The advancement of biopharmaceutical manufacturing, particularly continuous processing, has heightened the need for next-generation analytical tools approaching real-time monitoring of critical quality attributes (CQAs) and process parameters (CPPs). Current methods, primarily offline and labor-intensive, fail at delivering analytical information that can be used for process analytical technology (PAT) to control and optimize the manufacturing process, while also lacking the ability of multi-attribute monitoring, thus requiring a large number of samples (or sampling amount) to be collected. This work introduces the concept of PAT-on-a-chip, consisting of an integrated microfluidic platform designed to perform at-line analysis and characterization of cell culture samples in the context of monoclonal antibody (mAb) production. Specifically, a sample preparation-free miniaturized lectin-based assay was developed to measure levels of high mannose glycans and integrated with affinity-based assays to measure mAb titers and key impurities, namely Chinese hamster ovary (CHO) host cell proteins (HCP), within the same chip, resorting to a common colorimetric readout. The microfluidic chips were operated in a customized and integrated instrument comprising miniaturized photodiodes, connected to a graphical user interface for data recording and signal quantification. The PAT-on-a-chip unit allowed to achieve fit-for-purpose analyte quantification, while offering performance comparable to state-of-the-art offline analytical methods (Pearson R > 0.93), namely capillary electrophoresis with laser-induced fluorescence (CE-LIF) for glycan analysis, well plate immunoassays for CHO HCP and protein A HPLC for mAb titers, thus validating its potential to expand the modern PAT toolbox.

The broad availability of smartphones has provided new opportunities to develop less expensive, portable, and integrated point-of-care (POC) platforms. Here, a platform that consists of three main components is introduced: a portable housing, a centrifugal microfluidic disc, and a mobile phone. The mobile phone supplies the electrical power and serves as an analysing system. The low-cost housing made from cardboard serves as a platform to conduct tests. The electrical energy stored in mobile phones was demonstrated to be adequate for spinning a centrifugal disc up to 3000 revolutions per minute (RPM), a rotation speed suitable for majority of centrifugal microfluidics-based assays. For controlling the rotational speed, a combination of magnetic and acoustic tachometry using embedded sensors of the mobile phone was used. Experimentally, the smartphone-based tachometry was proven to be comparable with a standard laser-based tachometer. As a proof of concept, two applications were demonstrated using the portable platform: a colorimetric sandwich immunoassay to detect interleukin-2 (IL-2) having a limit of detection (LOD) of 65.17 ng/mL and a fully automated measurement of hematocrit level integrating blood-plasma separation, imaging, and image analysis that takes less than 5 mins to complete. The low-cost platform weighing less than 150 g and operated by a mobile phone has the potential to meet the REASSURED criteria for advanced diagnostics in resource limited settings.

Cytokines play a very important role in our immune system by acting as mediators to put up a coordinated defense against foreign elements in our body. Elevated levels of cytokines in the body can signal to an ongoing response of the immune system to some abnormality. Thus, the quantification of a panel of cytokines can provide valuable information regarding the diagnosis of specific diseases and state of overall health of an individual. Conventional Enzyme Linked Immunosorbent Assay (ELISA) is the gold-standard for quantification of cytokines, however the need for trained personnel and expensive equipment limits its application to centralized laboratories only. In this context, there is a lack of simple, low-cost and portable devices which can allow for quantification of panels of cytokines at point-of-care and/or resource limited settings.

Here, we report the development of a versatile, low-cost and portable bead-based centrifugal microfluidic platform allowing for multiplexed detection of cytokines with minimal hands-on time and an integrated colorimetric signal readout without the need for any external equipment. As a model, multiplexed colorimetric quantification of three target cytokines i.e., Tumor necrosis factor alpha (TNF-α), Interferon gamma (IFN-γ) and Interleukin-2 (IL-2) was achieved in less than 30 min with limits of detection in ng/mL range. The developed platform was further evaluated using spiked-in plasma samples to test for matrix interference. The ease of use, low-cost and portability of the developed platform highlight its potential to serve as a sample-to-answer solution for detection of cytokine panels in resource limited settings.

Advancements in medical diagnostics have allowed us to understand the underlying mechanism and treat the root cause for many diseases which had been causing morbidity and mortality up until this point in human history. Furthermore, many of the standard diagnostic procedures have now been transformed to provide answers at or near the point-of-care. However, the effects of these positive developments have not trickled down to the parts of our society which are considered underdeveloped and lack the necessary infrastructure and facilities. Diagnostics in such resource limited settings still lag behind and fail to provide the requisite healthcare. 

In order to translate the diagnostic solutions designed for central laboratories to resource limited settings, there are certain challenges that need to be addressed, such as portability, reduction in cost and ease-of-use, while keeping the sensitivity and specificity at the required level. The work presented in this thesis focuses on addressing some of these issues by using microfluidics to develop diagnostic platforms that are suitable to be used in resource limited settings. 

In paper I, a very low-cost and simple centrifugal microfluidic platform was developed to be used in settings which do not have a reliable supply of electricity. The platform uses a smartphone as a source of power and the sensors of the phone for speed control.

In paper II, a portable and low-cost diagnostic platform was developed for multiplexed detection of biomarkers based on centrifugal microfluidics. The platform uses colorimetric detection and a simple readout method which does not require a spectrophotometer for quantification.

In paper III, a platform was developed for COVID-19 diagnostics which combines centrifugal microfluidics with a novel bead-based strategy for signal enhancement. The platform uses fluorescent detection with a smartphone readout and has the capability to process up to 20 samples at the same time.

In paper IV, as a follow up of paper III, a more advanced platform was developed for COVID-19 diagnostics which allows the operator to carry out nucleic acid amplification in a completely automated manner, from adding the sample to getting the final result.

In paper V, an alternative method for detection of SARS-CoV-2 was developed using electrochemical biosensing. This work combines the electrochemical technique with a flexible printed circuit board for a rapid, amplification-free and label-free detection of target SARS-CoV-2 sequences.

Framsteg inom medicinsk diagnostik har gjort det möjligt att förstå och behandla många sjukdomar som tidigare varit en betydande orsak till dödlighet. Trots framstegen har inte alla delar av samhället haft tillgång dessa diagnostiska verktyg på samma sätt, särskilt i resursbegränsade miljöer i låg- och medelinkomstländer. Detta har lett till en ojämlik tillgång till sjukvård. Den senaste utvecklingen inom mikrofluidik möjliggör utveckling av så kallade patientnära analysverktyg till en fraktion av kostnaderna i traditionella labb-baserade tester. För applikationer i resursbegränsade miljöer krävs diagnostiska lösningar som är portabla, kostnadseffektiva och användarvänliga samtidigt som de har hög känslighet och specificitet. I denna avhandling har vi jobbat med framtagande av mikrofluidik-baserade diagnostiska plattformar som är lämpliga för resursbegränsade miljöer. Syftet är att kunna göra avancerade tester på platser där sjukvårdstjänster tidigare varit otillgängliga eller kostsamma att etablera. För att lösa de tekniska utmaningarna har flera nya tekniker utvecklats, bland annat en centrifugalmikrofluidik-baserad plattform.

Centrifugalmikrofluidik är en teknik för att hantera små mängder vätskor med hjälp av en roterande skiva, liknande CD/DVD skivor men i detta fall finns det mikrofluidiska kanaler mönstrade i skivan för att möjliggöra analys. När skivan roterar skapas centrifugalkraft som används för att flytta och manipulera vätskor i dessa kanaler för att utföra ett antal steg som är nödvändiga för att göra bioanalys av olika prover. Det finns olika applikationer för denna teknik inom biologi och vi har utvecklat olika typer av metoder i denna avhandling. I ett av projekten kombinerade vi centrifugalmikrofluidik med en mobiltelefon för att utveckla en diagnostisk plattform som använde mobiltelefonen som strömkälla, analysering av provresultat samt som sensor för att kontrollera rotationshastigheten av rotorn som driver disken med analysen. Dessutom använde vi oss av pappkartong för att montera rotorn och skivan där tanken är att slutanvändaren ska kunna montera ihop och använda en mobiltelefon för att utföra analysen i fältet. En annan plattform använde kolorimetrisk detektion av proteiner och en enkel avläsningsmetod integrerad på plattformen, som inte krävde en spektrofotometer för kvantifiering av proteinmängden på skivan. I ett tredje projekt utvecklades en plattform för COVID-19-diagnostik som kombinerade centrifugalmikrofluidik där skivan inkorporerar agaroskulor för signalförstärkning av nukleinsyror. Denna plattform använde fluorescensdetektion med avläsning av en smartphone. Skivan analyserar upp till 20 prover från COVID-19 patienter samtidigt och resultatet kan avläsas av en smartphone och hade förmågan att behandla upp till 20 prover samtidigt. Vi har vidareutvecklat metoden där vi inkorporerat en kamera istället för mobiltelefon för att automatisera bildanalysen och vi planerar att testa metoden på fält i olika länder i sub-Sahara Afrika inom en snar framtid. Slutligen utvecklades en alternativ metod för detektion av SARS-CoV-2 med hjälp av elektrokemisk biosensing. Denna metod använde ett flexibelt kretskort för en snabb detektion av SARS-CoV-2.

Sammanfattningsvis har vi inom denna avhandling utvecklat ett antal patientnära analysmetoder som riktar in sig på utmaningarna i resursbegränsade miljöer. Det är viktigt att utvecklar tekniker som kan användas i dessa miljöer, där infrastruktur och faciliteter är begränsade eller saknas helt. Med hjälp av den senaste teknikutvecklingen inom mikrofluidik tror vi att det är fullt möjligt att utveckla diagnostiska plattformar som är kostnadseffektiva och användas där de behövs som bäst, på ett innovativt sätt.

The up- and down- regulation of inflammatory biomarkers such as cytokines can be indicative of several diseases such as primary cancers and/or metastatic tumors, as well as less serious conditions. For point-of-care clinical applications, the detection of these biomarkers requires a combination of a sensitive assay and multiplexing capabilities, together with fit-for-purpose signal transduction strategies. Here, we report the development of a versatile and cost-effective integrated centrifugal microfluidic platform compatible with resource-limited settings using nanoporous microbeads for immunoaffinity-based profiling of cytokines. With an automated colorimetric readout at the end, the platform allows for profiling of cytokines in < 30 mins.

Since the COVID-19 outbreak was declared a pandemic by the World Health Organization (WHO) in March 2020, ongoing efforts have been made to develop sensitive diagnostic platforms. Detection of viral RNA provides the highest sensitivity and specificity for detection of early and asymptomatic infections. Thus, this work aimed at developing a label-free genosensor composed of graphene as a working electrode that could be embedded into a flex printed circuit board (FPCB) for the rapid, sensitive, amplification-free and label-free detection of SARS-CoV-2. To facilitate liquid handling and ease of use, the developed biosensor was embedded with a user-friendly reservoir chamber. As a proof-of-concept, detection of a synthetic DNA strand matching the sequence of ORF1ab was performed as a two-step strategy involving the immobilization of a biotinylated complementary sequence on a streptavidin-modified surface, followed by hybridization with the target sequence recorded by the differential pulse voltammetric (DPV) technique in the presence of a ferro/ferricyanide redox couple. The effective design of the sensing platform improved its selectivity and sensitivity and allowed DNA quantification ranging from 100 fg/mL to 1 mu g/mL. Combining the electrochemical technique with FPCB enabled rapid detection of the target sequence using a small volume of the sample (5-20 mu L). We achieved a limit-of-detection of 100 fg/mL, whereas the predicted value was similar to 33 fg/mL, equivalent to approximately 5 x 10(5) copies/mL and comparable to sensitivities provided by isothermal nucleic acid amplification tests. We believe that the developed approach proves the ability of an FPCB-implemented DNA sensor to act as a potentially simpler and more affordable diagnostic assay for viral infections in Point-Of-Care (POC) applications.

The demand for scalable, rapid and sensitive COVID-19 diagnostics is particularly pressing at present to help contain the spread of infection and prevent overwhelming the capacity of health systems. While high-income countries have managed to rapidly expand diagnostic capacities, such is not the case in resource-limited settings of low- to medium-income countries. We report the development of an integrated modular centrifugal microfluidic platform costing less than 250 USD to perform loop-mediated isothermal amplification (LAMP) of viral RNA directly from heat-inactivated nasopharyngeal swab samples. The platform was validated with a panel of 131 nasopharyngeal swab samples collected from symptomatic COVID-19 patients.

With its origin estimated around December 2019 in Wuhan, China, the ongoing SARS-CoV-2 pandemic is a major global health challenge. The demand for scalable, rapid and sensitive viral diagnostics is thus particularly pressing at present to help contain the rapid spread of infection and prevent overwhelming the capacity of health systems. While high-income countries have managed to rapidly expand diagnostic capacities, such is not the case in resource-limited settings of low- to medium-income countries. Aiming at developing cost-effective viral load detection systems for point-of-care COVID-19 diagnostics in resource-limited and resource-rich settings alike, we report the development of an integrated modular centrifugal microfluidic platform to perform loop-mediated isothermal amplification (LAMP) of viral RNA directly from heat-inactivated nasopharyngeal swab samples. The discs were pre-packed with driedn-benzyl-n-methylethanolamine modified agarose beads used to selectively remove primer dimers, inactivate the reaction post-amplification and allowing enhanced fluorescence detectionviaa smartphone camera. Sample-to-answer analysis within 1 hour from sample collection and a detection limit of approximately 100 RNA copies in 10 μL reaction volume were achieved. The platform was validated with a panel of 162 nasopharyngeal swab samples collected from patients with COVID-19 symptoms, providing a sensitivity of 96.6% (82.2-99.9%, 95% CI) for samples with Ct values below 26 and a specificity of 100% (90-100%, 95% CI), thus being fit-for-purpose to diagnose patients with a high risk of viral transmission. These results show significant promise towards bringing routine point-of-care COVID-19 diagnostics to resource-limited settings.

While diagnostics are critical for accurate and timely diagnosis, gold-standard diagnostic tests are commonly high- performance laboratory-based tests that require multi-step protocols for complex sample processing and highly trained personnel, both scarce in low-resource settings. Here, we address the need for an easy-to-use, rapid and reliable diagnostic testing pipeline by presenting a solution combining open-source modular automation and automation compatible microfluidics, easily adaptable to a pipeline for infectious diseases diagnosis. We demonstrate an automation compatible microfluidics pipeline for Neisseria meningitidis diagnosis by on-chip nucleic acids isolation and isothermal amplification, as well as pathogen detection on a paper-based microarray. 

The detection of panels of inflammatory biomarkers such as cytokines has potential for the rapid and specific diagnostic of several devastating diseases such as primary cancers and/or metastatic tumors, as opposed to less serious conditions. For point-of-care clinical applications, the detection of these biomarkers requires a combination of pg/mL sensitivities and multiplexing capabilities, coupled with fit-for-purpose signal transduction strategies. Here, we report the development of a versatile centrifugal microfluidic platform combined with nanoporous microbeads for immunoaffinity-based profiling of cytokines. The device allows sample and analyte multiplexing and detection limits below 1 ng/mL were achieved within 30 minutes, using colorimetric detection.