Combination of single vesicles and gold particles for label-free optical biosensing

Transcription

1 Combination of single vesicles and gold particles for label-free optical biosensing Li Wei Master Thesis: Dept. of Applied Physics, KTH, the Royal institute of technology Supervisor: Prof. Janos Vörös and Brigitte Städler Place: Laboratory of Biosensors and Bioelectronics, ETH Zurich, Switzerland 1

2 0. Abstract Table of content: 0. Abstract Introduction Materials and Methods Materials..10 A. Buffer...10 B. Polymers...10 C. Vesicles. 12 D. Gold colloids. 12 E. DNA..13 F. Streptavidin Quartz Crystal Microbalance with Dissipation (QCM-D) Fluorescence Recovery after Photobleaching(FRAP) Nanodrop spectrophotometer Results and Discussion Mobility measurements using FRAP Diffusion of lipid on bilayer tagged with DNA, vesicles, gold colloids Diffusion of tethered vesicle on lipid bilayers Mobility of lipids in POPC bilayers tagged with gold colloids Multilayer Formation Combining vesicles to immobilized vesicles via DNA Combining gold colloid to immobilized vesicles via DNA Analysis of reaction and UV spectrum of biomaterial using Nanodrop Gold colloids Reaction of gold colloids tethered to vesicles via DNA Conclusions Acknowledgements Outlook References 49 2

3 0. Abstract 0. Abstract A new method for optical biosensing is induced by combining vesicles and gold colloids in this project. Vesicles arrays as targets for drugs have the potential to host membrane proteins which are very important kind of proteins. Gold colloids which are attached to immobilized vesicles via DNA are sensitive to changes of the refractive index (n) of the medium in the range of Therefore gold colloids tethered to vesicles via DNA might serve as label-free optical biosensing of reactions between membrane proteins and other biomolecules. In this project, a specific binding could be achieved between polymer PPS-PEG coated gold colloids and vesicles through the DNA-DNA hybridization. Coverage of coating and absorptions of gold colloids tagged with DNA on different surfaces were investigated by Quartz Crystal Microbalance with Dissipation (QCM-D) experiments. Another aspect we were investigating in this project is mobility of the lipid upon bilayer incorporated with DNA, vesicles or gold colloids. FRAP measurements were performed in order to determine the diffusion coefficient of lipids in the membrane. It was found that tethered vesicles anchored through the maleimide-thiol linkage diffuse at similar rates as ones achored through cholesterol interaction. Further more, tagging of gold colloids to lipids bilayers slowed down the mobility of lipids. Finally, UV spectroscopy of the reaction between gold colloids and vesicles membrane is measured by NanoDrop spectrophotometer. But this instrument is not sensitive enough to study the wavelength shift of 1 nm which is caused by reaction between gold colloids and proteins or vesicles. Such a modification has the potential to be useful for biosensing, while the gold colloid is used as a label and vesicles serves as carriers for membrane proteins. Keywords ---Optical biosensing ---Poly (propylene sulfide-block-ethylene glycol ) ( PPS-PEG ) ---Gold colloids ---Vesicles 3

4 1. Introduction 1. Introduction Due to the importance of membrane proteins for discovering drug, vesicle arrays might become a very useful technology, because vesicles provide the nature environment for membrane proteins. Vesicles tagged with complementary cholesterol-terminated DNA are specifically coupled to the immobilized complexs of biotin-terminated DNA and streptavidin. [1]. The structure of vesicle arrays is showed in Figure 1.1: a Nb2o5 coated substrate is modified with PPL-g-PEGbiotein. BiotinDNA-Streptavidin complexes bind to the immobilized biotin on the surface. Complexes of cholesterol-tagged complementary DNAs and POPC vesicles are hybridized to the immobilized biotindna. Figure 1.1 schematics of vesicle arrays Vesicles arrays have few advantages than the latter work, a tethered bilayer lipid membrane (tblm) on electrolessly deposited gold film. [2]. The previous patterning approach is Molecular Assembly Patterning by Lift-off (MAPL) technique [3], which can be stored under ambient conditions without loss of performance. Furthermore, a 4

5 1. Introduction functional heterogeneous vesicle array is primarily used for the parallel screening for large amount of different target membrane molecules within a small area [4] and also solves the problem of reducing mobility of embedded proteins [5]. Mover over, it makes the incorporation of transmembrane proteins possible [6]. In this multistep surface modification process, coating surface with the polymer biotinylated poly( L-lysine)-g-poly(ethylene glycol)(ppl-g -PEGbiotin) is a well-know strategy for rendering surface protein resistant. [7] It adsorbs spontaneously from aqueous solution onto negative charged surfaces via electrostatic interactions. Furthermore, the PEG-chains in the polymer are functionalized with biotin to react selectively with a target molecule, such as streptavidin in this project. [8] The optical properties of noble metal colloidal particles such as gold or silver have been studied, because these particles are quite useful as functional materials for optical devices and biological sensors. Another reason for the interest of study is that gold colloids are used to replace the fluorohores due to their stability and ease of detection. [9] The absorbance spectrum of gold colloids exhibited a red shift in the peak wavelength ( λ max ) in proportion to the refractive index (n) of the medium in the range of and also implied a linear relationship between the n value and the absorbance at 550 nm. [10] Figure 1.2 shows the wavelength shift of absorption spectrum of gold colloids when proteins get into the sensitive area of gold colloids. At the same time, we can observe from Figure 1.3 that a change of the absorption amount at the peak (550 nm) depended in time of immersion. The absorption increases and then keeps stable at a certain amount after proteins get into the sensitive area of gold colloids. 5

6 1. Introduction Figure 1.2 UV spectra of gold colloids before proteins get into the sensitive area of gold colloids: dashed line. UV spectra of gold colloids after proteins get into the sensitive area of gold colloids: solid line.[5] Figure 1.3 Absorbance curve as a function of time for gold colloids at the peak (550 nm) when various proteins get into sensitive area: (a) Fru,(b) mixture of OVA and Fru, (c) mixture of OVA and Man, (d) mixture of OVA and GcNAc, (e) OVA. [4] For this property of gold colloids, the attachment of Gold colloids onto the membrane of vesicles through DNA linkage will be investigated for optical biosensing. Figure 1.4 shows the structure of this system with gold colloids attached onto the membrane of vesicles. Figure 1.4 Gold colloids are attached to the membrane of immobilized vesicles via DNA linkage for optical biosensing. 6

7 1. Introduction Three important aspects were studied. The specific binding between gold colloids and vesicles is investigated by Quartz Crystal Microbalance with Dissipation (QCM-D) experiments [11]. The mobility of lipids and vesicles was characterized using Fluorescence Recovery After Photobleaching (FRAP). Mobility of lipids was studied on the lipid bilayer which is formed by vesicles fusion, while cholesterol DNA and gold colloids were tagged. Also we use two different anchors, cholesterol-tagged DNA and maleimide-thiol reaction to achieve this combination by tethering liposomes to supported lipid bilayers. In J.J.Benkoski s work, cholesterol tagged DNAs serve as binding sites in proteins and vesicles-resistant lipid bilayer [12], and it was proved the diffusivity of vesicles was neither sensitive to the sized of the vesicles nor to the length of DNA [13][14]. In this part of work, 50 nm PC vesicles (intrinsic curvature nm 1 ) spontaneously absorb to solid surfaces, rupturing and fusing to form a supported lipid bilayer (SLB). [15] In H. Schonherr s paper, this process was studied by Atomic Force Microscopy, Figure 1.5. Bilayer formation via vesicle fusion was formed in four steps (1) vesicle adsorption, (2) deformation and fusion, (3) rupture of the fused vesicles, (4) SLB formation. [16] Figure 1.5 Bilayer formation via vesicle fusion was formed in four steps (1) vesicle adsorption, (2) deformation and fusion, (3) rupture of the fused vesicles, (4) SLB formation. [15] 7

8 1. Introduction In this work, we compared the diffusivity of vesicles with these two different anchors which are performed in two different process A/B. Process A: Maleimide vesicles (5 mg/ml) were mixed with tdna1 (0.1 nmol/ml) for an hour for reaction. Then Maleimide vesicles tagged with DNA (concentration: 5mg/ml) in buffer solution were pipetted into a glass slide in liquid cell. After vesicle fusion, a lipid bilayer with DNA tagged was formed on the glass. After rinsing with 2000 µl buffer H2, the upper solution of liquid cell was exchanged. With same concentration POPC vesicles mixed with complementary cholesterol DNA were injected into the liquid cell. POPC vesicles were tethered on the supported lipid bilayer via DNA hybridization. (Figure 1.6) Figure 1.6 Schematic of the tethered vesicle assembly process. Vesicles tagged with tdna1 exposed to a cleaned glass substrate to form a supported bilayer displaying mobile DNA on the surface. Subsequent incubation with fresh vesicle tagged with complementary cholesterol DNA cal results in the assembly of mobile tethered vesicles by hybridization and tethering. Process B: An opposite binding of vesicles and bilayers was also studied. First POPC vesicles were used to form bilayers. 0.1 nmol/ml cholesterol DNA cdnaal solution was injected into cell after rinsing. Subsequently maleimide vesicles tagged with tdnal were tethered. 8

9 1. Introduction Figure 1.7 Schematic of the tethered vesicle assembly process. Vesicles are exposed to a cleaned glass substrate to form a supported bilayer. After injected cholesterol DNA cdnaal, it is displaying mobile DNA on the surface. Subsequent interaction with fresh maleimide vesicles tagged with complementary tdna1 results in the assembly of mobile tethered vesicles by DNA-DNA hybridization. The UV spectrum of gold colloids was studied. By measuring absorption spectrum of gold colloids, we want to observe a wavelength shift at pick of the absorption maxima, where the plasmons are exited in the sensitive area because of the proximity of vesicles and other gold colloids. Figure 1.8 describes the absorption spectrum of 20 nm of gold colloids. Figures 1.8 Normalized adsorption spectra of 20 nm gold colloids 9

10 2. Materials and Methods 2. Materials and Methods 2.1 Materials A. buffer All the experiments were carried out in a 160 mm buffer solution, consisting of 10 mm 4-(2-hydroxyethyl)piperazine-1-ethane-sulfonic acid and 150 mm NaCl at ph=7.4. This is further in this work referred as Hepes 2 (H2) (MicroSelect, Fluka Chemie GmbH, Switzerland). Both buffers were made with ultrapure water, (Milli-Q gradient A 10 system, resistance 18 MΩcm, TOC < 4 ppb, Millipore Corporation, USA), stored at 4 C and filtered with a 0.2-μm pore filter (Sigma Aldrich Chemie GmbH, Germany) prior to use. B. Polymers Biotinylated Poly(L-lysine)-g-poly(ethylene glycol) (PLL-g-PEG) In this project, PLL-g-PEG/PEGbiotin with a poly(l-lysine) (PLL) backbone of 20 kda and poly(ethylene glycol) (PEG) side-chains of 2 kda with a grafting ratio of 3.5 was used to modify the biosensor surface for minimizing nonspecific binding. 50% of PEG side chains in PLL-g-PEG/PEGbiotin were biotinylated, and provided binding sites for Streptavidin as the basis for a specific biosensor. [8] It adsorbs spontaneously from aqueous solutions onto negatively charged surfaces via electrostatic interactions. It forms monolayers with densely packed PEG/PEGbiotin chains. (figure2.1) PLL-g-PEG/PEGbiotin coated surfaces were found to reduce nonspecific protein adsorption from serum and other proteins once the inter chain spacing between PEG/PEGbiotin groups became smaller than the radius of gyration, which is most efficient with a grafting ratio of 3.5. [7] 10

11 2. Materials and Methods Figure 2.1 (a) Molecular structure of the PLL-g-PEGcopolymer (b) Idealized scheme of the interfacial structure of a monolayer of PLL-g-PEG/PEGbiotin adsorbed on a metal oxide substrate, such as NbO. 2 5 Ref[7] Poly(propylene sulfide-block-ethylene glycol) (PPS-PEG) PPS-PEG is a diblock copolymer with a 2 kda PEG chain and a 2.2 kda PPS backbone, which has a similar function and structure with PPL-PEG. (Figure 2.2) It absorbs on gold surface through several stable thiol-sh linkages and forms proteinresistant adlayers. [17] In our experiments, it was used to coat gold colloids. Figure 2.2 structure of PPS-PEG diblock. 11

12 2. Materials and Methods C. vesicles 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine lipids (POPC; Avanti Polar Lipids, USA) dissolved in chloroform were stored at -20 C. For fluorescent vesicles, 1% (w/w) of 2-(12-(7-nitrobenz-2-oxa-1, 3-diyzol-4-yl) amino) dodecanoyl-1-hexadecanoyl-snglycero-3-phosphocholine (NBD-HPC; Molecular Probes, USA) was added to the lipid solution. Unilaminar lipid vesicles (stored at 4 C under N2 atmosphere) were prepared by evaporation of the solvent under nitrogen (1 h) followed by hydration in buffer (5 mg/ml) and extrusion through 50 nm filters (31 times). The pore size of the filter controlled the vesicle size. 50, 100, 200 and 400 nm filters produced vesicles with mean diameters of 50, 100, 200 and 400 nm, respectively. Maleimide vesicles were made in the same way, and 2wt% 1,2-Dipalmitoyl-sn-Glycero- 3-Phosphoethanolamine-N-[4-(P-maleimidophenyl) butyramide] (mal-pe) were added into the solution before drying under nitrogen. All vesicles in H2 are store in fridge at 4 C for less than 2 months. D. Gold colloids Gold colloids were purchased from Brit. Biocell, UK. The sizes were ranging from 5, 20, 50 to 100 nm. The concentrations of different sizes of gold colloids are showed below. The concentration of the gold colloid in water was wt%. This are 5 x gold particles for 5 nm, 7 x gold particles per ml for the 20 nm, 4.5 x gold particles per ml for the 50 nm and 5.6 x 10 9 gold particles per ml for the 100 nm gold colloid solution showed in table 2.1. Table 2.1. Concentrations of gold colloids in stock. Gold colloid particle size Size distribution Particles per ml 5nm <15% 5 x nm <15% 7 x nm <20% 4.5 x nm <20% 5.6 x

13 2. Materials and Methods DNA-tagging of gold colloids 2.5 ml different sizes of wt% gold colloids solution were mixed with 2.5 ml water and 10 μl tdnaal or long DNA tdna2 (5 nmol/ 20μl) for 5 nm and 20 nm gold colloids and 20 μl tdnaa1 or tdna2 DNA for 50 nm respectively. After 24 hours reaction, 30 mg NaCl and 40 mg phosphate buffer were added to solution. The solution was separated into 5 eppendorf tubes. Then the tubes were centrifuged (10 min, 14.5 rpm) and 200 ml H2 was add to the tubes after upper part of solution was taken out. PPS-PEG coating of DNA-tagged gold colloids The concentration of 1 mg/ml of PPS-PEG in methanol was used in our experiments to coat gold colloids tagged with tdnaa1 and long DNA tdna2. After mixing gold colloid tagged with DNA in H2 and PPS-PEG solution overnight, the solution was centrifuged (10 min, 14.5 rpm). Then methanol solution was taken out and gold colloids were diluted in H2 with a concentration of wt%. The scheme of coated gold colloids is showed in Figure 2.3. Figure 2.3 Coated gold colloids with DNA tdnaa1 and long DNA thb2. E. DNA DNA-strands were used as selective binding. Specific linkages were obtained by complementary DNA hybridization. DNA-strands modified with different heads and sequences used in this work are purchased from MedProbe, Norway. Biotinylated DNAstrands were used to bind to streptavidin, which is often used as an intermediate block in microarrays. Complementary DNA with cholesterol head were used to immobilize vesicles to streptavidin-modified surface, while a thiol group functionalized DNA can be 13

14 2. Materials and Methods used to attach gold colloids on the surface. DNA strands with thiol groups can also be used to tag maleimide vesicles. Reaction for this tagging is usually an hour. It takes 10 min for reaction between streptavidin and biotinylated DNA to get one or two DNA on one Streptavidin and also have some other free binding sites for binding to biotinylated PPL-PEG modified surface. Table 2.1.E Overview of DNA strands used in this project. Name DNA strands sequence Modification bdnaa1 5 -CCC-CCA-TGG-ATT-CGT-AA-3 5 modified with biotin bdnab1 5 -AAT-GCT-AAG-GTA-CCC-CC-3 5 modified with biotin bdnab2 5 -TAT-ACG-AGA-CTT-CCC-CC-3 3 modified with biotin cdnaa1 5 -CCC-CCT-AGT-TGT-GAC-GTA-CAT- 5 modified with TAC-GAT-TCC-AT-3 cholesterol cdnab1 5 -CCC-CCT-AGT-TGT-GAC-GTA-CAA- 5 modified with TAT-GCT-CTG-AA-3 cholesterol CDNA2 5 -TAT-ACG-AGA-CTT-CCC-CC-3 3 modified with cholesterol tdna2 Long 5 -AAG-TCT-CGT-ATA-CCT-GCA-AAT- 3 modified with thiol DNA ATC-TAA-TAC-CTT-AGC-ATT-CCC- CC-3 tdna1 5 -CC-CCC-TTA-CGA-TTC-CAT-3 5 modified with thiol F. Streptavidin Streptavidin proteins show 222 symmetry, with pairs of biotin-binding sites on opposite sides of the protein, making them favorable for use as intermediate building blocks. Streptavidin used in my project were obtained from Molecular Probes. The concentration of 20 μg/ml was used. 14

15 2. Materials and Methods 2.2 Quartz Crystal Microbalance with Dissipation (QCM-D) The QCM-D instrument (Q-Sense AB, Sweden) measures changes in the frequency (f) and dissipation factor (D) of an oscillating quartz crystal upon adsorption of a viscoelastic layer. [11] Immediately after cleaning, the crystal was mounted into the liquid-exchange cell of the instrument. The solution and the cell were temperature-stabilized at 24.9 ± 0.03 C. By using QCM-D, multistep surface modification was monitored and measured. First, PLL-g-PEGbiotin was adsorbed on a Nb 2 O 5 coated surface. Next, the biotindna- Streptavidin complexes were immobilized followed by hybridization with DNA-tagged vesicles. Cholesterol cdna was introduced, strengthening the interaction between the vesicles and surface. Finally, maleimide functionalized vesicles or gold colloids tagged with complementary thiolated DNA was attached to immobilized vesicles or gold colloids. The schematic of QCM-D instrument is described in Figure 2.4. Figure 2.4 the schematic of QCM instrument. The resonance frequency (f) of the crystal depends on the total oscillating mass, including water coupled to the oscillation. When a thin film is attached to the sensor crystal, the frequency decreases. If the film is thin and rigid the decrease in frequency is proportional to the mass of the film. In this way, the QCM operates as a very sensitive balance. 15

16 2. Materials and Methods 2.3 Fluorescence Recovery after Photobleaching (FRAP) Confocal Laser Scanning Microscope (CLSM) (Confocal ZeissLSM 510, Germany) was used to image lipid bilayers and also tethered vesicles onto bilayers. Bilayers were formed on cleaned glass slides which were fixed in liquid cells. To clean glass slides, the slides were immersed in 2 % SDS solution for 30 min and then rinsed with pure water and dried in nitrogen and finally cleaned in UV cleaner for 30mins. A ring was placed and fixed upon the glass slide, which contains approximately 800ul buffer in this ring. The walls of liquid cell confined buffer to a volume measuring 17.5 mm in diameter and 4.0 mm in height, as shown in Figure 2.5. The fluorescence lipids on the surface were observed using a 63 oil immersion objective. Figure 2.5 A ring placed atop stage defined the sample volume. Approximately 800ul of buffer was contained by the ring, and the fluorescent lipids on the surface were observed with a 63x oil immersion objective. In this projects, fluorescent vesicles with 1%(w/w) of 2-(12-(7-nitrobenz-2-oxa-1,3- diyzol-4-yl)amino)dodecanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBD-HPC; Molecular Probes, USA) was used to form bilayers. Supported lipid bilayers were formed upon glass slides. A liquid cell fixed the glass slide inside, and the cell contains the buffer to a volume approximately 800 μl mg/ml suspension of 50 nm POPC vesicles was added to the liquid cell. After waiting 30 min for bilayer formation, the suspension was exchanged with pure buffer. 16

17 2. Materials and Methods All diffusivity reported in this study were characterized by fluorescence recovery after photobleaching (FRAP). This widely used technique is performed by first bleaching a small spot on a fluorescent surface. We use laser with wavelength 488 nm to image and bleach lipid bilayers labeled by NBD-HPC. Unbleached molecules adjacent to the spot diffuse inward, leading to a recovery of the fluorescence within the spot. (Figure2.6). Figure 2.6 Example of a supported lipid bilayer which is recovery during a FRAP experiment. The characteristic time for fluorescence recovery ( τ ) varies as the square of the radius of the photobleached spot (w). [13] D τ 2 D = w /4D 17

18 2. Materials and Methods Using the software image J, a recovery curve was obtained by recording changes of intensity in the photobleached spot as a function of time. (Figure 2.7) Figure 2.7 Recovery curve of a lipid bilayer: Intensity of the photobleached spot as a function of time In our study, we usually used the slope of first three points in the recovery curve to estimate the diffusion coefficient of the lipids or vesicles. 18

19 2. Materials and Methods 2.4 NanoDrop Spectrophotometer The NanoDrop ND-1000 (figure 2.4.1) is a full spectrum ( nm) spectrophotometer that measures 1μl samples absorption spectrum from 220 nm to 750 nm with high accuracy and reproducibility. This eliminates It utilizes a patented sample retention technology that employs surface tension alone to hold the sample in place. Figure 2.8 (a) With the sample apparatus open, a droplet of sample is pipetted onto the measurement pedestal. (b) ND-100 Spectrophotometer for measuring absorbance. As a sample, 2 μl 20nm gold colloids solution in water was pipetted onto the end of a fiber optic cable (receiving fiber) after water was measured as a blank measurement. A second fiber optic cable (the source fiber) is then brought to contact with the liquid sample causing the liquid to bridge the gap between the fiber optic ends. Then a full a absorption spectrum of 20 nm gold colloids was measured. (Figure 2.9) 19

20 2. Materials and Methods Figure 2.9 UV absorption spectrum of 20 nm gold colloids in water In this project, gold colloids with different sizes and different concentrations were measured. 4 kinds of gold colloids with the sizes of 5 nm and 20 nm and 50 nm and 100 nm are measured. For original concentration of gold colloids in stock, there are 5 x gold particles for 5 nm, 7 x gold particles per ml for the 20 nm, 4.5 x gold particles per ml for the 50 nm and 5.6 x 10 9 gold particles per ml for the 100 nm gold colloid solution. We diluted all sizes of gold colloids to 2, 4, 8, 10, 12, 14, 16, 18, 20 times of the original volume of solution. Then we have gold colloids solution with lower and lower concentration. Then we measured absorption spectrums of gold colloids with different concentrations. 20

21 2. Materials and Methods Also some reactions between gold colloids and proteins were studied using NanoDrop. For an example, the reaction between gold colloids and vesicles via DNA-DNA hybridization was measure in such way: First, blank measurement was done by measuring 1μl buffer solution. Second, DNA-tagged gold colloids solution in buffer and vesicles solution in buffer was measured. Third, these two solutions were mixed together, and reaction time was record from the moment of mixing. 1μl of mixture was taken out after 0 min, 1 min, 2 min, 5 min, 10 min and 20 min and pipetted onto the end of a fiber optic cable (receiving fiber) to measure. 21

22 3. Results and Discussion 3. Results and Discussion 3.1 Mobility measurements using FRAP In this project, gold colloids are tethered to vesicle membranes via DNA-DNA hybridization. Once tethered, they should diffuse parallel to vesicles membranes to interact with membrane proteins or antibodies. The mobility of lipids in vesicle membranes in various conditions were investigated. It was studied on fluid supported bilayers which were formed by vesicle fusion onto a cleaned glass slide (The process is described in introduction) Diffusion of lipids on bilayer tagged with DNA Supported phospholipid bilayers are particularly attractive for use in biochemical assays because of their resistance to nonspecific binding and their ability to host biologically active molecules, such as antibodies, water-soluble proteins, and DNA. In this project, different concentrations of cholesterol tagged DNA were added into the liquid cell in which lipid bilayers were formed on glass slides. FRAP experiments were performed on lipid bilayers. To investigate the influence of DNA tagging to lipid bilayers to the mobility of lipids, we measured on DNA tagged lipid bilayers which were performed through three different way of tagging process. One is that different concentrations of DNA were added to several lipid bilayers respectively. The other two is that different concentrations of DNA were added to the same bilayer and one is rinsed after adding cholesterol DNA while the other is not rinsed. 1. A lipid bilayer was formed on glass slides from vesicle fusion. Diffusion coefficient of lipids was recorded by FRAP experiments. After that, the solution of cholesterol DNA (cdnab1) in H2 with a concentration of 0.1nmol/ml was added into liquid cell. After rinsing with 2000 μl H2, the upper solution in the cell was exchange with H2 after 10 min reaction. Then, the mobility of lipids when cholesterol DNA strands embed in bilayers spontaneous was recorded by FRAP experiments. Figure 3.1 below shows the recovery curve of POPC bilayer with 0.1 nmol/ml CDNAb1. 22

23 3. Results and Discussion Figure 3.1 The recovery curve of intensity of bleached spot as a function of time. Black line, on POPC bilayer. Red line, on POPC bilayer with cdnab1 In the same way, 0.2 nmol/ml cdnab1, 0.8 nmol/ml and 0.16 nmol/ml cdnab1 solution were added to a lipid bilayer respectively and the mobility of lipids in bilayers was measured before and after adding cholesterol DNA. Slopes in all curves were calculated from the first four points of the recovery curve. Table 3.1 shows the slopes of the recovery curve on POPC lipid bilayers with different concentrations of cholesterol DNA. By comparing slopes of lipid bilayers and lipid bilayers with different concentrations of cdnab1, increases caused by tagging cdnab1 described in percent are showed in the table. 23

24 3. Results and Discussion Table 3.1 cdnab1 Slopes of the recovery curve on lipid bilayers and on lipid bilayers with Concentration of Slopes on lipid bilayers Slopes in lipid Increases cdnab1 bilayers with cdna percent 0.1 nmol/ml % 0.2 nmol/ml % 0.4 nmol/ml % 0.8 nmol/ml % 0.16 nmol/ml % by From this table, we can observe tagging of DNA will not influence much to the mobility of lipids. With most of concentrations of cdnab1, the mobility of lipids in lipid bilayers were not changed by tagging cdnab1. The slope of the recovery curve of lipids increased by 27.6% in the lipid bilayer with 0.8 nmol/ml cdnaa1, but it is in the range of error of average slope of the recovery curve of a lipid bilayer. 2. Similarly a bilayer was formed and then 1 nmol/ml cdnab1 was added and FRAP experiments were done after rising. More cdnab1 was added to the same bilayer to increase the concentration of cdnab1. Then another FRAP experiments was done after adjusting to a new concentration of cdnab1. After every FRAP experiment, the upper solution of the liquid cell was taken out and rinsed with 2000 µl H2. Therefore the concentration of cdnab1 is changed by adding more cdnab1 to the same bilayer. In this way, we can compare the diffusion coefficient of lipids more efficiently and reasonably based on the same lipid bilayer. The recovery curves of all the FRAP experiments were plotted and compared in one graph, which is showed in Figure

25 3. Results and Discussion Figure 3.2 FRAP experiments were done when different concentration of cholesterol DNA strands embedded in the bilayer. The recovery curve of bleached spot were recorded with 0.1 nmol/ml, 0.2 nmol/ml, 0.4 nmol/ml, 0.8 nmol/ml, 1.6 nmol/ml, 2.4 nmol/ml cholesterol DNA. The slopes of the recovery curves are plotted and showed in the Table

26 3. Results and Discussion Table 3.2 Slopes of the recovery curve of lipid biliayers with different concentration of cdnab1. Concentration of cdnab1 slope 0.1 nmol/ml nmol/ml nmol/ml nmol/ml nmol/ml nmol/ml From the table, the slopes of the recovery curves on lipid bilayers with different concentrations of cdnab1 are almost the same. DNA strands are immobilized onto the lipid bilayer through the cholesterol head which is embedded spontaneously because of hydrophobic property. [13] But this connect is not stable and cholesterol DNA will dig into and out of the lipid bilayer according to the concentration change of DNA in the upper solution. Therefore, the process of rinsing after adding cholesterol DNA is not necessary and might not be proper. And concentration of cholesterol DNA embedded in the lipid bilay might be very low and keeps the same because of rinsing too much. In this case, the slope which estimates the diffusion coefficient of lipids remains the same on the lipid bilalyer with different concentrations of cdnab1. 2. The third way is proposed and carried out in similar process as in part 2. The different is described below. After adding cholesterol DNA to change the concentration, we gave up rinsing and pipetted solution out and then pipetted in the liquid cell to mixing the solution of cholesterol DNA on the formed bilayer. Results are below: 26

27 3. Results and Discussion Figure 3.3 After adding cholesterol DNA to change the concentration, we gave up rinsing and pipetted solution out and then pipetted in to mixing the solution of cholesterol DNA on the formed bilayer. In this way, FRAP experiments on bilayers with different concentration of cholesterol DNA were done. Table 3.3 Mobility of lipids in Bilayer tagged with cholesterol DNA (After rinsing ) Concentration of cdnab1 Slopes of the recovery curve of lipids in lipid bilayers tagged with cholesterol DNA 0.1 nmol/ml nmol/ml nmol/ml nmol/ml nmol/ml nmol/ml

28 3. Results and Discussion From the table 3.3, we found that the slopes of the recovery curve of lipids in lipid bilayer with different concentrations are similar, after we using a different process which is describe in part 3. And because of the hydrophobic property of cholesterol head of DNA, this way is more proper and more precise. However, the influence of concentration of cholesterol DNA is still not so obvious. Therefore, we believe that the concentration of tagged cholesterol DNA does not influence the mobility of lipids in the lipid bilayer Mobility of tethered vesicles on supported lipid bilayers In this part of work, we mainly study the mobility of tethered vesicles with different anchor groups for tagging to lipid bilayers. Two kinds of anchor groups were studied. One is maleimide vesicles with an anchor group of cholesterol head, and the other is POPC vesicles with an anchor group of maleimide-thiol connection. This work was done by comparing mobility of tethered vesicles which were performed by two processes A/B, which are described in introduction. After these two process, FRAP experiments were done on labeled vesicles while lipid bilayers with out label is not visible. The recovery curves of this two kinds of vesicles are plotted and compared in Figure 3.4 below. The red line is maleimide vesicles on the POPC bilayers with an anchor group of cholesterol head of DNA. The black line is POPC vesicles on the maleimide bilayer with an anchor group of maleimide-thiol connect. The recovery curve looks similar and the time taken to recover is almost the same too. And by more precise analysis, slopes of these two curves were compared and showed in Table

29 3. Results and Discussion Figure 3.4 In a selected spot, labeled vesicles were bleached using an iteration of 20 and intensity of this spot was recorded as a function of time. Red line, FRAP recovery curve of maleimide vesicles on the lipid bilayer. Black line, POPC vesicles on the maleimide bilayer. Table 3.4 Slopes of the recovery curves of two different kinds of vesicles. slope surface Iteration =20 Iteration =50 POPC vesicles Maleimide bilayer Maleimide vesicles POPC bilayer In this table, a conclusion that Maleimide vesicles on the POPC bilayer diffuse at a similar rate as POPC vesicles on Maleimide bilayer. The amount of slopes are different, but in the error rang of average slope of this kinds experiments. Thiol-maleimide lipids connection is more stable than cholesterol-lipid connection, which is hydrophobic binding and the cholesterol head of DNA goes in and out of membrane according to 29

30 3. Results and Discussion changes of DNA concentration in the solution. However, this difference in anchor group does not influence the mobility of tagged vesicles. Moreover, FRAP experiments were carried out on vesicles tethered on bilayers after 1 hours, 6 hours, and overnight to investigate the influence of reaction time to the mobility of vesicles. Then we have the follow result (Figure 3.5) Figure 3.5 These three groups of pictures about recovery of the photobleached spot were taken after different reaction time between POPC vesicles and the maleimide bilayer. (a). more than 15 hours (b). 6 hours (c). 1 hours In Figure 3.5, the recovery of the photobleached spot is very slow in the top group of images (a), while in the second and third group of images (b) (c) the recovery is much faster and the photobleached spot became almost like the first image taken before photobleaching in the last one. From this figure, we can get a conclusion that the diffusion coefficient of vesicles become smaller and smaller when we increase the reaction time. That means, more and more vesicles were absorbed on the surface. Therefore the vesicles move in a more and more crowd environment and diffuse slower and slower. 30

31 3. Results and Discussion Mobility of lipids in POPC bilayers tagged with gold colloids After a lipid bilayer was formed, FRAP experiment was done before and after adding cholesterol DNA. Then gold colloids were tethered to lipid bilayers, which were labeled by fluorescence, via DNA linkage. The FRAP experiments were done after different reaction time. Figure 3.6 and Figure 3.7 were recovery curves using different size of gold colloids. Figure 3.6 a bilayer was formed by vesicles fusion. Then FRAP experiments were done on this bilayer. After adding cdna (1 nmol/ml), another FRAP was done. Gold colloids were tether via DNA linkage and FRAP experiments after tagging were done after 2 hours reaction and overnight reaction. 31

32 3. Results and Discussion Figure 3.7 A bilayer was formed by vesicles fusion. Then FRAP experiments were done on this bilayer. After adding cdna (1 nmol/ml), another FRAP was done. Gold colloids were tethered via DNA linkage and FRAP experiments after tagging were done after 10mins, 1 hour, 3 hours reaction. Table 3.5 slopes of the recovery curves of lipids in lipids bilayers tagged with different sizes of gold colloids (5 nm and 20 nm). 5nm POPC bilayer POPC bilayer embedded with cdna slope (decreased : 5% ) 20nm POPC bilayer POPC bilayer embedded with cdna slope (increased : 5.8% ) POPC bilayer tagged with 5nm gold colloids, 1 hour (decreased : 7.8%) POPC bilayer tagged with 20nm gold colloids, 2 hour (decreased : 29.9%) 32

33 3. Results and Discussion From the table 3.5, we found small size of gold colloids influence less on diffusion coefficient of lipids in the lipid bilayers. To 20nm gold colloids, tagging of them will seriously slow down the mobility of lipids. From the Einstein relation, where k B k BT D = 6πηa is Boltzmann s constant, T is the temperature (298K), η is the viscosity of -3 2 water ( Ns/m ),[18] and a is the vesicle radius. The diffusion coefficient of gold colloids is in inverse proportion to the size of gold colloids. Therefore the gold colloids with a size of 20 nm diffuse slower than gold colloids with a size of 5 nm. Especially, when more and more gold colloids are tethered onto the lipid bilayer after a long reaction time, the 20 nm gold colloids will move in a very crowd environment compared to 5 nm gold colloids. And the diffusion of gold colloids will influence the diffusion of lipids in the lipid bilayer and cause a obvious decrease of the mobility of lipids. 33

34 3. Results and Discussion 3.2 Multilayer formation Combining vesicles to immobilized vesicles via DNA In this section, the absorptions of maleimide vesicles tagged with tdna1 to immobilized vesicles of different diameters were measured. The process of immobilizing vesicles to crystal surface is monitored in QCM experiments. A Nb2O5 -coated substrate is modified with PLL-g-PEG/PEG biotin. A frequency change of 40 ± 2 Hz and a dissipation change of 25 ± 4[ ]were measured. BiotinDNA-steptavidin complexes bind to the immobilized biotin on the PLL-g-PEG/PEG biotin modified surface. A frequency change of 25 ± 5 Hz and a 6 dissipation change of 5 ± 4[ 10 ]were measured. Complexes of cholesterol-tagged complementary DNA and POPC vesicles are hybridized to the immobilized biotindna. A frequency change of 130 ± 10 Hz and a dissipation change of 140 ± 20[ 10 6 ]were measured for 50 nm vesicles. The frequency change and dissipation change were slight bigger for a larger size of vesicles. Then maleimide vesicles were mixed with tdna1 for one hour for tagging. Adding one more step, maleimide vesicles tagged with tdna1 were inject into flow cell of QCM. The final change of frequency and dissipation were recorded and plotted as a function of the size of immobilized vesicles in Figure 3.9. The curves of frequency and dissipation change are show in Figure 3.8 below

35 3. Results and Discussion Figure 3.8 Changes in dissipation and normalized frequency (third overtone) of QCD-D measurements about absorptions of maleimide vesicles upon immobilized vesicles in H2 By using different size of immobilized vesicles, the absorptions of maleimide vesicles are very different. Figure 3.8 shows absorption of maleimide vesicles on 50 nm, 100 nm, 200 nm, 400 nm vesicles. 35

36 3. Results and Discussion Figures 3.9 In the flow cell of QCM-D, first, PPL-g-PEGbiotin was adsorbed on a SiO2 coated crystal. Next, the biotindna-streptavidin complexes were immobilized followed by hybridization with DNA-tagged vesicles. Cholesterol cdna were introduced, embedded in vesicles. Changes in the frequency (f) and dissipation factor (D) of an oscillation quartz crystal upon adsorption of viscoelastic layer in the last step( maleimide vesicles) were measured and recorded while 50 nm, 100 nm,200 nm, and 400 nm vesicles were immobilized on the surface respectively. From Figure 3.9, with icreasing vesicle diameter, the frequency change measured upon the addition of the second kind of vesicles was obviously increasing. However, the dissipation change did not show the same trend independent of the vesicles diameter. The reason of this result might be related to the intrinsic curvature of the vesicles. Vesicles with a large size have a small intrinsic curvature which makes the DNA-DNA hybridization easier. Besides, A large vesicle might be able to attach maleimide vesicles on the sides of it, not only on the top. Control experiments, in which complementary DNA strands embedded in immobilized vesicles were missing, were done and no absorption of maleimide vesicles was found. That means the bindings between vesicles were specific. 36

37 3. Results and Discussion Combining gold colloid to immobilized vesicles via DNA In this part, the attachment of gold colloids to immobilized vesicles is characterized by QCM-D experiments. Four kinds of gold colloids were used in this part of work. (a) gold colloids, (b) gold colloids tagged with tdna1, (c) gold colloids tagged with tdna1 coated with PPS-PEG, (d) gold colloids tagged with long tdna2 coated with PPS-PEG. The way to produce those three kinds of gold colloids was induced in the chapter material and methods. (2.1.D) Figure 3.10 Four kinds of gold colloids used in this project. (a) gold colloids, (b) gold colloids tagged with tdna1, (c) gold colloids tagged with tdna1 coated with PPS-PEG, (d) gold colloids tagged with long tdna2 coated with PPS-PEG Lipid bilayers were formed on a SiO 2 coated crystal in order to investigate reaction between membrane of vesicles and gold colloids. Figure 3.11 shows the process. A frequency change of formation of lipid bilayer on a SiO 2 coated crystal is 25 ± 5 Hz. After a lipid bilayer was formed, 3 kinds of gold colloids tagged with DNA with a concentration of wt% were added to lipid bilayers. 37

38 3. Results and Discussion Figures 3.11 Vesicles (0.25 mg/ml) were injected into the flow cell and a bilayer was formed on SiO2. Then control experiments were done by injecting gold colloids tagged with DNA, gold colloids with DNA and coated by PPS-PEG, gold colloids tagged with Long DNA and coated by PPS-PEG. The sizes of gold colloids used were 5nm, 20nm, 50nm. Frequency changes of crystal were recorded for different gold colloids. The frequency change and dissipation change caused by adding three kinds of DNA tagged gold colloids are zero. That means, there is no absorption of all kinds of gold colloids on the lipid bilayer. Therefore, non-specific absorption is not caused by vesiclegold colloids reaction. The absorptions of gold colloids tagged with DNA on follow modified surface were measured by QCM experiments (Figure 3.12) : (a) PPL-PEG/PEGbiotin coated surface, (b) immobilized streptavidin tagged with biotin DNA via interaction with biotin on PPL- PEG/PEGbiotin surface. (c) Vesicles tagged with the complementary cholesterol DNA l are hybridized to the surface-immobilized single-stranded DNAs. 38

39 3. Results and Discussion Figures 3.12 Using QCM, three kinds of surface were modified on Nb2O crystal. (a) PPL- 5 PEG/PEGbiotin coated surface, (b) immobilized streptavidin tagged with biotin DNA via immobilized biotin on surface. (c) Vesicles tagged with the complementary DNA via cholesterol are hybridized to the surface-immobilized single-stranded DNA. On these three surfaces, 20 nm gold colloids tagged with DNA were added. Changes in the frequency (f) and dissipation factor (D) of the surface modified crystal were recorded. From Figure 3.13, the frequency changes caused by adding gold colloids tagged with DNA (b in figure 3.12) on three different surface are all larger than 10 Hz. That means, gold colloids tagged with DNA absorb on these surfaces. Non-specific absorption can be observed on these three kinds of surface. For the PLL coated surface, the absorption should be zero. The reason of the absorption might be the complicated structure of the 39

40 3. Results and Discussion surface makes the gold colloids get stuck between side chains. For the surface of immobilized streptavidin and vesicles, the reason might be reaction between streptavidin and gold colloids. Figures 3.13 In QCM control experiments, vesicles tagged with the complementary DNA via cholesterol are hybridized to the surface-immobilized single-stranded DNAs. Then for kinds gold colloids were injected into the flow cell: (a) gold colloids in water, (b) gold colloids tagged with tdna1 in H2, (c) gold colloids with ta1 and coated by PPS-PEG, (d) gold colloids with Long DNA tdna2 and coated with PPS-PEG. The frequency changes of crystal were recorded and showed in the graph. Figure 3.14 shows the frequency change of absorption of three different kinds of gold colloids on immobilized vesicles. For gold colloids in water, the absorption is very small. For gold colloids tagged with DNA in H2, the absorption is biggest while the absorption of gold colloids tagged with DNA coated with PPS-PEG is very small. Therefore, we 40

41 3. Results and Discussion found out the coating of PPS-PEG is very efficient way of avoid non-specific binding of gold colloids to the surfaces. The coverage of coating was also monitored by QCM experiments. Biotin-DNA strands were immobilized onto the PPL-PEG/PEGbiotin modified surface through complexes of streptavidin-biotin. Gold colloids tagged with short and long complementary DNA were added. Then absorption of gold colloids with short DNA is zero while gold colloids with long DNA can be absorbed. In this case, a good coverage of coating is proved because that short DNA is totally covered by longer PEG side chain and can not reach complementary DNA on the surface. 41

42 3. Results and Discussion 3.3. NanoDrop Spectrophotometer Gold colloids In this part, the absorption spectrum of gold colloids was measured using NanoDrop Spectrophotometer. We tried to measure reaction between gold colloids and proteins, or between gold colloids and gold colloids using this technology. After diluting gold colloids in water to different concentration, we tried to find the limitation of detection of this instrument. And also the amount of absorption of pick in spectrum as a function of concentration was measured and analyzed. Using this optimized equation, we can get an idea about concentration of gold colloids when we know the amount of absorption in the pick of the absorption spectrum of gold colloids. Figures 3.14 the absorption spectrums of gold colloids solution using NanoDrop, original concentration of 20 nm gold colloids is wt%. diluted the solution 2, 4, 8,12,14,16 times. Figure 3.14 shows the absorption spectrum of 20 nmgold colloids. And all sizes of gold colloids are measured in the similar way. For all the gold colloids, the spectrum is almost similar to the background signal when the concentration is 20 times smaller than the original concentration. 42

43 3. Results and Discussion From Figure 3.14, the amount of absorption at the pick of the absorption spectrum was plotted as a function of the concentration. After analysis and calculation, a relationship between them was got in Table 3.6 Table 3.6 The relation between the amount of absorption at the pick of the spectrum and the concentration of gold colloids. Absorption = A + B * X particles per ml: Size of gold A error B error colloids 5 nm e e nm e e nm e e nm e e-13 The figure A should be zero in principle, but because of noise background of the measurement of this equipment, it might be below zero when we measure the background. 43

44 3. Results and Discussion Reaction between gold colloids and proteins. After analyzing data of NanoDrop Spectrophotometer, we found that this instrument is not sensitive enough to detect a wavelength shift of 1 nm. And the curve is not smooth at all and the measurement is not repeatable because of the special process of measurement. Figure 3.15 is a typical curve we got, and it shows it is not possible to observe a wavelength shift of 1 nm. 44

45 3. Results and Discussion Figures 3.15 Absorption spectrum of 20nm gold colloids and vesicles. Also, the two solution were mixed together, and measurements of absorption spectrum were done immediately, after 1,2,5,10,20 mins using NanoDrop. 45

46 4. Conclusions 4. Conclusions In this project, a new technology is established by combining vesicles with gold colloids. To optimize fundamental information for this system, three important aspects were researched. First, Specific binding between gold colloids and vesicles is accomplished by coating the gold colloids with PPS-PEG, which absorbs to gold surfaces and forms protein-resistant adlayer. And good coverage of coating is proved by QCM experiments. Second, the concentration of embedded DNA in lipid bilayers does not affect the mobility of lipids. Further more, the anchoring group does not affect the mobility of DNA-tagged vesicles. As an important issue for consideration of using which kind of gold colloids, the mobility of lipids in POPC bilayer tethered with large size of gold colloids is slowed down. Third, after measuring absorption spectrum of gold colloids by NanoDrop, we found out that NanoDrop is not sensitive enough to detect a wavelength shift of 1 nm. Therefore it is not a good instrument for analysis. 46

47 5. Acknowledgements 5. Acknowledgments I would like to appreciate all the members in Janos group (The laboratory of Biosensors and Bioelectronics in the Institute for Biomedical Engineering, ETH Zurich ) who gave me lot of help on different aspects to finish my master thesis. In this nice and friendly, welcoming group, I have learned a lot of things both from research and also the way people arranging their work. I would like to thank my supervisor Brigitte Stadler specially for her help and useful suggestions all the time. With her care and encouragement, I feel comfortable and confident in the office. I had a really great time work under her supervision. Without kindly help from all the members in this group, It will be much harder to finish this work. So, thank you very much, dear all! 47