Immobilization of Trypsin on Superparamagnetic Nanoparticles for

Mar 8, 2007 - Finally, immo- bilization of trypsin onto the aldehyde-functionalized magnetic nanoparticles was achieved through reaction of the aldehy...
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Immobilization of Trypsin on Superparamagnetic Nanoparticles for Rapid and Effective Proteolysis Yan Li, Xiuqing Xu, Chunhui Deng,* Pengyuan Yang, and Xiangmin Zhang* Department of Chemistry & Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, China Received March 8, 2007

Abstract: In this work, a novel and facile route was developed for the immobilization of enzyme on nanosized magnetic particles, and its application to fast protein digestion via a direct MALDI-TOF mass spectrometry analysis was demonstrated. At first, amine-functionalized magnetic particles with high magnetic responsivity and excellent dispersibility were prepared through a facile one-pot strategy. Then, magnetic nanoparticles were functionalized with numerous aldehyde(-CHO) groups by treating the as-synthesized, amine-functionalized magnetic nanoparticles with glutaraldehyde. Finally, immobilization of trypsin onto the aldehyde-functionalized magnetic nanoparticles was achieved through reaction of the aldehyde groups with amine groups of trypsin. The obtained trypsin-immobilized magnetic nanoparticles were conveniently applied for protein digestion. The digestion efficiency was demonstrated with peptide mapping analysis of three model proteins. The process of digestion is very facile due to the easy manipulation of magnetic nanoparticles. Complete protein digestion was achieved in a short time (5 min), without any complicated reduction and alkylation procedures. These results are expected to open up a new possibility for the proteolysis analysis as well as a new application of magnetic nanoparticles. Additionally, it is worth noting that, since the preparation and surface functionality of magnetic nanoparticles is lowcost and reproducible, the preparation method and application approach of the magnetic nanoparticles may find much potential in proteome research. Keywords: amine-functionalized magnetic particles • immobilized trypsin • digestion on magnetic nanoparticles

Introduction With the acceleration of complete genomes discovery, the challenge of proteomics is to identify and characterize proteins encoded by the genomes as efficiently and rapidly as possible.1 Proteolysis is the key step for positive sequencing in proteomics research integrated with MALDI-TOF MS. The conventional techniques of in-solution digestion of proteins offer limited sensitivity and are time-consuming procedures, affecting se* Corresponding authors, Prof. Dr. C. H. Deng and Prof. Dr. X. M. Zhang. E-mails: [email protected] (C.D.), [email protected] (X.Z.); tel, +86-21-65643983; fax, +86-21-65641740. 10.1021/pr070132s CCC: $37.00

 2007 American Chemical Society

verely the determination of comprehensive proteomic profiles.2,3 To solve this problem, immobilized enzyme has been widely utilized owning to their advantage of allowing the use of higher enzyme concentrations that lead to shorter digestion time, Furthermore, the immobilized enzyme could be isolated and removed from the protein digests prior to MS easily, eliminating or reducing the influence of the enzyme fragments on MS results. Additionally, the stability of enzyme toward chemical denaturants and organic solvents could be enhanced when immobilized on the solid supports.4-6 In recent years, several reports have demonstrated the feasibility of protein digestion using enzyme immobilized on various supports, such as glass,7 membrane,8,9 polymer,10 gel beads,11,12 sol-gel supports,13,14 porous silicon matrix,15-17 and porous monolithic materials.18-20 Magnetic polymer microspheres, due to unique magnetic responsivity and high dispersibility, are versatile supports for the immobilization of enzyme.21-24 Application of immobilized enzymes using magnetic polymer microspheres as carriers offers a distinct advantage over soluble enzymes, because they can be easily removed from the reaction mixture with the assistance of magnetic field, and can be used repeatedly. The storage properties and the pH stability of enzymes are often improved by immobilization.25-28 Therefore, immobilization of enzyme onto magnetic polymer microspheres can facilitates the separation and recycling of immobilized enzyme and the purification of product. In general, magnetic polymer microspheres are prepared by encapsulating inorganic magnetic particles (usually magnetite or maghemite) with organic polymers such as polystyrene and poly (alkyl acrylate) or with inorganic silica.29,30 However, the magnetic polymer microspheres reported in most of previous works showed poor responsivity to the applied magnetic field due to the low content of magnetic component in the magnetic polymer microspheres, which hampered their application for fast and efficient digestion of proteins. To solve this problem, in our previous work,30 we have synthesized magnetic microspheres composed of a large number of magnetite nanoparticles which led to strong magnetism. Additionally, after coating them with silica using a sol-gel approach based on the hydrolysis and condensation of tetraethyl orthosilicate (TEOS), the magnetic microspheres still retain high magnetic responsivity. Recently, we have immobilized trypsin onto these magnetic silica microspheres and utilized them for fabrication of an easily replaceable and regenerable on-chip enzymatic reactor.31 To immobilize trypsin, the metal chelating agent of iminodiacetic acid (IDA) was first reacted with glycidoxypropyltrimethoxysiJournal of Proteome Research 2007, 6, 3849-3855

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technical notes

Immobilization of Trypsin on Superparamagnetic Nanoparticles

Scheme 1. (a) Schematic Illustration of Trypsin Immobilization onto the Amine-Functionalized Magnetic Nanoparticles; (b) Process of Protein Digestion Using Trypsin-Immobilized Magnetic Nanoparticles

lane (GLYMO) and then grafted on the surface of the magnetic silica microspheres. The metal ion of copper and trypsin was subsequently adsorbed onto the surface through chelation. The method proved to be effective and has been successfully used for protein digestion. However, when using this method, three steps of surface modification on the magnetic microspheres were required prior to trypsin immobilization, which resulted in a complicated and time-consuming procedure. Isaac Koh et al.32 modified commercially available magnetic nanoparticles with aminopropyltriethoxy silane (APTES) and then activated them with glutaraldhyde prior to the immobilization of proteins on the nanoparticle surface. Their work offered a simpler way for preparation of protein-immobilized magnetic nanoparticles; however, five reaction steps are still needed. The work done by Nishimura et al.33 involves the in situ preparation of magnetic nanoparticles (mixture of Fe3O4/γ-Fe2O3) in the presence of trypsin at 4 °C by chemical coprecipitation of FeCl2 and FeCl3 using NH4OH as precipitator). This approach can directly lead to trypsin-modified magnetic nanoparticles; however because the synthesis temperature is too low, the obtained magnetic nanoparticles have poor crystallization as indicated by the X-ray diffraction spectrum. As a result, the magnetic nanoparticles possess poor magnetic response which may influence the practical application. Additionally, because the magnetic nanoparticles were synthesized in the trypsin aqueous solution, the location of trypsin molecules in the trypsinmodified magnetic nanoparticles is ill-defined. Therefore, the application of the trypsin-modified magnetic nanoparticles is quite questionable. In fact, until now, to our best knowledge, few works have been reported on the application of these functionalized magnetic nanoparticles. In this study, we report a novel and facile way for the preparation and application of trypsin immobilized magnetic nanoparticles with superparamagetism (Scheme 1). First, aminefunctionalized magnetic nanoparticles were prepared through a facile one-pot solvothermal synthetic strategy. Then, magnetic nanoparticles were functionalized with numerous alde3850

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hyde(-CHO) groups by treating the as-synthesized, aminefunctionalized magnetic nanoparticles with glutaraldehyde (GA). Finally, immobilization of trypsin onto the aldehydefunctionalized magnetic nanoparticles was achieved through reaction of the aldehyde groups with amine groups of trypsin. The obtained magnetic nanoparticles immobilized with trypsin were conveniently applied for protein digestion. The digestion efficiency was demonstrated with peptide mapping analysis of three model proteins.

Experimental Section Chemicals. (TPCK)-treated trypsin, cytochrome c (EC 232700-9), bovine serum albumin (BSA), myoglobin, and sodium cyanoborohydride (NaCNBH3) were purchased from Sigma Chemical (St. Louis, MO). Glycine and glutaraldehyde (25% (w/ v) aqueous solution) were from the Shanghai Chemical Reagent Co., Ltd. (Shanghai, China). All of other chemicals were of analytical grade and used as received without further purification. Water was purified using a Milli Q system (Millipore, Molsheim, France). Preparation of Amine-Functionalized Magnetic Nanoparticles. A solution of 1, 6-hexadiamine (3.6 g), anhydrous sodium acetate (4.0 g) and FeCl3‚6H2O (1.0 g) as a ferric source in ethylene glycol (30 mL) was stirred vigorously to acquire a transparent solution. The mixture was sealed in a Teflon-lined, stainless-steel autoclave and was heated at 200 °C for 6 h. The product, which settled at the bottom of the autoclave, was washed with hot water and ethanol (3 times) under ultrasonic conditions to remove the solvent and unbound 1,6-hexanediamine effectively, and then dried at 50 °C to gain the black powder. The product was separated from various solvents by using magnetic force during each step. Immobilization of Trypsin onto Amine-Functionalized Magnetic Nanoparticles. A total of 3 mg of amine-functionalized magnetic nanoparticles was transferred to a 1.5-mL Eppendorf tube, and the nanoparticles were retained by a

technical notes

Li et al.

Figure 2. The hysteresis loops of amine-functionalized magnetic nanoparticles, indicating that an MS value of about 45.0 emu/g.

Figure 1. (a) TEM image; (b) SEM image of the amine-functionalized magnetic nanoparticles.

magnet. The solution was removed, the nanoparticles were resuspended in 200 µL of coupling buffer (CB; 50 mM NH4OAc, pH 8.3, 1 mM CaCl2, and 1 mM MnCl2) and retained by a magnet again, and the solution was removed. The amine group of the nanoparticles was activated at room temperature under gentle rotation for 1.5 h by 200 µL of 5% glutaraldehyde solution in CB (pH ∼7.0). The nanoparticles were then retained by a magnet, and the solution was removed, followed by four washes each in 200 µL of CB. A total of 0.6 mg (TPCK)-treated trypsin was dissolved in 300 µL of CB containing 1% NaCNBH3, and the nanoparticles were incubated with the protein solution for 3 h under rotation. After removal of the (TPCK)-treated trypsin solution, the nanoparticles were incubated for 1 h with 200 µL of 0.75% glycine and 1% NaCNBH3 in CB. Finally the nanoparticles were washed four times in 200 µL of CB, before they were ready for use. Measurement for Amount of Trypsin Immobilized on Magnetic Nanoparticles. After the trypsin immobilization procedure was conducted, the magnetic nanoparticles were retained by a magnet, and the UV absorption value of the supernatant solution was measured at λ ) 280 nm to calculate the amount of trypsin immobilized on the magnetic nanoparticles. For accurate calculation of the amount immobilized, a calibration curve was obtained at λ ) 280 nm by the use of a series of standard protein solutions with different concentrations (buffered at pH 8.3). In-Solution Digestion of Standard Proteins. Three standard proteins, cytochrome c (Cyt-C), bovine serum albumin (BSA),

and myoglobin (MYO) were first denatured in 25 mM NH4HCO3 buffer containing 8 M urea for 1 h at 37 °C, followed by dilution with 25 mM NH4HCO3 (pH 8.3) buffer to the concentration of urea below 1 M. The in-solution digestion was performed by adding trypsin into the protein solution at a substrate-toenzyme ratio of 50:1, and the solution was incubated at 37 °C for 12 h. After digestion, 1 µL of formic acid was added into the solution to stop the reaction. Digestion of Standard Proteins Using Trypsin-Immobilized Magnetic Nanoparticles. The (TPCK)-treated trypsin-immobilized magnetic nanoparticles (6 mg) were suspended in 300 µL of 25 mM NH4HCO3 aqueous solution. Three standard proteins (20 µg for each protein), cytochrome c (Cyt-C), bovine serum albumin (BSA), and myoglobin (MYO) in 10 µL of 25 mM NH4HCO3 buffer solution (pH 8.3) were denatured in a 95 °C water bath for 15 min. Then, 10 µL of trypsin-immobilized magnetic nanoparticles solution was added into each Eppendorf tube. After incubation at 37 °C for 5 min, the trypsinimmobilized magnetic nanoparticles were retained by a magnet, and the supernatant was removed to another Eppendorf tube. The magnet used is a NdFeB magnet with a cylinder shape of 5 mm (D) × 3 mm (h), and surface magnetic field of 2, 100 G, which was purchased from Yingke (Beijing, China). MALDI-TOF-MS Process. Sample solutions were deposited on the MALDI target using dried droplet method. A total of 0.5 µL of sample solution was spotted onto the MALDI plate, and then another 0.5 µL of CHCA matrix solution (5 mg/mL and 0.1% TFA in 50% acetonitrile aqueous solution) was introduced. Positive ion MALDI-TOF mass spectra were acquired on 4700 Proteomics Analyzer (Applied Biosystem). Sample desorption was achieved using an Nd:YAG laser (355 nm) operated at a repetition rate of 200 Hz and acceleration voltage of 20 kV.

Results and Discussion The Preparation and Characterization of Amine-Functionalized Magnetic Nanoparticles. The amine-functionalized magnetic nanoparticles with high quality were synthesized by complexation, using FeCl3‚6H2O as a single iron source and 1, 6-hexadiamine as the ligand (see Preparation of AmineFunctionalized Magnetic Nanoparticles). Compared with the micron-sized magnetic microspheres or beads,30,31 our aminefunctionalized magnetic nanoparticles with mean diameter of Journal of Proteome Research • Vol. 6, No. 9, 2007 3851

Immobilization of Trypsin on Superparamagnetic Nanoparticles

technical notes

Figure 3. MALDI-TOF MS spectra of tryptic peptides originated from (a) myoglobin, (b) cytochrome c, and (c) bovine serum albumin digested with trypsin-immobilized magnetic nanoparticles. m, peptide from myoglobin; c, peptide from cytochrome c; b, peptide from bovine serum albumin.

50 nm possess several advantages, including (1) high surface/ volume ratio and good solubility which can provide a higher binding rate; (2) high content of magnetic component (Fe3O4) and nanometer sizes which endow them with high magnetic responsibility and superparamagnetism, both of which are critical to their practical application; (3) high dispersibility in liquid media due to their small size and the existence of large 3852

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amount of stabilizer (i.e., amine groups) on the their surface. Remarkably, the amine-functionalized magnetic nanoparticles derived from the facile one-pot synthesis process can readily bioconjugate with enzyme by using coupling agents such as carbodiimide and glutaraldehyde and, thus, cut short the complicated and time-consuming procedure of surface modification for enzyme immobilization.

technical notes Transmission electron microscope (TEM) observation indicates the as-prepared magnetic nanoparticles are about 50 nm with narrow size distribution (Figure 1a). Scanning electron microscope (SEM) image of the as-prepared magnetic nanoparticles are nearly spherical in shape and possess a smooth surface. The uniform and small sizes of the magnetic nanoparticles provide them with a high surface area-to-volume ratio (S/V), large density of binding sites for immobilization of enzyme, excellent dispersibility in water, fast motion with agitation, and rapid response toward magnetic field, which is of interest to their application. The magnetic property of assynthesized magnetic nanoparticles was investigated with a vibrating magnetometer. Figure 2 displays hysteresis loops of amine-functionalized magnetic nanoparticles, which indicates they possess a magnetic saturation (MS) value of about 45.0 emu/g. Notably, the remanence of these nanoparticles is zero once the applied magnetic field is removed, implying that these nanoparticles are superparamagnetic. The superparamagnetic properties of the magnetic nanoparticles is critical for their application in biomedical and bioengineering field, which prevents them from aggregation and enables them to redisperse rapidly when the magnetic field is removed.34 Figure S1 (Supporting Information) shows the Fourier transform infrared (FTIR) spectra of the amine-functionalized magnetic nanoparticles. The strong adsorption peak at 576 cm-1 is characteristic of the Fe-O vibrations, while the peak around 1626 cm-1 is ascribed to free 1, 6-hexadiamine, indicating the existence of the free -NH2 group on the aminefunctionalized nanoparticles. The FTIR characterization revealed that the magnetic nanocrystals functionalized with amine groups have been successfully prepared. The formation of hydrogen bonds between the amino groups and water may result in good water solubility which makes them easy for enzyme immobilization. Immobilization of Enzyme onto Magnetic Nanoparticles. To utilize the as-prepared, amine-functionalized magnetic nanoparticles for proteolysis analysis, they were further functionalized with aldehyde group followed by subsequential bioconjugation with enzyme. Among the arsenal of reagents used for protein immobilization, glutaraldehyde (GA) was finally selected in this study because it can react rapidly with primary amines, and thus with proteins.35,36 Since trypsin is capable of selectively cleaving proteins at arginine and lysine residues, which could provide typically peptides in a mass range compatible with MS for amino acid sequence determination, it is the most popular enzyme to fragment proteins (tryptic digestion) for their identification and characterization in proteomic studies. Therefore, trypsin was used as model enzyme to be immobilized onto magnetic nanoparticles. The reaction of GA with proteins is generally carried out at around neutral pH.37 In the case of trypsin immobilization onto magnetic nanoparticles, activation of the nanoparticles with GA was conducted at neutral pH, whereas protein coupling was carried out at an elevated pH (pH 8.3) to promote nucleophilic attack and improve immobilization, as maintained by Walt and Agayn.38 The formation of a Schiff base by elimination of water is a proposed mechanism of reaction of GA with primary amines. This is known to be reversible and could lead to gradual release of enzyme during prolonged exposure to buffer solutions, particularly at elevated pH. Therefore, reduction of Schiff base double bonds using a suitable reducing agent like NaCNBH3 has been proposed in order to produce a stable secondary amine that can tolerate pH variations. Finally, the

Li et al. Table 1. Identified Peptide Residues Digested with Trypsin-Immobilized Magnetic Nanoparticles protein myoglobin (AC P68082)a

MCb position

position

MW

1-16 17-31 17-42 32-42 32-45 48-56 64-77 64-78 79-96 80-96 103-118 119-133 134-139 134-145 146-153

1815.95 1606.89 2859.52 1271.69 1661.89 1086.58 1378.88 1506.98 1982.10 1854.00 1885.07 1502.71 748.46 1360.79 941.50

Seq. Cov.c Pep. Mat.d

BSA (AC P02769)a

cytochrome c (AC P00004)a

0 0 1 0 1 1 0 1 1 0 0 0 0 1 1

90% 15

9-22 14-22 26-38 28-38 28-39 39-53 40-53 40-55 56-72 61-72 61-73 80-86 89-99

MCb position

MW

1633.66 1018.47 1433.81 1168.65 1296.75 1598.83 1470.73 1712.89 2081.11 1495.77 1623.85 779.47 1350.76

Seq. Cov.c Pep. Mat.d

1 0 1 0 1 1 0 1 1 0 1 0 1

76% 13

MW

MCb

35-44 1249.65 1 45-65 2435.22 0 66-75 1163.66 0 89-105 1889.00 1 161-167 927.52 0 168-183 2045.05 1 221-228 918.53 1 223-232 1138.60 1 233-241 1001.62 1 249-263 1692.98 1 286-297 1386.65 0 347-359 1567.77 0 347-360 1723.87 1 360-371 1439.84 1 361-371 1283.74 0 402-412 1305.74 0 413-420 1011.44 0 413-433 2472.21 1 413-433 1479.82 0 421-433 2472.21 1 437-451 1639.98 1 438-451 1511.88 0 469-482 1667.86 0 483-489 841.48 0 499-507 1024.49 0 508-523 1823.92 0 529-544 1850.94 0 548-557 1142.73 1 549-557 1014.64 0 588-597 1050.40 0 Seq. Cov.c 46% Pep. Mat.d 30

a AC, Expasy accession number. b MC, number of missed cleavages. c Seq. Cov. ) sequence coverage. d Pep. Mat. ) peptides matched.

trypsin-immobilized magnetic nanoparticles were treated with glycine to deactivate the free (i.e., unreacted) aldehyde groups on the magnetic nanoparticles. The immobilization ability of magnetic nanoparticles for trypsin was studied by measuring the UV absorption value of the supernatant trypsin solution after the immobilization procedure, and the amount of trypsin immobilized on the magnetic nanoparticles was about 78 µg/ mg. Application of the Immobilized Enzyme on Magnetic Nanoparticles for Digestion. Three standard proteins, myoglobin (MYO, a protein known to be rather resistant to proteolysis, MW 16 900), cytochrome c (Cyt-C, MW 12 384), and bovine serum albumin (BSA, MW 66 000) were used to test the performance of trypsin-immobilized magnetic nanoparticles. The procedure of tryptic digestion using functionalized magnetic nanoparticles is depicted in Scheme 1. After digestion, the trypsin-immobilized magnetic nanoparticles can easily be separated from the digestion products by using a magnet. The resulted tryptic digests were then mass-analyzed by MALDITOF MS. Figure 3 displays the mass spectra of tryptic fragments generated from 5 min digestion using trypsin-immobilized magnetic nanoparticles. Many digest fragments were observed from the MS spectra. Detailed identification results were listed in Table 1. The observation corresponded to the detection of fragments containing 138 out of the 153 possible amino acids of MYO, 80 out of the 104 possible amino acids of Cyt-C, and 283 out of the 583 possible amino acids of BSA. The sequence coverages of 90% for MYO, 76% for Cyt-C, and 46% for BSA Journal of Proteome Research • Vol. 6, No. 9, 2007 3853

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Immobilization of Trypsin on Superparamagnetic Nanoparticles

Table 2. Summary of MALDI-TOF MS Results Obtained from 5-min Digestion with Trypsin-Immobilized Magnetic Nanoparticles Compared with 12-h In-Solution Digestion MYO

Cyt-C

BSA

protein

magnetic nanoparticles

in-solution

magnetic nanoparticles

in-solution

magnetic nanoparticles

in-solution

Amino acids identified Sequence coverage (%) Peptides matched

138 90 15

115 75 11

80 76 13

80 76 14

283 46 30

253 41 24

from the database were obtained. The identification results are comparable with or even better than those by in-solution digestion that required a complicated denaturing procedure and a reaction time of 12 h (Table 2). To demonstrate the ability of the approach for unambiguous protein identification, we carried out MS-MS fragmentation of isolated peptides to obtain sequence information. Figure S2 (Supporting Information) displays the MS/MS spectra of precursor ions of 1606.85, 1885.02, and 1502.67 marked with asterisk in Figure 3a. The spectra revealed that most of the b- and y-ions were produced from the precursor ion. The peptide sequence was confirmed by the presence of y-ions together with b-ions. Thus, the absence of several fragment ions had no influence on the determination of the peptide sequence. Additionally, according to the results illustrated above, it seems that there is no relationship between the protein substrate molecular weight and its digestion efficiency as indicated by the number of amino acids and sequence coverage detected. After protein digestion, the trypsin-immobilized magnetic nanoparticles were washed and stored at 4 °C to test the stability of the immobilized enzyme. The proteolytic reaction was done repeatedly with the magnetic nanoparticles during 1 week’s storage. Similar identification results witnessed by MALDI-TOF MS demonstrated that the stability of enzyme molecules and their bioactivity were preserved well when immobilized onto the magnetic nanoparticles. To further test the stability and reproducibility, 11 consecutive operations for Cyt-C digested with trypsin-immobilized magnetic nanoparticles were conducted, and resulted products were thereafter analyzed by MALDI-TOF MS (Figure 4). Between each operation, the trypsin-immobilized magnetic nanoparticles were washed with 25 mM NH4HCO3 buffer solution (pH 8.3) three times. As demonstrated in Figure 4, for the first 8 runs, the

sequence coverage maintains at around 75% with relative standard deviation (RSD) of 6.7%. At the ninth run, the sequence coverage decreased to 59% and further decreased to 42% at the 10th run and 30% at the 11th run, which indicates the loss of the enzyme activity.

4. Conclusions In summary, nanosized amine-functionalized magnetic particles with high magnetic responsivity and excellent dispersibility were prepared through a facile one-pot strategy and were successfully applied, for the first time, for protein digestion via a direct MALDI-TOF MS analysis. The process of digestion is very facile due to the easy manipulation of magnetic nanoparticles. Complete protein digestion was achieved in a short time (5 min), without any complicated reduction and alkylation procedures. These results are expected to open up a new possibility for the proteolysis analysis as well as a new application of magnetic nanoparticles. Additionally, it is worth noting that, since the preparation and surface functionality of magnetic nanoparticles is low-cost and reproducible, the preparation method and application approach of the magnetic nanoparticles may find much potential in proteome research.

Acknowledgment. The work was supported by grants from the 863 Project (No. 2006AA02Z4C5), Shanghai Basic Research Priorities Programme (No. 05dz19741), Natural Science Foundation of China (No. 39870451), and Shanghai Municipal Commission for Science and Technology (No. 0652nm006 and 0652nm018). Supporting Information Available: Figures showing the FT-IR spectra of amine-functionalized magnetic nanoparticles and MS/MS spectra of precursor ions marked with asterisk in Figure 3a. This material is available free of charge via the Internet at http://pubs.acs.org. References

Figure 4. Stability test of trypsin-immobilized magnetic nanoparticles. 3854

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