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A new method, based on the chloroauric acid-enhanced luminol chemiluminescence, is established for the chemiluminescent imaging detection of protein b...
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A Novel Probe Au(III) for Chemiluminescent Image Detection of Protein Blots on Nitrocellulose Membranes Jia Liu,† Xia Liu,† Willy R. G. Baeyens,‡ Joris R. Delanghe,§ and Jin Ouyang*,† College of Chemistry, Beijing Normal University, Beijing 100875, P. R. China, Department of Pharmaceutical Analysis, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, B-9000 Ghent, Belgium, and Department of Clinical Chemistry, Microbiology and Immunology, Ghent University Hospital, De Pintelaan 185, B-9000 Ghent, Belgium Received September 24, 2007

A new method, based on the chloroauric acid-enhanced luminol chemiluminescence, is established for the chemiluminescent imaging detection of protein blots on nitrocellulose membranes. After transferring to the nitrocellulose (NC) membranes, various proteins in human serum can be easily detected using this method. Simplicity and wide applicability are achieved, without the need of expensive antibodies or tedious immunoassay procedures. Furthermore, neither noxious materials nor radioactive pollution is produced. The successful detection of proteins is due to the binding of Au(III) to the protein blots and the chemiluminescent character of the enhanced luminol signal. As a novel chemiluminescent detection method, it offers significant biological analytical potentials in biochemistry and in molecular biology. Keywords: Chemiluminescent imaging • chloroauric acid • polyacrylamide gel electrophoresis • Western Blotting • human serum proteins

1. Introduction Polyacrylamide gel electrophoresis has been widely applied to the study of proteins in biological samples.1–6 After separation, proteins can be detected directly in polyacrylamide gels or after electroblotting onto membranes. Many bioanalytical assays shifted from the use of radioactive labels to nonisotopic detection systems. The hazards of working with radioactivity as well as the limited shelf life of the labels have motivated this change. Popular alternatives to radioactive assays are colorimetric, chemiluminescence (CL), or fluorescence detection.7 Proteins can be detected using organic dyes (Ponceau Red, Amido Black, Fast Green, Coomassie Brilliant Blue), metalbased stains (colloidal gold, silver stain), or by fluorescent labeling techniques (dansyl chloride, coumarine, fluorescamine, and derivatives). The commonly used organic dye is Coomassie Brilliant Blue (CBB), but this offers considerable less sensitivity than what is offered by colloidal gold or silver staining, which have been developed the past decade.7–10 Although these offer high sensitivity, it should be stated that colloidal metals consistently interfere with protein sequence analysis by Edman degradation as demonstrated by poor initial and repetitive sequencing yields. Since colloidal metals irreversibly stain proteins, subsequent evaluation by immunoblotting is neither feasible.11 Silver staining is considered a most complex protein detection process, depending on water quality * To whom correspondence should be addressed. Prof. Dr. Jin Ouyang, College of Chemistry, No. 44, Beijing Normal University, Beijing 100875, P. R. China. E-mail: mailto:[email protected]. Fax: 86-10-62799838. † Beijing Normal University. ‡ Ghent University. § Ghent University Hospital.

1884 Journal of Proteome Research 2008, 7, 1884–1890 Published on Web 03/26/2008

and requiring freshly prepared solutions and rigidly controlled reactions.12 Conventional fluorescent labels require modification of free amino, carboxyl, or sulfhydryl groups on proteins, often rendering them unsuitable for sequencing and mass spectrometry.7 CL was introduced recently in the area of biochemical research. One of the most attractive features of CL methods is their inherent sensitivity arising from the fact that the excited species is formed in the course of a specific chemical reaction. Thus, despite the relatively low quantum yields of CL reactions, CL-based methods provide lower detection limits than conventional fluorometry whose sensitivity is limited by the high background originating from the excitation light. Most proteins cannot produce a CL reaction, to the best of our knowledge. Therefore, to enhance detection, most analytical procedures rely upon derivatization where an appropriate probe is attached to the protein of interest. Appropriate probes can be found that exhibit CL properties.13 However, it has been reported that it is difficult to find CL probes which can undergo electrophoretic separation and that allow direct CL detection in a gel.14 Many efforts have been devoted to investigate a probe to detect proteins which cannot emit CL. Sensitivity of CL immunoanalysis has greatly improved by the CL reaction of luminol and peroxide probed by horseradish peroxidase.15 Previous studies carried out in our laboratories have indicated that direct CL imaging detection of serum proteins on nondenaturing one-dimensional polyacrylamide gel electrophoresis (1-D PAGE) can offer fast, convenient and inexpensive procedures.16–19 Proteins such as hemoglobin (Hb)18–21 and haptoglobin (Hp) with different phenotypes17 have been welldetected. However, these mentioned methods cannot be used 10.1021/pr700616u CCC: $40.75

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in denaturing electrophoresis and most proteins in serum cannot generate CL emission under the employed conditions. The ability to detect different proteins at extensive ranges may be limited. Therefore, CL probes for wide applicability are desirable. Gold, in the form of chloroauric(III) acid has been reported to greatly enhance the luminol-H2O2 CL signal.22–24 Moreover, various research work made clear that Au(III) can bind to different proteins.25–27 Thus, Au(III) may be employed as a catalyst in the CL imaging detection reaction. In this paper, we have developed a new CL probe, Au(III), based on its catalytic activity and binding ability. This metal complex is suitable for detection of proteins immobilized on membrane supports such as nitrocellulose membranes. There are several advantages to working with a membrane instead of the original gel: small amounts of samples are readily detected because they are easily accessible to probes and concentrated on the surface of the membrane, rather than spread throughout the thickness of the gel; a membrane is easier to handle than a gel. Proteins immobilized on solid phase supports are bound to the chloroaurate(III) ion through incubation in chloroauric acid. CL imaging detection of proteins on nitrocellulose membranes after PAGE or two-dimensional electrophoresis (2-DE) is achieved significantly; the protein detection after SDS-PAGE is also possible. On the basis of the obtained results, a simple model of CL enhancement with Au(III) assistance is proposed, enabling the detection of proteins, which cannot produce CL reactions.

Figure 1. Comparison of CL imaging with and without Au(III) as a probe. (A) Au(III)-enhanced CL imaging detection of the three major proteins on NC membrane. (B) Direct CL imaging detection after PAGE. Scheme 1. Schematic AuCl4– Binding to Proteins

Scheme 2. Possible Mechanism for the Luminol-H2O2-Au(III) CL System

2. Materials and Methods 2.1. Instruments. The electrophoresis system consisted of a DYY-6B and DYY-6C electrophoresis instrument, DYCZ-21 and DYCZ-24D vertical electrophoresis (Liuyi Instrument Factory, Beijing, China). The electrophoretic transfer system consisted of a DYY-6B electrophoresis instrument and a DYYIII 40B vertical transfer tank with built-in heat exchanger, electrode panels and gel cassettes. The developed X-ray films were scanned with a MICROTECK S700 scanner (MICROTECK, China). 2.2. Reagents. All reagents were of A.R. grade. Servalty carrier ampholytes (pH 4–6) were from Serva Feinbiochemica (Heidelberg/New York). Tris-hydroxymethyl aminomethane (Tris) and aminoacetic acid (glycine) were purchased from Sino-American Biotechnology Co. (Beijing, China); N,N-methylenebisacrylamide (BIS), H3PO4, NaOH, agar, H2O2 and trisodium citrate were obtained from Beijing Chemical Factory (Beijing, China); 3,3′,5,5′-tetrabromophenolsulfonphthalein (Bromophenol Blue) and acrylamide were purchased from Beijing DingGuo Biological Research Institute (Beijing, China). DTT, Tween 20 and tetramethylethylenediamine (TEMED) were obtained from Sigma (St. Louis, MO). 3-Aminophthalic hydrazide (luminol) was obtained from Acros Organics (NJ). CBBR250 was from Fluka (Switzerland). Human serum albumin (HSA) was obtained from Serva Feinbiochemica (Heidelberg/ New York); transferrin (Tf) and immunoglobin G (IgG) were purchased from Sigma (St. Louis, MO). Molecular-weight markers for proteins were purchased from Amersham Pharmacia Biotech. The protein mixture 250 µg/vial contains the following proteins: thyroglobulin, 76 µg, 669 kDa; ferritin, 50 µg, 440 kDa; catalase, 36 µg, 232 kDa; lactate dehydrogenase, 48 µg, 140 kDa; albumin, 40 µg, 66 kDa. NC membranes were purchased from Liuyi Instrument Factory (Beijing, China). HAuCl4 · 4H2O was obtained from Shanghai Reagent (Shanghai,

China). Purified water was prepared by passing house-distilled water through a Millipore Simplicity 185 water purification system. Fresh blood samples of healthy subjects were obtained from the affiliated hospital of the Beijing Normal University. After careful settlement for 1 h, the supernatant was extracted as serum with a mini-sample collector, and then centrifuged at 2500 rpm three times (each time 10 min). Sera were stored at -20 °C for 60 days. 2.3. One-Dimensional Electrophoresis Procedures. The nondenaturing one-dimensional electrophoresis was performed in a vertical discontinuous gel system, consisting of separating (7.5%, m/v) and stacking (4.0%, m/v) gels. Gel stock solution (30%, m/v): 29.2 g of acrylamide and 0.8 g of Bis were dissolved in 100 mL of distilled water, then filtrated. The separating gel solution was prepared by mixing 4.0 mL of gel stock solution, 4.0 mL of Tris-HCl (1.5 M, pH 8.80), 150 µL of (NH4)2S2O8 (10%, w/v), and 15 µL of TEMED, then diluting the mixture to 16.0 mL. For the stacking gel preparation, 1.33 mL of stock gel–solution was mixed with 2.5 mL of Tris-HCl (0.5 M, pH 6.80), 50 Journal of Proteome Research • Vol. 7, No. 5, 2008 1885

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Figure 2. Effect of luminol pH on CL intensity. (A) Luminol pH 6.8, then pH 13.0. (B) Luminol pH 8.3, then 13.0. (C) Luminol pH 9.3, then 13.0.

µL of (NH4)2S2O8 (10%, w/v), and 10 µL of TEMED, then the mixture was diluted with water to 10.0 mL. The solution of (NH4)2S2O8 was prepared daily to ensure the stability. Thirty microliters of serum was dissolved in 60 µL of Bromophenol Blue (0.02%, w/v), 60 µL of glycerol (20%, v/v) and 150 µL of H2O. The loading volume was 15 µL. The electrophoresis buffer was prepared by dissolving 3.0 g of Tris and 14.4 g of glycine in water, and adjusting the pH to 8.3. The voltage in the stacking gel was 130 V. When the sample entered the separating gel, the voltage was decresed to 100 V and continued until the Bromophenol Blue indicator moved to the bottom of the gels. SDS polyacrylamide gel electrophoresis was performed as described by Laemmi.28 The 4% stacking gel was overlaid on the 10% separating gel. Human serum was diluted 10 times with a buffer containing 60 mM Tris, pH 6.8, 25% glycerol, 2% SDS, 0.015 g/mL DTT and 0.1% Bromophenol Blue. The electrophoresis buffer for SDS gel consisted of 25 mM Tris, 192 mM glycine, and 0.1% SDS. The voltage in the stacking gels was of 130 V. When the sample entered the separation gels, the voltage was decreased to 110 V and kept constant for about 2 h. 2.4. Two-Dimensional Electrophoresis Procedures. Proteins were subjected to IEF in the absence of denaturants employing column gels (1 × 70 mm). Sample buffer for IEF consisted of 60 µL of Bromophenol Blue (0.02%, w/v), 60 µL of glycerol (20%, v/v) and 120 µL of H2O. The IEF gel solution was prepared with 0.6 mL of gel stock solution, 0.18 mL of carrier ampholytes, 15 µL of (NH4)2S2O8 (10%, m/v) and 3 µL of TEMED, then diluted with distilled water to 4.0 mL. The cathode electrode buffer in IEF was 0.1 M NaOH, and the anode was 0.03 M H3PO4. IEF was run at 200 V constant voltage for 30 min and continued at 400 V constant voltage for 15 h. After IEF, the focusing gels were transferred onto the seconddimension slab gels. The concentration for the second-dimension gel was 7.5% (w/v); it was prepared by mixing 4.0 mL of gel stock solution, 4.0 mL of Tris-HCl (1.5 M, pH 8.8), 150 µL of (NH4)2S2O8 (10%, m/v), 15 µL of TEMED and 8 mL of distilled water. Electrophoresis for the second-dimension gel was run at 125 V constant voltage. It was continued until the Bromophenol Blue indicator moved to the bottom of the slab gels (about 2.5 h). 2.5. Transfer of Proteins from Gel to Membrane. After separation of the protein sample on a polyacrylamide gel, the proteins are transferred to a membrane in a pattern replicating the separation seen on the gel. The NC membranes were immerged in the transfer buffer for at least 2 h before electrophoretic transfer. The gel and the membrane were mounted in a cassette and suspended vertically in a buffer1886

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Figure 3. Effect of different washing reagents on CL intensity; (A) 1000 mL of cool distilled water; (B) 1000 mL of 37 °C distilled water; (C) 500 mL of 0.012 M HCl and 500 mL of distilled water.

Figure 4. Comparison of Au(III)-enhanced CL detection, CBB-R250 staining and direct CL detection of human serum after nondenaturing 1-D PAGE. (A) Au(III)-enhanced CL imaging detection on NC membrane. (B) CBB-R250 staining. (C) Direct CL detection (Hp 2-2).

filled tank between electrode panels. Tris-glycine buffer was used (containing up to 20% methanol for SDS gel). The electrodes were parallel to the plane of the gel. The transfer was run at 300 mA and kept constant for about 2.5 h. 2.6. Procedure of Colloidal Gold Staining on NC Membrane. Colloidal gold was previously synthesized according to the procedure described by Grabar et al.29 After transfer, the NC membrane was blocked 30 min in PBS with 0.3% Tween 20 at 37 °C and washed three times 5 min in PBS with 0.3% Tween 20 at room temperature. After this, the membrane was washed three times 5 min in Milli-Q water. The membrane was incubated in gold colloidal solution which was prepared as described by Moeremans30 for 5–9 h and washed three times for 2 min in Milli-Q water. 2.7. Procedure of CL Detection on NC Membrane with Au(III) as a Probe. After transfer, the NC membrane was washed with distilled water, about 500 mL. Subsequently, it was incubated in HAuCl4 solution (0.01%, w/v). Then it was washed extensively with distilled water or with 0.012 M HCl. The washing reagent was optimized in Results and Discussion. The rinsed blot was spread on an even glass in a dark room. Hydrogen peroxide solution (1.2%, v/v) was blown onto the gel for 20 s, and luminol (1.0 × 10-3 M, pH ∼ 8.3) was blown onto the gel for 40 s; then pH ∼ 13.0 luminol (1.0 × 10-3 M) was insufflated onto the membrane for 20 s. Next the gel was quickly drained with filter paper, then covered with a sheet of

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Figure 5. Comparison of Au(III)-enhanced CL detection and CBB-R250 staining of human serum after native 2-D PAGE. (A) Au(III)enhanced CL detection on NC membrane. (B) CBB-R250 staining.

transparency film. Immediately, 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 films were scanned with a scanner and the results transferred to the computer. 2.8. Direct CL Imaging Detection in Gel. After removal from the electrophoresis mold, the polyacrylamide gel was washed with distilled water three times to avoid the interference of rudimental electrophoresis buffer. Then it was spread on an even glass in a dark room. Hydrogen peroxide solution (1.2%, v/v) was blown onto the gel for 20 s, and luminol (1.0 × 10-3 M, pH ∼ 13) was blown onto the gel for 1 min. Next, the gel was quickly drained with filter paper, then covered with a sheet of transparency film. Immediately after, 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 films were scanned with a scanner and the results transferred to the computer. When the traditional staining detection was used, after removal of the electrophoresis mold and washing, the gel was stained in CBB-R250 (0.1 g/100 mL) solution (VH2O/VCH3OH/VHAc ) 5:5:2) for 2 h. It was washed and submerged into methanol-acetic acid solution (VH2O/VCH3OH/VHAc ) 70:100:830) to destain for 10 h. The gel was scanned and the results transferred to the computer.

3. Results and Discussion 3.1. Comparison of CL Imaging with and without Au(III) as a Probe. To identify whether the Au(III) can be used as CL probe for protein detection, experiments were carried out by using HSA, Tf and IgG as protein models. It is well-known that these three proteins are major proteins in human serum, which cannot emit CL without a probe. After 1-D electrophoresis, these proteins were electrophoretically transferred to the NC membrane, through incubation in HAuCl4 solution; the NC membrane was detected by CL imaging. The results are illustrated in Figure 1A. It can be observed that the three major proteins can all be easily detected on NC membrane applying the CL imaging method. When using the direct CL imaging without Au(III) in the detection of these three proteins after 1-D electrophoresis, no signals can be obtained (Figure 1B). Because the reaction of luminol with H2O2 in alkaline solution in the absence of a catalyst produces only weak CL, it is assumed that the catalyst Au(III), which binds to proteins, may interact with the reactants or the intermediates of the reaction. The possible mechanisms are shown in Schemes 1 and 2.22,25 To analyze the different CL results, with and without Au(III) as a probe in the present study, and based on the literature reports on the CL characteristic of gold as mentioned above, it could be easily deduced that the only species responsible

Figure 6. Comparison of direct CL detection, Au(III)-enhanced CL detection and CBB-R250 staining of human serum after SDS PAGE. (A) Direct CL detection. (B) Au(III)-enhanced CL imaging detection on NC membrane. (C) CBB-R250 staining.

for the CL emission is Au(III) that hence can be used as CL probe in protein detection. 3.2. Optimization of the CL Imaging Conditions in Protein Detection. To obtain the optimized Au(III) CL imaging conditions, experiments were carried out by varying the pH of the luminol solution. Since the pH of the CL reagent has a substantial effect on CL performance,31,32 these experiments were of great importance. The pH values of the luminol solutions were adjusted to 6.8, 8.3, 9.3 and 13.0, respectively. According to our previous reports on direct CL imaging methods,16–19 a pH 13.0 solution was primarily applied to this experiment; however, the CL intensity reached the maximum value rapidly, then quickly decreased to the background; it decreased too fast so that the CL signals could not be recorded. Therefore, the pH was varied to test the optimized pH of luminol that can be suitable for an imaging assay. The pH of luminol was adjusted to 6.8, 8.3, 9.3, and 13.0. When using a pH of 6.8 for luminol, the CL signals were too weak and could hardly be imaged on the X-ray film. The situation with pH 8.3 and pH 9.3 solutions was somewhat better. The CL signals could be imaged; however, the required imaging time was relatively long (about 5 min) and the CL signals were not still strong enough. When the low pH solution was used followed by spraying the pH 13.0 luminol solution, the CL intensity was greatly enhanced compared to that when only spraying with low pH luminol onto the NC membrane; the CL signals can be easily imaged and only 5∼10 s are used for exposing. The results are shown in Figure 2. It can be observed that a proper luminol pH value of pH 8.3 and then pH 13.0 was chosen to obtain relatively higher CL light to be collected and lower background to be achieved under these conditions. Journal of Proteome Research • Vol. 7, No. 5, 2008 1887

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Figure 7. Comparison of the sensitivities of Au(III)-enhanced CL detection, colloidal gold staining and CBB-R250 staining using HSA, Tf and IgG. (A) Au(III)-enhanced CL detection; (B) colloidal gold staining; (C) CBB-R250 staining. Dilution ratio of the three proteins: (1) 1, (2) 1/2, (3) 1/4, (4) 1/8, (5) 1/16, (6) 1/32, (7) 1/64, (8) 1/128, (9) 1/256, (10) 1/512.

that the background decreased; the protein imaging was much clearer than in the former results. It can be concluded that the HCl was the most suitable washing reagent to remove the adsorbed Au(III) on NC membranes. It is presumed that the unbound AuCl4- on blank NC membrane can be more easily dissolved in Cl- solutions than in water; on the other hand, the low pH may accelerate the removal of AuCl4-.

Figure 8. Comparison of direct CL detection, CBB-R250 staining and Au(III)-enhanced CL detection of protein markers for nondenaturing gel after 1-D PAGE. (A) Direct CL imaging detection. (B) CBB-R250 staining. (C) Au(III)-enhanced CL.

3.3. Optimization of the Washing Reagent. During the procedure of labeling Au(III), when the NC membranes were incubated in 0.01% HAuCl4 solution, not only can the Au(III) bind to protein blots, but also the extra Au(III) could be adsorbed onto blank NC membrane. It is obvious that the nonspecific adsorptions induce high backgrounds and seriously interfere with the detection of the proteins. A thorough removal of the unbound reagents is required after the incubation in the procedure. This is done by performing series of washes. Hence, an appropriate washing reagent is to be selected. Three reagents were tested to wash the Au(III)-labeled NC membrane. The results are illustrated in Figure 3: it can be observed that the high background seriously interferes with the protein CL signals when using 1000 mL of cool distilled water in Figure 3A. When using 1000 mL of 37 °C water as the washing reagent, the background slightly decreased, as shown in Figure 3B. In Figure 3C, 500 mL of 0.12 M HCl was used as the washing reagent and then 500 mL of distilled water: it can be observed 1888

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Furthermore, to optimize the amount of washing reagent, an experiment was achieved using different amounts of HCl. Three different amounts (6 mL of HCl per cm2 NC, 12 mL of HCl per cm2 NC, 24 mL of HCl per cm2 NC) were tested. The results demonstrated that 12 mL of HCl per cm2 NC is appropriate. Less HCl amounts may induce higher backgrounds and more HCl would also remove the Au(III) bound to proteins. Therefore, it can be concluded that in the washing procedure, for an NC membrane, first using 500 mL of 0.012 M HCl (i.e., 12 mL of HCl per cm2 NC), then 500 mL of distilled water is appropriate to remove the Au(III) adsorbed on the blank NC membrane and to avoid the interference with protein detection. 3.4. Washing Step before the Procedure of Labeling AuIon. To identify whether the washing step with distilled water before labeling Au(III) is necessary, experiments were carried out with and without washing before labeling Au(III). After electrophoretic transfer, the NC membrane was directly impregnated with HAuCl4 solution without washing; it could be observed that the protein blots on the NC membrane are easily detectable by the Au(III)-enhanced direct CL imaging method. Moreover, when the NC membrane was washed with distilled water before labeling Au(III), the result was almost the same. It is presumed that the rudimental transfer buffer does not interfere with the procedure of incubation in HAuCl4 solution. 3.5. Application of CL Detection of Serum Samples with and without Au(III) as a Probe. To identify whether the Au(III)enhanced CL imaging detection can be used for complex samples, human serum, which was separated by 1-D PAGE and

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Au(III) for Chemiluminescent Image Detection transferred to the NC membrane, was detected by enhanced direct CL imaging and Au(III)-labeled CL imaging, separately. The results are shown in Figure 4. From Figure 4A,B, it can be observed that the 1-D maps obtained from the proposed CL imaging protocol are closer to the one from the CBB-R250 staining, but only Hp emitted CL light and several bands could be detected when using an optimized CL detection method as clarified in ref 16 (Figure 4C), where a large amount of proteins in human serum cannot emit CL signals. For Au(III)-enhanced CL imaging detection, an NC membrane was incubated in HAuCl4 solution, Au-ions bind to the proteins during this step. Through capturing the CL emission of AuCl4-, we can detect the bands of the proteins indirectly. In other words, whether the protein can generate CL emission or not, it still can be detected. The results of the experiments have proved their feasibility. Figure 5 demonstrates the ability of CL imaging method to detect a wide range of proteins in 2-D polyacrylamide gels. The CBB-R250 staining procedure is also carried out for comparison. This illustrates that the CL imaging method readily visualizes a wide range of proteins in 2-D gels. 3.6. Application in Denaturing Condition. SDS-PAGE is one of the most common variants of PAGE and is used to resolve proteins in biological samples according to their sizes. Since it was introduced by Shapiro et al.,33 it has been widely used in the estimation of protein size and purity.34 However, the direct CL imaging method failed to detect proteins in SDS gel. We attempted to spray luminol and hydrogen peroxide solution to the gel without Au(III) after SDS-PAGE, but the proteins did not generate any CL signals (as shown in Figure 6A). The reason may be that the complex of Hp-Hb which can generate CL emission in native PAGE was broken up during the procedure of denaturation in SDS-PAGE.35 However, when Au(III) was used as a probe to detect human serum proteins which had been separated by SDS-PAGE and transferred to NC membrane, a large amount of proteins can generate CL emission easily, the results are shown in Figure 6B. The Au(III)-enhanced CL imaging map matched the one of CBB-R250 staining (Figure 6C). The result revealed that the Au(III)-enhanced CL imaging detection could be successfully applied to SDS-PAGE. 3.7. Analytical Performance. To obtain the detection sensitivity, experiments were carried out using a mixture of the three proteins (HSA, Tf and IgG) with an original amount of 30 µg each. The mixture was 2-fold serially diluted to 1/512. The results are shown in Figure 7. It can be observed that the sensitivities were roughly equivalent for both detection methods: the Au(III)-enhanced CL detection and the colloidal gold staining (Figure 7A,B). However, colloidal gold staining required Tween 20 for stabilization of the colloidal particles. Nonionic surfactants partially remove proteins from transfer membranes resulting in lower overall yields of material for later characterization.36,37 In addition, colloidal gold interferes with protein subsequent evaluation by immunoblotting.11 We also evaluated the sensitivity of CBB-R250 staining which was less sensitive than Au(III)-enhanced CL detection (see Figure 7C). Moreover, the procedures of CL imaging did not need organic solvents as in the traditional CBB-R250 method; hence, the former did not produce environmental pollution. When comparing with the direct CL imaging method, it can be easily observed that only proteins containing heme-group or metallo-proteins can be detected by CL imaging without a probe such as ferritin and catalase (Figure 8A); the usefulness of this technique to detect different proteins at extensive ranges may be limited. When using the Au(III)-enhanced CL imaging

method, it can be observed that proteins which can be stained by the CBB-R250 staining method (Figure 8B) can also be detected by this proposed method (Figure 8C). It can be concluded that Au(III) can be applied as a CL probe for the detection of various proteins in complex samples.

4. Concluding Remarks The present study demonstrated Au(III)-enhanced CL imaging detection combined with PAGE on an NC membrane was an effective means to detect various proteins. Simplicity and wide applicability were achieved, without the need of expensive antibodies or immunoassay procedures. Notably, this was successfully applied to the detection of various proteins, which cannot yield CL signals. The successful detection of proteins is due to the binding of gold to the protein blots and the chemiluminescent character of the former. As a novel chemiluminescent detection method, it shows significant biological analytical potentials in biochemistry and in molecular biology.

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