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1.	Abbasi Aval, Negar, et al. (author) Assessing the Layer-by-Layer Assembly of Cellulose Nanofibrils and Polyelectrolytes in Pancreatic Tumor Spheroid Formation 2023 In: Biomedicines. - : MDPI AG. - 2227-9059. ; 11:11 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.
2.	Abbasi Aval, Negar, et al. (author) Evaluating the Impact of Positively Charged Polyelectrolyte Molecular Weightand Bilayer Number on Tumor Spheroid Formation in the Interaction with Negatively Charged Cellulose Nanofibrils in layer by layer assembly 2023 Other publication (other academic/artistic)
3.	Abbasi Aval, Negar, et al. (author) Influence of Decellularized Extra Cellular Matrix on 3D spheroids formed on Layer-by-Layer cellulose nanofibril/Polyelectrolytes coating as an in-vitro model for Hepatocellular Carcinoma Other publication (other academic/artistic)
4.	Abbasi Aval, Negar, et al. (author) Layer-by-Layer cellulose nanofibril coating for spheroid formation combined with decellularized extracellular matrix for 3D tumor modeling Other publication (other academic/artistic)
5.	Abbasi Aval, Negar, 1986- (author) Utilizing Biopolymers in 3D Tumor Modeling and Tumor Diagnosis 2023 Doctoral thesis (other academic/artistic)abstract Cancer represents a significant global public health challenge and ranks as the second mostcommon cause of death in the United States. The onset of cancer entails an initial phasewhere cells lose their polarity and disconnect from the normal basement membrane, allowingthem to form distinct three-dimensional (3D) configurations that interact with adjacent cellsand the surrounding microenvironment. Cells grown in 2D monolayers demonstrate differentgene expression patterns and different activation of signaling pathways compared to cellscultivated within the natural structure of tumor tissue of the same cell type. Multicellulartumor spheroids (MCTS) are extensively investigated as a well-studied model of organotypiccancer. These spheroids are formed by tumor cells, either alone or in combination with othercell types, and they can be created with or without the application of supportive scaffolds.The MCTSs are also considered promising models for preclinical assessments of chemosensitivity.However, the creation of these tumor spheroids presents challenges, as not alltumor cell lines can consistently form regular spheroids.Cellulose nanofibrils (CNF) have become essential as a sustainable and environmentallyfriendly material. For example, thin films, with inherent mechanical properties, and flexibility,offer versatility across various applications. Also known for its biocompatibility and non-toxicnature, native CNF is a natural option to use. Its fibrous structure closely mimics the collagenmatrix in human tissue, showing potential as an effective scaffold for 3D cell culture. In thisregard, an innovative Layer-by-Layer (LbL) coating technique using CNF-polyelectrolytebilayers was investigated to generate spheroids. This method constructs bilayers of CNFand polyelectrolytes and can coat various surfaces. In this thesis, the first focus was ondemonstrating the spheroid formation capability using low molecular weight polyelectrolytesin LbL assembly. Secondly, an investigation was conducted involving embedding of LbLgrownspheroids in a decellularized extracellular matrix (ECM) aiming to determine howECM, possessing suitable mechanical characteristics, could influence the cancer stem celltraits in spheroids. Thirdly, the thesis demonstrated the utilization of LbL for capturing andreleasing of circulating tumor cells. Lastly, the shift from using low molecular weightpolyelectrolytes in the LbL assembly to high molecular weight counterparts and analyzingthe differences in spheroid formation abilities to assess the underlying differences inmolecular weights of the polyelectrolytes was explored. All-in-all, employing the CNF-basedLbL surface coating strategy explored in the thesis has proven to be promising for thedevelopment of spheroid models closely resembling in vivo conditions and holds significantpotential for applications in drug development.
6.	Ahmadian, Afshin, et al. (author) SNP analysis by allele-specific extension in a micromachined filter chamber 2002 In: BioTechniques. - : Future Science Ltd. - 0736-6205 .- 1940-9818. ; 32:4, s. 748-754 Journal article (peer-reviewed)
7.	Akhtar, Ahmad Saleem, et al. (author) A portable and low-cost centrifugal microfluidic platform for multiplexed colorimetric detection of protein biomarkers 2023 In: Analytica Chimica Acta. - : Elsevier BV. - 0003-2670 .- 1873-4324. ; 1245 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.
8.	Akhtar, Ahmad Saleem, et al. (author) An integrated centrifugal microfluidic platform for multiplexed colorimetric immunodetection of protein biomarkers 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. 947-948 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.
9.	Akhtar, Ahmad Saleem, et al. (author) Centrifugal microfluidic platform comprising an array of bead microcolumns for the multiplexed colorimetric quantification of inflammatory biomarkers at the point-of-care 2019 In: 23rd International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2019. - : Chemical and Biological Microsystems Society. ; , s. 1230-1231 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.
10.	Akhtar, Ahmad Saleem (author) Centrifugal microfluidics-based point of care diagnostics at resource limited settings 2023 Doctoral thesis (other academic/artistic)abstract Advancements in medical diagnostics have allowed us to understand the underlying mechanism and treat the root cause for many diseases which had been causing morbidity and mortality up until this point in human history. Furthermore, many of the standard diagnostic procedures have now been transformed to provide answers at or near the point-of-care. However, the effects of these positive developments have not trickled down to the parts of our society which are considered underdeveloped and lack the necessary infrastructure and facilities. Diagnostics in such resource limited settings still lag behind and fail to provide the requisite healthcare. In order to translate the diagnostic solutions designed for central laboratories to resource limited settings, there are certain challenges that need to be addressed, such as portability, reduction in cost and ease-of-use, while keeping the sensitivity and specificity at the required level. The work presented in this thesis focuses on addressing some of these issues by using microfluidics to develop diagnostic platforms that are suitable to be used in resource limited settings. In paper I, a very low-cost and simple centrifugal microfluidic platform was developed to be used in settings which do not have a reliable supply of electricity. The platform uses a smartphone as a source of power and the sensors of the phone for speed control.In paper II, a portable and low-cost diagnostic platform was developed for multiplexed detection of biomarkers based on centrifugal microfluidics. The platform uses colorimetric detection and a simple readout method which does not require a spectrophotometer for quantification.In paper III, a platform was developed for COVID-19 diagnostics which combines centrifugal microfluidics with a novel bead-based strategy for signal enhancement. The platform uses fluorescent detection with a smartphone readout and has the capability to process up to 20 samples at the same time.In paper IV, as a follow up of paper III, a more advanced platform was developed for COVID-19 diagnostics which allows the operator to carry out nucleic acid amplification in a completely automated manner, from adding the sample to getting the final result.In paper V, an alternative method for detection of SARS-CoV-2 was developed using electrochemical biosensing. This work combines the electrochemical technique with a flexible printed circuit board for a rapid, amplification-free and label-free detection of target SARS-CoV-2 sequences.
11.	Akhtar, Ahmad Saleem, et al. (author) Fully automated centrifugal microfluidic platform for COVID-19 detection using computer vision-based readout 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.
12.	Aljadi, Zenib, et al. (author) A novel tool for clinical diagnosis of allergy operating a microfluidic immunoaffinity basophil activation test technique 2019 In: Clinical Immunology. - : ACADEMIC PRESS INC ELSEVIER SCIENCE. - 1521-6616 .- 1521-7035. ; 209 Journal article (peer-reviewed)abstract The Basophil Activation Test (BAT) is a valuable allergy diagnostic tool but is time-consuming and requires skilled personnel and cumbersome processing, which has limited its clinical use. We therefore investigated if a microfluidic immunoaffinity BAT (miBAT) technique can be a reliable diagnostic method. Blood was collected from allergic patients and healthy controls. Basophils were challenged with negative control, positive control (anti-FccRI), and two concentrations of a relevant and non-relevant allergen. CD203c and CD63 expression was detected by fluorescent microscopy and flow cytometry. In basophils from allergic patients the CD63% was significantly higher after allergen activation as compared to the negative control (p < .0001-p = .0004). Activation with non-relevant allergen showed equivalent CD63% expression as the negative control. Further, the miBAT data were comparable to flow cytometry. Our results demonstrate the capacity of the miBAT technology to measure different degrees of basophil allergen activation by quantifying the CD63% expression on captured basophils.
13.	Aljadi, Zenib, et al. (author) Activation of Basophils Is a New and Sensitive Marker of Biocompatibility in Hemodialysis 2014 In: Artificial Organs. - : Wiley. - 0160-564X .- 1525-1594. ; 38:11, s. 945-953 Journal article (peer-reviewed)abstract The hemodialysis procedure involves contact between peripheral blood and the surface of dialyzer membranes, which may lead to alterations in the pathways of innate and adaptive immunity. We aimed to study the effect of blood-membrane interaction on human peripheral basophils and neutrophils in hemodialysis with high- and low-permeability polysulfone dialyzers. The surface expression of CD203c (basophil selection marker) and CD63 (activation marker) after activation by the bacterial peptide formyl-methionyl-leucyl-phenylalanine (fMLP) or anti-Fc epsilon receptor I (Fc epsilon RI) antibody and the absolute number of basophils was investigated before and after hemodialysis with each of the dialyzers. Moreover, the expression on neutrophils of CD11b, the CD11b active epitope, and CD88 was analyzed in the same groups of individuals. The expression of CD63 in basophils following activation by fMLP was significantly higher in the patient group compared with that in healthy controls, but no differences were observed after activation by anti-Fc epsilon RI. During the hemodialysis procedure, the low-flux membrane induced up-regulation of CD63 expression on basophils, while passage through the high-flux membrane did not significantly alter the responsiveness. In addition, the absolute number of basophils was unchanged after hemodialysis with either of the dialyzers and compared with healthy controls. We found no significant differences in the expression of the neutrophil activation markers (CD11b, the active epitope of CD11b, and CD88) comparing the two different dialyzers before and after dialysis and healthy controls. Together, these findings suggest that alterations in basophil activity may be a useful marker of membrane bioincompatibility in hemodialysis.
14.	Aljadi, Zenib, et al. (author) Altered basophil function in patients with chronic kidney disease on hemodialysis 2017 In: Clinical Nephrology. - : DUSTRI-VERLAG DR KARL FEISTLE. - 0301-0430. ; 88:2, s. 86-96 Journal article (peer-reviewed)abstract Aims: Chronic kidney disease (CKD) leads to impairment of immune cell function. Given the potential role of basophils in the pathogenesis of CKD, we aimed to study the basophil responsiveness towards microbial antigen exposure, judged as adhesion molecule expression and degranulation, in CKD patients on hemodialysis. Materials and methods: We selected markers linked to two crucial biological phases: the transmigration and degranulation processes, respectively. For the transmigration process, we selected the adhesion molecules CD11b, active CD11b epitope, and CD62L and for the degranulation process CD203c (piecemeal degranulation marker), CD63 (degranulation marker), and CD300a (inhibitory marker of degranulation). We measured basophil responsiveness after stimulation of different activation pathways in basophils using lipopolysaccharide (LPS), peptidoglycan (PGN), formyl-methyinoyl-leucyl-phenylalanine (fMLP), and anti-FceRI-ab. Results: The expression of CD63 in basophils following activation by fMLP was significantly higher in the patient group compared to matched healthy controls, but no differences were observed after activation by anti-Fc.RI. CD300a expression was significantly higher in patients following activation by fMLP and anti-Fc.RI, and the active epitope CD11b expression was significantly higher in patients after LPS activation. In addition, we found that CD62L was not shed from the cell surface after activation with LPS and fMLP. A slight downregulation was noted after activation with anti-Fc.RI in healthy controls. Conclusion: Together, these data demonstrate that basophil functions related to adhesion and degranulation are altered in CKD patients on hemodialysis, which indicates a potential role for the basophil in the pathogenesis of complications related to infections.
15.	Aljadi, Zenib, et al. (author) 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 In: Macromolecular Bioscience. - : Wiley. - 1616-5187 .- 1616-5195. ; 22:10 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.
16.	Aljadi, Zenib, et al. (author) Microfluidic Immunoaffinity Basophil Activation Test for Point-of-Care Allergy Diagnosis 2019 In: Journal of Applied Laboratory Medicine (JALM). - : American Association for Clinical Chemistry. - 2475-7241 .- 2576-9456. ; 4:2, s. 152-163 Journal article (peer-reviewed)abstract Background: The flow cytometry-based basophil activation test (BAT) is used for the diagnosis of allergic response. However, flow cytometry is time-consuming, requiring skilled personnel and cumbersome processing, which has limited its use in the clinic. Here, we introduce a novel microfluidic-based immunoaffinity BAT (miBAT) method. Methods: The microfluidic device, coated with anti-CD203c, was designed to capture basophils directly from whole blood. The captured basophils are activated by anti-FceRI antibody followed by optical detection of CD63 expression (degranulation marker). The device was first characterized using a basophil cell line followed by whole blood experiments. Weevaluated the device with ex vivo stimulation of basophils in whole blood from healthy controls and patients with allergies and compared it with flow cytometry. Results: The microfluidic device was capable of capturing basophils directly from whole blood followed by in vitro activation and quantification of CD63 expression. CD63 expression was significantly higher (P = 0.0002) in on-chip activated basophils compared with nonactivated cells. The difference in CD63 expression on anti-FceRI-activated captured basophils in microfluidic chip was significantly higher (P = 0.03) in patients with allergies compared with healthy controls, and the results were comparable with flow cytometry analysis (P = 0.04). Furthermore, there was no significant difference of CD63% expression in anti-FceRI-activated captured basophils in microfluidic chip compared with flow cytometry. Conclusions: We report on the miBAT. This device is capable of isolating basophils directly from whole blood for on-chip activation and detection. The new miBAT method awaits validation in larger patient populations to assess performance in diagnosis and monitoring of patients with allergies at the point of care.
17.	Amasia, Mary, et al. (author) Modified DVD-drive as an integrated microfluidic system for precipitate-based detection of LAMP assay 2013 In: 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2013. ; , s. 1616-1618 Conference paper (peer-reviewed)abstract Loop-mediated isothermal amplification (LAMP) is a sensitive method for nucleic acid analysis, and has been demonstrated as a ideal technique for use in miniaturized microfluidic systems. While LAMP assays are often detected using absorbance or fluorescence, we demonstrate an integrated system for LAMP assays through the detection of precipitate formation using a modified commercial DVD drive. This integrated DVD drive system is able to automate the sedimentation process and scattering-based detection of accumulated precipitate, as well as maintain the constant temperature needed for LAMP analysis.
18.	Ardabili, Sahar, et al. (author) Dean flow-coupled inertial focusing for ultra-high-throughput particle filtration 2010 In: 14th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2010, MicroTAS 2010. - 9781618390622 ; , s. 1586-1588 Conference paper (peer-reviewed)abstract Particle manipulation represents an important and fundamental step prior to counting, sorting and detecting bio-particles. In this study, we report dean-coupled inertial focusing of particles in flows through a single curve microchannel at extremely high channel Reynold numbers (∼325). We found the lateral particle focusing position, xf to be fixed and largely independent of radius of curvature and whether particles are pre-focused (at equilibrium) entering the curvature or randomly distributed. Finally, using a single inlet, u-shaped, microchannel we demonstrate filtration of 10μm particles from 2 μm particles at throughputs several orders of magnitude higher than previously shown.
19.	Ardabili, Sahar, et al. (author) Epitope unmasking for improved immuno-magnetic isolation of Gram-negative bacteria Other publication (other academic/artistic)
20.	Ardabili, Sahar, 1982- (author) Microfluidic bases sample preparation for blood stream infections 2014 Doctoral thesis (other academic/artistic)abstract Microfluidics promises to re-shape the current health-care system by transferring diagnostic tools from central laboratories to close vicinity of the patient (point-of-care). One of the most important operational steps in any diagnostic platform is sample preparation, which is the main subject in this thesis. The goal of sample preparation is to isolate targets of interest from their surroundings. The work in this thesis is based on three ways to isolate bacteria: immune-based isolation, selective cell lysis, size-based separation.The first sample-preparation approach uses antibodies against lipopolysaccharides (LPS), which are surface molecules found on all gram-negative bacteria. There are two characteristics that make this surface molecule interesting. First, it is highly abundant: one bacterium has approximately a million LPS molecules on its cell-wall. Second, the molecule has a conserved region within all gram-negative bacteria, so using one affinity molecule to isolate disease-causing gram-negative bacteria is an attractive option, particularly from the point of view of sample preparation. The main challenge, however, is antigen accessibility. To address this, we have developed a treatment protocol that improves the capturing efficiency.The strategy behind selective cell lysis takes advantage of the differences between the blood-cell membrane and the bacterial cell-wall. These fundamental differences make it possible to lyse (destroy) blood-cells selectively while keeping the target of interest, here the bacteria, intact and, what is more important alive. Viability plays an important role in determining antibiotic susceptibility.Difference in size is another well-used characteristic for sample- separation. Inertial microfluidics can focus size-dependent particle at high flow-rates. Thus, particles of 10 µm diameter were positioned in precise streamlines within a curved channel. The focused particles can then be collected at defined outlets. This approach was then used to isolate white blood cells, which account for approximately 1% of the whole blood. In such a device particles of 2µm diameter (size of bacteria) would not be focused and thereby present at every outlet. To separate bacteria from blood elasto-inertial microfluidics was used. Here, e blood components are diverted to center of the channels while smaller bacteria remain in the side streams and can subsequently be separated.
21.	Bachmann, Till T., et al. (author) Expert guidance on target product profile development for AMR diagnostic tests 2023 In: BMJ Global Health. - : BMJ Publishing Group. - 2059-7908. ; 8:12 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.
22.	Baldasici, Oana, et al. (author) The transcriptional landscape of cancer stem-like cell functionality in breast cancer 2024 In: Journal of Translational Medicine. - : Springer Nature. - 1479-5876. ; 22:1, s. 530- 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.
23.	Banerjee, Indradumna, et al. (author) Analog particle position tuning in Elasto-inertial microfluidic flows Other publication (other academic/artistic)abstract We observe for the first time an analog trend in particle focusing in a high throughput weakly viscoelastic regime, where it is possible to tune particles into multiple intermediate focusing positions that lie between the "Segre-Silberberg annulus" and the center of a circular microcapillary. The "Segre-Silberberg annulus" (0.6 times the pipe radius), that describes particle equilibrium in a predominantly inertial flow, shrinks consistently closer to the center for increasing elasticity in extremely dilute PEO concentrations (ranging from 0.001 wt% to 0.05wt%). The experimental observations are supported by direct numerical simulations, where an Immersed Boundary Method is used to account for the presence of 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 analyze particle behavior at Reynolds number higher than what is allowed by the present experimental setup. In particular, we are able to report the entire migration trajectories of the particles as they reach their final equilibrium 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 a weakly viscoelastic regime, where neither inertia nor elasticity are able to mask each other's effect completely, thus leading to a number of intermediate focusing positions. The present study provides a new understanding into the mechanism of particle focusing in elasto-inertial flows and opens up new possibilities for exercising analog control in tuning the particle focusing positions.
24.	Banerjee, Indradumna, et al. (author) Analogue tuning of particle focusing in elasto-inertial flow 2021 In: Meccanica (Milano. Print). - : Springer Science and Business Media B.V.. - 0025-6455 .- 1572-9648. ; 56:7, s. 1739-1749 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.
25.	Banerjee, Indradumna, 1986-, et al. (author) Dynamics of Inertial migration of particles in straight channels 2017 Conference paper (peer-reviewed)abstract SUMMARYWe study numerically the entire migration dynamics of spherical and oblate particles in straight rectangular and square cross sectional ducts. The reported results can help in design of straight duct channel based microfluidic systems. KEYWORDS: Inertial microfluidics, Lateral migration, Oblate particles, Straight particles. INTRODUCTIONWe simulate spherical and oblate rigid particles in straight ducts of different aspect ratios using an Immersed Boundary Method. To the best of our knowledge, this is the first time not only the equilibrium position of particles is described, but also the entire migration dynamics of the particle from the initial to final position, including particle trajectory, velocity, rotation and orientation, are investigated. EXPERIMENTAL The fluid is considered incompressible and its motion is governed by the Navier Stokes and Continuity equations. The numerical approach employed is an Immersed Boundary Method (IBM) with two sets of grid points: an equispaced Eulerian mesh for the fluid flow, and Lagrangian grid points uniformly distributed on the surface of the particle. The flow is set up in square and rectangular cross section ducts with no slip and no penetration boundary conditions (Fig.1). RESULTS AND DISCUSSIONWe examine the lateral motion of spherical and oblate particles using the IBM method mentioned above. While simulating three different spheres in a square duct of duct width to sphere diameter ratio H/Ds= [3.5, 5, 10], we find that the particles focus at closest face-cantered equilibrium position from their point of introduction(Fig.2a). We also show the downstream length needed for a sphere to focus, focusing length, as a function of the distance from the vertical duct symmetry line and as a function of Reynolds number(Fig.2b and c respectively). Spherical particles in rectangular duct tend to move laterally toward the longer length wall and then slowly moves towards the equilibrium position at the face-centre along the long wall(fig.3a). We also observe that the focusing length is longer for spherical particles in a rectangular duct, about three times longer than that in square duct (fig. 3b). In case of an oblate particle flowing through a square duct, the lateral motion towards the face centred equilibrium position is similar to that of a sphere (fig.4a), however there is significant tumbling motion of the particle as it tries to reach equilibrium(fig.4b).In a rectangular duct of aspect ratio 2, the oblate particle reaches a steady configuration on the duct symmetry line at the center of the different faces (fig.5a). The focusing length surprisingly is shorter in a rectangular duct for an oblate particle in contrast to its focusing length in a square duct. This is attributed to the higher lateral velocity of the oblate in the second stage of the migration, that with negligible tumbling(fig.5b). The behavior of three oblate particles in a square duct of duct width to longer diameter ratio H/Ds= [3.5, 5, 10] is different compared to a sphere as the largest oblate tend to focus at the duct cross section diagonals compared to the other two which are at face centred equilibrium as in case of a sphere(fig.6a). We attribute this to the rotation rate of the larger particle which is initially increasing and then decreasing(fig.6b).When it comes to focusing lengths, the smaller particles need longer times to reach their final equilibrium(fig.6c). Another interesting behavior we see is the effect of Reynolds number, where it can be seen that the oblate particles show a tilt of 21 degrees when focusing at equilibrium at certain high Reynolds number (fig.7). CONCLUSIONThe results presented employ a highly accurate interface-resolved numerical algorithm, based on the Immersed Boundary Method to study the entire inertial migration of an oblate particle in both square and rectangular ducts and compare it with that of a single sphere. Currently, we apply a volume penalization method and polymeric drag component to the code to solve for viscoelastic effects in circular microcapillaries. ACKNOWLEDGEMENTSThis work was supported by the European Research Council Grant no. ERC-2013-CoG-616186, TRITOS and by the Swedish Research Council Grant no. VR 2014-5001, COST Action MP1305: Flowing matter, and computation time from SNIC. REFERENCES : Lashgari, Iman, et al. Journal of Fluid Mechanics 819 (2017): 540-561.
26.	Banerjee, Indradumna, et al. (author) Lab-on-DVD: Optical Disk Drive-Based Platforms for Point-of-Care Diagnostics 2018. - 2 In: Frugal Innovation in Bioengineering for the Detection of Infectious Diseases. - Switzerland : Springer. - 9783319666471 ; , s. 23-38 Book chapter (peer-reviewed)abstract There is a growing demand for simple, affordable, reliable and quality-assured point-of-care (POC) diagnostics for use in resource-limited settings. Among the top ten leading causes of death worldwide, three are infectious diseases, namely, respiratory infections, HIV/AIDS and diarrheal diseases (World Health Organization 2012). Although high-quality diagnostic tests are available, these are often not available to patients in developing countries. While recent development in microfluidics and “lab-on-a-chip” devices has the potential to spur the development of protocols and affordable instruments for diagnosis of infectious disease at POC, integration of complex sample preparation and detection into automated molecular and cellular systems remain a bottleneck for implementation of these systems at resource-limited settings. Towards this, we describe here how low-cost optical drives can, with minor modifications, be turned into POC diagnostic platforms. A DVD drive is essentially a highly advanced and low-cost optical laser-scanning microscope, with the capability to deliver high-resolution images for biological applications. Furthermore, the inherent centrifugal force on rotational discs is elegantly used for sample preparation and integration. Hence, the merging of low-cost optical disc drives with centrifugal microfluidics is feasible concept for POC diagnostics, specifically designed to meet the needs at resource-limited settings.
27.	Banerjee, Indradumna, 1986-, et al. (author) LDH based neonatal diagnostics on a low-cost slipdisc based sample preparation platform. 2016 Conference paper (peer-reviewed)abstract INTRODUCTIONSlipdisc is developed as a sample preparation platform based on slipchip technology [1], using a handwinded clockwork mechanism allowing sample processing from one spot to another with defined precision without the need for sophisticated tools or alignment (Fig.1). An ordinary smartphone or camera can be used to image and analyse the results making it an ideal tool for resource limited settings. Here, we demonstrate a bioassay for detecting LDH (Fig.2), a crucial enzyme found in all living cells which leaks out when the cellular membrane is damaged. This makes LDH a biomarker for several medical conditions in newborns, such as Ozkiraz-13, necrotizing enterocolitis (NEC), and Asphyxia.EXPERIMENTALFor assembling the slipdisc optically transparent, robust and disposable CD like polycarbonate discs were used with superhydrophobic coating on all except the embedded microfluidic channels. For the LDH assay, heparinized plasma samples were spiked with 7 different concentrations of the LDH enzyme (Lee Biosolutions, USA). These concentrations ranged from clinically normal to abnormal concentrations and used to construct a standard curve for LDH enzyme.RESULTS AND DISCUSSIONThe ability of the SlipDisc to quantify LDH enzyme levels from plasma samples was evaluated (Fig.3). Using 7 different concentrations, a standard curve with clinically relevant LDH concentrations was obtained (Fig4). Image and data analyses, including linear regression and Pearson’s correlation, were completed using Image processing tool in Matlab.CONCLUSIONWe demonstrate a low-cost neonatal diagnostics platform for the detection of LDH from plasma using a novel SlipDisc platform. The SlipDisc can further be modified to separate plasma from whole blood samples in order to fully integrate the assay. Its simple operation and smartphone based detection capabilities make it an ideal device for point-of-care neonatal diagnostics.
28.	Banerjee, Indradumna, et al. (author) MicroCAP 2018 Patent (pop. science, debate, etc.)abstract
29.	Banerjee, Indradumna, et al. (author) MicroCap : Microfluidic centrifuge assisted precipitation for DNA quantification on lab-on-DVD 2018 In: 22nd International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2018. - : Chemical and Biological Microsystems Society. - 9781510897571 ; , s. 1802-1805 Conference paper (peer-reviewed)abstract We report for the first time the MicroCAP technique, for rapid DNA detection and quantification, that does not require any purification or fluorescent labelling of DNA. The invention is based on DNA interacting with a detection dye (Gelred) to form a complex, that forms a visible precipitate within seconds of centrifugation. MicroCAP can be used for DNA quantification, when combined with the Lab-on-DVD with inbuilt centrifugation and sub-micron imaging resolution. We quantify PCR and LAMP assay products using MicroCAP on the integrated Lab-on-DVD platform, and demonstrate a detection limit of 10 ng/μl. Copyright
30.	Banerjee, Indradumna, et al. (author) MicroCAP: Microfluidic Centrifuge Assisted Precipitation for DNA Quantification on Lab-on-DVD 2018 Conference paper (peer-reviewed)abstract We report for the first time the MicroCAP technique, for rapid DNA detection and quantification, that does not require any purification or fluorescent labelling of DNA. The invention is based on DNA interacting with a detection dye (Gelred) to form a complex, that forms a visible precipitate within seconds of centrifugation. MicroCAP can be used for DNA quantification, when combined with the Lab-on-DVD with inbuilt centrifugation and sub- micron imaging resolution. We quantify PCR and LAMP assay products using MicroCAP on the integrated Lab-on- DVD platform, and demonstrate a detection limit of 10 ng/!".KEYWORDS: MicroCAP, DNA detection, Centrifuge,Precipitate, LAMP, PCR.INTRODUCTIONDetection of amplified DNA is often based on measurement of turbidity, fluorescence (after staining with a detec- tion dye) or absorbance. Commercially available instruments for DNA quantitation can be broadly divided into two categories: UV instruments based on absorbance (such as spectrophotometers, e.g. Nanodrop or Nanophotometer) and instruments based on measurement of a fluorescent dye (such as plate readers). One bottleneck in quantifying amplified DNA in a nucleic acid amplification test (NAAT) reaction, based on absorbance measurement technique, is the bias introduced due to the presence of the isothermal amplification buffer, dNTPs and other reagents. Each reagent or buffer may have an absorbance density at around 260 nm, elevating the apparent concentration measured by the device compared to the actual value. Hence, for most quantitation based NAATs, it is important to include an extra DNA purification step, which may result in non-negligible loss of the amplified product and increases the cost of the purification kit. Measurements based on fluorescence mostly use fluorescent dyes that are potentially hazardous for handling. In addition, fluorescence based quantitation methods require time consuming labelling and washing steps.In this report, we describe a new method, termed microfluidic centrifugation assisted precipitation (microCAP), involving quantification and detection of DNA based on precipitation of nucleic acids. The basis of the method is formation of a visible precipitate when GelRed, a nucleic acid intercalacting dye commonly used in gel electropho- resis, is mixed with DNA and centrifuged. A visible precipitate is formed after just a few seconds of centrifugation and enables rapid detection of the presence of DNA in a sample. To the best of our knowledge, the visible precipitate formed as a product of centrifuging GelRed mixed with DNA has not been reported before. We showed that the DNA GelRed complex is dense enough compared to water to precipitate upon centrifugation. Further, we extended the μCAP method to the Lab-on-DVD platform1 to quantify the DNA concentration from images generated using the optical DVD reader instrument. The modified DVD player was able to image the precipitate formed up to a detection limit of 10 ng/μl of DNA. For calibration of the images, known quantities of a purified PCR product were used to identify the relationship between the amounts of DNA and precipitate formed. We applied the method to quantify an unknown quantity of LAMP amplicons from a LAMP assay for a HIV-1B type genome containing plasmid on the Lab-on-DVD platform. A sensitivity limit of 10 ng/μl of DNA was achieved, comparable with that of a Nanophotometer.18 The results demonstrated that the method is able to quantitatively detect the presence of DNA in a sample in a few seconds without any purification step.EXPERIMENTALThe Lab-on-DVD system was employed for spinning and imaging the precipitate product using a modified DVD drive, as mentioned in our previous report.1 We began by dispensing the sample in the design chamber, adding GelRed dye (at a concentration of 4000X in water) and centrifuging the mixture at 1200 rpm. Figure 1a and 1bshow schematics of the DNA sample precipitation process conducted in test tubes and the DVD platform, respec- tively. We used known amounts of a PCR product to calibrate the quantity of precipitate to the DNA concentration. We used a HIV genome amplified from 50 ng of plasmid pNL4.3 using the primers 0776F and 6231R.2 To evaluate the sensitivity of DNA detection of our system, we used the amplified products from a LAMP assay. The sensitivity of LAMP primers was tested on DNA from pNL4.3 (a HIV-1B genome containing plasmid). A 25X LAMP primer mix was prepared according to Curtis et al.,3 using the same template DNA sequence, set of primers and DNA polymerase. Eight concentrations (each being 5 μl volume) of the HIV-1B genome containing plasmid (pNL4.3) were tested, starting from 1 ng/!" serially diluted to 1 fg/!". Two negative controls were also prepared, one without DNA and primers and one without primers. The total reaction volume was increased to 30 μl (instead of 25 μl used in Curtis et al.3) by multiplying every component volume in the reaction by a factor of 1.2. Fabrication of the multi- layer microfluidic Disc followed the same procedure as described in our previous report.1 The Lab-on-DVD system was used to generate images of the precipitation zone. To quantify the amount of precipitate, an image processing script was written in MATLAB software (Mathworks, USA).RESULTS AND DISCUSSIONMicroCAP was found to be suitable for determining the presence of DNA in a sample, We carried out the LAMP assay in Eppendorf tubes in an oven set at 65°C. After 45 minutes, 3 μl of 10,000X GelRed in water was added to two tubes of 30 μl volume each, one having an unknown concentration of LAMP amplified DNA and the other one with no DNA template as a control. After centrifugation for approximately 5 seconds, a visible precipitate was formed in the tube containing amplified DNA, whereas no precipitate was formed in the control tube (Fig. 2a). 10 μl volume of DNA was inserted into a U shaped channel of the DVD alongwith 1 μl of 10,000X GelRed in water, which was the same ratio of DNA sample to Gelred as used in the test tube. An imageable precipitate was observed in the Lab on DVD custom imaging software (fig.2b).A Matlab script was used for image analysis in which an original image(fig.3a) was transformed into a binary image (fig.3b) by defining a threshold pixel value, exploiting the difference in intensity of the precipitate from its background. The entire area to the left of the threshold line in the histogram (Fig. 3c), i.e. from value 0 to the threshold value (normally 90), was summed to estimate the total area of the precipitate.For DNA quantification, known concentrations of a PCR product was used for calibration. The initial concentration of purified PCR product was 129 ng/μl, measured with a Nanophotometer (in triplicates) after purification with a GeneJet PCR purification kit. The purified PCR product was subsequently diluted serially several times and each diluted concentration was measured again with the Nanophotometer (in triplicate). The measurements were then repeated with the Lab-on-DVD method. Fig. 4a shows four images recorded at four known concentrations together with their binary threshold images. Fig. 4b shows the precipitation area calculated from the images plotted against the known DNA concentrations, showing a linear relationship. 10 ng/μl was the lowest concentration detectable in the DVD images.For quantification of unknown quantities of nucleic acids, we carried out the LAMP assay on HIV-1B genome containing plasmid DNA using serial dilutions (10-fold dilutions from 1 ng/μl to 0.1 fg/μl) to evaluate the limit of detection (Fig.5). Two negative controls were also prepared, one comprising primers and no DNA template and second, no DNA template and no primers.Fig. 6 shows the precipitation area plotted against the starting concentration of DNA template. It shows that the amplification in the LAMP assay is not linear for all the starting concentrations of DNA template. The error bars in the figure show the standard deviation for a particular concentration. For a LAMP assay, which fluctuates somewhat in its yield of amplified prod- ucts, we believe that this error range is acceptable.The precipitation area was converted to an actual yield of DNA products for each of the concentrations. This conversion was based on the linear empirical equation generated from the calibration curve presented earlier in Fig. 4b, given by:y= 9.61x – 4.05 (1) Here, y denotes the precipitation area in arbitrary units while x denotes the DNA concentration.CONCLUSIONWe demonstrated an extremely fast visual DNA quantification method (μCAP) that can be made quantifiable on a Lab-on-DVD platform. The approach was based on DNA forming a precipitate upon centrifugation when in contact with the GelRed dye. Results using HIV-1B genome containing plasmid DNA revealed a detection limit of 0.01 pg/μl or total amount of 0.1 pg of starting DNA template, which is an acceptable standard for resource limited settings. The limit of detection of DNA with the Lab-on-DVD platform was found to be 10 ng/μl, which is almost comparable to the detection limits reported by commercially available instruments, such as the Nanophotometer. However, the μCAP method offers a distinct advantage over other state-of-the-art techniques as it does not require additional purification of the DNA. We believe the μCAP technique combined with the Lab-on-DVD platform provides a simple and low cost technology that can fulfil the need for a point-of-care device for DNA quantification.REFERENCES[1] H. Ramachandraiah, M. Amasia, J. Cole, P. Sheard, S. Pickhaver, C. Walker, V. Wirta, P. Lexow, R. Lione and A. Russom, "Lab-on-DVD: standard DVD drives as a novel laser scanning microscope for image based point of care diagnostics."Lab. Chip, 2013, 13, 1578–1585.[2] S. Grossmann, P. Nowak, and U. Neogi, “ Subtype-independent near full-length HIV-1 genome sequencing and assembly to be used in large molecular epide
31.	Banerjee, Indradumna, 1986-, et al. (author) Microfluidic Centrifugation Assisted Precipitation based DNA Quantification Other publication (other academic/artistic)abstract Nucleic acid amplification methods are increasingly being used to detect trace quantities of DNA in samples for various diagnostic applications. However, quantifying the amount of DNA from such methods often require time consuming purification, washing or labeling step. Here, we report a novel microfluidic centrifugation assisted precipitation (uCAP) method for single-step DNA quantification. The method is based on formation of a visible precipitate, that can be quantified, when an intercalating dye (GelRed) is added to DNA sample and centrifuged for few seconds. We describe the mechanism leading to the precipitation phenomenon. We utilize centrifugal microfluidics to precisely control the formation of visible and quantifiable mass. Using a standard CMOS sensor for imaging, we report a detection limit of 45 ng/ul. Furthermore, using an integrated Lab-on-DVD platform we recently developed, the detection limit was lowered to 10 ng/ul, which is comparable to current commercially available instruments for DNA quantification. As a proof of principle, we demonstrate the quantification of LAMP products for a HIV-1B type genome containing plasmid on the Lab-on-DVD platform. The simple DNA quantification system could facilitate advanced molecular diagnosis at point of care.
32.	Banerjee, Indradumna, et al. (author) Microfluidic centrifugation assisted precipitation based DNA quantification 2019 In: Lab on a Chip. - : Royal Society of Chemistry. - 1473-0197 .- 1473-0189. ; 19:9, s. 1657-1664 Journal article (peer-reviewed)abstract Nucleic acid amplification methods are increasingly being used to detect trace quantities of DNA in samples for various diagnostic applications. However, quantifying the amount of DNA from such methods often requires time consuming purification, washing or labeling steps. Here, we report a novel microfluidic centrifugation assisted precipitation (mu CAP) method for single-step DNA quantification. The method is based on formation of a visible precipitate, which can be quantified, when an intercalating dye (GelRed) is added to the DNA sample and centrifuged for a few seconds. We describe the mechanism leading to the precipitation phenomenon. We utilize centrifugal microfluidics to precisely control the formation of the visible and quantifiable mass. Using a standard CMOS sensor for imaging, we report a detection limit of 45 ng mu l(-1). Furthermore, using an integrated lab-on-DVD platform we recently developed, the detection limit is lowered to 10 ng mu l(-1), which is comparable to those of current commercially available instruments for DNA quantification. As a proof of principle, we demonstrate the quantification of LAMP products for a HIV-1B type genome containing plasmid on the lab-on-DVD platform. The simple DNA quantification system could facilitate advanced point of care molecular diagnostics.
33.	Banerjee, Indradumna, et al. (author) Particle focusing dynamics in extended elasto inertial flow 2018 In: 22nd International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2018. - : Chemical and Biological Microsystems Society. - 9781510897571 ; , s. 472-475 Conference paper (peer-reviewed)abstract We report the decoupled effects of inertial and viscous forces on particle focusing, the stability of particles, and particle trajectories to reach equilibrium position in an extended elasto inertial pressure driven flow, in a circular micro-capillary. We report numerically and experimentally for the first time, the existence of multiple stable equilibrium positions in the EEI regime, which was unobserved for flows previously studied at lower Reynolds number viscoelastic flows.
34.	Banerjee, Indradumna (author) Point of care microfluidic tool development for resource limited settings 2019 Doctoral thesis (other academic/artistic)abstract The development of point of care diagnostics using recent advances in microfluidics have the potential to transform health care in several ways, especially in resource limited settings with limited access to advanced health care infrastructure. However, translating a point of care device to reality is often a challenging task because of the complexities involved in integrating a number of diverse engineering concepts into an easy to use, accurate and portable device. This thesis focuses on miniaturization of crucial diagnostic laboratory tools, that can be used in a portable point of care format without compromising on the accuracy or performance. The first part of the thesis (Paper I-III) focuses on understanding and applying elasto-inertial microfluidics, which is a label-free and passive bio-particle sorting and separation method. A basic understanding of particle trajectories in both inertial (Paper I) and visco-elastic flows (Paper II) is established, followed by an investigation on the combined effects of inertia and elasticity (Paper III). The second part of the thesis (Paper IV-VI) focuses on developing integrated microfluidic platforms, each of which addresses different aspects of point of care diagnostic applications. The applications include neonatal diagnostics using a hand-driven Slipdisc technique (Paper IV), rapid nucleic acid quantification using a novel precipitate-based detection on a centrifugal microfluidics platform (Paper V), and hematocrit level measurement in blood using a portable lab-on- Disc platform operated by a mobile phone (Paper VI). The proof of concept microfluidic tools presented in the scope of this thesis have the potential to replace a number of functions of standard laboratory equipment, at a fraction of the price and without compromising performance. Hence, the different methods developed should contribute towards decentralization of medical testing laboratories, making healthcare accessible to one and all.
35.	Banerjee, I., et al. (author) Slipdisc : A versatile sample preparation platform 2015 In: MicroTAS 2015 - 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences. - : Chemical and Biological Microsystems Society. - 9780979806483 ; , s. 1256-1258 Conference paper (peer-reviewed)abstract We present "SlipDisc", a versatile sample preparation platform based on slipchip1 technology. The SlipDisc platform uses polycarbonate CDs and laser cut PSA instead of glass and a hand-winded mechanical clock mechanism to precisely manipulate minute amount of liquid. The innovative hand-winded mechanical "clockwork" that enables sample processing from one spot to another with defined precision. As a prof of principle of bioassay, we show HRP enzyme reacting with TMB substrate and a multilayer architecture used in manipulation of magnetic beads through an immiscible oil phase. Our long-term goal is to develop a sample-in-result-out multi-parametric bioanalytical SlipDisc platform specifically designed to need the needs at resource-limited settings for point of care molecular diagnostics.
36.	Banerjee, Indradumna, et al. (author) Slipdisc : A versatile sample preparation platform for point of care diagnostics 2017 In: RSC Advances. - : Royal Society of Chemistry. - 2046-2069. ; 7:56, s. 35048-35054 Journal article (peer-reviewed)abstract We report a microfluidic sample preparation platform called "Slipdisc" based on slipchip technology. Slipdisc is a rotational slipchip that uses a unique hand-wound clockwork mechanism for precise movement of specially fabricated polycarbonate discs. In operation, the microchannels and microchambers carved on the closely aligned microfluidic discs convert from continuous filled paths to defined compartments using the slip movement. The clockwork mechanism introduced here is characterised by a food dye experiment and a conventional HRP TMB reaction before measuring lactate dehydrogenase (LDH) enzyme levels, which is a crucial biomarker for neonatal diagnostics. The colorimetry based detection of LDH was performed with an unmodified camera and an image analysis procedure based on normalising images and observing changes in red channel intensity. The analysis showed a close to unity coefficient of determination (R2 = 0.96) in detecting the LDH concentration when compared with a standard Chemical Analyser, demonstrating the excellent performance of the slipdisc platform with colorimetric detection. The versatile point of care sample preparation platform should ideally be suited for a multitude of applications at resource-limited settings.
37.	Banerjee, Indradumna, 1986-, et al. (author) Three dimensional Slipdisc aimed at HIV viral load detection 2017 Conference paper (peer-reviewed)abstract In this study, we introduce a three dimensional Slipdisc1 based on slipchip technology, aimed to be used at detection of HIV-1 viral load in resource limited settings. This method is based on magnetic bead based isolation of RNA from sample, followed by release of RNA in an elution buffer, followed by amplification of the initial concentration of RNA in an elution buffer. The novelty is the extraction method which is a one step method and involves using magnetic beads attached to sample DNA to pass through an immiscible oil phase2 for one step washing.
38.	Bose, I., et al. (author) An integrated all foil based micro device for point of care diagnostic applications 2018 In: Sensors and actuators. B, Chemical. - : Elsevier B.V.. - 0925-4005 .- 1873-3077. ; 259, s. 917-925 Journal article (peer-reviewed)abstract Point-of-Care (POC) diagnostics often fail to meet the market requirements of low cost and advanced functionality, and are often limited to lateral flow based serological diagnostics with reduced sensitivity and specificity. We report here on an integrated microfluidic absorbance measurement device fabricated by roll-to-roll (R2R) compatible manufacturing processes, suitable for low cost POC systems. It is a device exclusively made of foils and takes external light from a low cost LED and converts the point light source to a homogeneous light via a foil based optical filter at the bottom of the device. The light is converted to an electrical signal by an amorphous organic semiconductor (OSC) material, integrated with screen-printed carbon finger on top of the device for electrical measurement. As a proof of principle, we demonstrate DNA hybridization assay, where the target DNA is coupled to magnetic beads for absorbance measurement. The device successfully distinguishes between matched and mismatched DNA hybridization and can differentiate between 1 μM, 50 nM and 2.5 nM DNA target concentrations. The inherent characteristics of the substrates and R2R fabrication concept significantly reduce the cost, making it suitable for POC applications at resource-limited settings.
39.	Bose, Indranil, et al. (author) DNA Analysis on integrated all foil based microdevices Other publication (other academic/artistic)
40.	Campinoti, Sara, et al. (author) Liver extracellular matrix and perfusion bioreactor culture promoting human amnion epithelial cell differentiation towards hepatocyte-like cells Other publication (other academic/artistic)
41.	Cruz, Javier, 1990- (author) Microfluidics for High-Pressure Inertial Focusing : Focusing, Separation and Concentration of Micro and Sub-micron Particles 2020 Doctoral thesis (other academic/artistic)abstract The birth of microsystems set the ground for technologies never imagined before, for it is not only the small size what characterizes the miniaturized systems, but unique phenomena arise in the micro scale. This thesis relates to one such unique phenomenon, inertial focusing, a phenomenon that occurs in microfluidic systems if very special conditions are met and that allows for fine manipulation of particles in fluid samples. This ability is key in a bigger picture: the analysis of complex fluids, where rare particles of interest may be present in very few numbers amongst a myriad of others, making the task difficult – if not impossible. A system exploiting inertial focusing allows, for instance, to focus, separate, isolate and concentrate such rare particles of interest, and even to transfer them to another fluid, thereby enabling/facilitating their detection and analysis. Examples of rare particles of interest in complex fluids are circulating tumor cells in blood, that give away the presence of cancer, extracellular vesicles also in blood, that contain biomarkers with physiological and pathological information about the patient, or bacteria in natural water, where the species present and their numbers are to be monitored for safety reasons and/or biological studies. This thesis covers the state of art physical principles behind the phenomenon and extends the understanding both in theory and applications. Specifically, the technology is extended to allow for manipulation of sub-micron particles, a range of interest as it comprises bacteria, viruses and organelles of eukaryotic cells. This was possible by an analysis of the balance of forces in play and by the integration of inertial focusing in high-pressure systems (up to 200 bar). In a second block, a very special line of inertial focusing is introduced and developed; inertial focusing in High Aspect Ratio Curved (HARC) microfluidics. These systems, engineered to rearrange the force field responsible for the particle manipulation, not only achieve excellent performances for focusing and concentration of particles, but also extreme resolution in their separation (mathematically unlimited; demonstrated experimentally for differences in size down to 80 nm). Perhaps more important than the performance, the systems are stable, intuitive and simpler to design, attributes that we hope will make the technology and its outstanding benefits more accessible to the community. With its remarkable performance, it would not come as a surprise if, in the near future, inertial focusing makes a strong impact on how analyses are performed nowadays and opens up for possibilities beyond the current state of the art.
42.	Damiati, Samar, et al. (author) Flex Printed Circuit Board Implemented Grapene-Based DNA Sensor for Detection of SARS-CoV-2 2021 In: IEEE Sensors Journal. - : Institute of Electrical and Electronics Engineers (IEEE). - 1530-437X .- 1558-1748. ; 21:12, s. 13060-13067 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.
43.	Dietvorst, Jiri, et al. (author) Bacteria Detection at a Single-Cell Level through a Cyanotype-Based Photochemical Reaction 2022 In: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 94:2, s. 787-792 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.
44.	Dånmark, Staffan, et al. (author) Development of a novel microfluidic device for long-term in situ monitoring of live cells in 3-dimensional matrices 2012 In: Biomedical microdevices (Print). - : Springer Science and Business Media LLC. - 1387-2176 .- 1572-8781. ; 14:5, s. 885-893 Journal article (peer-reviewed)abstract Using the latest innovations in microfabrication technology, 3-dimensional microfluidic cell culture systems have been developed as an attractive alternative to traditional 2-dimensional culturing systems as a model for long-term microscale cell-based research. Most microfluidic systems are based on the embedding of cells in hydrogels. However, physiologically realistic conditions based on hydrogels are difficult to obtain and the systems are often too complicated. We have developed a microfluidic cell culture device that incorporates a biodegradable rigid 3D polymer scaffold using standard soft lithography methods. The device permits repeated high-resolution fluorescent imaging of live cell populations within the matrix over a 4 week period. It was also possible to track cell development at the same spatial location throughout this time. In addition, human primary periodontal ligament cells were induced to produce quantifiable calcium deposits within the system. This simple and versatile device should be readily applicable for cell-based studies that require long-term culture and high-resolution bioimaging.
45.	Dånmark, Staffan, et al. (author) Development of Novel Microfluidic Device for Long-Term in situ Monitoring of Live Cells in 3-dimensional Matrices Other publication (other academic/artistic)
46.	Etcheverry, Sebastián, et al. (author) All fiber based micro-flow cytometer by combining optical fiber with inertial focusing 2016 In: 20th International Conference on Miniaturized Systems for Chemistry and Life Sciences (MicroTAS 2016). - : Chemical and Biological Microsystems Society. - 9780979806490 ; , s. 1655-1656 Conference paper (peer-reviewed)abstract Towards a portable point of care flow cytometry platform, we present here an integrated all optical fiber-based optofluidic system capable of counting and discriminating fluorescent particles and cells. The robust and compact device incorporates optical fibers and circular capillaries to build an all-fiber optofluidic device to enable counting particles based on their fluorescent and back-scatter light emission. Here, we combine this with inertial- and elasto-inertial microfluidics for sheathless particle and cell focusing for integrated detection with scattering and fluorescence detections - all necessary components of standard cytometers. We validated the system for cell counting based on scattering and fluorescence.
47.	Etcheverry, Sebastian, et al. (author) All silica fibre microflow cytometer Other publication (other academic/artistic)abstract Flow cytometry is currently the gold standard for analysis of cells in the medical laboratory and biomedical research. Fuelled by the need of point-of-care diagnosis, a significant effort has been made to miniaturize and reduce cost of flow cytometers. However, despite recent advances, current microsystems remain less versatile and much slower than their large-scale counterparts. In this work, an all-silica fibre microflow cytometer is presented that measures fluorescence and scattering from particles and cells. It integrates cell transport in circular capillaries and light delivery by optical fibres Single-stream cell focusing is performed by Elasto-inertial microfluidics to guarantee optical accuracy and sensitivity. The capability of this technique is extended to high flow rates (up to 800 µl/min), enabling throughput of 2500 particles/s. The robust, portable and low-cost system described here could be the basis for a point-of-care flow cytometer with a performance comparable to commercial systems.
48.	Etcheverry, Sebastián, et al. (author) Fluidic trapping and optical detection of microparticles with a functional optical fiber 2017 In: Optics Express. - : Optical Society of America. - 1094-4087. ; 25:26, s. 33657-33663 Journal article (peer-reviewed)abstract A fiber probe is presented that traps single micro-sized particles and allows detection of their optical properties. The trapping mechanism used is based on fluid suction with a micro-structured optical fiber that has five holes along its cladding. Proof-of-principle experiments with a diluted solution of fluorescently labeled particles are performed. The fiber probe presented here may find various applications in life-science and environmental monitoring. ©
49.	Etcheverry, Sebastián, et al. (author) High performance micro-flow cytometer based on optical fibres 2017 In: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 7:1 Journal article (peer-reviewed)abstract Flow cytometry is currently the gold standard for analysis of cells in the medical laboratory and biomedical research. Fuelled by the need of point-of-care diagnosis, a significant effort has been made to miniaturize and reduce cost of flow cytometers. However, despite recent advances, current microsystems remain less versatile and much slower than their large-scale counterparts. In this work, an all-silica fibre microflow cytometer is presented that measures fluorescence and scattering from particles and cells. It integrates cell transport in circular capillaries and light delivery by optical fibres. Single-stream cell focusing is performed by Elasto-inertial microfluidics to guarantee accurate and sensitive detection. The capability of this technique is extended to high flow rates (up to 800 mu l/min), enabling a throughput of 2500 particles/s. The robust, portable and low-cost system described here could be the basis for a point-of-care flow cytometer with a performance comparable to commercial systems.
50.	Etcheverry, Sebastian, et al. (author) Optical Fiber inertial focusing based micro Flowcytometer In: Nature Communications. - 2041-1723. Journal article (peer-reviewed)abstract Flow cytometry is a powerful method for analysis of cells and particles. Fueled by the need for point of care diagnostic applications, a significant effort has been made to miniaturize flow cytometry. However, despite recent advances, current microflow cytometers remain less versatile and much slower than their large-scale counterparts. Here, we present a portable all-silica optofluidic device that integrates particle focusing in flow through cylindrical silica capillaries and light delivery in optical fibers to simultaneously measure fluorescence and scattering from cells and particles at a rate of 2500 particles/s – a throughput comparable to conventional cytometers. Precise 3D cell focusing and ordering is accomplished using extended elasto-inertial focusing (EEF), a key enabler for eliminating the sheath fluid commonly employed in flow cytometry with maintained high throughput. We demonstrate simultaneously two-color fluorescence and scattering measurement of different sized particles and cells. This robust and low-cost optofluidic device, assembled without the need of clean-room facilities, is ideal suited for point of care applications.

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