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  • 1.
    Chondrogiannis, Georgios
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Fibre Technology. KTH Royal Institute of Technology.
    Sample-to-answer paper-based nucleic acid amplification tests2022Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    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.

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  • 2.
    Chondrogiannis, Georgios
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Fibre Technology.
    Khaliliazar, Shirin
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Fibre Technology.
    Toldrà Filella, Anna
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Fibre Technology.
    Reu, Pedro
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Fibre Technology.
    Hamedi, Mahiar
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Fibre Technology.
    Nitrocellulose-bound achromopeptidase for point-of-care nucleic acid tests2021In: Scientific Reports, E-ISSN 2045-2322, Vol. 11, no 1, article id 6140Article in journal (Refereed)
    Abstract [en]

    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.

  • 3.
    Chondrogiannis, Georgios
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology.
    Reu, Pedro
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology.
    Hamedi, Mahiar
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center.
    Paper-Based Bacterial Lysis Enables Sample-to-Answer Home-based DNA Testing2023In: Advanced Materials Technologies, E-ISSN 2365-709X, Vol. 8, no 4, p. 2201004-, article id 2201004Article in journal (Refereed)
    Abstract [en]

    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. 

  • 4.
    Chondrogiannis, Georgios
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Fibre Technology.
    Reu, Pedro
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Fibre Technology.
    Hamedi, Mahiar
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Fibre Technology.
    Paper‐Based Bacterial Lysis Enables Sample‐to‐Answer Home‐based DNATestingManuscript (preprint) (Other academic)
  • 5.
    Chondrogiannis, Georgios
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Fibre Technology.
    Toldrà Filella, Anna
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Fibre Technology.
    Hanze, Martin
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Fibre Technology.
    Paper‐based RNase digestion towards viral nucleic acid self‐testsManuscript (preprint) (Other academic)
  • 6.
    Khaliliazar, Shirin
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Fibre Technology.
    Chondrogiannis, Georgios
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Fibre Technology.
    Hamedi, Mahiar
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Fibre Technology.
    Electroanalytical Paper based Nucleic Acid Amplification Tests with Integrated Thread ElectrodesArticle in journal (Refereed)
  • 7.
    Khaliliazar, Shirin
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Fibre Technology.
    Ouyang, Liangqi
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Fibre Technology.
    Piper, Andrew
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Fibre Technology.
    Chondrogiannis, Georgios
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Fibre Technology.
    Hanze, Martin
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Fibre Technology.
    Herland, Anna
    KTH, School of Electrical Engineering and Computer Science (EECS), Intelligent systems, Micro and Nanosystems.
    Hamedi, Mahiar
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Fibre Technology.
    Electrochemical Detection of Genomic DNA Utilizing Recombinase Polymerase Amplification and Stem-Loop Probe2020In: ACS Omega, E-ISSN 2470-1343, Vol. 5, no 21, p. 12103-12109Article in journal (Refereed)
    Abstract [en]

    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.

  • 8.
    Khaliliazar, Shirin
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Fibre Technology.
    Toldrà, Anna
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Fibre Technology.
    Chondrogiannis, Georgios
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Fibre Technology.
    Hamedi, Mahiar
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Fibre Technology.
    Electroanalytical Paper-Based Nucleic Acid Amplification Biosensors with Integrated Thread Electrodes2021In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 93, no 42, p. 14187-14195Article in journal (Refereed)
    Abstract [en]

    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.

  • 9.
    Toldrà Filella, Anna
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Fibre Technology.
    Ainla, Alar
    International Iberian Nanotechnology Laboratory, Braga, 4715-330, Portugal.
    Khaliliazar, Shirin
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Fibre Technology.
    Landin, Roman
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Fibre Technology.
    Chondrogiannis, Georgios
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Fibre Technology.
    Hanze, Martin
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Fibre Technology.
    Reu, Pedro
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH).
    Hamedi, Mahiar
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Fibre Technology.
    Portable electroanalytical nucleic acid amplification tests using printed circuit boards and open-source electronics2022In: The Analyst, ISSN 0003-2654, E-ISSN 1364-5528, Vol. 147, no 19, p. 4249-4256Article in journal (Refereed)
    Abstract [en]

    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. 

  • 10.
    Toldrà Filella, Anna
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Fibre Technology.
    Chondrogiannis, Georgios
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Fibre Technology.
    Hamedi, Mahiar
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Fibre Technology.
    3D paper microfluidic devices for enzyme‐linked assays, and itsapplication to DNA analysisManuscript (preprint) (Other academic)
  • 11.
    Toldrà Filella, Anna
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Fibre Technology.
    Chondrogiannis, Georgios
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Fibre Technology.
    Hamedi, Mahiar
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Fibre Technology.
    A 3D paper microfluidic device for enzyme-linked assays: Application to DNA analysis2023In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 18, no 9, article id 2300143Article in journal (Refereed)
    Abstract [en]

    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.

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