*Article* **New Method for Preparing Small-Caliber Artificial Blood Vessel with Controllable Microstructure on the Inner Wall Based on Additive Material Composite Molding**

**Junchao Hu, Zhian Jian, Chunxiang Lu, Na Liu, Tao Yue , Weixia Lan and Yuanyuan Liu \***

School of Mechatronics Engineering and Automation, Shanghai University, Shanghai 200444, China; hujunchao@shu.edu.cn (J.H.); jianzhian@shu.edu.cn (Z.J.); 20721813@shu.edu.cn (C.L.); liuna\_sia@shu.edu.cn (N.L.); tao\_yue@shu.edu.cn (T.Y.); weixia\_lan@shu.edu.cn (W.L.) **\*** Correspondence: yuanyuan\_liu@shu.edu.cn

**Abstract:** The diameter of most blood vessels in cardiovascular and peripheral vascular system is less than 6 mm. Because the inner diameter of such vessels is small, a built-in stent often leads to thrombosis and other problems. It is an important goal to replace it directly with artificial vessels. This paper creatively proposed a preparation method of a small-diameter artificial vascular graft which can form a controllable microstructure on the inner wall and realize a multi-material composite. On the one hand, the inner wall of blood vessels containing direct writing structure is constructed by electrostatic direct writing and micro-imprinting technology to regulate cell behavior and promote endothelialization; on the other hand, the outer wall of blood vessels was prepared by electrospinning PCL to ensure the stability of mechanical properties of composite grafts. By optimizing the key parameters of the graft, a small-diameter artificial blood vessel with controllable microstructure on the inner wall is finally prepared. The corresponding performance characterization experimental results show that it has advantages in structure, mechanical properties, and promoting endothelialization.

**Keywords:** small caliber blood vessel; composite molding; micro-nano structure; tissue repair; 3D printing

#### **1. Introduction**

The incidence and mortality of cardiovascular diseases have been showing an upward trend year by year, which seriously affects human health [1]. Although a large number of vascular stents have been used clinically, most of them are built-in stent products for large blood vessels, and the treatment of small-diameter vascular diseases and functional defects is still a challenge [2,3]. Most of the blood vessels in the cardiovascular and peripheral vascular system are less than 6 mm in diameter [4]. Due to the small inner diameter of such blood vessels and the slow blood flow rate, built-in stents often lead to problems such as thrombosis. Direct replacement with artificial blood vessels is an important potential method [5,6].

The small-caliber blood vessels in the human body are not only small in diameter and thin in wall, but also have a complex layered structure, including an inner layer that supports cells and induces platelet adhesion and aggregation, as well as a middle layer and an outer layer that provide mechanical support [7–10]. In order to allow the artificial blood vessel to fuse with the host blood vessel after being transplanted into the body, and to quickly achieve the metabolic function of the natural blood vessel, the construction of a small-caliber artificial blood vessel must not only meet the bionic structure and mechanical properties, but also achieve rapid and effective endothelialization. This poses a challenge to both the material design and preparation process [11,12]. A large number of studies have shown that specific microstructures can guide the behavior and arrangement of cells. However, there are few studies that combine this type of research

**Citation:** Hu, J.; Jian, Z.; Lu, C.; Liu, N.; Yue, T.; Lan, W.; Liu, Y. New Method for Preparing Small-Caliber Artificial Blood Vessel with Controllable Microstructure on the Inner Wall Based on Additive Material Composite Molding. *Micromachines* **2021**, *12*, 1312. https://doi.org/10.3390/mi12111312

Academic Editors: Rui A. Lima, Susana Catarino and Graça Minas

Received: 8 October 2021 Accepted: 23 October 2021 Published: 26 October 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

with the construction of small-caliber blood vessels. At the same time, it must be able to take into account the multiple requirements of bionic small-caliber blood vessels in terms of structure and mechanical properties. In addition, in order to obtain a perfect small-caliber artificial blood vessel, the choice of materials is also very important [13–16]. Because a single material often cannot effectively take into account both biological functions and mechanical properties, natural biological materials combined with polymer composites have become a hot spot in current research [17–19]. However, there is still a lack of systematic composite molding process research, and the research on achieving controllable composites of different materials and structures is still very imperfect.

In this context, this research proposes an additive composite molding method that combines electrospinning [20–22] and electrostatic direct writing [23] micro-imprint technology. The specific process is as follows. As shown in Figure 1, the first major link includes the following three steps: First, the film structure is obtained by micro-imprinting the material, and, considering the needs of cell adhesion and growth, the film structure can also be imprinted into a surface with a specific microstructure. Then, electrostatically direct wiring forms an orderly arranged fiber structure and transfers the orderly arranged fiber structure to the film structure prepared in advance by embossing. On this basis, the second major link is the process of dynamically shaping the formed two-dimensional film structure into a tube through a tubular mold, which specifically includes the following two steps: one is based on a pre-designed and prepared tubular mold, using thin long tweezers extended from the tail of the mold, and the head stretches out to clamp the previously prepared base film and drag it into the tube. The film will be passively rolled into a tube due to the friction with the tube wall and the boundary effect. The diameter of the tube formed is the inner diameter of the mold. Second, under the drive of the three-axis motion platform, the nanofiber film is directly wrapped on the surface of the above-mentioned dynamically crimped tube through the electrospinning process, which can realize the shape of the dynamically formed tube, and it can also realize the on-demand optimization of the mechanical properties and biological properties of the overall pipe structure.

It should be pointed out that the effective realization of the process method proposed above is closely related to the materials selected and the structural parameters of the pipe to be prepared. First of all, in order to realize that the imprinted film is dynamically shaped into a tube, the mechanical properties of the material and the imprinted film thickness parameters need to be weighed and optimized. For this reason, this article chose a polyether ether ketone (PPDO) material with good biocompatibility, good melting characteristics, and ductility. It is a biodegradable and biocompatible aliphatic polyether ester. It has been approved by the FDA for use, and its degradation is mainly hydrolytic cleavage, which can form low-molecular-weight substances consistent with human metabolites, which can be metabolized or bioabsorbed by the human body [24,25]. Considering the need for cell adhesion growth in the later stage, the PPDO film prepared by imprinting can be surface functionalized by plasma treatment and dopamine soaking. Secondly, the ordered fibers of electrostatic direct writing can be effectively transferred to the imprinted twodimensional film structure, and it is necessary to comprehensively consider the interface energy competition between the direct writing material, the receiving interface, and the imprinting film. In order to solve this problem, this article proposes that the material selected for electrostatic direct writing and the embossed film material need to have different thermal melt ductility, so that effective transfer can be achieved by adjusting the temperature around the transfer device during the transfer process. In this paper, based on the temperature characteristics of PPDO, combined with the requirements of the electrostatic direct writing process, polycaprolactone (PCL) is selected for electrostatic direct writing. Taking into account the needs of cell adhesion and growth in the later stage, PCL materials can also be compounded with materials with good cell affinity such as gelatin.

materials with good cell affinity such as gelatin.

**Figure 1.** Process flow chart of preparation of small-diameter blood vessels. **Figure 1.** Process flow chart of preparation of small-diameter blood vessels.

It is not difficult to find that the process method proposed in this paper has good processing flexibility and can be controlled in the form of inner wall microstructure, pipe diameter, and wall thickness. The corresponding material selection and specific ratio can also be controlled, adjusted, and designed as needed. It is not difficult to find that the process method proposed in this paper has good processing flexibility and can be controlled in the form of inner wall microstructure, pipe diameter, and wall thickness. The corresponding material selection and specific ratio can also be controlled, adjusted, and designed as needed.

cell adhesion and growth in the later stage, PCL materials can also be compounded with

#### **2. Materials and Methods 2. Materials and Methods**

#### *2.1. Solution Preparation 2.1. Solution Preparation*

Preparation of pcl solution: 2.25 g pcl (Average Mn 80000, Sigma-Aldrich, Co., Haverhill, UK) particles were dissolved in 10 mL dichloromethane (Molecular weight 84.93, XiYaShiJi, Shanghai, China) and 5 mL dimethylformamide DMF (99.5%, Shanghai Aladdin Biochemical Technology Co., Ltd., Shanghai, China), and stirred under a magnetic stirrer (Shanghai MeiYingPu Instrument Manufacturing Co., Ltd., Shanghai, China) Preparation of pcl solution: 2.25 g pcl (Average Mn 80000, Sigma-Aldrich, Co., Haverhill, UK) particles were dissolved in 10 mL dichloromethane (Molecular weight 84.93, XiYaShiJi, Shanghai, China) and 5 mL dimethylformamide DMF (99.5%, Shanghai Aladdin Biochemical Technology Co., Ltd., Shanghai, China), and stirred under a magnetic stirrer (Shanghai MeiYingPu Instrument Manufacturing Co., Ltd., Shanghai, China) for 2 h at a concentration of 15% (*w*/*v*).

for 2 h at a concentration of 15% (*w*/*v*). Preparation of pcl-gelatin solution: 0.8 g of pcl particles were dissolved in 10 mL of trifluoroethanol (99.5%, Shanghai Aladdin Biochemical Technology Co., Ltd.), and stirred under a magnetic stirrer for 2 h. Subsequently, 0.2 g of gelatin (VetecTM reagent grade, Type A, Sigma-Aldrich, Co., St. Louis, MO, USA) was added and stirred for two hours at 37 degrees, and finally 38 ul of crosslinking agent was added and stirred for 2 h Preparation of pcl-gelatin solution: 0.8 g of pcl particles were dissolved in 10 mL of trifluoroethanol (99.5%, Shanghai Aladdin Biochemical Technology Co., Ltd.), and stirred under a magnetic stirrer for 2 h. Subsequently, 0.2 g of gelatin (VetecTM reagent grade, Type A, Sigma-Aldrich, Co., St. Louis, MO, USA) was added and stirred for two hours at 37 degrees, and finally 38 uL of crosslinking agent was added and stirred for 2 h at a concentration of 10% (*w*/*v*).

at a concentration of 10% (*w*/*v*). Dopamine solution preparation: 0.02 g dopamine (Shanghai KeLaMan Reagent Co., Ltd., Shanghai, China) powder was dissolved in 10 mL Tris buffer to prepare a dopamine Dopamine solution preparation: 0.02 g dopamine (Shanghai KeLaMan Reagent Co., Ltd., Shanghai, China) powder was dissolved in 10 mL Tris buffer to prepare a dopamine solution with a concentration of 2 mg/mL.

solution with a concentration of 2 mg/mL. ppdo: Poly(p-dioxanone) ppdo (Suzhou JIAYE Biotechnology Co., Ltd., Suzhou, China) material is granular. It is a kind of aliphatic polyester ether with good biodegradability and biocompatibility. Its unique ether ester structure gives the material high strength and good flexibility. The melting point is 109 °C. In this experiment, ppdo film PPDO: Poly(p-dioxanone) PPDO (Suzhou JIAYE Biotechnology Co., Ltd., Suzhou, China) material is granular. It is a kind of aliphatic polyester ether with good biodegradability and biocompatibility. Its unique ether ester structure gives the material high strength and good flexibility. The melting point is 109 ◦C. In this experiment, PPDO film was prepared under a hot machine (Qingdao Jinggang hot stamping equipment Co., Ltd., Qingdao, Shandong, China).

#### *2.2. Film Fabrication Approaches*

#### 2.2.1. Embossed Film Structure Preparation

A plan view of the groove structure is drawn on the drawing software CAD, and the mask plate is processed and prepared. First, a silicon wafer substrate containing a patterned structure is prepared, and the silicon wafer is cleaned then heated on a heating plate at 200 ◦C for 5 min to remove surface water molecules, followed by spin-coating su-8 2000 glue on the substrate with thick glue spin glue technology. The specific parameters of the rotation speed are: first accelerate to 500 r at an acceleration of 100 r/s and continue for 5 s, then adjust the speed to 2000 r for 30 s, and place it at 95 Heating on a hot plate at 95 ◦C for 1 min, the thickness of the su-8 2000 adhesive layer is about 0.6 mm.

The mask plate is attached to the adhesive layer and exposed with a lithography machine for 7 s, then heated on a 95 ◦C heating plate for 1 min, and finally immersed in a developer for 1 min for development and then dried to obtain a pattern-containing mold. The mold is placed in a petri dish, and 28 gpdms (SYLGARDTM 184) and 3 gpdms curing agent are mixed in a beaker and stirred with a glass rod for 3–5 min until milky white and the bubbles are small and uniform. Then it is poured into the petri dish and placed in a vacuum machine for 15 min, then placed in a fume hood for 8 h, and finally baked in an oven at 60 ◦C for 2 h to obtain a pdms mold with grooved microstructure. Same as above, the steps are repeated to prepare multiple ordinary pdms molds without patterns as auxiliary devices for the imprinting process.

Then, 1 g of PPDO (BaiMuDa, Nanjing, China) particles are placed evenly on the ordinary pdms mold, which are moved to the heating plate with the temperature kept at 120 degrees Celsius. They are heated for 5 min until the PPDO particles melt into a liquid state, and then the whole is transferred to the bottom of the imprinting machine. The PDMS mold containing the groove structure is put on the top to fit it, the air pump control valve adjusted to 0.3 MPa, the imprinting machine control switch turned on, and the hot plate squeezes the PPDO material downwards to adjust the temperature control and time module. The temperature is 120 ◦C and the duration is 30 min. After cooling, the PPDO base film containing the groove microstructure can be obtained.

#### 2.2.2. Ordered Fiber Structure Preparation

In order to be able to effectively analyze the influence of different material components on subsequent cell behavior, this paper designed two sets of samples, namely the pcl group and the pcl-gelatin group.

The prepared PCL solution is loaded into the syringe piston barrel and connected with the syringe on the micro pump actuator through a catheter. The spinning collector is fixed on the *XY*-axis platform of the three-axis motion platform and the syringe needle and the panel of the spinning collector are made to perpendicularly intersect, adjusting the *Z*-axis slider so that the distance between the end of the syringe needle and the collector is 5 mm. The positive pole of the high-voltage power supply is connected to the metal part of the syringe needle, the negative pole is connected to the metal part of the spinning collector, and the voltage between the two poles is set to 3200 V. The feed flow rate of the micro pump controller is set to 1 mL/h, the reciprocating speed of the needle with the *X*-axis platform of the three-axis motion platform is 0.25 m/s and the single stroke in the positive direction of the *X*-axis is 80 mm. Then, the positive *Y*-axis moves 50 um in the direction, and then moves in the negative direction of the *X*-axis. The distance is 50 um, and the reciprocating movement is repeated many times. With the deposition of pcl on the tin foil of the collector, the tin foil is finally removed to obtain the PCL direct writing fiber structure.

Loading the prepared PCL-gelatin solution into the syringe piston barrel, the flow rate of the micro pump solution is 1 mL/h, the distance between the end of the spinning syringe needle and the collector is 2 mm, and the needle size used is 23 g. The subsequent steps are similar to the previous PCL direct writing steps, and the pcl-gelatin direct writing fiber structure can be obtained.

#### 2.2.3. Preparation of Composite Film

Similar to the steps for preparing the embossed film, firstly, the PPDO base film is prepared by embossing with pdms chips that do not contain microstructures, and the preparation parameters remain the same as above. The PCL direct writing structure is placed together with the tin foil on the base of the imprinting machine. Covering the PPDO base film on the direct writing structure, the air pump control valve is adjusted to 0.3 MPa and the control switch of the imprinting machine is turned on. At this time, the hot plate will squeeze the PPDO film and the direct writing structure downwards. The temperature control and time module are adjusted to 40 ◦C and the duration is 30 min, and then it is taken out to obtain a composite film of PPDO and direct writing structure.

#### 2.2.4. Preparation of Artificial Blood Vessel

First, a cylindrical through hole with an inner diameter of 4 mm, an outer diameter of 5 mm, and a length of 20 mm is drawn in the solidworks three-dimensional drawing software, and the FDM software is imported to prepare the corresponding mold. Secondly, slender tweezers are used to insert from one end of the mold and extend the other end to clamp the middle of one end of the prepared film and drag it into the tube. Due to the inner wall of the tube, the film spontaneously curls and eventually rolls into a tube. In order to fully discuss the effects of microstructure and materials on cell behavior, the composite films constructed by the above three types of direct-write fibers were respectively crimped, and the film with only embossed groove structure was also selected for crimping. Finally, the three sets of tubular structures prepared above and the mold are connected with a shaft slightly less than 4 mm in diameter, are assembled on a rotating motor, and placed under a three-axis platform for electrospinning. Loading the prepared PCL solution into the syringe piston barrel, it is connected to the micro pump actuator, and the *Z*-axis slider is adjusted so that the distance between the end of the syringe needle and the collector is 100 mm. The voltage between the two poles is set to 7 kV. The feed flow rate of the micro pump controller is set to 1 mL/h, and the internal tubular structure is drawn out by 5 mm for every 5 min of spinning. When all of them are taken out, the blood vessel stent can be obtained; that is, the pcl, pcl-gelatin and PPDO with a groove structure inside the stent.

#### *2.3. Characterization of Vascular Grafts*

#### 2.3.1. Morphology Observation

In order to observe the guiding structure of the film sample and the macroscopic layered structure of the composite blood vessel, the blood vessels prepared in the imprint group, the pcl direct writing composite group film, and the pcl direct writing composite group were prepared respectively. The morphology of the film sample was detected by an optical microscope to observe the surface structure and morphology. The observation of blood vessels was mainly to observe the layered structure and overall size.

#### 2.3.2. Mechanical Properties

The prepared imprinted film, the film compounded with pcl direct writing fiber, the artificial blood vessel prepared with the imprinted film, and the artificial blood vessel prepared with the film compounded with pcl direct writing fiber were respectively subjected to an axial pull-up test. The prepared film has a size of 20 × 16 mm and a thickness of 0.3 mm, and the corresponding tube is prepared on the basis of the two films of the above specifications. All samples were covered with a pcl electrospun film after preparation, and the electrospinning parameters were all kept the same as described above.

The film and the tube sample are clamped on the universal testing machine. Taking the distance between the two clamps as the initial length, at room temperature, the test piece is stretched at a crosshead speed of 20 mm/min until it breaks. Assuming the incompressibility of the material, and considering the length and cross-sectional area, the load-displacement curve is calculated to determine the stress-strain relationship, using the formula ε = (LF − L1)/L1 to calculate the strain based on the initial length of the specimen (L1) and the tensile specimen length when the force (F) is applied to the specimen (LF). The tensile stress is calculated using σ = F/S, where S is the cross-sectional area of the sample. In this case, the cross-sectional area is calculated as S = tw, where t is the thickness of the stent and w is the width of the sample. Here, when stretched, it is divided into the film group (including the imprinted film and the pcl direct writing composite film) and the tubular graft group (including the imprinted blood vessel and the pcl direct writing composite blood vessel). At the same time, when the film group is stretched, it is divided into straight groove or direct writing structure stretch, and the corresponding tubular components are axial stretch and radial stretch.

#### 2.3.3. Suture Maintains Strength

Suture retention strength (SRS) is commonly used to measure the ability of a suture to adhere a graft to surrounding tissues. A universal testing machine is used to test the suture retention strength. Each is cut to obtain a film sample (length = 20 mm, width = 16 mm). Each sample is clamped in the test device at the edge of the film sample originally located. Using a 5-0 nylon surgical suture (Yangzhou Yuankang Medical Instruments Co., Ltd., Yangzhou, China), the other end of the sample is sutured to a distance of 2 mm from the end. The distance between the two needles is 2 mm. The suture is fixed on the hole in the self-made orifice plate, which is connected to the fixture of the test equipment. The suture is pulled out at an extension rate of 2 mm/s. SRS is calculated by dividing the maximum force recorded before the suture is pulled out by the number of sutures.

#### 2.3.4. In Vitro Cytocompatibility

The cells were selected from the same batch of endothelial cells (Human Umbilical Vein Endothelial Cells, HUVEC), and resuscitated in four bottles. After resuscitation, they were added to each sample dish for culture. Each culture sample dish was pre-added with 3 mL of culture medium (Lifeline Cell Technology, LLC, Lonza, Walkersvile, MD, USA). Finally, it was placed in a 37 degree incubator the culture medium changed every three days. During cell culture, the culture flask is pretreated first; that is, 50 mL gelatin solution with a concentration of 0.1% *w/v* is prepared, filtered with a syringe filter (0.22 u), and then used. Finally, the culture flask is filled with the filtered gelatin solution (10 cm diameter petri dish requires 2 mL of solution), until the bottom of the bottle is covered and left at room temperature for 5 min, and then suck gelatin solution is sucked from the petri dish, which can be used for endothelial cell resuscitation.

In the endothelial cell inoculation experiment, four groups were prepared: the PPDO film group without microstructure, PPDO imprint film group, pcl direct writing compound group, and pcl gelatin direct writing compound group. The sample preparation method of the composite group is as shown in the previous section, which is a composite of pcl or pcl gelatin direct writing structure and unpatterned PPDO base film. The sample size of each group is four, of which the unstructured PPDO film group is mainly used as a control group to compare and observe the influence of groove structure on cell behavior. The pcl direct writing compound group and the pcl gelatin direct writing compound group can be used for comparison and the influence of the material components forming the microstructure on the cell behavior can be analysed.

Before cell inoculation, the four groups of samples were treated with plasma for 90 s, and then the four groups of samples were immersed in dopamine solution, kept in the dark at room temperature for 24 h, then rinsed with deionized water three times, and finally placed in room temperature to air dry. Before cell seeding, all samples were irradiated with ultraviolet light on both sides for 30 min, and sealed and stored in a refrigerator at −20 ◦C.

The above-mentioned processed samples were inoculated and cultured with cells according to the groups, and the growth of cells on the samples at 1, 3, 7, and 14 days was recorded for each group of cells. The samples that need to observe the results of cell growth were stained with crystal violet to observe the staining and growth of the cells. The specific staining steps are as follows: first, the waste liquid of the sample is aspirated and DPBS

buffer is added for washing; then, the DPBS buffer is aspirated, 1 mL of paraformaldehyde solution is added, and it is left for 10–15 min. Then, the paraformaldehyde solution is aspirated and washed using the DPBS solution. Finally, crystal violet solution was added and the sample was immersed, placed on a shaker for 10 min, and finally the crystal violet solution was sucked out and cleaned, and the processed sample was placed under a microscope to observe the cell growth results. growth were stained with crystal violet to observe the staining and growth of the cells. The specific staining steps are as follows: first, the waste liquid of the sample is aspirated and DPBS buffer is added for washing; then, the DPBS buffer is aspirated, 1 mL of paraformaldehyde solution is added, and it is left for 10-15 min. Then, the paraformaldehyde solution is aspirated and washed using the DPBS solution. Finally, crystal violet solution was added and the sample was immersed, placed on a shaker for 10 min, and finally the crystal violet solution was sucked out and cleaned, and the processed sample was placed

Before cell inoculation, the four groups of samples were treated with plasma for 90 s, and then the four groups of samples were immersed in dopamine solution, kept in the dark at room temperature for 24 h, then rinsed with deionized water three times, and finally placed in room temperature to air dry. Before cell seeding, all samples were irradiated with ultraviolet light on both sides for 30 min, and sealed and stored in a refrig-

The above-mentioned processed samples were inoculated and cultured with cells according to the groups, and the growth of cells on the samples at 1, 3, 7, and 14 days was recorded for each group of cells. The samples that need to observe the results of cell

#### **3. Results** under a microscope to observe the cell growth results.

erator at −20 °C.

#### *3.1. Morphology of Vascular Grafts* **3. Results**

*Micromachines* **2021**, *12*, x FOR PEER REVIEW 7 of 14

Figure 2 shows the morphological observation results of the vascular graft. Figure 2A is the imprinted film, Figure 2B,C are the observation results under different magnification microscopes, Figure 2D is the pcl direct writing composite group film, Figure 2E,F are the observation results under different magnification microscopes, Figure 2G is the pcl Gelatin direct writing composite film, and Figure 2H,I are the observation results under different magnification microscopes. The inner diameter of the tubular structure is about 4 mm, as shown in Figure 2J. The layered structure of the stent remains intact, and the inner and outer membranes can be clearly seen, as shown in Figure 2K. Figure 2L is a macroscopic view of the composite tube. It can be seen that there is a composite direct writing structure on the inner wall. The thickness of the whole membrane is about 0.3 mm, which is close to the average thickness of human veins of 346 ± 121 um. *3.1. Morphology of Vascular Grafts* Figure 2 shows the morphological observation results of the vascular graft. Figure 2A is the imprinted film, Figure 2B,C are the observation results under different magnification microscopes, Figure 2D is the pcl direct writing composite group film, Figure 2E,F are the observation results under different magnification microscopes, Figure 2G is the pcl Gelatin direct writing composite film, and Figure 2H,I are the observation results under different magnification microscopes. The inner diameter of the tubular structure is about 4 mm, as shown in Figure 2J. The layered structure of the stent remains intact, and the inner and outer membranes can be clearly seen, as shown in Figure 2K. Figure 2L is a macroscopic view of the composite tube. It can be seen that there is a composite direct writing structure on the inner wall. The thickness of the whole membrane is about 0.3 mm, which is close to the average thickness of human veins of 346 ± 121 um.

**Figure 2.** Artificial blood vessel size and film structure: (**A**) is the PPDO imprinting group film structure prepared by imprinting with the pdms chip containing microstructures at 120 ◦C for 30 min at 120 ◦C. (**B**,**C**) is the imprinting group film at different magnifications. The microstructure diagram under the microscope (**D**) is a sample film composed of PPDO base film and PCL direct writing structure imprinted with a pdms chip that does not contain microstructures in an imprinting machine at 120 ◦C for 30 min. The temperature is 40 ◦C, and the printing time is 30 min. (**E**,**F**) is the microstructure of the PCL direct-write composite film under different magnification microscopes. (**G**) is the imprinting with the pdms chip without microstructure at 120 ◦C. The sample film composed of the PPDO base film prepared in 30 min and the PCL-gelatin direct writing structure meets the parameters of a temperature of 40 ◦C and an imprinting time of 30 min (**H**,**I**) for the PCL-gelatin direct writing composite film under different magnification microscopes. The microstructure diagram (**J**) is the overall size of the vascular graft (**K**) is the magnified diagram of the layered structure of the blood vessel (**L**) is the composite diagram of the direct writing structure of the inner wall of the vascular graft.

#### *3.2. Mechanical Properties*

The tensile test was carried out on the tensile machine to obtain the stress-strain curve of tensile strength and elongation at break, as shown in Figure 3. Since the difference between the pcl composite group and the pcl gelatin group is mainly in affecting cell growth, they are regarded as the same group for mechanical performance testing. In this experiment, the pcl composite group was selected for testing; the imprinting group with or without microstructures is similar. The situation is regarded as the same group for mechanical performance testing. In this experiment, a sample group with a micro-groove structure was selected for testing. Figure 3A,B are, respectively, the radial tensile and tubular axial tensile stress-strain curves of the film sample, and Figure 3D,E are the radial tensile and tubular axial tensile stress-strain curves of the tubular sample, respectively. *Micromachines* **2021**, *12*, x FOR PEER REVIEW 9 of 14 N; the maximum load of the imprint group sample is 19.420 N, and the final suture force is 4.855 N.

**Figure 3.** Tensile mechanics test: (**A**) is the drawing of the imprinted film and the pcl direct writing composite film in the vertical groove direction. The tensile strength of the composite stent is 18.154 MPa, and the tensile strength of the imprinted stent is 9.800 Mpa. (**B**) is the drawing of the imprinted film and the pcl direct-writing composite film stretching in the direction of the groove, the tensile strength of the composite stent is 27.784 MPa, and the tensile strength of the imprinted stent is 20.516 Mpa. (**C**) is the film tensile test graph. (**D**) is the drawing of the embossed tubular sample and the pcl direct-write composite tubular sample in the radial direction. The tensile strength of the composite stent is 3.279 MPa, and the tensile strength of the imprinted stent is 3.189 Mpa. (**E**) is the embossed tube. The sample and pcl direct-write composite tubular sample stretched along the axial direction, the tensile strength of the composite stent was 4.476 MPa, and the tensile strength of the imprinted group stent was 6.026 Mpa (**F**) for the tubular tensile experiment. **Figure 3.** Tensile mechanics test: (**A**) is the drawing of the imprinted film and the pcl direct writing composite film in the vertical groove direction. The tensile strength of the composite stent is 18.154 MPa, and the tensile strength of the imprinted stent is 9.800 Mpa. (**B**) is the drawing of the imprinted film and the pcl direct-writing composite film stretching in the direction of the groove, the tensile strength of the composite stent is 27.784 MPa, and the tensile strength of the imprinted stent is 20.516 Mpa. (**C**) is the film tensile test graph. (**D**) is the drawing of the embossed tubular sample and the pcl direct-write composite tubular sample in the radial direction. The tensile strength of the composite stent is 3.279 MPa, and the tensile strength of the imprinted stent is 3.189 Mpa. (**E**) is the embossed tube. The sample and pcl direct-write composite tubular sample stretched along the axial direction, the tensile strength of the composite stent was 4.476 MPa, and the tensile strength of the imprinted group stent was 6.026 Mpa (**F**) for the tubular tensile experiment.

For the film samples, the results of Figure 3A,B show that the tensile strength of the pcl composite group is worse than that of the imprinting group, and the corresponding elongation is not as good as the imprinting group. In the direction perpendicular to the groove, the tensile strength of the pcl composite stent is 18.154 MPa, and the tensile strength of the imprinted stent is 9.800 Mpa; in the direction of the groove, the tensile strength of the pcl composite stent is 27.784 MPa, and the tensile strength of the imprinted stent is 27.784 MPa. The tensile strength is 20.516 Mpa. Therefore, it can be found that both sets of samples meet the mechanical performance requirements of natural blood vessels, however, whether it is the stretching of the straight writing (imprinting) structure or the stretching of the vertical writing (imprinting) structure, the corresponding elongation of the composite group will be larger.

**Figure 4.** Suture retention experiment: (**A**) is the force\_displacement curve of the embossed film and the pcl direct\_write For the tubular sample, the results of Figure 3C,D show that the pcl composite stent and the PPDO imprinted stent have similar mechanical properties, and the imprinting group also has a larger elongation. In the radial direction, the tensile strength of the pcl composite stent is 3.279 MPa, and the tensile strength of the imprinted stent is 3.189 Mpa;

> Comprehensively looking at the stretch and stitching data, the composite group and the imprint group have similar mechanical performance test results. In fact, the basic film ppdo plays a major role. In terms of stitching, film-like stretch, and tubular stretch

properties, it can be seen that composite stents have certain advantages.

in the axial direction, the tensile strength of the pcl composite stent is 4.476 MPa, and the tensile strength of the imprinted stent. The intensity is 6.026 Mpa. Since the mechanical performance parameters of the ideal blood vessel are 2–3 Mpa in the radial direction and 4–6 Mpa in the axial direction, it can be found that the samples of the composite group and the imprint group basically meet the requirements of the ideal blood vessel. Comparing the mechanical properties of the film stretch, a comprehensive comparison shows that the composite blood vessel has a slight increase in mechanical properties while maintaining the mechanical requirements of the natural blood vessel. **Figure 3.** Tensile mechanics test: (**A**) is the drawing of the imprinted film and the pcl direct writing composite film in the vertical groove direction. The tensile strength of the composite stent is 18.154 MPa, and the tensile strength of the im-

N; the maximum load of the imprint group sample is 19.420 N, and the final suture force

The suture force of the graft is used to evaluate the sutureability of the graft implanted in the body. As can be seen in Figure 4, the stitching performance of the pcl composite stent is better than that of the imprinting group. The results show that: the maximum load of the pcl composite group sample is 23.552 N, and a total of four strands of nylon thread are used for testing during the experiment, so the final suture force is 5.89 N; the maximum load of the imprint group sample is 19.420 N, and the final suture force is 4.855 N. printed stent is 9.800 Mpa. (**B**) is the drawing of the imprinted film and the pcl direct-writing composite film stretching in the direction of the groove, the tensile strength of the composite stent is 27.784 MPa, and the tensile strength of the imprinted stent is 20.516 Mpa. (**C**) is the film tensile test graph. (**D**) is the drawing of the embossed tubular sample and the pcl direct-write composite tubular sample in the radial direction. The tensile strength of the composite stent is 3.279 MPa, and the tensile strength of the imprinted stent is 3.189 Mpa. (**E**) is the embossed tube. The sample and pcl direct-write composite tubular sample stretched along the axial direction, the tensile strength of the composite stent was 4.476 MPa, and the tensile strength of the imprinted group stent was 6.026 Mpa (**F**) for the tubular tensile experiment.

*Micromachines* **2021**, *12*, x FOR PEER REVIEW 9 of 14

is 4.855 N.

**Figure 4.** Suture retention experiment: (**A**) is the force\_displacement curve of the embossed film and the pcl direct\_write composite film suture retention experiment. (**B**) is the schematic diagram of the suture experiment. **Figure 4.** Suture retention experiment: (**A**) is the force\_displacement curve of the embossed film and the pcl direct\_write composite film suture retention experiment. (**B**) is the schematic diagram of the suture experiment.

Comprehensively looking at the stretch and stitching data, the composite group and the imprint group have similar mechanical performance test results. In fact, the basic film ppdo plays a major role. In terms of stitching, film-like stretch, and tubular stretch properties, it can be seen that composite stents have certain advantages. Comprehensively looking at the stretch and stitching data, the composite group and the imprint group have similar mechanical performance test results. In fact, the basic film PPDO plays a major role. In terms of stitching, film-like stretch, and tubular stretch properties, it can be seen that composite stents have certain advantages.

#### *3.3. Hydrophilic Results*

The contact angle is defined as the intersection of the material, water, and air along the surface of the material and the surface of the water droplet. The angle formed by the line, if the contact angle is greater than 90◦ , the material is judged to be hydrophobic, the larger the angle, the higher the hydrophobicity; if the contact angle is less than 90◦ , the material is judged to be hydrophilic, and the smaller the angle is, the material is judged to be hydrophilic. Before the four groups of samples were inoculated with cells, the PPDO base film was plasma treated. It can be seen from Figure 5 that before plasma treatment, the contact angle of the sample was 83◦ , but after plasma treatment, the hydrophilicity of the sample changed to 62◦ . The results show that the plasma treatment experiment can effectively improve the hydrophilicity of the PPDO film. Therefore, for cell seeding, such results are positive.

**Figure 5.** Hydrophilicity of ppdo film before and after plasma treatment: (**A**) before treatment (**B**) after treatment. **Figure 5.** Hydrophilicity of PPDO film before and after plasma treatment: (**A**) before treatment (**B**) after treatment.

The contact angle is defined as the intersection of the material, water, and air along the surface of the material and the surface of the water droplet. The angle formed by the line, if the contact angle is greater than 90°, the material is judged to be hydrophobic, the larger the angle, the higher the hydrophobicity; if the contact angle is less than 90°, the material is judged to be hydrophilic, and the smaller the angle is, the material is judged to be hydrophilic. Before the four groups of samples were inoculated with cells, the ppdo base film was plasma treated. It can be seen from Figure 5 that before plasma treatment, the contact angle of the sample was 83°, but after plasma treatment, the hydrophilicity of the sample changed to 62°. The results show that the plasma treatment experiment can effectively improve the hydrophilicity of the ppdo film. Therefore, for cell seeding, such

#### *3.4. Cell Viability 3.4. Cell Viability*

*3.3. Hydrophilic Results*

results are positive.

In order to study the stratified vascular inner layer membrane designed to be suitable for cell growth and cling, and thus its potential use as a component of vascular grafts, we evaluated the inner membranes of four groups of samples in vitro, and evaluated the cells on their respective substrates for activity, proliferation, and morphology. These four groups of samples were plasma treated and immersed in dopamine solution. In the experimental results on the first day, the orientation of the cells was observed (Figure 6). Secondly, in the 14-day experiment, the metabolic activity and proliferation of endothelial cells was observed (Figure 7). The results in Figure 6 show that there is no obvious regularity in cell growth in the unstructured ppdo film group. From the results of the other three groups of experiments, they have a certain effect on cell growth (Figure 6B). The cells in the pcl direct writing composite group have clinging growth in the direction perpendicular to the direct writing structure. Figure 6C,D has a certain direction in the direction of cell growth, and this kind of orientation is the prerequisite for the regular arrangement of cells in various tissues; it also plays an important role in maintaining In order to study the stratified vascular inner layer membrane designed to be suitable for cell growth and cling, and thus its potential use as a component of vascular grafts, we evaluated the inner membranes of four groups of samples in vitro, and evaluated the cells on their respective substrates for activity, proliferation, and morphology. These four groups of samples were plasma treated and immersed in dopamine solution. In the experimental results on the first day, the orientation of the cells was observed (Figure 6). Secondly, in the 14-day experiment, the metabolic activity and proliferation of endothelial cells was observed (Figure 7). The results in Figure 6 show that there is no obvious regularity in cell growth in the unstructured PPDO film group. From the results of the other three groups of experiments, they have a certain effect on cell growth (Figure 6B). The cells in the pcl direct writing composite group have clinging growth in the direction perpendicular to the direct writing structure. Figure 6C,D has a certain direction in the direction of cell growth, and this kind of orientation is the prerequisite for the regular arrangement of cells in various tissues; it also plays an important role in maintaining specific functions [26]. *Micromachines* **2021**, *12*, x FOR PEER REVIEW 11 of 14

**Figure 6.** Cell growth on the first day: (**A**) unstructured ppdo film group, (**B**) pcl direct writing compound group, (**C**) pcl gelatin direct writing compound group, and (**D**) ppdo imprinted film group. **Figure 6.** Cell growth on the first day: (**A**) unstructured PPDO film group, (**B**) pcl direct writing compound group, (**C**) pcl gelatin direct writing compound group, and (**D**) PPDO imprinted film group.

In addition, a macroscopic view of cell growth is shown in Figure 7. After the first day of inoculation, staining, and observation showed that the cells in the unstructured ppdo film group grew densely, while the other three groups also had cell attachment. On the third day, the number of cells in group A decreased, and the cells in group BCD were in a growing state. The results on the 7th day showed that the number of cells in the AD group continued to decline, while the number of cells in the BC group was larger. This difference may be due to the use of gelatin composite materials in group B, which is conducive to cell growth and clinging [27,28]; while group C has more groove structures than group A, which indicates that the introduced microstructure is conducive to cell In addition, a macroscopic view of cell growth is shown in Figure 7. After the first day of inoculation, staining, and observation showed that the cells in the unstructured PPDO film group grew densely, while the other three groups also had cell attachment. On the third day, the number of cells in group A decreased, and the cells in group BCD were in a growing state. The results on the 7th day showed that the number of cells in the AD group continued to decline, while the number of cells in the BC group was larger. This difference may be due to the use of gelatin composite materials in group B, which is conducive to cell growth and clinging [27,28]; while group C has more groove structures than group A, which indicates that the introduced microstructure is conducive to cell

number of cells is significantly reduced, which indicates that the gelatin composite material has better biocompatibility. The results on the 14th day showed that the growth of the three groups of ACD cells was not as ideal as that of group B, which also verified the

inferences made above.

growth; comparing group D samples with group B, there is less gelatin material, and the number of cells is significantly reduced, which indicates that the gelatin composite material has better biocompatibility. The results on the 14th day showed that the growth of the three groups of ACD cells was not as ideal as that of group B, which also verified the inferences made above. *Micromachines* **2021**, *12*, x FOR PEER REVIEW 12 of 14

**Figure 7.** Growth of cells inoculated in two weeks: (**A**) unstructured ppdo film group, (**B**) pcl gelaTable, (**C**) pcl gelatin direct writing compound group, and (**D**) pcl direct writing compound group. **Figure 7.** Growth of cells inoculated in two weeks: (**A**) unstructured PPDO film group, (**B**) pcl gelaTable, (**C**) pcl gelatin direct writing compound group, and (**D**) pcl direct writing compound group.

The preparation of small-diameter vascular grafts remains a challenge. Simulating

#### **4. Discussion**

**4. Discussion**

natural blood vessels should not only consider the requirements of small-diameter and tubular structures when constructing the microstructure surface, but also the flexibility requirements. On the other hand, the preparation of microstructures needs to take into account the thin-walled, layered, and other structures of natural blood vessels. These are essential for simulating natural blood vessels. To solve this problem, we designed a layered small-diameter vascular graft that mimics the structure of human blood vessels: an inner layer suitable for cell adhesion and an outer layer that provides mechanical properties. Here, the inner layer is made by micro-imprinting and electrostatic direct writing technology. The plasma and dopamine treatment of the ppdo base film and the groove structure of the inner wall provide the necessary growth conditions for cell attachment. The outer layer is obtained by electrospinning, which is a technology that can produce a shape and structure similar to the natural extracellular matrix (ECM), which provides mechanical properties for the overall vascular graft. The choice of pcl, gelatin, etc. as raw materials is based on their bioactive compatibility and electrospinning properties. In addition, these two materials have been tested to have good effects as vascular grafts. In addition to the morphology of the basic structure, good mechanical properties are also necessary for small-diameter vascular grafts. The mechanical properties of an ideal The preparation of small-diameter vascular grafts remains a challenge. Simulating natural blood vessels should not only consider the requirements of small-diameter and tubular structures when constructing the microstructure surface, but also the flexibility requirements. On the other hand, the preparation of microstructures needs to take into account the thin-walled, layered, and other structures of natural blood vessels. These are essential for simulating natural blood vessels. To solve this problem, we designed a layered small-diameter vascular graft that mimics the structure of human blood vessels: an inner layer suitable for cell adhesion and an outer layer that provides mechanical properties. Here, the inner layer is made by micro-imprinting and electrostatic direct writing technology. The plasma and dopamine treatment of the PPDO base film and the groove structure of the inner wall provide the necessary growth conditions for cell attachment. The outer layer is obtained by electrospinning, which is a technology that can produce a shape and structure similar to the natural extracellular matrix (ECM), which provides mechanical properties for the overall vascular graft. The choice of pcl, gelatin, etc. as raw materials is based on their bioactive compatibility and electrospinning properties. In addition, these two materials have been tested to have good effects as vascular grafts.

blood vessel are 2–3 Mpa in the radial direction and 4–6 Mpa in the axial direction, and the tensile strength of the vascular graft prepared by us meets the requirements in this respect. Another important mechanical property to consider is suture retention. The experiments in this article show that the mechanical properties of the composite group and the imprint group are similar to each other. In fact, the basic film ppdo plays a major role. It can be seen that composite stents have certain advantages in terms of stitching and film-like tensile properties. In terms of biocompatibility, in vitro cell culture experiments showed good cell compatibility. By recording the cell morphology under different ex-In addition to the morphology of the basic structure, good mechanical properties are also necessary for small-diameter vascular grafts. The mechanical properties of an ideal blood vessel are 2–3 Mpa in the radial direction and 4–6 Mpa in the axial direction, and the tensile strength of the vascular graft prepared by us meets the requirements in this respect. Another important mechanical property to consider is suture retention. The experiments in this article show that the mechanical properties of the composite group and the imprint group are similar to each other. In fact, the basic film PPDO plays a major role. It can be

seen that composite stents have certain advantages in terms of stitching and film-like tensile properties. In terms of biocompatibility, in vitro cell culture experiments showed good cell compatibility. By recording the cell morphology under different experimental groups and different growth periods, it is shown that the samples with PPDO-based film combined with pcl-gelatin direct writing structure are more in line with the expected cell growth effect. In short, in terms of mechanical properties, the composite group has advantages in stretchability and suture performance, so a comprehensive comparison of composite blood vessels is a prerequisite for preparation. In the in vitro cell culture experiment, additional pcl gelatin direct writing composite group samples were added for control. The experimental results show that the pcl-gelatin group has better biocompatibility. In summary, the pcl gelatin direct writing composite group samples meet our expectations.

#### **5. Conclusion and Future Work**

In this paper, a small-diameter graft is prepared by a combination of electrostatic spinning, electrostatic direct writing and micro-imprinting, which is mainly divided into imprinting group and composite group. The macroscopic structure of blood vessels is similar to that of natural blood vessels, and the microscopic structure can also achieve the expected effects of cell experiments. In terms of mechanical properties, for tensile properties (including axial and radial), the samples of the composite group and the imprint group basically meet the requirements of ideal blood vessels, and the tensile performance of the composite group is slightly enhanced. In terms of suturing performance, the composite group also has a slight advantage, so a comprehensive comparison of composite blood vessels is a prerequisite for preparation. In the HUVEC in vitro cell culture experiment, additional pcl gelatin direct writing composite group samples were added for control. HUVEC in vitro cell culture experiments, apoptosis and staining showed the compatibility of the graft, indicating that the pcl-gelatin group has better biocompatibility. To sum up, the pcl gelatin direct writing composite group samples meet our expectations, but there are still content that can be supplemented. For example, a variety of different patterns can be prepared on the inner wall of blood vessels to observe cell growth. Or, possibilities include preparing the outer wall of the blood vessel to control its thickness, observing the optimal mechanical properties, etc., which will become part of the continued research work in the future.

**Author Contributions:** Conceptualization, J.H. and Y.L.; methodology, J.H.; software, Z.J.; validation, C.L., N.L. and J.H.; formal analysis, C.L.; investigation, T.Y.; resources, W.L.; data curation, J.H.; writing—original draft preparation, J.H.; writing—review and editing, J.H. and Y.L.; visualization, W.L.; supervision, T.Y.; project administration, Y.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the grants from the National Natural Science Foundation of China (No. 61973206,61703265, 61803250,61933008), Shanghai Science and Technology Committee Rising-Star Program No. 19QA1403700.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


### *Article* **A Novel Microfluidic Device for Blood Plasma Filtration**

**Zaidon T. Al-aqbi 1,\*, Salim Albukhaty 2,\* , Ameerah M. Zarzoor <sup>3</sup> , Ghassan M. Sulaiman <sup>4</sup> , Khalil A. A. Khalil 5,6, Tareg Belali <sup>5</sup> and Mohamed T. A. Soliman <sup>5</sup>**


**Abstract:** The use of whole blood and some biological specimens, such as urine, saliva, and seminal fluid are limited in clinical laboratory analysis due to the interference of proteins with other small molecules in the matrix and blood cells with optical detection methods. Previously, we developed a microfluidic device featuring an electrokinetic size and mobility trap (SMT) for on-chip extract, concentrate, and separate small molecules from a biological sample like whole blood. The device was used to on-chip filtrate the whole blood from the blood cells and plasma proteins and then on-chip extract and separate the aminoglycoside antibiotic drugs within 3 min. Herein, a novel microfluidic device featuring a nano-junction similar to those reported in the previous work formed by dielectric breakdown was developed for on-chip filtration and out-chip collection of blood plasma with a high extraction yield of 62% within less than 5 min. The filtered plasma was analyzed using our previous device to show the ability of this new device to remove blood cells and plasma proteins. The filtration device shows a high yield of plasma allowing it to detect a low concentration of analytes from the whole blood.

**Keywords:** microfluidics; blood plasma filtration; chip extract; blood molecules

#### **1. Introduction**

Human blood plasma is one of the most convenient and the most important circulating biomarkers sources. Since this free-blood cells matrix has numerous clinically relevant analytes like metabolites, nucleic acids, and proteins, it has become a standard sample for the exclusion or diagnosis of several diseases [1,2]. Moreover, blood plasma is utilized in drug development trials, e.g., for drug monitoring and their metabolites, since it has the drug fraction most relevant to study the pharmacodynamic and pharmacokinetic influences of the drug [3,4]. Plasma transcriptome, proteome, and metabolome studies have increased the spectrum of the diagnostic target for different types of diseases from sepsis to cancer to Alzheimer's [5–7]. Further, foreign nucleic acids as well as antigens and antibodies present in plasma, allow the diagnosis of serious infectious diseases. Additionally, when carrying out plasma analysis in laboratories, blood plasma is useful in the analysis of glucose, total cholesterol, electrolyte concentration, lactate, etc. In clinical chemistry, blood plasma isolation is a necessary step performed, and for the development of miniaturized clinical diagnostic devices, beneficial sample preparation techniques are required [8]. Filtration of plasma from whole blood is very desirable in most cases. Venous blood samples centrifugation is the usual technique to prepare blood plasma with volumes that can be analyzed

**Citation:** Al-aqbi, Z.T.; Albukhaty, S.; Zarzoor, A.M.; Sulaiman, G.M.; Khalil, K.A.A.; Belali, T.; Soliman, M.T.A. A Novel Microfluidic Device for Blood Plasma Filtration. *Micromachines* **2021**, *12*, 336. https:// doi.org/10.3390/mi12030336

Academic Editors: Rui A. Lima and Nam-Trung Nguyen

Received: 22 February 2021 Accepted: 19 March 2021 Published: 22 March 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

using highly sensitive methods like LC-MS/MS (liquid chromatography−tandem mass spectrometry). The expansion of blood plasma separation (BPS) based on microfluidics, dealing only with small sample volumes, has rapidly grown in the field of clinical laboratory medicine [9]. This not only increases patient compliance, convenience, and comfort by reducing the amount of blood required to be pulled allowing point of care (POC) sample collection but also permits the analysis to be performed properly at a part of the duration and cost. Moreover, the potential of on-time repetitive sampling, automated parallelization, and portability are other benefits of performing different procedures at the microscale. Microsampling is a procedure for capturing small volumes of biological samples like whole blood from the human body to be analyzed in a minimally invasive method [10]. To deliver sample volumes sufficient to faithfully detect low concentration analytes from limited sample volumes is still the main challenge with microsampling, so a plasma sampling method must be in a high yield to arrive at reasonable levels in the detection of target analytes at low-concentrations and must give pure plasma without blood cells hemolysis or leakage to be clinically pertinent. Furthermore, extraction time is important to reduce the effect of coagulation, particularly when working with fresh samples, e.g., standard finger pricks. To achieve all these requirements, microfluidics is a favorable technology for manufacturing microminiaturized devices that studies fluids' behavior through micro-channels. There are two types of plasma separation techniques using microfluidics, passive and active. Passive separation techniques demand no external tools, making the devices smaller in size, easier to use, cheaper, and therefore suitable for POC uses [11] while active separation requires exertion of an exterior energy source, e.g., inertial, electric, or acoustic forces [12]. Passive separation techniques can be stored under pressure or driven by capillary forces [13–15]. Using capillary forces in plasma separation is more desirable, since it demands neither vacuum packaging, nor degassing of a suction material [16]. The mechanism of separation is generally based on sedimentation, size exception, or a combination of them [17]. Performing plasma separation using sedimentation gives a pure plasma at the time expense. Plasma separation during size-exclusion avoids the time limitation and permits a quick separation. It could be based on porous media like a filter membrane linked to a capillary channel or a membrane stack [18]. Other microfluidic devices for plasma separation have been demonstrated based on diffusion filter/microfilter [19], bends in micro-channels [20], acoustic waves [21], crossflow filtration [22], and dielectrophoresis [23,24]. Major research projects in recent decades have focused on microfluidics, drug, and gene nano delivery systems, tissue engineering, and biosensors due to cost-effectiveness and high performance [25–29]. Recently, we developed a microfluidic device featuring two nano-junctions with different sizes to form a size and mobility trap (SMT) for on-chip filtrate the whole blood from blood cells and plasma proteins, then on-chip extract, concentrate, and separate small molecules from whole blood [30]. The capability of the device was demonstrated for on-site therapeutic drug monitoring (TDM) of aminoglycoside antibiotic drugs within 3 min. However, this device was fabricated for on-chip filtration and analysis of small molecules from whole blood where the ability to collect the filtered blood for use in other applications is not possible through this device. Here, a novel microfluidic device with a nano-junction created by dielectric breakdown is developed for on-chip filtration and out-chip collection of whole blood to be used in other applications as a pure plasma.

#### **2. Materials and Methods**

#### *2.1. Chemicals and Sample Preparation*

Fluorescein from Sigma-Aldrich (Sydney, Australia) was prepared in Milli-Q water to get 200 µg/mL solution. Fluorescamine from Sigma-Aldrich was prepared in acetone to obtain 3 mg/mL as a stock solution. Bovine Serum Albumin (BSA) from Sigma-Aldrich was prepared in Milli-Q water 2 mg/mL stock solution and then labeled with Fluorescamine in a ratio 3:1 in borate buffer at pH = 9. Polydimethylsiloxane (PDMS) curing agent and elastomer were purchased from Dow Corning (Michigan, MI, USA). Sodium phosphate monobasic, disodium hydrogen phosphate, and sodium tetraborate were purchased from

Sigma-Aldrich (Sydney, Australia) and used for buffer preparation. All solutions were prepared by using Milli-Q water (18 MΩ, Millipore, North Ryde, Australia) purification system. The fresh finger-prick blood used in the experiments was obtained from healthy volunteers approved by the Tasmanian Health and Medical Human Research Ethics Committee, Office of Research Services, the University of Tasmania (Ethics Approval Ref is H0016575). phosphate monobasic, disodium hydrogen phosphate, and sodium tetraborate were purchased from Sigma-Aldrich (Sydney, Australia) and used for buffer preparation. All solutions were prepared by using Milli-Q water (18 MΩ, Millipore, North Ryde, Australia) purification system. The fresh finger-prick blood used in the experiments was obtained from healthy volunteers approved by the Tasmanian Health and Medical Human Research Ethics Committee, Office of Research Services, the University of Tasmania (Ethics Approval Ref is H0016575).

was prepared in Milli-Q water 2 mg/mL stock solution and then labeled with Fluorescamine in a ratio 3:1 in borate buffer at pH = 9. Polydimethylsiloxane (PDMS) curing agent and elastomer were purchased from Dow Corning (Michigan, MI, USA). Sodium

*Micromachines* **2021**, *12*, x FOR PEER REVIEW 3 of 10

#### *2.2. Device Fabrication*

Figure 1A presents an AutoCAD design of the device which was then 3D printed using an Eden 3D printer (Figure 1B) to produce a negative template of the device using the previously described process [31]. Then, PDMS was utilized to give the positive master embossing stamp by mixing 210 g in a mass ratio of 5:1 polymer to the elastomer. The PDMS mixture degassed for 15 min and left out for 30 min at room temperature and then poured onto the negative 3D printed template, and then allowed for curing in an oven at 70 ◦C for at least 12 h. The PDMS positive stamp was cut off and then taken away from the 3D printed template and allowed for thermal age in an oven for 30 min at 250 ◦C. Then, it used to hot emboss the poly (methyl methacrylate) (PMMA) channel plates (1.5 mm × 50 mm × 75 mm). The final PMMA chips were produced using a hot embossing procedure as reported in our previous work [30]. Briefly, PDMS positive stamp and the blank PMMA plate were placed between two 50 mm × 50 mm × 6 mm glass plates and place into the hot embosser (MTP-8, Tetrahedron, San Diego, CA, USA). Three steps were used in the embossing process. Step 1 was done by increasing the temperature from a rate of (92 ◦C/min) until the temperature reached (130 ◦C) and involved maintaining the pressure at 100 lbs. the next step was performed by enhancing the pressure up to 380 lbs at a rate of 75 lbs/min and maintaining the temperature at (130 ◦C). When the pressure goes up to 380 lbs, those conditions were held for 20 min. In the final step, the temperature was reduced at a rate of 15 ◦C /min until the temperature reached (60 ◦C) and involved maintaining the pressure at 380 lbs. The hot-embossed PMMA microchip device was subsequently bonded with single-sided adhesive tape (Tesa SE, Charlotte, NC, USA). An office laminator (Peach 3500, Peach, Switzerland) was then used and the PMMA channel plate and the adhesive tape were sandwiched between two 1 mm stainless steel plates at 20 ◦C temperature and 5 speed at 4 orientation, with 90-degree clockwise rotation at each pass. *2.2. Device Fabrication*  Figure 1A presents an AutoCAD design of the device which was then 3D printed using an Eden 3D printer (Figure 1B) to produce a negative template of the device using the previously described process [31]. Then, PDMS was utilized to give the positive master embossing stamp by mixing 210 g in a mass ratio of 5:1 polymer to the elastomer. The PDMS mixture degassed for 15 min and left out for 30 min at room temperature and then poured onto the negative 3D printed template, and then allowed for curing in an oven at 70 °C for at least 12 h. The PDMS positive stamp was cut off and then taken away from the 3D printed template and allowed for thermal age in an oven for 30 min at 250 °C. Then, it used to hot emboss the poly (methyl methacrylate) (PMMA) channel plates (1.5 mm × 50 mm × 75 mm). The final PMMA chips were produced using a hot embossing procedure as reported in our previous work [30]. Briefly, PDMS positive stamp and the blank PMMA plate were placed between two 50 mm × 50 mm × 6 mm glass plates and place into the hot embosser (MTP-8, Tetrahedron, San Diego, CA, USA). Three steps were used in the embossing process. Step 1 was done by increasing the temperature from a rate of (92 °C/min) until the temperature reached (130 °C) and involved maintaining the pressure at 100 lbs. the next step was performed by enhancing the pressure up to 380 lbs at a rate of 75 lbs/min and maintaining the temperature at (130 °C). When the pressure goes up to 380 lbs, those conditions were held for 20 min. In the final step, the temperature was reduced at a rate of 15 °C /min until the temperature reached (60 °C) and involved maintaining the pressure at 380 lbs. The hot-embossed PMMA microchip device was subsequently bonded with single-sided adhesive tape (Tesa SE, Charlotte, NC, USA). An office laminator (Peach 3500, Peach, Switzerland) was then used and the PMMA channel plate and the adhesive tape were sandwiched between two 1mm stainless steel plates at 20 °C temperature and 5 speed at 4 orientation, with 90-degree clockwise rotation at each pass.

**Figure 1.** (**A**) An AutoCAD design of the filtration device (**B**) photograph image of the negative 3D printed portable device using an Eden 3D printer (**C**) Photograph image of the hot embossed filtration poly (methyl methacrylate) (PMMA)/adhesive tape device filled with green food dye. Scale bar = 10 mm. (**D**) Zoomed-in image of the V channel and the filtration **Figure 1.** (**A**) An AutoCAD design of the filtration device (**B**) photograph image of the negative 3D printed portable device using an Eden 3D printer (**C**) Photograph image of the hot embossed filtration poly (methyl methacrylate) (PMMA)/adhesive tape device filled with green food dye. Scale bar = 10 mm. (**D**) Zoomed-in image of the V channel and the filtration channel (white box in panel C) filled with green food dye. Scale Bar = 200 µm. (**E**) Schematic of the microfluidic device (dimension not to scale) indicating terminating current and voltages used for generation of the nano-junction Sample (S), and Sample Waste (SW).

#### *2.3. Creation of a Nano-Junction*

The complete microchip device is a hybrid hot-embossed PMMA/adhesive tape with a photo of the device shown in Figure 1C and a zoomed-in image of the V-channel and the filtration channel in Figure 1D. The V-channel was 500 µm wide and the filtration channel was 50 µm. The V-channel tip was separated from the filtration channel by a 200 µm gap of PMMA to form the filtration nano-junction. This gap was chosen to allow for lengthy use of higher voltages through the filtration without the secondary breakdown's danger. To form the nanochannel, the V-channel and filtration channel were filled with the breakdown electrolyte, be composed of 10 mM phosphate buffer, pH 11. The filtration nano-junction was created by applying a high voltage of 5000 V to the V-channel whilst the filtration channel was kept grounded Figure 1E. Previously, the high voltage breakdown was 4000 V instead of 5000 V in this work, lower than what was performed in this work. This different voltage in the filtration PMMA devices could be regarding the difference in the gap distance between the separation channel in the double V device and the filtration channel here. The current limit was varied from 1 µA to 5 µA by utilizing an in-house adjustable power supply, controlling by LabView HV V.6 program (National Instrument, Austin, TX, USA) to realize the best repeatability. The V-channel and the filtration channel were cleaned and refilled with the experimental solutions.

#### *2.4. Device Operation and Experimental Practice*

Figure 2A,B show the operation of the filtration device, where the blood volume from a fresh finger prick was controlled by pipetting 50 µL of whole blood to the devices to ensure result comparability and evaluate the filtration method. Pipetting was carried out using an autopipette, and the experiments were performed at room temperature to reduce blood evaporation through the filtration process. A simple technique was used in this approach by pushing the whole blood using the autopipette from the V-channel to the filtration channel through the nanochannel and then a hand syringe-vacuum was used to collect the filtered plasma blood in a high yield. All experiments were performed with a Nikon Eclipse Ti−U inverted fluorescence microscope (Nikon Instruments Inc.) worked with NIS-Elements BR 3.10 software (Melville). A filter cube (Semrock, Rochester) composed of an excitation band-pass filter (488 ± 10 nm), emission filter (520 ± 10 nm), and dichroic mirror to deflect the broadband light source to a 20× objective was used to perform all the experiments. Fluorescence images were carried out using a high-definition color chargecoupled device camera (Digital Sight DS. Filc, Nikon, Japan). A photon multiplier tube (PMT) (Hamamatsu Photonics KK, Hamamatsu, Japan) linked to the microscope was used to record the electropherograms. The PMT was linked to an Agilent 35900E A/D box to allow data collection with the Chemstation software (Agilent Technologies, Waldbronn, Germany). An electrical potential was applied by using an in-house 4-channel (0–5 kV) dc power supply to each reservoir using a custom-designed interface connected to 2 platinum electrodes.

**Figure 2.** (**A**) Schematic of the operation of the microfluidic device (dimension not to scale) indicating the filtration process and (**B**) illustration of the filtration device concept. Nanochannel was formed by controlled dielectric breakdown of the tip of the V-channel and the filtration channel. Large molecules are blocked (Cells and Proteins) while small molecules pass the filtration channel. **Figure 2.** (**A**) Schematic of the operation of the microfluidic device (dimension not to scale) indicating the filtration process and (**B**) illustration of the filtration device concept. Nanochannel was formed by controlled dielectric breakdown of the tip of the V-channel and the filtration channel. Large molecules are blocked (Cells and Proteins) while small molecules pass the filtration channel.

#### **3. Results and Discussion 3. Results and Discussion**

#### *3.1. Filtration and Permeability Studies 3.1. Filtration and Permeability Studies*

Previously, we examined the relevance between the current limit and nano-junction permeability in the double V hot-embossed PMMA device through different breakdown experiments using different charge and size analytes. Briefly, using a current limit of 5 μA, the resulted nanochannels restricted blood cells (6–8 μm) [32], and R-phycoerythrin (RPE) (<10 nm in size) [33] from passing the separation channel while bovine serum albumin (BSA) (2–4 nm) [34] labeled-fluorescamine allowed to pass the separation channel. Reducing the current limit to 1 μA, blocked the BSA, but allowed the transport of anionic small molecules, such as fluorescein (1 nm) [34] and drugs. The SMT in our previous work is based on ions' favorable electrokinetic transport through the resulting nanochannels. The extraction nanochannel was created by applying for a 1 μA permit the transport small ions (<1000 Da) during the nano-junction, whilst blocking the transport of plasma proteins and blood cells. The concentration nanochannel between the V-sample channel and the separation channel was created by applying a 0.1 μA current limit to avoid target analytes transport but allows the transfer of small inorganic ions to make a sample desalting. Both the extraction and concentration nanochannels make the SMT in target analytes which can purify, concentrate, desalt, and separate the sample. Here, we build on our previous breakdown work and implement our new design. The results were similar to those reported in our previous work and summarized in Figure 3. We have formed two types of nano-junctions for the filtration process. First, since the current limit was set at 5 μA (Figure 3A), the produced nanochannels blocked blood cells from infiltrating the filtration channel, while allowing the transport of proteins like BSA into the filtration channel. For protein recovery assessment, the termination current was reduced to 1 μA (Figure 3B) which blocked the BSA but permitted the electrophoretic transport of small molecules Previously, we examined the relevance between the current limit and nano-junction permeability in the double V hot-embossed PMMA device through different breakdown experiments using different charge and size analytes. Briefly, using a current limit of 5 µA, the resulted nanochannels restricted blood cells (6–8 µm) [32], and R-phycoerythrin (RPE) (<10 nm in size) [33] from passing the separation channel while bovine serum albumin (BSA) (2–4 nm) [34] labeled-fluorescamine allowed to pass the separation channel. Reducing the current limit to 1 µA, blocked the BSA, but allowed the transport of anionic small molecules, such as fluorescein (1 nm) [34] and drugs. The SMT in our previous work is based on ions' favorable electrokinetic transport through the resulting nanochannels. The extraction nanochannel was created by applying for a 1 µA permit the transport small ions (<1000 Da) during the nano-junction, whilst blocking the transport of plasma proteins and blood cells. The concentration nanochannel between the V-sample channel and the separation channel was created by applying a 0.1 µA current limit to avoid target analytes transport but allows the transfer of small inorganic ions to make a sample desalting. Both the extraction and concentration nanochannels make the SMT in target analytes which can purify, concentrate, desalt, and separate the sample. Here, we build on our previous breakdown work and implement our new design. The results were similar to those reported in our previous work and summarized in Figure 3. We have formed two types of nano-junctions for the filtration process. First, since the current limit was set at 5 µA (Figure 3A), the produced nanochannels blocked blood cells from infiltrating the filtration channel, while allowing the transport of proteins like BSA into the filtration channel. For protein recovery assessment, the termination current was reduced to 1 µA (Figure 3B) which blocked the BSA but permitted the electrophoretic transport of small molecules (<1000 Da) such as fluorescein and drugs. Based on the transport results of different sized molecules (fluorescein 1 nm, BSA 2–4 nm, and Blood cell 4–6 µm), the size of fabricated nanochannels can be estimated with this method.

(<1000 Da) such as fluorescein and drugs. Based on the transport results of different sized molecules (fluorescein 1 nm, BSA 2–4 nm, and Blood cell 4–6 μm), the size of fabricated

nanochannels can be estimated with this method.

**Figure 3.** Screenshots presenting the restriction (left column) and passing (right column) of different molecules through nano-junctions formed under different conditions. (**A**) shows the use of 5 μA to create nanochannels which transported Bovine Serum Albumin (BSA) (blue, right) and restricted Blood Cells (left); (**B**) shows the use of 5 μA to create nanochannels which restricted BSA (blue, left) and transported fluorescein (green, right). The nanochannels formed using a breakdown electrolyte of 10 mM phosphate buffer, pH = 11, and terminating currents of 5 and 1 μA. Images on the left show blocked transport, while those on the right show the permeability of different molecules. Scale Bar = 200 μm. **Figure 3.** Screenshots presenting the restriction (left column) and passing (right column) of different molecules through nano-junctions formed under different conditions. (**A**) shows the use of 5 µA to create nanochannels which transported Bovine Serum Albumin (BSA) (blue, right) and restricted Blood Cells (left); (**B**) shows the use of 5 µA to create nanochannels which restricted BSA (blue, left) and transported fluorescein (green, right). The nanochannels formed using a breakdown electrolyte of 10 mM phosphate buffer, pH = 11, and terminating currents of 5 and 1 µA. Images on the left show blocked transport, while those on the right show the permeability of different molecules. Scale Bar = 200 µm.

#### *3.2. Analysis of Filtered Blood Plasma 3.2. Analysis of Filtered Blood Plasma*

The color of the plasma was properly similar to centrifuged plasma treated with EDTA, seemed clear without a pink trace in all filtered blood plasma samples which mentioned that the separation of plasma caused no or a little lysis of erythrocytes as clearly seen in Figure 3A. The purpose of the PMMA hot-embossed filtration device is to purify the whole blood from cells and plasma proteins and allow to pass small molecules like pharmaceuticals with filtered plasma. To evaluate the protein recovery assessment, centrifuged plasma relative to filtered plasma spiked with 5 ppm gentamicin and BSA was assessed for both venous blood samples treated with EDTA and filtered finger-prick blood. Two types of filtered plasma using our filtration device were used after applying 5 μA and 1 μA termination currents to create different sized nano-junctions comparing to an EDTA-treated centrifuged plasma. These all were analyzed using our previous SMT device after labeling the plasma with fluorescamine reagent and followed by their separation using electrophoresis. Electrophoresis is a separation approach in which particles, molecules, and ions are separated in a conducting liquid medium using an electric field. All primary amines found in the plasma including those on proteins, amino acids, urea, etc., will be reacted with fluorescamine to produce a fluorescent complex within seconds. Figure 4 shows the electrophoretic separation of filtered blood plasma labeled with fluorescamine after using two different nano-junctions. Filtering whole blood using a device with a termination current of 5 μA and analyzed with the double V-device gives a pure peak for gentamicin, but plasma proteins and BSA are observed. This indicates plasma proteins can pass through the nano-junctions, while blood cells cannot. In contrast, using a termination current of 1 μA gives pure separations due to the observing of small mole-The color of the plasma was properly similar to centrifuged plasma treated with EDTA, seemed clear without a pink trace in all filtered blood plasma samples which mentioned that the separation of plasma caused no or a little lysis of erythrocytes as clearly seen in Figure 3A. The purpose of the PMMA hot-embossed filtration device is to purify the whole blood from cells and plasma proteins and allow to pass small molecules like pharmaceuticals with filtered plasma. To evaluate the protein recovery assessment, centrifuged plasma relative to filtered plasma spiked with 5 ppm gentamicin and BSA was assessed for both venous blood samples treated with EDTA and filtered finger-prick blood. Two types of filtered plasma using our filtration device were used after applying 5 µA and 1 µA termination currents to create different sized nano-junctions comparing to an EDTA-treated centrifuged plasma. These all were analyzed using our previous SMT device after labeling the plasma with fluorescamine reagent and followed by their separation using electrophoresis. Electrophoresis is a separation approach in which particles, molecules, and ions are separated in a conducting liquid medium using an electric field. All primary amines found in the plasma including those on proteins, amino acids, urea, etc., will be reacted with fluorescamine to produce a fluorescent complex within seconds. Figure 4 shows the electrophoretic separation of filtered blood plasma labeled with fluorescamine after using two different nano-junctions. Filtering whole blood using a device with a termination current of 5 µA and analyzed with the double V-device gives a pure peak for gentamicin, but plasma proteins and BSA are observed. This indicates plasma proteins can pass through the nano-junctions, while blood cells cannot. In contrast, using a termination current of 1 µA gives pure separations due to the observing of small molecules without interfacing and also the removal of some larger proteins, allowing full quantitation of

some small molecules in the blood, with a relative standard deviation (RSD%) = 7.5% for gentamicin (n = 3 devices) and with a high recovery efficiency for Gentamicin 94%, and a linear calibration curve of gentamycin from blood is shown in Figure 5. In comparison to other microfluidic devices, the presented device display that the microchip enables the low-cost, rapid, and operationally straightforward separation of plasma from whole blood; also, it demands a small amount of blood to give results within less than 5 min. Therefore, the device provides a favorable solution for biochemical assays. = 7.5% for gentamicin (n = 3 devices) and with a high recovery efficiency for Gentamicin 94%, and a linear calibration curve of gentamycin from blood is shown in Figure 5. In comparison to other microfluidic devices, the presented device display that the microchip enables the low-cost, rapid, and operationally straightforward separation of plasma from whole blood; also, it demands a small amount of blood to give results within less than 5 min. Therefore, the device provides a favorable solution for biochemical assays.

cules without interfacing and also the removal of some larger proteins, allowing full quantitation of some small molecules in the blood, with a relative standard deviation (RSD%)

*Micromachines* **2021**, *12*, x FOR PEER REVIEW 7 of 10

**Figure 4.** Electropherograms show the analysis of filtered blood spiked with 5 ppm gentamicin and BSA after labeling with fluorescamine in size and mobility trap (SMT) device after using a current limit of 5 uA (red trace) and 1uA current limit (black trace). The background electrolyte (BGE) in the separation channel, was 100 mM phosphate buffer, pH 11.5, with 0.5% HPMC, while V-sample waste channel was 10 mM phosphate buffer, pH 11.5. Applied voltages used in SMT device for extraction and concentration were −200, −850, −600, and +650 V for 60s, and separation process were −250, +250, +2200, and −500 V at reservoirs B, S, BW, and SW, respectively. **Figure 4.** Electropherograms show the analysis of filtered blood spiked with 5 ppm gentamicin and BSA after labeling with fluorescamine in size and mobility trap (SMT) device after using a current limit of 5 uA (red trace) and 1uA current limit (black trace). The background electrolyte (BGE) in the separation channel, was 100 mM phosphate buffer, pH 11.5, with 0.5% HPMC, while V-sample waste channel was 10 mM phosphate buffer, pH 11.5. Applied voltages used in SMT device for extraction and concentration were −200, −850, −600, and +650 V for 60s, and separation process were −250, +250, +2200, and −500 V at reservoirs B, S, BW, and SW, respectively.

**References** 

3323–3346.

**Figure 5.** The linear calibration curve for Gentamicin from whole blood.

#### **Figure 5.** The linear calibration curve for Gentamicin from whole blood. **4. Conclusions**

**4. Conclusions**  In this work, we have introduced a new device for blood plasma separation using a piece of PMMA microfluidic device. The PMMA filtration device dominant dielectric breakdown enabling the pore size tuning of nano-junctions and thus their permeability for different sized molecules. The creation of nanochannels using dielectric breakdown is fast, simple, and does not depend on valves or pumps. This manner enabled the analysis of the filtered blood which was labeled with fluorescamine. The device is portable and can be offered for the implementation of blood plasma instead of centrifugation process and in POC devices as the device could be engineered into a hand-held portable device In this work, we have introduced a new device for blood plasma separation using a piece of PMMA microfluidic device. The PMMA filtration device dominant dielectric breakdown enabling the pore size tuning of nano-junctions and thus their permeability for different sized molecules. The creation of nanochannels using dielectric breakdown is fast, simple, and does not depend on valves or pumps. This manner enabled the analysis of the filtered blood which was labeled with fluorescamine. The device is portable and can be offered for the implementation of blood plasma instead of centrifugation process and in POC devices as the device could be engineered into a hand-held portable device and integrated with a hand-held reader for on-site testing and can tackle issues regarding sample storage, stability, and shipping and could be a fully separate plasma sampling device for POC.

sample storage, stability, and shipping and could be a fully separate plasma sampling device for POC. **Author Contributions:** conceptualization and methodology, Z.T.A.-a. and S.A.; formal analysis, A.M.Z., S.A., and G.M.S.; investigation and data curation, Z.T.A.-a., S.A., and G.M.S.; validation G.M.S., A.M.Z., and K.A.A.K.; visualization, T.B and M.T.A.S.; writing—original draft preparation, **Author Contributions:** Conceptualization and methodology, Z.T.A.-a. and S.A.; formal analysis, A.M.Z., S.A. and G.M.S.; investigation and data curation, Z.T.A.-a., S.A. and G.M.S.; validation G.M.S., A.M.Z. and K.A.A.K.; visualization, T.B. and M.T.A.S.; writing—original draft preparation, G.M.S. and K.A.A.K.; writing—review and editing, K.A.A.K., T.B. and M.T.A.S.; supervision, Z.T.A.-a. and G.M.S.; project administration, S.A. All authors have read and agreed to the published version of the manuscript.

and integrated with a hand-held reader for on-site testing and can tackle issues regarding

**Acknowledgments:** The authors extend their appreciation to the University of Misan, and Univer-

G.M.S. and K.A.A.K.; writing—review and editing, K.A.A.K., T.B., and M.T.A.S.; supervision, **Funding:** This research received no external funding.

Z.T.A.-a. and G.M.S.; project administration, S.A. All authors have read and agreed to the published version of the manuscript. **Acknowledgments:** The authors extend their appreciation to the University of Misan, and University of Technology, Baghdad for their technical support.

**Conflicts of Interest:** The authors declare no conflict of interest.

1. Kersaudy-Kerhoas, M.; Sollier, E. Micro-scale blood plasma separation: From acoustophoresis to egg-beaters. *Lab Chip* **2013**, *13*,

sity of Technology, Baghdad for their technical support.

**Funding:** This research received no external funding. **Conflicts of Interest:** The authors declare no conflict of interest.

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