The rise of antibiotic-resistant bacteria has intensified the need for innovative monitoring and treatment strategies. In this regard, the task of monitoring the state of biofilms formed by bacteria in real time is relevant. In this paper, the adaptive holographic interferometry method is used to construct a biosensor based on microresonance microweighing for monitoring the growth of bacterial biofilm. The sensitive element of the biosensor is a silicon Atomic Force Microscopy cantilever measuring 215×43×7 µm3, with a 100 nm thick gold coating. The cantilever is placed in a glass cuvette with a volume of 100 µl with two tubes for the inflow and outflow of liquid. Using a pulsed Nd:YAG laser (λ=532 nm; τ=5 ns; Ep=100 µJ), the cantilever's natural oscillations were excited and were recorded in an adaptive holographic interferometer using a CdTe:V photorefractive crystal.
Before the experiment, the oscillation frequency of the cantilever in water was 69.5±0.7 kHz. During the experiment, the resonant frequency of the cantilever was measured with a repetition rate of 20 Hz. The oscillations were recorded using an oscilloscope and then processed in MATLAB to obtain an FFT image of the recorded signal. The resonant peak corresponding to the cantilever oscillations was approximated to find the central frequency. In turn, the biofilm mass was calculated using a numerical model [1].
The bacterial cell suspension of E. coli K-12 strain XL1-Blue at an optical density of OD600 = 1 was fed into a cuvette housing a microcantilever sensor for 1 h at a flow rate of 1 ml/min. Following the initial incubation, fresh LB medium was continuously supplied into the cuvette at a rate of 0.2 ml/min. Over 6 hours, the change in cantilever frequency due to bacterial attachment was 10.3±0.7 kHz, which corresponds to a bacterial mass of 5.6±0.4 ng.
The proposed monitoring method can be used to test the effect of various agents on the process of biofilm formation. Due to the adaptive signal processing, there are no requirements for the precise alignment of light beams, and the sensitive element can have a complex shape and a low-reflective surface.
1 Efimov, T.A.; Rassolov, E.A.; Andryukov, B.G.; Zaporozhets, T.S.; Romashko, R.V. Calculation of resonant frequencies of silicon AFM cantilevers. J. Phys. Conf. Ser. 2020, 1439, 012017.
