Development of a Piezoelectric Actuator for Separation and Purification of Biological Microparticles

Round 1

Reviewer 1 Report

A novel method for the separation of microparticles in the biological suspensions by taking the advantatge of sonification process was proposed. The corresponding device and set-up have been built for studying the separation process. They found that the microparticles would quickly move to the nodal zones in 10-12s and they could be concentrated and separated. The frequency used was lower than the existing technologies, indicating less damage to the biological entities.

The manuscript could be publised after addressing the following questions:

1. I found the authors published a paper using ultrasonic standing waves to separate microparticles in Actuators in 2018 (separation of Microparticles from Suspension Utilizing Ultrasonic Standing Waves in a Piezoelectric Cylinder Actuator, Actuators 2018, 7(2), 14). Except the applied frequency, what are other differences? The authors have to clearly show their improvements in this paper.
2. Following question 1, the equations (1-4) are exactly the same as those in publised paper. They do not have to rewrite them here, but to cite them in the proper way.
3. The authors showed the pressure level in several figures (Figure 2, 3, 7). However, they didn’t tell how to calculate the pressure level in the equations. Morever, Figure 5(b) and 6(b) showed the frequency response. However, it is not clear what’s the physical meaning of frequency reaponse? Why the magnitute has the unit of um/s?
4. The second sentence in the “Discussion” -The microparticles in biological suspension were taken to act on [26] and ... What does “act on [26]” mean? I am confusing because something is acting on one reference? Please cite the reference in the proper way.

Author Response

Dear Reviewer,

We would like to thank you for reviewing our manuscript and for your interesting and relevant comments and suggestions. We have taken them into account as carefully as possible. Thorough answers to your comments are provided below.

A novel method for the separation of microparticles in the biological suspensions by taking the advantatge of sonification process was proposed. The corresponding device and set-up have been built for studying the separation process. They found that the microparticles would quickly move to the nodal zones in 10-12s and they could be concentrated and separated. The frequency used was lower than the existing technologies, indicating less damage to the biological entities.

The manuscript could be publised after addressing the following questions:

1. I found the authors published a paper using ultrasonic standing waves to separate microparticles in Actuators in 2018 (separation of Microparticles from Suspension Utilizing Ultrasonic Standing Waves in a Piezoelectric Cylinder Actuator, Actuators 2018, 7(2), 14). Except the applied frequency, what are other differences? The authors have to clearly show their improvements in this paper.

 Answer: Previous work presents a technology which enables the microparticles to be separated from suspension filled into a cylindrical ultrasonically excited piezo-actuator vessel. In the present work, the acoustic separation of microparticles is investigated in the frequency range from 10 kHz to 15 kHz, whereas in the 2018 publication the investigations were performed in the frequency band from 200 kHz to 345 kHz, i.e. about 20 times higher frequency, which may be dangerous for biocells, because the generated acoustic energy is just proportional to the square of the acoustic frequancy. The acoustic wave in this study propagates in the axial direction of the fluid container, whereas in the previous publication the direction is radial. The driving frequency, lower than ultrasonic in this study allows protecting the bioparticles from damage and developing technology for volume separation of bioparticles from constant stream of suspension.

2. Following question 1, the equations (1-4) are exactly the same as those in publised paper. They do not have to rewrite them here, but to cite them in the proper way.

Answer: It was cited [28], but it is expedient to leave the indicated formulas to the reader's orientation about the data characterizing the simulated suspension with bioparticles.

3. The authors showed the pressure level in several figures (Figure 2, 3, 7). However, they didn’t tell how to calculate the pressure level in the equations. Morever, Figure 5(b) and 6(b) showed the frequency response. However, it is not clear what’s the physical meaning of frequency reaponse? Why the magnitute has the unit of um/s?

Answer: The COMSOL Multiphysics software sets the pressure level. The Doppler laser Polytec 3D scanning vibrometer (type PSV-500-3D-HV, Polytec GmbH, Germany) was used to determine eigenmodes and to measure vibrovelocity (unit of µm/s) of the piezoelectric actuator (DSPTB) and of the container (CFC) wall. Frequency response is the dynamic output of the microparticles separation device in response to the vibrational excitation.

4.The second sentence in the “Discussion” -The microparticles in biological suspension were taken to act on [26] and ... What does “act on [26]” mean? I am confusing because something is acting on one reference? Please cite the reference in the proper way.

Answer: It was cited in a proper way: „The microparticles in biological suspension were taken to act as a substitute for red blood cells (before [26] and now [32]) demonstrate the validity of the proposed separation technology by experiment“.

Reviewer 2 Report

This manuscript describes the development of an acoustic based microparticle separation platform for large volume of samples. The authors performed numerical simulations and experimental validations. The paltform is portable and low cost. I would suggest the publication of this manuscript, while the authors should consider the following modifications.

1. Fig 7b is duplicate with Fig 3a. The authors should 
2. There is no quantitative data showing the separation performance. The only number mentioned by the authors claimed "The factor of purified/enriched microparticles was about 75 percent compared to the original sample". What is the definition of "factor of purified/enriched microparticles"? Please explain it. I would suggest the authors to quantitate their results using the common terms i.e. recovery rate, enrichment folder, particle concentration etc.
3. How is the microparticle different from biomicroparticles i.e platelets? And how feasible to implement this technique to applications including platelete separation?
4. What is the processing throughput? 
5. Fig 2a shows the acoustic pressure distribution, while Fig 2b and Fig 3a show a different distribution of acoustic pressure compared to Fig 2a. Please clearify and explain the data.
6. Please explain the meaning of the color mapping presented by Fig 5 and Fig 6.
7. Please add scale bars to some of the figures, i.e Fig 7a.

Author Response

Dear Reviewer,

We would like to thank you for reviewing our manuscript and for your interesting and relevant comments and suggestions. We have taken them into account as carefully as possible. Thorough answers to your comments are provided below.

This manuscript describes the development of an acoustic based microparticle separation platform for large volume of samples. The authors performed numerical simulations and experimental validations. The paltform is portable and low cost. I would suggest the publication of this manuscript, while the authors should consider the following modifications.

1. Fig 7b is duplicate with Fig 3a. The authors should

Answer: It was corrected, Fig. 7b was abandoned.

2. There is no quantitative data showing the separation performance. The only number mentioned by the authors claimed "The factor of purified/enriched microparticles was about 75 percent compared to the original sample". What is the definition of "factor of purified/enriched microparticles"? Please explain it. I would suggest the authors to quantitate their results using the common terms i.e. recovery rate, enrichment folder, particle concentration etc.

Answer: The term „ factor“ has been replaced by the common term „separation rate“. Here, we judge from different samples of the suspension affected by the acoustic field, whose microscopic images show differences in the concentration of microparticles and 75% means the enrichement rate of microparticles in suspension, the remaining 25% is a fluid. Fig. 8 depicts a comparison of the two samples of suspension from different clusters of purified and enriched microparticles, taken from the nodal and anti-nodal CFC zones vibrating on the fourth eigenmode. The area of microscopic views, acquired from video signals, is eąual in both images, but the distribution density of the particles is different. A commercial software package (NIS-Elements AR, Nikon, Japan) has been used to construct 2D microparticle images from the video signals, recorded with the Nikon Eclipse LV series microscope and PixeLINK PL-A662 camera. The percentages given are related to both modeling and experimentation at a time interval of 12s.

3. How is the microparticle different from biomicroparticles i.e platelets? And how feasible to implement this technique to applications including platelete separation?

Answer: Biomicroparticles i.e platelets diameter is 2–3µm as red blood cells - 8µm. Smaller diameter microparticles will accumulate in the lower nodal areas of the vibrating container wall.

4. What is the processing throughput? 

Answer: Processing throughput may be similar as with red blood cells.

5. Fig 2a shows the acoustic pressure distribution, while Fig 2b and Fig 3a show a different distribution of acoustic pressure compared to Fig 2a. Please clearify and explain the data.

Answer: Fig 2a shows resonating on fourth eigenmode CFC and sound pressure level (DB) of the fluid without microparticles, Fig 2b shows microparticles evenly distributed in the suspension at the initial time t = 0s and Fig 3a shows positions of the microparticles at time t = 12s and their motion trajectories in axial plane.

6. Please explain the meaning of the color mapping presented by Fig 5 and Fig 6.

Answer: When measuring the oscillations of the piezoelectric actuator (Fig.5a) and the container wall (Fig.6a) at 13.5 kHz with a Polytec 3D vibrometer, blue indicates the zones of minimum oscillations and yellow-red the zones of maximum oscillations of the excited standing wave.

7. Please add scale bars to some of the figures, i.e Fig 7a.

Answer: The dimensions of the container were added to the Fig. 7a.

Reviewer 3 Report

The authors present a platform for larger volume particle enrichment using acoustics. The platform consists of larger scale system to overcome the relatively smaller throughput achieved in microfluidic systems and operates at a lower frequency (as compared to typical acoustofluidic systems) due to potential hazardous effects on biological cells.

My major concerns with the manuscript in its current form is as below and should be addressed:

1) A major claim in this manuscript is that biocompatibility of systems in the MHz range is hazardous, thus, operation at lower frequencies are preferably. The biocompatibility of cells is not necessarily hazardous and are considered safe at higher frequencies, as exhibited in multiple published studies. For example:

• Proceedings of the National Academy of Sciences May 2020, 117 (20) 10976-10982; DOI: 10.1073/pnas.1917125117
• J Biomech Eng. Mar 2020, 142(3): 031005
• Wu et al. Microsystems & Nanoengineering (2019) 5:32
• Nat Commun 6, 8686 (2015). https://doi.org/10.1038/ncomms9686
• Lab Chip, 2019,19, 2435-2443
• Lab Chip, 2020,20, 1991-1998

However, this depends heavily on the amplitude, cell size and type. There are instances that untoward effects can be implicated on cells when exposed to an acoustic waves as detailed in the following reference:

• Adv. Sci. 2019, 6, 1902326. https://doi.org/10.1002/advs.201902326

Please amend the statements throughout the manuscript to reflect this, such that it doesn’t mislead the readers.

2) The authors do not show any results or discussion regarding acoustic streaming effects and is not taken into account within the models. This should be included or at the very least be described within the manuscript as the fluid wavelength (i.e. 0.11 m) considered here is significantly larger than the microparticles considered (i.e. 4 µm). I would expect a significant streaming influence on the particle trajectory.

The authors state the following on page 11

“Experiments have shown that increasing the amplitude of the oscillation at 13.5 kHz accelerates the separation process, but above a certain level, streaming becomes dominant, which disrupts the separated suspension phases.”

There are two issues with this sentence:

• Firstly, there is no data included in this manuscript that substantiates this claim. What level is indicated by certain level?
• Secondly, in my opinion, this isn’t true. As the excitation power increases, both, the acoustic radiation forces and the body forces increases commensurately. Therefore, a transition from radiation dominated to streaming dominated particle behaviour shouldn’t be expected (assuming, the system’s size and operational frequencies are held constant, the case in this manuscript). Please refer to the following references for both, Bulk acoustic wave and surface acoustic wave systems that detail the interplay between radiation and streaming dominated particle behaviour.

1. 1. Rev. E 85, 016327
   2. Lab Chip, 2012,12, 4617-4627
   3. Lab Chip, 2016,16, 3756-3766

3) The authors state the following on page 10:

“The microparticle separation in suspension by AW depended on their size, density, and compressibility.”

Once again, whilst this might be true and has been shown in other publications, there is no data to substantiate this claim within the manuscript. I would strongly encourage the authors to include data to demonstrate this statement.

4) Lastly, one of the author’s major claim of this manuscript is that the system is built for separation and purification of biologicals (in the title as well), however, there is no data that exhibits the use or efficacy of the system on biological matter throughout the manuscript. This should be addressed.

Author Response

Dear Reviewer,

We would like to thank you for reviewing our manuscript and for your interesting and relevant comments and suggestions. We have taken them into account as carefully as possible. Thorough answers to your comments are provided below.

The authors present a platform for larger volume particle enrichment using acoustics. The platform consists of larger scale system to overcome the relatively smaller throughput achieved in microfluidic systems and operates at a lower frequency (as compared to typical acoustofluidic systems) due to potential hazardous effects on biological cells.

My major concerns with the manuscript in its current form is as below and should be addressed:

1. A major claim in this manuscript is that biocompatibility of systems in the MHz range is hazardous, thus, operation at lower frequencies are preferably. The biocompatibility of cells is not necessarily hazardous and are considered safe at higher frequencies, as exhibited in multiple published studies. For example:

Proceedings of the National Academy of Sciences May 2020, 117 (20) 10976-10982; DOI: 10.1073/pnas.1917125117

J Biomech Eng. Mar 2020, 142(3): 031005

Wu et al. Microsystems & Nanoengineering (2019) 5:32

Nat Commun 6, 8686 (2015). https://doi.org/10.1038/ncomms9686

Lab Chip, 2019,19, 2435-2443

Lab Chip, 2020,20, 1991-1998

However, this depends heavily on the amplitude, cell size and type. There are instances that untoward effects can be implicated on cells when exposed to an acoustic waves as detailed in the following reference:

Adv. Sci. 2019, 6, 1902326. https://doi.org/10.1002/advs.201902326

Please amend the statements throughout the manuscript to reflect this, such that it doesn’t mislead the readers.

Answer: You are right, the new sentences in Introduction were added: „Despite the fact that some of the authors of the cited works claim that biocompatibility of systems in the MHz range is hazardous even at megahertz frequencies the variations and chronic thresholds in response are cell type‐specific and so safe operation ranges should be considered while developing acoustic based microfluidic platforms with reference to the cell type used [5, 23, 24, 25, 26, 27]“. All of the mentioned sources are associated with the separation of microparticles in microchannels, while we propose to process them in larger volume, concentrating in the nodal areas of the vibrating container.

2. The authors do not show any results or discussion regarding acoustic streaming effects and is not taken into account within the models. This should be included or at the very least be described within the manuscript as the fluid wavelength (i.e. 0.11 m) considered here is significantly larger than the microparticles considered (i.e. 4 µm). I would expect a significant streaming influence on the particle trajectory.

Answer: You are right, the new sentences in discussion were added: „As the microparticle separation process takes place by excitation of the piezoelectric actuator and the container in higher bending oscillation modes (frequency about 13.5 kHz), this allows, to a certain extent, the generation of a sufficiently high intensity acoustic field without streaming effect on the suspension. However, experiments have shown that at higher amplitudes of the oscillations of the piezoelectric actuator, a turbulent phenomenon occurs in the suspension, which, due to the resulting fluid streaming, begins to stir the fluid throughout the container volume, thus interfering with the acoustic standing wave effect on the suspension“.

The authors state the following on page 11

“Experiments have shown that increasing the amplitude of the oscillation at 13.5 kHz accelerates the separation process, but above a certain level, streaming becomes dominant, which disrupts the separated suspension phases.”

There are two issues with this sentence:

• Firstly, there is no data included in this manuscript that substantiates this claim. What level is indicated by certain level?
• Secondly, in my opinion, this isn’t true. As the excitation power increases, both, the acoustic radiation forces and the body forces increases commensurately. Therefore, a transition from radiation dominated to streaming dominated particle behaviour shouldn’t be expected (assuming, the system’s size and operational frequencies are held constant, the case in this manuscript). Please refer to the following references for both, Bulk acoustic wave and surface acoustic wave systems that detail the interplay between radiation and streaming dominated particle behaviour:

1. Rev. E 85, 016327
2. Lab Chip, 2012,12, 4617-4627
3. Lab Chip, 2016,16, 3756-3766

Answer: You are right, the new sentences in discussion were added: „Analogous to the results presented in [35, 36], we numerically simulated the distribution of red blood cells microparticles in acoustic pressure field level in the CFC volume at the initial time t = 0s in the three upper zones with a low pressure level which are perpendicular to the flow of fluid along the longitudinal axis, and the two lower zones which are not as shown in Fig.2(b). In Fig. 3(a), the position of the particles in the field of acoustic pressure at t = 12s and their trajectories are provided (all particles have the same diameter of 8 μm) where the microparticles "cling" to the inner surface of the CFC wall in eigenmode nodal zones. As a result of acoustic effects the microparticle movement was vertical near anti-node, horizontal at the node, and inclined in between. During the experiment the piezoelectric actuator was excited by an electrical signal with frequency of 13.5 kHz, the voltage amplitude of which was varied from 0 to 70V. During the study it was observed that the process of microparticle separation begins when a voltage of 30V was reached and further increasing the voltage to 50V a turbulent phenomenon occurs in the suspension, which, due to the resulting fluid streaming, begins to stir the fluid throughout the container volume, thus interfering with the acoustic standing wave effect on the suspension. As a result of the research the innovative piezoelectric microparticle separation device was developed”.

The indicated certain level was the 50V excitation voltage.

For detailing the interplay between radiation and streaming dominated particle behaviour the most important is a stable interface between vibrating surface of an actuator and fluid for bulk acoustic wave generation in suspension.

3. The authors state the following on page 10:

“The microparticle separation in suspension by AW depended on their size, density, and compressibility.”Once again, whilst this might be true and has been shown in other publications, there is no data to substantiate this claim within the manuscript. I would strongly encourage the authors to include data to demonstrate this statement.

Answer: Formula (3) gives the parameters (size, density, and compressibility) on which the acoustic radiation force depends: where ρ_p is the microparticle density; c is the speed of sound; f₁ is real-valued and depends only on the compressibility ratio between the microparticle and the fluid; f₂ is the dipole scattering coefficient, which is related to the microparticle translational motion and depends on the viscosity of the fluid; K is the bulk modulus of the fluid; K_p and V_p are the bulk modulus and volume of the microparticle.

4. Lastly, one of the author’s major claim of this manuscript is that the system is built for separation and purification of biologicals (in the title as well), however, there is no data that exhibits the use or efficacy of the system on biological matter throughout the manuscript. This should be addressed.

Answer: We have simulated the motion of red blood cells microparticles suspended in plasma and experimented with zeolite in artificial plazma..

The new sentences in experimental results were added: “Zeolite, the active ingredient, is micronized to the size of a red blood cell. This means the particles of zeolite are so fine that they can actually pass in between the body‘s cells. Zeolite was chosen because the experiment with the blood would be complicated due to the rapidly changing properties of red blood cells in the blood. Standard guidelines for handling blood samples indicate that plasma or serum should be separated from the cells as soon as possible (20–30 minutes) after the clot formation is complete to avoid clot-induced changes in the serum concentrations of the analytic liquid caused by coagulation. The agglutination of red blood cells can occur within a few minutes after finding blood outside the body“.

On the other hand, ethical permits are required to perform blood tests. Red blood cell size falls within the range of zeolite particle sizes.

Reviewer 4 Report

In this paper, the authors proposed a method for the separation of biological microparticles in suspension. It's meaningful, since it has the advantage of portable, low-cost and non-biodegradable procedures of energy-efficient separation/purification of microparticles in biological suspension. The manuscript also proved it a potential powerful tool in the application experiments. However, before publication, following issues should be well addressed.

In the experiment, the authors showed the effectiveness of the microparticle separation in biological suspension (Figure 8). However, at the same time we think the separation rate is also an important indicator to show the advantages of this technology. Thus, we suggest the authors list it. What’s the important factors of CFC's eigenmodes. The relationship between are also required to present.

Author Response

Dear Reviewer,

We would like to thank you for reviewing our manuscript and for your interesting and relevant comments and suggestions. We have taken them into account as carefully as possible.

Thorough answers to your comments are provided below.

In this paper, the authors proposed a method for the separation of biological microparticles in suspension. It's meaningful, since it has the advantage of portable, low-cost and non-biodegradable procedures of energy-efficient separation/purification of microparticles in biological suspension. The manuscript also proved it a potential powerful tool in the application experiments. However, before publication, following issues should be well addressed.

In the experiment, the authors showed the effectiveness of the microparticle separation in biological suspension (Figure 8). However, at the same time we think the separation rate is also an important indicator to show the advantages of this technology. Thus, we suggest the authors list it. What’s the important factors of CFC's eigenmodes. The relationship between are also required to present.

Answer: You are right, the new sentences in experimental results are included: “Figure 8 depicts a comparison of the two samples of suspension from different clusters of purified and enriched microparticles, taken from the nodal and anti-nodal CFC zones vibrating on the fourth eigenmode. The area of microscopic views, acquired from video signals, is eąual in both images, but the distribution density of the particles is different. A commercial software package (NIS-Elements AR, Nikon, Japan) has been used to construct 2D microparticle images from the video signals, recorded with the Nikon Eclipse LV series microscope and PixeLINK PL-A662 camera. The 75% separation rate is related to both modeling and experimentation at a time interval of 12 s“.

The coincidence of the higher bending modes of the container and the piezoelectric actuator at the frequency of 13.5 kHz generates an acoustic field of a standing wave of sufficient intensity in the suspension, in which the process of microparticle separation occurs. In this case, a sufficiently large container volume and a properly selected piezoelectric actuator allow to obtain high separation efficiencies compared to the microchannel separation process .

Round 2

Reviewer 3 Report

The authors have addressed my concerns raised satisfactorily.