Spectroelectrochemical Enzyme Sensor System for Acetaldehyde Detection in Wine.

A new spectroelectrochemical two-enzyme sensor system has been developed for the detection of acetaldehyde in wine. A combination of spectroscopy and electrochemistry improves the analytical features of the electrochemical sensor because the optical information collected with this system is only associated with acetaldehyde and avoids the interferents also present in wines as polyphenols. Spectroelectrochemical detection is achieved by the analysis of the optical properties of the K3[Fe(CN)6]/K4[Fe(CN)6] redox couple involved in the enzymatic process: aldehyde dehydrogenase catalyzes the aldehyde oxidation using ß-nicotinamide adenine dinucleotide hydrate (NAD+) as a cofactor and, simultaneously, diaphorase reoxidizes the NADH formed in the first enzymatic process due to the presence of K3[Fe(CN)6]. An analysis of the characteristic UV-vis bands of K3[Fe(CN)6] at 310 and 420 nm allows the detection of acetaldehyde, since absorption bands are only related to the oxidation of this substrate, and avoids the contribution of other interferents.

Detection of dithiocarbamate, chloronicotinyl and organophosphate pesticides by electrochemical activation of SERS features of screen-printed electrodes.

Enhancement of Raman intensity due to the electrochemical surface-enhanced Raman scattering (EC-SERS) effect is an interesting alternative to overcome the lack of sensitivity traditionally associated with Raman spectroscopy. Furthermore, activation of metallic screen-printed electrodes (SPEs) by electrochemical route leads to the reproducible generation of nanostructures with excellent SERS properties. EC-SERS procedure proposed in this work for the detection of several pesticides (thiram, imidacloprid and chlorpyrifos) with different nature, uses gold SPEs as SERS substrates, but also includes a preconcentration step as the initial and essential stage. Taking into account the small volume of solution employed, only 60 µL, the preconcentration cannot be performed for more than 15 min in order to ensure the proper contact of the solution with WE, RE and CE. Furthermore, selected temperature, 34 °C, is not very high to allow the exhaustive control of the drop volume. Optimization of preconcentration parameters (time and temperature) displays a crucial step, particularly in the detection of low concentrations of pesticides, because it will provide higher Raman intensity in EC-SERS experiments. After the initial step, gold SPEs were electrochemically activated by cyclic voltammetry, allowing the detection of very low concentration (µg·L-1) of pesticides due to the generation of fresh nanostructures with SERS effect.

Understanding the ECL interaction of luminol and Ru(bpy)32+ luminophores by spectro-electrochemiluminescence.

A detailed analysis of the ECL interaction between luminol and tris(2,2'-bipyridyl)dichlororuthenium(ii) (Ru(bpy)32+) is required before using them in ECL systems for multianalyte detection purposes. Spectro-electrochemiluminescence demonstrates that not only must the emission properties be considered, but also their additional optical characteristics are involved in the explanation of the interaction mechanism between these luminophores.

Screen-Printed Electrodes Modified with Metal Phthalocyanines: Characterization and Electrocatalysis in Chlorinated Media.

Metal phthalocyanines are well-known sensing phases with applications in different scientific fields due to their interesting properties. Detailed characterization by Raman spectroscopy was performed in order to study the shifting of the vibrational bands related to the coordination sphere of each metal phthalocyanine. In this work, a study involving the use of screen-printed electrodes (SPEs) with various metal phthalocyanines to electrochemically detect and quantify chlorine (Cl2) gas is presented. The Cl2 gas was generated in-situ via oxidation of the chloride present in form of aqueous salt solutions. The developed method offers not only the possibility to quantify chlorine, but also to discriminate among several chlorinated species due to the changes observed in the voltammetric profiles associated with the interaction between the specie assayed and the phthalocyanine metallic center. Optimization of detecting parameters was also performed to apply this procedure for the quantification of chlorine generated from commercial chlorine tablets. The development of this proof of concept shows interesting possibilities and easy-to-use applications with novel on metal phthalocyanines based SPE sensors.

Screen-Printed Electrodes Modified with Metal Nanoparticles for Small Molecule Sensing.

Recent progress in the field of electroanalysis with metal nanoparticle (NP)-based screen-printed electrodes (SPEs) is discussed, focusing on the methods employed to perform the electrode surface functionalization, and the final application achieved with different types of metallic NPs. The ink mixing approach, electrochemical deposition, and drop casting are the usual methodologies used for SPEs' modification purposes to obtain nanoparticulated sensing phases with suitable tailor-made functionalities. Among these, applications on inorganic and organic molecule sensing with several NPs of transition metals, bimetallic alloys, and metal oxides should be highlighted.

Spectroelectrochemical elucidation of B vitamins present in multivitamin complexes by EC-SERS.

Raman spectroelectrochemistry based on electrochemical surface-enhanced Raman scattering (EC-SERS) effect is an interesting alternative to overcome the lack of sensitivity of normal Raman spectroscopy. Electrochemical activation of metallic screen-printed electrodes (SPEs) leads to the reproducible generation of nanostructures with excellent SERS properties. In that way, gold SPEs circumvent the traditional reproducibility limitation and produce the enhancement of the Raman intensity to favor the detection of low concentrations. Furthermore, fingerprint features of Raman spectroscopy make possible the dynamic spectroelectrochemical analysis of B vitamins. The accuracy assignments of Raman bands associated with B1, B2, B3, B6 and B12 vitamins present in multivitamin complexes provides valuable information, allowing us not only the detection of B vitamin present in mixtures, but also to understand the interaction between vitamins and metallic SERS surfaces.

Resolution of mixed dyes by in situ near infrared (NIR) spectroelectrochemistry.

NIR spectroelectrochemistry has scarcely been used for deconvolving aqueous mixtures due to the water restriction in this spectral range. However, this work offers an interesting approach for the study of mixtures of molecules with similar electrochemical and spectroscopic behaviour by overcoming the limitations of this hybrid technique. As a proof of concept, the resolution of mixtures of two dyes with similar chemical structures demonstrates the usefulness of NIR spectroelectrochemistry.

Towards single-molecule in situ electrochemical SERS detection with disposable substrates.

Dynamic time-resolved Raman spectroelectrochemistry demonstrates the strong influence of nanostructuring and surface charge of in situ activated disposable substrates for SERS detection. Under specific conditions, a large enhancement factor and estimated calculations agree with the feasible detection of only a few molecules, approaching the limit of single-entity detection.

In Situ Spectroelectrochemical Monitoring of Dye Bleaching after Electrogeneration of Chlorine-Based Species: Application to Chloride Detection.

Spectroelectrochemical techniques are becoming increasingly versatile tools to solve a diverse range of analytical problems. Herein, the use of in situ real-time luminescence spectroelectrochemistry to quantify chloride ions is demonstrated. Utilizing the bleaching effect of chlorine-based electrogenerated products after chloride oxidation, it is shown that the fluorescence of the rhodamine 6G dye decreases proportionally to the initial chloride concentration in solution. A strong decrease of fluorescence is observed in acidic media compared to a lower decrease in alkaline media, which suggests that Cl2, favorably generated at low pH, could be the main species responsible for the fluorescence loss. This fact is corroborated with chronoamperometric measurements where the complete loss of fluorescence for the bulk solution is achieved. A fast mass transfer is needed to explain this behavior, in agreement with the generation of gaseous species such as Cl2. Chloride detection was performed in artificial sweat samples in less than 30 s with great accuracy. This electrochemical/optical combined approach allows us to quantify species that are difficult to measure by electrochemistry due to the inadequate resolution of their redox processes or being without significant optical properties.

Quenching of graphene quantum dots fluorescence by alkaline phosphatase activity in the presence of hydroquinone diphosphate.

In this work, a turn-off photoluminescent sensing proof-of-concept based on blue luminescent graphene quantum dots (GQDs) as the fluorescent probe was developed. For that purpose, GQDs optical response was related with the catalytic enzymatic activity of alkaline phosphatase (ALP), in the presence of hydroquinone diphosphate (HQDP). The hydrolysis of HQDP by ALP generated hydroquinone (HQ). The oxidation of HQ, enzymatically produced, to p-benzoquinone (BQ) resulted in the quenching of GQDs fluorescence (FL). Therefore, the developed luminescent sensing mechanism allowed the FL quenching with ALP activity to be related and thus quantified the concentration of ALP down to 0.5 nM of enzyme. This innovative design principle appears as a promising tool for the development of enzymatic sensors based on ALP labeling with fluorescent detection or even for direct ALP luminescent quantification in an easy, fast and sensitive manner.

Evaluation of electrochemical, UV/VIS and Raman spectroelectrochemical detection of Naratriptan with screen-printed electrodes.

Naratriptan, active pharmaceutical ingredient with antimigraine activity was electrochemically detected in untreated screen-printed carbon electrodes (SPCEs). Cyclic voltammetry and differential pulse voltammetry were used to carry out quantitative analysis of this molecule (in a Britton-Robinson buffer solution at pH 3.0) through its irreversible oxidation (diffusion controlled) at a potential of +0.75V (vs. Ag pseudoreference electrode). Naratriptan oxidation product is an indole based dimer with a yellowish colour (maximum absorption at 320nm) so UV-VIS spectroelectrochemistry technique was used for the very first time as an in situ characterization and quantification technique for this molecule. A reflection configuration approach allowed its measurement over the untreated carbon based electrode. Finally, time resolved Raman Spectroelectrochemistry is used as a powerful technique to carry out qualitative and quantitative analysis of Naratriptan. Electrochemically treated silver screen-printed electrodes are shown as easy to use and cost-effective SERS substrates for the analysis of Naratriptan.

Time-Resolved Luminescence Spectroelectrochemistry at Screen-Printed Electrodes: Following the Redox-Dependent Fluorescence of [Ru(bpy)3]2.

In this work, a compact instrument for time-resolved luminescence spectroelectrochemistry using low-cost disposable electrodes is reported. This instrument can be coupled with screen-printed electrodes via a specific cell and a reflection probe, which allows one to observe changes occurring at the electrode/solution interface. This approach allowed one to follow the fluorescence variation of electrofluorochromic species such as [Ru(bpy)3]2+ at screen-printed carbon electrodes. A strong correlation between the electrochemical processes and the fluorescence was found during potentiostatic or multipulsed amperometric measurements. A decrease of the fluorescence was observed when the [Ru(bpy)3]2+ was oxidized to [Ru(bpy)3]3+ and part of this fluorescence is recovered when [Ru(bpy)3]3+ was reduced to the initial species. Moreover, a significant increment of the fluorescence was found when the oxygen reduction reaction takes place, which also confirms its quenching effect. Finally, multipulsed amperometric detection was employed in order to obtain more information about the redox-dependent luminescence of [Ru(bpy)3]2+ finding a continuous quenching over time attributed to bleaching chlorine-based species.

Electroluminescence of [Ru(bpy)3]2+ at gold and silver screen-printed electrodes followed by real-time spectroelectrochemistry.

Real-time spectroelectrochemistry of [Ru(bpy)3]2+ electroluminescence showed a strong correlation with electrochemical processes occurring at metal screen-printed electrodes. Luminescence was quenched when the metal oxidation takes place, but it behaved differently when gold or silver were reduced, which suggests that changes in the structural characteristics of metallic electrodes play a decisive role in luminescence spectroelectrochemistry.

Enhanced detection of quantum dots by the magnetohydrodynamic effect for electrochemical biosensing.

In this work, we describe the use of a magnetoelectrochemical support for screen-printed electrodes to improve the anodic stripping voltammetry of cadmium due to the generated magnetohydrodynamic (MHD) effect. To create a significant MHD effect, Fe(iii) was added at mM concentrations to the solution. The reduction of Fe(iii) simultaneously with the cadmium deposition on the electrode surface allowed the production of a high cathodic current, which generated a large Lorentz force capable of exerting a convective effect on the solution in the presence of the magnetic field. This convective effect allowed the increase in the mass transfer in the quiescent solution, enhancing the deposition of cadmium as observed by an increased stripping peak current. The optimized method was applied to the detection of CdSe/ZnS quantum dots (QDs) in solution. Using the magnetoelectrochemical support, we were able to detect extremely low concentrations of QDs, with a detection limit of 100 amol of QDs (in particle number). The great performance shown by this system was evaluated in biosensing applications. Firstly, detection of biotin was carried out using a competitive bioassay between biotin and QD-labelled biotin, obtaining good analytical results (0.6 × 10-10 M as the limit of detection). Then, the magnetoelectrochemical support was tested in a more complex biosensor for the determination of anti-transglutaminase IgA antibodies, a celiac disease biomarker. This work shows that the improvement in the metal electrodeposition caused by the MHD effect can be used successfully for the development of disposable electrochemical biosensors with great performance using screen-printed electrodes.

Miniaturized analytical instrumentation for electrochemiluminescence assays: a spectrometer and a photodiode-based device.

Herein, a new miniaturized analytical instrumentation for electrochemiluminescence (ECL) assays is presented. A photodiode integrated in an ECL cell combined with a potentiostat/galvanostat, all integrated in a one-piece instrument (µSTAT ECL), was developed. In addition, a complementary micro-spectrometer integrated in a similar ECL cell for luminescence spectra recording is also proposed. Both cells are intended to be used with screen-printed electrodes and all the devices are portable and small sized. Their performance was corroborated with two innovative proofs-of-concept that centered on the luminol transduction chemistry: a first time reported ECL assay based on the enzymatic reaction between an indoxyl substrate and the enzyme alkaline phosphatase, and the electrochemiluminescence resonance energy transfer (ECL-RET) process triggered by the electro-oxidized luminol to the acceptor fluorescein. The photodiode system revealed to be more sensitive than the spectrometer device in collecting the light; however, with the latter, it is possible to discriminate different luminescent species according to their maximum wavelength emission, which is extremely useful for carrying out simple and simultaneous ECL multiplex analyzes. The spectrometer device works as an excellent accessory to couple with the µSTAT ECL instrument, complementing the experiments. Graphical abstract Schematic representation of the ECL-RET: from luminol-H2O2 system to fluorescein, the micro-spectrometer for the light collection and the 3D representation of the ECL-RET reaction.

Simultaneous detection of free and total prostate specific antigen on a screen-printed electrochemical dual sensor.

Voltammetric enzyme dual sensors for simultaneous determination of free and total prostate specific antigen (fPSA and tPSA) are described. Alkaline Phosphatase (AP) and a mixture solution of 3-indoxyl phosphate and silver ions were used as the enzymatic label and substrate, respectively. 8A6 or 5G6 antibodies specific for free and total PSA, respectively, were immobilized on different screen-printed electrodes (SPEs)--screen-printed carbon electrodes, screen-printed gold electrodes and screen-printed carbon electrodes modified with nanogold--in order to be able to select one of the surfaces as the most adequate one to develop the dual sensor. Screen-printed carbon electrodes modified with nanogold were the SPEs with the best analytical characteristics and lead to the most repeatable bioelectrodes, so they were selected for the development of the dual sensor. On Dualsensor-nAu electrodes, 8A6 antibody was immobilized on one working electrode and 5G6 antibody was immobilized on the other one by deposition of a drop of solution of each antibody and left overnight at 4 degrees C. Biotinylated anti-PSA antibody and streptavidin-AP conjugate were used as detection reagents, giving rise, to our knowledge, to the first simultaneous electrochemical biosensor for free and total PSA. The PSA dual sensor was used to monitor PSA production from three different cultures of human androgen-sensitive prostate tumor cells.

Electrochemical study and flow injection analysis of paracetamol in pharmaceutical formulations based on screen-printed electrodes and carbon nanotubes.

Acetaminophenol or paracetamol is one of the most commonly used analgesics in pharmaceutical formulations. Acetaminophen is electroactive and voltammetric mechanistic studies for the electrode processes of the acetaminophenol/N-acetyl-p-quinoneimine redox system are presented. Carbon nanotubes modified screen-printed electrodes with enhanced electron transfer properties are used for the study of the electrochemical-chemical oxidation mechanism of paracetamol at pH 2.0. Quantitative analysis of paracetamol by using its oxidation process (in a Britton-Robinson buffer solution pH 10.0) at +0.20 V (vs. an Ag pseudoreference electrode) on an untreated screen-printed carbon electrode (SPCE) was carried out. Thus, a cyclic voltammetric based reproducible determination of acetaminophen (R.S.D., 2.2%) in the range 2.5x10(-6) M to 1x10(-3) M, was obtained. However, when SPCEs are used as amperometric detectors coupled to a flow injection analysis (FIA) system, the detection limit achieved for paracetamol was 1x10(-7) M, one order of magnitude lower than that obtained by voltammetric analysis. The repeatability of the amperometric detection with the same SPCE is 2% for 15 successive injections of 10(-5) M acetaminophen and do not present any memory effect. Finally, the applicability of using screen-printed carbon electrodes for the electrochemical detection of paracetamol (i.e. for quality control analysis) was demonstrated by using two commercial pharmaceutical products.

Alkaline phosphatase-catalyzed silver deposition for electrochemical detection.

Alkaline phosphatase (AP) is one of the most used enzymatic labels for the development of ELISAs, immunosensors, DNA hybridization assays, etc. This enzyme catalyzes the dephosphorylation of a substrate into a detectable product usually quantified by optical or electrochemical measurements. This work is based on a substrate (3-indoxyl phosphate) that produces a compound able to reduce silver ions in solution into a metallic deposit, which is localized where the enzymatic label AP is attached. The deposited silver is electrochemically stripped into solution and measured by anodic stripping voltammetry. Its application to an enzymatic genosensor on streptavidin-modified screen-printed carbon electrodes for the detection of virulence nucleic acid determinants of autolysin gene, exclusively present on the genome of the human pathogen Streptococcus pneumoniae, is described. Compared with the direct voltammetric detection of indigo carmine, the anodic stripping voltammetry of silver ions is 14-fold more sensitive.

Genosensor based on a platinum(II) complex as electrocatalytic label.

Voltammetric genosensors on streptavidin-modified screen-printed carbon electrodes (SPCEs) for the detection of virulence nucleic acid determinants of pneumolysin (ply) and autolysin (lytA) genes, exclusively present on the genome of the human pathogen Streptococcus pneumoniae, were described. The oligonucleotide probes were immobilized on electrochemically pretreated SPCEs through the streptavidin/biotin reaction. After that, the hybridization reaction was carried out with labeled complementary targets on the electrode surface. The ply and lytA targets were labeled using the universal linkage system, which consists of the use of a platinum(II) complex that acts as coupling agent between targets and a, usually fluorescent, molecule label. In this case, the platinum(II) complex acts as a label itself because the analytical signal is achieved by measuring chronoamperometrically the current generated by the hydrogen evolution catalyzed by platinum. In nonstringent experimental conditions, these genosensors can detect 24.5 fmol of 20-mer oligonucleotide target and discriminate between a complementary oligo and an oligo with a three-base mismatch. In presence of 25% formamide in the hybridization buffer, a single-base mismatch on the oligonucleotide target can be detected.

Development of an immunosensor for the determination of rabbit IgG using streptavidin modified screen-printed carbon electrodes.

Voltammetric enzyme immunosensors based on the employment of streptavidin modified screen-printed carbon electrodes (SPCEs) for the detection of rabbit IgG, as a model analyte, were described. Alkaline phosphatase (AP) and 3-indoxyl phosphate (3-IP) were used as the enzymatic label and substrate, respectively. The adsorption of streptavidin was performed by deposition of a drop of a streptavidin solution overnight at 4 degrees C on the pre-oxidized surface of the SPCEs. The analytical characteristics of these sensors were evaluated using biotin conjugated to AP. The immunosensor devices were based on a specific reaction of rabbit IgG with its biotinylated antibodies, which were immobilised on the modified screen-printed carbon electrodes through the streptavidin:biotin reaction. The immunosensors were used for a direct determination of AP labelled rabbit IgG, and for free rabbit IgG detection using a sequential competitive immunoassay. A calibration curve in the range of 5 x 10(-11) to 1 x 10(-9)M of rabbit IgG was obtained with a estimated detection limit of 5 x 10(-11)M (7.0ng/ml). These immunosensors were stable for 5 months if they were stored at 4 degrees C.