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)

<p>Research in paper-based analytical devices has been increasing in recent years. Before technology transfer and market acceptance, these paper-based sensors have to be validated with field samples. In this study, we have made an attempt to evaluate the effectiveness of paper-based sensors to detect pesticides in real world samples. Generation 1 biosensor has been modified to be user friendly. There is no difference in the performance of generation 2 sensors; they detect pesticides based on colorimetric assay. The assay protocol involves first introducing the sample to the sensing zone by pipetting the water sample. Following an incubation period of 15min, the substrate end of the sensor is dipped into the sample to move paper bound indoxyl acetate (IDA) to the sensing region to initiate the enzyme-catalyzed hydrolysis of the substrate, resulting in the development of blue color. The presence of pesticide is indicated by either a decrease in color intensity or with no color development at all.</p> <p>To evaluate the effectiveness of biosensor in detecting pesticides in real world samples, a field study was conducted in four villages of southern India. Water samples from different aquatic environment including both surface water and ground water, were tested using generation 2 paper-based sensors. The paper-based sensors were capable of detecting organophosphorus pesticides in real world samples. The results were confirmed using GC-MS.</p> <p>The presence of higher concentration of dibutyl phthalate (in the range of 100uM to 10mM) in water can be a potential interference for the paper-based assay for the detection of pesticides in water. The paper-based biosensor assay platform can detect pesticides in the environmental samples and results have been validated by GC-MS. But for transfer of technology to the industry, further optimization is required to improve the stability of substrate to withstand atmospheric temperature fluctuations thus allowing the storage and shipment of the biosensor strips. Additionally to conduct reliable assays and obtain consistent results, the fabrication of biosensor strips needs to be improved to maintain the consistent volumes of bioinks impregnated on paper support.</p> / Master of Applied Science (MASc)

Lateral flow biosensors are analytical devices that detect biomaterials with physicochemical signals, such as optical signals. Unlike other biosensors, lateral flow biosensors are based on porous membranes, which use capillary force to transport biomaterials spontaneously. However, lateral flow biosensors are fabricated in batch mode, which means that membranes need to be cut from the rolls, pretreated, and assembled using a step-by-step process. Thus, there is a need for a more efficient manufacturing process. This thesis aims to accelerate the fabrication process by developing a method wherein the whole device is printed directly, including the printable substrates, as well as by developing a clog-free process for depositing expensive reagents. These novel printable porous media were developed using printing inks that contained various pigments and polymer binders. To this end, candidate formulations were screened from nine hundred inks formulations via wicking experiments. The results of these tests showed that the most promising formulations were based on calcium carbonates and latex polymers. This formulation was then used to develop printable porous media that can easily be printed into complex patterns, with changeable wicking speeds within each pattern. In addition, a bio- colorimetric assay of alkaline phosphates conducted on these porous media showed strong color signals that were comparable to the traditional membrane-based lateral flow strips. Clog-free printing processes were investigated by using a piezoelectric inkjet printer to print silica sols and six nanoparticle inks. The results of these tests showed that the vibration of the piezoelectric layer and the deposition of particles on the printhead surfaces induced clogging issues. Over time, the silica sols formed multilayer deposits on the print head surface, which subsequently detached due to the vibration of the piezoelectric layer. Consequently, these large sheets of silica clogged the nozzles during printing. This clogging issue was eliminated by adjusting the pH value of the silica sol inks to 3.1. The hydrophobic cationic polystyrene nanoparticles form a sub-monolayer on the printhead surface, which causes air entrainment and promotes air bubble adhesion into the interior of the print head surface when the piezoelectric layer deforms. Thus, alternate surface chemistries for the print head and ink particle surfaces may be required in order to print hydrophobic ink materials. Overall, this enhanced understanding of these clogging mechanisms helps to explain why printer performance varies when different particles are used. / Thesis / Doctor of Philosophy (PhD) / Many devices in our day-to-day lives incorporate lateral flow biosensors, for example, home pregnancy test kits. These tests allow users to obtain results within 30 minutes by simply applying a few droplets of urine onto a test strip. However, these biosensors are largely manufactured using manual processes: workers cut strips (also called substrates) from sheets, deposit reagents onto the strips, and then assemble the pretreated strips into devices. As such, these processes are time consuming and less productive. To accelerate the manufacturing process, we developed printable porous substrates and a clog-free printing process for depositing expensive reagents onto the substrates. Novel porous media can be flexibly printed into complex patterns using pigment- based inks. Moreover, the use of different pigments within the designed patterns enables these porous media to control wicking velocity. In addition to printable porous substrates, the research in this thesis shows that the manufacturing process can be improved by using piezoelectric inkjet printers. The use of these printers not only allows the expensive reagents to be precisely deposited onto the substrates, but it also offers a more cost-effective method of doing so. Finally, in order to ensure the printing process remained clog-free, we systematically investigated clogging mechanisms by printing with different polymers and nanoparticles.