Characterizing the Interaction of Chromatographic Beads with Yeast by Force Spectroscopy and XDLVO

Cristina Chiutu, Vikas Yelemane, Alexander Neveshkin, Marcelo Fernandez-Lahore, and Jürgen FritzJacobs University Bremen

IntroductionThis 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

SummaryHansenula 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 µmHeight 0.5-1.5 µm

Size 0.5 µmHeight 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.

• Type of ion exchanger: Strong cation• Functional group: Sulfonate R-SO3

−

• Matrix structure: polystyrene/divinyl benzene polymer

• Spherical, rigid, 30 µm monosized

• Type of ion exchanger: Strong anion• Functional group:

Quaternary ammonium -CH2N+(CH3)3

• Matrix structure: polystyrene/divinyl benzene polymer

• Spherical, rigid, 30 µm monosized

Source SBead

Source QBead

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 ComponentsTo 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 mannanon APTES