Abstract
Adenosine deaminase (ADA), able to catalyze the irreversible deamination of adenosine into inosine, can be found in almost all tissues and plays an important role in several diseases. In this work, we developed a label-free fluorescence method for the detection of adenosine deaminase activity and inhibition. In the presence of ADA, ATP has been shown to be hydrolyzed. The ATP aptamer was shown to form a G-quadruplex/thioflavin T (ThT) complex with ThT and exhibited an obvious fluorescence signal. However, the ATP aptamer could bind with ATP and exhibited a low fluorescence signal because of the absence of ADA. This assay showed high sensitivity to ADA with a detection limit of 1 U/L based on an SNR of 3 and got a good linear relationship within the range of 1–100 U/L with R2 = 0.9909. The LOD is lower than ADA cutoff value (4 U/L) in the clinical requirement and more sensitive than most of the reported methods. This technique exhibited high selectivity for ADA against hoGG I, UDG, RNase H and λexo. Moreover, this strategy was successfully applied for assaying the inhibition of ADA using erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) and, as such, demonstrated great potential for the future use in the diagnosis of ADA-relevant diseases, particularly in advanced drug development.
Highlights
Adenosine deaminase (ADA), a key hydrolytic enzyme in the purine metabolism, can catalyze the irreversible deamination of adenosine into inosine via removal of an amino group [1,2,3]
Overexpression of ADA may be closely related to hemolytic anemia [12], liver cancer, breast cancer, etc. [13]
Feng et al reported a fluorescence sensor for adenosine deaminase based on an adenosine-induced self-assembly of aptamer structures [20]
Summary
Adenosine deaminase (ADA), a key hydrolytic enzyme in the purine metabolism, can catalyze the irreversible deamination of adenosine (deoxyadenosine) into inosine (deoxyinosine) via removal of an amino group [1,2,3]. Various techniques have been used to study this enzyme type, including measuring the ammonia amount produced [14], high-performance liquid chromatography (HPLC) [15], colorimetric assay [16] and electrochemical aptasensors [17] Several limitations such as generally labor-intensive processes, complex sample preparations and low selectivity impede the overall applicability of these methods. Feng et al reported a fluorescence sensor for adenosine deaminase based on an adenosine-induced self-assembly of aptamer structures [20] All of these novel methods have been shown to be effective to assay ADA. These methods exhibited various disadvantages, including a time-consuming and complicated synthesis process of AgNCs or AuNCs, expensive fluorescence labeling and low sensitivity. Compared to currently reported methods [38,39,40], our assay provided high sensitivity and low cost
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