a,b
a,b a
b
Understanding the underlying, fundamental processes that govern the interaction of biological systems with surfaces is key to the development of biocompatible medical devices which lead to successful implantable devices. This in turn translates to improved healthcare, minimally invasive surgery, healthier and prolonged lives. Studies show that the performance of biomaterials depends on how proteins adsorb on the surface which mediates the interaction of living cells with surfaces [1].
There are different experimental methods for measuring the protein adsorption on surfaces [2], but the majority of techniques do not provide structural information about the adsorbed protein. Recently we described the used of fluorescence lifetime imaging microscopy (FLIM) in the measurement of Bovine
Serum Albumin (BSA) adsorbed onto different types of materials [3]. In the present work we report preliminary results from the adsorption of fluorescent labelled BSA (Fig. 1). BSA is covalently linked to fluorescein molecules (BSA-FITC), and also double labelled with tetramethylrhodamine (BSA-FITC-
TMR). Both proteins are allowed to adsorb onto solid hydrophilic and hydrophobic substrates (Fig. 2).
The measurements were made using an ALBA dual channel FLIM/FCS system from ISS (Fig. 3) [4].
FITC
FRET
TMR
Fig. 1: Serum Albumin Protein.
The BSA structure features:
• 67 kDa globular protein
• About 580 amino-acids (59 Lysine amino acids)
• 67 % alpha-helix without beta-sheets.
• Three homologous domains (I, II and III) divided into two sub-domains (A and B).
• 17 disulphide bridges and 9 loops.
Substrates:
O H O H
O H
S i
O H O H O H O H O H O H O H O H O H O H O H
Hydrophilic
S i S i S i
Hydrophobic
O H
S i
Fig. 2: Water contact angles.
PMT Ch1
PMT Ch2
Excitation beam Laser
Fig. 3 – FLIM system
• Olympus BX51 microscope.
• Water Immersion 60× (NA 1.2) objective.,
• 405 nm laser diode excitation.
• XYZ Piezo stage (5 nm step resolution).
• Microscope filters: BP filter 385-425nm (ex.), dichroic mirror 425nm, 405 nm stopline filter
+ LP 495 nm filter (em) ).
• Emission Filters: (BP filter 510-560 nm
(GREEN), dichroic mirror 570nm, BP filter
575-650 nm (RED) ).
• Image resolution of 0.78 pixel/
µ m.
• Pinhole size of 141 µm.
• Multi-frequency measurements used 30 frequencies (1 to 300 MHz).
• References: HPTS (5.4 ns), RhB (1.74 ns) and eosin Y (1.07 ns).
Fig. 6 shows the fluorescence spectra of labelled proteins overlaid on the bandpass spectra of the two filter sets used. Channel 1 detects the red emission from the TMR while Channel 2 detects green emission from
FITC (we ignore the overlap between the different emissions ). XZ Fluorescence lifetime imaging sections of a surface in contact with BSA-FITC solution shows a variation in the fluorescence lifetime (Fig 7). Similar images are recorded with the doubly labelled BSA-FITC-TMR, FITC and TMR are a common FRET pair (donor and acceptor in Foster Resonance Energy Transfer). An intense signal is observed on the hydrophobic side with short lifetime (blue pixels). This is in agreement with the average lifetime found from a multifrequency FLIM biexponential analysis of the surfaces, XY sections (Fig 8). On hydrophilic surfaces, the average lifetime is closest in value to the average lifetime of the protein in bulk solution. On hydrophobic surfaces there is a general reduction in average lifetime when proteins are adsorbed. This reduction is more dramatic for a thin protein film when the bulk solution is removed. Two factors which can be influence the lifetime changes are refractive index (RI) and protein fluorescence quenching. The RI effect on the lifetime of the labelled proteins was studied by measuring the lifetime in water-glycerol mixtures (1.33-1.44). The RI effect is smaller (11% in mixtures) than that observed on the surfaces, indicating that the lifetimes changes are due to other factors.
Fluorescence quenching by Tryptophan (Trp) residues can also cause decreases in lifetime. The efficiency of the process is based on the Trp - FITC distance, which depends on the structural conformation (REF).
Measuring the quenching contribution is currently underway.
For the adsorption of doubly labelled BSA onto hydrophobic surfaces, we observe a sharp increase in red fluorescence at the interface compared to green fluorescence when the intensity is probed at different z-axis positions (inset of Fig. 6), and this is not observed for hydrophilic substrates. Using the average lifetime obtained for BSA-FITC and BSA-FITC-TMR, the energy transfer efficiency and the average distance between
FITC and TMR can be calculated (Fig. 9). Furthermore, the fraction contribution of each channel to the total fluorescence emission detected can be easily obtained (Fig. 9).
The ET efficiency is high when the double labelled BSA is adsorbed on hydrophobic surfaces, where we also observe an increase in red emission and decrease of green emission at the hydrophobic interface. For BSA-
FITC-TMR adsorbed on hydrophilic surfaces, the ET efficiency and red/green emission are similar to the bulk solution. This indicates that BSA adsorbs strongly onto hydrophobic surfaces, and in the process changing from the native conformation present in solution. The modulation signal measured from BSA films on hydrophilic surfaces is too low for an accurate lifetime determination. Nevertheless, the intensity ratio between red and green emission of BSA-FITC-TMR films on hydrophilic surfaces is not significantly different from the bulk solution. We may therefore conclude that there is a significantly larger degree of structural change in BSA adsorbed onto on hydrophobic surfaces compared hydrophilic surfaces.
Fig. 4: Fixed (single use) chamber.
Fig. 5: Demountable (multiple use) chamber.
1
0.8
0.6
0.4
0.2
substrate + PS
Hydrophilic substrates were prepared as follows: a) 30 min sonication in alkaline detergent RBS 35, b) 3hr soak in piranha solution (3:7) v/v H
2
O
2
:H c) Sonication for 1 h in deionized water (DIW),
2
SO
4
, d) Dry overnight in air at 80ºC.
Hydrophilic surfaces were converted to hydrophobic substrates with the additional steps: e) Soaking dimethyldichlorosilane (2%) / trichloroethylene soln., f) Rinsing with trichloroethylene, methanol, and finally DIW.
A standard protein labelling protocol was used [5].
Protein solution (PS): 2 micromolar (approx.) BSA-FITC, and
BSA-FITC-TMR in PBS solution was added to a small volume limited by the imaging spacer on the surface material (fig. 4).
The system was equilibrated (~24 hrs. @ r.t.) before any measurement. In the preparation of protein films, a demountable chamber was used (fig. 5). DIW was added to avoid protein solution evaporation. PS was left in contact with the substrate for 24 hours at RT, and washed with PBS solution.
BSA-FITC-TRM
BSA-TMR)
BSA-FITC
+Filters
3750
3000
2250
1500
750
0
20
Ch1 ( Red )
Ch2 ( Green )
40 60 z-axis (micron)
80 100
0
480 505 530 555 580 605 wavelength (nm)
630 655 680
Fig. 6: Fluorescence spectra of labelled BSA. Inset the z-axis intensity profile of BSA-FITC-TMR on hydrophobic surface. * PMT spectral sensitive not included.
Intensity Lifetime
Fig. 7: Confocal Fluorescence intensity images (lhs) and FLIM (rhs). 100 MHz modulation for FLIM.
3.5
3
2.5
2
1.5
1
0.5
0
3.5
3
2.5
2
1.5
1
0.5
0
Bulk Bulk+lic Bulk+bic Lic
Bulk Bulk+lic Bulk+bic Lic
Bic
Bic
Fig. 8: Average fluorescence lifetimes of labelled proteins.
BSA-FITC-TRM
Blue channel
(Ch2)
BSA-FITC
BSA-FITC-TMR
Red channel
(Ch1)
BSA-TMR
BSA-FITC-TMR
1
0.8
0.6
0.4
0.2
0
100
80
60
40
20
0
Bulk Bulk+lic Bulk+bic Lic
Bulk Bulk+lic Bulk+bic Lic
Bic
Bic
Red
Green
R (Ro)
ET efficiency
Fig. 9: FRET in BSA-FITC-TMR adsorbed onto solid surfaces.
Based on lifetime and intensity ratio results, hydrophobic surfaces with large contact angle induce a larger degree of structural change in BSA, than that observed for hydrophilic surfaces with low contact angles.
Frequency Domain Fluorescence lifetime imaging provides sufficient contrast, to expose probe lifetime differences from the different protein micro-environments on surfaces.
1. “Serum Albumin” T.Peters Jr. Adv. Protein Chem.
37, 161-245 (1985). 2.“On the Adsorption of Proteins on Solid Surfaces, a Common but Very Complicated Phenonmenon.” K.Nakanishi, T.Sakiyama, and K. Imamura J. Biosc. Bioeng.
91 (3) 233-
244 (2001). 3. “Time-resolved fluorescence studies on bovine serum albumin denaturation process.” D.M. Togashi and A.G.
Ryder, Journal of Fluorescence, 16(2),153-160,(2006).
SFI (02/IN.1M231)