RESUMO
The inclusion of online, in situ biosensors in microfluidic cell cultures is important to monitor and characterize a physiologically mimicking environment. This work presents the performance of second-generation electrochemical enzymatic biosensors to detect glucose in cell culture media. Glutaraldehyde and ethylene glycol diglycidyl ether (EGDGE) were tested as cross-linkers to immobilize glucose oxidase and an osmium-modified redox polymer on the surface of carbon electrodes. Tests employing screen printed electrodes showed adequate performance in a Roswell Park Memorial Institute (RPMI-1640) media spiked with fetal bovine serum (FBS). Comparable first-generation sensors were shown to be heavily affected by complex biological media. This difference is explained in terms of the respective charge transfer mechanisms. Under the tested conditions, electron hopping between Os redox centers was less vulnerable than H2O2 diffusion to biofouling by the substances present in the cell culture matrix. By employing pencil leads as electrodes, the incorporation of these electrodes in a polydimethylsiloxane (PDMS) microfluidic channel was achieved simply and at a low cost. Under flow conditions, electrodes fabricated using EGDGE presented the best performance with a limit of detection of 0.5 mM, a linear range up to 10 mM, and a sensitivity of 4.69 µA mM-1 cm-2.
Assuntos
Técnicas Biossensoriais , Glucose , Glucose/metabolismo , Microfluídica , Polímeros/química , Peróxido de Hidrogênio , Glucose Oxidase/química , Oxirredução , Eletrodos , Técnicas de Cultura de Células em Três Dimensões , Técnicas Eletroquímicas , Enzimas Imobilizadas/químicaRESUMO
Fungal lectins have enormous biotechnological potential, but limited knowledge about their biochemical and biophysical features prevents their proper use. Herein, we report an innovative alternative to use Ganoderma applanatum lectin (GAL) as a glucose biorecognition element, after identifying the ideal electroanalytical conditions by machine learning studies performed with a homologous agglutinin from the same macrofungus. The research revealed that GAL has moderate resistance to pH (4-8) and temperature (20-60 °C) variations, but its hemagglutinating activity (376.5 HU mg-1 GAL at 20 °C) was better conserved under physiological conditions. Integrating electrochemical data and semi-empirical molecular modeling, biocompatible and electrostatically favorable conditions were found to immobilize the lectin on Prussian blue-modified glassy carbon electrode, after thermal activation of the metal-complex film. The glucose dose-response relationship obtained with the developed biosensor, defined as GAL/ta-PB/GCE, showed a typical Hill equation correlation, suggesting electrodic interactions represented by a sigmoidal mathematical function. GAL/ta-PB/GCE achieved remarkable electroanalytical performance, with emphasis on the detection limit (10.2 pM) and sensitivity (0.012 µA µM-1cm-2). The biosensor was successfully used to quantify glucose in pharmaceutical formulations, reiterating that the association of theoretical and experimental information drives important advances in bioelectrochemical studies.
Assuntos
Técnicas Biossensoriais , Ganoderma , Glucose , Lectinas/química , Eletroquímica , Eletrodos , Glucose Oxidase/químicaRESUMO
The fabrication of efficient organic electrochemical transistors (OECTs)-based biosensors requires the design of biocompatible interfaces for the immobilization of biorecognition elements, as well as the development of robust channel materials to enable the transduction of the biochemical event into a reliable electrical signal. In this work, PEDOT-polyamine blends are shown as versatile organic films that can act as both highly conducting channels of the transistors and non-denaturing platforms for the construction of the biomolecular architectures that operate as sensing surfaces. To achieve this goal, we synthesized and characterized films of PEDOT and polyallylamine hydrochloride (PAH) and employed them as conducting channels in the construction of OECTs. Next, we studied the response of the obtained devices to protein adsorption, using glucose oxidase (GOx) as a model system, through two different strategies: The direct electrostatic adsorption of GOx on the PEDOT-PAH film and the specific recognition of the protein by a lectin attached to the surface. Firstly, we used surface plasmon resonance to monitor the adsorption of the proteins and the stability of the assemblies on PEDOT-PAH films. Then, we monitored the same processes with the OECT showing the capability of the device to perform the detection of the protein binding process in real time. In addition, the sensing mechanisms enabling the monitoring of the adsorption process with the OECTs for the two strategies are discussed.
Assuntos
Técnicas Biossensoriais , Polímeros , Ligação Proteica , Polímeros/química , Glucose Oxidase/química , PoliaminasRESUMO
Ferrocene-based polymers as redox mediators are considered versatile and important in the study of glucose biosensors. Poly-L-lysine (PLL), as a cationic polymer, possesses good properties including biocompatibility, biodegradation and water solubility. In this work, PLL was modified with ferrocene carboxylate in a very simple way by activating the carboxyl group of Fc, which reacted with the amino groups of the polymer. The resulting product was analysed by FTIR. Performance as a redox mediator (Fc-PLL) with the enzyme glucose oxidase was tested by cyclic voltammetry and showed an increase in the oxidation current in the presence of glucose in PBS pH 7.4. Additionally, performance as a biosensor was evaluated by amperometry and gave a linear range of 0-10 mM, a limit of detection of 23 µM, a sensitivity of 6.55 µA/cm2 mM and high selectivity. To evaluate the charged regions of Fc-PLL/GOx on the electrode surface, analysis by scanning electrochemical microscopy showed remarkable activity. The Fc-PLL redox polymer as a glucose biosensor has been well accepted as this kind of material, and the results showed remarkable activity as an electron transfer mediator between the redox polymer and the GOx enzyme.
Assuntos
Técnicas Biossensoriais , Glucose , Técnicas Biossensoriais/métodos , Eletrodos , Enzimas Imobilizadas/química , Glucose/análise , Glucose Oxidase/química , Metalocenos , Oxirredução , Polilisina/metabolismo , Polímeros/químicaRESUMO
A biosensing membrane base on ferulic acid and glucose oxidase is synthesized onto a carbon paste electrode by electropolymerization via cyclic voltammetry in aqueous media at neutral pH at a single step. The developed biosensors exhibit a linear response from 0.082 to 34 mM glucose concentration, with a coefficient of determination R2 equal to 0.997. The biosensors display a sensitivity of 1.1 µAmM-1 cm-2, a detection limit of 0.025 mM, and 0.082 mM as glucose quantification limit. The studies reveal stable, repeatable, and reproducible biosensors response. The results indicate that the novel poly-ferulic acid membrane synthesized by electropolymerization is a promising method for glucose oxidase immobilization towards the development of glucose biosensors. The developed glucose biosensors exhibit a broader linear glucose response than other polymer-based glucose biosensors.
Assuntos
Técnicas Biossensoriais/métodos , Carbono/química , Ácidos Cumáricos/química , Técnicas Eletroquímicas/métodos , Glucose Oxidase/metabolismo , Glucose/análise , Polímeros/química , Técnicas Biossensoriais/normas , Eletrodos , Enzimas Imobilizadas , Glucose Oxidase/química , Limite de DetecçãoRESUMO
We report a straightforward route for the preparation of flexible, electrochemically stable and easily functionalizable poly(3,4-ethylenedioxythiophene) (PEDOT) composite films deposited on PET foils as biosensing platforms. For this purpose, poly(allylamine) hydrochloride (PAH) was blended with PEDOT to provide amine-bearing sites for further biofunctionalization as well as to improve the mechanical properties of the films. The conducting PEDOT-PAH composite films were characterized by cyclic voltammetry, UV-vis and Raman spectroscopies. An exhaustive stability study was carried out from the mechanical, morphological and electrochemical viewpoint. Subsequent sugar functionalization of the available amine groups from PAH allowed for the specific recognition of lectins and the subsequent self-assembly of glycoenzymes (glucose oxidase and horseradish peroxidase) concomitant with the prevention of non-specific protein fouling. The platforms presented good bioelectrochemical performance (glucose oxidation and hydrogen peroxide reduction) in the presence of redox mediators. The developed composite films constitute a promising option for the construction of all-polymer biosensing platforms with great potential owing to their flexibility, high transmittance, electrochemical stability and the possibility of glycosylation, which provides a simple route for specific biofunctionalization as well as an effective antifouling strategy.
Assuntos
Aspergillus niger/enzimologia , Técnicas Biossensoriais , Compostos Bicíclicos Heterocíclicos com Pontes/química , Técnicas Eletroquímicas , Proteínas Fúngicas/química , Glucose Oxidase/química , Glucose/análise , Membranas Artificiais , Poliaminas/química , Polímeros/química , Peroxidase do Rábano Silvestre/químicaRESUMO
Glucose and urea enzymatic biosensors were fabricated. One-step electrochemical immobilization process was used to produce thin polyaniline films with entrapped enzymes. Chronopotentiometric analysis, scanning electron microscopy, electrochemical impedance spectroscopy and optical reflectance spectroscopy were used to determine the structure-property relationship of the functionalized polymeric thin films. The device has a recognition stage connected to a potentiometric field-effect-transistor stage and is based on the measurement of microenvironment pH variation or locally produced ions. Optimization of biosensor fabrication and effective measurement conditions were performed. The optimized films presented sensitivity, linearity and detection range to glucose of 14.6 ± 0.4 mV/decade, 99.8% and from 10-4 M to 10-1 mol/L. Two different biosensors were produced based on the enzymatic reaction of urea with selectivity to ammonium or hydroxyl ions. For ammonium ion selective film, the sensor's parameters were 14.7 ± 0.9 mV/decade, 98.2% and from 10-5 to 10-1 mol/L. For the hydroxyl ion selective film, the same parameters were 7.4 ± 0.5 mV/decade, 98.1% and from 10-5 to 10-1 mol/L. The change in the oxidation state of the polymeric matrix explains: i) the large loss of functionality of glucose biosensor in time, ii) the conservation of functionality to the hydroxyl ions for urea biosensor and iii) the selectivity variation of the ammonium ion selective urea biosensor. The results indicate that the polymeric matrix has indeed changeable selectivity, what can be applied in different situations for biosensors production.
Assuntos
Técnicas Biossensoriais , Técnicas Eletroquímicas , Enzimas Imobilizadas/química , Glucose Oxidase/química , Glucose/análise , Membranas Artificiais , Ureia/análise , Compostos de Anilina/químicaRESUMO
We are reporting an original supramolecular architecture based on a rationally designed new nanohybrid with enhanced peroxidase-like activity and site-specific biorecognition properties using avidin-functionalized multi-walled carbon nanotubes (MWCNTs-Av) and Ru nanoparticles (RuNPs). The nanohybrid-electrochemical interface was obtained by drop-coating of MWCNTs-Av dispersion at glassy carbon electrodes (GCE) followed by solvent evaporation and further electrodeposition of RuNPs (50â¯ppm RuCl2 for 15â¯s at -0.600â¯V). The simultaneous presence of MWCNTs and RuNPs produces a synergic effect on the non-enzymatic catatalytic reduction of H2O2 and allows the quantification of H2O2 in a wide linear range (from 5.0â¯×â¯10-7â¯M to 1.75â¯×â¯10-3â¯M) with a low limit of detection (65â¯nM). The avidin residues present in MWCNTs-Av/RuNPs hybrid nanomaterial allowed the anchoring by bioaffinity of biotinylated glucose oxidase (biot-GOx) as proof-of-concept of the analytical application of MWCNTs-Av platform for biosensors development. The resulting nanoarchitecture behaves as a bienzymatic-like glucose biosensor with a competitive analytical performance: linear range between 2.0â¯×â¯10-5â¯M and 1.23â¯×â¯10-3â¯M, sensitivity of (0.343⯱â¯0.002) µA mM-1 or (2.60⯱â¯0.02) µA mM-1 cm-2, detection limit of 3.3⯵M, and reproducibility of 5.2% obtained with five different GCE/MWCNTs-Av/RuNPs/biot-GOx bioplatforms prepared the same day using the same MWCNTs-Av dispersion, and 9.1% obtained with nine biosensors prepared in different days with nine different MWCNTs-Av dispersions. The average concentrations of glucose in Gatorade®, Red bull® and Pepsi® with the biosensor demonstrated excellent agreement with those reported in the commercial beverages.
Assuntos
Avidina/química , Técnicas Biossensoriais/métodos , Nanopartículas/química , Nanotubos de Carbono/química , Rutênio/química , Aspergillus niger/enzimologia , Bebidas/análise , Materiais Biomiméticos/química , Biotinilação , Catálise , Técnicas Eletroquímicas/métodos , Glucose/análise , Glucose Oxidase/química , Peróxido de Hidrogênio/análise , Limite de Detecção , Nanopartículas/ultraestrutura , Nanotubos de Carbono/ultraestrutura , Peroxidase/químicaRESUMO
In this work, we detail the progress of a novel electrochemical disposable device, which has a relatively low cost and easy production, with a novel conductive ink, that consists of graphite and automotive varnish mixture, deposited over a self-adhesive paper, granting an easy production with relatively low cost. The electrode surface was characterized by scanning electron microscopy, X-ray powder diffraction and Fourier transforms infrared and Raman, cyclic voltammetry and electrochemical impedance spectroscopies. In addition, the proposed electrode was applied for individual electrochemical determination of dopamine and serotonin. The device achieved a linear response between 30 and 800⯵molâ¯L-1 and a limit of detection (LOD) of 0.13⯵molâ¯L-1, by square wave voltammetry for dopamine and a linear range from 6.0 to 100⯵molâ¯L-1, with a LOD of 0.39⯵molâ¯L-1, by differential pulse voltammetry for serotonin. Later, the working electrode was modified with glucose oxidase and dihexadecyl phosphate film in order to obtain a biosensor. At this stage, CV was applied to detect glucose in the range of 1.0-10⯵molâ¯L-1 and LOD of 0.21⯵molâ¯L-1. By three different techniques and analytes, the sensoring and biosensoring processes presented high reproducibility. The proposed adhesive electrode is easy to prepare, disposable, within non-restrictive nature, which allows an approach of a new device for electrochemical sensing and biosensing.
Assuntos
Técnicas Biossensoriais/instrumentação , Dopamina/análise , Glucose/análise , Neurotransmissores/análise , Papel , Serotonina/análise , Espectroscopia Dielétrica , Condutividade Elétrica , Técnicas Eletroquímicas , Eletrodos , Glucose Oxidase/química , Grafite/química , Tinta , Limite de Detecção , Organofosfatos/química , Reprodutibilidade dos TestesRESUMO
While the application of enzymes to synthetic and industrial problems continues to grow, the major development today is focused on multi-enzymatic cascades. Such systems are particularly attractive, because many commercially available enzymes operate under relatively similar operating conditions. This opens the possibility of one-pot operation with multiple enzymes in a single reactor. In this paper the concept of modules is introduced whereby groups of enzymes are combined in modules, each operating in a single reactor, but with the option of various operating strategies to avoid any complications of nonproductive interactions between the enzymes, substrates or products in a given reactor. In this paper the selection of modules is illustrated using the synthesis of the bulk chemical, gluconic acid, from lignocellulosic waste.