Rapid and Sensitive Detection of p53 Based on DNA-Protein Binding Interactions Using Silver Nanoparticle Films and Microwave Heating

Muzaffer Mohammed,Kadir Aslan

doi:10.5101/nbe.v6i3.p76-84

Abstract

Tumor detection can be carried out via the detection of proteins, such as p53, which is known to play vital role in more than 50% of all cancers affecting humans. Early diagnosis of tumor detection can be achieved by decreasing the lower detection limit of p53 bioassays. Microwave-accelerated bioassay (MAB) technique, which is based on the use of circular bioassay platforms in combination with microwave heating, is employed for the rapid and sensitive detection of p53 protein. Direct sandwich ELISA was constructed on our circular bioassay platforms based on DNA-protein binding interactions. Colorimetric and fluorescence based detection methods were used for room temperature bioassay (control bioassay; total bioassay time is 27 hours) and bioassay using microwave heating (i.e., the MAB technique; total bioassay time is 10 minutes). In the colorimetric based detection, a very high background signal due to the non-specific binding of proteins for the bioassay carried out at room temperature and a LLOD of 0.01 ng/mL for p53 was observed using the MAB technique. The LLOD for the fluorescence-based detection using the MAB technique was found to be 0.01 ng/mL. The use of circular bioassay platforms in the MAB technique results in microwave-induced temperature gradient, where the specific protein binding interactions are significantly accelerated; thereby reducing the background signal and the lower limit of detection of p53 protein.

Highlights

Since its discovery in SV40 transformed cells as T antigen associated protein in 1979, p53 protein is classified as a major tumor suppressor in mammalian tumors [1,2,3]
We present results of colorimetric and fluorescence based bioassays for p53 in buffer solution carried out on our circular bioassay platforms at room temperature and using the Microwave-accelerated bioassay (MAB) technique based on DNA-protein binding interactions
Our results show a significant increase in sensitivity of the p53 bioassay, with a LLOD of 0.01 ng/mL using the MAB technique with both colorimetric and fluorescence based detection methods

Summary

Introduction

Since its discovery in SV40 transformed cells as T antigen associated protein in 1979, p53 protein is classified as a major tumor suppressor in mammalian tumors [1,2,3]. In step 1, the attachment of thiolated DNA onto SNFs is accelerated due to both mass transfer and rapid thiol-silver interactions driven by microwaveinduced temperature gradient. We present results of colorimetric and fluorescence based bioassays for p53 in buffer solution carried out on our circular bioassay platforms at room temperature (i.e., gold standard bioassay) and using the MAB technique based on DNA-protein binding interactions. This bioassay setup can serve two purposes: i) Quantitative assessment of p53 protein from biological media; ii) Analysis of biological media for p53 autoantibodies (by use of secondary detector antibodies, not shown here). In comparison to commercially available ELISA kits, our method is ~100 times more sensitive (in terms of LLOD) and the total bioassay time is reduced from several hours to less than 10 min

Materials and methods

Methods

Findings

28 Bulk sensors Silver