Poster Presentation Cristina Chiutu Borstel 2015
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This study characterized the interaction between chromatographic beads and the yeast Hansenula polymorpha using atomic force microscopy. Both rigid and elastic beads were tested on H. polymorpha cells immobilized on glass in liquid. The negative Source S bead showed the most reproducible behavior over time. Interaction forces decreased with increasing salt concentration, indicating an electrostatic component. Cell elasticity also varied across growth phases but force spectra did not show a clear trend. Measurements on individual cells yielded higher elastic modulus values than using beads, possibly due to lower pressure over a larger contact area with beads.
![Atomic Force Spectroscopy between
Hansenula polymorpha and Ion Exchange Chromatographic Beads
Cristina Chiutu, Vikas Yelemane, Marcelo Fernandez-Lahore, and J端rgen Fritz
Jacobs University Bremen
Introduction
The design of ion exchange chromatographic beads plays a significant role in the performance, fluidity and classification of the
downstream processing and chromatography. Atomic Force Microscopy probes the interaction between beads and biomass, cells or
proteins at the nanoscale and can lead to a better understanding of the structure and functionality of various beads. In this project,
different types of beads, rigid and elastic, positive and negative, are attached on triangular soft cantilevers and characterised against a
negatively charged silicon surface via force spectroscopy with a Veeco PicoForce AFM. Furthermore, the beads are analysed in contact
with monocellular layer of Hansenula polymorpha (immobilized on Roti Bond glass) in buffer (with different NaCl concentration or on
various phases of cell life cycle). Elasticity of the cellular layer can be approximated by fitting the Hertz-Stanndon model. Hansenula
polymorpha is employed at commercial-scale in biotechnology for recombinant protein production.
Hansenula polymorpha Growth Phases
7 h (Lag) 12 h (Accelerated) 16 h (Exponential)
24 h (Late Exponential) 30 h (Stationary) 48 h (Late Stationary)
Approaching Hansenula polymorpha with Negative Beads
Beads-Silicon Measurements in Different NaCl Concentrations
Elasticity of Hansenula polymorpha in Liquid (Hertz-Stanndon fit) Summary and Outlook
Hansenula polymorpha cells were successfully immobilized on Roti Bond glass for liquid imaging
and force spectroscopy. Several types of chromatographic beads were investigated and it was
found that the rigid negative Source S bead is most reproducible and reliable to perform a larger
set of experiments on cells. Different salt concentrations affect the interaction between beads
and silicon/cells as well as cells in various phase of their life cycle leads to different result.
Elasticity of cellular layer can also be determined. Future measurements will be theoretically
characterized with the XDLVO theory calculations.
[1] R.R. Vennapusa et al., Separation Science and Technology, 2010, 45, 23352344.
[2] G. Helms et al., 2014, submitted.
[3] A. Touhami et al. Langmuir 2003, 19, 4539-4543.
Attractive interaction or force of adhesion of
the positive beads is significantly reduced in
buffer with increasing salt concentration.
This large magnitude for attraction force on
extend was measured only for a limited
number of Source Q beads.
For negative beads a reduction of the
electrostatic force is observed with
increasing salt concentration, emphasizing
the elastic component of the force.
(Air Imaging) 7 h: typical size 2 袖m, height 0.5 袖m, small buds growing
12 h: similar size, cells with more small buds
16 h: size of cells increases to 3 袖m and height to 0.8 袖m, bigger buds
24 h: cells 2-3 袖m, big buds, but some cells deflated
30 h: many big buds, many young cells
48 h: many young cells and many deflated
Hansenula polymorpha in Air
Hansenula polymorpha in Liquid
Detail Bud Scar (in Air)
Size 2-3 袖m
Height 0.5-0.8 袖m
Size 2-3 袖m
Height 1-1.5 袖m
Size 0.5 袖m
Height 0.2 袖m
Cristina Chiutu, 8th North German Biophysics Meeting, Jan. 2015, Borstel
Bead
Cantilever
Cell
Layer
Chromatographic Beads Characterization Imaging of Yeast Cells
Type of ion exchanger: Strong cation
Functional group: Sulfonate R-SO3
Matrix structure: polystyrene/divinyl benzene
polymer
Spherical, rigid, 30 袖m monosized
Type of ion exchanger: Strong anion
Functional group: Quaternary ammonium
-CH2N+(CH3)3
Matrix structure: polystyrene/divinyl benzene
polymer
Spherical, rigid, 30 袖m monosized
Type of ion exchanger: Strong cation
Functional group: Sulfopropyl
OCH2CHOHCH2O-CH2CH2CH2SO3
Matrix structure: 85-90% agarose (inert) and
10-15% tungsten carbide (inert)
Spherical, elastic (soft), 100-200 亮m
Source S
Bead
Source Q
Bead
Fastline
SP
Bead
Fastline
DEAE
Bead
Type of ion exchanger: Weak anion
Functional group: Diethylaminoethyl
OCH2CH2N+(C2H5)2H
Matrix structure: 85-90% agarose (inert) and
10-15% tungsten carbide (inert)
Spherical, elastic (soft), 100-200 亮m
Both rigid and elastic beads were characterized from interaction
with a negatively-charged silicon surface in water or phosphate buffer. The negative
beads demonstrated reproducibility and stability regarding the behaviour of electrostatic repulsion
and/or elastic forces over time, while the magnitude of attraction and adhesion forces of positive
beads decreases in time. Therefore, due to changes in time, the measurements on Hansenula polymorpha focused on the
interaction with negative beads. Data shown in this section are similar to other measurements on positive/negative beads
performed in our group by G. Helms [1,2].
Imaging on Hansenula polymorpha was performed with MSNL-10 cantilevers in liquid and air. This type of yeast showed good adhesion
to the Roti Bond glass (commercially purchased), therefore full monolayer of cells could be obtained. The cells were imaged with high
resolution of details such as bud scars or membrane structure (emphasized in the deflection image). However, in liquid the structure of
cell membrane can be imaged with poor resolution.
Interaction of negative beads (both rigid and elastic) with Hansenula polymorpha cells proved to be reproducible both in water and in phosphate buffer.
More difficult is the interpretation of spectra, since a separation between elastic and electrostatic forces is required considering the elasticity of both
beads and cells. The measurements on the same spot are fully reproducible, while elasticity or electrostatics varies significantly across the sample for
measurements in different points. In different salt concentrations the electrostatic component of force is reduced with increasing salt quantity, while
elasticity of cells seems to increase. Source S measurements present similar data to the Toyo SP bead experiments performed by G. Helms [2].
This experiment aims to
understand the behavior of
cells during the growth phases.
Images of cells and force
spectra were consecutively
recorded in phosphate buffer.
While images indicate
differences in size and
appearance of cells for each
phase, force spectra show a
change in slope for each phase,
but do not present a trend
or gradient with the phase as expected. One reason for this could
be that in later phases the sample is a mixture of young, old and
dead cells. On the other hand, various spots on sample may lead to
significant differences in the slope of spectra.
-100 0 100 200 300
Separation (nm)
Force(nN)
-7-6-5-4-3
Contact
Point
MSNL-10 cantilever on single cell
in water: 0.0627 MPa
0 100 200 300
Separation (nm)
Force(nN)
-4-3-2-10
Contact
Point
Source S bead on cellular layer
in water: 0.0021 MPa
For comparison and understanding of
spectra analysis with elasticity models,
measurements were performed with
MSNL-10 cantilever on individual cells.
Young modulus values resulted from curve
fitting are comparable to the values given
by A. Touhami [3] for measurements on
Saccharomices cerevisiae cells but slightly
softer. Measurements with beads lead to
smaller values for the Young modulus
resulted from higher elasticity of cellular
layer comparing to individual cell. This
could be explained in terms of pressure
decreasing with increasing contact area.](https://image.slidesharecdn.com/97cd88c6-290b-4332-8b4f-8febab1b571b-150324061959-conversion-gate01/85/Poster-Presentation-Cristina-Chiutu-Borstel-2015-1-320.jpg)
Poster Presentation Cristina Chiutu Borstel 2015
- 1. Atomic Force Spectroscopy between Hansenula polymorpha and Ion Exchange Chromatographic Beads Cristina Chiutu, Vikas Yelemane, Marcelo Fernandez-Lahore, and J端rgen Fritz Jacobs University Bremen Introduction The design of ion exchange chromatographic beads plays a significant role in the performance, fluidity and classification of the downstream processing and chromatography. Atomic Force Microscopy probes the interaction between beads and biomass, cells or proteins at the nanoscale and can lead to a better understanding of the structure and functionality of various beads. In this project, different types of beads, rigid and elastic, positive and negative, are attached on triangular soft cantilevers and characterised against a negatively charged silicon surface via force spectroscopy with a Veeco PicoForce AFM. Furthermore, the beads are analysed in contact with monocellular layer of Hansenula polymorpha (immobilized on Roti Bond glass) in buffer (with different NaCl concentration or on various phases of cell life cycle). Elasticity of the cellular layer can be approximated by fitting the Hertz-Stanndon model. Hansenula polymorpha is employed at commercial-scale in biotechnology for recombinant protein production. Hansenula polymorpha Growth Phases 7 h (Lag) 12 h (Accelerated) 16 h (Exponential) 24 h (Late Exponential) 30 h (Stationary) 48 h (Late Stationary) Approaching Hansenula polymorpha with Negative Beads Beads-Silicon Measurements in Different NaCl Concentrations Elasticity of Hansenula polymorpha in Liquid (Hertz-Stanndon fit) Summary and Outlook Hansenula polymorpha cells were successfully immobilized on Roti Bond glass for liquid imaging and force spectroscopy. Several types of chromatographic beads were investigated and it was found that the rigid negative Source S bead is most reproducible and reliable to perform a larger set of experiments on cells. Different salt concentrations affect the interaction between beads and silicon/cells as well as cells in various phase of their life cycle leads to different result. Elasticity of cellular layer can also be determined. Future measurements will be theoretically characterized with the XDLVO theory calculations. [1] R.R. Vennapusa et al., Separation Science and Technology, 2010, 45, 23352344. [2] G. Helms et al., 2014, submitted. [3] A. Touhami et al. Langmuir 2003, 19, 4539-4543. Attractive interaction or force of adhesion of the positive beads is significantly reduced in buffer with increasing salt concentration. This large magnitude for attraction force on extend was measured only for a limited number of Source Q beads. For negative beads a reduction of the electrostatic force is observed with increasing salt concentration, emphasizing the elastic component of the force. (Air Imaging) 7 h: typical size 2 袖m, height 0.5 袖m, small buds growing 12 h: similar size, cells with more small buds 16 h: size of cells increases to 3 袖m and height to 0.8 袖m, bigger buds 24 h: cells 2-3 袖m, big buds, but some cells deflated 30 h: many big buds, many young cells 48 h: many young cells and many deflated Hansenula polymorpha in Air Hansenula polymorpha in Liquid Detail Bud Scar (in Air) Size 2-3 袖m Height 0.5-0.8 袖m Size 2-3 袖m Height 1-1.5 袖m Size 0.5 袖m Height 0.2 袖m Cristina Chiutu, 8th North German Biophysics Meeting, Jan. 2015, Borstel Bead Cantilever Cell Layer Chromatographic Beads Characterization Imaging of Yeast Cells Type of ion exchanger: Strong cation Functional group: Sulfonate R-SO3 Matrix structure: polystyrene/divinyl benzene polymer Spherical, rigid, 30 袖m monosized Type of ion exchanger: Strong anion Functional group: Quaternary ammonium -CH2N+(CH3)3 Matrix structure: polystyrene/divinyl benzene polymer Spherical, rigid, 30 袖m monosized Type of ion exchanger: Strong cation Functional group: Sulfopropyl OCH2CHOHCH2O-CH2CH2CH2SO3 Matrix structure: 85-90% agarose (inert) and 10-15% tungsten carbide (inert) Spherical, elastic (soft), 100-200 亮m Source S Bead Source Q Bead Fastline SP Bead Fastline DEAE Bead Type of ion exchanger: Weak anion Functional group: Diethylaminoethyl OCH2CH2N+(C2H5)2H Matrix structure: 85-90% agarose (inert) and 10-15% tungsten carbide (inert) Spherical, elastic (soft), 100-200 亮m Both rigid and elastic beads were characterized from interaction with a negatively-charged silicon surface in water or phosphate buffer. The negative beads demonstrated reproducibility and stability regarding the behaviour of electrostatic repulsion and/or elastic forces over time, while the magnitude of attraction and adhesion forces of positive beads decreases in time. Therefore, due to changes in time, the measurements on Hansenula polymorpha focused on the interaction with negative beads. Data shown in this section are similar to other measurements on positive/negative beads performed in our group by G. Helms [1,2]. Imaging on Hansenula polymorpha was performed with MSNL-10 cantilevers in liquid and air. This type of yeast showed good adhesion to the Roti Bond glass (commercially purchased), therefore full monolayer of cells could be obtained. The cells were imaged with high resolution of details such as bud scars or membrane structure (emphasized in the deflection image). However, in liquid the structure of cell membrane can be imaged with poor resolution. Interaction of negative beads (both rigid and elastic) with Hansenula polymorpha cells proved to be reproducible both in water and in phosphate buffer. More difficult is the interpretation of spectra, since a separation between elastic and electrostatic forces is required considering the elasticity of both beads and cells. The measurements on the same spot are fully reproducible, while elasticity or electrostatics varies significantly across the sample for measurements in different points. In different salt concentrations the electrostatic component of force is reduced with increasing salt quantity, while elasticity of cells seems to increase. Source S measurements present similar data to the Toyo SP bead experiments performed by G. Helms [2]. This experiment aims to understand the behavior of cells during the growth phases. Images of cells and force spectra were consecutively recorded in phosphate buffer. While images indicate differences in size and appearance of cells for each phase, force spectra show a change in slope for each phase, but do not present a trend or gradient with the phase as expected. One reason for this could be that in later phases the sample is a mixture of young, old and dead cells. On the other hand, various spots on sample may lead to significant differences in the slope of spectra. -100 0 100 200 300 Separation (nm) Force(nN) -7-6-5-4-3 Contact Point MSNL-10 cantilever on single cell in water: 0.0627 MPa 0 100 200 300 Separation (nm) Force(nN) -4-3-2-10 Contact Point Source S bead on cellular layer in water: 0.0021 MPa For comparison and understanding of spectra analysis with elasticity models, measurements were performed with MSNL-10 cantilever on individual cells. Young modulus values resulted from curve fitting are comparable to the values given by A. Touhami [3] for measurements on Saccharomices cerevisiae cells but slightly softer. Measurements with beads lead to smaller values for the Young modulus resulted from higher elasticity of cellular layer comparing to individual cell. This could be explained in terms of pressure decreasing with increasing contact area.