Research Professor University of Pennsylvania, United States
Introduction: Monitoring flow velocity and patterns in situ in micro/nano devices is critical for many biomedical applications, including cell operations, tissue culture, material synthesis, and biosensors. Compared to Molecular Tagging Velocimetry, which requires aligning two laser sources to the microscope, Particle Image Velocimetry (PIV) can be operated on a regular fluorescence microscope by monitoring the trace of fluorescent tracer particles in the flow. Fluorescent polystyrene (PS) microspheres are commonly used as tracers in PIV. However, PS microspheres carry a large number of negative charges in the solution, making it impossible to track electrokinetic flow correctly due to electrophoresis. Moreover, PS microspheres adhere to the microchannel and the substrates monitored in the devices, which disturbs the function of the micro/nano devices. In the worst-case scenario, the micro/nano structure can be blocked by the PS microspheres. We solved these issues by replacing PS microspheres with fluorescent micelles. The fluorescent micelles we synthesized carry little charge across a pH range of 4 to 9 at a wide buffer concentration range, making them ideal for monitoring electrokinetic flow. Furthermore, as the micelles are dispersed in the liquid phase, they rarely adhere to the microchannel, can be easily washed away, and will never block the micro/nano structures. These improvements significantly enhance the viability and reliability of flow monitoring in micro/nano devices for biomedical applications.
Materials and
Methods: Poly(1,2-Butadiene)-b-Poly(Ethylene oxide) (PBD-PEO) is used to build the micelle scaffold. Two chain lengths, PBD2500-PEO1200 and PBD1200-PEO600, are selected to combine with three fluorescent dyes: 2,8-Diethyl-1,3,5,7-tetramethyl-9-phenylbipyrromethene difluoroborate (BODIPY dye), Cyanine3-maleimide (Cy3-m), and Cyanine3.5-dimethyl (Cy3.5), to construct the fluorescent micelles. BODIPY dye is neutral, while Cy3-m and Cy3.5 carry a +1 charge, with Cy3-m having a longer hydrophobic chain compared to Cy3.5.
The micelles are synthesized by drying a PBD-PEO and fluorescent dye mixture in chloroform and reconstructing it in 10 mM ammonium acetate buffer. This stock solution is diluted tenfold and centrifuged using Sartorius vivaclear centrifugal filters (pore size 0.8 µm) at 1000 rpm for half an hour. During centrifugation, a partial solution containing micelles smaller than 0.8 µm and free fluorescent dyes passes through the filter, while large micelle aggregates are deposited on the filter. The solution above the filters is collected, containing medium-sized micelles.
Two PS microspheres without surface modifications, FSEG004 (1.04 µm) from Bangs Laboratory Inc. and SPHERO™ Fluorescent Nile Red particles (0.87 µm) from Spherotech Inc., are tested for comparison with fluorescent micelles. The surface charge of the microspheres and micelles is characterized by zeta potential measurements using a Zetasizer Nano-ZS from Malvern Panalytical. PIV measurements are performed in 200 µm wide, 50 µm high PDMS microchannels with electroosmotic flow (EOF) induced by an electric field of 100 V/cm with 10 mM ammonium acetate buffer at pH 9. Image sequences were collected at 90 fps. PIV data analysis is conducted using Insight 4G software from TSI Incorporated.
Results, Conclusions, and Discussions: Six micelles are synthesized: PEO600-BODIPY, PEO1200-BODIPY, PEO600-Cy3-m, PEO1200-Cy3-m, PEO600-Cy3.5, and PEO1200-Cy3.5. Among these, BODIPY micelles have the least free fluorescent dye in the solution, providing the least background in fluorescent imaging. At pH 7 in 10 mM ammonium acetate buffer, PEO-BODIPY and PEO-Cy3-m are neutral with zeta potentials around -3.5 mV and 0 mV, respectively, while PEO-Cy3.5 carries a slightly positive charge of 15 mV. FSEG004 and Nile Red carry negative charges around -63 mV and -75 mV. When extending the pH range from 4 to 9, PEO-BODIPY and PEO-Cy3-m remain near neutral, whereas FSEG004 and Nile Red still carry large negative charges from -50 mV to -80 mV. When extending the concentration range from 0.1 mM to 100 mM, PEO1200-Cy3-m stays neutral, while PEO600-BODIPY carries -23 mV at 0.1 mV and remains neutral above 10 mM. PIV experiments are conducted in PDMS microchannels at pH 9 in 10 mM ammonium acetate by measuring EOF to examine the abilities of micelles and PS microspheres as tracers. FSEG004 (-61.5 ± 4.9 mV) and Nile Red (-80.3 ± 7.4 mV) move in the opposite direction of EOF due to their large negative charge, making it impossible to track EOF. The measured velocities of FSEG004 and Nile Red are -251 ± 20 µm/s and -245 ± 27 µm/s, respectively. In contrast, PEO600-BODIPY (-6.8 ± 4.9 mV) and PEO1200-Cy3-m (-8.6 ± 4.6 mV) follow EOF directions, with measured velocities of 211 ± 30 µm/s and 209 ± 18 µm/s, respectively. More experiments at pH 7, where the micelle zeta potential is around zero, will be conducted to correlate zeta potential and velocity. After the PIV experiments and repeated flushing with ethanol and water, microchannels tested with micelles stayed clean, while those tested with PS microspheres had many adhered particles. The micelles have little zeta potential change after one year stored in a 6°C fridge, proving their good stability. These findings highlight the superiority of fluorescent micelles over traditional PS microspheres in tracking electrokinetic flow, offering reliable and efficient alternatives for flow monitoring in micro/nano devices.
