Bile acids are physiologically important metabolites, which are synthesized in liver as the end products of cholesterol metabolism and then secreted into intestine. They are amphiphilic molecules which play a critical role in the digestion and absorption of fats and fat-soluble vitamins through emulsification. The concentration of bile acids is an indicator for liver function. Individual suffering from liver diseases has a sharp increase in bile acid concentrations. Hence, the concentration level of bile acids has long been used as a biomarker for the early diagnosis of intestinal and liver diseases. Conventional methods of bile acid detection such as chromatography-mass spectrometry and enzymatic reactions are complex and expensive. It is highly desired to have a simple, fast, and low-cost detection of bile acids that is available for self-testing or point-of-care testing. To achieve this goal, we develop a liquid crystal-based biosensor for the detection of bile acids. The sensor platform is based on the anchoring transition of liquid crystals (LCs) at the sodium dodecyl sulfate (SDS)-laden LC/aqueous interface for the detection of bile acids in aqueous solution. The first part of this dissertation focuses on the detection mechanism of bile acids. Our studies show that the displacement of SDS from the LC/aqueous interface by the competitive adsorption of bile acids induces a homeotropic-to-planar anchoring transition of the LC at the interface, providing an optical signature for the simple and rapid detection of bile acids. The adsorption of bile acids on the interface was found to follow Langmuir-Freundlich isotherm. The adsorption kinetics of different bile acids is compared. We find that both the number and position of hydroxyl groups of bile acids affect their adsorption kinetics. The different optical patterns of LC films formed by the adsorption of bile acids are also discussed. The second part of this dissertation studies the effect of solution conditions, surfactants, and liquid crystals on the detection limit of the LC-based biosensor for bile acids. Low pH and high ionic strength in the aqueous solution can reduce the electrostatic interaction between SDS and bile acids, which leads to a decreased detection limit. Surfactants with smaller headgroup and lower packing density also help to reduce the detection limit. To further reduce the detection limit, we investigate the effect of LC structures and find that LCs with a shorter chain length give lower detection limits. Also, by substituting a phenyl ring with a cyclohexane ring, we find that the detection limit is further reduced due to the decrease of the interaction between the phenyl rings of LCs. By mixing different LCs together, the detection limit can be linearly tuned from 160 μM to 1.5 μM, which is comparable to the traditional methods. But the LC-based biosensors have much simpler design and manufacture process. The third part of this dissertation is to apply this LC-based biosensor to the detection of urinary bile acids. We test the influence of several potential interfering species such as urea, creatinine, uric acid and ascorbic acid by conducting experiments in synthetic urine. By adjusting the concentration of SDS, we are able to eliminate the impact of those interfering species, and demonstrate that the LC-based biosensors can selectively detect urinary bile acids in human urine, suggesting its potential for screening liver dysfunctions. The final part of this dissertation is to investigate the application of LC-based biosensors in detecting the lipolysis process by porcine pancreatic lipase (PPL). It has been a long-standing argument over the role of bile salts on the activity of PPL. Thus, we study the time course of the hydrolysis of phospholipid L-dipalmitoylphosphatidylcholine (L-DPPC) by PPL at LC/aqueous interface. The hydrolysis of L-DPPC leads to a homeotropic-to-tilted anchoring transition of the LC at the interface, which allows the hydrolysis process to be monitored by a polarizing optical microscope. The microscopy image analysis reveals a lag-burst kinetics where a lag phase is followed by a burst phase. The effect of bile acids on these two phases is studied. We find that the activity of PPL both in the presence and absence of colipase can be improved by increasing the concentration of bile acids. The improvement becomes more distinct in the presence of colipase.

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The nitrogenous metabolite urea is an important biomarker that indicates cows’ nutritional intake. Today, determining the urea concentration from milk samples requires analysis techniques in a lab which is time-consuming and expensive. It would be favourable to have an on-site measurement method to achieve a fast detection allowing farmers to quickly adjust the feed of individual cows, which might make it possible to reduce costs, increase milk production, and/or reduce the amount of nitrogen emission to the environment. This thesis is a part of a collaboration project between Linköping University and the Swedish University of Agricultural Sciences named “On-farm measurement of milk urea - development of a sensor”. The thesis investigated the activity and stability of the urease in an electrochemical biosensor, in which the urease is immobilised via encapsulation in a gel of poly(carbamoyl sulfonate) on a screenprinted electrode coated with a metal catalyst (copper), a cation exchanger (Nafion) and a conductive polymer (polyaniline). The linear range of the biosensor was successfully extended up to 2500 μM urea with a diffusion barrier composed of chitosan and polyvinyl butyral, enabling higher urea concentrations measurement than without the barrier (680 μM). Reproducibility, reusability, and storage stability measurements of the urease immobilised electrodes were performed and evaluated. A comparison between free enzyme, immobilised enzyme on the electrode surface and immobilised enzyme on an electrode surface that had been stored for a few days, was conducted to determine urease activity and stability. In addition, five components of milk (casein, lactose, ammonium, iron, and ascorbic acid) were measured separately to evaluate their interferences during milk urea detection. Interference was observed in several cases. A final evaluation of the present electrochemical biosensor was done by analysis of real milk samples giving promising results for future development. / On-farm measurement of milk urea - development of a sensor

Diagnostics are critical tools to assist in the identification of pathogens, the assessment of medical conditions, and helping to inform therapeutic decisions. Nevertheless, commonly used molecular diagnostics often require sophisticated instruments and skilled technicians, and therefore can only be done in centralized, well-equipped laboratories, which leads to long turnaround times, increased costs, and limited accessibility. These limitations have motivated the development of rapid, low-cost, decentralized diagnostics that are more widely accessible, affordable, and suitable for point-of-care applications. Synthetic biology, by creating rationally designed biological components that can sense disease markers, provides innovative and promising diagnostic solutions to achieve highly sensitive and specific detection for targets of interest, while at the same time being time- and cost-efficient, field-deployable, and shelf-stable. This dissertation focuses on the development of novel biosensing elements and their diagnostic applications. First, I introduce the methods for the computational design of riboregulators using automated algorithms. Followed by that, I describe the development, optimization, and applications of toehold-switch-based platforms for the detection of coccidioides, noroviruses, and severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2). Next, I introduce the development of an ultra-specific riboregulator system termed single-nucleotide specific programmable riboregulators (SNIPRs) and their use for detecting different variants of concern of SARS-CoV-2. It is shown that riboregulators can be ideal solutions for various pathogen diagnostics with comparable accuracy and reduced cost. Lastly, I describe the use of peptide reporters derived from split protein systems to detect gene mutations. By incorporating peptide reporters into amplification primers, detection can be achieved by a quick isothermal amplification step and cell-free gene expression. Together, this research brings advancements in diagnostics based on riboregulators and cell-free systems that will increase the accessibility of these essential healthcare tools.

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<p>This thesis describes a new type of biosensor, which reports the presence of a target by generating a smell that can be easily detected by the human nose. This approach is radically different from, but complementary to, colorimetric based reporting and it paves the way for the development of multi-sensory biosensors that can be used in a variety of fields, such as diagnostic device, food processing and environmental monitoring</p> <p>Biosensors typically consist of two parts: a bio-recognition element and a signal transducer. The biorecognition element is the component that can specifically interact with its cognate target, while the transducer produces a signal that can be easily identified. The key element of the smell generating biosensor is the enzyme tryptophanase (TPase), which was used as the signal transducer. This enzyme uses either L-tryptophan or S-methyl-L-cysteine as substrates, to produce either indole or methyl mercaptan as final products- both molecules are easily detectable by the human nose. Proof-of-concept for this biosensor was achieved by performing an enzyme-linked immunosorbent assay (ELISA) on magnetic beads with detection of IgG from rabbit serum (the target) in a sample and reporting the presence of the target through the generation of a smell (either indole or methyl mercaptan, depending on the substrate used).</p> <p>The potential use of TPase for biosensing was further expanded by creating a bienzyme system that allows specifically detecting of adenosine-5’-triphosphate (ATP) and reporting its presence by generating a smell. This bienzyme system is based on the fact that TPase activity is greatly affected by the concentration of pyridoxal phosphate (PLP)- which acts as a cofactor that modulates enzyme activity. The enzyme pyridoxal kinase PKase catalyzes the phosphorylation of pyridoxal to PLP in the presence of ATP. The more ATP presents, the more PLP is produced per unit time. If this occurs in the presence of TPase, larger concentrations of ATP in samples will result in higher amounts and faster rates of PLP formation, leading to increased activity of TPase, hence faster generation of either indole or methyl mercaptan is achieved. This bienzyme was used for the detection of DNA molecules with a specific sequence as well as for the detection of microbial cells through smell generation.</p> <p>Most widely used biosensors require immobilization of the biologically active elements on a stable surface. Paper, being a cheap and easy accessible substrate, was used for fabrication of the olfactory-based biosensor. Poly(N-isopropylacrylamide-co-vinylacetic acid) (PNIPAM-VAA) microgels with functional groups present on their surface were modified by biotinylation and loaded with streptavidin/avidin (to be prepared as a platform for further biomolecule immobilization). The microgels were then used as a supporter for the bienzyme system on filter paper to construct a paper-based smell-generating biosensor, which opens the way for the creation of printable smell-reporting printable bio-inks.</p> / Doctor of Philosophy (PhD)