Proof of Concept for a Urine-Based Method for Monitoring Blood Phenylalanine for Patients with PKU

There is a great need for an at-home, simple, inexpensive, and noninvasive testing method to monitor the blood phenylalanine (Phe) levels of patients with PKU. PKU is a rare genetic disorder that affects approximately one out 15,000 people, which results in dangerously high levels of Phe in the blood. This project aims to show proof of concept for such a test, which is based upon a previously developed test method that was historically used to diagnose PKU from a urine sample. This redesigned test method represents the optimization of a little-understood reaction between Fe3+ ions and phenylpyruvic acid (PPA) that results in a color change. PPA is a metabolic byproduct of elevated blood Phe levels, thus enabling it to be used as an indicator of this genetic disease. The improved test increases sensitivity to the presence of PPA by two orders of magnitude over the previous method by including custom test substrates, controlled lighting conditions, and smart phone technology. The process by which the test substrates were developed is presented, as well as their performance for the quantification of PPA concentration in a urine sample. The additional accuracy of the refined method is primarily provided by the implementation of a custom-made device to control light conditions combined with the measurement of color using a smart phone to photograph test samples combined with an RGB color application for color quantification. The final test proposed is inexpensive, safe, and simple enough for a lay person to do at home. Further optimization and calibration to individuals with PKU will be needed for the test to be commercially viable

3D Printing of Dietary Products for the Management of Inborn Errors of Intermediary Metabolism in Pediatric Populations

© 2023 by the authors. Licensee MDPI, Basel, Switzerland. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY), https://creativecommons.org/licenses/by/4.0/The incidence of Inborn Error of Intermediary Metabolism (IEiM) diseases may be low, yet collectively, they impact approximately 6–10% of the global population, primarily affecting children. Precise treatment doses and strict adherence to prescribed diet and pharmacological treatment regimens are imperative to avert metabolic disturbances in patients. However, the existing dietary and pharmacological products suffer from poor palatability, posing challenges to patient adherence. Furthermore, frequent dose adjustments contingent on age and drug blood levels further complicate treatment. Semi-solid extrusion (SSE) 3D printing technology is currently under assessment as a pioneering method for crafting customized chewable dosage forms, surmounting the primary limitations prevalent in present therapies. This method offers a spectrum of advantages, including the flexibility to tailor patient-specific doses, excipients, and organoleptic properties. These elements are pivotal in ensuring the treatment’s efficacy, safety, and adherence. This comprehensive review presents the current landscape of available dietary products, diagnostic methods, therapeutic monitoring, and the latest advancements in SSE technology. It highlights the rationale underpinning their adoption while addressing regulatory aspects imperative for their seamless integration into clinical practice.Peer reviewe

Biochemical Testing

Clinical Correlation and Diagnosis highlights the improvements in methodological approaches for the purposes of disease diagnosis and health research. Chapters cover such topics as serum protein electrophoresis, urinary iodine measurement, blood collection tubes, semi-solid phase assay and advancement in analytical and bioanalytical techniques, and serological diagnostic tools for Zika virus, among other subjects. All these will not be possible without a proper laboratory management where this book also includes the Tissue Bank ATMP Production as a model. The chapters are expected to provide a new perspective in health science which may trigger a further exploration into the diagnostic and research field

Advances in Electronic-Nose Technologies Developed for Biomedical Applications

The research and development of new electronic-nose applications in the biomedical field has accelerated at a phenomenal rate over the past 25 years. Many innovative e-nose technologies have provided solutions and applications to a wide variety of complex biomedical and healthcare problems. The purposes of this review are to present a comprehensive analysis of past and recent biomedical research findings and developments of electronic-nose sensor technologies, and to identify current and future potential e-nose applications that will continue to advance the effectiveness and efficiency of biomedical treatments and healthcare services for many years. An abundance of electronic-nose applications has been developed for a variety of healthcare sectors including diagnostics, immunology, pathology, patient recovery, pharmacology, physical therapy, physiology, preventative medicine, remote healthcare, and wound and graft healing. Specific biomedical e-nose applications range from uses in biochemical testing, blood-compatibility evaluations, disease diagnoses, and drug delivery to monitoring of metabolic levels, organ dysfunctions, and patient conditions through telemedicine. This paper summarizes the major electronic-nose technologies developed for healthcare and biomedical applications since the late 1980s when electronic aroma detection technologies were first recognized to be potentially useful in providing effective solutions to problems in the healthcare industry

The development of smart-bandage technologies

Healthcare associated infections of wound sites are a complex problem with substantial effects on patient morbidity and financial ramifications to healthcare bodies. The increasing interest in novel diagnostic strategies and preventing infections have led to an incursion of research into the topic. Whilst most emphasis has been placed on preventing wound infections, the bacterial flora is an ever present risk to the compromised host. In contrast with the majority of research developing antibacterial smart-dressings, the research detailed within describes the development of in-situ electrochemical sensor assemblies suitable for incorporation within traditional or ‘smart’ wound dressings. Sensor developments have led to prototype construction of a multitude of sensing substrates capable of quantitative analyses for the identification of infection. The key developments contained within highlight both generic and organism-specific sensors which can reliably monitor key chemical components of a wound exudate to allow sampling-free infection diagnostics

Functionalization of Nanoparticles with Tyrosine Hydroxylase : Biotechnological and Therapeutic Implications

Tyrosine hydroxylase (TH) er et viktig enzym for nervesystemet, fordi det katalyserer det første steget i syntesen av dopamin, noradrenalin og adrenalin. Nivået på TH og dopamin synker hos Parkinsons pasienter pga. den gradvise celledøden i den hjernedelen som heter substantia nigra. Vanlig behandling går ut på å ta levodopa som er forløperen til dopamin, men effekten avtar etter hvert, og pasientene får alvorlige bivirkninger ved høye doser. Det trengs altså et bedre behandlingstilbud. En mulighet kan være å tilføre mer TH vha. en enzymerstatningsterapi som også vil gjenopprette dopaminnivået. Hovedformålet med denne avhandlingen er å finne ut hvordan TH kan bli brukt som biologisk medisin. Vi har derfor utviklet forskjellige nanopartikkel-baserte formuleringer som kan stabilisere og levere TH, og evaluert det terapeutiske potensiale til TH-lastede nanopartikler. Vi begynte med å produsere TH sammen med forskjellige fusjonspartnere og fikk et stabilt enzym som vi målte strukturen av. Så valgte vi porøst silisium og maltodekstrin nanopartikler som mulige bærere av TH. De første forsøkene med porøst silisium viste at det er en sammenheng mellom fotoluminescensen og frigjøringen av et modellprotein som kan være nyttig i sporing av legemiddelleveringen i kroppen. Videre fant vi at TH kunne bindes i nanopartikler av porøst silisium, men at dette førte til aggregering av TH. Maltodekstrin-nanopartikler kunne derimot absorbere store mengder TH samtidig som de forhindret eller forsinket TH aggregeringen. Vi observerte at disse nanopartiklene kunne levere TH til nerveceller og hjernevev og dermed økte den intracellulære TH aktiviteten. Alt i alt har denne avhandlingen gitt et godt innblikk i de strukturelle mekanismene og de funksjonelle forutsetningene som trengs for å kunne lage vellykkede nanopartikkel-baserte formuleringer av TH. TH-lastede nanopartikler har muligheten til å bli videreutviklet til enzymerstatningsterapi for sykdommer hvor det er for lite aktivt TH, som f.eks. ved Parkinsons sykdom.Tyrosine hydroxylase (TH) is important for neuronal function as it is the rate-limiting enzyme in the synthesis of dopamine, noradrenaline, and adrenaline. In Parkinson’s disease, the levels of TH and dopamine decrease, due to progressive loss of the dopaminergic neurons in a part of the midbrain called substantia nigra. Treatment is typically with the dopamine precursor, levodopa, but its pharmacological effect wears off, and the patients develop serious side effects, so there is a need for better treatment options. One alternative could be to replace the lacking TH with an enzyme replacement therapy and thereby restore the dopamine levels. The main goal of this thesis has been to investigate how TH can be pharmacologically developed into a potential biological drug. We have therefore studied different nanoparticle (NP)-based formulations to stabilize and deliver TH and evaluated the therapeutic potential of TH-loaded NPs. We started out by using fusion tags in the preparation of TH to obtain a stable enzyme of which we determined the full-length solution structure. Then we selected porous silicon and maltodextrin NPs as potential carriers of TH. Initial characterization revealed that the photoluminescent properties of porous silicon can be tuned to correlate with the release of a model protein, which can be useful in tracking of drug delivery. Furthermore, we found that TH loading in porous silicon NPs occurred through electrostatic interactions, but that it also induced TH aggregation. On the other hand, maltodextrin NPs absorbed large amounts of TH while preventing or delaying its aggregation. We observed functional delivery of TH by these NPs to neuronal cells and tissue, which significantly increased the intracellular TH activity. All in all, this thesis has given insights into the structural mechanisms and functional prerequisites necessary for successful formulations of TH with NPs, which shows the therapeutic potential of enzyme replacement therapy with TH-loaded NPs.Doktorgradsavhandlin

New insights into the structure and function of human phenylalanine hydroxylase : interallelic complementation, protein misfolding and mechanism of catalytic activation

Tese de doutoramento, Farmácia (Bioquímica), Universidade de Lisboa, Faculdade de Farmácia, 2011Phenylketonuria (PKU) is an autosomal recessive human inborn error of metabolism caused by dysfunction of the liver homotetrameric/homodimeric enzyme phenylalanine hydroxylase (hPAH), which results in increased levels of L-phenylalanine (L-Phe) in the blood and if untreated causes severe mental retardation. More than 550 mutations have been identified to date, with three quarters of PKU patients being compound heterozygous, leading to a high phenotypic diversity. Inconsistencies in some genotype-phenotype correlations with more severe phenotypes than the ones expected and lack of response to cofactor ((6R)-L-erythro-5,6,7,8- tetrahydrobiopterin (BH4)) supplementation in patients with two known responsive mutations have raised the possibility of interallelic complementation (IC). We have shown that mutations involved in inconsistencies (I65T, R261Q, R270K and V338M) display a negative IC, with reduced enzymatic activity when mimicking the heteroallelic state of compound heterozygous PKU patients. Moreover, using wild-type and truncated mutants forms we were able to isolate an heterotetrameric hPAH form, WT/ΔN102-hPAH, that resulted from the assembly of two homodimers, since heterodimers did not form. This hybrid species revealed kinetic and regulatory properties that were influenced by interactions between the two homodimers within the heterotetramer. Phenylketonuria is considered a protein misfolding disease with loss of function, as a large number of point mutations result in decreased stability, aggregation and accelerated protein degradation. The deleterious effects seem to particularly affect the R-domain of hPAH that contains an ACT module found in a large number of multimeric proteins with complex allosteric regulation. Here we have studied the effect of the missense mutation G46S in the R-domain that promotes self-association and fibril formation in vitro. The mutation seems to extend α-helix 1 and thus perturbs the α-β sandwich of the ACT module in the R-domain. We were able to modulate in vitro the mutant self-association process by chemical, pharmacological and molecular chaperones. The mutant G46S dimer also self-associates forming fibrils and an enhancement in the self-association was observed in the presence of WT dimer, due to hybrid formation (WT/G46S). However, only amorphous aggregates were observed and L-Phe binding precludes the self-association of the WT dimer, since the wild-type and mutant tetramer dimer equilibrium responds differently to the presence of L-Phe, thus explaining the phenotype of WT/G46S carriers, as none of the PKU heterozygous shows any clinical phenotype. The R-domain of full-length hPAH is particularly unstable and when isolated it self-associates even in its WT form. This self-association was inhibited stereospecifically by L-Phe, pointing to a regulatory binding site that has been disputed over the years. It seems now evident that the isolated R-domain is able to bind L-Phe as we and other groups have observed, but the full-length enzyme has lost this ability with the simultaneously acquiring of complex regulatory mechanisms. One of these regulatory mechanisms comprises the catalytic activation by the substrate. In the resting state hPAH is inhibited by the cofactor BH4. Binding of L-Phe triggers a series of conformational changes that resulted in the activation of the enzyme. Crystal structures of hPAH with bound cofactor and substrate have revealed a large displacement of Tyr138 in a highly flexible loop, from a surface position to a partial buried position at the active site upon substrate binding. Here we have found an important functional role for Tyr138 displacement in positioning substrates for catalysis and in the L-Phe triggered conformational isomerization that results in catalytic activation of the enzyme.A fenilcetonúria (PKU; OMIM 261600) é uma doença autossómica recessiva do metabolismo, causada por uma deficiente actividade da enzima homotetramérica/homodimérica fenilalanina hidroxilase (hPAH; EC 1.14.16.1). A enzima existe maioritariamente nos hepatócitos, onde é responsável pela hidroxilação da L-fenilalanina (L-Phe) em L-tirosina (L-Tyr) na presença do cofactor (6R)-L-eritro-5,6,7,8-tetrahidrobiopterina (BH4) e oxigénio. Nesta reacção ocorre simultaneamente a hidroxilação do cofactor, que posteriormente é regenerado pela acção da enzima dihidrobiopterina reductase. A fenilalanina hidroxilase é responsável pela homeostase da L-Phe, de forma a impedir o aumento dos níveis deste aminoácido, que são tóxicos para o cérebro, e simultaneamente evitar a sua completa depleção, comprometendo a síntese proteica. Deste modo, a enzima sofre uma regulação complexa, que envolve a activação catalítica pelo substrato L-Phe, com cooperatividade positiva e fosforilação do resíduo Ser16. A enzima é também regulada pelo cofactor BH4, que provoca a sua inibição e estabilização num estado inactivo, na ausência de L-Phe. Mais de 550 mutações foram identificadas no gene PAH humano, resultando em proteínas mutantes com baixa actividade e/ou estabilidade e consequente aparecimento da doença. Esta é caracterizada pelo aumento dos níveis de fenilalanina plasmática (hiperfenilalaninémia; HPA) e pela diminuição do teor de tirosina circulante. Na ausência de tratamento, a PKU resulta em atraso mental profundo. A terapêutica actual consiste na detecção da doença através do rastreio neonatal e na implementação imediata de uma dieta pobre em L-Phe, com controlo da tolerância ao aminoácido, a qual tem de ser mantida para a vida. Recentemente verificou-se que algumas mutações respondem à administração oral do cofactor BH4 (suplementação com BH4), especialmente as formas menos graves da doença, permitindo o aumento da actividade enzimática e consequente relaxamento da dieta. Este encontra-se já comercialmente disponível na forma sintética do isómero 6R do BH4 (cloreto de sapropterina, Kuvan). Devido à heterogeneidade da doença, mais de 75% dos doentes são heterozigóticos compostos (portadores de duas diferentes mutações). Em alguns destes doentes foram verificadas inconsistências tanto ao nível da correlação genotipo-fenótipo do récem-nascido HPA, quer ao nível da resposta à suplementação com BH4. A correlação genótipo/fenótipo bioquímico tem sido efectuada com base na actividade residual prevista (PRA), calculada pela média das actividades determinadas in vitro de cada uma das proteínas mutantes associadas ao genótipo do doente. Em alguns doentes heterozigóticos compostos foram descritos fenótipos mais severos do que os previstos pela determinação da PRA. Particularmente, correlações genótipo-fenótipo em doentes HPA Portugueses envolvendo as mutações I65T, R261Q, R270K e V338M revelaram inconsistências. No que diz respeito à suplementação com BH4, alguns heterozigóticos compostos não revelaram uma resposta positiva ao tratamento, embora as duas mutações presentes no seu genótipo se encontrem descritas como respondendo ao BH4 em doentes homozigóticos para as mesmas. Curiosamente, algumas dessas mutações estão relacionadas com as inconsistências detectadas ao nível de correlações genótipo-fenótipo (e.g. I65T e R261Q). De forma a explicar estas observações clínicas foi proposta a existência de fenómenos de complementação interalélica, a qual terá como base interacções entre as diferentes subunidades mutantes da enzima multimérica, originando uma proteína com menor actividade/estabilidade do que a prevista pela PRA (neste caso uma complementação inter-alélica negativa). De forma a estudar este fenómeno desenvolvemos sistemas de expressão procariótica de forma a produzir e isolar os possíveis híbridos. Utilizando um sistema de expressão dupla foi possível reproduzir bioquimicamente a situação de um heterozigótico composto (tetrâmeros, heterotetrâmeros e dímeros) com as combinações das mutações descritas anteriormente. Todas estas mutações revelaram uma diminuição da actividade enzimática quando comparada com a prevista pela PRA, demonstrando a existência de fenómenos de complementação inter-alélica negativa. Com a utilização da forma selvagem (WT) e mutantes de deleção num sistema bicistrónico foi possível isolar pela primeira vez um híbrido da hPAH, neste caso um heterotetrâmero WT/ΔN102-hPAH. Nestes estudos, a existência de heterodímeros foi excluída, explicando o facto da hPAH selvagem ser isolada como tetrâmeros e dímeros, mas nunca como monómeros. O heterotetrâmero isolado apresenta propriedades cinéticas e reguladoras que demonstram a existência de interacções entre os dois dímeros na proteína híbrida. Estes resultados foram corroborados pela formação de heterotetrâmeros a partir de homodímeros préformados da forma selvagem e de um mutante com uma elevada propensão para agregar (WT/G46S). A presença do mutante leva à agregação da proteína WT, processo este dependente da presença de L-Phe, pois o equilíbrio entre as diferentes formas oligoméricas responde de forma diferente à presença do substrato. A vasta maioria das mutações que afectam a proteína hPAH levam a uma diminuição da eficiência do folding proteico e/ou na redução da estabilidade da enzima, resultando na sua agregação e/ou rápida degradação. O domínio regulador da enzima revela uma reduzida estabilidade e o seu envolvimento no processo de misfolding tem sido evidenviado nos últimos anos. O estudo da mutação G46S, localizada no domínio regulador e que resulta na forma mais grave da doença, permitiu avaliar o processo de misfolding de um mutante envolvendo este domínio. Em estudos in vitro, a proteína mutante agrega, levando à formação de fibrilas. Este processo foi inibido parcialmente pela utilização de glicerol (chaperone químico), 3-amino-2- benzil-7-nitro-4-(2-quinolil)-1,2-dihidroisoquinolina-1-ona (um chaperone farmacológico recentemente proposto) e pelos chaperones moleculares Hsp70/Hsp40 e Hsp90. O domínio regulador da hPAH apresenta um módulo ACT, que ocorre em várias enzimas multiméricas com complexos modos de regulação. Os módulos ACT formam normalmente dímeros e estão envolvidos na ligação de aminoácidos nas interfaces entre as duas subunidades. A instabilidade do domínio regulador da hPAH, onde não é possível existir a formação de dímeros entre os módulos ACT, foi também analisada pelo estudo deste na sua forma isolada. Curiosamente, tanto o domínio regulador mutante como o selvagem revelam tendência para agregar. No entanto, a agregação é inibida estereo-especificamente na forma selvagem pela fenilalanina, indicando que o domínio regulador na forma isolada consegue ligar o substrato, embora na enzima full-length, essa capacidade tenha sido perdida. A ligação do substrato L-Phe ao sítio activo da enzima resulta num processo reversível de isomerização que ocorre numa escala de segundos a minutos. Este processo converte a enzima de um estado de baixa actividade/afinidade para um estado de alta actividade/afinidade, resultando na activação da enzima e na existência de cooperatividade positiva. Dado a inexistência da estrutura cristalográfica da enzima full-length, não foi possível até ao momento estabelecer o circuito de alterações conformacionais que levam à activação da enzima. No entanto, a existência de estruturas proteicas de formas truncadas do domínio catalítico da enzima em complexos binários (hPAH·(Fe(II)-cofactor) e ternários (hPAH·(Fe(II)-cofactor-substrato) permitiram verificar a existência de alterações conformacionais ao nível do domínio catalítico. Uma dessas alterações envolve o reposicionamento de um loop flexível da superfície da enzima para uma posição mais hidrofóbica no sítio activo da proteína. A maior alteração envolve o resíduo Tyr138, cuja cadeia lateral sofre uma deslocação de ∼21 Å. De forma a compreender a função desempenhada por este loop e em particular o papel desempenhado pela Tyr138, o resíduo foi mutado e o seu efeito avaliado ao nível do impacto na actividade enzimática e eficiência de coupling (co-hidroxilação de substrato e cofactor), bem como na isomerização da proteína. Estes resultados permitiram identificar que a Tyr138 desempenha um papel ao nível do reposicionamento dos substratos para catálise, mas sem afectar o coupling da reacção. No entanto, o resíduo Tyr138 também desempenha um papel regulador na isomerização conformacional desencadeada pela ligação do substrato no sítio activo, preenchendo mais uma lacuna no objectivo de compreender o processo de activação catalítica na fenilalanina hidroxilase humana.Fundação para a Ciência e a Tecnologia (FCT, SFRH/BD/19024/2004. POCI and FSE

Molecular Probes, Chemosensors, and Nanosensors for Optical Detection of Biorelevant Molecules and Ions in Aqueous Media and Biofluids

Synthetic molecular probes, chemosensors, and nanosensors used in combination with innovative assay protocols hold great potential for the development of robust, low-cost, and fast-responding sensors that are applicable in biofluids (urine, blood, and saliva). Particularly, the development of sensors for metabolites, neurotransmitters, drugs, and inorganic ions is highly desirable due to a lack of suitable biosensors. In addition, the monitoring and analysis of metabolic and signaling networks in cells and organisms by optical probes and chemosensors is becoming increasingly important in molecular biology and medicine. Thus, new perspectives for personalized diagnostics, theranostics, and biochemical/medical research will be unlocked when standing limitations of artificial binders and receptors are overcome. In this review, we survey synthetic sensing systems that have promising (future) application potential for the detection of small molecules, cations, and anions in aqueous media and biofluids. Special attention was given to sensing systems that provide a readily measurable optical signal through dynamic covalent chemistry, supramolecular host–guest interactions, or nanoparticles featuring plasmonic effects. This review shall also enable the reader to evaluate the current performance of molecular probes, chemosensors, and nanosensors in terms of sensitivity and selectivity with respect to practical requirement, and thereby inspiring new ideas for the development of further advanced systems

Aplicaciones fotónicas de nanomateriales de oro en el desarrollo de biosensores enzimáticos e inmunológicos

El objetivo de esta Tesis Doctoral es la aplicación de las propiedades ópticas de los nanomateriales de oro en el desarrollo de biosensores fotónicos para la determinación de biomarcadores clínicamente relevantes. Durante este trabajo, se han sintetizado, caracterizado e implementado varios nanomateriales de oro en diferentes esquemas de biodetección utilizando tanto enzimas como anticuerpos como elementos de reconocimiento biológico. De hecho, fue posible desarrollar nuevos esquemas de nanobiodetección con mejores características analíticas que las tradicionales, como sensibilidad mejorada y adaptabilidad en el punto de atención. En todos los casos, la gran selectividad alcanzada se basó en la selección de receptores biológicos apropiados para cada analito diana y la optimización de su unión orientada al nanomaterial correspondiente. Esto permitió asegurar la transducción más efectiva entre el reconocimiento biológico y los mecanismos fotónicos involucrados en cada caso.En concreto, en esta Tesis se desarrollaron nuevos nanobiosensores enzimáticos utilizando nanoclusters de oro como etiquetas luminiscentes Vis-NIR, evitando los inconvenientes de los fluoróforos convencionales, como la degradación química y la existencia de interferencias espectrales en muestras biológicas. Para ello, se exploraron dos líneas de investigación; a) la unión covalente de nanoclusters de oro cerca del sitio activo de una enzima redox para promover un fenómeno de transferencia de energía que permita la determinación fluorescente de los analitos objetivo, y b) aprovechar la extinción de la fluorescencia de los nanoclusters de oro por oxígeno para monitorear reacciones enzimáticas de la oxidorreductasa.También se propuso el diseño de nanomateriales de oro mediante síntesis dirigida y específica con ligandos de reconocimiento biológico (enzimas) para explorar dos conceptos sensoriales diferentes impulsados por NP-sintéticos: a) la síntesis in-situ de nanomateriales de oro mediante la reducción de residuos de la enzima yb) la formación de nanopartículas de oro aprovechando las propiedades redox de la enzima desencadenadas por su interacción con el sustrato. En todos los casos, la señal óptica del nanomaterial generado está relacionada con la concentración del analito objetivo.Finalmente, esta Tesis también ha explorado el desarrollo de inmuno-nanobiosensores plasmónicos fototérmicos, basados en el uso de nanoprismas de oro unidos a anticuerpos que pueden actuar como transductores fisicoquímicos del reconocimiento biológico del analito generando una señal térmica cuantificable. El uso de nanoprismas de oro como etiquetas térmicas se implementó en esquemas de biodetección comúnmente utilizados y, por lo tanto, bien aceptados en la clínica (ELISA: Enzyme-Linked ImmunoSorbent Assay y LFIA: lateral flow immunoassay). Esto permitió desarrollar un novedoso concepto térmico de estos esquemas tradicionales de biosensores (Thermo-LISA y Thermo-LFIA) no solo con mejores características analíticas sino también con posibilidades reales de transferencia al mercado en el corto-medio plazo.La optimización y evaluación de la respuesta analítica de los nanobiosensores desarrollados, ha permitido la determinación cuantitativa de analitos relevantes para la detección de cáncer (biomarcadores de cáncer gastrointestinal y de próstata), control de calidad de alimentos (aminas biogénicas) y para otras aplicaciones biomédicas como la determinación de neurotransmisores (acetilcolina) y aminoácidos (L-fenilalanina) relacionados con enfermedades específicas.The aim of this PhD Thesis is the application of the optical properties of gold nanomaterials in the development of photonic biosensors for the determination of clinically relevant biomarkers. During this work, various gold nanomaterials have been synthetized, characterized and implemented in different biosensing schemes using both enzymes and antibodies as biological recognition elements. In fact, it was possible to develop novel nanobiosensing schemes with better analytical characteristics than the traditional ones, such as improved sensitivity and point-of-care adaptability. In all cases, the great selectivity reached was based on the selection of appropriate biological receptors for each target analyte and the optimization of their oriented binding to the corresponding nanomaterial. This allowed ensuring the most effective transduction between the biological recognition and the photonic mechanisms involved in each case. In particular, in this Thesis, new enzymatic nanobiosensors were developed using gold nanoclusters as Vis-NIR luminescent labels, avoiding the drawbacks of conventional fluorophores, such as chemical degradation and the existence of spectral interferences in biological samples. For this, two lines of research were explored; a) the covalent binding of gold nanoclusters close to the active site of a redox enzyme to promote an energy transfer phenomena that allow the fluorescent determination of the target analytes, and b) to take advantage of the quenching of gold nanoclusters fluorescence by oxygen to monitor oxidoreductase enzymatic reactions. The design of gold nanomaterials by means of directed and specific synthesis with biological recognition ligands (enzymes) was also proposed to explore two different NP-synthetic driven sensory concepts: a) the in-situ synthesis of gold nanomaterials by means of the reducing residues of the enzyme and b) the formation of gold nanoparticles taking advantage of the redox properties of the enzyme triggered by its interaction with the substrate. In all cases, the optical signal of the generated nanomaterial is related to the concentration of the target analyte. Finally, this Thesis has also explored the development of photothermal plasmonic immuno-nanobiosensors, based on the use of gold nanoprism bound to antibodies which can act as physicochemical transducers of the biological recognition of the analyte generating a quantifiable thermal signal. The use of gold nanoprism as thermal labels was implemented in biosensing schemes commonly used and therefore well accepted in the clinic (ELISA: Enzyme-Linked ImmunoSorbent Assay, and LFIA: lateral flow immunoassay). This allowed the development of a novel thermal-based concept of these traditional biosensing schemes (Thermo-LISA and Thermo-LFIA) with not only better analytical characteristics but also with real possibilities of transference to the market in the short-medium term. The optimization and the evaluation of the analytical response of the developed nanobiosensors, has allowed the quantitative determination of relevant analytes for cancer detection (gastrointestinal and prostate cancer biomarkers), control food quality (biogenic amines) and for other biomedical applications such as the determination of neurotransmitters (acetylcholine), and amino acids (L-phenylalanine) related to specific diseases.<br /

2019 Oklahoma Research Day Full Program

Oklahoma Research Day 2019 - SWOSU Celebrating 20 years of Undergraduate Research Successes