A Multifunctional Double-Array Petals Flower-Shaped Microfluidic Chip Combining Affinity and Physical Properties in Isolation of CTCs

Comments 1: The manuscript describes the design of a double-array, petal-shaped microfluidic chip for the isolation of circulating tumor cells (CTCs) based on both affinity and physical properties. While the authors conducted a series of numerical analyses to theoretically validate high capture rates and purity, no experimental work regarding chip fabrication or CTC isolation was performed. Given that experimental validation using actual CTCs is feasible and essential for evaluating capture efficiency, the absence of such data significantly weakens the study. Furthermore, there are concerns regarding the manufacturability of the proposed structure, particularly due to the presence of narrow channels (5 µm), which may pose fabrication challenges. Therefore, it is strongly recommended that the authors fabricate the chips and perform experiments to evaluate CTC isolation performance before this work can be published.

Response 1: A more detailed and comprehensive explanation  in text have been provided. Thank you for pointing this out. We agree with this comment. Therefore, I/we have made changes as following,

I/we understand experimental validation using actual CTCs is feasible and essential for evaluating capture efficiency, and the absence of such data significantly weakens the study. However, no fabrication condition and research funding is available to carry out those experiments. I/we would include those manufacturability and experimental data when experiments condition is available.

Conclusion

Experimental validation using actual CTCs is feasible and essential for evaluating capture efficiency. Manufacturability and experimental data would be included when experiments condition is available.

Comments 1:

1. Introduction

The introduction would benefit from additional explanation regarding the use of microfluidics for CTCs detection. Why are they well-suited for this? The last paragraph of the introduction is identical to the paragraph in section 3, entitled “Discussion”. Aims and objectives should be stated clearly after the literature review in the introduction.

Response 1: We have described the additional explanation regarding the use of microfluidics for CTCs detection. They have been included in the Introduction part.  Thank you for pointing this out. I/We agree with this comment.  

Microfluidic chips are well suited in CTCs isolation based on cell-sized capture site, low chemical reagent consuming, and high throughput such as circular spiral chip. (Introduction)

The aims and objectives are to design a unique microfluidic chip to realize high capture efficiency without yielding isolation purity. Theoretical validation could be carried out with software to prove the feasibility. This is a necessary step before experimental fabrication and clinical assays to optimize the chip according to theoretical study. (Introduction)

We changed Discussion identical to last paragraph of Introduction in response 3. Please refer to. 

1. Comments

 2: General

There is no “Materials and Methods” section. In “Materials and Methods” section, all steps should be clearly described so that the study can be reproduced. The computational mesh can be included as figure in the simulation section or supporting information. Did you assign specific diameters for RBCs, WBCs or CTCs?  Equations of shear stress and capture efficiency could be presented. Did you assume laminar flow and no slip boundary condition? What is the residence time of cells in the device? More details are needed. After the introduction, there is a “Results and Discussion” section, followed by section 3 (Discussion) which repeats the last paragraph from the introduction. There are some typos: e.g. line 125. The working principle of the chip should be also described in the main text not only in the description of figure 3.

Response 2: Agree. We have accordingly done to emphasize this part. “Materials and Methods” section has been added. Thank you for pointing out. All steps had been clearly described so that the study can be reproduced. The computational mesh has been included. We did not assign specific diameters for RBCs, WBCs and CTCs. Equations of shear stress and capture efficiency was presented. Yes, we assume laminar flow and there is boundary condition. Since there were no cells in the device there is not residence time. Discussion has been modified not to be similar as the last paragraph in the Introduction.

The working principle of the chip was described in the main text instead in the description of figure 3.

Working principle of this multifunctional double-array petals flower-shaped microfluidic chip is described as following shown in Figure 3. It is combined both physical and affinity properties of CTCs isolation. When patient blood samples are introduced from center inlet antigen of CTCs such as EpCAM (Epithelial C Epithelial Cell Adhesion Molecule) bonded with antibody modified channel surfaces such as anti-EpCAM. CTCs are blocked by and have a collision with the protuberant parts, and then slow down and recline on the small hills. WBCs (White blood cells) and RBCs (Red blood cells) flow freely away from the central line.

The second structure is S-shaped concave microposts enclosed by parabolic circular microposts array CTCs. CTCs could be captured by grooves of concave triangle microposts, or the gaps of 5 μm formed by adjacent microposts. Relatively small sized and more deformable WBCs and RBCs could pass away. Parabolic circular array segregate CTCs once again.

For the third structure ellipse, inverted ellipse and circular microposts are organized close to outlet. Big elliptical microposts are utilized to function as flow diversion. Therefore, the diverting flow has to transverse out from the gaps formed by neighboring microposts. Blood cells flow easily away from outlet.

1.      Materials and Methods

Simulation

An example was given in following about how to perform the Comsol simulation.

Open the AutoCAD software and draw an ellipse centered at the origin with the major axis along Y-axis with length of 300 μm, and the minor axis along the X-axis with a length of 190 μm. Take the upper half of the ellipse, and draw seven circles with a diameter of 100 μm for each. Those circles are spaced at 8 μm apart, and with their centers all on the upper half of the ellipse. Draw a rectangle centered at the origin with a length of 900 μm and a width of 550 μm. Remove the elliptical portion and save the file as a dxf file. 

Open COMSOL software, click the Model Wizard, select 2D spatial dimensions, and in the physics field selection, choose Fluid Flow, Laminar Flow, and One-way Flow in sequence. Select Add and proceed to the Study Options, where you can choose the Steady-State option under General Studies. After setting the conditions locate the Geometry button in the Model Builder, right-click, and import the previously drawn dxf file. Verify that the imported model is correct. In the Model Builder, right-click the Laminar Flow option and add an inlet and outlet. Set the inlet to the lower short edge of the rectangle with a velocity of 4 μm/s, and the outlet to the upper short edge with a pressure of 0 Pa. Right-click the Mesh option in the Model Builder, select Free Quadrilateral Mesh, and choose the entire geometry as the domain. Click Build Selected Objects to complete the meshing. Finally, in the Model Builder, click the Study option, select Compute, and wait for the simulation results.

Figure 8 Computational mesh for simulation for the parabolic circular microposts array.

Isolation values

Capture efficiency (%)= CTCs captured in the chip/total CTCs introduced (including RBCs, WBCs, and CTCs)*100%

Or

Capture efficiency (%)= CTCs captured in the chip/(CTCs captured in the chip +CTCs flowed out)*100%

Isolation purity (%)= CTCs captured in the chip/total cells captured in the chip (including RBCs, WBCs, and CTCs)*100%

The core calculation formula for fluid shear stress is, which means that the magnitude of shear stress depends on the viscosity of the fluid and change of the speed of velocity.

 represents shear stress, which is the force generated by the friction between fluid layers, measured in pascals (Pa).

 represents dynamic viscosity, a physical quantity that reflects the viscosity of a fluid, measured in pascals second (Pa · s).

represents velocity gradient (also known as shear rate), describing the speed at which fluid velocity varies with distance, measured in seconds (s ⁻¹).