Enzyme mimics of bimetallic nanoclusters for detection of trypsin

Enzyme mimics of bimetallic nanoclusters for detection of trypsin | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Enzyme mimics of bimetallic nanoclusters for detection of trypsin Yong Zhu, Cuifeng Jiang, Min Huo, Zijie Wei, Wanting Han This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6373736/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Detection of trypsin is very important for human health. In this work, bovine serum albumin protected OsAg nanoclusters (BSA-OsAg NCs) were prepared. The ratio of Ag element in the nanoclusters could be regulated by controlling the reagent concentration. UV-Visible absorption spectra acquired from TMB(3,3',5,5'-Tetramethylbenzidine) to oxTMB were used to investigate the catalytic ability of the nanoclusters. The nanoclusters all displayed peroxidase-like activity. Especially, the BSA-OsAg NCs with 20% silver content showed the best catalytic ability. In addition, trypsin effectively increased the catalytic activity of BSA-OsAg NCs due to the interaction between trypsin and BSA. Based on this phenomenon, a novel method for selectively detection of trypsin was developed with a detection limit (LOD) of 5ng/mL. Promisingly, this method can be used for detection of trypsin in fetal bovine serum with acceptable recovery rate. BSA-OsAg bimetallic nanoclusters peroxidase-like activity trypsin Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Trypsin is a very important protease in the human internal environment. The occurrence of many diseases,such as pancreatitis [ 1 ] and metastasis of tumors [ 2 , 3 ] is related to its content.Therefore, a reliable method for detecting pancreatic protease is of great significance. Several detection methods for trypsin have been developed, such as fluorescence assay [ 4 , 5 ] , electrochemical assay [ 6 , 7 ] and surface enhanced Raman scattering [ 8 ] .Nevertheless, it is necessary to develop simpler and more convenient analytical methods for detecting trypsin concentration. Colorimetric method has become a good choice for solving this problem due to its advantage of simple operation and easy readability [ 9 – 11 ] . Meanwhile, nanozyme has become a strong tool for colorimetric analysis attributed to its color change from chromogenic substrate to its oxidized form [ 12 , 13 ] . Especially, noble metal nanoparticles Os has attracted attention for its significant higher peroxidase-like activity compared to other noble metal nanoparticles [ 14 ] . Os is an attractive precious metal with the highest density, low compressibility, high melting point, and bulk modulus [ 15 ] . However, exploration of Os nanoclusters is rarely reported [ 16 ] . In fact, nanoclusters (NCs) composed of several atoms with smaller size usually can exhibit special catalytic properties. Therefore, they can be involved in many applications in the field of sensing [ 17 ] .It is reported that Os@Mucin NCs exhibited tumor environment-responsive peroxidase-like activity [ 18 ] , BSA − Os NCs were found to possess intrinsic peroxidase-like activity,enable a BSA − Os NCs-based colorimetric sensor to detect H 2 O 2 from complex systems [ 19 ] .The laminarin-modulated osmium (laminarin-Os) nanoclusters had the selective peroxidase-like behavior under acidic conditions and were used for engineering colorimetric assay for hydroxyl radical [ 20 ] . These reports declared Os nanoclusters had peroxidase-like activity. Do bimetallic nanoclusters based on Os own better enzyme mimics? With this question in mind, we prepared OsAg bimetallic nanoclusters and investigated their catalytic property. Herein, BSA-OsAg NCs were prepared and applied for trypsin detection (as shown in Scheme 1). Among them, the BSA-OsAg NCs with a 20% silver content showed a smaller K m value and stronger affinity for the substrate TMB.In addition, based on the enhancement effect of trypsin on the peroxidase activity of BSA-OsAg NCs, the concentration of trypsin was detected using BSA-OsAg NCs with a 20% silver content. In order to explore its practical application effect, we found that the recovery rate in fetal bovine serum ranged from 86.8–106.5%, indicating high practicality. This indicates that this method has strong application prospects in environments without complex equipment. 2. Experimental 2.1. Chemicals K 2 OsCl 6 , NaOH, AgNO 3 , Rhodamine B (RhB), terephthalic acid (TA), bovine serum albumin (BSA) were purchased from Aladdin Industrial Corporation. Hydrogen peroxide (98%) and acetate buffer were purchased from Jiangsu Tongsheng Chemical Reagent Company. 3,3',5,5'-tetramethylbenzidine (TMB), trypsin, metal ions, amino acids and biomolecules were purchased from BBI Life Sciences. 2.2. Preparation of BSA-Os nanoclusters (Os NCs) 0.5mL 25mg/mL BSA aqueous solution and 0.5mL 20 mM K 2 OsCl 6 solution were added into a small centrifuge tube. Then, the pH of the total solution was adjusted to 12 by using 50µL 1M NaOH. After mixed thoroughly, the mixture was incubated in a water bath at 37℃ for 7 hours to obtain BSA-Os NCs. 2.3. Preparation of BSA-OsAg nanoclusters (OsAg NCs) with different silver contents 0.5mL 20mM K 2 OsCl 6 solution was mixed with 8 mM AgNO 3 solution to make the molar ratios of K 2 OsCl 6 and AgNO 3 as 4:1, 3:2, and 7:3, respectively. Then, the following procedure was the same as preparation of BSA-Os NCs. Finally, BSA-OsAg NCs with silver content of 20%, 40%, and 70% were obtained, respectively. 2.4. Enzyme-mimetic activities The peroxidase like activity (simulated enzyme activity) of OsAg NCs was evaluated by using chromogenic substrate TMB in the presence or absence of H 2 O 2 . Typically, 50µL TMB(8mM), 50µL H 2 O 2 (50mM), 10µL BSA-OsAg NCs, 350µL acetic acid buffer (pH = 4.8) and 500µL deionized water were mixed. The absorption spectrum of the mixed solution was measured after 10 minutes incubation. 2.5. Kinetics analysis The steady-state kinetics of BSA-OsAg NCs were investigated. In order to obtain the Michaelis constant ( K m ) and maximum reaction rate ( V max ) for H 2 O 2 , the same concentration of H 2 O 2 was used while changing the TMB (0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, 8 mM) at different concentrations. The absorption values obtained from UV measurements were used for calculation. On the other hand, in order to obtain the Michaelis constant ( K m ) and maximum reaction rate ( V max ) for TMB, UV absorption values were measured using the same concentration of TMB and different concentrations of H 2 O 2 (0.5, 1, 2, 5, 8, 10, 20, 30, 40, 50mM). 2.6. Mechanism investigation In order to verify the generation of hydroxyl radicals, a benzoic acid (TA) oxidation experiment was conducted. A concentration of 25 mM TA was prepared by using 0.1M NaOH solution. Then, 15µL TA, 150µL H 2 O 2 (50 mM) and 30µL BSA-OsAg NCs were added to 1.05mL of acetic acid buffer (pH = 4.8) and incubated in the dark for 1 hour. Finally, the fluorescence spectrum of the mixture was colloected. 2.7. Detection of trypsin Trypsin with different concentrations (5, 10, 50, 80, 100, 250, 500, 1000, 1500, 2000, 3000 ng/mL) were prepared. 50µL trypsin, 350µL buffer solution (pH = 4.8), 50µL H 2 O 2 (50 mM), 50µL TMB (8mM), 10µL BSA-OsAg NCs with 20% silver content and 650 µL deionized water were mixed and incubated for 5 minutes to obtain absorption spectra. 2.8.Characterization The morphology and microstructure was observed and analyzed by transmission electron microscope (TEM, JEM2100F). The absorption spectroscopy were read from UV–Vis (UV-2600).Fourier-transform infrared spectroscopy (FTIR) was recorded by using NEXUS-670. Fluorescence properties were explored with Fluorescence spectrometer (JASCO FP-6500). 3. Results and discussion 3.1. Characterization Firstly, the morphology and microstructure of BSA-OsAg NCs were characterized using TEM. As shown in Fig. 1 (a) - (c), the size of BSA-OsAg NCs (20%) is about 1nm. However, the size of BSA-OsAg NCs (40%) and (70%) are around 2 nm. Moreover, most nanoparticles sticked together and aggregated into large spheres for BSA-OsAg NCs (70%). Fourier transform infrared analysis was performed on individual BSA and BSA-OsAg NCs (Fig. 2 ).The FTIR spectra of BSA at 3300 ~ 3500cm − 1 is the N-H stretching vibration,1600 ~ 1700cm − 1 is a carbonyl stretching vibration and the amide Ⅰ band,1500 ~ 1600cm − 1 is the C-N stretching vibration, also amide Ⅱ band [ 21 ] . No obvious change for these band spectra was found in BSA-OsAg NCs, indicating that the influence of BSA-OsAg NCs on the structure of BSA is relatively small and will not damage the structure of BSA. 3.2.Enzyme-mimetic activities The simulated enzymatic properties of BSA-OsAg NCs were evaluated using the chromogenic substrate TMB, which will turn blue with appropriate catalysts. As shown in Fig. 3 (a), BSA-OsAg NCs(0%, 20%, 40%, 70%) + TMB + H 2 O 2 exhibited blue color and significant absorption at 652 nm, indicating that BSA-OsAg NCs possessed peroxidase like activity. It was also found that the color of solution with 70% silver content is relatively light compared to the other three, according with the lower absorption peak at 652 nm (Table S1 ). Based on this, it can be concluded that the peroxidase like activity of the BSA-OsAg NCs with 70% silver content is very low. Therefore, subsequent activity studies only focused on pure BSA-Os NCs, 20% silver, and 40% silver content BSA-OsAg NCs. In addition, BSA-OsAg NCs + TMB showed almost colorless, indicating BSA-OsAg do not exhibit oxidase-like activity. Most noble metal nanoparticles had both peroxidase and oxidase-like activity [ 22 – 24 ] . The specific activity of BSA-OsAg NCs could be applied for reaction of generation H 2 O 2 . For example, it could eliminate influence of O 2 on the activity when used for detection of uric acid, which will generate H 2 O 2 in the presence of uricase. Figure 3 (b) displayed all the absorbance value increased with time. However, OsAg NCs (20%) showed the biggest accelerated velocity and the highest absorbance after 20 min. 3.3.Kinetics analysis The steady-state kinetics were investigated and the results were shown in Fig. 4 . The Michaelis constant K m was calculated. The Lineweaver-Burk plot is obtained from the Michaelis-Menten equation, and K m and V max can be calculated by the double reciprocal of Lineweaver Burk. The smaller Km, the stronger the combination, and vice versa [ 25 ] . By using this method, the K m and V max of BSA-Os NCs with 20% silver content and 40% silver content were obtained. As shown in Table 1 , pure BSA-Os NCs with 20% silver content and 40% silver content have much lower K m to TMB compared to the pure Os NPs. It indicates that simulated enzymes have better reaction affinity for TMB. Comparing the three internally, it can be seen that BSA OsAg NCs with a 20% silver content have the lowest K m to TMB, indicating the best activity. Table 1 Comparison of the apparent Michaelis-Menten constant ( K m ) and maximum reaction rate ( V max ). Catalyst Substrate K m (mM) V max (10 − 7 M·s − 1 ) BSA-Os H 2 O 2 TMB 0.008 1.306 43.489 1.491 BSA-OsAg (20%Ag) H 2 O 2 TMB 5.54 0.121 7.966 9.494 BSA-OsAg (40%Ag) H 2 O 2 TMB 2.023 0.167 2.644 7.112 Os H 2 O 2 TMB 27.342 1.53 43.197 6.585 3.4. Catalytic mechanism Terephthalic acid (TA) can act as a capture molecule and react with ·OH to generate fluorescent 2-hydroxyterephthalic acid (TAOH) [ 26 ] . Therefore, we verified whether ·OH was generated in the reaction through TA oxidation experiments (Fig. 5 ). The fluorescence spectra of TA + H 2 O 2 and BSA-OsAg NCs with different silver contents excited at 310nm emitted unique fluorescence around 425nm, indicating that BSA-OsAg NCs generate ·OH and promote the reaction between H 2 O 2 and TA. 3.5. Detection of trypsin Detection of trypsin is of great importance for our health. We found the absorbance at 652nm increased upon addition of trypsin. Based on the enhancement effect of trypsin on the peroxidase activity of BSA-OsAg NCs, the concentration of trypsin was detected using BSA-OsAg NCs(20%). From Fig. 6 (a), it can be observed that as the concentration of trypsin increaseed, the absorption value at 652nm became higher. Figure 6 (b) shows the relationship between absorbance at 652nm and trypsin concentration. It can be seen that the absorbance shows an upward trend between 0-135ng/mL. There is a good linear relationship in the range of 0-100ng/mL with a detection limit (LOD) of 5ng/mL. Table 2 shows the comparison of different methods for trypsin detection. It can be seen this method has lower LOD compared to using other metal nanoparticles. Table 2 Comparison for the detection of trypsin Materials Linear range (ng/mL) LOD(ng/mL) Ref. BSA-stabilized Au NCs 10-50000 9 [ 27 ] SiQDs and silver nanoprisms 0–40 8 [ 28 ] Poly(thymine)-Templated Cu Nanoparticles 250-1000000 42 [ 1 ] Oligonucleotide-stabilized Ag Nanoclusters 700–4000 58.7 [ 29 ] AuNCs/CdTe QDs 20–500 12 [ 30 ] BSA-OsAg NCs 0-135 5 This work The selectivity experiment for trypsin detection was conducted and results showed in Fig. 6 (c,d). It can be seen that the absorption values of trypsin were significantly higher than other substances with the same concentration. In addition, to verify the feasibility of this method in practical application, the detection of trypsin in 10 times diluted fetal bovine serum was measured. As shown in Table 3 , the recovery rate of trypsin obtained was between 86.8% and 106.5%. These results indicate that it is feasible for this method to detect trypsin in practical application. Table 3 Detection of trypsin in fetal bovine serum. Samples Added (ng/mL) Measured (ng/mL) Recovery (%) RSD (%) 30 31.94 106.5 1.29 Trypsin 60 52.06 86.8 1.72 80 77.2 96.5 2.4 4. Conclusion Uniform bimetallic BSA-OsAg NCs (Ag 20%, 40%, 70%) were synthesized. Optical characterization revealed that BSA-OsAg NCs (20%) showed the strongest peroxidase-like activity with lower K m for TMB compared to other kinds of nanoclusters. BSA-OsAg NCs (70%) has the lowest catalytic ability. Based on the enhancement effect of trypsin on the peroxidase-like activity of BSA-OsAg NCs, a colorimetric assay for trypsin was proposed. This method exhibits excellent selectivity and practical application in fetal bovine serum. 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Technology","correspondingAuthor":false,"prefix":"","firstName":"Min","middleName":"","lastName":"Huo","suffix":""},{"id":451426108,"identity":"eff19cb5-6039-4935-b2eb-551d2e9c5234","order_by":3,"name":"Zijie Wei","email":"","orcid":"","institution":"Yancheng Institute of Technology","correspondingAuthor":false,"prefix":"","firstName":"Zijie","middleName":"","lastName":"Wei","suffix":""},{"id":451426109,"identity":"1d082c07-3f4b-45b8-a62d-4e88baa23d0d","order_by":4,"name":"Wanting Han","email":"","orcid":"","institution":"Yancheng Institute of Technology","correspondingAuthor":false,"prefix":"","firstName":"Wanting","middleName":"","lastName":"Han","suffix":""}],"badges":[],"createdAt":"2025-04-04 06:38:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6373736/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6373736/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82094195,"identity":"567d0c87-b867-417d-9ec9-7eccb10ae79a","added_by":"auto","created_at":"2025-05-06 17:02:47","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":426101,"visible":true,"origin":"","legend":"\u003cp\u003eTEM images of BSA-OsAg NCs (a) 20% (b) 40% (c) 70%\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6373736/v1/8190c4ae353141201ffe0489.png"},{"id":82094194,"identity":"2954ea41-1993-4869-a656-2232fd3d54ae","added_by":"auto","created_at":"2025-05-06 17:02:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":7466,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR spectra of BSA, BSA-OsAg NCs and BSA-OsAg NCs+Trypsin\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6373736/v1/0e9da5f2cf56d69ea64df783.png"},{"id":82094199,"identity":"a49bb51d-32a7-48ee-bdce-3457127df3d3","added_by":"auto","created_at":"2025-05-06 17:02:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":245264,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Absorption spectra of TMB oxidation in the presence of different substances, (b) change of A\u003csub\u003e652nm\u003c/sub\u003e over time for different substantces.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6373736/v1/5f7bce04bce74f8c5a507e5f.png"},{"id":82094779,"identity":"6c8aa65b-4abb-4029-9d48-c71ae66408df","added_by":"auto","created_at":"2025-05-06 17:10:47","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":117747,"visible":true,"origin":"","legend":"\u003cp\u003eSteady-state kinetic analysis with BSA-Os NCs with 20% silver content, Michaelis-Menten trend of (a) H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and (b) TMB, Lineweaver-Burk plot of (c) H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and (d) TMB.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6373736/v1/3123ea23491ec69e92cd5dc7.png"},{"id":82094201,"identity":"7df6a3f5-be8c-43b0-bfbc-1e67a5a2cfb2","added_by":"auto","created_at":"2025-05-06 17:02:47","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":5791,"visible":true,"origin":"","legend":"\u003cp\u003eThe fluorescence absorption values of TA oxidation excited at 310nm.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6373736/v1/c9f046416d391b985780d78b.png"},{"id":82094205,"identity":"84a016b6-bf1b-4186-b268-1f38f4f7e3be","added_by":"auto","created_at":"2025-05-06 17:02:48","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":181913,"visible":true,"origin":"","legend":"\u003cp\u003e(a) UV-Vis absorption spectrum with different concentrations of trypsin(b) relationship between absorption values at 652nm and concentration of trypsin (c) UV-Vis absorption spectrum with different substances (d) absorption values at 652 nm with different substances.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-6373736/v1/3cb196dbe0f441fc6aa81036.png"},{"id":82611013,"identity":"28294f0c-56a0-4fdf-b783-bfcaeae9acb9","added_by":"auto","created_at":"2025-05-13 10:47:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1714914,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6373736/v1/f659b3d0-5473-4915-90bb-06d62a43ef41.pdf"},{"id":82094778,"identity":"5828c625-e922-4272-b201-fe7e9109bec0","added_by":"auto","created_at":"2025-05-06 17:10:47","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1083184,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-6373736/v1/ef007cfd8bdf15551eac43cc.docx"},{"id":82094782,"identity":"8b8547d2-c882-4124-9a5d-ba6bafda0322","added_by":"auto","created_at":"2025-05-06 17:10:48","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":156162,"visible":true,"origin":"","legend":"","description":"","filename":"graphicalabstract.docx","url":"https://assets-eu.researchsquare.com/files/rs-6373736/v1/e744f88263c79c1c55ce510f.docx"},{"id":82094780,"identity":"7b727f7e-73c5-4802-8c03-71ebfa4a8f1c","added_by":"auto","created_at":"2025-05-06 17:10:47","extension":"jpeg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":141655,"visible":true,"origin":"","legend":"\u003cp\u003eScheme1 illustration for the detection of trypsin concentration by using BSA-OsAg NCs.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6373736/v1/259e155020fd91739a3453e6.jpeg"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eEnzyme mimics of bimetallic nanoclusters for detection of trypsin\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eTrypsin is a very important protease in the human internal environment. The occurrence of many diseases,such as pancreatitis\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e and metastasis of tumors\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e is related to its content.Therefore, a reliable method for detecting pancreatic protease is of great significance. Several detection methods for trypsin have been developed, such as fluorescence assay\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e, electrochemical assay\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e and surface enhanced Raman scattering\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e.Nevertheless, it is necessary to develop simpler and more convenient analytical methods for detecting trypsin concentration. Colorimetric method has become a good choice for solving this problem due to its advantage of simple operation and easy readability\u003csup\u003e[\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMeanwhile, nanozyme has become a strong tool for colorimetric analysis attributed to its color change from chromogenic substrate to its oxidized form\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Especially, noble metal nanoparticles Os has attracted attention for its significant higher peroxidase-like activity compared to other noble metal nanoparticles\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. Os is an attractive precious metal with the highest density, low compressibility, high melting point, and bulk modulus\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. However, exploration of Os nanoclusters is rarely reported\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. In fact, nanoclusters (NCs) composed of several atoms with smaller size usually can exhibit special catalytic properties. Therefore, they can be involved in many applications in the field of sensing\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e.It is reported that Os@Mucin NCs exhibited tumor environment-responsive peroxidase-like activity\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e, BSA\u0026thinsp;\u0026minus;\u0026thinsp;Os NCs were found to possess intrinsic peroxidase-like activity,enable a BSA\u0026thinsp;\u0026minus;\u0026thinsp;Os NCs-based colorimetric sensor to detect H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e from complex systems\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e.The laminarin-modulated osmium (laminarin-Os) nanoclusters had the selective peroxidase-like behavior under acidic conditions and were used for engineering colorimetric assay for hydroxyl radical\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. These reports declared Os nanoclusters had peroxidase-like activity. Do bimetallic nanoclusters based on Os own better enzyme mimics? With this question in mind, we prepared OsAg bimetallic nanoclusters and investigated their catalytic property.\u003c/p\u003e \u003cp\u003eHerein, BSA-OsAg NCs were prepared and applied for trypsin detection (as shown in Scheme 1). Among them, the BSA-OsAg NCs with a 20% silver content showed a smaller \u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e value and stronger affinity for the substrate TMB.In addition, based on the enhancement effect of trypsin on the peroxidase activity of BSA-OsAg NCs, the concentration of trypsin was detected using BSA-OsAg NCs with a 20% silver content. In order to explore its practical application effect, we found that the recovery rate in fetal bovine serum ranged from 86.8\u0026ndash;106.5%, indicating high practicality. This indicates that this method has strong application prospects in environments without complex equipment.\u003c/p\u003e "},{"header":"2. Experimental","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Chemicals\u003c/h2\u003e \u003cp\u003eK\u003csub\u003e2\u003c/sub\u003eOsCl\u003csub\u003e6\u003c/sub\u003e, NaOH, AgNO\u003csub\u003e3\u003c/sub\u003e, Rhodamine B (RhB), terephthalic acid (TA), bovine serum albumin (BSA) were purchased from Aladdin Industrial Corporation. Hydrogen peroxide (98%) and acetate buffer were purchased from Jiangsu Tongsheng Chemical Reagent Company. 3,3',5,5'-tetramethylbenzidine (TMB), trypsin, metal ions, amino acids and biomolecules were purchased from BBI Life Sciences.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Preparation of BSA-Os nanoclusters (Os NCs)\u003c/h2\u003e \u003cp\u003e0.5mL 25mg/mL BSA aqueous solution and 0.5mL 20 mM K\u003csub\u003e2\u003c/sub\u003eOsCl\u003csub\u003e6\u003c/sub\u003e solution were added into a small centrifuge tube. Then, the pH of the total solution was adjusted to 12 by using 50\u0026micro;L 1M NaOH. After mixed thoroughly, the mixture was incubated in a water bath at 37℃ for 7 hours to obtain BSA-Os NCs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Preparation of BSA-OsAg nanoclusters (OsAg NCs) with different silver contents\u003c/h2\u003e \u003cp\u003e0.5mL 20mM K\u003csub\u003e2\u003c/sub\u003eOsCl\u003csub\u003e6\u003c/sub\u003e solution was mixed with 8 mM AgNO\u003csub\u003e3\u003c/sub\u003e solution to make the molar ratios of K\u003csub\u003e2\u003c/sub\u003eOsCl\u003csub\u003e6\u003c/sub\u003e and AgNO\u003csub\u003e3\u003c/sub\u003e as 4:1, 3:2, and 7:3, respectively. Then, the following procedure was the same as preparation of BSA-Os NCs. Finally, BSA-OsAg NCs with silver content of 20%, 40%, and 70% were obtained, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Enzyme-mimetic activities\u003c/h2\u003e \u003cp\u003eThe peroxidase like activity (simulated enzyme activity) of OsAg NCs was evaluated by using chromogenic substrate TMB in the presence or absence of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Typically, 50\u0026micro;L TMB(8mM), 50\u0026micro;L H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e(50mM), 10\u0026micro;L BSA-OsAg NCs, 350\u0026micro;L acetic acid buffer (pH\u0026thinsp;=\u0026thinsp;4.8) and 500\u0026micro;L deionized water were mixed. The absorption spectrum of the mixed solution was measured after 10 minutes incubation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Kinetics analysis\u003c/h2\u003e \u003cp\u003eThe steady-state kinetics of BSA-OsAg NCs were investigated. In order to obtain the Michaelis constant (\u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e) and maximum reaction rate (\u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e) for H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, the same concentration of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was used while changing the TMB (0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, 8 mM) at different concentrations. The absorption values obtained from UV measurements were used for calculation. On the other hand, in order to obtain the Michaelis constant (\u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e) and maximum reaction rate (\u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e) for TMB, UV absorption values were measured using the same concentration of TMB and different concentrations of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (0.5, 1, 2, 5, 8, 10, 20, 30, 40, 50mM).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Mechanism investigation\u003c/h2\u003e \u003cp\u003eIn order to verify the generation of hydroxyl radicals, a benzoic acid (TA) oxidation experiment was conducted. A concentration of 25 mM TA was prepared by using 0.1M NaOH solution. Then, 15\u0026micro;L TA, 150\u0026micro;L H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (50 mM) and 30\u0026micro;L BSA-OsAg NCs were added to 1.05mL of acetic acid buffer (pH\u0026thinsp;=\u0026thinsp;4.8) and incubated in the dark for 1 hour. Finally, the fluorescence spectrum of the mixture was colloected.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Detection of trypsin\u003c/h2\u003e \u003cp\u003eTrypsin with different concentrations (5, 10, 50, 80, 100, 250, 500, 1000, 1500, 2000, 3000 ng/mL) were prepared. 50\u0026micro;L trypsin, 350\u0026micro;L buffer solution (pH\u0026thinsp;=\u0026thinsp;4.8), 50\u0026micro;L H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (50 mM), 50\u0026micro;L TMB (8mM), 10\u0026micro;L BSA-OsAg NCs with 20% silver content and 650 \u0026micro;L deionized water were mixed and incubated for 5 minutes to obtain absorption spectra.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8.Characterization\u003c/h2\u003e \u003cp\u003eThe morphology and microstructure was observed and analyzed by transmission electron microscope (TEM, JEM2100F). The absorption spectroscopy were read from UV\u0026ndash;Vis (UV-2600).Fourier-transform infrared spectroscopy (FTIR) was recorded by using NEXUS-670. Fluorescence properties were explored with Fluorescence spectrometer (JASCO FP-6500).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Characterization\u003c/h2\u003e \u003cp\u003eFirstly, the morphology and microstructure of BSA-OsAg NCs were characterized using TEM. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(a) - (c), the size of BSA-OsAg NCs (20%) is about 1nm. However, the size of BSA-OsAg NCs (40%) and (70%) are around 2 nm. Moreover, most nanoparticles sticked together and aggregated into large spheres for BSA-OsAg NCs (70%).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFourier transform infrared analysis was performed on individual BSA and BSA-OsAg NCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).The FTIR spectra of BSA at 3300\u0026thinsp;~\u0026thinsp;3500cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is the N-H stretching vibration,1600\u0026thinsp;~\u0026thinsp;1700cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is a carbonyl stretching vibration and the amide Ⅰ band,1500\u0026thinsp;~\u0026thinsp;1600cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is the C-N stretching vibration, also amide Ⅱ band\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. No obvious change for these band spectra was found in BSA-OsAg NCs, indicating that the influence of BSA-OsAg NCs on the structure of BSA is relatively small and will not damage the structure of BSA.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.2.Enzyme-mimetic activities\u003c/h2\u003e \u003cp\u003eThe simulated enzymatic properties of BSA-OsAg NCs were evaluated using the chromogenic substrate TMB, which will turn blue with appropriate catalysts. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(a), BSA-OsAg NCs(0%, 20%, 40%, 70%)\u0026thinsp;+\u0026thinsp;TMB\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e exhibited blue color and significant absorption at 652 nm, indicating that BSA-OsAg NCs possessed peroxidase like activity. It was also found that the color of solution with 70% silver content is relatively light compared to the other three, according with the lower absorption peak at 652 nm (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Based on this, it can be concluded that the peroxidase like activity of the BSA-OsAg NCs with 70% silver content is very low. Therefore, subsequent activity studies only focused on pure BSA-Os NCs, 20% silver, and 40% silver content BSA-OsAg NCs. In addition, BSA-OsAg NCs\u0026thinsp;+\u0026thinsp;TMB showed almost colorless, indicating BSA-OsAg do not exhibit oxidase-like activity. Most noble metal nanoparticles had both peroxidase and oxidase-like activity\u003csup\u003e[\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. The specific activity of BSA-OsAg NCs could be applied for reaction of generation H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. For example, it could eliminate influence of O\u003csub\u003e2\u003c/sub\u003e on the activity when used for detection of uric acid, which will generate H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in the presence of uricase. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(b) displayed all the absorbance value increased with time. However, OsAg NCs (20%) showed the biggest accelerated velocity and the highest absorbance after 20 min.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.3.Kinetics analysis\u003c/h2\u003e \u003cp\u003eThe steady-state kinetics were investigated and the results were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The Michaelis constant \u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e was calculated. The Lineweaver-Burk plot is obtained from the Michaelis-Menten equation, and \u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e and \u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e can be calculated by the double reciprocal of Lineweaver Burk. The smaller Km, the stronger the combination, and vice versa\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBy using this method, the \u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e and \u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e of BSA-Os NCs with 20% silver content and 40% silver content were obtained.\u003c/p\u003e \u003cp\u003eAs shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, pure BSA-Os NCs with 20% silver content and 40% silver content have much lower \u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e to TMB compared to the pure Os NPs. It indicates that simulated enzymes have better reaction affinity for TMB. Comparing the three internally, it can be seen that BSA OsAg NCs with a 20% silver content have the lowest \u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e to TMB, indicating the best activity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of the apparent Michaelis-Menten constant (\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e) and maximum reaction rate (\u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCatalyst\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSubstrate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e(mM)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e (10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e M\u0026middot;s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBSA-Os\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003cp\u003eTMB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.008\u003c/p\u003e \u003cp\u003e1.306\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e43.489\u003c/p\u003e \u003cp\u003e1.491\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBSA-OsAg (20%Ag)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003cp\u003eTMB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.54\u003c/p\u003e \u003cp\u003e0.121\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.966\u003c/p\u003e \u003cp\u003e9.494\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBSA-OsAg (40%Ag)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003cp\u003eTMB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.023\u003c/p\u003e \u003cp\u003e0.167\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.644\u003c/p\u003e \u003cp\u003e7.112\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003cp\u003eTMB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27.342\u003c/p\u003e \u003cp\u003e1.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e43.197\u003c/p\u003e \u003cp\u003e6.585\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Catalytic mechanism\u003c/h2\u003e \u003cp\u003eTerephthalic acid (TA) can act as a capture molecule and react with \u0026middot;OH to generate fluorescent 2-hydroxyterephthalic acid (TAOH)\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. Therefore, we verified whether \u0026middot;OH was generated in the reaction through TA oxidation experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The fluorescence spectra of TA\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and BSA-OsAg NCs with different silver contents excited at 310nm emitted unique fluorescence around 425nm, indicating that BSA-OsAg NCs generate \u0026middot;OH and promote the reaction between H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and TA.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Detection of trypsin\u003c/h2\u003e \u003cp\u003eDetection of trypsin is of great importance for our health. We found the absorbance at 652nm increased upon addition of trypsin. Based on the enhancement effect of trypsin on the peroxidase activity of BSA-OsAg NCs, the concentration of trypsin was detected using BSA-OsAg NCs(20%). From Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e (a), it can be observed that as the concentration of trypsin increaseed, the absorption value at 652nm became higher. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e(b) shows the relationship between absorbance at 652nm and trypsin concentration. It can be seen that the absorbance shows an upward trend between 0-135ng/mL. There is a good linear relationship in the range of 0-100ng/mL with a detection limit (LOD) of 5ng/mL. Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the comparison of different methods for trypsin detection. It can be seen this method has lower LOD compared to using other metal nanoparticles.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison for the detection of trypsin\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMaterials\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLinear range (ng/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLOD(ng/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRef.\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBSA-stabilized Au NCs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10-50000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSiQDs and silver nanoprisms\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u0026ndash;40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePoly(thymine)-Templated Cu Nanoparticles\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e250-1000000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOligonucleotide-stabilized Ag\u003c/p\u003e \u003cp\u003eNanoclusters\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e700\u0026ndash;4000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e58.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAuNCs/CdTe QDs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20\u0026ndash;500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBSA-OsAg NCs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0-135\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eThis work\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe selectivity experiment for trypsin detection was conducted and results showed in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e(c,d). It can be seen that the absorption values of trypsin were significantly higher than other substances with the same concentration.\u003c/p\u003e \u003cp\u003eIn addition, to verify the feasibility of this method in practical application, the detection of trypsin in 10 times diluted fetal bovine serum was measured. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, the recovery rate of trypsin obtained was between 86.8% and 106.5%. These results indicate that it is feasible for this method to detect trypsin in practical application.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDetection of trypsin in fetal bovine serum.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSamples\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAdded (ng/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMeasured (ng/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRecovery (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRSD (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e31.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e106.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.29\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTrypsin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e52.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e86.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.72\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e77.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e96.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eUniform bimetallic BSA-OsAg NCs (Ag 20%, 40%, 70%) were synthesized. Optical characterization revealed that BSA-OsAg NCs (20%) showed the strongest peroxidase-like activity with lower \u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e for TMB compared to other kinds of nanoclusters. BSA-OsAg NCs (70%) has the lowest catalytic ability. Based on the enhancement effect of trypsin on the peroxidase-like activity of BSA-OsAg NCs, a colorimetric assay for trypsin was proposed. This method exhibits excellent selectivity and practical application in fetal bovine serum. In conclusion, this study revealed that bimetallic nanoclusters with appropriate proportion could achieve better catalytic ability. It provides some ideas for the design of excellent nanozyme.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declared that they have no conflicts of interest to this work.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis work was supported by Natural Science Foundation of China (21605128).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eL. J. Ou, X. Y. Li, L. J. Li, H. W. Liu, A. M. Sun, K. J. Liu (2015).A sensitive assay for trypsin using poly(thymine)-templated copper nanoparticles as fluorescent probes. \u003cem\u003eAnalyst\u003c/em\u003e.\u003cstrong\u003e140\u003c/strong\u003e, 1871-1875.https://10.1039/c4an01994f.\u003c/li\u003e\n\u003cli\u003eS. He, L. Yang, P. Balasubramanian, S. Li, H. Peng, Y. Kuang, H. Deng, W. 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Pan (2023).Improved sensitivity of gold nanoclusters toward trypsin under synergistic adsorption of CdTe quantum dots. \u003cem\u003eMICROCHEM J\u003c/em\u003e.\u003cstrong\u003e187\u003c/strong\u003e.https://10.1016/j.microc.2023.108457.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Scheme 1","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e\n"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"BSA-OsAg, bimetallic nanoclusters, peroxidase-like activity, trypsin","lastPublishedDoi":"10.21203/rs.3.rs-6373736/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6373736/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDetection of trypsin is very important for human health. In this work, bovine serum albumin protected OsAg nanoclusters (BSA-OsAg NCs) were prepared. The ratio of Ag element in the nanoclusters could be regulated by controlling the reagent concentration. UV-Visible absorption spectra acquired from TMB(3,3',5,5'-Tetramethylbenzidine) to oxTMB were used to investigate the catalytic ability of the nanoclusters. The nanoclusters all displayed peroxidase-like activity. Especially, the BSA-OsAg NCs with 20% silver content showed the best catalytic ability. In addition, trypsin effectively increased the catalytic activity of BSA-OsAg NCs due to the interaction between trypsin and BSA. Based on this phenomenon, a novel method for selectively detection of trypsin was developed with a detection limit (LOD) of 5ng/mL. Promisingly, this method can be used for detection of trypsin in fetal bovine serum with acceptable recovery rate.\u003c/p\u003e","manuscriptTitle":"Enzyme mimics of bimetallic nanoclusters for detection of trypsin","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-06 17:02:43","doi":"10.21203/rs.3.rs-6373736/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2e956ffc-f4ed-483f-8f4c-fda90e58bedb","owner":[],"postedDate":"May 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-05-13T10:38:55+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-06 17:02:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6373736","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6373736","identity":"rs-6373736","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}