Highly sensitive and multiplexed platforms for allergy diagnostics

Thesis (Ph.D.)--Boston UniversityAllergy is a disorder of the immune system caused by an immune response to otherwise harmless environmental allergens. Currently 20% of the US population is allergic and 90% of pediatric patients and 60% of adult patients with asthma have allergies. These percentages have increased by 18.5% in the past decade, with predicted similar trends for the future. Here we design sensitive, multiplexed platforms to detect allergen-specific IgE using the Interferometric Reflectance Imaging Sensor (IRIS) for various clinical settings. A microarray platform for allergy diagnosis allows for testing of specific IgE sensitivity to a multitude of allergens, while requiring only small volumes of patient blood sample. However, conventional fluorescent microarray technology is limited by i) the variation of probe immobilization, which hinders the ability to make quantitative, assertive, and statistically relevant conclusions necessary in immunodiagnostics and ii) the use of fluorophore labels, which is not suitable for some clinical applications due to the tendency of fluorophores to stick to blood particulates and require daily calibration methods. This calibrated fluorescence enhancement (CaFE) method integrates the low magnification modality of IRIS with enhanced fluorescence sensing in order to directly correlate immobilized probe (major allergens) density to allergen-specific IgE in patient serum. However, this platform only operates in processed serum samples, which is not ideal for point of care testing. Thus, a high magnification modality of IRIS was adapted as an alternative allergy diagnostic platform to automatically discriminate and size single nanoparticles bound to specific IgE in unprocessed, characterized human blood and serum samples. These features make IRIS an ideal candidate for clinical and diagnostic applications, such a POC testing. The high magnification (nanoparticle counting) modality in conjunction with low magnification of IRIS in a combined instrument offers four significant advantages compared to existing sensing technologies: IRIS i) corrects for any variation in probe immobilization, ii) detects proteins from attomolar to nanomolar concentrations in unprocessed biological samples, iii) unambiguously discriminates nanoparticles tags on a robust and physically large sensor area, iv) detects protein targets with conjugated nanoparticle tags (~40nm diameter), which minimally affect assay kinetics compared to conventional microparticle tagging methods, and v) utilizes components that make the instrument inexpensive, robust, and portable. This platform was successfully validated on patient serum and whole blood samples with documented allergy profiles (ImmunoCAP®, ThermoFisher Scientific)

Instrument design and optimization of interferometric reflectance imaging sensors for in vitro diagnostics

Thesis (Ph.D.)--Boston UniversityIn the field of drug discovery and disease diagnostics, protein microarrays have generated much enthusiasm for their high-throughput monitoring of biomarkers; however, this technology has yet to translate from research laboratories to commercialization. The hindrance is the considerable uncertainty and skepticism regarding data obtained. The disparity in results from different laboratories performing identical tests is attributed to a lack of assay quality control. Unlike DNA microarrays, protein microarrays have a higher level of bioreceptor immobilization variability and non-specific binding because of the more complex molecular structure and broader physiochemical properties. Traditional assay detection modalities, such as fluorescence microscopy and surface plasmon resonance, are unable to overcome both of these sources of variation. This dissertation describes the hardware and software design and biological validation of three complementary platforms that overcome bioreceptor variability and non-specific binding for diagnostics. In order to quantify the bioreceptor quality, a label-free, nondestructive, low cost, and high-throughput interferometric sensor has been developed as a quality control tool. The quality control tool was combined with a wide-field fluorescence imaging system to improve fluorescence experimental repeatability. Lastly, a novel high-throughput and label-free platform for quality control and specific protein microarray detection is described. This platform overcomes the additional complexities and time required with labeled assays by discriminating between specific and nonspecific detection by including sizing of individual binding events. Protein microarrays may one day emerge as routine clinical laboratory tests; however, it is important that the proper quality control procedures are in place to minimize erroneous results. These platforms provide reliable and repeatable protein microarray measurements for new advancements in disease diagnostics with the potential for drug discovery

ELISA in the multiplex era: potentials and pitfalls

Multiplex immunoassays confer several advantages over widely adopted singleplex immunoassays including increased efficiency at a reduced expense, greater output per sample volume ratios and higher throughput predicating more resolute, detailed diagnostics and facilitating personalised medicine. Nonetheless, to date, relatively few protein multiplex immunoassays have been validated for in vitro diagnostics in clinical/point-of-care settings. This review article will outline the challenges, which must be ameliorated prior to the widespread integration of multiplex immunoassays in clinical settings: (i) biomarker validation; (ii) standardisation of immunoassay design and quality control (calibration and quantification); (iii) availability, stability, specificity and cross-reactivity of reagents; (iv) assay automation and the use of validated algorithms for transformation of raw data into diagnostic results. A compendium of multiplex immunoassays applicable to in vitro diagnostics and a summary of the diagnostic products currently available commercially are included, along with an analysis of the relative states of development for each format (namely planar slide based, suspension and planar/microtitre plate based) with respect to the aforementioned issues

Pursuing precision in medicine and nutrition: the rise of electrochemical biosensing at the molecular level

In the era that we seek personalization in material things, it is becoming increasingly clear that the individualized management of medicine and nutrition plays a key role in life expectancy and quality of life, allowing participation to some extent in our welfare and the use of societal resources in a rationale and equitable way. The implementation of precision medicine and nutrition are highly complex challenges which depend on the development of new technologies able to meet important requirements in terms of cost, simplicity, and versatility, and to determine both individually and simultaneously, almost in real time and with the required sensitivity and reliability, molecular markers of different omics levels in biofluids extracted, secreted (either naturally or stimulated), or circulating in the body. Relying on representative and pioneering examples, this review article critically discusses recent advances driving the position of electrochemical bioplatforms as one of the winning horses for the implementation of suitable tools for advanced diagnostics, therapy, and precision nutrition. In addition to a critical overview of the state of the art, including groundbreaking applications and challenges ahead, the article concludes with a personal vision of the imminent roadmap.The financial support of PID2019-103899RBI00 (Spanish Ministerio de Ciencia e Innovación), and PMP22/00084, PI17CIII/00045, PI20CIII/00019 and PI22/00727 (AES-ISCIII) cofounded with FEDER funds Research Projects and the TRANSNANOAVANSENS-CM Program from the Comunidad de Madrid (Grant S2018/NMT-4349) are gratefully acknowledged. Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature.S

Consumer-friendly food allergen detection : moving towards smartphone-based immunoassays

In this critical review, we provide a comprehensive overview of immunochemical food allergen assays and detectors in the context of their user-friendliness, through their connection to smartphones. Smartphone-based analysis is centered around citizen science, putting analysis into the hands of the consumer. Food allergies represent a significant worldwide health concern and consumers should be able to analyze their foods, whenever and wherever they are, for allergen presence. Owing to the need for a scientific background, traditional laboratory-based detection methods are generally unsuitable for the consumer. Therefore, it is important to develop simple, safe, and rapid assays that can be linked with smartphones as detectors to improve user accessibility. Smartphones make excellent detection systems because of their cameras, embedded flash functions, portability, connectivity, and affordability. Therefore, this review has summarized traditional laboratory-based methods for food allergen detection such as enzyme-linked-immunosorbent assay, flow cytometry, and surface plasmon resonance, and the potential to modernize these methods by interfacing them with a smartphone readout system, based on the aforementioned smartphone characteristics. This is the first review focusing on smartphone-based food-allergen detection methods designed with the intention of being consumer-friendly. [Figure not available: see fulltext.

Development of Universal Biosensing Platforms Based on CRISPR/Cas12a systems

CRISPR/Cas technologies possess the promising potential to affect biosensing field by providing a sensitive, precise, rapid, versatile and cost-effective method for diverse target detections. This thesis focusses on the development of CRISP/Cas12a based biosensing platforms for nucleic acid and protein detection. Two distinct CRISPR/Cas based diagnostic methods were developed. The first developed method is a plasmonic CRISPR Cas12a assay for colorimetric detection of viral nucleic acid. This assay generates colorimetric signals for nucleic acid amplicons by combining the unique target-induced collateral cleavage activity of Cas12a with plasmon coupling of DNA functionalized gold nanoparticles. The practical applications of this assay were successfully demonstrated through the nucleic acid detection of hepatitis B virus (HBV) and Grapevine Red-Blotch Virus (GRBV). The second developed method is a universal proximity CRISPR Cas12a assay for ultrasensitive detection of nucleic acids and proteins. The target recognition is achieved through proximity binding rather than direct CRISPR/Cas 12a recognition, allows the flexible assay design and expansion to target diverse targets. This assay was successfully adapted to detect nucleic acids and antibodies in both buffer and diluted human serum

IgE allergy diagnostics and other relevant tests in allergy, a World Allergy Organization position paper

Correction: Volume14 Issue7 Article Number100557 DOI10.1016/j.waojou.2021.100557Currently, testing for immunoglobulin E (IgE) sensitization is the cornerstone of diagnostic evaluation in suspected allergic conditions. This review provides a thorough and updated critical appraisal of the most frequently used diagnostic tests, both in vivo and in vitro. It discusses skin tests, challenges, and serological and cellular in vitro tests, and provides an overview of indications, advantages and disadvantages of each in conditions such as respiratory, food, venom, drug, and occupational allergy. Skin prick testing remains the first line approach in most instances; the added value of serum specific IgE to whole allergen extracts or components, as well as the role of basophil activation tests, is evaluated. Unproven, non-validated, diagnostic tests are also discussed. Throughout the review, the reader must bear in mind the relevance of differentiating between sensitization and allergy; the latter entails not only allergic sensitization, but also clinically relevant symptoms triggered by the culprit allergen.Peer reviewe

IgE allergy diagnostics and other relevant tests in allergy, a World Allergy Organization position paper

Currently, testing for immunoglobulin E (IgE) sensitization is the cornerstone of diagnostic evaluation in suspected allergic conditions. This review provides a thorough and updated critical appraisal of the most frequently used diagnostic tests, both in vivo and in vitro. It discusses skin tests, challenges, and serological and cellular in vitro tests, and provides an overview of indications, advantages and disadvantages of each in conditions such as respiratory, food, venom, drug, and occupational allergy. Skin prick testing remains the first line approach in most instances; the added value of serum specific IgE to whole allergen extracts or components, as well as the role of basophil activation tests, is evaluated. Unproven, non-validated, diagnostic tests are also discussed. Throughout the review, the reader must bear in mind the relevance of differentiating between sensitization and allergy; the latter entails not only allergic sensitization, but also clinically relevant symptoms triggered by the culprit allergen.info:eu-repo/semantics/publishedVersio

Biogratings: Diffractive Transducers for Biosensing in Photonic Platforms

Tesis por compendio[ES] El desarrollo científico y tecnológico de las últimas décadas ha dado lugar a sistemas sensores capaces de obtener, procesar y transmitir información sobre multitud de aspectos físicos y químicos, y utilizarla para mejorar aspectos clave de multitud de áreas de nuestra sociedad. Los sensores químicos son dispositivos compactos y miniaturizados capaces de ofrecer soluciones alternativas a las técnicas de análisis instrumental convencionales. En especial, los biosensores han adquirido gran relevancia por los avances que han supuesto para sectores estratégicos como el diagnóstico clínico, la industria alimentaria y el medio ambiente. Los biosensores ópticos se basan en interacciones entre la luz y la materia para transducir eventos de bioreconocimiento y presentan prestaciones importantes como la estabilidad, inmunidad a estímulos externos y versatilidad en el desarrollo de aproximaciones sin marcaje (label-free). Este último aspecto suele aprovechar fenómenos nanoscópicos y su desarrollo se encuentra muy ligado al progreso de la nanociencia y nanotecnología. Un aspecto clave en el biosensado sin marcaje consiste en descubrir y desarrollar nuevas estrategias de transducción. En este sentido, aunque se encuentren aun en una etapa temprana de desarrollo, los biosensores difractivos presentan un gran potencial en términos de simplicidad, miniaturización, y capacidad para minimizar señales no deseadas fruto de interacciones no específicas, entre otros aspectos.[CA] El desenvolupament científic i tecnològic de les últimes dècades ha donat lloc a sistemes sensors capaços d'obtindre, processar i transmetre informació sobre multitud d'aspectes físics i químics, i utilizar-la per a millorar aspectes clau de multitud d'arees de la nostra societat. Els sensors químics són dispositius compactes i miniaturitzats capaços d'oferir solucions alternatives a les tècniques d'analisi instrumental convencionals. Especialment, els biosensors han adquirit gran rellevància pels avanços que han suposat per als sectors estratègics com el diagnòstic clínic, la industria alimentària i el medi ambient. Els biosensors òptics es basen en interaccions entre la llum i la matèria per a transduir esdeveniments de bioreconèixement i presenten prestacions importants com estabilitat, immunitat a estímuls externs i versatilitat en el desenvolupament d'aproximacions sense marcatge (label-free). Aquest últim aspecte sol aprofitat fenòmens nanoscòpics i el seu desenvolupament es troba molt lligat al progrés de la nanociència i nanotecnologia. Un aspecte clau en el biosensat sense marcatge consisteix a descobrir i desenvolupar noves estratègies de transducció. En aquest sentit, encara que es troben fins i tot en una etapa primerenca de desenvolupament, els biosensors difractius presenten un gran potencial en termes de simplicitat, miniaturització, i capacitat per a minimitzar senyals no desitjats fruit d'interaccions no específiques, entre altres aspectes.[EN] The scientific and technological progress in recent decades has given rise to sensor systems capable of obtaining, processing, and transmitting information on a multitude of physical and chemical aspects and using it to improve key aspects of many areas of our society. Chemical sensors are compact, miniaturized devices capable of offering alternative solutions to conventional instrumental analysis techniques. In particular, biosensors have become highly relevant due to the progress they have brought to strategic sectors such as clinical diagnostics, the food industry, and the environment. Optical biosensors rely on interactions between light and matter to transduce biosensing events and provide important features such as stability, immunity to external stimuli, and versatility in the development of label-free approaches. This last aspect usually exploits nanoscopic phenomena and its development in closely linked to the progress in nanoscience and nanotechnology. A key aspect of label-free biosensing is the discovery and development of new transduction strategies. In this regard, although they are at an early stage of development, diffractive biosensors offer great potential in terms of simplicity, miniaturization, and the ability to minimize unwanted signals from non-specific interactions, among other aspects.This work was financially supported by the Ministerio de Ciencia e Innovación/Agencia Estatal de Investigación (MCIN/AEI/10.13039/501100011033) co-funded by the European Union “ERDF A way of making Europe” (PID2019-110713RB-I00, TED2021-132584B-C21, PID2019-110877GB-I00), Ministerio de Economía y Competitividad (TEC2016-80385-P), Generalitat Valenciana (PROMETEO/2019/048 PROMETEO/2020/094, PROMETEO/2021/015, IDIFEDER/2021/046). A.J.D. ackowledges the FPI-UPV 2017 grant program. The authors acknowledge Instituto de Microelectrónica de Barcelona CNM-CSIC for the support in the fabrication of the measured chip samples on the Multiproject CNM-VLC silicon nitride technology platform.Juste Dolz, AM. (2023). Biogratings: Diffractive Transducers for Biosensing in Photonic Platforms [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/194251Compendi

The role of IL-33 and ST2 in allergic airways disease

Asthma is a chronic disease characterised by variable airflow obstruction, bronchial hyperresponsiveness and airways inflammation. At an immunological level Th2 inflammation and the presence of activated eosinophils and mast cells are key features of asthma. ST2, the receptor for the novel cytokine IL-33, is expressed upon Th2 lymphocytes and mast cells but its role in clinical and experimental asthma remains unclear. IL-33 has been shown to induce local and systemic eosinophilia when administered to the peritoneum of mice. In this thesis I have set out to test the hypothesis that the activation of mast cells by IL-33 acting on cell surface ST2 plays a critical role in allergic airways inflammation. I began by studying the function of ST2 on mast cells in vitro. I found that ST2 was expressed at an early stage of development, and correlated closely with the expression of the stem cell factor receptor (c-kit), a marker present on mast cells from a progenitor stage. Despite this mast cells generated form ST2 gene deleted mice proliferated and matured normally. When mast cells were activated by IL-33, acting in an ST2-dependent manner, pro-inflammatory cytokines and chemokines were released that have potential roles in asthma, specifically IL-6, IL-13, MIP-1α and MCP-1. To extend these findings I looked at the role of ST2 in allergic airways inflammation. I first optimised and validated an ovalbumin and adjuvant based ‘short’ twelve day model of murine asthma and demonstrated that ST2 gene deletion results in attenuated eosinophilic inflammation. In addition to being ST2 dependent it is possible that this adjuvant based short model is mast cell dependent, unlike longer adjuvant based models which are mast cell and ST2 independent. Therefore I went on to study an adjuvant-free model of asthma which has been demonstrated to be mast cell dependent. In this adjuvant-free model of asthma the airway inflammation was attenuated in ST2 gene deficient mice compared with wild type mice, while AHR was unaffected. There was an associated reduction in IgE production and thoracic lymph node recall Th2 cytokine responses. I then examined the effect of ST2 activation in the lungs. When IL-33 was administered directly to the airways of naïve mice it induced the features of experimental asthma. There was extensive eosinophilic inflammation within the lung tissue and airspaces. The Th2 cytokines IL-5 and IL-13, and the eosinophil chemoattractant chemokines eotaxin-1 and eotaxin-2 were detected at increased concentrations. Significant airways hyperresponsiveness was also generated. Using ST2 gene deleted mice I demonstrated that these effects were ST2 specific. Although I have shown that mast cells are activated by IL-33 in vitro, I used mast cell deficient mice to demonstrate that the eosinophilic inflammation generated by IL-33 is unaffected by the absence of mast cells. These data show that IL-33 can induce in the lungs the cardinal pathological characteristics of asthma, and that it appears to act upstream of other important mediators such as IL-13 and the eotaxins. Furthermore the IL-33 receptor ST2 is required in an adjuvant free model of asthma, which is more akin to human disease. Placing these findings in the context of recent evidence that IL-33 is released by structural cells in response to damage or injury suggests that IL-33 may play a key role in initiating the immunological features of clinical asthma. As a consequence of this position in the hierarchy of inflammation IL-33 offers a promising direct target for novel biological therapies in asthma