Hydrazone chemistry assisted DNAzyme for the analysis of double targets

Anzhi Sheng a, Lihong Su a, Mohammed Jalalah b, M. S. Al-Assiri b, Farid A. Harraz *bc and Juan Zhang *a
aCenter for Molecular Recognition and Biosensing, School of Life Sciences, Shanghai University, Shanghai 200444, P. R. China. E-mail: juanzhang@shu.edu.cn
bPromising Centre for Sensors and Electronic Devices (PCSED), Advanced Materials and Nano-Research Centre, Najran University, P.O. Box: 1988, Najran 11001, Saudi Arabia. E-mail: faharraz@nu.edu.sa
cNanomaterials and Nanotechnology Department, Central Metallurgical Research and Development Institute (CMRDI), P.O. 87 Helwan, Cairo 11421, Egypt

Received 3rd December 2019 , Accepted 9th December 2019

First published on 10th December 2019


In this work, a hydrazone chemistry assisted DNAzyme has been designed and constructed. The introduction of hydrazone chemistry increases the versatility of DNAzymes. With superior catalytic capability, the hydrazone chemistry assisted DNAzyme has been successfully applied for the analysis of double targets. Taking 5-hydroxymethylfurfural (HMF) and lipopolysaccharide (LPS) as samples, the hydrazone chemistry assisted DNAzyme can be used for the detection of different combinations of targets. Moreover, because hydrazone chemistry is popular in nature, this work may also provide a new insight for the development of DNAzymes and their multifunctionality.


As a catalyst, DNAzymes can catalyze various reactions and they are single-stranded DNA molecules generated via in vitro selection.1–3 With high chemical and biological stability, DNAzymes can be synthesized conveniently, which will be beneficial for therapeutic and other biotechnological applications.4,5 For example, DNAzymes can be used as an ideal candidate for the development of amplified sensing platforms6,7 due to their properties including the flexibility in encoding recognition functions into DNAzyme sequences,8 the intrinsic cyclic biotransformations, and the reduced nonspecific absorptions. Nucleic acid sequences including an intact catalytic core can be utilized for signal amplification systems such as rolling circle amplification.5,9 Moreover, the split enzyme strands have also been explored for the detection of various molecules.10–13 In these research studies, the formation of the whole enzyme strand can be induced through base complementary pairing, which can be adjusted directly and indirectly by the introduction of a target.10–13

Except for non-covalent effect forces such as base complementary pairing, there are all kinds of covalent forces, which can originate from chemoselective ligation. As a kind of chemoselective ligation, hydrazone reaction happens between a hydrazide and a carbonyl with the equilibrium constant (>108 M−1).14,15 It has been applied in many research fields including sensors,16–19 dynamic combinatorial chemistry,20–22 metal and covalent organic frameworks,23,24 metallo-assemblies,25,26 and molecular switches.27–29 Regarding the design of split enzyme strands, the introduction of hydrazone ligation will undoubtedly enrich the multifunction of DNAzymes and further extend their application in different fields including therapy, biotechnology, biosensing and so on.

In this work, we design the split 8–17 DNAzyme1 assisted by hydrazone ligation and explore its application for different signal outputs. Meanwhile, a hydrazone ligation assisted DNAzyme has been successfully applied for the detection of double targets including lipopolysaccharides (LPS) and 5-hydroxymethylfurfural (HMF). Among them, LPS induces microcirculation disturbance, septic shock, fever reaction and diarrhea30 and HMF is an index of heat treatment and an indicator of deteriorative changes.31,32 Therefore, it is of great interest to analyze LPS and HMF in medical and food products.

The construction of the hydrazine chemistry assisted DNAzyme and the corresponding catalyzed reaction has been illustrated in Fig. 1(A). The enzyme strand is split into two parts, i.e. enzyme strand I (ESI) and enzyme strand II (ESII). Among them, ESI is modified with the hydrazine group at its terminal end and ESII is linked with the aldehyde group. Under mild conditions, the aldehyde group can react with the hydrazine group to form a hydrazone bond, resulting in the formation of the whole enzyme strand (WES). A new band appears for the mixture of ESI and ESII (Fig. S2, ESI) and the quasi-molecular ion [M − 2H + Na] peak at m/z 15000.7 can be found for WES in the ESI-MS spectrum (Fig. S3, ESI), suggesting the successful synthesis of WES. The formed WES can further hybridize with the substrate strand (SS) labelled with a fluorescent probe at one end and a quenching molecule at the other end. In the presence of Mg2+ ions, the SS strand is cleaved into two segments, leading to the dissociation of the hybridization strand and the recovery of quenched fluorescence.


image file: c9cc09389c-f1.tif
Fig. 1 (A) Schematic illustration for the hydrazone chemistry assisted DNAzyme. (B1) Fluorescence spectrum obtained through the cleavage of the substrate chain by the hydrazone chemistry assisted DNAzyme. (B2) Fluorescence spectrum obtained through the cleavage of the substrate chain by the full length DNAzyme strand (FES). (C1) Reaction rate versus the concentration of substrate chain for the hydrazone chemistry assisted DNAzyme. (C2) Reaction rate versus the concentration of substrate chain for the full length DNAzyme. (D1) Dynamic curve of the reaction catalyzed by the hydrazone chemistry assisted DNAzyme. (D2) Dynamic curve of the reaction catalyzed by the full length DNAzyme. (E) The measurements of kcat and KM from the Lineweaver–Burk plot for WES and FES.

We further study the influence of hydrazone chemistry on the catalytic activity of the DNAzyme and the corresponding results are shown in Fig. 1(B)–(D). Almost the same Cy3 fluorescence intensity can be found for the hydrazone chemistry assisted DNAzyme compared with that of the full length DNAzyme (Fig. 1(B1) and (B2)). This result well signifies that hydrazone ligation has a negligible influence on the catalytic activity of the DNAzyme dependent on metal ions. Moreover, the dynamic experiments suggest that the reactions catalyzed by the hydrazone chemistry assisted DNAzyme and the full length DNAzyme, respectively, conform to the first-order dynamic mode (Fig. 1(D1) and (D2)). Along with the increased reaction time, the concentration of the reactant gradually decreases and then remains unchanged. Inversely, the concentration of the product gradually increases and subsequently reaches a plateau. For the hydrazone chemistry assisted DNAzyme, half of the reactant (2.2 μM) can be transferred into the product at 2.4 min. For the full length DNAzyme, half of the reactant (2.5 μM) can be consumed at 2.5 min. Furthermore, similar values of the Michaelis constants (Km) and the rate constants (kcat) as well as the ratio of kcat/Km can be obtained for the hydrazone chemistry assisted DNAzyme, in comparison with those of the full length DNAzyme (Fig. 1(E)). These results well signify that the hydrazone chemistry assisted DNAzyme has an excellent catalytic capacity to cut ribonucleoside acid. The introduction of hydrazone ligation will undoubtedly increase the flexibility and the versatility of nucleases.

The mechanism for the analysis of double targets by using the hydrazone chemistry assisted DNAzyme is illustrated in Fig. 2(A). Without any target, ESI can react with ESII to form WES, which can catalyze the cleavage of SS in the presence of Mg2+ ions, resulting in the recovery of Cy3 fluorescence ((a), Fig. 2(A)). Meanwhile, the free probe strand (PS) does not exist in the supernatant after magnetic separation ((a), Fig. 2(A)). In the presence of only HMF which contains an aldehyde group in its molecular structure, HMF can competitively react with ESI due to its small molecular weight, so as to inhibit the formation of WES and the corresponding recovery of Cy3 fluorescence ((b), Fig. 2(A)). At the same time, no HMF occurs in the supernatant ((b), Fig. 2(A)). LPS can bind with LPS aptamer (LPSA) around magnetic beads (MB), leading to the release of PS into the solution. After magnetic separation, FAM fluorescence can be observed with only LPS ((c), Fig. 2(A)). Meanwhile, the formed WES can catalyze the cleavage of SS, thereby recovering Cy3 fluorescence ((c), Fig. 2(A)). In the presence of both HMF and LPS, the formation of WES will be prevented by HMF and SS cannot be cleaved, while PS release can occur as a result of binding between LPS and LPSA.


image file: c9cc09389c-f2.tif
Fig. 2 (A) Schematic illustration of the mechanism for the analysis of double targets by using the hydrazone chemistry assisted DNAzyme. (B) Fluorescence spectra obtained (a) in the absence of both HMF and LPS, (b) in the presence of only HMF, (c) in the presence of only LPS and (d) in the presence of both HMF and LPS. (C) Normalized fluorescence intensities of FAM versus different combinations of HMF and LPS. (D) Normalized fluorescence intensities of Cy3 versus different combinations of HMF and LPS.

As shown in Fig. 2(B), only one emission peak at 560 nm can be observed without both HMF and LPS (curve a). It can be explained for the cleavage of SS catalyzed by WES originating from the linkage of ESI with ESII through a hydrazone bond. There is no fluorescence peak found with only HMF (curve b, Fig. 2(B)). HMF can not only inhibit the cleavage of SS and the recovery of the corresponding Cy3 fluorescence, but also has no ability to induce the release of PS from the magnetic interface into the solution. Two evident peaks at 520 nm and 560 nm appear in the presence of only LPS (curve c, Fig. 2(B)). For one thing, LPS can induce the conformational change of LPSA and the following dissociation of PS from the double strand, resulting in the appearance of FAM fluorescence in the supernatant after magnetic separation. For another thing, LPS has no effect on the formation of WES, which can catalyze the cleavage of SS, so Cy3 fluorescence can be recovered. In the presence of both HMF and LPS, only one emission peak at 520 nm can be given (curve d, Fig. 2(B)). This can be attributed to the induced effect of LPS on the release of PS and the inhibition effect of HMF on the cleavage of SS. These results are in good agreement with our prediction, suggesting that the analysis of double targets is feasible.

The experimental conditions including ratio between ESII and ESI, Mg2+ concentration, pH value, time and temperature for hydrazone reaction, have been optimized (Fig. S4, ESI). Under optimized conditions, the fluorescence intensities have been further normalized to rapidly analyse HMF and LPS. Fig. 2(C) and (D) depict the normalized fluorescence intensities against different combinations of HMF and LPS. FAM fluorescence intensity lower than 0.45 and Cy3 intensity higher than 0.35 suggests that there is no occurrence of both HMF and LPS in the sample. If the fluorescence intensities of FAM and Cy3 are separately lower than 0.45 and 0.35, the sample contains only HMF. If the fluorescence intensities of FAM and Cy3 are higher than 0.45 and 0.35, respectively, there is only LPS in the sample. When both HMF and LPS appear, the fluorescence intensity of FAM will be higher than 0.45 and Cy3 intensity will be lower than 0.35. Therefore, the analysis of double targets can be quickly carried out by detecting the fluorescence intensities.

Dual targets including LPS and HMF have been quantitatively detected by using the hydrazone chemistry assisted DNAzyme and the corresponding results have been given in Fig. 3. With the increase of LPS concentration from 0.02 ng mL−1 to 8000 ng mL−1, the fluorescence intensities of FAM gradually increase and then reach a plateau, but the Cy3 intensities are relatively unchanged (Fig. 3(A)). It can be explained for the reason that the increased LPS can induce the increasing release of PS, resulting in a stepwise increase of FAM fluorescence intensities, and PS can be totally released when the LPS concentration reaches 2000 ng mL−1 (Fig. 3(B)). Moreover, LPS has a negligible influence on the formation of WES and its corresponding catalysis capability to cleave SS, so the Cy3 fluorescence intensities almost remain unchanged. In order to quantitatively analyze LPS, the ratio of the two-fluorescence emission peak intensities (IFAM/ICy3) has been calculated. It can be found that IFAM/ICy3 increases linearly with the logarithm of LPS concentrations from 0.08 ng mL−1 to 2000 ng mL−1 (inset, Fig. 3(B)), which is wider than the value reported previously.33,34 Meanwhile, a linear fitting equation of IFAM/ICy3 = 1.22253 + 0.29825 × log[thin space (1/6-em)]CLPS can be obtained with a correlation coefficient (R2) of 0.9917, where IFAM/ICy3 is the ratio between FAM fluorescence emission intensity and Cy3 intensity, and CLPS is LPS concentration. The lowest detection limit has been calculated to be 0.0796 ng mL−1, which is lower than that of the previous reports.33,35,36 Moreover, the experiments have been carried out more than three times in the LPS concentration range from 0.08 ng mL−1 to 2000 ng mL−1 and the mean relative standard deviation (RSD) value has been calculated to be 2.32%. This signifies that the established method exhibits acceptable repeatability and precision.


image file: c9cc09389c-f3.tif
Fig. 3 (A) Fluorescence spectra obtained with the addition of different concentrations of LPS. (B) Ratio between FAM fluorescence intensity and Cy3 fluorescence intensity (IFAM/ICy3) against different concentrations of LPS. Inset: Linear relationship between IFAM/ICy3 and the logarithm of LPS concentration. (C) Fluorescence spectra obtained with the addition of different concentrations of HMF. (D) Ratio between Cy3 fluorescence intensity and FAM fluorescence intensity (ICy3/IFAM) against different concentrations of HMF. Inset: Linear relationship between ICy3/IFAM and the logarithm of HMF concentration.

As shown in Fig. 3(C), with increasing concentration of HMF, the FAM and Cy3 fluorescence intensities decrease. This can be attributed to the reason that compared with the nucleic acid strand ESII, HMF is superior to competitively react with ESI due to its small molecular weight. So, the number of the formed WES will reduce, leading to the decreasing cleavage of SS and recovery of Cy3 fluorescence intensity. When the HMF concentrations increase from 0.01 μM to 5 μM, the ratios of the two-fluorescence emission peak intensities (ICy3/IFAM) linearly decrease. The linear fitting equation of ICy3/IFAM = 1.66098 − 2.92775 × log[thin space (1/6-em)]CHMF can be obtained with R2 of 0.99041, where CHMF is the HMF concentration. Meanwhile, the minimum detection limit of 0.0403 μM can be obtained and it is lower than the values reported previously (Fig. 3(D)).37–39 In addition, the RSD value has been calculated to be 3.02% by using the slopes of three regression equations with HMF concentrations from 0.01 μM to 5 μM. These results well demonstrate the good reproducibility and accuracy of the established method.

To further evaluate the analytical reliability and practical applicability of the established method, LPS and HMF with different concentrations have been added into different beverages and the content has been tested using our established method. As shown in Tables S2 and S3 (ESI), the recoveries are between 90% and 112%, and the relative errors are within 7%. This confirms that the established method based on the hydrazone chemistry assisted DNAzyme has good anti-interference ability, and it can be applied for the detection of LPS and HMF in real samples.

In summary, the enzyme strand of the DNAzyme has been split into two segments to construct the hydrazone chemistry assisted DNAzyme. In comparison with the full length DNAzyme, the hydrazone chemistry assisted DNAzyme exhibits admirable catalytic capability. Subsequently, the hydrazone chemistry assisted DNAzyme has been further designed for the analysis of HMF and LPS. The analysis of different combinations of double targets can be well realized with dual output of the fluorescence signals. Moreover, the concentrations of HMF and LPS can be quantitatively detected with a wide detection range and a low detection limit. In addition, the current-established method has been successfully applied for the analysis of HMF and LPS in soft drink samples.

The authors would like to acknowledge the support of the National Natural Science Foundation of China (Grant No. 31671923), Shanghai Pujiang Program (Grant No. 18PJD016) and the Ministry of Education, Kingdom of Saudi Arabia (PCSED-003-18).

Conflicts of interest

There are no conflicts to declare.

Notes and references

  1. S. W. Santoro and G. F. Joyce, Proc. Natl. Acad. Sci. U. S. A., 1997, 94, 4262–4266 CrossRef CAS PubMed.
  2. M. Liu, D. Chang and Y. Li, Acc. Chem. Res., 2017, 50, 2273–2283 CrossRef CAS PubMed.
  3. J. Feng, Z. Xu, F. Liu, Y. Zhao, W. Yu, M. Pan, F. Wang and X. Liu, ACS Nano, 2018, 12, 12888–12901 CrossRef CAS PubMed.
  4. Q. Wang, D. Zhang, M. Cheng, J. He and K. Liu, Acta Pharm. Sin. B, 2012, 2, 28–31 CrossRef CAS.
  5. J. J. Zhang, Catalysts, 2018, 8, 550 CrossRef.
  6. Q. Wu, H. Wang, K. Gong, J. Shang, X. Liu and F. Wang, Anal. Chem., 2019, 91, 10172–10179 CrossRef CAS PubMed.
  7. M. Hollenstein, Curr. Opin. Chem. Biol., 2019, 52, 93–101 CrossRef CAS PubMed.
  8. S. Kumar, S. Jain, N. Dilbaghi, A. S. Ahluwalia, A. A. Hassan and K. H. Kim, Trends Biochem. Sci., 2019, 44, 190–213 CrossRef CAS PubMed.
  9. S. A. McManus and Y. F. Li, J. Am. Chem. Soc., 2013, 135, 7181–7186 CrossRef CAS PubMed.
  10. E. Mokany, S. M. Bone, P. E. Young, T. B. Doan and A. V. Todd, J. Am. Chem. Soc., 2010, 132, 1051–1059 CrossRef CAS PubMed.
  11. B. Tian, Y. Y. Han, E. Wetterskog, M. Donolato, M. F. Hansen, P. Svedlindh and M. Stromberg, ACS Sens., 2018, 3, 1884–1891 CrossRef CAS PubMed.
  12. S. F. Bakshi, N. Guz, A. Zakharchenko, H. Deng, A. V. Tumanov, C. D. Woodworth, S. Minko, D. M. Kolpashchikov and E. Katz, J. Am. Chem. Soc., 2017, 139, 12117–12120 CrossRef CAS PubMed.
  13. L. M. Lu, X. B. Zhang, R. M. Kong, B. Yang and W. H. Tan, J. Am. Chem. Soc., 2011, 133, 11686–11691 CrossRef CAS PubMed.
  14. D. Anouk, D. Sjoerd, T. M. Hackeng and P. E. Dawson, J. Am. Chem. Soc., 2006, 128, 15602–15603 CrossRef PubMed.
  15. D. K. Kolmel and E. T. Kool, Chem. Rev., 2017, 117, 10358–10376 CrossRef CAS PubMed.
  16. J. Zhang, X. Wang, T. Chen, C. Feng and G. Li, Anal. Chem., 2017, 89, 13245 CrossRef CAS PubMed.
  17. S. Xin and A. Ivan, Chem. Soc. Rev., 2014, 45, 1963–1981 Search PubMed.
  18. J. Zhang, Y. Liu, J. Lv and G. Li, Nano Res., 2015, 8, 920–930 CrossRef CAS.
  19. H. Y. Lee, X. Song, H. Park, M. H. Baik and D. Lee, J. Am. Chem. Soc., 2010, 132, 12133–12144 CrossRef CAS PubMed.
  20. P. T. Corbett, L. Julien, V. Laurent, K. R. West, W. Jean-Luc, J. K. M. Sanders and O. Sijbren, Chem. Rev., 2006, 106, 3652–3711 CrossRef CAS PubMed.
  21. J. Yinghua, Y. Chao, R. J. Denman and Z. Wei, Chem. Soc. Rev., 2013, 42, 6634 RSC.
  22. I. Cvrtila, H. Fanlo-Virgos, G. Schaeffer, G. M. Santiago and S. Otto, J. Am. Chem. Soc., 2017, 139, 12459–12465 CrossRef CAS PubMed.
  23. D. N. Bunck and W. R. Dichtel, J. Am. Chem. Soc., 2013, 135, 14952–14955 CrossRef CAS PubMed.
  24. Z. Xiao-Ping, W. Yuan and L. Dan, J. Am. Chem. Soc., 2013, 135, 16062–16065 CrossRef PubMed.
  25. J. R. Nitschke, Chem. Soc. Rev., 2014, 43, 1798–1799 RSC.
  26. D. G. Reuven, H. Li, I. I. Harruna and X. Q. Wang, J. Mater. Chem., 2012, 22, 15689–15694 RSC.
  27. S. C. Burdette, Nat. Chem., 2012, 4, 695–696 CrossRef CAS PubMed.
  28. D. Ray, J. T. Foy, R. P. Hughes and I. Aprahamian, Nat. Chem., 2012, 4, 757–762 CrossRef CAS PubMed.
  29. S. Pramanik and I. Aprahamian, J. Am. Chem. Soc., 2016, 138, 15142–15145 CrossRef CAS PubMed.
  30. S. E. Kim, W. Su, M. Cho, Y. Lee and W. S. Choe, Anal. Biochem., 2012, 424, 12–20 CrossRef CAS PubMed.
  31. A. Theobald, A. Müller and E. Anklam, J. Agric. Food Chem., 1998, 46, 1850–1854 CrossRef CAS.
  32. U. M. Shapla, M. Solayman, N. Alam, M. I. Khalil and S. H. Gan, Chem. Cent. J., 2018, 12, 35 CrossRef CAS PubMed.
  33. P. Xie, L. Zhu, X. Shao, K. Huang, J. Tian and W. Xu, Sci. Rep., 2016, 6, 29524 CrossRef CAS PubMed.
  34. W. J. Shen, Y. Zhuo, Y. Q. Chai and R. Yuan, Anal. Chem., 2015, 87, 11345–11352 CrossRef CAS PubMed.
  35. S. J. Ding, B. W. Chang, C. C. Wu, C. J. Chen and H. C. Chang, Electrochem. Commun., 2007, 9, 1206–1211 CrossRef CAS.
  36. G. Priano, D. Pallarola and F. Battaglini, Anal. Biochem., 2007, 362, 108–116 CrossRef CAS PubMed.
  37. Y. Guan, X. Wu and H. Meng, J. Dairy Sci., 2013, 96, 4885–4890 CrossRef CAS PubMed.
  38. J. K. de Andrade, E. Komatsu, H. Perreault, Y. R. Torres, M. R. da Rosa and M. L. Felsner, Food Chem., 2016, 190, 481–486 CrossRef CAS PubMed.
  39. J. K. de Andrade, C. K. de Andrade, E. Komatsu, H. Perreault, Y. R. Torres, M. R. da Rosa and M. L. Felsner, Food Chem., 2017, 228, 197–203 CrossRef PubMed.

Footnote

Electronic supplementary information (ESI) available. See DOI: 10.1039/c9cc09389c

This journal is © The Royal Society of Chemistry 2020