A paper microfluidic device capable of conducting enzyme-linked assays is presented: a microfluidic enzyme-linked paper analytical device (μEL-PAD). The system exploits a wash-free sandwich coupling to form beads/analyte/enzyme complexes, which are subsequently added to the vertical flow device composed of wax-printed paper, waxed nitrocellulose membrane and absorbent/barrier layers. The nitrocellulose retains the bead complexes without disrupting the flow, enabling for an efficient washing step. The entrapped complexes then interact with the chromogenic substrate stored on the detection paper, generating a color change on it, quantified with an open-source smartphone software. This is a universal paper-based technology suitable for high-sensitivity quantification of many analytes, such as proteins or nucleic acids, with different enzyme-linked formats. Here, the potential of the μEL-PAD is demonstrated to detect DNA from Staphylococcus epidermidis. After generation of isothermally amplified genomic DNA from bacteria, Biotin/FITC-labeled products were analyzed with the μEL-PAD, exploiting streptavidin-coated beads and antiFITC-horseradish peroxidase. The μEL-PAD achieved a limit of detection (LOD) and quantification <10 genome copies/μL, these being at least 70- and 1000-fold lower, respectively, than a traditional lateral flow assay (LFA) exploiting immobilized streptavidin and antiFITC-gold nanoparticles. It is envisaged that the device will be a good option for low-cost, simple, quantitative, and sensitive paper-based point-of-care testing.

Nucleic acid amplification testing (NAAT) is the gold standard for infectious disease diagnostics. Currently NAATs are mainly limited to centralized laboratories, while paper-based antigen tests are used for rapid home-based diagnostics. DNA extraction, the initial sample preparation step in NAATs, remains a bottleneck that hinders its development toward home-based kits. This step requires the use of compounds detrimental to the enzymes in downstream DNA amplification. Here, this work overcomes this bottleneck by immobilizing the enzyme achromopeptidase (ACP) on nitrocellulose, to both store and enable the separation of the enzymes from the other steps. This work provides proof-of-concept that immobilized ACP is effective at lysis and release of amplifiable DNA from gram-positive Staphylococcus epidermidis and enables the use of the lysate directly for DNA amplification, without the need for heat deactivation of the enzyme. This sample preparation method requires only incubation at 37 °C and mild agitation, which allows to implement it with fully disposable and affordable equipment. Consequently, this work enables to combine the paper-based DNA extraction method with the isothermal recombinase polymerase amplification (RPA) followed by lateral flow detection to demonstrate a sample-to-answer NAAT packaged as an instrument free self-test kit expanding the capabilities of home-testing beyond antigen tests. 

The realization of electrochemical nucleic acid amplification tests (NAATs) at the point of care (POC) is highly desirable, but it remains a challenge given their high cost and lack of true portability/miniaturization. Here we show that mass-produced, industrial standardized, printed circuit boards (PCBs) can be repurposed to act as near-zero cost electrodes for self-assembled monolayer-based DNA biosensing, and further integration with a custom-designed and low-cost portable potentiostat. To show the analytical capability of this system, we developed a NAAT using isothermal recombinase polymerase amplification, bypassing the need of thermal cyclers, followed by an electrochemical readout relying on a sandwich hybridization assay. We used our sensor and device for analytical detection of the toxic microalgae Ostreopsis cf. ovata as a proof of concept. This work shows the potential of PCBs and open-source electronics to be used as powerful POC DNA biosensors at a low-cost. 

Nucleic Acid Amplification Tests (NAATs) with PCR technology to amplify DNA, are the golden standard for infectious disease diagnostics, but they require benchtop instruments and trained users to be performed. For this reason, we all had to send PCR test to centralized laboratories during the Covid-19 pandemic. A year into the pandemic, home-based antigen paper-based tests became available for Covid, but these were not as sensitive, so PCR tests had to be used still. This development emphasized the need for technologies that enable NAATs with superior sensitivity to be performed at home. There are three technological advanced that could make such tests possible: 1)  Paper based devices, called paper microfluidics, have been developed to enable more advanced steps of testing without laboratory equipment. These paper-based system incorporate advanced functionality and multiple reaction steps. 2) New DNA amplification techniques, called isothermal amplification, have been developed which, contrary to PCR, can be run without a thermocycler, enabling DNA amplification to be carried out even inside a paper.  3) Several methods to detect DNA have been shown using paper.

One step that is still largely unsolved in NAATs is the sample preparation step, hindering the development of fully paper-based NAATs. In sample preparation, nucleic acids are extracted from bacteria or virus, usually using reagents harmful to DNA amplification. These steps are thereofore complicated and require several washing steps and heating, and are therefore difficult to integrate into paper.

In this thesis, we used a simple, cost-effective, and scalable method to incorporate sample preparation in paper, thus taking NAATs towards point of care. We solve this problem by immobilizing enzymes that are used for sample preparation on nitrocellulose paper. The immobilized enzymes remain functional and can be used for biochemical reactions, while they are strongly bound to the paper. This method enables the separation of these enzymes from the sample, protecting downstream sensitive reactions of DNA amplification and eliminates the need for high temperature deactivation or washing steps. Specifically, we show that the enzyme achromopeptidase can do cell lysis from the Staphylococcus epidermidis bacteria, a common pathogenic gram-positive bacterium, and use its DNA in further reaction to perform a sample-to-answer paper-based NAAT. These NAATs employed a low temperature amplification step called Recombinase Polymerase Amplification (RPA) and DNA detection with a lateral flow strip.

We further show the enzyme proteinase K, also immobilized on paper, can digest RNase in saliva samples, an enzyme that breaks down RNA leading to false-negative results. This results enabled an easy sample preparation step towards saliva viral DNA self-testing.

Finally, in this work we developed a paper microfluidic system that can carry out an enzyme-linked oligonucleotide assay, which demonstrated much higher sensitivity in detecting amplified DNA than conventional lateral flow assays. In summary, these results provide solutions towards high-performing, affordable and instrument-free paper-based NAATs home-testing.

DNA tester också kallade NAAT tester, utförs idag med PCR-teknik, som först detekterar en specifik DNA och sedan replikerar den för detektion av infektionssjukdomar. PCR tester kräver dock instrument och utbildade personal. Av denna anledning var vi alla tvungna att skicka PCR-test till centraliserade laboratorier under COVID-19-pandemin. Ett år in i pandemin blev antigen pappersbaserade tester tillgängliga för hemtestning, men dessa var fortfarande inte tillräckligt känsliga och PCR-testerna behövde fortfarande användas. Denna utveckling visade behovet av att tillgängliggöra NAAT för hemmabruk. Det har på senare tid gjorts tre tekniska framsteg som för oss närmare sådana tester:_1) Pappersbaserade tester, så kallade pappers mikrofluidik, har utvecklats, för att möjliggöra mer avancerade tester. Dessa pappersbaserade system innehåller avancerad funktionalitet och flera reaktionssteg. 2) Nya DNA-amplifieringstekniker som kallas isotermisk amplifiering har utvecklats. Dessa kan i motsats till PCR köras utan maskiner, under konstant temperatur, och därmed blir DNA-amplifiering möjlig att utföra även inuti ett papper. 3) Flera metoder för att detektera DNA-metoder har visats med papper.

Ett steg som fortfarande inte är helt löst är provberedningssteget för NAAT. I provberedningen extraheras nukleinsyror från bakterier eller virus, vanligtvis genom användning av reagenser som är förstörr DNA-amplifiering. Provbredning är därför komplicerade. Dom kräver flera tvättsteg och uppvärmning och är därför svåra att integrera i papper.

I det här arbetet använde vi en enkel, kostnadseffektiv och skalbar metod för att integrera provberedning i papper. Våra resultat tar oss ett steg närmare DNA självtestning. Vi löser problemet med provbredning genom att immobilisera relevanta enzymer på nitrocellulosapapper. Dessa immobiliserade enzymer förblir funktionella och kan sedan utföra biokemiska reaktioner, samtidigt som de är starkt bundna till papperet. Denna metod möjliggör separation av provbredningssenzymer från, andra känsliga reaktioner såsom DNA-amplifiering. Samtidigt elimineras behovet av högtemperaturdeaktivering eller tvättsteg. Vi visar specifikt att enzymet akromopeptidas kan ta sönder cellväggen för att extrahera DNA hos Staphylococcus-epidermidis bakterien, en vanlig patogen, och vi kan sedan direkt använda bakteriens DNA i ytterligare reaktion för att utföra en prov-till-svar pappersbaserad DNA test. Dessa tester använder lågtemperatur för amplifieringssteget med en teknik som kallas Recombinase Polymerase Amplification (RPA) och vi utför DNA-detektion med en paperbaserad flödesremsa.

Vi visar vidare att enzymet proteinas K också kan immobiliseras på papper för att sedan förstora RNas:er, ett enzym som bryter ner RNA och som finns i humana salivprover. Möjligheten att använda papper för att ta bort RNAs från saliv öppnar vägen mot självtestning av salivvirus-DNA, som influenza virus.

Slutligen, utvecklade vi ett pappersmikrofluidik system som kunde utföra en enzymkopplad oligonukleotid test. Denna test visar mycket högre känslighet för detektering av amplifierat DNA än vad en kommersiell paperbaserad flödesremsa visar. 

Sammanfattningsvis har denna avhandlig visat några olika lösningar som för oss närmare prisvärda och instrumentfria pappersbaserade DNA tester för hemanvändning.

Nucleic acid amplification tests (NAATs) are very sensitive and specific methods, but they mainly rely on centralized laboratories and therefore are not suitable for point-of-care testing. Here, we present a 3D microfluidic paper-based electrochemical NAAT. These devices use off-the-shelf gold plasma-coated threads to integrate electroanalytical readouts using ex situ self-assembled monolayer formation on the threads prior to assembling into the paper device. They further include a sandwich hybridization assay with sample incubation, rinsing, and detection steps all integrated using movable stacks of filter papers to allow time-sequenced reactions. The devices use glass fiber substrates for storing recombinase polymerase amplification reagents and conducting the isothermal amplification. We used the paper-based device for the detection of the toxic microalgae Ostreopsis cf. ovata. The NAAT, completed in 95 min, attained a limit of detection of 0.06 pM target synthetic DNA and was able to detect 1 ng/mu L O. cf. ovata genomic DNA with negligible cross-reactivity from a closely related microalgae species. We think that the integration of thread electrodes within paper-based devices paves the way for digital one-time use NAATs and numerous other advanced electroanalytical paper- or textile-based devices.

Enzymes are the cornerstone of modern biotechnology. Achromopeptidase (ACP) is a well-known enzyme that hydrolyzes a number of proteins, notably proteins on the surface of Gram-positive bacteria. It is therefore used for sample preparation in nucleic acid tests. However, ACP inhibits DNA amplification which makes its integration difficult. Heat is commonly used to inactivate ACP, but it can be challenging to integrate heating into point-of-care devices. Here, we use recombinase polymerase amplification (RPA) together with ACP, and show that when ACP is immobilized on nitrocellulose paper, it retains its enzymatic function and can easily and rapidly be activated using agitation. The nitrocellulose-bound ACP does, however, not leak into the solution, preventing the need for deactivation through heat or by other means. Nitrocellulose-bound ACP thus opens new possibilities for paper-based Point-of-Care (POC) devices.

Nucleic acid tests integrated into digital point-of-care (POC) diagnostic systems have great potential for the future of health care. However, current methods of DNA amplification and detection require bulky and expensive equipment, many steps, and long process times, which complicate their integration into POC devices. We have combined an isothermal DNA amplification method, recombinase polymerase amplification, with an electrochemical stem-loop (S-L) probe DNA detection technique. By combining these methods, we have created a system that is able to specifically amplify and detect as few as 10 copies/mu L Staphylococcus epidermidis DNA with a total time to result of 70-75 min.