Development of a Fluorescence Assay for Rapid Detection of Alkaline Phosphatase Activity in Fresh Milk Based on Inner Filter Effect of Carbon Dots

JIA Baozhu; HE Zhenxi; HUANG Xinru; QIU Zhijing; GUO Linyuan; YUAN Yupei; WANG Biman; LUO Lin

doi:10.13386/j.issn1002-0306.2022110075

Volume 44 Issue 17

Aug. 2023

Turn off MathJax

Article Contents

Abstract

References

Science and Technology of Food Industry > 2023 > 44(17): 334-341. > DOI: 10.13386/j.issn1002-0306.2022110075

JIA Baozhu, HE Zhenxi, HUANG Xinru, et al. Development of a Fluorescence Assay for Rapid Detection of Alkaline Phosphatase Activity in Fresh Milk Based on Inner Filter Effect of Carbon Dots[J]. Science and Technology of Food Industry, 2023, 44(17): 334−341. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2022110075.

Citation:

PDF (6448 KB)

Development of a Fluorescence Assay for Rapid Detection of Alkaline Phosphatase Activity in Fresh Milk Based on Inner Filter Effect of Carbon Dots

1.
College of Biological and Food Engineering, Guangdong University of Education, Guangzhou 510303, China
2.
Guangdong Provincial Key Laboratory of Food Quality and Safety, College of Food, South China Agricultural University, Guangzhou 510642, China

More Information

Received Date: November 08, 2022
Available Online: July 04, 2023

Graphical Abstract

Abstract

Abstract

This work aimed at developing a fluorescence assay for rapid and sensitive determination of alkaline phosphatase (ALP) activity in fresh milk using nitrogen doped carbon dots (N-CDs) as fluorescence probe. Green-emissive N-CDs were synthesized by using ethylenediamine as the nitrogen source and catechol as the carbon source through a hydrothermal method. The obtained N-CDs were characterized by transmission electron microscopy (TEM), UV-vis spectroscopy, X-ray photoelectron spectroscopy (XPS), and Fourier transform infrared (FT-IR) spectra. Control experiments were used to verify the feasibility for constructing the fluorescent assay using the N-CDs. Single-factor experiments were conducted to optimize the pH of working buffer, the concentration of disodium p-nitrophenyl phosphate (PNPP) and enzymatic reaction time for ALP detection. The results indicated that the as-synthesized N-CDs were successfully prepared with all the characterization results being consistent with those in previous works. The optimal working conditions for this assay were as follows: Tris-HCl (20 mmol/L, pH10) as working buffer, 1 mmol/L of PNPP as enzymatic substrate, incubation for 50 min. Under the optimal conditions, it was found that fluorescence intensity of N-CDs linearly corelated with ALP concentration from 0.01 to 25 U/L with correlation coefficients of 0.995. The linear regression equation was Y=15397.05X+70344.46 with a limit of detection (LOD) of 0.001 U/L (S/N=3). Recoveries for two kinds of fresh milk were in the range from 100.1% to 107.2%, and the coefficients of variations were less than 14.3%, which indicated that the proposed method had good accuracy and reliability. This study provides an accurate and efficient method for the detection of ALP in fresh milk.
- nitrogen-doped carbon dots (N-CDs),
- fluorescent probe,
- inner filter effect (IFE),
- alkaline phosphatase,
- fresh milk

FullText(HTML)

References (29)

References

[1]	刘悦, 覃炎峰, 李荷. 土壤磷酸酯酶基因的筛选、克隆及酶学性质分析[J]. 河南农业科学,2014,43(3):70−74. [LIU Y, TAN Y F, LI H. Screening, cloning and enzymatic characterization of phosphohydrolase from soil[J]. Journal of Henan Agricultural Sciences,2014,43(3):70−74. LIU Y, TAN Y F, LI H. Screening, cloning and enzymatic characterization of phosphohydrolase from soil[J]. Journal of Henan Agricultural Sciences, 2014: 43（3）: 70−74.
[2]	MILLAN J L. Alkaline phosphatases: Structure, substrate specificity and functional relatedness to other members of a large superfamily of enzymes[J]. Purinergic Signal,2006,2(2):335−341. doi: 10.1007/s11302-005-5435-6
[3]	翁子帅. 乳及乳制品消毒杀菌关键技术探究[J]. 产业科技创新,2019,1(27):28−29. [WENG Z S. Exploration of key technologies for disinfection and sterilization of milk and dairy products[J]. Industrial Technology Innovation,2019,1(27):28−29. WENG Z S. Exploration of key technologies for disinfection and sterilization of milk and dairy products[J]. Industrial Technology Innovation, 2019, 1(27): 28-29.
[4]	LORENZEN P C, MARTIN D, CLAWIN-RADECKER I, et al. Activities of alkaline phosphatase, gamma-glutamyltransferase and lactoperoxidase in cow, sheep and goat's milk in relation to heat treatment[J]. Small Ruminant Res,2010,89(1):18−23. doi: 10.1016/j.smallrumres.2009.11.013
[5]	WILLIAMS R S, HOY W A. The viability of B. tuberculosis (bovinus) on pasture land, in stored faeces and in liquid manure[J]. Journal of Hygiene,1930,30(4):413−419.
[6]	洪红. 乳品中碱性磷酸酶活度的比色测定[J]. 中国乳品工业,2004,32(4):34−36. [HONG H. Colorimetric method for determination of alkaline phosphatase activity in dairy products[J]. China Diary Industry,2004,32(4):34−36. doi: 10.3969/j.issn.1001-2230.2004.04.011 HONG H. Colorimetric method for determination of alkaline phosphatase activity in dairy products[J]. China Diary Industry, 2004, 32(4): 34-36. doi: 10.3969/j.issn.1001-2230.2004.04.011
[7]	RONALD S, CAYLEY H, PREETHA B, et al. A portable chemiluminescence assay of alkaline phosphatase activity to monitor pasteurization of milk products[J]. Journal of Food Protection,2019,82(12):2119−2125. doi: 10.4315/0362-028X.JFP-19-153
[8]	ALBILLOS S M, REDDY R, SALTER R. Evaluation of alkaline phosphatase detection in dairy products using a modified rapid chemiluminescent method and official methods[J]. Journal of Food Protection,2011,74(7):1144−1154. doi: 10.4315/0362-028X.JFP-10-422
[9]	SCINTU M F, DAGA E, LEDDA A. Evaluation of spectrophotometric and fluorometric methods for alkaline phosphatase activity determination in ewe's milk[J]. Journal of Food Protection,2000,63(9):1258−1261. doi: 10.4315/0362-028X-63.9.1258
[10]	ANGELINO P D, CHRISTEN G L, PENFIELD M P, et al. Residual alkaline phosphatase activity in pasteurized milk heated at various temperatures-measurement with the fluorophos and Scharer rapid phosphatase tests[J]. Journal of Food Protection,1999,62(1):81−85. doi: 10.4315/0362-028X-62.1.81
[11]	SERRA B, MORALES M D, REVIEJO A J, et al. Rapid and highly sensitive electrochemical determination of alkaline phosphatase using a composite tyrosinase biosensor[J]. Analytical Biochemistry,2005,336(2):289−294. doi: 10.1016/j.ab.2004.10.039
[12]	CHEN Q M, LI S Q, LIU Y, et al. Size-controllable Fe-N/C single-atom nanozyme with exceptional oxidase-like activity for sensitive detection of alkaline phosphatase[J]. Sensors and Actuators B-Chemical,2020,305:127511. doi: 10.1016/j.snb.2019.127511
[13]	SCHARER H. Laboratory control of milk under war conditions: the rapid phosphatase test[J]. American Journal of Public Health and the Nation's Health,1943,33(4):396−398. doi: 10.2105/AJPH.33.4.396
[14]	杨梅, 王欢, 孙彤, 等. 顺序注射荧光法测定生鲜牛奶中碱性磷酸酶[J]. 辽宁师范大学学报(自然科学版),2012,35(3):354−357. [YANG M, WANG H, SUN T, et al. Determination of alkaline phosphatase in milk products using sequential injection-fluorometry method[J]. Journal of Liaoning Normal University (Natural Science Edition),2012,35(3):354−357. YANG M, WANG H, SUN T, et al. Determination of alkaline phosphatase in milk products using sequential injection-fluorometry method[J]. Liaoning: Journal of Liaoning Normal University (Natural Science Edition), 2012: 35(3): 354-357.
[15]	SU B, XU H, XIE G, et al. Generation of a nanobody-alkaline phosphatase fusion and its application in an enzyme cascade-amplified immunoassay for colorimetric detection of alpha fetoprotein in human serum[J]. Molecular and Biomolecular Spectroscopy,2021,262:120088. doi: 10.1016/j.saa.2021.120088
[16]	LI H X, YAN X, KONG D S, et al. Recent advances in carbon dots for bioimaging applications[J]. Nanoscale Horizons,2020,5(2):218−234. doi: 10.1039/C9NH00476A
[17]	LI H X, YAN X, LU G Y, et al. Carbon dot-based bioplatform for dual colorimetric and fluorometric sensing of organophosphate pesticides[J]. Sensors and Actuators B-Chemical,2018,260:563−570. doi: 10.1016/j.snb.2017.12.170
[18]	LI H, SU D, GAO H, et al. Design of red emissive carbon dots: robust performance for analytical applications in pesticide monitoring[J]. Analytical Chemistry,2020,92(4):3198−3205. doi: 10.1021/acs.analchem.9b04917
[19]	YAN X, SONG Y, ZHU C, et al. MnO₂ nanosheet-carbon dots sensing platform for sensitive detection of organophosphorus pesticides[J]. Analytical Chemistry,2018,90(4):2618−2624. doi: 10.1021/acs.analchem.7b04193
[20]	CHEN S, YU Y L, WANG J H. Inner filter effect-based fluorescent sensing systems: A review[J]. Analytica Chimica Acta,2018,999:13−26. doi: 10.1016/j.aca.2017.10.026
[21]	ZHU R, HUANG W, MA X, et al. Nitrogen-doped carbon dots-V2O5 nanobelts sensing platform for sensitive detection of ascorbic acid and alkaline phosphatase activity[J]. Analytica Chimica Acta,2019,1089:131−143. doi: 10.1016/j.aca.2019.08.061
[22]	LUO S Z, YANG J Y, JIA B Z, et al. Multicolorimetric and fluorometric dual-modal immunosensor for histamine via enzyme-enabled metallization of gold nanorods and inner filter effect of carbon dots[J]. Food Control,2022,137:108941. doi: 10.1016/j.foodcont.2022.108941
[23]	YANG Q, WANG X Y, PENG H L, et al. Ratiometric fluorescence and colorimetry dual-mode assay based on manganese dioxide nanosheets for visual detection of alkaline phosphatase activity[J]. Sensor Actuat B-Chem,2020,302:127176. doi: 10.1016/j.snb.2019.127176
[24]	LUO L, LIN S Q, WU Z Y, et al. Nanobody-based fluorescent immunoassay using carbon dots anchored cobalt oxyhydroxide composite for the sensitive detection of fenitrothion[J]. Journal of Hazardous Materials,2022,439:129701. doi: 10.1016/j.jhazmat.2022.129701
[25]	LUO L, JIA B Z, WEI X Q, et al. Development of an inner filter effect-based fluorescence immunoassay for the detection of acrylamide using 9-xanthydrol derivatization[J]. Sensors and Actuators B-Chemical,2021,332:129561. doi: 10.1016/j.snb.2021.129561
[26]	马金虎, 薛福来, 王一帆, 等. 草鱼肝胰脏碱性磷酸酶的分离纯化及性质的研究[J]. 水产科学,2014,33(8):498−502. [MA J H, XUE F L, WANG Y F, et al. Purification and characterization phosphatase from grass carp (Ctenopharyngodon idellus C)[J]. Fisheries Science,2014,33(8):498−502. doi: 10.3969/j.issn.1003-1111.2014.08.007 MA J H, XUE FL, WANG Y F, et al. Purification and characterization phosphatase from grass carp (Ctenopharyngodon idellusC) [J]. Fisheries Science, 2014, 33(8): 498-502. doi: 10.3969/j.issn.1003-1111.2014.08.007
[27]	余同, 张营, 赵欣平, 等. 牛小肠碱性磷酸酶部分性质研究[J]. 四川大学学报(自然科学版),2009,46(6):1838−1844. [YU T, ZHANG Y, ZHAO X P, et al. Some characterization of alkaline phosphatase from bovine intestine[J]. Journal of Sichuan University (Natural Science Edition),2009,46(6):1838−1844. YU T, ZHANG Y, ZHAO XP, et al. Some characterization of alkaline phosphatase from bovine intestine[J]. Sichuan: Journal of Sichuan University (Natural Science Edition), 2009, 46(6): 1838-1844.
[28]	YU L, SHI Z, FANG C, et al. Disposable lateral flow-through strip for smartphone-camera to quantitatively detect alkaline phosphatase activity in milk[J]. Biosensors & Bioelectronics,2015,69:307−315.
[29]	SHRESTHA S, PUDASAINI S, GIRI B. Towards the development of paper analytical devices for testing alkaline phosphatase, starch, and urea in milk[J]. International Dairy Journal,2022,134:105470. doi: 10.1016/j.idairyj.2022.105470