Copper(II)−Alizarin Red S Complex as an Efficient Chemiluminescent

Nov 4, 2008 - Beijing Normal University. ... Ghent University Hospital. ... Li, Zhao, Zhang, Luo, Corkins, Cole, Wang, Xiao, Bi, Pang, McElroy, Bird, ...
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Copper(II)-Alizarin Red S Complex as an Efficient Chemiluminescent Probe for the Detection of Human Serum Proteins after Polyacrylamide Gel Electrophoresis Zhenzhen Wang,† Xia Liu,† Willy R. G. Baeyens,‡ Joris R. Delanghe,§ and Jin Ouyang*,† College of Chemistry, Beijing Normal University, Beijing, P. R. China, Department of Pharmaceutical Analysis, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium, and Department of Clinical Chemistry, Microbiology and Immunology, Ghent University Hospital, Ghent, Belgium Received May 20, 2008

A novel chemiluminescent probe, copper(II)-Alizarin Red S (ARS) complex, for the detection of human serum proteins after polyacrylamide gel electrophoresis (PAGE) is described. The detection is based on the binding of the copper(II)-ARS complex to proteins and the catalytic activity of copper(II) in the luminol-hydrogen peroxide chemiluminescence (CL) system. Various proteins are directly detected in polyacrylamide gels, avoiding tedious transferring procedures. In the present study, the possible reaction mechanism and sensitivity evaluation are analyzed. The experimental conditions such as solution concentration, complex ratio, and washing reagents are likewise optimized. The proposed method offers simple, fast, and sensitive detection of serum proteins. As a novel chemiluminescent detection method, it shows significant analytical potential in biochemistry. Keywords: Copper(II)-Alizarin Red S complex • Chemiluminescence imaging • Probe • Human serum proteins • CBB staining

1. Introduction The analysis of human serum proteins is of major importance in biochemistry and in clinical applications since it can provide information for the (early) diagnosis of diseases. Polyacrylamide gel electrophoresis (PAGE) implies common methods for separating human serum proteins. After separation, the detection methods can be roughly divided into the following categories: (i) organic dyes stains; (ii) silver stains; (iii) fluorescent stains; (iv) chemiluminescence (CL) immunoanalysis.1 Coomassie Brilliant Blue (CBB) represents a traditional and prevalent organic dye staining method for protein detection, but it needs gel staining and destaining, which has some limits.2 Silver staining may offer very high sensitivity; however, it is a multistep procedure, and the staining reaction has no end point.3,4 Fluorescent staining techniques may be nearly as sensitive as silver staining. The SYPRO family, such as SYPRO Ruby, Red, and Orange have emerged in laboratory practice.5-8 However, these fluorescent dyes are very expensive, and special equipment is required to detect the fluorescence signal. Additionally, UV light sometimes used for this illumination is rather hazardous. Chemiluminescence immunoanalysis (CLIA) techniques have been developed recently, and they represent highly selective techniques for protein detection. Using the latter, the electrophoresis pattern is being transferred to some protein-binding membranes to * Corresponding author. E-mail: [email protected]. Fax: +86-1062799838. † Beijing Normal University. ‡ Ghent University. § Ghent University Hospital. 10.1021/pr800365n CCC: $40.75

 2008 American Chemical Society

form a “Western blot”.9-11 Since the transfer efficiency of different proteins varies, the replicated pattern may be inaccurate when representing the original electropherogram, particularly for large proteins and small peptides. In recent years, a direct CL imaging technique was developed for determining human serum proteins after PAGE, wherein several CL probes have been reported: using hemoglobin (Hb) or ammonium persulfate as probes to catalyze the luminolH2O2 CL reaction, some proteins in human serum and metalloenzymes in rat liver cytosol could be detected.12-16 Sensitivity of the detection technique was improved. However, only the proteins containing a metal ion or metal complexes could generate CL emission under these conditions, hence more investigation was devoted to extend the detection range: [Ag(NH3)2]+ has been reported as a probe for CL imaging, catalyzing the luminol-K2S2O8 CL system.17 This technique could detect more proteins; however, it needs special ammonia-controlled equipment for preparing NH3. Copper(II) has high catalytic activity and high sensitivity for the luminol-H2O2 CL reaction system.18-22 However, copper(II) cannot directly make the proteins emit CL because it cannot bind with these proteins. Some dyes could form a complex with copper(II), which could also bind the proteins.23 Hence the dyes can act as bridges connecting copper(II) and protein to form a copper(II)-dye-protein complex. Through capturing the CL emission of copper(II), the corresponding proteins could consequently be detected. In the present study, Alizarin Red S (1,2-dihydroxyanthraquinone-3-sulfonate, ARS) is a dye which is used for binding proteins and copper(II) under appropriate conditions.24 A possible reaction mechanism is Journal of Proteome Research 2008, 7, 5075–5081 5075 Published on Web 11/04/2008

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Figure 1. Presumed reaction mechanism of ARS and copper(II) binding with proteins catalyzing the luminol-hydrogen peroxide system.

shown in Figure 1. Utilizing the catalytic activity of copper(II) to the luminol-H2O2 system and the binding ability of ARS to proteins, copper(II)-ARS is used as a probe applied in the CL imaging detection of human serum proteins. The detection range is significantly extended since various proteins which bind with the complex can be detected. In this study, the experimental conditions were optimized, and the detection limit of the system was analyzed. The sensitivities for some proteins are superior to those in direct CL imaging and CBB staining methods. This detection technique offers several advantages: (1) it is simple, fast, and noncontaminative; (2) it offers high sensitivity, low background, and clear resolution; and (3) the procedure does not require special instruments.

2. Materials and Methods 2.1. Materials. All reagents were of analytical reagent grade. Acrylamide, N,N′-methylenebisacrylamide (Bis), ammoniumpersulfate, tris(hydroxymethyl)aminomethane (Tris), N,N,N′,N′tetramethylethylenediamine (TEMED), and aminoacetic acid (glycine) were from Sino-American Biotechnology Co. (Beijing, China). Alizarin Red S (C14H7NaO7S · H2O) was purchased from Beijing Reagents Co. (Beijing, China). NaOH, glycerine, bromophenol blue, hydrogen peroxide, sodium acetate, acetic acid, and ethylenediamine tetracetic acid (EDTA) were purchased from Beijing Chemical Factory (Beijing, China). Carrier ampholytes (pH 3-10) and Coomassie Brilliant Blue R-250 (CBBR250) were purchased from Fluka (Switzerland). 3-Aminophthalic hydrazide (luminol) was from Acros Organics (New Jersey, USA). MilliQ water (Millipore, Bedford, MA) was used to prepare the solutions. The human serum samples from healthy patients were obtained from Xinjingke Company (Beijing, China) and the affiliated Hospital of Beijing Normal University. 2.2. Instruments. The electrophoresis system consisted of a DYY-6C and DYY-6B electrophoresis instrument, DYCZ-21 and DYCZ-24D vertical electrophoresis tanks (Liuyi Instrument Factory, Beijing, China). Quantitation of the developed X-ray films and the protein bands was performed with a Microtek 5076

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Wang et al. ScanMaker s700 scanner (Founder, Shanghai, China). The binding interactions were detected using a GBC Cintra 10 UV-visible spectrometer and an RF-5301PC fluorescence spectrometer. The intensity of protein bands in X-ray films was analyzed by a gel documentation and analysis system (UVPEC3). 2.3. Protein Electrophoresis. Nondenaturing 1-D and 2-D PAGE was carried out as previously described.17 Briefly, the 1-D PAGE was performed in a vertical discontinuous gel system, consisting of separating (7.5%, m/v) and stacking (4.0%, m/v) gels. The voltage in the stacking gels was 120 V. When the sample entered the separation gels, the voltage was turned down to 100 V and kept constant for 2 h. For 2-D PAGE, serum proteins were first subjected to IEF employing column gels. IEF was run at 200 V for 30 min and continued at a 400 V constant voltage for 15 h. After that, gels were transferred onto the second-dimension slab gels (7.5%, m/v). The voltage was set at 110 V for 3 h. 2.4. Detection of Proteins. 2.4.1. Direct CL Imaging Detection. After separation, the gel was taken out of the mold and washed with deionized water. Then the gel was sprayed with the optimized concentrations of hydrogen peroxide (1.2%, w/v) and luminol (1.0 × 10-3 mol L-1) solutions in a dark room until protein signals appeared.12,14,16 X-ray films (Fuji) were used to collect the CL signals. The film was exposed for about 1 min and developed for about 2 min. Next, it was submerged into the fixing agent for a few minutes. After developing and drying, the X-ray film was scanned, and the data were transferred to the computer. 2.4.2. CBB-R250 Staining Detection. After separation, the gel was stained in CBB-R250 (0.1%, w/v) solution at room temperature for 2 or 3 h, the solution being prepared by dissolving 0.1 g of CBB-R250 in the mixture of 35 mL of CH3OH, 10 mL of acetic acid, and 55 mL of water, followed by filtration. The gel was destained by ethanol/acetic acid solution (10%/ 7% in distilled water) for about 8-10 h. 2.4.3. Copper(II)-ARS Complex Labeled Staining and CL Imaging Detection. Copper(II)-ARS complexes were prepared by the dye ARS and CuSO4 solutions. An amount of 0.018 g of ARS was dissolved in 25 mL of CH3COOH-CH3COONa buffer (0.002 M, pH 3.70-4.50), 20 mL of CuSO4 (0.005-0.1 M) complexing with this solution. When the gel was taken out of the system, it was washed with fixation solution containing 50% ethanol/10% acetic acid (v/v), then deionized water, and stained in the complex solution for 30-50 min until the protein bands could be observed. To eliminate the superfluous copper(II), complexing reagent EDTA (0.05 M) was used for washing the gels. Before CL imaging detection, the gel was washed with deionized water. Then, hydrogen peroxide (1.2%, w/v) and luminol (1.0 × 10-3 mol L-1) were sprayed onto the gel in a dark room. An X-ray film was exposed to the gel for 1 min, then developed for 1 min, and submerged into the fixing agent for 2 min. The staining gels and CL imaging film were scanned with a scanner, and the results were transferred to the computer.

3. Results and Discussion 3.1. Comparison of Direct CL Detection, CBB-R250 Staining, Copper(II)-ARS Complex Staining, Complex-Labeled CL Imaging Detection, and Copper(II) Detection in 1-D PAGE. Nondenaturing PAGE offered a comprehensive understanding to examine the physiological properties and biological functions of individual proteins from serum.25,26 In the present

Copper(II)-ARS Complex as Efficient Chemiluminescent Probe

Figure 2. Five detection methods used after 1-D PAGE. (A) Direct CL imaging detection (Hp 2-1). (B) CBB-R250 staining detection. (C) Alizarin Red S-copper(II) complex staining detection. (D) Alizarin Red S-copper(II) complex-labeled CL detection. (E) Copper(II) detection (after interaction with copper(II), washing with EDTA, the same as method D). An amount of 15 µL of serum was dissolved in 135 µL of sample buffer. Loading volume: 15 µL.

study, human serum samples were detected by direct CL imaging, CBB-R250 staining, copper(II)-ARS complex staining, copper(II)-ARS complex-labeled CL imaging, and copper(II) detection after nondenaturing 1-D PAGE. The results of 1-D PAGE are shown in Figure 2. When comparing the five detection methods, the results appeared quite different. From the direct CL imaging results, it can be seen that only proteins of haptoglobin (Hp) could be detected in 1-D PAGE (Figure 2A) because of the Hp-Hb complex enhancing the CL system.12 Besides this method, CBBR250 staining and copper(II)-ARS complex staining methods were also used to detect the human serum proteins. CBB-R250 staining showed a relatively low sensitivity, the background of the gel always being difficult to destain, which caused the protein bands to be unclear for observation (Figure 2B). The copper(II)-ARS complex staining was fast and easy to produce; however, the sensitivity was too low, and the background color of the gel was similar to that of the protein bands (Figure 2C). However, the copper(II)-ARS complex-labeled CL probe could avoid the shortcoming of the direct CL imaging method, the CBB-R250, and the copper(II)-ARS complex staining methods. A great number of proteins can generate CL emission applying this probe (Figure 2D). When only using copper(II) for detection, the experimental conditions were the same as the copper(II)-ARS complex-labeled CL method, and when EDTA was used after the gel interaction with copper(II), the result was no CL signals (Figure 2E). 3.2. Optimization of the Experimental Conditions. 3.2.1. Optimization of CL Reagent Concentrations. To obtain optimal imaging results, the effects of luminol and H2O2 concentration were examined by a spectrofluorophotometer. The luminol concentration was changed from 1.0 × 10-4 to 1.0 × 10-2 M. The signal was increased gradually when the luminol concentration increased from 1.0 × 10-4 to 1.0 × 10-3 M. Concentrations higher than 1.0 × 10-3 M did not continuously improve the CL intensity. Therefore, 1.0 × 10-3 M luminol was chosen for further experiments. The concentration of H2O2 was optimized within the range from 0.3% (v/v) to 4.8% (v/v). The optimal H2O2 concentration was found to be 1.2% (v/v). Higher concentrations caused a high background and decreased the S/N ratio. 3.2.2. Optimization of pH Condition of the ARS Solution. ARS can bind the proteins when the pH of the ARS solution

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Figure 3. Effect of pH conditions for Alizarin Red S upon CL intensity. (A) pH 3.35, (B) pH 3.70, (C) pH 4.00, (D) pH 4.14, (E) pH 4.50. Alizarin Red S concentration: 0.002 M. Copper(II) concentration: 0.005 M. An amount of 15 µL of serum was dissolved in 135 µL of sample buffer. Loading volume: 15 µL.

lies under the pI of the proteins. When pH < pI, the proteins are electropositive, and they can bind the electronegative SO32on the ARS.27 The interaction is dependent on electrostatic interaction forces.28 Hence, to bind the ARS onto more proteins, the pH of the solution should be optimized. Some experiments were carried out to obtain optimal CL imaging results. The signal intensity was different when the pH was gradually changed from 3.35 to 4.50. Figure 3A illustrated that less protein bands were detected below pH 3.70. However, when the pH value was higher than 3.70, the background increased, and protein bands became unclear. Thus, the optimal pH was installed at 3.70. 3.2.3. Optimization of the Complex Ratio of ARS and Copper(II). In the structure of ARS, there are several functional groups including phenolic hydroxyl (-OH) and carbonylic groups (-CdO). These groups can form a hexahydric ring complex with copper(II) under appropriate solution conditions (Figure 1). The ratio of ARS and copper(II) could affect the imaging results. In the present assay, the ratio of ARS and copper(II) was tested in the range of 2:1 to 1:3 (2:1, 1:1, 2:3, 1:2, 2:5, and 1:3). When the ratio of ARS and copper(II) was lower than 1:2, superfluous copper(II) causes the low signal/ noise ratio (S/N ratio) because of the high lighting background. When higher than 1:2, the signals of the proteins were not clear, and the S/N decreased. Therefore, the best complex ratio was considered to be 1:2. 3.2.4. Optimization of CuSO4 Solution Concentration. When the complex ratio of ARS and copper(II) was fixed on 1:2, the concentration of copper(II) affects the results of CL imaging. High concentrations of copper(II) cause high lighting backgrounds which could affect the S/N ratio. The concentration of copper(II) selected for the assay was from 0.1 to 0.005 M. When copper(II) concentration was higher than 0.01 M (Figures 4A and B), the background of the gel was enhanced, and the S/N was reduced. When lower than 0.01 M (Figures 4C and D), the S/N will be improved. The CL imaging results learned that the optimal concentration of copper(II) was 0.005 M. 3.2.5. Using EDTA for Eliminating the Free Copper(II) Ion on the Gels. Because of the high catalytic activity of the copper(II) ion in the luminol-H2O2 CL system,19,29 the background intensity of the gels will increase when the noncomplexing copper(II) ion absorbs on the blank gels, which would seriously interfere with the detection of the protein bands. Hence, the complexing agent was applied for removing the free copper(II) ion. EDTA was a prevalent agent for this purpose. In this study, 100 mL of EDTA solution was used for washing each gel, and a Journal of Proteome Research • Vol. 7, No. 12, 2008 5077

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Figure 4. Effect of the concentration of CuSO4 on CL emission. (A) 0.1 M, (B) 0.05 M, (C) 0.01 M, and (D) 0.005 M. Alizarin Red S concentration: 0.002 M. The ratio of Alizarin Red S and copper(II) was 1:2. An amount of 15 µL of serum was dissolved in 135 µL of sample buffer. Loading volume: 15 µL.

Figure 5. Effect of complexing agent EDTA concentration for washing the superfluous copper(II) upon CL intensity. (A) 0.00025 M, (B) 0.0005 M, (C) 0.00125 M, (D) 0.0025 M, and (E) 0.005 M. The washing volume of EDTA for each gel was 200 mL. Alizarin Red S concentration: 0.002 M. Copper(II) concentration: 0.005 M. Alizarin Red S/CuSO4 ) 1:2.

series of EDTA concentrations was tested. From 2.5 × 10-4 M to 1.25 × 10-3 M, the S/N increased (Figure 5A-C). When the concentration of EDTA was higher than 1.25 × 10-3 M, the signal of the CL imaging result could hardly be found (Figure 5D and E). Therefore, according to a serial assay, the optimal concentration of EDTA was determined at 1.25 × 10-3 M. 3.3. Discussion of the Possible Reaction Mechanism. 3.3.1. Binding Interaction between Copper(II)-ARS Complex and Proteins. Because of the structure of ARS, some functional groups could form a hexahydric ring complex with copper(II) under the appropriate conditions.24 The ARS part of the complex could bind the proteins when the solution pH is below the isoelectric point (pI) of the proteins, the latter taking positive charges and binding the electronegative sulfonic group of the dye.23 To confirm the binding interaction between the copper(II)-ARS complex and the proteins, experiments for HSA and the copper(II)-ARS complex interaction were carried out using UV-spectrophotometric and spectrofluorescence measurements. The UV absorptions of compounds at different wavelength are shown in Figure 6A, confirming the binding interactions between ARS-copper(II) and HSA. The main absorption of HSA was 280 nm; the absorption of ARS (pH 3.70) was at 420 nm; the absorption of copper(II)-ARS (pH 3.70, ARS/copper(II) ratio, 1:2) was at 500 nm; and ARS-copper(II)HSA complex was at 530 nm. The fluorescence spectra of HSA in the absence and presence of copper(II)-ARS in pH 3.70 5078

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Figure 6. Binding interaction between the copper(II)-ARS complex and proteins. (A) UV absorption of the reaction compounds. Absorption of ARS (6 × 10-5 M), copper-ARS, HSA (2 × 10-5 M), and copper(II)-ARS-HSA complex. (B) Fluorescence emission spectra upon excitation at 280 nm (pH 3.70). (a) and 2.0 × 10-6 M HSA and (b)-(g) 2.0 × 10-6 M HSA in the presence of ARS-copper(II) (0.4 × 10-5 M, 0.6 × 10-5 M, 0.8 × 10-5 M, 1.0 × 10-5 M, 2.0 × 10-5 M, 5 × 10-5 M), complex ratio: 1:2. (C) Stern-Volmer plots of the HSA-ARS-copper(II) system (λex ) 280 nm, CHSA ) 2.0 × 10-6 M).

buffer were measured with an excitation wavelength at 280 nm, their representative spectra being shown in Figure 6B (a-g). Data from the fluorescence experiments can be analyzed using a modified Stern-Volmer equation.30,31 1 1 1 ) + IF0 - IF IF0 K · IF0 · Cq where IF0 is the fluorescence intensity in the absence of an external quencher; IF is the difference in fluorescence in the

Copper(II)-ARS Complex as Efficient Chemiluminescent Probe

Figure 7. Copper(II)-ARS complex catalyzing effect in the CL system. Results of CL imaging after gel immersion in (A) ARS solution, (B) CuSO4 solution, and the (C) copper(II)-ARS complex solution.

presence of the quencher copper(II)-ARS at different concentrations Cq; and K is the binding constant. The plots of 1/(IF0 - IF) versus 1/Cq (Figure 6C) yield 1/IF0 as the intercept and 1/KIF0 as the slope. The binding constant K obtained herein was of 1.59 × 105 L/mol, which confirmed the binding interaction between HSA and copper(II)-ARS. 3.3.2. Comparison of ARS, Copper(II), and Copper(II)-ARS Complex Solution for Catalyzing the CL System. The copper(II)ARS complex could catalyze the luminol-H2O2 CL reaction because of the activity of the copper(II) ion.18,20,21 To identify whether the copper(II)-ARS complex affects the catalysis for producing luminol CL emission, some experiments were carried out to confirm this suggestion (Figure 7). After electrophoresis, the gels were taken out of the mold and then respectively immerged in ARS solution, CuSO4 solution, and copper(II)-ARS complex solution. The results showed that the gel in dye ARS solution did not emit CL signals (Figure 7A). The gel in CuSO4 solution had a high CL background, and the protein CL signals could not be recorded by the X-ray films (Figure 7B). The gel in the complex solution could generate the relatively lasting CL emission which could be captured by the X-ray films (Figure 7C). The results of the CL imaging confirm that the copper(II)-ARS complex plays a key role in the catalytic effect for producing luminol CL emission. 3.4. Comparison of Four Detection Methods in 2-D PAGE. In this study, human serum samples were detected by direct CL imaging, CBB-R250 staining, copper(II)-ARS complex staining, and copper(II)-ARS complex-labeled CL imaging after 2-D PAGE, respectively. The results are shown in Figure 8. From the direct CL imaging results, it can be seen that only a few proteins could be detected by 2-D PAGE16 and that not all the proteins emit CL radiation (Figure 8A). CBB-R250 staining and copper(II)-ARS complex-labeled CL methods provide approximate results, but the latter one could detect more protein spots and has a much higher resolution which are shown in the rectangle regions (Figures 8B and D). The copper(II)-ARScomplex staining was easy to obtain, but the sensitivity was too low, and the protein points were difficult to distinguish

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Figure 8. Four detection methods used after 2-D PAGE. (A) Direct CL imaging detection (Hp 2-1), (B) CBB-R250 staining detection, (C) copper(II)-ARS complex staining detection, (D) copper(II)-ARS complex-labeled CL detection. An amount of 20 µL of human serum was dissolved in 200 µL of sample buffer. Loading volume: 100 µL of sample was used in the first-dimension separation.

Figure 9. Alizarin Red S-copper(II) complex-labeled CL probe detecting the mixture sample of IgG, Tf, and HSA. (A) Direct CL imaging detection. (B) Alizarin Red S-copper(II) complex-labeled CL detection. (C) CBB-R250 staining detection. 50 µL, 2 mg/mL HSA, Tf, and IgG mixture sample was dissolved in 50 µL of sample buffer. Loading volume: 15 µL.

(Figure 8C). Hence, the copper(II)-ARS complex-labeled CL probe presents some advantages. 3.5. Detection of IgG, Tf, and HSA Using a Copper(II)-ARS Complex-Labeled CL Probe. In human serum proteins, albumin (HSA), transferrin (Tf), and immunoglobulin G (IgG) take important parts in health and relate to many diseases.32-35 However, these proteins could not be detected by the direct CL imaging method because they do not catalyze the luminol-H2O2 CL system emitting CL radiation (Figure 9A). Using the copper(II)-ARS complex-labeled CL probe method in the reaction system, HSA, Tf, and IgG could be labeled by this probe and emitted CL signals, which could be captured by the X-ray film (Figure 9B). The signals of the proteins could be well matched with the CBB-R250 staining method (Figure Journal of Proteome Research • Vol. 7, No. 12, 2008 5079

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Figure 10. Comparison of the sensitivities of CBB-R250 staining, ARS-copper(II) complex-labeled CL imaging, and silver staining. Serial dilutions of human serum loaded onto gels (1) 1/5, (2) 1/10, (3) 1/20, (4) 1/40, (5) 1/80, (6) 1/100, (7) 1/160, (8) 1/200, (9) 1/300, and (10)1/400. (A) CBB-R250 staining detection, (B) ARS-copper(II) complex-labeled CL probe detection, and (C) silver staining.

9C). From Figure 9, it can be seen that the copper(II)-ARS complex-labeled CL detection method offers many advantages. It detects more proteins than in the direct CL imaging method, which extends the imaging range. It also causes a shorter reaction procedure than in the CBB-R250 staining method, the results of the CL imaging films being easy to preserve. 3.6. Sensitivity Evaluation. The detection sensitivity was investigated by serial dilutions of healthy human serum. The pure serum was diluted using the sample buffer containing glycerine solution and deionized water by 1/5 to 1/400, and 15 µL of prepared sample was loaded. The results of CBB-R250 staining, copper(II)-ARS complex-labeled CL imaging, and silver staining are shown in Figure 10. It can be easily seen that the sensitivity of copper(II)-ARS complex-labeled CL imaging is superior to that of CBB-R250 staining (Figures 10A and B), taking haptoglobin protein as an example, when considering the concentration of haptoglobin in the serum around 1.33 mg/mL, the detection limit of the complex probe method being approximately 0.125 µg, which is superior to CBB-R250 staining. We also evaluated the sensitivity of silver staining which was generally more sensitive than both methods mentioned above (Figure 10C). However, for the detection of some proteins such as haptoglobin, the copper(II)-ARS complexlabeled CL imaging was more sensitive. Additionally, the resolution of silver staining was relatively low, especially the first few lanes. The intensities of protein bands in the X-ray film of Figure 10B were analyzed by the Gel Documentation and Analysis System (UVP-EC3). Taking an average HSA concentration as 40 mg/mL in healthy adults’ serum, the calibration curve is shown in Figure 11. The linear dynamic range of HSA in copper(II)-ARS complex-labeled CL imaging was 1.5-120 µg (R2 ) 0.992), and the RSD value 8.7% is based on n ) 4. 5080

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Figure 11. Calibration curve of HSA in copper(II)-ARS complexlabeled CL imaging.

4. Concluding Remarks We have built up a novel CL probe copper(II)-Alizarin Red S complex, which has been successfully applied for the detection of human serum proteins after PAGE. This novel method is capable to detect more proteins than the direct CL imaging method, the mechanism being the metal-ARS complex labeling the proteins and catalyzing the luminol-H2O2 CL system. Comparing the CBB-R250 staining and the direct CL imaging methods, the present complex probe technique demonstrated higher sensitivity. Furthermore, this approach offers rapid, convenient, inexpensive, and noncontaminative features. These special characteristics may contribute to new possibilities for human serum analysis, offering a potential technique to define serum biomarkers for early disease diagnosis. Besides this ARS-copper(II) complex, other metal-dye complex CL labeled probes are likewise expected to be used for detecting human serum proteins after PAGE.

Acknowledgment. The authors gratefully acknowledge the support from the National Nature Science Foundation of China (20675010).

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Copper(II)-ARS Complex as Efficient Chemiluminescent Probe

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PR800365N

Journal of Proteome Research • Vol. 7, No. 12, 2008 5081