Recombinant Laccase: II. Medical Biosensor

Abstract

Langmuir-Blodgett (LB) technology was used to build a high-sensitivity enzyme-based biosensor for medical purposes. Recombinant fungal laccase from Rigidoporous lignosus, as previously described, was used to catalyze a widely used antidepressant in a micromolar range, namely, clomipramine. The topological properties of the laccase thin film were characterized via LB π-A isotherm and AFM (mean roughness 8.22 nm, compressibility coefficient 37.5 m/N). The sensitivity of the biosensor was investigated via UV spectroscopy, and linearity was found in the absorbance peak shift at 400 nm at drug concentration varying up to 20 uM. The enzyme kinetics was subsequently investigated with potentiometric and amperometric measurements, and we found electronic transfer of at least 1 electron, k(s) 0.57 s(-1), diffusion coefficient 3 × 10(-6) cm(2)/s, K(cat) 6825.92 min(-1), K(M) 4.1 uM, K(cat)/K(M) 2.8 × 10(7) mol(-1) s(-1), sensitivity of 440 nA/uM, maximum velocity 1706.48 nA/s, and response time less than 5 s. The amperometric and potentiometric measurements were repeated after a month, confirming the stability of the biosensor.

Determination of Protein-Protein Interaction for Cancer Control via Mass Spectrometry and Nanoconductimetry of NAPPA SNAP Arrays: An Overview

“…This, taken together with the unique properties of proteins obtained with Langmuir-Blodgett (LB)-based crystallography [31][32][33][34][35][36][37][38] that can enable new strategies for drug design (work in progress), makes the goal of cancer control tremendously ambitious.…”

Ab Initio Semi-Quantitative Analysis of Micro-Beam Grazing-Incidence Small-Angle X-Ray Scattering (Μ-GISAXS) during Protein Crystal Nucleation and Growth

“…LB nanobiotemplate enables scientists to finely manipulate molecules and proteins, and allows the development and design of highly ordered nanobiopatterns and sensitive nanobiosensors [15][16][17].…”

Prototypes of Newly Conceived Inorganic and Biological Sensors for Health and Environmental Applications

“…We used inorganic nanocomposites, such as matrices of calcium oxide [2], and biological ones such as Nucleic Acid Programmable Protein Arrays (NAPPA) [3,4] along with Langmuir-Blodgett (LB) multi-layers of proteins of primary interest such as laccase [5,6]. Special attention was paid to the detection of substances significant for both the environment (such as carbon dioxide) and medicine (drugs and cancer) by means of a wide variety of detection methods (amperometric, conductometric, and nanogravimetric).…”

“…For what concerns the two health prototypes [3,5], one biosensor of new conception was realized by coupling the traditional quartz crystal microbalance with frequency monitoring (QCM_F) [7] with the quartz crystal microbalance with dissipation monitoring (QCM_D) [8,9], and with an innovative protein cell-free expression system named Nucleic Acid Programmable Protein Arrays (NAPPA) [10,11], that allowed us to immobilize on the quartz surface, as sensing molecule, any kind of protein [3]. In the QCM_D sensor [12] each quartz contained 4×4 NAPPA spots of 300-micron square and functional proteins were synthesized in situ directly from printed cDNAs (complementary DNAs) just before the assay [4,12].…”