Switchable coatings for Biomedical applications

Bellingham, Washingto

Multiplex giant magnetoresistive biosensor microarrays identify interferon-associated autoantibodies in systemic lupus erythematosus.

High titer, class-switched autoantibodies are a hallmark of systemic lupus erythematosus (SLE). Dysregulation of the interferon (IFN) pathway is observed in individuals with active SLE, although the association of specific autoantibodies with chemokine score, a combined measurement of three IFN-regulated chemokines, is not known. To identify autoantibodies associated with chemokine score, we developed giant magnetoresistive (GMR) biosensor microarrays, which allow the parallel measurement of multiple serum antibodies to autoantigens and peptides. We used the microarrays to analyze serum samples from SLE patients and found individuals with high chemokine scores had significantly greater reactivity to 13 autoantigens than individuals with low chemokine scores. Our findings demonstrate that multiple autoantibodies, including antibodies to U1-70K and modified histone H2B tails, are associated with IFN dysregulation in SLE. Further, they show the microarrays are capable of identifying autoantibodies associated with relevant clinical manifestations of SLE, with potential for use as biomarkers in clinical practice

Biodétection de Legionella pneumophila par biocapteur à photocorrosion digitale à base de peptide antimicrobien

La détection de bactéries pathogènes par culture microbienne est lente, nécessite un milieu de culture spécifique pour garantir la croissance de certaines souches bactériennes fastidieuses telle que Legionella pneumophila (L. pneumophila) et en plus pourrait ne pas déceler les bactéries viables mais non cultivables mais restant dangereuse en termes de pathogénicité. Par conséquent, l’usage de biocapteurs pour la détection de L. pneumophila serait, potentiellement, une approche attrayante permettant une détection précise et rapide. Cependant, la sensibilité et la spécificité des biocapteurs dépendent fortement des molécules de bioreconnaissance utilisées. Jusqu'à présent, différents ligands tels que les anticorps, les enzymes, les acides nucléiques fonctionnels (aptamères) et les bactériophages ont été utilisés comme éléments de bioreconnaissance. En raison de leur haute spécificité, Les anticorps de mammifères ont été largement employés pour le développement de divers biocapteurs. Cependant, les anticorps sont connus pour souffrir de la variabilité des lots produits et d'une stabilité limitée, ce qui réduit l'usage et la constance des performances des biocapteurs à base d'anticorps. Au cours des dernières années, les peptides antimicrobiens (PAM) ont été de plus en plus investigués pour des applications thérapeutiques en plus d’être considérés comme des ligands de bioreconnaissance prometteurs en raison de leur grande stabilité et leurs fortes réactivités aux bactéries. Dans le but d’améliorer les performances du biocapteur à DIP, notre hypothèse reposait sur l’usage de bioarchitectures à base de PAM à courte séquence pour une capture efficace des bactéries et une détection considérablement améliorée en raison du transfert de charge plus facilitée vers dans la biopuce à base de semiconducteur III-V. Dans la première phase du projet, nous avons évalué un biocapteur à DIP consistant en une puce d’arséniure de gallium/arséniure de gallium aluminium (GaAs/AlGaAs) fonctionnalisée par le warnericine RK pour la détection directe in situ de L. pneumophila dans l’eau. Nous avons démontré une détection linéaire de L. pneumophila pour des concentrations allant de 103 à 106 CFU/mL. De plus, le nombre relativement important d'interfaces constituant la bioarchitecture d’un tel biocapteur pourrait affecter sa reproductibilité et sa sensibilité. Dans ce cas, la couche de bioreconnaissance est plus mince (~ 2 nm) permettant une distance plus courte entre les bactéries et la surface du biocapteur, ce qui pourrait jouer un rôle important dans la promotion du transfert de charge entre les bactéries et la biopuce, et ainsi nous avons pu démontrer une détection efficace de L. pneumophila à une concentration de 2 x 102 CFU/mL. Cette configuration a permis d’atteindre des LODs de 50 et 100 UFC/mL, respectivement pour de légionnelle dans du PBS et collectées d’échantillons d’eau de tour de refroidissement. Nous avons observé une détection sélective de L. pneumophila sérogroupe 1 (SG1) comparé au sérogroupe 5 (SG 5). Les biocapteurs à photocorrosion digitale (DIP) en configuration sandwich PAM et Ab pourraient être une approche prometteuse pour développer un biocapteur à faible coût, hautement sensible et spécifique pour la détection rapide de L. pneumophila dans l’eau.Abstract: Culture based detection of pathogenic bacteria is time consuming, and needs specific culture medium to identify bacterial strains such as Legionella pneumophila (L. pneumophila) which does not flourish in typical growth medium. Culture based methods cannot detect viable but unculturable bacteria. Therefore, the detection of L. pneumophila with biosensors potentially could be an attractive approach enabling accurate and rapid detection. The sensitivity and specificity of biosensors depend critically on the biorecognition probes employed for the detection. Until now, different elements such as antibodies, enzymes, functional nucleic acids (aptamers) and bacteriophages have been utilized as biorecognition elements. Due to high specificity of antibodies, and the advanced technology of their production, mammalian antibodies have been widely investigated for the development of various biosensors. However, mammalian antibodies are known to suffer from batch-to-batch variation, as well as limited stability, which could reduce the consistent utility of the proposed biosensors. In recent years, antimicrobial peptides (AMPs) have been increasingly investigated for their therapeutic applications. At the same time, AMPs are considered as promising biorecognition ligands due to their high stability and multiple niches for capturing bacteria. The hypothesis was that AMP-based bioarchitectures allows for highly efficient capturing of bacteria, and the short length of the AMP would significantly enhance detection due to limited obstructive charge transfer in the charge sensing biosensor. In the first phase of the project, we investigated a warnericin RK AMP functionalized gallium arsenide/aluminum gallium arsenide (GaAs/AlGaAs) photonic biosensor for direct detection of L. pneumophila in water environments. This approach allowed for detecting a low to high concentration of L. pneumophila (103 to 106 CFU/mL) with a 103 CFU/mL limit of detection (LOD). In addition, a relatively large number of interfaces constituting the architecture of such biosensors could affect their reproducibility and sensitivity. A thinner biorecognition layer (~2 nm) resulted in a shorter distance between bacteria and the biosensor surface, which played important role in promoting charge transfer between bacteria and biochip. L. pneumophila was detected at concentrations as low of 2 x 102 CFU/mL. This configuration allowed the detection sensitivity of L. pneumophila as low as 50 CFU/mL and 100 CFU/mL in clean water and water originated from cooling tower, respectively, along with the selective detection of whole cell L. pneumophila serogroup 1 (SG1) and serogroup 5 (SG5). The proposed AMP and Ab conjugated sandwich architecture with digital photocorrosion (DIP) biosensors is a promising approach for developing low cost, highly sensitive and specific biosensors for rapid detection of L. pneumophila in water environments

A unified platform for experimental and computational biology

PhD ThesisIn natural sciences, the correct engineering of a system’s chemical, biological and physical properties may allow it to sustain life. Bioengineering cells is probably one of the most complex challenges of biological research; yet, the little we do know about the nature of life is sufficient to guide scientific research, and to explore the elements beyond the apparent simple proliferation of living cells. Although Mendel first characterised the concept of genetic heredity over 150 years ago, we only recently became able to perform tailored genetic modification of living organisms. The development of digital technologies, in particular, has positively influenced the quality and reproducibility of experimental results emerging from biological assays. However, the use of any equipment may require the need for a specific expertise in order to perform a given experimental procedure. Therefore, multidisciplinary research can bring benefits to all fields of science by helping the development of analytical methods that cross the boundaries of individual disciplines. This emerges as a systematic view of scientific problems, and relies on the adequation and integration of results from different research areas. Nevertheless, there is a complex interface between hard sciences that often creates a gap between experimental and theoretical models. In this thesis, we explored synthetic biology approaches and created a unified platform to fill this gap. We propose the first barcoding platform (Bac2code) that allows the identification and the tracking of bacterial strains. In order to facilitate communication between researchers, we developed a barcode system in DNA that physically links bacteria to their genetic description. We designed DNA barcodes as bioorthogonal elements, elaborated a universal cloning strategy to integrate these sequences in Gram-negative and Gram-positive bacteria, and demonstrated their stability over time. Through a generic protocol, any barcoded strain can later be identified via a single sequencing read. With the engineering of a synthetic circuit library, we built a biorepository of genetic constructs for our barcoding platform. These biological devices were optimised based on the closest achievable interface between experimental biology and viii computational results. Following their characterisation, and in the context of intercellular communication, we studied the behaviour of small cohorts of bioengineered cells at the microscale in microfluidics. We pushed the biological and physical boundaries of engineering techniques to the maximum, in order to observe physiological changes between bacteria separated by distances down to 20µm. However, we also showed that we reached a technological barrier, where even the use of nanoscale features was found insufficient to maintain cells isolated under high cellular density. Yet, microfluidics remains a remarkable technology, and we propose the expansion of barcoding methods to automated systems, which would allow serial barcode integration and documentation retrieval at any one time. Here, we developed and tested a barcoding method to ensure the cohesion of experimental and computational biology resources. We demonstrated its use by the in vitro assembly and the in vivo or in silico characterisation of a series of genetic circuits via different techniques. The research output of this thesis is realised as a step forward in interdisciplinary studies, and is now being adapted to reach a larger community of users as a startup companyEngineering and Physical Sciences Research Council and Newcastle University’s School of Computing Science

Optimisation of the isolation and identification of circulating melanoma cells

Although melanoma is largely curable when detected in its earliest stages, it can metastasise to other tissues, drastically reducing survival rates. The most recent therapies used to treat metastatic melanoma are effective long-term in only 11 to 33% of patients. Our ability to monitor treatment failure is limited. New prognostic markers are urgently required to allow monitoring of treatment response and disease progression. Circulating tumour cells (CTCs) are released into the bloodstream by the tumours within a patient, this being a key step in melanoma spread. Since CTCs can be detected in the blood of metastatic melanoma patients, these cells can be used as a “liquid biopsy”, providing critical insight into each person’s melanoma biology. Melanoma CTCs have been described as very heterogeneous, hindering their isolation via commonly used CTC capturing methods. To address this, microfluidic devices have been developed to isolate viable CTCs from blood, independently of their marker expression. This study aimed to determine the effectiveness of two different microfluidic devices (Slanted and A5) in recovering melanoma cell lines, and their potential use in the isolation of CTCs from the blood of metastatic melanoma patients. It also aimed to study additional cancer or melanoma specific markers to be used in immunostaining protocols for detection of CTCs after microfluidic enrichment. The optimal isolation procedure was identified as two rounds of enrichment with the Slanted spiral device, after which we obtained a 3-log depletion of white blood cells and a recovery of over 60% when cells from two melanoma cell lines were spiked into blood samples from healthy volunteers. In addition, we optimised the detection of CTCs using four melanoma markers (gp100, Melan-A, s100 and MCSP) combined in a multimarker immunocytochemistry staining protocol. The optimised enrichment and detection procedures were validated in a cohort of ten metastatic melanoma patients. Results showed that 40% of the patients had one or more CTCs in their blood (1-4 CTCs/8 mL of blood). Furthermore, three additional markers (Vimentin, RANK, and ABCB5) were trialled so as to increase detection of highly heterogeneous melanoma CTCs in samples that have been processed through the Slanted microfluidic device. The improved enrichment and detection of CTCs in the blood of melanoma patients using the methods developed as part of this study will facilitate the molecular, genomic and functional characterisation of melanoma CTCs. This will ultimately improve our understanding of the biology of melanoma CTCs and their role in metastatic spread and treatment response

Loop mediated isothermal amplification based detection of equine respiratory pathogens using a portable, smartphone-based setup

New tools are needed to enable rapid detection, identification, and reporting of infectious viral and microbial pathogens in a wide variety of point-of-care applications that impact human and animal health. We report the design, construction, and characterization of a multiplexed platform for multiplexed analysis of disease-specific DNA sequences that utilizes a smartphone camera as the sensor in conjunction with a handheld “cradle” that interfaces the phone with a silicon-based microfluidic chip embedded within a credit card-sized cartridge. Utilizing specific nucleic acid sequences for four equine respiratory pathogens as representative examples, we demonstrate the ability of the system to utilize a single 15 µL droplet of test sample to perform selective positive/negative determination of target sequences, including integrated experimental controls, in approximately 30 minutes. Our approach utilizes loop mediated isothermal amplification (LAMP) reagents pre-deposited into distinct lanes of the microfluidic chip, which, when exposed to target nucleic acid sequences from the test sample, generates fluorescent products that, when excited by appropriately selected light emitting diodes (LEDs) are visualized and automatically analyzed by a software application running on the smartphone microprocessor. The system achieves detection limits comparable to those obtained by laboratory-based methods and instruments. Assay information is combined with information from the cartridge and the patient to populate a cloud-based database for epidemiological reporting of test results