Biosensors and Bioelectronics 67 (2015) 497–502
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A droplet-based fluorescence polarization immunoassay (dFPIA) platform for rapid and quantitative analysis of biomarkers Jae-Won Choi a,1, Gil-Jung Kim a,1, Sangmin Lee b, Joonwon Kim b, Andrew J. deMello c, Soo-Ik Chang a,n a
Department of Biochemistry, Chungbuk National University, Cheongju 361-763, Republic of Korea Department of Mechanical Engineering, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea c Department of Chemistry and Applied Biosciences, ETH Zürich, Vladimir Prelog Weg 1, Zürich CH-8093, Switzerland b
art ic l e i nf o
a b s t r a c t
Article history: Received 5 June 2014 Received in revised form 30 August 2014 Accepted 4 September 2014 Available online 8 September 2014
Herein, we describe for the first time the integration of pneumatic micro-pumps with droplet-based microfluidic systems as basic platform for the rapid detection and quantitation of biomarkers. Specifically, we combine this microfluidic platform with fluorescence polarization detection to identify and quantify the potent blood vessel inducing protein bovine angiogenin within cow's milk in highthroughput. The droplet-based fluorescence polarization immunoassay is successful in accurately determining the concentration (4.84 7 1.21 μg/mL) of bovine angiogenin in cow's milk, affords a 10 fold reduction in dead volumes when compared to conventional droplet-based microfluidic experiments and requires an total sample volume of less than 1 nL. & 2014 Elsevier B.V. All rights reserved.
Keywords: Droplet-based microfluidics Biomarker Fluorescence polarization Immunoassay Angiogenin Milk
1. Introduction A biomarker defines a measureable characteristic that may be used as an indicator of a biological state in normal biological and pathogenic processes, and of pharmacologic responses to therapeutic drug administration (Hawkridge and Muddiman, 2009; Mayeux, 2004; Younes and Berry, 2012). The detection of biomarkers in foods is now recognized to be of high clinical importance due to an incomplete understanding of nutrient intake from foods and health problems related to food sensitivities and allergies (Borchers et al., 2010; Schroeter et al., 2006; Schubert-Ullrich et al., 2009; Vandevijvere et al., 2012). Accordingly, the development of robust methods for high-throughput detection and quantitation of biomarkers is of high importance. Methods for biomarker analysis can be broadly classified as being either heterogeneous or homogeneous in nature. Heterogeneous assays typically require analyte immobilization, multiple washing steps, and the physical separation of target bound complexes from non-target species prior to detection (Tachi et al., 2009; Tian et al., 2012). Traditionally, lateral flow assays (LFAs) and enzyme-linked immunosorbent assays (ELISAs) have n
Corresponding author. Fax: þ 82 43 267 2306. E-mail address: [email protected] (S.-I. Chang). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bios.2014.09.013 0956-5663/& 2014 Elsevier B.V. All rights reserved.
been most commonly used to detect biomarkers in the human sera, urine, tears and food products (Kiening et al., 2005; Van Coillie et al., 2004; Wen et al., 2005a, 2005b). Unfortunately, such methods possess drawbacks such as poor limits of detection and needs of hundreds microliter. Mass spectrometry-based methods have also been used for biomarker analysis, however they require expensive and sophisticated instruments and ionization of samples (Agrawal et al., 2013; Panchaud et al., 2012). To overcome such drawbacks, protein microarrays and surface plasmon resonance (SPR) have been successfully used to quantify biomarkers in cow's milk and human sera (Hochwallner et al., 2010; Jung et al., 2009; Uludag and Tothill, 2012). However, they have limited applicability in high-throughput biomarker screens since the immobilization of antibodies, washing steps, and the separation of bound analytes from antibodies is a lengthy and complex process. To overcome these drawbacks fluorescence polarization immunoassays (FPIAs) have been increasingly used for biomarker detection (Tachi et al., 2009; Tian et al., 2012; Wang et al., 2011; Xu et al., 2011). FPIAs are homogeneous immunoassays, rapid and simple to implement and do not require antibody immobilization or washing steps. They are based on the competition of unlabelled analytes and fluorophore-labelled analytes for antibody binding sites and thus do not require separation of any kind.
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To demonstrate robust high-throughput detection and quantitation of biomarkers, we have integrated FPIA with a dropletbased microfluidics. Droplet-based (or segmented flow) microfluidic systems have been shown to overcome many of the problematic issues, such as Taylor dispersion, cross-contamination and slow mixing, encountered in continuous-flow microfluidics (Huebner et al., 2008). Significantly, picoliter-volume droplets can be generated at kilohertz (kHz) frequencies with individual droplets acting as isolated micro-reactors (Choi et al., 2012a, 2012b; Elvira et al., 2013; Song et al., 2006). To this end, dropletbased microfluidic systems have been used to perform highthroughput biological analysis in a range of genomic, proteomic, and cellomic applications (Bardiya et al., 2014; Guo et al., 2012; Therberge et al., 2010). Key examples include protein-protein interactions analysis (Choi et al., 2012a, 2012b; Srisa-Art et al., 2009), enzyme activity assays (Bui et al., 2011; Najah et al., 2013), in vitro protein expression (Huebner et al., 2009; Wu et al., 2009) and protein crystallization (Yamaguchi et al., 2013; Zheng et al., 2003). In a recent study, we reported the use of droplet-based microfluidics and FP detection for protein-protein interaction analysis (Choi et al., 2012a, 2012b). Although successful, in probing the interaction between purified antigen and antibody, specific protein biomarkers from clinical samples and/or foods were not quantified. In this study, we report, for the first time, a new platform for high-throughput detection and quantitative analysis of protein biomarkers using droplet-based fluorescence polarization immunoassay (dFPIA). Importantly, the incorporation of integrated pneumatic micro-pumps system with the dropletbased microfluidic systems obviates the need syringe-pumps or large dead-volume connections(Choi et al., 2014). It should be noted that some studies incorporating pneumatic micro-pumps within microfluidic systems have recently been reported (Sun et al., 2013; Zeng et al., 2013). However, the system described herein possesses additional desirable features; most notably complete pump integration (removing the need for external syringe pumps), simple operation via single step actuation and facile device fabrication. As a model system, we probe and quantify bovine angiogenin (bANG) from cow's milk using dFPIA. bANG is a 14.6 kDa protein and is essential to angiogenesis in normal growth processes such as development, wound healing, and neural protection (Thiyagarajan et al., 2012; Trouillon et al., 2011). In addition, bANG belongs to the RNase protein superfamily and is well known to exist in blood and milk (Chang et al., 1997; Komolova and Fedorova, 2002). bANG can be used as a biomarker for the quality control of cow's milk and commercial milk products.
2. Materials and methods 2.1. Materials Phosphate buffered saline (PBS, pH 7.4) was purchased from Amresco (Solon, OH, USA). Bovine serum albumin (BSA) was purchased from GenDEPOT (Barker, TX, USA). Fluorinert FC-40, mineral oil, and fluorescein isothiocyanate (FITC) were purchased from Sigma-Aldrich (St. Louis, MO, USA). ABIL EM 90 surfactant was purchased from Evonik Industries AG (Essen, Germany). An Alexa Fluor 488 protein labelling kit was purchased from Life Technologies (Carlsbad, CA, USA). A PD-10 desalting column was purchased from GE Healthcare (Wauwatosa, WI, USA). Polydimethylsiloxane (PDMS, Sylgard 184 silicone elastomer kit) was purchased from Dow Corning (Midland, MI, USA). Teflon AF 1600 was purchased from DuPont (Wilmington, DE, USA). All materials and chemicals were used as received.
2.2. Preparation of protein samples The Escherichia coli strain Rosetta (DE3)pLysS, which carries the pET-angiogenin plasmid, was used to express bovine angiogenin (bANG). Specific expression and purification procedures are described elsewhere (Jang et al., 2004).The bANG was labelled with Alexa Fluor 488 (AF488) using AF488 protein labelling kits according manufacturer's protocol. The serum for anti-bovine angiogenin antibody (anti-bANG Ab) was obtained from AbClon (Seoul, Republic of Korea). Anti-bANG Ab was purified by affinity chromatography using bANG-conjugated agarose beads from AbClon. The column was washed with PBS (pH 7.4) and antibody eluted using 0.1 M glycine (pH 2.8). To increase pH, each fraction was collected in tubes containing 1 M Tris–HCl (pH 8.0). The collected fractions were dialyzed into PBS to exchange buffer. Raw cow's milk samples were obtained from dairy cattle (Cheongju, Republic of Korea). Fat was removed from raw milk by centrifugation (5000 g, 4 °C, 10 min) to keep the same condition as the standard solution. 2.3. Microfluidic device fabrication To reduce the dead volumes associated with syringe pumpbased droplet microfluidics a novel microfluidic device integrating pneumatic micro-pumps was fabricated. This microfluidic device is composed of three layers (PDMS channel, glass, and flexible PDMS membrane) as shown in Fig. S1. The top layer is structured in PDMS and contains a microfluidic channel for droplet generation. This was fabricated by pouring a 10:1 (w/w) mixture of PDMS pre-polymer and curing agent (Dow Corning, Midland, MI, USA) onto a patterned Si-wafer (LG Siltron, Gumi, Republic of Korea). After curing on a hot plate at 70 °C for 2 h, the PDMS substrate was peeled off the master and inlet/outlet holes punched using a 1.5 mm diameter disposable punch. The middle layer, used to define the liquid chamber, was formed using a glass slide (Matsunani, Tokyo, Japan). An 8 mm diameter hole was drilled in the glass substrate using a MAXNC milling machine (Gilbert, AZ, USA). The bottom layer defined a flexible PDMS membrane for fluid actuation. This was formed from a 10:1 (w/w) mixture of PDMS pre-polymer and curing agent, which was spin coated (750 rpm for 50 s) on a flat Teflon-coated substrate. The entire construct was then heated on a hot plate for 15 min at 90 °C. Bonding of the three layers was achieved by treatment with an oxygen plasma. After bonding, 2 mm diameter polyurethane tube (SMC, Noblesville, IN, USA) was inserted into the inlet of microfluidic device, and a 200 μL pipette tip inserted into the outlet of the microfluidic device. Specific dimensions of the microfluidic device are described in Fig. S2. 2.4. Device operation The microfluidic device consists of two liquid chambers that deliver aqueous sample and oil to a junction where microdroplets are generated and a single outlet. 30 μL aqueous samples were loaded into each liquid chamber manually. Negative pressure ( 70 kPa) was applied to the thin PDMS membrane using inhouse pressure controller, and then positive pressure (5 kPa) was applied to the thin PDMS membrane below the each liquid chamber. The PDMS membrane motivates fluid within the microfluidic channel whilst simultaneously closing each inlet hole. Meanwhile, oil flows into the microfluidic channel due to negative pressure (up to 80 kPa) applied to the outlet and permits droplet generation. Diagram about device operation was shown in Fig. S3.
J.-W. Choi et al. / Biosensors and Bioelectronics 67 (2015) 497–502
2.5. Optical instrumentation A detailed description of the optical setup used for fluorescence polarization measurements is described elsewhere (Choi et al., 2012a, 2012b). Briefly, the fluorescence detection system consists of 488 nm diode laser (10 mW, World Star Tech, Canada), inverted fluorescence microscope (IX71, Olympus, Japan), filter cube set for Alexa Fluor 488 (FITC-3540B, Semrock, USA), dual polarization system, and an electron multiplying-charge coupled device (EMCCD, Princeton Instruments, USA). Using the system shown in Fig. S4, polarized fluorescence emission originating from sample contained within a segmented flow could be measured through 20 objective. All EM-CCD images were acquired and processed using WinSpec/32 (Princeton Instruments, Trenton, NJ, USA) with a 1 ms exposure time in binning mode.
3. Results and discussion 3.1. Performance characterization of pneumatic micro-pumps for droplet-based microfluidics application Detection and quantitation of bovine angiogenin (bANG) from cow's milk was initially performed within 1 nL droplets using a droplet-based fluorescence polarization immunoassay (dFPIA). Specifically, the competitive inhibition between unlabelled bovine angiogenin (ANG) and Alexa Fluor 488-labelled bovine angiogenin (AF488-bANG) against anti-angiogenin antibody (anti-bANG Ab) was analyzed. The bANG from cow's milk was detected and quantified using a competitive inhibition standard curve. A schematic of the microfluidic system used for dFPIA measurements is provided in Fig. 1. The integrated micro-pump is used to deliver two different aqueous and oil phases to a microchannel T-junction where droplets form. To demonstrate the efficacy of the general approach microdroplets were formed using mineral oil containing 2% ABIL EM 90 as the continuous phase and FITC
499
(80 μM) and bovine serum albumin (0.1 mg/mL) in phosphatebuffered saline (PBS, pH 7.4) as the discret phase. Initially 30 μL of each phase were loaded into the left and right chambers by applying a negative pressure of 70 kPa to the flexible PDMS membrane. Subsequently, a steady positive pressure of 5 kPa was applied to the membrane to motivate the fluids into the microchannel whilst simultaneously closing each inlet hole as shown in Fig. S1. Fig. 2a illustrates the formation of monodisperse droplets at the T-junction using a 1:1 volume ratio of FITC and PBS solutions. To further assess the performance of the system, various droplet sizes and droplet generation frequencies were accessed by control of the applied pressures. As shown in Fig. 2b, droplet size (reported through the droplet length) can be varied between 100 and 300 μm by varing the negative pressure ( 50 to 80 kPa) against a fixed positive pressure of 5 kPa. In this way, the droplet generation frequency could also be varied between 2 and 15 Hz (data not shown). 3.2. Droplet-based fluorescence polarization immunoassay Subsequently, competitive inhibition of the interaction between AF488-bANG and anti-bANG Ab by unlabelled bANG was probed. Unlabelled bANG was used as a competitive inhibitor for the AF488-bANG/anti-bANG Ab interaction. As the concentration of unlabelled bANG increases, the fluorescence polarization reduces since unlabelled bANG act as competitor of AF488-bANG as shown in Fig. S5a. Based on this principle, the fluorescence polarization will decrease when AF488-bANG is replaced by unlabelled bANG in cow's milk as shown in Fig. S5b, and as the concentration of cow's milk increases, the fluorescence polarization is reduced as shown in Fig. S5c. To calibrate such a dependency, 30 μL of off-chip mixed AF488-bANG and unlabelled bANG were loaded via the left sample inlet, and 30 μL of anti-bANG Ab loaded via the right sample inlet (Fig. 1). In this experiment, the final concentration of unlabelled bANG was varied between 0.49
Fig. 1. Schematic illustration of droplet-based fluorescence polarization immunoassay (dFPIA) using the integrated pneumatic micro-pumps for the rapid detection and quantitation of protein biomarkers. Bovine angiogenin (bANG) from cow's milk was used as a biomarker model for dFPIA.
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Fig. 3. Standard curve for angiogenin quantitation from cow's milk using dropletbased fluorescence polarization immunoassay (dFPIA). Normalized fluorescence polarization was plotted as the concentration of the unlabelled bovine angiogenin (bANG). The linear region of competitive inhibition curve is shown in the inset.
3.3. Bovine angiogenin quantitation from cow's milk through dFPIA system
Fig. 2. The formation of monodisperse droplets at the T-junction (A) and various droplet sizes by control of the applied pressures (B) using the microfluidic device used for droplet-based fluorescence polarization immunoassay (dFPIA). (A) For the droplet generation, 0.1 mg/mL bovine serum albumin (BSA) in phosphate buffered saline (PBS, pH 7.4) was injected through left inlet and fluorescein isothiocyanate (FITC) was injected through right inlet, respectively. Scale bar, 100 μm. (B) All droplets were generated at each negative pressure ( 50 to 80 kPa) against fixed positive pressure (5 kPa).
and 2000 nM against a fixed final concentration of 15 nM AF488bANG and 70 nM anti-bANG Ab. In all the experiments, 1 nL microdroplets were generated at 5 Hz using a negative pressure of 70 kPa against positive pressure of 5 kPa. The fluorescence polarization (P) values from competitive inhibition assay in droplet-based microfluidics were calculated from IV and IH using the following formula:
P = (IV − IH)/(IV + IH)
(1)
where IV defines the vertical fluorescence intensity and IH the horizontal fluorescence intensity. The variation in polarization due to the interaction between AF488-bANG and anti-bANG Ab can be modeled using the following function:
P = Pmin +
(Pmax − Pmin) (1 + 10([Unlabelled
bANG] − Log IC50)
)
(2)
Here Pmax is the maximal polarization value obtained from the competitive inhibition curve containing no unlabelled bANG, Pmin is the minimal fluorescence polarization value obtained from the competitive inhibition curve containing the maximal concentration of unlabelled bANG, and IC50 defines the half of maximal inhibitory concentration value of unlabelled bANG. Fig. 3 shows the competitive inhibition curve using unlabelled bANG. An IC50 value of 51.37 72.85 nM, a Pmax of 0.96 70.01 mP and a Pmin of 0.04 70.01 mP were extracted through a nonlinear least-sqaures fit of the experimental data (R2 ¼0.99 and power (p)¼ 1.3).
To extend our experimental approach for the high-throughput quantitation of biomarkers, inhibition of the interaction between AF488-bANG and anti-bANG Ab by cow's milk instead of unlabelled bANG was assessed. 30 μL of off-chip mixed AF488-bANG and cow's milk were loaded via the left sample inlet, and 30 μL of anti-bANG Ab of loaded via the right sample inlet (Fig. 1). It should be noted that the cow's milk sample was serially diluted to find an adequate quantitation point against a fixed final concentration of 15 nM AF488-bANG and 70 nM anti-bANG Ab. Table 1 illustrates the normalized FP values of competitive inhibition for each diluted milk sample. From this analysis, we selected and averaged the fluorescence polarization value from 4-folds to 16-folds dilution points of milk for bANG quantitation since it is located in the linear region of competitive inhibition curve as shown in the inset of Fig. 3. To quantify bANG from cow's milk by dFPIA, we used the analysis model previously reported by Jung et al. (2009) to quantify C-reactive protein (CRP) in human sera using a heterogeneous protein microarray method. Here the normalized fluorescence polarization value (y) is expressed as,
y = 100 ×
(Pmax − Px) (Pmax − Pmin)
(3)
where Px is the fluorescence polarization value obtained from the competitive inhibition curve containing x mole/dm3 of unlabelled bANG.The concentration of bANG in milk can then be determined
Table 1 FP values of diluted milk using dFPIA. Dilution factors of milk
FP values (mP)
1/2 1/4 1/8 1/16 1/32
0.117 0.03 0.29 7 0.02 0.54 7 0.02 0.677 0.01 0.89 7 0.02
J.-W. Choi et al. / Biosensors and Bioelectronics 67 (2015) 497–502
Table 2 Comparison between ELISA and FPIA for quantitation of bovine angiogenin from cow's milk. Platforms
Concentration of bANG
ELISA (μg/mL)
FPIA (μg/mL)
Chang et al. Rustam'ian (1997) et al. (1999)
Bulk
Droplet
3.80–7.50
4.03 7 0.51
4.84 71.21
2.09–4.85
(Grant no. K20904000004-13A0500-00410) and BioNano HealthGuard Research Center as Global Frontier Project (Grant no. 2013M3A6B2078957). In addition, we are grateful to Mr. Kyu-Wan Kim at Chungbuk National University for preparation of the bovine angiogenin and Mr. Hojin Kim at Pohang University of Science and Technology for fabrication of the microfluidic chips.
Appendix A. Supplementary information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.09.013.
by,
⎡ (y − y ) ⎤1/ p 2 x = x 0⎢ 1 − 1⎥ ⎢⎣ (y − y2 ) ⎥⎦
501
(4)
where x is the bANG concentration in cow's milk, x0 the bANG concentration at half of the maximal normalized fluorescence polarization value, y1 the minimal normalized fluorescence polarization value, y2 the maximal normalized fluorescence polarization value, y is the normalized fluorescence polarization value of cow's milk, and p is the power value. In established equation, y1 and y2 value were 0.04 and 0.96, respectively and p value was 1.3. Using this approach, the detection and quantitation of bANG from cow's milk using the dFPIA platform was successful. The concentration of bANG in cow's milk was determined to be 4.84 71.21 μg/mL using dFPIA, which is comparable to bulk FPIA measurements of 4.03 7 0.51 μg/mL (the standard curve for quantitation in bulk are shown in Fig. S6). These results also compare well with the reported concentrations of bANG from cow's milk as shown in Table 2 (Chang et al., 1997; Rustam'ian et al., 1999).
4. Conclusion Herein, we have demonstrated for the first time the use of a fluorescence polarization immunoassay (FPIA) and droplet-based microfluidic system for the rapid detection and quantitation of specific protein biomarker from a raw biological sample. Specifically, bovine angiogenin was successfully detected and quantified in cow's milk within a short time period and with high precision. The integration of pneumatic micro-pumps with our dropletbased microfluidic system afforded a 10 fold reduction in associated dead volumes when compared to conventional (syringe pump-based) droplet-based microfluidic experiments. In addition, the droplet-based fluorescence polarization immunoassay (dFPIA) requires an analytical volume of less than 1 nL, which represents a reduction of 5 orders of magnitude when compared to bulk FPIA systems. A significant advantage of the dFIPA platform in quantifying biomarkers is the homogeneous nature of the method. This contrasts with conventional heterogeneous approaches based on ELISA, SPR and microarrays, which all involve immobilization, washing and separation steps. Importantly this means that the described dFPIA platform is suitable for performing diagnostic measurements of clinical samples without the need for sample purification or separation. In addition, the dFIPA platform can be used as a new tool for the quality control of food and environmental samples.
Acknowledgements This work was supported by National Research Foundation of Korea (NRF) Grant funded by the Ministry of Science, ICT and Future Planning (MSIP) of Korea through Global Research Laboratory Program
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
A droplet-based fluorescence polarization immunoassay (dFPIA) platform for rapid and quantitative analysis of biomarkers Jae-Won Choi a,1, Gil-Jung Kim a,1, Sangmin Lee b, Joonwon Kim b, Andrew J. deMello c, Soo-Ik Chang a,n a
Department of Biochemistry, Chungbuk National University, Cheongju 361-763, Republic of Korea Department of Mechanical Engineering, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea c Department of Chemistry and Applied Biosciences, ETH Zürich, Vladimir Prelog Weg 1, Zürich CH-8093, Switzerland b
art ic l e i nf o
a b s t r a c t
Article history: Received 5 June 2014 Received in revised form 30 August 2014 Accepted 4 September 2014 Available online 8 September 2014
Herein, we describe for the first time the integration of pneumatic micro-pumps with droplet-based microfluidic systems as basic platform for the rapid detection and quantitation of biomarkers. Specifically, we combine this microfluidic platform with fluorescence polarization detection to identify and quantify the potent blood vessel inducing protein bovine angiogenin within cow's milk in highthroughput. The droplet-based fluorescence polarization immunoassay is successful in accurately determining the concentration (4.84 7 1.21 μg/mL) of bovine angiogenin in cow's milk, affords a 10 fold reduction in dead volumes when compared to conventional droplet-based microfluidic experiments and requires an total sample volume of less than 1 nL. & 2014 Elsevier B.V. All rights reserved.
Keywords: Droplet-based microfluidics Biomarker Fluorescence polarization Immunoassay Angiogenin Milk
1. Introduction A biomarker defines a measureable characteristic that may be used as an indicator of a biological state in normal biological and pathogenic processes, and of pharmacologic responses to therapeutic drug administration (Hawkridge and Muddiman, 2009; Mayeux, 2004; Younes and Berry, 2012). The detection of biomarkers in foods is now recognized to be of high clinical importance due to an incomplete understanding of nutrient intake from foods and health problems related to food sensitivities and allergies (Borchers et al., 2010; Schroeter et al., 2006; Schubert-Ullrich et al., 2009; Vandevijvere et al., 2012). Accordingly, the development of robust methods for high-throughput detection and quantitation of biomarkers is of high importance. Methods for biomarker analysis can be broadly classified as being either heterogeneous or homogeneous in nature. Heterogeneous assays typically require analyte immobilization, multiple washing steps, and the physical separation of target bound complexes from non-target species prior to detection (Tachi et al., 2009; Tian et al., 2012). Traditionally, lateral flow assays (LFAs) and enzyme-linked immunosorbent assays (ELISAs) have n
Corresponding author. Fax: þ 82 43 267 2306. E-mail address: [email protected] (S.-I. Chang). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bios.2014.09.013 0956-5663/& 2014 Elsevier B.V. All rights reserved.
been most commonly used to detect biomarkers in the human sera, urine, tears and food products (Kiening et al., 2005; Van Coillie et al., 2004; Wen et al., 2005a, 2005b). Unfortunately, such methods possess drawbacks such as poor limits of detection and needs of hundreds microliter. Mass spectrometry-based methods have also been used for biomarker analysis, however they require expensive and sophisticated instruments and ionization of samples (Agrawal et al., 2013; Panchaud et al., 2012). To overcome such drawbacks, protein microarrays and surface plasmon resonance (SPR) have been successfully used to quantify biomarkers in cow's milk and human sera (Hochwallner et al., 2010; Jung et al., 2009; Uludag and Tothill, 2012). However, they have limited applicability in high-throughput biomarker screens since the immobilization of antibodies, washing steps, and the separation of bound analytes from antibodies is a lengthy and complex process. To overcome these drawbacks fluorescence polarization immunoassays (FPIAs) have been increasingly used for biomarker detection (Tachi et al., 2009; Tian et al., 2012; Wang et al., 2011; Xu et al., 2011). FPIAs are homogeneous immunoassays, rapid and simple to implement and do not require antibody immobilization or washing steps. They are based on the competition of unlabelled analytes and fluorophore-labelled analytes for antibody binding sites and thus do not require separation of any kind.
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To demonstrate robust high-throughput detection and quantitation of biomarkers, we have integrated FPIA with a dropletbased microfluidics. Droplet-based (or segmented flow) microfluidic systems have been shown to overcome many of the problematic issues, such as Taylor dispersion, cross-contamination and slow mixing, encountered in continuous-flow microfluidics (Huebner et al., 2008). Significantly, picoliter-volume droplets can be generated at kilohertz (kHz) frequencies with individual droplets acting as isolated micro-reactors (Choi et al., 2012a, 2012b; Elvira et al., 2013; Song et al., 2006). To this end, dropletbased microfluidic systems have been used to perform highthroughput biological analysis in a range of genomic, proteomic, and cellomic applications (Bardiya et al., 2014; Guo et al., 2012; Therberge et al., 2010). Key examples include protein-protein interactions analysis (Choi et al., 2012a, 2012b; Srisa-Art et al., 2009), enzyme activity assays (Bui et al., 2011; Najah et al., 2013), in vitro protein expression (Huebner et al., 2009; Wu et al., 2009) and protein crystallization (Yamaguchi et al., 2013; Zheng et al., 2003). In a recent study, we reported the use of droplet-based microfluidics and FP detection for protein-protein interaction analysis (Choi et al., 2012a, 2012b). Although successful, in probing the interaction between purified antigen and antibody, specific protein biomarkers from clinical samples and/or foods were not quantified. In this study, we report, for the first time, a new platform for high-throughput detection and quantitative analysis of protein biomarkers using droplet-based fluorescence polarization immunoassay (dFPIA). Importantly, the incorporation of integrated pneumatic micro-pumps system with the dropletbased microfluidic systems obviates the need syringe-pumps or large dead-volume connections(Choi et al., 2014). It should be noted that some studies incorporating pneumatic micro-pumps within microfluidic systems have recently been reported (Sun et al., 2013; Zeng et al., 2013). However, the system described herein possesses additional desirable features; most notably complete pump integration (removing the need for external syringe pumps), simple operation via single step actuation and facile device fabrication. As a model system, we probe and quantify bovine angiogenin (bANG) from cow's milk using dFPIA. bANG is a 14.6 kDa protein and is essential to angiogenesis in normal growth processes such as development, wound healing, and neural protection (Thiyagarajan et al., 2012; Trouillon et al., 2011). In addition, bANG belongs to the RNase protein superfamily and is well known to exist in blood and milk (Chang et al., 1997; Komolova and Fedorova, 2002). bANG can be used as a biomarker for the quality control of cow's milk and commercial milk products.
2. Materials and methods 2.1. Materials Phosphate buffered saline (PBS, pH 7.4) was purchased from Amresco (Solon, OH, USA). Bovine serum albumin (BSA) was purchased from GenDEPOT (Barker, TX, USA). Fluorinert FC-40, mineral oil, and fluorescein isothiocyanate (FITC) were purchased from Sigma-Aldrich (St. Louis, MO, USA). ABIL EM 90 surfactant was purchased from Evonik Industries AG (Essen, Germany). An Alexa Fluor 488 protein labelling kit was purchased from Life Technologies (Carlsbad, CA, USA). A PD-10 desalting column was purchased from GE Healthcare (Wauwatosa, WI, USA). Polydimethylsiloxane (PDMS, Sylgard 184 silicone elastomer kit) was purchased from Dow Corning (Midland, MI, USA). Teflon AF 1600 was purchased from DuPont (Wilmington, DE, USA). All materials and chemicals were used as received.
2.2. Preparation of protein samples The Escherichia coli strain Rosetta (DE3)pLysS, which carries the pET-angiogenin plasmid, was used to express bovine angiogenin (bANG). Specific expression and purification procedures are described elsewhere (Jang et al., 2004).The bANG was labelled with Alexa Fluor 488 (AF488) using AF488 protein labelling kits according manufacturer's protocol. The serum for anti-bovine angiogenin antibody (anti-bANG Ab) was obtained from AbClon (Seoul, Republic of Korea). Anti-bANG Ab was purified by affinity chromatography using bANG-conjugated agarose beads from AbClon. The column was washed with PBS (pH 7.4) and antibody eluted using 0.1 M glycine (pH 2.8). To increase pH, each fraction was collected in tubes containing 1 M Tris–HCl (pH 8.0). The collected fractions were dialyzed into PBS to exchange buffer. Raw cow's milk samples were obtained from dairy cattle (Cheongju, Republic of Korea). Fat was removed from raw milk by centrifugation (5000 g, 4 °C, 10 min) to keep the same condition as the standard solution. 2.3. Microfluidic device fabrication To reduce the dead volumes associated with syringe pumpbased droplet microfluidics a novel microfluidic device integrating pneumatic micro-pumps was fabricated. This microfluidic device is composed of three layers (PDMS channel, glass, and flexible PDMS membrane) as shown in Fig. S1. The top layer is structured in PDMS and contains a microfluidic channel for droplet generation. This was fabricated by pouring a 10:1 (w/w) mixture of PDMS pre-polymer and curing agent (Dow Corning, Midland, MI, USA) onto a patterned Si-wafer (LG Siltron, Gumi, Republic of Korea). After curing on a hot plate at 70 °C for 2 h, the PDMS substrate was peeled off the master and inlet/outlet holes punched using a 1.5 mm diameter disposable punch. The middle layer, used to define the liquid chamber, was formed using a glass slide (Matsunani, Tokyo, Japan). An 8 mm diameter hole was drilled in the glass substrate using a MAXNC milling machine (Gilbert, AZ, USA). The bottom layer defined a flexible PDMS membrane for fluid actuation. This was formed from a 10:1 (w/w) mixture of PDMS pre-polymer and curing agent, which was spin coated (750 rpm for 50 s) on a flat Teflon-coated substrate. The entire construct was then heated on a hot plate for 15 min at 90 °C. Bonding of the three layers was achieved by treatment with an oxygen plasma. After bonding, 2 mm diameter polyurethane tube (SMC, Noblesville, IN, USA) was inserted into the inlet of microfluidic device, and a 200 μL pipette tip inserted into the outlet of the microfluidic device. Specific dimensions of the microfluidic device are described in Fig. S2. 2.4. Device operation The microfluidic device consists of two liquid chambers that deliver aqueous sample and oil to a junction where microdroplets are generated and a single outlet. 30 μL aqueous samples were loaded into each liquid chamber manually. Negative pressure ( 70 kPa) was applied to the thin PDMS membrane using inhouse pressure controller, and then positive pressure (5 kPa) was applied to the thin PDMS membrane below the each liquid chamber. The PDMS membrane motivates fluid within the microfluidic channel whilst simultaneously closing each inlet hole. Meanwhile, oil flows into the microfluidic channel due to negative pressure (up to 80 kPa) applied to the outlet and permits droplet generation. Diagram about device operation was shown in Fig. S3.
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2.5. Optical instrumentation A detailed description of the optical setup used for fluorescence polarization measurements is described elsewhere (Choi et al., 2012a, 2012b). Briefly, the fluorescence detection system consists of 488 nm diode laser (10 mW, World Star Tech, Canada), inverted fluorescence microscope (IX71, Olympus, Japan), filter cube set for Alexa Fluor 488 (FITC-3540B, Semrock, USA), dual polarization system, and an electron multiplying-charge coupled device (EMCCD, Princeton Instruments, USA). Using the system shown in Fig. S4, polarized fluorescence emission originating from sample contained within a segmented flow could be measured through 20 objective. All EM-CCD images were acquired and processed using WinSpec/32 (Princeton Instruments, Trenton, NJ, USA) with a 1 ms exposure time in binning mode.
3. Results and discussion 3.1. Performance characterization of pneumatic micro-pumps for droplet-based microfluidics application Detection and quantitation of bovine angiogenin (bANG) from cow's milk was initially performed within 1 nL droplets using a droplet-based fluorescence polarization immunoassay (dFPIA). Specifically, the competitive inhibition between unlabelled bovine angiogenin (ANG) and Alexa Fluor 488-labelled bovine angiogenin (AF488-bANG) against anti-angiogenin antibody (anti-bANG Ab) was analyzed. The bANG from cow's milk was detected and quantified using a competitive inhibition standard curve. A schematic of the microfluidic system used for dFPIA measurements is provided in Fig. 1. The integrated micro-pump is used to deliver two different aqueous and oil phases to a microchannel T-junction where droplets form. To demonstrate the efficacy of the general approach microdroplets were formed using mineral oil containing 2% ABIL EM 90 as the continuous phase and FITC
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(80 μM) and bovine serum albumin (0.1 mg/mL) in phosphatebuffered saline (PBS, pH 7.4) as the discret phase. Initially 30 μL of each phase were loaded into the left and right chambers by applying a negative pressure of 70 kPa to the flexible PDMS membrane. Subsequently, a steady positive pressure of 5 kPa was applied to the membrane to motivate the fluids into the microchannel whilst simultaneously closing each inlet hole as shown in Fig. S1. Fig. 2a illustrates the formation of monodisperse droplets at the T-junction using a 1:1 volume ratio of FITC and PBS solutions. To further assess the performance of the system, various droplet sizes and droplet generation frequencies were accessed by control of the applied pressures. As shown in Fig. 2b, droplet size (reported through the droplet length) can be varied between 100 and 300 μm by varing the negative pressure ( 50 to 80 kPa) against a fixed positive pressure of 5 kPa. In this way, the droplet generation frequency could also be varied between 2 and 15 Hz (data not shown). 3.2. Droplet-based fluorescence polarization immunoassay Subsequently, competitive inhibition of the interaction between AF488-bANG and anti-bANG Ab by unlabelled bANG was probed. Unlabelled bANG was used as a competitive inhibitor for the AF488-bANG/anti-bANG Ab interaction. As the concentration of unlabelled bANG increases, the fluorescence polarization reduces since unlabelled bANG act as competitor of AF488-bANG as shown in Fig. S5a. Based on this principle, the fluorescence polarization will decrease when AF488-bANG is replaced by unlabelled bANG in cow's milk as shown in Fig. S5b, and as the concentration of cow's milk increases, the fluorescence polarization is reduced as shown in Fig. S5c. To calibrate such a dependency, 30 μL of off-chip mixed AF488-bANG and unlabelled bANG were loaded via the left sample inlet, and 30 μL of anti-bANG Ab loaded via the right sample inlet (Fig. 1). In this experiment, the final concentration of unlabelled bANG was varied between 0.49
Fig. 1. Schematic illustration of droplet-based fluorescence polarization immunoassay (dFPIA) using the integrated pneumatic micro-pumps for the rapid detection and quantitation of protein biomarkers. Bovine angiogenin (bANG) from cow's milk was used as a biomarker model for dFPIA.
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Fig. 3. Standard curve for angiogenin quantitation from cow's milk using dropletbased fluorescence polarization immunoassay (dFPIA). Normalized fluorescence polarization was plotted as the concentration of the unlabelled bovine angiogenin (bANG). The linear region of competitive inhibition curve is shown in the inset.
3.3. Bovine angiogenin quantitation from cow's milk through dFPIA system
Fig. 2. The formation of monodisperse droplets at the T-junction (A) and various droplet sizes by control of the applied pressures (B) using the microfluidic device used for droplet-based fluorescence polarization immunoassay (dFPIA). (A) For the droplet generation, 0.1 mg/mL bovine serum albumin (BSA) in phosphate buffered saline (PBS, pH 7.4) was injected through left inlet and fluorescein isothiocyanate (FITC) was injected through right inlet, respectively. Scale bar, 100 μm. (B) All droplets were generated at each negative pressure ( 50 to 80 kPa) against fixed positive pressure (5 kPa).
and 2000 nM against a fixed final concentration of 15 nM AF488bANG and 70 nM anti-bANG Ab. In all the experiments, 1 nL microdroplets were generated at 5 Hz using a negative pressure of 70 kPa against positive pressure of 5 kPa. The fluorescence polarization (P) values from competitive inhibition assay in droplet-based microfluidics were calculated from IV and IH using the following formula:
P = (IV − IH)/(IV + IH)
(1)
where IV defines the vertical fluorescence intensity and IH the horizontal fluorescence intensity. The variation in polarization due to the interaction between AF488-bANG and anti-bANG Ab can be modeled using the following function:
P = Pmin +
(Pmax − Pmin) (1 + 10([Unlabelled
bANG] − Log IC50)
)
(2)
Here Pmax is the maximal polarization value obtained from the competitive inhibition curve containing no unlabelled bANG, Pmin is the minimal fluorescence polarization value obtained from the competitive inhibition curve containing the maximal concentration of unlabelled bANG, and IC50 defines the half of maximal inhibitory concentration value of unlabelled bANG. Fig. 3 shows the competitive inhibition curve using unlabelled bANG. An IC50 value of 51.37 72.85 nM, a Pmax of 0.96 70.01 mP and a Pmin of 0.04 70.01 mP were extracted through a nonlinear least-sqaures fit of the experimental data (R2 ¼0.99 and power (p)¼ 1.3).
To extend our experimental approach for the high-throughput quantitation of biomarkers, inhibition of the interaction between AF488-bANG and anti-bANG Ab by cow's milk instead of unlabelled bANG was assessed. 30 μL of off-chip mixed AF488-bANG and cow's milk were loaded via the left sample inlet, and 30 μL of anti-bANG Ab of loaded via the right sample inlet (Fig. 1). It should be noted that the cow's milk sample was serially diluted to find an adequate quantitation point against a fixed final concentration of 15 nM AF488-bANG and 70 nM anti-bANG Ab. Table 1 illustrates the normalized FP values of competitive inhibition for each diluted milk sample. From this analysis, we selected and averaged the fluorescence polarization value from 4-folds to 16-folds dilution points of milk for bANG quantitation since it is located in the linear region of competitive inhibition curve as shown in the inset of Fig. 3. To quantify bANG from cow's milk by dFPIA, we used the analysis model previously reported by Jung et al. (2009) to quantify C-reactive protein (CRP) in human sera using a heterogeneous protein microarray method. Here the normalized fluorescence polarization value (y) is expressed as,
y = 100 ×
(Pmax − Px) (Pmax − Pmin)
(3)
where Px is the fluorescence polarization value obtained from the competitive inhibition curve containing x mole/dm3 of unlabelled bANG.The concentration of bANG in milk can then be determined
Table 1 FP values of diluted milk using dFPIA. Dilution factors of milk
FP values (mP)
1/2 1/4 1/8 1/16 1/32
0.117 0.03 0.29 7 0.02 0.54 7 0.02 0.677 0.01 0.89 7 0.02
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Table 2 Comparison between ELISA and FPIA for quantitation of bovine angiogenin from cow's milk. Platforms
Concentration of bANG
ELISA (μg/mL)
FPIA (μg/mL)
Chang et al. Rustam'ian (1997) et al. (1999)
Bulk
Droplet
3.80–7.50
4.03 7 0.51
4.84 71.21
2.09–4.85
(Grant no. K20904000004-13A0500-00410) and BioNano HealthGuard Research Center as Global Frontier Project (Grant no. 2013M3A6B2078957). In addition, we are grateful to Mr. Kyu-Wan Kim at Chungbuk National University for preparation of the bovine angiogenin and Mr. Hojin Kim at Pohang University of Science and Technology for fabrication of the microfluidic chips.
Appendix A. Supplementary information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.09.013.
by,
⎡ (y − y ) ⎤1/ p 2 x = x 0⎢ 1 − 1⎥ ⎢⎣ (y − y2 ) ⎥⎦
501
(4)
where x is the bANG concentration in cow's milk, x0 the bANG concentration at half of the maximal normalized fluorescence polarization value, y1 the minimal normalized fluorescence polarization value, y2 the maximal normalized fluorescence polarization value, y is the normalized fluorescence polarization value of cow's milk, and p is the power value. In established equation, y1 and y2 value were 0.04 and 0.96, respectively and p value was 1.3. Using this approach, the detection and quantitation of bANG from cow's milk using the dFPIA platform was successful. The concentration of bANG in cow's milk was determined to be 4.84 71.21 μg/mL using dFPIA, which is comparable to bulk FPIA measurements of 4.03 7 0.51 μg/mL (the standard curve for quantitation in bulk are shown in Fig. S6). These results also compare well with the reported concentrations of bANG from cow's milk as shown in Table 2 (Chang et al., 1997; Rustam'ian et al., 1999).
4. Conclusion Herein, we have demonstrated for the first time the use of a fluorescence polarization immunoassay (FPIA) and droplet-based microfluidic system for the rapid detection and quantitation of specific protein biomarker from a raw biological sample. Specifically, bovine angiogenin was successfully detected and quantified in cow's milk within a short time period and with high precision. The integration of pneumatic micro-pumps with our dropletbased microfluidic system afforded a 10 fold reduction in associated dead volumes when compared to conventional (syringe pump-based) droplet-based microfluidic experiments. In addition, the droplet-based fluorescence polarization immunoassay (dFPIA) requires an analytical volume of less than 1 nL, which represents a reduction of 5 orders of magnitude when compared to bulk FPIA systems. A significant advantage of the dFIPA platform in quantifying biomarkers is the homogeneous nature of the method. This contrasts with conventional heterogeneous approaches based on ELISA, SPR and microarrays, which all involve immobilization, washing and separation steps. Importantly this means that the described dFPIA platform is suitable for performing diagnostic measurements of clinical samples without the need for sample purification or separation. In addition, the dFIPA platform can be used as a new tool for the quality control of food and environmental samples.
Acknowledgements This work was supported by National Research Foundation of Korea (NRF) Grant funded by the Ministry of Science, ICT and Future Planning (MSIP) of Korea through Global Research Laboratory Program
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