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Competitive immunoassay of progesterone by microchip electrophoresis with chemiluminescence detection
Journal of Chromatography B, 936 (2013) 74–79
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Competitive immunoassay of progesterone by microchip electrophoresis with chemiluminescence detection Fanggui Ye, Jinwen Liu, Yong Huang, Shutin Li, Shulin Zhao ∗ Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources (Ministry of Education), College of Chemistry and Chemical Engineering, Guangxi Normal University, Guilin 541004, China
a r t i c l e
i n f o
Article history: Received 9 June 2013 Accepted 1 August 2013 Available online 8 August 2013 Keywords: Chemiluminescence detection Competitive immunoassay Human serum Microchip electrophoresis Progesterone
a b s t r a c t A sensitive and rapid homogeneous immunoassay method based on microchip electrophoresischemiluminescence detection (MCE-CL) using luminol-hydrogen peroxide as chemiluminescence system catalyzed by horseradish peroxidase (HRP) was developed for the determination of progesterone (P). The assay was based on the competitive immunoreactions between HRP-labeled P antigen (HRP-P) and P with a limited amount of anti-P mouse monoclonal antibody (Ab), and MCE separation of free HRP-P and HRP-P-Ab immunocomplex followed by CL detection. The effect of various factors such as conditions for the CL reaction, MCE and incubation time for the immunoreactions were examined and optimized. Under optimal assay conditions, the MCE separation was accomplished within 80 s. The linear range of detection for P was 8–800 nM with a detection limit of 3.8 nM (signal/noise ratio = 3). This present method has been applied to determine P in human serum samples from normal and pregnant women. The result indicates that the proposed MCE-CL based homogeneous immunoassay method can serve as an alternative tool for clinical assay of P. © 2013 Published by Elsevier B.V.
1. Introduction Progesterone (P) is one of the hormones in human bodies that stimulates and regulates various functions, and plays an important role in maintaining pregnancy. It helps to prepare women body for conception and pregnancy, and regulates the monthly menstrual cycle [1]. The dynamic monitoring of P in human serum is important to reproductive system and investigation of the mechanism of various steroid prophylactics and anti-early-pregnancy drugs [2]. In addition, it has been indicated that P also have neuroprotective effect after traumatic brain injury [3]. Currently, the most common methods used to quantify P are chromatography and immunoassay methods. Chromatography methods such as LC–MS [4] and GC–MS [5] have been developed as reference methods for determination of P in human serum, which have advantages of high accuracy, selectivity, reproducibility and sensitivity. Immunoassays used in clinical practice including radioimmunoassay [6], enzyme linked immuno absorbent assay
∗ Corresponding author at: College of Chemistry and Chemical Engineering, Guangxi Normal University, Guilin 541004, China. Tel.: +86 773 5856104; fax: +86 773 5832294. E-mail addresses: [email protected] (F. Ye), [email protected] (J. Liu), [email protected] (Y. Huang), [email protected] (S. Li), [email protected] (S. Zhao). 1570-0232/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.jchromb.2013.08.002
(ELISA) [7], fluoroimmunoassay [8], chemiluminescent enzyme immunoassay (CL-EIA) [9,10] and time-resolved fluorescence immunoassay [11] have also been reported for the evaluation of P in human serum. By far, the most immunoassays for P are heterogeneous, i.e. either antibody or antigen is immobilized at the solid phase. Heterogeneous immunoassays in general have low limits of detection (nM to pM), but they require a multi-step work flow (e.g. primary antibody incubation, washing, secondary antibody incubation etc.), and are therefore time-consuming [12]. Microchip electrophoresis (MCE) has become a very attractive separation technique for chemical and biological analyses [13]. It have various advantages such as reduced sample and reagent consumption, high separation speed and efficiency, short analysis time, simple operation, and easy integration and automation, which make it unequally suitable for biological and clinical analysis. Immunoassay is one application that makes use of these advantages. Many types of immunoassays on MCE have been applied in clinical diagnoses and biochemical studies. Over the past decade, MCE-enhanced immunoassays have been developed to determine cortisol [14], theophylline [15], matrix metalloproteinase-8 [16], goat anti-human IgG [17], B-type natriuretic peptide [18] and staphylococcal enterotoxin B [19]. Laser-induced fluorescence (LIF) detection is the most sensitive detection scheme available for MCE immunoassay. However, LIF detection requires relatively large and expensive apparatus systems. Chemiluminescence (CL) detection offers advantages including high sensitivity, simple optical
F. Ye et al. / J. Chromatogr. B 936 (2013) 74–79
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Fig. 1. Schematic diagram of the layout of the glass/PDMS microchip. S, sample reservoir; B, buffer reservoir; SW, sample waste reservoir; BW, buffer waste reservoir; R, the oxidizer reagent reservoir.
structure, low background noise and relatively simple and inexpensive instrumentation. CL is particularly suitable for integration on microfluidic devices. MCE-CL-enhanced immunoassays have been successfully used for analysis of rat IgG [20], human serum albumin [21], immunosuppressive acidic protein [22], atrazine [23], testosterone [24] and thyroxine [25]. Herein, we report on the development of a homogeneous immunoassay coupled with MCE-CL to quantify P in human serum. The assay was based on competitive immunoreactions between horseradish peroxidase (HRP)-labeled P antigen (HRP-P) and P with a limited amount of anti-P mouse monoclonal antibody (Ab), and MCE separation of free HRP-P and HRP-P-Ab immunocomplex followed by CL detection. It is well known that HRP catalyzes the luminol-H2 O2 CL reaction and greatly enhances the CL emission [26]. Thus, two peaks for free HRP-P and HRP-P-Ab immunocomplex were observed in electropherogram. Both HRP-P and the HRP-P-Ab immunocomplex were sensitively detected. Because of the effective MCE separation, the CL analytical signal was less prone to sample matrix interference. The conditions for MCE separation and CL detection were investigated in detail, and the quantification of P in human serum from normal and pregnant women was demonstrated.
introduction channel (250 m) was 65 m, the depth of all microchannels was 25 m, and the length of double T was 60 m. 2.2. Human serum sample preparation Human serum samples were kindly provided by the No. 5 People’s Hospital (Guilin, China). A 250 L of human serum sample was collected in a 1.5 mL vial, then 500 L trichloroacetic acid was added and votexed for 5 min. The mixture solution was centrifuged for 20 min at 12,000 rpm. The supernatant was transferred to another 1.5 mL vial and evaporated to dryness under N2 . The residue was reconstituted in 250 L 20 mM borate solution and kept at 4 ◦ C before analysis. 2.3. Immunoreaction To carry out the immunoreaction, 20 L different concentrations of P or serum sample, 20 L 5.0 × 10−7 M HRP-P and 20 L 2.5 × 10−7 M Ab were mixed and diluted to 100 L with phosphate buffer (pH 7.4), and incubated for 35 min at 37 ◦ C. The whole procedure is illustrated in the following equations: HRP-P + P + Ab − HRP-P-Ab + HRP-P + P-Ab + P
2. Experimental 2.1. Reagents and apparatus HRP-P, P and Ab was purchased from Zhengzhou Biocell Biotechnology Co., Ltd. (Zhengzhou, China). Tween 20 was obtained from Shanghai Chemical Reagents Corporation (Shanghai, China). Luminol was purchased from Fluka (Buchs, Switzerland). Paraiodophenol (PIP), hydrogen peroxide (H2 O2 ) were obtained from Taopu Chemicals (Shanghai, China). All other chemicals used in this work were of analytical grade. The P and Ab solutions were prepared by dissolving the reagents in 20 mM phosphate buffer (pH 7.4). The electrophoretic buffer and oxidizer solution were prepared according to method described previously [27]. Briefly, the electrophoretic buffer was 10 mM phosphate buffer (pH 10.2) containing 1.2 mM luminol and 0.08% (w/w) Tween 20. The oxidizer solution was 80 mM H2 O2 and 1 mM PIP in 40 mM NaHCO3 solution at pH 8.7. All solutions were filtered through 0.22-m membrane filters prior to use. Water was purified by employing a Milli-Q plus equip from Millipore (Bedford, MA, USA), and used throughout the work. A homemade MCE-CL system used in this work was similar to that described in our previous work [27]. The fabrication of the double “T” microchip was described previously [28] and illustrated in Fig. 1. The width of all microchannels except the oxidizer
where Ab is anti-P antibody added at a limited amount, HRP-P was added at a fixed amount, and P is free progesterone. HRP-P competes with P in the solution for binding to a limited amount of Ab. The concentration of P in the solution is directly proportional to that of free HRP-P, but inversely proportional to that of the HRP-P-Ab. Therefore, P in the sample can be measured by monitoring the CL intensity from free HRP-P after separation from the immunocomplex. 2.4. MCE-CL procedure The microchannels were rinsed sequentially with 0.1 M NaOH, water and electrophoresis buffer for 10 min each. Prior to electrophoresis, all reservoirs were filled with the electrophoretic buffer. Vacuum was applied to the reservoir BW in order to fill the separation channel with the electrophoretic buffer. Then, the electrophoretic buffer solutions in reservoir S and reservoirs R were replaced by sample solution and oxidizer solution, respectively. Sample injection was performed by applying 450 V to S for 15 s with SW grounded, whereas B was set at 280 V, BW was set at 350 V, and R was left floating. For MCE separation, 2500 V was applied to B and 1500 V was applied to both S and SW with BW grounded. At the same time, 500 V was applied to R. The analytes were transported into the separation channel toward BW and then were mixed with the oxidizer solution at the junction of the oxidizer introduction
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Fig. 2. The effect of the oxidizer solution pH on CL intensity. Electrophoretic buffer was 10 mM phosphate buffer (pH = 10.2) containing 0.08% Tween 20 and 1.2 mM luminol. The oxidizer solution was 40 mM NaHCO3 containing 80 mM H2 O2 and 1 mM PIP at different pH values.
Fig. 3. The effect of luminol concentration on CL intensity. Electrophoretic buffer was 10 mM phosphate buffer (pH = 10.2) containing 0.08% Tween 20 and luminol at different concentrations. The oxidizer solution was 40 mM NaHCO3 (pH = 8.7) containing 80 mM H2 O2 and 1 mM PIP.
3.2. Optimization of MCE separation condition channel and the separation channel, producing CL emission that was detected by PMT.
3. Results and discussion 3.1. Optimization of CL detection Enhancing the sensitivity of CL detection is paramount for successful homogeneous competitive immunoassay. Besides the setup of the CL detector, CL sensitivity is mainly dependent on the CL reaction and MCE conditions, which need to be systematically optimized. Therefore, the effects of pH value of the oxidizer solution (CL reaction medium), luminol and PIP concentrations on the separation and detection were investigated. The oxidizer solution pH affected significantly the effective electric charge of the analytes. Generally, the luminol-H2 O2 CL immunoreaction was carrying out in alkalescence medium [25]. Therefore, the effect of pH value of the oxidizer solution was investigated in a pH range of 8.0–9.5. The results were illustrated in Fig. 2. The CL intensity increased gradually with the increase of pH value up to 8.7, where the maximum CL signal was reached. Further increasing of pH value resulted in a decrease in CL signal. Therefore, the oxidizer solution was buffered at 8.7 for a maximum enhanced CL signal. Fig. 3 shows the effect of luminol concentration. A concentration ranged from 0.5 to 2.0 mM was tested. As can be seen from this figure, the CL intensity first increased and then decreased with the increase of luminol concentration. The maximum CL intensity was obtained when the concentration of luminol was 1.2 mM. Therefore, a luminol solution at this concentration was chosen for subsequent studies. Keeping luminol concentration at 1.2 mM and pH value of the oxidizer solution at 8.7, PIP concentration in a range of 0.2–1.2 mM was tested. As can be seen from Fig. 4, the CL intensity increased with increasing PIP concentration in the range of 0.2–1.0 mM, whereas it decreased with further increasing PIP concentration from 1.0 to 1.2 mM. A concentration of 1.0 mM PIP was used for further experiments.
Effective separation of HRP-P and HRP-P-Ab complex is a key step for the detection of P in this work. However, it was found that adsorption of proteins by the glass channel surface significantly deteriorated the separation of HRP-P from the HRP-P-Ab complex. To overcome this difficulty, a popular surfactant, Tween 20, was added into running buffer, and the influence of Tween 20 concentration ranging from 0.04 to 0.12% (w/w) on the separation was investigated. The results indicate the resolutions (Rs ) between HRPP and HRP-P-Ab complex all increased with increasing Tween 20 concentrations. Conversely, the migration times of HRP-P and its immunocomplex increase with the increase of Tween 20 content. Taking into consideration the Rs and speed of analysis, 0.08% (w/w) Tween 20 was used to further optimize separation conditions. The pH of the electrophoretic buffer has an important effect on the channel-wall surfaces characteristics of microchip and the
Fig. 4. The effect of PIP concentration on CL intensity. Electrophoretic buffer was 10 mM phosphate buffer (pH = 10.2) containing 0.08% Tween 20 and 1.2 mM luminol. The oxidizer solution was 40 mM NaHCO3 (pH = 8.7) containing 80 mM H2 O2 and PIP at different concentrations.
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Fig. 6. The effect of incubation time on the CL intensity of the immunocomplex. The concentration of HRP-P and Ab are 5 × 10−7 M, 2.5 × 10−7 M respectively. Fig. 5. The effect of electrophoretic buffer pH on resolution. Electrophoretic buffer was 10 mM phosphate buffer at different pH value containing 0.08% (w/w) Tween 20 and 1.2 mM luminol. The oxidizer solution was 40 mM NaHCO3 (pH 8.7) containing 80 mM H2 O2 and 1 mM PIP.
effective electric charge of the analytes. In this work, the phosphate buffer was used as background electrolyte for the separation of HRP-P and its immunocomplex, and pH of phosphate buffer were optimized. It can be seen from Fig. 5, the Rs of HRP-P and its immunocomplex increases with the increase of electrophoretic buffer pH value from 9.6 to 10.2, while they decrease with further increasing of running buffer pH from 10.2 to 10.6. Therefore, a running buffer with a pH value of 10.2 was used for further experiments. 3.3. Effect of incubation time The incubation time had a great influence on the immunocomplex formation. Therefore, the incubation time of HRP-P and Ab was studied. In this experiment, seven identical samples containing 5 × 10−7 M HRP-P and 2.5 × 10−7 M Ab were incubated for different lengths of time, and then the solution was injected into the MCE system to obtain an optimal incubation time (Fig. 6). It can be seen that CL intensity of HRP-P-Ab complex increases with increasing the incubation time. When the incubation time was over 35 min, the CL intensity of HRP-P-Ab complex was constant. The results showed that the suitable incubation time was 35 min. According to the experiment results described above, the best conditions for the determination of P were confirmed as following: electrophoretic buffer was 10 mM borate buffer (pH 10.2) containing 1.2 mM luminol and 0.08% (w/w) Tween 20; oxidizer solution was 80 mM H2 O2 and 1 mM PIP in 40 mM NaHCO3 solution at pH 8.7; incubation time was 35 min. Under this optimized condition, the typical electropherograms for the competitive immunoassay of P were shown in Fig. 7. It can be seen from the electropherograms that HRP-P and HRP-P-Ab immunocomplex were baseline separated within 80 s. In these electropherograms, trace a was obtained from a solution containing only HRP-P, and trace b from a solution containing both HRP-P and HRP-P-Ab. A new peak at a longer migration time that was from the HRP-P-Ab complex was observed. Traces c and d were obtained from a solution containing P at 1.0 × 10−7 M, HRP-P and HRP-P-Ab, and a solution containing P at 2.5 × 10−7 M, HRP-P and HRP-P-Ab. Compared with trace b, the CL signal (peak height) from HRP-P-Ab complex decreased. These
results indicate the competitive binding of P to the Ab. Because of the effective MCE separation, the CL analytical signal was less prone to sample matrix interference, which makes the current assay very selective and useful for clinical sample analysis.
3.4. Analytical figures of merit The present competitive immunoassay method based on MCECL was evaluated in terms of the response linearity, limit of detection (LOD) and reproducibility under the optimized conditions. Seven-point calibration curves were prepared by using P standard solutions ranging from 8.0 to 800.0 nM. The CL signal
Fig. 7. Electropherograms from separating the competitive immunoreaction solutions. (a) 5.0 × 10−7 M HRP-P solution; (b) mixture of 5.0 × 10−7 M HRP-P and 2.5 × 10−7 M Ab; (c) mixture of 1.0 × 10−7 M P, 5.0 × 10−7 M HRP-P, and 2.5 × 10−7 M Ab; (d) mixture of 2.5 × 10−7 M P, 5.0 × 10−7 M HRP-P, and 2.5 × 10−7 M Ab. Electrophoretic buffer was 10 mM phosphate buffer (pH 10.2) containing 0.08% (w/w) Tween 20 and 1.2 mM luminol. The oxidizer solution was 40 mM NaHCO3 (pH 8.7) containing 80 mM H2 O2 and 1 mM PIP.
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Table 1 Assay results and recovery of P in human serum samples. Sample
Found (10−9 M)
RSD (%, n = 7)
Added (10−9 M)
Total found (10-9 M)
Recovery (%)
1 2 3 4 5
756 25.8 45.7 672.6 413.2
4.3 3.8 1.8 2.5 3.2
10.0 10.0 10.0 200.0 200.0
84.2 33.8 54.2 875.3 611.4
98.1 92.3 96.7 100.4 99.5
1–3, normal. 4–5, pregnancy.
(peak height) from free HRP-P was used for quantification of P. A linear regression was represented by the equation: H = 7.6884C + 1.0104 where H is the relative peak height (in V) and C is the P concentration (nM). The linear range of the calibration curve was from 8.0 to 800.0 nM (R = 0.9968), and the LOD (S/N = 3) for P was 3.8 nM. The LOD is similar to those of many reported immunoassays methods for P, such as CL-EIA (0.08 ng/mL) [10], ELISA (0.11 ng/mL) [29], and CL-IA (0.1 ng/mL) [30]. These data illustrate that present MCE-CL system has a good linearity and high sensitivity. Assay reproducibility was studied by analyzing a standard solution containing 100 nM
P seven times and recording the migration times and peak heights. The relative standard deviations (RSDs) of the migration times and peak heights were 4.2%, 2.6% for HRP-P and 3.5%, 2.2% for HRP-P-Ab, respectively. 3.5. Analysis of P in human serum The contents of P in the human serum from normal and pregnant women were determined according to the procedure mentioned above. The electropherograms of a normal woman serum sample and a pregnant woman serum sample were shown in Fig. 8. Table 1 summarizes the analytical results. P was detected at a significantly higher level in pregnant women serum samples than in the normal women serum samples. These results for P level in the normal women serum samples are in accordance with those obtained by a procedure based on LC/MS/MS [4]. In order to verify this assay, the standard addition experiments were carried out according to the procedure mentioned above. The results obtained are also summarized in Table 1. The recoveries of the method were from 92.3% to 100.4%, and the RSDs were between 1.8% and 4.3% (n = 7). 4. Conclusions A new MCE-CL based competitive immunoassay method for the sensitive and selective determination of P in human serum was developed. The efficient separation of HRP-P and HRP-P-Ab complex was rapidly achieved with good reproducibility. The assay was successfully used for the determination of P contents in serum from normal women and pregnant women. Assay results illustrate that the levels of P in the pregnant women were significantly elevated over that of the normal women. When compared to current immunoassays such as CLEIA and ELISA, the MCE-CL method was sensitive and simple, and can meet the demands in clinical measurement. Acknowledgements This work was supported by the National Natural Science Foundations of China (No. 21065002, 21175030), and the Natural Science Foundations of Guangxi Province (No. 2012GXNSFAA053024) as well as BAGUI Scholar Program and the project of Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources (Guangxi Normal University), Ministry of Education of China. References [1] [2] [3] [4] [5]
Fig. 8. Electropherograms obtained from the separation of the serum samples from normal women (A, diluted 5 times) and pregnant women (B, diluted 10 times). MCE conditions were as in Fig. 7.
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Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Competitive immunoassay of progesterone by microchip electrophoresis with chemiluminescence detection Fanggui Ye, Jinwen Liu, Yong Huang, Shutin Li, Shulin Zhao ∗ Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources (Ministry of Education), College of Chemistry and Chemical Engineering, Guangxi Normal University, Guilin 541004, China
a r t i c l e
i n f o
Article history: Received 9 June 2013 Accepted 1 August 2013 Available online 8 August 2013 Keywords: Chemiluminescence detection Competitive immunoassay Human serum Microchip electrophoresis Progesterone
a b s t r a c t A sensitive and rapid homogeneous immunoassay method based on microchip electrophoresischemiluminescence detection (MCE-CL) using luminol-hydrogen peroxide as chemiluminescence system catalyzed by horseradish peroxidase (HRP) was developed for the determination of progesterone (P). The assay was based on the competitive immunoreactions between HRP-labeled P antigen (HRP-P) and P with a limited amount of anti-P mouse monoclonal antibody (Ab), and MCE separation of free HRP-P and HRP-P-Ab immunocomplex followed by CL detection. The effect of various factors such as conditions for the CL reaction, MCE and incubation time for the immunoreactions were examined and optimized. Under optimal assay conditions, the MCE separation was accomplished within 80 s. The linear range of detection for P was 8–800 nM with a detection limit of 3.8 nM (signal/noise ratio = 3). This present method has been applied to determine P in human serum samples from normal and pregnant women. The result indicates that the proposed MCE-CL based homogeneous immunoassay method can serve as an alternative tool for clinical assay of P. © 2013 Published by Elsevier B.V.
1. Introduction Progesterone (P) is one of the hormones in human bodies that stimulates and regulates various functions, and plays an important role in maintaining pregnancy. It helps to prepare women body for conception and pregnancy, and regulates the monthly menstrual cycle [1]. The dynamic monitoring of P in human serum is important to reproductive system and investigation of the mechanism of various steroid prophylactics and anti-early-pregnancy drugs [2]. In addition, it has been indicated that P also have neuroprotective effect after traumatic brain injury [3]. Currently, the most common methods used to quantify P are chromatography and immunoassay methods. Chromatography methods such as LC–MS [4] and GC–MS [5] have been developed as reference methods for determination of P in human serum, which have advantages of high accuracy, selectivity, reproducibility and sensitivity. Immunoassays used in clinical practice including radioimmunoassay [6], enzyme linked immuno absorbent assay
∗ Corresponding author at: College of Chemistry and Chemical Engineering, Guangxi Normal University, Guilin 541004, China. Tel.: +86 773 5856104; fax: +86 773 5832294. E-mail addresses: [email protected] (F. Ye), [email protected] (J. Liu), [email protected] (Y. Huang), [email protected] (S. Li), [email protected] (S. Zhao). 1570-0232/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.jchromb.2013.08.002
(ELISA) [7], fluoroimmunoassay [8], chemiluminescent enzyme immunoassay (CL-EIA) [9,10] and time-resolved fluorescence immunoassay [11] have also been reported for the evaluation of P in human serum. By far, the most immunoassays for P are heterogeneous, i.e. either antibody or antigen is immobilized at the solid phase. Heterogeneous immunoassays in general have low limits of detection (nM to pM), but they require a multi-step work flow (e.g. primary antibody incubation, washing, secondary antibody incubation etc.), and are therefore time-consuming [12]. Microchip electrophoresis (MCE) has become a very attractive separation technique for chemical and biological analyses [13]. It have various advantages such as reduced sample and reagent consumption, high separation speed and efficiency, short analysis time, simple operation, and easy integration and automation, which make it unequally suitable for biological and clinical analysis. Immunoassay is one application that makes use of these advantages. Many types of immunoassays on MCE have been applied in clinical diagnoses and biochemical studies. Over the past decade, MCE-enhanced immunoassays have been developed to determine cortisol [14], theophylline [15], matrix metalloproteinase-8 [16], goat anti-human IgG [17], B-type natriuretic peptide [18] and staphylococcal enterotoxin B [19]. Laser-induced fluorescence (LIF) detection is the most sensitive detection scheme available for MCE immunoassay. However, LIF detection requires relatively large and expensive apparatus systems. Chemiluminescence (CL) detection offers advantages including high sensitivity, simple optical
F. Ye et al. / J. Chromatogr. B 936 (2013) 74–79
75
Fig. 1. Schematic diagram of the layout of the glass/PDMS microchip. S, sample reservoir; B, buffer reservoir; SW, sample waste reservoir; BW, buffer waste reservoir; R, the oxidizer reagent reservoir.
structure, low background noise and relatively simple and inexpensive instrumentation. CL is particularly suitable for integration on microfluidic devices. MCE-CL-enhanced immunoassays have been successfully used for analysis of rat IgG [20], human serum albumin [21], immunosuppressive acidic protein [22], atrazine [23], testosterone [24] and thyroxine [25]. Herein, we report on the development of a homogeneous immunoassay coupled with MCE-CL to quantify P in human serum. The assay was based on competitive immunoreactions between horseradish peroxidase (HRP)-labeled P antigen (HRP-P) and P with a limited amount of anti-P mouse monoclonal antibody (Ab), and MCE separation of free HRP-P and HRP-P-Ab immunocomplex followed by CL detection. It is well known that HRP catalyzes the luminol-H2 O2 CL reaction and greatly enhances the CL emission [26]. Thus, two peaks for free HRP-P and HRP-P-Ab immunocomplex were observed in electropherogram. Both HRP-P and the HRP-P-Ab immunocomplex were sensitively detected. Because of the effective MCE separation, the CL analytical signal was less prone to sample matrix interference. The conditions for MCE separation and CL detection were investigated in detail, and the quantification of P in human serum from normal and pregnant women was demonstrated.
introduction channel (250 m) was 65 m, the depth of all microchannels was 25 m, and the length of double T was 60 m. 2.2. Human serum sample preparation Human serum samples were kindly provided by the No. 5 People’s Hospital (Guilin, China). A 250 L of human serum sample was collected in a 1.5 mL vial, then 500 L trichloroacetic acid was added and votexed for 5 min. The mixture solution was centrifuged for 20 min at 12,000 rpm. The supernatant was transferred to another 1.5 mL vial and evaporated to dryness under N2 . The residue was reconstituted in 250 L 20 mM borate solution and kept at 4 ◦ C before analysis. 2.3. Immunoreaction To carry out the immunoreaction, 20 L different concentrations of P or serum sample, 20 L 5.0 × 10−7 M HRP-P and 20 L 2.5 × 10−7 M Ab were mixed and diluted to 100 L with phosphate buffer (pH 7.4), and incubated for 35 min at 37 ◦ C. The whole procedure is illustrated in the following equations: HRP-P + P + Ab − HRP-P-Ab + HRP-P + P-Ab + P
2. Experimental 2.1. Reagents and apparatus HRP-P, P and Ab was purchased from Zhengzhou Biocell Biotechnology Co., Ltd. (Zhengzhou, China). Tween 20 was obtained from Shanghai Chemical Reagents Corporation (Shanghai, China). Luminol was purchased from Fluka (Buchs, Switzerland). Paraiodophenol (PIP), hydrogen peroxide (H2 O2 ) were obtained from Taopu Chemicals (Shanghai, China). All other chemicals used in this work were of analytical grade. The P and Ab solutions were prepared by dissolving the reagents in 20 mM phosphate buffer (pH 7.4). The electrophoretic buffer and oxidizer solution were prepared according to method described previously [27]. Briefly, the electrophoretic buffer was 10 mM phosphate buffer (pH 10.2) containing 1.2 mM luminol and 0.08% (w/w) Tween 20. The oxidizer solution was 80 mM H2 O2 and 1 mM PIP in 40 mM NaHCO3 solution at pH 8.7. All solutions were filtered through 0.22-m membrane filters prior to use. Water was purified by employing a Milli-Q plus equip from Millipore (Bedford, MA, USA), and used throughout the work. A homemade MCE-CL system used in this work was similar to that described in our previous work [27]. The fabrication of the double “T” microchip was described previously [28] and illustrated in Fig. 1. The width of all microchannels except the oxidizer
where Ab is anti-P antibody added at a limited amount, HRP-P was added at a fixed amount, and P is free progesterone. HRP-P competes with P in the solution for binding to a limited amount of Ab. The concentration of P in the solution is directly proportional to that of free HRP-P, but inversely proportional to that of the HRP-P-Ab. Therefore, P in the sample can be measured by monitoring the CL intensity from free HRP-P after separation from the immunocomplex. 2.4. MCE-CL procedure The microchannels were rinsed sequentially with 0.1 M NaOH, water and electrophoresis buffer for 10 min each. Prior to electrophoresis, all reservoirs were filled with the electrophoretic buffer. Vacuum was applied to the reservoir BW in order to fill the separation channel with the electrophoretic buffer. Then, the electrophoretic buffer solutions in reservoir S and reservoirs R were replaced by sample solution and oxidizer solution, respectively. Sample injection was performed by applying 450 V to S for 15 s with SW grounded, whereas B was set at 280 V, BW was set at 350 V, and R was left floating. For MCE separation, 2500 V was applied to B and 1500 V was applied to both S and SW with BW grounded. At the same time, 500 V was applied to R. The analytes were transported into the separation channel toward BW and then were mixed with the oxidizer solution at the junction of the oxidizer introduction
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Fig. 2. The effect of the oxidizer solution pH on CL intensity. Electrophoretic buffer was 10 mM phosphate buffer (pH = 10.2) containing 0.08% Tween 20 and 1.2 mM luminol. The oxidizer solution was 40 mM NaHCO3 containing 80 mM H2 O2 and 1 mM PIP at different pH values.
Fig. 3. The effect of luminol concentration on CL intensity. Electrophoretic buffer was 10 mM phosphate buffer (pH = 10.2) containing 0.08% Tween 20 and luminol at different concentrations. The oxidizer solution was 40 mM NaHCO3 (pH = 8.7) containing 80 mM H2 O2 and 1 mM PIP.
3.2. Optimization of MCE separation condition channel and the separation channel, producing CL emission that was detected by PMT.
3. Results and discussion 3.1. Optimization of CL detection Enhancing the sensitivity of CL detection is paramount for successful homogeneous competitive immunoassay. Besides the setup of the CL detector, CL sensitivity is mainly dependent on the CL reaction and MCE conditions, which need to be systematically optimized. Therefore, the effects of pH value of the oxidizer solution (CL reaction medium), luminol and PIP concentrations on the separation and detection were investigated. The oxidizer solution pH affected significantly the effective electric charge of the analytes. Generally, the luminol-H2 O2 CL immunoreaction was carrying out in alkalescence medium [25]. Therefore, the effect of pH value of the oxidizer solution was investigated in a pH range of 8.0–9.5. The results were illustrated in Fig. 2. The CL intensity increased gradually with the increase of pH value up to 8.7, where the maximum CL signal was reached. Further increasing of pH value resulted in a decrease in CL signal. Therefore, the oxidizer solution was buffered at 8.7 for a maximum enhanced CL signal. Fig. 3 shows the effect of luminol concentration. A concentration ranged from 0.5 to 2.0 mM was tested. As can be seen from this figure, the CL intensity first increased and then decreased with the increase of luminol concentration. The maximum CL intensity was obtained when the concentration of luminol was 1.2 mM. Therefore, a luminol solution at this concentration was chosen for subsequent studies. Keeping luminol concentration at 1.2 mM and pH value of the oxidizer solution at 8.7, PIP concentration in a range of 0.2–1.2 mM was tested. As can be seen from Fig. 4, the CL intensity increased with increasing PIP concentration in the range of 0.2–1.0 mM, whereas it decreased with further increasing PIP concentration from 1.0 to 1.2 mM. A concentration of 1.0 mM PIP was used for further experiments.
Effective separation of HRP-P and HRP-P-Ab complex is a key step for the detection of P in this work. However, it was found that adsorption of proteins by the glass channel surface significantly deteriorated the separation of HRP-P from the HRP-P-Ab complex. To overcome this difficulty, a popular surfactant, Tween 20, was added into running buffer, and the influence of Tween 20 concentration ranging from 0.04 to 0.12% (w/w) on the separation was investigated. The results indicate the resolutions (Rs ) between HRPP and HRP-P-Ab complex all increased with increasing Tween 20 concentrations. Conversely, the migration times of HRP-P and its immunocomplex increase with the increase of Tween 20 content. Taking into consideration the Rs and speed of analysis, 0.08% (w/w) Tween 20 was used to further optimize separation conditions. The pH of the electrophoretic buffer has an important effect on the channel-wall surfaces characteristics of microchip and the
Fig. 4. The effect of PIP concentration on CL intensity. Electrophoretic buffer was 10 mM phosphate buffer (pH = 10.2) containing 0.08% Tween 20 and 1.2 mM luminol. The oxidizer solution was 40 mM NaHCO3 (pH = 8.7) containing 80 mM H2 O2 and PIP at different concentrations.
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Fig. 6. The effect of incubation time on the CL intensity of the immunocomplex. The concentration of HRP-P and Ab are 5 × 10−7 M, 2.5 × 10−7 M respectively. Fig. 5. The effect of electrophoretic buffer pH on resolution. Electrophoretic buffer was 10 mM phosphate buffer at different pH value containing 0.08% (w/w) Tween 20 and 1.2 mM luminol. The oxidizer solution was 40 mM NaHCO3 (pH 8.7) containing 80 mM H2 O2 and 1 mM PIP.
effective electric charge of the analytes. In this work, the phosphate buffer was used as background electrolyte for the separation of HRP-P and its immunocomplex, and pH of phosphate buffer were optimized. It can be seen from Fig. 5, the Rs of HRP-P and its immunocomplex increases with the increase of electrophoretic buffer pH value from 9.6 to 10.2, while they decrease with further increasing of running buffer pH from 10.2 to 10.6. Therefore, a running buffer with a pH value of 10.2 was used for further experiments. 3.3. Effect of incubation time The incubation time had a great influence on the immunocomplex formation. Therefore, the incubation time of HRP-P and Ab was studied. In this experiment, seven identical samples containing 5 × 10−7 M HRP-P and 2.5 × 10−7 M Ab were incubated for different lengths of time, and then the solution was injected into the MCE system to obtain an optimal incubation time (Fig. 6). It can be seen that CL intensity of HRP-P-Ab complex increases with increasing the incubation time. When the incubation time was over 35 min, the CL intensity of HRP-P-Ab complex was constant. The results showed that the suitable incubation time was 35 min. According to the experiment results described above, the best conditions for the determination of P were confirmed as following: electrophoretic buffer was 10 mM borate buffer (pH 10.2) containing 1.2 mM luminol and 0.08% (w/w) Tween 20; oxidizer solution was 80 mM H2 O2 and 1 mM PIP in 40 mM NaHCO3 solution at pH 8.7; incubation time was 35 min. Under this optimized condition, the typical electropherograms for the competitive immunoassay of P were shown in Fig. 7. It can be seen from the electropherograms that HRP-P and HRP-P-Ab immunocomplex were baseline separated within 80 s. In these electropherograms, trace a was obtained from a solution containing only HRP-P, and trace b from a solution containing both HRP-P and HRP-P-Ab. A new peak at a longer migration time that was from the HRP-P-Ab complex was observed. Traces c and d were obtained from a solution containing P at 1.0 × 10−7 M, HRP-P and HRP-P-Ab, and a solution containing P at 2.5 × 10−7 M, HRP-P and HRP-P-Ab. Compared with trace b, the CL signal (peak height) from HRP-P-Ab complex decreased. These
results indicate the competitive binding of P to the Ab. Because of the effective MCE separation, the CL analytical signal was less prone to sample matrix interference, which makes the current assay very selective and useful for clinical sample analysis.
3.4. Analytical figures of merit The present competitive immunoassay method based on MCECL was evaluated in terms of the response linearity, limit of detection (LOD) and reproducibility under the optimized conditions. Seven-point calibration curves were prepared by using P standard solutions ranging from 8.0 to 800.0 nM. The CL signal
Fig. 7. Electropherograms from separating the competitive immunoreaction solutions. (a) 5.0 × 10−7 M HRP-P solution; (b) mixture of 5.0 × 10−7 M HRP-P and 2.5 × 10−7 M Ab; (c) mixture of 1.0 × 10−7 M P, 5.0 × 10−7 M HRP-P, and 2.5 × 10−7 M Ab; (d) mixture of 2.5 × 10−7 M P, 5.0 × 10−7 M HRP-P, and 2.5 × 10−7 M Ab. Electrophoretic buffer was 10 mM phosphate buffer (pH 10.2) containing 0.08% (w/w) Tween 20 and 1.2 mM luminol. The oxidizer solution was 40 mM NaHCO3 (pH 8.7) containing 80 mM H2 O2 and 1 mM PIP.
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Table 1 Assay results and recovery of P in human serum samples. Sample
Found (10−9 M)
RSD (%, n = 7)
Added (10−9 M)
Total found (10-9 M)
Recovery (%)
1 2 3 4 5
756 25.8 45.7 672.6 413.2
4.3 3.8 1.8 2.5 3.2
10.0 10.0 10.0 200.0 200.0
84.2 33.8 54.2 875.3 611.4
98.1 92.3 96.7 100.4 99.5
1–3, normal. 4–5, pregnancy.
(peak height) from free HRP-P was used for quantification of P. A linear regression was represented by the equation: H = 7.6884C + 1.0104 where H is the relative peak height (in V) and C is the P concentration (nM). The linear range of the calibration curve was from 8.0 to 800.0 nM (R = 0.9968), and the LOD (S/N = 3) for P was 3.8 nM. The LOD is similar to those of many reported immunoassays methods for P, such as CL-EIA (0.08 ng/mL) [10], ELISA (0.11 ng/mL) [29], and CL-IA (0.1 ng/mL) [30]. These data illustrate that present MCE-CL system has a good linearity and high sensitivity. Assay reproducibility was studied by analyzing a standard solution containing 100 nM
P seven times and recording the migration times and peak heights. The relative standard deviations (RSDs) of the migration times and peak heights were 4.2%, 2.6% for HRP-P and 3.5%, 2.2% for HRP-P-Ab, respectively. 3.5. Analysis of P in human serum The contents of P in the human serum from normal and pregnant women were determined according to the procedure mentioned above. The electropherograms of a normal woman serum sample and a pregnant woman serum sample were shown in Fig. 8. Table 1 summarizes the analytical results. P was detected at a significantly higher level in pregnant women serum samples than in the normal women serum samples. These results for P level in the normal women serum samples are in accordance with those obtained by a procedure based on LC/MS/MS [4]. In order to verify this assay, the standard addition experiments were carried out according to the procedure mentioned above. The results obtained are also summarized in Table 1. The recoveries of the method were from 92.3% to 100.4%, and the RSDs were between 1.8% and 4.3% (n = 7). 4. Conclusions A new MCE-CL based competitive immunoassay method for the sensitive and selective determination of P in human serum was developed. The efficient separation of HRP-P and HRP-P-Ab complex was rapidly achieved with good reproducibility. The assay was successfully used for the determination of P contents in serum from normal women and pregnant women. Assay results illustrate that the levels of P in the pregnant women were significantly elevated over that of the normal women. When compared to current immunoassays such as CLEIA and ELISA, the MCE-CL method was sensitive and simple, and can meet the demands in clinical measurement. Acknowledgements This work was supported by the National Natural Science Foundations of China (No. 21065002, 21175030), and the Natural Science Foundations of Guangxi Province (No. 2012GXNSFAA053024) as well as BAGUI Scholar Program and the project of Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources (Guangxi Normal University), Ministry of Education of China. References [1] [2] [3] [4] [5]
Fig. 8. Electropherograms obtained from the separation of the serum samples from normal women (A, diluted 5 times) and pregnant women (B, diluted 10 times). MCE conditions were as in Fig. 7.
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