Optical Detection of Human Papillomavirus Type 16 and Type 18 by Sequence Sandwich Hybridization With Oligonucleotide-Functionalized Au Nanoparticles

Abstract

The importance of detecting and subtyping human papillomaviruses (HPVs) in clinical and epidemiological studies has been well addressed. In detecting the most common types of HPV, type 16 (HPV-16) and type 18 (HPV-18), in the cervical mucous of patients in a simple and rapid manner, the assay of a label-free colorimetric DNA sensing method based on sequence sandwich hybridization with oligonucleotide-functionalized Au nanoparticles (AuNPs) was fabricated in this study. Specific oligonucleotide probes were designed for the sequence detection within the L1 gene of HPV-16 and HPV-18, and the probes were capped onto AuNPs, as AuNP probes. The target HPV sequences in clinical specimens were obtained by an asymmetric polymerase chain reaction (PCR) with universal primers, which can amplify the target sequences from several HPV serotypes, including HPV-16 and HPV-18. The DNA sandwich hybridization between the target sequences and the specific AuNP probes was performed at a temperature closer to the theoretical melting temperature of the DNA hybridization. Next, the procedure of increasing salt concentration and cooling the hybridizing solution was immediately utilized to discriminate the target sequences of HPV-16 or HPV-18. If the target sequences were not complementary to sequences of AuNP probes, the AuNPs would aggregate because no duplex DNA formation occurred such that the color of the reaction solution changed from red to purple. If the AuNP probes were a perfect match to the target sequences and a full DNA sandwich hybridization occurred, the reaction solution maintained its red color. A total of 70 mucous specimens from patients with cervical intraepithelial neoplasia were tested by the AuNP probes sandwich hybridization. The results show that there were 33, 16, 5, and 16 cases detected with HPV-16, HPV-18, both HPV-16 and HPV-18 (HPV-16/HPV-18), and neither HPV-16 nor HPV-18, respectively.

Optical assays based on colloidal inorganic nanoparticles

“…Chen et al established a colorimetric technique based on the immobilization of two thiol probes on the surface of 13 nm AuNPs. In this nanobiosensor , when amplified genomic DNA and probe sequences were mixed, color variations could not be observed because of the too wide gap between the probes attached to the nanoparticles and the target DNA [109]. Influenza virus A (IAV) is one RNA virus composed of single-stranded RNA (ssRNA) and two membrane proteins HA and NA.…”

Gold Nanoparticles for DNA/RNA-Based Diagnostics

“…Earlier methods used enzymatic amplification techniques, such as PCR, asymmetric PCR, and NASBA [14][15][16][17][18][19]. Recently, Zagorovsky and Chan reported on the integration of a multicomponent DNA-responsive DNAzyme with colorimetric detection using AuNPs, allowing for nonenzymatic signal amplification [20].…”

Investigation of the DNA target design parameters for effective hybridization-induced aggregation of particles for the sequence-specific detection of DNA

“…Included in these are Raman spectroscopy, 7,8 electrical stimulation, 9,10 electrophoresis 11 and optical detection. These range from simplistic, cost-effective methods like colorimetric assays, 12,13 to the more complex microfabrication-dependent systems like cantilever deflection. 14 Effective DNA hybridization is essential for sensitive detection, regardless of the platform.…”

“…Fluorescence is the detection method of choice for many highly selective assays that aim to detect a single base mismatch at concentrations of 50 nM, 20 10 nM 21 and most recently 0.26 fM. 22 A number of label-free methods for SPM detection have also been reported, including resonator arrays, 23 silicon nanowires 24 and colorimetric detection with gold nanoparticles, 12,[25][26][27][28] and these have been associated with limits of detection (LODs) of 1.95 nM, 1 nM and 50 fM, respectively. However, they involve relatively lengthy assay times (1-4 hours), are destructive to the sample, and require cumbersome instrumentation, and/or specialized fabrication techniques.…”