A carbon nanotube integrated microfluidic device for blood plasma extraction

Blood is a complex fluid consisting of cells and plasma. Plasma contains key biomarkers essential for disease diagnosis and therapeutic monitoring. Thus, by separating plasma from the blood, it is possible to analyze these biomarkers. Conventional methods for plasma extraction involve bulky equipment, and miniaturization constitutes a key step to develop portable devices for plasma extraction. Here, we integrated nanomaterial synthesis with microfabrication, and built a microfluidic device. In particular, we designed a double-spiral channel able to perform cross-flow filtration. This channel was constructed by growing aligned carbon nanotubes (CNTs) with average inter-tubular distances of ~80 nm, which resulted in porosity values of ~93%. During blood extraction, these aligned CNTs allow smaller molecules (e.g., proteins) to pass through the channel wall, while larger molecules (e.g., cells) get blocked. Our results show that our device effectively separates plasma from blood, by trapping blood cells. We successfully recovered albumin -the most abundant protein inside plasma- with an efficiency of ~80%. This work constitutes the first report on integrating biocompatible nitrogen-doped CNT (CNxCNT) arrays to extract plasma from human blood, thus widening the bio-applications of CNTs.

In this paper, we integrated CNT synthesis with microfabrication techniques to construct a CNT-based microfluidic device. This microdevice effectively separates plasma from blood by performing cross-flow filtration. Our work now expands applications of CNTs in point-of-care blood analysis.

Design and Manufacturing of the Plasma Extraction Device
We designed a double spiral microdevice to continuously separate plasma from blood by using cross flow filtration as illustrated in Fig. 1. This microdevice was constructed by integrating porous aligned nitrogen doped multi-walled CNT (CN x CNT) channel and a polydimethylsiloxane (PDMS) top cover. Inside this microdevice, we constructed a porous microfluidic channel by aligning CN x CNTs to form a membrane. The microdevice has one inlet and two outlets (i and ii). Blood samples were then loaded from the inlet port. Human whole blood was obtained from consented donors at the General Clinical Research Center of Penn State, following to an institutional review board-approved protocol. The samples were drawn into 10-mL Ethylenediaminetetraacetic acid (EDTA) K 2 -tubes (Vacutainer; Becton Dickinson). The blood flowing through the device was then collected by the outlet (i). Note that outlet (ii) was connected to a vacuum source to extract and collect the plasma. When blood was transported within the double spiral channel, plasma diffuses through CN x CNTs and it is collected at the outlet (ii).
We constructed this microdevice by integrating techniques of chemical vapor deposition (CVD) synthesis and microfabrication 38 . The process steps are shown in Fig. 2. We patterned iron thin films using the photoresist lift-off process. First, we spin-coated photoresists, LOR-5A, and SPR3012 in sequence at 4,000 RPM (Fig. 2a). Subsequently, we exposed the photoresist by an exposer (MA-6, SUSS contact aligner) to define a double spiral pattern (Fig. 2b). Next, we deposited iron catalyst thin films of 5 nm in thickness using an e-beam evaporator (Fig. 2c) and soaked it inside a solvent (Remover-PG, MicroChem) overnight (Fig. 2d). We then diced patterned substrates into a 1 cm × 1 cm device using a dicing saw. To grow CN x CNTs on individual devices, we used CVD with benzylamine as a precursor (Fig. 2e). The precursor was generated by an ultrasonic nebulizer and transported by argon/ hydrogen gas into two furnaces in series at 825 °C with a flow rate of 2.5 L/min. On this patterned substrate, we grew CN x CNTs of 60 µm in height. Then, we built a microfluidic device by bonding a top cover made of PDMS (Fig. 2f). The mass of an assembled device was only 1.5 gram, which is three orders of magnitude lighter than a conventional centrifuge used for plasma extraction. This PDMS top cover was fabricated by micro-molding with fluidics accesses and a chamber of 50 µm in height. We defined the chamber dimensions by employing an SU-8 mold and by puncturing three fluidic access ports: one inlet and two outlets. The chamber height was slightly lower than the CN x CNT channel wall to achieve better sealing between PDMS and the top of the A-CN x CNTs. To enhance bonding, surfaces of both PDMS top cover and A-CN x CNT channel walls were treated with an oxygen plasma. Before processing the blood samples, we flushed microdevices by 0.5% Tween-20 and phosphate-buffered saline (PBS) sequentially.

Results
Characterization of a double spiral microdevice. We apply a homogeneous membrane (3D filter) of aligned CN x CNTs. This membrane has to be biocompatible, porous, and to exhibit controllable inter-tubular distances. We used scanning electron microscopy (SEM) and Raman spectroscopy to characterize the structural properties of our CN x CNTs.
As explained above, the miniaturized microdevices consist of aligned CN x CNTs. Figure 3a shows one patterned microdevice after growing CN x CNTs for 30 minutes. Figure 3b confirms that CN x CNTs only grow on the pre-patterned iron thin film and form a microfluidic channel wall. This double spiral channel has a wall thickness of 100 µm and a channel width of 100 µm (Fig. 3c). Figure 3d depicts aligned CN x CNTs of uniform height (~60 µm). Figure 3e demonstrates that the height variation is below 5 µm. This particular array exhibits a ~80 nm inter-tubular distance (Fig. 3f,g). Controlling the inter-tubular distance is critical for allowing plasma and other smaller moieties such as proteins to pass through while blocking larger particles (>1 µm), including platelets and cells that are contained in blood. Controlling the height and inter-tube distances of CN x CNTs is very important for the design of our micro-fluidic device. In this context, we grew CN x CNTs for 10, 20, 30, and 40 minutes and measured the diameter, density, and inter-tubular distance (ITD) from cross-sectional SEM images (Fig. 4a). In Fig. 4b, the height of the aligned CN x CNTs increases with synthesis time. The height of CN x CNTs reaches 61.1 ± 10.1 µm after 40 minutes of synthesis. For nanotube diameter measurements, the images were taken under 6 × 10 4 magnification. A total number of 200 CNTs were measured for each synthesis. For the density measurements, we counted the number of CNTs per unit length. Both the diameter and density of aligned CN x CNTs were independent of each synthesis experiment. The aligned CN x CNTs possess an average diameter of 26.5 ± 1.2 nm and a density of 1.7 × 10 9 ± 7.4 × 10 8 counts/cm (Fig. 4c). Similarly, we measured ITD of CN x CNTs to be 80.0 ± 7.3 nm (Fig. 4d). The ITD was measured from bottom sections of the CN x CNT arrays, where tubes were better aligned in the vertical direction compared to the upper sections 3,39 . Next, we employed a geometrical model to describe the dimensions of the CNT arrays 35 . This model assumes that the orientation of the aligned CN x CNT structure follows a cylindrical model with uniform density and diameter. For the model of cylindrical pillars, the bulk porosity of the cylindrical array is described below where ∅ is the porosity, P is the inter-tubular distance, and D is the diameter of the cylindrical pillar 40 : As shown in Fig. 4d, the porosity was calculated using density and diameter measurements extracted from Fig. 4c. The result showed that the porosity of aligned CN x CNTs reached average values as high as 93.8 ± 0.3%. In comparison with other types of porous membranes, this high porosity of the CN x CNTs can significantly increase the extraction efficiency (see below). Raman spectroscopy is a well-established tool to characterize CNTs in a non-destructive manner 41 . Raman spectroscopy measures the degree of crystallinity of CNTs, chirality in the case of single-walled nanotubes, defects, etc. 42,43 . In our study, we used Raman microscopy (using a Renishaw, InVia Raman microscopy) to characterize our synthesized CN x CNTs. Raman spectra were recorded using a 514 nm laser excitation for 30 seconds under 50X magnification. The laser power used for the measurements was 10 µW. The Raman spectrum showed the D-band centered at 1352 cm −1 , G-band at 1578 cm −1 , and D′-band at 2659 cm −1 , respectively (Fig. 5a). The intensity ratio of D-band to G-band is ca. 0.7, a value significantly higher than that obtained for un-doped CNTs. Since the D-band is a second order feature originated by structural defects in CNTs, the high I D /I G ratio suggests structural disorder, such as the presence of nitrogen dopants and vacancies within the tubes lattice (Fig. 5b) 44 . Furthermore, the characteristic bamboo-like morphology of CNTs shown in the inset of Fig. 5a provides a clear signature for nitrogen doping 29,45-47 . As indicated above, the successful incorporation of nitrogen dopants in the lattice is vital for enhancing the bio-compatibility of CNTs, but the exact mechanism of such enhancement requires further investigation 29,31,48 . Plasma extraction and albumin measurement. To characterize the plasma extraction performance, we measured the albumin concentration from different extractions (BCP Albumin Assay Kit #MAK 125, Sigma-Aldrich). Albumin is the most abundant protein inside plasma and serves as a biomarker of different diseases [49][50][51][52] . During extractions, we monitored blood flow in real-time under a bright-field optical microscope to track red blood cells. Blood samples were drawn from a healthy donor into 10-mL EDTA coated tubes (Vacutainer; Becton Dickinson). Samples in EDTA tubes were processed within 24 hours. Before processing blood samples, we wet a microdevice by flushing a surfactant (0.5% Tween-20, #P9416 Sigma-Aldrich) until all the air inside a microdevice was replaced. Subsequently, we introduced PBS with a rate of 100 µL/min for five minutes to wash off the residual surfactant inside the microdevices. After flushing, we turned on a vacuum source connected to the outlet and started removing the residual PBS inside the microdevice. We transported blood samples into a microdevice at a rate of 100 µL/min. As demonstrated in Fig. 6a, we observed that red blood cells were transported and confined inside a double spiral channel. Simultaneously, plasma diffused through A-CN x CNT channels; yet blood cells were still confined inside the double spiral channel without leaking or clogging. Time-lapse images shown in Fig. 6a reveal the initial stage of blood plasma is displacing the air inside the spiral channel at a speed of ~24 µm/sec (Fig. 6b). The filtrate plasma was then collected at outlet-ii. We measured albumin concentration using BCG Albumin Assay Kit (Sigma-Aldrich MAK124). The albumin concentrations of the original sample and outlet were 52.0 ± 2.1 mg/mL and 42.1 ± 4.1 mg/mL, respectively. Therefore, this device recovers 80.1 ± 5.4% of the albumin within the extracted plasma. The successful extraction of plasma is attributed to unique high porosity (>90%).

Conclusions
We successfully constructed a microfluidic device with a porous channel wall consisting of aligned CN x CNTs. This porous channel exhibits a high porosity (>90%) with a nanometer scale inter-tubular distance (~80 nm). This channel separates micron-scale blood cells, such as leukocytes (diameter: 12~17 µm) and erythrocytes (diameter: 3~5 µm) from nano-scale biomarkers, such as albumin (diameter: ~5 nm). These aligned tubes allowed an effective separation of micron-sized particles within whole blood, with a recovery rate of ~80%, averaged from at least 20 devices. Also, this miniaturized device is disposable and three orders of magnitude lighter in weight than conventional centrifuges. This novel portable microdevice now allows point-of-care diagnostics by extracting plasma containing proteins of key biomarkers.

Materials and Methods
Fabrication of iron catalyst thin film and CNT growth. Detailed information is described in our previous report 34 . In short, the iron catalyst thin film was deposited by e-beam evaporation and further patterned by a lift-off process. The CN x CNT was synthesized by AACVD using Benzylamine as a precursor. The deposition was performed at 825 °C for 30 minutes, under argon and 15% hydrogen flow of 2.5 L/min.   Device assembly and experimental setup. As described in our previous study 34 , the PDMS mold was manufactured by using a commercialized kit (Sylgard 184, Dow Corning). Before bonding, RF oxygen plasma (M4L, PVA TePla Inc) was applied to activate both the PDMS and CN x CNT surfaces. After bonding, the microdevices were baked at 85 °C for four hours.