Real-time monitoring of formaldehyde-induced DNA–lysozyme cross-linking with piezoelectric

Abstract

A novel method for monitoring, in real time, the formaldehyde (FA)-induced DNA–protein cross-linking process with the piezoelectric quartz crystal impedance (PQCI) technique is proposed. The method was used to monitor FA-induced DNA–lysozyme cross-link formation. Lysozyme was directly immobilized on the silver electrode surface of a piezoelectric quartz crystal by adsorption. The lysozyme-coated piezoelectric sensor was in contact with FA and DNA solutions. The time courses of the resonant frequency and equivalent circuit parameters of the sensor during the cross-linking were simultaneously obtained and are discussed in detail. On the basis of the feature of the multi-dimensional information provided by the PQCI technique, it was concluded that the observed frequency decrease could be mainly ascribed to the mass increase resulting from the cross-linking. According to the frequency decrease with time, the kinetics of the cross-linking process were quantitatively studied. A piezoelectric response model for the cross-linking was theoretically derived. Fitting the experimental data to the model, the kinetic parameters, such as the binding and dissociation rate constants (k₁ and k₋₁) and the cross-linking equilibrium constant (K_a), were determined. At 37 °C, the k₁, k₋₁ and K_a values obtained were 7.0 (±0.1) × 10⁻⁵ (μg ml⁻¹)⁻¹ s⁻¹, 6.6 (±0.1) × 10⁻³ s⁻¹ and 1.06 (±0.02) × 10⁻² (μg ml⁻¹)⁻¹, respectively.