Analytica Chimica Acta 865 (2015) 53–59
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Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
In situ fabrication of cleavable peptide arrays on polydimethylsiloxane and applications for kinase activity assays Huang-Han Chen, Yu-Chieh Hsiao, Jie-Ren Li, Shu-Hui Chen * Department of Chemistry, National Cheng Kung University, No.1 College Road, Tainan 701, Taiwan
H I G H L I G H T S
G R A P H I C A L A B S T R A C T
A novel approach for in situ fabrication of cleavable peptide arrays on polydimethylsiloxane (PDMS). The first report of peptide synthesis on PDMS. Use of the PDMS peptide array for developing sensitive chip-based kinase activity bioassays. The on-chip synthesized peptides can be cleaved for MS detection.
A R T I C L E I N F O
A B S T R A C T
Article history: Received 14 October 2014 Received in revised form 23 January 2015 Accepted 28 January 2015 Available online 30 January 2015
Polydimethylsiloxane (PDMS) is widely used for microfabrication and bioanalysis; however, its surface functionalization is limited due to the lack of active functional groups and incompatibility with many solvents. We presented a novel approach for in situ fabrication of cleavable peptide arrays on polydimethylsiloxane (PDMS) via tert-butyloxycarbonyl (t-Boc)/trifluoroacetic acid (TFA) chemistry using gold nanoparticles (AuNPs) as the anchor and a disulfide/amine terminated hetero-polyethylene glycol as the cleavable linker. The method was fine tuned to use reagents compatible with the PDMS. Using 5-mer pentapeptide, Trp5, as a model, step-by-step covalent coupling during the reaction cycles was monitored by Attenuated total reflectance-Fourier transform infrared spectrometer (ATR-FTIR), X-ray photoelectron spectroscopy (XPS), or atomic force microscopy (AFM), and further confirmed by mass spectrometry (MS) detection of the cleaved peptides. Using such a method, heptapeptides of the PKA substrate, LRRASLG (Kemptide), and its point mutated analogs were fabricated in an array format for comparative studies of cAMP-dependent protein kinase (PKA) activity. Based on on-chip detection, Kemptide sequence exhibited the highest phosphorylation activity, which was detected to a 1.5-time lesser extent for the point mutated sequence (LRRGSLG) containing the recognition motif (RRXS), and was nearly undetectable for another point mutated sequence (LRLASLG) that lacked the recognition motif. These results indicate that the reported fabrication method is able to yield highly specific peptide sequences on PDMS, leading to a highly motif-sensitive enzyme activity assay. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Polydimethylsiloxane Gold nanoparticle Cleavable peptide array Kinase assays
1. Introduction
* Corresponding author. Tel.: +886 6 2757575x65339; fax: +886 6 2740552. E-mail addresses: [email protected] (H.-H. Chen), [email protected] (Y.-C. Hsiao), [email protected] (J.-R. Li), [email protected] (S.-H. Chen). http://dx.doi.org/10.1016/j.aca.2015.01.040 0003-2670/ ã 2015 Elsevier B.V. All rights reserved.
Due to inherent limitations, which include physicochemical incompatibility with several solvents [1], instability owing to hydrophobic recovery, and the lack of functional groups for surface functionalization, polydimethylsiloxane (PDMS) is not considered a useful substrate for chemical reactions. Whereas, PDMS is one of the
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most widely used materials for microfabrication and bioanalysis and it has several advantages including low cost, high optical transparency, and an easy and flexible fabrication method [2–5]. Nanowells can be fabricated to fit commercial readers for an enzyme-linked immunosorbent assay (ELISA) by simple molding techniques [6]; data can be directly read using commercially available instruments. Moreover, PDMS can better isolate individual spots to prevent cross contamination, which is troublesome on cellulose or glass chips [7– 10]. Three aspects of compatibility including the swelling of PDMS in a solvent, the partitioning of solutes between a solvent and PDMS, and the dissolution of PDMS oligomers in a solvent were proposed to be considered for conducting chemical reactions on the PDMS surface [1]. Based on these, there are still a broad range of compatible solvents which can be used for chemical reactions on PDMS [1]. In recent years, permanently stable surface functionalization on PDMS has been demonstrated by us [11] and other groups [12–16] and further applied to develop PDMS-based array assays [17,18]. In situ oligonucleotide synthesis on PDMS has also been reported using conventional phosphoramidite chemistry [19]. These findings indicate that PDMS holds great promise as an alternative substrate for the development of various biochemical assays. In this study, we report, to our knowledge, the first in situsynthesized peptide array on PDMS and its application for PKA assays. We proposed a surface functionalization scheme on PDMS that uses gold nanoparticles (AuNPs) as the anchor and thiol/amine terminated hetero-polyethylene glycol for the attachment of the first amino acid followed by in situ peptide synthesis via tertbutyloxycarbonyl (t-Boc)/trifluoroacetic acid (TFA) chemistry. Cleavable disulfide groups could also be added as part of the linker for off-line detection and characterization of products by mass spectrometry (MS). Various peptide sequences and their analogs were designed in an array format on a chip to demonstrate a selective and sensitive enzymatic assay for cAMP-dependent protein kinase (PKA). 2. Materials and methods
mixture was degassed in a vacuum for 30 min. The degassed PDMS mixture was poured on a stainless steel or glass template and then cured at 70 C for four hours. Once peeled from the template, the resulting 12 8 array pattern (2.5 mm id and 0.5 mm height for each spot in a space of 5 cm width 4 cm length) of the PDMS substrate was used as the grid for solution printing. The bare PDMS array chip with transferred 12 8 grids was then pipetted with 5 mL HAuCl4 aqueous solution (1% v/v in DI water) and incubated at room temperature for 48 h to form AuNPs on the surface [20,21]. 2.3. Functionalization and in situ peptide synthesis on AuNPs-coated PDMS arrays The AuNP-coated PDMS array was first functionalized to form exposed amine groups for the attachment of the first amino acid. A volume of 5 mL of a mixture composed of SH-PEG-NH2 (1 mM) and SH-PEG-OCH3 (1 mM) in DI-water with a molar ratio of 1:9 was pipetted onto each spot and incubated at 4 C overnight followed by washes with DI water. When necessary, 5 mL of 2-iminothiolane hydrochloride (2 mM in DI water) and PDA (1 mM in phosphate buffer) was sequentially spotted onto the array and incubated at room temperature for 2 and 48 h, respectively, to form a disulfide linker. For peptide synthesis, 5 mL of the mixture containing t-BocAA-OH (60 mM in DMF) and DIC (1% v/v in DMF) was spotted onto the surface to react with the exposed amine group at room temperature for 2 h. The unbound t-Boc-AA-OH was washed away by DMF and the t-Boc protection group of the bound amino acid was removed by incubation with 5 mL of 50% TFA at room temperature for half an hour to expose the amine group. Each spot surface was washed with de-ionized water and DMF and confirmed to be neutralized (pH 7) before proceeding to the next coupling reaction. The three-step reaction including coupling, deprotection, and neutralization was repeated for several cycles until the desired sequence was synthesized. 2.4. Attenuated total reflectance-Fourier transform infrared spectrometer (ATR-FTIR) characterization
2.1. Regents and apparatus The Sylgard 184 kit, containing vinyl-terminated PDMS base and curing agent, was acquired from Dow Corning (Midland, MI, USA). HAuCl43H2O was obtained from Alfa Aesar (Johnson Matthey Co., London, UK). SH-PEG-OCH3 (MW 750) was obtained from Rapp Polymere GmbH (Tübingen, Germany). SH-PEG-NH2 and Pyridine dithioethylamine (PDA) were obtained from Nanocs (New York, USA). 2-Iminothiolane hydrochloride, Cysteamine, BocTrp-OH, Boc-Gly-OH, Boc-Leu-OH, Boc-Ser-OH, Boc-Ala-OH, BocArg-OH, Boc-Ser(Bzl)-OH, Boc-Arg(Tos)-OH and tris(2-carboxyethyl) phosphine hydrochloride (TCEP-HCl) were obtained from Sigma (St. Louis, MO, USA). N,N0 -Diisopropylcarbodiimide (DIC) and trifluoroacetic acid (TFA) were obtained from Alfa Aestar. N,NDimethylformamide (DMF) and Acetonitrile (ACN) were obtained from J.T. Baker (Canada). Phosphate buffered saline (PBS) was obtained from Pierce (Rockford, IL, USA). Protein kinase A (PKA) kinase activity kit was obtained from Enzo Life Sciences (New York, USA). The Enhanced Chemiluminescent Luminol Reagent-kit (ECL) was obtained from PerkinElmer Life Sciences (Boston, MA, USA) for detection, and the emission was captured by a digital imaging system (UVP Bio-Imaging Systems, CA, USA). Milli-Q-purified de-ionized water was used for preparing solutions. 2.2. Fabrication of the gold nanoparticles (AuNPs)-coated PDMS arrays To form the PDMS prepolymer, the PDMS oligomer was mixed with curing agent at a weight ratio of 10:1, and the resulting
The ATR-FTIR instrument (PerkinElmer, Spectrum RX1, CA, USA) was used to characterize the in situ peptide synthesis on PDMS. On each spot, 20 scans were collected at a resolution of 1 cm1 and the background signals acquired from the spot without synthesized peptides were subtracted. All PDMS plates were dried with a stream of nitrogen before the measurements. 2.5. Atomic force microscope (AFM) characterization AFM investigations were carried out on a MultiMode AFM with Nanoscope-V controller (Bruker, Santa Barbara, CA, USA). Rectangular silicon nitride cantilevers with a force constant of 0.4 N m1 and an average resonance frequency of 130 kHz was used for imaging with PeakForce-Quantitative Nanomechanical Mapping (PFQNM) mode in air (ScanAsyst-Air-HR, Bruker AFM Probes Americas, Camarillo, CA, USA). All digital images were processed with the NanoScope Analysis (version 1.4, Bruker, Santa Barbara, CA, USA). The PFQNM mode, a new AFM mode developed by Bruker, was applied to measure elastic modulus and other mechanical properties of the sample surface. The foundation of elastic modulus mapping with PFQNM mode is the ability of the system to acquire and analyze the individual force curves produced during the imaging process. PFQNM mode enables to measure the instantaneous force on the AFM probe, in order to separate the contributions from different material properties such as adhesion, modulus, dissipation, and deformation. The PFQNM procedure begins from calibration of the AFM probe. Deflection sensitivity was determined on a sapphire surface, and the tip radius and
H.-H. Chen et al. / Analytica Chimica Acta 865 (2015) 53–59
spring constant were calibrated respectively on the PDMS standard surface (2.5 GPa). The Young’s moduli measurements after the calibration procedure completed. 2.6. X-ray photoelectron spectroscopy (XPS) characterization All XPS measurements were carried out on a ULVAC-PHI 5000 VersaProbe (PHI, Tokyo, Japan) system in Al Ka mode. All PDMS surfaces were dried with a stream of nitrogen before the measurements.
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in kinase assay dilution buffer) and 3 mL of kinase assay dilution buffer was pipetted onto PDMS arrays. After a 90 min incubation at 30 C, 5 mL of rabbit polyclonal antibody specific for phosphorylated PKA substrate and 5 mL of anti-rabbit IgG-HRP (0.4 mg mL1 in PBST buffer) were added in sequence. Finally, 5 mL of ECL reagent was added to enable the detection of emitted chemiluminescence by a BioSpectrum imaging system (UVP, Bio-Imaging Systems, CA, USA). 3. Results and discussion
2.7. Mass spectrometry (MS) characterization
3.1. Surface functionalization
Since a relatively large volume is required for off-line detection, the PDMS substrate was fabricated using ELISA plate (96 wells 100 mL for each well) as the molding template following the same procedure described previously. After the reaction cycle for peptide synthesis, 80 mL of TCEP-HCl (1 mM in deionized water pH 4.5) was added and incubated at room temperature for half an hour to cleave the disulfide linkage. A volume of 50 mL of the cleaved solution was directly infused into electro-spray ionization (ESI) quadrupole time-of-flight (Q-TOF) MS (Waters, Xevo G2-S, USA), at a flow rate of 10 mL min1. MS conditions were set up as the following: positive ionization mode with 2800 V ionization source voltage and 100 C source temperature. Liquid nitrogen was used as the drying and nebulizing gas.
The substrates used for in situ peptide synthesis should confer stability to facilitate the coupling reaction, and the surface must be functionalized to enable the attachment of the first amino acid. As depicted in Steps A–D of Fig. 1, the PDMS substrate was first coated with AuNPs synthesized in situ via the silane redox reaction [20,21], followed by a thiol/amine terminated hetero-PEG and disulfide linker (if required) to result in a free amine functional group. As indicated by the CCD images shown in Fig. 2A and B (left), a 10:1 mass ratio of the curing agent to the monomer led to the formation of AuNPs with a homogeneous pink color, consistent with previous reports [20,21]. Using AFM (Fig. 2A and B), the spot boundary clearly showed a stiff inner and soft outer surface due to the formation of metallic AuNPs inside the array spot. In situsynthesized AuNPs on PDMS was used as the anchoring layer for subsequent covalent binding with PEGs. According to Fig. 2C, the surface coverage of AuNPs in a microscale was very high (>90%). To reduce steric hindrance, we mixed the bi-functional PEG, HS-PEGNH2, with the shorter mono-functional PEG, HS-PEG-OCH3. Thus,
2.8. The PKA kinase assay The mixture containing 0.75 mL of active PKA (15 ng in kinase assay dilution buffer), 1.25 mL of adenosine triphosphate (1.25 mg
[(Fig._1)TD$IG]
Fig. 1. Schematics of the surface functionalization and step-by-step peptide synthesis via tert-butyloxycarbonyl (t-Boc)/trifluoroacetic acid (TFA) chemistry.
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[(Fig._2)TD$IG]
H.-H. Chen et al. / Analytica Chimica Acta 865 (2015) 53–59
Fig. 2. Surface functionalization and peptide synthesis on PDMS. Charge-coupled device (CCD) images and AFM modulus images reveal the boundary of the spot coated with AuNPs (A) and the spot further topped with the first tryptophan (B). The blue squares indicate the location where each enlarged boundary was characterized by AFM. The AFM modulus images of the spots attached with no (C), 2 (D), and 5 (E) tryptophan(s) are also shown. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
only PEG with an amine end (HS-PEG-NH2) can bind to the carboxylate group; PEG with a methoxy end (HS-PEG-OCH3) acts as a spacer to create room for coupling. The molar ratio between the functional and spacer PEGs was examined to fine-tune the conditions and a ratio of 1:9 (HS-PEG-NH2:HS-PEG-OCH3) was determined to be optimal for the experiment (Supplement 1 Optimization for PEG modifications). The modification of PEG was evidenced by C—H stretching (2879 cm1) and C—O stretching (1107 cm1) bands detected by ATR-FTIR (Fig. 3A). The terminal amine was further converted to a cleavable disulfide linkage via a subsequent reaction with 2-iminothiolane hydrochloride followed by PDA (Step C–D of Fig. 1). The conjugation of PDA was evidenced by the S2p core disulfide band (164 eV) detected by XPS (Fig. 3B) as well as by the cleaved [HS-C2H4-NH2]+ ion (m/z 78.0354) detected by MS (Supplement 2 MS detection of the cleaved thiol). This design led to a terminally exposed free amine group for the attachment of the first amino acid. 3.2. In situ peptide synthesis on PDMS Currently, two protecting groups, t-Boc and 9-fluorenylmethyloxycarbonyl (Fmoc), are commonly used in solid-phase peptide synthesis. However, deprotection of the a-amino Fmoc group requires a base such as piperidine that is incompatible with PDMS due to dissolution. In contrast, t-Boc uses acids such as TFA, which is compatible with PDMS, for deprotection [19]. In addition, t-Boc protecting strategies are useful for reducing peptide aggregation during synthesis owing to the formation of a positively charged amino group. This is particularly suitable for PDMS because its hydrophobic nature is likely to cause protein adsorption and aggregation. Thus, t-Boc/TFA chemistry was adopted for in situ peptide synthesis on PDMS. The reaction scheme (Steps E–H of Fig. 1) was tested for the synthesis of a 5-mer model peptide poly-tryptophan (Trp)5. The
C-termini of the first Boc protected amino acid Boc-Trp-OH were covalently bound to the free amine on the surface by carbodiimide DIC reaction to initiate the coupling cycle, which included three steps: (1) coupling reaction with the protected amino acid, (2) deprotection by 50% TFA, (3) neutralization by a series of washes with DI water and DMF. DMF was used as the solvent since it is compatible with PDMS. As shown in Fig. 2A and B (right), the AFM modulus image showed that the spot boundary changed from stiff/ soft to soft/stiff upon covalent binding of the first tryptophan. Moreover, sequential decreases in the surface stiffness were detected as the coupling reaction proceeded from no tryptophan (Fig. 2C) to 2 tryptophans (Fig. 2D) and eventually to 5 tryptophans (Fig. 2E). The averaged elastic modulus values varied from 6.4 MPa (no tryptophan), 3.9 MPa (2 tryptophans), to 1.7 MPa (5 tryptophans) with the increase in the numbers of tryptophans. Local stiff and soft domains of peptide layer(s) were clearly identified with the AFM modulus imaging. The surface becomes softer as the peptide grows. The ATR-FTIR spectra acquired during the reaction cycle (Fig. 3C) also clearly showed that the amide stretching (3435 cm1) and bending (1660 and 1505 cm1) peaks gradually increased with the number of the coupling cycles. Contributions of these bands by the trapped reagents were thought to be unlikely since chemicals must be deeply trapped inside PDMS beyond the probing range of the reflected IR. As shown, the inner PEG band (Fig. 3A) became undetectable (Fig. 3C) when the PEG layer was topped by peptides. The products were cleaved by the reducing agent TCEP-HCl and characterized by MS. As shown in Fig. 3D, although many ions in TCEP-HCl solution were detected by electrospray ionization (ESI)-MS, the monoisotopic peak (m/z = 1030.7582) of the final product ion, HS-C2H4-Trp5, could only be detected from the product solution (inset of Fig. 3D). Intermediate products, Trp1–5-mers, cleaved after each reaction cycle were also collected and examined by MS. The ion intensity was normalized by that of the reducing agent, TCEP-HCl
[(Fig._3)TD$IG]
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Fig. 3. In situ peptide synthesis on PDMS. (A) ATR-FTIR spectra of the PDMS surface modified by AuNPs (top) in situ synthesized on the surface and followed by PEGs conjugation (bottom). (B) XPS spectra (the S2p core disulfide band) detected from the PDMS surface modified by AuNPs (bottom), followed by PEGs (middle), and then PDA (top). (C) ATR-FTIR spectra (amide bands) of the PDMS surface after 1–5 (from the top to the bottom) Trp coupling cycles. (D) MS spectra (m/z 200–1200) detected from the Trp5 product solution (top) cleaved by TCEP-HCl (bottom) after 5 synthesis cycles. The insets show the enlarged monoisotopic peak profile of Trp5 sodium adduct (m/z 1030.7582) and the star indicates overlap of its third isotopic peak with another precursor ion. (C) Normalized ion intensities of the products (Trp1–5-mers) from the cleaved solutions after each coupling cycle using the ion intensity of the reducing agent TCEP-HCl (m/z = 251.0685) as the normalization factor.
(m/z = 251.0685), concurrently detected from the same solution for relative quantification. As shown in Fig. 3E, individual products of Trp1–5 were mainly synthesized after each corresponding reaction cycle, i.e., Trp1 after one reaction cycle, Trp2 after 2 reaction cycles etc. Incomplete products such as Trp1, and Trp4 were still detected from the final product solution after 5 reaction cycles but these short peptides were estimated to be less than 10%. We also noticed some Boc-protected ions in the final product (data not shown); however, their intensities were relatively low (
Contents lists available at ScienceDirect
Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
In situ fabrication of cleavable peptide arrays on polydimethylsiloxane and applications for kinase activity assays Huang-Han Chen, Yu-Chieh Hsiao, Jie-Ren Li, Shu-Hui Chen * Department of Chemistry, National Cheng Kung University, No.1 College Road, Tainan 701, Taiwan
H I G H L I G H T S
G R A P H I C A L A B S T R A C T
A novel approach for in situ fabrication of cleavable peptide arrays on polydimethylsiloxane (PDMS). The first report of peptide synthesis on PDMS. Use of the PDMS peptide array for developing sensitive chip-based kinase activity bioassays. The on-chip synthesized peptides can be cleaved for MS detection.
A R T I C L E I N F O
A B S T R A C T
Article history: Received 14 October 2014 Received in revised form 23 January 2015 Accepted 28 January 2015 Available online 30 January 2015
Polydimethylsiloxane (PDMS) is widely used for microfabrication and bioanalysis; however, its surface functionalization is limited due to the lack of active functional groups and incompatibility with many solvents. We presented a novel approach for in situ fabrication of cleavable peptide arrays on polydimethylsiloxane (PDMS) via tert-butyloxycarbonyl (t-Boc)/trifluoroacetic acid (TFA) chemistry using gold nanoparticles (AuNPs) as the anchor and a disulfide/amine terminated hetero-polyethylene glycol as the cleavable linker. The method was fine tuned to use reagents compatible with the PDMS. Using 5-mer pentapeptide, Trp5, as a model, step-by-step covalent coupling during the reaction cycles was monitored by Attenuated total reflectance-Fourier transform infrared spectrometer (ATR-FTIR), X-ray photoelectron spectroscopy (XPS), or atomic force microscopy (AFM), and further confirmed by mass spectrometry (MS) detection of the cleaved peptides. Using such a method, heptapeptides of the PKA substrate, LRRASLG (Kemptide), and its point mutated analogs were fabricated in an array format for comparative studies of cAMP-dependent protein kinase (PKA) activity. Based on on-chip detection, Kemptide sequence exhibited the highest phosphorylation activity, which was detected to a 1.5-time lesser extent for the point mutated sequence (LRRGSLG) containing the recognition motif (RRXS), and was nearly undetectable for another point mutated sequence (LRLASLG) that lacked the recognition motif. These results indicate that the reported fabrication method is able to yield highly specific peptide sequences on PDMS, leading to a highly motif-sensitive enzyme activity assay. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Polydimethylsiloxane Gold nanoparticle Cleavable peptide array Kinase assays
1. Introduction
* Corresponding author. Tel.: +886 6 2757575x65339; fax: +886 6 2740552. E-mail addresses: [email protected] (H.-H. Chen), [email protected] (Y.-C. Hsiao), [email protected] (J.-R. Li), [email protected] (S.-H. Chen). http://dx.doi.org/10.1016/j.aca.2015.01.040 0003-2670/ ã 2015 Elsevier B.V. All rights reserved.
Due to inherent limitations, which include physicochemical incompatibility with several solvents [1], instability owing to hydrophobic recovery, and the lack of functional groups for surface functionalization, polydimethylsiloxane (PDMS) is not considered a useful substrate for chemical reactions. Whereas, PDMS is one of the
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H.-H. Chen et al. / Analytica Chimica Acta 865 (2015) 53–59
most widely used materials for microfabrication and bioanalysis and it has several advantages including low cost, high optical transparency, and an easy and flexible fabrication method [2–5]. Nanowells can be fabricated to fit commercial readers for an enzyme-linked immunosorbent assay (ELISA) by simple molding techniques [6]; data can be directly read using commercially available instruments. Moreover, PDMS can better isolate individual spots to prevent cross contamination, which is troublesome on cellulose or glass chips [7– 10]. Three aspects of compatibility including the swelling of PDMS in a solvent, the partitioning of solutes between a solvent and PDMS, and the dissolution of PDMS oligomers in a solvent were proposed to be considered for conducting chemical reactions on the PDMS surface [1]. Based on these, there are still a broad range of compatible solvents which can be used for chemical reactions on PDMS [1]. In recent years, permanently stable surface functionalization on PDMS has been demonstrated by us [11] and other groups [12–16] and further applied to develop PDMS-based array assays [17,18]. In situ oligonucleotide synthesis on PDMS has also been reported using conventional phosphoramidite chemistry [19]. These findings indicate that PDMS holds great promise as an alternative substrate for the development of various biochemical assays. In this study, we report, to our knowledge, the first in situsynthesized peptide array on PDMS and its application for PKA assays. We proposed a surface functionalization scheme on PDMS that uses gold nanoparticles (AuNPs) as the anchor and thiol/amine terminated hetero-polyethylene glycol for the attachment of the first amino acid followed by in situ peptide synthesis via tertbutyloxycarbonyl (t-Boc)/trifluoroacetic acid (TFA) chemistry. Cleavable disulfide groups could also be added as part of the linker for off-line detection and characterization of products by mass spectrometry (MS). Various peptide sequences and their analogs were designed in an array format on a chip to demonstrate a selective and sensitive enzymatic assay for cAMP-dependent protein kinase (PKA). 2. Materials and methods
mixture was degassed in a vacuum for 30 min. The degassed PDMS mixture was poured on a stainless steel or glass template and then cured at 70 C for four hours. Once peeled from the template, the resulting 12 8 array pattern (2.5 mm id and 0.5 mm height for each spot in a space of 5 cm width 4 cm length) of the PDMS substrate was used as the grid for solution printing. The bare PDMS array chip with transferred 12 8 grids was then pipetted with 5 mL HAuCl4 aqueous solution (1% v/v in DI water) and incubated at room temperature for 48 h to form AuNPs on the surface [20,21]. 2.3. Functionalization and in situ peptide synthesis on AuNPs-coated PDMS arrays The AuNP-coated PDMS array was first functionalized to form exposed amine groups for the attachment of the first amino acid. A volume of 5 mL of a mixture composed of SH-PEG-NH2 (1 mM) and SH-PEG-OCH3 (1 mM) in DI-water with a molar ratio of 1:9 was pipetted onto each spot and incubated at 4 C overnight followed by washes with DI water. When necessary, 5 mL of 2-iminothiolane hydrochloride (2 mM in DI water) and PDA (1 mM in phosphate buffer) was sequentially spotted onto the array and incubated at room temperature for 2 and 48 h, respectively, to form a disulfide linker. For peptide synthesis, 5 mL of the mixture containing t-BocAA-OH (60 mM in DMF) and DIC (1% v/v in DMF) was spotted onto the surface to react with the exposed amine group at room temperature for 2 h. The unbound t-Boc-AA-OH was washed away by DMF and the t-Boc protection group of the bound amino acid was removed by incubation with 5 mL of 50% TFA at room temperature for half an hour to expose the amine group. Each spot surface was washed with de-ionized water and DMF and confirmed to be neutralized (pH 7) before proceeding to the next coupling reaction. The three-step reaction including coupling, deprotection, and neutralization was repeated for several cycles until the desired sequence was synthesized. 2.4. Attenuated total reflectance-Fourier transform infrared spectrometer (ATR-FTIR) characterization
2.1. Regents and apparatus The Sylgard 184 kit, containing vinyl-terminated PDMS base and curing agent, was acquired from Dow Corning (Midland, MI, USA). HAuCl43H2O was obtained from Alfa Aesar (Johnson Matthey Co., London, UK). SH-PEG-OCH3 (MW 750) was obtained from Rapp Polymere GmbH (Tübingen, Germany). SH-PEG-NH2 and Pyridine dithioethylamine (PDA) were obtained from Nanocs (New York, USA). 2-Iminothiolane hydrochloride, Cysteamine, BocTrp-OH, Boc-Gly-OH, Boc-Leu-OH, Boc-Ser-OH, Boc-Ala-OH, BocArg-OH, Boc-Ser(Bzl)-OH, Boc-Arg(Tos)-OH and tris(2-carboxyethyl) phosphine hydrochloride (TCEP-HCl) were obtained from Sigma (St. Louis, MO, USA). N,N0 -Diisopropylcarbodiimide (DIC) and trifluoroacetic acid (TFA) were obtained from Alfa Aestar. N,NDimethylformamide (DMF) and Acetonitrile (ACN) were obtained from J.T. Baker (Canada). Phosphate buffered saline (PBS) was obtained from Pierce (Rockford, IL, USA). Protein kinase A (PKA) kinase activity kit was obtained from Enzo Life Sciences (New York, USA). The Enhanced Chemiluminescent Luminol Reagent-kit (ECL) was obtained from PerkinElmer Life Sciences (Boston, MA, USA) for detection, and the emission was captured by a digital imaging system (UVP Bio-Imaging Systems, CA, USA). Milli-Q-purified de-ionized water was used for preparing solutions. 2.2. Fabrication of the gold nanoparticles (AuNPs)-coated PDMS arrays To form the PDMS prepolymer, the PDMS oligomer was mixed with curing agent at a weight ratio of 10:1, and the resulting
The ATR-FTIR instrument (PerkinElmer, Spectrum RX1, CA, USA) was used to characterize the in situ peptide synthesis on PDMS. On each spot, 20 scans were collected at a resolution of 1 cm1 and the background signals acquired from the spot without synthesized peptides were subtracted. All PDMS plates were dried with a stream of nitrogen before the measurements. 2.5. Atomic force microscope (AFM) characterization AFM investigations were carried out on a MultiMode AFM with Nanoscope-V controller (Bruker, Santa Barbara, CA, USA). Rectangular silicon nitride cantilevers with a force constant of 0.4 N m1 and an average resonance frequency of 130 kHz was used for imaging with PeakForce-Quantitative Nanomechanical Mapping (PFQNM) mode in air (ScanAsyst-Air-HR, Bruker AFM Probes Americas, Camarillo, CA, USA). All digital images were processed with the NanoScope Analysis (version 1.4, Bruker, Santa Barbara, CA, USA). The PFQNM mode, a new AFM mode developed by Bruker, was applied to measure elastic modulus and other mechanical properties of the sample surface. The foundation of elastic modulus mapping with PFQNM mode is the ability of the system to acquire and analyze the individual force curves produced during the imaging process. PFQNM mode enables to measure the instantaneous force on the AFM probe, in order to separate the contributions from different material properties such as adhesion, modulus, dissipation, and deformation. The PFQNM procedure begins from calibration of the AFM probe. Deflection sensitivity was determined on a sapphire surface, and the tip radius and
H.-H. Chen et al. / Analytica Chimica Acta 865 (2015) 53–59
spring constant were calibrated respectively on the PDMS standard surface (2.5 GPa). The Young’s moduli measurements after the calibration procedure completed. 2.6. X-ray photoelectron spectroscopy (XPS) characterization All XPS measurements were carried out on a ULVAC-PHI 5000 VersaProbe (PHI, Tokyo, Japan) system in Al Ka mode. All PDMS surfaces were dried with a stream of nitrogen before the measurements.
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in kinase assay dilution buffer) and 3 mL of kinase assay dilution buffer was pipetted onto PDMS arrays. After a 90 min incubation at 30 C, 5 mL of rabbit polyclonal antibody specific for phosphorylated PKA substrate and 5 mL of anti-rabbit IgG-HRP (0.4 mg mL1 in PBST buffer) were added in sequence. Finally, 5 mL of ECL reagent was added to enable the detection of emitted chemiluminescence by a BioSpectrum imaging system (UVP, Bio-Imaging Systems, CA, USA). 3. Results and discussion
2.7. Mass spectrometry (MS) characterization
3.1. Surface functionalization
Since a relatively large volume is required for off-line detection, the PDMS substrate was fabricated using ELISA plate (96 wells 100 mL for each well) as the molding template following the same procedure described previously. After the reaction cycle for peptide synthesis, 80 mL of TCEP-HCl (1 mM in deionized water pH 4.5) was added and incubated at room temperature for half an hour to cleave the disulfide linkage. A volume of 50 mL of the cleaved solution was directly infused into electro-spray ionization (ESI) quadrupole time-of-flight (Q-TOF) MS (Waters, Xevo G2-S, USA), at a flow rate of 10 mL min1. MS conditions were set up as the following: positive ionization mode with 2800 V ionization source voltage and 100 C source temperature. Liquid nitrogen was used as the drying and nebulizing gas.
The substrates used for in situ peptide synthesis should confer stability to facilitate the coupling reaction, and the surface must be functionalized to enable the attachment of the first amino acid. As depicted in Steps A–D of Fig. 1, the PDMS substrate was first coated with AuNPs synthesized in situ via the silane redox reaction [20,21], followed by a thiol/amine terminated hetero-PEG and disulfide linker (if required) to result in a free amine functional group. As indicated by the CCD images shown in Fig. 2A and B (left), a 10:1 mass ratio of the curing agent to the monomer led to the formation of AuNPs with a homogeneous pink color, consistent with previous reports [20,21]. Using AFM (Fig. 2A and B), the spot boundary clearly showed a stiff inner and soft outer surface due to the formation of metallic AuNPs inside the array spot. In situsynthesized AuNPs on PDMS was used as the anchoring layer for subsequent covalent binding with PEGs. According to Fig. 2C, the surface coverage of AuNPs in a microscale was very high (>90%). To reduce steric hindrance, we mixed the bi-functional PEG, HS-PEGNH2, with the shorter mono-functional PEG, HS-PEG-OCH3. Thus,
2.8. The PKA kinase assay The mixture containing 0.75 mL of active PKA (15 ng in kinase assay dilution buffer), 1.25 mL of adenosine triphosphate (1.25 mg
[(Fig._1)TD$IG]
Fig. 1. Schematics of the surface functionalization and step-by-step peptide synthesis via tert-butyloxycarbonyl (t-Boc)/trifluoroacetic acid (TFA) chemistry.
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[(Fig._2)TD$IG]
H.-H. Chen et al. / Analytica Chimica Acta 865 (2015) 53–59
Fig. 2. Surface functionalization and peptide synthesis on PDMS. Charge-coupled device (CCD) images and AFM modulus images reveal the boundary of the spot coated with AuNPs (A) and the spot further topped with the first tryptophan (B). The blue squares indicate the location where each enlarged boundary was characterized by AFM. The AFM modulus images of the spots attached with no (C), 2 (D), and 5 (E) tryptophan(s) are also shown. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
only PEG with an amine end (HS-PEG-NH2) can bind to the carboxylate group; PEG with a methoxy end (HS-PEG-OCH3) acts as a spacer to create room for coupling. The molar ratio between the functional and spacer PEGs was examined to fine-tune the conditions and a ratio of 1:9 (HS-PEG-NH2:HS-PEG-OCH3) was determined to be optimal for the experiment (Supplement 1 Optimization for PEG modifications). The modification of PEG was evidenced by C—H stretching (2879 cm1) and C—O stretching (1107 cm1) bands detected by ATR-FTIR (Fig. 3A). The terminal amine was further converted to a cleavable disulfide linkage via a subsequent reaction with 2-iminothiolane hydrochloride followed by PDA (Step C–D of Fig. 1). The conjugation of PDA was evidenced by the S2p core disulfide band (164 eV) detected by XPS (Fig. 3B) as well as by the cleaved [HS-C2H4-NH2]+ ion (m/z 78.0354) detected by MS (Supplement 2 MS detection of the cleaved thiol). This design led to a terminally exposed free amine group for the attachment of the first amino acid. 3.2. In situ peptide synthesis on PDMS Currently, two protecting groups, t-Boc and 9-fluorenylmethyloxycarbonyl (Fmoc), are commonly used in solid-phase peptide synthesis. However, deprotection of the a-amino Fmoc group requires a base such as piperidine that is incompatible with PDMS due to dissolution. In contrast, t-Boc uses acids such as TFA, which is compatible with PDMS, for deprotection [19]. In addition, t-Boc protecting strategies are useful for reducing peptide aggregation during synthesis owing to the formation of a positively charged amino group. This is particularly suitable for PDMS because its hydrophobic nature is likely to cause protein adsorption and aggregation. Thus, t-Boc/TFA chemistry was adopted for in situ peptide synthesis on PDMS. The reaction scheme (Steps E–H of Fig. 1) was tested for the synthesis of a 5-mer model peptide poly-tryptophan (Trp)5. The
C-termini of the first Boc protected amino acid Boc-Trp-OH were covalently bound to the free amine on the surface by carbodiimide DIC reaction to initiate the coupling cycle, which included three steps: (1) coupling reaction with the protected amino acid, (2) deprotection by 50% TFA, (3) neutralization by a series of washes with DI water and DMF. DMF was used as the solvent since it is compatible with PDMS. As shown in Fig. 2A and B (right), the AFM modulus image showed that the spot boundary changed from stiff/ soft to soft/stiff upon covalent binding of the first tryptophan. Moreover, sequential decreases in the surface stiffness were detected as the coupling reaction proceeded from no tryptophan (Fig. 2C) to 2 tryptophans (Fig. 2D) and eventually to 5 tryptophans (Fig. 2E). The averaged elastic modulus values varied from 6.4 MPa (no tryptophan), 3.9 MPa (2 tryptophans), to 1.7 MPa (5 tryptophans) with the increase in the numbers of tryptophans. Local stiff and soft domains of peptide layer(s) were clearly identified with the AFM modulus imaging. The surface becomes softer as the peptide grows. The ATR-FTIR spectra acquired during the reaction cycle (Fig. 3C) also clearly showed that the amide stretching (3435 cm1) and bending (1660 and 1505 cm1) peaks gradually increased with the number of the coupling cycles. Contributions of these bands by the trapped reagents were thought to be unlikely since chemicals must be deeply trapped inside PDMS beyond the probing range of the reflected IR. As shown, the inner PEG band (Fig. 3A) became undetectable (Fig. 3C) when the PEG layer was topped by peptides. The products were cleaved by the reducing agent TCEP-HCl and characterized by MS. As shown in Fig. 3D, although many ions in TCEP-HCl solution were detected by electrospray ionization (ESI)-MS, the monoisotopic peak (m/z = 1030.7582) of the final product ion, HS-C2H4-Trp5, could only be detected from the product solution (inset of Fig. 3D). Intermediate products, Trp1–5-mers, cleaved after each reaction cycle were also collected and examined by MS. The ion intensity was normalized by that of the reducing agent, TCEP-HCl
[(Fig._3)TD$IG]
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Fig. 3. In situ peptide synthesis on PDMS. (A) ATR-FTIR spectra of the PDMS surface modified by AuNPs (top) in situ synthesized on the surface and followed by PEGs conjugation (bottom). (B) XPS spectra (the S2p core disulfide band) detected from the PDMS surface modified by AuNPs (bottom), followed by PEGs (middle), and then PDA (top). (C) ATR-FTIR spectra (amide bands) of the PDMS surface after 1–5 (from the top to the bottom) Trp coupling cycles. (D) MS spectra (m/z 200–1200) detected from the Trp5 product solution (top) cleaved by TCEP-HCl (bottom) after 5 synthesis cycles. The insets show the enlarged monoisotopic peak profile of Trp5 sodium adduct (m/z 1030.7582) and the star indicates overlap of its third isotopic peak with another precursor ion. (C) Normalized ion intensities of the products (Trp1–5-mers) from the cleaved solutions after each coupling cycle using the ion intensity of the reducing agent TCEP-HCl (m/z = 251.0685) as the normalization factor.
(m/z = 251.0685), concurrently detected from the same solution for relative quantification. As shown in Fig. 3E, individual products of Trp1–5 were mainly synthesized after each corresponding reaction cycle, i.e., Trp1 after one reaction cycle, Trp2 after 2 reaction cycles etc. Incomplete products such as Trp1, and Trp4 were still detected from the final product solution after 5 reaction cycles but these short peptides were estimated to be less than 10%. We also noticed some Boc-protected ions in the final product (data not shown); however, their intensities were relatively low (
