DNAzyme Amplified Aptasensing Platform for Ochratoxin A Detection Using a Personal Glucose Meter

Abstract

Aptamer-based sensors have emerged as a major platform for detecting small-molecular targets, because aptamers can be selected to bind these small molecules with higher affinity and selectivity than other receptors such as antibodies. However, portable, accurate, sensitive, and affordable detection of these targets remains a challenge. In this work, we developed an aptasensing platform incorporating magnetic beads and a DNAzyme for signal amplification, resulting in high sensitivity. The biosensing platform was constructed by conjugating a biotin-labeled aptamer probe of small-molecular targets such as toxins and a biotinlabeled substrate strand on magnetic beads, and the DNAzyme strand hybridized with the aptamer probe to block the substrate cleavage activity. The specific binding of the small-molecular target by the aptamer probe can replace the DNAzyme strand and then induce the hybridization between the DNAzyme strand and substrate strand, and the iterative signal amplification reaction of hydrolysis and cleavage of the substrate chain occurs in the presence of a metal ion cofactor. Using invertase to label the substrate strand, the detection of small molecules of the toxin is successfully transformed into the measurement of glucose, and the sensitive analysis of small molecules such as toxins can be realized by using the household portable glucose meter as a readout. This platform is shown to detect ochratoxin, a common toxin in food, with a linear detection range of 5 orders of magnitude, a low detection limit of 0.88 pg/mL, and good selectivity. The platform is easy to operate and can be used as a potential choice for quantitative analysis of small molecules, at home or under point-of-care settings. Moreover, by changing and designing the aptamer probe and the arm of DNAzyme strand, it can be used for the analysis of other analytes.

Self-Protected DNAzyme Walker with a Circular Bulging DNA Shield for Amplified Imaging of miRNAs in Living Cells and Mice

“…To generate an amplified signal output, some protein enzyme-mediated isothermal signal amplification methods, such as rolling circle amplification (RCA) , and isothermal circular strand displacement polymerization (ICSDP), have been employed for miRNA detection. However, protein enzymes are susceptible to changes in the surrounding microenvironment and often exhibit a compromised catalytic activity in cellular milieu. , Recently, DNAzyme-mediated catalytic (DNAzymatic) reaction, as an emerging class of protein enzyme-free signal amplification methods, has been explored. ,− Among them, RNA-cleaving DNAzymes are of particular interest for the detection of miRNAs due to their excellent activity and flexible programmability. − However, the application of DNAzymes in the intracellular detection of miRNAs still faces two major issues. First, owing to the highly negative charge backbone and hydrophilic nature of the nucleobases, it is difficult for DNAzymes to traverse the lipophilic cell membrane.…”

Antibody–Invertase Fusion Protein Enables Quantitative Detection of SARS-CoV-2 Antibodies Using Widely Available Glucometers

“… 26 To avoid the steric issues that likely interfere with efficient chemical conjugation methods, invertase has been conjugated to a small nucleic acid aptamer. 27 , 28 Just one group has reported the direct, site-specific, enzyme-mediated conjugation of invertase to an antibody single-chain fragment (scFv), 29 and though this method had the advantage of ensuring a consistent ratio of invertase to the detection molecule, conjugation efficiency was found to be very low.…”

Programmable, Universal DNAzyme Amplifier Supporting Pancreatic Cancer-Related miRNAs Detection

“…For example, Lu et al [35] developed a signal amplification strategy based on DNAzyme catalytic cutting and the utilization of CHA to detect low concentrations of endogenous metal ions in cells. Furthermore, they also took advantage of DNAzyme to establish an aptamer sensing platform based on the combination of magnetic beads and DNAzyme for the highly sensitive detection of small molecules such as toxins [36,37]. Based on this, there is the potential to construct a universal DNAzyme amplifier through integration of the programmable nature of DNA nanodevices and the catalytic capability of DNAzyme, which can be conveniently applied for the detection of a wide range of pancreatic cancer-related miRNAs through reasonable sequence design.…”