Triglyceride-Tethered Membrane Lipase Sensor

Abstract

Sensors that can quickly measure the lipase activity from biological samples are useful in enzyme production and medical diagnostics. However, current lipase sensors have limitations such as requiring fluorescent labels, pH control of buffer vehicles, or lengthy assay preparation. We introduce a sparsely tethered triglyceride substrate anchored off of a gold electrode for the impedance sensing of real-time lipase activity. The tethered substrate is self-assembled using a rapid solvent exchange technique and can form an anchored bilayer 1 nm off the gold electrode. This allows for an aqueous reservoir region, providing access to ions transported through membrane defects caused by triglyceride enzymatic hydrolysis. Electrical impedance spectroscopy techniques can readily detect the decrease in resistance caused by enzymatically induced defects. This rapid and reliable lipase detection method can have potential applications in disease studies, monitoring of lipase production, and as point-of-care diagnostic devices.

Membrane-Disruptive Effects of Fatty Acid and Monoglyceride Mitigants on E. coli Bacteria-Derived Tethered Lipid Bilayers

“…In more detail, Table 1 summarizes the full set of electrochemical parameters that were obtained by fitting the EIS data to the equivalent circuit model and confirmed that the fitted parameter values were in good agreement with expected ranges. More specifically, the fitted G m signal for the E. coli tBLM platform was around 1.36 ± 0.45 µS, which agrees well with recently studied tBLM platforms composed of biologically relevant two-chain phospholipids or three-chain triglycerides [39]. Of note, the fitted C m signal was 0.83 ± 0.17 µF/cm 2 , which is within the range reported for tBLM platforms composed exclusively of two-chain phospholipids (~1.2-1.4 µF/cm 2 ) or three-chain triglycerides (~0.6 µF/cm 2 ).…”

“…More specifically, the fitted G m signal for the E. coli tBLM platform was around 1.36 ± 0.45 μS, which agrees well with recently studied tBLM platforms composed of biologically relevant two-chain phospholipids or three-chain triglycerides [ 39 ]. Of note, the fitted C m signal was 0.83 ± 0.17 μF/cm 2 , which is within the range reported for tBLM platforms composed exclusively of two-chain phospholipids (~1.2–1.4 μF/cm 2 ) or three-chain triglycerides (~0.6 μF/cm 2 ).…”

“…In addition, the Q s parameter represents the imperfect capacitance in the reservoir region, whereas the ∝ s parameter is defined as the CPE dimensional constant and describes the contribution of restricted ion diffusion in the reservoir region. The fitted values of both parameters agree well with those obtained for tBLM platforms composed of biologically relevant phospholipids [ 39 ], while the electrolyte resistance (R e ) is also consistent with the ionic strength of the buffer composition used in this study. We also fabricated E. coli tBLM platforms from more purified polar lipid extracts, which had a similar lipid composition to the total extract and had been prepared by further precipitating the total lipid extract with acetone, followed by extraction with diethyl ether.…”

“…v2.0.58), which can fit the EIS data (frequency-dependent impedance magnitude and phase) to an equivalent circuit model by a procedure based on the Levenberg–Marquardt algorithm [ 43 ]. The chosen equivalent circuit model represents the tBLM as a resistor and capacitor that is in series with a constant phase element (CPE) that describes the imperfect capacitance of the gold electrode interface [ 53 ] (ionic reservoir region) and with a resistor that describes the impedance of the bulk electrolyte solution, as previously described [ 39 ]. The use of a CPE in the equivalent circuit model has been justified in the context of a heterogenous distribution of conductive elements (e.g., defects) within the membrane [ 43 , 54 , 55 ].…”