Effect of pH on the Interaction of Gold Nanoparticles with DNA and Application in the Detection of Human p53 Gene Mutation
© to the authors 2008
Received: 22 October 2008
Accepted: 25 November 2008
Published: 10 December 2008
Gold nanoparticles (GNPs) are widely used to detect DNA. We studied the effect of pH on the assembly/disassembly of single-stranded DNA functionalized GNPs. Based on the different binding affinities of DNA to GNPs, we present a simple and fast way that uses HCl to drive the assembly of GNPs for detection of DNA sequences with single nucleotide differences. The assembly is reversible and can be switched by changing the solution pH. No covalent modification of DNA or GNP surface is needed. Oligonucleotide derived from human p53 gene with one-base substitution can be distinguished by a color change of the GNPs solution or a significant difference of the maximum absorption wavelength (λmax), compared with wildtype sequences. This method enables detection of 10 picomole quantities of target DNA.
Gold nanoparticles (GNPs) coupled with biomolecules are of great current interest because of their biomedical applications. GNPs have been used to detect DNA with high sensitivity and selectivity . Mutations, single nucleotide polymorphisms (SNPs), chromosomal translocations, gene expression, and pathogens from clinical samples can be easily detected [2–5]. The first reports on GNPs–DNA complex were published in the August issue of Nature in 1996. Two groups of CA Mirkin and AP Alivisatos described the organization of GNPs with DNA oligonucleotides [6, 7]. Mirkin et al. mixed two batches of 13-nm gold particles, each attached by thiolated non-complementary oligonucleotides. When a complementary oligonucleotide duplex was added to the solution, the nanoparticles assembled into aggregates, which provoked a red-to-blue color change accomplished by a red-shift of the surface plasmon band. The disadvantage of this method is the requirements of two sets of oligonucleotide probes and thiol-modification of the probe DNA, which is expensive and time-consuming .
The optical property of DNA-linked gold nanoparticle assemblies was developed for sequence-specific DNA detection. Li and Rothberg invented another non-cross-linking method, where GNPs aggregation was induced by an increasing salt concentration. This method took advantage of the preferential nonspecific binding of single-stranded oligonucleotides over double-stranded ones to the surface of GNPs and devised a label-free way, which requires no covalent modification of the DNA. The presence of non-complementary probes could prevent aggregation and the solution remains red; while complementary probes could not prevent gold nanoprobe aggregation resulting in a color change from red to blue. This simple assay has been applied to clinical samples of genomic DNA that screen for SNPs associated with long QT syndrome [9, 10].
Target oligonucleotide sequences
HAuCl4, sodium tris-citrate, NaOH and HCl were purchased from Xilong Chemical (Guangdong, China). All reagents were of analytical grade. Single-stranded DNA (ssDNA) was synthesized by Shanghai Sangon Biological and Engineering Company with the following sequences (Table 1). Milli-Q water (18.2 MΩ) was used in all of the experimental processes.
GNPs were prepared in water as follows. An aqueous solution of sodium tris-citrate (5 mL, 1%) was mixed with HAuCl4(1 mL, 1%) solution in a conical flask and sealed with parafilm. The reaction solution was first boiled in a 700-watt microwave oven on high power for 1 min, and then kept heating on medium power for 5 min.
GNPs (10 nM) were mixed with 50 pmol ssDNA and incubated at room temperature for 10 min. The pH of aqueous solutions was adjusted by adding 1 M HCl or 1 M NaOH. 50 μL cuvettes (Beckman) were used to hold the samples, and all UV–visible spectra were collected on a Beckman H800 UV–vis spectrophotometer with 1 nm resolution.
Zeta Potentials and Particle Size Measurement
The determination of zeta potential was carried out using Zetasizer Nano-ZS (Malvern Instruments Ltd. UK). The zeta potentials of GNPs and GNPs binding with ssDNA at different pH values were measured. Each data point for zeta potential was an average of at least 15 measurements.
Transmission Electron Microscopy (TEM)
Samples for TEM characterization were prepared by placing a drop of sample solution on a carbon coated copper grid and dried at 37 °C. TEM measurements were made in a JEM-2100 transmission electron microscopy operated at an accelerating voltage of 200 kV.
Results and Discussion
Tumor suppressor gene p53 (MIM 191170) is the most frequently mutated gene in human cancer. Detection of p53 abnormalities is very important for cancer diagnosis and early therapy [12, 13]. In the present work we developed a simple colorimetric assay using unmodified GNPs to detect 12 of the most frequent point mutations in exon 5, 7, and 8 of human p53 gene. The target mutant ssDNA with one-base substitution can be differentiated clearly from the wildtype oligonucleotide.
We improved the conventional method to synthesize GNPs by citrate reduction of HAuCl4 under microwave [14, 15]. The average particle size was 13 nm in diameter measured by TEM. The surface plasmon band (λmax) was 520 nm. The initial colloidal solution of GNPs or ssDNA-coated GNPs was wine red at pH 7.0. A characteristic blue color of naked GNPs was shown in alkaline condition (pH > 12.0). With addition of HCl or NaOH (pH 2.0–12.0), GNPs solution turned light pinkish, at pH < 2.0, the solution first became blue, then turned colorless instantaneously, and, finally, a black precipitation settled to the bottom. If GNPs were incubated with single-stranded DNA, addition of HCl caused a color change to blue or purple, while addition of NaOH (pH 7.0–12.0) did not change the solution color. The absorption peak red-shifted to different long-wave-length regions according to different DNA sequences (pH < 4.0 or pH > 12.0). And the redshift was accompanied by the attenuation of its intensity, which clearly indicated the occurrence of aggregation.
This work demonstrated that adsorption of ssDNA on GNPs could effectively stabilize the colloid against HCl-induced fusion. In addition, we presented an easy and inexpensive way for single-base mismatch detection. Based on the electrostatic interaction of DNA and GNPs upon addition of HCl, ssDNA sequence can be easily distinguished from sequence with single-base mismatch by measuring λmaxof the two systems. Comparing with other nanoparticle-based DNA detection assays, our method has the following advantages: (1) no need of complicated modification of GNPs or DNA, (2) no additional DNA probes are required, (3) no need of signal amplification or temperature control. Only three components exist in our system: GNPs, target oligonucleotide, and HCl. We successfully applied this method to detect 12 point mutations derived from human p53 gene. Since this methodology is limited to a single color change, two individual reactions are required for comparison-a wildtype sequence and a mutant sequence. This colorimetric method should have the potential for genetic tests.
This work is supported by the Science and Technology Innovation Project of Fujian Province for Young Scientific Researchers, China (No. 2006F3128) and the Open Fund of State Key Laboratory for Physical Chemistry of Solid Surfaces, Xiamen University (No. 200602).
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