Full length histone H3 conjugated electrochemical biosensor for extracellular proteolytic Cathepsin L activity detection

Background

CTSL is a cysteine protease overexpressed in several cancers, and its extracellular form, reported to be 10 times more active than the lysosomal form, takes part in matrix degradation during metastasis. Antibody-based assays measure the amount of CTSL rather than its activity, fluorometric substrates have not established a detection limit and need high cell densities, and photoaffinity probes depend on light-sensitive labels. Earlier FETs for enzyme activity conjugated the substrate on the FET itself, limiting the number of measurements per device.

Design and approach

BFLH3, a 136-amino-acid substrate, is attached through streptavidin and biotin to an indium tin oxide (ITO) extended gate (EG) prepared with (3-aminopropyl)triethoxysilane and glutaraldehyde, and the EG carries a polydimethylsiloxane (PDMS) block with four wells and is connected to the top gate of a dual-gate silicon-on-insulator FET. CTSL cleaves BFLH3 mainly after amino acid 21; because biotin is on the N terminus, cleavage releases the larger 12.97 kDa portion of the 16.3 kDa protein. The released charge at pH 7 is given as +13.2 in the introduction and the FET characterization section and as +13.4 in the section comparing FET with fluorometry. The resulting change in EG surface charge is read as a shift in the back-gate voltage sweep. Because the EG is replaceable, one FET can be used for many measurements.

How it was evaluated

The FET was characterized by drain current under top-gate voltage steps and in pH 4, 7 and 10 buffers, and BFLH3 conjugation was checked by atomic force microscopy and bicinchoninic acid (BCA) assay. CTSL from human liver was tested from 0.1 ng/mL to 10 μg/mL in CTSL buffer and compared with the fluorometric substrate magic red-FRFR (MR-FRFR) both in a plate assay and as an EG coating. Culture media of LNCaP (prostate adenocarcinoma, metastatic lesion) and PANC-1 (pancreatic cancer, used as a negative control) cells grown at 10^4 cells/well in 96-well plates were measured with self-normalization against RPMI 1640 and DMEM and quantified against the same media spiked with 0.5 and 5 ng/mL CTSL, and compared with ELISA and western blotting of cell lysates.

Key results

Raising the top-gate voltage from 1 V to 5 V increased the drain current by roughly two orders of magnitude, and the device showed a pH sensitivity of 173.33 mV pH−1, above the Nernst limit of 59.6 mV pH−1. After streptavidin and BFLH3 conjugation, the root-mean-square roughness of the EG decreased from 6.184 nm to 4.323 nm and 3.786 nm and the median height rose from 4.435 nm to 6.331 nm and 9.104 nm. BCA absorbance saturated at 10^13 molecules/well (2.8 × 10^13 molecules per cm2, corresponding to 1.12 mg/mL of BFLH3), and this loading was used thereafter.

In CTSL buffer the FET reached a detection limit of 1 ng/mL, compared with 10 μg/mL for MR-FRFR fluorometry and 1.7 ng/mL reported for ELISA; the authors note that most cancer cell lysates tested contained 3.34-12.80 ng/mL of CTSL. With 100 ng/mL CTSL, an MR-FRFR-coated EG showed no significant difference from buffer, which the authors relate to the small 0.34 kDa, +1 fluorophore released, whereas the BFLH3-coated EG shifted by 0.25 V after 30 min compared with 0.01 V for buffer.

In culture media, the voltage shift fell from 1.50 V for RPMI 1640 to 0.23 V for LNCaP medium and from 1.05 V for DMEM to 0.55 V for PANC-1 medium; buffering with 50 mM HEPES limited pH drift to 7.2-7.6, corresponding to 69.33 mV. Calibration with the spiked media gave 7.29 ng of extracellular CTSL for LNCaP and 2.44 ng for PANC-1, while ELISA of cell lysates gave 10.45 ng and 5.52 ng, respectively. The FET quantified CTSL from media of 10^4 cells/well, compared with a cell detection limit of 10^5 cells/well for the fluorometric method, and western blotting and ELISA both showed roughly two-fold higher CTSL in LNCaP than in PANC-1.

Open questions

The authors report an inherent instability of the FET when switching from one EG to the next and therefore took the measurements for each condition from the four chambers of a single EG. The cell-media experiments are presented as a proof of concept in two cultured cell lines, and the FET measures extracellular CTSL whereas ELISA and western blotting measure whole cell lysates.

How it connects to other research

Antibody-functionalized FETs such as the silicon nanowire sensor for salivary interleukin 8 report how much of a target protein binds, whereas here the measured quantity is enzymatic activity: CTSL cleaves an immobilized substrate and the loss of the charged fragment changes the gate potential.