Rozprawy doktorskie na temat „Paper-based diagnostics”

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemical Engineering, 2019
Cataloged from PDF version of thesis.
Includes bibliographical references.
The timely diagnosis and treatment of disease in resource-constrained settings requires the development of robust point-of-care (POC) diagnostics, which provide accurate results and can be employed by users with minimal medical training and limited access to basic infrastructure. One of the most common POC diagnostic formats is the immunochromatographic rapid diagnostic test, which traditionally uses nitrocellulose-immobilized IgG antibodies as binding proteins for the capture of disease biomarkers from patient samples. However, these antibodies are expensive to produce and structurally complex, and are prone to thermal denaturation. In contexts where continuous cold chain storage may be infeasible, the resulting loss in binding activity can manifest as diminished assay sensitivity, leading to adverse clinical outcomes and eroding patient trust in the diagnostic format.
In the interest of replacing diagnostic antibodies with a more cost-effective, robust class of binding proteins, this thesis explores the development of thermostable affinity reagents based on the hyperthermophilic scaffold protein rcSso7d. Given its native microbial host, minimalist structure, and high wild-type melting temperature (98°C), rcSso7d represents a viable alternative to antibodies in in vitro POC assays. To assess the applicability of the rcSso7d scaffold in this context, protein engineering techniques were used to rapidly select analyte-specific binding variants from a yeast surface display library of >109 members. A high-affinity rcSso7d binder was identified, produced in high yield in a bacterial host, and readily purified in a single chromatographic step.
The in vitro activity and thermal stability of this engineered binder were characterized in the context of a low-cost, paper-based assay, and significant improvements in stability and production economics were observed for rcSso7d-based assays, relative to assays featuring a representative antibody reagent. Additionally, general strategies were developed to improve the diagnostic performance of paper-based assays employing rcSso7d-based reagents. In one instance, chimeric protein constructs in which rcSso7d variants are fused to a cellulose-binding domain were found to bind to unmodified cellulose in an oriented fashion and with high efficiency. This substrate anchoring approach permits the rapid, high-density immobilization of the rcSso7d species in paper-based assays, and yields significant sensitivity enhancement by enabling both the depletion of the soluble analyte from the sample, and the processing of large sample volumes within clinically relevant timescales.
Detection reagents incorporating rcSso7d binders were also developed, using novel fusion constructs which enabled in vivo labelling while preserving analyte binding activity. These techniques were applied in the context of a urine-based tuberculosis biomarker, and may one day permit the development of multiplexed assays targeting a suite of these analytes. Such a development would enable point-of-care diagnostic testing without requiring the production of sputa, facilitating disease detection in otherwise inaccessible patient populations (e.g. children under five, the elderly, and immunocompromised patients).
"People who have financially supported this thesis: the NIH Biotechnology Training Program, the Tata Center for Technology and Design, the Deshpande Center, the Sandbox program, and the Singapore- MIT Alliance for Research and Technology"
by Eric Alexander Miller.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Chemical Engineering

The purpose of this thesis project was to demonstrate the ability to utilize the capabilities of aptamers so that they may act as capture reagents for paper microfluidic devices. Several characterization experiments were conducted as a precursor before the final experimentation was performed. Paper characterization, manufacturing protocols for printing and heating, as well as 3D chip fabrication were all performed and analyzed. The results confirmed that the control of fluid through a 3D microfluidic device based in nitrocellulose is possible. For the biochemistry portion of this thesis report, antibodies and aptamers were chosen to react with IgE, an antibody that is present in high concentrations in the urine of patients diagnosed with respiratory distress. Antibody chips were successfully created as a baseline lateral flow assay for comparison to new aptamer detector reagents. The aptamer experiments were able to demonstrate that it is possible to utilize the capabilities of aptamers so that they may behave as capture reagents in paper microfluidic devices. Overall, the experiments performed were extremely supportive of the ability to develop the field of paper microfluidics with the use of aptamers so that patient populations across the globe can be more accurately and effectively diagnosed.

This thesis describes methods for improving sensitivity in rapid singleplex and multiplex microarray assays. The assays utilize the optical characteristics of colloidal gold nanoparticles for the colorimetric detection of proteins. Multiplexed detection in sandwich immunoassays is limited by cross-reactivity between different detection antibodies. The cross-reactivity between antibodies can contribute to increased background noise - decreasing the Limit-of-Detection of the assay - or generate false positive signals. Paper I shows improved assay sensitivity in a multiplexed vertical flow assay by the application of ultrasonic energy to the gold nanoparticles functionalized with detection antibodies. The ultrasonication of the antibody conjugated gold nanoparticles resulted in a 10 000 fold increase in sensitivity in a 3-plex assay. COMSOL Multiphysics was used to simulate the acoustical energy of the probe used in Paper I for obtaining an indication of the size and direction of the forces acting upon the functionalized gold nanoparticles. In Paper II, it was studied if different gold nanoparticle conjugation methods and colorimetric signal enhancement of the gold nanoparticle conjugates could influence the sensitivity of a paper-based lateral flow microarray assay, targeting cardiac troponin T for the rapid diagnostics of acute myocardial infarction. Ultrasonication and signal enhancement of the detection gold nanoparticles has the potential of improving the sensitivity of paper based assays and expanding their potential future applications.

QC 20170911

Paper (and other cellulose-based materials such as cotton thread and fabrics) are underexploited as materials for the construction of “high-tech” and “lab-on-a-chip” devices. One major drawback of paper is its tendency to absorb water from the environment and, with wetting, to change its mechanical properties; other challenges relate to control over the attachment of molecules (e.g. antibodies, DNA) and cells on its surface, and to the addition of electronic function. The goal of this thesis is to develop paper as a substrate for a range of applications— microfluidics, substrates for electronic systems and MEMS, low-cost diagnostics, cell biology, and optics. The approach involves chemically modifying the surface of the paper to provide new functions without altering any of its defining properties: mechanical flexibility, foldability, light weight, gas permeability, and low cost. The first part of my thesis describes the modification of paper by silanization with organosilanes such as alkyl- and fluoroalkyl trichlorosilanes in the gas phase. Here, silanization is used to lower the surface free energy of the paper and to minimize the tendency of paper to absorb liquids and vapors, and especially water. Chapter 1 and Appendix 3 demonstrate that the combination of long fluoroalkyl chains of grafted siloxanes with the micro-scale roughness and porosity of paper yielded a material that is omniphobic (both hydrophobic and oleophobic), while preserving the properties of mechanical flexibility and low resistance to transport of gas of the untreated paper. Appendix 3 shows that features of omniphobic paper can be used to construct microtiter plates and liquid-filled gas sensors using standard paper folding techniques, while Appendix 4 shows that new type of microfluidic device fabricated by carving microchannels into the surface of omniphobic paper. The resulting devices have open, unobstructed channels (with dimensions as small as 45 μm) and thus exhibit fluid dynamics similar to conventional PDMS-based microfluidics, but are much lighter and have the potential to be much less expensive than PDMS-based devices. The second part of my thesis is focused on engineering the surface of paper to enable efficient immobilization of capture and target molecules for bioanalysis. In one approach, described in Appendix 5, we exploit the ease with which the surface chemistry of paper (i.e. the surface of the cellulose fibers making up the paper) can be modified, in order to enhance the immobilization of antibodies and antigens on the surface of the paper via hydrophobic interactions, while preventing the wicking of the fluids into the paper substrate. As an application in low-cost diagnostics, we describe a low-cost electrochemical device for ELISA intended for use in resource-limited settings. In a second approach, described in Chapter 2, we developed of an efficient procedure for assembling microarrays of ssDNA and proteins on paper, at the lowest practical cost. This method starts with the synthesis of DNA oligonucleotides covalently linked to paper, and proceeds to generate ssDNA arrays that, through hybridization with complementary strands of DNA, are capable of simultaneously capturing DNA, DNA-conjugated protein antigens, and DNA-conjugated antibodies. The third part of my thesis describes the simple, inexpensive fabrication of electrodes for paper-based electrochemical systems. A first method describes, in Appendix 6, the development of inkjet printing as a method for high resolution printing of conductive patterns on omniphobic “RF” paper, both to extend its promise as a substrate for paper electronics, and to enable us to integrate it into our program in low-cost, paper based diagnostics. A second method, described in Chapter 3, circumvents the need for printing, and instead focuses on the fabrication and reconfiguration of simple, versatile, and inexpensive electroanalytical devices in which conventional stainless-steel pins—in unmodified form or after coating with a carbon paste—are used as electrodes.
Chemistry and Chemical Biology

We report on the use of laser direct-write techniques for the fabrication of point-of-care paper-based diagnostic sensors. These include laser-based deposition, laser ablation and laser-induced photo-polymerisation. Firstly, Laser Induced Forward Transfer (LIFT) was employed to deposit biomolecules from a donor film onto paper receivers. Paper was chosen as the ideal receiver because of its inherent properties which make it an efficient and suitable platform for point-of-care diagnostic sensors. Both enzyme-tagged and untagged antibodies were LIFT-printed and their viability was confirmed via a colorimetric enzyme-linked immunosorbent assay (ELISA). Secondly, we report on the laser-based structuring of paper-based fluidic devices. Laser-scanning the paper defines the areas that will be polymerised, thus creating barriers that keep the liquid solutions contained. Complicated devices are easy to fabricate and the flexibility of this technique allows for unique patterns, making it appropriate for rapid prototyping but also for large-scale production. Furthermore, the laser patterning technique allows control of the depth or degree of polymerisation, thereby allowing the liquid to wick through but also imposition of flow delays. Finally, the use of lasers for the fabrication of a 'master' which can be used for casting a PDMS mould for applications in micro-contact printing. The combination of the above mentioned techniques represent the platform technology for the rapid, precise and versatile laser-based fabrication of diagnostic point-of-care sensors.

The purpose of this thesis was to demonstrate device functionality of 3-D paper-based, multiplex platforms, µPADs, without the use of coupling agents between layers. Previously, these platforms were fabricated with double-sided tape and cellulose powder to try to augment proper fluid routing, but difficulties with this method occurred. An acrylic housing unit with strategically placed pressure tabs was designed to aid horizontal and vertical fluid routing through the platform, thus eliminating the inconsistencies associated with coupling agents. Channel characterization studies, a COMSOLTM simulation, and development time studies were performed to aid device design and demonstrate device functionality. The implementation of this µPAD platform as a diagnostic instrument was validated via lateral flow immunoassays utilizing both biotinylated antibodies and biotinylated aptamers as capture reagents. Successful detection of the target analyte, IgE, as well as successful fluid routing through multiple layers of membrane was demonstrated by immunoassays performed on 3-D, multiplex platforms. Another important result determined the aptamers’ ability to detect IgE to be statistically the same as the antibodies’ ability; thus confirming aptamers as viable capture reagent alternatives to antibodies in lateral flow assays. Overall, this research project was performed to develop and validate via experiment a prototype paper-based microfluidic diagnostic device, µPAD, with the capability to detect multiple biomarkers on one platform.

La prevenció i el control de les malalties transmissibles depenen, en gran mesura, de la detecció ràpida i eficaç. Els mètodes convencionals per a la detecció d'un patogen, com ara el cultiu microbiològic, generalment requereixen molt de temps, són laboriosos, necessiten personal qualificat i no són aptes com a eines de diagnòstic en el punt d'atenció. El desenvolupament de mètodes de diagnòstic ràpid en el marc dels criteris ASSURED, de l'anglès (A) Affordable, (SS) Sensitive i Didàctiques, (O) User-friendly, (R) Rapid and Robust, (I) Equipment free, and (d) Deliverable to those who need it, Affordable, descrits per l'Organització Mundial de la Salut (OMS), es troben en l'actualitat sota intens estudi. Per tant, la present tesi aborda el disseny i desenvolupament d'estratègies, mètodes i materials per millorar les prestacions analítiques i simplificar el procediment en proves de diagnòstic ràpid, incloses noves estratègies de preconcentració en fase sòlida, mètodes d'amplificació i materials avançats, així com la seva integració en diferents plataformes (principalment biosensors basats en detecció electroquímica i proves en paper amb lectura òptica). En tots els casos, les aplicacions seleccionades es centren en malalties transmissibles, inclosos els patògens transmesos pels aliments i els micobacteris. Amb aquesta finalitat, es comparen dues plataformes basades en paper en diferents configuracions (flux lateral i vertical) en termes de rendiment analític per a la detecció de Mycobacterium. Per aconseguir una millora addicional en el límit de detecció, s'estudia la preconcentració prèvia dels bacteris per separació immunomagnètica. En segon lloc, s'avaluen i es comparen en termes del seu rendiment analític la detecció simultània de Salmonella i E. coli mitjançant flux lateral d'àcid nucleic amb lectura visual i genosensors electroquímics. Si bé aquests mètodes requereixen PCR de doble etiquetatge per a l'amplificació, es poden adaptar fàcilment a termocicladors portàtils que funcionen amb bateries per poder ser realitzats en entorns amb recursos limitats per satisfer les demandes de diagnòstic ASSURED. A més, també es presenta en aquesta tesi la síntesi de polímers magnètics impresos molecularment, per tal de reemplaçar les partícules magnètiques biològicament modificades, i prenent com a model la detecció de biotina i de molècules biotinilades. A més, es realitza la caracterització del material mitjançant diferents tècniques analítiques i es compara, en tots els casos, amb el polímer no imprès. Aquest material biomimètic mostra un gran potencial per a la preconcentració i detecció d'una àmplia gamma d'analits. Malgrat tot els progressos, les tècniques d'amplificació d'àcid nucleic segueixen essent necessàries per assolir els límits de detecció requerits en algunes malalties transmissibles. En aquest sentit, les tècniques d'amplificació isotèrmiques són bons candidats per dur a terme proves de diagnòstic en entorns on la PCR pot ser una barrera. En concret, es descriu en aquest treball la detecció d' E.coli mitjançant un genosensor electroquímic basat en l'amplificació isotèrmica. En aquest cas, s'optimitza la lectura electroquímica per voltamperometria d'ona quadrada en elèctrodes d'un sol ús comparant dues estratègies de marcatge del producte amplificat. És important ressaltar que totes aquestes estratègies apunten a ser utilitzades com a eines per millorar les proves de diagnòstic ràpid en entorns de baixos recursos, per interrompre la cadena d'infecció de malalties transmissibles i permetre, per tant, un tractament precoç.
La prevención y el control de las enfermedades transmisibles dependen, en gran medida, de la detección rápida y eficaz. Los métodos convencionales para la detección de un patógeno, como el cultivo microbiológico, generalmente requieren mucho tiempo, son laboriosos, necesitan personal cualificado y no son aptos como herramientas de diagnóstico en el punto de atención. El desarrollo de métodos de diagnóstico rápido en el marco de los criterios ASSURED, del inglés (A) Affordable, (SS) Sensitive and Specific, (U) User-friendly, (R) Rapid and Robust, (E) Equipment free, and (D) Deliverable to those who need it, Affordable, descritos por la Organización Mundial de la Salud (OMS), se encuentran en la actualidad bajo intenso estudio. Por lo tanto, la presente tesis aborda el diseño y desarrollo de estrategias, métodos y materiales para mejorar las prestaciones analíticas y simplificar el procedimiento en pruebas de diagnóstico rápido, incluidas nuevas estrategias de preconcentración en fase sólida, métodos de amplificación y materiales avanzados, así como su integración en diferentes plataformas (principalmente biosensores basados en detección electroquímica y pruebas en papel con lectura óptica). En todos los casos, las aplicaciones seleccionadas se centran en enfermedades transmisibles, incluidos los patógenos transmitidas por los alimentos y las micobacterias. Con este fin, se comparan dos plataformas basadas en papel en diferentes configuraciones (flujo lateral y vertical) en términos del rendimiento analítico para la detección de Mycobacterium. Para lograr una mejora adicional en el límite de detección, se estudia la preconcentración previa de las bacterias por separación inmunomagnética. En segundo lugar, se evalúan y se comparan en términos de su rendimiento analítico la detección simultánea de Salmonella y E. coli mediante flujo lateral de ácido nucleico con lectura visual y genosensores electroquímicos. Si bien estos métodos requieren PCR de doble etiquetado para la amplificación, se pueden adaptar fácilmente a termocicladores portátiles que funcionan con baterías para poder ser realizados en entornos con recursos limitados para satisfacer las demandas de diagnóstico ASSURED. Además, también se presenta en esta disertación la síntesis de polímeros magnéticos impresos molecularmente, con el objeto de reemplazar las partículas magnéticas biológicamente modificadas, y tomando como modelo la detección de biotina y moléculas biotiniladas. Además, se realiza la caracterización del material mediante diferentes técnicas analíticas y se compara, en todos los casos, con el polímero no impreso. Este material biomimético muestra un gran potencial para la preconcentración y detección de una amplia gama de analitos. A pesar de todo este progreso, las técnicas de amplificación de ácido nucleico siguen siendo necesarias para alcanzar los límites de detección requeridos en algunas enfermedades transmisibles. Las técnicas de amplificación isotérmica son buenos candidatos para llevar pruebas de diagnóstico en entornos donde la PCR puede ser una barrera. En concreto, se describe en esta disertación la detección de E. coli mediante un genosensor electroquímico basada en la amplificación isotérmica. En este caso, se optimiza la lectura electroquímica por voltamperometría de onda cuadrada en electrodos desechables comparando dos estrategias de marcaje del producto amplificado. Es importante resaltar que todas estas estrategias apuntan a ser utilizadas como herramientas para mejorar las pruebas de diagnóstico rápido en entornos de bajos recursos, para interrumpir la cadena de infección de enfermedades transmisibles y permitir, por tanto, un tratamiento precoz.
The prevention and control of communicable disease rely, to a large extent, on effective and early detection approaches. Conventional methods for the detection of a pathogen, such as microbiological culture, are usually time-consuming, laborious, need skilled personnel and are non-amenable to point-of-care diagnostic tools. The development of rapid diagnostic methods in the framework of the ASSURED criteria as (A) Affordable, (SS) Sensitive and Specific, (U) User-friendly, (R) Rapid and Robust, (E) Equipment free, and (D) Deliverable to those who need it, outlined by the World Health Organization (WHO), are under intensive study. Therefore, the present dissertation addresses the design and development of strategies, methods and materials to improve the analytical performance and to simplify the analytical procedure in rapid diagnostic tests, including novel solid-phase preconcentration strategies, amplification methods and advanced materials, as well as their integration in different platforms (mainly biosensors based on electrochemical detection and paper-based strips for optical readout). In all instances, the applications selected are focused on communicable diseases, including foodborne pathogens and mycobacteria. Therefore, two paper-based platforms in different configurations (nucleic acid lateral and vertical flow) are compared in terms of the analytical performance for the detection of Mycobacterium. In order to achieve a further improvement in the limit of detection, the preconcentration of the bacteria is performed by immunomagnetic separation. Secondly, the simultaneous detection of Salmonella and E. coli by nucleic acid lateral flow with visual readout and electrochemical genosensing are evaluated and compared in terms of their analytical performance. Although these methods required double-tagging PCR for amplification, portable, battery-powered thermocyclers can easily be adapted for resource-constrained settings to meet the demands for ASSURED diagnosis. Furthermore, the synthesis of Magnetic Molecularly Imprinted Polymers, in order to replace biological-modified magnetic particles is also presented in this dissertation, taking as a model the detection of biotin and biotinylated molecules with outstanding performance. Moreover, the characterization of the material is performed by different analytical techniques and compared, in all instances, with the non-imprinted polymer. This biomimetic material shows a great potential for the preconcentration and detection of a huge range of analytes. Despite all these progress, nucleic acid amplification techniques are still necessary to reach the challenging limits of detection required in some communicable disease. Isothermal amplification techniques are good candidates to bring sensitive diagnostic tests in places where the PCR can be a barrier. In detail, the electrochemical genosensing of E. coli based on isothermal amplification is also described in this dissertation. In this approach, the electrochemical readout by square-wave voltammetry on disposable electrodes is optimized comparing two different labelling approaches. It is important to highlight that all these strategies aim to be used as tools for the improvement of rapid diagnostic test in low resource settings, to interrupt the chain of infection of communicable diseases and enabling the rapid treatment.

N,N′-methylenebisacrylamide-crosslinked poly(N-isopropylacrylamide), also known as P(NIPAM), was developed as a fluid delivery system for use with microfluidic paper-based analytical devices (microPADs). MicroPADs are postage-stamp-sized devices made out of paper that can be used as platforms for low-cost, simple-to-use point-of-care diagnostic assays. P(NIPAM) is a thermally responsive polymer that absorbs aqueous solutions at room temperature and will expel the solutions to microPADs when heated. The fluid delivery characteristics of P(NIPAM) were assessed, and P(NIPAM) was able to deliver multiple solutions to microPADs in specific sequences or simultaneously in a laminar-flow configuration. P(NIPAM) was then shown to be suitable for delivering four classes of reagents to microPADs: small molecules, enzymes, antibodies and DNA. P(NIPAM) successfully delivered a series of standard concentrations of glucose (0 – 5 mM) to microPADs equipped to perform a colorimetric glucose assay. The results of these tests were used to produce an external calibration curve, which in turn was used to determine accurately the concentrations of glucose in sample solutions. P(NIPAM) successfully delivered fluorescein-labeled IgG and fluorescein-labeled oligonucleotides (20 base pairs) to microPADs in a variety of concentrations. P(NIPAM) also successfully delivered horseradish peroxidase (HRP) to microPADs, and it was determined that HRP could be stored in P(NIPAM) for 35 days with minimal loss in activity. The combination of P(NIPAM) with microPADs will allow for more complex assays to be performed with minimal user input, will facilitate the preparation of external calibration curves in the field, and may be useful in extending the shelf life of microPADs by stabilizing reagents.

A funcionalização da matriz celulósica é um ponto essencial para o aprimoramento dos dispositivos microfluídicos baseados em papel (µPADs). Ela permite minimizar o preparo de amostras e a interferência do usuário, principais fontes de erro no processo analítico. A oxidação da celulose durante uma hora com m-periodato de sódio e a imobilização química de enzimas a partir da formação de bases de Schiff (iminas), via a adição direta da enzima ao substrato oxidado sem a necessidade de outras etapas, é um processo rápido e de baixo custo, apresentando grande potencialidade de aplicação nos dispositivos microfluídicos em papel. A enzima glicose oxidase imobilizada na celulose, com a adição do estabilizante trealose, apresentou elevada atividade catalítica - de 31,9 ± 5,5 mmol L-1 para a enzima não imobilizada a 14,8 ± 2,0 mmol L-1 para a enzima imobilizada e com o estabilizante - além de apresentar maior homogeneidade de sinal, condições desejáveis em testes rápidos em papel. A confecção de dispositivos em papel via impressão em cera alia rapidez e baixo custo de produção, e o arranjo em camadas para originar dispositivos tridimensionais (3D) permite ampliar as funcionalidades dos dispositivos em duas dimensões, tal como o tratamento individualizado de camadas e o armazenamento de reagentes no próprio dispositivo. O método da adição de padrão para obtenção de curvas analíticas no próprio microchip em papel surge como alternativa às curvas analíticas externas, minimizando a manipulação e o preparo de amostras. O uso do ácido 2,2’-azino-bis (3-etilbenzotiazolina-6-sulfônico) - ABTS como indicador redox para as reações enzimáticas e o método de adição de padrão nos µPADs apresentou boa correlação com um modelo de crescimento e saturação de Michaelis-Menten (r2 = 0,8723) na faixa de 0 a 10 mmol L-1, e a utilização da faixa linear para quantificação de glicose (0 a 3 mmol L-1) apresentou grande correlação linear com a concentração estimada pelas curvas de adição de padrão (r2 = 0,959), demonstrando a potencialidade do método. A união da tecnologia desses dispositivos em papel com a de um software automatizado de reconhecimento de imagens (PAlizer) torna instantânea a obtenção de resultados, eliminando-se a necessidade de intervenção humana no processo, tornando os testes em papel mais robustos, reprodutíveis e rápidos. Com o contínuo aperfeiçoamento das funcionalidades e potencialidades dos dispositivos microfluídicos em papel espera-se que os testes diagnósticos de baixo custo atinjam àqueles que deles necessitam, contribuindo para a saúde da população.
Functionalization of a cellulosic matrix is essential for the success of the paper-based microfluidic analytical devices (µPADs). It allows minimization of sample preparation and user interference, both being major sources of errors in the analytical process. Cellulose oxidation with sodium m-periodate during one hour and the direct chemical immobilization of enzymes on it by Schiff-base (imines) formation, which is made by direct insertion of the enzyme on the oxidized substrate without subsequent steps, is a fast and low cost process of immobilization, presenting great potential of application in paper-based microfluidic analytical devices. The glucose oxidase enzyme immobilized on cellulose, with the addition of trehalose stabilizer presented enhanced catalytic activity - from 31.9 ± 5.5 mmol L-1 for the non-immobilized enzyme to 14.8 ± 2.0 mmol L-1 for the immobilized enzyme with the stabilizing agent - also presenting greater signal homogeneity, which are ideal characteristics in a paper-based rapid test. Wax printing is a simple, inexpensive and fast method by which micro-devices can be fabricated. Additionally, the stacking of layers originating tridimensional devices (3D) allow for the improvement of functionalities of 2-dimensional ones, such as individualized layer treatment and reagent storage at different layers in the same device. Standard addition to analytical curves in paper-based microchips is an alternative to external analytical curves, minimizing handling/sample preparation. The use of 2,2\'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid - ABTS redox indicator with the enzymatic reactions and the standard addition method in µPADs presented a good correlation in a growth and saturation Michaelis-Menten model (r2 = 0.8723), in the range of 0 to 10 mmol L-1, and the usage of the linear range to the glucose quantification (0 to 3 mmol L-1) presented a high linear correlation with the estimated concentration from the standard addition curves (r2 = 0.959), showing the potentiality of the method. The coupling of such paper-based devices to automated image analysis software, such as \'PAlizer\', turns the data acquisition process instantaneous, eliminating the need of human intervention during the process, making it more robust, reproducible and rapid. Expectations lie in improving the devices functions and potential so that these low-cost diagnostic devices can one day reach those who need them, contributing significantly to public health.

Clinically tractable diagnostics must be low-cost, rapid, sensitive, easy to use, and adaptable to new targets. With its rational design, synthetic biology holds promise for developing diagnostic technologies that can address these needs. In particular, progress in synthetic biology has led to improved circuit-building abilities and a large collection of biomolecular sensors. However, these technologies fundamentally require transcription and translation, limiting their applicability to cellular contexts In vitro cell-free expression systems that contain transcription and translation machinery provide the environment necessary for biologically-based technologies to function independently of living cells. Our lab recently developed a paper-based system for cell-free gene expression, which utilizes cell-free extracts that are freeze-dried on to paper and other porous substrates to allow for long-term preservation of synthetic circuits at room temperature. Our platform represents a scalable, cost-effective technology that is easy to use and is compatible with synthetic biology tools. In this dissertation, I present several advancements to this diagnostic platform that are geared towards improving the system’s clinical tractability. In the context of developing a diagnostic for Zika virus that could be deployed in low-resource settings, I demonstrate improvements to diagnostic sensitivity and rapid sample processing that allow for detection of low femtomolar quantities of active virus directly from blood plasma samples. I also describe preliminary results towards a streamlined one-pot amplification-sensing reaction, and propose the development of a paper-based diagnostic for antibiotic susceptibility testing.

The disproportionate burden of infectious disease and lack of appropriate diagnostic tools in the developing world suggest that future health technology development efforts need to more effectively target these resource-limited settings. Microfluidic systems, like lab-on-a-chip technologies, offer the potential to miniaturize the large, complex processes performed in first-world laboratories onto a portable chip for use in remote settings. The problem with these systems is that they require equipment for fluidic handling and many other aspects of diagnostic assays such as sample preparation and analyte detection. The notion of “paperfluidics” has garnered much attention due to paper’s natural ability to wick fluids through capillary action without the need for pumps or other equipment. This and many other qualities of paper make it well suited for point-of-care diagnostics. Paper diagnostics have successfully been employed to detect the presence of antigens or small molecules in clinical samples; however, the detection of many disease targets relies on the much higher sensitivity and specificity of molecular diagnostics achieved via nucleic acid amplification tests (NAAT). The work presented in this dissertation describes the design and development of a paperfluidic sample-to-answer NAAT platform. Preliminary work focused on the development of separate NAAT modules for the extraction, amplification, and detection of nucleic acids from clinical samples directly within a paper matrix. A paper-based assay was developed, using Influenza A (H1N1) as a model system, for the extraction and purification of RNA directly from patient nasopharyngeal specimens, in situ isothermal amplification, and immediate lateral flow detection of amplified products. We then integrate these paper-based NAAT modules onto a single paperfluidic chip in a modular, foldable system that allows for fully-integrated fluidic handling from sample to answer. We showcase the full functionality of the chip by extracting, amplifying and detecting human papillomavirus (HPV) 16 DNA directly from crude cervical specimens in less than 1 hour, for early point-of-care detection of cervical cancer. The chip is made entirely of paper and adhesive sheets, making it low-cost, portable, and disposable, offering the potential for use in very remote settings and increasing access to screening to those most in need.

Swift recognition of disease-causing pathogens at the point-of-care enables life-saving treatment and infection control. However, current rapid diagnostic devices often fail to detect the low concentrations of pathogens present in the early stages of infection, causing delayed and even incorrect treatments. Rapid diagnostics that require multiple steps and/or elevated temperatures to perform have a number of barriers to use at the point-of-care and in the field, and despite efforts to simplify these platforms for ease of use, many still require diagnostic-specific training for the healthcare professionals who use them. Most nucleic acid amplification assays require hours to perform in a sterile laboratory setting that may be still more hours from a patient’s bedside or not at all feasible for transport in remote or low-resourced areas. The cold-chain storage of reagents, multistep sample preparation, and costly instrumentation required to analyze samples has prohibited many nucleic acid detection and antibody-based assays from reaching the point-of-care. There remains a critical need to bring rapid and accessible pathogen identification technologies that determine disease status and ensure effective treatment out of the laboratory.

Paper-based diagnostics have emerged as a portable platform for antigen and nucleic acid detection of pathogens but are often limited by their imperfect control of reagent incubation, multiple complex steps, and inconsistent false positive results. Here, I have developed mechanisms to economically improve thermal incubations, automate dried reagent flow for multistep assays, and specifically detect pathogenic antigens while improving final output sensitivity on paper-based devices. First, I characterize miniaturized inkjet printed joule-heaters (microheaters) that enable thermal control for pathogen lysis and nucleic acid amplification incubation on a low-cost paper-based device. Next, I explore 2-Dimensional Paper Networks as a means to automate multistep visual enhancement reactions with dried reagents to increase the sensitivity and readability of nucleic acid detection with paper-based devices. Lastly, I aim to create a novel Reverse-Transcription Recombinase Polymerase Reaction mechanism to amplify and detect a specific region of the Spike protein domain of SARS-CoV-2. This will allow the rapid detection of SARS-CoV-2 infections to aid in managing the current COVID-19 pandemic. In the future, these tools could be integrated into a rapid diagnostic test for SARS-CoV-2 and other pathogens, ultimately improving the accessibility and sensitivity of rapid diagnostics on multiple fronts.

Molecular diagnostic techniques like polymerase chain reaction (PCR) revolutionized the world of healthcare. Instead of waiting for infectious agents to physically grow as in conventional culture-based tests, genetic material of pathogens could be used as a biomarker to detect their presence. Though PCR has deeply penetrated and greatly benefited the field of disease diagnosis, infrastructure required to perform PCR testing makes it unaffordable and hence inaccessible for majority of world population. The COVID-19 pandemic has strongly highlighted many such grave limitations of existing molecular diagnostic systems, which are rampant in all kinds of healthcare settings. More importantly, obstacles in timely, accurate, and affordable diagnosis of infections make infectious diseases the leading cause of mortality in low-income economies. The aim of my research has been to develop an ASSURED (affordable, sensitive, specific, user-friendly, rapid and robust, equipment free and deliverable to end users) molecular diagnostic tool for infectious disease diagnosis that fits well in the purview of WHO prescribed criteria for developing point-of-care (POC) diagnostic tools. While the diagnostic tool is a platform technology, adaptable to different infectious diseases, the first application has been demonstrated for tuberculosis (TB) diagnosis because it is the deadliest infectious disease today. The developed prototype called fluorescent isothermal paper-and-plastic nucleic acid amplification test (FLIPP-NAAT) uses loop-mediated isothermal amplification (LAMP) for DNA amplification and the only ancillary equipment required for testing is a laboratory incubator. A very low-cost imaging box for filter-free end-point fluorescence detection of amplified DNA using a cell phone has also been developed. To best of our knowledge, this work is the first demonstration of a paper-based NAAT for TB starting from gDNA as template and for testing clinical patient samples using a paper-based NAAT. TB testing for 30 clinical samples demonstrated a clinical sensitivity of 100% and clinical specificity of 68.75%. Material cost for a 12-test zone device is $0.88 and reagent cost per reaction is $0.43. Since clinical specificity of FLIPP-NAAT required improvement, a new detection strategy has hence been proposed where lateral flow detection replaces fluorescence-based detection. Proof-of-concept demonstrations have been made to confirm correct diagnosis of patient samples which were detected as false positives with FLIPP-NAAT. Consistent efforts were also made to enable dry storage of NAAT chemistry in paper pads to develop a shelf-stable product. In addition to developing FLIPP-NAAT, a first of its kind stoichiometric and pseudo-kinetic model has also been developed for LAMP. This is the first model which can predict concentrations of different amplicon types and help researchers design informed experiments for sequence-specific DNA detection strategies. In conclusion, my PhD research reports significant advancement in development of a paper-based ASSURED diagnostic tool and demonstrates its successful application for TB diagnosis. Furthermore, contributions have been made to improve the fundamental understanding of technologies used in building ASSURED molecular diagnostics.

In the year of 2020, an international pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has afflicted tens of millions of people’s life also disrupting global economics. Diagnostic testing is an important part of ensuring public health until a vaccine that has been shown to be safe and effective is made available to the general public. Most tests for detecting COVID-19 utilize quantitative polymerase chain reaction (qPCR) assays, which is a specific and relatively simple quantitative assay that could provide adequate sensitivity for diagnosing early infection. Although powerful, these lab-based molecular assays have a significant lag time, usually several days before receiving results. To satisfy the needs of different purposes (diagnostics, screening, and surveillance), a unified approach is impractical. This thesis presents an alternative testing method supporting the current procedure of point of care (POC) testing and in community testing. This paper-based test overcomes the limitations of current testing methods by utilizing reverse-transcription loop-mediated isothermal amplification (RT-LAMP) and receiving the result on-site by a color change in the presence of the virus within 60 minutes. The test utilizes untreated freshly collected saliva, a less invasive specimen, as the sample and possesses a limit of detection (LoD) of 200 copies of virus per microliter of whole saliva with an analytical sensitivity of 97% and analytical specificity of 100%. The test requires minimal operator training and could be fabricated on a large-scale using roll-to-roll methods. Since the test is based on nucleic acids, the testing platform itself lends to further applications including food safety monitoring, animal diagnostic, etc. simply by changing the specific primers.

Breast cancer (BCa) is one of the most common and deadly diseases in women worldwide. In 2018, 2.1 million cases were diagnosed with BCa from which 626,679 women died. Therefore, an early diagnosis is imperative for treatment and a cure success rate. Recent studies have concluded that exosomes can be used as biomarkers, since they participate in the communication between cells, carrying genetic information from the mother cells. Common methods of detection of exosomes as Enzyme-Linked Immunosorbent Assays (ELISA) are used, however, large amounts of highly concentrated sample, special preparation and labelling processes are required. As an alternative, Raman Spectroscopy stands out as a low-cost, simple and fast detection method that leads to a less invasive real sample collection and sample preparation. Low sample volumes are needed due to surface enhancement signal (SERS). However commercial SERS substrate for these measures present high cost and low shelf-life. In this work, Ag-core-Au-shell bimetallic nanoparticles were directly synthesized on paper substrates (Whatman and Office paper) through two-stage successive ionic layer absorption and reaction (SILAR) techniques and tested as SERS substrates. Enhancement factors (EF) from 104 to 105 were reached and, for both substrates the Limit of Detection (LOD) was calculated as 10-11 M R6G. Non-tumoral (MCF-10A) and tumoral (MDA-MB-231) exosomes from breast cells were tested on the optimized substrates and Raman spectra were analysed by a statistical method called PCA (Principal Component Analysis). The data was successfully grouped with 95% confidence confirming its potential as a low-cost, label-free point-of-care test chip for the early diagnosis of breast cancer diseases.

碩士
國立臺灣大學
化學工程學研究所
101
Paper-based sensors are rapidly immerging to meet the needs for point-of-care diagnose. Due to its low cost, portable, widely available and easy to operate properties, it has attracted lots of attention in biochemical analysis field. In this work, paper 96 well plate was fabricated via wax printing method to build up a multiple diagnostic system. horseradish peroxidase (HRP enzyme) was conjugated with monoclonal antibody to vascular endothelial grow factor (VEGF antibody), and the quantification of the enzyme was carried out on paper 96 well plate. The influence of temperature and conjugated antibody was also investigated. To test the feasibility using this system for drug screening, HRP-linked VEGF antibody was encapsulated in alginate microspheres via an air pressure driven nozzle and used as a model drug. And the encapsulation efficiency of VEGF antibody was reveal by this paper system. Also, through the recognition of VEGF antibody toward VEGF protein, we perform paper-based ELISA to prove the immuno bioactivity of this system. Another part in our study is to investigate the enzymatic reaction on paper fiber, which is an inhomogeneous condition. Meanwhile, we want to accomplish some common diagnostic trials on paper system. Firstly, glucose and bovine serum albumin (BSA protein) assay was carried out on paper 96 well plate. Afterwards, aspartate transaminase (AST) and alkaline phosphatase (ALP) assay was carried out with different immobilization order. Quantitative analysis was achieved by photographing and processing by imaging software. The Michaelis constant for paper plate and plastic plate was calculated by measuring the initial reaction rate of each group. We observed a 60% increase in Michaelis constant for HRP and glucose oxidase group and no significant difference in ALP group. The quantification of four colorimetric reactions can be successfully carried out on this paper plate system. The result acquired can be distinguished by naked eye and digitalized by imaging software. In addition, the cost for a single trial was highly reduced. With these preliminary data, we believe this paper based system can provide a novel alternative for diagnose purpose in developing countries. And with further modifications, this paper-based system can have a wide potential application in long tern health management, food quality control and environment monitoring.

碩士
國立中興大學
生醫工程研究所
102
Tuberculosis has always been considered one of the most serious infectious disease, with the advancement of science and medical technology, people continuously find new ways of diagnosing tuberculosis from old theories, but still unable to develop a real-time, convenient and effective diagnostics. In this study, a colorimetric sensing strategy employing gold nanoparticles and a paper assay platform has been developed for tuberculosis diagnosis. Unmodified gold nanoparticles and single-stranded detection oligonucleotides are used to achieve rapid diagnosis, through the theory of DNA hybridization, single-stranded detection oligonucleotides and mycobacterium tuberculosis (MTB) will combine together to become a MTB-detection probe, and the addition of high-concentration sodium chloride solution will induce gold nanoparticles aggregate and make the colloid color change which is regarded as the mechanism to proceed the diagnosis of tuberculosis. This method avoids using complicated and time-consuming thiolated or other surface-modified probe preparation process. Besides combining with paper assay platform can also eliminate the use of sophisticated equipment for data analysis, the color variance for multiple detection results was simultaneously collected and concentrated on cellulose paper with the data readout transmitted for cloud computing via a smartphone. The results show that the 2.6 nM tuberculosis mycobacterium target sequences extracted from patients can easily be detected, and the turnaround time after the human DNA is extracted from clinical samples is approximately 1 hour. We believe the proposed platform possesses the potential for tuberculosis diagnosis monitoring in resource constrained settings.

Cystic Fibrosis (CF) is an inherited disorder affecting more than 70000 people worldwide, especially Caucasian populations with a carrier prevalence of 1/3000. Currently, it has no cure but an early diagnosis remains a critical issue. Sweat chloride test has been the gold standard to diagnose CF since the affected present sweat chloride concentrations ≥ 60 mM. In this work, a planar electrochromic “point-of-care” device, based on tungsten trioxide nanoparticles produced by microwave assisted hydrothermal synthesis, was developed as a first approach for CF diagnostic testing especially in resource-limited environments. For electrodes patterning, a CO2 laser technology was used in a PET/ITO sheet. The device presents a design that allows the NaCl-based electrolyte deposition, used as artificial sweat, only on time of usage directly on the nanoparticles or in a paper pad. By applying an operating voltage of -3 V, the nanoparticles change their optical properties according to NaCl concentration, presenting a blue colouration with different intensities for different NaCl concentrations. The device was able to differentiate between a positive and negative diagnosis, with a colouration time of only 1 min, using an RGB analysis with a B/R ratio of 1.37±0.03 for 60 mM of NaCl, and a low power consumption.

abstract: The fundamental building blocks for constructing complex synthetic gene networks are effective biological parts with wide dynamic range, low crosstalk, and modularity. RNA-based components are promising sources of such parts since they can provide regulation at the level of transcription and translation and their predictable base pairing properties enable large libraries to be generated through in silico design. This dissertation studies two different approaches for initiating interactions between RNA molecules to implement RNA-based components that achieve translational regulation. First, single-stranded domains known as toeholds were employed for detection of the highly prevalent foodborne pathogen norovirus. Toehold switch riboregulators activated by trigger RNAs from the norovirus RNA genome are designed, validated, and coupled with paper-based cell-free transcription-translation systems. Integration of paper-based reactions with synbody enrichment and isothermal RNA amplification enables as few as 160 copies/mL of norovirus from clinical samples to be detected in reactions that do not require sophisticated equipment and can be read directly by eye. Second, a new type of riboregulator that initiates RNA-RNA interactions through the loop portions of RNA stem-loop structures was developed. These loop-initiated RNA activators (LIRAs) provide multiple advantages compared to toehold-based riboregulators, exhibiting ultralow signal leakage in vivo, lacking any trigger RNA sequence constraints, and appending no additional residues to the output protein. Harnessing LIRAs as modular parts, logic gates that exploit loop-mediated control of mRNA folding state to implement AND and OR operations with up to three sequence-independent input RNAs were constructed. LIRA circuits can also be ported to paper-based cell-free reactions to implement portable systems with molecular computing and sensing capabilities. LIRAs can detect RNAs from a variety of different pathogens, such as HIV, Zika, dengue, yellow fever, and norovirus, and after coupling to isothermal amplification reactions, provide visible test results down to concentrations of 20 aM (12 RNA copies/µL). And the logic functionality of LIRA circuits can be used to specifically identify different HIV strains and influenza A subtypes. These findings demonstrate that toehold- and loop-mediated RNA-RNA interactions are both powerful strategies for implementing RNA-based computing systems for intracellular and diagnostic applications.
Dissertation/Thesis
Doctoral Dissertation Biochemistry 2019

abstract: This work describes efforts made toward the development of a compact, quantitative fluorescence-based multiplexed detection platform for point-of-care diagnostics. This includes the development of a microfluidic delivery and actuation system for multistep detection assays. Early detection of infectious diseases requires high sensitivity dependent on the precise actuation of fluids. Methods of fluid actuation were explored to allow delayed delivery of fluidic reagents in multistep detection lateral flow assays (LFAs). Certain hydrophobic materials such as wax were successfully implemented in the LFA with the use of precision dispensed valves. Sublimating materials such as naphthalene were also characterized along with the implementation of a heating system for precision printing of the valves. Various techniques of blood fractionation were also investigated and this work demonstrates successful blood fractionation in an LFA. The fluid flow of reagents was also characterized and validated with the use of mathematical models and multiphysics modeling software. Lastly intuitive, user-friendly mobile and desktop applications were developed to interface the underlying Arduino software. The work advances the development of a system which successfully integrates all components of fluid separation and delivery along with highly sensitive detection and a user-friendly interface; the system will ultimately provide clinically significant diagnostics in a of point-of-care device.
Dissertation/Thesis
Masters Thesis Biomedical Engineering 2018