Quantitative electrochemical detection of cathepsin B activity in breast cancer cell lysates using carbon nanofiber nanoelectrode arrays toward identification of cancer formation.

The proteolytic activity of cathepsin B in complex breast cell lysates has been measured with alternating current voltammetry (ACV) using ferrocene (Fc)-labeled-tetrapeptides immobilized on nanoelectrode arrays (NEAs) fabricated with vertically aligned carbon nanofibers (VACNFs). Four types of breast cells have been tested, including normal breast cells (HMEC), transformed breast cells (MCF-10A), breast cancer cells (T47D), and metastatic breast cancer cells (MDA-MB-231). The detected protease activity was found increased in cancer cells, with the MDA-MB-231 metastatic cancer cell lysate showing the highest cathepsin B activity. The equivalent cathepsin B concentration in MDA-MB-231 cancer cell lysate was quantitatively determined by spiking recombinant cathepsin B into the immunoprecipitated MDA-MB-231 lysate and the HMEC whole cell lysate. The results illustrated the potential of this technique as a portable multiplex electronic device for cancer diagnosis and treatment monitoring through rapid profiling the activity of specific cancer-relevant proteases. FROM THE CLINICAL EDITOR: Breast cancer is the most common cancer in women. In this report, the authors applied the technique of nanoelectrode arrays to try to detect and compare cathepsin B activities in normal and breast cancer cells. It was found that protease activity correlated positively with the degree of malignancy cancer cells. Taking this further, this technique may be useful for rapid diagnosis of cancer in the future.

Integration of a nanostructured dielectrophoretic device and a surface-enhanced Raman probe for highly sensitive rapid bacteria detection.

This work reports a synergistic approach to the concentration, detection and kinetic monitoring of pathogens through the integration of nanostructured dielectrophoresis (DEP) with nanotag-labelled Surface Enhanced Raman Spectroscopy (SERS). A nanoelectrode array made of embedded Vertically Aligned Carbon Nanofibers (VACNFs) at the bottom of a microfluidic chip was used to effectively capture and concentrate nanotag-labelled E. coli DHα5 cells into a 200 µm × 200 µm area on which a Raman laser probe was focused. The SERS nanotags were based on iron oxide-gold (IO-Au) core-shell nanoovals (NOVs) of ∼50 nm size, which were coated with a QSY21 Raman reporter and attached to E. coli through specific immunochemistry. The combination of the greatly enhanced Raman signal by the SERS nanotags and the effective DEP concentration significantly improved the detection limit and speed. The SERS signal was measured with both a confocal Raman microscope and a portable Raman probe during DEP capture, and was fully validated with fluorescence microscopy measurements under all DEP conditions. The SERS measurements were sensitive enough to detect a single bacterium. A concentration detection limit as low as 210 cfu ml(-1) using a portable Raman system was obtained with a DEP capture time of only ∼50 s. These results demonstrate the potential to develop a compact portable system for rapid and highly sensitive detection of specific pathogens. This system is reusable, requires minimum sample preparation, and is amenable to field applications.

Syntheses, neural protective activities, and inhibition of glycogen synthase kinase-3ß of substituted quinolines.

A new series of fifteen 5-, 6-, and 8-appended 4-methylquinolines were synthesized and evaluated for their neural protective activities. Selected compounds were further examined for their inhibition of glycogen synthase kinase-3ß (GSK-3ß) and protein kinase C (PKC). Two most potent analogs, compounds 3 and 10, show nanomolar protective activities in amyloid ß-induced MC65 cells and enzymatic inhibitory activities against GSK-3ß, but poor PKC inhibitory activities. Using normal mouse model, the distribution of the most potent analog 3 in various tissues and possible toxic effects in the locomotors and inhibition of liver transaminases activities were carried out. No apparent decline of locomotor activity and no inhibition of liver transaminases were found. The compound appears to be safe for long-term use in Alzheimer's disease mouse model.

Quantitative electrochemical detection of cathepsin B activity in complex tissue lysates using enhanced AC voltammetry at carbon nanofiber nanoelectrode arrays.

The proteolytic activity of a cancer-related enzyme cathepsin B is measured with alternating current voltammetry (ACV) using ferrocene (Fc) labeled tetrapeptides attached to nanoelectrode arrays (NEAs) fabricated with vertically aligned carbon nanofibers (VACNFs). This combination enables the use of high AC frequencies (~1kHz) with enhanced electrochemical signals. The specific proteolysis of the Fc-peptide by cathepsin B produces decay in the ACV peak current versus the reaction time. The exponential component of the raw data can be extracted and defined as the "extracted proteolytic signal" which allows consistent quantitative analyses using a heterogeneous Michaelis-Menten model. A "specificity constant" kcat/KM = (3.68 ± 0.50) × 10(4)M(-1)s(-1) for purified cathepsin B was obtained. The detections of cathepsin B activity in different concentrations of whole lysate of human breast tissue, tissue lysate spiked with varied concentrations of cathepsin B, and the tissue lysate after immunoprecipitation showed that there is ~13.4 nM higher cathepsin B concentration in 29.1 µg mL(-1) of whole tissue lysate than the immunoprecipitated sample. The well-defined regular VACNF NEAs by e-beam lithography show a much faster kinetics for cathepsin B proteolysis with kcat/KM = 9.2 × 10(4)M(-1)s(-1). These results illustrate the potential of this technique as a portable multiplex electronic system for cancer diagnosis by rapid protease profiling of serum or blood samples.

Electrochemical Protease Biosensor Based on Enhanced AC Voltammetry Using Carbon Nanofiber Nanoelectrode Arrays.

We report an electrochemical method for measuring the activity of proteases using nanoelectrode arrays (NEAs) fabricated with vertically aligned carbon nanofibers (VACNFs). The VACNFs of ~150 nm in diameter and 3 to 5 µm in length were grown on conductive substrates and encapsulated in SiO2 matrix. After polishing and plasma etching, controlled VACNF tips are exposed to form an embedded VACNF NEA. Two types of tetrapeptides specific to cancer-mediated proteases legumain and cathepsin B are covalently attached to the exposed VACNF tip, with a ferrocene (Fc) moiety linked at the distal end. The redox signal of Fc can be measured with AC voltammetry (ACV) at ~1 kHz frequency on VACNF NEAs, showing distinct properties from macroscopic glassy carbon electrodes due to VACNF's unique interior structure. The enhanced ACV properties enable the kinetic measurements of proteolytic cleavage of the surface-attached tetrapeptides by proteases, further validated with a fluorescence assay. The data can be analyzed with a heterogeneous Michaelis-Menten model, giving "specificity constant" kcat /Km as (4.3 ± 0.8) × 104 M-1s-1 for cathepsin B and (1.13 ± 0.38) × 104 M-1s-1 for legumain. This method could be developed as portable multiplex electronic techniques for rapid cancer diagnosis and treatment monitoring.