Dialysis and column chromatography for biomass pyrolysis liquids separation.

In the current study, a novel approach for separating value-added chemicals from pine wood residues' pyrolysis liquids (bio-oil) was effectively carried out. It combined two separation techniques used for the first time in this field: dialysis with water, methanol and acetone, and column chromatography with Amberlite™ XAD7 resin. This strategy made it possible to separate bio-oil into four fractions: (1) pyrolytic lignin, which can be utilized in the synthesis of resins, foams, electrodes, asphalt, etc. (2) acid-rich fraction, with particular relevance to the chemical industry, (3) antioxidant fraction, containing phenolic compounds, with a lot of interest for pharmaceutical and nutraceutical industry, and (4) a final fraction containing the most non-polar chemicals from bio-oil. Thus, it was possible to develop a process that allows the obtention of bioproducts from woody biomass, a residue obtained in significant quantities in the management of non-profitable forests, making a step forward within the context of circular economy and bioeconomy.

Employing the metabolic "branch point effect" to generate an all-or-none, digital-like response in enzymatic outputs and enzyme-based sensors.

Here, we demonstrate a strategy to convert the graded Michaelis-Menten response typical of unregulated enzymes into a sharp, effectively all-or-none response. We do so using an approach analogous to the "branch point effect", a mechanism observed in naturally occurring metabolic networks in which two or more enzymes compete for the same substrate. As a model system, we used the enzymatic reaction of glucose oxidase (GOx) and coupled it to a second, nonsignaling reaction catalyzed by the higher affinity enzyme hexokinase (HK) such that, at low substrate concentrations, the second enzyme outcompetes the first, turning off the latter's response. Above an arbitrarily selected "threshold" substrate concentration, the nonsignaling HK enzyme saturates leading to a "sudden" activation of the first signaling GOx enzyme and a far steeper dose-response curve than that observed for simple Michaelis-Menten kinetics. Using the well-known GOx-based amperometric glucose sensor to validate our strategy, we have steepen the normally graded response of this enzymatic sensor into a discrete yes/no output similar to that of a multimeric cooperative enzyme with a Hill coefficient above 13. We have also shown that, by controlling the HK reaction we can precisely tune the threshold target concentration at which we observe the enzyme output. Finally, we demonstrate the utility of this strategy for achieving effective noise attenuation in enzyme logic gates. In addition to supporting the development of biosensors with digital-like output, we envisage that the use of all-or-none enzymatic responses will also improve our ability to engineer efficient enzyme-based catalysis reactions in synthetic biology applications.

Amperometric bienzymatic biosensor for L-lactate analysis in wine and beer samples.

A novel amperometric bienzymatic biosensor has been developed based on the incorporation of Lactate Oxidase (LOx) and Horseradish Peroxidase (HRP) into a carbon nanotube/polysulfone membrane by the phase inversion technique onto screen-printed electrodes (SPEs). In order to improve the sensitivity and reduce the working potential, experimental conditions have been optimized and ferrocene has also been incorporated into the membrane as a redox mediator of the enzymatic reactions, which allows the reduction of H(2)O(2) at -100 mV. Measurements were carried out in phosphate buffer solution at pH 7.5 and under batch conditions. The biosensor response time to L-lactate was only 20 s and showed a good reproducibility (RSD 2.7%). Moreover, the detection limit was 0.05 mg L(-1) of l-lactate with a linear interval range from 0.1 mg L(-1) to 5 mg L(-1). Finally, the biosensor has been applied to the determination of l-lactic acid in different wine and beer samples. Then, the results obtained with the biosensor were compared with the ones obtained using, as a reference method, a commercial kit based on spectrophotometric measurements, obtaining an excellent agreement between the results, validating our approach.

The effect of reactor scale on biochars and pyrolysis liquids from slow pyrolysis of coffee silverskin, grape pomace and olive mill waste, in auger reactors.

Several studies have addressed the potential biorefinery, through small-scale pyrolysis, of coffee silverskin (CSS), grape pomace (GP) and olive mill waste (OMW), which are respectively the main solid residues from coffee roasting, wine making and olive oil production processes. However, increasing the scale of reactor to bring these studies to an industrial level may affect the properties, and hence applications, of the resulting products. The aim of this study is therefore to perform pilot scale experiments to compare and verify the results of analytical study (TGA) and bench scale reactor runs, in order to understand the fundamental differences and create correlations between pyrolysis runs at different scales. To this end, pyrolysis liquids and biochars from the slow pyrolysis of CSS, GP and OMW, performed using different scale auger reactors (15 kg/h and 0.3 kg/h), have been analysed (TGA, pH, density, proximate and ultimate analyses, HHV, FTIR, GCMS) and compared. The results showed no major differences in biochars when the temperature and the solid residence time were fixed. However, regarding pyrolysis liquids, compounds from the lab reactor were more degraded than pilot plant ones, due to, in this case, the vapour residence time was longer. Regarding the properties of the pyrolysis products, GP 400 °C biochars showed the best properties for combustion; CSS biochars were especially rich in nitrogen, and 400 °C GP and OMW pyrolysis liquids showed the highest number of phenolics. Hence, this study is considered a first step towards industrial scale CSS, GP and OMW pyrolysis-based biorefinery.

Production of antioxidants and other value-added compounds from coffee silverskin via pyrolysis under a biorefinery approach.

The coffee roasting industry produces about 0.4 Mt of coffee silverskin (CSS) per year, the only residue generated from the roasting process that is mostly disposed as industrial waste. The aim of this study is to convert CSS into value-added products by intermediate pyrolysis, transforming the waste into a resource within an integrated biorefinery perspective. To this end, bio-oils and biochars from the intermediate pyrolysis of CSS at 280 °C, 400 °C and 500 °C have been studied. GC-MS analysis showed that bio-oils were composed of value-added products such as caffeine, acetic acid, pyridine and phenolics, the latter being the most interesting due to their antioxidant properties. Total phenolic content and antioxidant capacity of the samples were determined through Folin-Ciocalteu (FC) and DPPH methods, revealing an increase in phenolics in bio-oils compared to CSS extract directly from the feedstock. The bio-oil with the highest phenolic content and antioxidant properties was produced at 280 °C and contained 6.09 and 3.02 mg of gallic acid equivalents /g of bio-oil determined by FC and DPPH methods, respectively. This represents a global potential of up to 487 and 242 tones of gallic acid equivalents per year, considering the FC results and DPPH respectively. The resulting 280 °C biochar presented significant calorific values (22 MJ/kg), indicating its potential use as an energy source. Hence, CSS pyrolysis converts a waste into a by-product and a resource, increasing the environmental benefits and contributing to the circular economy and bioeconomy.

Toward a fast, easy, and versatile immobilization of biomolecules into carbon nanotube/polysulfone-based biosensors for the detection of hCG hormone.

The aim of this study was the fabrication and characterization of biomembranes by the phase inversion (PI) method followed by their subsequent casting onto screen-printed electrodes (SPE) for biomedical applications. The combination of multiwalled carbon nanotubes (MWCNT) as a transducer with polysulfone (PSf) polymer enables easy incorporation of biological moieties (hormones or antibodies), providing a 3D composite with high electrochemical response to corresponding analytes. Antibody/MWCNT/PSf biosensors were characterized by confocal scanning laser microscopy (CSLM), scanning electron microscopy (SEM), and electrochemical methods. For biomedical purposes, human chorionic gonadotropin (hCG) hormone was tested by competitive immunoassay. The detection limit was determined to be 14.6 mIU/mL with a linear range up to 600 mIU/mL. We concluded that the easy and fast incorporation of biomolecules by the PI method, as well as their stability and distribution throughout the 3D polysulfone composite, are testament to the utility for the versatile fabrication of biosensors for clinical diagnosis.

Evaluation of different strategies for the development of amperometric biosensors for L-lactate.

Two strategies were investigated for the development of lactate biosensors based on sol-gel matrixes and polysulfone composite films, both containing L-lactate dehydrogenase (LDH). Firstly, reagentless disposable screen-printed electrodes (SPE's) with Meldola's Blue (MB) and the cofactor NAD(+) inside a sol-gel matrix were prepared. These showed relatively low sensitivities (260 microA/M). Secondly, mediator-modified-polysulfone-graphite composite films deposited over both cylindrical epoxy-graphite and SPE's. These electrodes showed enhanced performance characteristics: improved sensitivity (80 mA/M), detection limit (0.87 microM) and reproducibility (2%). Reagentless electrodes, incorporating NAD(+) in the polysulfone film, had a decreased sensitivity, although better than that achieved by the sol-gel electrodes. While sol-gel electrodes showed a linear range between 1.25 x 10(-4) and 2.48 x 10(-3)M, the epoxy-graphite composite electrodes based on polysulfone composite films allowed the detection of lactate at a linear range of lower concentrations from 1 x 10(-6) to 1.2 x 10(-5)M. Finally, the performance of the LDH-MB-polysulfone-composite film-based SPE's in a flow system was studied. Short response times were obtained (t<30s). Furthermore, repeatability and reproducibility values were notably improved, especially when working with electrodes covered with a polyamide layer prepared with N-(2-aminoethyl)-piperazine.

Carbon nanotube/polysulfone screen-printed electrochemical immunosensor.

The simple and efficient method for preparing sensitive carbon nanotube/polysulfone/RIgG immunocomposite is described. The membrane of the modified disposable screen-printed electrochemical immunosensor is based on phase inversion method. Carbon nanotube/polysulfone membrane acts both as reservoir of immunological material and transducer while offering high surface area, high toughness and mechanical flexibility. The comparison with graphite/polysulfone/RIgG immunosensors shows a much higher sensitivity for those prepared with carbon nanotubes coupled with polysulfone (PSf). The membrane was characterized by scanning electron microscopy/energy dispersive X-ray analysis (SEM/EDX), laser profilometer and by atomic force microscopy (AFM). The purity of the materials was evaluated by thermogravimetric analysis (TGA). The roughness value is doubled when MWCNTs are used instead of graphite into the PSf membranes and the incorporation of antibodies enhances the dispersion of the carbon with the polymeric membrane reducing the roughness in all cases. This biosensor was based on the competitive assay between free and labelled anti-RIgG for the available binding sites of immobilized rabbit IgG (RIgG). The RIgG was incorporated into the polysulfone membrane by a phase inversion method. Horse radish peroxidase (HRP) enzyme was used as label and hydroquinone as mediator. The detection limit for competitive assay was determined to be 1.66 microg/ml. the linear range of anti-RIgG from 2 to 5 microg/ml and the C(50) was found at 3.56 microg/ml. The sensitivity is five times higher for MWCNT than for graphite electrodes, showing lower unspecific adsorption.

New antibodies immobilization system into a graphite-polysulfone membrane for amperometric immunosensors.

Polysulfone membrane is used for the first time for the preparation of electrochemical immunosensors. A disposable immunosensor based on a porous conductor polymer graphite-polysulfone-electrode has been developed using a phase inversion technique for the determination of anti-rabbit IgG (anti-RIgG) as a model analyte. To construct the sensor, a conductor membrane was deposited on the surface of working graphite-epoxy composite (GEC) electrode. The membrane was characterized by SEM. This sensor was based on the competitive assay between free and labeled anti-RIgG for the available binding sites of immobilized rabbit IgG (RIgG). Incubation parameters were optimized in this work. The immunological reaction was detected using an enzymatic-labeling procedure (HRP enzyme) combined with the amperometric detection using H(2)O(2) as substrate and hydroquinone as mediator. This sensor shows stability during a week and a good reproducibility. The current was monitored amperometrically at -0.1 V versus SCE and this method showed a linear range of the anti-RIgG from 1 to 6 microg/ml. The detection limit was determined to be 0.77 microg/ml.

New redox mediator-modified polysulfone composite films for the development of dehydrogenase-based biosensors.

This work presents polysulfone membranes as new materials for the development of compact dehydrogenase-based biosensors. Composite films were prepared by mixing polysulfone with graphite and were deposited on epoxy-graphite composite electrodes. Redox mediators were successfully immobilized in the composite film leading to highly reproducible biosensors, without leakage of the immobilized species. This results in a more reliable analytical system as, at the same time, problems of electrode fouling related to the detection of the coenzyme nicotinamide adenine dinucleotide (NADH) on which is based the amperometric detection of dehydrogenase-based biosensors are avoided. Scanning electron microscopy was used to study the morphological characteristics of the surface and the cross-section of the polysulfone-graphite composite films. Several procedures to immobilize enzymes in these membranes were demonstrated. Glutamate dehydrogenase (GlDH) was immobilized as an example of dehydrogenase enzyme, in this case for the development of an ammonium biosensor. High sensitivity, good selectivity, wide linear ranges and short response times were obtained for the optimized sensors and biosensors. Their good performance combined with the simplicity of the construction method, make the polysulfone-graphite composite films attractive matrices for the development of new enzyme-based biosensors, especially those based on dehydrogenase enzymes.

Comparative study of electron mediators used in the electrochemical oxidation of NADH.

A comparison of the behavior of six different redox mediators for the electrocatalysis of the oxidation of NADH, which are widely used in the construction of dehydrogenase-based biosensors, is reported. The redox mediators were, potassium hexacyanoferrate (II), Meldola's Blue (MB), dichlorophenolindophenol (DCPIP), p-benzoquinone (p-BQ), o-phenylenediamine (o-PDA) and 3,4-dihydroxybenzaldehyde (3,4-DHB). After incorporating each of them in the sensor system following four different strategies (in solution, entrapped in epoxy-composites, adsorbed or electropolymerized on the electrode surface), several aspects regarding repeatability and reproducibility were considered and compared.

Amperometric biosensor for the determination of histamine in fish samples.

A bienzymatic biosensor employing diamine oxidase (DOx) and horseradish peroxidase (HRP) for the detection of histamine in fish samples has been developed and optimized in this work. These enzymes have been co-immobilized into a polysulfone/carbon nanotubes/ferrocene membrane by means of phase inversion technique onto screen-printed electrodes. The electrochemical measurements have been carried out in phosphate buffer solution at pH 8.0 in batch mode and low applied potential (-50 mV vs. Ag/AgCl, KCl 0.1 M) to minimize the interferences. Developed biosensor exhibits high sensitivity (1.9×10(7) nA(-1)), low limit of detection (1.7×10(-7) M), high storage stability and excellent reproducibility, obtaining a linear interval range from 3×10(-7) to 2×10(-5) M. Finally, applicability of the biosensor to the estimation of histamine content in different fish samples has been assessed; obtaining a good correlation between results obtained with the biosensor and those obtained with the reference method (ELISA) in case of sardines, mackerel and greater weever.

Detection of biomarkers with carbon nanotube-based immunosensors.

A facile and capable method of preparation of sensitive carbon nanotube (CNT)/polysulfone/RIgG immunosensor is discussed in this chapter. The immunosensor is based on the modification of disposable screen-printed electrodes by phase inversion method. CNT/polysulfone membrane acts as the reservoir of immunomolecules as well as a transducer. This configuration offers large surface area, elevated porosity, and mechanical flexibility. The comparison with graphite/polysulfone/RIgG immunosensors shows a significantly improved sensitivity for those prepared with CNTs coupled with polysulfone (PSf). The immunosensing scheme is based on the competitive assay between free and labeled anti-RIgG for the available binding sites of immobilized rabbit IgG (RIgG). The RIgG is incorporated into the PSf immunosensor using a phase inversion method. Horseradish peroxidase enzyme is used as label and hydroquinone as electrochemical mediator. The limit of detection for competitive assay is 1.66 microg/mL. The sensitivity is six times higher for MWCNT-based than for graphite-based electrodes.

The first isoform-selective protein biosensor: a metallothionein potentiometric electrode.

The construction of a biosensor membrane by embedding mammalian Zn(7)-MT1 complexes as ionophores in a polysulfone matrix resulted in precise, accurate and, significantly, selective electrodes for MT1 quantification, so that the presence of other mammalian MT isoforms did not interfere in MT1 measurement.

Electrochemical activation of carbon nanotube/polymer composites.

Electrochemical activation of carbon nanotube/polysulfone composite electrodes for enhanced heterogeneous electron transfer is studied. The physicochemical insight into the electrochemical activation of carbon nanotube/polymer composites was provided by transmission electron microscopy, Raman spectroscopy, electrochemical impedance spectroscopy, and cyclic voltammetry. Dopamine, ascorbic acid, NADH, and ferricyanide are used as a model redox system for evaluating the performance of activated carbon nanotube/polymer composite electrodes. We demonstrate that polymer wrapping of carbon nanotubes is subject to defects and to partial removal during activation. Such tunable activation of electrodes would enable on-demand activation of electrodes for satisfying the needs of sensing or energy storage devices.

Carbon nanotube/polysulfone soft composites: preparation, characterization and application for electrochemical sensing of biomarkers.

Multi-walled carbon nanotube/polysulfone soft composites (MWCNT/PSf) prepared via phase inversion are a novel platform for electrochemical and electroanalytical purposes with practical applications in the design of screen-printed electrodes for electrochemical sensing. We present here a thorough characterization of the morphological, physical, chemical and electrochemical properties of this material. These composites constitute a robust mesoporous network with high specific surface area, which is beneficial for trapping bioanalytes and increasing the electrochemical sensitivity. We highlight the advantages of these soft composites by comparing them with analogous graphite composites.

Novel potentiometric sensors based on polysulfone immobilized metallothioneins as metal-ionophores.

We show here the use of immobilized metal-binding biomolecules for metal analysis by using novel potentiometric sensors. To this end and as a model, Ag(+)-ISEs were developed using polysulfone matrix embedding metallothioneins as ionophores (mouse MT1 (P1) or sea urchin SpMTA (P2)). Polysulfone, a porous polymer that was not used until the present in potentiometric biosensors, has the advantage of being compatible with biological materials. Also, the phase inversion procedure allows protein incorporation into the membrane with minima alterations, since it always remains in the aqueous phase. Construction of these biosensors required small amounts of protein; they can be dry-stored and have long lifetimes. They exhibited linear responses with slopes of ca. 61mV per decade within the 10(-5) to 10(-2)M Ag(+) concentration range, detection limits of about 10(-5)M, and worked in the 2-to-8 pH range. Except for Hg(2+), the Pb(2+), Zn(2+), Cd(2+), Cu(2+) cations do not interfere with Ag(+) determination. Significantly, different affinities of Pb(2+) and Zn(2+) towards P1- and P2-ISE were found, in good correlation with the higher affinity of these cations towards SpMTA than to MT1. Consequently, the distinct metal-binding features of each MT are conserved and determine the differential properties of their biosensors. These results open a broad range of possibilities for the use of proteins as ionophores in what could be considered a new type of potentiometric biosensor if their response mechanism is taken into account.

Carbon nanotube/polysulfone composite screen-printed electrochemical enzyme biosensors.

The fabrication, evaluation and attractive performance of multiwall carbon nanotube(MWCNT)/polysulfone biocomposite membrane modified thick-film screen-printed electrochemical biosensors are reported. The fabricated carbon nanotube/polysulfone (CNT/PS) strips combine the attractive advantages of carbon nanotube materials, polysulfone matrix and disposable screen-printed electrodes. Such thick-film carbon nanotubes/polysulfone sensors have a well defined performance, are mechanically stable, and exhibit high electrochemical activity. Furthermore, biocompatibility of CNT/PS composite allows easy incorporation of biological functional moiety of horseradish peroxidase by phase inversion technique. The comparison of graphite with MWCNT as conductor material is described in this paper. The proposed H(2)O(2) biosensor exhibited a linear range (applied potential, -0.2 V) from 0.02 to 0.5 mM and a K(M)(app) of 0.71 mM.

Evaluation of different mediator-modified screen-printed electrodes used in a flow system as amperometric sensors for NADH.

This work presents a comparative study between two different methods for the preparation of mediator-modified screen-printed electrodes, to be used as detectors in a reliable flow injection system for the determination of the nicotinamide adenine dinucleotide (NADH) coenzyme. The best strategy was selected for the final development of compact biosensors based on dehydrogenase enzymes. For the first immobilisation strategy, different redox mediators were electropolymerised onto the SPE surface. The second immobilisation strategy was carried out using polysulfone-graphite composites, which were deposited by screen-printing technology onto the screen-printed electrode (SPE) surface. Both methods achieved an effective and reliable incorporation of redox mediators to the SPE configuration. Finally, a flow system for ammonium determination was developed using a glutamate dehydrogenase (GlDH)-Meldola's Blue (MB)-polysulfone-composite film-based biosensor. The stability of the redox mediators inside the composite films as well as the negligible fouling effect observed on the electrode surface improve the repeatability and reproducibility of the sensors, important features for continuous analysis in flow systems. Furthermore, the optimised bio/sensors, incorporated in a flow injection system, showed good sensitivities and short response times. Such a good analytical performance together with the simple and fast sensor construction are interesting characteristics to consider the polysulfone-composite films as attractive electrochemical transducer materials for the development of new dehydrogenase-based SPEs.