0 1 2 3 4
0,4 ml / min 2,6 ml / min 4,2 ml / min
Relative adsorption
Flow velocity
Figure 6.3:Antibody immobilization as function of different flow rates in microfluidic devices. The binding signal is given as the relative adsorption as function of different flow rates. The data presented are the average of three independent experiments, each performed in triplicate.
6.3 Measurement using luminescent particles
In Chapter 4, proposed microstructures were simulated to analyze how the shape of the channel can influence the flow velocity profile. From the simulation results, the channel with integrated column structures was selected as the optimal design because of the particular fluid flow velocity profile, low flow resistance and increased surface. In order to evaluate the immobilization per-formance, flow visualization was used as a qualitative measure of immobilization performance using a confocal laser scanning microscope.
For this analysis, microfluidic devices with parallel straight channels, meander shaped channel structures and with structured columns with a channel height of 150µm were fabricated because of their uniform and constant velocity magni-tude in comparison with the other channel heights evaluated in this dissertation (see section 4.4). The measurement setup with utilized components are shown in figure 6.4.
The LSM is established as a valuable instrument for obtaining non-invasive and high resolution images. To visualize the interaction of very small particles along the microfluidic structures, distilled water was mixed with FITC particles (Flu-oresceinisothiocyanate, marked latex particles) having a diameter of 977nm.
Then, the resulting fluid flow (0.05mg/mL FITC nanoparticles in distilled water) was induced via pressure driven flow throughout the microfluidic chip.
The portable micro pump mp6 was utilized for this purpose (see section 7.3).
According to the characteristic curve of this micro pump , the inlet flow rate was increased to 5mL/min reaching its maximum value. This value was obtained by applying a voltage of 250V at 100Hz to the pump controller [Bar10].
Chapter 6 Characterization of Fabricated Microfluidic Devices
FI TC nanoparticles (Marked latex
particles) PC
LSM I mage 4.2
DI -Water
DI -Water + FI TC nanoparticles PUMP
I nlet
Outlet Microfluidic Holder
LSM (Laser Scanning
Microscope)
Figure 6.4:Schematic measurement system using FITC particles with a diameter of 977nm. The red arrows indicate the fluid direction. The trajectory of FITC along the microchannel was observed by using laser scanning microscope. The series of images captured during the flow visualization were then analyzed using the LSM Image 4.2 Viewer software. This measurement system was implemented at the Jacobs University Bremen.
After required equipment states were defined and fluid connections were made, the fluid flow was pumped and fluorescence development was simultaneously visualized and photographed through LSM. The probes were observed on three different channels with corresponding excitations and emissions lights.
1. Nano particles FITC dependent green fluorescence was excited using a 488nm laser and detected through a long-pass filter LP 505nm.
2. DI water dependent red fluorescence was excited using a 543nm laser and visualized using a long-pass filter LP 560nm.
3. SU-8 structures dependent blue fluorescence was excited with a 633nm laser and detected through a long-pass filter LP 650nm.
Figure 6.5 shows the particle distribution within the microfluidic devices with parallel shaped channels, meander shaped channels and with structured columns, which have a channel height of 150µm. During the measurement of the fluo-rescent signal, circulatory paths or flow vortices did not take place. A homoge-neous distribution of nanoparticles was observed as expected from simulation results by using microfluidic devices with a channel height of 150µm (see section 4.4). In spite of this uniform flow profile and respectively particle distribution, the interaction between marked nanoparticles and SU-8 walls was not observed in all microfluidic devices; thus reducing the possibility of particle adhesion. According to the experiments using marked particles, the amount of immobilized particles was markedly low within microfluidic devices with
6.3 Measurement using luminescent particles
parallel and meander shaped channels. In contrast to that, a large amount of immobilized particles was observed within the channel with integrated columns as shown in figure 6.5. The particle adhesion observed within microfluidic devices with structured columns was attributed to the particular flow profile generated by the staggered arrangement of the structured columns. In this case, the flow velocity decreases considerably when approaching the column surface.
Although laminar flow is also present in this microfluidic device, the direction of marked particles was strongly influenced by the presence of the structured columns. The advantageous trajectory and the resulting immobilization of the particles are then associated to the stagnation point. Some particles flowed between the columns, whereas particles flowing in direction to the column surface were caught by the stagnation zone. Because of the very low velocities encountered in this region, marked nanoparticles could not change the flow direction and thus, they had sufficient time to bind covalently to the column surface. The nanoparticles moving between the columns continued flowing until approaching the next column surface. The visual observation of marked nanoparticles within the channel with integrated columns demonstrated that a large amount of FITC particles could be adhered in the column region.
Stagnation point took place in the vicinity of the structured columns being an optimal mechanism for increasing cell adhesion. In accordance with the experimental results presented in this section, the microfluidic device with integrated columns is the best device for the current application.
(a) (b) (c)
Figure 6.5:LSM micrographs of FITC flowing within the microfluidic devices. The probes were visualized on three channels with corresponding excitations and emissions: FITC (488 nm, LP 505 nm), green color; fluid (543 nm, LP 560 nm), red color; SU-8 structures (633 nm, LP 650 nm), blue color.
The images were analyzed using the LSM Image 4.2 Viewer software.
The images were taken after approximately 450 sec. After this time, any significant change was observed. Distribution of FITC particles within microfluidic devices with a channel height of 150µm (a) parallel shaped channels (b) meander shaped channels (c) structured columns.
Chapter 6 Characterization of Fabricated Microfluidic Devices