Nanogold-Functionalized DNAzyme Concatamers with Redox-Active Intercalators for Quadruple Signal Amplification of Electrochemical Immunoassay

Abstract

A novel and in situ amplified immunoassay strategy with quadruple signal amplification was designed for highly efficient electrochemical detection of low-abundance proteins (carcinoembryonic antigen, CEA, as a model) by using nanogold-functionalized DNAzyme concatamers with redox-active intercalators. To construct such an in situ amplification system, streptavidin-labeled gold nanoparticles (AuNP-SA) were initially used for the labelling of initiator strands (S0) and detection antibody (mAb2) with a large ratio (mAb2-AuNP-S0), and then two auxiliary DNA strands S1 and S2 were designed for in situ propagation of DNAzyme concatamers with the hemin/G-quadruplex format. The quadruple signal amplification was implemented by using the avidin-biotin chemistry, nanogold labels, DNA concatamers, and DNAzymes. In the presence of target CEA, the sandwiched immunocomplex was formed between the immobilized primary antibodies on the electrode and the conjugated detection antibodies on the mAb2-AuNP-S0. The carried S0 initiator strands could progress a chain reaction of hybridization events between alternating S1/S2 DNA strands to form a nicked double-helix. Upon addition of hemin, the hemin-binding aptamers could be bound to form the hemin/G-quadruplex-based DNAzymes. The formed double-helix DNA polymers could cause the intercalation of numerous electroactive methylene blue molecules. During the electrochemical measurement, the formed DNAzymes could catalyze the reduction of H2O2 in the solution to amplify the electrochemical signal of the intercalated methylene blue. Under optimal conditions, the electrochemical immunoassay exhibited a wide dynamic range of 1.0 fg mL(-1) to 20 ng mL(-1) toward CEA standards with a low detection limit of 0.5 fg mL(-1). Intra-assay and inter-assay coefficients of variation (CV) were less than 8.5% and 11.5%, respectively. No significant differences at the 0.05 significance level were encountered in the analysis of 14 clinical serum specimens between the developed immunoassay and commercialized electrochemiluminescent (ECL) method for detection of CEA.

Native and Synthetic G-quartet-based DNAzyme Systems – Artificial Enzymes for Biotechnological Applications

“…Wang is based on the same concept as the Freeman's one, but using a 93-mer nucleotide telomerase substrate primer which leads to a detection limit as low as 0.1 aM (= 10 -18 ) able to detect telomerase activity from HeLa cells. [156] h. Detection of antibodies to develop ELISA-type immunoassays [157,158] or for immunohistochemistry assays are also extremely promising. G-quadruplex DNAzyme enables the visualization of the prostate-specific antigen (PSA), a high-potential tumor marker, directly in solid tissue sections.…”

Dna功能化金纳米及其应用

“…这种利用靶标改变电导率的检测方法还能 够应用于单碱基错配 [174] 、汞离子 [175] 、铅离子 [176] 、 腺苷 [177] 、组氨酸 [178] 和甲基化酶的检测 [179] . 另外, 还 可以通过延长 DNA [180,181] 、在 DNA 中嵌入钌氨配合 物 [66,182] 或利用金纳米的催化作用 [183] 等方式增强电 导率的改变, 进一步提高检测灵敏度 [124,182] . AuNP@ DNA 结合电化学的检测方法有着检测速度快、灵敏 度高、选择性好及成本低廉等优点, 适于溶液中痕量 靶标的分析检测.…”

An enzyme-free and label-free fluorescent biosensor for small molecules by G-quadruplex based hybridization chain reaction

“…HCR not only provides an enzyme-free signal amplification method but also involves a unique assembly process, which works under mild conditions with no specific demand for ionic strength, pH or temperature. Nowadays, many studies combined the amplification capability of HCR with various sensing platforms and showed promising results for the detection of biomolecules such as DNA [8][9][10][11][12][13][14][15][16][17][18][19][20][21][22], microRNA [23,24], proteins [25][26][27][28][29][30][31], cells [32,33]. However, to this day there are few reports for the detection of small molecules [34][35].…”