M. Tech. Polymer Technology / Type II diabetes afflicts more than 300 million people worldwide. The pursuit for improved targeted drug delivery systems has led to the development of highly improved biomaterials with enhanced biocompatibility and biodegradability properties. Hydrogels are of particular interest for drug delivery applications due to their ability to address targeted drug delivery, in addition to their good biocompatibility, tunable network structure needed to control the diffusion of drugs and their ability to imbibe drugs within their mesh network structure. Hydrogels are promising candidates for advanced anti-diabetic applications. They were prepared by application of free-radical polymerization of acrylamide (AAm) in the presence of partially and thermally reduced graphene oxide (rGO) and wheat protein isolate (WPI). The incorporation of two (or more) different drugs onto a single delivery vehicle and the realization of combination therapy is a challenging, just as it is an important aspect for smart drug delivery. Thus, the development of dual drug delivery systems that can control the release behaviours of each drug is highly pertinent. This project aims to develop a dual drug delivery system with smart polymers, exploiting stimuli responses to be utilized as a carrier vehicle to aid in proffering a cure for diabetes. Also, it aims at proffering a solution to the lingering issue of combination therapy; by comparing the effect of the test drugs individually and in combination as anti-diabetic drugs.

Thesis (Ph. D.)--University of Oregon, 2007. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 109-114). Also available for download via the World Wide Web; free to University of Oregon users.

The purpose of this project was to identify and evaluate nucleotide sequences enhancing translation of proteins in Chinese hamster ovary (CHO) cells. Candidate sequences were placed in the 5´-untranslated region (UTR) or 3´ UTR respectively and evaluated in a CHO-based expression system with a fluorescent Fc-fusion protein as a model protein.Five plasmid vectors were constructed, two of which designed to have a randomized nucleotide library in their 5´ and 3´ UTR respectively, and three of which designed to hold varying repeats of a known enhancing translation (ET) sequence in their 5´ or 3´ UTR. The plasmid constructs were transfected into CHO cells and the protein expression was analyzed both by fluorescence intensity in single cells using flow cytometry and in bulk by monoclonal antibody titer analysis based on Protein A affinity.The main result is that both flow cytometry and titer analysis indicate that insertion of five repeats of the ET in the 5´UTR has a negative effect on protein expression as compared to the control which had no ET repeats. Results related to the insertion of three ETs in the 5´ UTR were ambiguous. The titer analysis indicated that it had a negative effect on the protein expression compared to the control which had no ET repeats, whereas the flow cytometry results suggest that the effect is negligible. Transfection of library plasmids was unsuccessful; hence no library expression analysis results were achieved. Due to the time constraints of the project, the reason for the unsuccessful transfection of library plasmids was not investigated, but the LTX transfection method is stated as a highly plausible cause.Based on the outcome of this study, two recommendations for future work are suggested. The first one is to continue the focus on UTR sequences in terms of library screening, and to improve the method of transfecting library plasmid constructs into CHO cells using lipofection. The second suggestion for further studies is to test different UTR sequence lengths without involving potential ETs, to rule out the effect and positions of the ETs and investigate the expressional effect of UTR length solely.

Chronic obstructive pulmonary disease (COPD) is a high mortality disease, second to stroke and ischemic heart disease. This non-curable disease progressively exacerbates, leading to high personal and societal economic impact, reduced quality of life and often death. General treatment plans for COPD risk mistreating the individuals’ condition. To be effective, the treatment should be individualized following the practices of precision medicine. The aim of this thesis was to develop a data driven algorithm and system with visualization to assess individual COPD risk. With MRI body composition profile measurements, it is possible to accurately assess propensity of a multitude of metabolic conditions, such as coronary heart disease and type 2 diabetes.  The algorithm and system has been developed using Wolfram Language and R within the Wolfram Mathematica framework. The algorithm calculates individualized virtual control groups metabolically similar to the patient’s body composition and spirometric profile. Using UK Biobank data, our tool was used to assess patient COPD propensity using an individual-specific virtual control group with AUROC 0.778 (female) and 0.758 (men). Additionally, the tool was used to identify new body composition profiles related to COPD and associated comorbid conditions.

Made available in DSpace on 2015-07-13T12:10:10Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-08-01. Added 1 bitstream(s) on 2015-07-13T12:25:41Z : No. of bitstreams: 1 000837513.pdf: 748713 bytes, checksum: f6c3f2e57d1d839e2a01a84e58c8b82b (MD5) / Sistemas de liberação de fármacos tornaram-se importantes no campo da farmacologia e da biomedicina, uma vez que podem reduzir a dose terapêutica através da liberação local e evitar possíveis efeitos da droga durante sua passagem pelo organismo. Biomembranas de látex natural extraído da Hevea brasiliensis têm mostrado interessantes resultados na área da biomedicina por apresentarem proteínas que estimulam a angiogênese, acelerararem processos de cicatrização, constituirem próteses e pelo bom desempenho como matriz para liberação de fármacos, extratos vegetais, nanopartículas e proteínas. Este trabalho apresenta a elaboração e a utilização de um novo sistema, dito dinâmico, onde a liberação se dá sob fluido circulante e utilizando biomembranas de látex natural como carreador da liberação. A fim de complementar as propriedades cicatrizantes do látex, foi escolhido o cetoprofeno como modelo de fármaco, devido à sua ação antiinflamatória, analgésica e antipirética, de uso veterinário e humano. As biomembranas foram produzidas misturando látex natural com uma solução de cetoprofeno pelo método de deposição, e secas à temperatura ambiente. Um novo sistema dinâmico para liberação de fármacos com fluxo circulante foi implementado, onde foram comparados resultados de liberações estáticas (sem fluxo) e dinâmicas, em mesmas condições, a fim de se obter a influência do fluxo na liberação. As biomembranas foram caracterizadas por Microscopia Eletrônica de varredura (MEV), espectroscopia no infravermelho por transformada de Fourier (FTIR) e teste mecânico de tração. Os resultados de MEV mostram a existência de fármaco superficial nas membranas e os resultados de FTIR mostram que não há interação entre o biomaterial e o fármaco, porém o cetoprofeno torna a biomembra mais frágil com a redução do modulo de Young, da porcentagem de alongamento... / Drug delivery systems have become important in the field of pharmacology and biomedicine, as they may reduce the therapeutic dose through the local release, avoiding possible effects during its passage through the body. Biomembranes made of natural latex extracted from Hevea brasiliensis has shown interesting results in biomedicine, by presenting proteins that stimulate angiogenesis, wound healing, constitute prostheses and good performance as a matrix for release of drugs, plant extracts, nanoparticles and proteins. This paper presents the development and use of a new system, said dynamic, where the release occurs from circulating fluid and using natural latex biomembranes as carrier for the releases. In order to supplement its healing properties, ketoprofenwas chosen as model drug because of its anti-inflammatory, analgesic and antipyretic properties, in the veterinary and human use. Biomembranes were produced by mixing natural rubber latex with a ketoprofen solution by the casting, and dried at room temperature. A new dynamic system for drug delivery with circulating flow was implemented and the static (no flow) and dynamic releases were compared in order to obtain the influence of the flow in the release. The biomembranes were characterized by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and mechanical testing. The SEM results showed the existence of the drug in the surface membranes. The FTIR results showed that there is no interaction between the drug and biomaterial, although the incorporation of ketoprofen makes the biomembranes more fragile by reducing the Young's modulus, elongation at break and tensile strength, indicating that the drug is only interleaved in the matrix polymer. These results indicate differences between static and dynamic release, since the dynamic released 30% more drug than static. Mathematical modeling showed that the initial release...

A amiodarona é um dos fármacos mais usados para o tratamento de arritmias cardíacas, tanto ventriculares como supraventriculares. Apesar de sua eficácia, o uso da amiodarona está associado a vários efeitos adversos, incluindo a toxicidade pulmonar. O mecanismo pelo qual a amiodarona causa lesão nas células pulmonares humanas não é inteiramente conhecido, mas estudos em cultura de células hepáticas humanas e pulmonares de ratos têm sugerido que a disfunção mitocondrial e o estresse oxidativo têm um papel importante na citotoxicidade da amiodarona. Os compostos fenólicos, incluindo catequina e epicatequina são amplamente distribuídos na natureza e conhecidos por sua capacidade de reduzir o estresse oxidativo. Além disso, alguns compostos fenólicos são capazes de modular a atividade mitocondrial. Em vista disso, o objetivo deste trabalho foi avaliar a capacidade dos compostos fenólicos catequina e epicatequina em a disfunção mitocondrial e os danos oxidativos causados pela amiodarona em células de fibroblasto de pulmão humano (MRC-5). Para atingir os objetivos as células MRC-5 foram tratadas com diferentes concentrações de catequina e epicatequina e após foram expostas a amiodarona 100 μM. A disfunção mitocondrial foi determinada através da atividade do complexo I da cadeia de transporte de elétrons e a biossíntese de ATP usando kits específicos. A viabilidade celular foi avaliada através do ensaio de 3-[4,5- dimetiltiazol 2-il]-2,5 difenil brometo de tetrazolina. A atividade das enzimas superóxido dismutase e catalase foram determinadas espectrofotometricamente. Os danos oxidativos a lipídeos e proteínas foram verificados através dos ensaios de substâncias reativas ao acido tiobarbitúrico e a proteínas carboniladas, respectivamente, e os níveis de óxido nítrico foram avaliados usando o método de Griess. Os resultados mostraram que a amiodarona inibiu a atividade do complexo I da cadeia de transporte de elétrons em 53% e a biossíntese de ATP em 9,5% e tanto a catequina como a epicatequina foram capazes de evitar estes efeitos em todas as concentrações (5, 10, 20 μM) testadas. Verificou-se que a amiodarona reduziu a atividade das enzimas superóxido dismutase e catalase (indicando produção de superóxido e peróxido de hidrogênio) e aumentou os danos oxidativos a lipídeos e proteínas. Os compostos fenólicos catequina e epicatequina foram capazes de minimizar as alterações no metabolismo redox induzidos pela amiodarona e aumentar a viabilidade nas células MRC-5. Catequina e epicatequina reduziram a depleção de óxido nítrico causada pela amiodarona. Este trabalho mostrou, pela primeira vez, que o mecanismo de toxicidade da amiodarona em células MRC-5 está associado à disfunção mitocondrial, principal causa de geração de dano oxidativo celular e que estes efeitos tóxicos são em parte reduzidos pela catequina e epicatequina. Embora outros estudos sejam necessários, estes dados abrem novas perspectivas para estudos visando o desenvolvimento de medicamentos que minimizem os efeitos tóxicos da amiodarona. / Submitted by Ana Guimarães Pereira (agpereir@ucs.br) on 2015-12-09T15:32:21Z No. of bitstreams: 1 Dissertacao Luciana Fernandes Silva Santos.pdf: 1790981 bytes, checksum: 4f3e8dbb7bc255b525503174b7e1a3d2 (MD5) / Made available in DSpace on 2015-12-09T15:32:21Z (GMT). No. of bitstreams: 1 Dissertacao Luciana Fernandes Silva Santos.pdf: 1790981 bytes, checksum: 4f3e8dbb7bc255b525503174b7e1a3d2 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, CAPES. / Amiodarone is among the most widely used drugs for the treatment of ventricular and supraventricular cardiac arrhythmias. However, the use of amiodarone is associated with several side effects including pulmonary toxicity. The mechanism of amiodarone toxicity is not well known, but studies in human liver cells and rats lung cells have been suggested that mitochondrial dysfunction and oxidative stress play important role in the amiodarone cytotoxicity. Phenolic compounds, including catechin and epicatechin are widespread in nature and known for their ability to reduce oxidative stress. In addition, some phenolic compounds are able to modulate mitochondrial activity. Therefore, the objective of this study was to evaluate the ability of phenolic compounds catechin and epicatechin to minimize the mitochondrial dysfunction and oxidative damage induced by amiodarone in human lung fibroblast cells (MRC-5). To achieve the objectives, MRC-5 cells were treated with different concentrations of catechin and epicatechin and then amiodarone 100 μM. Mitochondrial dysfunction was determined by the activity of complex I of the electron transport chain and ATP biosynthesis using specific kits. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The activity of the enzymes superoxide dismutase and catalase were determined spectrophotometrically. The oxidative damage to lipids and proteins have been verified through the test substances reactive to the thiobarbituric acid and carbonyl protein, respectively, and nitric oxide levels were evaluated using the Griess method. The results showed that amiodarone inhibit 53% of the activity of complex I of the electron transport chain and 9.5% of ATP biosynthesis and both catechin and epicatechin were able to avoid these effects in all concentrations (5 10, 20 mM) tested. It was found that amiodarone reduced the superoxide dismutase and catalase activities (indicating the production of radicals superoxide and hydrogen peroxide) and increased oxidative damage to lipids and proteins. Phenolic compounds catechin and epicatechin were able to minimize alterations in the redox metabolism and increase in viability of MRC-5 cells. Furthermore, catechin and epicatechin reduced nitric oxide depletion caused by amiodarone. This study showed, for the first time, that toxicity of amiodarone in human lung cultured cells is associated, at least, in part, with mitochondrial dysfunction which was avoided by catechin and epicatechin. Although further studies are needed, these data open new perspectives for studies aiming the development of drugs that minimize the toxic effects of amiodarone.

Orientador: Rondinelli Donizetti Herculano / Banca: Catarina dos Santos / Banca: André Gonzaga dos Santos / Resumo: Sistemas de liberação de fármacos tornaram-se importantes no campo da farmacologia e da biomedicina, uma vez que podem reduzir a dose terapêutica através da liberação local e evitar possíveis efeitos da droga durante sua passagem pelo organismo. Biomembranas de látex natural extraído da Hevea brasiliensis têm mostrado interessantes resultados na área da biomedicina por apresentarem proteínas que estimulam a angiogênese, acelerararem processos de cicatrização, constituirem próteses e pelo bom desempenho como matriz para liberação de fármacos, extratos vegetais, nanopartículas e proteínas. Este trabalho apresenta a elaboração e a utilização de um novo sistema, dito dinâmico, onde a liberação se dá sob fluido circulante e utilizando biomembranas de látex natural como carreador da liberação. A fim de complementar as propriedades cicatrizantes do látex, foi escolhido o cetoprofeno como modelo de fármaco, devido à sua ação antiinflamatória, analgésica e antipirética, de uso veterinário e humano. As biomembranas foram produzidas misturando látex natural com uma solução de cetoprofeno pelo método de deposição, e secas à temperatura ambiente. Um novo sistema dinâmico para liberação de fármacos com fluxo circulante foi implementado, onde foram comparados resultados de liberações estáticas (sem fluxo) e dinâmicas, em mesmas condições, a fim de se obter a influência do fluxo na liberação. As biomembranas foram caracterizadas por Microscopia Eletrônica de varredura (MEV), espectroscopia no infravermelho por transformada de Fourier (FTIR) e teste mecânico de tração. Os resultados de MEV mostram a existência de fármaco superficial nas membranas e os resultados de FTIR mostram que não há interação entre o biomaterial e o fármaco, porém o cetoprofeno torna a biomembra mais frágil com a redução do modulo de Young, da porcentagem de alongamento... / Abstract: Drug delivery systems have become important in the field of pharmacology and biomedicine, as they may reduce the therapeutic dose through the local release, avoiding possible effects during its passage through the body. Biomembranes made of natural latex extracted from Hevea brasiliensis has shown interesting results in biomedicine, by presenting proteins that stimulate angiogenesis, wound healing, constitute prostheses and good performance as a matrix for release of drugs, plant extracts, nanoparticles and proteins. This paper presents the development and use of a new system, said dynamic, where the release occurs from circulating fluid and using natural latex biomembranes as carrier for the releases. In order to supplement its healing properties, ketoprofenwas chosen as model drug because of its anti-inflammatory, analgesic and antipyretic properties, in the veterinary and human use. Biomembranes were produced by mixing natural rubber latex with a ketoprofen solution by the casting, and dried at room temperature. A new dynamic system for drug delivery with circulating flow was implemented and the static (no flow) and dynamic releases were compared in order to obtain the influence of the flow in the release. The biomembranes were characterized by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and mechanical testing. The SEM results showed the existence of the drug in the surface membranes. The FTIR results showed that there is no interaction between the drug and biomaterial, although the incorporation of ketoprofen makes the biomembranes more fragile by reducing the Young's modulus, elongation at break and tensile strength, indicating that the drug is only interleaved in the matrix polymer. These results indicate differences between static and dynamic release, since the dynamic released 30% more drug than static. Mathematical modeling showed that the initial release... / Mestre

Our increasing understanding of disease mechanisms coupled with technological advances has facilitated the generation of pharmaceutical proteins, which are able to address yet unmet medical needs. Diseases that were fatal in the past can now be treated with novel biological medications improving and prolonging life for many patients. Pharmaceutical protein production is, however, a complex undertaking, which is by no means problem-free. The demand for more complex proteins and the realization of the importance of post-translational modifications have led to an increasing use of mammalian cells for protein expression. Despite improvements in design and production, the costs required for the development of pharmaceutical proteins still are far greater than those for conventional, small molecule drugs. To render such treatments affordable for healthcare suppliers and assist in the implementation of precision medicine, further progress is needed. In five papers this thesis describes strategies and methods that can help to advance the development and manufacturing of pharmaceutical proteins. Two platforms for antibody engineering have been developed and evaluated, one of which allows for efficient screening of antibody libraries whilst the second enables the straightforward generation of bispecific antibodies. Moreover, a method for epitope mapping has been devised and applied to map the therapeutic antibody eculizumab’s epitope on its target protein. In a second step it was shown how this epitope information can be used to stratify patients and, thus, contribute to the realization of precision medicine. The fourth project focuses on the cell line development process during pharmaceutical protein production. A platform is described combining split-GFP and fluorescence-activated droplet sorting, which allows for the efficient selection of highly secreting cells from a heterogeneous cell pool. In an accompanying study, the split-GFP probe was improved to enable shorter assay times and increased sensitivity, desirable characteristics for high-throughput screening of cell pools. In summary, this thesis provides tools to improve design, development and production of future pharmaceutical proteins and as a result, it makes a contribution to the goal of implementing precision medicine through the generation of more cost-effective biopharmaceuticals for well-characterized patient groups. / <p>QC 20170828</p>

A amiodarona é um dos fármacos mais usados para o tratamento de arritmias cardíacas, tanto ventriculares como supraventriculares. Apesar de sua eficácia, o uso da amiodarona está associado a vários efeitos adversos, incluindo a toxicidade pulmonar. O mecanismo pelo qual a amiodarona causa lesão nas células pulmonares humanas não é inteiramente conhecido, mas estudos em cultura de células hepáticas humanas e pulmonares de ratos têm sugerido que a disfunção mitocondrial e o estresse oxidativo têm um papel importante na citotoxicidade da amiodarona. Os compostos fenólicos, incluindo catequina e epicatequina são amplamente distribuídos na natureza e conhecidos por sua capacidade de reduzir o estresse oxidativo. Além disso, alguns compostos fenólicos são capazes de modular a atividade mitocondrial. Em vista disso, o objetivo deste trabalho foi avaliar a capacidade dos compostos fenólicos catequina e epicatequina em a disfunção mitocondrial e os danos oxidativos causados pela amiodarona em células de fibroblasto de pulmão humano (MRC-5). Para atingir os objetivos as células MRC-5 foram tratadas com diferentes concentrações de catequina e epicatequina e após foram expostas a amiodarona 100 μM. A disfunção mitocondrial foi determinada através da atividade do complexo I da cadeia de transporte de elétrons e a biossíntese de ATP usando kits específicos. A viabilidade celular foi avaliada através do ensaio de 3-[4,5- dimetiltiazol 2-il]-2,5 difenil brometo de tetrazolina. A atividade das enzimas superóxido dismutase e catalase foram determinadas espectrofotometricamente. Os danos oxidativos a lipídeos e proteínas foram verificados através dos ensaios de substâncias reativas ao acido tiobarbitúrico e a proteínas carboniladas, respectivamente, e os níveis de óxido nítrico foram avaliados usando o método de Griess. Os resultados mostraram que a amiodarona inibiu a atividade do complexo I da cadeia de transporte de elétrons em 53% e a biossíntese de ATP em 9,5% e tanto a catequina como a epicatequina foram capazes de evitar estes efeitos em todas as concentrações (5, 10, 20 μM) testadas. Verificou-se que a amiodarona reduziu a atividade das enzimas superóxido dismutase e catalase (indicando produção de superóxido e peróxido de hidrogênio) e aumentou os danos oxidativos a lipídeos e proteínas. Os compostos fenólicos catequina e epicatequina foram capazes de minimizar as alterações no metabolismo redox induzidos pela amiodarona e aumentar a viabilidade nas células MRC-5. Catequina e epicatequina reduziram a depleção de óxido nítrico causada pela amiodarona. Este trabalho mostrou, pela primeira vez, que o mecanismo de toxicidade da amiodarona em células MRC-5 está associado à disfunção mitocondrial, principal causa de geração de dano oxidativo celular e que estes efeitos tóxicos são em parte reduzidos pela catequina e epicatequina. Embora outros estudos sejam necessários, estes dados abrem novas perspectivas para estudos visando o desenvolvimento de medicamentos que minimizem os efeitos tóxicos da amiodarona. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, CAPES. / Amiodarone is among the most widely used drugs for the treatment of ventricular and supraventricular cardiac arrhythmias. However, the use of amiodarone is associated with several side effects including pulmonary toxicity. The mechanism of amiodarone toxicity is not well known, but studies in human liver cells and rats lung cells have been suggested that mitochondrial dysfunction and oxidative stress play important role in the amiodarone cytotoxicity. Phenolic compounds, including catechin and epicatechin are widespread in nature and known for their ability to reduce oxidative stress. In addition, some phenolic compounds are able to modulate mitochondrial activity. Therefore, the objective of this study was to evaluate the ability of phenolic compounds catechin and epicatechin to minimize the mitochondrial dysfunction and oxidative damage induced by amiodarone in human lung fibroblast cells (MRC-5). To achieve the objectives, MRC-5 cells were treated with different concentrations of catechin and epicatechin and then amiodarone 100 μM. Mitochondrial dysfunction was determined by the activity of complex I of the electron transport chain and ATP biosynthesis using specific kits. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The activity of the enzymes superoxide dismutase and catalase were determined spectrophotometrically. The oxidative damage to lipids and proteins have been verified through the test substances reactive to the thiobarbituric acid and carbonyl protein, respectively, and nitric oxide levels were evaluated using the Griess method. The results showed that amiodarone inhibit 53% of the activity of complex I of the electron transport chain and 9.5% of ATP biosynthesis and both catechin and epicatechin were able to avoid these effects in all concentrations (5 10, 20 mM) tested. It was found that amiodarone reduced the superoxide dismutase and catalase activities (indicating the production of radicals superoxide and hydrogen peroxide) and increased oxidative damage to lipids and proteins. Phenolic compounds catechin and epicatechin were able to minimize alterations in the redox metabolism and increase in viability of MRC-5 cells. Furthermore, catechin and epicatechin reduced nitric oxide depletion caused by amiodarone. This study showed, for the first time, that toxicity of amiodarone in human lung cultured cells is associated, at least, in part, with mitochondrial dysfunction which was avoided by catechin and epicatechin. Although further studies are needed, these data open new perspectives for studies aiming the development of drugs that minimize the toxic effects of amiodarone.

Immunoassays are widely used for detection of antigens in a wide range of applications including assays in pharmaceutical development. Immunoassays are continuously improved in many aspects including automatization, miniaturization and extending the dynamic range. The need to measure low abundance molecules are challenging and the need to improve the sensitivity is desired. The Gyrolab technology is a miniaturized immunoassay performed in an automated system covering a broad concentration range. In order to  extend the sensitivity, the technology is combined with Binding Oligo Ladder Detection (BOLD) amplification. The technology behind BOLD or Exazym ® utilizes a DNA primer, a polymerase, and a template (RNA) to generate a ladder-like modified DNA strand. Antibodies with affinity for the polymerized DNA:RNA hybrid strand (duplex) conjugated with reporter molecules are introduced to the system, resulting in an increased number of signal-generating molecules associated with each bound analyte molecule. In this thesis, the development of an ultra-sensitive immunoassay is pursued by applying Exazym ® add-on reagents to the Gyrolab platform, comparing performance with the standard Gyrolab sandwich assay and other commercially available high-performing TNF-α assays. The work includes characterization of a wide range of reaction variables involved in the BOLD signal amplification process including hybridization, polymerization, and detection of a synthetic oligonucleotide. The breakthrough involves the introduction of Allophycocyanin (APC) as a fluorescent conjugate, significantly improving sensitivity and signal-to-noise ratios. The BOLD amplified sensitivity for the TNF-α assay approaches levels seen in ultra-sensitive biomarker assays like Erenna ® and Simoa®. Exazym® technology on the Gyrolab platform allows highly sensitive biomarker assays with minimal sample volume and a 1–2-hour run-time. The study marks substantial progress in achieving ultra-sensitive biomarker assays on the Gyrolab platform through BOLD signal amplification. The use of APC-conjugated detection reagents holds promise for future optimization studies.