Christos Kokkinos

Disposable mercury-free cell-on-a-chip devices with integrated microfabricated electrodes for the determination of trace nickel(II) by adsorptive stripping voltammetry.

This work reports the fabrication of disposable three-electrode cells with integrated sputtered metal-film electrodes. The working electrode was a bismuth-film electrode (BiFE) while the reference and counter electrodes were made of Ag and Pt, respectively. The deposition of the metal layers was carried out by sputtering of the respective metals on a silicon substrate while the exact geometry of the electrodes was defined via a metal mask placed on the substrate during the deposition process. Initially, the electrodes were characterised by cyclic voltammetry. The utility of these devices was tested for the trace determination of Ni(II) by square wave adsorptive stripping voltammetry (SWAdSV) after complexation with dimethylglyoxime (DMG). The experimental variables (the presence of oxygen, the DMG concentration, the preconcentration potential, the accumulation time and the SW parameters), as well as potential interferences, were investigated. Using the selected conditions, the 3sigma limit of detection was 100 ng L(-1) for Ni(II) (for 90 s of preconcentration) and the relative standard deviation for Ni(II) was 2.3% at the 10 microg L(-1) level (n=8). Finally, the method was applied to the determination of Ni(II) in a certified river water sample.

Determination of trace cobalt(II) by adsorptive stripping voltammetry on disposable microfabricated electrochemical cells with integrated planar metal-film electrodes

This work reports the determination of trace Co(II) by adsorptive stripping voltammetry on disposable three-electrode cells with on-chip metal-film electrodes. The heart of the sensors was a bismuth-film electrode (BiFE) with Ag and Pt planar strips serving as the reference and counter electrodes, respectively. Metals were deposited on a silicon chip by sputtering while the areas of the electrodes were patterned via a metal mask. Co(II) was determined by square wave adsorptive stripping voltammetry (SWAdSV) after complexation with dimethylglyoxime (DMG). The experimental variables (the DMG concentration, the preconcentration potential, the accumulation time and the SW parameters), as well as potential interferences, were investigated. Using the selected conditions, the 3 sigma limit of detection was 0.09 microg l(-1) of Co(II) (for 90s of preconcentration) and the relative standard deviation for Co(II) was 3.8% at the 2 microg l(-1) level (n=8). The method was applied to the determination of Co(II) in a certified river water sample. These mercury-free electrochemical devices present increased scope for field analysis and mu-TAS applications.

Microfabricated Tin–Film Electrodes for Protein and DNA Sensing Based on Stripping Voltammetric Detection of Cd(II) Released from Quantum Dots Labels

A novel disposable microfabricated tin-film electrochemical sensor was developed for the detection of proteins and DNA. The sensor was fabricated on a silicon wafer through photolithography to define the sensor geometry followed by tin sputtering. A sandwich-type immunoassay with biotinylated reporter antibody was employed for the determination of prostate-specific antigen (PSA) in human serum samples. For the detection of C533G mutation of the RET gene, biotinylated oligonucleotide probes were used. The biotinylated biomolecular probes were labeled with streptavidin (STV)-conjugated CdSe/ZnS quantum dots (QDs); quantification of the analytes was performed through acidic dissolution of the QDs and stripping voltammetric detection of the Cd(II) released. The proposed QD-based electrochemical sensor overcomes the limitations of existing voltammetric sensors and provides a mercury-free sensing platform with scope for mass-production and further potential for application in clinical diagnostics.

Microfabricated disposable lab-on-a-chip sensors with integrated bismuth microelectrode arrays for voltammetric determination of trace metals

This work reports the fabrication of disposable three-electrode cells with integrated metal-film electrodes. The devices were fabricated by a multi-step micro-fabrication approach combining sputtering for the deposition of metals and the dielectric material (SiO(2)) on the surface of a silicon wafer and photolithography for the definition of the geometry of the sensors. The working electrode was a microelectrode array consisting of bismuth microdisks while the reference and counter electrode strips were made of Ag and Pt, respectively. The utility of these devices was tested for the trace determination of Pb(II) and Cd(II) by anodic stripping voltammetry and Ni(II) by adsorptive stripping voltammetry. The detection of these trace metals was carried out in unstirred and undeoxygenated solutions exhibiting sub-μg L(-1) limits of detection and enhanced analytical characteristics compared to conventional bismuth-film electrodes.

Disposable integrated bismuth citrate-modified screen-printed immunosensor for ultrasensitive quantum dot-based electrochemical assay of C-reactive protein in human serum.

A novel immunosensor based on graphite screen-printed electrodes (SPEs) modified with bismuth citrate was developed for the voltammetric determination of C-reactive protein (CRP) in human serum using quantum dots (QDs) labels. The sandwich-type immunoassay involved physisorption of CRP capture antibody on the surface of the sensor, sequential immunoreactions with CRP and biotinylated CRP reporter antibody and finally reaction with streptavidin-conjugated PbS QDs. The quantification of the target protein was performed with acidic dissolution of the PbS QDs and anodic stripping voltammetric detection of the Pb(II) released. Detection was performed at bismuth nanodomains formed on the sensor surface during the electrolytic preconcentration step, as bismuth citrate was reduced to metallic bismuth simultaneously with the deposition of Pb on the surface of the immunosensor. Under optimal conditions, the response was linear over the range 0.2-100 ng mL(-1) CRP and the limit of detection was 0.05 ng mL(-1) CRP. Since the modified SPE serves as both the biorecognition element and the QDs reader, the analytical procedure is simplified, the drawbacks of existing electroplated immunosensors are minimized while the proposed disposable sensing platform provides convenient, low-cost and ultrasensitive detection of proteins and wider scope for mass-production.

Disposable Nafion-modified micro-fabricated bismuth-film sensors for voltammetric stripping analysis of trace metals in the presence of surfactants

This work is a study of the analytical utility of Nafion-modified microfabricated bismuth film electrodes (BiFEs) for the determination of Pb(II) and Cd(II) by anodic stripping voltammetry (ASV) in the presence of surfactants. Micro-fabricated BiFEs were prepared by depositing a thin film of bismuth on the surface of a silicon substrate by sputtering while the two-dimensional geometry of the final sensors was defined by photolithography. The BiFEs were further drop-coated with a Nafion film. These devices were applied to the determination of Pb(II) and Cd(II) by square wave ASV (SWASV) in the presence of Triton X-100 (a non-ionic surfactant), cetyltrimethylammonium bromide (CTAB) (a cationic surfactant) and sodium dodecyl sulphate (SDS) (an anionic surfactant). It was found that the presence of Nafion afforded an increase in sensitivity and the tolerance against surfactants but these properties were severely influenced by both the thickness of the Nafion film and the nature of the interfering surfactant. Using a Nafion of 0.4 μm thickness and 120 s of preconcentration, the repeatability (expressed as the % relative standard deviation on the same sensor (n=8)) at the 20 μg l(-1) level was 3.8% for Pb(II) and 3.1% for Cd(II) and the limits of detection were 0.5 μg l(-1) for Cd(II) and Pb(II). The sensors were applied to Cd(II) and Pb(II) determination in a certified lake-water sample.

Lab-on-a-membrane foldable devices for duplex drop-volume electrochemical biosensing using quantum dot tags

This work describes a new type of integrated lab-on-a-membrane foldable device suitable for on-site duplex electrochemical biosensing using drop-size sample volumes. The devices are fabricated entirely by screen-printing on a nylon membrane and feature two assay zones which are located symmetrically on either side of a three-electrode voltammetric cell with a bismuth citrate-loaded graphite working electrode. After the completion of two spatially separated drop-volume competitive immunoassays on the assay zones using biotinylated antibodies labeled with streptavidin-conjugated Pb- and Cd-based quantum dots (QDs), respectively, the QD labels are dissolved releasing Pb(II) and Cd(II) in the assay zones. Then, the two assay zones are folded over, and they are brought in contact with the voltammetric cell for simultaneous anodic stripping voltammetric (ASV) determination of Pb(II) and Cd(II) at the bismuth nanostructured layer formed on the working electrode by reduction of the bismuth citrate during the preconcentration step. The fabrication of the devices is discussed in detail, and their operational characteristics are exhaustively studied. In order to demonstrate their applicability to the analysis in complex matrices, duplex ASV-QDs-based determination of bovine casein and bovine immunoglobulin G is carried out in milk samples yielding limits of detection of 0.04 μg mL(-1) and 0.02 μg mL(-1), respectively. The potential of the devices to detect milk adulteration is further demonstrated. These new membrane devices enable duplex biosensing with distinct advantages over existing approaches in terms of cost, fabrication, and operational simplicity and rapidity, portability, sample size, disposability, sensitivity, and suitability for field analysis.

Emerging trends in biosensing using stripping voltammetric detection of metal-containing nanolabels – A review

Over the last years, nanomaterials have found many applications in the development of electrochemical biosensors. Among other functions, metal nanoparticles (NPs) and quantum dots (QDs) (semiconducting nanocrystals composed of metal salts) are increasingly being used as voltammetric labels in affinity biosensing. Labeling is based on the attachment of the label(s) on the target biomolecules or on a biorecognition reporting probe. After an appropriate specific affinity interaction between the target and the reporting probe, the metallic nanolabels are converted to the respective cations which are quantified by a voltammetric technique. The very use of metal-containing nanoprobes as labels provides a first amplification step since each nanoprobe can release a very significant number of detectable cations. When anodic stripping voltammetry (ASV) (in which a preconcentration step precedes the actual voltammetric scan) is further employed as the detection format, ultra-sensitive bioassays can be developed. The present paper reviews the emerging trends in affinity biosensing using ASV detection of metal-containing nanolabels. It provides a critical discussion of recent developments in ASV transduction and electrodes, novel strategies for signal enhancement, approaches for multiplexed detection as well as fluidics, paper-based and lab-on-a-chip devices.

Flexible microfabricated film sensors for the in situ quantum dot-based voltammetric detection of DNA hybridization in microwells.

A new flexible miniaturized integrated device was microfabricated for the in situ ultrasensitive voltammetric determination of DNA mutation in a microwell format, using quantum dots (QDs) labels. The integrated device consisted of thin Bi, Ag, and Pt films (serving as the working, reference, and counter electrode, respectively) deposited by sputtering on a flexible polyimide substrate. A DNA assay was employed in microwell format, where an immobilized complementary oligonucleotide probe hybridized with the biotinylated target oligonucleotide followed by reaction with streptavidin-conjugated PbS QDs. After the acidic dissolution of the QDs, the flexible sensor was rolled and inserted into the microwell and the Pb(II) released was determined in situ by anodic stripping voltammetry. Since the analysis took place directly in the microwell, the volume of the working solution was only 100 μL and the target DNA could be detected at a concentration down to 1.1 fmol L(-1). The proposed flexible microdevice addresses the restrictions of conventional rigid electrodes while it provides a low cost integrated transducer for the ultrasensitive detection of important biomolecules.

Paper-Based Microfluidic Device with Integrated Sputtered Electrodes for Stripping Voltammetric Determination of DNA via Quantum Dot Labeling

This work reports a microfabricated electrochemical paper-based analytical device (ePAD) for the voltammetric determination of DNA. The device is patterned by wax-printing on paper and features a circular assay zone connected to an inlet zone and a sink via grooved microfluidic channels for accelerated flow rate. An electrochemical cell with integrated electrodes is formed on the reverse side of the paper by sputtering of thin metal films (Sn, Pt and Ag as the working, counter and reference electrode, respectively). Proof-of-principle of the ePAD for biosensing is demonstrated for a DNA assay involving attachment of capture DNA, hybridization with biotinylated target oligonucleotide and labeling with streptavidin-conjugated CdSe/ZnS quantum dots (QDs). After the acidic dissolution of the QDs, the released Cd(II) is quantified by anodic stripping voltammetry (ASV) at the Sn-film working electrode. Thanks to the synergistic effects of QDs amplification, the inherent sensitivity of ASV and the excellent detection capabilities of the Sn-film working electrode for Cd(II), the target DNA can be detected at levels as low as 0.11 pmol L-1 using sample volumes as low as 1 μL. The developed microfluidic ePAD costs only 0.11