An ultrasensitive and specific electrochemiluminescence (ECL) biosensor has been designed for the p53 DNA sequence, which is based on cascade signal amplification of nicking endonuclease assisted target recycling and hyperbranched rolling circle amplification (HRCA). First of all, biotin modified hairpin capture DNA (HP) probe was immobilized on the surface of streptavidin magnespheres paramagnetic particles (PMPs). Target DNA hybridized with the loop portion of the HP probe, therefore unfolding HP to form a double-stranded DNA (dsDNA) containing the specific nicking site of the nicking endonuclease. Then, the nicking endonuclease recognized the specific nicking site and cleaved the HP into two pieces, liberating target DNA and the complementary sequence piece for the padlock probe. The intact target DNA would initiate the next cycle of hybridization and cleavage, thereby releasing multiple complementary sequences for the padlock probes. The liberated complementary sequences hybridized with the padlock probes, subsequently inducing the HRCA reaction and generating numerous dsDNA segments. Herein, Ru(phen)3(2+) was embedded into dsDNA and worked as ECL signal reporter. The reaction products were eventually pretreated by dialysis tube with the cutoff membrane to remove the residual Ru(phen)3(2+) in the solution for the following ECL measurements. Using this cascade amplification strategy, an ultrasensitive p53 DNA sequence detection method was developed with a wide linear range from 0.05 to 100 fM and a low detection limit of 0.02 fM. Moreover, this cascade amplified ECL biosensor had specific recognition capacity for noncomplementary and single- and double-base mismatched DNA. The proposed ECL biosensor might have a great potential in biomedical research and clinic analysis.
A simple, sensitive and selective fluorescence biosensor for determination of DNA using CuS particles based on click chemistry is reported. Biotin-modified capture DNA was modified on Streptavidin MagneSphere Paramagnetic Particles (PMPs) and hybridized with target DNA (hepatitis B virus DNA had been chosen as an example), then bound target DNA was hybridized with DNA-CuS particles and formed a sandwich like structure. CuS particles on the sandwich structures can be destroyed by acid to form Cu(II), and Cu(II) can be reduced to Cu(I) by sodium ascorbate, which in turn catalyzes the reaction between a weak-fluorescent 3-azido-7-hydroxycoumarin and propargyl alcohol to form a fluorescent 1,2,3-triazole compound. Using this method, target DNA concentration can be determined by a change in the fluorescence intensity of the system. It is found that the fluorescence increase factor has a direct linear relationship to the logarithm of target DNA concentrations in the range of 0.1 to 100 nM, and the detection limit is 0.04 nM (S/N = 3). The proposed sensor not only allows high sensitivity and good reproducibility, but also has a good selectivity to single-nucleotide mismatches.
The detection of hydrogen sulfide (H2S) is important due to its role in the diagnosis of many diseases since H2S is involved in the protection of neurons from oxidative stress and neuronal transmission modulation in the brain.
Telomerase is one of the most common markers of human malignant tumors, such as uterine, stomach, esophageal, breast, colorectal, laryngeal squamous cell, thyroid, bladder, and so on. It is necessary to develop some sensitive but convenient detection methods for telomerase activity determination. In this study, a label-free and ultrasensitive electrochemiluminescence (ECL) biosensor has been fabricated to detect the activity of telomerase extracted from HeLa cells. Thiolated telomerase substrate (TS) primer was immobilized on the gold electrode surface through gold-sulfur (Au-S) interaction and then elongated by telomerase specifically. Then, it was hybridized with complementary DNA to form double-stranded DNA (dsDNA) fragments on the electrode surface, and Ru(phen)3 (2+) has been intercalated into the dsDNA grooves to act as the ECL probe. The enhanced ECL intensity has a linear relationship with the number of HeLa cells in the range of 5∼5000 and with a detection limit of 2 HeLa cells. The proposed ECL biosensor has high specificity to telomerase in the presence of common interferents. The relative standard deviations (RSDs) were <5 % at 100 HeLa cells. The proposed method provides a convenient approach for telomerase-related cancer screening or diagnosis.
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