Aptamer-Based Sandwich Assay for Measurement of Thymidine Kinase 1 in Serum of Cancerous Patients

Abstract

Thymidine kinase 1 (TK1) is traditionally a serum biomarker that is elevated in the early stages of malignancies. The diagnostic and prognostic role of TK1 for screening and monitoring human malignancies has recently been investigated. Anti-human TK1 aptamers were selected through 12 iterative rounds of systematic evolution of ligands by exponential enrichment from a DNA library. The aptamer pool of round 12 was amplified, and the polymerase chain reaction product was cloned on the TA vector. Of the 85 colonies obtained, 52 were identified as positive clones. These aptamers were screened for TK1 with surface plasmon resonance, where apta37 and apta69 showed the highest affinity for TK1. The TK1_apta37 and TK1_apta69 aptamers were used in a sandwich assay platform and successfully detected TK1 in the concentration range of 54–3500 pg mL–1. Clinical samples from 60 cancerous patients were also tested with this assay system and compared using the conventional antibody-based enzyme-linked immunosorbent assay kit. The aptamer sandwich assay demonstrated a dynamic range for TK1 at clinically relevant serum levels, covering subpicogram per milliliter concentrations. The new approach offers a simple and robust method for detecting serum biomarkers that have low and moderate abundance. The results of this study demonstrate the screening capability of the aptamer sandwich assay platform and its potential applicability to the point-of-care testing system.

Biosensors Made of Synthetic Functional Nucleic Acids Toward Better Human Health

“…A large proportion of aptasensors that were described in 2018 and 2019 were for general cancer biomarkers, meaning they were biomarkers that were useful for multiple cancer types. Biomarkers that were examined include PDGF-BB (LODs, 0.13 nM, 0.08 ng/mL, 0.52 nM, and 3.2 pM), − VEGF (LODs, 0.3 fM and 12 pM), − CD70 (LOD, 14 cells/mL), neutrophil gelatinase-associated lipocalin (NGAL), nucleolin, ,,, ATP (LOD, 0.01 pM), , epidermal growth factor receptor (EGFR, LODs, 5.64 fg/mL, 0.7 ng/mL, and 0.1 ng/mL), − MCF-7 cells (LOD, 61 cells/mL), telomerase (LOD, 100 cells), thymidine kinase 1 (LOD, 54 pg/mL), miRNA let-7a (LOD, 5.12 aM), cytochrome C (LOD, 25.90 nM), lysozyme (LOD, 0.94 nM), mucin 1 (LOD, 0.13 ng/mL), , epithelial cell adhesion molecule (EpCAM, LODs, 10 pM and 20 pg/mL), , N -glycolylneuraminic acid (Neu5Gc, LOD, 10 ng/mL), and CD63 exosome surface protein (LODs, 32 exosomes/μL, 73 exosomes/μL, 203 exosomes/μL). − The usefulness of many of these biosensors was evaluated in complex matrices, such as whole blood, serum, or cell lysate, or in the presence of whole cells. The strength of each of these biosensors is their diverse application to multiple cancers.…”

Two-Dimensional Hybridization Chain Reaction Strategy for Highly Sensitive Analysis of Intracellular mRNA

“…mRNA translation and decay are the key steps of post-transcriptional gene regulation, which enable RNA quality control and rapid changes in gene expression. − Tremendous amount of researches have been conducted, which involve the subcellular localization of mRNAs and their dynamic interactions with other components. , In addition, abnormal expressions of certain mRNAs are revealed to be associated with the development of many diseases . For example, human thymidine kinase 1 (TK1), a cell proliferation-related enzyme, has aberrant expression during carcinogenesis . TK1 mRNA is thus considered as a promising marker to probe tumor burden and progression .…”

“…9 For example, human thymidine kinase 1 (TK1), a cell proliferation-related enzyme, has aberrant expression during carcinogenesis. 10 TK1 mRNA is thus considered as a promising marker to probe tumor burden and progression. 11 As a result, highly sensitive detection and imaging of specific mRNA inside cells are critical tasks that can help better understand the mechanism of mRNA functions and clinical diagnosis of related diseases.…”