| 1. |
- Iyengar, Sharath Narayana
(author)
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Novel microfluidic based sample preparation methods for rapid separation and detection of viable bacteria from blood for sepsis diagnostics
- 2021
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Doctoral thesis (other academic/artistic)abstract
- Sepsis is a serious medical condition characterized by a whole-body inflammatory response caused by bloodstream infection. The final stage of sepsis can lead to septic shock, multiple organ failure, and death. In early sepsis, the concentration of bacteria in the bloodstream is typically low, making diagnosis challenging. Rapid diagnosis of sepsis is crucial as there is an exponential increase in mortality for every hour delay in the appropriate antibiotics administration. Common culture-based methods fail in fast bacteria determination as it takes up to 24-72 hr. On the other hand, recent rapid nucleic acid-based diagnostic methods are prone to false positives from human DNA mainly due to a lack of efficient sample preparation methods. This Ph.D. work was aimed at the development of novel sample preparation methods for rapid and efficient separation and identification of bacteria from blood for sepsis diagnostics. To address this, two different approaches were explored. In the first approach, a label-free, size-based, passive elasto-inertial microfluidics (visco-elastic flows) method was developed (Paper I and II). Initially, behavior of particles were studied in solution containing polyethylene oxide (PEO) using different spiral designs (Paper I). By using the knowledge from paper I, a spiral design was used to preposition the particles at the outer wall of the inlet using PEO as sheath and we showed that a particle of a certain size remains fully focused at the outer wall throughout the channel length. The optimized parameters were extended to demonstrate that when bacteria is spiked into diluted blood, blood remains fully focused at the outer wall throughout the channel length while smaller bacteria differentially migrate towards the inner wall for rapid separation. Using E.coli spiked into the diluted blood sample, bacteria separation is demonstrated at an efficiency of 82 to 90% depending on the blood dilution using a single spiral chip (Paper-II). The second approach (Paper III) involves a selective cell lysis method where lysis buffer composition is optimized to selectively lyse blood cells in 5 min while maintaining bacterial viability. The lysed blood cells were filtered through a filter paper to capture viable bacteria. The captured bacteria on the filter paper was detected using Prussian blue (PB) colorimetric analysis. In PB color-based assay, viable bacteria metabolically reduce iron (III) complexes, initiating a photo-catalytic cascade toward PB formation on the filter paper visible to the naked eye. Using this approach it was possible to detect bacteria by the naked eye. This approach was also further optimized to perform antibiotic susceptibility testing to determine the minimum inhibitory concentration (MIC). Furthermore, as a step towards rapid genomic analysis, a novel method combining ITP-RCA (Isotachophoresis – Rolling Circle Amplification) was studied and optimized for real-time amplification (RCA), focusing and detection of bacterial DNA in a microfluidic channel (Paper IV). In this study we demonstrate rapid and increased sensitivity of bacterial DNA detection. This method has a huge potential to accelerate the time needed for DNA based analysis for infectious diseases. All in all, the ability of these sample preparation methods for rapid and effective separation and detection of key pathogens in blood will help in decreasing the time of sepsis diagnosis and aid towards efficient phenotypic or genotypic analysis.
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| 2. |
- Abbasi Aval, Negar, et al.
(author)
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Assessing the Layer-by-Layer Assembly of Cellulose Nanofibrils and Polyelectrolytes in Pancreatic Tumor Spheroid Formation
- 2023
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In: Biomedicines. - : MDPI AG. - 2227-9059. ; 11:11
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Journal article (peer-reviewed)abstract
- Three-dimensional (3D) tumor spheroids are regarded as promising models for utilization as preclinical assessments of chemo-sensitivity. However, the creation of these tumor spheroids presents challenges, given that not all tumor cell lines are able to form consistent and regular spheroids. In this context, we have developed a novel layer-by-layer coating of cellulose nanofibril–polyelectrolyte bilayers for the generation of spheroids. This technique builds bilayers of cellulose nanofibrils and polyelectrolytes and is used here to coat two distinct 96-well plate types: nontreated/non-sterilized and Nunclon Delta. In this work, we optimized the protocol aimed at generating and characterizing spheroids on difficult-to-grow pancreatic tumor cell lines. Here, diverse parameters were explored, encompassing the bilayer count (five and ten) and multiple cell-seeding concentrations (10, 100, 200, 500, and 1000 cells per well), using four pancreatic tumor cell lines—KPCT, PANC-1, MiaPaCa-2, and CFPAC-I. The evaluation includes the quantification (number of spheroids, size, and morphology) and proliferation of the produced spheroids, as well as an assessment of their viability. Notably, our findings reveal a significant influence from both the number of bilayers and the plate type used on the successful formation of spheroids. The novel and simple layer-by-layer-based coating method has the potential to offer the large-scale production of spheroids across a spectrum of tumor cell lines.
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| 3. |
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| 4. |
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| 5. |
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| 6. |
- Akhtar, Ahmad Saleem, et al.
(author)
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A portable and low-cost centrifugal microfluidic platform for multiplexed colorimetric detection of protein biomarkers
- 2023
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In: Analytica Chimica Acta. - : Elsevier BV. - 0003-2670 .- 1873-4324. ; 1245
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Journal article (peer-reviewed)abstract
- 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.
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| 7. |
- Akhtar, Ahmad Saleem, et al.
(author)
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An integrated centrifugal microfluidic platform for multiplexed colorimetric immunodetection of protein biomarkers in resource-limited settings
- 2021
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In: Proceedings MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. - : Chemical and Biological Microsystems Society. ; , s. 947-948
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Conference paper (peer-reviewed)abstract
- 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.
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| 8. |
- Akhtar, Ahmad Saleem, et al.
(author)
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Centrifugal microfluidic platform comprising an array of bead microcolumns for the multiplexed colorimetric quantification of inflammatory biomarkers at the point-of-care
- 2019
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In: 23rd International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2019. - : Chemical and Biological Microsystems Society. ; , s. 1230-1231
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Conference paper (peer-reviewed)abstract
- 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.
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| 9. |
- Akhtar, Ahmad Saleem, et al.
(author)
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Fully automated centrifugal microfluidic platform for COVID-19 detection using computer vision-based readout
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Other publication (other academic/artistic)abstract
- COVID-19 pandemic made it evident that the world is unprepared for effectively tackling a pandemic resulting from an infectious disease. The conventional diagnostic methods for detection of infectious diseases were limited to centralized laboratories. As the burden of testing increased with the spread of the disease, the centralized testing facilities were strained for resources and personnel. These problems were further exacerbated in low- and middle-income countries where the health and transport infrastructure are not very well developed. To overcome this reliance on centralized testing and to facilitate decentralized testing, focus was shifted towards development of novel point-of-care diagnostic methods. We report the development of a fully automated centrifugal microfluidic platform that uses loop mediated isothermal amplification (LAMP) combined with computer vision-based readout for COVID-19 detection. The integrated platform allows sample to answer analysis at the push of a single button and can process 26 samples in 40 minutes. The platform performs a completely automated assay protocol involving heating, rotation and detection without the need for user intervention. A limit of detection of approximately 100 RNA copies in 10 µL reaction was achieved using RNA fragments spiked in water and similar results were obtained for artificial saliva samples.
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| 10. |
- Aljadi, Zenib, et al.
(author)
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Layer-by-Layer Cellulose Nanofibrils : A New Coating Strategy for Development and Characterization of Tumor Spheroids as a Model for In Vitro Anticancer Drug Screening
- 2022
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In: Macromolecular Bioscience. - : Wiley. - 1616-5187 .- 1616-5195. ; 22:10
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Journal article (peer-reviewed)abstract
- Three-dimensional multicellular spheroids (MCSs) are complex structure of cellular aggregates and cell-to-matrix interaction that emulates the in-vivo microenvironment. This research field has grown to develop and improve spheroid generation techniques. Here, we present a new platform for spheroid generation using Layer-by-Layer (LbL) technology. Layer-by-Layer (LbL) containing cellulose nanofibrils (CNF) assemble on a standard 96 well plate. Various bi-layer numbers, multiple cell seeding concentration, and two tumor cell lines (HEK 293 T, HCT 116) are utilized to generate and characterize spheroids. The number and proliferation of generated spheroids, the viability, and the response to the anti-cancer drug are examined. The spheroids are formed and proliferated on the LbL-CNF coated wells with no significant difference in connection to the number of LbL-CNF bi-layers; however, the number of formed spheroids correlates positively with the cell seeding concentration (122 ± 17) and (42 ± 8) for HCT 116 and HEK 293T respectively at 700 cells ml−1. The spheroids proliferate progressively up to (309, 663) µm of HCT 116 and HEK 293T respectively on 5 bi-layers coated wells with maintaining viability. The (HCT 116) spheroids react to the anti-cancer drug. We demonstrate a new (LbL-CNF) coating strategy for spheroids generation, with high performance and efficiency to test anti-cancer drugs.
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| 11. |
- Bachmann, Till T., et al.
(author)
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Expert guidance on target product profile development for AMR diagnostic tests
- 2023
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In: BMJ Global Health. - : BMJ Publishing Group. - 2059-7908. ; 8:12
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Journal article (peer-reviewed)abstract
- Diagnostics are widely considered crucial in the fight against antimicrobial resistance (AMR), which is expected to kill 10 million people annually by 2030. Nevertheless, there remains a substantial gap between the need for AMR diagnostics versus their development and implementation. To help address this problem, target product profiles (TPP) have been developed to focus developers’ attention on the key aspects of AMR diagnostic tests. However, during discussion between a multisectoral working group of 51 international experts from industry, academia and healthcare, it was noted that specific AMR-related TPPs could be extended by incorporating the interdependencies between the key characteristics associated with the development of such TPPs. Subsequently, the working group identified 46 characteristics associated with six main categories (ie, Intended Use, Diagnostic Question, Test Description, Assay Protocol, Performance and Commercial). The interdependencies of these characteristics were then identified and mapped against each other to generate new insights for use by stakeholders. Specifically, it may not be possible for diagnostics developers to achieve all of the recommendations in every category of a TPP and this publication indicates how prioritising specific TPP characteristics during diagnostics development may influence (or not) a range of other TPP characteristics associated with the diagnostic. The use of such guidance, in conjunction with specific TPPs, could lead to more efficient AMR diagnostics development.
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| 12. |
- Baldasici, Oana, et al.
(author)
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The transcriptional landscape of cancer stem-like cell functionality in breast cancer
- 2024
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In: Journal of Translational Medicine. - : Springer Nature. - 1479-5876. ; 22:1, s. 530-
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Journal article (peer-reviewed)abstract
- BACKGROUND: Cancer stem-like cells (CSCs) have been extensively researched as the primary drivers of therapy resistance and tumor relapse in patients with breast cancer. However, due to lack of specific molecular markers, increased phenotypic plasticity and no clear clinicopathological features, the assessment of CSCs presence and functionality in solid tumors is challenging. While several potential markers, such as CD24/CD44, have been proposed, the extent to which they truly represent the stem cell potential of tumors or merely provide static snapshots is still a subject of controversy. Recent studies have highlighted the crucial role of the tumor microenvironment (TME) in influencing the CSC phenotype in breast cancer. The interplay between the tumor and TME induces significant changes in the cancer cell phenotype, leading to the acquisition of CSC characteristics, therapeutic resistance, and metastatic spread. Simultaneously, CSCs actively shape their microenvironment by evading immune surveillance and attracting stromal cells that support tumor progression. METHODS: In this study, we associated in vitro mammosphere formation assays with bulk tumor microarray profiling and deconvolution algorithms to map CSC functionality and the microenvironmental landscape in a large cohort of 125 breast tumors. RESULTS: We found that the TME score was a significant factor associated with CSC functionality. CSC-rich tumors were characterized by an immune-suppressed TME, while tumors devoid of CSC potential exhibited high immune infiltration and activation of pathways involved in the immune response. Gene expression analysis revealed IFNG, CXCR5, CD40LG, TBX21 and IL2RG to be associated with the CSC phenotype and also displayed prognostic value for patients with breast cancer. CONCLUSION: These results suggest that the characterization of CSCs content and functionality in tumors can be used as an attractive strategy to fine-tune treatments and guide clinical decisions to improve patients therapy response.
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| 13. |
- Banerjee, Indradumna, et al.
(author)
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Analogue tuning of particle focusing in elasto-inertial flow
- 2021
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In: Meccanica (Milano. Print). - : Springer Science and Business Media B.V.. - 0025-6455 .- 1572-9648. ; 56:7, s. 1739-1749
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Journal article (peer-reviewed)abstract
- We report a unique tuneable analogue trend in particle focusing in the laminar and weak viscoelastic regime of elasto-inertial flows. We observe experimentally that particles in circular cross-section microchannels can be tuned to any focusing bandwidths that lie between the “Segre-Silberberg annulus” and the centre of a circular microcapillary. We use direct numerical simulations to investigate this phenomenon and to understand how minute amounts of elasticity affect the focussing of particles at increasing flow rates. An Immersed Boundary Method is used to account for the presence of the particles and a FENE-P model is used to simulate the presence of polymers in a Non-Newtonian fluid. The numerical simulations study the dynamics and stability of finite size particles and are further used to analyse the particle behaviour at Reynolds numbers higher than what is allowed by the experimental setup. In particular, we are able to report the entire migration trajectories of the particles as they reach their final focussing positions and extend our predictions to other geometries such as the square cross section. We believe complex effects originate due to a combination of inertia and elasticity in the weakly viscoelastic regime, where neither inertia nor elasticity are able to mask each other’s effect completely, leading to a number of intermediate focusing positions. The present study provides a fundamental new understanding of particle focusing in weakly elastic and strongly inertial flows, whose findings can be exploited for potentially multiple microfluidics-based biological sorting applications.
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| 14. |
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| 15. |
- Damiati, Samar, et al.
(author)
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Flex Printed Circuit Board Implemented Grapene-Based DNA Sensor for Detection of SARS-CoV-2
- 2021
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In: IEEE Sensors Journal. - : Institute of Electrical and Electronics Engineers (IEEE). - 1530-437X .- 1558-1748. ; 21:12, s. 13060-13067
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Journal article (peer-reviewed)abstract
- 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.
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| 16. |
- Dietvorst, Jiri, et al.
(author)
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Bacteria Detection at a Single-Cell Level through a Cyanotype-Based Photochemical Reaction
- 2022
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In: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 94:2, s. 787-792
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Journal article (peer-reviewed)abstract
- The detection of living organisms at very low concentrations is necessary for the early diagnosis of bacterial infections, but it is still challenging as there is a need for signal amplification. Cell culture, nucleic acid amplification, or nano-structure-based signal enhancement are the most common amplification methods, relying on long, tedious, complex, or expensive procedures. Here, we present a cyanotype-based photochemical amplification reaction enabling the detection of low bacterial concentrations up to a single-cell level. Photocatalysis is induced with visible light and requires bacterial metabolism of iron-based compounds to produce Prussian Blue. Bacterial activity is thus detected through the formation of an observable blue precipitate within 3 h of the reaction, which corresponds to the concentration of living organisms. The short time-to-result and simplicity of the reaction are expected to strongly impact the clinical diagnosis of infectious diseases.
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| 17. |
- Etcheverry, Sebastián, et al.
(author)
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Optofludics in microstructured fibers combining particle elasto-inertial focusing and fluorescence
- 2016
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In: 2016 CONFERENCE ON LASERS AND ELECTRO-OPTICS (CLEO). - Washington, D.C. : IEEE conference proceedings. - 9781943580118
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Conference paper (peer-reviewed)abstract
- Optofluidics is exploited in an all-fiber component to detect and identify through fluorescence particles flowing at high rate and inertially focused in a capillary. The system represents a first step towards an in-fiber flow cytometer.
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| 18. |
- Harish, Achar Vasant, et al.
(author)
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Fiber Based Optofluidic Micro-Flow Cytometer Collecting Side-Scattered Light
- 2020
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In: Conference Proceedings - Lasers and Electro-Optics Society Annual Meeting-LEOS. - : Institute of Electrical and Electronics Engineers (IEEE).
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Conference paper (peer-reviewed)abstract
- A compact fiber capillary based microflow cytometer capable of detecting side-scattered-light is demonstrated by using a 45° angle-cleaved metal coated optical fiber tip.
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| 19. |
- Harish, Achar Vasant, et al.
(author)
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Lab-in-a-fiber microfluidic cytometer for point-of-care biomedical diagnostics
- 2021
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In: Proceedings MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. - : Chemical and Biological Microsystems Society. ; , s. 261-262
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Conference paper (peer-reviewed)abstract
- We describe a prototype of an all-fiber microfluidic cytometer capable of analyzing particles ranging from 2-20 µm in diameter. The portable all-fiber cytometer is fabricated using different diameter silica capillaries for flow of microparticles and optical fibers to deliver and collect light. As a proof of concept, we analyze mixed population of beads of three different sizes and classify them with our all-fiber cytometer.
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| 20. |
- Harish, Achar Vasant, et al.
(author)
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Optofluidic Fiber Component to Separate Micron-Sized Particles using Elasto-Inertial Focusing
- 2020
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In: Conference Proceedings - Lasers and Electro-Optics Society Annual Meeting-LEOS. - : Institute of Electrical and Electronics Engineers Inc..
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Conference paper (peer-reviewed)abstract
- Using various fiber capillaries with different diameters and multiple holes we develop an optofluidic component capable of separating micron-sized beads emulating cells and bacteria, exploiting particle focusing in a viscoelastic fluid and analyzed optically.
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| 21. |
- Iyengar, Sharath Narayana, et al.
(author)
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High resolution and rapid separation of bacteria from blood using elasto‐inertial microfluidics
- 2021
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In: Electrophoresis. - : Wiley. - 0173-0835 .- 1522-2683. ; 42:23, s. 2538-2551
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Journal article (other academic/artistic)abstract
- Improved sample preparation has the potential to address unmet needs for fast turnaroundsepsis tests. In this work, we report elasto-inertial based rapid bacteria separation from diluted blood at high separation efficiency. In viscoelastic flows, we demonstrate novel findings where blood cells prepositioned at the outer wall entering a spiral device remain fullyfocused throughout the channel length while smaller bacteria migrate to the opposite wall.Initially, using microparticles, we show that particles above a certain size cut-off remainfully focused at the outer wall while smaller particles differentially migrate toward the inner wall. We demonstrate particle separation at 1 μm resolution at a total throughput of1 mL/min. For blood-based experiments, a minimum of 1:2 dilution was necessary to fullyfocus blood cells at the outer wall. Finally, Escherichia coli spiked in diluted blood were continuously separated at a total flow rate of 1 mL/min, with efficiencies between 82 and 90%depending on the blood dilution. Using a single spiral, it takes 40 min to process 1 mLof blood at a separation efficiency of 82%. The label-free, passive, and rapid bacteria isolation method has a great potential for speeding up downstream phenotypic and genotypicanalysis.
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| 22. |
- Iyengar, Sharath Narayana, et al.
(author)
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Rapid detection of viable bacteria in whole blood for early sepsis diagnosis and suseptibility testing
- 2021
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In: Proceedings MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. - : Chemical and Biological Microsystems Society. ; , s. 791-792
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Conference paper (peer-reviewed)abstract
- Sepsis is a serious medical condition characterized by a whole-body inflammatory state caused by infection. Here, we present a sepsis method for rapid detection of bacteria from whole blood in less than 5h, combining selective blood cell lysis and a sensitive colorimetric based detection method. Selective cell lysis buffer allows selective rupture of blood cells (5 min), while maintaining bacteria 100% viable. Viable bacteria metabolically reduce iron (III) complexes, initiating a photo-catalytic cascade toward Prussian Blue formation visible to the naked eye. The method is finally validated for antibiotic susceptibility testing.
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| 23. |
- Iyengar, Sharath Narayana, et al.
(author)
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Toward Rapid Detection of Viable Bacteria in Whole Blood for Early Sepsis Diagnostics and Susceptibility Testing
- 2021
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In: ACS Sensors. - : American Chemical Society (ACS). - 2379-3694. ; 6:9, s. 3357-3366
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Journal article (peer-reviewed)abstract
- Sepsis is a serious bloodstream infection where the immunity of the host body is compromised, leading to organ failure and death of the patient. In early sepsis, the concentration of bacteria is very low and the time of diagnosis is very critical since mortality increases exponentially with every hour after infection. Common culture-based methods fail in fast bacteria determination, while recent rapid diagnostic methods are expensive and prone to false positives. In this work, we present a sepsis kit for fast detection of bacteria in whole blood, here achieved by combining selective cell lysis and a sensitive colorimetric approach detecting as low as 10(3) CFU/mL bacteria in less than 5 h. Homemade selective cell lysis buffer (combination of saponin and sodium cholate) allows fast processing of whole blood in 5 min while maintaining bacteria alive (100% viability). After filtration, retained bacteria on filter paper are incubated under constant illumination with the electrochromic precursors, i.e., ferricyanide and ferric ammonium citrate. Viable bacteria metabolically reduce iron(III) complexes, initiating a photocatalytic cascade toward Prussian blue formation. As a proof of concept, we combine this method with antibiotic susceptibility testing to determine the minimum inhibitory concentration (MIC) using two antibiotics (ampicillin and gentamicin). Although this kit is used to demonstrate its applicability to sepsis, this approach is expected to impact other key sectors such as hygiene evaluation, microbial contaminated food/beverage, or UTI, among others.
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| 24. |
- Kalm, Frida, et al.
(author)
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Adhesion molecule cross-linking and cytokine exposure modulate IgE- and non-IgE-dependent basophil activation
- 2021
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In: Immunology. - : Wiley. - 0019-2805 .- 1365-2567. ; 162:1, s. 92-104
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Journal article (peer-reviewed)abstract
- Basophils are known for their role in allergic inflammation, which makes them suitable targets in allergy diagnostics such as the basophil activation test (BAT) and the microfluidic immunoaffinity basophil activation test (miBAT). Beside their role in allergy, basophils have an immune modulatory role in both innate immunity and adaptive immunity. To accomplish this mission, basophils depend on the capability to migrate from blood to extravascular tissues, which includes interactions with endothelial cells, extracellular matrix and soluble mediators. Their receptor repertoire is well known, but less is known how these receptor–ligand interactions impact the degranulation process and the responsiveness to subsequent activation. As the consequences of these interactions are crucial to fully appreciate the role of basophils in immune modulation and to enable optimization of the miBAT, we explored how basophil activation status is regulated by cytokines and cross-linking of adhesion molecules. The expression of adhesion molecules and activation markers on basophils from healthy blood donors was analysed by flow cytometry. Cross-linking of CD203c, CD62L, CD11b and CD49d induced a significant upregulation of CD63 and CD203c. To mimic in vivo conditions, valid also for miBAT, CD62L and CD49d were cross-linked followed by IgE-dependent activation (anti-IgE), which caused a reduced CD63 expression compared with anti-IgE activation only. IL-3 and IL-33 priming caused increased CD63 expression after IgE-independent activation (fMLP). Together, our data suggest that mechanisms operational both in the microfluidic chip and in vivo during basophil adhesion may impact basophil anaphylactic and piecemeal degranulation procedures and hence their immune regulatory function.
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| 25. |
- Kalm, Frida, et al.
(author)
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Development and clinical testing of a microfluidic immunoaffinity basophil activation test for point-of-care allergy diagnosis
- 2019
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In: 23rd International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2019. - : Chemical and Biological Microsystems Society. ; , s. 657-658
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Conference paper (peer-reviewed)abstract
- The Basophil Activation Test (BAT) is a valuable allergy diagnostic tool but is time-consuming and requires skilled personnel, which has limited its clinical use. We therefore developed and clinically tested a microfluidic immunoaffinity BAT (miBAT) technique where we captured basophils directly from whole blood followed by in vitro activation and quantification of activation markers. For the first time basophils captured from whole blood, from both allergic patients and healthy donors, have been activated using allergens.
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| 26. |
- Khati, Vamakshi, et al.
(author)
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3D Bioprinting of Multi-Material Decellularized Liver Matrix Hydrogel at Physiological Temperatures
- 2022
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In: Biosensors. - : MDPI AG. - 2079-6374. ; 12:7
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Journal article (peer-reviewed)abstract
- Bioprinting is an acclaimed technique that allows the scaling of 3D architectures in an organized pattern but suffers from a scarcity of appropriate bioinks. Decellularized extracellular matrix (dECM) from xenogeneic species has garnered support as a biomaterial to promote tissue-specific regeneration and repair. The prospect of developing dECM-based 3D artificial tissue is impeded by its inherent low mechanical properties. In recent years, 3D bioprinting of dECM-based bioinks modified with additional scaffolds has advanced the development of load-bearing constructs. However, previous attempts using dECM were limited to low-temperature bioprinting, which is not favorable for a longer print duration with cells. Here, we report the development of a multi-material decellularized liver matrix (dLM) bioink reinforced with gelatin and polyethylene glycol to improve rheology, extrudability, and mechanical stability. This shear-thinning bioink facilitated extrusion-based bioprinting at 37 degrees C with HepG2 cells into a 3D grid structure with a further enhancement for long-term applications by enzymatic crosslinking with mushroom tyrosinase. The heavily crosslinked structure showed a 16-fold increase in viscosity (2.73 Pa s(-1)) and a 32-fold increase in storage modulus from the non-crosslinked dLM while retaining high cell viability (85-93%) and liver-specific functions. Our results show that the cytocompatible crosslinking of dLM bioink at physiological temperatures has promising applications for extended 3D-printing procedures.
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| 27. |
- Khati, Vamakshi, et al.
(author)
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A tunable decellularized liver-based hybrid bioink
- 2021
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In: MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. - : Chemical and Biological Microsystems Society. ; , s. 281-282
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Conference paper (peer-reviewed)abstract
- Decellularized extracellular matrix is a tissue-specific biomaterial that recapitulates the complexity of the inherent tissue environment to elicit cellular response. Here, a multi-material decellularized liver (dLM)-based bioink with gelatin is developed and polyethylene glycol crosslinking is used to enhance the viscoelasticity of the dLM. We evaluated the necessity of a post-printing secondary cross-linker mushroom tyrosinase to improve robustness and long term stability. We further demonstrate it's biocompatibility using liver specific gene analysis of HepG2 cells.
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| 28. |
- Khati, Vamakshi, et al.
(author)
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Development of robust sacrificial support construct with decellularized liver extracellular matrix
- 2022
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In: MicroTAS 2022. - : Chemical and Biological Microsystems Society. ; , s. 432-433
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Conference paper (peer-reviewed)abstract
- 3D bioprinting is an acclaimed technology to develop architecturally significant tissue models, however most bioinks require a secondary support structure to create a clinically relevant sized model. In this work, we develop a 3D sacrificial support structure of polyvinyl alcohol (PVA) with decellularized liver extracellular matrix (dECM) bioink with HepG2 cells. The PVA backbone imparts its 3D structure to dECM and dissolves in cell culture media. We evaluated the PVA dissolution using refractometry and microscopic observation. We further tested the crosslinking of dECM with a PEG-based crosslinker using scanning electron microscopy (SEM). Alamar blue assay and gene expression analysis results demonstrated an increase in cell proliferation within the 3D structure.
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| 29. |
- Khati, Vamakshi, et al.
(author)
-
Indirect 3D Bioprinting of a Robust Trilobular Hepatic Construct with Decellularized Liver Matrix Hydrogel
- 2022
-
In: Bioengineering. - : MDPI AG. - 2306-5354. ; 9:11, s. 603-603
-
Journal article (peer-reviewed)abstract
- The liver exhibits complex geometrical morphologies of hepatic cells arranged in a hexagonal lobule with an extracellular matrix (ECM) organized in a specific pattern on a multi-scale level. Previous studies have utilized 3D bioprinting and microfluidic perfusion systems with various biomaterials to develop lobule-like constructs. However, they all lack anatomical relevance with weak control over the size and shape of the fabricated structures. Moreover, most biomaterials lack liver-specific ECM components partially or entirely, which might limit their biomimetic mechanical properties and biological functions. Here, we report 3D bioprinting of a sacrificial PVA framework to impart its trilobular hepatic structure to the decellularized liver extracellular matrix (dLM) hydrogel with polyethylene glycol-based crosslinker and tyrosinase to fabricate a robust multi-scale 3D liver construct. The 3D trilobular construct exhibits higher crosslinking, viscosity (182.7 ± 1.6 Pa·s), and storage modulus (2554 ± 82.1 Pa) than non-crosslinked dLM. The co-culture of HepG2 liver cells and NIH 3T3 fibroblast cells exhibited the influence of fibroblasts on liver-specific activity over time (7 days) to show higher viability (90–91.5%), albumin secretion, and increasing activity of four liver-specific genes as compared to the HepG2 monoculture. This technique offers high lumen patency for the perfusion of media to fabricate a densely populated scaled-up liver model, which can also be extended to other tissue types with different biomaterials and multiple cells to support the creation of a large functional complex tissue.
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| 30. |
- Kumar, Tharagan, et al.
(author)
-
High throughput viscoelastic particle focusing and separation in spiral microchannels
- 2021
-
In: Scientific Reports. - : Springer Nature. - 2045-2322. ; 11:1
-
Journal article (peer-reviewed)abstract
- Passive particle manipulation using inertial and elasto-inertial microfluidics have received substantial interest in recent years and have found various applications in high throughput particle sorting and separation. For separation applications, elasto-inertial microfluidics has thus far been applied at substantial lower flow rates as compared to inertial microfluidics. In this work, we explore viscoelastic particle focusing and separation in spiral channels at two orders of magnitude higher Reynolds numbers than previously reported. We show that the balance between dominant inertial lift force, dean drag force and elastic force enables stable 3D particle focusing at dynamically high Reynolds numbers. Using a two-turn spiral, we show that particles, initially pinched towards the inner wall using an elasticity enhancer, PEO (polyethylene oxide), as sheath migrate towards the outer wall strictly based on size and can be effectively separated with high precision. As a proof of principle for high resolution particle separation, 15 mu m particles were effectively separated from 10 mu m particles. A separation efficiency of 98% for the 10 mu m and 97% for the 15 mu m particles was achieved. Furthermore, we demonstrate sheath-less, high throughput, separation using a novel integrated two-spiral device and achieved a separation efficiency of 89% for the 10 mu m and 99% for the 15 mu m particles at a sample flow rate of 1 mL/min-a throughput previously only reported for inertial microfluidics. We anticipate the ability to precisely control particles in 3D at extremely high flow rates will open up several applications, including the development of ultra-high throughput microflow cytometers and high-resolution separation of rare cells for point of care diagnostics.
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| 31. |
- Kumar, Tharagan, et al.
(author)
-
Lab-in-a-fiber-based integrated particle separation and counting
- 2023
-
In: Lab on a Chip. - : Royal Society of Chemistry. - 1473-0197 .- 1473-0189. ; 23, s. 2286-
-
Journal article (peer-reviewed)abstract
- An all-fiber integrated device capable of separating and counting particles is presented. A sequence of silica fiber capillaries with various diameters and longitudinal cavities are used to fabricate the component for size-based elasto-inertial passive separation of particles followed by detection in an uninterrupted continuous flow. Experimentally, fluorescent particles of 1 μm and 10 μm sizes are mixed in a visco-elastic fluid and fed into the all-fiber separation component. The particles are sheathed by an elasticity enhancer (PEO - polyethylene oxide) to the side walls. Larger 10 μm particles migrate to the center of the silica capillary due to the combined inertial lift force and elastic force, while the smaller 1 μm particles are unaffected, and exit from a side capillary. A separation efficiency of 100% for the 10 μm and 97% for the 1 μm particles is achieved at a total flow rate of 50 μL min−1. To the best of our knowledge, this is the first time effective inertial-based separation has been demonstrated in circular cross-section microchannels. In the following step, the separated 10 μm particles are routed through another all-fiber component for counting and a counting throughput of ∼1400 particles per min is demonstrated. We anticipate the ability to combine high throughput separation and precise 3D control of particle position for ease of counting will aid in the development of advanced microflow cytometers capable of particle separation and quantification for various biomedical applications.
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| 32. |
- Kumar, Tharagan, et al.
(author)
-
Lab-in-a-fiber optofluidic device for separation and detection of micron-sized particles
- 2021
-
In: MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. - : Chemical and Biological Microsystems Society. ; , s. 699-700
-
Conference paper (peer-reviewed)abstract
- An all-fiber component capable of sorting and counting microparticles based on size is presented. A sequence of silica fiber capillaries were used to fabricate the component for separation and detection. The portable, lab-scale “all-fiber” device was fabricated by assembling different silica fiber capillaries and optical fibers in a Vytran glass processing station. We report elasto-inertial microfluidics based particle migration and focusing and demonstrate high separation efficiency between 10 µm (100%) and 1 µm (97%) microparticles. The separated 10 µm particles were further analyzed for counting in the integrated fiberoptics component at a speed of ~1400 particles/min.
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| 33. |
- Kumar, Tharagan, et al.
(author)
-
Optofluidic Fiber Component for Separation and counting of Micron-Sized Particles
- 2021
-
Other publication (other academic/artistic)abstract
- n all-fiber separation component capable of sorting and counting micron-sized particles based on size is presented. A sequence of silica fiber capillaries with various diameters and longitudinal cavities were used to fabricate the component for separation and detection in an uninterrupted flow. Fluorescence microparticles of 1 μm and 10 μm sizes are mixed in a visco-elastic fluid and infused into the all-fiber separation component. Elasto-inertial forces focus the larger particle to the center of the silica capillary, while the smaller microparticles exit from a side capillary. Analysis of the separated particles at the output showed a separation efficiency of 100% for the 10 μm and 97% for the 1 μm particles. In addition, the counting of the larger particles is demonstrated in the same flow. The separated 10 μm particles are further routed through another all-fiber component for counting. A counting speed of ~1400 particles/min and with the variation in amplitude of 10% is achieved. A combination of separation and counting can be powerful tool may find several applications in biology and medicine, such as separation and analysis of exosomes, bacteria, and blood cell sub-populations.
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| 34. |
- Lapins, Noa, et al.
(author)
-
A smartphone powered centrifugal microfluidic platform for point-of-care diagnostics in resource limited settings
-
Other publication (other academic/artistic)abstract
- The broad availability of smartphones has provided new opportunities to develop less expensive, portable and integrated point-of-care (POC) platforms. To date, many point-of-care devices have been developed that employ the computing power and the optical sensing capabilities available in smartphones. 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 that captures and processes the test images. The housing made from cardboard serves as a platform to conduct tests and ensures the portability and rigidity of the platform while being extremely low-cost. 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 bioassays. For controlling the rotational speed without the need for external circuitry, 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) and a fully automated measurement of hematocrit level integrating blood-plasma separation, imaging and image analysis. The low-cost platform weighing less than 150 grams operated by a mobile phone has the potential to meet the REASSURED criteria for advanced diagnostics in resource limited settings.
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| 35. |
- Lapins, Noa, et al.
(author)
-
Automated blood plasma separation and metering for clinical settings and centrifugal microfluidics devices
- 2020
-
In: MicroTAS 2020 - 24th International Conference on Miniaturized Systems for Chemistry and Life Sciences. - : Chemical and Biological Microsystems Society. ; , s. 378-379
-
Conference paper (peer-reviewed)abstract
- To date, blood plasma analytes are frequently being used as accepted biomarkers for disease [1]. In order to efficiently detect these analytes, the sample is preferred to be completely free of cells. The lack of a generic integrated blood plasma separation technique has long been one of the obstructions for the large-scale adaptation of microfluidic-based sample-to-answer diagnostic assays, especially for larger sample volumes. Here, we present a robust, scalable, low-cost and secure technology that automatically measures, and dispenses sequentially and proportionally a given volume of blood plasma inside a centrifuge tube.
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| 36. |
- Parker, Helen E., et al.
(author)
-
A Lab-in-a-Fiber optofluidic device using droplet microfluidics and laser-induced fluorescence for virus detection
- 2022
-
In: Scientific Reports. - : Nature Research. - 2045-2322. ; 12:1
-
Journal article (peer-reviewed)abstract
- Microfluidics has emerged rapidly over the past 20 years and has been investigated for a variety of applications from life sciences to environmental monitoring. Although continuous-flow microfluidics is ubiquitous, segmented-flow or droplet microfluidics offers several attractive features. Droplets can be independently manipulated and analyzed with very high throughput. Typically, microfluidics is carried out within planar networks of microchannels, namely, microfluidic chips. We propose that fibers offer an interesting alternative format with key advantages for enhanced optical coupling. Herein, we demonstrate the generation of monodisperse droplets within a uniaxial optofluidic Lab-in-a-Fiber scheme. We combine droplet microfluidics with laser-induced fluorescence (LIF) detection achieved through the development of an optical side-coupling fiber, which we term a periscope fiber. This arrangement provides stable and compact alignment. Laser-induced fluorescence offers high sensitivity and low detection limits with a rapid response time making it an attractive detection method for in situ real-time measurements. We use the well-established fluorophore, fluorescein, to characterize the Lab-in-a-Fiber device and determine the generation of ∼ 0.9 nL droplets. We present characterization data of a range of fluorescein concentrations, establishing a limit of detection (LOD) of 10 nM fluorescein. Finally, we show that the device operates within a realistic and relevant fluorescence regime by detecting reverse-transcription loop-mediated isothermal amplification (RT-LAMP) products in the context of COVID-19 diagnostics. The device represents a step towards the development of a point-of-care droplet digital RT-LAMP platform. © 2022, The Author(s).
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| 37. |
- Parker, Helen E., et al.
(author)
-
Digital detection and quantification of SARS-CoV-2 in a droplet microfluidic all-fiber device
- 2021
-
In: Proceedings MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. - : Chemical and Biological Microsystems Society. ; , s. 1047-1048
-
Conference paper (peer-reviewed)abstract
- Silica fibers and capillaries offer opportunities for compact integration of optics with microfluidics while adding advantages such as; flexibility within a high aspect ratio format, uniaxial arrangements, and measurement-at-a-distance. Here, we describe droplet microfluidics-based nucleic acid detection of SARS-CoV-2 in a lab-in-a-fiber platform. The fiber component integrates three modules with key functions: droplet generation, incubation, and fluorescence detection. Within the scope of this work, we developed the component specifically to target the quantification of SARS-CoV-2 viral RNA through reverse-transcription loop-mediated isothermal amplification (RT-LAMP). The all-fiber component could successfully generate uniform droplets and differentiate pre-amplified positive LAMP reaction from negative sample.
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| 38. |
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| 39. |
- Parker, Helen E., et al.
(author)
-
Viral detection and quantification in a digital droplet microfluidic lab-in-a-fiber device
- 2021
-
In: Proceedings of SPIE - The International Society for Optical Engineering. - : SPIE. - 9781510643802
-
Conference paper (peer-reviewed)abstract
- In this work, we present the design and fabrication of a fiber device that performs digital droplet microfluidics for molecular diagnostics. A variety of fibers and capillaries were used to build three connected modules dedicated to droplet generation, incubation, and fluorescence detection which enables a uniaxial arrangement. This is in contrast to the traditional 2-dimensional lab-on-a-chip architecture. We characterize our fiber device using a fluorescein dilution series. Our observed detection limit is on the order of 10 nM fluorescein. We demonstrate our all-fiber device for the fluorescence readout after loop-mediated isothermal amplification (LAMP) of synthetic SARS-CoV-2. Our results suggest that this fiber device can successfully distinguish between positive and negative samples in molecular diagnostics. We propose that our fiber device offers benefits over microfluidic chip techniques such as easier optical integration, much simpler sample loading, and faster diagnosis with high specificity and sensitivity. Keywords: All-fiber device, microfluidics, optofluidics, loop-mediated isothermal amplification (LAMP), molecular diagnostics, SARS-CoV2.
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| 40. |
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| 41. |
- Pinto, Ines Fernandes, et al.
(author)
-
Microfluidic Cartridge for Bead-Based Affinity Assays
- 2024
-
In: Methods in Molecular Biology. - : Springer Nature. - 1064-3745 .- 1940-6029. ; 2804, s. 127-138
-
Journal article (peer-reviewed)abstract
- Within the vast field of medical biotechnology, the biopharmaceutical industry is particularly fast-growing and highly competitive, so reducing time and costs associated to process optimization becomes instrumental to ensure speed to market and, consequently, profitability. The manufacturing of biopharmaceutical products, namely, monoclonal antibodies (mAbs), relies mostly on mammalian cell culture processes, which are highly dynamic and, consequently, difficult to optimize. In this context, there is currently an unmet need of analytical methods that can be integrated at-line in a bioreactor, for systematic monitoring and quantification of key metabolites and proteins. Microfluidic-based assays have been extensively and successfully applied in the field of molecular diagnostics; however, this technology remains largely unexplored for Process Analytical Technology (PAT), despite holding great potential for the at-line measurement of different analytes in bioreactor processes, combining low reagent/molecule consumption with assay sensitivity and rapid turnaround times.Here, the fabrication and handling of a microfluidic cartridge for protein quantification using bead-based affinity assays is described. The device allows geometrical multiplexed immunodetection of specific protein analytes directly from bioreactor samples within 2.5 h and minimal hands-on time. As a proof-of-concept, quantification of Chinese hamster ovary (CHO) host cell proteins (HCP) as key impurities, IgG as product of interest, and lactate dehydrogenase (LDH) as cell viability marker was demonstrated with limits of detection (LoD) in the low ng/mL range. Negligible matrix interference and no cross-reactivity between the different immunoassays on chip were found. The results highlight the potential of the miniaturized analytical method for PAT at reduced cost and complexity in comparison with sophisticated instruments that are currently the state-of-the-art in this context.
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| 42. |
- Pinto, Ines Fernandes, et al.
(author)
-
Multiplexed Microfluidic Cartridge for At-Line Protein Monitoring in Mammalian Cell Culture Processes for Biopharmaceutical Production
- 2021
-
In: ACS Sensors. - : American Chemical Society (ACS). - 2379-3694. ; 6:3, s. 842-851
-
Journal article (peer-reviewed)abstract
- The biopharmaceutical market has been rapidly growing in recent years, creating a highly competitive arena where R&D is critical to strike a balance between clinical safety and profitability. Toward process optimization, the recent development and adoption of new process analytical technologies (PAT) highlight the dynamic complexity of mammalian/human cell culture processes, as well as the importance of fine-tuning and modeling key metabolites and proteins. In this context, simple, rapid, and cost-effective devices allowing routine at-line monitoring of specific proteins during process development and production are currently lacking. Here, we report the development of a versatile microfluidic protein analysis cartridge allowing the multiplexed bead-based immunodetection of specific proteins directly from complex mixtures with minimal hands-on time. Colorimetric quantification of Chinese hamster ovary (CHO) host cell proteins as key impurities, monoclonal antibodies as target biopharmaceuticals, and lactate dehydrogenase as a marker of cell viability was achieved with limits of detection in the 1-10 ng/mL range and analysis times as short as 30 min. The device was further demonstrated for the monitoring of a Rituximab-producing CHO cell bioreactor over the course of 8 days, providing comparable recoveries to standard enzyme-linked immunosorbent assay (ELISA) kits. The high sensitivity combined with robustness to matrix interference highlights the potential of the device to perform at-line measurements spanning from the bioreactor to the downstream processing.
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| 43. |
- Pinto, Ines F., et al.
(author)
-
Single-Step Quantification of Specific Nucleic Acid Sequences in Microfluidics Using a Multilabeled Hybrid DNA Duplex
- 2021
-
In: MicroTAS 2021. - : Chemical and Biological Microsystems Society. ; , s. 757-758
-
Conference paper (peer-reviewed)abstract
- The rapid and specific detection of nucleic acid sequences is highly demanded for several applications including pathogen diagnostics, quality control of biopharmaceutical products and forensics. Nucleic acid amplification methods based on mixtures of primers and polymerase enzymes such as polymerase chain reaction (PCR) and other isothermal methods are typically the standard approach. Here, using SARS-CoV-2 ORF1ab sequence as a model, we report the development of a simple enzyme-free and single-step competitive hybridization method allowing the specific detection of any type of nucleic acid sequence (ss/dsDNA or RNA) within 15 min with 89% sequence homology and sensitivity in the pM-range.
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| 44. |
- Ramachandraiah, Harisha, et al.
(author)
-
Extended elasto-inertial microfluidics for high throughput separation in low aspect ratio spiral microchannels
- 2020
-
In: 21st International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2017. - : Chemical and Biological Microsystems Society. ; , s. 1401-1402
-
Conference paper (peer-reviewed)abstract
- Manipulation of particles and cells in viscoelastic fluids has received substantial interest because this phenomenon provides high-quality focusing. Here we present an enhanced particle focusing and separation in spiral channels, at a ten-fold increase of Reynolds number as compared to current state of the art elasto-inertial microfluidics and report stable particle focusing in spiral low aspect ratio channels at flow rates two magnitudes higher than that previously reported at a high throughput of 2 mL/min is demonstrated with an separation efficiency of 99% for the 15-micron and 91% for the 10-micron particles is demonstrated.
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| 45. |
- Ramachandraiah, Harisha, et al.
(author)
-
Layer-by-layer system based on cellulose nanofibrils for capture and release of cells in microfluidic device
- 2020
-
In: Proceedings 21st International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2017. - : Chemical and Biological Microsystems Society. ; , s. 796-797
-
Conference paper (peer-reviewed)abstract
- Layer-by-layer (LBL) technique facilitates the production of the thin coating of cellulose onto polymeric surfaces and modified to form affinity based cell capture surface. We demonstrate an efficiently capture and release of cells, the release is done by selectively degrading the cellulose layers using enzyme and cells can be collected without losing cell viability.
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| 46. |
- Soares, Ruben R. G., et al.
(author)
-
Circle-to-circle amplification coupled with microfluidic affinity chromatography enrichment for in vitro molecular diagnostics of Zika fever and analysis of anti-flaviviral drug efficacy
- 2021
-
In: Sensors and actuators. B, Chemical. - : Elsevier BV. - 0925-4005 .- 1873-3077. ; 336
-
Journal article (peer-reviewed)abstract
- Sensitive viral diagnostic methods are increasingly in demand to tackle emerging epidemics. The Zika virus (ZIKV) is particularly relevant in tropical resource limited settings (RLS) and is associated with intermittent epidemics such as the recent 2016 ZIKV outbreak in South America, wherein Zika fever was classified by WHO as a public health emergency of international concern. Thus, there is an urgent need for widespread Zika fever diagnostics and efficient drug therapies. ZIKV diagnostics are typically performed using RT-qPCR in centralized laboratories. While extremely sensitive, RT-qPCR requires rapid heating-cooling cycles, combined with continuous fluorescence measurements to allow quantification, implying high costs and limiting availability of molecular diagnostics in RLS. Here, we report isothermal amplification of ZIKV cDNA using padlock probes followed by two rounds of Rolling Circle Amplification (RCA), termed as circle-to-circle amplification (C2CA), combined with a microfluidic affinity chromatography enrichment (mu ACE) platform. This platform allowed the detection of <17 vRNA copies per reaction mixture, equivalent to similar to 3 aM, showed a positive correlation with RT-qPCR in both average (r = 0.80) and discrete (r = 0.95) signal modes, and was validated for drug efficiency tests using in vitro infected peripheral blood mononuclear cells from 3 healthy donors. This performance shows significant promise towards highly sensitive, albeit simple and cost-effective point-of-care viral diagnostics.
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| 47. |
- Soares, Ruben R. G., et al.
(author)
-
Point-of-care isothermal nucleic acid amplification platform for COVID-19 diagnostics in resource-limited settings
- 2021
-
In: Proceedings MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. - : Chemical and Biological Microsystems Society. ; , s. 863-864
-
Conference paper (peer-reviewed)abstract
- 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.
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| 48. |
- Soares, Ruben R. G., et al.
(author)
-
Sample-to-answer COVID-19 nucleic acid testing using a low-cost centrifugal microfluidic platform with bead-based signal enhancement and smartphone read-out
- 2021
-
In: Lab on a Chip. - : Royal Society of Chemistry (RSC). - 1473-0197 .- 1473-0189. ; 21:15, s. 2932-2944
-
Journal article (peer-reviewed)abstract
- 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.
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| 49. |
- Soares, Ruben R. G., et al.
(author)
-
Towards point-of-care HIV diagnostics using dual-labelled rolling circle products for efficient capture and detection in a microfluidic device
- 2019
-
In: 23rd International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2019. - : Chemical and Biological Microsystems Society. ; , s. 734-735
-
Conference paper (peer-reviewed)abstract
- HIV infections are devastating in resource-limited settings, where portable, fit-for-purpose and affordable diagnostic devices would allow effective monitoring of infection spread and aid therapeutics. Here, a rolling circle amplification (RCA)-based assay using multiple specific padlock probes (PLP) targeting a conserved pol gene of HIV-1 subtype B is presented. These PLPs were designed to allow (1) fluorescence detection of the rolling circle products (RCP), as well as (2) their specific capture in a microfluidic device resorting to streptavidin-biotin interactions. The device provided detection limits as low as 10 fM and allowed the detection of HIV in infected 293T cell culture supernatants.
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| 50. |
- Tanriverdi, Selim, et al.
(author)
-
Elasto-inertial focusing and particle migration in high aspect ratio microchannels for high-throughput separation
- 2024
-
In: Microsystems and Nanoengineering. - : Springer Nature. - 2055-7434. ; 10:1
-
Journal article (peer-reviewed)abstract
- The combination of flow elasticity and inertia has emerged as a viable tool for focusing and manipulating particles using microfluidics. Although there is considerable interest in the field of elasto-inertial microfluidics owing to its potential applications, research on particle focusing has been mostly limited to low Reynolds numbers (Re<1), and particle migration toward equilibrium positions has not been extensively examined. In this work, we thoroughly studied particle focusing on the dynamic range of flow rates and particle migration using straight microchannels with a single inlet high aspect ratio. We initially explored several parameters that had an impact on particle focusing, such as the particle size, channel dimensions, concentration of viscoelastic fluid, and flow rate. Our experimental work covered a wide range of dimensionless numbers (0.05 < Reynolds number < 85, 1.5 < Weissenberg number < 3800, 5 < Elasticity number < 470) using 3, 5, 7, and 10 µm particles. Our results showed that the particle size played a dominant role, and by tuning the parameters, particle focusing could be achieved at Reynolds numbers ranging from 0.2 (1 µL/min) to 85 (250 µL/min). Furthermore, we numerically and experimentally studied particle migration and reported differential particle migration for high-resolution separations of 5 µm, 7 µm and 10 µm particles in a sheathless flow at a throughput of 150 µL/min. Our work elucidates the complex particle transport in elasto-inertial flows and has great potential for the development of high-throughput and high-resolution particle separation for biomedical and environmental applications. (Figure presented.)
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