Analytical Chemistry

Transcription

1 Supplemental Information Analytical Chemistry Microfluidic system for rapid detection of α-thalassemia-1 deletion using saliva samples Kang-Yi Lien 1, Chien-Ju Liu 2, Pao-Lin Kuo 3 and Gwo-Bin Lee 1,2,4* 1 Institute of Nanotechnology and Microsystems Engineering, 2 Department of Engineering Science, 3 Department of Obstetrics and Gynecology, Medical College, National Cheng Kung University, Tainan 701, Taiwan 4 Medical Electronics and Device Technology Center, Industrial Technology Research Institute, Hsinchu 310, Taiwan 42 mm 27 mm Figure S-1. A photograph of the integrated microfluidic platform. The dimensions of the microfluidic chip are measured to be 42 mm 27 mm. Floating block structure Top layer: thick-film PDMS structure Pneumatic micropump Second layer: thin-film PDMS membrane Circular microcoils array Self-compensated PCR module (a) Bottom layer: glass substrate Pressure-relief structure Micro temperature sensor Pt pattern Au pattern (b-1) Pneumatic micro pump structure (b-2) Figure S-2. (a) An exploded view of the microfluidic system composed of three layers, namely a glass substrate, a thin-film PDMS membrane and the thick PDMS structure. (b) SEM images of each element: (b-1) circular microcoil array, (b-2) SU-8 mold of the normally-closed pneumatic micropump and the (b-3) (b-4) S1

2 pressure-relief structure, (b-3) microheaters, and (b-4) a micro temperature sensor. 1. Design and fabrication of the integrated system An exploded view of the microfluidic chip made of one glass substrate and two layers of polydimethylsiloxane (PDMS, Dow Corning Corp., USA) structures is schematically shown in Figure S-2(a). The bottom layer is a bio-compatible soda-lime glass substrate (G-Tech Optoelectronics Corp., Taiwan) patterned with electrodes including a circular microcoil array, two self-compensated microheaters and micro temperature sensors. The second layer is a thin-film PDMS membrane with a thickness of 150 µm replicated from a SU-8 (MicroChem, USA) master mold to form the air chambers of the pneumatic micropumps. Another thick-film PDMS layer is placed on top of the thin PDMS layer to form microfluidic channels and the floating block structures. The microfluidic chip is assembled by bonding the two PDMS structures and the glass substrate together utilizing an oxygen plasma treatment. Note that Scanning electron microscope (SEM) images of each component are shown in Figure S-2 (b). Compressed air inlet Top view Normally-closed microvalve Compressed air inlet Cross-sectional view Air chamber Normally-closed microvalve Microfluidic channel Thin-film PDMS membrane (a) Pressure-relief chamber Membrane deflected upwards to drive the fluid (b) (c) Microvalve open (d) (e) Figure S-3. Schematic illustration of the top- and cross-sectional views, respectively, of the working principle of the normally-closed pneumatic micropump. S2

3 2. gdna extraction module The gdna extraction module consists of normally-closed pneumatic micropumps, multiple microfluidic channels, a circular microcoil array, a sample loading chamber, a waste collection chamber and PCR reaction chambers. A new normally-closed pneumatic micropump consisting of three underlying connected pneumatic air chambers, an individual air chamber as a pressure-relief structure, three elastic, thin-film, PDMS membranes and a PDMS-based floating block structure were designed for sample delivery and backflow prevention [20]. Figure S-3 shows the working principle of the normally-closed pneumatic micropump from the top- and cross-sectional views, respectively. The sample fluids in the microchannel can be driven in a specific direction based on the deflections of PDMS membranes actuated by three interconnected air chambers to generate a peristaltic-like effect for driving the fluid forward. The time-phased expansion of the PDMS membranes underneath the flow microchannel is achieved when the compressed air fills up the interconnected air chambers sequentially and generates a peristaltic effect which drives the fluid along the microchannel (Figures S-3(a)-(c)). A floating block structure is used as a normally-closed microvalve inside the microchannel and plays an important part in increasing the pumping rate of the flow. The thin-film PDMS membrane and another air chamber placed underneath the microchannel allow the deflection of the PDMS membrane so that the fluid can flow through the valve structure in the microchannel (Figures S-3(d)-(e)). Note that the normally-closed microvalve is not bonded with the thin-film PDMS membrane so that backflow is prevented. Electromagnetic valves (EMVs, SMC Inc., S070M-5BG-32, Japan) are used to control the microfluidic pumps by regulating the operated frequency of the EMVs that causes the thin-film PDMS membranes to expand under the supplied compressed air pressure. In addition, the circular microcoil arrays made of copper (Cu) resistors have also been integrated into the gdna extraction module. The required magnetic field can be generated to attract the magnetic beads onto the surface of the extraction chamber for purification so that the other suspended substances can be washed away when the washing buffer flows through the chamber. A local magnetic field is produced within the extraction chamber when a direct current (DC) is supplied to the microcoil array. 3. Self-compensated PCR module The thermal uniformity of the temperature distribution within a PCR reaction chamber is crucial in performing accurate thermal cycling, particularly in maintaining the uniform temperature distribution inside a chamber with a larger reaction area. Therefore, a self-compensated PCR module integrated with two array-type microheaters with symmetrical layouts and three temperature sensors are adopted to S3

4 improve the temperature uniformity in the reaction chambers for rapid nucleic acid amplification of genes [21]. The design parameters of the self-compensated PCR module are shown in Figures S-4. The platinum (Pt) resistors are first deposited as microheaters and temperature sensors, followed by a patterned gold (Au) metallization as the electrical leads of the temperature sensors and the array-type microheaters. Without using additional control circuits and fabrication processes, the PCR module is modified with the surrounding heating grids which are used as compensating heaters for the edge areas. Hence, the self-compensated PCR module has two-dimensional thermal compensation of the temperature distribution near the edges of the reaction area. Note that multiple PCR reaction chambers are both used to perform nucleic acids amplification for genetic identification. Two different sets of specific primers, including one for the detection gene of the normal α-globin gene and another for the genetic gene with α-thalassemia deletion, are used in PCR reaction chambers A and B, respectively. Both of the fluorescent signals from the amplified genes can be detected by the optical detection module. Meanwhile, an application specific integrated circuit (ASIC) controller is used to control the micropumps and the PCR module by providing digital signals to regulate the EMV and the on-chip microheaters. Therefore, the temperature field and thermal cycling of a PCR process can be precisely controlled by adjusting the input power of the microheaters in the PCR module. Au pattern Pt pattern Micro temperature sensor 45 Self-compensated heating girds Heating elements Micro temperature sensor Unit: µm Figure S-4. Design parameters of the self-compensated PCR module. Note that the array-type microheaters with symmetrical layouts and three temperature sensors are adopted to improve the temperature uniformity in the reaction chambers 4. Optical detection module S4

5 The external optical detection module is designed for rapid detection of the fluorescent signals from the amplified nucleic acids of the genes. The fluorescent reporter dye is labeled on the 5 -end of a specific TaqMan DNA probe. It is excited and detected by the optical detection module when the 5 -nucleotide of the probe is cleaved and is released by DNA polymerase during the extension step of a PCR process. The module is composed of a PMT device (R928, Hamamatsu, Japan), a mercury lamp (MODEL C-SHG1, Nikon Corp., Japan), a set of optical components including three fluorescence filters (Nikon B-2A, Nikon Corp., Japan), one collimation lens and one objective lens (Nikon LU Plan 10x/0.30 A, Nikon Corp., Japan). The light source from the mercury lamp is first directed through a band-pass (BP) filter (470/20BP, Nikon Corp., Japan) and is used to excite the reporter dye in the amplified PCR products at an excitation wavelength of 488 nm. The emitted fluorescent signals from the reporter dye are then directed through a long-pass filter (505LP, Nikon Corp., Japan), followed by filtering out other fluorescent signals excited from the PCR product utilizing another BP filter (522/16 BP, Nikon Corp., Japan). Only signals with wavelengths ranging from 506 nm to 538 nm can pass through and be detected by the PMT. With this approach, high-sensitivity detection of the genes can be achieved and the corresponding genetic disease is analyzed in a shorter period of time by utilizing the optical detection module. 5. Standard procedure for saliva samples collection 25 healthy adult volunteers were recruited and each individual provided ~1 ml of saliva sample. The subjects were advised not to food, drink or even smoke for at least 1 hour prior to saliva collection. The collected saliva was thoroughly mixed by vortexing in the 1.5-ml eppendorf tube, followed by extracted and loaded 100-µl saliva samples into the developed microfluidic system utilizing pipettes. 6. Detailed operation conditions of the on-chip gdna extraction and molecular diagnosis gdna extraction materials and process: The detailed procedure of on-chip gdna extraction, PCR process and optical analysis is described and listed in the Table S-1. Briefly, 100 µl of saliva samples, a 200 µl solution of silica-coated, DNA-specific magnetic beads (diameter=4.5-µm, concentration= beads/ml, Dynabeads DNA DIRECT Universal, Invitrogen Corporation, USA) containing cell lysis buffer (Invitrogen Corporation, USA) with 2 µl of Proteinase K (Viogene, USA) are loaded into the reaction chambers, respectively. The magnetic beads solution are then transported into the DNA extraction chamber at a pumping rate of 200 µl/min. Note that a flow disturbance is generated inside the chamber to gently mix the bio-samples S5

6 and the magnetic beads. Next, 100 µl of a washing buffer (10 washing buffer (100mM Tris-HCl, ph=7.5), Invitrogen Corporation, USA) is loaded into the sample loading chamber and the washing process is completed by continuously pumping the buffer through the extraction chamber while the magnetic complex is still restrained by the generated magnetic field. The other substances are washed away and blocked within the waste collection chamber by the normally-closed valve. Next, the re-suspended buffer (10mM Tris-HCl, ph=8.0, Invitrogen Corporation, USA) with a volume of 125 µl is loaded into the sample loading chamber and pumped into the DNA extraction chamber for gdna elution for another 3 minutes. The gdna is then released from the magnetic beads and re-suspended into the buffer solution, followed by transporting them into the subsequent PCR reaction chambers. The entire process of gdna extraction can be completed in approximately 10 minutes in an automatic manner and the detail information of the on-chip operation protocol can be found in Table S-1 in the supplemental information. PCR materials and process: Extracted gdna with a volume of 3 µl is transported in parallel into the subsequent multiple PCR reaction/optical detection chambers (A and B), respectively, followed by pumping 22 µl of PCR reaction mixture along with 10 µl of mineral oil which are pre-loaded in the PCR reagent chambers into the PCR reaction chambers. Note that the 10-µl mineral oil is used to avoid the evaporation of PCR reaction mixture during the amplification process. A fluorescent-based PCR technique is employed for the detection of genetic diseases by using a TaqMan DNA probe, which is a dual-labelled fluorochrome DNA segment (i.e. a reporter fluorochrome (carboxyfluorescein, FAM) labeled on the 5 -end nucleotide and a quencher fluorochrome (minor groove binder, MGB) labeled on 3 -end nucleotide) that will emit specific fluorescent signals from the 5 -end reporter dye by the Taq DNA polymerase during the PCR process [23]. Note that two specific primer sets are used to verify the α-thalassemia deletion in the multiple PCR reaction chambers. The primer set of S1 (5 -GTGTTCTCAGTATTGGAGGGAA-3 )/S2 (5 -GACACGCTTCCAATACGCTTA-3 ) with a 287-bp fragment is designed for the wild-type α-globin gene alleles in the PCR reaction chamber A, while a primer set of S1/S3 (5 -CTACTGCAGCCTTGAACTCC-3 ) with a 194-bp fragment is used for the detection of α-thalassemia-1 SEA-type deletions in the PCR reaction chamber B [29]. The amplification process is carried out in a total reaction volume of 25 µl containing the PCR reagent (12.5 µl of the 2 TaqMan PCR master mix (Applied Biosystems, CA, USA), 10 µm of each primer (S1/S2 or S1/S3), 1 µm of the TaqMan probe (5 -FAM-AACTCGGTCGTCCCCAC-MGB-3 ) with 2.5-µl distilled water and 3-µl purified gdna. The PCR process then starts with an initial pre-heating step at 95.0 C for 5 min, followed by denaturation at 95.0 C for 20s, annealing at 60.0 C for 25s and S6

7 extension 72.0 C for 25s for 30 cycles, followed by an additional 72.0 C for 5 min for DNA extension in the final cycle. Finally, the fluorescent signals from the target amplified gene indicating genetic deletion is immediately detected by the optical detection module. Table S-1. Detailed protocols of the on-chip operation process Biological process Operation step gdna 1) Load saliva samples into extraction DNA extraction chamber Sample loading 2) Load silica-coated magnetic particles solution (containing cell lysis buffer) into sample loading chamber 3) Load Proteinase K into sample loading chamber 4) Load PCR reaction mixture (with mineral oil) into PCR reagent chamber A/B Volume 100 µl 200 µl 2 µl 22-µl reagent and 10-µl mineral oil for each chamber Operation conditions Time Incubation Separation /Washing process 1) Gentle mixing is generated by pumping magnetic particle solution into DNA extraction chamber 2) Wait for five minutes for cell lysis and gdna binding 3) Load washing buffer into sample loading chamber at the same time 1) A magnetic field (65G) can be generated in DNA extraction chamber by applying a DC current into circular microcoils array 2) Wait for 1 minute for DNA-bound magnetic complex to be attracted onto the bottom of DNA extraction chamber 3) Continuously pump washing buffer through DNA extraction chamber 300 µl 300 µl/min 150 ma 100 µl/min 30 sec 5 min 1 min 30 sec 4) Load re-suspended buffer 125 µl S7

8 Elution into sample loading chamber 1) Pump re-suspended buffer into DNA extraction chamber 2) Wait for 3 minute for gdna to be released from magnetic 200 µl/min 30 sec 3 min PCR process Reagent transportation particles 1) Transport eluted gdna into each PCR reaction chamber 2) Transport PCR reaction mixture (with mineral oil) into PCR reaction chamber 3 µl for each chamber 22-µl reagent and 10-µl mineral oil for each chamber 200 µl/min 200 µl/min 1 sec 10 sec Nucleic acid amplification 1) Perform PCR process 45 min Optical detection Fluorescent signal analysis 1) Place the system into optical detection module 2) Switch the filter to collect signals emitted from optical detection chamber A 15 sec 3) Switch the filter to collect signals emitted from optical detection chamber B 15 sec Note that the estimated price of the integrated microfluidic system can be divided into three major parts, namely the cost of the integrated microfluidic chip, the reagent for DNA extraction and PCR, and the instrument for digital controller and optical detection module. The prices of each set are 1.5, 2.5 and 1500 US dollars, respectively. 7. Optical signals from the optical detection module The detection limit of the end-point microfluidic PCR platform has also been explored. The optical signals from the amplified PCR products are shown in Table S-2. The concentrations of samples from number 1 to number 9 are ng/µl, ng/µl, 2.25 ng/µl, 0.32 ng/µl, pg/µl, pg/µl, 2.56 pg/µl, 0.51 pg/µl, and 0.11 pg/µl, respectively. The fluorescent signals of the positive and negative patients are measured and analyzed by using the optical detection module and the excited fluorescent signals are converted into digital signals by using a 24-bit A/D (Model ; Scientific Information Service, Taiwan) and recorded by the computer. From the experimental results, the detection limit of the developed system is found to be ~36.00 pg of total gdna (3 µl of gdna with the concentration of pg/µl) and the S/N ratio of the detected fluorescent signal is about 8.69 (sample 6). Therefore, the detection limit of the developed system is determined to be pg/µl. S8

9 900 Fluorescent intensity (mv) Positive patient with α-thalassemia Negative patient without α-thalassemia Sample number Figure S-5. Detection limit of the developed system. gdna with a concentration of pg/µl (sample 6) can be successfully amplified and the fluorescent intensity can be detected with a high sensitivity up to 90% by the developed system. (Note that the smooth curve presented in the figure is splines) Table S-2. Raw data of the detection limit of the developed system Sample number Used amount of gdna conc. (ng/µl) Fluorescent signal (mv) Positive patient Negative patient Signal-to-noise ratio (S/N) S9