972 Yan-ming Liu1 Lin Mei1 Ying-ying Liu1 Min Zhou1 Ke-jing Huang1 Yong-hong Chen2 Shu-wei Ren2 1 College
of Chemistry and Chemical Engineering, Xinyang Normal University, Xinyang, P. R. China 2 Xinyang Central Hospital, Xinyang, P. R. China
Received October 28, 2013 Revised November 29, 2013 Accepted November 30, 2013
Electrophoresis 2014, 35, 972–977
Research Article
Highly sensitive capillary electrophoretic immunoassay of rheumatoid factor in human serum with gold nanoparticles enhanced chemiluminescence detection A new CE-based immunoassay method for the determination of rheumatoid factor was developed using chemiluminescent reaction of luminol and hydrogen peroxide catalyzed by gold nanoparticles (AuNPs). In this method, AuNPs were synthesized and conjugated with anti-RF (antibody, Ab) to form tagged Ab (AuNPs-Ab, Ab* ), which subsequently linked to limited amount of RF (antigen, Ag) to produce Ab* –Ag complex by a noncompetitive immunoreaction. AuNPs were used to label antibody and amplify chemiluminescent signal. Under the optimized conditions, the mixture of free Ab* and Ab* –Ag complex was well separated and detected. This method yields a wide linear range of 0.01–20 g/mL with a correlation coefficient of 0.997, and the detection limit of RF reaches 5.95 ng/mL (ca. 6.0 pmol/L, S/N = 3). The proposed method was successfully applied for the quantification of RF in human sera from patients with rheumatoid arthritis. This highly sensitive and selective method could be developed into a promising and useful technique for biological molecules determination in clinical analysis. Keywords: Capillary electrophoresis / Chemiluminescence / Gold nanoparticles / Immunoassay / Rheumatoid factor DOI 10.1002/elps.201300524
1 Introduction Rheumatoid arthritis (RA) is a systemic autoimmune disease that is usually a chronic disease and characterized especially by pain, stiffness, inflammation, swelling, and eventually causing irreversible joint destruction leading to severe disability [1]. Presenting rheumatoid factor (RF) in sera, a characteristic autoantibody associated with RA, is a pivotal mark in categorizing suspected-RA patients into seropositive or seronegative groups [2, 3]. Eighty-five percent of RA patients with RF seropositive mean that the RF concentration in sera is significantly elevated above that of healthy persons [4]. Generally, RF is only serological diagnosis index in clinical diagnosis of RA. Thus, the detection of RF concentration in sera is vital in discovery and early diagnosis of RA. The latex fixation test and enzyme-linked immunosorbent assay are frequently used to determine RF content [5, 6]. Immunoassay (IA) with high selectivity is known as one of
Correspondence: Professor Yan-ming Liu, College of Chemistry and Chemical Engineering, Xinyang Normal University, 237 Chang’an Road, Xinyang 464000, P. R. China E-mail: [email protected] Fax: +86-376-6392889
Abbreviations: AuNP, gold nanoparticle; CEIA, capillary electrophoretic immunoassay; CL, chemiluminescence; IA, Immunoassay; ITT, immunity transmission turbidity; RA, rheumatoid arthritis; RF, rheumatoid factor
C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
the most important analytical tool to determine analytes in clinical diagnosis [7, 8]. However, these assays are usually sample/reagent-consuming and involve multiple steps, such as incubations and rinsing [9]. Therefore, the development of new IA method that has high efficacy, sensitivity, and low costs for determination of RF concentration is very essential in clinical medicine. In the past decade, capillary electrophoretic immunoassay (CEIA) has been proved to be a powerful technique for the separation and analysis of biological compounds [10]. CEIA can rapidly separate antibody and antigen/antigen–antibody complexes using small amounts of sample and reagent, while requiring highly sensitive detector for the detection of trace analytes from complex biological compound. Chemiluminescence (CL) is a highly sensitive method of detection with simple optical structure and low background nature and has been widely used in analytical science [11]. Recently, CEIA coupled with CL detection (CEIA–CL) has been developed into a potential and promising analytical tool for the determination of bioactive molecule in biomedical and clinical application. When combined with the high separation efficiency of CE, the high sensitivity of CL, and the high specificity of IA, CEIA–CL can assist in further developments in complex biological compound assays and diagnostic clinical analysis [12]. It has been successfully applied to detect bone morphogenic protein-2 in rat vascular smooth muscle cells [13], tumor marker carbohydrate antigen 125 [14], and carcinoembryonic antigen [15] in sera, and sympathomimetic drug clenbuterol in urine [16]. www.electrophoresis-journal.com
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Nowadays developments in nanomaterials have kindled widespread interest in numerous bioassays [17–19]. Among them, gold nanoparticles (AuNPs) with special physical and chemical properties are attracting strong interest in the fundamental sciences and being widely investigated for their potential use in a range of biological applications [20–22]. The functionalized AuNPs linking to biological molecules such as proteins [23, 24], drugs [25], enzymes [26], and nucleic acids [27, 28], provide interesting tools for biological analysis. Due to AuNPs with excellent catalytic effect for the CL intensity of luminol–H2 O2 reaction, AuNPs as CL label for CEIA offer a promising alternative to conventional methodology. To the best of our knowledge, AuNPs labeled antibody with CEIA–CL method for the analysis of RF has not been reported. In this work, a new CEIA coupled with CL detection method has been developed by using AuNPs as label of antiRF for sensitive detection of RF. This assay was based on the catalytic effects of AuNPs on the luminol–hydrogen peroxide reaction. The conditions for CE separation and CL detection were investigated in detail. The proposed method was demonstrated in the quantitative analysis of RF in human sera.
2 Materials and methods 2.1 Materials Human RF and anti-RF were purchased from Santa Cruz Biotechnology (California, US). Hydrogen tetrachloroaurate (III) trihydrate (HAuCl4 ·3H2 O, Au 49.5% min) and PVP, Mr = 1 300 000) were obtained from Alfa Aesar (A Johnson Matthey Company, Ward Hill, MA, USA). BSA was from Siobio Biotechnology (Shanghai, China). Luminol was purchased from Yacoo Chemical Factory (Suzhou, China). All reagents used were of analytical grade. The pure water (18.2 M⍀·cm) used was processed with an Ultrapure Water System (Kangning Water Treatment Solution Provider, China). All solutions were stored in the refrigerator at 4°C and filtered through a 0.22 m cellulose acetate membrane filters (Shanghai Xingya Purification Material Factory, China) before use. 2.2 Instrumentation The basic design of the CE–CL system has been previously described [29]. Briefly, a high-voltage supply (0–30 kV, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, China) was used to drive the electrophoresis. A 50 cm × 75 m id uncoated fused silica capillary (Hebei Yongnian Ruifeng Chromatographic Apparatus, China) was used for the separation. One end of the separation capillary was burned off 5 cm polyimide coating. After etching with 40% hydrofluoric acid for 2.5 h, this end of capillary was inserted into a 20 cm × 530 m id reaction capillary. The outlet of the separation capillary was located at the detection window, which was made by burning 1 cm of the polyimide of the reaction capillary and placed in front of the PMT (CR 120, Bin C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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song Photonics, Beijing, China). The pair of 40 cm × 320 m id capillary was used to introduce postcolumn CL reagents to a tee reservoir from two vials located 20 cm above it. The CL reagents were delivered by gravity through the reagentintroducing capillaries, mixed and flowed coaxially along the separation capillary. The CL reaction took place at the separation capillary outlet when analyte eluted out from the separation capillary. The ground electrode was also put into one joint of the tee. CL emission was collected with a BPCL ultraweak luminescence analyzer (Institute of Biophysics, Chinese Academy of Science, Beijing, China) and then recorded and processed with a computer using BPCL software. Transmission electron microscopy images were obtained by using a JEM-2100F transmission electron microscope (JEOL, Tokyo, Japan). Absorption spectra were recorded by an UVmini-1240 UV-vis spectrophotometer (Shimadzu, Kyoto, Japan). 2.3 Preparation of gold nanoparticles AuNPs production by sodium citrate reduction of HAuCl4 in water was performed according to the literature [30]. Briefly, 50 mL of 0.01% HAuCl4 solution was added to a 100 mL beaker and brought to a rolling boil. Next, 0.5 mL of 2% sodium citrate was quickly added to the rapidly stirred boiling solution. The formation of AuNPs can be observed by a change in color from pale-yellow to wine-red. The colloids were stirred for another 15 min and cooled to room temperature. After cooling, the synthesized AuNPs was filtered through a 0.22 m cellulose membrane and stored at 4°C. Transmission electron microscopy images confirmed that the average diameter of AuNPs used for labeling is about 36 nm. 2.4 Preparation of AuNPs-antibody conjugate Binding of anti-RF to AuNPs was performed according to previously published report [31]. In brief, 500 L of pH 8.5 colloidal AuNPs were incubated with 44 L of 0.3 mg/mL anti-RF solution for 30 min at room temperature under agitation. Afterwards, 5% BSA was added to a final concentration of 1% with stirring for 5 min. Then the conjugate was centrifuged at 12 000 rpm for 30 min. The red sediment was the anti-RF-AuNPs conjugate, which was resuspended in 0.5 mL of 0.04 mol/L pH 7.4 PBS (containing 1% BSA and 0.05% Tween 20) with the supernatant being removed. 2.5 Immune reaction procedure The IA protocol is noncompetitive format and the immunoreaction was conducted as follows: RF (Ag) + AuNPs-anti-RF (Ab∗ , excess) → RF-AuNPs-anti-RF (Ag-Ab∗ ) + AuNPs-anti-RF (Ab∗ , free) www.electrophoresis-journal.com
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Ten microliters of RF standard or serum sample was mixed with 10 L of AuNPs labeled anti-RF in a 200 L microcentrifuge tube and then diluted with 0.01 mol/L pH 7.4 PBS to 50 L. After incubating at 37°C for 1 h, the mixture was analyzed by CE as described below.
2.6 CE procedure The new capillary was preconditioned sequentially by flushing with 2.0 mol/L NaOH–CH3 OH, 1.0 mol/L NaOH, water, and electrophoretic buffer for 20 min before the first use. At the beginning of each day, the separation capillary was reconditioned by flushing with 0.1 mol/L NaOH, water, and electrophoretic buffer for 3–5 min successively. After three consecutive injections, the separation capillary was flushed with 0.1 mol/L NaOH, water, and electrophoretic buffer for 2 min, respectively. The electrophoretic buffer was 7 mmol/L Na2 B4 O7 with 0.40 mmol/L luminol and 1.0% PVP at pH 11.0. The postcolumn CL reagents, one containing 40 mmol/L H2 O2 and another containing 20 mmol/L pH 12.0 NaHCO3 , were separately introduced into the reaction capillary from two vials. The voltage of PMT for collecting the CL signal was set at −800 V. The sample was introduced in the separation capillary by electrokinetic injection at 10 kV for 6 s. CE separation was performed at 20 kV. The peak area was used in analysis.
2.7 Preparation of human serum samples Human serum samples from patients were provided by Xinyang Central Hospital (Xinyang, China). The fresh blood samples were immediately centrifuged for 10 min at 1200 rpm to remove erythrocytes. Subsequently, the supernatant was diluted 20-fold by using 0.01 mol/L pH 7.4 PBS as diluent buffer and analyzed.
3 Results and discussion 3.1 Determination of the minimum quantity of antibody for AuNPs labeling The citrate stabilized AuNPs suspension could be very stable due to the electrostatic repulsions between negatively charged AuNPs. However, when the salt solution (e.g. 10% NaCl) was added into the suspension, AuNPs may cause aggregation due to the interaction between the cations and the negatively charged AuNPs. When proteins (e.g. antibody) are added to AuNPs suspension, the proteins can be adsorbed to the surface of AuNPs, preventing the AuNPs from flocculation. [32, 33]. A flocculation test was often carried out to determine the minimum quantity of antibody for AuNPs labeling [34, 35]. C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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In this experiment, an increasing volume (0–65 L) of antiRF at the fixed concentration of 0.3 mg/mL were added to 500 L of AuNPs under agitation and incubated for 30 min. Then 50 L of 10% NaCl was added to the mixture. UVvis absorption spectrum of each mixture was recorded after 30 min at room temperature to assess the minimal quantity of antibody. In the present study, with the increasing quantity of antiRF, the absorbance at maximum wavelength (max ) of AuNPs begins to blue shift and maintains constant when the volume of anti-RF is above 40 L, which indicates that 40 L is the minimal quantity needed to stabilize 500 L AuNPs. Therefore, the quantity of the antibody used for labeling in this work is 44 L of 0.3 mg/mL anti-RF, which is 10% more than the minimum amount required to ensure that the quantity of antibody is enough for the stability of the anti-RF-AuNPs conjugate.
3.2 Optimization of CE separation conditions Since concentration and pH of the electrophoretic buffer greatly influence the ionization of the silanols on the capillary wall and ionic strength of the buffer, changing these in the buffer are the most direct and important strategy for optimizing CE separation. In this experiment, the Na2 B4 O7 was used as the electrophoretic buffer. The effect of the Na2 B4 O7 concentration on CL intensity and resolution was examined in the range 1–13 mmol/L, as shown in Fig. 1A. The highest CL intensity and the best resolution were achieved at 7 and 9 mmol/L, respectively. Although the resolution was the best at 9 mmol/L, the CL intensity was not the highest. When the CL intensity was the highest, the free Ab* and the Ab* –Ag complex could be baseline separated (the resolution was 1.51). Therefore, 7 mmol/L was selected as the optimum Na2 B4 O7 concentration. The effects of buffer pH were investigated with pH ranging from 10.1 to 11.9 (Fig. 1B). The results indicate that the CL intensity and resolution reach the maximum at pH 11.0 and then decreased. Therefore, pH 11.0 was used. In order to decrease the adsorption of protein on the capillary inner wall, PVP was used as a capillary inner wall dynamic coating reagent for analyte separation. The effects of PVP concentration on the CL intensity and resolution were investigated and the results were shown in Fig. 1C. The CL intensity and resolution were increased with the increase in the PVP concentration. When the PVP concentration reached 1.0%, the highest CL intensity and satisfactory separation (the resolution was 1.53) can be obtained. Moreover, the migration time became longer and the peaks became broader above 1.0%. As a result, 1.0% PVP was chosen. Effects of separation voltage on the CL intensity and resolution were also studied. By changing the separation voltage from 14 to 26 kV, the CL intensity increased and the resolution decreased with separation voltage increase (Fig. 1D). Meanwhile, the Joule heats existent in the running buffer www.electrophoresis-journal.com
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Figure 1. Effect of Na2 B4 O7 concentration (A), buffer pH (B), PVP concentration (C), and separation voltage (D) on CL intensity and resolution. Conditions: the electrophoretic buffer, 7 mmol/L Na2 B4 O7 with 0.40 mmol/L luminol and 1.0% PVP at pH 11.0; the postcolumn CL reagents, 40 mmol/L H2 O2 and 20 mmol/L pH 12.0 NaHCO3 ; PMT voltage, −800 V; electrokinetic injection with 10 kV for 6 s; separation voltage, 20 kV; separation capillary, 50 cm × 75 m id.
system also increased. When the separation voltage was 20 kV, the resolution was calculated to be 1.57. Therefore, 20 kV was set.
3.3 Optimization of the CL detection conditions In this CL reaction, the concentrations of luminol and H2 O2 have a great influence on CL intensity. To obtain the optimal luminol concentration, CL intensity was detected at different luminol concentration ranging from 0.1 to 0.7 mmol/L (Fig. 2A). It was found that a concentration of 0.4 mmol/L luminol produced maximum CL intensity. Thus 0.4 mmol/L luminol was chosen. The effect of H2 O2 concentration was investigated. As can be seen in Fig. 2B, the CL intensity rapidly increased in the range from 10 to 40 mmol/L. When H2 O2 concentration was higher than 40 mmol/L, the CL intensity decreased. Therefore, 40 mmol/L H2 O2 was selected. The concentration and pH of NaHCO3 , used as postcolumn buffer, also have an important effect on CL intensity. The effect of NaHCO3 concentration was evaluated from 5 to 35 mmol/L. It can be seen from Fig. 2C that the CL intensity increased with the concentration from 5 to 20 mmol/L and then decreased. Hence, 20 mmol/L was chosen. Because the CL reaction of luminol occurs in basic medium, the effect of NaHCO3 pH was investigated over a pH range from 10.0 to 13.0 and the results were shown in Fig. 2D. The CL intensity was found to increase with the increasing NaHCO3 pH and reached its apex when the NaHCO3 pH was 12.0. Thus, pH 12.0 was used. C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 2. Effects of luminol (A), H2 O2 (B), NaHCO3 (C) concentration, and NaHCO3 pH (D) on CL intensity. Other conditions were as described in the legend of Fig. 1.
3.4 Detection of human RF by the proposed method Under the optimized conditions, the typical electropherograms of 0 and 1.0 g/mL human RF are shown in Fig. 3A and B, respectively. We examined the linearity, detection limit, and precision of the present method for human RF analysis. The calibration curve was calculated plotting the peak area values of Ab* –Ag complex against the RF concentrations. The linear range of the calibration curve was from 0.01 to 20 g/mL (R = 0.997), and the detection limit was 5.95 ng/mL (ca. 6.0 pmol/L, S/N = 3). The precisions (measured by RSD, www.electrophoresis-journal.com
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Electrophoresis 2014, 35, 972–977 Table 1. Analytical results of eleven human sera samples (n = 3)
Number of human sera samples
Figure 3. Electropherograms of human RF in standard solution. (A) 0 g/mL human RF; (B) 1.0 g/mL human RF. Peak 1, free Ab*; peak 2, immune complex; peak X, unknown compounds. Other conditions were as described in the legend of Fig. 1.
RF content (g/mL)
1 2 3 4 5 6 7 8 9 10 11
This method (RSD, %)
ITT
6.76 (3.1) 14.1 (3.7) 8.98 (3.7) 28.6 (6.4) 8.89 (4.5) 10.9 (2.3) 8.03 (4.1) 5.11 (5.4) 8.34 (6.1) 37.2 (6.7) 49.1 (5.8)
7.55 13.54 9.33 25.75 9.26 10.66 7.71 4.87 8.00 36.91 44.78
4 Concluding remarks
Figure 4. Electropherograms of human RF in sera samples from two patients with RA. (A) The serum sample of patient A; (B) the serum sample of patient B. Peak 1, free Ab*; peak 2, immune complex; peak X, unknown compounds. Other conditions were as described in the legend of Fig. 1.
n = 5) were examined by assaying 2.5 g/mL human RF within a day (intraday) and in 3 days (interday). The RSDs of the migration time were 1.7% for intraday and 3.4% for interday, respectively. The RSDs of the peak area were 3.2% for intraday and 5.2% for interday, respectively.
3.5 Analysis of RF in human sera So far, RF has been widely used as a biomarker for clinical diagnosis of RA. The contents of RF in the sera from two patients with RA were determined and the electropherograms were shown in Fig. 4. As seen in Fig. 4, the peak of Ab* –Ag complex can be observed obviously in the sera of patients with RA. In order to verify this method, the contents of RF in sera from 11 patients were determined by the proposed method (X) and immunity transmission turbidity (ITT) on Roche clinical chemistry analyzer (Y) employed in the Xinyang Central Hospital as control. The results of the 11 samples determined by the two methods were listed in Table 1. Two methods coincided well with a regression equation of Y = 0.9485 X + 0.3726 and a correlation coefficient of 0.995. The results indicate that the proposed method has potential application in the clinical analysis. C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
This study presents a sensitive and rapid CEIA method combined with CL detection using AuNPs as label of antibody for the determination of RF in the human serum. This method has many outstanding features including high efficiency, high selectivity, and high practicality. The CEIA method with the noncompetitive immunoreactions can be accomplished in a homogeneous solution with one-step operation. AuNPs show excellent catalytic property to luminol CL reaction and good labeling property to protein molecules, making it possible to detect the analytes at lower concentrations. Besides, AuNPs exhibit the advantage of better stability over the enzyme labels, and the whole procedure is uncomplicated and does not affect the biochemical activity of the labeled compound. The analytical results of proposed method coincided well with that of ITT. The results demonstrate that the current CE–IA–CL method maybe served as an alternative tool for determination of other biomarker (e.g. tumor marker) in the early disease diagnosis. This work was supported by the National Natural Science Foundation of China (21075106, 21375114). The authors have declared no conflict of interest.
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of Chemistry and Chemical Engineering, Xinyang Normal University, Xinyang, P. R. China 2 Xinyang Central Hospital, Xinyang, P. R. China
Received October 28, 2013 Revised November 29, 2013 Accepted November 30, 2013
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Research Article
Highly sensitive capillary electrophoretic immunoassay of rheumatoid factor in human serum with gold nanoparticles enhanced chemiluminescence detection A new CE-based immunoassay method for the determination of rheumatoid factor was developed using chemiluminescent reaction of luminol and hydrogen peroxide catalyzed by gold nanoparticles (AuNPs). In this method, AuNPs were synthesized and conjugated with anti-RF (antibody, Ab) to form tagged Ab (AuNPs-Ab, Ab* ), which subsequently linked to limited amount of RF (antigen, Ag) to produce Ab* –Ag complex by a noncompetitive immunoreaction. AuNPs were used to label antibody and amplify chemiluminescent signal. Under the optimized conditions, the mixture of free Ab* and Ab* –Ag complex was well separated and detected. This method yields a wide linear range of 0.01–20 g/mL with a correlation coefficient of 0.997, and the detection limit of RF reaches 5.95 ng/mL (ca. 6.0 pmol/L, S/N = 3). The proposed method was successfully applied for the quantification of RF in human sera from patients with rheumatoid arthritis. This highly sensitive and selective method could be developed into a promising and useful technique for biological molecules determination in clinical analysis. Keywords: Capillary electrophoresis / Chemiluminescence / Gold nanoparticles / Immunoassay / Rheumatoid factor DOI 10.1002/elps.201300524
1 Introduction Rheumatoid arthritis (RA) is a systemic autoimmune disease that is usually a chronic disease and characterized especially by pain, stiffness, inflammation, swelling, and eventually causing irreversible joint destruction leading to severe disability [1]. Presenting rheumatoid factor (RF) in sera, a characteristic autoantibody associated with RA, is a pivotal mark in categorizing suspected-RA patients into seropositive or seronegative groups [2, 3]. Eighty-five percent of RA patients with RF seropositive mean that the RF concentration in sera is significantly elevated above that of healthy persons [4]. Generally, RF is only serological diagnosis index in clinical diagnosis of RA. Thus, the detection of RF concentration in sera is vital in discovery and early diagnosis of RA. The latex fixation test and enzyme-linked immunosorbent assay are frequently used to determine RF content [5, 6]. Immunoassay (IA) with high selectivity is known as one of
Correspondence: Professor Yan-ming Liu, College of Chemistry and Chemical Engineering, Xinyang Normal University, 237 Chang’an Road, Xinyang 464000, P. R. China E-mail: [email protected] Fax: +86-376-6392889
Abbreviations: AuNP, gold nanoparticle; CEIA, capillary electrophoretic immunoassay; CL, chemiluminescence; IA, Immunoassay; ITT, immunity transmission turbidity; RA, rheumatoid arthritis; RF, rheumatoid factor
C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
the most important analytical tool to determine analytes in clinical diagnosis [7, 8]. However, these assays are usually sample/reagent-consuming and involve multiple steps, such as incubations and rinsing [9]. Therefore, the development of new IA method that has high efficacy, sensitivity, and low costs for determination of RF concentration is very essential in clinical medicine. In the past decade, capillary electrophoretic immunoassay (CEIA) has been proved to be a powerful technique for the separation and analysis of biological compounds [10]. CEIA can rapidly separate antibody and antigen/antigen–antibody complexes using small amounts of sample and reagent, while requiring highly sensitive detector for the detection of trace analytes from complex biological compound. Chemiluminescence (CL) is a highly sensitive method of detection with simple optical structure and low background nature and has been widely used in analytical science [11]. Recently, CEIA coupled with CL detection (CEIA–CL) has been developed into a potential and promising analytical tool for the determination of bioactive molecule in biomedical and clinical application. When combined with the high separation efficiency of CE, the high sensitivity of CL, and the high specificity of IA, CEIA–CL can assist in further developments in complex biological compound assays and diagnostic clinical analysis [12]. It has been successfully applied to detect bone morphogenic protein-2 in rat vascular smooth muscle cells [13], tumor marker carbohydrate antigen 125 [14], and carcinoembryonic antigen [15] in sera, and sympathomimetic drug clenbuterol in urine [16]. www.electrophoresis-journal.com
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Nowadays developments in nanomaterials have kindled widespread interest in numerous bioassays [17–19]. Among them, gold nanoparticles (AuNPs) with special physical and chemical properties are attracting strong interest in the fundamental sciences and being widely investigated for their potential use in a range of biological applications [20–22]. The functionalized AuNPs linking to biological molecules such as proteins [23, 24], drugs [25], enzymes [26], and nucleic acids [27, 28], provide interesting tools for biological analysis. Due to AuNPs with excellent catalytic effect for the CL intensity of luminol–H2 O2 reaction, AuNPs as CL label for CEIA offer a promising alternative to conventional methodology. To the best of our knowledge, AuNPs labeled antibody with CEIA–CL method for the analysis of RF has not been reported. In this work, a new CEIA coupled with CL detection method has been developed by using AuNPs as label of antiRF for sensitive detection of RF. This assay was based on the catalytic effects of AuNPs on the luminol–hydrogen peroxide reaction. The conditions for CE separation and CL detection were investigated in detail. The proposed method was demonstrated in the quantitative analysis of RF in human sera.
2 Materials and methods 2.1 Materials Human RF and anti-RF were purchased from Santa Cruz Biotechnology (California, US). Hydrogen tetrachloroaurate (III) trihydrate (HAuCl4 ·3H2 O, Au 49.5% min) and PVP, Mr = 1 300 000) were obtained from Alfa Aesar (A Johnson Matthey Company, Ward Hill, MA, USA). BSA was from Siobio Biotechnology (Shanghai, China). Luminol was purchased from Yacoo Chemical Factory (Suzhou, China). All reagents used were of analytical grade. The pure water (18.2 M⍀·cm) used was processed with an Ultrapure Water System (Kangning Water Treatment Solution Provider, China). All solutions were stored in the refrigerator at 4°C and filtered through a 0.22 m cellulose acetate membrane filters (Shanghai Xingya Purification Material Factory, China) before use. 2.2 Instrumentation The basic design of the CE–CL system has been previously described [29]. Briefly, a high-voltage supply (0–30 kV, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, China) was used to drive the electrophoresis. A 50 cm × 75 m id uncoated fused silica capillary (Hebei Yongnian Ruifeng Chromatographic Apparatus, China) was used for the separation. One end of the separation capillary was burned off 5 cm polyimide coating. After etching with 40% hydrofluoric acid for 2.5 h, this end of capillary was inserted into a 20 cm × 530 m id reaction capillary. The outlet of the separation capillary was located at the detection window, which was made by burning 1 cm of the polyimide of the reaction capillary and placed in front of the PMT (CR 120, Bin C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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song Photonics, Beijing, China). The pair of 40 cm × 320 m id capillary was used to introduce postcolumn CL reagents to a tee reservoir from two vials located 20 cm above it. The CL reagents were delivered by gravity through the reagentintroducing capillaries, mixed and flowed coaxially along the separation capillary. The CL reaction took place at the separation capillary outlet when analyte eluted out from the separation capillary. The ground electrode was also put into one joint of the tee. CL emission was collected with a BPCL ultraweak luminescence analyzer (Institute of Biophysics, Chinese Academy of Science, Beijing, China) and then recorded and processed with a computer using BPCL software. Transmission electron microscopy images were obtained by using a JEM-2100F transmission electron microscope (JEOL, Tokyo, Japan). Absorption spectra were recorded by an UVmini-1240 UV-vis spectrophotometer (Shimadzu, Kyoto, Japan). 2.3 Preparation of gold nanoparticles AuNPs production by sodium citrate reduction of HAuCl4 in water was performed according to the literature [30]. Briefly, 50 mL of 0.01% HAuCl4 solution was added to a 100 mL beaker and brought to a rolling boil. Next, 0.5 mL of 2% sodium citrate was quickly added to the rapidly stirred boiling solution. The formation of AuNPs can be observed by a change in color from pale-yellow to wine-red. The colloids were stirred for another 15 min and cooled to room temperature. After cooling, the synthesized AuNPs was filtered through a 0.22 m cellulose membrane and stored at 4°C. Transmission electron microscopy images confirmed that the average diameter of AuNPs used for labeling is about 36 nm. 2.4 Preparation of AuNPs-antibody conjugate Binding of anti-RF to AuNPs was performed according to previously published report [31]. In brief, 500 L of pH 8.5 colloidal AuNPs were incubated with 44 L of 0.3 mg/mL anti-RF solution for 30 min at room temperature under agitation. Afterwards, 5% BSA was added to a final concentration of 1% with stirring for 5 min. Then the conjugate was centrifuged at 12 000 rpm for 30 min. The red sediment was the anti-RF-AuNPs conjugate, which was resuspended in 0.5 mL of 0.04 mol/L pH 7.4 PBS (containing 1% BSA and 0.05% Tween 20) with the supernatant being removed. 2.5 Immune reaction procedure The IA protocol is noncompetitive format and the immunoreaction was conducted as follows: RF (Ag) + AuNPs-anti-RF (Ab∗ , excess) → RF-AuNPs-anti-RF (Ag-Ab∗ ) + AuNPs-anti-RF (Ab∗ , free) www.electrophoresis-journal.com
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Ten microliters of RF standard or serum sample was mixed with 10 L of AuNPs labeled anti-RF in a 200 L microcentrifuge tube and then diluted with 0.01 mol/L pH 7.4 PBS to 50 L. After incubating at 37°C for 1 h, the mixture was analyzed by CE as described below.
2.6 CE procedure The new capillary was preconditioned sequentially by flushing with 2.0 mol/L NaOH–CH3 OH, 1.0 mol/L NaOH, water, and electrophoretic buffer for 20 min before the first use. At the beginning of each day, the separation capillary was reconditioned by flushing with 0.1 mol/L NaOH, water, and electrophoretic buffer for 3–5 min successively. After three consecutive injections, the separation capillary was flushed with 0.1 mol/L NaOH, water, and electrophoretic buffer for 2 min, respectively. The electrophoretic buffer was 7 mmol/L Na2 B4 O7 with 0.40 mmol/L luminol and 1.0% PVP at pH 11.0. The postcolumn CL reagents, one containing 40 mmol/L H2 O2 and another containing 20 mmol/L pH 12.0 NaHCO3 , were separately introduced into the reaction capillary from two vials. The voltage of PMT for collecting the CL signal was set at −800 V. The sample was introduced in the separation capillary by electrokinetic injection at 10 kV for 6 s. CE separation was performed at 20 kV. The peak area was used in analysis.
2.7 Preparation of human serum samples Human serum samples from patients were provided by Xinyang Central Hospital (Xinyang, China). The fresh blood samples were immediately centrifuged for 10 min at 1200 rpm to remove erythrocytes. Subsequently, the supernatant was diluted 20-fold by using 0.01 mol/L pH 7.4 PBS as diluent buffer and analyzed.
3 Results and discussion 3.1 Determination of the minimum quantity of antibody for AuNPs labeling The citrate stabilized AuNPs suspension could be very stable due to the electrostatic repulsions between negatively charged AuNPs. However, when the salt solution (e.g. 10% NaCl) was added into the suspension, AuNPs may cause aggregation due to the interaction between the cations and the negatively charged AuNPs. When proteins (e.g. antibody) are added to AuNPs suspension, the proteins can be adsorbed to the surface of AuNPs, preventing the AuNPs from flocculation. [32, 33]. A flocculation test was often carried out to determine the minimum quantity of antibody for AuNPs labeling [34, 35]. C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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In this experiment, an increasing volume (0–65 L) of antiRF at the fixed concentration of 0.3 mg/mL were added to 500 L of AuNPs under agitation and incubated for 30 min. Then 50 L of 10% NaCl was added to the mixture. UVvis absorption spectrum of each mixture was recorded after 30 min at room temperature to assess the minimal quantity of antibody. In the present study, with the increasing quantity of antiRF, the absorbance at maximum wavelength (max ) of AuNPs begins to blue shift and maintains constant when the volume of anti-RF is above 40 L, which indicates that 40 L is the minimal quantity needed to stabilize 500 L AuNPs. Therefore, the quantity of the antibody used for labeling in this work is 44 L of 0.3 mg/mL anti-RF, which is 10% more than the minimum amount required to ensure that the quantity of antibody is enough for the stability of the anti-RF-AuNPs conjugate.
3.2 Optimization of CE separation conditions Since concentration and pH of the electrophoretic buffer greatly influence the ionization of the silanols on the capillary wall and ionic strength of the buffer, changing these in the buffer are the most direct and important strategy for optimizing CE separation. In this experiment, the Na2 B4 O7 was used as the electrophoretic buffer. The effect of the Na2 B4 O7 concentration on CL intensity and resolution was examined in the range 1–13 mmol/L, as shown in Fig. 1A. The highest CL intensity and the best resolution were achieved at 7 and 9 mmol/L, respectively. Although the resolution was the best at 9 mmol/L, the CL intensity was not the highest. When the CL intensity was the highest, the free Ab* and the Ab* –Ag complex could be baseline separated (the resolution was 1.51). Therefore, 7 mmol/L was selected as the optimum Na2 B4 O7 concentration. The effects of buffer pH were investigated with pH ranging from 10.1 to 11.9 (Fig. 1B). The results indicate that the CL intensity and resolution reach the maximum at pH 11.0 and then decreased. Therefore, pH 11.0 was used. In order to decrease the adsorption of protein on the capillary inner wall, PVP was used as a capillary inner wall dynamic coating reagent for analyte separation. The effects of PVP concentration on the CL intensity and resolution were investigated and the results were shown in Fig. 1C. The CL intensity and resolution were increased with the increase in the PVP concentration. When the PVP concentration reached 1.0%, the highest CL intensity and satisfactory separation (the resolution was 1.53) can be obtained. Moreover, the migration time became longer and the peaks became broader above 1.0%. As a result, 1.0% PVP was chosen. Effects of separation voltage on the CL intensity and resolution were also studied. By changing the separation voltage from 14 to 26 kV, the CL intensity increased and the resolution decreased with separation voltage increase (Fig. 1D). Meanwhile, the Joule heats existent in the running buffer www.electrophoresis-journal.com
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Figure 1. Effect of Na2 B4 O7 concentration (A), buffer pH (B), PVP concentration (C), and separation voltage (D) on CL intensity and resolution. Conditions: the electrophoretic buffer, 7 mmol/L Na2 B4 O7 with 0.40 mmol/L luminol and 1.0% PVP at pH 11.0; the postcolumn CL reagents, 40 mmol/L H2 O2 and 20 mmol/L pH 12.0 NaHCO3 ; PMT voltage, −800 V; electrokinetic injection with 10 kV for 6 s; separation voltage, 20 kV; separation capillary, 50 cm × 75 m id.
system also increased. When the separation voltage was 20 kV, the resolution was calculated to be 1.57. Therefore, 20 kV was set.
3.3 Optimization of the CL detection conditions In this CL reaction, the concentrations of luminol and H2 O2 have a great influence on CL intensity. To obtain the optimal luminol concentration, CL intensity was detected at different luminol concentration ranging from 0.1 to 0.7 mmol/L (Fig. 2A). It was found that a concentration of 0.4 mmol/L luminol produced maximum CL intensity. Thus 0.4 mmol/L luminol was chosen. The effect of H2 O2 concentration was investigated. As can be seen in Fig. 2B, the CL intensity rapidly increased in the range from 10 to 40 mmol/L. When H2 O2 concentration was higher than 40 mmol/L, the CL intensity decreased. Therefore, 40 mmol/L H2 O2 was selected. The concentration and pH of NaHCO3 , used as postcolumn buffer, also have an important effect on CL intensity. The effect of NaHCO3 concentration was evaluated from 5 to 35 mmol/L. It can be seen from Fig. 2C that the CL intensity increased with the concentration from 5 to 20 mmol/L and then decreased. Hence, 20 mmol/L was chosen. Because the CL reaction of luminol occurs in basic medium, the effect of NaHCO3 pH was investigated over a pH range from 10.0 to 13.0 and the results were shown in Fig. 2D. The CL intensity was found to increase with the increasing NaHCO3 pH and reached its apex when the NaHCO3 pH was 12.0. Thus, pH 12.0 was used. C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 2. Effects of luminol (A), H2 O2 (B), NaHCO3 (C) concentration, and NaHCO3 pH (D) on CL intensity. Other conditions were as described in the legend of Fig. 1.
3.4 Detection of human RF by the proposed method Under the optimized conditions, the typical electropherograms of 0 and 1.0 g/mL human RF are shown in Fig. 3A and B, respectively. We examined the linearity, detection limit, and precision of the present method for human RF analysis. The calibration curve was calculated plotting the peak area values of Ab* –Ag complex against the RF concentrations. The linear range of the calibration curve was from 0.01 to 20 g/mL (R = 0.997), and the detection limit was 5.95 ng/mL (ca. 6.0 pmol/L, S/N = 3). The precisions (measured by RSD, www.electrophoresis-journal.com
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Electrophoresis 2014, 35, 972–977 Table 1. Analytical results of eleven human sera samples (n = 3)
Number of human sera samples
Figure 3. Electropherograms of human RF in standard solution. (A) 0 g/mL human RF; (B) 1.0 g/mL human RF. Peak 1, free Ab*; peak 2, immune complex; peak X, unknown compounds. Other conditions were as described in the legend of Fig. 1.
RF content (g/mL)
1 2 3 4 5 6 7 8 9 10 11
This method (RSD, %)
ITT
6.76 (3.1) 14.1 (3.7) 8.98 (3.7) 28.6 (6.4) 8.89 (4.5) 10.9 (2.3) 8.03 (4.1) 5.11 (5.4) 8.34 (6.1) 37.2 (6.7) 49.1 (5.8)
7.55 13.54 9.33 25.75 9.26 10.66 7.71 4.87 8.00 36.91 44.78
4 Concluding remarks
Figure 4. Electropherograms of human RF in sera samples from two patients with RA. (A) The serum sample of patient A; (B) the serum sample of patient B. Peak 1, free Ab*; peak 2, immune complex; peak X, unknown compounds. Other conditions were as described in the legend of Fig. 1.
n = 5) were examined by assaying 2.5 g/mL human RF within a day (intraday) and in 3 days (interday). The RSDs of the migration time were 1.7% for intraday and 3.4% for interday, respectively. The RSDs of the peak area were 3.2% for intraday and 5.2% for interday, respectively.
3.5 Analysis of RF in human sera So far, RF has been widely used as a biomarker for clinical diagnosis of RA. The contents of RF in the sera from two patients with RA were determined and the electropherograms were shown in Fig. 4. As seen in Fig. 4, the peak of Ab* –Ag complex can be observed obviously in the sera of patients with RA. In order to verify this method, the contents of RF in sera from 11 patients were determined by the proposed method (X) and immunity transmission turbidity (ITT) on Roche clinical chemistry analyzer (Y) employed in the Xinyang Central Hospital as control. The results of the 11 samples determined by the two methods were listed in Table 1. Two methods coincided well with a regression equation of Y = 0.9485 X + 0.3726 and a correlation coefficient of 0.995. The results indicate that the proposed method has potential application in the clinical analysis. C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
This study presents a sensitive and rapid CEIA method combined with CL detection using AuNPs as label of antibody for the determination of RF in the human serum. This method has many outstanding features including high efficiency, high selectivity, and high practicality. The CEIA method with the noncompetitive immunoreactions can be accomplished in a homogeneous solution with one-step operation. AuNPs show excellent catalytic property to luminol CL reaction and good labeling property to protein molecules, making it possible to detect the analytes at lower concentrations. Besides, AuNPs exhibit the advantage of better stability over the enzyme labels, and the whole procedure is uncomplicated and does not affect the biochemical activity of the labeled compound. The analytical results of proposed method coincided well with that of ITT. The results demonstrate that the current CE–IA–CL method maybe served as an alternative tool for determination of other biomarker (e.g. tumor marker) in the early disease diagnosis. This work was supported by the National Natural Science Foundation of China (21075106, 21375114). The authors have declared no conflict of interest.
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