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Article
Rational Design of Peptide-Functionalized Poly(methacrylic acid) Brushes for On-Chip Detection of Protease Biomarkers Yeping Wu, Muhammad Naeem Nizam, Xiaokang Ding, and Fu-Jian Xu ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00584 • Publication Date (Web): 29 Aug 2017 Downloaded from http://pubs.acs.org on September 10, 2017
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ABSTRACT There is an increasing demand in developing new materials and approaches for rapid and sensitive detection of protease biomarkers. Herein the poly(methacrylic acid) (PMAA)
brushes
synthesized
via
surface-initiated
atom
transfer
radical
polymerization (ATRP) from silica nanoparticles were flexibly functionalized with different fluorescein-labeled peptides, serving as the substrates for protease assay. To facilitate the point-of-care detection of protease, polyacrylamide gel pad arrays were fabricated to allow permeation of fluorescein-labeled peptide fragments cleaved from the PMAA brushes. This experimental setup enables on-chip protease assay with adequate limit of detection (LOD) for detecting trypsin in buffer solution (3.9 pM) or in serum (1.4 nM), and good specificity for differentiation of trypsin and chymotrypsin. By using this experimental setup, matrix metalloproteinase-2 and matrix metalloproteinase-9 can be detected with LODs of 2.5 nM and 3.3 nM, respectively. Moreover, by introducing an adamantine (Ad) motif to the side-chain of peptide fragment and β-cyclodextrin (β-CD) groups to the gel pad matrix, 2.2-fold lower of LOD was achieved for detection of trypsin (1.8 pM) due to the supramolecular selfassembly of Ad and β-CD. Given the advances in the ease of sample handling, this rational design of peptide-functionalized PMAA brushes could be useful for on-chip detection of protease biomarkers or the screening of protease inhibitors.
KEYWORDS: polymer brush; poly(methacrylic acid); gel pad array; protease assay. 2
INTRODUCTION Poly(methacrylic acid) (PMAA) brushes have been investigated as the scaffold for the engineering of advanced biomaterials.1-3 PMAA brushes are highly swelling in aqueous solution due to the ionization of carboxyl groups, resulting in their responsive abilities to pH and ionic strength of solution.4-6 Meanwhile, the abundance of carboxyl groups on PMAA brushes allows versatile conjugation strategies for the immobilization of functional molecules.7-9 For example, PMAA (or PMAA– containing) brushes have been utilized for enzyme immobilization,10 controlled release and delivery,11 cell adhesion and proliferation,12 antibacterial applications,13 and direction of the growth of inorganic materials.14 More recently, PMAA brushes have attracted much attention in developing novel biosensors attributed to their robustness, flexibility, and three-dimensional architectures that facilitate the immobilization of multilayer biomolecules.15-17 However, the potentials of PMAA brushes for detection and quantification of protease activities have not been fully exploited. Proteases are able to catalyze the hydrolysis of the amide bonds in proteins or peptides, and thereafter trigger series of cascading biochemistry reactions to regulate numerous biological and physiological processes.18-21 The dysfunction or overactivity of proteases are associated with many pathological processes, such as arthritis, cirrhosis, Alzheimer disease, cardiovascular disease, and tumor invasion and metastasis.22 Therefore, a variety of techniques were developed for the detection and quantification of the protease activities.23-25 Although the high performance liquid 3
chromatography26 or mass spectroscopy27-29 –based techniques are able to detect the peptide fragments cleaved by the proteases, they are limited by their bulky and complicated instrumentation. Alternatively, several state-of-the-art protease assays were
reported
based
on
conjugated
polymers,30-33
aggregation
of
gold
nanoparticles,34-37 quantum dots,38-41 Förster resonance energy transfer,43,44 and aggregation–induced emission effect.42 However, the above strategies still suffer from disadvantages such as insufficient sensitivity or tedious synthetic work. As a result, developing of novel approaches for highly sensitive protease assay is still needed. Recently, our group demonstrated that the peptide–functionalized PMAA brushes on silica particles can be used as the sensitive substrate for protease assay.45 This strategy is very promising because of the low limit of detection (LOD) (1.4 pM for trypsin). However, this protease assay requires multiple centrifugation and spin filtration steps. To achieve rapid and high throughput protease assays, an on-chip protease assay was demonstrated by employing polyethylene glycol (PEG) gel array to differentiate the small peptide fragments and full-length peptides.46 However, this on-chip protease assay suffers from the relatively high background signal caused by the permeability of full-length peptides into the gel matrix. Meanwhile, the leaching of small peptide fragments from the gel matrix in the washing steps also would deteriorate the performance of the protease assay. Herein, we designed series of peptide-functionalized PMAA brushes and different polyacrylamide gel pad arrays for effective on-chip detection of protease biomarkers (Scheme 1). Because the peptides were covalently immobilized on silica 4
particles, they cannot permeate into the gel matrix, resulting in low background signal. The pore sizes of the gel pad arrays fabricated with polyacrylamide were large enough to allow fast permeating of peptide fragments cleaved from the full-length peptide sequence (Scheme 1a). More importantly, we also proposed to introduce β-cyclodextrin (β-CD) into the gel matrix to reduce the leaching of the peptide fragments. The cleaved peptide fragments modified with adamantane (Ad) motif can be easily captured by the β-CD units in the gel matrix for improved LOD of the protease
assay
(Scheme
1b).
By
combining
the
advantages
of
the
peptide-functionalized PMAA brushes and the gel pad arrays, the present work would provide a new approach for rapid and high throughput detection of protease biomarkers.
EXPERIMENTAL SECTION Materials. p-Aminophenylmercuric acetate (APMA) was purchased from J&K Scientific. Ammonia solution (25 % w/w) was purchased from Tianjin Chemical Co. (China). Anhydrous dimethyl sulfoxide (DMSO), hydrogen peroxide solution (30% w/w) and chloroform were purchased from Beijing Chemical Works (China). Heptane, β-cyclodextrin (β-CD), N-(3-aminopropyl)methacrylamide hydrochloride (APMAc) and 1,1'-carbonyldiimidazole (CDI) were purchased from Energy Chemical (Shanghai, China).
2,2-Dimethoxy-2-phenyl-acetophenone
(DMPA,
Irgacure
651),
acrylamide/bisacrylamide (40% w/v, 19:1), N,N,N′,N′-tetramethylethylenediamine (TEMED), 3-(trichlorosilyl)propyl methacrylate (TPM), triethylamine (TEA), poly(ethylene glycol) diacrylate (PEG-DA, average Mn 575 Da), dimethyl sulfoxide-d6, trypsin (from bovine pancreas, type II, activity, 10000 BAEE U mg−1), α-chymotrypsin (from bovine pancreas, type II, activity: 40 U mg−1), matrix metalloproteinase-2 (MMP-2, human recombinant, expressed in mouse NSO cells), and matrix metalloproteinase-9 (MMP-9, human recombinant, expressed in mouse NSO
cells)
were
purchased
CGGGGGRGGK-FITC
from
Sigma-Aldrich
(P1),
(China).
The
CGGGGGWGGK-FITC
peptides (P2),
KKGGPLGLAGGK-FITC (P3), CGGGGGRGK(Ad)GK-FITC (P4) and GGK-FITC (Purity>95%) were synthesized by Scilight Peptide Co. (China). Peptide Functionalization of PMAA–Grafted Silica Particles. The PMAA– grafted silica particles were synthesized via surface-initiated ATRP, and the PMAA brushes were functionalized with peptides (P1–P4) using EDC/NHS chemistry, 6
SiO2-g-PMAA-P4, respectively. The details for the ATRP reaction and peptide functionalization were described in our earlier work.45 The ATRP reaction was performed at the optimum monomer concentration of 432 mg/mL. Fabrication of Gel Pad Array Chip. The fabrication of gel pad array chip can be found elsewhere.47 Firstly, the glass slides were cleaned using 5.0% v/v Decon-90 solution (an alkalic detergent)48 and treated with basic piranha solution (saturated ammonia solution:30% H2O2=3:1, v/v) for 30 min to promote the surface silanol groups.47 After washing the glass slides with copious amounts of deionized water and blowing dry under a stream of nitrogen gas, the glass slides were immersed in a mixture of heptane and chloroform (4:1, v/v) containing 5 mM of TPM. After the incubation at room temperature (~25 °C) for 2 h, the glass slides were rinsed with heptane and deionized water respectively to remove unreacted TPM. Secondly, aqueous solution containing 10% v/v of acrylamide/bisacrylamide precursors (40% w/v, 19:1), 0.5% w/w of DMPA, and 1.2% v/v of TEMED was drawn into the gap between the TPM-modified glass slide and a quartz photomask (see the Supporting Information). The TPM-modified glass slide and quartz photomask were separated by using two strips of aluminium foil spacers (~25 µm), and secured with two binder clips. Thereafter, the solution was exposed to 365 nm UV light (CEAULIGHT®, CEL-LED100) for 15 min with a power of 4 mW cm-2. After UV shining, the glass slide was washed with 0.05% v/v PBST solution (PBS buffer solution containing 0.05% v/v Tween-20) and deionized water to remove non-polymerized precursors, and blown 7
dry under a stream of nitrogen gas. To fabricate the micropillar rings, the glass slide bearing the polyacrylamide gel pad arrays was carefully aligned to the photomask with the patterns of micropillar ring arrays, placing each gel pad array unit in the center of each micropillar ring. The glass slide and the photomask were separated by using two strips of 8-folded aluminium foil spacers (~200 µm), and the PEG-DA monomer containing 2% w/w DMPA was drawn into the gap formed between the glass slide and the photomask. After the PEG-DA monomer was exposed to UV light (365 nm, 4 mW cm-2) for 6 s, the glass slide was washed with ethanol and deionized water to remove unreacted monomers, and blown dry under a stream of nitrogen gas. To fabricate β-CD containing gel pad arrays, the acrylated β-cyclodextrin (CD-Ac) was synthesized (see the Supporting Information). An aqueous solution containing 10% v/v of acrylamide/bisacrylamide precursors (40% w/v, 19:1), 5% w/w of CD-Ac, 0.5% w/w of DMPA, and 1.2% v/v of TEMED was used for photo polymerization. On-chip Protease Assay. For the trypsin assay, 40 µL of Tris buffer solution (50 mM, pH 8.0) containing 5 mg/mL of SiO2-g-PMAA-P1 and different concentrations of trypsin (0-100 nM) was placed in a 37 °C oven. After 2 h of trypsin digestion, 2 µL of the mixture solution was pipetted into each PEG micropillar ring and incubated at 37 °C in a humidified petri dish to prevent drying of the solution. After 1 h, the chip was briefly washed using 0.05% v/v PBST solution for three times to remove the unreacted SiO2-g-PMAA-P1. (Note: prolonged washing of the chip would result in deteriorated detection limit due to the leaching of the fluorescein-labeled peptide 8
fragments.) After dried under a stream of nitrogen gas, the fluorescent images of the gel pad arrays were captured by using a fluorescent microscope (DMI 3000B, Leica) with an exposures time of 10 s. The fluorescent images were converted to RGB values by using ImageJ software (1.46r). For the specificity test, the protease samples of trypsin and chymotrypsin were mixed with either SiO2-g-PMAA-P1 or SiO2-g-PMAA-P2. The final concentrations of trypsin, chymotrypsin, and peptide-functionalized silica particles (SiO2-g-PMAA-P1 or SiO2-g-PMAA-P2) were 25 nM, 40 µM, and 5 mg/mL, respectively. The rest of the experimental procedures were in accordance with the on-chip trypsin assay as described above. For the trypsin assay in serum, 4 µL of bovine serum containing different amount of trypsin was added into 36 µL of Tris buffer solution containing 5 mg/mL of SiO2-g-PMAA-P1. For the MMP assay, MMP-2 or MMP-9 were preactivated by incubating with 1 mM of p-aminophenylmercuric acetate at 37 °C for 16 h. Then, the activated MMP-2 or MMP-9 were incubated with SiO2-g-PMAA-P3 (5 mg/mL) in buffer solution (50 mM Tris-HCl pH 7.5, 10 mM calcium chloride, 150 mM sodium chloride, 0.05% v/v Brij 35). The rest of the experimental procedures were the same as the on-chip trypsin assay described above. For the enhanced protease assay, different amounts of trypsin were mixed with SiO2-g-PMAA-P4 (5 mg/mL) in Tris buffer solution (50 mM, pH 8.0), while the gel pad arrays containing β-CD groups were used to promote the capture of cleaved peptide fragments containing Ad motifs. The exposure time for the image capturing is 1 s for all enhanced trypsin assays. 9
Trypsin Inhibition Assay. 1 µL of benzamidine hydrochloride (BH) solution was added into 50 µL of Tris buffer (50 mM, pH 8.0) containing 50 nM of trypsin, to achieve the final concentration of BH ranging from 0 to 2 mM. After incubating at 4 °C for 30 min, another 50 µL of Tris buffer (50 mM, pH 8.0) containing 10 mg/mL of SiO2-g-PMAA-P1 was added into the inhibited trypsin solution. The rest of the procedures were the same as described in the above trypsin assay.
RESULTS AND DISCUSSION The synthesis and characterization of the PMAA-grafted silica particles and subsequent functionalization of peptides (P1 to P4, Scheme 1) followed an established protocol as described in our earlier work.45 On-chip Trypsin Assay. We first fabricated a 4×10 gel pad array chip, each unit of which consists a subarray of a 17×17 polyacrylamide gel pads to allow the permeation of peptide fragments cleaved by protease, and micropillar ring to confine liquid samples (Scheme 1a). Trypsin belongs to the serine protease family and play important roles in food digestion and disease development.46 As a result, trypsin was chosen as the target protease molecule, and PMAA-grafted silica particles functionalized with P1 (containing the cleavage site of R) was used as substrate. Figure 1a shows the fluorescent images of the gel pad arrays (5×5 arrays cropped from the original images) when the concentration of trypsin ranges from 0 nM to 50 nM. In general, the fluorescent intensities of the gel pad arrays increases with the increasing concentration of trypsin, showing that the cleaved peptide fragments are able to be trapped in the matrix of polyacrylamide gel pads. Interestingly, we notice that the fluorescent intensities at the edges of the square-shaped gel pads are higher than that in the interior. This nonuniform fluorescence distribution was caused by the simultaneously diffusion of peptide fragments into the gel matrix in both vertical direction (from top to bottom) and lateral directions (see the Supporting Information). Although the diffusion of peptide fragments in all directions resulted in faster permeation of peptide fragments into the gel matrix, the nonuniform fluorescence 11
distribution caused difficulties in quantification. To address this problem, we plotted the fluorescent intensity profiles of one row of 5 gel pads. Figure 1b shows that in the absence of trypsin, the fluorescent intensities of each gel pad is minimal (the black line). When the trypsin concentration is 0.25 nM, the fluorescent intensities increases significantly, and 5 plateaus representing the 5 gel pads in a row can be easily distinguished (the red line). With the increasing concentration of trypsin, the fluorescent intensities of the gel pads increases accordingly. To avoid the interferences from the signals between edges of the gel pads, we randomly picked 5 square-shaped gel pads from the 5×5 array, and quantified the average fluorescent intensity of each gel pad (n=5, see the Supporting Information). Figure 1c shows that when the trypsin concentration is 2.0 nM or below, the fluorescent intensities of the gel pads increase linearly. When the trypsin concentration is 50 nM or higher, the fluorescent intensities reach a plateau because of the over-exposure of the fluorescent images. The inset of Figure 1c shows the linear fitting of the fluorescent intensities with the trypsin concentrations from 0 nM to 2.0 nM. The limit of detection (LOD) is determined to be 3.9 pM by using LOD = 3 SD/slope, where the SD is the standard deviation of the fluorescent intensities of 10 individual gel pads with 0 nM of trypsin.49 This LOD is 307-fold lower than a recently reported on-chip protease assay, mostly owing to (i) the high loading capacity of peptides on the PMAA-grafted silica particles; (ii) the lower background signal due to the larger particle size (~200 nm) that prevent the permeation of fluorescein-labeled peptide substrates into the gel pad matrix; and (iii) the larger poresize of the polyacrylamide gel matrix and the patterned architecture of 12
the gel pad arrays lead to higher permeability of the peptide fragments and shorter incubation time. Moreover, this on-chip protease assay does not require the delicate tuning of permeability of the gel matrix to differentiate the small peptide fragments from the full-length peptide substrates. This feature would benefit the reproducibility of the protease assay as well as the quality control for the scale-up production of the chips. To explore the feasibility for detection and quantification of trypsin in real samples, non-diluted bovine serum was spiked with different amount of trypsin, and samples were subjected to the on-chip protease assay after proteolytic digestion. Figure 2a shows the fluorescent images of the gel pad arrays after incubating with the proteolytically digested solution. In the absence of trypsin, no fluorescence is observed on the gel pad array. When the trypsin concentration in serum are 25 nM and 50 nM, the fluorescence of the gel pads is significantly weaker than that in buffer solution. Figure 2b shows the fluorescent intensities of the gel pad arrays with different trypsin concentration. When the trypsin concentration of is 100 nM or below, the fluorescent intensities of the gel pads increase in a linear range, with a slope of 0.37 a.u./nM. The LOD for the detection of trypsin in serum is 1.4 nM, which is 359-fold higher than that in buffer solution. This is probably caused by the blocking of the peptide substrates by the serum proteins, or the competitive cleavages of the peptide substrates with serum proteins.45 13
Specificity Test. Chymotrypsin also belongs to the serine protease family, and it selectively cleaves the peptide bonds of hydrophobic amino acid residues such as tyrosine (Y), phenylalanine (F) and tryptophan (W). As a result, the PMAA-grafted silica particles functionalized with P2 (containing the cleavage site of W) was used as substrate. Figure 3 shows the fluorescent images of the gel pad arrays when SiO2-g-PMAA-P1 and SiO2-g-PMAA-P2 are digested by 25 nM of trypsin and 40 µM of chymotrypsin, respectively. The concentration of chymotrypsin is much higher than trypsin because of its lower proteolytic activity (40 U/mg) compared to trypsin (10000 U/mg), as provided by the supplier. As shown in Figure 3, when SiO2-g-PMAA-P1 was used as substrate, strong fluorescent signal can be observed in the gel pad array when the P1 was digested by trypsin. In contrast, the fluorescent signal of the gel pad array was minimal when the SiO2-g-PMAA-P1 was incubated with chymotrypsin, and vice versa in the case of SiO2-g-PMAA-P2 being used as substrate. These results proved good specificity of the on-chip protease assay. Detection of MMP-2 and MMP-9. Matrix metalloproteinases (MMPs) are involved in many normal physiological processes by breaking down the extracellular matrix (ECM). However, dysfunction or overactivity of MMPs (e.g. MMP-2 and MMP-9) are correlated with the development of many diseases such as such as arthritis, tumor metastasis, and cardiovascular diseases. To explore the feasibility for the detection of MMP-2 and MMP-9 by using the gel pad arrays, a peptide sequence of P3 containing the cleavage site for MMP-2 and MMP-9 was synthesized and 14
immobilized to PMAA-grafted silica particles. Figure 4a shows the fluorescent images of the gel pad arrays when the SiO2-g-PMAA-P3 was digested by MMP-2. Similar to the trypsin assay as we described above, the fluorescent signal of the gel pad arrays increases with the increasing concentration of MMP-2. By plotting the fluorescent intensities with the MMP-2 concentration, we noticed the fluorescent intensities increase linearly when the concentration of MMP-2 is 20 nM or below, and a linear fitting gives a slope of 0.983 a.u./nM, with a R2 value of 0.997, as shown in Figure 4b. The LOD of the MMP-2 assay is 2.5 nM, which is 641-fold higher than the LOD for trypsin assay. One possible explanation is that the cleavage site of the PLGLAG in the peptide sequence is more difficult to access the pocket of MMP-2, due to the higher steric interactions of PLGLAG for MMP-2 than that of R for trypsin. Likewise, Figure 5 shows the fluorescent images of the gel pad arrays after the SiO2-g-PMAA-P3 was digested by MMP-9, and the plotting of their fluorescent intensities as a function of concentrations of MMP-9. In the case of MMP-9 assay, it is notable that the slope of the linear range is 0.597 a.u./nM, which is 39.3% lower than that of MMP-2 assay, suggesting a lower proteolytic activity of MMP-9 to the substrate of SiO2-g-PMAA-P3. Consequently, the LOD for MMP-9 is 3.3 nM, which is 32% higher than the LOD of MMP-2. Inhibition Assay. Screening of protease inhibitors is extensively demanded in the development of new drugs to suppress the overexpression of the protease activity. 15
To explore the capability of the gel pad arrays for the screening of protease inhibitors, the trypsin solution was incubated with benzamidine hydrochloride (BH), a known potent trypsin inhibitor, before subjected to SiO2-g-PMAA-P1 to digest the peptide substrate. Figure 6a shows the fluorescent images of the gel pad arrays in the inhibition assay. With the increasing concentration of BH, the fluorescent intensities decreased due to the inhibited proteolytic activity of trypsin. To quantify the inhibition efficiency of BH, the fluorescent images of the gel pad arrays were converted to intensity profiles by using ImageJ software, as shown in Figure 6b. The fluorescent intensities of the gel pad arrays decrease rapidly when the concentration of BH is 0.5 mM or below. In the cases of BH concentration higher than 1.0 mM, the fluorescent intensities of the gel pad arrays decrease slowly, indicating the saturation of the inhibited proteolytic activity of trypsin. For example, when the concentration of BH is 2.0 mM, the average fluorescent intensity is 35.4±0.6 a.u., which is in accordance with 0.9 nM of trypsin when it is correlated to the inset figure of Figure 1c. Since the initial concentration of trypsin is 50 nM, we can estimate 98.2% of the proteolytic activity of trypsin have been inhibited. Enhanced Trypsin Assay on β-Cyclodextrin Containing Gel Pad Arrays. Although the gel pad arrays have been demonstrated as a promising platform for on-chip protease assay, the leaching of peptide fragments from the gel pad matrix in the washing steps is still challenging. To address this problem, we developed an enhanced trypsin assay by incorporating β-CD groups in the gel pad matrix while 16
introducing an Ad motif to the side-chain of peptide fragment (Scheme 1b). In the past, cyclodextrins have been used to fabricate hydrogel biomaterials.50-54 Herein, as a “proof-of-concept” demonstration, the monomer of acrylated β-CD (CD-Ac) was synthesized by CDI activation of hydroxyl groups on β-CD, and followed by the amidation reaction with APMA. The 1H NMR spectrum of CD-Ac shows that approximately 1.06 acryl groups have been tethered to β-CD (see the Supporting Information). Next, 5% w/w of CD-Ac was spiked into the precursor solution of acrylamide/bisacrylamide and copolymerized to fabricate β-CD containing gel pad arrays. (Note: higher doping concentration of CD-Ac would result in broken gel pad arrays after polymerization.) We noticed that the fluorescent intensities of the gel pad array containing β-CD groups were significantly enhanced due to the supramolecular interaction between Ad and β-CD, and we have to tune the exposure time to 1 s to avoid over-exposure of the fluorescent images. Figure 7 shows that the fluorescent intensities of the gel pad arrays containing β-CD increase linearly when the concentration of trypsin is 0.5 nM or below, with a slope of 82.93 a.u./nM. It is notable that this slope is 2.3 times higher than that in plain polyacrylamide gel pad arrays. Consequently, the LOD of the enhanced trypsin assay can reach 1.8 pM, which is comparable among the other state-of-art trypsin assays.55-58 As only 2 µL of the liquid sample is required in a typical experiment, 3.6 amol (10-18 mol) of trypsin can be detected by using the on-chip trypsin assay.
CONCLUSIONS In conclusion, series of peptide-functionalized PMAA brushes were successfully synthesized for on-chip detection of protease biomarkers on different polyacrylamide gel pad arrays. The gel pad arrays demonstrated adequate LOD for trypsin in buffer solution (LOD=3.9 pM) or in serum (LOD=1.4 nM), and good specificity to differentiate trypsin and chymotrypsin. Meanwhile, we have proved the gel pad arrays can also be employed to detect other protease biomarkers such as MMP-2 (LOD=2.5 nM) and MMP-9 (LOD=3.3 nM). Moreover, we have demonstrated an enhanced on-chip protease assay by incorporating supramolecular interactions between β-CD groups in the gel pad matrix and the Ad motifs in the peptide fragments. In the case of enhanced protease assay, the capturing of the peptide fragments has been greatly improved, resulting in a 2.2-fold lower of LOD for detection of trypsin (1.8 pM). The rational design of peptide-functionalized PMAA brushes was proven as an efficient substrate for protease assay. In addition, the high-throughput nature of on-chip gel pad arrays allowed simultaneous detection of different protease biomarkers for disease diagnosis or screening of protease inhibitors for drug development. These features shed light on the development of novel on-chip diagnostic devices for “point-of-care” detection of protease biomarkers, especially in resource-limited areas.
ACKNOWLEDGMENTS This work was partially supported by National Key Research and Development Program of China (grant number 2016YFC1100404), National Natural Science Foundation of China (grant numbers 21504006, 51573014 and 51325304), and Beijing Natural Science Foundation (grant number L160004). ASSOCIATED CONTENT Supporting Information. The design of photomasks for the fabrication of gel pad arrays, photographs of a glass slide bearing 4×10 arrays of polyacrylamide gel pad units and PEG micropillar rings, schematic illustration of the diffusion of peptide fragments into the gel matrix, and the synthesis and 1H NMR characterization of acrylated β-cyclodextrin (CD-Ac).
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Scheme 1. Schematic illustration of (a) protease assay on gel pad array and (b) enhanced protease assay on β-CD containing gel pad array. The cleavage sites in peptide sequences to trypsin, chymotrypsin, or MMP-2/MMP-9 are highlighted in red color.
Figure 1. (a) Fluorescent images of the on-chip protease assay in buffer solution with different concentrations of trypsin ranging from 0 nM to 50 nM. Arrays of 5×5 gel pads were cropped from the original images, and analyzed by using ImageJ software. The scale bars are 50 µm. (b) Fluorescent intensities when plotting one row of 5 polyacrylamide gel pads with different trypsin concentrations ranging from 0 nM to 2.0 nM. (c) Fluorescent intensities of the polyacrylamide gel pads with different trypsin concentrations (n=5). The inset shows the linear range of the protease assay.
Figure 2. (a) Fluorescent images of the on-chip protease assay in bovine serum with different concentrations of trypsin ranging from 0 nM to 500 nM. Arrays of 5×5 gel pads were cropped from the original images, and analyzed by using ImageJ software (scale bar: 50 µm). (b) Fluorescent intensities of the polyacrylamide gel pads with different trypsin concentrations in bovine serum (n=5).
Figure 4. (a) Fluorescent images of the on-chip protease assay when the SiO2-g-PMAA-P3 was digested by different concentrations of MMP-2. Arrays of 5×5 gel pads were cropped from the original images, and analyzed by using ImageJ software (scale bar: 50 µm). (b) Plotting of fluorescent intensities of the gel pads as a function of MMP-2 concentrations in buffer solution (n=5).
Figure 5. (a) Fluorescent images of the on-chip protease assay when the SiO2-g-PMAA-P3 was digested by different concentrations of MMP-9. Arrays of 5×5 gel pads were cropped from the original images, and analyzed by using ImageJ software (scale bar: 50 µm). (b) Plotting of fluorescent intensities of the gel pads as a function of MMP-9 concentrations in buffer solution (n=5).
Figure 6. (a) Fluorescent images of the gel pad arrays (scale bar: 50 µm) and (b) plotting of fluorescent intensities as a function of BH concentrations in the protease inhibition assay (n=5).
Figure 7. Enhanced on-chip protease assay using β-CD containing gel pad arrays: (a) fluorescent images of the gel pad arrays (scale bar: 50 µm) and (b) fluorescent intensities of the gel pad arrays as a function of trypsin concentrations (n=5).
Title: Rational Design of Peptide-Functionalized Poly(methacrylic acid) Brushes for On-Chip Detection of Protease Biomarkers Authors: Yeping Wu, Muhammad Naeem Nizam, Xiaokang Ding, and Fu-Jian Xu
