In situ encapsulation of horseradish peroxidase in zeolitic imidazolate framework–8 enables catalyzing luminol reaction under near-neutral conditions for sensitive chemiluminescence determination of cholesterol


HRP@ZIF–8 nanocomposite was prepared by in situ encapsulation of horseradish peroxidase (HRP) in the frame of zeolitic imidazolate framework–8 (ZIF–8) with a simple one-pot method. The HRP@ZIF–8 nanocomposite displays outstanding thermal stability and efficiently catalyzes the chemiluminescence (CL) reaction of luminol with hydrogen peroxide (H2O2) under near-neutral pH condition (pH 7–8). This CL system has a good response to H2O2 with a linear range of 0.1–100.0 μmol L−1. The limit of detection (LOD) is 0.06 μmol L−1 H2O2. By marriage with cholesterol oxidase, cholesterol is determined with a linear range from 0.1 to 100.0 μmol L−1 and a LOD of 0.04 μmol L−1. The relative standard deviations (RSD) are 1.7% and 2.5%, respectively, in 11 repeated measurements of 50.0 μmol L−1 solutions of H2O2 and cholesterol, indicating excellent precision of the method. The method shows good selectivity and has been applied to the determination of total cholesterol in real serum samples. No significant difference has been observed between the results obtained by this method and the cholesterol oxidase–peroxidase coupling method.

Schematic presentation of in situ one-pot synthesis of horseradish peroxidase@zeolitic imidazolate framework–8 (HRP@ZIF–8) nanocomposite and chemiluminescence determination of cholesterol with HRP@ZIF–8 catalyzing luminol–H2O2 system.

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  1. 1.

    Cicero AFG, Rosticci M, Baronio C, Morbini M, Parini A, Grandi E, D’Addato S, Borghi C (2014) Serum LDL cholesterol levels and new onset of arterial hypertension: an 8–year follow–up. Eur J Clin Investig 44:926–932.

    CAS  Article  Google Scholar 

  2. 2.

    Su-Min J, Seulggie C, Kyuwoong K, Min KS, Gyeongsil L, Yong PS, Yeon-Yong K, Sik SJ, Jae-Moon Y, Min PS (2018) Effect of change in total cholesterol levels on cardiovascular disease among young adults. J Am Heart Assoc 7:e008819.

    CAS  Article  Google Scholar 

  3. 3.

    Silvente-Poirot S, Poirot M (2014) Cholesterol and cancer, in the balance. Science 343:1445–1446.

    Article  PubMed  Google Scholar 

  4. 4.

    Lin T, Zhong L, Chen H, Li Z, Song Z, Guo L, Fu F (2017) A sensitive colorimetric assay for cholesterol based on the peroxidase–like activity of MoS2 nanosheets. Microchim Acta 184:1233–1237.

    CAS  Article  Google Scholar 

  5. 5.

    Dhawane M, Deshpande A, Jain R, Dandekar P (2019) Colorimetric point–of–care detection of cholesterol using chitosan nanofibers. Sensors Actuators B Chem 281:72–79.

    CAS  Article  Google Scholar 

  6. 6.

    He Y, Niu X, Shi L, Zhao H, Li X, Zhang W, Pan J, Zhang X, Yan Y, Lan M (2017) Photometric determination of free cholesterol via cholesterol oxidase and carbon nanotube supported Prussian blue as a peroxidase mimic. Microchim Acta 184:2181–2189.

    CAS  Article  Google Scholar 

  7. 7.

    Chang H-C, Ho JA (2015) Gold nanocluster–assisted fluorescent detection for hydrogen peroxide and cholesterol based on the inner filter effect of gold nanoparticles. Anal Chem 87:10362–10367.

    CAS  Article  PubMed  Google Scholar 

  8. 8.

    Hassanzadeh J, Khataee A, Eskandari H (2018) Encapsulated cholesterol oxidase in metal–organic framework and biomimetic Ag nanocluster decorated MoS2 nanosheets for sensitive detection of cholesterol. Sensors Actuators B Chem 259:402–410.

    CAS  Article  Google Scholar 

  9. 9.

    Pramanik K, Sarkar P, Bhattacharyay D, Majumdar P (2018) One step electrode fabrication for direct electron transfer cholesterol biosensor based on composite of polypyrrole, green reduced graphene oxide and cholesterol oxidase. Electroanalysis 30:2719–2730.

    CAS  Article  Google Scholar 

  10. 10.

    Maluin FN, Sharifah M, Rattanarat P, Siangproh W, Chailapakul O, Issam AM, Manan NSA (2016) Synthesis of PANI/hematite/PB hybrid nanocomposites and fabrication as screen printed paper based sensors for cholesterol detection. Anal Methods 8:8049–8058.

    CAS  Article  Google Scholar 

  11. 11.

    Timofeeva II, Vakh CS, Bulatov AV, Worsfold PJ (2018) Flow analysis with chemiluminescence detection: recent advances and applications. Talanta 179:246–270.

    CAS  Article  PubMed  Google Scholar 

  12. 12.

    Hong L, Liu AL, Li GW, Chen W, Lin XH (2013) Chemiluminescent cholesterol sensor based on peroxidase–like activity of cupric oxide nanoparticles. Biosens Bioelectron 43:1–5.

    CAS  Article  PubMed  Google Scholar 

  13. 13.

    Hassanzadeh J, Khataee A (2018) Ultrasensitive chemiluminescent biosensor for the detection of cholesterol based on synergetic peroxidase–like activity of MoS2 and graphene quantum dots. Talanta 178:992–1000.

    CAS  Article  PubMed  Google Scholar 

  14. 14.

    Khan P, Idrees D, Moxley MA, Corbett JA, Ahmad F, von Figura G, Sly WS, Waheed A, Hassan MI (2014) Luminol–based chemiluminescent signals: clinical and non–clinical application and future uses. Appl Biochem Biotechnol 173:333–355.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Li Q, Zhang L, Li J, Lu C (2011) Nanomaterial–amplified chemiluminescence systems and their applications in bioassays. TrAC–Trend Anal Chem 30:401–413.

    CAS  Article  Google Scholar 

  16. 16.

    Zhang Z, Lai J, Wu K, Huang X, Guo S, Zhang L, Liu J (2018) Peroxidase–catalyzed chemiluminescence system and its application in immunoassay. Talanta 180:260–270.

    CAS  Article  PubMed  Google Scholar 

  17. 17.

    Lemos MA, Oliveira JC, Saraiva JA (2000) Influence of pH on the thermal inactivation kinetics of horseradish peroxidase in aqueous solution. LWT–Food Sci Technol 33:362–368.

    CAS  Article  Google Scholar 

  18. 18.

    Fatima A, Husain Q, Khan RH (2007) A peroxidase from bitter gourd (Momordica charantia) with enhanced stability against organic solvent and detergent: a comparison with horseradish peroxidase. J Mol Catal B Enzym 47:66–71.

    CAS  Article  Google Scholar 

  19. 19.

    Zhang C, Yan K, Hu C, Zhao Y, Chen Z, Zhu X, Möller M (2015) Encapsulation of enzymes in silica nanocapsules formed by an amphiphilic precursor polymer in water. J Mater Chem B 3:1261–1267.

    CAS  Article  PubMed  Google Scholar 

  20. 20.

    Xiang B, He K, Zhu R, Liu Z, Zeng S, Huang Y, Nie Z, Yao S (2016) Self–assembled DNA hydrogel based on enzymatically polymerized DNA for protein encapsulation and enzyme/DNAzyme hybrid cascade reaction. ACS Appl Mater Interfaces 8:22801–22807.

    CAS  Article  PubMed  Google Scholar 

  21. 21.

    Anik Ü, Timur S, Dursun Z (2019) Metal organic frameworks in electrochemical and optical sensing platforms: a review. Microchim Acta 186:196.

    CAS  Article  Google Scholar 

  22. 22.

    Hu Y, Dai L, Liu D, Du W, Wang Y (2018) Progress & prospect of metal–organic frameworks (MOFs) for enzyme immobilization (enzyme/MOFs). Renew Sust Energ Rev 91:793–801.

    CAS  Article  Google Scholar 

  23. 23.

    Feng D, Liu T-F, Su J, Bosch M, Wei Z, Wan W, Yuan D, Chen Y-P, Wang X, Wang K, Lian X, Gu Z-Y, Park J, Zou X, Zhou H-C (2015) Stable metal–organic frameworks containing single–molecule traps for enzyme encapsulation. Nat Commun 6:5979.

    CAS  Article  PubMed  Google Scholar 

  24. 24.

    Li P, Moon S-Y, Guelta MA, Lin L, Gómez-Gualdrón DA, Snurr RQ, Harvey SP, Hupp JT, Farha OK (2016) Nanosizing a metal–organic framework enzyme carrier for accelerating nerve agent hydrolysis. ACS Nano 10:9174–9182.

    CAS  Article  PubMed  Google Scholar 

  25. 25.

    Gkaniatsou E, Sicard C, Ricoux R, Benahmed L, Bourdreux F, Zhang Q, Serre C, Mahy J-P, Steunou N (2018) Enzyme encapsulation in mesoporous metal–organic frameworks for selective biodegradation of harmful dye molecules. Angew Chem Int Ed 57:16141–16146.

    CAS  Article  Google Scholar 

  26. 26.

    Zhang J, Tan Y, Song W-J (2020) Zeolitic imidazolate frameworks for use in electrochemical and optical chemical sensing and biosensing: a review. Microchim Acta 187:234.

    CAS  Article  Google Scholar 

  27. 27.

    Liang K, Coghlan CJ, Bell SG, Doonan C, Falcaro P (2016) Enzyme encapsulation in zeolitic imidazolate frameworks: a comparison between controlled co–precipitation and biomimetic mineralisation. Chem Commun 52:473–476.

    CAS  Article  Google Scholar 

  28. 28.

    Zhang Y, Wang YN, Sun XT, Chen L, Xu ZR (2017) Boron nitride nanosheet/CuS nanocomposites as mimetic peroxidase for sensitive colorimetric detection of cholesterol. Sensors Actuators B Chem 246:118–126.

    CAS  Article  Google Scholar 

  29. 29.

    Park KS, Ni Z, Côté AP, Choi JY, Huang R, Uribe-Romo FJ, Chae HK, O’Keeffe M, Yaghi OM (2006) Exceptional chemical and thermal stability of zeolitic imidazolate frameworks. Proc Natl Acad Sci U S A 103:10186–10191.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Lyu F, Zhang Y, Zare RN, Ge J, Liu Z (2014) One–pot synthesis of protein–embedded metal–organic frameworks with enhanced biological activities. Nano Lett 14:5761–5765.

    CAS  Article  PubMed  Google Scholar 

  31. 31.

    Chen G, Huang S, Kou X, Wei S, Huang S, Jiang S, Shen J, Zhu F, Ouyang G (2019) A convenient and versatile amino–acid–boosted biomimetic strategy for the nondestructive encapsulation of biomacromolecules within metal–organic frameworks. Angew Chem Int Ed 58:1463–1467.

    CAS  Article  Google Scholar 

  32. 32.

    Wang L, Zhi W, Lian D, Wang Y, Han J, Wang Y (2019) HRP@ZIF–8/DNA hybrids: functionality integration of ZIF–8 via biomineralization and surface absorption. ACS Sustain Chem Eng 7:14611–14620.

    CAS  Article  Google Scholar 

  33. 33.

    Zhang FF, Zou HY, Lan J, Wang H, Huang CZ (2016) Catalytic chemiluminescent detection of cholesterol in serum with Cu2−xSe semiconductor nanoparticles. Anal Bioanal Chem 408:8771–8778.

    CAS  Article  PubMed  Google Scholar 

  34. 34.

    Lin KL, Yang T, Zhang FF, Lei G, Zou HY, Li YF, Huang CZ (2017) Luminol and gold nanoparticle–co–precipitated reduced graphene oxide hybrids with long–persistent chemiluminescence for cholesterol detection. J Mater Chem B 5:7335–7341.

    CAS  Article  PubMed  Google Scholar 

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Financial support from the Natural Science Foundation of Shaanxi Province (2020JM–035) is gratefully acknowledged.

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Correspondence to Yinhuan Li.

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Xu, X., Zhao, Y., Tan, H. et al. In situ encapsulation of horseradish peroxidase in zeolitic imidazolate framework–8 enables catalyzing luminol reaction under near-neutral conditions for sensitive chemiluminescence determination of cholesterol. Microchim Acta 187, 346 (2020).

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  • Chemiluminescence
  • Metal organic frameworks
  • Horseradish peroxidase
  • Enzyme immobilization
  • Cholesterol