Design and development of a multifunctional fluidic sensor platform for particle and cellular characterisation utilising resistive pulse sensing

The characterisation of particulates suspended in a liquid often involves the determination of one or more of their physical characteristics such as their size, shape, concentration, and charge. Micro and nanoparticles are becoming more present in many areas such as nanomedicines, aquatic environments, as well as in food and drinks. Within these applications the particles are contained within an array of matrixes which can make their characterisation difficult and time consuming using traditional methods such as dynamic light scattering. Resistive Pulse Sensing (RPS) is a technique that allows for the rapid analysis of these samples without time-consuming sample preparation. The technique provides a particle-by-particle analysis of the sample which gives a highly accurate characterisation of the sample.The aim of this thesis was to produce a microfluidic platform which incorporates multiple RPS sensors within the same device. Additive manufacturing was selected as this would allow for a rapid design process where the device can be prototyped without high costs or time intensive fabrication methods. By having multiple pores in series, a sample with a large size range can be measured within a single device. In RPS sensing the minimum and maximum sizes detectable are related to the dimensions of the pore used. Therefore, samples which contain large analytes can cause blockages in smaller pores, or smaller analytes can remain undetected in a pore which is too large. This makes exploratory work more difficult to achieve as an element of the analytes size must be known in order to tune the sensor for analysis. The research within aims to characterise the whole size range of a sample within a single measurement. The integration of RPS into microfluidics allows for multiple pores within a single device as well as easier sample handling and integration into existing systems.The sensor platform developed in this thesis is an additively manufactured (AM) device produced via dynamic light processing (DLP) with reusable components and was equipped with two RPS sensors. The first RPS sensor can detect particles in size ranges of 2-30 µm, which is a significant improvement over the previous generation of AM RPS. By using a lid, the sensing region of the pore could be adjusted to dimensions which were not possible to print using the equipment in this thesis. Therefore, this method can be used to overcome the resolution limit imposed by a 3D printer which is a core limitation of AM RPS. The first RPS sensor was then applied to a range of applications, these studies included measuring microplastics in tea, and the detection and shape analysis of algae.Two tea bag manufacturers were selected and the size and concentration of the microplastics was measured with HCT giving a modal and mean size of 22 ± 0.6 µm and 21 ± 2 µm respectively with a total concentration of 6.52x104 particles per 10 mL of solution. The next manufacturer, LMHG, had a modal and mean size of 21 ± 2 µm and 22 ± 2 µm respectively and a total concentration of 7.76x106 particles per 10 mL of solution. During this investigation it was noted that the direction of the pulses depended on the concentration of the electrolyte they were immersed in. At low concentrations, 0.25 mM, the pulses were conductive yet at higher concentrations, >50 mM, these pulses became resistive. This is suspected to be due to the high surface area of the particles generating a large diffuse double layer at low electrolyte concentrations, when the particle traverses the pore the conductivity of the pore is temporarily increased due to this large double layer in comparison to the bulk solution. Whereas high concentrations of electrolyte the particles are always resistive in comparison to the bulk solution. The second application focused on discriminating between two shapes of algae in comparison to a known calibrant. The algae were compared to the calibrant via a model which broke each pulse into splines in order to compare them across a dataset. Once the model was created, the algae could be compared, and could successfully detect 87 % of the spherical algae and 86% of the rod shaped algae. The focus of the thesis then shifted onto fabricating solid-state pores to be embedded into the platform as the second sensor for particle detection. Three methods of fabrication were used: dielectric breakdown, focused ion beam and heated thermoplastic puncture. Dielectric breakdown had a low success rate with only 14 % of pores being able to detect a particle. Focused ion beam had a higher rate of 21 % for all pore sizes but this increased to 40 % when considering pores larger than 600 nm, the pores here were able to detect particles as small as 158 nm and had an LOD of 1.68x107 particles per mL. The final method was heated thermoplastic puncture, this could only produce large micropores which were not able to detect particles smaller than 1 µm. The arrays produced by this method contained pores of various sizes which were not useful for this project. As the focused ion beam pores proved the most successful these progressed onto the next stage. Embedding the pores into the platform required the creation of a pore holder, a design based around the HPLC fittings was the most successful. Due to wettability issues with the silicon nitride, alternative materials were used including polyurethane micropores and glass pipettes. The polyurethane pores proved successful however were unable to detect particles smaller than 2 µm and did not fit the required sub-µm criteria. The glass pipettes were more successful being able to detect particles as small as 158 nm to 20 µm depending on the size of pipette used.The glass pipettes had a limit of detection of 1.89x103 particles per mL. The next test was to demonstrate if the printed pore and the pipette could work in tandem. Both pores were able to detect the same sample of particles successfully demonstrating the device is able to use both pores in series. The final experiment was to see if a sample containing large and small particles would cause the glass pipette to block. A sample containing 2, 40 and 80 µm particles was used and only the 2 µm particles were detected, the baseline was also stable during this indicating that the larger particles did not interact with the pore. Overall, this thesis has designed and successfully demonstrated a dual pore RPS sensor platform capable of detecting a wide range of analyte sizes within the same sample. This represents a significant advantage over previous RPS sensors which only use one pore and therefore have a restrictive maximum and minimum size they can detect.