Colorimetric Detection of DNA Using Unmodified Metallic Nanoparticles and Peptide Nucleic Acid Probes

We have developed a colorimetric assay for DNA detection based on the aggregation of unmodified metallic nanoparticles. Charge neutral peptide nucleic acids (PNA) are used as a coagulant of citrate anion-coated particles and as hybridization probe. In the absence of a complementary target DNA, free PNA molecules in solution induce aggressive particle aggregation because of the removal of charge repulsion as a result of PNA coating on nanoparticles. When a complementary DNA is present and PNA-DNA complexes are formed, the particles remain stable because the negative charges of the DNA strands in the complexes adsorbed on the particle surface ensure sufficient charge repulsions. In this method, no probe immobilization is needed and PNA-DNA hybridization occurs in a homogeneous phase. The assay results are displayed as rapidly as the changes in color and/or in UV-vis adsorption spectra of the colloidal solutions. We have validated the assay principle using gold- and silver-nanoparticles (AuNPs and AgNPs), with the involvement of a shorter (13 mer) and a longer (22 mer) probe sequences. A specific DNA can be detected in the presence of at least 10 times of interference DNA, and the detection limit is at a DNA/PNA ratio of 0.05. When NaCl is added to accelerate the particle aggregation, the selectivity is further improved, and single-base-mismatch discrimination is achieved. A two-component assay using a mixture of AuNPs and AgNPs has also been constituted, aiming to improve the result accuracy by making use of the multiple aggregation signatures from the two types of particles. For single-base-mismatch discrimination, the AgNPs offer a higher sensitivity than AuNPs by showing more obvious spectra and color alternation, and the two-component assay offers three parameters in the UV-vis adsorption spectra.