Publications by authors named "Aman Russom"

43 Publications

Bacteria Detection at a Single-Cell Level through a Cyanotype-Based Photochemical Reaction.

Anal Chem 2022 Jan 21;94(2):787-792. Epub 2021 Dec 21.

Instituto de Microelectrónica de Barcelona (IMB-CNM, CSIC), Bellaterra (Barcelona) 08193, Spain.

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 nanostructure-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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http://dx.doi.org/10.1021/acs.analchem.1c03326DOI Listing
January 2022

High resolution and rapid separation of bacteria from blood using elasto-inertial microfluidics.

Electrophoresis 2021 12 23;42(23):2538-2551. Epub 2021 Sep 23.

KTH Royal Institute of Technology, Division of Nanobiotechnology, Department of Protein Science, Science for Life Laboratory, Solna, Sweden.

Improved sample preparation has the potential to address unmet needs for fast turnaround sepsis 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 fully focused 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 remain fully 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 of 1 mL/min. For blood-based experiments, a minimum of 1:2 dilution was necessary to fully focus 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 mL of 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 genotypic analysis.
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http://dx.doi.org/10.1002/elps.202100140DOI Listing
December 2021

Toward Rapid Detection of Viable Bacteria in Whole Blood for Early Sepsis Diagnostics and Susceptibility Testing.

ACS Sens 2021 09 19;6(9):3357-3366. Epub 2021 Aug 19.

Division of Nanobiotechnology, Department of Protein Science, Science for Life Laboratory, KTH Royal Institute of Technology, Stockholm 17165, Sweden.

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 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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http://dx.doi.org/10.1021/acssensors.1c01219DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8477386PMC
September 2021

Knowing more from less: miniaturization of ligand-binding assays and electrophoresis as new paradigms for at-line monitoring and control of mammalian cell bioprocesses.

Curr Opin Biotechnol 2021 10 7;71:55-64. Epub 2021 Jul 7.

KTH Royal Institute of Technology, Department of Industrial Biotechnology, School of Engineering Sciences in Chemistry, Biotechnology and Health, Stockholm, Sweden; AdBIOPRO, Competence Centre for Advanced BioProduction by Continuous Processing, KTH, Stockholm, Sweden. Electronic address:

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http://dx.doi.org/10.1016/j.copbio.2021.06.018DOI Listing
October 2021

Sample-to-answer COVID-19 nucleic acid testing using a low-cost centrifugal microfluidic platform with bead-based signal enhancement and smartphone read-out.

Lab Chip 2021 08 11;21(15):2932-2944. Epub 2021 Jun 11.

KTH Royal Institute of Technology, Division of Nanobiotechnology, Department of Protein Science, Science for Life Laboratory, Solna, Sweden. and AIMES - Center for the Advancement of Integrated Medical and Engineering Sciences at Karolinska Institutet and, KTH Royal Institute of Technology, Stockholm, Sweden.

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 dried n-benzyl-n-methylethanolamine modified agarose beads used to selectively remove primer dimers, inactivate the reaction post-amplification and allowing enhanced fluorescence detection via a 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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http://dx.doi.org/10.1039/d1lc00266jDOI Listing
August 2021

High throughput viscoelastic particle focusing and separation in spiral microchannels.

Sci Rep 2021 04 19;11(1):8467. Epub 2021 Apr 19.

Division of Nanobiotechnology, Department of Protein Science, Science for Life Laboratory, KTH Royal Institute of Technology, Solna, Sweden.

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 µm particles were effectively separated from 10 µm particles. A separation efficiency of 98% for the 10 µm and 97% for the 15 µm 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 µm and 99% for the 15 µm 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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http://dx.doi.org/10.1038/s41598-021-88047-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8055915PMC
April 2021

Multiplexed Microfluidic Cartridge for At-Line Protein Monitoring in Mammalian Cell Culture Processes for Biopharmaceutical Production.

ACS Sens 2021 03 16;6(3):842-851. Epub 2021 Mar 16.

KTH Royal Institute of Technology, Division of Nanobiotechnology, Department of Protein Science, Science for Life Laboratory, 171 21 Solna, Sweden.

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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http://dx.doi.org/10.1021/acssensors.0c01884DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8034812PMC
March 2021

Multi-layer assembly of cellulose nanofibrils in a microfluidic device for the selective capture and release of viable tumor cells from whole blood.

Nanoscale 2020 Nov;12(42):21788-21797

KTH Royal Institute of Technology, Division of Nanobiotechnology, Department of Protein Science, Science for Life Laboratory, Solna, Sweden.

According to reports by the World Health Organization (WHO), cancer-related deaths reached almost 10 million in 2018. Nearly 65% of these deaths occurred in low- to middle-income countries, a trend that is bound to increase since cancer diagnostics are not currently considered a priority in resource-limited settings (RLS). Thus, cost-effective and specific cancer screening and diagnostics tools are in high demand, particularly in RLS. The selective isolation and up-concentration of rare cells while maintaining cell viability and preventing phenotypic changes is a powerful tool to allow accurate and sensitive downstream analysis. Here, multi-layer cellulose nanofibril-based coatings functionalized with anti-EpCAM antibodies on the surface of disposable microfluidic devices were optimized for specific capture of target cells, followed by efficient release without significant adverse effects. HCT 116 colon cancer cells were captured in a single step with >97% efficiency at 41.25 μL min-1 and, when spiked in whole blood, an average enrichment factor of ∼200-fold relative to white blood cells was achieved. The release of cells was performed by enzymatic digestion of the cellulose nanofibrils which had a negligible impact on cell viability. In particular, >80% of the cells were recovered with at least 97% viability in less than 30 min. Such performance paves the way to expand and improve clinical diagnostic applications by simplifying the isolation of circulating tumor cells (CTCs) and other rare cells directly from whole blood.
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http://dx.doi.org/10.1039/d0nr05375aDOI Listing
November 2020

Adhesion molecule cross-linking and cytokine exposure modulate IgE- and non-IgE-dependent basophil activation.

Immunology 2021 01 29;162(1):92-104. Epub 2020 Oct 29.

Department of Clinical Science and Education, Karolinska Institutet, Södersjukhuset, Stockholm, Sweden.

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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http://dx.doi.org/10.1111/imm.13268DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7730031PMC
January 2021

Sub-attomole detection of HIV-1 using padlock probes and rolling circle amplification combined with microfluidic affinity chromatography.

Biosens Bioelectron 2020 Oct 26;166:112442. Epub 2020 Jul 26.

Division of Nanobiotechnology, Department of Protein Science, Science for Life Laboratory, KTH Royal Institute of Technology, Solna, Sweden. Electronic address:

Despite significant progress in diagnostics and disease management during the past decades, human immunodeficiency virus (HIV) infections are still responsible for nearly 1 million deaths every year, mostly in resource-limited settings. Thus, novel, accurate and cost-effective tools for viral load monitoring become crucial to allow specific diagnostics and the effective monitoring of the associated antiviral therapies. Herein, we report an effective combination of a (1) padlock probe (PLP)-mediated rolling circle amplification (RCA) bioassay and an (2) agarose bead-based microfluidic device for the affinity chromatography-based capture and detection of RCA products (RCPs) pre-labelled simultaneously with biotin and an organic fluorophore. This method allowed the efficient capture of ~1 μm-sized RCPs followed by their quantification either as discrete signals or an average fluorescence signal, thus being compatible with both high-resolution imaging for maximum sensitivity as well as simpler optical detection setups. A limit of detection < 30 fM was obtained for HIV-1 synthetic target with just a single round of RCA, comparable to recently reported procedures requiring technically complex amplification strategies such as hyperbranching and/or enzymatic digestion/amplification. Furthermore, targeting a set of five conserved regions in the HIV-1 gag gene, the method could specifically detect HIV-1 in 293T cell culture supernatants, as well as a set of 11 HIV-1 NIH reference samples with four different subtypes. The reported method provides simplicity of operation, unique versatility of signal transduction (i.e. average or discrete signals), and potential coupling with previously reported miniaturized photodetectors. These combined features hold promise for bringing RCA-based molecular diagnostics closer to the point-of-care.
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http://dx.doi.org/10.1016/j.bios.2020.112442DOI Listing
October 2020

Frequency dependent multiphase flows on centrifugal microfluidics.

Lab Chip 2020 02 3;20(3):514-524. Epub 2020 Jan 3.

Division of Nanobiotechnology, Department of Protein Sciences, Science for Life Laboratory, KTH Royal Institute of Technology, Stockholm, Sweden.

The simultaneous flow of gas and liquids in large scale conduits is an established approach to enhance the performance of different working systems under critical conditions. On the microscale, the use of gas-liquid flows is challenging due to the dominance of surface tension forces. Here, we present a technique to generate common gas-liquid flows on a centrifugal microfluidic platform. It consists of a spiral microchannel and specific micro features that allow for temporal and local control of stratified and slug flow regimes. We investigate several critical parameters that induce different gas-liquid flows and cause the transition between stratified and slug flows. We have analytically derived formulations that are compared with our experimental results to deliver a general guideline for designing specific gas-liquid flows. As an application of the gas-liquid flows in enhancing microfluidic systems' performance, we show the acceleration of the cell growth of E. coli bacteria in comparison to traditional culturing methods.
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http://dx.doi.org/10.1039/c9lc00924hDOI Listing
February 2020

A novel tool for clinical diagnosis of allergy operating a microfluidic immunoaffinity basophil activation test technique.

Clin Immunol 2019 12 24;209:108268. Epub 2019 Oct 24.

Department of Clinical Science and Education, Karolinska Institutet, Stockholm, Sweden; Sachs´ Children and Youth Hospital, Södersjukhuset, Stockholm, Sweden. Electronic address:

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-FcεRI), 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.
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http://dx.doi.org/10.1016/j.clim.2019.108268DOI Listing
December 2019

Microfluidic Immunoaffinity Basophil Activation Test for Point-of-Care Allergy Diagnosis.

J Appl Lab Med 2019 09 9;4(2):152-163. Epub 2019 May 9.

Division of Nanobiotechnology, Department of Protein Science, Science for Life Laboratory, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, Stockholm, Sweden;

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-FcεRI 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. We evaluated 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 ( = 0.0002) in on-chip activated basophils compared with nonactivated cells. The difference in CD63 expression on anti-FcεRI-activated captured basophils in microfluidic chip was significantly higher ( = 0.03) in patients with allergies compared with healthy controls, and the results were comparable with flow cytometry analysis ( = 0.04). Furthermore, there was no significant difference of CD63% expression in anti-FcεRI-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.
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http://dx.doi.org/10.1373/jalm.2018.026641DOI Listing
September 2019

A rapid smartphone-based lactate dehydrogenase test for neonatal diagnostics at the point of care.

Sci Rep 2019 06 26;9(1):9301. Epub 2019 Jun 26.

Division of Nanobiotechnology, Department of Protein Science, Science for Life Laboratory, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, Stockholm, Sweden.

There is a growing recognition of the importance of point-of-care tests (POCTs) for detecting critical neonatal illnesses to reduce the mortality rate in newborns, especially in low-income countries, which account for 98 percent of reported neonatal deaths. Lactate dehydrogenase (LDH) is a marker of cellular damage as a result of hypoxia-ischemia in affected organs. Here, we describe and test a POC LDH test direct from whole blood to provide early indication of serious illness in the neonate. The sample-in-result-out POC platform is specifically designed to meet the needs at resource-limited settings. Plasma is separated from whole blood on filter paper with dried-down reagents for colorimetric reaction, combined with software for analysis using a smartphone. The method was clinically tested in newborns in two different settings. In a clinical cohort of newborns of Stockholm (n = 62) and Hanoi (n = 26), the value of R using Pearson's correlation test was 0.91 (p < 0.01) and the R = 0.83 between the two methods. The mean LDH (±SD) for the reference method vs. the POC-LDH was 551 (±280) U/L and 552 (±249) U/L respectively, indicating the clinical value of LDH values measured in minutes with the POC was comparable with standardized laboratory analyses.
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http://dx.doi.org/10.1038/s41598-019-45606-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6595069PMC
June 2019

A micro-dispenser for long-term storage and controlled release of liquids.

Nat Commun 2019 01 14;10(1):189. Epub 2019 Jan 14.

Division of Nanobiotechnology, Department of Protein Sciences, Science for Life Laboratory, KTH Royal Institute of Technology, Stockholm, 17165, Sweden.

The success of lab-on-a-chip systems may depend on a low-cost device that incorporates on-chip storage and fluidic operations. To date many different methods have been developed that cope separately with on-chip storage and fluidic operations e.g., hydrophobic and capillary valves pneumatic pumping and blister storage packages. The blister packages seem difficult to miniaturize and none of the existing liquid handling techniques despite their variety are capable of proportional repeatable dispensing. We report here on an inexpensive robust and scalable micro-dispenser that incorporates long-term storage and aliquoting of reagents on different microfluidics platforms. It provides long-term shelf-life for different liquids enables precise dispensing on lab-on-a-disc platforms and less accurate but proportional dispensing when operated by finger pressure. Based on this technology we introduce a method for automation of blood plasma separation and multi-step bioassay procedures. This micro-dispenser intends to facilitate affordable portable diagnostic devices and accelerate the commercialization of lab-on-a-chip devices.
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http://dx.doi.org/10.1038/s41467-018-08091-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6331589PMC
January 2019

Silica bead-based microfluidic device with integrated photodiodes for the rapid capture and detection of rolling circle amplification products in the femtomolar range.

Biosens Bioelectron 2019 Mar 18;128:68-75. Epub 2018 Dec 18.

Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University, SE-171 65 Solna, Sweden. Electronic address:

The rapid and sensitive detection of specific nucleic acid sequences at the point-of-care (PoC) is becoming increasingly in demand for a variety of emergent biomedical applications ranging from infectious disease diagnostics to the screening of antimicrobial resistance. To meet such demand, considerable efforts have been invested towards the development of portable and integrated analytical devices combining microfluidics with miniaturized signal transducers. Here, we demonstrate the combination of rolling circle amplification (RCA)-based nucleic acid amplification with an on-chip size-selective trapping of amplicons on silica beads (~8 nL capture chamber) coupled with a thin-film photodiode (200 × 200 µm area) fluorescence readout. Parameters such as the flow rate of the amplicon solution and trapping time were optimized as well as the photodiode measurement settings, providing minimum detection limits below 0.5 fM of targeted nucleic acids and requiring only 5 μL of pre-amplified sample. Finally, we evaluated the analytical performance of our approach by benchmarking it against a commercial instrument for RCA product (RCP) quantification and further investigated the effect of the number of RCA cycles and elongation times (ranging from 10 to 120 min). Moreover, we provide a demonstration of the application for diagnostic purposes by detecting RNA from influenza and Ebola viruses, thus highlighting its suitability for integrated PoC systems.
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http://dx.doi.org/10.1016/j.bios.2018.12.004DOI Listing
March 2019

Bioanalytical advantages of a novel recombinant apyrase enzyme in ATP-based bioluminescence methods.

Anal Chim Acta 2018 Sep 26;1025:118-123. Epub 2018 Apr 26.

Division of Nanobiotechnology, Department of Protein Science, Science for Life Laboratory, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, Stockholm, Sweden. Electronic address:

Ultrasensitive measurements of intracellular ATP (intATP) based on the firefly luciferase reactions are frequently used to enumerate bacterial or mammalian cells. During clinical applications, extracellular ATP (extATP) should be depleted in biological samples since it interferes with intATP and affects the quantification of bacteria. The extATP can be eliminated by ATP-degrading enzymes but complete hydrolysis of extATP remains a challenge for today's commercial enzymes. The catalytic efficiency of ATP-degrading enzymes depends on enzyme characteristics, sample composition and the ability to deplete diphosphates, triphosphates and their complexes generated during the reaction. This phenomenon restricts the usage of bioluminescence-based ATP methods in clinical diagnostics. In light of this, we have developed a recombinant Shigella flexneri apyrase (RSFA) enzyme and analysed its ATP depletion potential with five commercial biochemical sources including potato apyrase, acid phosphatase, alkaline phosphatase, hexokinase and glycerol kinase. The RSFA revealed superior activity by completely eliminating the extracellular ATP and ATP-complexes, even in biological samples like urine and serum. Therefore, our results can potentially unwrap the chemical and bio-analytical applications of ATP-based bioluminescence tests to develop highly sensitive point-of-care diagnostics.
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http://dx.doi.org/10.1016/j.aca.2018.04.054DOI Listing
September 2018

Transcriptomics and Targeted Proteomics Analysis to Gain Insights Into the Immune-control Mechanisms of HIV-1 Infected Elite Controllers.

EBioMedicine 2018 Jan 12;27:40-50. Epub 2017 Dec 12.

Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden; Science for Life Laboratory, Division of Proteomics and Nanobiotechnology, KTH Royal Institute of Technology, Solna, Stockholm, Sweden. Electronic address:

A small subset of HIV-1 infected individuals, the "Elite Controllers" (EC), can control viral replication and restrain progression to immunodeficiency without antiretroviral therapy (ART). In this study, a cross-sectional transcriptomics and targeted proteomics analysis were performed in a well-defined Swedish cohort of untreated EC (n=19), treatment naïve patients with viremia (VP, n=32) and HIV-1-negative healthy controls (HC, n=23). The blood transcriptome identified 151 protein-coding genes that were differentially expressed (DE) in VP compared to EC. Genes like CXCR6 and SIGLEC1 were downregulated in EC compared to VP. A definite distinction in gene expression between males and females among all patient-groups were observed. The gene expression profile between female EC and the healthy females was similar but did differ between male EC and healthy males. At targeted proteomics analysis, 90% (29/32) of VPs clustered together while EC and HC clustered separately from VP. Among the soluble factors, 33 were distinctive to be statistically significant (False discovery rate=0.02). Cell surface receptor signaling pathway, programmed cell death, response to cytokine and cytokine-mediated signaling seem to synergistically play an essential role in HIV-1 control in EC.
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http://dx.doi.org/10.1016/j.ebiom.2017.11.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5828548PMC
January 2018

Altered basophil function in patients with chronic kidney disease on hemodialysis
.

Clin Nephrol 2017 Aug;88(8):86-96

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-FcεRI-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-FcɛI. CD300a expression was significantly higher in patients following activation by fMLP and anti-FcɛI, 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-FcɛI 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.
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http://dx.doi.org/10.5414/CN108992DOI Listing
August 2017

Quantitative humoral profiling of the HIV-1 proteome in elite controllers and patients with very long-term efficient antiretroviral therapy.

Sci Rep 2017 04 6;7(1):666. Epub 2017 Apr 6.

Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institutet, Huddinge, Stockholm, Sweden.

A major challenge in evaluating the success of HIV eradication approaches is the need for accurate measurement of persistent HIV during effective antiretroviral therapy (ART). Previous studies have reported that the anti-HIV antibody assay "luciferase immuno-precipitation systems (LIPS)" can distinguish HIV-infected individuals harboring different sizes of the viral reservoirs. We performed antibody profiling of HIV-1 proteomes using LIPS in viremic progressors (n = 38), elite controllers (ECs; n = 19) and patients with fully suppressive long-term antiretroviral therapy (ART) (n = 19) (mean 17 years). IgG was quantified against six HIV-1 fusion proteins: p24, gp41, RT, Tat, integrase and protease. Lower antibody levels to all six-fusion proteins were observed in long-term ART patients compared to viremics (p < 0.05). In contrast ECs had lower antibody levels only against Tat and Integrase (p < 0.05). Principal component analysis and cluster-network analysis identified that 68% (13/19) of the long-term ART patients clustered together with 26% (5/19) ECs. The remaining ECs clustered together with the viremics indicating non-homogeneity among the ECs. The low anti-HIV levels in the long-term treated patients may indicate a restricted remaining viral replication. In contrast, the higher levels in ECs suggest a continuous viral expression with a limited concomitant release of extracellular virus.
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http://dx.doi.org/10.1038/s41598-017-00759-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5429677PMC
April 2017

Elasto-inertial microfluidics for bacteria separation from whole blood for sepsis diagnostics.

J Nanobiotechnology 2017 Jan 4;15(1). Epub 2017 Jan 4.

Division of Proteomics & Nano-biotechnology, School of Biotechnology, Royal Institute of Technology KTH, SciLifeLab Tomtebodavägen 23, 17165, Solna, Sweden.

Background: Bloodstream infections (BSI) remain a major challenge with high mortality rate, with an incidence that is increasing worldwide. Early treatment with appropriate therapy can reduce BSI-related morbidity and mortality. However, despite recent progress in molecular based assays, complex sample preparation steps have become critical roadblock for a greater expansion of molecular assays. Here, we report a size based, label-free, bacteria separation from whole blood using elasto-inertial microfluidics.

Results: In elasto-inertial microfluidics, the viscoelastic flow enables size based migration of blood cells into a non-Newtonian solution, while smaller bacteria remain in the streamline of the blood sample entrance and can be separated. We first optimized the flow conditions using particles, and show continuous separation of 5 μm particles from 2 μm at a yield of 95% for 5 µm particle and 93% for 2 µm particles at respective outlets. Next, bacteria were continuously separated at an efficiency of 76% from undiluted whole blood sample.

Conclusion: We demonstrate separation of bacteria from undiluted while blood using elasto-inertial microfluidics. The label-free, passive bacteria preparation method has a great potential for downstream phenotypic and molecular analysis of bacteria.
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http://dx.doi.org/10.1186/s12951-016-0235-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5210221PMC
January 2017

Microfluidic-Based Bacteria Isolation from Whole Blood for Diagnostics of Blood Stream Infection.

Methods Mol Biol 2017 ;1547:175-186

Science for Life Laboratory, Division of Proteomics and Nanobiotechnology, KTH Royal Institute of Technology, PO Box 1031, 17121, Solna, Stockholm, Sweden.

Bacterial blood stream infection (BSI) potentially leads to life-threatening clinical conditions and medical emergencies such as severe sepsis, septic shock, and multi organ failure syndrome. Blood culturing is currently the gold standard for the identification of microorganisms and, although it has been automated over the decade, the process still requires 24-72 h to complete. This long turnaround time, especially for the identification of antimicrobial resistance, is driving the development of rapid molecular diagnostic methods. Rapid detection of microbial pathogens in blood related to bloodstream infections will allow the clinician to decide on or adjust the antimicrobial therapy potentially reducing the morbidity, mortality, and economic burden associated with BSI. For molecular-based methods, there is a lot to gain from an improved and straightforward method for isolation of bacteria from whole blood for downstream processing.We describe a microfluidic-based sample-preparation approach that rapidly and selectively lyses all blood cells while it extracts intact bacteria for downstream analysis. Whole blood is exposed to a mild detergent, which lyses most blood cells, and then to osmotic shock using deionized water, which eliminates the remaining white blood cells. The recovered bacteria are 100 % viable, which opens up possibilities for performing drug susceptibility tests and for nucleic-acid-based molecular identification.
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http://dx.doi.org/10.1007/978-1-4939-6734-6_14DOI Listing
February 2018

Acoustic micro-vortexing of fluids, particles and cells in disposable microfluidic chips.

Biomed Microdevices 2016 08;18(4):71

Department of Applied Physics, Royal Institute of Technology, KTH-Albanova, SE-106 91, Stockholm, Sweden.

We demonstrate an acoustic platform for micro-vortexing in disposable polymer microfluidic chips with small-volume (20 μl) reaction chambers. The described method is demonstrated for a variety of standard vortexing functions, including mixing of fluids, re-suspension of a pellet of magnetic beads collected by a magnet placed on the chip, and lysis of cells for DNA extraction. The device is based on a modified Langevin-type ultrasonic transducer with an exponential horn for efficient coupling into the microfluidic chip, which is actuated by a low-cost fixed-frequency electronic driver board. The transducer is optimized by numerical modelling, and different demonstrated vortexing functions are realized by actuating the transducer for varying times; from fractions of a second for fluid mixing, to half a minute for cell lysis and DNA extraction. The platform can be operated during 1 min below physiological temperatures with the help of a PC fan, a Peltier element and an aluminum heat sink acting as the chip holder. As a proof of principle for sample preparation applications, we demonstrate on-chip cell lysis and DNA extraction within 25 s. The method is of interest for automating and chip-integrating sample preparation procedures in various biological assays.
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http://dx.doi.org/10.1007/s10544-016-0097-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4956691PMC
August 2016

Ultra-High-Throughput Sample Preparation System for Lymphocyte Immunophenotyping Point-of-Care Diagnostics.

J Lab Autom 2016 Oct 26;21(5):706-12. Epub 2016 Feb 26.

Science for Life Laboratory, Division of Proteomics and Nanobiotechnology, KTH Royal Institute of Technology, Stockholm, Sweden

Point-of-care (POC) microfluidic devices often lack the integration of common sample preparation steps, such as preconcentration, which can limit their utility in the field. In this technology brief, we describe a system that combines the necessary sample preparation methods to perform sample-to-result analysis of large-volume (20 mL) biopsy model samples with staining of captured cells. Our platform combines centrifugal-paper microfluidic filtration and an analysis system to process large, dilute biological samples. Utilizing commercialization-friendly manufacturing methods and materials, yielding a sample throughput of 20 mL/min, and allowing for on-chip staining and imaging bring together a practical, yet powerful approach to microfluidic diagnostics of large, dilute samples.
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http://dx.doi.org/10.1177/2211068216634003DOI Listing
October 2016

Microfluidic-based isolation of bacteria from whole blood for sepsis diagnostics.

Biotechnol Lett 2015 Apr 21;37(4):825-30. Epub 2014 Nov 21.

Science for Life Laboratory, Division of Proteomics and Nanobiotechnology, KTH Royal Institute of Technology, Solna, PO Box 1031, 17121, Stockholm, Sweden.

Blood-stream infections (BSI) remain a major health challenge, with an increasing incidence worldwide and a high mortality rate. Early treatment with appropriate antibiotics can reduce BSI-related morbidity and mortality, but success requires rapid identification of the infecting organisms. The rapid, culture-independent diagnosis of BSI could be significantly facilitated by straightforward isolation of highly purified bacteria from whole blood. We present a microfluidic-based, sample-preparation system that rapidly and selectively lyses all blood cells while it extracts intact bacteria for downstream analysis. Whole blood is exposed to a mild detergent, which lyses most blood cells, and then to osmotic shock using deionized water, which eliminates the remaining white blood cells. The recovered bacteria are 100% viable, which opens up possibilities for performing drug susceptibility tests and for nucleic-acid-based molecular identification.
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http://dx.doi.org/10.1007/s10529-014-1734-8DOI Listing
April 2015

Dean flow-coupled inertial focusing in curved channels.

Biomicrofluidics 2014 May 24;8(3):034117. Epub 2014 Jun 24.

Division of Proteomics and Nanobiotechnology, Science for Life Laboratory, KTH Royal Institute of Technology , Stockholm, Sweden.

Passive particle focusing based on inertial microfluidics was recently introduced as a high-throughput alternative to active focusing methods that require an external force field to manipulate particles. In inertial microfluidics, dominant inertial forces cause particles to move across streamlines and occupy equilibrium positions along the faces of walls in flows through straight micro channels. In this study, we systematically analyzed the addition of secondary Dean forces by introducing curvature and show how randomly distributed particles entering a simple u-shaped curved channel are focused to a fixed lateral position exiting the curvature. We found the lateral particle focusing position to be fixed and largely independent of radius of curvature and whether particles entering the curvature are pre-focused (at equilibrium) or randomly distributed. Unlike focusing in straight channels, where focusing typically is limited to channel cross-sections in the range of particle size to create single focusing point, we report here particle focusing in a large cross-section area (channel aspect ratio 1:10). Furthermore, we describe a simple u-shaped curved channel, with single inlet and four outlets, for filtration applications. We demonstrate continuous focusing and filtration of 10 μm particles (with >90% filtration efficiency) from a suspension mixture at throughputs several orders of magnitude higher than flow through straight channels (volume flow rate of 4.25 ml/min). Finally, as an example of high throughput cell processing application, white blood cells were continuously processed with a filtration efficiency of 78% with maintained high viability. We expect the study will aid in the fundamental understanding of flow through curved channels and open the door for the development of a whole set of bio-analytical applications.
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http://dx.doi.org/10.1063/1.4884306DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4162445PMC
May 2014

Activation of basophils is a new and sensitive marker of biocompatibility in hemodialysis.

Artif Organs 2014 Nov 9;38(11):945-53. Epub 2014 Apr 9.

Unit of Clinical Immunology and Allergy, Department of Medicine, Karolinska University Hospital Solna, Karolinska Institute, Stockholm, Sweden; Division of Proteomics and Nanobiotechnology, School of Biotechnology, Royal Institute of Technology, Stockholm, Sweden.

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-Fcε receptor I (FcεRI) 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-FcεRI. 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.
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http://dx.doi.org/10.1111/aor.12297DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4257079PMC
November 2014

Modulated fluorescence of colloidal quantum dots embedded in a porous alumina membrane.

J Phys Chem B 2013 Nov 31;117(45):14151-6. Epub 2013 Oct 31.

Science for Life Laboratory, Department of Applied Physics, Royal Institute of Technology , SE-106 91 Stockholm, Sweden.

The fluorescence spectrum of CdSe core-CdS/ZnS shell colloidal quantum dots (QDs) embedded in porous alumina membrane was studied. Small peaks, superimposed on the principal QD fluorescence spectrum, were observed. Finite-difference time-domain simulation indicates that the QD point radiation emitting from within the membrane is strongly modulated by the photonic band structure introduced by the membrane pores, leading to the observed fine spectral features. Moreover, the principal QD fluorescence peak red-shifted when the optical excitation power was increased, which is attributed to QD material heating due to emitted phonons when the photoexcited electron and hole relax nonradiatively from high-energy states to the ground exciton state before fluorescence.
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http://dx.doi.org/10.1021/jp409132eDOI Listing
November 2013

Genotyping of single nucleotide polymorphisms by melting curve analysis using thin film semi-transparent heaters integrated in a lab-on-foil system.

Lab Chip 2013 Jun;13(11):2075-82

Fraunhofer EMFT, Hansastrasse 27d, 80686 Munich, Germany.

The recent technological advances in micro/nanotechnology present new opportunities to combine microfluidics with microarray technology for the development of small, sensitive, single-use, point-of-care molecular diagnostic devices. As such, the integration of microarray and plastic microfluidic systems is an attractive low-cost alternative to glass based microarray systems. This paper presents the integration of a DNA microarray and an all-polymer microfluidic foil system with integrated thin film heaters, which demonstrate DNA analysis based on melting curve analysis (MCA). A novel micro-heater concept using semi-transparent copper heaters manufactured by roll-to-roll and lift-off on polyethylene naphthalate (PEN) foil has been developed. Using a mesh structure, heater surfaces have been realized in only one single metallization step, providing more efficient and homogenous heating characteristics than conventional meander heaters. A robust DNA microarray spotting protocol was adapted on Parylene C coated heater-foils, using co-polymer poly(DMA-NAS-MAPS) to enable covalent immobilization of DNA. The heaters were integrated in a microfluidic channel using lamination foils and MCA of the spotted DNA duplexes showed single based discrimination of mismatched over matched target DNA-probes. Finally, as a proof of principle, we perform MCA on PCR products to detect the Leu7Pro polymorphism of the neutropeptide Y related to increased risk of Type II diabetes, BMI and depression.
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http://dx.doi.org/10.1039/c3lc50171jDOI Listing
June 2013

Lab-on-DVD: standard DVD drives as a novel laser scanning microscope for image based point of care diagnostics.

Lab Chip 2013 Apr;13(8):1578-85

Science for Life Laboratory, Div. of Proteomics and Nanobiotechnology, KTH Royal Institute of Technology, Stockholm, Sweden.

We present a novel "Lab-on-DVD" system and demonstrate its capability for rapid and low-cost HIV diagnostics by counting CD4+ cells isolated from whole blood. We show that a commercial DVD drive can, with certain modifications, be turned into an improved DVD-based laser scanning microscope (DVD-LSM). The system consists of a multi-layered disposable polymer disc and a modified commercial DVD reader with rotational control for sample handling, temperature control for optimized bioassay, a photodiode array for detection, and software for signal processing and user interface - all the necessary components required for a truly integrated lab-on-a-chip system, with the capability to deliver high-resolution images down to 1 μm in size. Using discs modified with antibodies, we specifically captured CD4+ cells from whole blood, demonstrating single cell resolution imaging. The novel integrated DVD platform with sub-micron image resolution brings, for the first time, affordable cellular diagnostic testing to the point-of-care and should be readily applicable at resource-limited settings.
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http://dx.doi.org/10.1039/c3lc41360hDOI Listing
April 2013
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