Magnetically-assisted impedimetric detection of bacteria using phage-modified carbon microarrays.

This study presents an investigation on the possibility of improving the detection limit of bacteria with an inexpensive electrochemical, impedimetric sensor platform, by integrating the sensor with magnetic manipulation. The approach uses T4 bacteriophage coated Dynabeads to selectively capture and concentrate E. coli K12 cells from samples, to increase the sensitivity of detection at the surface of functionalized screen-printed carbon microarrays. Fluorescence and flow cytometry measurements indicate that the surface modification of the magnetic beads, with phages, and binding with the bacteria, were successful. Integration of the screen-printed carbon-based impedimetric sensor, with a magnetic manipulation system, was found to improve the sensitivity of the device, decreasing the limit of detection of E. coli K12 from 10(4) to 10(3) cfu/mL. We have also demonstrated that this approach provides for more specific detection of bacteria, enabling the operator to account for non-specific adsorption, and detection of bacteria in more complex (real) samples (milk).

Design of a universal biointerface for sensitive, selective, and multiplex detection of biomarkers using surface plasmon resonance imaging.

This paper reports on the sensitive, selective, and simultaneous detection of four protein biomarkers involved in metastasis of various cancers, namely Fas, angiopoietin-2 (Ang-2), human epidermal growth factor receptor 2 (HER2), and matrix metallopeptidase-9 (MMP-9) using an antibody-conjugated quantum dot (QD) chip and surface plasmon resonance imaging (SPRi) biosensors. Initially, a self-assembled monolayer film of l-cysteine, using glutaraldehyde as a linker and QDs as signal enhancement moieties, was employed to immobilize anti-Fas for the detection of Fas as a model protein in buffer. The biointerface was characterized using confocal microscopy, atomic force microscopy, and scanning electron microscopy to provide evidence of uniform surface coverage by the QDs. The SPRi signal was enhanced 100-fold to achieve a detection limit of 25 pg mL(-1) after applying biotinylated detection antibody-conjugate streptavidin-modified QDs. Secondly, this signal amplification strategy was applied to sequentially detect Fas, HER2, MMP-9, and Ang-2 at low concentrations on a protein-microprinted/gold-coated SPRi chip. The results showed the absence of cross-reactivity among these proteins and the feasibility of the approach for multiplex detection of biomarkers as required for the accurate diagnosis of various diseases.

Carbon microarrays for the direct impedimetric detection of Bacillus anthracis using Gamma phages as probes.

A direct and efficient impedimetric method is presented for the detection of Bacillus anthracis Sterne vegetative cells, using Gamma phages as probes attached to screen-printed carbon electrode microarrays. The carbon electrodes were initially functionalized through cyclic-voltammetric reduction of a nitro-aryl diazonium moiety, followed by further reduction of nitro groups to amino groups, and finally by treatment with glutaraldehyde. Functionalization (probe immobilization) using Gamma phages was verified by XPS and TOF-SIM experiments. The Gamma phage-modified microarrays were then used to detect B. anthracis Sterne bacteria in aqueous electrolyte media. Faradaic impedimetric detection of bacteria in KCl solution containing the ferri/ferro cyanide redox couple shows a gradual increase in Z' (real impedance) values, taken from the extrapolation of the linear portion of Nyquist plots in the low frequency range, for sensors placed in contact with increasing concentrations of B. anthracis. ΔZ' values vary from 700 to 5300 Ohms for bacteria concentrations ranging from 10(2) to 10(8) cfu mL(-1). These shifts in Z' are attributed to a decrease in diffusion controlled charge transfer to the electrode surface following capture of intact B. anthracis. No significant ΔZ' was observed for control experiments using E. coli. K12 as a non-specific target, even at a concentration of 10(8) cfu mL(-1).

Bacteriophage-modified microarrays for the direct impedimetric detection of bacteria.

A novel method is presented for the specific and direct detection of bacteria using bacteriophages as recognition receptors immobilized covalently onto functionalized screen-printed carbon electrode (SPE) microarrays. The SPE networks were functionalized through electrochemical oxidation in acidic media of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) by applying a potential of +2.2 V to the working electrode. Immobilization of T4 bacteriophage onto the SPEs was achieved via EDC by formation of amide bonds between the protein coating of the phage and the electrochemically generated carboxylic groups at the carbon surface. The surface functionalization with EDC, and the binding of phages, was verified by time-of-flight secondary ion mass spectrometry. The immobilized T4 phages were then used to specifically detect E. coli bacteria. The presence of surface-bound bacteria was verified by scanning electron and fluorescence microscopies. Impedance measurements (Nyquist plots) show shifts of the order of 10(4) Omega due to the binding of E. coli bacteria to the T4 phages. No significant change in impedance was observed for control experiments using immobilized T4 phage in the presence of Salmonella. Impedance variations as a function of incubation time show a maximum shift after 20 min, indicating onset of lysis, as also confirmed by fluorescence microscopy. Concentration-response curves yield a detection limit of 10(4) cfu/mL for 50-microL samples.