Latent analysis of unmodified biomolecules and their complexes in solution with attomole detection sensitivity


The study of biomolecular interactions is central to an understanding of function, malfunction and therapeutic modulation of biological systems, yet often involves a compromise between sensitivity and accuracy. Many conventional analytical steps and the procedures required to facilitate sensitive detection, such as the incorporation of chemical labels, are prone to perturb the complexes under observation. Here we present a ‘latent’ analysis approach that uses chemical and microfluidic tools to reveal, through highly sensitive detection of a labelled system, the behaviour of the physiologically relevant unlabelled system. We implement this strategy in a native microfluidic diffusional sizing platform, allowing us to achieve detection sensitivity at the attomole level, determine the hydrodynamic radii of biomolecules that vary by over three orders of magnitude in molecular weight, and study heterogeneous mixtures. We illustrate these key advantages by characterizing a complex of an antibody domain in the solution phase and under physiologically relevant conditions.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Latent labelling enables the development of native microfluidic analysis systems.
Figure 2: Precise control of reaction time enables quantitative, fluorogenic protein labelling before detection.
Figure 3: Sizing proteins, heterogeneous mixtures and protein complexes.


  1. 1

    Balch, W. E., Morimoto, R. I., Dillin, A. & Kelly, J. W. Adapting proteostasis for disease intervention. Science 319, 916–919 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. 2

    Suh, E. H. et al. Stilbene vinyl sulfonamides as fluorogenic sensors of and traceless covalent kinetic stabilizers of transthyretin that prevent amyloidogenesis. J. Am. Chem. Soc. 135, 17869–17880 (2013).

    CAS  Article  Google Scholar 

  3. 3

    Aguzzi, A. & Calella, A. M. Prions: protein aggregation and infectious diseases. Phys. Rev. 89, 1105–1152 (2009).

    CAS  Google Scholar 

  4. 4

    Campioni, S. et al. The presence of an air–water interface affects formation and elongation of α-synuclein fibrils. J. Am. Chem. Soc. 136, 2866–2875 (2014).

    CAS  Article  Google Scholar 

  5. 5

    Adamcik, J. et al. Understanding amyloid aggregation by statistical analysis of atomic force microscopy images. Nature Nanotech. 5, 423–428 (2010).

    CAS  Article  Google Scholar 

  6. 6

    Chiti, F. & Dobson, C. M. Protein misfolding, functional amyloid, and human disease. Annu. Rev. Biochem. 75, 333–366 (2006).

    CAS  Article  Google Scholar 

  7. 7

    Arkin, M. R. & Whitty, A. The road less traveled: modulating signal transduction enzymes by inhibiting their protein–protein interactions. Curr. Opin. Chem. Biol. 13, 284–290 (2009).

    CAS  Article  Google Scholar 

  8. 8

    Wells, J. A. & McClendon, C. L. Reaching for high-hanging fruit in drug discovery at protein–protein interfaces. Nature 450, 1001–1009 (2007).

    CAS  Article  Google Scholar 

  9. 9

    Berggård, T., Linse, S. & James, P. Methods for the detection and analysis of protein–protein interactions. Proteomics 7, 2833–2842 (2007).

    Article  Google Scholar 

  10. 10

    Uetz, P. et al. A comprehensive analysis of protein–protein interactions in Saccharomyces cerevisiae. Nature 403, 623–627 (2000).

    CAS  Article  Google Scholar 

  11. 11

    Ho, Y. et al. Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry. Nature 415, 180–183 (2002).

    CAS  Article  Google Scholar 

  12. 12

    Yu, X., Xu, D. & Cheng, Q. Label-free detection methods for protein microarrays. Proteomics 6, 5493–5503 (2006).

    CAS  Article  Google Scholar 

  13. 13

    Zuiderweg, E. R. P. Mapping protein–protein interactions in solution by NMR spectroscopy. Biochemistry 41, 1–7 (2001).

    Article  Google Scholar 

  14. 14

    Hanlon, A. D., Larkin, M. I. & Reddick, R. M. Free-solution, label-free protein–protein interactions characterized by dynamic light scattering. Biophys. J. 98, 297–304 (2010).

    CAS  Article  Google Scholar 

  15. 15

    Lee, T. T. & Yeung, E. S. High-sensitivity laser-induced fluorescence detection of native proteins in capillary electrophoresis. J. Chromatogr. A 595, 319–325 (1992).

    CAS  Article  Google Scholar 

  16. 16

    Bornhop, D. J. et al. Free-solution, label-free molecular interactions studied by back-scattering interferometry. Science 317, 1732–1736 (2007).

    CAS  Article  Google Scholar 

  17. 17

    Jungbauer, L. M., Yu, C., Laxton, K. J. & LaDu, M. J. Preparation of fluorescently-labeled amyloid-beta peptide assemblies: the effect of fluorophore conjugation on structure and function. J. Mol. Recogn. 22, 403–413 (2009).

    CAS  Article  Google Scholar 

  18. 18

    Herling, T. W. et al. Integration and characterization of solid wall electrodes in microfluidic devices fabricated in a single photolithography step. Appl. Phys. Lett. 102, 184102–4 (2013).

    Article  Google Scholar 

  19. 19

    Bruus, H. Theoretical Microfluidics (Oxford Master Series in Physics, Oxford Univ. Press, 2008).

    Google Scholar 

  20. 20

    Brody, J. P. & Yager, P. Diffusion-based extraction in a microfabricated device. Sens. Actuat. A 58, 13–18 (1997).

    CAS  Article  Google Scholar 

  21. 21

    Weigl, B. H. & Yager, P. Microfluidic diffusion-based separation and detection. Science 283, 346–347 (1999).

    Article  Google Scholar 

  22. 22

    Kamholz, A. E., Weigl, B. H., Finlayson, B. A. & Yager, P. Quantitative analysis of molecular interaction in a microfluidic channel: the T-sensor. Anal. Chem. 71, 5340–5347 (1999).

    CAS  Article  Google Scholar 

  23. 23

    Hatch, A. et al. A rapid diffusion immunoassay in a T-sensor. Nature Biotechol. 19, 461–465 (2001).

    CAS  Article  Google Scholar 

  24. 24

    Kamholz, A. E., Schilling, E. A. & Yager, P. Optical measurement of transverse molecular diffusion in a microchannel. Biophys. J. 80, 1967–1972 (2001).

    CAS  Article  Google Scholar 

  25. 25

    Knowles, T., Devlin, G., Dobson, C. & Welland, M. in Methods in Molecular Biology Vol. 752 (eds Hill, A. F., Barnham, K. J., Bottomley, S. P. & Cappai, R.) 137–145 (Humana, 2011).

    Google Scholar 

  26. 26

    Gao, D., Liu, H., Jiang, Y. & Lin, J.-M. Recent advances in microfluidics combined with mass spectrometry: technologies and applications. Lab Chip 13, 3309–3322 (2013).

    CAS  Article  Google Scholar 

  27. 27

    Roth, M. Fluorescence reaction for amino acids. Anal. Chem. 43, 880–882 (1971).

    CAS  Article  Google Scholar 

  28. 28

    Benson, J. R. & Hare, P. o-Phthalaldehyde fluorogenic detection of primary amines in the picomole range. comparison with fluorescamine and ninhydrin. Proc. Natl Acad. Sci. USA 72, 619–622 (1975).

    CAS  Article  Google Scholar 

  29. 29

    Simons, S. S. & Johnson, D. F. The structure of the fluorescent adduct formed in the reaction of o-phthalaldehyde and thiols with amines. J, Am. Chem. Soc. 98, 7098–7099 (1976).

    CAS  Article  Google Scholar 

  30. 30

    Sternson, L. A., Stobaugh, J. F. & Repta, A. J. Rational design and evaluation of improved o-phthalaldehyde-like fluorogenic reagents. Anal. Biochem. 144, 233–246 (1985).

    CAS  Article  Google Scholar 

  31. 31

    Jacobson, S. C., Koutny, L. B., Hergenroeder, R., Moore, A. W. & Ramsey, J. M. Microchip capillary electrophoresis with an integrated postcolumn reactor. Anal. Chem. 66, 3472–3476 (1994).

    CAS  Article  Google Scholar 

  32. 32

    Otzen, D. Protein–surfactant interactions: a tale of many states. Biochim. Biophys. Acta 1814, 562–591 (2011).

    CAS  Article  Google Scholar 

  33. 33

    Stobaugh, J., Repta, A., Sternson, L. & Garren, K. Factors affecting the stability of fluorescent isoindoles derived from reaction of o-phthalaldehyde and hydroxyalkylthiols with primary amines. Anal. Biochem. 135, 495–504 (1983).

    CAS  Article  Google Scholar 

  34. 34

    Lindroth, P. & Mopper, K. High performance liquid chromatographic determination of subpicomole amounts of amino acids by precolumn fluorescence derivatization with o-phthaldialdehyde. Anal. Chem. 51, 1667–1674 (1979).

    CAS  Article  Google Scholar 

  35. 35

    Svedas, V. J., Galaev, I. J., Borisov, I. L. & Berezin, I. V. The interaction of amino acids with o-phthalaldehyde: a kinetic study and spectrophometric assay of the reaction product. Anal. Biochem. 101, 188–195 (1980).

    CAS  Article  Google Scholar 

  36. 36

    Horrocks, M. H. et al. Single-molecule measurements of transient biomolecular complexes through microfluidic dilution. Anal. Chem. 85, 6855–6859 (2013).

    CAS  Article  Google Scholar 

  37. 37

    Waters, J. C. Accuracy and precision in quantitative fluorescence microscopy. J. Cell Biol. 185, 1135–1148 (2009).

    CAS  Article  Google Scholar 

  38. 38

    De Genst, E. J. et al. Structure and properties of a complex of alpha-synuclein and a single-domain camelid antibody. J. Mol. Biol. 402, 326–343 (2010).

    CAS  Article  Google Scholar 

  39. 39

    Jha, A. K., Colubri, A., Freed, K. F. & Sosnick, T. R. Statistical coil model of the unfolded state: resolving the reconciliation problem. Proc. Natl Acad. Sci. USA, 102, 13099–13104 (2005).

    CAS  Article  Google Scholar 

  40. 40

    Ulmer, T. S., Bax, A., Cole, N. B. & Nussbaum, R. L. Structure and dynamics of micelle-bound human α-synuclein. J. Biol. Chem. 280, 9595–9603 (2005).

    CAS  Article  Google Scholar 

  41. 41

    Dedmon, M. M., Lindorff-Larsen, K., Christodoulou, J., Vendruscolo, M. & Dobson, C. M. Mapping long-range interactions in α-Synuclein using spin-label NMR and ensemble molecular dynamics simulations. J. Am. Chem. Soc. 127, 476–477 (2005).

    CAS  Article  Google Scholar 

  42. 42

    Morar, A. S., Olteanu, A., Young, G. B. & Pielak, G. J. Solvent-induced collapse of α-synuclein and acid-denatured cytochrome c. Prot. Sci. 10, 2195–2199 (2001).

    CAS  Article  Google Scholar 

  43. 43

    Weinreb, P. H., Zhen, W., Poon, A. W., Conway, K. A. & Lansbury, P. T. NACP, a protein implicated in Alzheimer's disease and learning, is natively unfolded. Biochemistry 35, 13709–13715 (1996).

    CAS  Article  Google Scholar 

  44. 44

    Lord, R. S., Gubensek, F. & Rupley, J. A. Insulin self-association. Spectrum changes and thermodynamics. Biochemistry 12, 4385–4392 (1973).

    CAS  Article  Google Scholar 

  45. 45

    Bocian, W. et al. Structure of human insulin monomer in water/acetonitrile solution. J. Biomol. NMR 40, 55–64 (2008).

    CAS  Article  Google Scholar 

  46. 46

    Lin, M. & Larive, C. Detection of insulin aggregates with pulsed-field gradient nuclear magnetic resonance spectroscopy. Anal. Biochem. 229, 214–220 (1995).

    CAS  Article  Google Scholar 

  47. 47

    Muyldermans, S. Nanobodies: natural single-domain antibodies. Annu. Rev. Biochem. 82, 775–797 (2013).

    CAS  Article  Google Scholar 

  48. 48

    De Genst, E., Messer, A. & Dobson, C. M. Antibodies and protein misfolding: from structural research tools to therapeutic strategies. Biochim. Biophys. Acta 1844, 1907–1919 (2014).

    CAS  Article  Google Scholar 

  49. 49

    De Genst, E. & Dobson, C. M. in Methods in Molecular Biology Vol. 911 (eds Saerens, D. & Mulydermans, S.), 533–558 (2012).

    CAS  Article  Google Scholar 

  50. 50

    Guilliams, T. et al. Nanobodies raised against monomeric α-synuclein distinguish between fibrils at different maturation stages. J. Mol. Biol. 425, 2397–2411 (2013).

    CAS  Article  Google Scholar 

Download references


The authors acknowledge the European Research Council, Biotechnology and Biological Sciences Research Council, Wellcome Trust, Newman Foundation, Winston Churchill Foundation and Elan Pharmaceuticals for financial support. E.D.G. was supported by the Medical Research Council (G1002272). The authors thank J. Steyaert at the Free University of Brussels for sharing the NbSyn1a clone.

Author information




T.P.J.K. and C.M.D. supervised the research. E.V.Y., L.R., M.V., C.M.D. and T.P.J.K. conceived and designed the experiments. E.V.Y. performed the experiments. E.V.Y. and T.M. analysed the data. E.J.D.G., P.A. and S.L. contributed materials and/or analysis tools. E.V.Y., C.M.D. and T.P.J.K. wrote the paper, and all authors commented on the paper.

Corresponding authors

Correspondence to Christopher M. Dobson or Tuomas P. J. Knowles.

Ethics declarations

Competing interests

Part of the work described here has been the subject of a patent application filed by Cambridge Enterprise Ltd, a fully owned subsidiary of the University of Cambridge (now licensed to Fluidic Analytics, of which C.M.D. is a scientific advisor and T.P.J.K. is a board member).

Supplementary information

Supplementary information

Supplementary information (PDF 821 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Yates, E., Müller, T., Rajah, L. et al. Latent analysis of unmodified biomolecules and their complexes in solution with attomole detection sensitivity. Nature Chem 7, 802–809 (2015).

Download citation

Further reading


Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing