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Ramachandraiah, HarishaORCID iD iconorcid.org/0000-0001-5199-0663
Publications (10 of 12) Show all publications
Kjellander, M., Billinger, E., Ramachandraiah, H., Boman, M., Lind, S. B. & Johansson, G. (2018). A flow-through nanoporous alumina trypsin bioreactor for mass spectrometry peptide fingerprinting. Journal of Proteomics, 172, 165-172
Open this publication in new window or tab >>A flow-through nanoporous alumina trypsin bioreactor for mass spectrometry peptide fingerprinting
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2018 (English)In: Journal of Proteomics, ISSN 1874-3919, E-ISSN 1876-7737, Vol. 172, p. 165-172Article in journal (Refereed) Published
Abstract [en]

Mass spectrometry-based proteomics benefits from efficient digestion of protein samples. In this study, trypsin was immobilized on nanoporous anodized alumina membranes to create an enzyme reactor suitable for peptide mass fingerprinting. The membranes were derivatized with 3-aminopropyltriethoxysilane and the amino groups were activated with carbonyldiimidazole to allow coupling of porcine trypsin via c-amino groups. The function was assessed using the artificial substrate Na-Benzoyl-L-arginine 4-nitroanilide hydrochloride, bovine ribonuclease A and a human plasma sample. A 10-membrane flow-through reactor was used for fragmentation and MS analysis after a single pass of substrate both by collection of product and subsequent off-line analysis, and by coupling on-line to the instrument. The peptide pattem allowed correct identification of the single target protein in both cases, and of > 70 plasma proteins in single pass mode followed by LC-MS analysis. The reactor retained 76% of the initial activity after 14 days of storage and repeated use at room temperature. Significance: This manuscript describes the design of a stable enzyme reactor that allows efficient and fast digestion with negligible leakage of enzyme and enzyme fragments. The high stability facilitates the use in an online-setup with MS detection since it allows the processing of multiple samples within an extended period of time without replacement.

Place, publisher, year, edition, pages
Elsevier, 2018
Keywords
Nanoporous aluminum oxide, Immobilization, Trypsin, Peptide mass fingerprinting, On-line digestion, ESI-TOF-MS, Enzyme stability
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-223261 (URN)10.1016/j.jprot.2017.09.008 (DOI)000423655300016 ()28942014 (PubMedID)2-s2.0-85032355956 (Scopus ID)
Funder
Swedish Research Council, 2005-3308Magnus Bergvall Foundation, 2015-01200 201601675Åke Wiberg Foundation, M14-0127Carl Tryggers foundation , CST 15:57
Note

QC 20180216

Available from: 2018-02-16 Created: 2018-02-16 Last updated: 2018-02-16Bibliographically approved
Faridi, M. A., Ramachandraiah, H., Banerjee, I., Ardabli, S., Zelenin, S. & Russom, A. (2017). Elasto-inertial microfluidics for bacteria separation from whole blood for sepsis diagnostics. Journal of Nanobiotechnology, 15, Article ID 3.
Open this publication in new window or tab >>Elasto-inertial microfluidics for bacteria separation from whole blood for sepsis diagnostics
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2017 (English)In: Journal of Nanobiotechnology, ISSN 1477-3155, E-ISSN 1477-3155, Vol. 15, article id 3Article in journal (Refereed) Published
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. 

Place, publisher, year, edition, pages
BioMed Central, 2017
Keywords
Micro particle separation, Elasto-inertial microfluidics, Sepsis, Sample preparation
National Category
Medical Biotechnology
Identifiers
urn:nbn:se:kth:diva-200300 (URN)10.1186/s12951-016-0235-4 (DOI)000391073000001 ()2-s2.0-85008198016 (Scopus ID)
Projects
RAPP_ID
Funder
EU, European Research Council, 115153
Note

QC 20170124

Available from: 2017-01-24 Created: 2017-01-24 Last updated: 2019-02-28Bibliographically approved
Etcheverry, S., Faridi, M. A., Ramachandraiah, H., Kumar, T., Margulis, W., Laurell, F. & Russom, A. (2017). High performance micro-flow cytometer based on optical fibres. Scientific Reports, 7, Article ID 5628.
Open this publication in new window or tab >>High performance micro-flow cytometer based on optical fibres
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2017 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 5628Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
Nature Publishing Group, 2017
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-211606 (URN)10.1038/s41598-017-05843-7 (DOI)000405677200013 ()2-s2.0-85025168074 (Scopus ID)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscienceSwedish Research CouncilKnut and Alice Wallenberg FoundationSwedish Childhood Cancer Foundation
Note

QC 20170814

Available from: 2017-08-14 Created: 2017-08-14 Last updated: 2017-10-17Bibliographically approved
Ramachandraiah, H., Svahn Andersson, H. & Russom, A. (2017). Inertial microfluidics combined with selective cell lysis for high throughput separation of nucleated cells from whole blood. RSC Advances (47), 29505-29514
Open this publication in new window or tab >>Inertial microfluidics combined with selective cell lysis for high throughput separation of nucleated cells from whole blood
2017 (English)In: RSC Advances, ISSN 2046-2069, E-ISSN 2046-2069, no 47, p. 29505-29514Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
RSC Publishing, 2017
Keywords
inertial microfluidics, nucleated cells, selective cell lysis
National Category
Medical Engineering
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-203241 (URN)10.1039/C7RA02992F (DOI)2-s2.0-85021694374 (Scopus ID)
Funder
Swedish Childhood Cancer FoundationEU, FP7, Seventh Framework ProgrammeSwedish Research Council
Note

QC 20170320

Available from: 2017-03-14 Created: 2017-03-14 Last updated: 2018-09-19Bibliographically approved
Zelenin, S., Ramachandraiah, H., Faridi, M. A. & Russom, A. (2017). Microfluidic-based bacteria isolation from whole blood for diagnostics of blood stream infection. In: Methods in Molecular Biology: Microchip Diagnostics. Paper presented at Methods in Molecular Biology (pp. 175-186). Springer
Open this publication in new window or tab >>Microfluidic-based bacteria isolation from whole blood for diagnostics of blood stream infection
2017 (English)In: Methods in Molecular Biology: Microchip Diagnostics, Springer, 2017, p. 175-186Conference paper, Published 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.

Place, publisher, year, edition, pages
Springer, 2017
Keywords
Bacteremia, Blood stream infection, Diagnostic, Microfluidics, Pathogens, Point-of-care diagnostics, Sepsis
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-202244 (URN)10.1007/978-1-4939-6734-6_14 (DOI)2-s2.0-85008466301 (Scopus ID)
Conference
Methods in Molecular Biology
Note

Funding text: This work was supported by the European Commission (projects FP7 InTopSens and IMI RAPP-ID). QC 20170222

Available from: 2017-03-06 Created: 2017-03-06 Last updated: 2017-03-06Bibliographically approved
Pettersson, T., Ramachandraiah, H. & Russom, A. (2017). Nanocellulose mediated layer-by-layer chip modification for cellular in-vitro diagnostics. Paper presented at 253rd National Meeting of the American-Chemical-Society (ACS) on Advanced Materials, Technologies, Systems, and Processes, APR 02-06, 2017, San Francisco, CA. Abstracts of Papers of the American Chemical Society, 253
Open this publication in new window or tab >>Nanocellulose mediated layer-by-layer chip modification for cellular in-vitro diagnostics
2017 (English)In: Abstracts of Papers of the American Chemical Society, ISSN 0065-7727, Vol. 253Article in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
American Chemical Society (ACS), 2017
National Category
Chemical Sciences
Identifiers
urn:nbn:se:kth:diva-243616 (URN)000430568503375 ()
Conference
253rd National Meeting of the American-Chemical-Society (ACS) on Advanced Materials, Technologies, Systems, and Processes, APR 02-06, 2017, San Francisco, CA
Note

QC 20190206

Available from: 2019-02-06 Created: 2019-02-06 Last updated: 2019-08-20Bibliographically approved
Banerjee, I., Salih, T., Ramachandraiah, H., Erlandsson, J., Pettersson, T., Araújo, A. C., . . . Russom, A. (2017). Slipdisc: A versatile sample preparation platform for point of care diagnostics. RSC Advances, 7(56), 35048-35054
Open this publication in new window or tab >>Slipdisc: A versatile sample preparation platform for point of care diagnostics
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2017 (English)In: RSC Advances, ISSN 2046-2069, E-ISSN 2046-2069, Vol. 7, no 56, p. 35048-35054Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
Royal Society of Chemistry, 2017
Keywords
Chemical analysis, Chemical detection, Clocks, Colorimetric analysis, Colorimetry, Coefficient of determination, Colorimetric detection, Lactate dehydrogenase, Micro-chambers, Point of care, Point of care diagnostic, Red channels, Sample preparation, Microfluidics
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:kth:diva-219077 (URN)10.1039/c7ra05209j (DOI)000405811400015 ()2-s2.0-85025080747 (Scopus ID)
Note

QC 20171201

Available from: 2017-12-01 Created: 2017-12-01 Last updated: 2019-11-06Bibliographically approved
Etcheverry, S., Faridi, M. A., Ramachandraiah, H., Margulis, W., Laurell, F. & Russom, A. (2016). All fiber based micro-flow cytometer by combining optical fiber with inertial focusing. In: 20th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2016: . Paper presented at 20th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2016, 9 October 2016 through 13 October 2016 (pp. 1655-1656). Chemical and Biological Microsystems Society
Open this publication in new window or tab >>All fiber based micro-flow cytometer by combining optical fiber with inertial focusing
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2016 (English)In: 20th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2016, Chemical and Biological Microsystems Society , 2016, p. 1655-1656Conference paper, Published 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.

Place, publisher, year, edition, pages
Chemical and Biological Microsystems Society, 2016
Keywords
Cell counting, Flow cytometry, Inertial focusing, Optical fibers
National Category
Physical Sciences
Identifiers
urn:nbn:se:kth:diva-207530 (URN)2-s2.0-85014148814 (Scopus ID)9780979806490 (ISBN)
Conference
20th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2016, 9 October 2016 through 13 October 2016
Note

QC 20170531

Available from: 2017-05-31 Created: 2017-05-31 Last updated: 2018-02-27Bibliographically approved
Banerjee, I., Salih, T., Ramachandraiah, H., Erlandsson, J., Petterson, T., Silva, A., . . . Russom, A. (2016). LDH based neonatal diagnostics on a low-cost slipdisc based sample preparation platform.. In: : . Paper presented at Microsystems workshop, May 17-18,2016, Lund, Sweden.
Open this publication in new window or tab >>LDH based neonatal diagnostics on a low-cost slipdisc based sample preparation platform.
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2016 (English)Conference paper, Poster (with or without abstract) (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.

National Category
Engineering and Technology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-255527 (URN)
Conference
Microsystems workshop, May 17-18,2016, Lund, Sweden
Note

QC 20190819

Available from: 2019-07-30 Created: 2019-07-30 Last updated: 2019-08-19Bibliographically approved
Etcheverry, S., Faridi, A., Ramachandraiah, H., Margulis, W., Laurell, F. & Russom, A. (2016). Optofludics in microstructured fibers combining particle elasto-inertial focusing and fluorescence. In: 2016 CONFERENCE ON LASERS AND ELECTRO-OPTICS (CLEO): . Paper presented at Conference on Lasers and Electro-Optics (CLEO), JUN 05-10, 2016, San Jose, CA. IEEE conference proceedings
Open this publication in new window or tab >>Optofludics in microstructured fibers combining particle elasto-inertial focusing and fluorescence
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2016 (English)In: 2016 CONFERENCE ON LASERS AND ELECTRO-OPTICS (CLEO), IEEE conference proceedings, 2016Conference paper, Published paper (Refereed)
Abstract [en]

Optofluidics is exploited in an all-fiber component to detect and identify through fluorescence particles flowing at high rate and inertially focused in a capillary. The system represents a first step towards an in-fiber flow cytometer.

Place, publisher, year, edition, pages
IEEE conference proceedings, 2016
Series
Conference on Lasers and Electro-Optics, ISSN 2160-9020
National Category
Physical Sciences
Identifiers
urn:nbn:se:kth:diva-201282 (URN)000391286403491 ()2-s2.0-85010651492 (Scopus ID)978-1-9435-8011-8 (ISBN)
Conference
Conference on Lasers and Electro-Optics (CLEO), JUN 05-10, 2016, San Jose, CA
Note

QC 20170215

Available from: 2017-02-15 Created: 2017-02-15 Last updated: 2017-05-17Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0001-5199-0663

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