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DNAzyme-Au nanoprobe coupled with graphene-oxide–loaded hybridization chain reaction signal amplification for fluorometric determination of alkaline phosphatase

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Abstract

A sensing platform is presented for the determination of alkaline phosphatase (ALP) activity based on the cooperation of DNAzyme-Au spherical nucleic acid nanoprobe with the graphene-oxide–loaded hybridization chain reaction (HCR/GO) system to achieve good detection sensitivity and specificity. This assay takes advantage of the strong affinity of pyrophosphate (PPi) to Cu2+ ions and the fact that ALP can hydrolyze pyrophosphate (PPi) to release free Cu2+ ions. In the presence of ALP, the released Cu2+ can promote the Cu2+-dependent DNAzyme to cleave the substrate that generates a shorter DNA fragment, which is responsible for further triggering the HCR/GO system to form a long fluorescence dsDNA and thereby giving an amplified fluorescence signal. Linear calibration range was obtained from 0.2 to 10 U L−1, and the limit of detection (LOD) is about 0.14 U L−1. The feasibility of the proposed method was validated by spiking ALP standards in bovine serum. The recovery ranged from 97.2 to 104.6%, and a coefficient of variation (CV) of less than 8% (n = 3) was obtained. This assay strategy was also applied to evaluate the ALP inhibitor efficiency, which indicates that the assay has potential for drug screening.

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References

  1. Siller AF, Whyte MP (2018) Alkaline phosphatase: discovery and naming of our favorite enzyme. J Bone Miner Res 33:362–364

    Article  Google Scholar 

  2. Millán JL (2006) Alkaline phosphatases. Purinergic Signal 2:335–341

    Article  Google Scholar 

  3. Han Y, Chen J, Li Z, Chen H, Qiu H (2020) Recent progress and prospects of alkaline phosphatase biosensor based on fluorescence strategy. Biosens Bioelectron 148:111811

    Article  CAS  Google Scholar 

  4. Zhao WW, Xu JJ, Chen HY (2015) Photoelectrochemical bioanalysis: the state of the art. Chem Soc Rev 44:729–741

    Article  CAS  Google Scholar 

  5. Song Z, Kwok RTK, Zhao E, He Z, Hong Y, Lam JWY, Liu B, Tang BZ (2014) A ratiometric fluorescent probe based on ESIPT and AIE processes for alkaline phosphatase activity assay and visualization in living cells. ACS Appl Mater Inter 6:17245–17254

    Article  CAS  Google Scholar 

  6. Tang Z, Chen H, He H, Ma C (2019) Assays for alkaline phosphatase activity: progress and prospects. TrAC-Trend Anal Chem 113:32–43

    Article  CAS  Google Scholar 

  7. Niu X, Ye K, Wang L, Lin Y, Du D (2019) A review on emerging principles and strategies for colorimetric and fluorescent detection of alkaline phosphatase activity. Anal Chim Acta 1086:29–45

    Article  CAS  Google Scholar 

  8. Jin C, He J, Zou J et al (2019) Phosphorylated lipid-conjugated oligonucleotide selectively anchors on cell membranes with high alkaline phosphatase expression. Nat Commun 10:1–7

    Article  Google Scholar 

  9. Zhang L, Nie JF, Wang HL, Yang JH, Wang BY, Zhang Y, Li JP (2017) Instrument-free quantitative detection of alkaline phosphatase using paper-based devices. Anal Methods 9:3375–3379

  10. Sun J, Zhao J, Bao X, Wang Q, Yang X (2018) Alkaline phosphatase assay based on the chromogenic interaction of diethanolamine with 4-aminophenol. Anal Chem 90:6339–6345

    Article  CAS  Google Scholar 

  11. Park KS, Lee CY, Park HG (2014) A sensitive dual colorimetric and fluorescence system for assaying the activity of alkaline phosphatase that relies on pyrophosphate inhibition of the peroxidase activity of copper ions. Analyst 139:4691–4695

    Article  CAS  Google Scholar 

  12. Ma JL, Yin BC, Wu X, Ye BC (2016) Copper-mediated DNA-scaffolded silver nanocluster on-off switch for detection of pyrophosphate and alkaline phosphatase. Anal Chem 88:9219–9225

    Article  CAS  Google Scholar 

  13. Kong RM, Fu T, Sun NN, Qu FL, Zhang SF, Zhang XB (2013) Pyrophosphate-regulated Zn2+-dependent DNAzyme activity: an amplified fluorescence sensing strategy for alkaline phosphatase. Biosens Bioelectron 50:351–355

    Article  CAS  Google Scholar 

  14. Das P, Chandar NB, Chourey S, Agarwalla H, Ganguly B, Das A (2013) Role of metal ion in specific recognition of pyrophosphate ion under physiological conditions and hydrolysis of the phosphoester linkage by alkaline phosphatase. Inorg Chem 52:11034–11041

    Article  CAS  Google Scholar 

  15. Kang W, Ding Y, Zhou H, Liao Q, Yang X, Yang Y, Jiang J, Yang M (2015) Monitoring the activity and inhibition of alkaline phosphatase via quenching and restoration of the fluorescence of carbon dots. Microchim Acta 182:1161–1167

    Article  CAS  Google Scholar 

  16. Wang F, Zhang C, Xue Q, Li H, Xian Y (2017) Label-free upconversion nanoparticles-based fluorescent probes for sequential sensing of Cu2+, pyrophosphate and alkaline phosphatase activity. Biosens Bioelectron 95:21–26

    Article  CAS  Google Scholar 

  17. Zhou W, Saran R, Liu J (2017) Metal sensing by DNA. Chem Rev 117:8272–8325

    Article  CAS  Google Scholar 

  18. Yin BC, Zuo P, Huo H, Zhong X, Ye BC (2010) DNAzyme self-assembled gold nanoparticles for determination of metal ions using fluorescence anisotropy assay. Anal Biochem 401:47–52

    Article  CAS  Google Scholar 

  19. Wu P, Hwang K, Lan T, Lu Y (2013) A DNAzyme-gold nanoparticle probe for uranyl ion in living cells. J Am Chem Soc 135:5254–5257

    Article  CAS  Google Scholar 

  20. Tang Z, Zhang H, Ma C, Gu P, Zhang G, Wu K, Chen M, Wang K (2018) Colorimetric determination of the activity of alkaline phosphatase based on the use of Cu (II)-modulated G-quadruplex-based DNAzymes. Microchim Acta 185:109

    Article  Google Scholar 

  21. Peng H, Newbigging AM, Wang Z, Tao J, Deng W, Le XC, Zhang H (2018) DNAzyme-mediated assays for amplified detection of nucleic acids and proteins. Anal Chem 90:190–207

    Article  CAS  Google Scholar 

  22. Peng H, Newbigging AM, Reid MS et al (2019) Signal amplification in living cells: a review of microRNA detection and imaging. Anal Chem 92:292–308

    Article  Google Scholar 

  23. Zhang Y, Fan J, Nie J, Le S, Zhu W, Gao D, Yang J, Zhang S, Li J (2015) Timing readout in paper device for quantitative point-of-use hemin/G-quadruplex DNAzyme-based bioassays. Biosens Bioelectron 73:13–18

    Article  CAS  Google Scholar 

  24. Gao R, Xu L, Hao C, Xu C, Kuang H (2019) Circular polarized light activated chiral satellite nanoprobes for the imaging and analysis of multiple metal ions in living cells. Angew Chem 131:3953–3957

    Article  Google Scholar 

  25. Hong M, Xu L, Xue Q, Li L, Tang B (2016) Fluorescence imaging of intracellular telomerase activity using enzyme-free signal amplification. Anal Chem 88:12177–12182

    Article  CAS  Google Scholar 

  26. Si H, Sheng R, Li Q, Feng J, Li L, Tang B (2018) Highly sensitive fluorescence imaging of Zn2+ and Cu2+ in living cells with signal amplification based on functional DNA self-assembly. Anal Chem 90:8785–8792

    Article  CAS  Google Scholar 

  27. He S, Song B, Li D, Zhu C, Qi W, Wen Y, Wang L, Song S, Fang H, Fan C (2010) A graphene nanoprobe for rapid, sensitive, and multicolor fluorescent DNA analysis. Adv Funct Mater 20:453–459

    Article  CAS  Google Scholar 

  28. Cutler JI, Auyeung E, Mirkin CA (2012) Spherical nucleic acids. J Am Chem Soc 134:1376–1391

    Article  CAS  Google Scholar 

  29. Yang Y, Zhong S, Wang K, Huang J (2019) Gold nanoparticle based fluorescent oligonucleotide probes for imaging and therapy in living systems. Analyst 144:1052–1072

    Article  CAS  Google Scholar 

  30. Shen C, Li X, Rasooly A, Guo L, Zhang K, Yang M (2016) A single electrochemical biosensor for detecting the activity and inhibition of both protein kinase and alkaline phosphatase based on phosphate ions induced deposition of redox precipitates. Biosens Bioelectron 85:220–225

    Article  CAS  Google Scholar 

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Acknowledgments

The authors are thankful for the support of this work by the National Natural Science Foundation of China (Grant No. 21575165), the Hunan Provincial Science and Technology Plan Project, China (No. 2019TP1001), and the Innovation-Driven Project of Central South University (2020CX002).

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Authors and Affiliations

  1. Department of Chemistry and the MOE Key Laboratory of Spectrochemical Analysis and Instrumentation, College of Chemistry and Chemical Engineering and State Key Lab of Marine Environmental Science, Xiamen University, Xiamen, 361005, China

    Zhengxian Lv & Qiuquan Wang

  2. Hunan Provincial Key Laboratory of Micro & Nano Materials Interface Science, College of Chemistry and Chemical Engineering, Central South University, Changsha, 410083, China

    Minghui Yang

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  1. Zhengxian Lv
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  2. Qiuquan Wang
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  3. Minghui Yang
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Correspondence to Qiuquan Wang or Minghui Yang.

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Lv, Z., Wang, Q. & Yang, M. DNAzyme-Au nanoprobe coupled with graphene-oxide–loaded hybridization chain reaction signal amplification for fluorometric determination of alkaline phosphatase. Microchim Acta 188, 7 (2021). https://doi.org/10.1007/s00604-020-04681-1

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  • DOI: https://doi.org/10.1007/s00604-020-04681-1

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