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Clinical Chemistry 54: 116-123, 2008. First published November 12, 2007; 10.1373/clinchem.2007.091801
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Right arrow Automation and Analytical Techniques
(Clinical Chemistry. 2008;54:116-123.)
© 2008 American Association for Clinical Chemistry, Inc.

Automation and Analytical Techniques

Nanogold Catalysis–Based Immunoresonance-Scattering Spectral Assay for Trace Complement Component 3

Zhiliang Jiang1,2,a, Wenxing Huang2, Jiangping Li1, Mingshun Li2, Aihui Liang1, Shengsen Zhang2 and Bing Chen2

1 Key Laboratory of New Processing Technology for Nonferrous Metals and Materials of Education Ministry, Department of Material and Chemical Engineering, Guilin University of Technology, Guilin, China, 2 School of Environment and Resource, Guangxi Normal University, Guilin, China.

aAddress correspondence to this author at: Zhiliang Jiang, School of Environment and Resource, Guangxi Normal University, Guilin 541004, China. Fax +86-0773-5846201; e-mail zljiang{at}mailbox.gxnu.edu.cn or zljiang{at}glite.edu.cn.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Background: Complement component 3 (C3) is an essential bridge linking innate immunity and adaptive immunity. We describe an immunonanogold catalytic resonance-scattering (RS) technique for assaying C3 in serum.

Methods: We used nanogold to label goat antihuman C3 antibody to obtain an immunonanogold RS probe for C3. The immune reaction between nanogold-labeled antibodies and antigens was carried out in Na2HPO4–sodium citrate buffer, pH 5.6, containing polyethylene glycol. After centrifuging the particle suspension, we used RS to monitor the catalytic effect of nanogold-labeled anti-C3 in the supernatant on the chlorauric acid–hydroxylamine (HAuCl4–NH2OH) particle reaction and used electron microscopy to monitor particle shape. We assayed 36 human serum samples with the immunonanogold catalytic RS assay and immunoturbidimetry.

Results: Nanogold-labeled anti-C3 had a marked catalytic effect on the reaction of HAuCl4 and NH2OH to form particles, which exhibit a maximum RS peak at 585 nm. The decrease in RS intensity, {Delta}IRS, of the nanocatalytic system was proportional to C3 concentration from 5.0 to 160.0 ng/L. The detection limit for the C3 assay was 1.52 ng/L. Results obtained with serum samples agreed with those obtained with an immunoturbidimetric method. A linear regression analysis of 28 nonpathologic serum samples revealed a correlation coefficient of 0.960, with mean (SD) slope and intercept values of 0.787 (0.0218) g/L and 0.28 (0.026) g/L C3, respectively.

Conclusion: The immunonanogold catalytic RS assay showed high sensitivity and good selectivity for measuring C3 in human serum. This method may become useful for diagnosing certain diseases, such as hepatitis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
The 3rd component of complement, C3,1 is mainly synthesized by macrophages and the liver (1). It is an essential bridge linking innate immunity and adaptive immunity and is an indicator of inflammation (2). C3 deficiency is associated with such abnormalities as nephritis, hepatitis, liver cirrhosis, and acute inflammation (3).

Various immunoassays, including immunonephelometry, radial immunodiffusion, RIA, ELISA, and solid-substrate room-temperature phosphorescence immunoassay, have been used to measure C3 (4)(5)(6); each technique has its own advantages and disadvantages (7). In recent years, gold nanoparticles, with their inherent advantages of easy preparation, high electron density, good biocompatibility, and novel optical properties (8), have been used in biochips, biosensors, and biomacromolecule labeling (9)(10). Gold nanoparticles have also been used for tag amplification (11)(12). Holgate et al. (13) and Danscher and Norgaard (11) first established the silver-enhancement process, in which gold nanoparticles promote the reduction of silver ions and their deposition on the surface of gold particles, thereby enlarging the gold particles. This approach has successfully been used for immunogold silver staining (or "silver enhancement") techniques in histochemistry, cytochemistry, and antibody or antigen detection (14)(15). Lower limits of detection have been reported (in the ng/L range) (16), much lower than C3 concentrations measured with radioactive, fluorescence, and enzyme/colorimetric methods (17). The silver-enhancement technique, however, is sensitive to pH, natural light, and chloride ion (18). A recently proposed gold-enhancement approach (19), which is similar to silver enhancement, has resolved these problems. Ma and Sui (20) used the gold-enhancement technique to enlarge immunogold particles immobilized on nitrocellulose strips and were able to detect 10 ng/L human IgG by visual means. Su (21) described a gold-enhancement assay for human IgG that has an ultraviolet–visible spectral range and a 0.1-ng/L lower detection limit; however, this gold-enhancement technique required the use of an antibody fixed to a solid support.

The resonance-scattering (RS) technique, with its advantages of simplicity and sensitivity, has been successfully used to measure trace concentrations of inorganic and organic compounds (22)(23). To improve the selectivity, we have combined immunoreaction and nanogold-labeled immunoreaction with RS for the assay of trace amounts of protein (24). To further improve the sensitivity of this approach, we propose an ultrasensitive, selective, and simple assay for C3 that couples the catalytic effect of immunonanogold, the nanogold-labeling technique, and the RS effect of the gold particles.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
reagents and apparatus
Chlorauric acid (HAuCl4 · 4H2O) was obtained from National Pharmaceutical Group Chemical Reagents Company, China. Gold nanoparticles approximately 10 nm in diameter were prepared by an improved sodium citrate procedure (25). C3 (3400 mg/L) and goat antihuman C3 antibody (1000 mg/L; production no. 05080101) were purchased from Jiemen Biological Technology Cooperation Company. Human sera were supplied by the No. 5 Hospital of Guilin. We prepared McIlvaine buffer solutions (Na2HPO4–citric acid) between pH 2.2 and pH 8.0 with solutions of 0.20 mol/L Na2HPO4 and 0.10 mol/L citric acid and prepared Sorensen buffer solutions (HCl–sodium citrate) between pH 1.17 and pH 4.8 with 0.10 mol/L HCl and 0.10 mol/L sodium citrate. We prepared fresh solutions of 200.0 mg/L HAuCl4 with HAuCl4 · 4H2O and 556.0 mg/L hydroxylamine hydrochloride (NH2OH · HCl). We also prepared 100 g/L KCl, 0.20 mol/L K2CO3, and 300-g/L solutions of polyethylene glycol (PEG)-6000, PEG-4000, PEG-10000, and PEG-20000. All reagents were of analytical grade, and water was doubly distilled.

We used a model RF-540 spectrofluorometer (Shimadzu), a model TU-1901 ultraviolet–visible spectrophotometer (Purkinje General Instrument Limited Company), a model 79–1 magnetic heat agitator (Zhongda Instrumental Plant), a model H-600 transmission electron microscope (Electronic Stock Limited Company), a model JSM-6380LV scanning electron microscope (Electronic Stock Limited Company), a model SK8200LH ultrasonic reactor (ultrasonic energy, 450 W; ultrasonic frequency, 59 kHz; Kedao Ultrasonic Instrument Limited Company), and a model YGL-16G centrifuge (Anting Science Instrumental Plant).

preparation of immunonanogold probe for C3
Influence of pH on gold labeling of anti-C3.
The combination of nanogold with protein was accomplished via hydrophobic interaction, van der Waals, and intermolecular forces, and the successful combination of these forces depends on the pH. We tested the effect of varying pH on the RS intensity of gold-labeling anti-C3. We transferred 1.0 mL nanogold solution into each calibration test tube, varied the pH from 4.0 to 8.5 by adding 0.2 mol/L K2CO3 or 0.1 mol/L HCl, and then added 30.0 µL anti-C3 antibody (1000 mg/L) into each test tube. After incubating the tubes for 5 min at room temperature, we pipetted 0.10 mL 100 g/L KCl into each test tube. After 10 min, we diluted each tube to 3.0 mL with water and recorded the RS intensity at 580 nm (I580 nm) on the spectrofluorometer. When the pH was <6.5, the anti-C3 antibody could not stabilize nanogold; consequently, the I580 nm was enhanced because the gold nanoparticles had aggregated. When the pH was 6.5–7.5, the I580 nm remained approximately constant. We interpreted this observation as indicating that anti-C3 antibody coating of the gold nanoparticles had prevented KCl from aggregating the nanoparticles. A nanogold solution pH of 7.5 was thus considered suitable for the experiment.

Selection of the anti-C3 amount.
Different amounts of anti-C3 antibody (2.5–30.0 µg) were added to 1.0 mL of nanogold solution, pH 7.5. After 5 min, we added 0.10 mL of 100 g/L KCl solution, mixed the tube well, diluted the solution to 3.0 mL 10 min later, and measured the I580 nm. The results indicated that 20.0 µg anti-C3 was the minimum for stabilizing a 1.0-mL nanogold solution. This anti-C3 amount was chosen for labeling 1.0 mL of nanogold solution.

Preparation of nanogold-labeled anti-C3.
We added 0.10 mL of 300 g/L PEG-20000 (300 mL/L) to 100 mL of nanogold solution as a stabilizer and adjusted the pH to 7.5. While stirring the solution with a magnetic stirrer at a low speed, we slowly added 1.0 mg anti-C3 antibody to the nanogold solution and continued stirring for 15 min. The solution containing 57.9 mg/L nanogold was stored at 4 °C.

optimized C3 assay procedure
To an assay tube we added 0.20 mL Na2HPO4–citric acid buffer (pH 5.6), 0.50 mL of 57.9 mg/L (calculated by the gold concentration) immunonanogold-labeled anti-C3, a volume of C3 or 10 µL of sample that had been diluted 40-fold with Na2HPO4–citric acid buffer (pH 5.6), and 0.60 mL of 300 g/L PEG-6000. We then diluted the tube to 3.0 mL with water, mixed it well, and incubated the tube in an ultrasonic reactor for 15 min at 28 °C. The solution was then centrifuged at 14 650g for 10 min to separate the immunogold complex. The supernatant was removed and stored in a refrigerator at 4 °C until use.

To 10-mL graduated test tubes we successively added 0.20 mL HCl–sodium citrate buffer (pH 2.97), 3.0 µL supernatant, 0.80 mL of 200.0 mg/L HAuCl4, and 0.40 mL of 556.0 mg/L NH2OH · HCl, diluted the solution to 3.0 mL, and placed the tubes in a bath at 37 °C for 3 min. Suitable volumes of the prepared solutions were then transferred into a quartz cell. We chose a low sensitivity setting on the detector and a longitudinal-coordinate scale of 4 and recorded the RS spectrum of the system by synchronously scanning the excitation wavelength and emission wavelength (excitation wavelength = emission wavelength). We then recorded the RS intensity at 585 nm (I585 nm) and measured the I585 nm of the blank solution without C3 [(I585 nm)b]. We then calculated the decrease in RS intensity ({Delta}IRS) as (I585 nm)b – I585 nm.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
principle
The catalytic properties of nanoparticles are influenced by their size, morphology, and composition. We first used the RS technique to study the catalytic effect of several nanocatalysts, such as Au, Pt, Pd, Ag, Fe3O4, TiO2, and ZnO, on the particle reaction between HAuCl4 and NH2OH (Table 1 ). We found no catalytic effect of TiO2 and ZnO nanoparticles on the reaction, and the nanogold system provided the widest linear range (Table 1 ). Furthermore, nanogold preparation is simple, and it has commonly been used as a label for the detection of biorecognition events, such as DNA hybridization or the formation of the antigen–antibody complex, and for the analysis of the catalytic functions of nucleic acids. Thus, nanogold was chosen to prepare the immuno-RS spectral probe used in this study.


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  		Table 1. Relationship between nanocatalyst concentration (c) and decrease in RS intensity ({Delta}IRS).

The immunoreactions between C3 and the immunonanogold probe formed the immunogold complex, for which the enhanced RS intensity could be used to detect C3, but the sensitivity was not satisfactory. In this study, we used the immunonanogold catalytic effect on the particle reaction between HAuCl4 and NH2OH to improve the sensitivity in a new C3 assay. The potential of Auatom/AuI (aq) is known to be –1.5 V vs a normal hydrogen electrode, but the potential of Aumetal/AuI (aq) is +1.68 V vs a normal hydrogen electrode (26). NH2OH was a weak reducing agent under acidic conditions, with a potential of +1.35 V vs a normal hydrogen electrode. According to the surface autocatalytic principle (27), NH2OH cannot reduce HAuCl4. In fact, HAuCl4 was first adsorbed onto the surface of immunonanogold in the supernatant, and HAuCl4 was then catalytically reduced to gold on the seeding catalyst of immunonanogold. Results showed that NH2OH · HCl reduction of HAuCl4 was slow in the absence of nanocatalyst. When nanocatalyst was added, the gold surfaces dramatically accelerated the reaction. Consequently, no new particle formation occurred by nucleation in solution (19); the added HAuCl4 went into the production of larger gold particles (Fig. 1 ), which greatly enhanced RS intensity. The production of the immunonanogold complex increased as the C3 concentration increased, the immunonanogold in the supernatant decreased spontaneously after centrifugation, and the RS intensity of the immunonanogold catalytic system decreased. We established a linear relationship between {Delta}IRS and C3 concentration.


Figure 1
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  		Figure 1. Principle of immunonanogold catalytic enhancement.

The "n" and "m" in the Fig. represent the reaction coefficient of anti-C3 and C3, respectively.

transmission and scanning electron microscopy
When we prepared spherical 10-nm gold nanoparticles with the improved sodium citrate procedure, we found the immunonanogold particles to have good dispersion and to retain their original size (Fig. 2A ); the immunonanogold complex formed and aggregated (Fig. 2B ). According to this procedure, the immunonanogold catalytic system consists of larger gold particles (mean diameter, 220 nm; Fig. 2C ). We characterized the particles in the immunonanogold catalytic system by scanning electron microscopy with an accelerated voltage of 10 kV (Fig. 2D ); the mean diameter was also approximately 220 nm, and no small particles were detected.


Figure 2
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  		Figure 2. Electron microscopy (EM) images of immunonanogold.

Transmission EM (TEM) images of immunonanogold (A) and the immunonanogold complex (B). TEM (C) and scanning EM (D) images of HCl–sodium citrate buffer (pH 2.97) with 3.0 µL of 0.025 mg/L C3 immunonanogold complex supernatant, 53.33 mg/L HAuCl4, and 74.13 mg/L NH2OH · HCl.

ultraviolet absorbance spectra
For gold nanoparticles of 10, 30, 50, and 70 nm, the strongest absorbance peaks were seen at 518 nm, 522 nm, 524 nm, and 539 nm, respectively, a result consistent with the Mie theory of a redshift with increasing diameter. On binding of anti-C3, a modest redshift of approximately 2 nm in the peak of maximum absorbance was found, compared with bare nanogold particles approximately 10 nm in diameter. When immunoreactions between the C3 protein and nanogold-labeled anti-C3 took place, a new absorbance band was recorded at longer wavelengths. The peak of maximum absorbance was centered at 522 nm. This behavior appeared to be due to surface modification, as well as to the electrolyte, which could affect the plasmon band because of a shielding effect and a change in the dielectric constant (28).

An examination of ultraviolet spectra showed that the HAuCl4–NH2OH reaction was very slow in the absence of nanocatalysis, with an absorbance peak at 307 nm owing to HAuCl4. The fact that almost no absorbance values were detected at wavelengths longer than 480 nm demonstrated that no larger immunogold particles were formed. When the immunonanogold particle was increasing in size, we observed a new, wider absorbance peak with a maximum at 789 nm, and the absorbance value increased dramatically. This observation demonstrated that there were larger gold particles in the catalytic system.

rs spectra
The scattering signals of C3, anti-C3, and the anti-C3–C3 immunocomplex systems were weak. The immunonanogold complex system had an RS peak at 580 nm that can be used to assay C3, but the sensitivity at this wavelength is not high. The immunonanogold catalytic system exhibited the strongest RS peak at 585 nm. The peak at 470 nm was because the light source has a maximum emission at this wavelength (Fig. 3 ). With the addition of C3, the immunonanogold in the supernatant decreased, and the I585 nm signal decreased. {Delta}IRS was linearly related to C3 concentration. Therefore, we chose 585 nm as the assay wavelength.


Figure 3
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  		Figure 3. RS spectra of the immunonanogold catalytic systems.

HCl–sodium citrate buffer (pH 2.97) containing 53.33 mg/L HAuCl4, 74.13 mg/L NH2OH · HCl, and 9.65 µg/L nanogold-labeled anti-C3 antibody, plus 0 ng/L C3 (a), 10 ng/L C3 (b), 40 ng/L C3 (c), 80 ng/L C3 (d), or 120 ng/L C3 (e).

optimization of the nanogold-labeled immunoreaction
The results of our experiments to optimize immunoreaction conditions showed that 0.20 mL of Na2HPO4–citric acid buffer (pH 5.6), 9.67 mg/L nanogold-labeled anti-C3 antibody, and an ultrasonic incubation time of 15 min were the best conditions for the system. We also studied the effects of different concentrations of PEG-6000, PEG-4000, PEG-10000, and PEG-20000 on {Delta}IRS and found that the use of 60 g/L PEG-6000 yielded a maximal {Delta}IRS. Our results with these conditions showed that the increased I580 nm for the C3-nanogold-labeled anti-C3 antibody immunoreaction system was proportional to C3 in the range of 0.00833–0.200 mg/L, with a detection limit of 0.0028 mg/L C3, which was calculated according to the following equation:

Formula
where DL is the detection limit, 3 is the factor at the 95% confidence level, Sb is the SD of the blank measurements (n = 20), and K is the slope of the calibration curve.

selection of immunonanogold catalysis conditions
Effect of centrifugation speed and time.
We optimized the factors contributing to the immunonanogold catalytic effect to maximize sensitivity and to minimize assay time. With a fixed centrifugation time of 20 min, {Delta}IRS increased with centrifugation speeds between 10 170g and 14 650g and tended to be stable, whereas {Delta}IRS decreased as the speed was increased between 17 190g and 19 940g. With the centrifugation speed fixed at 14 650g, we then varied the centrifugation time from 5–25 min, and found that {Delta}IRS increased with centrifugation time in the range of 5–10 min and reached a maximum at 10 min. {Delta}IRS decreased gradually as the centrifugation time was prolonged beyond 10 min. We therefore chose 14 650g for 10 min as the centrifugation conditions.

Effect of pH.
In our tests of the influence of pH on {Delta}IRS we found that {Delta}IRS increased in HCl–sodium citrate buffer in the pH range of 1.93–2.97. The results indicated that a pH of 2.97 was optimal, and we chose to use 0.20 mL of this buffer in the assay.

Effect of NH2OH · HCl and HAuCl4 concentrations.
We tested the influence of NH2OH · HCl and HAuCl4 on {Delta}IRS and found an optimal immunogold catalytic reaction depended on the appropriate ratio of NH2OH· HCl and HAuCl4 concentrations. When the NH2OH ·HCl concentration was increased beyond 74.13 mg/L, {Delta}IRS increased slowly, but the blank value increased accordingly. {Delta}IRS was maximal at 74.13 mg/L NH2OH · HCl. Similarly, {Delta}IRS was maximal at 53.33 mg/L HAuCl4.

Effect of reaction temperature and time.
The effect of reaction temperatures between 25 °C and 47 °C on {Delta}IRS was studied. {Delta}IRS increased with increasing temperature to a maximum at 37 °C; higher temperatures yield higher blank values. We selected a reaction temperature of 37 °C. We also tested the effect of reaction time at 28 °C (room temperature) and at 37 °C. At times under 7 min, the growth of immunonanogold particles was slower at room temperature than at 37 °C. At 37 °C, {Delta}IRS increased rapidly within 3 min and then reached a plateau. Consequently, we used 3 min at 37 °C as the best compromise between sensitivity and speed.

Selection of supernatant amount.
With nanogold-labeled immunocomplex supernatant containing 0.025 mg/L C3 as an example, we found that {Delta}IRS increased linearly with supernatant volume in the range of 0.5–3.0 µL. For volumes between 4.0 and 10.0 µL, {Delta}IRS increased slightly and displayed a small tendency to decrease. We therefore used a 3.0-µL volume of supernatant solution for further experiments.

analytical performance
According to the procedure and using these conditions, we recorded {Delta}IRS values for different C3 concentrations.{Delta}IRS was proportional to C3 concentration in the range of 5.0–160.0 ng/L. The regression equation was: y = 0.490x + 2.68, where y is {Delta}IRS and x is the C3 concentration (R = 0.9971). A detection limit of 1.52 ng/L C3 was calculated as the mean plus 3 SDs for measurements of a blank solution (n = 20). The sensitivity and selectivity of this assay were superior to those of other C3 assays (Table 2 ) (29)(30)(31)(32)(33). Our assay is highly sensitive compared with the described nanogold-enhancement assays (14)(15)(20)(34)(35)(36)(37)(38) (see Table 1 in the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/54/1). Thus, we were able to use only a microamount of sample that had been diluted 40-fold.


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  		Table 2. Comparison of C3 assays.

influence of interfering substances
We examined the influence of potentially interfering substances on the C3 assay. When the C3 concentration was 100 ng/L with a relative error of ±5%, the examined substances did not interfere appreciably with the assay (see Table 2 in the online Data Supplement).

comparison study
We analyzed 36 serum samples both with this new assay and by immunoturbidimetry (32). Twenty-eight of the serum samples were nonpathologic (Fig. 4 ; also see Table 3 in the online Data Supplement). The mean (SD) C3 concentration measured with this assay was 1.29 (0.035) g/L for undiluted samples (10 µL of a sample diluted 40-fold was used to bring the concentration within the linear range of the assay, 5.0–160 ng/L). The linear regression analysis revealed a correlation coefficient of 0.960 and slope and intercept values of 0.787 (0.0218) g/L and 0.28 (0.026) g/L, respectively. The results obtained with both assays were consistent with published reference intervals for serum (32). We tested 8 abnormal serum samples (sample nos. 29–36 in Table 3 of the online Data Supplement) with this new assay and by immunoturbidimetry; C3 concentrations in these samples exceeded the reference range (0.90–1.80 g/L).


Figure 4
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  		Figure 4. Linear regression analysis of the immunoturbidity results and the results for the immunonanogold RS assay.

In conclusion, we were able to measure C3 concentrations in serum samples with this new approach and obtained satisfactory results.


   Acknowledgments
 
Grant/funding Support: The National Natural Science Foundation of China (grant nos. 20667001 and 20365001), the Natural Science Foundation of Guangxi (grant no. 0728213), and the Foundation of New Century Ten-Hundred-Thousand Talents of Guangxi supported this research.

Financial Disclosures: None declared.

Acknowledgments: We thank greatly the National Natural Science Foundation of China, the Natural Science Foundation of Guangxi and Foundation of New Century Ten-Hundred-Thousand Talents of Guangxi for supporting this work.


   Footnotes
 
1 Nonstandard abbreviations: C3, complement component 3; RS, resonance scattering; PEG, polyethylene glycol; {Delta}IRS, decrease in RS intensity.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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