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  • 1.
    Ciftci, Sibel
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Neumann, Felix
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Abdurahman, Samir
    Appelberg, Sofia
    Mirazimi, Ali
    Madaboosi, Narayanan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Multiplexed rolling circle amplification detection of Ebola, Zika and Dengue towards point-of-care diagnosticsManuscript (preprint) (Other academic)
    Abstract [en]

    Emerging tropical viruses have caused serious outbreaks during the recent years, such as Ebola virus (EBOV) in 2014 and the most recent 2018-19 outbreak in Congo. Immediate diagnostic attention is demanded, and most importantly at the point-of-care in resource-limited settings. The performance and the operational parameters of conventional EBOV testing are limited by either their sensitivity, specificity, or both, and often do not cover other tropical disease viruses. We present a padlock probe (PLP)-based rolling circle amplification (RCA) method for the detection of EBOV from cell culture isolates as well as clinical samples obtained from patients of West Africa outbreak. For this, a set of PLPs, separately targeting the vRNA and cRNA of all the seven genes of EBOV, were used in the RCA and validated on virus isolates from cell culture. The assay was then translated for testing clinical samples, and simultaneous duplex detection of both EBOV vRNA and cRNA was demonstrated. For increased sensitivity, the RCA products were enriched on a simple and pump-free microfluidic chip. As PLPs and RCA are inherently mulitplexable, we demonstrate the extension of the probe panel to the simultaneous detection of the tropical viruses Ebola, Zika and Dengue. The simple, rapid, specific and multiplexable isothermal assay developed for tropical virus detection suits the point-of-care needs, bringing RCA a step closer to bedside diagnostics.

  • 2.
    Ciftci, Sibel
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Neumann, Felix
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Hernández-Neuta, Iván
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Hakhverdyan, Mikhayil
    Bálint, Ádám
    Herthnek, David
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Madaboosi, Narayanan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    A novel mutation tolerant padlock probe design for multiplexed detection of hypervariable RNA viruses2019In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, article id 2872Article in journal (Refereed)
    Abstract [en]

    The establishment of a robust detection platform for RNA viruses still remains a challenge in molecular diagnostics due to their high mutation rates. Newcastle disease virus (NDV) is one such RNA avian virus with a hypervariable genome and multiple genotypes. Classical approaches like virus isolation, serology, immunoassays and RT-PCR are cumbersome, and limited in terms of specificity and sensitivity. Padlock probes (PLPs) are known for allowing the detection of multiple nucleic acid targets with high specificity, and in combination with Rolling circle amplification (RCA) have permitted the development of versatile pathogen detection assays. In this work, we aimed to detect hypervariable viruses by developing a novel PLP design strategy capable of tolerating mutations while preserving high specificity by targeting several moderately conserved regions and using degenerate bases. For this, we designed nine padlock probes based on the alignment of 335 sequences covering both Class I and II NDV. Our PLP design showed high coverage and specificity for the detection of eight out of ten reported genotypes of Class II NDV field isolated strains, yielding a detection limit of less than ten copies of viral RNA. Further taking advantage of the multiplex capability of PLPs, we successfully extended the assay for the simultaneous detection of three poultry RNA viruses (NDV, IBV and AIV) and combined it with a paper based microfluidic enrichment read-out for digital quantification. In summary, our novel PLP design addresses the current issue of tolerating mutations of highly emerging virus strains with high sensitivity and specificity.

  • 3.
    Ciftci, Sibel
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Neumann, Felix
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Paulraj, Thomas
    Crespo, Gaston
    Madaboosi, Narayanan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    The sweet detection of rolling circle amplification: Glucose-based electrochemical detection of virus nucleic acidManuscript (preprint) (Other academic)
    Abstract [en]

    Infectious diseases remain a constant threat on a global scale by recurring pandemics. Rapid and portable diagnostics hold the promise to tackle the spreading of diseases and decentralizing healthcare to point-of-care needs. Ebola, a hypervariable RNA virus causing fatalities of up to 90% for recent outbreaks in Africa, demands immediate attention for bedside diagnostics. Nucleic acid amplification technology (NAAT) has proven to be a powerful tool for the control of outbreak with high sensitivity and specificity. However, NAAT is mostly based on amplification methods that require specialized instrumentation and trained personnel, such as PCR with sophisticated detectors. Here, we present an isothermal padlock probe-based assay for the detection of pathogens coupled with a glucose oxidase (GOx)-based electrochemical approach as the read-out. The assay design is based on rolling circle amplification (RCA) upon magnetic beads, connecting the RCA products (RCPs) via streptavidin-biotin bridges to GOx needed for the electrochemical measurement with externally provided glucose. The RCPs forming on the surface of beads are imaged using scanning electron microscopy, and the presence of the GOx to the RCP complex is confirmed using atomic force microscopy. Parameters such as the choice of buffers, concentrations of glucose and GOx and measurement time were optimized, as well as the mode of addition of glucose was tested. 125 μg/mL of GOx with 5 mM glucose using PBS as washing buffer, monitored for 15 min were chosen as the optimized conditions. The effect of temperature was tested and found to be critical at 37 °C for enhanced performance of the sensor. Finally, we evaluate the analytical performance of our sensor system by using cell culture isolate and clinical samples of Ebola virus. The study provides a proof-of-concept of simple and portable molecular diagnostics for emerging pathogens, beneficial especially for resource-limited settings. 

  • 4.
    Hernández-Neuta, Iván
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Neumann, Felix
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Brightmeyer, J.
    Tis, T. Ba
    Madaboosi, Narayanan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Wei, Q.
    Ozcan, A.
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Smartphone-based clinical diagnostics: towards democratization of evidence-based health care2019In: Journal of Internal Medicine, ISSN 0954-6820, E-ISSN 1365-2796, Vol. 285, no 1, p. 19-39Article, review/survey (Refereed)
    Abstract [en]

    Recent advancements in bioanalytical techniques have led to the development of novel and robust diagnostic approaches that hold promise for providing optimal patient treatment, guiding prevention programs and widening the scope of personalized medicine. However, these advanced diagnostic techniques are still complex, expensive and limited to centralized healthcare facilities or research laboratories. This significantly hinders the use of evidence-based diagnostics for resource-limited settings and the primary care, thus creating a gap between healthcare providers and patients, leaving these populations without access to precision and quality medicine. Smartphone-based imaging and sensing platforms are emerging as promising alternatives for bridging this gap and decentralizing diagnostic tests offering practical features such as portability, cost-effectiveness and connectivity. Moreover, towards simplifying and automating bioanalytical techniques, biosensors and lab-on-a-chip technologies have become essential to interface and integrate these assays, bringing together the high precision and sensitivity of diagnostic techniques with the connectivity and computational power of smartphones. Here, we provide an overview of the emerging field of clinical smartphone diagnostics and its contributing technologies, as well as their wide range of areas of application, which span from haematology to digital pathology and rapid infectious disease diagnostics.

  • 5.
    Neumann, Felix
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Hernández-Neuta, Iván
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Grabbe, Malin
    Madaboosi, Narayanan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Albert, Jan
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Padlock Probe Assay for Detection and Subtyping of Seasonal Influenza2018In: Clinical Chemistry, ISSN 0009-9147, E-ISSN 1530-8561, Vol. 64, no 12, p. 1704-1712Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Influenza remains a constant threat worldwide, and WHO estimates that it affects 5% to 15% of the global population each season, with an associated 3 to 5 million severe cases and up to 500000 deaths. To limit the morbidity and the economic burden of influenza, improved diagnostic assays are needed. METHODS: We developed a multiplexed assay for the detection and subtyping of seasonal influenza based on padlock probes and rolling circle amplification. The assay simultaneously targets all 8 genome segments of the 4 circulating influenza variants-A(H1N1), A(H3N2), B/Yamagata, and B/Victoria-and was combined with a prototype cartridge for inexpensive digital quantification. Characterized virus isolates and patient nasopharyngeal swabs were used for assay design and analytical validation. The diagnostic performance was assessed by blinded testing of 50 clinical samples analyzed in parallel with a commercial influenza assay, Simplexa (TM) Flu A/B & RSV Direct. RESULTS: The assay had a detection limit of 18 viral RNA copies and achieved 100% analytical and clinical specificity for differential detection and subtyping of seasonal circulating influenza variants. The diagnostic sensitivity on the 50 clinical samples was 77.5% for detecting influenza and up to 73% for subtyping seasonal variants. CONCLUSIONS: We have presented a proof-of-concept padlock probe assay combined with an inexpensive digital read-out for the detection and subtyping of seasonal influenza strains A and B. The demonstrated high specificity and multiplexing capability, together with the digital quantification, established the assay as a promising diagnostic tool for seasonal influenza.

  • 6.
    Neumann, Felix
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Madaboosi, Narayanan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Hernández-Neuta, Iván
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Salas, Jeanpierre
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Ahlford, Annika
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Devyser AB, Sweden.
    Mecea, Vasile
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    QCM mass underestimation in molecular biotechnology: Proximity ligation assay for norovirus detection as a case study2018In: Sensors and actuators. B, Chemical, ISSN 0925-4005, E-ISSN 1873-3077, Vol. 273, p. 742-750Article in journal (Refereed)
    Abstract [en]

    The development of piezoelectric mass-sensitive devices is based on the shift in resonance frequency that is proportional to the deposited mass. However, this holds true only for small, rigid masses, while it can result in mass underestimation for heavy, non-rigid masses. In this work, we demonstrate this 'missing mass' phenomenon by measurement of high molecular weight biomolecules on a Quartz Crystal Microbalance (QCM) platform. For this, we present a model bioassay consisting of a sandwich-type proximity ligation assay for the detection of norovirus-like particles, and its real-time build-up on QCM as an experimental evidence. Upon combination with a localized QCM platform, we explain the pronounced slipping effect in multilayer biological systems resulting in energy dissipation and subsequent mass underestimation. This helps in pointing out the limitations of mega-gravity field sensors for molecular diagnostics where absolute quantification of pathogen load becomes indispensable towards biosensing applications.

  • 7. Soares, Ruben R. G.
    et al.
    Neumann, Felix
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Caneira, Catarina R. F.
    Madaboosi, Narayanan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Ciftci, Sibel
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Hernández-Neuta, Iván
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Pinto, Ines F.
    Santos, Denis R.
    Chu, Virginia
    Russom, Aman
    Conde, Joao P.
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Silica bead-based microfluidic device with integrated photodiodes for the rapid capture and detection of rolling circle amplification products in the femtomolar range2019In: Biosensors & bioelectronics, ISSN 0956-5663, E-ISSN 1873-4235, Vol. 128, p. 68-75Article in journal (Refereed)
    Abstract [en]

    The rapid and sensitive detection of specific nucleic acid sequences at the point-of-care (PoC) is becoming increasingly in demand for a variety of emergent biomedical applications ranging from infectious disease diagnostics to the screening of antimicrobial resistance. To meet such demand, considerable efforts have been invested towards the development of portable and integrated analytical devices combining microfluidics with miniaturized signal transducers. Here, we demonstrate the combination of rolling circle amplification (RCA)-based nucleic acid amplification with an on-chip size-selective trapping of amplicons on silica beads (similar to 8 nL capture chamber) coupled with a thin-film photodiode (200 x 200 mu m area) fluorescence readout. Parameters such as the flow rate of the amplicon solution and trapping time were optimized as well as the photodiode measurement settings, providing minimum detection limits below 0.5 fM of targeted nucleic acids and requiring only 5 mu L of pre-amplified sample. Finally, we evaluated the analytical performance of our approach by benchmarking it against a commercial instrument for RCA product (RCP) quantification and further investigated the effect of the number of RCA cycles and elongation times (ranging from 10 to 120 min). Moreover, we provide a demonstration of the application for diagnostic purposes by detecting RNA from influenza and Ebola viruses, thus highlighting its suitability for integrated PoC systems.

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