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  • 1.
    Aljadi, Zenib
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. Karolinska Inst, Sweden.
    Nopp, Anna
    Winqvist, Ola
    Russom, Aman
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. Karolinska Inst, Sweden.
    Hylander, Britta
    Jacobson, Stefan H.
    Lundahl, Joachim
    Altered basophil function in patients with chronic kidney disease on hemodialysis2017In: Clinical Nephrology, ISSN 0301-0430, Vol. 88, no 2, p. 86-96Article in journal (Refereed)
    Abstract [en]

    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.

  • 2.
    Banerjee, Indradumna
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Aralaguppe, S. G.
    Karolinska Inst, Dept Lab Med, Div Clin Microbiol, Stockholm, Sweden..
    Lapins, Noa
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Zhang, W.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. Karolinska Inst, Dept Lab Med, Div Clin Microbiol, Stockholm, Sweden..
    Kazemzadeh, Amin
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Sönnerborg, A.
    Karolinska Inst, Dept Lab Med, Div Clin Microbiol, Stockholm, Sweden..
    Neogi, Ujjwal
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. Karolinska Inst, Dept Lab Med, Div Clin Microbiol, Stockholm, Sweden..
    Russom, Aman
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Microfluidic centrifugation assisted precipitation based DNA quantification2019In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 19, no 9, p. 1657-1664Article in journal (Refereed)
    Abstract [en]

    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.

  • 3.
    Banerjee, Indradumna
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Aralaguppe, Shambhu Prasad
    Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institute, Sweden.
    Lapins, Noa
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Kazemzadeh, Amin
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Sönneborg, Anders
    Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institute, Sweden.
    Neogi, Ujjwal
    Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institute, Sweden.
    Russom, Aman
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    MicroCAP: Microfluidic Centrifuge Assisted Precipitation for DNA Quantification on Lab-on-DVD2018Conference paper (Refereed)
    Abstract [en]

    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.

    INTRODUCTION

    Detection 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.

    EXPERIMENTAL

    The 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 1b

    show 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 DISCUSSION

    MicroCAP 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.

    CONCLUSION

    We 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. [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. [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 epidemiological studies and clinical man- agement.” Journal of the International AIDS Society, 2015,18(1), 20035.

    3. [3]  K. A. Curtis, D. L. Rudolph, I. Nejad, J. Singleton, A. Beddoe, B. Weigl, P. LaBarre and S. M. Owen, "Rapid detection of HIV-1 by reverse-transcription, loop-mediated isothermal amplification (RT- LAMP)." PLoS ONE, , DOI:10.1371/journal.pone.0031432.

    CONTACT

    *A. Russom; phone: +46-87909863; aman@kth.se

  • 4.
    Banerjee, Indradumna
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Ganeshappa Aralaguppe, Shambhu Prasad
    Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institute, Sweden..
    Lapins, Noa
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Zhang, Wang
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institute, Sweden..
    Kazemzadeh, Amin
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Sönnerborg, Anders
    Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institute, Sweden..
    Neogi, Ujjwal
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology. Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institute, Sweden..
    Russom, Aman
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Microfluidic Centrifugation Assisted Precipitation based DNA QuantificationManuscript (preprint) (Other academic)
    Abstract [en]

    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.

  • 5.
    Banerjee, Indradumna
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Rosti, Marco E.
    KTH, School of Engineering Sciences (SCI), Mechanics.
    Kumar, Tharagan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Brandt, Luca
    KTH, School of Engineering Sciences (SCI), Mechanics.
    Russom, Aman
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Particle focusing dynamics in extended elasto inertial flow2018Conference paper (Refereed)
    Abstract [en]

    Elasto Inertial microfluidics has been exploited recently for a number of industrial and biological applications. Recently, we experimentally showed that it is possible to achieve single stream focusing of particles even at higher flow rates in the elasto inertial regime, relevant to flow cytometry applications, and , based on this concept, built a silica fibre based micro flow cytometer.1 However, the physics behind the focusing of particles is still poorly understood, specially for combinations of higher Reynolds (Re) and Weissenberg numbers(Wi).

    In the present study, for the first time, we seek to understand both experimentally and with numerical simulations, particle focusing across elasticity regimes. We vary the concentration of PEO (200 ppm to upto 10000 ppm) in PBS solution at sufficiently high flow rates of 100!l/min or above. We introduce a parameter, focusing bandwidth (F) to evaluate the extent of single stream focusing of 15 !m particles in a 75 !m diameter circular channel. Fig.1 shows the flow setup(fig.1a) along with images demonstrating the focused (fig.1b) and unfocused cases(fig.1c), as well as how F is calculated(fig.1d). We evaluate particle focusing by identifying the flow conditions for each concentration that leads to the minimum value of F. Fig.2 shows the variation of the focusing bandwidth(fig.2a) when changing PEO concentration, and the variation in Re along with Wi (fig.2b) and Elasticity number(El). The results show that for identical mass flow conditions across the different regimes the focusing bandwidth slowly shifts to a narrow single stream with increasing elasticity. We validated our experimental results as well as gained new insights into particle focusing with 3D numerical simulations based on a FENE P model. We studied the decoupled effects of Reynolds number and Weissenberg number on particle focusing, as well as the particle trajectories and migration dynamics as the particles reach equilibrium. Interestingly, enough we find a combination of high Re(Re=400) and sufficiently high Wi(Wi=3) for which the particles achieve a single stream focusing (fig.3a). The entire dynamics of particle migration in a circular cross section is also shown (in fig.3b) by changing Wi for a constant Re(Re=200). It can be seen that the particle goes through a longer amount of oscillations to reach its final equilibrium position as Wi is increased. Fig.4a shows the equilibrium position of the particle moving closer to the center with an increase in Wi at the same Re(Re=200). However, in the Non Newtonian cases, the particle has a slight oscillatory behaviour as it reaches its equilibrium position as compared to the Newtonian one. We introduced the particle at two different positions(at Re=200, We=0 and 1) and observed the same equilibrium positions in both cases (Fig.4b).

  • 6.
    Banerjee, Indradumna
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Rosti, Marco Edoardo
    KTH, School of Engineering Sciences (SCI), Mechanics.
    Kumar, Tharagan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Brandt, Luca
    KTH, School of Engineering Sciences (SCI), Mechanics.
    Russom, Aman
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Analog particle position tuning in Elasto-inertial microfluidic flowsManuscript (preprint) (Other academic)
    Abstract [en]

    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.

  • 7.
    Banerjee, Indradumna
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Rosti, Marco Eduardo
    KTH, School of Engineering Sciences (SCI), Mechanics. KTH, Centres, SeRC - Swedish e-Science Research Centre. KTH, School of Engineering Sciences (SCI), Centres, Linné Flow Center, FLOW.
    Niazi Ardekani, Mehdi
    KTH, School of Engineering Sciences (SCI), Mechanics. KTH, Centres, SeRC - Swedish e-Science Research Centre. KTH, School of Engineering Sciences (SCI), Centres, Linné Flow Center, FLOW.
    Kumar, Tharagan
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Lashgari, Iman
    KTH, School of Engineering Sciences (SCI), Mechanics. KTH, School of Engineering Sciences (SCI), Centres, Linné Flow Center, FLOW. KTH, Centres, SeRC - Swedish e-Science Research Centre.
    Brandt, Luca
    KTH, School of Engineering Sciences (SCI), Mechanics. KTH, School of Engineering Sciences (SCI), Centres, Linné Flow Center, FLOW. KTH, Centres, SeRC - Swedish e-Science Research Centre.
    Russom, Aman
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Dynamics of Inertial migration of particles in straight channels2017Conference paper (Refereed)
    Abstract [en]

    SUMMARY

    We 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.

     

    INTRODUCTION

    We  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 DISCUSSION

    We 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).

     

    CONCLUSION

    The 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.

     

    ACKNOWLEDGEMENTS

    This 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.

  • 8.
    Banerjee, Indradumna
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Russom, Aman
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Lab-on-DVD: Optical Disk Drive-Based Platforms for Point-of-Care Diagnostics2018In: Frugal Innovation in Bioengineering for the Detection of Infectious Diseases / [ed] AK Chavali, R Ramji, Switzerland: Springer, 2018, 2, p. 23-38Chapter in book (Refereed)
    Abstract [en]

    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.

  • 9.
    Banerjee, Indradumna
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Russom, Aman
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    MicroCAP2018Patent (Other (popular science, discussion, etc.))
    Abstract [en]

  • 10.
    Banerjee, Indradumna
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Salih, Tagrid
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Ohlander, Anna
    Russom, Aman
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Three dimensional Slipdisc aimed at HIV viral load detection2017Conference paper (Refereed)
    Abstract [en]

    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.

  • 11.
    Banerjee, Indradumna
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Salih, Tagrid
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Ramachandraiah, Harisha
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Erlandsson, J.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Surface and Corrosion Science.
    Petterson, T.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Surface and Corrosion Science.
    Silva, AC
    Karlsson, M
    Russom, Aman
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    LDH based neonatal diagnostics on a low-cost slipdisc based sample preparation platform.2016Conference paper (Refereed)
    Abstract [en]

    INTRODUCTION

    Slipdisc 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.

    EXPERIMENTAL

    For 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 DISCUSSION

    The 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.

    CONCLUSION

    We 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.

  • 12.
    Banerjee, Indradumna
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Salih, Tagrid
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Ramachandraiah, Harisha
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Erlandsson, Johan
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Fibre Technology.
    Pettersson, Torbjörn
    KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center. KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    Araújo, A. C.
    Karlsson, M.
    Russom, Aman
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Slipdisc: A versatile sample preparation platform for point of care diagnostics2017In: RSC Advances, ISSN 2046-2069, E-ISSN 2046-2069, Vol. 7, no 56, p. 35048-35054Article in journal (Refereed)
    Abstract [en]

    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.

  • 13. Bose, I.
    et al.
    Ohlander, Anna
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Kutter, C.
    Russom, Aman
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    An integrated all foil based micro device for point of care diagnostic applications2018In: Sensors and actuators. B, Chemical, ISSN 0925-4005, E-ISSN 1873-3077, Vol. 259, p. 917-925Article in journal (Refereed)
    Abstract [en]

    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. 

  • 14. Bose, Indranil
    et al.
    Ohlander, Anna
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Kutter, Christoph
    Russom, Aman
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    DNA Analysis on integrated all foil based microdevicesManuscript (preprint) (Other academic)
  • 15.
    Etcheverry, Sebastian
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Laser Physics.
    Faridi, Asim
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Ramachandraiah, Harisha
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Kumar, Tharagan
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Margulis, Walter
    KTH, School of Engineering Sciences (SCI), Applied Physics, Laser Physics.
    Laurell, Fredrik
    KTH, School of Engineering Sciences (SCI), Applied Physics, Laser Physics.
    Russom, Aman
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    All silica fibre microflow cytometerManuscript (preprint) (Other academic)
    Abstract [en]

    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. 

  • 16.
    Etcheverry, Sebastian
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Faridi, Muhammad Asim
    KTH. mafaridi@kth.se.
    Ramachandraiah, Harisha
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Margulis, Walter
    Laurell, Fredrik
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Russom, Aman
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Optical Fiber inertial focusing based micro FlowcytometerIn: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723Article in journal (Refereed)
    Abstract [en]

    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.

  • 17.
    Etcheverry, Sebastián
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Laser Physics. RISE Acreo AB, Sweden.
    Faridi, Muhammad Asim
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Ramachandraiah, Harisha
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Kumar, T.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Margulis, Walter
    KTH, School of Engineering Sciences (SCI), Applied Physics, Laser Physics. RISE Acreo AB, Sweden.
    Laurell, Fredrik
    KTH, School of Engineering Sciences (SCI), Applied Physics, Laser Physics.
    Russom, Aman
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    High performance micro-flow cytometer based on optical fibres2017In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 5628Article in journal (Refereed)
    Abstract [en]

    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.

  • 18.
    Etcheverry, Sebastián
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics. Dept. of Fiber Optics, Acreo Swedish ICT AB, Sweden .
    Faridi, Muhammad Asim
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Ramachandraiah, Harisha
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Margulis, W.
    Laurell, Fredrik
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Russom, Aman
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    All fiber based micro-flow cytometer by combining optical fiber with inertial focusing2016In: 20th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2016, Chemical and Biological Microsystems Society , 2016, p. 1655-1656Conference paper (Refereed)
    Abstract [en]

    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.

  • 19.
    Etcheverry, Sebastián
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics. Department of Fiber Optics, RISE Acreo AB, Stockholm, Sweden.
    Russom, Aman
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Laurell, Fredrik
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Margulis, Walter
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Trapping and optical identification of microparticles in a liquid with a functional optical fiber probe2018In: Optics InfoBase Conference Papers, Optical Society of America, 2018Conference paper (Refereed)
    Abstract [en]

    A fiber probe traps single micrometer-particles by fluid suction into a hollow microstructure and enables optical identification by the fluorescence light collected in a fiber core. The probe finds applications in life-science and environmental monitoring.

  • 20.
    Faridi, M. A.
    et al.
    KTH, School of Biotechnology (BIO). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Ramachandraiah, H.
    KTH, School of Biotechnology (BIO).
    Iranmanesh, I. S.
    KTH, School of Biotechnology (BIO). KTH, School of Engineering Sciences (SCI), Applied Physics.
    Grishenkov, Dmitry
    KTH, School of Technology and Health (STH), Medical Engineering, Medical Imaging.
    Wiklund, Martin
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Russom, Aman
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Microbubble assisted cell sorting by acoustophoresis2016In: 20th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2016, Chemical and Biological Microsystems Society , 2016, p. 1677-1678Conference paper (Refereed)
    Abstract [en]

    Polymer shelled gas microbubbles (MBs) are used to sort cells in a microfluidic chip under acoustic standing waves (SW). When particles are subjected to SW based on their acoustic contrast factor (ACF) they migrate to nodes (positive contrast factor particles; PACP) or antinodes (negative acoustic contrast particles; NACP)[1]. We have bounded functionalized MBs with cells such that, they can be selectively migrated to antinodes under SW and sorted from unbounded cell both in no flow and flow conditions. Here we demonstrate acoustic mediated microbubble tagged cell sorting with 75% efficiency.

  • 21.
    Faridi, Muhammad Asim
    et al.
    KTH. mafaridi@kth.se.
    Iranmanesh, Ida Sadat
    KTH.
    Ramachandraiah, Harisha
    Vanderleyden, Els
    Dubruel, Peter
    Wiklund, Martin
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Russom, Aman
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Glass Capillary based cavity resonator for particle trapping study and bacteria up-concentrationIn: Biomedical microdevices (Print), ISSN 1387-2176, E-ISSN 1572-8781Article in journal (Refereed)
    Abstract [en]

    We have performed particle aggregation characterization on the basis of their material and suspending

    medium in a capillary-based cavity resonator used for acoustophoresis. We have investigated the experimental

    aggregation time of 5μm polystyrene and silica particles, size of aggregate, number of trapped particles and upconcentration

    factor in water, 0.01M phosphate buffered saline (PBS) and 0.005M PBS at 1.97MHz and with

    actuation voltages between 4, 8 and 12Vpp. We have found that there is little difference between using water and

    PBS as suspension medium, approximately 5-10% longer trapping times with PBS compared with water.

    However we get approx. 5.5 times faster trapping time for silica than for polystyrene. It is also observed and

    calculated that silica particle aggregates have 3.4 times larger area than the polystyrene aggregates using the same

    starting particle concentrations, revealing similar amount of difference in trapped number of particles. The upconcentration

    factor for silica is also about 3.2 times higher than that of polystyrene due to larger aggregate area

    of silica particles. Based on theoretical predictions and experimental characterization of the particle aggregation

    pattern, we note that the particle-particle interaction force contribution to the total acoustic radiation force is more

    pronounced for silica than for polystyrene. Finally as a proof of principle for biomedical sample preparation

    application we demonstrate the capillary-based silica particles mediated bacteria acoustophoretic upconcentration.

    This setup could potentially be utilized not only for sample preparation application but also for

    bead based affinity immunoassays.

  • 22.
    Faridi, Muhammad Asim
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab. mafaridi@kth.se.
    Ramachandraiah, Harisha
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Banerjee, Indradumna
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Ardabli, Sahar
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Zelenin, Sergey
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Russom, Aman
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Elasto-inertial microfluidics for bacteria separation from whole blood for sepsis diagnostics2017In: Journal of Nanobiotechnology, ISSN 1477-3155, E-ISSN 1477-3155, Vol. 15, article id 3Article in journal (Refereed)
    Abstract [en]

    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 sepa- rated. 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 continu- ously 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. 

  • 23.
    Faridi, Muhammad Asim
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab. mafaridi@kth.se.
    Ramachandraiah, Harisha
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Iranmanesh, Ida Sadat
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Grishenkov, Dmitry
    KTH, School of Technology and Health (STH), Medical Engineering, Medical Imaging.
    Wiklund, Martin
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Russom, Aman
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    MicroBubble Activated Acoustic Cell Sorting: BAACSIn: Biomedical microdevices (Print), ISSN 1387-2176, E-ISSN 1572-8781Article in journal (Refereed)
    Abstract [en]

    Acoustophoresis, the ability to acoustically manipulate particles and cells inside a microfluidic channel, is a critical enabling technology for cell-sorting applications. However, one of the major impediments for routine use of acoustophoresis at clinical laboratory has been the reliance on the inherent physical properties of cells for separation. Here, we present a microfluidic-based microBubble-Activated Acoustic Cell Sorting (BAACS) method that rely on the specific binding of target cells to microbubbles conjugated with specific antibodies on their surface for continuous cell separation using ultrasonic standing wave. In acoustophoresis, cells being positive acoustic contrast particles migrate to pressure nodes. On the contrary we show that air-filled polymer-shelled microbubbles being strong negative acoustic contrast particles migrate to pressure antinodes at acoustic pressure amplitudes as low as 60 kPa. As a proof of principle, using the BAACS strategy, we demonstrate the separation of cancer cell line in a suspension with better than 75% efficiency. Moreover, 100% of the microbubble-cell conjugates migrated to the anti-node. Hence a better upstream affinity-capture has the potential to provide higher sorting efficiency. The BAACS technique may potentially provide a simplistic approach for similar sized selective isolation of cells, and is suited for applications in point of care.

  • 24.
    Faridi, Muhammad Asim
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab. mafaridi@kth.se.
    Ramachandraiah, Harisha
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Iranmanesh, Ida Sadat
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Grishenkov, Dmitry
    KTH, School of Technology and Health (STH), Medical Engineering, Medical Imaging.
    Wiklund, Martin
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Russom, Aman
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    MicroBubble Activated Acoustic Cell Sorting: BAACS2017In: Biomedical microdevices (Print), ISSN 1387-2176, E-ISSN 1572-8781, Vol. 19, no 2, article id 23Article in journal (Refereed)
    Abstract [en]

    Acoustophoresis, the ability to acoustically manipulate particles and cells inside a microfluidic channel, is a critical enabling technology for cell-sorting applications. However, one of the major impediments for routine use of acoustophoresis at clinical laboratory has been the reliance on the inherent physical properties of cells for separation. Here, we present a microfluidic-based microBubble-Activated Acoustic Cell Sorting (BAACS) method that rely on the specific binding of target cells to microbubbles conjugated with specific antibodies on their surface for continuous cell separation using ultrasonic standing wave. In acoustophoresis, cells being positive acoustic contrast particles migrate to pressure nodes. On the contrary we show that air-filled polymer-shelled microbubbles being strong negative acoustic contrast particles migrate to pressure antinodes at acoustic pressure amplitudes as low as 60 kPa. As a proof of principle, using the BAACS strategy, we demonstrate the separation of cancer cell line in a suspension with better than 75% efficiency. Moreover, 100% of the microbubble-cell conjugates migrated to the anti-node. Hence a better upstream affinity-capture has the potential to provide higher sorting efficiency. The BAACS technique may potentially provide a simplistic approach for similar sized selective isolation of cells, and is suited for applications in point of care.

  • 25.
    Faridi, Muhammad Asim
    et al.
    KTH.
    Shahzad, Adnan Faqui
    KTH.
    Russom, Aman
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Wiklund, Martin
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Milliliter scale acoustophoresis based bioparticle processing platform2018In: ASME 2018 16th International Conference on Nanochannels, Microchannels, and Minichannels, ICNMM 2018, ASME Press, 2018Conference paper (Refereed)
    Abstract [en]

    Bioparticles such as mammalian cells and bacteria can be manipulated directly or indirectly for multiple applications such as sample preparation for diagnostic applications mainly up-concentration, enrichment & separation as well as immunoassay development. There are various active and passive microfluidic particle manipulation techniques where Acoustophoresis is a powerful technique showing high cell viability. The use of disposable glass capillaries for acoustophoresis, instead of cleanroom fabricated glass-silicon chip can potentially bring down the cost factor substantially, aiding the realization of this technique for real-world diagnostic devices. Unlike available chips and capillary-based microfluidic devices, we report milliliter-scale platform able to accommodate 1ml of a sample for acoustophoresis based processing on a market available glass capillary. Although it is presented as a generic platform but as a demonstration we have shown that polystyrene suspending medium sample can be processed with trapping efficiency of 87% and the up-concentration factor of 10 times in a flow through manner i.e., at 35µl/min. For stationary volume accommodation, this platform practically offers 50 times more sample handling capacity than most of the microfluidic setups. Furthermore, we have also shown that with diluted blood (0.6%) in a flow-through manner, 82% of the white blood cells (WBCs) per ml could be kept trapped. This milliliter platform could potentially be utilized for assisting in sample preparation, plasma separation as well as a flow-through immunoassay assay development for clinical diagnostic applications.

  • 26.
    Halvorsen, Cecilia Pegelow
    et al.
    Karolinska Inst, Dept Clin Res & Educ, Sodersjukhuset, Stockholm, Sweden.;Sachs Children & Youth Hosp, Neonatal Unit, Stockholm, Sweden..
    Olson, Linus
    Karolinska Inst, Dept Womens & Childrens Hlth, Stockholm, Sweden.;Karolinska Inst, Dept Publ Hlth Sci, Stockholm, Sweden.;TRAC Sweden Vietnam, Hanoi, Vietnam..
    Araujo, Ana Catarina
    Calmark Sweden AB, Stockholm, Sweden..
    Karlsson, Mathias
    Calmark Sweden AB, Stockholm, Sweden.;Uppsala Univ, Dept Med Sci Biomed Struct & Funct, Uppsala, Sweden..
    Nguyen, Trang Thi
    Khu, Dung T. K.
    TRAC Sweden Vietnam, Hanoi, Vietnam.;Vietnam Natl Childrens Hosp, Neonatal Intens Care Unit, Hanoi, Vietnam..
    Le, Ha T. T.
    Vietnam Natl Childrens Hosp, Neonatal Intens Care Unit, Hanoi, Vietnam.;Res Inst Child Hlth, Hanoi, Vietnam..
    Nguyen, Hoa T. B.
    Vietnam Natl Childrens Hosp, Neonatal Intens Care Unit, Hanoi, Vietnam.;Res Inst Child Hlth, Hanoi, Vietnam..
    Winbladh, Birger
    Karolinska Inst, Dept Clin Res & Educ, Sodersjukhuset, Stockholm, Sweden..
    Russom, Aman
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    A rapid smartphone-based lactate dehydrogenase test for neonatal diagnostics at the point of care2019In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, article id 9301Article in journal (Refereed)
    Abstract [en]

    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-inresult- 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-2 = 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.

  • 27.
    Kazemzadeh, Amin
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Eriksson, Anders
    KTH, School of Engineering Sciences (SCI), Mechanics.
    Madou, Marc
    Univ Calif Irvine, Dept Mech & Aerosp Engn, Irvine, CA 92697 USA..
    Russom, Aman
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    A micro-dispenser for long-term storage and controlled release of liquids2019In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 10, no 1, article id 189Article in journal (Refereed)
    Abstract [en]

    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.

  • 28.
    Kazemzadeh, Amin
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Lapins, Noa
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Banerjee, Indradumna
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Akhtar, Ahmad Saleem
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Russom, Aman
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Mobile-LabDisc for Point-of-Care DiagnosticsManuscript (preprint) (Other academic)
    Abstract [en]

    At resource limited settings, point of care devices require a very low-cost, robust and easy to use platform that is preferably capable of automating and multiplexing intricate bioassays. We report on a mobile lab-disc platform that is specifically designed to meet the needs at resource- limited settings. It uses a smartphone as an electrical power source and a disposable, rigid and portable casing made of cardboard that securely accommodate the entire lab-disc system rotor, lightning and wiring and other accessories. The mobile lab-disc is light, less-expensive and functional at places where the electrical power infrastructure is not available. We show that the electrical energy stored in most mobile phones can be used for spinning a lab-Disc at up to 5500 rpm, a speed sufficient for most of the required functional steps in a bioassay including ELISA. We develop individual components of the mobile lab-disc system by experimentally conducting colorimetric assays using HRP and sandwich immunoassay. Finally, the full potential of the mobile lab-disc for integrating and multiplexing bioassays is demonstrated by measuring the hematocrit level in whole blood. The mobile phone-operated process integrates sample preparation i.e., blood-plasma separation, imaging and image analysis. The total cost of our prototype system for the tests, excluding the phone is ~$5, assuming that a lab-disc unit is worth $1.

  • 29.
    Ohlander, Anna
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Bose, Indranil
    Njenda, Duncan
    Zelenin, Sergey
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Neogi, Ujjwal
    Kutter, Christoph
    Russom, Aman
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lab-on-foil based portable microPCR for point-of-care nucleic acid testing of HIV-1Manuscript (preprint) (Other academic)
  • 30.
    Ohlander, Anna
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Zelenin, Sergey
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Huygens, Flavia
    Kutter, Christoph
    Russom, Aman
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Staphylococcus Aureus subtyping and detection of MRSA on a microfluidic lab-on-Foil deviceManuscript (preprint) (Other academic)
  • 31. Pavankumar, A. R.
    et al.
    Zelenin, Sergey
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lundin, A.
    Schulte, T.
    Rajarathinam, K.
    Rebellato, P.
    Ardabili, Sahar
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Salas, J.
    Achour, A.
    Russom, Aman
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Bioanalytical advantages of a novel recombinant apyrase enzyme in ATP-based bioluminescence methods2018In: Analytica Chimica Acta, ISSN 0003-2670, E-ISSN 1873-4324, Vol. 1025, p. 118-123Article in journal (Refereed)
    Abstract [en]

    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.

  • 32.
    Pettersson, Torbjörn
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Ramachandraiah, Harisha
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Russom, Aman
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Nanocellulose mediated layer-by-layer chip modification for cellular in-vitro diagnostics2017In: Abstracts of Papers of the American Chemical Society, ISSN 0065-7727, Vol. 253Article in journal (Other academic)
  • 33.
    Ramachandraiah, Harisha
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Kugiejko, Karol
    KTH, School of Biotechnology (BIO).
    Pettersson, Torbjörn
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    Heuchel, Rainer
    Karolinska Institutet.
    Löhr, Matthias
    karolinska Institute.
    Hedhammar, My
    KTH, School of Biotechnology (BIO), Protein Technology.
    Russom, Aman
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Microfluidic based circulating tumor cell isolation and release from whole blood of pancreatic cancer patients using bio-functionalized recombinant spider silkManuscript (preprint) (Other academic)
    Abstract [en]

    A bio-functionalized microsystem was developed for the capture and release of cancer cells from whole blood. Effective isolation and purification of circulating tumor cells from whole blood provides important capability for clinical application and biological research. Here, we demonstrate a single step surface modification procedure for a microfluidic device based on self-assembly of recombinant spider silk harbouring an affinity domain for antibody binding. The surfaces of microfluidic devices were conjugated/equipped with anti-EpCAM antibody for selective isolation of pancreatic cancer cells from spiked whole blood and finally circulating tumor cells from pancreatic cancer patients. Moreover, a protease-cleavage site in the recombinant spider silk proteins provides the unique option to release the captured cancer cells on command from the device without compromising the cell’s viability. Our approach offers a simple, easy and robust surface modification process with a 85% cancer cell capture efficiency. Subsequent addition of a site-specific protease results in the release of 95% of captured cells from the bio functionalized microfluidic systems. 

  • 34.
    Ramachandraiah, Harisha
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Pettersson, Torbjörn
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    Russom, Aman
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Layer-by-layer system based cellulose nanofibrils for capture and release of cells in microfluidic deviceManuscript (preprint) (Other (popular science, discussion, etc.))
    Abstract [en]

    Selective isolation of cells, without inducing any phenotypic changes and maintaining cell viability will preserve the information necessary for down stream analysis. Here we present an ultra thin coating on the surface of disposable microfluidic device based on cellulose nanofibrils, that is modified to capture cells and for later release. Layer-by-layer technique facilitates the production of the thin coating of cellulose onto polymeric surfaces and modified to form affinity based cell capture surface. We demonstrate an  efficiently capture and release of cells, the release is done by selectively degrading 

  • 35.
    Ramachandraiah, Harisha
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Svahn Andersson, Helene
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Russom, Aman
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Inertial microfluidics combined with selective cell lysis for high throughput separation of nucleated cells from whole blood2017In: RSC Advances, ISSN 2046-2069, E-ISSN 2046-2069, no 47, p. 29505-29514Article in journal (Refereed)
    Abstract [en]

    The ability to rapidly analyze and extract information from peripheral blood cells has the potential of providing a wealth of new information about immune function and general health of the patient. In spite of the tremendous progress achieved in the field of leukocyte analysis, one of the major impediments for routine analysis is the enrichment of cell populations from heterogeneous sources such as blood, as the currently used techniques tend to be laborious. Moreover, the isolation of small and transient cell populations in blood, like circulating tumor cells during cancer metastasis, is even more challenging. Here, we report an integrated device for label-free continuous flow separation of nucleated cells from unprocessed whole blood at high throughput. The method utilizes exposure to hypotonic buffer to completely remove red blood cells and at the same time a size increase of nucleated cells for inertial focusing and separation in spiral microchannel. Using an integrated device with two outlets, we isolated total leukocytes at a high yield of 99%. Furthermore cancer cells spiked into whole blood could be separated at a yield of 88% while 80% of leukocyte could be depleted into separate outlet by simply changing the resistance between the two outlets. Finally, using a three-outlet integrated device, we demonstrate fractionation of leukocyte into subpopulation. The device continuously separates granulocytes at a purity of 86%, monocyte at a purity of 43% and lymphocytes at a purity of 91% simultaneously. Finally, a cell activation study of the immune system using blood from healthy subjects, stimulated ex vivo with lipopolysaccharides (LPS), confirmed that the high operational flow rate of the device does not alter the activation levels of leukocytes or introduce artifacts. Hence, the simple, high-throughput and low-cost integrated device requiring neither external force fields nor mechanical parts to operate should readily be applicable to sort nucleated cells as stand-alone and/or as integrated lab-on-a-chip devices with high-throughput requirements.

  • 36.
    Soares, Ruben R. G.
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Neumann, Felix
    Stockholm Univ, Dept Biochem & Biophys, Sci Life Lab, SE-17165 Solna, Sweden..
    Caneira, Catarina R. F.
    Inst Engn Sistemas & Comp Microsistemas & Nanotec, Lisbon, Portugal.;IN Inst Nanosci & Nanotechnol, Lisbon, Portugal..
    Madaboosi, Narayanan
    Stockholm Univ, Dept Biochem & Biophys, Sci Life Lab, SE-17165 Solna, Sweden..
    Ciftci, Sibel
    Stockholm Univ, Dept Biochem & Biophys, Sci Life Lab, SE-17165 Solna, Sweden..
    Hernandez-Neuta, Ivan
    Stockholm Univ, Dept Biochem & Biophys, Sci Life Lab, SE-17165 Solna, Sweden..
    Pinto, Ines F.
    Inst Engn Sistemas & Comp Microsistemas & Nanotec, Lisbon, Portugal.;IN Inst Nanosci & Nanotechnol, Lisbon, Portugal.;Univ Lisbon, Inst Super Tecn, IBB Inst Bioengn & Biosci, Lisbon, Portugal..
    Santos, Denis R.
    Inst Engn Sistemas & Comp Microsistemas & Nanotec, Lisbon, Portugal.;IN Inst Nanosci & Nanotechnol, Lisbon, Portugal.;Univ Lisbon, Inst Super Tecn, IBB Inst Bioengn & Biosci, Lisbon, Portugal..
    Chu, Virginia
    Inst Engn Sistemas & Comp Microsistemas & Nanotec, Lisbon, Portugal.;IN Inst Nanosci & Nanotechnol, Lisbon, Portugal..
    Russom, Aman
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Conde, Joao P.
    Inst Engn Sistemas & Comp Microsistemas & Nanotec, Lisbon, Portugal.;IN Inst Nanosci & Nanotechnol, Lisbon, Portugal.;Univ Lisbon, Inst Super Tecn, Dept Bioengn, Lisbon, Portugal..
    Nilsson, Mats
    Stockholm Univ, Dept Biochem & Biophys, Sci Life Lab, SE-17165 Solna, Sweden..
    Silica bead-based microfluidic device with integrated photodiodes for the rapid capture and detection of rolling circle amplification products in the femtomolar range2019In: Biosensors & bioelectronics, ISSN 0956-5663, E-ISSN 1873-4235, Vol. 128, p. 68-75Article in journal (Refereed)
    Abstract [en]

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

  • 37.
    Zelenin, Sergey
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Ramachandraiah, Harisha
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Faridi, Muhammad Asim
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Russom, Aman
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Microfluidic-based bacteria isolation from whole blood for diagnostics of blood stream infection2017In: Methods in Molecular Biology: Microchip Diagnostics, Springer, 2017, p. 175-186Conference paper (Refereed)
    Abstract [en]

    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. © Springer Science+Business Media LLC 2017.

  • 38.
    Zhang, Wang
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology. karolinska institutet.
    Aljadi, Zenib
    Neogi, Ujjwal
    Russom, Aman
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Microfluidic based immunoaffinity mononuclear leukocytes isolation from whole bloodManuscript (preprint) (Other academic)
    Abstract [en]

    CD4+ T cells, monocyte/macrophages and natural killer cells are believed to be the main source for HIV-1 reservoirs in peripheral blood. However, despite the potential these subsets of providing a wealth of new information about immune function and host pathology, current HIV latency studies are often based on PBMCs or only CD4+ T cells, mainly due to the lack of appropriate cell subset isolation methods. We present here a microfluidic chip-based method to capture and enrich the three mononuclear cells sub-population peripheral leukocyte sub-populations: CD4+ lymphocytes, natural killer cells and monocytes; using a single source of whole blood (volume < 200 μL) on a single integrated platform, within a time frame of 20 min. The single step isolation method can be used for downstream proteomics and genomics analysis to study the aberrations in these cell types’ functions in critical diseases such as HIV.

  • 39.
    Zhang, Wang
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Ambikan, Anoop T.
    Sperk, Maike
    van Domselaar, Robert
    Nowak, Piotr
    Noyan, Kajsa
    Russom, Aman
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Sonnerborg, Anders
    Neogi, Ujjwal
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Transcriptomics and Targeted Proteomics Analysis to Gain Insights Into the Immune-control Mechanisms of HIV-1 Infected Elite Controllers2018In: EBioMedicine, E-ISSN 2352-3964, Vol. 27, p. 40-50Article in journal (Refereed)
    Abstract [en]

    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 naive 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 SIGLEC1were 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.

  • 40.
    Zhang, Wang
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Karolinska Inst, Sweden.
    Morshed, Mohammed M.
    Noyan, Kajsa
    Russom, Aman
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Sonnerborg, Anders
    Neogi, Ujjwal
    Quantitative humoral profiling of the HIV-1 proteome in elite controllers and patients with very long-term efficient antiretroviral therapy2017In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 666Article in journal (Refereed)
    Abstract [en]

    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.

1 - 40 of 40
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