Live-cell targeting of his-tagged proteins by multivalent N-nitrilotriacetic acid carrier complexes.

Selective and fast labeling of proteins in living cells is a major challenge. Live-cell labeling techniques require high specificity, high labeling density, and cell permeability of the tagging molecule to target the protein of interest. Here we report on the site-specific, rapid, and efficient labeling of endogenous and recombinant histidine-tagged proteins in distinct subcellular compartments using cell-penetrating multivalent chelator carrier complexes. In vivo labeling was followed in real time in living cells, demonstrating a high specificity and high degree of colocalization in the crowded cellular environment.

Amperometric immunosensor for carcinoembryonic antigen in colon cancer samples based on monolayers of dendritic bipodal scaffolds.

Detection of proteins that signal the presence or recurrence of cancer is a powerful therapeutic tool for effective early diagnosis and treatment. Carcinoembryonic antigen (CEA) has been extensively studied as a tumor marker in clinical diagnosis. We report on the development of an amperometric biosensor for the detection of CEA based on the immobilization of anti-CEA monoclonal antibody on a novel class of bipodal thiolated self-assembled monolayers containing reactive N-hydroxysuccinimide (NHS) ester end groups. The current variations showed a linear relationship with the concentration of CEA over the range of 0-200 ng/mL with a sensitivity of 3.8 nA x mL x ng(-1) and a detection limit of 0.2 ng/mL, which is well below the commonly accepted concentration threshold (5 ng/mL) used in clinical diagnosis. Real time and accelerated stability studies of the reporter antibody under various storage conditions demonstrated that the enzymatic activity and antibody affinity of the conjugate is retained for long periods of time in commercial stabilizing buffers such as StabilGuard Biomolecule Stabilizer, and a prediction of the stability trends was carried out using the kinetic and thermodynamic parameters obtained from the Arrhenius equation. The developed immunosensor as well as a commercially available enzyme-linked immunosorbent assay (ELISA) kit were successfully applied to the detection of CEA in serum samples obtained from colon cancer patients, and an excellent correlation of the levels of CEA measured was obtained. Ongoing work is looking at the incorporation of the developed biosensor into a platform for multiplexed simultaneous detection of several breast cancer related biomarkers.

Design and testing of a packaged microfluidic cell for the multiplexed electrochemical detection of cancer markers.

We present the rapid prototyping of electrochemical sensor arrays integrated to microfluidics towards the fabrication of integrated microsystems prototypes for point-of-care diagnostics. Rapid prototyping of microfluidics was realised by high-precision milling of polycarbonate sheets, which offers flexibility and rapid turnover of the desired designs. On the other hand, the electrochemical sensor arrays were fabricated using standard photolithographic and metal (gold and silver) deposition technology in order to realise three-electrode cells comprising gold counter and working electrodes as well as silver reference electrode. The integration of fluidic chips and electrode arrays was realised via a laser-machined double-sided adhesive gasket that allowed creating the microchannels necessary for sample and reagent delivery. We focused our attention on the reproducibility of the electrode array preparation for the multiplexed detection of tumour markers such as carcinoembryonic antigen and prostate-specific antigen as well as genetic breast cancer markers such as estrogen receptor-alpha, plasminogen activator urokinase receptor, epidermal growth factor receptor and erythroblastic leukemia viral oncogene homolog 2. We showed that by carefully controlling the electrode surface pre-treatment and derivatisation via thiolated antibodies or short DNA probes that the detection of several key health parameters on a single chip was achievable with excellent reproducibility and high sensitivity.

Integrated microfluidic platform for the electrochemical detection of breast cancer markers in patient serum samples.

A microsystem integrating electrochemical detection for the simultaneous detection of protein markers of breast cancer is reported. The microfluidic platform was realized by high precision milling of polycarbonate sheets and features two well distinguishable sections: a detection zone incorporating the electrode arrays and the fluid storage part. The detection area is divided into separate microfluidic chambers addressing selected electrodes for the measurement of samples and calibrators. The fluidic storage part of the platform consists of five reservoirs to store the reagents and sample, which are interfaced by septa. These reservoirs have the appropriate volume to run a single assay per cartridge and are manually filled. The liquids from the reservoirs are actuated by applying a positive air pressure (i.e.via a programmable syringe pump) through the septa and are driven to the detection zone via two turning valves. The application of the realised platform in the individual and simultaneous electrochemical detection of proteic cancer markers with very low detection limits are demonstrated. The microsystem has also been validated using real patient serum samples and excellent correlation with ELISA results obtained.

Electron permeable self-assembled monolayers of dithiolated aromatic scaffolds on gold for biosensor applications.

Self-assembled monolayers (SAMs) of thiolated compounds are formed by the spontaneous chemisorption of thiolate groups on metal surfaces. In biosensors, they are most commonly used to covalently immobilize a biorecognition molecule onto the surface of the transducer, thus offering the possibility of controlling the orientation, distribution, and spacing of the sensing element while reducing nonspecific interactions. In this paper, self-assembled monolayers of dithiolated derivatives of 3,5-dihydroxybenzyl alcohol containing carboxyl and hydroxyl end groups have been prepared on gold surfaces and characterized by cyclic voltammetry and electrochemical impedance spectroscopy. Impedance measurements revealed that SAM formation is essentially completed after 3-5 h of exposure by observing the successive blocking of the faradic response of ferricyanide anion due to the adsorption of the dithiol molecules. The surface coverage of these molecules, estimated by reductive desorption experiments, was in the range of (1.1-2.8) x 10-10 mol/cm2. To demonstrate the potential of the dithiol SAM, a model system for detection of a tumor marker, prostate-specific antigen (PSA), was developed. The carboxyl groups of the SAM were succinimide-activated, and an anti-PSA antibody was covalently immobilized via amide bonds. The modified SAM was used for the label-free detection of prostate-specific antigen using EIS with a detection limit of 9 ng/mL. The results described here demonstrate that this kind of dithiol-modified SAM can be used as supports in electrochemical biosensors and the results are explained in terms of the structural features of these dithiols.