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1.
Chem Commun (Camb) ; 57(71): 8989-8992, 2021 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-34486607

RESUMO

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/química
2.
Photochem Photobiol Sci ; 20(6): 831-841, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34091863

RESUMO

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 , Oxirredução
3.
Anal Chim Acta ; 1165: 338550, 2021 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-33975699

RESUMO

Chemiluminescence (CL) assay is a promising point-of-care testing (POCT) technology due to the fast response, high sensitivity, and easy miniaturization. The application and performance of CL POCT method were highly dependent on the CL reaction. Herein, based on the CL reaction between luminol and in-situ generated K3Fe(CN)6, a low-cost, enzyme-free, and label-free CL POCT method was explored via a portable and handheld luminometer to detect telomerase activity. Telomerase elongated telomere substrate (TS) primer to form (TTAGGG)n repeats which hybridize with multiple short DNAs. The intercalation of SYBR Green I (SGI) into double-stranded DNA (dsDNA) generated singlet oxygen under the irradiation of LED light source. Singlet oxygen was then employed for in-situ oxidation of K4Fe(CN)6 to K3Fe(CN)6, which could react with luminol to generate a strong CL intensity. Thus, telomerase activity could be specifically, sensitively, and label-free detected. The detection limit was down to 98 HeLa cells. The detection process was very simple, and the cost was about $0.01 for each measurement. Furthermore, telomerase activity was detectable in human serum samples, with spike recoveries from 96% to 105%. According to our knowledge, it is the first effort to develop a low-cost, label-free and enzyme-free CL method with good repeatability for detecting biomarker based on the analyte-triggered and in-situ generated K3Fe(CN)6/luminol CL reaction.


Assuntos
Técnicas Biossensoriais , Telomerase , Ferricianetos , Células HeLa , Humanos , Luminescência , Medições Luminescentes , Luminol , Telomerase/metabolismo
4.
Sci Rep ; 11(1): 7663, 2021 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-33828136

RESUMO

Modern day hospital treatments aim at developing electrochemical biosensors for early diagnosis of diseases using unconventional human bio-fluids like sweat and saliva by monitoring the electron transfer reactions of target analytes. Such kinds of health care diagnostics primarily avoid the usage of human blood and urine samples. In this context, here we have investigated the electron transfer reaction of a well-known and commonly used redox probe namely, potassium ferro/ferri cyanide by employing artificially simulated bio-mimics of human sweat and saliva as unconventional electrolytes. Typically, electron transfer characteristics of the redox couple, [Fe(CN)6]3-/4- are investigated using electrochemical techniques like cyclic voltammetry and electrochemical impedance spectroscopy. Many different kinetic parameters are determined and compared with the conventional system. In addition, such electron transfer reactions have also been studied using a lyotropic liquid crystalline phase comprising of Triton X-100 and water in which the aqueous phase is replaced with either human sweat or saliva bio-mimics. From these studies, we find out the electron transfer reaction of [Fe(CN)6]3-/4- redox couple is completely diffusion controlled on both Au and Pt disc shaped electrodes in presence of sweat and saliva bio-mimic solutions. Moreover, the reaction is partially blocked by the presence of lyotropic liquid crystalline phase consisting of sweat and saliva bio-mimics indicating the predominant charge transfer controlled process for the redox probe. However, the rate constant values associated with the electron transfer reaction are drastically reduced in presence of liquid crystalline phase. These studies are essentially carried out to assess the effect of sweat and saliva on the electrochemistry of Fe2+/3+ redox couple.


Assuntos
Técnicas Biossensoriais , Técnicas Eletroquímicas , Ferricianetos , Ferrocianetos , Espectroscopia Dielétrica , Transporte de Elétrons , Oxirredução , Saliva/química , Suor/química
5.
Theranostics ; 11(7): 3213-3228, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33537083

RESUMO

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 fármacos
6.
Food Chem ; 338: 127851, 2021 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-32836002

RESUMO

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/normas
7.
Food Chem ; 339: 127881, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-32866703

RESUMO

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/química
8.
ACS Sens ; 5(11): 3540-3546, 2020 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-33064465

RESUMO

Photoanode sensing platforms with remarkable photoelectrochemical (PEC) response and satisfying visible-light absorption have become the most promising detection systems. Nevertheless, their inevitable electrophilic character limits their expansion in the bioassay because of reductive substances in serum or other body fluids that can severely interfere with the photocurrent to be read. To solve it, a PEC platform-assembled dual-active electrode is designed to realize the separation of biological monitoring from the photoanode. The ferricyanide ([Fe(CN)6]3-)-mediated redox cycle is first proposed to meet the gain and loss electron requirements of the PEC system. It can avoid the self-reaction in the electrolyte caused by the addition of a traditional electron donor and acceptor, for instance, ascorbic acid and hydrogen peroxide. As a consequence, the traditional counter electrode (Pt wire) is replaced by Fe2O3/AgInS2 heterojunction, which can amplify the PEC response of the cathode to meet the requirement of trace analysis. An aptasensor fabricated by the above strategies exhibits convincing data for 17ß-estradiol (E2) detection from which a wide detection range is obtained in 10 fg/mL to 1 µg/mL with a detection limit of 2.74 fg/mL (S/N = 3). These advanced elements show a rosy prospect for environmental monitoring and point-of-care biomarker diagnosis.


Assuntos
Técnicas Biossensoriais , Técnicas Eletroquímicas , Eletrodos , Ferricianetos , Oxirredução
9.
Free Radic Biol Med ; 160: 458-470, 2020 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-32828952

RESUMO

Deferoxamine (DFO), an iron chelator, is used therapeutically for the removal of excess iron in multiple clinical conditions such as beta thalassemia and intracerebral hemorrhage. DFO is also used as an iron chelator and hypoxia-mimetic agent in in vivo and in vitro basic research. Here we unexpectedly discover DFO to be a nitric oxide (NO) precursor in experiments where it was intended to act as an iron chelator. Production of NO from aqueous solutions of DFO was directly observed by ozone-based chemiluminescence using a ferricyanide-based assay and was confirmed by electron paramagnetic resonance (EPR). DFO also produced NO following exposure to ultraviolet light, and its incubation with sheep adult and fetal blood resulted in considerable formation of iron nitrosyl hemoglobin, as confirmed by both visible spectroscopy and EPR. These results suggest that experiments using DFO can be confounded by concomitant production of NO, and offer new insight into some of DFO's unexplained clinical side effects such as hypotension.


Assuntos
Desferroxamina , Quelantes de Ferro , Animais , Ferricianetos , Óxido Nítrico , Ovinos , Raios Ultravioleta
10.
Talanta ; 216: 120960, 2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-32456941

RESUMO

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/metabolismo
11.
ACS Appl Mater Interfaces ; 12(25): 28915-28922, 2020 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-32460478

RESUMO

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 Estática
12.
Nat Commun ; 11(1): 2427, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32415193

RESUMO

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 Fluorescência
13.
Mater Sci Eng C Mater Biol Appl ; 108: 110388, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31923985

RESUMO

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 Fourier
14.
Chemosphere ; 244: 125511, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31809936

RESUMO

Respiration substrate (RS)-less isolation method was developed for enhancing the sensitivity of acute toxicity assessment of heavy metal ions. RS was removed from the first step of previous isolation method, which was an effective strategy for improving acute toxicity assessment. 50% inhibiting concentration (IC50) values of Cu2+, Cd2+, Zn2+, Hg2+ and Ni2+ were 0.39 mg L-1, 5.99 mg L-1, 3.99 mg L-1, 0.23 mg L-1 and 5.74 mg L-1, respectively. Beyond that, the complicacy of organic toxicants assessments was investigated by choosing 3,5-dichlorophenol (DCP) as model toxicant. Biofilm sensor, morphology method and suspended microbes-based methods including one-pot method, RS-isolation method, RS-less isolation method, RS-less isolation method with added potassium ferricyanide (+F), were compared. The sensitivity to DCP can be ranked as morphology method > suspended microbes-based methods > biofilm method. The difference of the present results implicated that the methodological interference, leading in different detection mechanisms of these methods. The relative investigations can provide theoretical guidance for developing comprehensive detection methods of pollutants.


Assuntos
Testes de Toxicidade Aguda/métodos , Ferricianetos , Íons , Metais Pesados , Fenóis/toxicidade , Poluentes Químicos da Água/análise
15.
Biosens Bioelectron ; 150: 111876, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31748195

RESUMO

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 2.56 × 10-3 cm2/s (ν = 100 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 , Transdutores
16.
Anal Chim Acta ; 1095: 219-225, 2020 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-31864626

RESUMO

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 µM, 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/química
17.
Talanta ; 206: 120192, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31514850

RESUMO

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 300  µm bacteria-containing PES membrane, using non-solvent induced phase separation (membrane thickness ≈ 300 µm). With this method, up to 2.3 × 106 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 9.2 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 Testes
18.
Anal Chem ; 91(23): 15293-15299, 2019 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-31674761

RESUMO

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.


Assuntos
Técnicas Biossensoriais , Carbono/química , Técnicas Eletroquímicas , Grafite/química , NAD/química , Oxirredutases/química , Carbono/metabolismo , Eletrodos , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Etanol/análise , Etanol/metabolismo , Ferricianetos/química , Ferricianetos/metabolismo , Formiatos/análise , Formiatos/metabolismo , Grafite/metabolismo , Concentração de Íons de Hidrogênio , Ácido Láctico/análise , Ácido Láctico/metabolismo , NAD/metabolismo , Oxirredutases/metabolismo , Prata/química
19.
Mikrochim Acta ; 186(12): 828, 2019 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-31754797

RESUMO

The autosomal recessive-hyper immunoglobulin E syndromes (AR-HIES) are inherited inborn primary immunodeficiency disorders caused mainly by mutations in the dedicator of cytokinesis 8 (DOCK8) gene. A method is described for the selection of DNA aptamers against DOCK8 protein. The selection was performed by using a gold electrode as the solid matrix for immobilization of DOCK8. This enables voltammetric monitoring of the bound DNA after each selection cycle. After eight rounds of selection, high affinity DNA aptamers for DOCK8 were identified with dissociation constants (Kds) ranging from 3.3 to 66 nM. The aptamer which a Kd of 8.8 nM was used in an aptasensor. A gold electrode was modified by self-assembly of the thiolated aptamer, and the response to the DOCK8 protein was detected by monitoring the change in the electron transfer resistance using the ferro/ferricyanide system as a redox probe. The aptasensor works in the 100 pg.mL-1 to 100 ng.mL-1 DOCK8 concentration range, has a detection limit of 81 pg.mL-1 and good selectivity over other proteins in the serum. Graphical abstractSchematic representation of an electrochemical screening protocol for the selection of DNA aptamer against dedicator of cytokinesis 8 protein using electrode as solid support for target immobilization.


Assuntos
Aptâmeros de Nucleotídeos/química , Fatores de Troca do Nucleotídeo Guanina/análise , Proteínas Imobilizadas/análise , Técnicas Biossensoriais , Espectroscopia Dielétrica , Dimerização , Técnicas Eletroquímicas/métodos , Eletrodos , Ferricianetos/química , Ouro/química , Limite de Detecção , Oxirredução , Técnica de Seleção de Aptâmeros/métodos , Sensibilidade e Especificidade , Compostos de Sulfidrila/química , Propriedades de Superfície
20.
Artigo em Inglês | MEDLINE | ID: mdl-31561902

RESUMO

Immune Assist (IA) is produced from extract of six species of medical mushrooms: Agaricus blazei - Cordyceps sinensis - Grifola frondosa - Ganoderma lucidum - Coriolus versicolor - Lentinula edodes. The genoprotective potential of IA was evaluated for the first time. Significant antigenotoxic effects were detected in human peripheral blood cells against H2O2 induced DNA damage, in the pretreatment and in the posttreatment. The most efficient concentration of IA in pretreatment was 500 µg/mL, while in posttreatment it was the concentration of 250 µg/mL. Kinetics of attenuation of H2O2 induced DNA damage in posttreatment with the optimal concentration of IA showed significant decrease in the number of damaged cells at all time periods (15-60 min), reaching the greatest reduction after 15 and 45 min. Remarkable ·OH scavenging properties and moderate reducing power, together with the modest DPPH scavenging activity, could be responsible for the great attenuation of DNA damage after 15 min of exposure to IA, while reduction of DNA damage after 45 min could be the result in additional stimulation of the cell's repair machinery. Our results suggest that IA displayed antigenotoxic and antioxidant properties. A broader investigation of its profile in biological systems is needed.


Assuntos
Agaricales/química , Antioxidantes/farmacologia , Células Sanguíneas/efeitos dos fármacos , Ensaio Cometa , Dano ao DNA/efeitos dos fármacos , Radicais Livres/toxicidade , Extratos Vegetais/farmacologia , Adulto , Células Sanguíneas/química , DNA/sangue , DNA/efeitos dos fármacos , Feminino , Ferricianetos/toxicidade , Humanos , Peróxido de Hidrogênio/toxicidade , Radical Hidroxila/toxicidade , Técnicas In Vitro , Oxidantes/toxicidade , Extratos Vegetais/toxicidade , Análise de Célula Única , Adulto Jovem
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