Biosensors and Bioelectronics 53 (2014) 330–335

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An automatic enzyme immunoassay based on a chemiluminescent lateral flow immunosensor Hyou-Arm Joung a, Young Kyoung Oh b, Min-Gon Kim a,b,n a b

School of Physics and Chemistry, Gwangju Institute of Science and Technology, Gwangju 500-712, Republic of Korea INGIbio Co. Ltd., R&D Center, 206, APRI, 123 Chemdan-gwagiro, Buk-gu, Gwangju 500-712, Republic of Korea

art ic l e i nf o

a b s t r a c t

Article history: Received 22 July 2013 Received in revised form 1 October 2013 Accepted 3 October 2013 Available online 16 October 2013

Microfluidic integrated enzyme immunosorbent assay (EIA) sensors are efficient systems for point-ofcare testing (POCT). However, such systems are not only relatively expensive but also require a complicated manufacturing process. Therefore, additional fluidic control systems are required for the implementation of EIAs in a lateral flow immunosensor (LFI) strip sensor. In this study, we describe a novel LFI for EIA, the use of which does not require additional steps such as mechanical fluidic control, washing, or injecting. The key concept relies on a delayed-release effect of chemiluminescence substrates (luminol enhancer and hydrogen peroxide generator) by an asymmetric polysulfone membrane (ASPM). When the ASPM was placed between the nitrocellulose (NC) membrane and the substrate pad, substrates encapsulated in the substrate pad were released after 5.37 0.3 min. Using this delayed-release effect, we designed and implemented the chemiluminescent LFI-based automatic EIA system, which sequentially performed the immunoreaction, pH change, substrate release, hydrogen peroxide generation, and chemiluminescent reaction with only 1 sample injection. In a model study, implementation of the sensor was validated by measuring the high sensitivity C-reactive protein (hs-CRP) level in human serum. & 2013 Elsevier B.V. All rights reserved.

Keywords: Enzyme immunoassay Lateral flow immunoassay Chemiluminescence Asymmetric polysulfone membrane Automatic enzyme immunoassay C-reactive protein

1. Introduction The immunoassay is a biochemical test that is commonly used to measure the concentration of target molecules. The enzyme immunoassay (EIA) is a well-established in-vitro diagnostic technique using enzyme-labeled antibodies (Lequin, 2005; Wisdom, 1976). In clinical chemistry, EIA systems, such as the enzymelinked immunosorbent assay (ELISA), are the most widely used analytic tools for determining various biomarkers, including the Creactive protein (CRP), troponin, and cytokines in body fluids (Heeschen et al., 1999; Laurent et al., 1985; Leng et al., 2008), due to their ability to produce highly specific and sensitive results within 5 h. EIA-based microfluidic immunosensors have received increased attention from researchers and have recently been commercialized (e.g. i-STATs, Abbott Laboratories., USA) (Hervás et al., 2012; Yakovleva et al., 2002; Zang et al., 2012). By using a microfluidic system, the EIA makes it possible to obtain faster and simpler measurements automatically, even with very small sample amounts. Therefore, microfluidic integrated EIA sensors are suitable for point-of-care testing (POCT). However, as a disposable n Corresponding author at: School of Physics and Chemistry, Gwangju Institute of Science and Technology, Gwangju 500-712, Republic of Korea. Tel.: þ 82 62 715 3330; fax: þ 82 62 715 3419. E-mail address: [email protected] (M.-G. Kim).

0956-5663/$ - see front matter & 2013 Elsevier B.V. All rights reserved.

sensor, microfluidic systems are not only relatively expensive, but also require a complicated manufacturing process (Yager et al., 2006). For these reasons, cheaper and simpler EIA-based POCT sensors are required for efficient on-site diagnostics. The lateral flow immunosensor (LFI) strip is one of the most widely used POCT systems for various types of detections (Ngom et al., 2010; Posthuma-Trumpie et al., 2009; Wongsrichanalai et al., 2007; Zuk et al., 1985) due to its rapid assay time, cost effectiveness, and ease of use. The conventional LFI strip uses antibody-labeled gold nanoparticles as a signal indicator, and fluorescent materials such as fluorescein, Q-dot, and europium are also used for obtaining better quantitative and sensitive results (Choi et al., 2004; Li et al., 2010; Xia et al., 2009). However, the EIA system is nonetheless difficult to implement in the LFI strip sensor because it requires additional steps during measurement, such as washing and injecting. Therefore, additional fluidic control systems are required for the implementation of EIA in an LFI strip sensor. For example, an ELISA-on-a-chip system has been reported for the detection of cardiac troponin I (cTnI) by using a cross flow of enzyme substrate solutions through a plastic fluidic channel (Cho et al., 2006). For this reason, no automatic EIA-based LFI strip immunosensor without additional fluidic control has yet been reported. In this study, we developed a novel chemiluminescence LFI strip-based automatic EIA system using a delayed-release technique that could delay the release of enzyme substrates and change

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the reaction pH during the sample flow without the need for additional operations or complex manufacturing processes. To date, several types of horseradish peroxidase (HRP) catalyzed chemiluminescence LFI (CLFI) strip sensors have been reported (Cho et al., 2009; Kim et al., 2010; Mirasoli et al., 2012). However, these strip sensors require a substrate (luminol and hydrogen peroxide) addition step for generation of the chemiluminescent signal because of the following 3 problems. First, the optimal pH conditions differ for the immune response (neutral pH) and chemiluminescence (pH 8.5–9.5) (Dotsikas and Loukas, 2007). Therefore, the strip sensor pH needs to be changed from neutral to alkaline during the sample flow. Second, hydrogen peroxide has storage and stability problems. Third, if HRP-labeled detection antibodies are mixed with the substrates before the end of the immune reaction, an undesired signal will be produced. Therefore, the chemiluminescence reaction should occur after the sandwich immune reaction is complete. To overcome these problems, we designed the new LFI strip to sequentially control the immunoassay, delayed release of substrates with pH change, hydrogen peroxide generation, and the luminol reaction.

2. Materials and methods 2.1. Materials C-reactive protein (CRP)-free serum (90R-100), surfactant 10G (95R-103), CK-MB (30-AC66), troponin I (30-AT43), proBNP (30CCP1149) and myoglobin (30-AM20) were purchased from Fitzgerald Industries International (Acton, MA). CRP was purchased from Wako Chemicals (309–51191; Osaka, Japan), and anti-CRP polyclonal antibody and monoclonal antibody-HRP conjugate were purchased from Abcam Inc. (Cambridge, MA, USA). The nitrocellulose (NC) membrane was purchased from Millipore (HFB02404; Billerica, MA). The sample pad (P/N BSP-133-20) and asymmetric polysulfone membrane (ASPM, Vivid plasma separation-GX, USA) were purchased from Pall Co. (Port Washington, NY). Choline oxidase (ChOx) was purchased from TOYOBO Co., Ltd. Luminol, choline chloride, p-coumaric acid, p-iodophenol, polyvinylpyrrolidone (PVP55K), sucrose, human serum albumin, thrombin and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO). All buffers and reagent solutions were prepared with water purified using the Brema water purification system. 2.2. Preparation of the substrate pad A solution containing 100 μL luminol (0.5 M in 50 mM NaOH), 30 μL choline chloride (1 M in DW), and 1 μL p-coumaric acid (0.5 M in dimethylformamide (DMF)) was mixed with 870 μL 0.1 M carbonate buffer (pH 9.2). After mixing, the 75 μL mixed solution was loaded on the pad (50  3.8 mm2) and was subsequently dried in a dry oven at 65 1C for 30 min. A solution-treated pad was attached to the middle of a piece of sealing tape (50  15 mm2) and was cut to obtain a 3.8  3.8-mm2 section by using a cutter. All solution components were optimized for the chemiluminescence lateral flow immunosensor (LFI). 2.3. Preparation of detection, capture, and control antibody solutions 2.3.1. Detection antibody solution The solution containing 5 μL anti-CRP monoclonal antibodyHRP (2 mg mL  1) was mixed with 95 μL of 2% (v/v) surfactant 10G, 3.75% (w/v) PVP 55 K, and 0.5% (w/v) sucrose-added 1X pH 7.2 phosphate-buffered saline (PBS) which is composed of 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4 and 1.4 mM KH2PO4.


2.3.2. Capture and control antibody solutions The solution containing 5 μL anti-CPR polyclonal antibody (10 mg mL  1) for capture or 5 μL anti-mouse IgG (1 mg mL  1) was mixed with 45 μL choline oxidase (100 unit mL  1)-added PBS. 2.4. Dispensing of the detection, capture, and control antibody solutions An NC membrane (0.25  30 cm2) was used to immobilize the capture and control antibodies and to load the detection antibodyHRP conjugate to different zones by using a dispenser (DCI100; Zeta Corporation, Kyunggi-do, South Korea); the detection antibody (2.5 μL cm  1) and capture and control antibodies (1 μL cm  1 for both) were added consecutively. The distance between the test and control zones was approximately 3 mm, and the distance between the detection antibody and the test zone was approximately 5 mm. The dispensed NC membrane was dried for 1 h in a desiccator at room temperature. 2.5. Preparation of the chemiluminescence LFI (CLFI) strip The strip was composed of 6 parts, including the sample pad, inter pad, substrate pad, asymmetric membrane, antibody-dispensed NC membrane, and absorbent pad. An absorbent pad (1.5  30 cm2) was attached to the top of the NC membrane, and the inter pad (5 mm  30 cm) and sample pad (1.5  30 cm2) were consecutively assembled on a plastic adhesive backing (60  300 mm2). The assembled strips (3.8 mm in width) were cut with a cutter device. After cutting, the asymmetric membrane (5  3.8 mm2) and substrate pad were stacked on the NC membrane between the inter pad and the antibody-HRP loaded line. The larger pore side of the asymmetric membrane made contact with the substrate pad. 2.6. Optimization of luminol, p-coumaric acid, choline chloride, and choline oxidase concentrations 2.6.1. Luminol One microliter of 20 μg mL  1 Ab-HRP was spotted on the NC membrane and dried at 37 1C in a dry oven for 15 min. Various concentrations of luminol in pH 9.2 0.1 M carbonate buffer were loaded on the substrate pad and dried at 65 1C in a dry oven for 30 min. The luminol-stored substrate pads were attached to the Ab-HRP-spotted NC strip, and 100 μL of 1 mM hydrogen peroxideadded CRP free serum was loaded. The concentration of 50 mM showed the highest signal intensity (Figure S2A). The strip preparation process was performed as described above. 2.6.2. p-coumaric acid p-coumaric acid was dissolved in DMF (0.5 M). Various concentrations of p-coumaric acid were prepared on substrate pads containing 50 mM luminol. Strip preparation and measurement were performed in the same manner as described for the luminol optimization steps, but with a different Ab-HRP concentration (0.2 μg mL  1). The signal (I) was compared to that of the no enhancer condition (I0). The concentration of 0.5 mM showed the highest signal intensity (Figure S2B). 2.6.3. Choline chloride One microliter of 2 μg mL  1 Ab-HRP in 100 units mL  1 choline oxidase-added PBS was spotted on the NC membrane. Various concentrations of choline chloride-added substrate pads with 50 mM luminol and 0.5 mM p-coumaric acid were prepared. The test strip was prepared in the same way as described above in section (a). The signal intensity was saturated at concentrations of more than 10 mM choline chloride (Figure S2C).


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2.6.4. Choline oxidase Various concentrations of choline oxidase were spotted on strips with 2 μg mL  1 Ab-HRP in PBS and dried. The 50 mM luminol, 0.5 mM p-coumaric acid, and 30 mM choline chloride in pH 9.2 carbonate buffer were dried on a substrate pad. The test strip was prepared in the same way as described above in section (a). The concentration of 100 unit mL  1 choline oxidase showed the highest signal/background ratio (Figure S2D). 2.7. Optimization of PVP and surfactant 10G (S-10G) concentrations 2.7.1. PVP screening The 2 μL solutions containing 10 μg mL  1 Ab-HRP and 1% (w/v) PVPs of various molecular weights (10 K, 29 K, 40 K, 55 K, and 360 K) in PBS were spotted on the NC membrane of the strip and dried at 37 1C in a dry oven for 15 min. After drying, 100 μL CRPfree serum was loaded on the sample pad. After 10 min, the sample pad and absorbent pad were removed, and 30 μL luminol solution (1 mM luminol, 0.5 mM p-coumaric acid, and 1 mM hydrogen peroxide in 0.1 M carbonate buffer (pH 9.2)) was loaded on the NC membrane of the strip. The chemiluminescence signal intensity ratio between the Ab-HRP spotting area (S) and the background (B) were compared; 55 K PVP showed the lowest S/B (Figure S4A). 2.7.2. Surfactant 10G The 2 μL solutions containing of 10 μg mL  1 Ab-HRP with 1% PVP (55 K) and various volumes (%) of S-10G were spotted on the NC membrane of the strip. All experimental steps were carried out in the same manner as described above for PVP screening. The 2% S10G showed the lowest S/B ratio at the spotting zone (Figure S4b). 2.7.3. PVP The 2 μL 10 μg mL  1 Ab-HRP solutions, containing 2% (v/v) S-10G and various weights (%) of PVP (55 K) and the 1 μL 1 mg mL  1

capture antibody in PBS, were spotted on different zones of the NC membrane of strips. After drying, the 100 μL CRP-free serum, containing no or 10 ng mL  1 CRP, was loaded on the sample pad. After 10 min, the sample and absorbent pads were removed, and the chemiluminescence signal was measured. To determine the optimal PVP concentration, we compared the chemiluminescence signal intensity obtained with 0 (I0) and 10 (I) ng mL  1 CRP (Figure S4C). 2.8. Analysis of CRP and selectivity evaluation The sample solutions containing various CRP concentrations or other serum proteins were prepared in a CRP-free human serum solution. In selectivity evaluation, the concentration of CRP, myoglobin, CK-MB, troponin I and proBNP were adjusted to 100 ng mL  1. In case of albumin and thrombin, the concentration was adjusted to 1 mg mL  1. The 100 μL sample solutions prepared were incubated on the LFI strip sensor. The signal was measured using the ChemiDoc system (Bio-Rad), and analyses were conducted using the Image Lab 4.0 software.

3. Results and discussions 3.1. Basic concept of LFI strip-based EIA system Fig. 1 shows a schematic illustration of the chemiluminescence LFI strip-based automatic EIA system. This system consists of 3 parts (Fig. 1A). Part I involves the delayed-release of luminol, choline chloride (hydrogen peroxide generator), and p-coumaric acid (luminol signal enhancer) (Ramos et al., 2001). Part II involves the separation of the HRP-labeled detection antibody from its substrates. Instead of using a conjugate pad, we coated the HRPlabeled detection antibody, which is located between the substrate pad and test line, on the NC membrane with polyvinylpyrrolidone (PVP) and Surfactant 10G (S-10G). PVP is a hydrophilic polymer

Fig. 1. Schematic illustrations of the structure (A) and overall reaction process (B) of the chemiluminescence LFI strip-based EIA system.

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Fig. 2. Experimental schemes and images for time-dependent absorption (A) and release (B) effects using various membrane types, including no membrane, ASPM, and NC. (C) Color intensities of the substrate pad over the absorption of samples. (D) Chemiluminescence intensities of the test line over the release of the substrates from the substrate pad.

and is known to inhibit protein adsorption activity (Matsuda et al., 2008). Therefore, the detection antibody was not adsorbed on the NC membrane, and the nonspecific signal was minimized by the S-10G (Figure S3 and S4). When the samples flowed into the NC membrane, the detection antibodies flowed to the test and control lines along with the target antigens. As a result of the immune reaction, antigen and detection antibody complexes bound with capture antibodies and also un-reacted detection antibodies were washed out within 5 min. Therefore, we were able to perform the chemiluminescence reaction automatically after immune reactions were complete. Part III involves automatic generation of hydrogen peroxide during the immune reaction. In this part of the system, hydrogen peroxides were generated by the enzyme reaction of immobilized choline oxidase (ChOx) on the capture and control lines. ChOx is suitable for use in a chemiluminescence system as a hydrogen peroxide generator because the maximum activity pH of the choline chloride oxidation reaction is similar to that of the chemiluminescence reaction (approximately pH 8.5). As a result of the oxidation of choline chloride, hydrogen peroxides were generated on the test and control lines. Therefore, the chemiluminescence reaction occurred proportionally to the antigen concentrations. The overall reaction steps are described in detail in Fig. 1B. 3.2. A delayed-release effect of chemiluminescence substrates In our previous study, we discovered interesting fluid flow characteristics of the asymmetric polysulfone membrane (ASPM), which is the plasma separation membrane whose pore sizes depend on hydrophilic properties (Oh et al., 2013). Horizontal flow at the small pore side was found to be stronger than that at the large pore side. In contrast, vertical flow was stronger at the large pore side due to the capillary action. Therefore, the samples could not flow laterally at the large pore side of the ASPM. In part I of Fig. 1A, the ASPM and

substrate pad were stacked at the center of the NC membrane, and then the small pore side of the ASPM made contact with the NC membrane. We predicted that if the aqueous samples flowed to the NC membrane, a proportion of the samples would be absorbed to the substrate pad through the ASPM, and after a certain period of time, the substrates would be released to the NC membrane. To confirm this delayed-release effect, we performed two experiments: the absorption of a color material in the substrate pad and the release of substrates from the substrate pad. Fig. 2 shows the timedependent sample absorption and substrates release. Fig. 2A and C shows the color changes in the substrate pad due to the absorption of loaded samples. After injection of 100 μL color dye (0.1 mg mL  1 sulforhodamine 6G in phosphate-buffered saline (PBS) with 1% S-10G), we measured color changes in the substrate pad for three cases: (i) the ASPM was inserted between the NC membrane and the substrate pad with the small-pore side in contact with the NC membrane, (ii) the NC membrane was inserted, or (iii) no membrane was inserted. The results showed that the color of the substrate pad was saturated after approximately 4 min for ASPM, 3 min for NC, and 2 min for no membrane, which indicated that the sample absorption was delayed on using the ASPM. Fig. 2B and D show the chemiluminescence signals by release of substrates from the substrate pad. The HRP labeled-antibody (1 μg mL  1) and choline oxidase (ChOx; 100 U/mL) were immobilized in the NC membrane between the absorbent pad (see Figs. S1 and S2) and the substrate pad, which encapsulated the luminol (50 mM), choline chloride (30 mM), and p-coumaric acid (0.5 mM). After 100 μL PBS buffer was loaded on the sample pad, the chemiluminescence signal was monitored. As shown in Fig. 2D, in the no membrane case, the chemiluminescence signal was observed after approximately 2 min, sharply increased until 7 min, and then rapidly decreased. In the NC test, the signal was observed after 2–3 min, increased up to 15 min, and then decreased. In the ASPM test, the signal appeared after approximately 5 min, gradually increased till 10 min, and was maintained until the end of


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measurement (up to 25 min) with the lowest standard deviations. This indicated that the ASPM inserted between the NC membrane and the substrate pad functioned to delay the release of substrates from the substrate pad. The times required to produce the initial chemiluminescence signal were calculated as 0.7970.07 min for the no membrane case and 2.0170.47 min for the NC membrane case. In these cases, the release of substrates started before complete wetting of the solution in the substrate pad. This suggested that these 2 cases could not completely separate the absorption and release. In contrast, the ASPM could release substrates after the end of the absorption. Therefore, if the HRP-labeled detection antibodies flowed to the absorbent pad within 5 min, the substrates were not mixed with HRP as a result of immune reaction, with the exception of the test or control line-bound antibodies. Thus, the specific chemiluminescence signal was generated on the test or control line. For comparison, the asymmetric membrane was placed in the opposite direction; because the capillary force was acting in the opposite direction, the signal intensity significantly decreased (data not shown). 3.3. Automatic pH change during the measurement As mentioned above, the reaction pH must be changed during the sample flow when using an automatic chemiluminescence LFI system. All substrates were mixed with 0.1 M carbonate buffer (pH 9.2) so that the buffer components were released with the substrates. Therefore, when substrates are released, the pH of the NC membrane should be changed from neutral to alkaline. To confirm the pH change, we treated the NC membrane with 0.05% α-naphtholbenzein (containing 1% S10G and 50% ethanol), which is a pH indicator that changes the color from yellow to blue between pH 8.2 and 10.0. After drying the NC membrane, the PBS buffer was loaded on the sample pad, and time-dependent pH changes were observed on the NC membrane. As shown in Fig. 3, we clearly observed a pH change from neutral to alkaline on the NC membrane after 5 min, which showed a similar shape as that of the release test. Based on this result, we confirmed that the reaction pH changed automatically to an alkaline pH during the sample flow with the release of the substrates. 3.4. Measurement of hs-CRP in human serum For the performance evaluation, we measured the content of the high sensitivity C-reactive protein (hs-CRP) in human serum. The hs-CRP is now recognized as a major cardiovascular risk factor and as a secondary target for statin therapy (Ridker, 2010). For this reason, several large-scale prospective studies have demonstrated that hs-CRP is a strong independent predictor of future myocardial infarctions and strokes in apparently healthy people (Guijarro, 2001). Fig. 4 shows the results of the hs-CRP measurement.

Fig. 3. Plot and image (inset) of time-dependent color-intensity changes in the α-naphtholbenzein (pH indicator, pH 8.2–10.0)-treated NC membrane.

Fig. 4. Image (A) and plot (B) of change in chemiluminescence signals depending on the concentration of hs-CRP.

The chemiluminescence signal was measured over 10–12 min after sample injections with various hs-CRP concentrations in human serum. We obtained similar detection sensitivity (limit of detection, LOD, 1.05 ng mL  1) and a wider dynamic range (detection range: 1–10,000 ng mL  1) compared to the results of our previous hs-CRP measurements using a conventional LFI system (detection range 10–1000 ng mL  1) (Oh et al., 2013). In addition, we performed the selectivity evaluation with other serum proteins. As a result, the undesirable signals were not observed in tested proteins except in the CRP spiked sample (Fig. S5).

4. Conclusions In conclusion, we developed a novel delayed substrate release technique by using the fluid characteristics of the ASPM in an LFI strip, and used the chemiluminescence LFI strip-based automatic EIA system without using an additional fluidic system or complex production process. The use of the delayed release technique enabled automatic implementation of multiple EIA steps such as pH change, delayed release of secondary reagents, and separation of immune and chemiluminescence reactions. To demonstrate the application of the developed method for biological/complex sample measurement, we used the chemiluminescence-based EIA system for CRP measurement. We showed that the developed method provides quantitative measurement with a wide measuring range without using a light source. In addition, compared to the conventional LFI strip sensor method, the developed system can provide several advantages such as the use of a single-step measurement, cost-effectiveness, and the ease of mass production. Until now, complex microfluidic devices or multi-step injection methods have been used for the implementation of EIA in LFI strip biosensors. However, compared to the LFI strip sensor, the other methods require the use of complex mass production systems and have relative higher costs and multiple measurement steps. To the best of our knowledge, no research has been reported on automatic strip sensing methods for multi-reactions without any fluidic control system. Furthermore, we think that this method overcomes the limitations of the conventional LFI strip biosensor. Finally, this system expands the scope of EIA, which we believe to be the most significant achievement of the present study.

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Acknowledgements This work was financially supported by the R&D Joint Venture Program, NLRL Program (2011-0028915) and Public welfare & Safety Research Program (2010-0020776) through the National Research Foundation of Korea (NRF) funded by the Korean government (MEST). Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at References Cho, I.H., Paek, E.H., Kim, Y.K., Kim, J.H., Paek, S.H., 2009. Anal. Chim. Acta 632 (2), 247–255. Cho, J.-H., Han, S.-M., Paek, E.-H., Cho, I.-H., Paek, S.-H., 2006. Anal. Chem. 78 (3), 793–800. Choi, S., Choi, E.Y., Kim, H.S., Oh, S.W., 2004. Clin. Chem. 50 (6), 1052–1055. Dotsikas, Y., Loukas, Y.L., 2007. Talanta 71 (2), 906–910. Guijarro, C., 2001. Circulation 104 (22), E127. Heeschen, C., Goldmann, B.U., Langenbrink, L., Matschuck, G., Hamm, C.W., 1999. Clin. Chem. 45 (10), 1789–1796. Hervás, M., López, M.A., Escarpa, A., 2012. TrAC Trends Anal. Chem. 31 (0), 109–128.


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An automatic enzyme immunoassay based on a chemiluminescent lateral flow immunosensor.

Microfluidic integrated enzyme immunosorbent assay (EIA) sensors are efficient systems for point-of-care testing (POCT). However, such systems are not...
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