Biosensor for a Rapid and Sensitive Detection and Quantification of Nuclease Activity

Abstract

Nucleases are family of hydrolytic enzymes which cleave the phosphodiesteric bonds between the nucleotides producing 3'-phospho or 5'-phospho end products. These enzymes can be classified as endo- and exo-nucleases, cleaving inside of a polynucleotide chain or at the ends respectively, however, some enzymes may have both activities. Typically, some nucleases prefer double-stranded nucleic acid (e.g. DNase I), while other cleave single-stranded (e.g. Nuclease P1) or RNA-DNA hybrid (RNase H) polynucleotides. Deoxyribonucleases (DNases) are important objects in biotechnology presenting both as tools for manipulations with DNA1 as well as contaminants where nuclease-like activity is undesired.2 In addition, data emerges indicating the importance of DNase activity detection for medical applications, i.e., DNases may be related with various diseases and could be used to monitor the progress of various cancers3 , 4 as well as could be a rapid and fast biomarker of bacterial infections.5 Typically, DNases are being detected and quantified using classical approach: fluorescence detection or gel electrophoresis techniques. Though those methods are highly sensitive and reliable, they are costly, time-consuming as well as require toxic markers such as ethidium bromide or radiolabeled molecules. As alternative methods novel detection techniques for DNase detection were reported, mostly based on fluorescence or chemiluminescence detection incorporating nanomaterials for signal enhancement.6 In addition, electrochemical biosensors, provide simple and inexpensive platform for biomolecule detection while maintaining a high degree of accuracy and sensitivity. However, to this day the absolute majority of electrochemical biosensors are being developed from small biomolecules, e.g., glucose, glycerol, lactate while the development of large marcomolecules, i.e., proteins or enzymes is still lagging. In a case of DNase detection, a few biosensors have been reported. Sato et al. reported electrochemical biosensor for DNase I detection based on ferrocene-modified DNA immobilized on the electrode surface with the detection limit (LOD) up to 10–2 U mL–1. Ding and Qin have reported a potentiometric biosensor for the detection of DNAses based on polycation-sensitive membrane, and for DNase I the LOD was 0.45 U mL–1.7 Despite advances, the developed electrochemical biosensors for DNase detection still lack sensitivities comparable to fluorescence- or radiolabeling-based detection methods and reliability required for the analysis of real samples.In this work we present a rapid and sensitive bioelectrochemical device for the determination of nuclease activity in various fluids. The device system consists of a sensor electrode, a special design DNA target to maximize the nuclease cleavage rate, signal analysis algorithm and the supporting electronics. The developed sensor allows to determine DNase activity in range 0.0003–0.03 U mL–1 with the limit of detection up to 0.00034 U mL–1 over 15 min measurement (calibrated against DNase I standard) as well as has a high storage stability. The system was also implemented using inexpensive single-used electrodes. Moreover, the sensor was tested measuring nuclease activity in real unmodified human saliva samples and demonstrated high accuracy compared to the industry standard DNaseAlert™ QC System. The developed technology could significantly improve nuclease quality control processes in pharma/biotech industry and give new insights into nuclease importance for medical applications. References (1) Pan, Y.; Xiao, L.; Li, A. S. S.; Zhang, X.; Sirois, P.; Zhang, J.; Li, K. Mol Biotechnol 2013, 55 (1), 54–62.(2) Senavirathne, G.; Liu, J.; Lopez, M. A.; Hanne, J.; Martin-Lopez, J.; Lee, J.-B.; Yoder, K. E.; Fishel, R. Nat Methods 2015, 12 (10), 901–902.(3) Patel, P. S.; Patel, B. P.; Rawal, R. M.; Raval, G. N.; Patel, M. M.; Patel, J. B.; Jha, F. P.; Patel, D. D. Tumor Biol 2000, 21(2), 82–89.(4) Balian, A.; Hernandez, F. J. Biomark Res 2021, 9 (1), 86.(5) Flenker, K. S.; Burghardt, E. L.; Dutta, N.; Burns, W. J.; Grover, J. M.; Kenkel, E. J.; Weaver, T. M.; Mills, J.; Kim, H.; Huang, L.; Owczarzy, R.; Musselman, C. A.; Behlke, M. A.; Ford, B.; McNamara, J. O. Molecular Therapy 2017, 25 (6), 1353–1362.(6) Mozioğlu, E.; Akgoz, M.; Kocagöz, T.; Tamerler, C. Anal. Methods 2016, 8 (20), 4017–4021.(7) Ding, J.; Qin, W. Biosensors and Bioelectronics 2013, 47, 559–565. Figure. The general operation scheme of nuclease biosensor. (A) In a case a sample does not contain nuclease the Target nucleotide is not cleaved and its hybridization rate is high. (B) In a case sample contains nucleases the Target nucleotide is cleaved and its hybridization rate is lower and relates to the nuclease activity. Figure 1