Ultrasound particle manipulation & mid-infrared spectroscopy towards bacteria sensing in water

Abstract

This thesis was carried out as part of the H2020 research project WaterSpy. The task set out in the description of action was to use ultrasound (US) particle manipulation and attenuated total reflection infrared (ATR IR) spectroscopy for monitoring bacteria in water. The initial idea was to trap bacteria using US standing waves (USW) around 2 MHz followed by measurement using mid-IR ATR spectroscopy. Several generations of acoustic traps were designed and tested, resulting in a device completely made out of aluminum only relying on a piezo disc as US source. This acoustic trap was thermoelectrically stabilized exploiting the thermal conductivity of aluminum enabling stable operation at the desired temperature (37°C) and high US intensities. In the built acoustic traps ATR elements, which were required for mid-IR spectrum acquisition, also served as reflectors for USW generation. During this thesis 3D printing was used for rapid prototyping. Besides its usage for developing auxiliary equipment crucial for protecting electric contacts or its implementation in the liquid handling system, several different ATR fixtures were manufactured with this technique. By combining US particle manipulation and ATR IR spectroscopy it was demonstrated that US-enhanced direct measurement of Escherichia coli (E. coli) can be performed at high concentrations. It was found that pushing the bacteria conglomerates into the evanescent field at the ATR element is not straightforwardly doable by simply switching the US frequency as suggested by previous studies using yeast. Thus, an US-assisted assay as alternative to the direct measurement approach was explored. For this purpose, a sequential injection analysis system was linked to the acoustic trap housing the ATR element to perform complex liquid handling sequences. In contrast to recording mid-IR spectra of E. coli directly, the activity of the enzyme alkaline phosphatase (AP) conjugated to antibodies used to label the bacteria was monitored. In a first step, AP was conjugated to beads. After trapping those beads and supplying them with the enzyme substrate p-nitrophenylphosphate in a fully automated way, the enzymatic conversion into the products p-nitrophenol and phosphate could be monitored via ATR IR spectroscopy. It was found that bead trapping can be done in a highly reproducible manner and acoustically retained beads could be reused several times. With later generations of acoustic traps straightforward bacteria handling was demonstrated. During the development of the US-assisted assay it was found that antibody labelling in the acoustic trap led to unspecific binding of antibody resulting in poor reproducibility. By decoupling both procedures reproducible quantification of bacteria in water was possible down to 1.95 x 106 bacteria mL−1. The optimized acoustic trap was characterized by image processing and electrical impedance measurements to find the optimum working frequency. Paired with the higher US intensities enabled by thermal stabilization, flow rates during bacteria trapping experiments significantly higher than the ones usable in previous generation acoustic traps were achievable (1.17 μL s−1), thus enabling higher sample throughput.Furthermore, a novel technique for recording ATR IR spectra based on mid-IR lasers was developed during this thesis. By combining an extended tunable quantum cascade laser (QCL-XT) with a dedicated mid-IR balanced detection module the capabilities of the novel sensing scheme termed “polarimetric balanced detection” was demonstrated. It was showcased that by exploiting unequal effective thicknesses of parallel and perpendicular polarized light in an ATR measurement configuration for balanced detection the stability of the setup could be improved.