Heating promoted super sensitive electrochemical detection of p53 gene based on alkaline phosphatase and nicking endonuclease Nt.BstNBI-assisted target recycling amplification strategy at heated gold disk electrode.

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.

Plasmonic Au nanocube enhanced SERS biosensor based on heated electrode and strand displacement amplification for highly sensitive detection of Dam methyltransferase activity.

In this work, a novel "turn-on" mode Au nanocubes (AuNCs) enhanced surface-enhanced Raman scattering (SERS) biosensing platform coupled with heated Au electrode (HAuE) and strand displacement amplification (SDA) strategy was proposed for highly sensitive detection of DNA adenine methylation (Dam) Methyltransferase (MTase) activity. The Dam MTase and DpnI enzyme activities were significantly increased by elevating the HAuE surface temperature, resulting in the rapid production of template DNA for later SDA. During the SDA process, the released single-stranded DNA (ssDNA) could be amplified exponentially, and its concentration was positively related to the Dam MTase activity. The plasmonic AuNCs in SERS tags could provide significant SERS enhancement due to their "lightning rod" effect resulting from the sharp feature of the edges and corners of AuNCs. Because of these factors, the proposed biosensors exhibited high sensitivity in detecting the Dam MTase activity. The limit of detection was estimated to be 8.65 × 10-5 U mL-1, which was lower than that in most of the sensors for detection of Dam MTase activity in the literature. This SERS biosensor could also be used to screen inhibitors of Dam MTase and had the potential for detecting Dam MTase activity in real biological samples.

AuNanostar@4-MBA@Au Core-Shell Nanostructure Coupled with Exonuclease III-Assisted Cycling Amplification for Ultrasensitive SERS Detection of Ochratoxin A.

The "turn-on" mode surface-enhanced Raman scattering (SERS) aptasensor for ultrasensitive ochratoxin A (OTA) detection was developed based on the SERS "hot spots" of AuNanostar@4-MBA@Au core-shell nanostructures (AuNS@4-MBA@Au) and exonuclease III (Exo III)-assisted target cycle amplification strategy. Compared with conventional gold nanoparticles, AuNS@4-MBA@Au provides a much higher SERS enhancement factor because AuNS exhibits a larger surface roughness and the lightning rod effect, as well as an excellent electromagnetic field between the AuNS core and the Au shell, which contribute to the superstrong SERS signal. Meanwhile, Exo III-assisted target cycle amplification can be used as an effective method for the further amplified detection of OTA. Additionally, the utilization of streptavidin magnesphere paramagnetic particles offers a green, economical, and facile technology for the accumulation and separation of the signal probe AuNS@4-MBA@Au from solution. All these factors lead to a significant enhancement of detectable signals and superhigh sensitivity. As a result, the limit of detection as low as 0.25 fg mL-1 could be achieved, which was lower than that in the other reported literatures on SERS methods for OTA detection as we know. The developed SERS aptasensor also provides a promising tool for foodstuff detection.