Lab exercise instruction Acoustophoresis

Transcription

1 Lab exercise instruction Acoustophoresis Theory The fundamental theory for acoustic force interactions in an acoustic standing wave field is rather complex. To give a general picture we can observe the simplified equation for the acoustic radiation force, F rad, on a particle in a standing wave field, Eq. 1&2. The radiation force depends on several factors as tabulated below Table 1.!!!"# = 4!Φ(!,!)!!!!!" sin (2!") (Eq 1) Φ!,! =!!!!!!!!!!!!!!!!!!!! (Eq 2) k a E ac Φ(κ,ρ) ρ p ρ 0 κ p κ 0 Wavenumber Particle radius Acoustic energy density Acoustic contrast factor Particle density Fluid density Particle compressibility Fluid compressibility (Table 1) The physical parameters that govern the resulting radiation force on a particle are the volume, density and compressibility of the particle in relation to the surrounding fluid. As can be noted, the radiation force also scales with the frequency through the wavenumber, eq. 3.! =!!! =!!"! (Eq 3) The acoustic contrast factor Φ, eq 2, decides whether a particle will migrate to the pressure node or antinode of the acoustic standing wave. A particle that has a positive contrast factor will migrate to the pressure node and a negative contrast factor will yield particles in the pressure antinode. In general rigid particles have a positive contrast factor while gas bubbles, oil droplets and other particles with a lower densitythan the surrounding bufferhave a negative contrast factor and migrate toward the antinode (it should be noted that there are exemptions from this rule of thumb) TL

2 EEMN25. Lab-on-a-chip in Biomedicalapplications The flow system Figure 1. Overview of the flow system including the silicon chip manifold (top) Our sample enters the pre-focusing channel to the far left and is subsequently split into two streams (red) that enter the separation channel along the sidewalls. A clean buffer (light blue) enters in the centre of the separation channel. Questions to answer before the lab exercise Q 1.What is the purpose of the pre-focusing? TL

3 Q 2.What ratio between the height and width of the channel is required to accomplish the prefocusing using only one piezoelectric transducer? Q 3.What is the purpose of the wash buffer? The chip has two outlets, one central through which particles that have migrated into the central flow exit and one for particles that follow the flow along the side walls and further into the side outlets in the outlet flow splitter. The fluid flow is monitored at the in- and outlets by flow sensors. Data from these are used to control the pressure in the fluid containers such that the desired flow values are obtained. The entire flow system is operated via a LabView control algorithm. Q 4.How does the flow through the separation channel depend on the driving pressure? TL

4 Lab exercise 1 dimensionalfocusing 1. The separation channel is ca 375 µm wide. What frequency should you set to obtain a half wavelength resonance across the channel width? Assume water as the carrier buffer and a speed of sound in water of 1500 m/s. 2. Tune the frequency and amplitude by studying the focusing in the separation channel such that you obtain the best possible particle focusing at minimal amplitude across the piezoactuator. What frequency and what amplitude did you obtain? 3. What happens if you run at a too high amplitude? Why? 4. What bandwidth do you have when focusing is satisfactory? 5. How is the pre-focusing affected by increased flow rate? 2 dimensional pre-focusing 6. In the pre-focusing step we want to generate two particle bands that branch of in each side of the flow splitter. The channel is 300 µm wide. Around which frequency would you start searching for the desired resonance? 7. Tune the pre-focusing frequency and transducer amplitude by studying the focusing in the pre-focusing channel such that you obtain full focusing at minimal transducer amplitude. What frequency and what amplitude did you obtain? TL

5 8. Turn the pre-focusing on and off while running the system. Observe the particle streams at the outlet of the separation channel. What differences do you see when pre-focusing is on and off respectively? Discuss the outcome with the supervisor. Separation of 5µm and 7µm particles 9. Use the frequency and amplitude settings you got from the previous experiments for the 2 dimensional pre-focusing in the 300 µm channel and the 1 dimensional focusing in the 375 µm channel and now try to tune the system such that you find a condition where the 7 µm particles exit in the centre outlet and the 5 µm particles go in the side outlet. 10. Once you are satisfied with the settings to accomplish a good separation of the 5 and 7 µm particles, change the sample outlet tubes and collect clean fractions. Analyse the results with flow cytometry. How good was the separation? Calculate the purity of each fraction. Ask the supervisor to get the FACS data for your report. 11. If time allows you may want to do the corresponding separation experiment without the prefocusing active. Particle washing You ll get a sample containing white polystyrene particles (PS) in a salt buffer (sodium chloride) with a blue colour compound (Evans Blue) and a colourless salt buffer in the centre inlet. 12. Perform an acoustophoretic wash sequence of the white particles, i.e. make a buffer exchange where you move the white particles from the blue fluid to the clear fluid in the centre of the channel. 13. Change the wash fluid in the centre inlet from sodium chloride to MQ water. What happens? 14. Explain the difference in the outcome of the wash experiments TL