ABSTRACT
Human epidermal growth factor receptor 2 (HER2), a common proto-oncogene, is overexpressed in a subset of breast cancer patients. It is essential to track HER2 expression for early breast cancer diagnosis. Herein, a ratiometric electrochemical biosensor for detection of HER2 based on activators generated by electron transfer for atom transfer radical polymerisation (AGET ATRP) and hairpin DNA was developed. Specifically, hairpin DNA was first self-assembled on the gold electrode by Au-S bond. Upon capturing HER2, the stem-loop structure of hairpin DNA was unfolded, the signal value of methylene blue (MB) decreased as it moved away from the electrode surface. cDNA was linked with HER2 by complementary base pairing to introduce amino group. Then, the initiator 2-bromo-2-methylpropionic acid (BMP) were connected to the amino group on the cDNA to activate ARGET ATRP. The detection performance of biosensors for HER2 was explored by the ratio signal between two signal molecules. Under optimal conditions, this ratiometric electrochemical biosensor shows good selectivity and stability with a wide detection range of 1-1 × 106 pM and a detection limit of 78.47 fM. Furthermore, the biosensor exhibits satisfactory anti-interference ability due to the hairpin DNA and dual signal system, and has promising application prospects in the detection of other DNA disease markers.
Subject(s)
Biosensing Techniques , Electrochemical Techniques , Proto-Oncogene Mas , Receptor, ErbB-2 , Biosensing Techniques/methods , Receptor, ErbB-2/genetics , Humans , Electrochemical Techniques/methods , Electrodes , Gold/chemistry , Limit of Detection , Polymerization , DNA/chemistry , DNA/geneticsABSTRACT
Alkaline phosphatase (ALP), an important hydrolase involved in dephosphorylation, is a common clinical indicator of many diseases. In the present study, we constructed a novel electrochemical sensor using amifostine as the substrate of ALP and activators regenerated by electron transfer atom transfer radical polymerization (ARGET ATRP) as a signal amplification strategy for sensitive determination of ALP activity. In particular, in the presence of ALP, the phosphate group of amifostine was hydrolyzed to form a sulfhydryl group, which could attach to a gold electrode via a sulfur-gold bond. Then, the initiator α-bromophenylacetic acid (BPAA) was linked to the hydrolysis product of amifostine through an amide bond, resulting in the production of electroactive polymer chains on the gold electrode by the monomer ferrocenylmethyl methacrylate (FMMA) via ARGET ATRP. Under optimal parameters, the electrochemical sensor demonstrated a limit of detection (LOD) of 1.71 mU mL-1 with a linear range of 5-100 mU mL-1. In addition to satisfactory selectivity, the potential application of this approach for ALP activity detection in human serum samples was demonstrated. Due to its efficiency, simplicity of operation, and cost-effectiveness, the proposed electrochemical sensor has great promise as a universal method for ALP assays and inhibitor screening.
Subject(s)
Amifostine , Biosensing Techniques , Alkaline Phosphatase , Biosensing Techniques/methods , DNA/chemistry , Electrochemical Techniques/methods , Gold/chemistry , Humans , Limit of DetectionABSTRACT
A novel fluorescence assay is proposed through activators regenerated by electron transfer atom transfer radical polymerization (ARGET ATRP) strategy for alkaline phosphatase (ALP) activity detection. First of all, 2-bromo-2-methylpropionic acid (BMP) was employed as the initiator to modify on the surface of the magnetic nanoparticle (Fe3O4-MNP) by amide bonding. Then, ascorbic acid (AA) produced by ALP catalyzed the phosphate group removal from L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (AAPS), which underwent a redox reaction with Cu(II) and the product Cu(I) triggered the ARGET ATRP reaction. Finally, a strong fluorescent signal could be detected at 514 nm due to numerous fluorescent monomers being grafted to the Fe3O4-MNPs surface (Ex = 490 nm, Em = 514 nm). Under optimal experimental conditions, the linear range of this fluorometric assay for ALP activity was 1-80 mU mL-1, and the detection limit was 0.68 mU mL-1. The method exhibited excellent selectivity and satisfactory results were obtained in the inhibition rate and human serum experiments. Therefore, this ALP activity detection strategy has great potential for clinically relevant disease detection and drug screening. A novel fluorescence strategy for alkaline phosphatase activity detection based on the dephosphorylation property of alkaline phosphatase and ARGET ATRP reaction.
