Markus Håkansson

Heterogeneous and homogeneous electrochemiluminoimmunoassays of hTSH at disposable oxide-covered aluminum electrodes

Heterogeneous and homogeneous immunoassays of human thyroid stimulating hormone (hTSH) were developed on immunometric basis using aromatic Tb(III) chelates as electrochemiluminescent labels and varied types of disposable oxide-covered aluminum electrodes as the solid phase of the immunoassays. The long luminescence lifetime of the present labels allows the use of time-resolved electrochemiluminescence detection and provide the low detection limits of these labels and, thus, sensitive immunoassays. The primary antibody of immunometric immunoassays was coated upon aluminum oxide surface by physical absorption. In homogeneous immunoassays using 66l cell and 15 min incubation time, a linear calibration range of 0.25-324U/ml was obtained by applying only a single cathodic excitation pulse in the detection step of the assay.

Electrochemiluminoimmunoassay of hTSH at disposable oxide-coated n-silicon electrodes

Abstract Cathodic pulse polarisation of thin insulating film-coated electrodes enables the generation of electrochemiluminescence (ECL) by tunnel emission of hot electrons from the Fermi level of the conductor material of the conductor–insulator–aqueous electrolyte solution junction to the solutes at the vicinity of the electrode surface and probably also to the conduction band of water. The latter process can generate hydrated electrons as strongly reducing slightly longer-lived cathodic intermediate s , which are known to be able to induce chemiluminescence (CL) of various types of luminophores having very different photophysical and chemical properties. The generation of the above-mentioned cathodic primary species provides good possibilities to use many types of luminophores as label molecules in sensitive immuno and DNA-probing assays. This paper introduces an electrochemiluminoimmunoassay (ECLIA) for human thyroid stimulating hormone (hTSH) at oxide-coated n-silicon electrodes and demonstrates the suitability of silicon electrodes covered with thermally grown silicon dioxide film as disposable working electrodes (WEs) in sensitive time-resolved ECL (tr-ECL) measurements in aqueous solution. The label chelate can be detected almost down to picomolar level and the calibration curve of the chelate covers more than five orders of magnitude of chelate concentration. Also the calibration curve of the immunometric hTSH assay was found to be linear over a wide range of hTSH concentration, the detection limit of the hormone being below 1 mU l −1 (4 pmol l −1 ).

Time-resolved electrochemiluminescence of platinum(II) coproporphyrin

Cathodic pulse polarisation of oxide-covered aluminium electrodes can generate electrochemiluminescence (ECL) from metalloporphyrins. This is based on the tunnel emission of hot electrons into aqueous electrolyte solution, which probably results in the generation of hydrated electrons as reducing mediators. These tunnel emitted electrons allow the production of highly reactive radicals, such as sulfate radicals from peroxodisulfate ions, which can induce strong redox luminescence from various organic chemiluminophores including metalloporphyrins. The work presented here illustrates the generation of ECL from platinum(II) coproporphyrin (PtCP) and its bovine serum albumin (BSA) conjugate. This allows the detection of these molecules below nanomolar concentrations and over several orders of magnitude of concentration. The relatively long luminescence lifetime of PtCP allows discrimination from the background ECL signal using time resolved measurements, leading to higher sensitivity and the detection of PtCP-BSA indicates the potential use of metalloporphyrins as labels in ECL-based bioassays such as immunoassays and DNA-binding assays.

Chemiluminescence of luminol induced by dissolution of oxide-covered aluminum in alkaline aqueous solution

One-electron reduction of oxygen, hydrogen peroxide, potassium peroxodisulphate and potassium peroxodiphosphate was studied during the dissolution of oxide-covered aluminum in alkaline aqueous solution. The production of free oxidizing radicals was monitored by luminol chemiluminescence (CL). It was observed superoxide, hydroxyl, sulphate and phosphate radicals can be generated by the present method. In addition, luminol can be detected below nanomolar level, the linear logarithmic calibration range covering several orders of magnitude of concentration. The metallic aluminum and low-valent aluminum ions are the primary reductants of the system. The electron transfer to the solution is proposed to occur by tunneling through a thin insulating aluminum oxide film at the solid/electrolyte interface in moderately alkaline solutions with simultaneous dissolution of the forming oxide film. In a highly alkaline solution, it is more probable that the oxidation of aluminum species occurs in direct contact of the metallic aluminum with the aqueous solution. In the latter case, short-lived solvated low-valent aluminum ions, hydrogen atom and its deprotonated form, the hydrated electron, can exist as reducing mediators in the chemical reactions in the close vicinity of the dissolving solid/electrolyte interface. Luminol was also observed to exhibit CL under purely reducing conditions produced by a presently unknown excitation pathway.

Electrogenerated chemiluminescence induced by sequential hot electron and hole injection into aqueous electrolyte solution.

Hole injection into aqueous electrolyte solution is proposed to occur when oxide-coated aluminum electrode is anodically pulse-polarized by a voltage pulse train containing sufficiently high-voltage anodic pulses. The effects of anodic pulses are studied by using an aromatic Tb(III) chelate as a probe known to produce intensive hot electron-induced electrochemiluminescence (HECL) with plain cathodic pulses and preoxidized electrodes. The presently studied system allows injection of hot electrons and holes successively into aqueous electrolyte solutions and can be utilized in detecting electrochemiluminescent labels in fully aqueous solutions, and actually, the system is suggested to be quite close to a pulse radiolysis system providing hydrated electrons and hydroxyl radicals as the primary radicals in aqueous solution without the problems and hazards of ionizing radiation. The analytical power of the present excitation waveforms are that they allow detection of electrochemiluminescent labels at very low detection limits in bioaffinity assays such as in immunoassays or DNA probe assays. The two important properties of the present waveforms are: (i) they provide in situ oxidation of the electrode surface resulting in the desired oxide film thickness and (ii) they can provide one-electron oxidants for the system by hole injection either via F- and F(+)-center band of the oxide or by direct hole injection to valence band of water at highly anodic pulse amplitudes.

Miniature Tunneloxide Electrodes on Silicon for Aqueous Hot Electron Electrochemistry and Electrochemiluminecscence Studies

The basic principles of cathodic hot electron-induced electrochemiluminescence (HECL) and hot electron (HE) injection into aqueous electrolyte solution are shortly discussed. The applicability of miniaturized oxide-coated silicon electrodes as working electrodes in detection of electrochemiluminescent labels by HECL is studied. In addition, the fabrication processes of these tunnel oxide electrodes are described, and an immunoassay is used as an example of a real bioaffinity assay carried out using oxide-coated silicon electrodes.