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Versatile Functionalization of Poly(methacrylic acid) Brushes with Series of Proteolytically Cleavable Peptides for Highly Sensitive Protease Assay Yeping Wu, Anzhi Wang, Xiaokang Ding, and Fu-Jian Xu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b12033 • Publication Date (Web): 13 Dec 2016 Downloaded from http://pubs.acs.org on December 21, 2016
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Versatile Functionalization of Poly(methacrylic acid) Brushes with Series of Proteolytically Cleavable Peptides for Highly Sensitive Protease Assay Yeping Wu,a,b,c† Anzhi Wang,a,b,c† Xiaokang Ding,a,b,c,* and Fu-Jian Xua,b,c* a
State Key Laboratory of Chemical Resource Engineering, Beijing University of
Chemical Technology, Beijing 100029 China b
Key Laboratory of Carbon Fiber and Functional Polymers (Beijing University of
Chemical Technology), Ministry of Education, Beijing 100029 China c
Beijing Laboratory of Biomedical Materials, Beijing Advanced Innovation Center for
Soft Matter Science and Engineering, Beijing University of Chemical Technology, Beijing 100029 China
ABSTRACT The development of new materials for fast and sensitive protease assay is in demand for timely diagnosis of diseases, such as cardiovascular disease, cancers, and Alzheimer disease. Herein, poly(methacrylic acid) (PMAA) brushes were synthesized from the surfaces of silica nanoparticles via surface-initiated atom transfer radical polymerization (ATRP), and functionalized with series of proteolytically cleavable peptides for highly sensitive protease assay. Upon the proteolytic cleavage of the peptides, a short peptide fragment with fluorescent tag (GGK-FITC) is released to the solution, which can be easily detected with a bench-top fluorescence microscope. The grafting densities of PMAA brushes and peptides can be readily tuned by controlling the monomer concentrations of sodium methacrylate in the ATRP reaction. Owing to the three dimensional architecture of PMAA brushes, the loading amount of peptides can reach 21.4% of the total weight of functionalized silica particles (22.4 peptides/nm2), which is much higher than direct immobilization on silica nanoparticles without polymer brushes. Because of the high loading density of peptides, the limit of detection (LOD) of trypsin can reach 1.4 pM in buffer solution or 2.6 nM in non-diluted serum. By rational design of peptide substrates, the peptide-functionalized PMMA brushes can be readily expanded to detect other proteases, such as matrix metalloproteinase-2 (MMP-2), a virtual biomarker for many cancers, with an LOD of 1.1 pM. The proteolytically cleavable peptide-functionalized PMAA brushes offer a starting point for fast and sensitive protease assay. Keywords: polymer brush; ATRP; PMAA; peptide; protease assay 2
INTRODUCTION Polymer brushes are stretched polymer chains with one end attached to a surface.1-4 The physical and chemical properties of polymer brushes can be flexibly tailored by adjusting monomer types, polymerization conditions, and functionalization of biomolecules.5,6 These features of polymer brushes have been exploited for the engineering of cell adhesive surfaces,7,8 antibacterial surfaces,9,10 drug/gene delivery,11,12 and biosensors.13,14 Polymer brushes containing carboxyl acid groups, primary amino groups, epoxy groups, and hydroxyl groups are suitable for immobilization of biomolecules.6 Compared to immobilization of biomolecules on surfaces modified with self-assembled monolayers, it is easy to achieve higher loading density of biomolecules by using polymer brushes due to their three-dimensional architecture.5 In particular, poly(methacrylic acid) (PMAA) is one common anionic polyelectrolyte polymer with plenty of carboxyl acid groups which are widely used for biomolecule immobilization under mild conditions.15 Moreover, the carboxyl acid groups also result in highly swelling of PMAA brushes in a buffer solution, which confers good dispersity of nano/sub-micro particles in aqueous solution due to electrostatic repulsion and steric interactions. Although PMAA brushes are often utilized in immunoassays,14,16,17 they have not been exploited for fast quantification of protease activities through the immobilization of proteolytically cleavable peptides. Proteases exist ubiquitously in all living forms and play important roles in regulating numerous biological and physiological processes (e.g. food digestion, 3
blood clotting, cell apoptosis and disease development).18-20 Many diseases such as cardiovascular disease,21-23 Alzheimer disease24,25 and cancers26,27 are correlated with dysfunction or over-activity of proteases. Therefore, monitoring of protease activity is important for both disease diagnosis and screening of protease inhibitors. In comparison with direct detecting protease molecules by immunoassays, the quantification of protease activity is more challenging, because the differentiation of protease-cleaved peptide fragments usually requires bulky and expensive instruments, such as high performance liquid chromatography,28 gel electrophoresis,29,30 or mass spectrometry.31,32 Alternatively, a peptide sequence labeled with a pair of fluorophore and quencher molecules can be employed as the substrate for protease assay through fluorescence resonance energy transfer (FRET) effect.33 Once the peptide substrate is cleaved by protease, the fluorophore/quencher pair is separated and the fluorescence property
will
be
restored
in
solution.
However,
such
dual-labelling
of
fluorophore/quencher pair to peptide molecules is often tedious and expensive. Currently, several colorimetric protease assays have been reported based on the aggregation of gold nanoparticles (AuNPs)34-36 or the formation of peptide-metal ion complex.37 Such detection procedures are simple and the protease activities can be easily readout by naked-eye. Nevertheless, colorimetric protease assays are susceptible to ionic strength and matrix proteins in samples. The limit-of-detection (LOD) of most colorimetric protease assays is in the range of nanomolars, which is still unsatisfactory for clinical application.37 To achieve lower LOD of a protease assay, it is essential to control the density of 4
functional groups and surface loading amount of peptides. Atom transfer radical polymerization (ATRP) is one of the most prevalent controlled/living radical polymerization techniques for versatile synthesis of polymer brushes from solid surfaces.38-40 The grafting density of polymer brushes via ATRP is highly controllable and the reaction can be carried out in aqueous solution under mild conditions. Herein, we proposed a versatile strategy to functionalize PMAA brushes with series of proteolytically cleavable fluorescein-labeled peptides for highly sensitive protease assay (Scheme 1). This strategy employed peptide-functionalized polymer brushes as substrates for sensitive protease assay, which can avoid tedious and expensive dual-labeling of fluorophores and quenchers. The proof-of-concept of this strategy is firstly proved by using trypsin and chymotrypsin as model proteases with low LODs. Such protease assay also can be flexibly expanded to detect other proteases, such as matrix metalloproteinase-2 (MMP-2), a virtual cancer biomarker. In addition, the effect of PMAA grafting densities and peptide loading capacities are also investigated. This study would provide useful information on how to explore functionalized PMAA brushes for rapid and highly sensitive protease assay.
EXPERIMENTAL SECTION Materials. Silica particles (~200 nm) were purchased from Aladdin (Beijing). Sodium
methacrylate
(NaMA),
3-aminopropyl
triethoxysilane
(APTES),
2-(N-morpholino)ethanesulfonic acid hydrate (MES), glycine, ninhydrin hydrate, potassium iodate, and D-fructose were purchased from Energy Chemical (Shanghai, China). Calcium chloride anhydrous, sodium chloride, ethylenediamine tetraacetic acid
disodium
dichloromethane
salt were
(EDTA),
sodium
purchased
from
hydroxide,
ethanol,
Beijing
Chemical
1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide
methanol,
hydrochloride
Co.
and
(China). (EDC),
n-hydroxysuccinimide (NHS) and Tween-20 were obtained from TCI (Shanghai). Copper (I) bromide (CuBr) and Copper (II) bromide (CuBr2) were purchased from Alfa Aesar (China). Trypsin (from bovine pancreas, type II, activity, 10000 BAEE Umg-1), α-chymotrypsin (from bovine pancreas, type II, activity: 40 Umg-1), matrix metalloproteinase-2 (MMP-2), 2,2′-bipyridyl (bpy), α-bromoisobutyryl bromide (BIBB), triethylamine (TEA) and ethanolamine (EA) were purchased from Sigma-Aldrich (China). Sodium dodecyl sulfate (SDS) was obtained from Beijing Modern Eastern Fine Chemical Co. (China). Phosphate buffered saline (PBS) buffer (pH 7.4) was reconstituted from PBS salt package purchased from Solarbio (China). Tris was purchased from Amresco (U.S.A.). Peptides CGGGGGRGGK-FITC (P1), CGGGGGWGGK-FITC (P2), KKGGPLGLAGGK-FITC (P3), and GGK-FITC (Purity>95%) were synthesized by Scilight Peptide Co. (China). The CuBr2 impurities in CuBr was removed by using dry acetic acid under a blanket of nitrogen gas, and the 6
CuBr precipitate was rinsed with copious of dry methanol using vacuum filtration. The other chemicals were used as received. Immobilization of ATRP Initiator. The immobilization of ATRP initiator on the surface of silica particles can be found elsewhere.12 Briefly, the surface of silica particles were cleaned by soaking in 5.0% (v/v) Decon-90 solution (an alkalic detergent) under gentle rocking. After 2 h, the silica particles were sonicated for 15 min and rinsed with copious deionized water. After drying in an oven at 100 oC for 3 h, the silica particles were ground to break down the large aggregated clots before use. For amino-functionalization, silica particles (SiO2, 1.5 g) were added into 150 mL of alcoholic solution (ethanol/water = 9/1, v/v) containing 1.0% (v/v) of APTES with constant stirring at room temperature. After 6 h, 750 µL of TEA was added, and the reaction continued for another 12 h. The APTES-modified silica particles (SiO2-NH2) were centrifuged at 8000 rpm, and the pellets were washed with ethanol for 3 times to remove unreacted reagents. SiO2-NH2 was dried under vacuum for 30 min and then heated in a 130 oC oven for 3 h to promote crosslinking of silane. To immobilize the ATRP initiator, 1.5 g of SiO2-NH2 and 1.8 mL of TEA were introduced into 18 mL of dichloromethane in a 50-mL round-bottom flask. Next, the round-bottom flask was kept in an ice-bath, where 1.8 mL of BIBB was added dropwise. (Warning: BIBB is highly corrosive and reacts strongly with water vapor in air. It should be handled with extreme caution in a fume hood.) After stirring for 30 min, the ice-bath was removed and the reaction continued for another 3 h at room temperature. The resultant product of SiO2-Br was centrifuged at 12,000 rpm and the 7
pellets were washed with a mixture of methanol and water (1/1 by volume) for five times to remove unreacted reagents. SiO2-Br was finally dried at 100 oC. Synthesis of PMAA Brushes. The synthesis of PMAA-grafted silica nanoparticles (SiO2-g-PMAA) was carried out via surface-initiated ATRP using SiO2-Br based on our earlier work.12 Briefly, different amounts of NaMA and 400 mg of SiO2-Br were introduced into 25 mL of water in a nitrogen-purged round-bottom flask, and the ATRP reaction was performed in the typical CuBr/CuBr2/bpy system. After 1 h, the reaction was terminated, and SiO2-g-PMAA was precipitated by addition of 150 mL of methanol. To remove copper ions, SiO2-g-PMAA was washed for five times with aqueous solution containing 1 mM of EDTA and subsequently rinsed for another three times with deionized water. (Note: complete removal of copper ions is especially important for successful detection of MMP-2.) SiO2-g-PMAA was placed in a lyophilizer for 48 h to dry the product. Functionalization of PMAA Brushes with Peptides. To activate the carboxyl groups of PMAA brushes, 405 mg of SiO2-g-PMAA was added into 13.5 mL of MES buffer (0.1 M, pH 6.8) containing 200 mM of EDC and 500 mM of NHS. After stirring for 30 min, the activated SiO2-g-PMAA particles were centrifuged (12,000 rpm) and washed with PBS buffer (pH 7.4) for three times to remove excess reactants. To immobilize peptides, the activated SiO2-g-PMAA was re-suspended in 1.2 mL of PBS buffer containing 1 mg/mL of P1 (or P2, P3) to react for 1 h at room temperature with constant stirring. The reaction was kept in dark to avoid photobleaching of fluorescein. Then, the SiO2-g-PMAA-peptide was washed twice using 0.05% PBST 8
(PBS buffer containing 0.05% v/v of Tween-20) and rinsed with PBS buffer to remove non-immobilized peptides. Next, the excess of active sites were blocked by adding 9 mL of aqueous solution containing 1 mM of ethanolamine (pH 8.5). After 30 min, the SiO2-g-PMAA-peptide was thoroughly washed with 0.1 wt% SDS aqueous solution
and
PBS
buffer
to
remove
unreacted
reagents.
The
final
SiO2-g-PMAA-peptide was obtained after lyophilization (Note: it is important to thoroughly remove unreacted peptides. Otherwise, the background signal is high for subsequent protease assay). Protease Assay. Different amounts of trypsin were added into 300 µL of Tris buffer (50 mM, pH 8.0) containing 5 mg/mL of SiO2-g-PMAA-P1 to achieve the final trypsin concentrations between 0 to 20 nM. The proteolytic reaction was performed in a water bath (37 oC) with constant stirring. After 2 h, the mixture was centrifuged at 12,000 rpm for 10 min, and the supernatant was transferred to a spin-filtration tube (MWCO = 10 kDa, Millipore) and spun at 12,000 rpm for 3 min to separate peptide fragments. The leachate collected from spin-filtration containing peptide fragments (GGK-FITC) was drawn into a rectangular glass tube (dimension: 0.2 × 2.0 × 50 mm, VitroCom) by capillary force. The fluorescent images of the leachate solution were captured by using a fluorescent microscope (DMI 3000B, Leica) equipped with mercury discharge lamp (ebq 100-04, Leica). The exposure time was 5.1 s for all fluorescence images. The fluorescence intensity of each image was converted to RGB values by using ImageJ (1.46r), and the values obtained from green channel were used for data plotting. To study the effect of the loading amounts of peptides on LOD, the 9
loading amounts of the peptides were tuned by using 0.5 and 2.0 mg/mL of P1. To mimic a protease assay in serum, trypsin was added into non-diluted bovine serum to achieve a final concentration between 0 to 100 nM. The final concentration of SiO2-g-PMAA-P1 was 5 mg/mL for all the samples. After 2 h incubation at 37 oC, the leachates were collected and the fluorescent images were captured as described above. For the detection of MMP-2, the assay was performed in a buffer solution (50 mM Tris-HCl pH 7.5, 10 mM calcium chloride, 150 mM sodium chloride, 0.05% v/v Brij 35) containing 5 mg/mL of SiO2-g-PMAA-P3. The MMP-2 was pre-activated in a solution containing 1 mM of p-aminophenylmercuric acetate (37 oC, 16 h). Other assay procedures were similar to those described above. For the specificity test, the protease samples of trypsin (14,408 U/mg) or chymotrypsin
(40
U/mg)
were
mixed
with
either
SiO2-g-PMAA-P1 or
SiO2-g-PMAA-P2 in Tris buffer (50 mM, pH 8.0). The final concentrations of trypsin and chymotrypsin were 10 nM and 4 µM, respectively. The final concentration of SiO2-g-PMAA-peptide was 5 mg/mL. The proteolytic reaction was kept at 37 oC with constant stirring. After 2 h, the peptide fragments were separated by using spin filtration and their fluorescent images were captured using a fluorescent microscope. For the kinetics study, the mixture of trypsin and SiO2-g-PMAA-P1 was prepared as described above. After a period of time, the leachate solution was collected and the corresponding fluorescent image was captured. The concentration of GGK-FITC in the leachate solution was determined from a calibration curve using customly synthesized GGK-FITC. The rate of the proteolytic reaction (v, µM/min) was 10
determined from the slope of GGK-FITC concentration versus the reaction time. The kinetic parameters of Michaelis constant (Km) and the maximum reaction rate (vmax) were determined from the Lineweaver–Burk plotting, where the concentration of trypsin was fixed at 10 nM. Protease Inhibition Assay. Different amounts of benzamidine hydrochloride (BH) were added into Tris buffer (50 mM, pH 8.0) containing 10 nM of trypsin. Next, SiO2-g-PMAA-P1 was added into the above solution to achieve a final concentration of 5 mg/mL. Then, the protease reaction was performed as described above. After 2 h, the remaining proteolytic activity of trypsin was examined by measuring the fluorescence intensity of the leachate solution. The inhibition efficiency (IE) of BH was defined by the following equation,37 IE=(C1,t - C2,t)/(C0 - C2,t) ×100%
(Eq. 1)
where C1,t and C2,t are the uncleaved peptide concentrations with and without addition of BH after trypsin digestion, while C0 is the initial peptide concentration before trypsin digestion. C0, C1,t, and C2,t were determined from the fluorescence intensity by using a calibration curve of P1. The IC50 of BH toward trypsin is defined as the concentration of BH where half of the protease activity of trypsin is inhibited. Characterizations. The sizes of silica particles were measured by using Zetasizer (Malvern Nano-ZS90). The surface density of amino groups on APTES-modified silica nanoparticles was determined by using ninhydrin assay as described elsewhere.41 Briefly, 250 µL of aqueous solution containing 1 mg/mL of SiO2-NH2 was mixed with 250 µL of freshly prepared PBS buffer (pH 7.4) containing 11
20 mg/mL of ninhydrin and 12 mg/mL of fructose. The solution was heated to 100 oC in an oil bath for 20 min. After the solution was cooled to room temperature, 200 µL of aqueous solution containing 45 mg/mL of potassium iodate was added to the sample. After centrifugation, the absorbance of the supernatant at 562 nm was measured using a UV-vis spectrophotometer (Shimadzu UV-2600). The amount of amino groups was calibrated by using a standard aqueous solution of glycine (0 to 8 µg/mL). Triplicate samples were measured to calculate the average absorbance and the standard deviation. Similarly, the remaining of amino groups on SiO2-Br was also determined to estimate the surface density of bromoisobutyryl initiators. The amount of organic coatings of silica particles was determined by using thermogravimetric analyzer (TGA, Netzsch, Tarsus) with a heating rate of 10 oC/min from room temperature to 1000 oC. In the TGA experiments, the samples were purged with a flow of compressed air to remove decomposed organic components. The X-ray photoelectron spectroscopy (XPS) experiments were performed as described elsewhere to determine the elemental compositions of the silica particle surfaces.12
RESULTS AND DISCUSSION As shown in Scheme 1, the PMAA brushes are firstly synthesized via surface-initiated ATRP from silica particles which serve as solid supports to facilitate the separation process by centrifugation. The PMAA brushes are subsequently functionalized
with
series
of
fluorescein-labeled
peptides
containing
the
proteolytically cleavable sites. Once the peptide is cleaved, the peptide fragments containing fluorescein are released to the solution. After spin-filtration, the peptide fragments in solution are collected and the protease activity can be quantified by measuring the fluorescent intensity of the leachate. Details of each step reaction and materials characterization are discussed below. Peptide-Functionalized PMAA Polymer Brushes. The detailed synthetic routes of PMAA polymer brushes from silica nanoparticles (SiO2-g-PMAA) are illustrated in Scheme 1. The diameter of plain silica nanoparticles is 227.6±24.7 nm (Malvern Nano-ZS90, n=5). After silica nanoparticles are modified with amino groups via the silanization reaction of APTES, the ninhydrin assay shows that the surface density of amino groups is 3.6 amines/nm2 (based on the silica particle density of 2.6 g/cm3 provided from the product information), which is similar to that (2.5 amines/nm2) reported earlier.42 Once the bromoisobutyryl initiators are introduced onto the silica nanoparticles via amidation reaction, the surface density of remaining amino groups reduces to 1.3 amines/nm2. Thus, the surface density of bromoisobutyryl groups is about 1.2 bromoisobutyryl groups/nm2. Figure 1 shows the XPS spectra of SiO2, SiO2-NH2, SiO2-Br, and SiO2-g-PMAA, respectively. In 13
comparison with the plain silica particles (Figure 1a), the presence of amino groups (N 1s, 399 eV) and bromoisobutyryl groups (Br 3d, 67 eV) was further confirmed by the corresponding XPS spectra of SiO2-NH2 (Figure 1b and 1b′) and SiO2-Br (Figure 1c and 1c′).43 After the ATRP reaction, the XPS spectrum of SiO2-g-PMAA (Figure 1d) shows a drastic increase in the peak of C 1s (284 eV), due to the grafting of PMAA brushes on the surface of silica nanoparticles. Meanwhile, significant decrease in the peak signals of Si 2p (100 eV) and Si 2s (152 eV) is observed, because the surface of silica is covered by the PMAA brushes. The above results suggest that PMAA brushes are successfully tethered to the surface of silica nanoparticles. To quantify the amount of PMAA brushes, the weight of organic components on silica nanoparticles was determined by using TGA. Figure 2 shows that only 3.7% and 10.0% of weight loss are observed after the silica nanoparticles are modified with APTES and bromoisobutyryl groups, respectively, which is similar to our earlier result.12 After a typical ATRP reaction where the monomer concentration is 432 mg/mL, over 40.2% of weight loss is observed, implying that PMAA brushes take 33.3% of the total weight (see Supporting Information). The grafted PMAA polymer brushes
are
used
as
the
scaffold
for
coupling
peptides.
The
peptide
CGGGGGRGGK-FITC (P1) was immobilized on the PMAA brushes via EDC/NHS chemistry. For the peptide functionalized silica particles (SiO2-g-PMAA-P1) (when P1 concentration is 1.0 mg/mL), 53.0% of the initial weight is lost (Figure 2), suggesting that the immobilized peptide takes 21.4% of the total weight (see Supporting 14
Information). Different weight ratios of PMAA and peptides are obtained from the TGA data, when different amounts of NaMA are added in the ATRP reactions. Figure 3a shows that the weight ratio of PMAA brushes is less than 3.0% of the total weight when the monomer concentration of NaMA is below 216 mg/mL. With increasing the initial concentrations of NaMA to be 324 mg/mL and 432 mg/mL, the corresponding weight ratios of PMAA brushes increase significantly to be around 16.7% and 33.3%. When the concentration of NaMA is further increased to be 648 mg/mL, the amount of PMAA brushes increases mildly, probably due to the increasing chain termination caused by bimolecular coupling or disproportionation reactions. Figure 3a also shows the weight ratio of peptides immobilized on silica nanoparticles is closely related to the amount of PMAA brushes. To better understand their relationship, the surface density of peptide (Γpeptide) is calculated based on the diameter of the silica nanoparticles. Figure 3b shows that the surface density of peptide increases in a linear relationship with the amount of grafted PMAA brushes. As shown in Figure 3, when the monomer concentration is 432 mg/mL or above, the amounts of PMAA brushes and peptides change slightly. Thus, the monomer concentration is fixed at 432 mg/mL in the following protease assay experiments. As mentioned above (Figure 2), at such concentration, the immobilized peptide of SiO2-g-PMAA-P1 takes 21.4% of the total weight. This loading amount of peptide, due to the three dimensional architecture of the PMAA brushes, is significantly higher than that (~2.1%) on the reported silica 15
nanoparticles without involving PMAA brushes.44 Protease Assay. Trypsin was first used as the model protease in this work. The proteolytic reaction of trypsin cleaves the P1 peptide at the carboxyl terminal of arginine (R), resulting in the release of GGK-FITC which can be detected by using fluorescence microscopes. Figure 4 shows the fluorescent images and plotting profiles of the protease assay performed in Tris buffer. When the trypsin concentration is 5.0 nM or below, the fluorescence intensity increases in a linear relationship. When the trypsin concentration is 10 nM or above, the fluorescence intensity reaches a plateau due to the saturation of fluorescence intensity in the solution. The inset of Figure 4b′ shows the linear fitting of the fluorescence intensity when the trypsin concentration ranges from 0 to 1.0 nM. The sensitivity (the slope of Figure 4b′) for the trypsin assay is 62.7 a.u./nM. The LOD is determined to be 19.6 pM (LOD=3.3 SD/sensitivity, where SD is the standard deviation of 10 measurements of blank samples).37 More interestingly, when 0.05 % (v/v) of Tween-20, a nonionic surfactant, is added into the Tris buffer (pH 8.0), the sensitivity for the trypsin assay significantly increases to be 166.5 a.u./nM, and the resultant LOD can reach 1.4 pM (Figures 4c, 4d, and 4d′). Such LOD is 857-fold lower than a recent gel-array fluorescent protease assay where free peptides were employed as the protease substrate.45 Although the mechanism is still unknown, the polyethylene glycol units in Tween-20 may prevent the non-specific adsorption of trypsin onto the PMAA brushes, which may hinder proteolytic activities. We also found that the LOD of the protease assay is related to 16
the loading amount of the peptides on silica particles. The loading amount of peptide can be easily tuned by controlling the initial concentrations of peptide. Figure 5 shows that the LOD for detecting trypsin decreases significantly from 130.6 pM to 20.6 pM with increasing the loading amount of peptides (Mpeptide/Msilica), where Msilica and Mpeptide are the weights of the silica particles and loaded peptides, respectively. The Mpeptide/Msilica ratios were calculated from the TG data with the initial concentrations of peptide (0.5 and 2.0 mg/mL of P1). Table 1 summarizes the performance of the protease assays in the literatures. In comparison with the reported methods, the LOD of this work is much lower.
Measuring protease activities in the matrix of serum is challenging. Abundant serum proteins usually deteriorate the performance of protease assays, which constrains the practical application at clinical sites. To mimic the protease assay in serum, non-diluted bovine serum was spiked with different trypsin concentrations. The fluorescence intensity of the protease assay performed in bovine serum is much weaker than that in buffer solution. Figure 6 shows that the sensitivity of the protease assay in serum is 0.3 a.u./nM, which is about 200-fold lower than that in buffer solution. The LOD of protease assay in serum is 2.6 nM, which is 132-fold higher than the LOD in buffer solution (without Tween 20). The probable reason is the competitive cleavages of the peptide substrates within non-diluted serum. Nevertheless, the LOD of trypsin in serum is still 6.8-fold lower than that reported recently.45 17
Specificity Test. To test the specificity of the fluorescence protease assay, another peptide CGGGGGWGGK-FITC (P2) with a chymotrypsin cleavage site of tryptophan (W) was immobilized to PMAA brushes under the reaction condition similar to P1. The silica nanoparticles functionalized with P1 or P2 were mixed with 10 nM of trypsin or 4 µM of chymotrypsin, respectively. Figure 7 shows that strong fluorescence is observed when trypsin is mixed with SiO2-g-PMAA-P1. However, no obvious fluorescence is observed when trypsin is mixed with SiO2-g-PMAA-P2, due to the lack of cleavage site of trypsin in the sequence of P2, and vice versa. The above results suggest the good specificity of SiO2-g-PMAA-P1 in the protease assay. Proteolytic Kinetics of Trypsin. To study the proteolytic reaction kinetics, the amount of peptide fragments (GGK-FITC) cleaved by trypsin is calibrated by using a standard solution containing custom synthesized GGK-FITC. Figure 8a shows that no GGK-FITC is cleaved from SiO2-g-PMAA-P1 within 120 min in the absence of trypsin. For the samples with the trypsin concentrations of 1, 2.5, and 5 nM, the GGK-FITC concentration increases linearly due to the cleavage of P1 by trypsin. When the trypsin concentration increases to be 20 nM, the GGK-FITC concentration increases linearly within the first 30 min and then reaches a plateau, suggesting that the reaction is complete. Figure 8b shows the Lineweaver–Burk plots of trypsin with different concentrations of P1. The reaction rate (v, µM/min) was calculated from the slope of [GGK-FITC] vs time when different concentrations of P1 are used as 18
substrates (Table 2). The kinetic parameters of Michaelis constant (Km=11.8 mM) and the maximum reaction rate (vmax=0.2 µM min-1) can be determined from the Lineweaver–Burk plots. The turnover number (kcat= 20 min-1) is determined by kcat=vmax/[E]0, where [E]0 is the trypsin concentration (10 nM). This turnover number is significantly lower than that reported in our earlier work (310 min-1), where free peptides are used as substrates.37 Although the immobilization of peptides may hinder the access of trypsin to the peptide substrates, the current study is still advantageous because of the high sensitivity for detecting fluorescence. Besides, a proteolytic inhibition assay further confirms the validity of the protease assay, and shows the possibility for the screening of protease inhibitors (see Supporting Information).
Detection of MMP-2. To explore the feasibility using peptide-functionalized PMAA brushes for detecting protease biomarkers, the PMAA brushes were functionalized with KKGGPLGLAGGK-FITC (P3). P3 contains the cleavage site of PLGLAG for MMP-2.46 Two lysine (K) motifs were added to the N-terminal of the peptide sequence to facilitate the functionalization of PMAA brushes. Figure 9a shows the fluorescent images of the leachate collected from spin-filtration. The fluorescent intensities of the samples increase with the increasing MMP-2 concentrations. Figure 9b shows that the fluorescence intensity increases linearly when the MMP-2 concentration is 20 nM or below. The sensitivity is 5.6 a.u./nM (Figure 9b′), and the corresponding LOD is 1.1 pM. This above results indicate that the peptide-functionalized PMAA brushes are able to detect MMP-2. 19
CONCLUSIONS PMAA brushes have been successfully synthesized and functionalized with series of proteolytically cleavable peptides for highly sensitive protease assay. The densities of grafted PMAA brushes and peptides can be finely tuned by controlling the monomer concentrations of NaMA. Owing to the three dimensional architecture of PMAA brushes, the loading amount of peptides can reach 21.4% of the total weight of functionalized silica particles. With such high loading density of peptides, the LOD for trypsin assay can reach 1.4 pM in buffer solution (containing 0.05% v/v Tween 20) and 2.6 nM in serum. Moreover, the peptide-functionalized PMMA brushes can be flexibly extended to detect other proteases, such as MMP-2, with the LOD of 1.1 pM. By rational design of peptide substrates, peptide-functionalized PMMA brushes are potentially useful for the fabrication of sensitive protease assay devices. This study provides a starting point for rapid and sensitive protease assay in clinical sites equipped with a bench-top fluorescent microscope and a palm-sized mini centrifuge. ASSOCIATED CONTENT Supporting Information Calculation of the mass ratio of PMAA brushes and peptides and trypsin inhibition assay. ACKNOWLEDGMENT This work was partially supported by NNSFC (National Natural Science Foundation of China, grant numbers 21504006, 51573014, 51325304, and 51521062), the Opening Project of State Key Laboratory of Polymer Materials Engineering (Sichuan University) (Grant number sklpme2015-4-23), the Higher Education and 20
High-quality and World-call Universities (JC1501), and Collaborative Innovation Center for Cardiovascular Disorders, Beijing Anzhen Hospital Affiliated to the Capital Medical University.
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Table 1. Comparison of the performance of the protease (trypsin) assays Method
Material
LOD
Reference
LRETa
UCNPsb
50 pM
21
FRETc
Donor-acceptor pair
2 pM
33
Fluorescence
Fluorescence-conjugated peptide
1.2 nM
45
Colorimetric
AuNPsd
0.5 nM
36
Colorimetric
Peptide/metal ion complex
16.7 nM
37
GEe
Fluorescence-conjugated peptide
0.2 nM
29
GE
Peptide-functionalized AuNPs
1.2 µM
30
Fluorescence
Peptide-functionalized PMAA brushes
1.4 pM
This work
a
Luminescence resonance energy transfer; bUp-conversion nanoparticles; c Fluorescence resonance energy transfer; dGold nanoparticles; eGel electrophoresis
Figure 1. XPS spectra of (a) SiO2, (b) SiO2-NH2, (c) SiO2-Br, and (d) SiO2-g-PMAA. The insets of (b′) and (c′) show the core-level spectra of N 1s (399 eV) and Br 3d (67 eV), respectively.
Figure 2. Thermogravimetric analysis of SiO2, SiO2-NH2, SiO2-Br, SiO2-g-PMAA and SiO2-g-PMAA-P1. The concentration of sodium methacrylate monomer is 432 mg/mL in the ATRP reaction, and the concentration of P1 is 1.0 mg/mL for peptide functionalization.
Figure 3. (a) The weight ratio of PMAA and P1 immobilized on silica particles by adjusting the concentrations of NaMA: (1) 108 mg/mL, (2) 216 mg/mL, (3) 324 mg/mL, (4) 432 mg/mL, and (5) 648 mg/mL. (b) The surface density of immobilized peptide regarding to the grafting amount of PMAA brushes.
Figure 4. Fluorescent images and fluorescence intensities in the trypsin assay performed in (a,b) Tris buffer and (c,d) Tris buffer containing 0.05 % v/v of Tween-20, where the insets of (b′) and (d′) show the linear range of the trypsin assay. The error bars represent the standard deviation of three parallel measurements. In each experiment, 5 mg/mL of SiO2-g-PMAA-P1 was mixed with different amounts of trypsin to achieve the desired final concentration.
Figure 5. Protease assays with different loading amounts (a: Mpeptide/Msilica=0.66; b: Mpeptide/Msilica=0.76) of peptides on the PMAA-grafted silica particles.
Figure 6. Trypsin assay performed in serum. The inset shows the linear range of the protease assay. The error bars represent the standard deviation of three parallel measurements. In each experiment, 5 mg/mL of SiO2-g-PMAA-P1 was mixed with different amounts of trypsin to achieve the desired final concentration.
Figure 8. Proteolytic kinetics of trypsin. (a) Release of GGK-FITC fragments by the enzymatic cleavage of SiO2-g-PMAA-P1 in the presence of different concentrations of trypsin. (b) Lineweaver–Burk plots of trypsin with different concentrations of P1, where the trypsin concentration is fixed at 10 nM. The error bars represent the standard deviation of three parallel measurements.
Figure 9. (a) Fluorescent images and (b) plotting of the fluorescence intensity profiles for detecting MMP-2. The inset of (b′) shows the linear range of the protease assay. The error bars represent the standard deviation of three parallel measurements. In each experiment, 5 mg/mL of SiO2-g-PMAA-P3 was mixed with different amounts MMP-2 to achieve desired the final concentration.
