Sample Scanner Laser Position Sensitive Photodiode Tip Cantilever AFM working in IFJ atomic force microscopy FNFN FLFL

 scanner linearization  spring constant  A–B signal  geometry:  = 10º  tip – shape Hoh et al. force spectroscopy

scanner linearization scanner photodiode laser

without protein (conA) nominal value = 7 kHz, k = 0.01 N/m measured value = 5.8 kHz, k = N/m resonant frequency of a thermally excited cantilever with protein (conA) 0.3 mg/ml = 5.8 kHz, k = N/m 1 mg/ml = 5.4 kHz, k = N/m Sader et al. !

k = 0.03 N/m RT F min  11 pN

probe Standard TGT01 Si 3 N 4 + APTES (4%) + glutaraldehyde (GL; 2.5%) Si 3 N 4 + APTES (4%) + GL (2.5%) + conA (0.3 g/ml) R = 54 +/- 7 nm R = 275 +/- 10 nm

 probe size  protein concentration  immobilization procedure Moy et al. Grandbois et al. number of molecules on probe Single molecular pair

bond typeenergy [kJ/mol] bond length [nm] force [nN] covalent250 (for S–S) noncovalentionic Van der Waals hydrogen rough estimation of the bond strength + hydrophobic forces

Kim et al. Distribution of vitronectin receptors on a living MC3T3-E1 cell (murine osteoblastic cell)

Grandbois et al. AFM tip functionalized with Helix pomatia N-acetylgalactosamine in membrane of group A of RBC mixed red blood cells

No of events Force [nN]

Tees et al.

Lee et al. E b  [J] k off [s -1 ]x b [Å] AGD – α IIb β 3 RGD – α IIb β

No of events Force [nN] 5 s 0.3 s retraction velocity 3.5 μm/s ConA-CaY

How to check what it is measured ? not functionalized AFM probe measurements of known ligand-receptor pair HCV 29 cells silicon nitride tip ConA–PC-3ConA–ASAConA–CaY Force [pN] 116    39

CaY – Con A + 1 mg/ml Con A non-specific interaction interaction between ligand – receptor pair blocking of the binding sites CaY– Con A free amount of ligand in solution all or certain number of binding sites can be blocked

different types of interaction characteristic for cancerous cells HCV 29 non–malignant transitional epithelial cells of ureter T24 transitional cell cancer of urine bladder AFM, contact mode

SNA PHA-L ConA lectins sialic acid N-acetylglucosamine mannose, glucose carbohydrates

binding force Cell line Lectin Binding force [pN] HCV29ConA 43.3   3.4 PHA-L 59.9   7.1 SNA   5.5 T24ConA   18.1 PHA-L   8.2 SNA 76.2   10.9

verification 50 µg/ml ConA

conclusions AFM allows detecting molecular interaction on a surface of living cell The spatial arrangement of functional carbohydrate groups on cell surface was attributed to the density of all types of the carbohydrate structures (mannose, N-acetylglucosamine, sialic acids). The maximum range of force distribution (presented in histograms up to 1.2 nN), the size of the adhesion spot (i.e. one single point on the distribution map ~ 0.95 μm 2 ), the number of bonds (2–3 for cancerous cells) suggested that ligands present on a surface of T24 cells formed groups composed of several single carbohydrate chains involved in adhesion process in the lectin recognition.

Institute of Medical Biochemistry Medical College Jagiellonian University Piotr Laidler Joanna Dulińska Maryla Łabędź The Henryk Niewodniczański Institute of Nuclear Physics Polish Academy of Sciences Zbigniew Stachura Małgorzata Lekka Janusz Lekki Jan Styczeń PhD students Joanna Gryboś Kateryna Lebed Grażyna Pyka Atomic Force Microscop y

Histogram Autocorrelation function bin size large number of data force between single pair: CaY-ConA F = 960 +/- 110 pN

more complex interactions F  const single interaction F  const m =  m 2  = m · F + F 0 F 0 – non-specific force F · F 0 f 1 (F,  F 2, F 0 )·  f 2 (F,  F 2, F 0 )