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  • 1.
    Afrasiabi, Roodabeh
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Material Physics, MF.
    Söderberg, Lovisa
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Jönsson, Håkan
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Björk, Per
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology (closed 20130101).
    Linnros, Jan
    KTH, School of Information and Communication Technology (ICT), Materials- and Nano Physics.
    Integration of a Droplet-Based Microfluidic System and Silicon Nanoribbon FET Sensor2016In: Micromachines, ISSN 2072-666X, E-ISSN 2072-666X, Vol. 7, no 134Article in journal (Refereed)
    Abstract [en]

    We present a novel microfluidic system that integrates droplet microfluidics with a silicon nanoribbon field-effect transistor (SiNR FET), and utilize this integrated system to sense differences in pH. The device allows for selective droplet transfer to a continuous water phase, actuated by dielectrophoresis, and subsequent detection of the pH level in the retrieved droplets by SiNR FETs on an electrical sensor chip. The integrated microfluidic system demonstrates a label-free detection method for droplet microfluidics, presenting an alternative to optical fluorescence detection. In this work, we were able to differentiate between droplet trains of one pH-unit difference. The pH-based detection method in our integrated system has the potential to be utilized in the detection of biochemical reactions that induce a pH-shift in the droplets.

  • 2.
    Antypas, H.
    et al.
    Karolinska Inst, Dept Neurosci, Swedish Med Nanosci Ctr, Stockholm, Sweden..
    Veses-Garcia, M.
    Karolinska Inst, Dept Neurosci, Swedish Med Nanosci Ctr, Stockholm, Sweden..
    Weibull, Emelie
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Svahn Andersson, Helene
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Richter-Dahlfors, A.
    Karolinska Inst, Dept Neurosci, Swedish Med Nanosci Ctr, Stockholm, Sweden..
    A universal platform for selection and high-resolution phenotypic screening of bacterial mutants using the nanowell slide2018In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 18, no 12, p. 1767-1777Article in journal (Refereed)
    Abstract [en]

    The Petri dish and microtiter plate are the golden standard for selection and screening of bacteria in microbiological research. To improve on the limited resolution and throughput of these methods, we developed a universal, user-friendly platform for selection and high-resolution phenotypic screening based on the nanowell slide. This miniaturized platform has an optimal ratio between throughput and assay complexity, holding 672 nanowells of 500 nl each. As monoclonality is essential in bacterial genetics, we used FACS to inoculate each nanowell with a single bacterium in 15 min. We further extended the protocol to select and sort only bacteria of interest from a mixed culture. We demonstrated this by isolating single transposon mutants generated by a custom-made transposon with dual selection for GFP fluorescence and kanamycin resistance. Optical compatibility of the nanowell slide enabled phenotypic screening of sorted mutants by spectrophotometric recording during incubation. By processing the absorbance data with our custom algorithm, a phenotypic screen for growth-associated mutations was performed. Alternatively, by processing fluorescence data, we detected metabolism-associated mutations, exemplified by a screen for -galactosidase activity. Besides spectrophotometry, optical compatibility enabled us to perform microscopic analysis directly in the nanowells to screen for mutants with altered morphologies. Despite the miniaturized format, easy transition from nano- to macroscale cultures allowed retrieval of bacterial mutants for downstream genetic analysis, demonstrated here by a cloning-free single-primer PCR protocol. Taken together, our FACS-linked nanowell slide replaces manual selection of mutants on agar plates, and enables combined selection and phenotypic screening in a one-step process. The versatility of the nanowell slide, and the modular workflow built on mainstream technologies, makes our universal platform widely applicable in microbiological research.

  • 3.
    Banerjee, Indradumna
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Point of care microfluidic tool development for resource limited settings2019Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The development of point of care diagnostics using recent advances in microfluidics have the potential to transform health care in several ways, especially in resource limited settings with limited access to advanced health care infrastructure. However, translating a point of care device to reality is often a challenging task because of the complexities involved in integrating a number of diverse engineering concepts into an easy to use, accurate and portable device. This thesis focuses on miniaturization of crucial diagnostic laboratory tools, that can be used in a portable point of care format without compromising on the accuracy or performance. The first part of the thesis (Paper I-III) focuses on understanding and applying elasto-inertial microfluidics, which is a label-free and passive bio-particle sorting and separation method. A basic understanding of particle trajectories in both inertial (Paper I) and visco-elastic flows (Paper II) is established, followed by an investigation on the combined effects of inertia and elasticity (Paper III). The second part of the thesis (Paper IV-VI) focuses on developing integrated microfluidic platforms, each of which addresses different aspects of point of care diagnostic applications. The applications include neonatal diagnostics using a hand-driven Slipdisc technique (Paper IV), rapid nucleic acid quantification using a novel precipitate-based detection on a centrifugal microfluidics platform (Paper V), and hematocrit level measurement in blood using a portable lab-on- Disc platform operated by a mobile phone (Paper VI). The proof of concept microfluidic tools presented in the scope of this thesis have the potential to replace a number of functions of standard laboratory equipment, at a fraction of the price and without compromising performance. Hence, the different methods developed should contribute towards decentralization of medical testing laboratories, making healthcare accessible to one and all.

  • 4.
    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

  • 5.
    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.

  • 6.
    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).

  • 7.
    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.

  • 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.
    Björk, Sara
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Droplet microfluidics for screening and sorting of microbial cell factories2019Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Cell factories are cells that have been engineered to produce a compound of interest, ranging from biopharmaceuticals to biofuels. With advances in metabolic engineering, the number of cell factory variants to evaluate has increased dramatically, necessitating screening methods with increased throughput. Microfluidic droplets, which can be generated, manipulated and interrogated at very high throughput, are isolated reaction vessels at the single cell scale. Compartmentalization maintains the genotype-phenotype link, making droplet microfluidics suitable for screening of extracellular traits such as secreted products and for screening of microcolonies originating from single cells.

     

    In Paper I, we investigated the impact of droplet microfluidic incubation formats on cell culture conditions and found that syringe and semi open incubation resulted in different metabolic profiles. Controlling culture conditions is key to cell factory screening, as product formation is influenced by the state of the cell.

     

    In Paper II and III, we used droplet microfluidics to perform screening campaigns of interference based cell factory variant libraries. In Paper II, two S. cerevisiae RNAi libraries were screened based on amylase secretion, and from the sorted fraction genes linked to improved protein secretion could be identified. In paper III, we screened a Synecosystis sp. CRISPRi library based on lactate secretion. The library was sorted at different time points after induction, followed by sequencing to reveal genes enriched by droplet sorting.

     

    In Paper IV, we developed a droplet microcolony-based assay for screening intracellular triacylglycerol (TAG) in S. cerevisiae, and showed improved strain separation compared to flow cytometry in a hypothetical sorting scenario. By screening microcolonies compartmentalized in droplets, we combine the throughput of single cell screening methods with the reduced impact of cell-to-cell noise in cell ensemble analysis.

  • 11.
    Björk, Sara
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Jönsson, Håkan
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Microfluidics for cell factory and bioprocess development2019In: Current Opinion in Biotechnology, ISSN 0958-1669, E-ISSN 1879-0429, Vol. 55, p. 95-102Article in journal (Refereed)
    Abstract [en]

    Bioindustry is expanding to an increasing variety of food, chemical and pharmaceutical products, each requiring rapid development of a dedicated cell factory and bioprocess. Microfluidic tools are, together with tools from synthetic biology and metabolic modeling, being employed in cell factory and bioprocess development to speed up development and address new products. Recent examples of microfluidics for bioprocess development range from integrated devices for DNA assembly and transformation, to high throughput screening of cell factory libraries, and micron scale bioreactors for process optimization. These improvements act to improve the biotechnological engineering cycle with tools for building, testing and evaluating cell factories and bioprocesses by increasing throughput, parallelization and automation.

  • 12.
    Björk, Sara M.
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Sjostrom, Staffan L.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Jönsson, Håkan N.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Metabolite profiling of microfluidic cell culture conditions for droplet based screening2015In: Biomicrofluidics, ISSN 1932-1058, E-ISSN 1932-1058, Vol. 9, no 4, article id 044128Article in journal (Refereed)
    Abstract [en]

    We investigate the impact of droplet culture conditions on cell metabolic state by determining key metabolite concentrations in S. cerevisiae cultures in different microfluidic droplet culture formats. Control of culture conditions is critical for single cell/clone screening in droplets, such as directed evolution of yeast, as cell metabolic state directly affects production yields from cell factories. Here, we analyze glucose, pyruvate, ethanol, and glycerol, central metabolites in yeast glucose dissimilation to establish culture formats for screening of respiring as well as fermenting yeast. Metabolite profiling provides a more nuanced estimate of cell state compared to proliferation studies alone. We show that the choice of droplet incubation format impacts cell proliferation and metabolite production. The standard syringe incubation of droplets exhibited metabolite profiles similar to oxygen limited cultures, whereas the metabolite profiles of cells cultured in the alternative wide tube droplet incubation format resemble those from aerobic culture. Furthermore, we demonstrate retained droplet stability and size in the new better oxygenated droplet incubation format.

  • 13.
    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.

  • 14. Fornell, Anna
    et al.
    Nilsson, Johan
    Jonsson, Linus
    Periyannan Rajeswari, Prem Kumar
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Jönsson, Håkan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Tenje, Maria
    Particle enrichment in droplet acoustofluidics2016In: Micronano System Workshop (MSW 2016), Lund, Sweden, May 17-18 2016, 2016Conference paper (Refereed)
  • 15. Fornell, Anna
    et al.
    Nilsson, Johan
    Jonsson, Linus
    Periyannan Rajeswari, Prem Kumar
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Jönsson, Håkan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Tenje, Maria
    Particle enrichment in two-phase microfluidic systems using acoustophoresis2016In: Acoustofluidics 2016, Kongens Lyngby, Denmark, September 22-23 2016, 2016Conference paper (Refereed)
  • 16. Hammond, Maria
    et al.
    Homa, Felix
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Ettema, Thijs J. G.
    Jönsson, Håkan
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Picodroplet partitioned whole genome amplification of low biomass samples preserves genomic diversity for metagenomic analysis2016In: Microbiome, ISSN 0026-2633, E-ISSN 2049-2618, Vol. 4, article id 52Article in journal (Refereed)
    Abstract [en]

    Background: Whole genome amplification (WGA) is a challenging, key step in metagenomic studies of samples containing minute amounts of DNA, such as samples from low biomass environments. It is well known that multiple displacement amplification (MDA), the most commonly used WGA method for microbial samples, skews the genomic representation in the sample. We have combined MDA with droplet microfluidics to perform the reaction in a homogeneous emulsion. Each droplet in this emulsion can be considered an individual reaction chamber, allowing partitioning of the MDA reaction into millions of parallel reactions with only one or very few template molecules per droplet. Results: As a proof-of-concept, we amplified genomic DNA from a synthetic metagenome by MDA either in one bulk reaction or in emulsion and found that after sequencing, the species distribution was better preserved and the coverage depth was more evenly distributed across the genomes when the MDA reaction had been performed in emulsion. Conclusions: Partitioning MDA reactions into millions of reactions by droplet microfluidics is a straightforward way to improve the uniformity of MDA reactions for amplifying complex samples with limited amounts of DNA.

  • 17. Huang, M.
    et al.
    Jönsson, Håkan
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology. Novo Nordisk Foundation Center for Biosustainability.
    Nielsen, J.
    High-throughput microfluidics for the screening of yeast libraries2018In: Synthetic Metabolic Pathways: Methods and Protocols, Humana Press, 2018, Vol. 1671, p. 307-317Chapter in book (Refereed)
    Abstract [en]

    Cell factory development is critically important for efficient biological production of chemicals, biofuels, and pharmaceuticals. Many rounds of the Design–Build–Test–Learn cycles may be required before an engineered strain meeting specific metrics required for industrial application. The bioindustry prefer products in secreted form (secreted products or extracellular metabolites) as it can lower the cost of downstream processing, reduce metabolic burden to cell hosts, and allow necessary modification on the final products, such as biopharmaceuticals. Yet, products in secreted form result in the disconnection of phenotype from genotype, which may have limited throughput in the Test step for identification of desired variants from large libraries of mutant strains. In droplet microfluidic screening, single cells are encapsulated in individual droplet and enable high-throughput processing and sorting of single cells or clones. Encapsulation in droplets allows this technology to overcome the throughput limitations present in traditional methods for screening by extracellular phenotypes. In this chapter, we describe a protocol/guideline for high-throughput droplet microfluidics screening of yeast libraries for higher protein secretion. This protocol can be adapted to screening by a range of other extracellular products from yeast or other hosts.

  • 18. Jeong, Yunjin
    et al.
    Svedberg, Gustav
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Réu, Pedro
    Lee, Yongju
    Song, Seo Woo
    Na, Hunjong
    Lee, Amos Chungwon
    Choi, Yeongjae
    Gantelius, Jesper
    Andersson Svahn, Helene
    Kwon, Sunghoon
    Solid-phase PCR on graphically encoded microparticles for multiplexed colorimetric detection of bacterial DNAManuscript (preprint) (Other academic)
  • 19.
    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.

  • 20.
    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.

  • 21.
    Langer, Krzysztof
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Jönsson, Håkan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Rapid production and recovery of cell spheroids by automated droplet microfluidics2019In: bioRxivArticle in journal (Refereed)
    Abstract [en]

    Droplet microfluidics enables high throughput cell processing, analysis and screening by miniaturizing the reaction vessels to nano- or pico-liter water-in oil droplets, but like many other microfluidic formats, droplet microfluidics have not been interfaced with or automated by laboratory robotics. Here we demonstrate automation of droplet microfluidics based on an inexpensive liquid handling robot for the automated production of human scaffold-free cell spheroids, using pipette actuation and interfacing the pipetting tip with a droplet generating microfluidic chip. In this chip we produce highly mono-disperse 290μm droplets with diameter CV of 1.7%. By encapsulating cells in these droplets, we produce cell spheroids in droplets and recover them to standard formats at a throughput of 85000 spheroids per microfluidic circuit per hour. The viability of the cells in spheroids remains high after recovery only decreased by 4% starting from 96% after 16 hours incubation in nanoliter droplets. Scaffold-free cell spheroids and 3D tissue constructs recapitulate many aspects of functional human tissue more accurately than 2D or single cell cultures, but assembly methods for spheroids, e.g. hanging drop micro-plates, has had limited throughput. The increased throughput and decreased cost of our method enables spheroid production at the scale needed for lead discovery drug screening and approaches the cost where these micro tissues could be used as building blocks for organ scale regenerative medicine.

  • 22.
    Langer, Krzysztof
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Jönsson, Håkan
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Rapid Production and Recovery of Cell Spheroids by Automated Droplet Microfluidics2019In: SLAS TECHNOLOGYArticle in journal (Refereed)
    Abstract [en]

    The future of the life sciences is linked to automation and microfluidics. As robots start working side by side with scientists, robotic automation of microfluidics in general, and droplet microfluidics in particular, will significantly extend and accelerate the life sciences. Here, we demonstrate the automation of droplet microfluidics using an inexpensive liquid-handling robot to produce human scaffold-free cell spheroids at high throughput. We use pipette actuation and interface the pipetting tip with a droplet-generating microfluidic device. In this device, we produce highly monodisperse droplets with a diameter coefficient of variation (CV) lower than 2%. By encapsulating cells in these droplets, we produce cell spheroids in droplets and recover them to standard labware containers at a throughput of 85,000 spheroids per microfluidic circuit per hour. The viability of the cells in spheroids remains high throughout the process and decreases by >10% (depending on the cell line used) after a 16 h incubation period in nanoliter droplets and automated recovery. Scaffold-free cell spheroids and 3D tissue constructs recapitulate many aspects of functional human tissue more accurately than 2D or single-cell cultures, but assembly methods for spheroids (e.g., hanging drop microplates) have limited throughput. The increased throughput and decreased cost of our method enable spheroid production at the scale needed for lead discovery drug screening, and approach the cost at which these microtissues could be used as building blocks for organ-scale regenerative medicine.

  • 23.
    Periyannan Rajeswari, Prem Kumar
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Söderberg, Lovisa M.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Yacoub, Alia
    Leijon, Mikael
    Andersson Svahn, Helene
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Jönsson, Håkan
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Multiple pathogen biomarker detection using an encoded bead array in droplet PCR2017In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 139, p. 22-28Article in journal (Refereed)
    Abstract [en]

    We present a droplet PCR workflow for detection of multiple pathogen DNA biomarkers using fluorescent color coded Luminex beads. This strategy enables encoding of multiple singleplex droplet PCRs using a commercially available bead set of several hundred distinguishable fluorescence codes. This workflow provides scalability beyond the limited number offered by fluorescent detection probes such as TaqMan probes, commonly used in current multiplex droplet PCRs. The workflow was validated for three different Luminex bead sets coupled to target specific capture oligos to detect hybridization of three microorganisms infecting poultry: avian influenza, infectious laryngotracheitis virus and Campylobacter jejuni. In this assay, the target DNA was amplified with fluorescently labeled primers by PCR in parallel in monodisperse picoliter droplets, to avoid amplification bias. The color codes of the Luminex detection beads allowed concurrent and accurate classification of the different bead sets used in this assay. The hybridization assay detected target DNA of all three microorganisms with high specificity, from samples with average target concentration of a single DNA template molecule per droplet. This workflow demonstrates the possibility of increasing the droplet PCR assay detection panel to detect large numbers of targets in parallel, utilizing the scalability offered by the color-coded Luminex detection beads.

  • 24.
    Reu, Pedro
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Svedberg, Gustav
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Hässler, Lars
    KTH, School of Industrial Engineering and Management (ITM), Machine Design (Dept.).
    Möller, Björn
    KTH, School of Industrial Engineering and Management (ITM), Machine Design (Dept.).
    Svahn Andersson, Helene
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Gantelius, Jesper
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    A 61% lighter cell culture dish to reduce plastic waste2019In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 14, no 4, article id e0216251Article in journal (Refereed)
    Abstract [en]

    Cell culture is a ubiquitous and flexible research method. However, it heavily relies on plastic consumables generating millions of tonnes of plastic waste yearly. Plastic waste is a major and growing global concern. Here we describe a new cell culture dish that offers a culture area equivalent to three petri dishes but that is on average 61% lighter and occupies 67% less volume. Our dish is composed of a lid and three thin containers surrounded by a light outer shell. Cell culture can be performed in each of the containers sequentially. The outer shell provides the appropriate structure for the manipulation of the dish as a whole. The prototype was tested by sequentially growing cells in each of its containers. As a control, sequential cultures in groups of 3 petri dishes were performed. No statistical differences were found between the prototype and the control in terms of cell number, cell viability or cell distribution.

  • 25. Siedler, S.
    et al.
    Kumar Khatri, Narendar
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Zsohár, A.
    Kjærbølling, I.
    Vogt, M.
    Hammar, Petter
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Nielsen, C. F.
    Marienhagen, J.
    Sommer, M. O. A.
    Jönsson, Håkan
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Development of a Bacterial Biosensor for Rapid Screening of Yeast p-Coumaric Acid Production2017In: ACS Synthetic Biology, E-ISSN 2161-5063, Vol. 6, no 10, p. 1860-1869Article in journal (Refereed)
    Abstract [en]

    Transcription factor-based biosensors are used to identify producer strains, a critical bottleneck in cell factory engineering. Here, we address two challenges with this methodology: transplantation of heterologous transcriptional regulators into new hosts to generate functional biosensors and biosensing of the extracellular product concentration that accurately reflects the effective cell factory production capacity. We describe the effects of different translation initiation rates on the dynamic range of a p-coumaric acid biosensor based on the Bacillus subtilis transcriptional repressor PadR by varying its ribosomal binding site. Furthermore, we demonstrate the functionality of this p-coumaric acid biosensor in Escherichia coli and Corynebacterium glutamicum. Finally, we encapsulate yeast p-coumaric acid-producing cells with E. coli-biosensing cells in picoliter droplets and, in a microfluidic device, rapidly sort droplets containing yeast cells producing high amounts of extracellular p-coumaric acid using the fluorescent E. coli biosensor signal. As additional biosensors become available, such approaches will find broad applications for screening of an extracellular product.

  • 26.
    Svedberg, Gustav
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Novel planar and particle-based microarrays for point-of-care diagnostics2019Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Point-of-care assays are easy-to-use, portable and inexpensive tests that can

    be used to aid diagnostics by measuring levels of disease-specific molecules

    in settings where access to advanced laboratory equipment and trained

    personnel are limited, such as at the patient's bedside or in low resource

    parts of developing countries. In order to achieve high multiplexing

    capacities, such assays can be based on planar microarrays consisting of

    spots immobilized on a flat surface or on particle-based microarrays based

    on populations of encoded particles. The aim of the work presented in this

    thesis is to develop new point-of-care amenable planar and particle-based

    microarrays that allow for highly multiplexed assays while maintaining low

    sample-to-result times, complexity and instrumentation requirements.

    Paper I demonstrates the use graphically encoded particles for colorimetric

    detection of autoantibodies using a consumer-grade flatbed scanner. Four

    graphical characters on the surface of each particle allows for millions of

    codes and the use of gold nanoparticles as a detection label allows both the

    code and the signal intensity to be read out in a single image.

    Paper II describes a signal enhancement method that increases the

    sensitivity of gold nanoparticle detection on planar microarrays. Using this

    method, detection of allergen-specific IgE can be carried out using a

    consumer-grade flatbed scanner instead of a more expensive fluorescence

    scanner without sacrificing assay performance.

    Paper III demonstrates the use of an isothermal DNA amplification method

    for detection of adenoviral DNA on a paper-based microarray. Using an

    isothermal amplification method eliminates the need for a thermocycler,

    reducing the instrumentation required for such detection.

    Paper IV shows the use of solid-phase PCR to amplify bacterial DNA directly

    on the surface of particles. This strategy reduces assay time by eliminating

    the need for separate amplification and hybridisation steps.

  • 27. Wang, Guokun
    et al.
    Björk, Sara
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Huang, Mingtao
    Liu, Quanli
    Campbell, Kate
    Nielsen, Jens
    Jönsson, Håkan
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Petranovic, Dina
    RNAi expression tuning, microfluidic screening, and genome recombineering for improved protein production in Saccharomyces cerevisiae2019In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 116, no 19, p. 9324-9332Article in journal (Refereed)
    Abstract [en]

    The cellular machinery that supports protein synthesis and secretion lies at the foundation of cell factory-centered protein production. Due to the complexity of such cellular machinery, the challenge in generating a superior cell factory is to fully exploit the production potential by finding beneficial targets for optimized strains, which ideally could be used for improved secretion of other proteins. We focused on an approach in the yeast Saccharomyces cerevisiae that allows for attenuation of gene expression, using RNAi combined with high-throughput microfluidic single-cell screening for cells with improved protein secretion. Using direct experimental validation or enrichment analysis-assisted characterization of systematically introduced RNAi perturbations, we could identify targets that improve protein secretion. We found that genes with functions in cellular metabolism (YDC1, AAD4, ADE8, and SDH1), protein modification and degradation (VPS73, KTR2, CNL1, and SSA1), and cell cycle (CDC39), can all impact recombinant protein production when expressed at differentially down regulated levels. By establishing a workflow that incorporates Cas9-mediated recombineering, we demonstrated how we could tune the expression of the identified gene targets for further improved protein production for specific proteins. Our findings offer a high throughput and semirational platform design, which will improve not only the production of a desired protein but even more importantly, shed additional light on connections between protein production and other cellular processes.

  • 28.
    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.

1 - 28 of 28
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