| 1. |
- Ben Aissa, Alejandra, et al.
(författare)
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Electrochemical Genosensing of E. coli Based on Padlock Probes and Rolling Circle Amplification
- 2021
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Ingår i: Sensors. - : MDPI AG. - 1424-8220. ; 21:5
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Tidskriftsartikel (refereegranskat)abstract
- Isothermal amplification techniques are emerging nowadays for the rapid and accurate detection of pathogenic bacteria in low resource settings, where many infectious diseases are endemic, and the lack of reliable power supply, trained personnel and specialized facilities pose critical barriers for timely diagnosis. This work addresses the detection of E. coli based on DNA isothermal amplification performed on magnetic particles (MPs) followed by electrochemical genosensing on disposable electrodes by square-wave voltammetry. In this approach, the bacterial DNA is preconcentrated using a target-specific magnetic probe and then amplified on the MPs by rolling circle amplification (RCA). Two different electrochemical readout methods for the RCA amplicons are tested. The first one relied on the labelling of the magnetic RCA product with a digoxigenin probe followed by the incubation with antiDIG-HRP antibody as electrochemical reporter. In the second case, the direct detection with an HRP-probe was performed. This latter strategy showed an improved analytical performance, while simultaneously avoiding the use of thermocyclers or bulky bench top equipment.
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| 2. |
- Ciftci, Sibel, et al.
(författare)
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A novel mutation tolerant padlock probe design for multiplexed detection of hypervariable RNA viruses
- 2019
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 3. |
- Ciftci, Sibel, et al.
(författare)
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Digital Rolling Circle Amplification-Based Detection of Ebola and Other Tropical Viruses
- 2020
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Ingår i: Journal of Molecular Diagnostics. - : Elsevier BV. - 1525-1578 .- 1943-7811. ; 22:2, s. 272-283
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Tidskriftsartikel (refereegranskat)abstract
- Emerging tropical viruses have caused serious outbreaks during the recent years, such as Ebola virus (EBOV) in 2014 and the most recent in 2018 to 2019 in Congo. Thus, immediate diagnostic attention is demanded at the point of care in resource-limited settings, because the performance and the operational parameters of conventional EBOV testing are Limited. Especially, their sensitivity, specificity, and coverage of other tropical disease viruses make them unsuitable for diagnostic at the point of care. Here, a padlock probe (PLP)-based rolling circle amplification (RCA) method for the detection of EBOV is presented. For this, a set of PLPs, separately targeting the viral RNA and complementary RNA of all seven EBOV genes, was used in the RCA assay and validated on virus isolates from cell culture. The assay was then translated for testing clinical samples, and simultaneous detection of both EBOV RNA types was demonstrated. For increased sensitivity, the RCA products were enriched on a simple and pump-free microfluidic chip. Because PLPs and RCA are inherently multiplexable, we demonstrate the extension of the probe panel for the simultaneous detection of the tropical viruses Ebola, Zika, and Dengue. The demonstrated high specificity, sensitivity, and multiplexing capability in combination with the digital quantification rendered the assay a promising diagnostic tool toward tropical virus detection at the point of care.
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| 4. |
- Ciftci, Sibel, 1987-, et al.
(författare)
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Multiplexed rolling circle amplification detection of Ebola, Zika and Dengue towards point-of-care diagnostics
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 5. |
- Ciftci, S., et al.
(författare)
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The sweet detection of rolling circle amplification : Glucose-based electrochemical genosensor for the detection of viral nucleic acid
- 2020
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Ingår i: Biosensors & bioelectronics. - : Elsevier Ltd. - 0956-5663 .- 1873-4235. ; 151
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Tidskriftsartikel (refereegranskat)abstract
- Herein, an isothermal padlock probe-based assay for the simple and portable detection of pathogens coupled with a glucose oxidase (GOx)-based electrochemical readout is reported. Infectious diseases remain a constant threat on a global scale, as in recurring pandemics. Rapid and portable diagnostics hold the promise to tackle the spreading of diseases and decentralising 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. The design of the demonstrated assay consists of a rolling circle amplification (RCA) technique, responsible for the generation of nucleic acid amplicons as RCA products (RCPs). The RCPs are generated on magnetic beads (MB) and subsequently, connected via streptavidin-biotin bonds to GOx. The enzymatic catalysis of glucose by the bound GOx allows for an indirect electrochemical measurement of the DNA target. The RCPs generated on the surface of the MB were confirmed by scanning electron microscopy, and among other experimental conditions such as the type of buffer, temperature, concentration of GOx, sampling and measurement time were evaluated for the optimum electrochemical detection. Accordingly, 125 μg mL−1 of GOx with 5 mM glucose using phosphate buffer saline (PBS), monitored for 1 min were selected as the ideal conditions. Finally, we assessed the analytical performance of the biosensing strategy by using clinical samples of Ebola virus from patients. Overall, this work provides a proof-of-concept bioassay for simple and portable molecular diagnostics of emerging pathogens using electrochemical detection, especially in resource-limited settings.
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| 6. |
- Ciftci, Sibel, 1987-, et al.
(författare)
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The sweet detection of rolling circle amplification : Glucose-based electrochemical detection of virus nucleic acid
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 7. |
- Guo, Maoxiang, et al.
(författare)
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Cross-membrane electrical detection of DNA
- 2017
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Ingår i: Proceedings 21st International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2017. - : The Chemical and Biological Microsystems Society (CBMS).
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Konferensbidrag (refereegranskat)abstract
- We introduce out-of-plane metallic nanowire formation on DNA templates, which are stretched through a porous membrane by applying a receding meniscus interface. We demonstrate the direct electrical detection of DNA using these gold nanowire bridges between the membrane’s opposite surfaces. Such a simple electrical readout can be extended for biosensor applications, thanks to the high specificity and multiplexing offered by Rolling Circle Amplification (RCA).
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| 8. |
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| 9. |
- Guo, Maoxiang, et al.
(författare)
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Efficient DNA-assisted synthesis of trans-membrane gold nanowires
- 2018
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Ingår i: microsystems and nanoengineering. - : Springer Science and Business Media LLC. - 2055-7434. ; 4
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Tidskriftsartikel (refereegranskat)abstract
- Whereas electric circuits and surface-based (bio) chemical sensors are mostly constructed in-plane due to ease of manufacturing, 3D microscale and nanoscale structures allow denser integration of electronic components and improved mass transport of the analyte to (bio) chemical sensor surfaces. This work reports the first out-of-plane metallic nanowire formation based on stretching of DNA through a porous membrane. We use rolling circle amplification (RCA) to generate long single-stranded DNA concatemers with one end anchored to the surface. The DNA strands are stretched through the pores in the membrane during liquid removal by forced convection. Because the liquid-air interface movement across the membrane occurs in every pore, DNA stretching across the membrane is highly efficient. The stretched DNA molecules are transformed into trans-membrane gold nanowires through gold nanoparticle hybridization and gold enhancement chemistry. A 50 fM oligonucleotide concentration, a value two orders of magnitude lower than previously reported for flat surface-based nanowire formation, was sufficient for nanowire formation. We observed nanowires in up to 2.7% of the membrane pores, leading to an across-membrane electrical conductivity reduction from open circuit to <20 Omega. The simple electrical read-out offers a high signal-to-noise ratio and can also be extended for use as a biosensor due to the high specificity and scope for multiplexing offered by RCA.
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| 10. |
- Hernández-Neuta, Iván, et al.
(författare)
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Smartphone-based clinical diagnostics : towards democratization of evidence-based health care
- 2019
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Ingår i: Journal of Internal Medicine. - : Wiley. - 0954-6820 .- 1365-2796. ; 285:1, s. 19-39
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Forskningsöversikt (refereegranskat)abstract
- 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.
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