1) The researchers studied the interactions between chromatographic beads and yeast cells at the nanoscale using force spectroscopy and XDLVO theory. 2) They found that the attraction or repulsion between positively and negatively charged beads and yeast cells or silicon surfaces decreased with increasing salt concentration in the buffer solution. 3) Preliminary results replacing the yeast cell layer with individual cell wall components like mannan showed attraction between mannan and negatively charged beads, but combining multiple components will better mimic the yeast cell surface.

1. Characterizing the Interaction of
Chromatographic Beads with Yeast by Force Spectroscopy and XDLVO
Cristina Chiutu, Vikas Yelemane, Alexander Neveshkin, Marcelo Fernandez-Lahore, and Jürgen Fritz
Jacobs University Bremen
Introduction
This project aims to quantify the interactions between biomass and chromatographic beads occuring in different chromatographic
methods. Characterizing chromatographic beads at the nanoscale as well as a detailed analysis of their interaction with biomass may
lead to an improved performance of chromatographic methods in biotechnology. Here we directly measure the forces between single
chromatographic beads and a cellular layer of yeast. We probe the interaction of commercial Source S (negatively charged) and Source Q
(positively charged) beads with a layer of Hansenula polymorpha in phosphate buffer at different salt concentration. The results from
force spectroscopy are compared against XDLVO calculations obtained from contact angle and zeta potential measurements. To simplify
the measurements in the long term, we aim to replace the cellular layer by a solid surface which is functionalized with the major
components of the yeast cell wall (mannan, glucan, and mannoproteins).
7 h (Lag Phase) 12 h (Accelerated) 24 h (Late Exponential)
Bead-Hansenula polymorpha Interaction in Different NaCl Concentrations
Bead-Silicon Interaction in Different NaCl Concentrations
Summary
Hansenula polymorpha cells were successfully immobilized on Roti Bond glass for imaging in liquids and for force spectroscopy. Several types of chromatographic beads were investigated, and it was found that the rigid Source beads were
most reproducible and reliable to perform a larger set of force measurements on cells. Interactions of chromatographic beads and yeast have also been studied using XDLVO theory. We calculated interaction energies and forces by XDLVO for
Source Q & S beads with Hansenula polymorpha cells at various salt concentrations. Surface energies were calculated from contact angles (Lifshitz-van der Waals and acid-base component) of cell and adsorbents, and their surface charge
was determined by zeta potential measurements (electrostatic component). From XDLVO calculations we observed that as the ionic strength of the solution increases, the interactions become weaker. XDLVO results corroborate the results
from AFM measurements and both methods show a good qualitative agreement.
[1] R.R. Vennapusa et al., Separation Science and Technology, 2010, 45, 2335–2344.
Attractive interaction or
force of adhesion of
positively charged beads to
negatively charged silicon is
significantly reduced in
buffer with increasing salt
concentration.
Preparation:
To get reproducible results
beads had to be
equilibrated and cleaned in
50 mM PB and afterwards
thoroughly washed in
water.
Detail Bud Scars
Size 2-3 µm
Height
0.5-1.5 µm
Size 0.5 µm
Height 0.2 µm
Cristina Chiutu, Bremen Life Sciences Meeting, May 2015, Bremen
Bead
Cantilever
Cell
Layer
Chromatographic Beads Characterization Imaging of Yeast Hansenula polymorpha Cells
Chromatographic beads were firstly characterized by their interaction
with a negatively-charged silicon surface in water or phosphate buffer.
The beads showed good reproducibility and stability regarding
electrostatic repulsion or attraction.
The SEM image shows a bead glued to an AFM cantilever. The AFM
image shows the bead surface with an average roughness of ~100 nm.
AFM measurements were done with a Veeco Multimode / PicoForce
AFM in air (imaging) and liquid (force spectroscopy and imaging).
For AFM imaging Hansenula polymorpha were immobilized on a glass surface and investigated in air and liquid. This type of yeast
showed good adhesion to Roti Bond cover glass (Carl Roth GmbH) so that a monolayer of cells on glass could be obtained. When imaging
in air the AFM could resolve details such as bud scars, membrane structure, or changes due to cellular growth phases. Imaging in liquid
revealed less cellular details.
Interaction of negative Source S beads with Hansenula polymorpha cells proved to be reproducible both in water and in phosphate buffer.
Measurements on the same location were fully reproducible, while force curves varied significantly at different points on the sample due to different
electrostatic and elastic contribution of the cellular layer. A detailed separation of elastic and electrostatic contributions to the interaction forces is still
difficult. Attractive interactions of positive Source Q beads are more difficult to investigate due to strong adhesion forces.
The observed reduction of electrostatic componenst with increasing salt concentrations is qualitatively supported by XDLVO calculations. However, the
range of electrostatic decay observed in AFM is much larger than that predicted by theory.
Source S
Bead
Source Q
Bead
Source S XDLVO
XDLVO XDLVO
The change of electrostatic
repulsion between beads
and silicon in dependence
of buffer and ionic
strength is most clearly
observed between
measurements in pure
water and high salt
concentrations.
Source QSource S
Interaction between Beads and Yeast Cell Wall Components
To simplify the interaction measurements
between beads and yeast cells, we aim to
immobilize the major cell wall
components of yeast (the sugars mannan
and glucan, and mannoproteins) on a
solid surface to mimic the surface of
yeast.
To produce a mannan terminated surface
we silanized a silicon surface with
aminosilane (APTES) and then incubated
it with a high concentration of mannan.
Thickness of silane and sugar layers were
checked by ellipsometry. First AFM measurenments were done with Source S beads and
immobilized layers of mannan.
Preliminary results show
first a repulsion but then an
adhesion force between
Source S beads and a
mannan layer.
Comparison to our yeast cell
measurements above
indicate that different cell
wall components have to be
combined on a surface to
mimic the yeast cell wall
layer.
Exp.Exp.
Exp. Exp.
Source Q XDLVO
on mannan
on APTES