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1.
Anal Bioanal Chem ; 411(21): 5481-5488, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31236648

RESUMO

The mechanism behind the variation in the photoluminescence (PL) of a MoS2 nanohybrid material with pH was investigated. Highly fluorescent MoS2 quantum dots dispersed across MoS2 nanosheets (MoS2 QDNS) were synthesized by a hydrothermal route in the presence of NaOH. Upon reducing the pH from 13 to 6.5, the PL intensity was markedly quenched. The removal of dangling sulfur atoms by adding mineral acids could be a plausible mechanism for this PL quenching, together with the inner filter effect and Förster resonance energy transfer due to the resulting species. A label-free turn-on fluorescence sensor for H2O2 was developed using this hybrid material. The PL of the acidified MoS2 QDNS at pH 6.5 increased (i.e., recovered) linearly with the concentration of H2O2. The dynamic range of the sensor was found to be 2-94 µM with a limit of detection (LOD) of 2 µM. This sensing strategy was also extended for the detection of glucose by appending glucose oxidase (GOx) as a catalyst. In the presence of GOx, glucose oxidizes to gluconic acid and H2O2, so the original level of glucose can be estimated by determining the H2O2 present. The absence of a complicated enzyme immobilization step is the prime advantage of the present glucose sensor. The current work exemplifies the utility of MoS2-based nanoparticle systems in the biological sensor domain. Graphical abstract.


Assuntos
Dissulfetos/química , Peróxido de Hidrogênio/análise , Concentração de Íons de Hidrogênio , Molibdênio/química , Transferência Ressonante de Energia de Fluorescência , Glucose Oxidase/análise , Limite de Detecção , Medições Luminescentes
2.
Anal Chim Acta ; 1021: 95-102, 2018 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-29681289

RESUMO

Coupling infrared (IR) spectroscopy to microfluidic devices provides a powerful tool for characterizing complex chemical and biochemical reactions. Examples of microfluidic devices coupled with infrared spectroscopy have been limited, however, largely due to the difficulties associated with fabricating systems in common infrared transparent materials like CaF2. Recent reports have shown that polydimethylsiloxane (PDMS) can be used as an IR transparent substrate when fabricated with thin layers. The use of soft lithography with PDMS expands the library of possible designs that can be achieved for IR measurements in microfluidics. In initial reports with thin PDMS, the target analytes were small molecules; however, IR spectroscopy offers a powerful tool to study protein structure and reactions. Here, a PDMS microfluidic device compatible with IR spectroscopy was fabricated by means of spin-coating of PDMS pre-polymer to obtain thin PDMS microfluidic features. The device was comprised of only PDMS and IR absorption of PDMS was significantly minimized due to the thickness (∼40 µm) of the PDMS layer. The use of thin PDMS allowed for measuring the amide I and II vibrational bands of proteins that have been difficult to measure in other microfluidic devices. To demonstrate the power of the system, the microfluidic device was successfully used to measure the enzyme kinetics as one class of important biochemical reactions with broad use in a variety of fields from medicine to biotechnology. As a model, the reaction of glucose oxidase with glucose was tracked by following the formation of gluconic acid. Michaelis-Menten kinetics from the device were compared with bulk solution measurements and found to be in good agreement.


Assuntos
Dimetilpolisiloxanos/química , Glucose Oxidase/análise , Técnicas Analíticas Microfluídicas , Gluconatos/química , Gluconatos/metabolismo , Glucose/química , Glucose/metabolismo , Glucose Oxidase/metabolismo , Cinética , Técnicas Analíticas Microfluídicas/instrumentação , Espectrofotometria Infravermelho/instrumentação
3.
Biosens Bioelectron ; 105: 65-70, 2018 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-29355780

RESUMO

The fluorescent CuNCs/ZIF-8 nanocomposites were facilely prepared by mixing the PEI protected CuNCs with the precursors of ZIF-8, and characterized by UV-vis absorption, fluorescence, FT-IR, TEM, XRD and XPS. The quantum yield of the CuNCs/ZIF-8 is 15 times that of the PEI-CuNCs. Furthermore, the CuNCs/ZIF-8 possesses better stability and higher fluorescence response due to protective and confinement effects of MOFs. It was found that H2O2 could cause much more quenching of fluorescent CuNCs/ZIF-8 than that of PEI-CuNCs, which might result from enriching H2O2 by ZIF-8. The CuNCs/ZIF-8 can be designed as a fluorescence probe to selectively and sensitively detect H2O2 with a linear range from 0.01 to 1.5µM and a LOD of 0.01µM, while those with PEI-CuNCs are 0.5-30µM and 0.50µM, respectively. Through formation of CuNCs/ZIF-8 hybrid, the sensitivity for the detection of H2O2 increases by nearly 50-fold, which makes CuNCs/ZIF-8 a desirable probe to detect H2O2 content in human serum samples. Also, we successfully demonstrated the potential application of the CuNCs/ZIF-8 for screening and evaluating activities of oxidase using glucose oxidase as a model. The glucose oxidase activity can be detected in a range of 0.1-10mU/L with a LOD of 0.1mU/L.


Assuntos
Técnicas Biossensoriais/métodos , Cobre/química , Ensaios Enzimáticos/métodos , Corantes Fluorescentes/química , Glucose Oxidase/análise , Peróxido de Hidrogênio/sangue , Nanocompostos/química , Glucose Oxidase/metabolismo , Humanos , Peróxido de Hidrogênio/análise , Limite de Detecção , Polietilenoimina/química , Espectrometria de Fluorescência/métodos
4.
Anal Bioanal Chem ; 410(6): 1775-1783, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29279991

RESUMO

To immobilize enzymes at the surface of a nanoparticle-based electrochemical sensor is a common method to construct biosensors for non-electroactive analytes. Studying the interactions between the enzymes and nanoparticle support is of great importance in optimizing the conditions for biosensor design. This can be achieved by using a combination of analytical methods to carefully characterize the enzyme nanoparticle coating at the sensor surface while studying the optimal conditions for enzyme immobilization. From this analytical approach, it was found that controlling the enzyme coverage to a monolayer was a key factor to significantly improve the temporal resolution of biosensors. However, these characterization methods involve both tedious methodologies and working with toxic cyanide solutions. Here we introduce a new analytical method that allows direct quantification of the number of immobilized enzymes (glucose oxidase) at the surface of a gold nanoparticle coated glassy carbon electrode. This was achieved by exploiting an electrochemical stripping method for the direct quantification of the density and size of gold nanoparticles coating the electrode surface and combining this information with quantification of fluorophore-labeled enzymes bound to the sensor surface after stripping off their nanoparticle support. This method is both significantly much faster compared to previously reported methods and with the advantage that this method presented is non-toxic. Graphical abstract A new analytical method for direct quantification of the number of enzymes immobilized at the surface of gold nanoparticles covering a glassy carbon electrode using anodic stripping and fluorimetry.


Assuntos
Aspergillus niger/enzimologia , Enzimas Imobilizadas/análise , Corantes Fluorescentes/análise , Glucose Oxidase/análise , Ouro/química , Nanopartículas Metálicas/química , Técnicas Biossensoriais , Eletrodos , Espectrometria de Fluorescência
5.
Biosens Bioelectron ; 87: 339-344, 2017 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-27573301

RESUMO

In this work, we proposed a novel and facile method to monitor oxidase activities based on size-selective fluorescent quantum dot (QD)@metal-organic framework (MOF) core-shell nanocomposites (CSNCPs). The CSNCPs were synthesized from ZIF-8 and CdTe QDs in aqueous solution in 40min at room temperature with stirring. The prepared CdTe@ZIF-8 CSNCPs , which have excellent water dispersibility and stability, displays distinct fluorescence responses to hole scavengers of different molecular sizes (e.g., H2O2, substrate, and oxidase) due to the aperture limitation of the ZIF-8 shell. H2O2 can efficiently quench the fluorescence of CdTe@ZIF-8 CSNCPs over a linearity range of 1-100nM with a detection limit of 0.29nM, whereas large molecules such as substrate and oxidase have very little effect on its fluorescence. Therefore, the highly sensitive detection of oxidase activities was achieved by monitoring the fluorescence quenching of CdTe@ZIF-8 CSNCPs by H2O2 produced in the presence of substrate and oxidase, which is proportional to the oxidase activities. The linearity ranges of the uricase and glucose oxidase activity are 0.1-50U/L and 1-100U/L, respectively, and their detection limits are 0.024U/L and 0.26U/L, respectively. Therefore, the current QD@MOF CSNCPs based sensing system is a promising, widely applicable means of monitoring oxidase activities in biochemical research.


Assuntos
Compostos de Cádmio/química , Ensaios Enzimáticos/métodos , Glucose Oxidase/metabolismo , Nanocompostos/química , Compostos Organometálicos/química , Telúrio/química , Urato Oxidase/metabolismo , Técnicas Biossensoriais/métodos , Corantes Fluorescentes/química , Glucose Oxidase/análise , Limite de Detecção , Nanocompostos/ultraestrutura , Pontos Quânticos/química , Pontos Quânticos/ultraestrutura , Espectrometria de Fluorescência/métodos , Urato Oxidase/análise
6.
Biosens Bioelectron ; 86: 90-94, 2016 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-27336616

RESUMO

A low invasive type glucose sensor, which has a sensing region at the tip of a fine pointed electrode, was developed for continuous glucose monitoring. Platinum-iridium alloy electrode with a surface area of 0.045mm(2) was settled at the middle of pointed PEEK (Polyetheretherketone) tubing and was employed as sensing electrode. Electrodeposition of glucose oxidase in the presence of surfactant, Triton X-100, was performed for high-density enzyme immobilization followed by the electropolymerization of o-phenylenediamine for the formation of functional entrapping and permselective polymer membrane. Ag/AgCl film was coated on the surface of PEEK tubing as reference electrode. Amperometric responses of the prepared sensors to glucose were measured at a potential of 0.60V (vs. Ag/AgCl). The prepared electrode showed the sensitivity of 2.55µA/cm(2) mM with high linearity of 0.9986, within the glucose concentration range up to 21mM. The detection limit (S/N=3) was determined to be 0.11mM. The glucose sensor properties were evaluated in phosphate buffer solution and in vivo monitoring by the implantation of the sensors in rabbit, while conventional needle type sensors as a reference were used. The results showed that change in output current of the proposed sensor fluctuated similar with one in output current of the conventional needle type sensors, which was also in similar accordance with actual blood sugar level measured by commercially glucose meter. One-point calibration method was used to calibrate the sensor output current.


Assuntos
Técnicas Biossensoriais/instrumentação , Automonitorização da Glicemia/instrumentação , Glicemia/análise , Condutometria/instrumentação , Eletrodos , Glucose Oxidase/química , Glicemia/química , Enzimas Imobilizadas/química , Desenho de Equipamento , Análise de Falha de Equipamento , Glucose Oxidase/análise , Humanos , Cetonas/química , Agulhas , Polietilenoglicóis/química , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
7.
Scand J Clin Lab Invest ; 76(4): 304-8, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26956540

RESUMO

Background and aim This study was conducted to investigate the association between serum nitric oxide metabolites (NOx) and the risk of chronic kidney disease (CKD). Methods We recruited 3462 and 2504 participants of the Tehran Lipid and Glucose Study (TLGS), for a cross-sectional and prospective analysis, respectively. Serum NOx concentrations were measured at baseline (2006-2008), and demographics, anthropometrics and biochemical variables were evaluated at baseline and again after 3 years (2009-2011). Estimated glomerular filtration rate (eGFR) and CKD were defined. Association between serum NOx and CKD in the cross-sectional phase and the predictability of NOx in CKD occurrence were assessed using multivariable logistic regression models with adjustment for confounders. Results Mean age of participants was 45.0 ± 15.9 years at baseline and 40.5% were male. The prevalence of CKD was 17.9% (13.4% in men, 21.0% in women) at baseline, at which point, marginally significant odds of CKD in the highest tertile of serum NOx in men (OR = 1.53, 95% CI = 0.96-2.45, p for trend = 0.047) and a significant odds of CKD in women (OR = 2.48, 95% CI = 1.76-3.49, p for trend = 0.001) were observed. After a 3-year follow-up, in women, risk of CKD was higher in the highest compared to the lowest NOx tertiles (OR = 1.86, 95% CI = 1.10-3.14, p for trend = 0.032) but no significant association was observed in men. Conclusion Serum NOx level was found to be an independent predictor of CKD in women; it could be a valuable surrogate for prediction of renal dysfunction in women and help to identify high-risk subjects.


Assuntos
Glicemia/análise , Lipídeos/sangue , Óxido Nítrico/sangue , Insuficiência Renal Crônica/fisiopatologia , Adulto , Idoso , Bioestatística , HDL-Colesterol/análise , HDL-Colesterol/sangue , Colorimetria , Feminino , Taxa de Filtração Glomerular , Glucose Oxidase/análise , Glucose Oxidase/sangue , Humanos , Irã (Geográfico) , Lipídeos/análise , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Óxido Nítrico/análise , Fatores de Risco , Espectrofotometria
8.
J Pharm Biomed Anal ; 117: 551-9, 2016 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-26498392

RESUMO

Paper-based sensors gained almost explosive attention during the last few years. A large number of systems, often destined to resource limited settings is based on enzymatic reactions. Choice of an adequate immobilization method could significantly prolong the shelf-life of such sensors, especially in applications, where exposure to high temperatures during storage and transport is more than a threat. We are seeking to compare a variety of immobilization methods based on different phenomena (adsorption, entrapment in gel, microencapsulation, covalent linkage), with total of 33 methods tested. Glucose oxidase was used as a model enzyme. Enzymatic activity of immobilized samples was accompanied for a period of 24 weeks considering two sets of samples, one stored in 4 °C and other in ambient temperature.


Assuntos
Técnicas Biossensoriais/métodos , Técnicas Biossensoriais/normas , Enzimas Imobilizadas/análise , Glucose Oxidase/análise , Papel , Adsorção , Concentração de Íons de Hidrogênio
9.
Environ Entomol ; 44(6): 1531-43, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26454474

RESUMO

As sessile organisms, plants have evolved different methods to defend against attacks and have adapted their defense measures to discriminate between mechanical damage and herbivory by insects. One of the ways that plant defenses are triggered is via elicitors from insect oral secretions (OS). In this study, we investigated the ability of second-instar (L2) spruce budworm [SBW; Choristoneura fumiferana (Clemens)] to alter the volatile organic compounds (VOCs) of four conifer species [Abies balsamea (L.) Mill., Picea mariana (Miller) B.S.P., Picea glauca (Moench) Voss, Picea rubens (Sargent)] and found that the emission profiles from all host trees were drastically changed after herbivory. We then investigated whether some of the main elicitors (fatty acid conjugates [FACs], ß-glucosidase, and glucose oxidase) studied were present in SBW OS. FACs (glutamine and glutamic acid) based on linolenic, linoleic, oleic, and stearic acids were all observed in varying relative quantities. Hydroxylated FACs, such as volicitin, were not observed. Enzyme activity for ß-glucosidase was also measured and found present in SBW OS, whereas glucose oxidase activity was not found in the SBW labial glands. These results demonstrate that SBW L2 larvae have the ability to induce VOC emissions upon herbivory and that SBW OS contain potential elicitors to induce these defensive responses. These data will be useful to further evaluate whether these elicitors can separately induce the production of specific VOCs and to investigate whether and how these emissions benefit the plant.


Assuntos
Abies/química , Herbivoria , Mariposas/fisiologia , Picea/química , Compostos Orgânicos Voláteis/análise , Abies/fisiologia , Animais , Glândulas Exócrinas/enzimologia , Ácidos Graxos/análise , Glucose Oxidase/análise , Larva/química , Larva/fisiologia , Mariposas/química , Mariposas/enzimologia , Boca/química , Boca/metabolismo , Picea/fisiologia , beta-Glucosidase/análise
10.
Lab Chip ; 15(22): 4314-21, 2015 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-26394820

RESUMO

In this paper, we present a novel approach to enhance the sensitivity of microfluidic biosensor platforms with self-assembled magnetic bead chains. An adjustable, more than 5-fold sensitivity enhancement is achieved by introducing a magnetic field gradient along a microfluidic channel by means of a soft-magnetic lattice with a 350 µm spacing. The alternating magnetic field induces the self-assembly of the magnetic beads in chains or clusters and thus improves the perfusion and active contact between the analyte and the beads. The soft-magnetic lattices can be applied independent of the channel geometry or chip material to any microfluidic biosensing platform. At the same time, the bead-based approach achieves chip reusability and shortened measurement times. The bead chain properties and the maximum flow velocity for bead retention were validated by optical microscopy in a glass capillary. The magnetic actuation system was successfully validated with a biotin-streptavidin model assay on a low-cost electrochemical microfluidic chip, fabricated by dry-film photoresist technology (DFR). Labelling with glucose oxidase (GOx) permits rapid electrochemical detection of enzymatically produced H2O2.


Assuntos
Técnicas Biossensoriais/instrumentação , Técnicas Eletroquímicas/instrumentação , Campos Magnéticos , Técnicas Analíticas Microfluídicas/instrumentação , Microesferas , Glucose Oxidase/análise , Glucose Oxidase/metabolismo , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/metabolismo , Estreptavidina/química
11.
Diabetes Technol Ther ; 17(9): 635-48, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26110670

RESUMO

BACKGROUND: Adherence to established standards (e.g., International Organization for Standardization [ISO] 15197) is important to ensure comparable and sufficient accuracy of systems for self-monitoring of blood glucose (SMBG). Accuracy evaluation was performed for different SMBG systems available in Europe with three reagent lots each. MATERIALS AND METHODS: Test procedures followed the recently published revision ISO 15197:2013. Comparison measurements were performed with a glucose oxidase (YSI 2300 STAT Plus™ glucose analyzer; YSI Inc., Yellow Springs, OH) and a hexokinase (cobas Integra(®) 400 Plus analyzer; Roche Instrument Center, Rotkreuz, Switzerland) method. Compliance with ISO 15197:2013 accuracy criteria was determined by calculating the percentage of results within ±15% or within ±0.83 mmol/L of the comparison measurement results for glucose concentrations at and above or below 5.55 mmol/L, respectively, and by calculating the percentage of results within consensus error grid Zones A and B. RESULTS: Seven systems showed with all three tested lots that 95-100% of the results were within the accuracy limits of ISO 15197:2013 and that 100% of results were within consensus error grid Zones A and B, irrespective of the comparison method used. Regarding results of individual lots, slight differences between the glucose oxidase method and the hexokinase method were found. Accuracy criteria of ISO 15197:2003 (±20% for concentrations ≥4.2 mmol/L and±0.83 mmol/L for concentrations <4.2 mmol/L) were fulfilled by eight systems with all three lots and by one system with two lots. CONCLUSIONS: In this study, seven systems complied with the accuracy criteria of ISO 15197:2013. The results also indicate that the comparison measurement method/system is important, as it may have a considerable impact on accuracy data obtained for a system.


Assuntos
Automonitorização da Glicemia/instrumentação , Glicemia/análise , Diabetes Mellitus/sangue , Automonitorização da Glicemia/normas , Glucose Oxidase/análise , Hexoquinase/análise , Humanos , Fitas Reagentes , Padrões de Referência , Reprodutibilidade dos Testes
12.
J Agric Food Chem ; 62(20): 4571-6, 2014 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-24766280

RESUMO

The glucose oxidase/peroxidase assay (GOP) is a coupled enzymatic assay commonly used in measuring glucose concentrations in biological sciences and food chemistry, particularly for quantification of α-glucosidase activity. However, we found that the GOP assay is prone to interference, especially from reducing substances such as polyphenolic compounds, which are commonly found in botanical materials. To establish the scope and limitation of the assay in measuring α-glucosidase inhibition activity, we systematically investigated the structural features of the polyphenolic compounds that can lead to false positives. Utilizing sodium dodecyl sulfate (SDS) as surrogate for the meriquinone intermediate formed during the reaction, we measured the reactivity of this redox active intermediate toward common flavonoids. Our results show that flavonoids with o-dihydroxy groups in the B-ring cause strong interference and that compounds with little DPPH scavenging activity do not have interference. Our results highlight the need for checking the suitability of the GOP assay first before it is applied in measuring α-glucosidase inhibition activity. In addition, when the literature data on α-glucosidase inhibition activity of botanical extracts or polyphenolic compounds using GOP assay are interpreted, potential false positives due to interference on the assay will need to be taken into consideration.


Assuntos
Inibidores Enzimáticos/química , Glucose Oxidase/análise , Glucose/química , Peroxidases/análise , Polifenóis/química , Ensaios Enzimáticos , Glucose Oxidase/antagonistas & inibidores , Oxirredução , Peroxidases/antagonistas & inibidores
14.
J Med Food ; 17(2): 290-3, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24192110

RESUMO

Although hydrogen peroxide (H2O2) is one of the major antibacterial factors in most honeys, it does not accumulate in medical-grade manuka honey. The goal of this study was to investigate the effect of artificially added methylglyoxal (MGO) on H2O2 accumulation in natural non-manuka honeys. H2O2 concentrations in the honey solutions were determined using a fluorimetric assay. Two, the most potent H2O2 producers honeydew honeys were mixed with MGO at final concentrations of 250, 500, and 1000 mg/kg, and incubated for 4 days at 37°C. Subsequently, H2O2 concentrations were determined in 50% (wt/vol) MGO supplemented honey solutions. In vitro crosslinking of the enzyme glucose oxidase (GOX) after incubation with MGO was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Tested honeys at a concentration of 50% (wt/vol) accumulated up to 495.8±9.1 µM H2O2 in 24 h. The most potent producers were the two honeydew honeys, whose 50% solutions accumulated 306.9±6.8 and 495.8±9.1 µM H2O2, respectively. Levels of H2O2 increased significantly over time in both honey solutions. Contrary to this, the MGO-treated honeys generated significantly lower amounts of H2O2 (P<.001), and this reduction was dose dependent. In addition, MGO-treated GOX formed high molecular weight adducts with increasing time of incubation accompanied by loss of its enzymatic activity. High levels of MGO in manuka honey, by modifying the enzyme GOX, might be responsible for suppressing H2O2 generation. These data highlight the detrimental effect of MGO on significant proteinaceous components of manuka honey.


Assuntos
Inibidores Enzimáticos/análise , Proteínas Fúngicas/antagonistas & inibidores , Glucose Oxidase/antagonistas & inibidores , Mel/análise , Peróxido de Hidrogênio/análise , Aldeído Pirúvico/análise , Aspergillus niger/enzimologia , Proteínas Fúngicas/análise , Proteínas Fúngicas/metabolismo , Glucose Oxidase/análise , Glucose Oxidase/metabolismo , Peróxido de Hidrogênio/metabolismo
15.
Opt Express ; 22(25): 30571-8, 2014 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-25607004

RESUMO

We demonstrate a novel glucose sensor based on an optical fiber grating with an excessively tilted index fringe structure and its surface modified by glucose oxidase (GOD). The aminopropyltriethoxysilane (APTES) was utilized as binding site for the subsequent GOD immobilization. Confocal microscopy and fluorescence microscope were used to provide the assessment of the effectiveness in modifying the fiber surface. The resonance wavelength of the sensor exhibited red-shift after the binding of the APTES and GOD to the fiber surface and also in the glucose detection process. The red-shift of the resonance wavelength showed a good linear response to the glucose concentration with a sensitivity of 0.298 nm·(mg/ml)-1 in the very low concentration range of 0.0~3.0mg/ml. Compared to the previously reported glucose sensor based on the GOD-immobilized long period grating (LPG), the 81° tilted fiber grating (81°-TFG) based sensor has shown a lower thermal cross-talk effect, better linearity and higher Q-factor in sensing response. In addition, its sensitivity for glucose concentration can be further improved by increasing the grating length and/or choosing a higher-order cladding mode for detection. Potentially, the proposed techniques based on 81°-TFG can be developed as sensitive, label free and micro-structural sensors for applications in food safety, disease diagnosis, clinical analysis and environmental monitoring.


Assuntos
Técnicas Biossensoriais/instrumentação , Tecnologia de Fibra Óptica/instrumentação , Glucose Oxidase/análise , Glucose/análise , Fibras Ópticas , Refratometria/instrumentação , Transdutores , Desenho de Equipamento
16.
Anal Chem ; 85(12): 6107-12, 2013 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-23697395

RESUMO

The display of glucose oxidase (GOx) on yeast cell surface using a-agglutinin as an anchor motif was successfully developed. Both the immunochemical analysis and enzymatic assay showed that active GOx was efficiently expressed and translocated on the cell surface. Compared with conventional GOx, the yeast cell surface that displayed GOx (GOx-yeast) demonstrated excellent enzyme properties, such as good stability within a wide pH range (pH 3.5-11.5), good thermostability (retaining over 94.8% enzyme activity at 52 °C and 84.2% enzyme activity at 56 °C), and high d-glucose specificity. In addition, direct electrochemistry was achieved at a GOx-yeast/multiwalled-carbon-nanotube modified electrode, suggesting that the host cell of yeast did not have any adverse effect on the electrocatalytic property of the recombinant GOx. Thus, a novel electrochemical glucose biosensor based on this GOx-yeast was developed. The as-prepared biosensor was linear with the concentration of d-glucose within the range of 0.1-14 mM and a low detection limit of 0.05 mM (signal-to-noise ratio of S/N = 3). Moreover, the as-prepared biosensor is stable, specific, reproducible, simple, and cost-effective, which can be applicable for real sample detection. The proposed strategy to construct robust GOx-yeast may be applied to explore other oxidase-displaying-system-based whole-cell biocatalysts, which can find broad potential application in biosensors, bioenergy, and industrial catalysis.


Assuntos
Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos , Glucose Oxidase/análise , Glucose/análise , Leveduras/química , Leveduras/enzimologia , Enzimas , Glucose/metabolismo , Glucose Oxidase/metabolismo , Propriedades de Superfície
17.
Nanotechnology ; 24(21): 215101, 2013 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-23619092

RESUMO

In this study, an enzymatic glucose biosensor based on a three-dimensional gold nanodendrite (GND) modified screen-printed electrode was developed. The GNDs were electrochemically synthesized on the working electrode component of a commercially available screen-printed electrode using a solution acquired by dissolving bulk gold in aqua regia as the precursor. The 3D GND electrode greatly enhanced the effective sensing area of the biosensor, which improved the sensitivity of glucose detection. Actual glucose detections demonstrated that the fabricated devices could perform at a sensitivity of 46.76 µA mM⁻¹ cm⁻² with a linear detection range from 28 µM-8.4 mM and detection limit of 7 µM. A fast response time (∼3 s) was also observed. Moreover, only a 20 µl glucose oxidase is required for detection owing to the incorporation of the commercially available screen-printed electrode.


Assuntos
Técnicas Biossensoriais/instrumentação , Condutometria/instrumentação , Glucose Oxidase/química , Glucose/análise , Ouro/química , Nanopartículas Metálicas/química , Nanopartículas Metálicas/ultraestrutura , Microeletrodos , Nanotecnologia/instrumentação , Cristalização/métodos , Desenho de Equipamento , Análise de Falha de Equipamento , Glucose/química , Glucose Oxidase/análise , Teste de Materiais , Tamanho da Partícula , Propriedades de Superfície
18.
J Sep Sci ; 36(7): 1169-75, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23495135

RESUMO

Lectin-functionalized monolithic columns were prepared within polyether ether ketone (PEEK) columns (150 × 4.6 mm id) via transition metal-catalyzed ring-opening metathesis polymerization of norborn-2-ene (NBE) and trimethylolpropane-tris(5-norbornene-2-carboxylate) (CL) using the first-generation Grubbs initiator RuCl2 (PCy3 )2 (CHPh) (1, Cy = cyclohexyl) in the presence of a macro- and microporogen, i.e. of 2-propanol and toluene. Postsynthesis functionalization was accomplished via in situ grafting of 2,5-dioxopyrrolidin-1-yl-bicyclo[2.2.1]hept-5-ene-2-carboxylate to the surface of the monoliths followed by reaction with α,ω-diamino-poly(ethyleneglycol). The pore structure of the poly(ethyleneglycol)- derivatized monoliths was investigated by electron microscopy and inverse-size exclusion chromatography, respectively. The amino-poly(ethyleneglycol) functionalized monolithic columns were then successfully used for the immobilization of lectin from Lens culinaris hemagglutinin. The thus prepared lectin-functionalized monoliths were applied to the affinity chromatography-based purification of glucose oxidase. The binding capacity of Lens culinaris hemagglutinin-immobilized monolithic column for glucose oxidase was found to be 2.2 mg/column.


Assuntos
Cromatografia de Afinidade , Glicoproteínas/química , Lectinas/química , Catálise , Glucose Oxidase/análise , Glucose Oxidase/química , Glicoproteínas/análise , Cetonas/química , Microscopia Eletrônica de Varredura , Estrutura Molecular , Polietilenoglicóis/química , Polimerização , Ligação Proteica , Reprodutibilidade dos Testes
19.
J Nanobiotechnology ; 11: 6, 2013 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-23425592

RESUMO

BACKGROUND: Generation-3 (Gen-3) biosensors and advanced enzyme biofuel cells will benefit from direct electron transfer to oxidoreductases facilitated by single-walled carbon nanotubes (SWNTs). METHODS: Supramolecular conjugates of SWNT-glucose oxidase (GOx-SWNT) were produced via ultrasonic processing. Using a Plackett-Burman experimental design to investigate the process of tip ultrasonication (23 kHz), conjugate formation was investigated as a function of ultrasonication times (0, 5, 60 min) and functionalized SWNTs of various tube lengths (SWNT-X-L), (X = -OH or -COOH and L = 3.0 µm, 7.5 µm). RESULTS: Enzyme activity (KM, kcat, kcat/KM, vmax and n (the Hill parameter)) of pGOx (pristine), sGOx (sonicated) and GOx-SWNT-X-L revealed that sonication of any duration increased both KM and kcat of GOx but did not change kcat/KM. Functionalized tubes had the most dramatic effect, reducing both KM and kcat and reducing kcat/KM. UV-vis spectra over the range of 300 to 550 nm of native enzyme-bound FAD (λmax at 381 and 452 nm) or the blue-shifted solvated FAD of the denatured enzyme (λmax at 377 and 448 nm) revealed that ultrasonication up to 60 minutes had no influence on spectral characteristics of FAD but that the longer SWNTs caused some partial denaturation leading to egress of FAD. Circular dichroism spectral analysis of the 2° structure showed that sonication of any duration caused enrichment in the α-helical content at the sacrifice of the unordered sequences in GOx while the presence of SWNTs, regardless of length and/or functionality, reduced the ß-sheet content of pristine GOx. Surface profiling by white light interferometry revealed that ultrasonication produced some aggregation of GOx and that GOx effectively debundled the SWNT. CONCLUSIONS: Supramolecular conjugates formed from shorter, -OH functionalized SWNTs using longer sonication times (60 min) gave the most favored combination for forming bioactive conjugates.


Assuntos
Glucose Oxidase/análise , Nanotubos de Carbono/análise , Materiais Biocompatíveis/análise , Materiais Biocompatíveis/química , Técnicas Biossensoriais , Dicroísmo Circular , Transporte de Elétrons , Glucose Oxidase/química , Nanotubos de Carbono/química , Ultrassom/métodos
20.
Chem Commun (Camb) ; 48(90): 11133-5, 2012 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-23044599

RESUMO

Based on selective pore-opening in the presence of protease, we have developed a novel signal amplification assay for multiple proteases detection and their inhibition using protein-capped mesoporous scaffolding as the substrate.


Assuntos
Peptídeo Hidrolases/análise , Flavina-Adenina Dinucleotídeo/química , Glucose Oxidase/análise , Mercúrio/química , Nanopartículas/química , Fluoreto de Fenilmetilsulfonil/química , Porosidade , Dióxido de Silício/química
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