Skip to main content
SpringerLink
Log in

Real-time quantification of protein expression and translocation at individual cell resolution using imaging-dish-based live cell array

  • Research Paper
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

Cell populations represent intrinsically heterogeneous systems with a high level of spatiotemporal complexity. Monitoring and understanding cell-to-cell diversity is essential for the research and application of intra- and interpopulation variations. Optical analysis of live cells is challenging since both adherent and nonadherent cells change their spatial location. However, most currently available single-cell techniques do not facilitate treatment and monitoring of the same live cells over time throughout multistep experiments. An imaging-dish-based live cell array (ID-LCA) has been developed and produced for cell handling, culturing, and imaging of numerous live cells. The dish is composed of an array of pico scale cavities—pico wells (PWs) embossed on its glass bottom. Cells are seeded, cultured, treated, and spatiotemporally measured on the ID-LCA, while each cell or small group of cells are locally constrained in the PWs. Finally, predefined cells can be retrieved for further evaluation. Various types of ID-LCAs were used in this proof-of-principle work, to demonstrate on-ID-LCA transfection of fluorescently tagged chimeric proteins, as well as the detection and kinetic analysis of their induced translocation. High variability was evident within cell populations with regard to protein expression levels as well as the extent and dynamics of protein redistribution. The association of these parameters with cell morphology and functional parameters was examined. Both the new methodology and the device facilitate research of the translocation process at individual cell resolution within large populations and thus, can potentially be used in high-throughput fashion.

ᅟ

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

Abbreviations

3D:

Three dimension

BF:

Bright field

CV:

Coefficient of variation

DRIE:

Deep reactive ion etching

FC:

Flow cytometry

FDA:

Fluorescein diacetate

FI:

Fluorescence intensity

HTS:

High-throughput screening

ID-LCA:

Imaging-dish-based live cell array

MC:

Microcavity

NPs:

Nanoparticles

PCR:

Polymerase chain reaction

PDMS:

Polydimethylsiloxane

PWs:

Picowells

ROI:

Region of interest

SD:

Standard deviation

TL:

Translocation level

UV:

Ultraviolet

References

  1. Wang D, Bodovitz S (2010) Single cell analysis: the new frontier in “omics”. Trends Biotechnol 28:281–290. doi:10.1016/j.tibtech.2010.03.002

    Article  CAS  Google Scholar 

  2. Kalisky T, Blainey P, Quake SR (2011) Genomic analysis at the single-cell level. Annu Rev Genet 45:431–445. doi:10.1146/annurev-genet-102209-163607

    Article  CAS  Google Scholar 

  3. Tang F, Lao K, Surani MA (2011) Development and applications of single-cell transcriptome analysis. Nat Methods 8:S6–S11. doi:10.1038/nmeth.1557

    Article  CAS  Google Scholar 

  4. Rubakhin SS, Romanova EV, Nemes P, Sweedler JV (2011) Profiling metabolites and peptides in single cells. Nat Methods 8:S20–S29. doi:10.1038/nmeth.1549

    Article  CAS  Google Scholar 

  5. Borland LM, Kottegoda S, Phillips KS, Allbritton NL (2008) Chemical analysis of single cells. Annu Rev Anal Chem (Palo Alto, Calif) 1:191–227. doi:10.1146/annurev.anchem.1.031207.113100

    Article  CAS  Google Scholar 

  6. Trouillon R, Passarelli MK, Wang J, Kurczy ME, Ewing AG (2013) Chemical analysis of single cells. Anal Chem 85:522–542. doi:10.1021/ac303290s

    Article  CAS  Google Scholar 

  7. Lecault V, White AK, Singhal A, Hansen CL (2012) Microfluidic single cell analysis: from promise to practice. Curr Opin Chem Biol 16:381–390. doi:10.1016/j.cbpa.2012.03.022

    Article  CAS  Google Scholar 

  8. Walling MA, Shepard JRE (2011) Cellular heterogeneity and live cell arrays. Chem Soc Rev 40:4049–4076. doi:10.1039/c0cs00212g

    Article  CAS  Google Scholar 

  9. Lindström S, Andersson-Svahn H (2011) Miniaturization of biological assays—overview on microwell devices for single-cell analyses. Biochim Biophys Acta 1810:308–316. doi:10.1016/j.bbagen.2010.04.009

    Article  Google Scholar 

  10. Mazutis L, Gilbert J, Ung WL, Weitz DA, Griffiths AD, Heyman JA (2013) Single-cell analysis and sorting using droplet-based microfluidics. Nat Protoc 8:870–891. doi:10.1038/nprot.2013.046

    Article  CAS  Google Scholar 

  11. Lin Y, Trouillon R, Safina G, Ewing AG (2011) Chemical analysis of single cells. Anal Chem 83:4369–4392. doi:10.1021/ac2009838

    Article  CAS  Google Scholar 

  12. Wlodkowic D, Khoshmanesh K, Sharpe JC, Darzynkiewicz Z, Cooper JM (2011) Apoptosis goes on a chip: advances in the microfluidic analysis of programmed cell death. Anal Chem 83:6439–6446. doi:10.1021/ac200588g

    Article  CAS  Google Scholar 

  13. Lindström S, Andersson-Svahn H (2010) Overview of single-cell analyses: microdevices and applications. Lab Chip 10:3363–3372. doi:10.1039/c0lc00150c

    Article  Google Scholar 

  14. Raj A, van Oudenaarden A (2008) Nature, nurture, or chance: stochastic gene expression and its consequences. Cell 135:216–226. doi:10.1016/j.cell.2008.09.050

    Article  CAS  Google Scholar 

  15. Huh D, Paulsson J (2011) Non-genetic heterogeneity from stochastic partitioning at cell division. Nat Genet 43:95–100. doi:10.1038/ng.729

    Article  CAS  Google Scholar 

  16. Cohen AA, Geva-Zatorsky N, Eden E, Frenkel-Morgenstern M, Issaeva I, Sigal A, Milo R, Cohen-Saidon C, Liron Y, Kam Z, Cohen L, Danon T, Perzov N, Alon U (2008) Dynamic proteomics of individual cancer cells in response to a drug. Science 322:1511–1516. doi:10.1126/science.1160165

    Article  CAS  Google Scholar 

  17. Spencer SL, Gaudet S, Albeck JG, Burke JM, Sorger PK (2009) Non-genetic origins of cell-to-cell variability in TRAIL-induced apoptosis. Nature 459:428–432. doi:10.1038/nature08012

    Article  CAS  Google Scholar 

  18. Nelson DE, Ihekwaba AEC, Elliott M, Johnson JR, Gibney CA, Foreman BA, Nelson G, See V, Horton CA, Spiller DG, Edwards SW, McDowell HP, Unitt JF, Sullivan E, Grimley R, Benson N, Broomhead D, Kell DB, White MRH (2004) Oscillations in NF-kappaB signaling control the dynamics of gene expression. Science 306:704–708. doi:10.1126/science.1099962

    Article  CAS  Google Scholar 

  19. Blake WJ, KAErn M, Canton CR, Collins JJ (2003) Noise in eukaryotic gene expression. Nature 422:633–637. doi:10.1038/nature01546

    Article  CAS  Google Scholar 

  20. Kuwai T, Nakamura T, Kim S-J, Sasaki T, Kitadai Y, Langley RR, Fan D, Hamilton FIJ (2008) Intratumoral heterogeneity for expression of tyrosine kinase growth factor receptors in human colon cancer surgical specimens and orthotopic tumors. Am J Pathol 172:358–366. doi:10.2353/ajpath.2008.070625

    Article  Google Scholar 

  21. Rubakhin SS, Lanni EJ, Sweedler JV (2013) Progress toward single cell metabolomics. Curr Opin Biotechnol 24:95–104. doi:10.1016/j.copbio.2012.10.021

    Article  CAS  Google Scholar 

  22. Wier WG, Cannell MB, Berlin JR, Marban E, Lederer WJ (1987) Cellular and subcellular heterogeneity of [Ca2+]i in single heart cells revealed by fura-2. Science 235:325–328

    Article  CAS  Google Scholar 

  23. Ormerod MG (2004) Cell-cycle analysis of asynchronous populations. Methods Mol Biol 263:345–354. doi:10.1385/1-59259-773-4:345

    Google Scholar 

  24. Tay S, Hughey JJ, Lee TK, Lipniacki T, Quake SR, Covert MW (2010) Single-cell NF-kappaB dynamics reveal digital activation and analogue information processing. Nature 466:267–271. doi:10.1038/nature09145

    Article  CAS  Google Scholar 

  25. Woods NM, Cuthbertson KS, Cobbold PH (1986) Repetitive transient rises in cytoplasmic free calcium in hormone-stimulated hepatocytes. Nature 319:600–602. doi:10.1038/319600a0

    Article  CAS  Google Scholar 

  26. Monk NAM (2003) Oscillatory expression of Hes1, p53, and NF-kappaB driven by transcriptional time delays. Curr Biol 13:1409–1413

    Article  CAS  Google Scholar 

  27. Sims CE, Allbritton NL (2007) Analysis of single mammalian cells on-chip. Lab Chip 7:423–440. doi:10.1039/b615235j

    Article  CAS  Google Scholar 

  28. McGrath KE, Bushnell TP, Palis J (2008) Multispectral imaging of hematopoietic cells: where flow meets morphology. J Immunol Methods 336:91–97. doi:10.1016/j.jim.2008.04.012

    Article  CAS  Google Scholar 

  29. Nilsson J, Evander M, Hammarström B, Laurell T (2009) Review of cell and particle trapping in microfluidic systems. Anal Chim Acta 649:141–157. doi:10.1016/j.aca.2009.07.017

    Article  CAS  Google Scholar 

  30. Lindström S, Larsson R, Svahn HA (2008) Towards high-throughput single cell/clone cultivation and analysis. Electrophoresis 29:1219–1227. doi:10.1002/elps.200700536

    Article  Google Scholar 

  31. Love JC, Ronan JL, Grotenbreg GM, van der Veen AG, Ploegh HL (2006) A microengraving method for rapid selection of single cells producing antigen-specific antibodies. Nat Biotechnol 24:703–707. doi:10.1038/nbt1210

    Article  CAS  Google Scholar 

  32. Lew V, Nguyen D, Khine M (2011) Shrink-induced single-cell plastic microwell array. J Lab Autom 16:450–456. doi:10.1016/j.jala.2011.06.003

    Article  Google Scholar 

  33. Frisk TW, Khorshidi MA, Guldevall K, Vanherberghen B, Önfelt B (2011) A silicon-glass microwell platform for high-resolution imaging and high-content screening with single cell resolution. Biomed Microdevices 13:683–693. doi:10.1007/s10544-011-9538-2

    Article  Google Scholar 

  34. Shirasaki Y, Nakahara A, Shimura N, Yamagishi M, Mizuno J, Ohara O, Shoji S (2011) Single cell real time secretion assay using amorphous fluoropolymer microwell array. 2011 16th Int. Solid-State Sensors, Actuators Microsystems Conf. IEEE, pp 755–758

  35. Yamamura S, Kishi H, Tokimitsu Y, Kondo S, Honda R, Rao SR, Omori M, Tamiya E, Muraguchi A (2005) Single-cell microarray for analyzing cellular response. Anal Chem 77:8050–8056. doi:10.1021/ac0515632

    Article  CAS  Google Scholar 

  36. Rettig JR, Folch A (2005) Large-scale single-cell trapping and imaging using microwell arrays. Anal Chem 77:5628–5634. doi:10.1021/ac0505977

    Article  CAS  Google Scholar 

  37. Kantlehner M, Kirchner R, Hartmann P, Ellwart JW, Alunni-Fabbroni M, Schumacher A (2011) A high-throughput DNA methylation analysis of a single cell. Nucleic Acids Res 39:e44. doi:10.1093/nar/gkq1357

    Article  CAS  Google Scholar 

  38. Kobayashi K, Kajiwara E, Ishikawa M, Mimura H, Oka H, Ejiri Y, Hosoda M, Chiba K (2013) Cytotoxic effects of benzbromarone and its 1′-hydroxy metabolite in human hepatocarcinoma FLC4 cells cultured on micro-space cell culture plates. Drug Metab Pharmacokinet 28:265–268

    Article  CAS  Google Scholar 

  39. Lindström S, Eriksson M, Vazin T, Sandberg J, Lundeberg J, Frisén J, Andersson-Svahn H (2009) High-density microwell chip for culture and analysis of stem cells. PLoS One 4:e6997. doi:10.1371/journal.pone.0006997

    Article  Google Scholar 

  40. Friedman M, Lindström S, Ekerljung L, Andersson-Svahn H, Carlsson J, Brismar H, Gedda L, Frejd FY, Ståhl S (2009) Engineering and characterization of a bispecific HER2 x EGFR-binding affibody molecule. Biotechnol Appl Biochem 54:121–131. doi:10.1042/BA20090096

    Article  CAS  Google Scholar 

  41. Lindström S, Hammond M, Brismar H, Andersson-Svahn H, Ahmadian A (2009) PCR amplification and genetic analysis in a microwell cell culturing chip. Lab Chip 9:3465–3471. doi:10.1039/b912596e

    Article  Google Scholar 

  42. Deutsch M, Deutsch A, Shirihai O, Hurevich I, Afrimzon E, Shafran Y, Zurgil N (2006) A novel miniature cell retainer for correlative high-content analysis of individual untethered non-adherent cells. Lab Chip 6:995–1000. doi:10.1039/b603961h

    Article  CAS  Google Scholar 

  43. Markovitz-Bishitz Y, Tauber Y, Afrimzon E, Zurgil N, Sobolev M, Shafran Y, Deutsch A, Howitz S, Deutsch M (2010) A polymer microstructure array for the formation, culturing, and high throughput drug screening of breast cancer spheroids. Biomaterials 31:8436–8444. doi:10.1016/j.biomaterials.2010.07.050

    Article  CAS  Google Scholar 

  44. Zurgil N, Afrimzon E, Deutsch A, Namer Y, Shafran Y, Sobolev M, Tauber Y, Ravid-Hermesh O, Deutsch M (2010) Polymer live-cell array for real-time kinetic imaging of immune cells. Biomaterials 31:5022–5029. doi:10.1016/j.biomaterials.2010.02.035

    Article  CAS  Google Scholar 

  45. James CD, Moorman MW, Carson BD, Branda CS, Lantz JW, Manginell RP, Martino A, Singh AK (2009) Nuclear translocation kinetics of NF-kappaB in macrophages challenged with pathogens in a microfluidic platform. Biomed Microdevices 11:693–700. doi:10.1007/s10544-008-9281-5

    Article  Google Scholar 

  46. Springs SL, Diavolitsis VM, Goodhouse J, McLendon GL (2002) The kinetics of translocation of Smac/DIABLO from the mitochondria to the cytosol in HeLa cells. J Biol Chem 277:45715–45718. doi:10.1074/jbc.C200524200

    Article  CAS  Google Scholar 

  47. Goldstein JC, Waterhouse NJ, Juin P, Evan GI, Green DR (2000) The coordinate release of cytochrome c during apoptosis is rapid, complete and kinetically invariant. Nat Cell Biol 2:156–162. doi:10.1038/35004029

    Article  CAS  Google Scholar 

  48. Goldstein JC, Muñoz-Pinedo C, Ricci J-E, Adams SR, Kelekar A, Schuler M, Tsien RY, Green DR (2005) Cytochrome c is released in a single step during apoptosis. Cell Death Differ 12:453–462. doi:10.1038/sj.cdd.4401596

    Article  CAS  Google Scholar 

  49. Furger C, Derick S, Boutin JA, Nosjean O (2009) Image-free assessment of protein translocation in live cells. Curr Opin Pharmacol 9:650–656. doi:10.1016/j.coph.2009.07.002

    Article  CAS  Google Scholar 

  50. Chapnik E, Rivkin N, Mildner A, Beck G, Pasvolsky R, Metzl-Raz E, Birger Y, Amir G, Tirosh I, Porat Z, Israel LL, Lellouche E, Michaeli S, Lellouche JP, Izraeli S, Jung S, Hornstein E. (2014) miR-142 orchestrates a network of actin cytoskeleton regulators during megakaryopoiesis. Elife e01964. Doi: 10.7554/eLife.01964

  51. Israel LL, Buchman K, Lellouche E, Michaeli S, Lellouche, JP, Magnetic inorganic iron-based nanoparticles—generalities and use in drug delivery, PCT Patent Application PCT/IL2014/050064 (January 19, 2014)

  52. Kronfeld I, Kazimirsky G, Lorenzo PS, Garfield SH, Blumberg PM, Brodie C (2000) Phosphorylation of protein kinase Cdelta on distinct tyrosine residues regulates specific cellular functions. J Biol Chem 275:35491–35498. doi:10.1074/jbc.M005991200

    Article  CAS  Google Scholar 

  53. Schenborn ET, Goiffon V (2000) Calcium phosphate transfection of mammalian cultured cells. Methods Mol Biol 130:135–145

    CAS  Google Scholar 

  54. Kajimoto T, Shirai Y, Sakai N, Yamamoto T, Matsuzaki H, Kikkawa U, Saito N (2004) Ceramide-induced apoptosis by translocation, phosphorylation, and activation of protein kinase Cdelta in the Golgi complex. J Biol Chem 279:12668–12676. doi:10.1074/jbc.M312350200

    Article  CAS  Google Scholar 

  55. Thomas P, Smart TG (2005) HEK293 cell line: a vehicle for the expression of recombinant proteins. J Pharmacol Toxicol Methods 51:187–200. doi:10.1016/j.vascn.2004.08.014

    Article  CAS  Google Scholar 

  56. Wurm F, Bernard A (1999) Large-scale transient expression in mammalian cells for recombinant protein production. Curr Opin Biotechnol 10:156–159

    Article  CAS  Google Scholar 

  57. Zhao M, Xia L, Chen G-Q (2012) Protein kinase cδ in apoptosis: a brief overview. Arch Immunol Ther Exp (Warsz) 60:361–372. doi:10.1007/s00005-012-0188-8

    Article  CAS  Google Scholar 

  58. Goerke A, Sakai N, Gutjahr E, Schlapkohl WA, Mushinski JF, Haller H, Kolch W, Saito N, Mischak H (2002) Induction of apoptosis by protein kinase C delta is independent of its kinase activity. J Biol Chem 277:32054–32062. doi:10.1074/jbc.M203734200

    Article  CAS  Google Scholar 

  59. Blass M, Kronfeld I, Kazimirsky G, Blumberg PM, Brodie C (2002) Tyrosine phosphorylation of protein kinase Cdelta is essential for its apoptotic effect in response to etoposide. Mol Cell Biol 22:182–195

    Article  CAS  Google Scholar 

  60. Banan A, Fields JZ, Farhadi A, Talmage DA, Zhang L, Keshavarzian A (2002) Activation of delta-isoform of protein kinase C is required for oxidant-induced disruption of both the microtubule cytoskeleton and permeability barrier of intestinal epithelia. J Pharmacol Exp Ther 303:17–28. doi:10.1124/jpet.102.037218

    Article  CAS  Google Scholar 

  61. Li L, Lorenzo PS, Bogi K, Blumberg PM, Yuspa SH (1999) Protein kinase Cdelta targets mitochondria, alters mitochondrial membrane potential, and induces apoptosis in normal and neoplastic keratinocytes when overexpressed by an adenoviral vector. Mol Cell Biol 19:8547–8558

    CAS  Google Scholar 

  62. Lee Y-J, Soh J-W, Dean NM, Cho C-K, Kim TH, Lee S-J, Lee Y-S (2002) Protein kinase Cdelta overexpression enhances radiation sensitivity via extracellular regulated protein kinase 1/2 activation, abolishing the radiation-induced G(2)-M arrest. Cell Growth Differ 13:237–246

    CAS  Google Scholar 

  63. Lai J-M, Hsieh C-L, Chang Z-F (2003) Caspase activation during phorbol ester-induced apoptosis requires ROCK-dependent myosin-mediated contraction. J Cell Sci 116:3491–3501. doi:10.1242/jcs.00660

    Article  CAS  Google Scholar 

  64. Coleman ML, Sahai EA, Yeo M, Bosch M, Dewar A, Olson MF (2001) Membrane blebbing during apoptosis results from caspase-mediated activation of ROCK I. Nat Cell Biol 3:339–345. doi:10.1038/35070009

    Article  CAS  Google Scholar 

  65. Mills JC, Stone NL, Erhardt J, Pittman RN (1998) Apoptotic membrane blebbing is regulated by myosin light chain phosphorylation. J Cell Biol 140:627–636

    Article  CAS  Google Scholar 

  66. Gomel R, Xiang C, Finniss S, Lee HK, Lu W, Okhrimenko H, Brodie C (2007) The localization of protein kinase Cdelta in different subcellular sites affects its proapoptotic and antiapoptotic functions and the activation of distinct downstream signaling pathways. Mol Cancer Res 5:627–639. doi:10.1158/1541-7786.MCR-06-0255

    Article  CAS  Google Scholar 

  67. Brodie C, Blumberg PM (2003) Regulation of cell apoptosis by protein kinase c delta. Apoptosis 8:19–27

    Article  CAS  Google Scholar 

  68. Retrieval of fluorescent stained cells from PW array chip. http://www.jsc.ph.biu.ac.il/video-demonstrations. Accessed Aug 2013

Download references

Acknowledgments

This study was endowed by the Bequest of Moshe-Shimon and Judith Weisbrodt. EGFP-PKCδ vector was a generous gift from Prof. Chaya Brodie's laboratory at Bar-Ilan University.

Author information

Authors and Affiliations

  1. The Biophysical Interdisciplinary Schottenstein Center for the Research and Technology of the Cellome, Department of Physics Building 214, Bar-Ilan University, Ramat Gan, 5290002, Israel

    Orit Ravid-Hermesh, Naomi Zurgil, Yana Shafran, Maria Sobolev, Moti Galmidi, Yoav Badihi & Mordechai Deutsch

  2. Laboratory of Nanoscale Materials and Systems, Department of Chemistry, Building 211, Bar-Ilan University, Ramat Gan, 5290002, Israel

    Liron Limor Israel & Jean Paul Lellouche

  3. The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, 5290002, Israel

    Emmanuel Lellouche & Shulamit Michaeli

  4. Nanomaterials Research Center, Institute of Nanotechnology & Advanced Materials, Bar-Ilan University, Ramat Gan, 5290002, Israel

    Liron Limor Israel, Jean Paul Lellouche, Emmanuel Lellouche & Shulamit Michaeli

Authors
  1. Orit Ravid-Hermesh
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Naomi Zurgil
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yana Shafran
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Maria Sobolev
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Moti Galmidi
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yoav Badihi
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Liron Limor Israel
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jean Paul Lellouche
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Emmanuel Lellouche
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Shulamit Michaeli
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Mordechai Deutsch
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mordechai Deutsch.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(PDF 449 kb)

ESM 2

(MPG 1.43 mb)

ESM 3

(MPG 1.51 mb)

ESM 4

(MPG 838 kb)

ESM 5

(MPG 624 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ravid-Hermesh, O., Zurgil, N., Shafran, Y. et al. Real-time quantification of protein expression and translocation at individual cell resolution using imaging-dish-based live cell array. Anal Bioanal Chem 406, 7085–7101 (2014). https://doi.org/10.1007/s00216-014-8157-1

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-014-8157-1

Keywords

Search

Navigation