Metronidazole To improve the performance of the immunoassay

To improve the performance of the immunoassay, various nanomaterials including carbon nanospheres [9], gold nanoparticles [10] and transition metal compounds [11] were used to immobilize natural enzymes as the label. Natural enzymes, such as horseradish peroxidase (HRP) [12] and glucose oxidase (GOD) [13] toward the oxidation and reduction of the substrate, or alkaline phosphatase (ALP) [14] toward the hydrolysis of the substrate, possess excellent Metronidazole transfer and biological catalysis. However, such enzymes are usually composed of protein subunits or RNA subunits, thus are unavoidably subjected to some inherent drawbacks including rigorous catalysis condition, expensive extraction or separation, and protein denaturation [15]. Therefore, they are still limited in the wide application. Recently, researchers have continuously explored novel mimic enzymes such as cyclodextrins, calixarenes, crown ethers and porphyrins [16]. Especially, nanozymes, a class of nanomaterials with enzymatic activity including Fe3O4 [17], Fe2O3 [18], Co3O4 [19], CuO [20], V2O5 [21], MnO2 [22] and CeO2 [23], have emerged. Due to their large specific surface area, surface active centers and biocompatibility, their catalytic activities are far greater than natural enzymes. In addition, such nanozymes can be easily mass-produced with inorganic compounds but not easily deactivated in a harsh environment. Thus, nanozymes have been widely used in biomedicine, biosensing, and bioimage [24]. Especially, some nanozymes have been reported with a peroxidase activity to catalyze the oxidation of hydrogen peroxide with 3,3′,5,5′-tetramethylbenzidine (TMB) [25] or amplex-red [26], or the color reaction of 2, 2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) with o-phenylenediamine [27]. Additionally, DNAzyme, as another kind of functional single-strand DNA or RNA with unique properties such as high catalytic ability, versatility, and designability, has been used in Pb2+-specific DNAzyme, Mg2+-specific DNAzyme [28], Cu2+-specific DNAzyme [29] and Ca2+-specific DNAzyme [30]. Specifically, with the aid of K+, a guanine-rich DNA sequence can be folded into a special structure to form G-quadruplex and then associated with hemin to generate G-quadruplex/hemin with similar peroxidase activities. Due to its distinct properties including low cost, high stability, and electrochemical property, it has been widely applied in biosensors [31]. The recent development of nucleic acid circuits, such as hybridization chain reaction (HCR), exponential amplification reaction, and rolling circle amplification [32], can enable the sufficient replication of DNA sequences. Particularly, as the autonomous self-assembly of two single DNA strands, HCR is an in-situ isothermal amplification technique to form long and repetitive DNA sequences, which can provide abundant signals for biosensing [33]. Herein, by using double-integrated mimic enzymes from copper hydroxide nanozyme and G-quadruplex/hemin DNAzyme, a visual immunoassay was developed to detect MC-LR with high sensitivity and selectivity. In this immunoassay, the microplates were modified with flaky nickel silicate-coated silica nanospheres (SiO2@Ni Silicate) to immobilize antigens, while Cu(OH)2 nanocages were used to capture the secondary antibody for the recognition of immunoreaction and DNA primer for the propagation. Both Cu(OH)2 nanozyme and G-quadruplex/hemin DNAzyme showed the peroxidase activity to ABTS with the aid of H2O2, which can improve the visual signal greatly. By using a competitive immunoassay, MC-LR was successfully detected with convenient and sensitive visualization, which is critical for practical MC-LR detection.
Experimental
Results and discussion
Conclusion In summary, based on double-integrated artificial enzyme from copper hydroxide nanozyme and G-quadruplex/hemin DNAzyme, we proposed an ultrasensitive and enzyme-free immunosensor to detect MC-LR with a low detection limit of 6 ng/L. These excellent performances were ascribed to the following features. First, core-shell silica/nickel silicates with large surface area were used as the substrate to capture sufficient MC-LR antigens. Second, Cu(OH)2 nanocages can be easily tuned with ammonium to achieve the optimal and stable catalytic ability. Third, HCR signal amplification strategy was used to form multiple G-quadruplex/hemin DNAzymes. Finally, such double-integrated artificial enzyme showed a stable and catalytic ability to H2O2 and ABTS, where the color change of ABTS can realize the visual detection of MC-LR. The successful characterization of this work may offer researchers to engage in materials for more convenient and visual detection of pollutants, pesticides or other organic molecules.