Article pubs.acs.org/ac
Lab-on-a-Disc for Fully Integrated Multiplex Immunoassays Jiwoon Park, Vijaya Sunkara, Tae-Hyeong Kim, Hyundoo Hwang, and Yoon-Kyoung Cho* School of Nano-Bioscience and Chemical Engineering, Ulsan National Institute of Science & Technology (UNIST), 100 Banyeon-ri, Eonyang-eup, Ulju-gun, Ulsan 689-798, Republic of Korea S Supporting Information *
ABSTRACT: This paper presents a cost-effective, rapid, and fully automated lab-on-a-disc for simultaneous detection of multiple protein biomarkers in raw samples such as whole blood or whole saliva. For the diagnosis of cardiovascular disease, here, a novel centrifugal microfluidic layout was designed to conduct the simultaneous detection of high sensitivity C-reactive protein, cardiac troponin I, and Nterminal pro-B type natriuretic peptide based on a bead-based sandwich type enzyme-linked immunosorbent assay (ELISA). Three reaction chambers are initially interconnected for the common processes such as sample injection, incubation, and washing and then isolated on-demand for the independent processes such as substrate incubation and final detection. The assay performances such as the limit of detection and the dynamic range were comparable with those of the conventional ELISA despite the significant reduction of the minimum sample volume (200 μL), the amount of washing buffer (700 μL), and the total process time (20 min).
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utilized as labels for multiplexed immunoassays.15,21,24 The barcoded carriers have also attracted great attention. For example, QDs-conjugated microbeads,22,23,29 barcoded hydrogel microparticles,30 silica colloidal crystal beads,28,31 and barcoded chips6 have been used as a carrier for the multiplexed immunoassay to differentiate multiple analytes. In addition, chips with 3D structures such as pillars12 or micro/nanosized beads have been utilized as well.23,32,33 While the previous reports could achieve significantly enhanced detection sensitivity, the final readout systems often require more sophisticated and expensive hardware and/or software to differentiate multiple detection signals in either spatiotemporal or spectroscopic domain. Furthermore, it has been rare to achieve the full integration of the total analysis starting from real samples such as whole blood to the final detection for the POC applications.4,5,34 Lab-on-a-disc is a unique microfluidic platform in which the integration of biological assays is relatively simple utilizing centrifugal pumping force to transfer liquid samples.13,35−42 Previously, we have demonstrated a cost-effective, rapid, and fully integrated sandwich type immunoassay on a disc.37 The polystyrene (PS) beads modified with antibodies and all other necessary reagents for the ELISA were preloaded on a disc. The only manual step for the user was to inject 100 μL of whole blood on the disc and insert it to the analyzer, which is equipped with a motor for the centrifugal pumping, a laser for the valve actuation, and a detection system composed of a photodiode and a light-emitting diode (LED) for the absorbance measurement. The fully integrated bead-based
he quantitative analysis of protein biomarkers plays a prominent role in biomedical diagnostics.1−5 Today’s immunoassays are routinely performed in typical 96-well microtiter plates. Even though there are automated instruments using robotic liquid handling systems available, manual type of enzyme-linked immunosorbent assay (ELISA) is still the most popular in small laboratories where simple and automated point-of-care (POC) system is desired. The miniaturized bioanalytical devices utilizing microfluidics and/or nanotechnologies have showed great advantages compared to the conventional systems, e.g., less consumption of reagents, smaller sample volume required, shorter assay time, smaller device size, and higher detection sensitivity.3−13 Because most protein biomarkers are not highly specific to a target disease, simultaneous analysis of multiple biomarkers could provide a more accurate diagnostic result.2,14 Various kinds of assay platforms have been proposed to realize the multiplexed immunoassay using either spatial localization3,6−8,15−20 or multiple labels21−24 to discriminate between different analytes. For example, multiple kinds of biospecific probes have been patterned on 2D surfaces such as glass slides,6,25 poly-(methylmethacrylate),7 digital versatile disc,17 silicon nitride,20,26 silicon nanowires,20 and gold.16,18,27 Massively parallel analysis of multiple biomarkers on the spotted protein microarrays has been achieved by detecting the signal by various technologies including fluorescence,6,20 optical waveguide,16,26 charge-coupled device (CCD) camera,7 and chemiluminescence,28 electrical,19 electrochemical, and surface plasmon resonance18 measurements. In order to achieve faster reaction kinetics, suspension assays utilizing multiple bioconjugated labels or 3D encoded carriers have been developed as well. For example, the semiconductor quantum dots (QDs) with various emission spectra have been © 2012 American Chemical Society
Received: August 6, 2011 Accepted: January 25, 2012 Published: January 25, 2012 2133
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Figure 1. (A) A photograph shows the fabricated disc. One disc has two analyses units: one for the unknown sample and the other for the positive control sample analysis. (B) A CCD image shows the main reaction chambers preloaded with PS beads. In the photo image, the NO-LIFM (#8− #10) between main chambers (I−III) is closed, and each chamber is isolated. The schematic diagram of the multiplexed bead-based sandwich type ELISA is also shown. (C) The disc design shows the detailed microfluidic layout. The blue and the red circles with numbers are normally open-laser irradiated ferrowax microvalves (NO)-LIFM and normally closed-laser irradiated ferrowax microvalves (NC)-LIFM, respectively. The number indicates the order of the valve operation. (D) The schematic diagram showing the multiple actuations of a NO-LIFM. The open state of the valve can be changed to the closed state and back to the open state again on demand.
different kinds of beads premodified with the corresponding biomarkers.
immunoassay on a disc could be accomplished within 30 min starting from injection of whole blood, and the assay performances such as limit of detection (LOD) and dynamic range were as good as the conventional ELISA. In this report, a novel centrifugal microfluidic device for the multiplexed immunoassay is developed. The three reaction chambers preloaded with beads modified with different biomarkers are initially interconnected during the common reaction steps but isolated on-demand for the independent detection step. Utilizing the reversible actuation of the laser irradiated ferrowax microvalves (LIFM),43 the open channel could be closed by laser irradiation and then the channel could be reverted back to the open state again by applying the laser on the valve position. As a result, the centrifugal microfluidic layout could be much simpler and the total numbers of valves as well as the footprint of the disc were significantly reduced. For example, 3 targets could be analyzed using 45 valves in the previous report37 while 6 targets can be tested in the current design using 32 valves on the disc with same diameter, 12 cm. As a model system, simultaneous detection of three protein biomarkers, high-sensitive C-reactive protein (hsCRP), cardiac troponin I (cTnI), and N-terminal pro-B type natriuretic peptide (NT-proBNP) is demonstrated for the diagnosis of cardiovascular disease (CVD) which is the most common cause of death in the Western countries. As Zethelius et al. also reported,2 the use of the multiple biomarkers, e.g., combination of hsCRP, cTnI, and NT-proBNP, may enhance the predictive value for the risk factor significantly. Using the proposed device, the simultaneous detection of three biomarkers could be accomplished within 20 min in a fully automated fashion. The LOD and the dynamic range were comparable with those reached by ELISA. It is also noteworthy that the same disc can be used for other kinds of multiplexed immunoassay panels, e.g., cancer or infectious disease detection, by simply utilizing
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EXPERIMENTAL SECTION Reagents. Monoclonal mouse antihuman hsCRP (clone # C5; Hytest Ltd., Finland), monoclonal mouse anti-cTnI (clone # 19C7; Hytest Ltd., Finland), and monoclonal mouse antihuman NT-proBNP (clone # 15F11; Hytest Ltd., Finland) in a coating buffer (each concentration of 10 μg/mL in 100 mM bicarbonate/carbonate, pH 9.6; Junsei Chemicals Co., Ltd., Japan) were used as the capture antibodies coated on the PS beads (diameter = 2 mm; Hoover’s Inc., TX). Horseradish peroxidase (HRP) conjugated goat polyclonal anti-hsCRP (Abcam plc., MA), mouse monoclonal anti-cTnI (clone # 16A11; Abcam plc., MA), and mouse monoclonal anti-proBNP (clone # 24E11; Abcam plc., MA) were used as detection proteins to detect hsCRP (Abcam plc., MA), cTnI (Fitzgerald, MA), and NT-proBNP (Abcam plc., MA), respectively. As a blocking solution, 1% BSA-1× PBS (99% purified Bovine Albumin; Bio Basic Inc., Canada) was used. The washing solution (0.05% Tween # 20 in PBS at pH 7.4; AbD Serotec, UK.) and the 3,3′,5,5′-tetramethylbenzidine (TMB) solution (AbD Serotec, UK.) were used as received. The stop solution composed of 0.2 M sulfuric acid (Matsunoen Chemicals Ltd., Japan) was prepared in the lab. Target-free serum (Fetal Bovine Serum-Charcoal Stripped; GeneTex Inc., CA) was used to achieve the calibration curves for the serum-based immunoassays. Fabrication of Lab-on-a-Disc. The detail of the disc fabrication and the valve formation is reported elsewhere.13,36,37,43 In brief, the microfluidic channels and chambers are usually fabricated on the bottom disc by CNCmicromachining (3D modeling machine; M&I CNC Lab, Korea). The top disc has injection holes and the ferrowax loading chambers. The ferrowax is printed on the loading 2134
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Figure 2. The CCD images of the spinning disc. After the sample is separated by centrifugation (A), the supernatant such as plasma is transferred and mixed with immunoreagents containing three kinds of horseradish peroxidase (HRP) conjugated detection antibodies (B). Then, the solution is transferred to the three main reaction chambers preloaded with PS beads conjugated with capture antibodies (C). After the binding reaction, the plasma residue is removed to the waste chamber (D). The first (E) and the second (F) washing buffers are introduced to the mixing chamber to wash away the residues (G). Then, the valves between the main chambers are closed, and the substrate solution (TMB) is transferred to each main reaction chamber (H). After the incubation, the solution is moved to the detection chamber and mixed with the preloaded stopping solution for the final absorbance measurements (I).
Multiplex Immunoassay on a Lab-on-a-Disc. Figure 1A shows the picture of the fabricated lab-on-a-disc containing antibody coated PS beads in the main chambers. The fluidic layout is designed for the analysis of three kinds of biomarkers from two samples using bead-based ELISA, i.e., up to 6 biomarkers per disc. Figure 1B shows the basic scheme of the bead-based sandwich type ELISA. In brief, the capture antibody coated beads are incubated with the mixture of the target antigen and three kinds of HRP-conjugated detecting antibodies. Then, the beads are washed, the substrate solution is introduced, and the absorbance was measured after mixing with the stopping solution. In this work, the main reaction chambers, chamber I, II, and III, are preloaded with beads premodified with capture antibodies for hsCRP, cTnI, and NT-proBNP, respectively. Figure 1C shows the detailed design of the fluidic channels and the chambers. All of the required reagents such as the antibody-coated beads, the mixture of immunoreagents, the washing buffer, the substrate solution, and the stopping solution are preloaded on the disc before the application of the real sample such as whole blood or whole saliva. During the disc operation, the multiple microvalves are actuated to control the fluidic transfer sequentially. The valves in red circles are the normally closed LIFM (NC-LIFM), and others in blue circles are the normally open LIFM (NO-LIFM). The detailed explanation for the valve actuation can be found elsewhere.43 In brief, the nanoparticles mixed in the paraffin wax act as nanoheaters upon the laser irradiation, and the wax is instantaneously melted. The disc stops in order to align the
chambers by the custom-designed wax-dispensing machine (Hanra Precision Eng. Co. Ltd., Korea). Before the bonding of two plates, PS beads modified with capture antibody were added in the main reaction chambers. The top and bottom plates were bonded using double-sided adhesive tape (DFM 200 clear 150 POLY H-9 V-95, FLEXcon, USA) prepared by cutting plotter (Graphtec CE3000-60 MK2, Graphtec Corporation, Japan). Immunoassay on 96 Well Plates. The same kinds of reagents were used for the immunoassay done in conventional 96 well plate (96 Well Clear Flat Bottom Polystyrene High Bind Microplate, Corning Inc., USA). The coating condition for the capture antibody was the same as for the PS beads. The capture antibody solution (100 μL) in the same concentration and the same coating buffer described above were introduced to each well and incubated at room temperature (RT, 22−25 °C) for overnight (16−24 h). After removal of the solution, 300 μL of blocking solution was added and incubated at room temp for 1 h and the plate was washed 3 times with 300 μL of washing buffer. Then, 100 μL of each target solution was added and incubated at RT for 2 h followed by 3 times of washing using 300 μL of the washing buffer. Then, 100 μL of detection antibody solution was added and incubated at RT for 2 h and washed 3 times with the washing buffer. Then, 100 μL of TMB solution was added and then incubated at RT for 20 min, and finally, 50 μL of the stop solution was added to each well. Then, the absorbance of the solution was measured by a plate reader spectrophotometer (Envision 2104 multilabel reader, PerkinElmer, MA) at 450 nm. 2135
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the plasma separation was normally completed within 60−120 s for the blood samples from normal healthy donors, 3 min was used for more robust operation. It is noteworthy that the same design of the disc can be used for any sample such as whole blood, whole saliva, and whole urine where the initial sample preparation step is target separation by centrifugation. In this report, we tested both whole saliva and whole blood samples using the same disc with the same spin protocol. Upon laser irradiation at the valve #1, 200 μL of the supernatant plasma, transferred into the detecting antibody storage chamber, is well-mixed with 200 μL of the prestored mixture of the immunoreagents, anti-hsCRP (500 ng/mL), anti-cTnI (1 μg/mL), and anti-NT-proBNP (1 μg/mL), by spinning the disc in mixing mode for 5 s. (See Figure 2B.) The mixing mode during which the disc repeats the cycle of acceleration and deceleration (±20 Hz/s) promotes more efficient binding between proteins in the solution and the capture antibodies on the beads surface. After opening the valve #2, the mixture is transferred into the three interconnected reaction chambers (See Figure 2C) and incubated for 10 min in mixing mode, and the residual solution is removed into the waste chamber by opening the valve #3. (See Figure 2D.) When the time for the mixing is reduced to 5 min, the final optical density values were reduced to about 92% of the data obtained at 10 min incubation time, which reduce the total assay time from 20 to 15 min. After closing the valve #4, the washing buffer (350 μL) is transferred into the reaction chambers by opening the valve #5. (See Figure 2E.) After washing the beads, the waste solution is removed to the waste chamber by opening the valve #4 followed by sealing the chamber by closing the valve #6. (See Figure 2F.) After repeating the washing step, transfer of the washing buffer by opening the valve #7, and removal of the residue by opening the valve #6, one more time, the channel to the waste chamber is closed by the actuation of the valve #8. (See Figure 2G.) Finally, each reaction chamber is sealed and isolated by closing the valves #9 and #10. The beads are reacted with the TMB solution (100 μL each) by opening the valves #11, #12, and #13. (See Figure 2H.) After 5 min of incubation, the solution is transferred into each detection chamber preloaded with the stopping solution (50 μL each) by opening the valves #14, #15, and #16. (See Figure 2I.) Finally, the transmittance is measured at 450 nm on the detection chamber using the builtin LED and the photodiode, and the absorbance is calculated by comparing the value measured at the control chamber.13
laser to the valve position. Using this principle, NC-LIFM, NOLIFM, and reversible LIFM have been developed. In order to close the channel, the laser is irradiated at the ferrowax-loading chamber noted as “a” in Figure 1D. Then, the molten ferrowax flows to the thinnest gap and then is instantaneously solidified to seal the channel. In order to change the closed channel back to the open state again, the laser is now focused on the position “b” as shown in Figure 1D. Then, the molten ferrowax wets the nearby surfaces, and the closed channel is reverted back to the open state. Figure 2 shows the image of the disc during the full process of ELISA starting from raw samples such as whole blood. For clear visualization, food dye solutions with multiple colors were used. (See Movie 1 in the Supporting Information.) The details of the centrifugal visualization system can be found elsewhere.13,37 In brief, the images were captured during the spinning using a CCD camera and a strobe light. The detail spin program is given in Table 1. As the disc is inserted into the Table 1. Spin Program for Multiplex Immunoassay spin no.
speed (rpm)
time (s)
1 2
3600 2400
180 5
3 4
±1200 2400
5 5
5 6 7
±1200 2400 2400
600 5 8
8 9 10
±1200 2400 2400
20 5 8
11 12
±1200 2400
20 5
13 14
2400
5 12
15 16
±1200 2400
300 12
17 total time
5 ∼20 min
operation sample preparation open valve #1 to transfer the sample into the reservoir containing multiple detecting Ab mix sample and detecting Ab open valve #2 to transfer mixture into reaction chambers mix beads, sample, and detecting Ab open valve #3 to remove reaction mixture close valve #4 and open valve #5 to transfer first washing buffer mix beads and washing buffer 1 open valve #4 to remove first washing buffer close valve #6 and open valve #7 to transfer second washing buffer mix beads and washing buffer 2 open valve #6 to remove second washing buffer close valve #8, #9, #10 open valve #11, #12, #13 to transfer TMB solution to each chamber mix beads and TMB solution open valve #14, #15, #16 to transfer the each solution to the corresponding detection chamber absorbance detection
blood analyzer,13 the disc spins at 3600 rpm for 3 min for the separation of the red blood cell. (See Figure 2A.) Even though
Figure 3. Immunoassay results performed on lab-on-a-disc using hsCRP (A), cTnI (B), and NT-proBNP (C) spiked in PBS buffer and whole saliva. The results obtained from antigen spiked to whole saliva were similar to those spiked to PBS buffer. 2136
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Figure 4. The cross-reactivity among the different antibodies and antigens were systematically evaluated. (A) The beads coated with hsCRP, cTnI, and NT-proBNP capture antibody were mixed with whole saliva spiked with each corresponding target protein, and only the case mixed with the matched target antigen showed the binding signal. The multiplex immunoassay result was almost identical as the single immunoassay for all of the cases: hsCRP (0−50 ng/mL) (B), cTnI (0−100 ng/mL) (C), and NT-proBNP (0−100 ng/mL) (D). In the multiplex ELISA, the detecting antibody was the mixture of three HRP conjugated hsCRP, cTnI, and NT-proBNP antibodies, and the sample was the mixture of the three target proteins spiked in whole saliva. The concentration of the nonmatched target protein was fixed at 20 ng/mL.
Figure 5. Comparison of the ELISA result performed on lab-on-a-disc vs on conventional 96 well plate shows higher absorbance signal for the labon-a-disc; hsCRP (0−50 ng/mL) (A), cTnI (0−100 ng/mL) (B), and NT-proBNP (0−100 ng/mL) (C). The concentration of the nonmatched target protein was fixed at 20 ng/mL. The ELISA on 96 well plate was done using 300 μL of whole saliva spiked with target protein and conventional manual protocol suggested by the manufacturer (over 3 h), while the bead-based ELISA on lab-on-a-disc was done using 200 μL of sample and the fully automated proposed protocol (20 min).
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RESULTS AND DISCUSSION Immunoassay on a Disc Using Whole Saliva. Saliva offers intrinsic advantages in sample collection compared to the blood because it is less invasive, painless, and convenient. Recently, it has been reported that saliva contains locally produced as well as serum-derived biomarkers that might be useful for the diagnostics in future.9,11,44 However, the salivary diagnostics in microfluidic chip could be very challenging because viscosity is very high and the target concentration is very low. In order to test the sample preparation of the saliva on a disc, whole saliva (250 μL) was introduced on a disc. After centrifugation at 3600 rpm for 2 min, the supernatant saliva (200 μL) was transferred to the next chamber containing detecting antibodies and used for the following steps in the immunoassay on the disc. The same disc shown in Figure 1 was used, and the simple centrifugation step was enough to remove
mucus and to reduce the viscosity. As shown in Figure 3, the results obtained from antigen spiked to whole saliva were similar to those spiked to PBS buffer. Test for Cross-Reactivity. No cross-reactivity among different sets of antigens and antibodies is an essential criterion to achieve good selectivity in the multiplex immunoassay. To confirm this, each batch of the beads coated with different capture antibody, e.g., hsCRP, cTnI, and NT-proBNP, were incubated with each target antigen spiked whole saliva and the mixture of the three kinds of the corresponding detecting antibodies. The beads coated with hsCRP capture antibody had high signal for the matched target only, and no significant binding for other two targets, cTnI (20 ng/mL) and NTproBNP (20 ng/mL). Likewise, cTnI and NT-proBNP coated beads were also tested, and the result indicates that there is no significant cross reactivity among nonmatched target proteins. (See Figure 4A.) Furthermore, as shown in Figure 4B−D, the 2137
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Figure 6. Calibration curves for the detection of hsCRP, cTnI, and NT-proBNP spiked in protein-free serum (A−C) and in whole saliva (D−F) performed on lab-on-a-disc. The concentration of the nonmatched target protein was fixed at 20 ng/mL. In order to obtain the signals at low concentration, the target-free serum was used instead of whole blood. Each data points is the average of at least 4 independent measurements done using different discs. Inset graph shows the dynamic range.
obtain the signals at low concentration, the target-free serum was used instead of whole blood. The dynamic range and the limit of detection of the immunoassay performed on lab-on-adisc were as good as the data obtained by the conventional ELISA for each target as summarized in Table 2.
multiplex ELISA results were almost identical to the single immunoassay confirming that there is no cross-reactivity among different kinds of target proteins. In the multiplex ELISA, the immunoreagents were the mixture of three HRP conjugated hsCRP, cTnI, and NT-proBNP antibodies and the sample was the mixture of the three target proteins spiked in whole saliva. Conventional ELISA versus Lab-on-a-Disc. The absorbance signals obtained by the fully automated bead-based ELISA on a disc were compared with those obtained by the conventional manual assay on 96 well plates as shown in Figure 5. For all the tested biomarkers, the signal intensity from lab-on-a-disc was about 1.5 times higher than that of the conventional method even though the sample volume and the total assay time were significantly reduced. For example, the proposed bead-based ELISA on a disc was done using 200 μL of sample, 750 μL of washing buffer, and the fully automated 20 min long protocol, while 300 μL of sample, over 2.4 mL of washing buffer and over a 3 h long manual protocol, was used for the conventional ELISA. In the proposed bead-based ELISA, 10 PS beads were used per one test. The total surface area of 10 beads is approximately 125.6 mm2 which is only about ∼1.3 times larger compared to the surface area of 1 well of the conventional 96 well plate. However, the 3D mixing of fluids around the beads stored in a mixing chamber of a spinning disc is much more efficient compared to the 2D mixing on 96 well plates. During both acceleration and deceleration steps of the disc in the mixing mode, the beads inside of the mixing chamber are efficiently agitated. The more efficient mixing could be the major reason why the absorbance signals were significantly higher even though sample volume and the reaction time were significantly reduced. In addition, the use of a larger sized ball, diameter of 2 mm in this work, has additional practical advantage compared to a few micrometer-sized beads. For example, it is easy to handle to have the better reproducibility in terms of the surface area. Calibration Curves of Multiplex Immunoassay on Labon-a-Disc. Figure 6 shows the calibration curves for the detection of hsCRP, cTnI, and NT-proBNP spiked in targetfree serum (6A−C) and in whole saliva (6D−F)). In order to
Table 2. Comparison of the Immunoassay Performance Measured by Lab-on-a-Disc and Conventional ELISA Kit
sample type sample volume (μL) time operation limit of hsCRP detection cTnI c (ng/mL) NTproBNP dynamic range hsCRP (ng/mL)d cTnI NTproBNP
conventional ELISAa
lab-on-a-disc
lab-on-a-disc
whole saliva 300b 2−3 h manual 0.27 0.46
whole saliva 200
