Abstract:Abstract:We report here the in vitro selection of DNA aptamers for electric eel acetylcholinesterase (AChE). One selected aptamer sequence (R15/19) has a high affinity towards the enzyme (K d = 157 ± 42 pM). Characterization of the aptamer showed its binding is not affected by low ionic strength (20 mM), however significant reduction in affinity occurred at high ionic strength (1.2 M). In addition, this aptamer does not inhibit the catalytic activity of AChE that we exploit through immobilization of the DNA … Show more
“…AChE (280 kDa, pl of 5.0–5.3), is a key enzyme at the neuromuscular junction in controlling the concentration of neurotransmitter, acetylcholine in mammals. AChE was an ideal point for comparison to thrombin due to its substantially larger molecular size (280 kDa vs. 37.5 kDa) and a reported recognition aptamer sequence with high affinity ( K d = 14 ± 1 pM) at an ionic strength comparable to our experimental conditions 49 , 50 . Fig.…”
The capability to screen a range of proteins at the single-molecule level with enhanced selectivity in biological fluids has been in part a driving force in developing future diagnostic and therapeutic strategies. The combination of nanopore sensing and nucleic acid aptamer recognition comes close to this ideal due to the ease of multiplexing, without the need for expensive labelling methods or extensive sample pre-treatment. Here, we demonstrate a fully flexible, scalable and low-cost detection platform to sense multiple protein targets simultaneously by grafting specific sequences along the backbone of a double-stranded DNA carrier. Protein bound to the aptamer produces unique ionic current signatures which facilitates accurate target recognition. This powerful approach allows us to differentiate individual protein sizes via characteristic changes in the sub-peak current. Furthermore, we show that by using DNA carriers it is possible to perform single-molecule screening in human serum at ultra-low protein concentrations.
“…AChE (280 kDa, pl of 5.0–5.3), is a key enzyme at the neuromuscular junction in controlling the concentration of neurotransmitter, acetylcholine in mammals. AChE was an ideal point for comparison to thrombin due to its substantially larger molecular size (280 kDa vs. 37.5 kDa) and a reported recognition aptamer sequence with high affinity ( K d = 14 ± 1 pM) at an ionic strength comparable to our experimental conditions 49 , 50 . Fig.…”
The capability to screen a range of proteins at the single-molecule level with enhanced selectivity in biological fluids has been in part a driving force in developing future diagnostic and therapeutic strategies. The combination of nanopore sensing and nucleic acid aptamer recognition comes close to this ideal due to the ease of multiplexing, without the need for expensive labelling methods or extensive sample pre-treatment. Here, we demonstrate a fully flexible, scalable and low-cost detection platform to sense multiple protein targets simultaneously by grafting specific sequences along the backbone of a double-stranded DNA carrier. Protein bound to the aptamer produces unique ionic current signatures which facilitates accurate target recognition. This powerful approach allows us to differentiate individual protein sizes via characteristic changes in the sub-peak current. Furthermore, we show that by using DNA carriers it is possible to perform single-molecule screening in human serum at ultra-low protein concentrations.
“…The DNA aptamer sequence was ordered from Sangon Biotech (Shanghai, China). The AChE aptamer sequence information is as follows: 5′‐GGT TGA CTG TAG CTC TGG CAG ACG TAG TGT GAA GGT ACC‐(CH 2 ) 3 ‐SH‐3′.…”
A feasible label-free electrochemiluminescence (ECL) aptasensor that uses an Au-nanoparticle-functionalized g-C 3 N 4 nanohybrid (Au-g-C 3 N 4 NH) as the luminophore was constructed for highly sensitive acetylcholinesterase (AChE) detection. The sensor was fabricated by successively modifying a glassy carbon electrode with Au-g-C 3 N 4 NH and thiol-modified AChE-specific aptamers. In the presence of AChE, the ECL signal decreased significantly, because AChE could hydrolyze the substrate acetylthiocholine to generate acetic acid, which could react with the co-reactant triethylamine (Et 3 N), leading to evident consumption of the coreactant. The ECL response of the aptasensor was linearly proportional to the concentration of AChE ranging from 0.1 pg/ mL to 10 ng/mL, with a detection limit of 42.3 fg/mL (S/N = 3). This novel ECL sensing strategy demonstrated a highly sensitive and selective method for AChE detection and was expected to possess potential applications in clinical diagnosis and biomedical technology.
“…Compared to antibodies, the capability to be selected against cytotoxic or very small compounds even under non-physiological parameters chosen with respect to the final application and with dissociation constants in a picomolar range, is advantageous. While in the past decade aptamers have been investigated in pharmacological/toxicological analytics of tissues [107], blood [108], water [109,110], food [111,112] or in therapeutically studies against cancer [113], infections [114] or in gene therapy [115], today, there are definite efforts of their use in upstream processing, e.g., of his-tagged proteins. Aptamers are frequently applied in an immobilized mode whereby nanoscaled/gold particles [117] or graphite layers [118] mediate the solid support.…”
For several thousand years, biotechnology and its associated technical processes have had a great impact on the development of mankind. Based on empirical methods, in particular for the production of foodstuffs and daily commodities, these disciplines have become one of the most innovative future issues. Due to the increasing detailed understanding of cellular processes, production strains can now be optimized. In combination with modern bioprocesses, a variety of bulk and fine chemicals as well as pharmaceuticals can be produced efficiently. In this article, some of the current trends in biotechnology are discussed.
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