A label-free near-infrared fluorescent assay for the determination of deoxyribonuclease I activity based on malachite green/G-quadruplexes

Abstract

Owing to the biological and clinical significance of deoxyribonuclease I (DNase I), it is highly desirable to develop near-infrared (NIR) fluorescent assays for the determination of DNase I activity. Here we report a label-free NIR fluorescent assay for selective determination of DNase I activity based on malachite green (MG)/G-quadruplexes. In the presence of Na(+) or K(+), single stranded DNA (ssDNA) is able to form a G-quadruplex structure, thus to increase the rigidity of MG structure and result in a remarkable NIR fluorescence. As DNase I is capable of cleaving all types of DNA indiscriminately to release nucleotide products, the G-quadruplexes are cleaved into oligonucleotides in the presence of DNase I. As a result, the rigidity of MG structure is reduced, and the NIR fluorescence of the solution decreases with increase of DNase I activity, providing a useful platform for low-cost, label-free and convenient detection of DNase I activity. Under the optimum conditions, the proposed label-free NIR fluorescent assay gave a detection limit of 1 u mL(-1), and a relative standard deviation of 3.2% for eleven replicate detections of 50 u mL(-1) DNase I. The proposed assay was applied to the determination of DNase I activity in spiked human urine samples with recoveries from 99.1 to 109.0%.

Expanding the Toolbox for Label-Free Enzyme Assays: A Dinuclear Platinum(II) Complex/DNA Ensemble with Switchable Near-IR Emission

“…The high cost and synthetic challenges encountered in developing fluorophore-labelled DNA probes render label-free assays more convenient and cost-effective alternatives. Most existing label-free DNAse I assays are based on “turn-off” fluorescence signaling, which can lead to reduced sensitivity and false positive responses [28,29,30,31,32,33]. Consequently, “turn-on” luminescence bioassays are more attractive, but only one label-free turn-on DNAse I assay has been reported.…”

Salt-Assisted Graphene Oxide Dispersive Solid Phase Microextraction for Sensitive Detection of Malachite Green and Crystal Violet by HPLC

“…In the past decades, various techniques for detection of MG and CV have been reported, such as spectrophotometric method [7], fluorescence [8], surface-enhanced Raman scattering [9], enzyme-linked-immunosorbent assay (ELISA) [10], electrochemical aptasensor [11], capillary electrophoresis (CE) [12,13], as well as high-performance liquid chromatography [14,15]. The spectrometric, electrochemical approaches cannot be used for simultaneous detection of MG and CV, and usually suffered from serious matrix effect.…”

Chiral supernanostructures for ultrasensitive endonuclease analysis

“…However, these methods have the disadvantages of being insensitive, time-consuming, requiring sophisticated instruments or being laborious. To address these problems, novel detection strategies such as uorescent assay, [14][15][16][17][18] electrochemical methods [19][20][21] and colorimetric assay [22][23][24] have been developed. Of these strategies, the colorimetric method is simple and low-cost, but the sensitivity is relatively low.…”

“…In addition, low DNase I activity can initiate human or animal systemic lupus erythematosus and is associated with stomach or colon cancer, while high DNase I activity is connected with breast cancer. 18,44 Thus, it is an urgent demand for developing a simple, sensitive and fast strategy to assay DNase I activity. The sensing principle presented here was based on the changes of the CD spectrum when the disassembly of chiral pyramids took place using different amounts of DNase I.…”