An ultrasensitive electrochemical biosensor for detecting p53 gene was fabricated based on heated gold disk electrode coupling with endonuclease Nt.BstNBI-assisted target recycle amplification and alkaline phosphatase (ALP)-based electrocatalytic signal amplification. For biosensor assembling, biotinylated ssDNA capture probes were first immobilized on heated Au disk electrode (HAuDE), then combined with streptavidin-alkaline phosphatase (SA-ALP) by biotin-SA interaction. ALP could catalyze the hydrolysis of ascorbic acid 2-phosphate (AAP) to produce ascorbic acid (AA). While AA could induce the redox cycling to generate electrocatalytic oxidation current in the presence of ferrocene methanol (FcM). When capture probes hybridized with p53, Nt.BstNBI would recognize and cleave the duplexes and p53 was released for recycling. Meanwhile, the biotin group dropt from the electrode surface and subsequently SA-ALP could not adhere to the electrode. The signal difference before and after cleavage was proportional to the p53 gene concentration. Furthermore, with electrode temperature elevated, the Nt.BstNBI and ALP activities could be increased, greatly improving the sensitivity and efficiency for p53 detection. A detection limit of 9.5 × 10 −17 M could be obtained (S/N = 3) with an electrode temperature of 40 °C, ca. four magnitudes lower than that at 25 °C.
An Yaqian,Yu Zhiqi,Liu Di,Han Lirong,Zhang Xian,Xin Xuelian,Li Cuiping
The detection of methyltransferase (MTase) activity is of great significance in methylation-related disease diagnosis and drug screening. Herein, a HpaII-assisted and linear amplification-enhanced exponential amplification strategy is proposed for sensitive and label-free detection of M.SssI MTase activity. The P1 probe contains self-complementary sequence 5′-CTAGCCGGCTAG-3′ at 3′-terminal. After denaturation and annealing, P1 probes hybridize with itself to generate P1 duplexes. M.SssI MTase induces methylation of cytosine at 5′-CG-3′ in P1 duplexes, and thus, HpaII fails to cleave at 5′-CCGG-3′ due to methylation sensitivity, leaving P1 duplex intact. Then, these intact P1 duplexes are extended along 3′-terminal through Vent (exo − ) DNA polymerase to generate dsDNA, which is recognized and nicked at the recognition sites by Nt.BstNBI, releasing two copies of primer X. Primer X hybridizes with X′ at the amplification template T1 (X′-Y′-X′) and then serves as primers to trigger the exponential amplification reaction (EXPAR). The point of inflection (POI) values of real-time fluorescence curves is linearly correlated with the logarithm of M.SssI MTase concentration in the range of 0.125 \(-\) 8 U mL −1 with a low detection limit of 0.034 U mL −1 . In the absence of M.SssI, P1 duplexes are cut by HpaII and separated into ssDNA under the executed temperature of EXPAR and thus unable to trigger the amplification. The strategy provides good selectivity against other types of MTases and protein and is able to detect M.SssI activity in human serum. Furthermore, the analytical method has the generality and can be extended to the analysis of other types of DNA MTases. Graphical Abstract
Di Liu,Xuelian Chen,Yaping Li,Guiping Sun,Jingwen Gao,Hailiang Nie,Cuiping Li
The abuse of 17β-estradiol (E2) in breeding and the excretion into the environment by human beings result in exposure of E2 to humans. Excessive levels of E2 can cause an adverse effect on the human endocrine systems. Therefore, an efficient and sensitive method for the analysis of E2 is required. Herein, based on the specific capture of aptamer, the digestion of Exonuclease III, and efficient amplification of isothermal exponential amplification reaction (EXPAR), a highly sensitive and selective strategy is established for E2 analysis. E2 binds to the aptamer due to their high affinity, preventing the complementary DNA (cDNA) of the aptamer from hybridizing with it. Thus, the cDNA remains unbound and freely available in the solution. Then, Exonuclease III digests the cDNA-aptamer complex, leaving the free cDNA intact. cDNA serves as primers to trigger EXPAR with the help of Bst 2.0 WarmStart DNA polymerase and Nt.BstNBI nicking enzyme, achieving sensitive detection of E2. Without E2, cDNA hybridizes with the aptamer to form double-stranded DNA, which is degraded by Exonuclease III. As a result, the amplification reaction fails to initiate. The approach shows a linear range of 100 aM-10 pM, spanning 5 orders of magnitude, with the limit of detection as low as 36 aM. In addition, the selectivity and reproducibility are great, and the recoveries in tap water and milk ranges from 99.4 % to 117.3 %, suggesting good practicability. This method may also provide a new possibility for sensitive detection of other small-molecule targets via using the corresponding aptamers.
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