Enhancing the peroxidase activity and decreasing the protease activity of ficin with rational modification and its application to one-step colorimetric detection of glucose.

Ficin has dual enzyme activity, i.e., protease and peroxidase-like activity. In some respects, its application is limited by the protease activity of ficin. Herein, we used tris (2-carboxyethyl) phosphine hydrochloride (TCEP) to break the three pairs of disulfide bonds of ficin, and then blocked the free thiol groups with N-ethylmaleimide (NEM) to synthesize ficin-TN. The results showed that ficin-TN had increased peroxidase-like activity and reduced protease activity. According to this phenomenon, we have exploited a colorimetric method with high sensitivity and selectivity for the one-step detection of glucose. Comparing with ficin, ficin-TN has wider detection range (0.1-300 µM) and lower detection limit (88 nM), and our method is simpler and more timesaving than other two-step methods. Furthermore, the actual appliances of ficin-TN for glucose detection in human serum have been illustrated with satisfied result, suggesting that its promising utilization in various fields.

Ficin encapsulated in mesoporous metal-organic frameworks with enhanced peroxidase-like activity and colorimetric detection of glucose.

Ficin has been reported to possess peroxidase activity, but its applications in some respects have been limited because of its relatively low activity. Herein, a mesoporous metal-organic framework, PCN-333(Fe), was synthesized, which was selected to encapsulate ficin to form ficin@PCN-333(Fe). Compared with ficin, the peroxidase-like activity of ficin@PCN-333(Fe) toward 3,3',5,5'-tetramethylbenzidine (TMB) oxidation was about 3 times increase in the presence of H2O2, and followed classical Michaelis-Menten model. The kinetic parameters showed that stronger affinity and higher catalytic constant (Kcat) of ficin@PCN-333(Fe) to both TMB and H2O2 compared with ficin, and Kcat of ficin@PCN-333(Fe) was increased by 3.65 folds and 3.59 folds for TMB and H2O2, respectively. Taking advantages of higher catalytic property of ficin@PCN-333(Fe), we developed a colorimetric method with high sensitivity and selectivity to detect glucose, which displayed a good linear response toward glucose in the range of 0.5-180 µM with a limit of detection of 97 nM. Furthermore, ficin@PCN-333(Fe) has been proven to successfully detect glucose in human serum, implying its great potentialities and wide applications as peroxidase mimics.

Enhancing the peroxidase-like activity of ficin by rational blocking thiol groups for colorimetric detection of biothiols.

The peroxidase-like activity of ficin is relatively low, which limits its application. It was found that thiol groups of ficin could inhibit its peroxidase-like activity. So, two procedures, i.e., direct blocking with N-ethylmaleimide (NEM), or using tris (2-carboxyethyl) phosphine hydrochloride (TCEP) to interrupt disulfide bonds then blocking thiol groups with NEM, were applied to block thiol groups of ficin, ficin-NEM (ficin-N) and ficin-TCEP-NEM (ficin-TN) were produced, respectively. The blocking of thiol groups accelerated the peroxidase activity dramatically. The peroxidase catalytic activity of ficin-N and ficin-TN toward the peroxidase substrate 3,3',5,5'-tetramethylbenzidine (TMB) oxidation by H2O2 was about 2.5-fold and 5-fold increase compared with ficin, respectively, which accompanied a color change from colorless to blue and followed classic Michaelis-Menten model. The kinetic parameters indicated that higher affinity of ficin-N (Km = 0.31) and ficin-TN (Km = 0.39) to H2O2 compared with ficin (Km = 0.58), and ficin-TN had the highest Kcat which increased by 6.5 times and 4.5 times for TMB and H2O2, respectively. According to these findings, a colorimetric method with high sensitivity for the detection of biothiols was developed due to sulfhydryl compounds inhibited the peroxidase activity of ficin. Comparing with ficin and ficin-N, ficin-TN had the widest detection range (0.01-16 µM) and the lowest detection limit (3 nM). The practical applications of ficin-TN for biothiol determination in human serum samples have been demonstrated with satisfactory results. Ficin-N and ficin-TN are promising to apply to the bioanalysis.

One-pot synthesis of a composite consisting of the enzyme ficin and a zinc(II)-2-methylimidazole metal organic framework with enhanced peroxidase activity for colorimetric detection for glucose.

An enzyme-metal organic framework (MOF) composite with saucer-like structure was prepared via one-pot synthesis from ficin (a cysteine proteolytic enzyme with POx activity), zinc(II) ions and 2-methylimidazole. The composites exhibit a 2.5-fold higher catalytic activity and stronger affinity for substrates compared to free ficin. This was exploited to design a colorimetric assay for the determination of glucose. The addition of glucose oxidase causes the formation of H2O2 which is catalytically oxidized by ficin to form a blue coloration that can be measured at 652 nm. The assay has a 0.12 µM detection limit and excellent selectivity. It was successfully applied to the determination of glucose in diluted serum samples. Graphical abstract Schematic presentation of the synthesis process and the enhanced peroxidase activity of ficin@MOF composites. Ficin was immobilized in a new saucer-like shape of Ficin@MOF composites, which showed higher peroxidase activity than free ficin. Scheme and graphical abstract contains poor quality of text.We have attach a new picture with 600 dpi as scheme and graphical abstract.

Competitive method for fluorescent dopamine detection in cerebrospinal fluid based on the peroxidase-like activity of ficin.

Dopamine (DA), a catecholamine neurotransmitter, is considered to be an important indicator for mental diseases detection in the clinic. In this study, a novel fluorescent sensing platform consisting of the ficin-H2O2-tyramine system for determining DA in cerebrospinal fluids (CSF) was established. The proposed method is based on the fact that ficin, a mimetic peroxidase, can catalyze H2O2 decomposition into OH radicals, which can oxidize non-fluorescent tyramine into fluorescent dityramine. When DA was introduced, DA can compete with tyramine for OH and resulting in the oxidation reaction of tyramine inhibited along with the fluorescence intensity of the system decreased, which provides a unique strategy for fluorescence detection of DA. Under optimal conditions, the fluorescence intensity decreased linearly with the DA level over a wide concentration range from 0.05 to 12.0 µM (R2 = 0.995) with a detection limit of 46 nM (3σ/k). More importantly, the proposed sensing approach exhibits high sensitivity, good selectivity and has been successfully applied to DA sensing in complex biological samples, which made it hold great potential for DA determination in chemical and biological analytical applications.

Enhancing the peroxidase-like activity of ficin via heme binding and colorimetric detection for uric acid.

Ficin, a classical sulfhydryl protease, was found to possess intrinsic peroxidase-like activity. In this paper, we have put forward a novel strategy to improving the peroxidase-like activity of ficin through binding heme. Heme-ficin complexes were successfully obtained by simple one-step syntheticism. The results demonstrated that the catalytic activity and efficiency of heme-ficin complexes were about 1.7 times and 3 times higher than those of native ficin, respectively. Taking advantages of the high peroxidase-like activity, the heme-ficin complexes were used for colorimetric determination of uric acid with a low detection limit of 0.25 µM. Based on the excellent selectivity and sensitivity, we detected the concentration of uric acid in human serum successfully. On the basis of these findings, the heme-ficin complexes are promising for wide applications in various fields. Thus we not only optimized the peroxidase-like activity of the ficin, but also established a new strategy for development of artificial enzyme mimics by mimicking the architecture of the active site in horseradish peroxidase.