglobulina G/metabolismo , Proteína Estafilocócica A/metabolismo , Eletrodos , Ferricianetos/química , Ferrocianetos/química , Ouro/química , Humanos , Imunoglobulina G/química , Microscopia de Força Atômica , Oxirredução , Ligação Proteica , Proteína Estafilocócica A/químicaRESUMO
Electronic information can be transmitted to cells directly from microelectronics via electrode-activated redox mediators. These transmissions are decoded by redox-responsive promoters which enable user-specified control over biological function. Here, we build on this redox communication modality by establishing an electronic eCRISPR conduit of information exchange. This system acts as a biological signal processor, amplifying signal reception and filtering biological noise. We electronically amplify bacterial quorum sensing (QS) signaling by activating LasI, the autoinducer-1 synthase. Similarly, we filter out unintended noise by inhibiting the native SoxRS-mediated oxidative stress response regulon. We then construct an eCRISPR based redox conduit in both E. coli and Salmonella enterica. Finally, we display eCRISPR based information processing that allows transmission of spatiotemporal redox commands which are then decoded by gelatin-encapsulated E. coli. We anticipate that redox communication channels will enable biohybrid microelectronic devices that could transform our abilities to electronically interpret and control biological function.
Assuntos
Sistemas CRISPR-Cas , Engenharia Genética/métodos , Oxirredução , Eletroquímica , Eletrodos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Ferricianetos/química , Regulação Bacteriana da Expressão Gênica , Estresse Oxidativo , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Piocianina/química , Percepção de Quorum , Regulon , Salmonella enterica/metabolismo , Espectrometria de FluorescênciaRESUMO
A novel ferricyanide/Prussian blue (PB) assay for total antioxidant capacity (TAC) determination was developed exploiting the formation of PB nanoparticles in the presence of polyvinylpyrrolidone (PVP) as stabilizer. This improved method, named as "nanoparticle-based ferricyanide/Prussian blue assay (PBNP)", was applied to the TAC measurement of Cynara Scolymus L. (globe artichoke). The calibration results of the novel (PBNP) method were compared with those of a similar nanoparticle PB method performed in the absence of PVP, and of a sodium dodecyl sulfate-modified and acid-optimized ferricyanide reference assay. Compared to similar common Fe(III)-based TAC assays, much higher molar absorptivities, pointing out higher response to different kinds of antioxidants, were obtained with PBNP for all tested antioxidants, and lower LOD and LOQ values were achieved for thiols. As an additional advantage, methionine, not responding to other electron-transfer based TAC reagents, could be measured. PBNP could detect various antioxidants with one-two orders-of-magnitude lower LOD values than those of widely used TAC assays like CUPRAC and Folin-Ciocalteau well correlating with the proposed assay.
Assuntos
Antioxidantes/metabolismo , Cynara scolymus/metabolismo , Ferricianetos/química , Ferrocianetos/química , Nanopartículas/química , Antioxidantes/análise , Calibragem , Cynara scolymus/química , Povidona/química , Povidona/metabolismoRESUMO
Biological ion channels can realize delicate mass transport under complicated physiological conditions. Artificial nanochannels can achieve biomimetic ion current rectification (ICR), gating, and selectivity that are mostly performed in pure salt solutions. Synthetic nanochannels that can function under mixed ion systems are highly desirable, yet their performances are hard to be compared to those under pure systems. Seeking out the potential reasons by investigating the effect of mixed-system components on the ion-transport properties of the constructed nanochannels seems necessary and important. Herein, we report the effect of anions with different charges and sizes on the ICR properties of positively charged nanochannels. Among the investigated anions, the low-valent anions showed no impact on the ICR direction, while the high-valent component ferrocyanide [Fe(CN)64-] caused significant ICR inversion. The ICR inversion mechanism is evidenced to result from the adsorption of Fe(CN)64--induced surface charge reversal, which relates to solution concentration, pH conditions, and nanochannel sizes and applies to both aminated and quaternized nanochannels that are positively charged. Noticeably, Fe(CN)64- is found to interfere with the transport of protein molecules in the nanochannel. This work points out that the ion species from mixed systems would potentially impact the intrinsic ICR properties of the nanochannels. Replacing highly charged counterions with organic components would be promising in building up future nanochannel-based mass transport systems running under mixed systems.
Assuntos
Ânions/química , Nanotecnologia/métodos , Eletroquímica/métodos , Ferricianetos/química , Eletricidade EstáticaRESUMO
In the present research, a nanoaptasensor is proposed for electrochemical measurement of chloramphenicol (CAP). To this purpose, the nanocomposite prepared from graphene oxide and functionalized with (3-Aminopropyl) triethoxysilane/silver nanoparticles to the abbreviated AgNPs/[NH2-Si]-f-GO, was utilized to modify the glassy carbon electrode (GCE). Furthermore, the modified electrode was also investigated using the electrochemical methods such as electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The AgNPs/[NH2-Si]-f-GO nanocomposite was investigated by UV-Vis spectrophotometry. Fourier transform infrared (FT-IR) spectrometry and transmission electron microscopy (TEM). Moreover, [Fe(CN)6]3-/4 solution in the role of an electrochemical probe was applied. The AgNPs/[NH2-Si]-f-GO nanocomposite was confirmed as a good layer to covalent immobilization of aptamer (Apt) onto the GCE surface. In this sense, the DPV was used as a sensitive electrochemical technique for the measurement of CAP with an appropriate linear concentration range which was found to be between 10 pM and 0.2 µM and, with a low limit of detection, it equaled 3.3 pM. CAP which was identified in the presence of other usual antibiotics existed in the real samples.
Assuntos
Aptâmeros de Nucleotídeos/química , Cloranfenicol/análise , Técnicas Eletroquímicas/métodos , Nanopartículas Metálicas/química , Animais , Antibacterianos/análise , Espectroscopia Dielétrica/métodos , Técnicas Eletroquímicas/instrumentação , Eletrodos , Ferricianetos/química , Contaminação de Alimentos/análise , Grafite/química , Mel/análise , Limite de Detecção , Microscopia Eletrônica de Transmissão , Leite/química , Nanocompostos/química , Propilaminas/química , Reprodutibilidade dos Testes , Silanos/química , Prata/química , Espectrofotometria Ultravioleta , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Herein we present a general and turn-on strategy for enzymatic bioassays on the basis of redox state dependent emission of gold nanoclusters (AuNCs). The photoluminescence of AuNCs was quenched obviously by the oxidative ferricyanide while unaffected by its corresponding reduced state, i.e., ferrocyanide. The distinctive quenching abilities for AuNCs by the redox couple (ferricyanide/ferrocyanide) enabled their utility as new fluorescent sensing platforms to detect redox-related phenomena. The proposed protocols were conducted by using the model oxidoreductases of glucose oxidase (GOx) and the enzyme cascade of lactate dehydrogenase (LDH)/diaphorase to catalytically convert ferricyanide to ferrocyanide, which switched on fluorescence of the detection systems. The detection limit for glucose and lactate was found to be as low as 0.12 and 0.09⯵M, respectively. This work features the first use of the redox couple of ferricyanide/ferrocyanide in fluorescent bioanalysis, which enables versatile, signal on and highly sensitive/selective detections as compared to the state of the art fluorescently enzymatic sensing platforms. Importantly, considering the significance of ferricyanide/ferrocyanide involves in numerous other oxidoreductases mediated biocatalysis, this protocol has wide versatility that enables combination with oxidoreductases related reactions for biosensing.
Assuntos
Corantes Fluorescentes/química , Glucose/análise , Ácido Láctico/análise , Nanopartículas Metálicas/química , Espectrometria de Fluorescência/métodos , Animais , Bovinos , Ferricianetos/química , Fluorescência , Glucose/química , Glucose Oxidase/química , Ouro/química , Humanos , L-Lactato Desidrogenase/química , Ácido Láctico/química , Limite de Detecção , NADH Desidrogenase/química , Oxirredução , Soroalbumina Bovina/químicaRESUMO
In biosensors fabrication, entrapment in polymeric matrices allows efficient immobilization of the biorecognition elements without compromising their structure and activity. When considering living cells, the biocompatibility of both the matrix and the polymerization procedure are additional critical factors. Bio-polymeric gels (e.g. alginate) are biocompatible and polymerize under mild conditions, but they have poor stability. Most synthetic polymers (e.g. PVA), on the other hand, present improved stability at the expense of complex protocols involving chemical/physical treatments that decrease their biological compatibility. In an attempt to explore new solutions to this problem we have developed a procedure for the immobilization of bacterial cells in polyethersulfone (PES) using phase separation. The technology has been tested successfully in the construction of a bacterial biosensor for toxicity assessment. Biosensors were coated with a 300â¯â¯µm bacteria-containing PES membrane, using non-solvent induced phase separation (membrane thicknessâ¯≈â¯300⯵m). With this method, up to 2.3â¯×â¯106â¯cells were immobilized in the electrode surface with an entrapment efficiency of 8.2%, without compromising cell integrity or viability. Biosensing was performed electrochemically through ferricyanide respirometry, with metabolically-active entrapped bacteria reducing ferricyanide in the presence of glucose. PES biosensors showed good stability and reusability during dry frozen storage for up to 1 month. The analytical performance of the sensors was assessed carrying out a toxicity assay in which 3,5-dichlorophenol (DCP) was used as a model toxic compound. The biosensor provided a concentration-dependent response to DCP with half-maximal effective concentration (EC50) of 9.2â¯ppm, well in agreement with reported values. This entrapment methodology is susceptible of mass production and allows easy and repetitive production of robust and sensitive bacterial biosensors.
Assuntos
Técnicas Biossensoriais/métodos , Clorofenóis/toxicidade , Escherichia coli/isolamento & purificação , Polímeros/química , Sulfonas/química , Testes de Toxicidade/métodos , Sobrevivência Celular/efeitos dos fármacos , Células Imobilizadas/efeitos dos fármacos , Células Imobilizadas/metabolismo , Técnicas Eletroquímicas/métodos , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Ferricianetos/química , Ferricianetos/metabolismo , Glucose/metabolismo , Membranas Artificiais , Oxirredução , Reprodutibilidade dos TestesRESUMO
Preparation and electrochemical interrogation of a novel redox active progesterone derivative progesterone thiosemicarbazone (PATC) is presented here together with an investigation into its suitability as conjugate in progesterone hormone immunosensing. PATC synthesis involved a condensation reaction between progesterone acetate and thiosemicarbazone hydrochloride. Voltammetric and pulse techniques confirmed the redox behaviour of the new compound with concentration and scan rate dependant irreversible behaviour evident at glassy carbon and gold transducers - ko (standard heterogeneous rate constant) was 2.56â¯×â¯10-3â¯cm2/s (νâ¯=â¯100â¯mV/s in non-aqeuous media). Bioaffinity studies towards anti-progesterone antibodies involved a competitive ELISA format (optical) which confirmed recognition of the new progesterone derivative. Electrochemical impedance spectroscopy was employed as an interrogation technique in order to establish optimum binding and surface conditions for progesterone antigen-antibody interaction with the assistance of a redox probe (potassium hexacyanoferrate).
Assuntos
Anticorpos Imobilizados/química , Técnicas Biossensoriais/métodos , Progesterona/análise , Carbono/química , Espectroscopia Dielétrica/métodos , Ensaio de Imunoadsorção Enzimática , Ferricianetos/química , Ouro/química , Imunoensaio/métodos , Oxirredução , Progesterona/análogos & derivados , TransdutoresRESUMO
A facile approach for the construction of reagent-free electrochemical dehydrogenase-based biosensors is presented. Enzymes and cofactors (NAD+ and Fe(CN)63-) were immobilized by modification of screen-printed carbon electrodes with graphene oxide (GO) and an additional layer of cellulose acetate. The sensor system was exemplarily optimized for an l-lactate electrode in terms of GO concentration, working potential, and pH value. The biosensor exhibited best characteristics at pH 7.5 in 100 mM potassium phosphate buffer at an applied potential of +0.250 V versus an internal pseudo Ag reference electrode. Thereby, sensor performance was characterized by a linear working range from 0.25 to 4 mM and a sensitivity of 0.14 µA mM-1. The detection principle was additionally evaluated with three other dehydrogenases (d-lactate dehydrogenase, alcohol dehydrogenase, and formate dehydrogenase, respectively). The developed reagentless biosensor array enabled simultaneous and cross-talk free determination of l-lactate, d-lactate, ethanol, and formate.