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Technical Briefs |
Department of Chemistry, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Tsinghua University, Beijing, People’s Republic of China;
aAddress correspondence to this author at: Department of Chemistry, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Tsinghua University, Beijing, 100084, People’s Republic of China; fax 86-10- 62795290; e-mail jhli{at}mail.tsinghua.edu.cn
Abstract
Background: Au(III) catalyzed luminol chemiluminescence (CL) is classic in luminescence analysis. Recently, spherical gold nanoparticles (Au-NPs) were found displaying far stronger catalytic activity on luminol CL than that of Au(III). Some methods based on Au-NPs probes have been developed for DNA detection or immunoassay. However, more complicated labeling or stripping procedures are often inescapable in these protocols.
Methods: We synthesized specially shaped, irregular gold nanoparticles (IGNPs) and found their catalytic efficiency on luminol CL to be 100-fold greater than that of spherical Au-NPs. Using the IGNPs-functionalized DNA oligomers and the IGNPs-modified anti-IgG as in situ chemiluminescent probes, we established sandwich-type analytic methods for rapid, simple, selective, and sensitive sequence-specific DNA detection and for human plasma IgG immunoassay, respectively. We used 12 clinical human plasma samples to examine the precision and accuracy of the proposed method for IgG content determination.
Results: Calibration curves for the oligonucleotide [
I = 15.73 + 27.55 (DNA) x 1010 (mol/L); R2 = 0.9936] and IgG [I = 48.84 + 30.23 (IgG) x 1010 (mol/L); R2 = 0.9964] show good correlation, demonstrating the linear response over the concentrations tested (0.04–10 nmol/L for DNA, 0.05–10 nmol/L for IgG). The limit of detection, calculated based on 50 µL of a solution of calibrators, was 13 pmol/L for DNA and 17 pmol/L for IgG, with a signal-to-noise ratio of 3. We obtained good intra-and interassay reproducibility. The IgG contents in 12 human plasma samples obtained by the proposed method are identical with the data of clinical laboratory.
Conclusions: We developed a simple and sensitive method for in situ amplified chemiluminescence detection of sequence-specific DNA and immunoassay of IgG by use of highly active, specially shaped, irregular gold nanoparticles (IGNPs) as label and confirmed by clinical samples test. This method has many desirable features including rapid detection, selectivity, and little required instrumentation. This new protocol may be quite promising, with potentially broad applications for clinical immunoassays and DNA hybridization analysis.
The detection of sequence-specific DNA is of great importance because of its application in diagnosis of pathogenic and genetic diseases (1)(2)(3). Various labeled probes based on radioactive, electrochemical, fluorescent, and electrochemiluminescent principles have been developed to establish corresponding detection methods through target hybridization technique (4)(5)(6)(7)(8)(9). Chemiluminescence, with the advantages of high sensitivity and wide linear range, has also been applied to DNA hybridization analysis. Niazov et al. (10), and Pavlov et al. (11), recently reported 2 methods for detecting polynucleotides that used DNAzyme or DNAzyme-Au nanoparticles (Au-NPs), conjugates functionalized with 5'-(alkanethiol)-capped oligonucleotides as the label to catalyze luminol chemiluminescence. Immunoglobulin G (IgG) is the integrant testing object for immune detection. It is often used as model for the study of biomolecular affinity interaction. Fan et al. (12) described a chemiluminescent immunoassay of IgG labeled with spherical nanogold, and a stripping technique was introduced to transfer nanogold into Au(III) to catalyze luminol chemiluminescence (CL).
Au(III) catalyzed luminol chemiluminescence is classic in luminescence analysis. An excellent biological tag, colloidal gold has been used extensively to label a broad range of biological receptors and has been applied to surface-enhanced Raman scattering, surface plasmon resonance, and immunoblotting (13)(14)(15)(16). Recently, spherical Au-NPs were found to display stronger catalytic activity on luminol CL than on Au(III) (17). As reported in this study, we synthesized specially shaped, irregular gold nanoparticles (IGNPs) and investigated their catalytic activity on luminol CL reaction. An increase of catalytic efficiency on luminol CL over 100-fold than that of spherical Au-NPs made it an ideal amplified catalytic CL label to trace sequence-specific DNA and immune analytes in clinical diagnosis (Fig. 1
). Compared with DNAzyme label, the present label method offers the advantage of simple label process and high stability. And compared with that of spherical nanogold label and stripping CL detection, the proposed label provides in situ detection with high sensitivity and no requirement for stripping procedure.
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We prepared 
25-nm–sized spherical Au-NPs stabilized with citrate according to a well-known method (18), and we synthesized the specially shaped irregular gold nanoparticles (IGNPs) briefly, as follows: a 150-mL round-bottom flask containing 50 mL (final volume) aqueous solution of 0.1 mmol/L chloroauric acid (HAuCl4, analytical grade) was first prepared. Second, 5 mL of 0.17% starch was added into the flask and purged by oxygen with argon for 10 min. Third, 1 mL of 1 mmol/L D-glucose purged with argon was introduced to the prepared solution. Finally, the solution kept stirring at 40 °C for 24 h. The reaction mixtures finally turned light violet. The concentration of the Au-NPs was determined by the absorbance spectra at 519 nm. After confirmation by transmission electron microscopy images (Hitachi Model H-800), UV-Vis spectra (Shimadzu UV-21005), and X-ray diffraction pattern (Bruker D8), we obtained the specially shaped, irregular gold nanoparticles (IGNPs) with an mean length of 50 nm and a narrowing width from 25 to 30 nm along its longitudinal axis (see Fig. S1 in the online Data Supplement that accompanies the online version of this Technical brief at http://www.clinchem.org/content/vol52/issue9).
First, we investigated the catalytic effect of as-prepared IGNPs on CL of luminol-H2O2 reaction with a flow-injection CL setup (Model MPI-B multiparameters chemical analysis system, Xi’an Remax Analytical Instrument Co., Xi’an, China), and compared with that of spherical Au-NPs and Au(III). The results show that the catalytic effect of the IGNPs is far stronger than that of spherical Au-NPs and Au(III) under the same conditions, listed in Table 1
. The detection limit for IGNPs was 1 x 10–11 mol/L when the luminol (in 0.01 mol/L NaOH) and H2O2 were fed at an optimized concentration of 0.2 mmol/L and 0.01 mol/L, respectively. On the basis of the finding, we then proposed to use the IGNPs as a simple and effective amplified catalytic CL label, and we expected to find it useful in bioanalysis applications. Sequence-specific DNA and IgG were then chosen as model compounds to establish corresponding sensitive CL detection protocols.
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The following sequences of DNA oligomers: (a), 5'-HS(CH2)6-ATGGGCGGAATGAAC-3'; (b), 5'-CTCGCACCGTCCACCTTTGTTCATTCCGCCCAT-3'; (b1), 5'-CTCGCACCGTCCACCTTTCCCCTTCTTGTTCCC-3'; and (c), 5'-GGTGGACGGTGCGAG-(CH2)6SH-3', synthesized by Shanghai Sangon (Shanghai, China) and purified using the polyacrylamide gel electrophoresis method were used in the present work. Sequence (c)-modified IGNPs probes were prepared similar to literature method (19). We first pretreated the gold substrates with "piranha" solution (98% H2SO4:H2O2, 7:3 by volume) before use and rinsed with ultra-purified water. Then, the self-assembled monolayers of HS-ssDNA surfaces were formed by immersing the gold substrates (0.3 mm x 3 mm x 35 mm) in a 250 nmol/L HS-ssDNA solution (Sequence 1) in 0.4 mol/L phosphate buffer solution (PBS; pH 7.3) for 3 h. Before analysis or hybridization, we rinsed each sample thoroughly with deionized water. We washed the resulting plate with the PBS and then treated it with 1-mercaptohexanol, 1 mmol/L in 0.1 mol/L PBS, pH 7.4, for 1 h. The resulting monolayer-functionalized surface was treated with different concentrations of the complementary analyte DNA (Sequence 2) in 0.1 mol/L PBS for 3 h to yield the double-stranded DNA assembly on the surface. The surface was then allowed to hybridize with a solution of Sequence-modified (3) IGNPs in 0.1 mol/L PBS, 40 °C, for 3 h. At last the surfaces were rinsed thoroughly with 0.1 mol/L PBS and ultra-purified water to remove the unbound DNA-IGNPs probe. The process for formation of SH-DNA/Target DNA/DNA-IGNPs sandwich-type complex is schematically presented in Fig. 1(A)
. Random sequence (2A) was used as noncomplementary comparison.
Human IgG and sheep antihuman IgG obtained from Dingguo Biological Technology Co. were used for the CL immunoassay. Similar to the method described in the literature (20), we prepared antihuman IgG-IGNPs conjugate). Sandwich-type conjugate of (antihuman IgG)/IgG/(antihuman IgG-IGNPs) was assembled on a gold substrate as follows: We immersed the pretreated gold substrate mentioned above in 1,9-nonanedithiol ethanol solution to form the SAMs of 1,9-nonanedithiol. The thiol functionalized gold substrates were dipped into the spherical gold colloid solution in darkness. The spherical nanogold-modified gold substrates were further immersed in 0.5 mL PBS at pH 7.4 with 0.1 g/L of the antihuman IgG and incubated overnight at 4 °C to immobilize more antihuman IgG on the substrate (or antiIgG/spherical nanogold/SH(CH2)9SH/gold substrate). We then thoroughly rinsed the gold substrates with 0.02 mol/L PBS (pH 7.4) to remove the weakly absorbed anti-IgG. Bovine serum albumin (BSA) in 0.02 mol/L PBS (pH 7.4) then was introduced to saturate the possible bare spherical nanogold for 30 min and rinsed with 0.02 mol/L PBS (pH 7.4). Next the substrates were dipped into a pH 7.4 PBS containing various concentrations of IgG at 37 °C for 30 min, and then rinsed by 0.02 mol/L PBS (pH 7.4) solution to remove unbound IgG. (21) Finally it was immersed in antihuman IgG-IGNPs conjugate solution at 37 °C for 30 min, and then rinsed by 0.02 mol/L PBS (pH 7.4) to remove unbound antihuman IgG-IGNPs conjugate. The processes of the immobilization of antibody onto the gold substrate and the formation of sandwich-type (anti-IgG)/IgG/(anti-IgG-IGNPs) are shown in Fig. 1
(B).
After the formation of the sandwich-type complexes of SH-DNA/target DNA/DNA-IGNPs and (anti-IgG)/IgG/(anti-IgG-IGNPs), the assembled gold substrates were located in a colorless glass tube directly facing to the window of PMT of CL detector, and connecting into the flow lines of the flow-injection CL setup. 0.2 mmol/L luminol (in 0.01 mol/L NaOH) and 0.01 mol/L H2O2 were injected into the system, which were catalyzed by IGNPs probe to produce enhanced CL. The concentrations of target DNA and IgG are quantified via the relative CL intensity. Dilutions of calibrator target DNA sequence and human IgG were used for testing the analytical performance of the proposed method.
Calibration curves for the oligonucleotide (I = 15.73 + 27.55 [DNA] x 1010 (mol/L); R2 = 0.9936) and IgG (I = 48.84 + 30.23 [IgG] · 1010 (mol/L); R2 = 0.9964) show good correlation, demonstrating the linear response over the concentrations tested (0.04–10 nmol/L for DNA, 0.05–10 nmol/L for IgG) (Fig.S3) The limit of detection, calculated based on 50-µL of a solution of calibrators, was 13 pmol/L for DNA and 17 pmol/L for IgG, with a signal-to-noise ratio of 3. We calculated the reproducibility of DNA detection and IgG immunoassay by analyzing the calibrator target oligonucleotide (0.5, 1, and 8 nmol/L) and IgG (0.5, 1, and 8 nmol/L) for 5 times consecutively. We evaluated the intraassay imprecision of the method by analyzing the same concentration samples 7 times consecutively and the interassay imprecision by analyzing the same concentration samples on 5 consecutive days. Before the test, we performed aliquots of the sample, and all the calibrator samples of DNA and IgG were stored in a refrigerator at 4 °C when not in use. Precision tests indicated good repeatability of our method for CL intensity (see Table S1 in the online Data Supplement). Moreover, we obtained good intra- and interassay reproducibility (see Table S1 in the online Data Supplement).
Control experiments showed that almost no enhanced CL signals were recorded when we substituted different concentrations of sequence (Fig 2A; 0.1, 1, 10, and 100 nmol/L) for sequence (2) in DNA hybridization and used different concentrations of BSA (0.1, 1, 10, 100 nmol/L) for immunoassay instead of IgG. The experiments also revealed that very weak, enhanced light was emitted from the system after this protocol in the absence of sequence 2 or in the absence of the DNA-functionalized IGNPs (sequence 3), or in the absence of IgG or in the absence of IGNPs-functionalized anti-IgG. These results clearly imply that the association of the DNA-functionalized IGNPs or IGNPs-functionalized anti-IgG to the surface was essential to generate the enhanced light signals and that no substantial nonspecific adsorption was observed in the systems.
For comparison, we also used the spherical gold nanoparticles as label instead of IGNPs to carry out DNA detection and IgG immunoassay following the same protocols. The results show that the detection limit was 0.67 nmol/L for DNA and 0.50 nmol/L for IgG (S/n = 3) when spherical gold nanoparticles were used. It revealed that the proposed IGNPs labeling method possessed the advantages of more sensitivity and wider linear range compared with that of spherical gold nanoparticles labeling in this case.
We tested the performance of the proposed IGNPs-labeled CL immunoassay method by measuring IgG content in 12 human plasma samples, freshly obtained from Tsinghua University Hospital. Before the test, the samples were diluted appropriately step by step to be in the linear range of the proposed method. The samples were stored in refrigerator at 4 °C when not in use. The results obtained in clinical laboratory by rate-scattering turbidimetry with a Model ARRAY360 Specific Protein Analyzer (Beckman) were used for comparison. It was found that the values got from the proposed method was identical to that of the clinic method (r = 0.991, P <0.0001) [see Table S2, Fig. S4(A) in the online Data Supplement] Bland-Altman analysis also implied that there was no substantial difference between the 2 methods for IgG immunoassay. [Fig. S4(B) in the online Data Supplement]. Recovery experiments of calibrator human IgG and target oligonucleotide sequence 2 from enhanced human plasma samples also indicated the excellent accuracy and precision of the proposed method (see Table S3 in the online Data Supplement).
In conclusion, we developed a simple and sensitive method for in situ amplified chemiluminescence detection of sequence-specific DNA and immunoassay of IgG using highly active IGNPs as labels and confirmed by clinical samples test. This method has many desirable features including rapid detection, good selectivity, and little required instrumentation. From the analytical chemistry point of view, this new protocol will be quite promising for broad potential applications in clinic immunoassay and DNA hybridization analysis.
Acknowledgments
This work was supported financially by National Science Foundation of China (No. 20125513, No. 20435010), by the Foundation for the Author of National Excellent Doctoral Dissertation of the People’s Republic of China, and by the 38th Postdoctoral Science Foundation of the People’s Republic of China.
References
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