Blood Plasma Separation Using a Fidget-Spinner - Analytical

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Blood Plasma Separation Using a Fidget-Spinner Chao-Hsuan Liu, Chung-An Chen, Shi-Jia Chen, Tsung-Ting Tsai, Chin-Chou Chu, Chien-Cheng Chang, and Chien-Fu Chen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04860 • Publication Date (Web): 11 Dec 2018 Downloaded from http://pubs.acs.org on December 12, 2018

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Analytical Chemistry

Blood Plasma Separation Using a Fidget-Spinner Chao-Hsuan Liu,† Chung-An Chen,† Shi-Jia Chen,† Tsung-Ting Tsai,‡ Chin-Chou Chu,† Chien-Cheng Chang,†,* and Chien-Fu Chen†,* †Institute

of Applied Mechanics, National Taiwan University, Taipei, Taiwan of Orthopaedic Surgery, Bone and Joint Research Center, Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Taoyuan 333, Taiwan ‡Department

ABSTRACT: In this study, we present a simple, hand-powered, and electricity-free centrifuge platform based on a commercially available “fidget-spinner.” The centrifugal force provided by this inexpensive and easy-to-use toy is sufficient to separate whole blood, producing a plasma yield rate and purity of 30% and 99%, respectively, separated in as little as 4–7 minutes. We verified the separated plasma by performing a paper-based HIV-1 p24 capsid protein enzyme-linked immunosorbent assay, which achieved a recovery rate of up to 98%, indicating the plasma features extremely low matrix interference effects. These results demonstrate the reliability of the platform for practical use, in addition to greatly reducing the overall cost and time of analysis while retaining detection precision, making it suitable for medical applications in resource-limited regions of the world.

Blood can be divided into two components: plasma and blood cells. Plasma is mainly composed of water and proteins, but also contains bacteria, viruses, metabolites, and other substances.1-3 Despite its complexity, blood is still extensively used in clinical diagnostics, as the contents can reflect a person’s health. However, plasma must be separated from blood prior to analysis since blood cells will affect test results.1,2,4 Traditionally, blood plasma separation is executed by centrifugation, which utilizes the gravity field generated by high speed rotation to sediment blood cells.1,5 Although centrifuges can produce extremely pure plasma samples, these instruments are expensive and require electricity to operate, which may not be feasible in resource limited regions of the world.3,6 Sending blood samples to centralized medical centers in cities can be a solution, but the transportation costs and the need to preserve samples can also be significant hurdles. Additionally, the waiting time required may cause patients to delay treatment. Finally, invasive venous blood extraction often hinders people from seeking medical care, especially as large sample volumes are required for detection processes, such as polymerase chain reaction (PCR) and enzyme-linked immunosorbent assays (ELISA).7,8 Ideally, plasma separation and analysis should be completed on site using non-invasive sampling methods to simplify biomedical tests, limit wasted resources, and provide immediate diagnosis for prompt treatment.9 As an alternative to centrifugation, researchers have proposed microfluidic-based blood plasma separation methods, including microfluidic chips and paper filtration platforms, to achieve onsite blood/plasma separation.1 Microfluidic chips exploit special flow fields and channel designs to manipulate the motion of particles (e.g., blood cells) in fluid to reach the goal of separation. For example, a dual-elbow structured microchannel design was shown to produce a special flow field that can generate a sedimentation phenomenon to separate blood cells and plasma.10 Another approach is to concentrate

and separate different sizes of particles by taking advantage of their different inertial forces within the microchannel as a result of the micro-structural design.11 Such microfluidic chips can achieve microscale or continuous separation, however, they require expensive syringe pumps and electricity to drive and control the fluid. Moreover, the micro-channel can become blocked by the blood flow, making a dilution step necessary, which lengthens and complicates the separation process. An alternative to traditional microfluidic platforms involves centrifugation on chip-based systems to separate microscale blood and plasma samples. The most classical example is the rotating disk design.5 This CD-shaped chip contains several chambers, which can obtain quantitative plasma separation at certain rotation speeds. But this method still relies on an electrical motor to generate rotation, and the biomedical applicability of the separated plasma has not been verified. Other methods have been proposed that rely on the centrifugation phenomenon. For example, the Whitesides group proposed an egg-beater centrifuge separation method, in which they were able to generate enough centrifugal force to separate blood plasma by rotating the egg-beater by hand12 Similarly, Saad Bhamla et al.13 used an ancient toy called a whirligig to run a centrifuge that could provide high speed rotation and achieve blood separation within a few minutes. The egg-beater and whirligig are examples of low-cost, widely-available, handpowered platforms that are suitable for resource-limited regions of the world. However, such hand-held devices do not have a stationary component in their design and require continuous driving force, which may exhaust the user and prevent reproducible results. In this study, we utilize a “fidget-spinner” to provide continuous centrifugal force with just the flick of a finger for facile and consistent plasma separation. A fidget-spinner is a toy that consists of a ball bearing at the center of a multi-lobed flat structure made from metal or plastic, designed to spin along its axis (Scheme 1). The rotating speed needed in microscale

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Scheme 1. Schematic illustration of the centrifuge fidget-spinner for plasma separation, validated using a p-ELISA test. The whole blood is pre-loaded in narrow PE tubing, which is then attached to the fidget-spinner for centrifugal separation. The separated plasma and red blood cells (RBCs) are then isolated by cutting the PE tubing, at which point the plasma fraction can be loaded onto the sample wells of a p-ELISA test for biomedical detection. blood plasma separation can be provided by rotating the fidgetspinner by hand (made easy through the ball-bearing design of the toy) and excluding any possible interface contamination due to the absence of a pump.14 We demonstrate this device can separate highly pure plasma (99%) within 4–7 min from ultralow blood sample volumes that can be obtained with just a finger-prick (~5 to 15 μL). We chose to validate the separated plasma using a paperbased ELISA technique (p-ELISA) to make the entire separation and detection process more feasible in resource limited areas. By pre-infiltrating the cellulose paper with ELISA reactants, we can readily test for the presence of a targeted analyte through colorimetric results that can be easily read using just the camera on a smartphone.15-17 Our analysis of HIV-1 p24 spiked blood using a p-ELISA test demonstrated the utility of the fidget-spinner separated plasma, with a 98% recovery rate compared to standard solutions. This inexpensive, user-friendly device provides high purity separation of blood samples, which in conjunction with the p-ELISA test, could be transformative for biomedical testing in developing parts of the world.

MATERIALS AND METHODS Materials. Human immunodeficiency virus type 1 (HIV-1) p24 / capsid protein p24 ELISA pair set (mouse anti-HIV-1 p24, recombinant HIV-1 p24, mouse anti-HIV-1 p24-HRP) was purchased from Sino Biological (Beijing, China). Phosphate buffered saline (PBS) and phosphate buffered saline Tween 20 (PBS Tween 20) were purchased from Protech. Bovine serum albumin (BSA), TMB, and Whatman chromatography paper were purchased from Sigma-Aldrich (St. Louis, MO). Ultrapure water (18.2 mΩ•cm) was filtered through a Milli-Q system (Millipore, Milford, MA). Red Blood Cell Imaging of Whole Blood. The human blood sample used in this work was approved and issued by the institutional review board of Chang Gung Memorial Hospital, Taoyuan, Taiwan. Whole blood samples were provided by a healthy volunteer and diluted 1000x before imaging to reduce overlapping red blood cells under the microscope. We pipetted 3 µL of the diluted blood onto a slide and covered it with a cover slip. Using an optical microscope (IX73, Olympus, Tokyo, Japan) and a CCD camera (Panda, PCO Imaging, Kelheim, Germany), we observed and calculated the plasma purity at different locations. Fidget-Spinner Sample Preparation. Whole blood was injected into 50 mm long pieces of PE tubing, which were then

sealed by melting the ends of the tubing and crimping them shut with pliers. The samples were immediately tested after preparation. One sample was fixed to each of the three prongs of the fidget-spinner using scotch tape so that the tubing was parallel with the direction of the rotation radius. The rotation radius was defined as the distance between the rotation center and the mass center of the blood in the tubing. We spun the fidget-spinner by hand and measured the rotation speed with a digital tachometer, which is capable of recording the maximum, minimum, and last rotating speed. Rotation radii and blood sample volumes of 30 to 50 mm (5 mm intervals) and 5 to 15 µL (2.5 µL intervals) were studied. Additionally, we refrigerated the blood samples in eppendorf tubes at 4° C and conducted the fidget spinning centrifugation process every 2 days, analyzing the separation results to observe the influence of the preservation time. After removing the tubing from the spinner, we can isolate the separated plasma by simply cutting the tubing to divide the fractions for further testing. HIV-1 p24 p-ELISA Test. A 96-well pattern was waxprinted onto cellulose paper and baked to define hydrophilic wells with melted wax. All the ELISA detection steps were performed within the hydrophilic wells. We first added 3 µL of capture antibody mouse anti-HIV-1 p24 2 µg/mL in PBS onto the well areas, followed by heating the paper substrate for 1 min at 40 °C. The blocking process was performed by adding 3 µL of 5% BSA (w/w) in PBS and baked at 40 °C for 1 min. Then we added recombinant HIV-1 p24 protein in PBS and whole blood samples at different concentrations to the sample wells and baked at 40 °C for 1 min, respectively. 3 µL of detection antibody mouse anti-HIV-1 p24HRP (0.64 µg/mL) was then added and baked for 2 min. We then wash non-specific antigens and antibodies with 27.5 mL PBS Tween 20. The paper was then baked until it was completely dry and 3 µL of TMB was added to generate a color change. The colorimetric results were observed and recorded once per min using a USB digital microscope (UPG650, Upmost, Taiwan) and a smartphone (iPhone, Apple, CA). Image RGB analysis was carried out using ImageJ software (National Institutes of Health, Bethesda, MD) and a selfdeveloped smartphone app. More details please see supporting information. Recovery Rate Estimation Curve. To establish an estimation curve for obtaining the measured value of the spiked blood sample, we used standardly centrifuged plasma as a reference matrix. Fresh whole blood was centrifuged with a centrifuge machine at 3000 r.p.m. for 10 min. The plasma supernatant was extracted into sterile eppendorf tubes and


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spiked with p24 protein at concentrations mentioned previously. Unlike the plasma sample spiked prior to separation, these samples were considered to contain 100% p24 protein, and the estimation curve was obtained with these samples through the p-ELISA process and RGB analysis.

shows 99% purity for all rotation radii with small error bars, which

RESULTS AND DISCUSSION By simply rotating the spinner using the force applied by a single index finger, the device forces the red blood cells to sediment at the outer end of the tubing, leaving the purified plasma at the other end (Video S1). To determine the optimized separation conditions, we studied the effects of rotation speed, the placement of the samples on the fidget-spinner, the sample volume, and the blood preservation time to determine whether this simple design could be applied to microscale blood plasma separation. Rotation Speed. The rotation speed is a critical variable in traditional centrifugation. First, we tested the fidget-spinner’s maximum rotation speed with sample loading and the corresponding separation effect in terms of yield and % purity. Yield represents the percentage of plasma volume separated from the whole blood volume. Purity refers to the extent of RBCs remaining in the separated plasma, commonly defined as 1-(RBCs in plasma/RBCs in whole blood). In this research we used optical microscope to capture and calculate the number of RBCs per certain area in plasma and whole blood. The spinning force was applied with one finger while the other hand fixed the rotation axis. One rotation cycle was defined as the number of rotations that occurred from the start of the spinning process until the device came to a natural stop. Figure 1a shows the spinner achieved an average rotation speed of 1200 rpm, which is high enough to complete microscale blood plasma separation.12 Figure 1b shows that it takes ~80 seconds for the fidget-spinner to reach a natural stop when the starting rotation speed is 1200 rpm. Sample Tube Position on the Fidget-Spinner. Centrifugal force is proportional to the rotation radius, according to the centrifugal force formula: 𝐹𝑐 = 𝑚𝜔2𝑟 (1) in which m is the mass of the reference object, 𝜔 is the rotation speed, and r is the rotation radius. Therefore, a larger radius will generate a higher centrifugal force and purer plasma as a result. We fixed the PE sample tubing at different rotation radii (30–50 mm, distance between the rotation center and sample mass center) on the fidget-spinner and measured the resulting separation efficacy, represented by the plasma yield and purity. On average, the plasma yield began to saturate after the 7th rotation cycle for the different rotation radii (Figure 2a), and can reach ~30% in 3 to 5 rotation cycles. The highest plasma yield generated after 10 rotation cycles was ~30% (3–4 µL plasma) for a rotation radius of 40 mm (Figure 2b). This is interesting because we would expect the larger 50 mm radius to have a higher separation efficiency, but saturation occurring at 45 mm is reasonable due to the fixed hematocrit ratio of the blood sample (i.e., the volume ratio between all blood cells and the whole blood sample), which ultimately will limit the yield. We also evaluated the plasma purity achieved for the different radii using an optical microscope to count the number of red blood cells that remained in the separated plasma fraction. Figure 2c

Figure 1. Rotation speed tests of the fidget-spinner (N = 5). (a) The average maximum rotation speed of the sample-loaded fidget-spinner was 1200 rpm. (b) The rotating speed of the loaded fidget-spinner from starting rotation to natural stop shows an exponential decrease. A rotation cycle takes 80 s to complete at an initial speed of 1200 rpm. clearly indicates the efficacy of this separation method. However, rotation radii of over 40 mm tended to result in segmentation of the blood sample in the tubing because of the overloaded centrifugal force, leading to a reduced quantity of usable plasma that can be removed (Figure 2c, inset). Based on the plasma quantity, purity, and convenience of use, we chose 40 mm as the optimal rotation radius for a standard fidgetspinner. Relation between Sample and Plasma Volume. In addition to contributing to the mass m in the centrifugal force equation, the sample volume is closely related to the separated plasma volume. To determine optimal processing conditions, we studied various sample volumes ranging from 5 to 15 μL, corresponding to the typical amount that can be obtained by a finger-prick, and observed how the sample volume affected the separation results (Figure 3a). The highest plasma yield we observed was for a sample volume of 10 μL. Meanwhile, the plasma volume starts to saturate at ~3 μL for the 10 μL sample volume, which is a sufficient quantity for subsequent analysis (Figure 3b). Additionally, the plasma purity remains 99% after separation, independent of the sample volume (Figure 3c). Based on these results, we considered 10 μL to be the optimal sample volume due to the sufficient amount of plasma that can be separated, the high plasma purity, and the fact that 10 μL can



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be easily obtained through a simple finger-prick, for both adults and infants.1

Figure 2. Test results of different rotation radii, ranging from 30 to 50 mm (N = 5). (a) The relation between the plasma yield and the number of rotation cycles shows a saturating trend regardless of rotation radius. (b) Plasma yield and (c) purity after 10 rotation cycles of different rotation radii. The inset images of (c) show real images of blood samples separated using different rotation rad.

Figure 3. Test results of different blood sample volumes, ranging from 5 to 15 μL (N = 5). (a) Terminal plasma yield for different blood sample volumes after 10 rotations. (b) The separated plasma volume, calculated from (a). (c) Purity of the plasma separated from different sample volumes. > 99% purity was obtained for all samples. Influence of Blood Sample Preservation Time. The influences of sample preservation conditions and time must be considered for practical applications, and therefore we also tested preserved blood samples (Figure 4). Our findings indicate that the yield decreases with increasing preservation time, with a significant decrease occurring in the first 7 days. However, we could ensure a 25% yield for blood samples that had been extracted within 3 days of separation (Figure 4a). A yield of over 30% is recommended to achieve a sufficient plasma volume for detection (3 μL) based on a sample volume of 10 μL. In addition, we observed hemolysis for sample preservation times exceeding 11 days. After 19 days storage, the fidget-spinning centrifuge was unable to separate any plasma, possibly due to the increased viscosity of the blood as it degraded over time (Figure 4c), causing the friction in the blood fluid to be greater than the centrifugal force on the blood cells. However, the purity of the separated plasma samples showed little to no dependence on the storage time, as 99% purity could still be obtained after 17 days cold storage (Figure 4b). Thus, conducting the separation within 3 days from blood extraction is recommended. HIV-1 p24 P-ELISA Spiked Test: Standard and Plasma. To affirm the applicability of the plasma separated by our fidget-spinner centrifuge for disease detection, we chose p24 capsid protein as a detection target, which is a biomarker of HIV-1. p24 is more advantageous in early diagnosis than other

HIV biomarkers because of its detectability during the window period.18-20 We verified the applicability of the separated plasma using a p-ELISA test (Figure 5a). First, we used p24 spiked PBS buffer solution to eliminate matrix interferences to determine the optimized testing parameters (2 μg/mL capture antibody, 0.64 μg/mL detection antibody, PBS Tween 20 washing buffer, and 5 mins reaction time). Then, we made p24 standard solutions and conducted the p-ELISA (sandwich) test on wax-printed cellulose paper. Upon binding between the antigen and antibodies, the 3,3',5,5'tetramethylbenzidine (TMB) enzyme linked to the antibody causes a color change. By imaging the colorimetric results of the test and analyzing the red, green, blue (RGB) values using a smartphone and a remote computer, we were able to construct a calibration curve (Figure S1). At the optimized conditions, the B/R values ranged from 1.23 to 1.94, with a linear range of 0.1 to 10 ng/mL and limit of detection of 0.03 ng/mL (R2 = 0.9918). Compared with standard p24 ELISA tests, the limit of detection of the paper-based platform used in this work is relatively high.18 The possible reasons for this include the relatively short reaction time, low reagent volume, and minimal interference from the cellulose. These factors might lead to the instability of the detection results but can be improved with surface modification and other techniques.


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Figure 4. (a) The plasma yield and (b) purity as a function of the blood sample preservation time at 4° C. (N = 5) (c) Images of the plasma separation for different preservation times and as a function of rotation cycles. Obvious hemolysis (a rupturing phenomenon of red blood cells, accompanied with the release of hemoglobin) was observed by the 11th day, at which point the plasma is no longer suitable for detection. Plasma Validation using the p24 spiked p-ELISA Test. Next, we used p24-spiked human blood to simulate a real sample test. With the optimized parameters previously established using p24 standard solutions, we tested the spiked plasma obtained from the fidget-spinner centrifuge (Figure 5b). The plasma calibration curve shows the B/R values ranged from 1.176 to 1.907, with a linear range from 0.1 to 10 ng/mL and a limit of detection of 0.03 ng/mL (R2 = 0.9852). The results in Figure 5b are very similar to the standard curve shown in Figure S1, with the same linear range and signal difference. The large error bars of the plasma curve are due to the internal interferences of the plasma content, as expected. The limit of detection of our work lies in the clinical patients’ p24 concentration ranges.19 With this we can conclude that the fidget spinner separation can actually be used in clinical applications. These results show that the applicability of the fidget-spinner-separated plasma for biomedical testing. Recovery Rate of the Fidget-spinner Centrifuge. We also determined the recovery rate of the fidget-spinner platform. The recovery rate refers to the difference in the amount of analyte measured between the spiked sample and the standard, which represents the antigen quantity difference between the fidgetspinner and standard centrifuged plasma. Knowing the recovery rate helps researchers to understand the interferences of the sample matrix relative to internal substances. The recovery rate can be calculated as follows:

𝑅% =

(𝑆𝑆𝑅 ― 𝑆𝑅) × 100% 𝑆𝐴

(2)

in which SSR is the measured spiked concentration, SR is the unspiked concentration, and SA is the real spiked concentration.

In this work, the unspiked concentration equals zero, and hence the recovery rate can be considered as the ratio of the measured concentration to the known spiked concentration, which ideally would be 100% if there was no interference from the matrix. We used the p-ELISA results of the plasma produced from a standard centrifuge spiked with HIV-1 p24 protein as the comparison to the fidget spinning centrifuged plasma, establishing a concentration estimation curve within the linear range. The measured concentrations of the fidget spinning centrifuged plasma were calculated with this estimation curve, and the recovery rate can thus be obtained. Figure S2a displays the estimation curve of the standard centrifuged spiked plasma, which features an R2 = 0.9993 and a linear relationship described by the equation y = 0.2004x + 1.4259. The SSR values were obtained with the recovery rate formula (equation 2), enabling us to calculate the recovery rate. Using this platform, we achieved a peak recovery rate of 98% at a p24 protein concentration of 10 ng/mL, with the lowest value still being 89% (Figure S2b), which is in agreement with the literature and demonstrates the fidget-spinner separated plasma features little interference in detection.21 These recovery results show that our simple, inexpensive, and rapid fidgetspinner platform can achieve equally sensitive detection results as other studies, demonstrating its potential for clinical applications.20 Compared with related studies, this platform can reach the high rotation speeds required for microscale centrifugation12 without electricity or a continuous driving force, accompanied with relatively or equally high plasma yield purities6. Additionally, reasonable detection results were obtained, and the low base interference of this platform was confirmed by the recovery rate.20



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Figure 5. p-ELISA test of the p24 antigen. (a) The p-ELISA test operating procedure. (b) Calibration curve of p24 spiked plasma separated by the fidget-spinner platform and the resulting colorimetric readout (N = 5). 2628-E-002-007-MY3), and the Higher Education Sprout Program at National Taiwan University.

CONCLUSIONS In this work, we developed a hand-powered, electricity-free centrifugation platform based on a fidget-spinner that can complete blood plasma separation within a few minutes. After practicing for 5 min, it is easy for everyone to achieve rotation speed higher than 1000 rpm (Figure S3). With the optimized centrifuge parameters, 3 μL plasma of 99% purity can be obtained within 3 to 5 rotation cycles and easily observed and confirmed by the naked eye. To validate that the produced plasma can be used for biomedical analysis, we chose HIV-1 p24 capsid protein as a detection target in a spiked plasma pELISA test. After optimization, we achieved a reasonable limit of detection of 0.03 ng/mL at an optimized reaction time of 5 min and a recovery rate as high as 98% for the separated plasma. The materials and facilities used in this platform are all relatively inexpensive and widely available, enabling rapid plasma separation to be achieved in resource-limited regions, like similar egg-beater and paperfuge devices (Table S1). In addition to paper-based tests, we envision that the fidgetspinner could be combined with other analytical devices or integrated with other diagnostic functions into a single platform.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author * Chien-Fu Chen: [email protected] * Chien-Cheng Chang: [email protected]

Notes The authors declare no competing interest.

ACKNOWLEDGMENTS This research was supported by the Ministry of Science and Technology, Taiwan (MOST 106-2221-E-002-139-MY2 and 107-

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Blood Plasma Separation Using a Fidget-Spinner - Analytical

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