Credible and Facile Fluorometric Detection of Soybean Trypsin Inhibitor Activity with a Water-Soluble Poly(diphenylacetylene) Derivative

Abstract

Herein, a novel method for fluorometric detection of soybean trypsin inhibitor (SBTI) activity based on a water-soluble poly(diphenylacetylene) derivative was reported. Fluorescence quenching of the polymer via p-nitroaniline, produced from the trypsin-catalyzed decomposition of N-benzoyl-DL-arginine-4-nitroanilide hydrochloride (L-BAPA), was well described using the Stern–Volmer equation. SBTI activity was quantitatively assessed based on changes in the fluorescence intensity of the polymer. This strategy has several advantages, such as high sensitivity and ease of operation. Moreover, its applicability to other biochemical analyses is promising.

Keywords

Fluorometric detection / Soybean trypsin inhibitor / Inhibition activity / Fluorescence quenching

References

[1] Avilés-Gaxiola S, Chuck-Hernández C, Serna Saldívar SO Inactivation methods of trypsin inhibitor in legumes: a review. J Food Sci, 2018, 83(1): 17-29.

[2] Birk Y The bowman-birk inhibitor. Trypsin- and chymotrypsin-inhibitor from soybeans. Int J Pept Protein Res, 1985, 25(2): 113-131.

[3] Ge G, Guo W, Zheng J, et al. Effect of interaction between tea polyphenols with soymilk protein on inactivation of soybean trypsin inhibitor. Food Hydrocoll, 2021, 111.

[4] Li Z, Zhao C, Li Z, et al. Reconstructed mung bean trypsin inhibitor targeting cell surface GRP78 induces apoptosis and inhibits tumor growth in colorectal cancer. Int J Biochem Cell Biol, 2014, 47: 68-75.

[5] Shamsi TN, Parveen R, Fatima S Characterization, biomedical and agricultural applications of protease inhibitors: a review. Int J Biol Macromol, 2016, 91: 1120-1133.

[6] Burshtein G, Itin C, Tang JCY, et al. The combined effect of permeation enhancement and proteolysis inhibition on the systemic exposure of orally administrated peptides: salcaprozate sodium, soybean trypsin inhibitor, and teriparatide study in pigs. Int J Pharm X, 2021, 3

[7] Kong X, Li Y, Liu X Purification of soybean Kunitz trypsin inhibitor and the mechanism of its passivation by lysine and disulfide bond modifications. Food Biosci, 2023, 55.

[8] Bendre AD, Ramasamy S, Suresh CG Analysis of Kunitz inhibitors from plants for comprehensive structural and functional insights. Int J Biol Macromol, 2018, 113: 933-943.

[9] Zhang Y, Chang SKC Trypsin inhibitor activity, phenolic content and antioxidant capacity of soymilk as affected by grinding temperatures, heating methods and soybean varieties. LWT, 2022, 153.

[10] Li C, Li W, Zhang Y, et al. Comparison of physicochemical properties of recombinant buckwheat trypsin inhibitor (rBTI) and soybean trypsin inhibitor (SBTI). Protein Expr Purif, 2020, 171.

[11] Wang Y, Li Y, Wu J, et al. Development of an immunochromatographic strip test for the rapid detection of soybean Bowman-Birk inhibitor. Food Agric Immunol, 2019, 30: 1202-1211.

[12] Liu K Trypsin inhibitor assay: expressing, calculating, and standardizing inhibitor activity in absolute amounts of trypsin inhibited or trypsin inhibitors. J Am Oil Chem Soc, 2021, 98: 355-373.

[13] Xu L, Song Y, Liu L, et al. Sandwich ELISA and immunochromatographic strip of Kunitz trypsin inhibitor using sensitive monoclonal antibodies. Food Agric Immunol, 2016, 27: 772-782.

[14] GB/T 21498—2008. People’s Republic of China, Determination of trypsin inhibitor activity of soya products.

[15] Xiao CW, Wood CM, Robertson P, et al. Protease inhibitor activities and isoflavone content in commercial soymilks and soy-based infant formulas sold in Ottawa. Canada J Food Compos Anal, 2012, 25(2): 130-136.

[16] Li H, Shang Q, Zhang L, et al. Europium nanospheres based ultrasensitive fluorescence immunosensor for aflatoxin B1 determination in feed. Talanta, 2024, 270.

[17] Gao X, Sun JZ, Tang BZ Reaction-based AIE-active fluorescent probes for selective detection and imaging. Isr J Chem, 2018, 58: 845-859.

[18] Pucci A Mechanochromic fluorescent polymers with aggregation-induced emission features. Sensors, 2019, 19(22): 4969.

[19] Hussain S, Zhu C, Yue Z, et al. Rational design of signal amplifying fluorescent conjugated polymers for environmental monitoring applications: recent advances and perspectives. Coord Chem Rev, 2024, 499.

[20] Zhang Y, He Z, Shen X, et al. A multiple-responsive fluorescence probe based on water-soluble fluorescent conjugated polymer for biomolecules detection. ACS Appl Polym Mater, 2023, 5: 2877-2886.

[21] Alizadeh T, Sharifi AR, Ganjali MR A new bio-compatible Cd²⁺-selective nanostructured fluorescent imprinted polymer for cadmium ion sensing in aqueous media and its application in bio imaging in Vero cells. RSC Adv, 2020, 10(7): 4110-4117.

[22] Wang Y, Zhang Y, Liu B Conjugated polyelectrolyte based fluorescence turn-on assay for real-time monitoring of protease activity. Anal Chem, 2010, 82(20): 8604-8610.

[23] Liu R, Tan Y, Zhang C, et al. A real-time fluorescence turn-on assay for trypsin based on a conjugated polyelectrolyte. J Mater Chem B, 2013, 1(10): 1402-1405.

[24] Sathiyaraj M, Pavithra K, Thiagarajan V Azine based AIEgens with multi-stimuli response towards picric acid. New J Chem, 2020, 44(20): 8402-8411.

[25] Qin W, Xua C, Zhao Y, et al. Recent progress in small molecule fluorescent probes for nitroreductase. Chin Chem Lett, 2018, 29: 1451-1455.

[26] Tong J, Hu T, Qin A, et al. Deciphering the binding behaviours of BSA using ionic AIE-active fluorescent probes. Faraday Discuss, 2017, 196: 285-303.

[27] Liu K, Hu Z A novel conjugated polymer consists of benzimidazole and benzothiadiazole: synthesis, photophysics properties, and sensing properties for Pd²⁺. J Polym Sci, 2020, 58: 831-842.

[28] Zhang XA, Qin A, Tong L, et al. Synthesis of functional disubstituted polyacetylenes bearing highly polar functionalities via activated ester strategy. ACS Macro Lett, 2012, 1(1): 75-79.

[29] Zhang XA, Chen MR, Zhao H, et al. A facile synthetic route to functional poly(phenylacetylene)s with tunable structures and properties. Macromolecules, 2011, 44(17): 6724-6737.

[30] Chen M, Wang X, Yang F, et al. Azobenzene functionalized poly(diphenylacetylene):polymer synthesis and tunable fluorescent emission. J Polym Sci, 2022, 61: 659-670.

[31] Tarrío JJ, Rodríguez R, Fernández B, et al. Dissymmetric chiral poly(diphenylacetylene)s: secondary structure elucidation and dynamic luminescence. Angew Chem Int Ed, 2022, 61(9

[32] Sona S, Hirose D, Kurihara Y, et al. Thermally stable and rewritable circularly polarized luminescent helical poly(diphenylacetylene)s: stabilization of macromolecular helicity memory via reversible ion-pair formation. J Mater Chem C, 2023, 11(4): 1271-1277.

[33] Lee WE, Oh C, Kang I, et al. Diphenylacetylene polymer nanofiber mats fabricated by freeze drying: preparation and application for explosive sensors. Macromol Chem Phys, 2010, 211: 1900-1908.

[34] Kwak G, Lee WE, Kim WH, et al. Fluorescence imaging of latent fingerprints on conjugated polymer films with large fractional free volume. Chem Commun, 2009, 16: 2112-2114.

[35] Yoon JH, Jin YJ, Sakaguchi T, et al. Visualization of sweat fingerprints on various surfaces using a conjugated polyelectrolyte. ACS Appl Mater Interfaces, 2016, 8(36): 24025-24029.

[36] Lee WE, Jin YJ, Kim SI, et al. Fluorescence turn-on response of a conjugated polyelectrolyte with intramolecular stack structure to biomacromolecules. Chem Commun, 2013, 49(84): 9857-9859.

[37] Li Y Improvement method of determination trypsin inhibitor activity in soybean products. China Food Addit, 2022, 33: 212-218.

[38] Yin S, Jia C, Tian H, et al. Determination of trypsin inhibitor activity of soybean milk. J Food Saf Qual, 2016, 7: 1000-1005.

[39] Hackler LR, VAN BUREN JP, Steinkraus KH, et al. Effect of heat treatment on nutritive value of soymilk protein fed to weanling rats. J Food Sci, 1965, 30(4): 723-728.