RESUMO
Polyaniline (PANI) films are promising candidates for electronic nose-based IoT applications, but device performances are influenced by fabrication parameters and ambient conditions. Affinities of different PANI composites to analytes for gas sensing applications remain elusive. In this study, we investigate the material properties in detail for two different dopant systems: F4TCNQ and carbon black. Using a reproducibility-driven approach, we investigate different dopant concentrations in regard to their sensitivity and specificity towards five relevant markers for breath cancer diagnosis. We benchmark the system using ammonia measurements and evaluate limits of detection. Furthermore, we provide statistical analysis on reproducibility and pave the way towards machine learning discrimination via principal component analysis. The influence of relative humidity on sensor hysteresis is also investigated. We find that F4TCNQ-doped PANI films show improved reproducibility compared to carbon black-doped films. We establish and quantify a tradeoff between sensitivity, reproducibility, and environmental stability by the choice of dopant and concentrations ratios.
Assuntos
Amônia , Fuligem , Amônia/análise , Compostos de Anilina , Reprodutibilidade dos TestesRESUMO
By combining surface plasmon resonance (SPR) and electrolyte gated field-effect transistor (EG-FET) methods in a single analytical device we introduce a novel tool for surface investigations, enabling simultaneous measurements of the surface mass and charge density changes in real time. This is realized using a gold sensor surface that simultaneously serves as a gate electrode of the EG-FET and as the SPR active interface. This novel platform has the potential to provide new insights into (bio)adsorption processes on planar solid surfaces by directly relating complementary measurement principles based on (i) detuning of SPR as a result of the modification of the interfacial refractive index profile by surface adsorption processes and (ii) change of output current as a result of the emanating effective gate voltage modulations. Furthermore, combination of the two complementary sensing concepts allows for the comparison and respective validation of both analytical techniques. A theoretical model is derived describing the mass uptake and evolution of surface charge density during polyelectrolyte multilayer formation. We demonstrate the potential of this combined platform through the observation of layer-by-layer assembly of PDADMAC and PSS. These simultaneous label-free and real-time measurements allow new insights into complex processes at the solid-liquid interface (like non-Fickian ion diffusion), which are beyond the scope of each individual tool.
RESUMO
Mutations in Toll-like receptor (TLR) and myeloid differentiation primary response 88 (MYD88) genes have been found in chronic lymphocytic leukemia (CLL) at low frequency. We analyzed the incidence, clinicobiological characteristics, and outcome of patients with TLR/MYD88 mutations in 587 CLL patients. Twenty-three patients (3.9%) had mutations, 19 in MYD88 (one with concurrent IRAK1 mutation), 2 TLR2 (one with concomitant TLR6 mutation), 1 IRAK1, and 1 TLR5. No mutations were found in IRAK2 and IRAK4. TLR/MYD88-mutated CLL overexpressed genes of the nuclear factor κB pathway. Patients with TLR/MYD88 mutations were significantly younger (83% age ≤50 years) than those with no mutations. TLR/MYD88 mutations were the most frequent in young patients. Patients with mutated TLR/MYD88 CLL had a higher frequency of mutated IGHV and low expression of CD38 and ZAP-70. Overall survival (OS) was better in TLR/MYD88-mutated than unmutated patients in the whole series (10-year OS, 100% vs 62%; P = .002), and in the subset of patients age ≤50 years (100% vs 70%; P = .02). In addition, relative OS of TLR/MYD88-mutated patients was similar to that in the age- and gender-matched population. In summary, TLR/MYD88 mutations identify a population of young CLL patients with favorable outcome.
Assuntos
Leucemia Linfocítica Crônica de Células B/diagnóstico , Leucemia Linfocítica Crônica de Células B/genética , Mutação , Fator 88 de Diferenciação Mieloide/genética , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/mortalidade , Masculino , Pessoa de Meia-Idade , Prognóstico , Transdução de Sinais/genética , Adulto JovemRESUMO
An artificial bilayer lipid membrane system is presented, featuring the oriented encapsulation of membrane proteins in a functionally active form. Nickel nitrilo-triacetic acid-functionalized silica nanoparticles, of a diameter of around 25 nm, are used to attach the proteins via a genetically engineered histidine tag in a uniform orientation. Subsequently, the proteins are reconstituted within a phospholipid bilayer, formed around the particles by in situ dialysis to form so-called proteo-lipobeads (PLBs). With a final size of about 50 nm, the PLBs can be employed for UV/vis spectroscopy studies, particularly of multiredox center proteins, because the effects of light scattering are negligible. As a proof of concept, we use cytochrome c oxidase (CcO) from P. denitrificans with the his tag genetically engineered to subunit I. In this orientation, the P side of CcO is directed to the outside and hence electron transfer can be initiated by reduced cytochrome c (cc). UV/vis measurements are used in order to determine the occupancy by CcO molecules encapsulated in the lipid bilayer as well as the kinetics of electron transfer between CcO and cc. The kinetic data are analyzed in terms of the Michaelis-Menten kinetics showing that the turnover rate of CcO is significantly decreased compared to that of solubilized protein, whereas the binding characteristics are improved. The data demonstrate the suitability of PLBs for functional cell-free bioassays of membrane proteins.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/química , Bicamadas Lipídicas/química , Proteínas de Membrana/química , Nanopartículas/química , Fosfolipídeos/química , Dióxido de Silício/química , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Bicamadas Lipídicas/síntese química , Modelos Moleculares , Paracoccus denitrificans/enzimologia , Tamanho da Partícula , Fosfolipídeos/síntese química , Propriedades de SuperfícieRESUMO
As a surrogate of live cells, proteo-lipobeads are presented, encapsulating functional membrane proteins in a strict orientation into a lipid bilayer. Assays can be performed just as on live cells, for example using fluorescence measurements. As a proof of concept, we have demonstrated proton transport through cytochrome c oxidase.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/química , Bicamadas Lipídicas/química , Sefarose/química , Biomimética , Fluorescência , Corantes Fluorescentes/químicaRESUMO
An olfactory biosensor based on a reduced graphene oxide (rGO) field-effect transistor (FET), functionalized by the odorant-binding proteinâ 14 (OBP14) from the honey bee (Apis mellifera) has been designed for the inâ situ and real-time monitoring of a broad spectrum of odorants in aqueous solutions known to be attractants for bees. The electrical measurements of the binding of all tested odorants are shown to follow the Langmuir model for ligand-receptor interactions. The results demonstrate that OBP14 is able to bind odorants even after immobilization on rGO and can discriminate between ligands binding within a range of dissociation constants from K(d)=4â µM to K(d)=3.3â mM. The strongest ligands, such as homovanillic acid, eugenol, and methyl vanillate all contain a hydroxy group which is apparently important for the strong interaction with the protein.
Assuntos
Abelhas/química , Técnicas Biossensoriais , Grafite/química , Odorantes/análise , Óxidos/química , Receptores Odorantes/química , Transistores Eletrônicos , Animais , Elétrons , OxirreduçãoRESUMO
The widespread availability of statistical packages has undoubtedly helped hematologists worldwide in the analysis of their data, but has also led to the inappropriate use of statistical methods. In this article, we review some basic concepts of survival analysis and also make recommendations about how and when to perform each particular test using SPSS, Stata and R. In particular, we describe a simple way of defining cut-off points for continuous variables and the appropriate and inappropriate uses of the Kaplan-Meier method and Cox proportional hazard regression models. We also provide practical advice on how to check the proportional hazards assumption and briefly review the role of relative survival and multiple imputation.
Assuntos
Leucemia Linfocítica Crônica de Células B/mortalidade , Guias de Prática Clínica como Assunto , Software , Idoso , Intervalo Livre de Doença , Feminino , Humanos , Estimativa de Kaplan-Meier , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Modelos de Riscos Proporcionais , Curva ROCRESUMO
A novel multivariable system, combining a transistor with fiber optic-based surface plasmon resonance spectroscopy with the gate electrode simultaneously acting as the fiber optic sensor surface, is reported. The dual-mode sensor allows for discrimination of mass and charge contributions for binding assays on the same sensor surface. Furthermore, we optimize the sensor geometry by investigating the influence of the fiber area to transistor channel area ratio and distance. We show that larger fiber optic tip diameters are favorable for electronic and optical signals and demonstrate the reversibility of plasmon resonance wavelength shifts after electric field application. As a proof of principle, a layer-by-layer assembly of polyelectrolytes is performed to benchmark the system against multivariable sensing platforms with planar surface plasmon resonance configurations. Furthermore, the biosensing performance is assessed using a thrombin binding assay with surface-immobilized aptamers as receptors, allowing for the detection of medically relevant thrombin concentrations.
Assuntos
Técnicas Biossensoriais , Fibras Ópticas , Técnicas Biossensoriais/métodos , Eletrodos , Tecnologia de Fibra Óptica/métodos , Trombina/análiseRESUMO
Whether advances in treatment are prolonging survival of patients with chronic lymphocytic leukemia (CLL) is unclear. We analyzed presentation patterns and survival over time in 929 patients followed from 1980 to 2008 at the Hospital Clinic of Barcelona. The 5- and 10-year relative survival (adjusted for the expected survival in the general population) was estimated in patients seen in 2 periods of time: 1980-1994 (n = 451) and 1995-2004 (n = 365). We found that CLL shortens life expectancy in all age groups independently of clinical features at diagnosis. Nevertheless, survival is improving, particularly in some groups of patients. Thus, relative survival was significantly higher in the 1995-2004 cohort than in the 1980-1994 group both at 5 years (incidence rate ratio [IRR] = 0.46; P = .004) and 10 years (IRR = 0.65; P = .007) from diagnosis. The improved survival was largely due to a decrease in CLL-attributable mortality in patients younger than 70 years in Binet stage B or C at diagnosis (IRR = 0.40; P = .001 at 5 years; IRR = 0.33; P < .001 at 10 years). These results suggest that newer treatments are changing the prognosis of CLL, particularly in younger patients with advanced disease, whereas no improvement is yet observed in older subjects or those with lower-risk disease.
Assuntos
Leucemia Linfocítica Crônica de Células B/mortalidade , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Intervalo Livre de Doença , Feminino , Hospitais Públicos , Humanos , Leucemia Linfocítica Crônica de Células B/terapia , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Espanha , Taxa de SobrevidaRESUMO
The inflammasomes are macromolecular cytosolic complexes involved in the production of interleukin-1beta (IL-1beta) and IL-18 in response to several pathogen-derived stimuli. Such interleukins have been implicated in the origin of severe allogeneic stem cell transplant (allo-SCT) complications. We analyzed the relationship between the interindividual variability in inflammasome protein-encoding genes in donors and patients and clinical outcome after allo-SCT. Fourteen common genetic variants in 5 genes of the inflammasome, namely, NLRP1, NLRP2, NLRP3, CARD8, and CASP5, were genotyped in 133 human leukocyte antigen-identical sibling pairs undergoing allo-SCT. In the multivariate analysis, donor variants in NLRP2 and NLRP3 were the most important prognostic factors for the clinical outcome after allo-SCT. Thus, donor TT genotype at rs10925027 in NLRP3 was associated with disease relapse (odds ratio (OR) = 6.3, P = 1 x 10(-7)), and donor GG genotype at rs1043684 in NLRP2 was associated with nonrelapse mortality (OR = 4.4, P = 6 x 10(-4)) and overall survival (OR = 3.1, P = .001). In addition, patient AA genotype at rs5862 in NLRP1 was associated with nonrelapse mortality (OR = 2.8, P = .005) and overall survival (OR = 2.0, P = .009). These results suggest that inflammasome genetic variants are important prognostic factors for the outcome of allo-SCT. If validated in larger studies, including unrelated allo-SCT, NLRPs genotype would become an important factor in donor selection.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Transporte/genética , Variação Genética , Antígenos HLA , Irmãos , Transplante de Células-Tronco , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adolescente , Adulto , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Proteínas de Transporte/metabolismo , Caspases/genética , Caspases/metabolismo , Intervalo Livre de Doença , Feminino , Genótipo , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/metabolismo , Neoplasias Hematológicas/mortalidade , Humanos , Interleucina-18 , Interleucina-1beta/metabolismo , Masculino , Pessoa de Meia-Idade , Proteína 3 que Contém Domínio de Pirina da Família NLR , Proteínas NLR , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Valor Preditivo dos Testes , Recidiva , Estudos Retrospectivos , Taxa de Sobrevida , Transplante HomólogoRESUMO
BACKGROUND: The concept of "accelerated" chronic lymphocytic leukemia is frequently used by both pathologists and clinicians. However, neither histological criteria to define this form of chronic lymphocytic leukemia nor its clinical correlates and prognostic impact have been formally defined in large series of patients. DESIGN AND METHODS: Tissue biopsies from 100 patients with chronic lymphocytic leukemia were analyzed for the size of proliferation centers and their proliferation rate as assessed by mitosis count and Ki-67 immunostaining. Histological patterns were correlated with main clinico-biological features and outcome. RESULTS: A suspicion of disease transformation was the main reason for carrying out tissue biopsy, which was performed at a median time of 14 months (range, 0 to 204 months) after the diagnosis of chronic lymphocytic leukemia. The biopsy showed histological transformation to diffuse large B-cell lymphoma in 22 cases. In the remaining 78 patients, the presence of expanded proliferation centers (broader than a 20x field) and high proliferation rate (either >2.4 mitoses/proliferation center or Ki-67 >40%/proliferation center) predicted a poor outcome and were selected to define a highly proliferative group. Thus, 23 patients with either expanded proliferation centers or high proliferation rate were considered as having "accelerated" chronic lymphocytic leukemia. These patients displayed particular features, including higher serum lactate dehydrogenase levels and more frequently elevated ZAP-70 than "non-accelerated" cases. The median survival from biopsy of patients with "non-accelerated" chronic lymphocytic leukemia, "accelerated" chronic lymphocytic leukemia and transformation to diffuse large B-cell leukemia was 76, 34, and 4.3 months, respectively (P<0.001). CONCLUSIONS: The presence of expanded and/or highly active proliferation centers identifies a group of patients with "accelerated" chronic lymphocytic leukemia characterized by an aggressive clinical behavior.
Assuntos
Proliferação de Células , Leucemia Linfocítica Crônica de Células B/patologia , Linfonodos/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biópsia , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/diagnóstico , Masculino , Pessoa de Meia-Idade , Prognóstico , Adulto Jovem , Proteína-Tirosina Quinase ZAP-70/análiseRESUMO
OBJECTIVE: The Mannan-binding lectin (MBL) pathway involves recognition of fungal surfaces by MBL and cleavage of C2 and C4 by MBL-associated serine protease (namely, MASP-2). Recent data show that MBL pathway deficiency might result not only from polymorphisms of the MBL2 gene but also of MASP2. The aim of the study was to assess whether polymorphisms of these genes are associated with invasive fungal infections (IFIs) following allogeneic stem cell transplantation (allo-SCT). METHODS: The promoter and the exon 1 of MBL2 and the exon 3 of MASP2 were sequenced in 106 donor-recipient pairs from HLA-identical sibling allo-SCTs performed in a single institution. RESULTS: Ten percent of the donors and 11% of the recipients carried the MBL-low (O/O, LXA/O) genotypes; 7% of the donors and 3% of the recipients were heterozygous for the MASP2 Asp105Gly variant. Factors associated with a higher probability of IFIs were donor's MBL-low genotype (38% vs 12%, p = 0.01), recipient's MASP2 variant (67% vs 14%, p = 0.01), and acute graft-versus-host disease (GVHD) grades II to IV (27% vs 11%, p = 0.04); in the multivariate analysis MBL-low genotype (relative risk [RR] 7.3, p = 0.003), MASP2 variant (RR 6.4, p = 0.002), and acute GVHD II to IV (RR 3.8, p = 0.02) retained independent prognostic value. CONCLUSION: These results show for the first time that polymorphisms responsible for not only MBL but also MASP-2 deficiency are independent predictive factors for IFI after allo-SCT.
Assuntos
Lectina de Ligação a Manose da Via do Complemento/genética , Lectina de Ligação a Manose/genética , Serina Proteases Associadas a Proteína de Ligação a Manose/genética , Micoses/genética , Polimorfismo Genético , Transplante de Células-Tronco , Doença Aguda , Adulto , Complemento C2/genética , Complemento C2/metabolismo , Complemento C4/genética , Complemento C4/metabolismo , Análise Mutacional de DNA/métodos , Éxons/genética , Feminino , Predisposição Genética para Doença , Genótipo , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/metabolismo , Neoplasias Hematológicas/complicações , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/terapia , Humanos , Masculino , Lectina de Ligação a Manose/metabolismo , Serina Proteases Associadas a Proteína de Ligação a Manose/metabolismo , Pessoa de Meia-Idade , Micoses/etiologia , Micoses/metabolismo , Valor Preditivo dos Testes , Prognóstico , Regiões Promotoras Genéticas/genética , Transplante de Células-Tronco/efeitos adversos , Doadores de Tecidos , Transplante HomólogoRESUMO
We present the construction of layer-by-layer (LbL) assemblies of polyethylenimine and urease onto reduced-graphene-oxide based field-effect transistors (rGO FETs) for the detection of urea. This versatile biosensor platform simultaneously exploits the pH dependency of liquid-gated graphene-based transistors and the change in the local pH produced by the catalyzed hydrolysis of urea. The use of an interdigitated microchannel resulted in transistors displaying low noise, high pH sensitivity (20.3µA/pH) and transconductance values up to 800 µS. The modification of rGO FETs with a weak polyelectrolyte improved the pH response because of its transducing properties by electrostatic gating effects. In the presence of urea, the urease-modified rGO FETs showed a shift in the Dirac point due to the change in the local pH close to the graphene surface. Markedly, these devices operated at very low voltages (less than 500mV) and were able to monitor urea in the range of 1-1000µm, with a limit of detection (LOD) down to 1µm, fast response and good long-term stability. The urea-response of the transistors was enhanced by increasing the number of bilayers due to the increment of the enzyme surface coverage onto the channel. Moreover, quantification of the heavy metal Cu2+(with a LOD down to 10nM) was performed in aqueous solution by taking advantage of the urease specific inhibition.
Assuntos
Técnicas Biossensoriais/instrumentação , Grafite/química , Óxidos/química , Polieletrólitos/química , Transistores Eletrônicos , Ureia/sangue , Canavalia/enzimologia , Enzimas Imobilizadas/química , Desenho de Equipamento , Humanos , Limite de Detecção , Ureia/análise , Urease/químicaRESUMO
Proteo-lipobeads (PLBs) are investigated as cell-free model systems to encapsulate membrane proteins such as ion channels and transporters. PLBs are based on nickel nitrile tri-acetic acid (Ni-NTA)-functionalized agarose beads, onto which membrane proteins (MP) are bound via histidine(his)-tag. Composite beads thus obtained (subsequently called proteobeads) are dialyzed in the presence of lipid micelles to form PLBs. As an example we employed cytochrome c oxidase from P. denitrificans with a his-tag fused to the C-terminus of subunitI. In this orientation the P side of CcO faces the outside of the PLB and hence protons are released to the outer aqueous phase, when electron transfer is initiated by light excitation of Ru complexes. Proton release kinetics was probed by fluorescence microscopy using the pH-sensitive sensor molecule fluorescein DHPE inserted into the lipid layer. In order to monitor the generation of membrane potentials we performed a FLIPR assay on the CcO embedded in PLBs using the FRET pair CC2-DMPE/DiSBAC2(3). The combined results show that PLBs can be used as a model system designed to quantify the kinetic parameters of membrane proteins. In addition, the FLIPR assay demonstrates the feasibility of PLBs for high throughput screening applications.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/química , Paracoccus denitrificans/enzimologia , Proteolipídeos/química , Sefarose/química , Portadores de Fármacos/química , Liberação Controlada de Fármacos , Transporte de Elétrons , Corantes Fluorescentes/química , Ensaios de Triagem em Larga Escala/métodos , Cinética , Bicamadas Lipídicas/química , Potenciais da Membrana , Microesferas , Propriedades de SuperfícieRESUMO
His-tag technology is employed to bind membrane proteins, such as the bc1 complex and the reaction center (RC) from Rhodobacter sphaeroides, to spherical as well as planar surfaces in a strict orientation. Subsequently, the spherical and planar surfaces are subjected to in situ dialysis to form proteo-lipobeads (PLBs) and protein-tethered bilayer membranes, respectively. PLBs based on Ni-nitrileotriacetic acid-functionalized agarose beads that have diameters ranging from 50 to 150 µm are used to assess proton release and membrane potential parameters by confocal laser-scanning microscopy. The pH and potential transients are thus obtained from bc1 activated by the RC. To assess the turnover of bc1 excited by the RC in a similar setting, we used the planar surface of an attenuated total reflection crystal modified with a thin gold layer to carry out time-resolved surface-enhanced IR absorption spectroscopy triggered by flash lamp excitation. The experiments suggest that both proteins interact in a cyclic manner in both environments. The activity of the proteins seems to be preserved in the same manner as that in chromatophores or reconstituted in liposomes.
Assuntos
Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Rhodobacter sphaeroides/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/química , Concentração de Íons de Hidrogênio , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Lipossomos/química , Lipossomos/metabolismo , Tamanho da Partícula , Rhodobacter sphaeroides/química , Propriedades de SuperfícieRESUMO
In the following we give a short summary of examples for biosensor concepts in areas in which reduced graphene oxide-based electronic devices can be developed into new classes of biosensors, which are highly sensitive, label-free, disposable and cheap, with electronic signals that are easy to analyze and interpret, suitable for multiplexed operation and for remote control, compatible with NFC technology, etc., and in many cases a clear and promising alternative to optical sensors. The presented areas concern sensing challenges in medical diagnostics with an example for detecting general antibody-antigen interactions, for the monitoring of toxins and pathogens in food and feed stuff, exemplified by the detection of aflatoxins, and the area of smell sensors, which are certainly the most exciting development as there are very few existing examples in which the typically small and hydrophobic odorant molecules can be detected by other means. The example given here concerns the recording of a honey flavor (and a cancer marker for neuroblastoma), homovanillic acid, by the odorant binding protein OBP 14 from the honey bee, immobilized on the reduced graphene oxide gate of an FET sensor.
Assuntos
Técnicas Biossensoriais , Eletrônica , Grafite/química , Óxidos/química , Transistores Eletrônicos , Anticorpos , Antígenos , Inocuidade dos Alimentos , Odorantes , Ligação Proteica , Sensibilidade e Especificidade , Toxinas BiológicasRESUMO
Time-resolved surface-enhanced IR-absorption spectroscopy (tr-SEIRAS) has been performed on cytochrome c oxidase from Rhodobacter sphaeroides. The enzyme was converted electrochemically into the fully reduced state. Thereafter, in the presence of oxygen, the potential was switched to open circuit potential (OCP). Under these conditions, the enzyme is free to undergo enzymatic oxidation in the absence of an external electric field. Tr-SEIRAS was performed using the step-scan technique, triggered by periodic potential pulses switching between - 800mV and OCP. Single bands were resolved in a broad band in the amide I region using phase sensitive detection. Amplitudes of these bands were analyzed as a function of time. Time constants in the ms time scale were considered in terms of conformational changes of the protein secondary structures associated with the enzymatic turnover of the protein.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Rhodobacter sphaeroides/enzimologia , Transporte de Elétrons , Complexo IV da Cadeia de Transporte de Elétrons/química , Proteínas Imobilizadas/química , Proteínas Imobilizadas/metabolismo , Cinética , Modelos Moleculares , Conformação Proteica , Espectrofotometria InfravermelhoRESUMO
Herein, we present a fast and sensitive biosensor for detection of Ochratoxin A (OTA) in a red wine that utilizes gold nanoparticle-enhanced surface plasmon resonance (SPR). By combining an indirect competitive inhibition immunoassay and signal enhancement by secondary antibodies conjugated with gold nanoparticles (AuNPs), highly sensitive detection of low molecular weight compounds (such as OTA) was achieved. The reported biosensor allowed for OTA detection at concentrations as low as 0.75 ng mL(-1) and its limit of detection was improved by more than one order of magnitude to 0.068 ng mL(-1) by applying AuNPs as a signal enhancer. The study investigates the interplay of size of AuNPs and affinity of recognition elements affecting the efficiency of the signal amplification strategy based on AuNP. Furthermore, we observed that the presence of polyphenolic compounds in wine samples strongly interferes with the affinity binding on the surface. To overcome this limitation, a simple pre-treatment of the wine sample with the binding agent poly(vinylpyrrolidone) (PVP) was successfully applied.
Assuntos
Ouro/química , Nanopartículas Metálicas/química , Ocratoxinas/análise , Ressonância de Plasmônio de Superfície , Vinho/análiseRESUMO
We present an experimental and theoretical characterization for reduced Graphene-Oxide (rGO) based FETs used for biosensing applications. The presented approach shows a complete result analysis and theoretically predictable electrical properties. The formulation was tested for the analysis of the device performance in the liquid gate mode of operation with variation of the ionic strength and pH-values of the electrolytes in contact with the FET. The dependence on the Debye length was confirmed experimentally and theoretically, utilizing the Debye length as a working parameter and thus defining the limits of applicability for the presented rGO-FETs. Furthermore, the FETs were tested for the sensing of biomolecules (bovine serum albumin (BSA) as reference) binding to gate-immobilized anti-BSA antibodies and analyzed using the Langmuir binding theory for the description of the equilibrium surface coverage as a function of the bulk (analyte) concentration. The obtained binding coefficients for BSA are found to be same as in results from literature, hence confirming the applicability of the devices. The FETs used in the experiments were fabricated using wet-chemically synthesized graphene, displaying high electron and hole mobility (µ) and provide the strong sensitivity also for low potential changes (by change of pH, ion concentration, or molecule adsorption). The binding coefficient for BSA-anti-BSA interaction shows a behavior corresponding to the Langmuir adsorption theory with a Limit of Detection (LOD) in the picomolar concentration range. The presented approach shows high reproducibility and sensitivity and a good agreement of the experimental results with the calculated data.