CHAPTER 2. Fully automated, on-site isolation of cfDNA from whole blood on a disc
2.3 A fully automated cfDNA isolation on a disc
2.3.1 Experimental details
As shown in Figure 2.2a, the experimental set-up for the centrifugal microfluidic control was designed and used to control the linear stage motor (EDB2000-28V24-S, ERAETECH) for the positioning of the valve actuator as well as the main motor (EDB2000- 56V24/48-S, ERAETECH) for the spinning of the disc.
The actuation principle of the reversible valve is schematically shown in Figure 2.2b. To close the channel, the valve actuator equipped with an electromagnet (N pole facing down) in contact with the S pole of a neodymium magnet is aligned on top of the valve position, and then the polarity of the electromagnet is switched so that the repulsive magnetic force between the S poles of the electromagnet and the permanent magnet moves the push pin down to squeeze the elastomer such that it can block the fluidic channel. Then, the polarity of the electromagnet switches back to the N pole such that the attractive force between the electromagnet and the permanent magnet can return the permanent magnet back to the valve actuator housing. To open the microfluidic channel, the valve actuator moves down such that the bottom tip of the actuator housing is pushing the inner rim of the valve adaptor, and the pushpin is thereby moved upwards to a prefixed position.
Even though the reversible actuations of the elastomer-based valves were quite common in conventional microfluidic chips,129-132 it was extremely rare in centrifugal microfluidic devices.8, 10 We previously reported the reversible and thermally stable actuation of 3D printed ‘push & twist’-type diaphragm valves integrated on a spinning disc, which have critical advantages of thermal stability, vapor-tightness, robustness, and reversible operation.22, 133 In this work, the ID valves are modified, injection-molded (Figure 2.3) so that the actuation does not require ‘push & twist’ type action. Instead, alignment of the actuator on top of the valve and electromagnetic switch were enough to have reversible actuation of the valves. In addition, the fabrication step is simplified as shown in Figure 2.3 and the operating system is upgraded and miniaturized as shown in Figure 2.2a.
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Figure 2.1. Schematic illustration showing workflow of cfDNA-based liquid biopsy
46 Device fabrication
The disc was designed using 3D CAD software, and is composed of two PC (I-Components Co. Ltd.) plates as the body and a PDMS (Dow Corning) layer as an adhesive. A CNC milling machine (Promill Smart 3530, Protek) was used to mill the PC plates according to the design. A 10 : 1 mixture of a PDMS pre-polymer and a curing agent was spun-coated onto the PC plate (0.2 mm in thickness), and cured in the oven at 65 °C for 4 h. A cutting plotter (CE3000-60, Graphtec) was used to generate the microfluidic channel on the PDMS sheet as the adhesive material for aminosilane-mediated bonding.134 3-Aminopropyltriethoxysilane (APTES, Sigma-Aldrich Corp.) was used to treat the PC plate for the disc assembly as shown in Figure 2.3. An aqueous solution of 1% v/v APTES was prepared by mixing with deionized (DI) water and stirred at room temperature (RT) for 20 min. Both milled PC plates and patterned PDMS layers were cleaned with isopropyl alcohol and 70% ethanol, and then treated with oxygen plasma (60 W, Cute Plasma System) for 90 s. The oxygen plasma-activated surface of the PC plate was treated with 1% APTES solution for 20 min. The APTES-treated surface was then washed with DI water and dried by using N2 gas over a short time. Both the plasma-activated PDMS and ATPES-treated PC plates were kept in contact at RT for 10 min to complete the disc assembly.
The fabrication protocols used in this study is for the manufacturing of the lab-scale prototype device, which can be further improved in a large-scale production stage. One of the drawbacks of the current design is that the users need to add the reagents manually, which can be replaced by pre-stored reagents so that the users perform only one-step of manual operation; adding blood samples.135-137 In this study, the disc was CNC milled and bonded with the APTES bonding (Figure 2.3b). Even though the bonding strength is good and does not give bonding failure, it needs to be improved for mass production. The valves are injection-molded and attached on the disc to simplify the manufacturing steps. However, we believe experts in industry can further improve the current fabrication method.
Furthermore, the device may carry internal control DNA so that the DNA amplification data can be used as positive control for quality control purpose.
cfDNA purification
The fragmented short DNA was prepared by PCR with a pair of primers (5-ACA AAT TTA ACA GCT AAA GAG T-3 and 5-TAG ACA ACG ATG TTT TTA ACA-3) using genomic DNA of Staphylococcus aureus to mimic the 300 bp cfDNA. PCR amplification was performed using the following protocol: 95 °C for 2 min; 40 cycles of 95 °C for 15 s, 52 °C for 30 s, and 72 °C for 30 s, with 100 μL of final PCR volume: 10 μL of 10× PCR buffer (25 mM MgCl2, Solgent), 12 μL of dNTP mixture (10 mM, Solgent), 10 μL of the set of forward and reverse primers (0.5 μM, Macrogen), 20 μL
47
Figure 2.2. Fully automated cfDNA isolation disc and its operation system. a) Schematic diagram (left) and photo image (right) of a point-of-care-type system to operate a lab-on-a-disc for fully automated cfDNA isolation. b) Illustrations showing the injection-molded ID valves when the valve actuator opens (left) or closes (right) the microfluidic channel. By setting the polarity of the electromagnet to be repulsive, the permanent magnet can move down to squeeze the elastomer to block the channel or reversibly be pulled back to the original position by attractive magnetic force by simply changing the polarity of the electromagnet. To open the channel, the valve actuator housing pushes the inner rim of the valve adaptor, releasing the push pin to the top position and thereby opening the microfluidic channel. c) Top view of a photo image of the lab-on-a-disc with full integration of the total process of cfDNA isolation from whole blood, i.e., plasma separation, protein lysis, cfDNA binding, multiple steps of washing, and elution of the cfDNA, performed within 30 min. d) Schematic illustration of the cross-sectional view showing the principles of cfDNA isolation using trapped silica beads.
48
of 5× PCR additives (Solgent), 37 μL of DI water, 6 μL of Taq polymerase (2.5 U/μL, Solgent), and 10 μL of template DNA. Human serum was purchased from Sigma-Aldrich (H4522). The synthetic short DNA (300 bp) was spiked into human serum and used for further analysis.
For optimization experiments using synthetic short DNA spiked in human serum, the reagents such as proteinase K solution and buffers (AL, AW1, AW2, AE) were from a commercially available QIAamp DNA Blood Mini Kit (51106, Qiagen). All procedures followed the manufacturer's instructions.
For the clinical sample analysis, QIAamp Circulating Nucleic Acid Kit, cat. no. 55114 (Qiagen), was used and the same buffer solutions (proteinase K, ACL, ACB, ACW1, ACW2, and AVE) were used for the disc experiments with the same recommended volume. To perform the disc operation, reagents and samples were preloaded; 3 mL of whole blood, 100 μL of proteinase K, 800 μL of lysis buffer (ACL), 1800 μL of binding buffer (ACB), 600 μL of washing buffer 1 (ACW1), 750 μL of washing buffer 2 (ACW2), 750 μL of ethanol, 200 μL of elution buffer (AVE), and 400 mg of the silica beads (dia. 100 μm). The detailed operation steps used for the Disc operation and the recommended protocol for the commercial product are compared in Table 2.1, which requires 30 min and 78 min for the Disc and QIAamp Circulating Nucleic Acid Kit, cat. no. 55114 (Qiagen), respectively.
Clinical samples
This study was reviewed and approved by the Institutional Review Board (IRB) of Pusan National University Hospital (PNUH; PNUHIRB-017), and Ulsan National Institute of Science and Technology (UNIST; UNISTIRB-13-002-A), Republic of Korea. Signed informed consent was obtained from all participants and the peripheral blood samples were collected through an IRB consent process. The experiments were performed in accordance with the regulations and guidelines established by these committees. Blood samples were collected into vacutainer tubes (BD Vacutainer) with ethylenediaminetetraacetic acid to prevent blood coagulation. Collected blood samples were processed within 6 h.
Real-time qPCR
To quantify the yield of DNA eluted from the spiked experiment, real-time qPCR was performed using a TaqMan Gene Expression Master Mix kit (4369016, Applied Biosystems Inc.) with a real-time PCR instrument (QuantStudio 6 Flex, Applied Biosystems Inc.) using the following protocol:
50 °C for 2 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 30 s, with 10 μL of final PCR volume: 5 μL of TaqMan Gene Expression Master Mix, 1 μL each of a pair of primers (forward, reverse), 1 μL of probe, 2 μL of DI water, and 1 μL of eluted DNA sample. The primers and
49
Figure 2.3. Schematic illustration of disc assembly and APTES-mediated covalent bonding. a) Lateral view of the disc showing top and bottom plates made of polycarbonate that are assembled by APTES-mediated covalent bonding with the PDMS layer; the injection-molded ID valves are assembled on the bonded disc to prepare the lab-on-a-disc b) (i) Uncured PDMS was uniformly deposited onto a backing substrate by spin coating (700 rpm, 60 sec) and then cured in a drying oven (65 °C, 4 h). (ii) The cured thin PDMS sheet was patterned according to the design by using a plotter.
(iii) Peeling off the un-patterned area. (iv) The patterned PDMS layer was modified by O2 plasma treatment. (v-vi) The top PC plate was modified by O2 plasma treatment and APTES treatment. (vii) The patterned PDMS layer was stamped to the modified PC plate. (viii) Removal of the backing substrate. (ix-x) The milled bottom PC plate was modified by O2 plasma treatment and APTES treatment. (xi) The surface of the stamped PDMS layer was modified by O2 plasma treatment. (xii) Finally, the two surface-modified PC plates were assembled together by PDMS adhesive support.
50
probe were purchased from Macrogen, and the concentrations of the stock solution of each primer and probe were 10 μM. The sequence for the forward primer was 5′-TAA AGA GTT TGG TGC CTT TAC AGA-3′, that for the reverse primer was 5′-TTA ACT CAT CAT AGT GGC CAA CAG TTT-3′, and that for the probe was 5′-TAG CAT GCC ATA CAG TCA TTT CAC GC-3′.
Droplet digital PCR
To detect the L858R and T790M mutations from the eluted cfDNA samples, ddPCR was performed using a ddPCR Supermix for Probes (BR186-3023, Bio-Rad) and a ddPCR instrument (QX200, Bio-Rad) with the following protocol: 95 °C for 10 min, followed by 45 cycles of 95 °C for 30 s and 56 °C for 1 min, with 20 μl of final PCR volume: 10 μL of ddPCR Supermix for Probe, no UTP, 1 μL of 20× primer/probe assay (FAM and HEX) for mutants and wild type, and 8 μL of eluted cfDNA sample. The target primers and probe were purchased from Bio-Rad. Analysis of ddPCR data to quantify the copy number of the mutants was performed using QuantaSoft software (Bio-Rad).
Genomic DNA of A549 cells was used as a negative control and to decide the cut-off for mutant calling.
2.3.2 Results and discussion
Fully automated cfDNA purification from whole blood on a disc
A custom-designed, table-top-sized operation system to control the spin program and valve actuation was developed (Figure 2.2a and 2.2b). The actuation mechanism for multiple valves on the spinning disc is similar to our previously reported laser-actuated ferrowax microvalves20 or ‘push &
twist’-type diaphragm valves.133 However, the current electromagnetic control of the injection-molded, individually addressable diaphragm (ID) valves significantly simplifies the thermally stable, robust, and reversible actuation of the valves, which enables full automation of complex biological reactions on a disc using a small, point-of-care-type operation system.
The disc is designed to have eleven liquid storage chambers connected by channels with ten reversible ID valves for automatic control of sequential transfer of liquid samples (Figure 2.2c and Figure 2.3). The total process of cfDNA enrichment from whole blood including plasma separation, protein lysis, binding cfDNA on silica beads, washing, and elution could be fully integrated on the disc.
The silica beads with 100 μm diameter were trapped in the mixing chamber and used as the substrate for binding of cfDNA (Figure 2.2d).
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Table 2.1. Comparison between disc and manual protocol
Disc
Commercial kit
(QIAamp Circulating Nucleic Acid Kit, Qiagen).
Step Operation Time
(min) Reagent: volume Operation Time
(min) Reagent: volume Plasma
separation Spin (3600 rpm) 5 Plasma: 1 mL
Centrifuge (1,900 g, 16,000g,
4 °C)
20 Plasma: 1 mL
Lyse
Spin (2400 rpm) Mix, &
Incubation (60 °C, 5 min)
10.8
Proteinase K:
0.1 mL Lysis buffer:
0.8 mL
Pipetting &
Incubation (60 °C, 30 min)
31
Proteinase K:
0.1 mL Lysis buffer:
0.8 mL
Bind Spin (2400 rpm)
& Mix 8 Binding buffer:
1.8mL
Pipetting &
Incubation (on ice, 5 min)
& Centrifuge (10,000 g)
5.5 Binding buffer:
1.8mL
Wash Spin (2400 rpm, 3600 rpm) & Mix 4.3
Washing buffer 1 (ACW1): 0.6 mL Washing buffer 2
(ACW2):
0.75 mL EtOH: 0.75 mL
Centrifuge (6,000 g), Dry at 56 °C for
10min
20
Washing buffer 1 (ACW1): 0.6 mL Washing buffer 2
(ACW2):
0.75 mL EtOH: 0.75 mL Elute Spin (2400 rpm)
& Mix 1.3 Elution buffer:
0.2 mL
Centrifuge (20,000
g) 1 Elution buffer:
0.2 mL
29.4 min 77.5 min
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The total process of cfDNA enrichment from whole blood on the spinning disc was imaged by using a custom-designed visualization system equipped with a motor, a CCD camera, and a strobe light (Figure 2.4).32, 138 The reagents and operation steps are summarized in Tables 2.2, 2.3, respectively.
First, plasma samples (>1 mL) are obtained from whole blood (>3 mL) by spinning the discs at 3600 rpm for 5 min (Figure 2.4a). For the robust operation, the plasma chamber volume was designed to accommodate broad range of hematocrit values of typical adults; males (42–54%) and women (38–
46%). The typical plasma volume is larger than 46%, which is much larger than the current design of 30%. The plasma prepared in the chamber positioned near the center of the rotation is sequentially transferred to chambers located in the radially outwards direction. Notably, the depth of the blood cell storage chamber, 5.5 mm, is much deeper than the interface, 500 μm, connecting to the plasma storage chamber such that the sediment blood cells remain in the storage chamber during the deceleration to minimize contamination (Figure 2.5a,b). Furthermore, tilted groove walls with a slanted angle were introduced in the blood sample chamber to expedite the plasma separation process (Figure 2.5c). The shortened sedimentation distance owing to the tilted wall generates regions with a higher cell fraction where cells move downward faster, whereas the clear liquid, plasma, flows upward in low cell density regions.30
After the plasma separation step, the disc stops, valves #1 and #2 are opened, and then the disc rotates with spin step #2 to transfer 1 mL of plasma sample to chamber 3, preloaded with lysis buffer, through chamber 2 separately containing pre-loaded proteinase K solution (see details of the operation conditions in Table 2.3). The plasma solution is then well mixed with proteinase K and lysis buffer using the mixing mode (Figure 2.6) and incubated at 60 °C for 5 min to promote the effective release of cfDNA by lysing the residual proteins, lipids, and DNases (Figure 2.4b). Next, the lysate solution is mixed with the binding buffer (Figure 2.4c) and then added into the bead chamber (Figure 2.4d) for binding of cfDNA on silica beads. The detailed operation is as follows. Valve #3 is opened and the binding buffer stored in chamber 4 is added to the lysate to increase the absorption strength of nucleic acids on the surface of the silica beads. After valve #4 is opened, the lysate solution (approximately 650 μl) is transferred into the silica bead chamber and incubated for 1 min in mixing mode. After the binding step, the solution in the bead chamber is transferred to the waste chamber by spinning the disc in the closed and open states of valves #4 and #5, respectively. The binding reaction is repeated 5 more times until the total lysate solution is processed. We note that the reversible nature of the ID valves is essential to accomplish efficient binding of cfDNA from the large volume (3.7 mL) of plasma lysate solution.
Next, the cfDNA-bound silica beads are washed by two washing buffers and ethanol to eliminate impurities (Figure 2.4e). Finally, the cfDNA is eluted by adding the elution buffer into the bead chamber.
After an agitation step, the eluted cfDNA sample is transferred to the eluent eluent chamber (Figure 2.4f). The total operation could be finished within 30 min.
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Figure 2.4. Snapshot images of the spinning disc during the total process of cfDNA purification from whole blood. a) Disc spinning for plasma separation; b) valve #1 and #2 opening for proteinase K addition and mixing the plasma with lysis buffer; c) valve #3 opening and mixing the binding buffer with lysate; d) valve #4 opening and valve #5 closing, mixing of the binding mixture with silica beads for 1 min, and repeating for a total of six times to process 1 ml of plasma sample; e) opening valve #6 to wash the DNA-bound beads, and then opening valves #7 and #8 for additional sequential washing; f) closing valve #5 and opening valve #9 to transfer the elution buffer and mix the elution buffer with DNA-bound beads, and then opening valve #10 to transfer the eluted DNA sample. Whole blood and colored dyes were used for easy visualization. Numbered circles indicate the status of the valve (blue: open, red: closed). Solid yellow arrows indicate the flow of fluids, and dashed yellow arrows indicate the mixing mode of the disc.
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Table 2.2. Description of the reagents loaded in 11 chambers on the disc
Chamber Reagents Reagent volume (μL)
C1 Blood 3000
C2 Proteinase K 100
C3 Lysis buffer 800
C4 Binding buffer (BB) 1800
C5 Silica beads 400 mg
C6 Waste storage 5800
C7 Washing buffer 1
(WB1) 600
C8 Washing buffer 2
(WB2) 750
C9 Ethanol 750
C10 Elution buffer 200
C11 Eluent 200
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Table 2.3. Operation steps for fully automated cfDNA purification
Step
Spin speed (rpm)
Time (s) Operation 1 Plasma
separation 3600 300 Plasma separation from whole blood 2
Lyse
2400 20
Open valve #1 and #2 to transfer the plasma sample (C1) to lysis buffer chamber(C3) through the chamber containing proteinase K (C2)
3 Mix 20 Mix plasma sample with lysis buffer
4 * 300 Incubate at 60 °C
5 2400 10 Open valve #3 to transfer the binding buffer (C4) into lysis buffer chamber (C3)
6 Mix** 300 Mix lysate with binding buffer (BB) and incubate at RT 7
8 9
Bind
2400 10 Open valve #4 to transfer the lysate-BB mixture into silica beads (C5)
Mix 60 Mix the lysate-BB mixture with silica beads 2400 10
Close valve #4 and open valve #5 to remove the lysate-BB mixture to waste chamber (C6)
Repeat step 7 ~ 9 for 6 times 10
Wash
2400 10 Close valve #5 and open valve #6 to transfer washing buffer 1 (WB1, C7) into beads chamber (C5)
11 Mix 10 Mix beads with WB1
12 2400 10 Open valve #5 to transfer WB1 into waste chamber (C6) 13 2400 10 Close valve #5 and open valve #7 to transfer washing buffer 2
(WB2, C8) into beads chamber (C5)
14 Mix 10 Mix beads with WB2
15 2400 10 Open valve #5 to transfer WB2 into waste chamber (C6)
16 2400 10 Close valve #5 and open valve #8 to transfer ethanol (C9) into beads chamber (C5)
17 Mix 10 Mix beads with ethanol
18 3600 180 Open valve #5 to transfer ethanol into waste chamber (C6) and dry the beads
19 Elute
2400 10 Close valve#5 and open valve #9 to transfer elution buffer (EB, C10) into beads chamber (C5)
20 Mix 60 Mix beads with EB
21 3600 10 Open valve #10 to transfer eluted DNA sample into eluent chamber (C11)
< 30 min
* The temperature of the stage beneath the disc were increased to 60 °C.
* Mix is the spin program with acceleration rate of 30 rad/s2 as shown in Figure 2.6
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Figure 2.5. Geometric configurations for effective plasma separation. a) Top view of the blood chamber showing the embankment positioned between the plasma storage chamber (volume: 1.0 ml) and the blood cell storage chamber (volume: 2.0 ml), and tilted groove walls with slanted angle;
b) Upper: lateral view of the blood chamber indicating the dimensions with each radial position;
bottom: photographs of the interface between the plasma storage chamber and the blood cell storage chamber taken immediately after the plasma separation process, showing that the narrower depth of interface prevents contaminants (blood cells); c) Still images at 5 min during the plasma separation process showing that tilted groove walls expedite blood cell sedimentations.