Poly(methyl methacrylate

Nov 19, 2009 - To whom correspondence should be addressed. (H.M.) Tel/Fax: ... Fax: 86-10-62656765. E-mail: ... We report a strategy to fabricate PDMS...
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Biomacromolecules 2009, 10, 3335–3340

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Electrospinning of Poly(dimethylsiloxane)/Poly(methyl methacrylate) Nanofibrous Membrane: Fabrication and Application in Protein Microarrays Dayong Yang,† Xing Liu,† Yu Jin,‡ Ying Zhu,† Dongdong Zeng,† Xingyu Jiang,*,‡ and Hongwei Ma*,† Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou 215125, China, and CAS Key Lab for Biological Effects of Nanomaterials and Nanosafety, National Center for NanoScience and Technology, Beijing 100190, China Received August 21, 2009; Revised Manuscript Received October 25, 2009

Fabrication of poly(dimethylsiloxane) (PDMS)/poly(methyl methacrylate) (PMMA) nanofibers is critical to harness the advantage of nanostructured membrane applied in protein microarrays. Electrospinning (ES) of PDMS nanofibers is challenging because of the relatively low molecular weight of PDMS prepolymer. We report a strategy to fabricate PDMS/PMMA nanofibers via ES by introducing carrier polymer PMMA into PDMS solutions to supplement the deficiency of chain entanglements in the PDMS prepolymer. The prepared PDMS/PMMA nanofibrous membrane (PDMS/PMMA NFM) was successfully used as substrates for protein microarrays. The results of immunoassays showed the superior performance of PDMS/PMMA NFM as 3D substrate for protein microarrays; the limit-of-detection (LOD) on PDMS/PMMA NFM was 32 times lower than that on nitrocellulose membrane. The realization of ES PDMS extends the scope of ES materials from thermoplastic polymers to thermosetting materials. Given the simplicity, low cost, and high efficiency of ES technology, we believe that PDMS/PMMA NFM is a promising 3D substrate for protein microarrays.

1. Introduction In this report, we realize the electrospinning (ES) of thermosetting poly(dimethylsiloxane) (PDMS) by using poly(methyl methacrylate) (PMMA) as the carrier polymer and apply the ES mats as 3D substrate for protein microarrays. ES is a simple yet effective technology to fabricate nanofibers, and the resulting mats have found uses in various fields.1–7 Traditionally, only polymer solutions were applicable for ES because it was necessary to have chain entanglements that prevent solution from breaking into droplets and produce nanofibers.8–15 Recently, ES of small molecules has drawn special attention because it extends ES mats from polymers to small entities that have special functionality. Several groups have succeeded in ES of small molecules by creating entanglements among small molecules.16–18 As a pioneer example, McKee et al. fabricated lecithin into nonwoven electrospun membranes.16 Lecithin is a phospholipid of molecular weight only at 530 g/mol. At concentrations >35% by weight (i.e., entanglement concentration), however, lecithin formed cylindrical micelles that were entangled in a fashion similar to polymers, and, subsequently, fibers formed. Singh et al. reported ES of peptide diphenylalanine (molecular weight 241 g/mol).17 This peptide self-assembled into nanotubes at high concentrations (17.5%), where π-π interactions served as molecular entanglements that were vital in stabilizing the fiber structures. Differently, Uyar et al. electrospun cyclodextrin into nanofibers by using poly(ethylene oxide) as the carrier polymer.18 Although these advances are significant, there are remaining challenges to be addressed. For instance, although * To whom correspondence should be addressed. (H.M.) Tel/Fax: 86512-62872539. E-mail: [email protected]. (X.J.) Tel: 86-1082545558. Fax: 86-10-62656765. E-mail: [email protected]. † Chinese Academy of Sciences. ‡ National Center for NanoScience and Technology.

these small molecules were successfully fabricated into nanofibers, these fibers made of small molecules would dissolve into solutions, resulting in poor stability. Herein, we report a strategy to fabricate a widely used thermosetting materials composed of prepolymers with limited molecular weight, PDMS, into nanofibers via ES, which could be subsequently thermocured into cross-linked fibers. These stable fibers were further tested as a 3D substrate for protein microarrays. The main reason why we choose PDMS as the substrate material for protein microarrays is that PDMS shows special properties that cater for the current needs of protein microarrays. One of the challenges in protein microarrays is to develop an appropriate substrate to improve the limit-of-detection (LOD).19–21 Different materials and methods for surface modification have been tested to achieve this goal. Glass slides with different surface functional groups were the most commonly used 2D substrates. To improve LOD, quasi-3D or 3D films have been developed, such as polymer brushes,22 poly(vinylidene fluoride) (PVDF)23 and nitrocellulose (NC)24 membrane, polyacrylamide gel pads,25 and PDMS microwells.26 However, these 3D systems had only limited improvement and had inherent weakness. For example, PVDF and NC membrane showed a high level of intrinsic background and nonspecific adsorption of proteins, and PDMS microwells were expensive to fabricate. It is therefore necessary to explore new, easy-to-fabricate, and cost-effective methods to prepare 3D substrate with large specific surface areas. PDMS is a suitable candidate for protein microarrays because PDMS exhibits special properties. PDMS is a hydrophobic material that adsorbs proteins from solutions in a manner similar to polystyrene of Petri dish and titer plates.27,28 Compared with other materials for protein microarrays such as BoroFloat glass, PDMS exhibits the lowest autofluorescence;29 moreover, the development of microfluidics endows PDMS with much diversified surface modification strategies.30,31 Because

10.1021/bm900955p CCC: $40.75  2009 American Chemical Society Published on Web 11/19/2009

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we have previously demonstrated that ES nanofibrous polycarbonate membrane showed superior performance in microfluidic immunoassay,7 the aim of this article is to utilize ES technology to prepare PDMS/PMMA nanofibrous membrane (PDMS/ PMMA NFM) as a 3D substrate for protein microarray to improve LOD.

Yang et al. Scheme 1. (A) Illustration of Process for Preparing PDMS Matrix Nanofibers; (B,C) Molecular Formula of PDMS Prepolymer and Curing Agent, Respectivelya

2. Experimental Section 2.1. Materials and Reagents. PDMS (Sylgard 184) was commercially available from Dow Corning and consisted of liquid components (a mixture of catalyst Pt and prepolymer dimethylsiloxane with vinyl groups) and curing agent (prepolymer dimethylsiloxane with vinyl groups and Si-H groups). PMMA was from Aldrich with Mw ) 350 000. DMF and THF were from Sinopharm Chemical Reagent. Rabbit IgG and FITC-labeled goat-antirabbit IgG were from Beijing Zhong Shan-Golden Bridge Biological Technology, China. All reagents were used directly without any purification. 2.2. Preparation and Characterization of PDMS/PMMA Solutions. We dissolved a measured amount of PMMA in DMF/THF at 50 °C and obtained a transparent solution. After cooling PMMA solutions to room temperature, we added a calculated amount of PDMS together with the curing agent (10:1) into the solution to achieve a designed PDMS/PMMA weight ratio. Viscosity was measured by a digital viscometer (SNB-1, Shanghai Nirun Intelligent Technology, China). All measurements were conducted at room temperature corresponding to the condition of ES (25 °C). 2.3. Electrospinning. We employed a DC high-voltage generator (Spellman SL150) to produce voltages ranging from 0 to 50 kV. We loaded the solution in a 5 mL syringe with a flat needle. A syringe pump was used to feed the polymer solution. A flat metal needle was connected to the syringe at the bottom of the syringe. The anode of the high-voltage generator was connected to the metal needle, and the cathode was connected to aluminum foil. The aluminum foil also acted as the collector. After the application of high voltage, the solution was charged with positive charges and erupted from the orifice of the metal needle. The ES fibers flew to the cathode and deposited on the collector as a mat, and at the same time, solvent evaporated. All experiments were conducted at 25 °C. 2.4. Scanning Electron Microscopy (SEM). We used SEM (FEI, Quanta 400 FEG) to observe the morphology of the electrospun mats. All samples were sputter-coated with gold prior to observation with SEM (resulting in an Au coating of about 10 nm). 2.5. X-ray Photoelectron Spectroscopy (XPS). We used XPS (AXIS Ultra by Kratos Analytical, U.K.) to determine the surface composition of ES fibers. Monochromatic Al KR X-rays (1486.7 eV) were employed. The X-ray source was 2 mm nominal X-ray spot size operating at 15 kV, 8.9 mA for both survey and high-resolution spectra. Survey spectra, from 0 to 1200 eV binding energy (BE), were recorded at 160 eV pass energy with an energy step of 1.0 eV and a dwell time of 100 ms. High-resolution spectra were recorded at 20 eV pass energy with an energy step of 0.1 eV and a dwell time of 1.2 s, with a typical average of 12 scans. The operating pressure of the spectrometer was typically ∼10-9 mbar. For quantitative XPS measurements, a survey scan was first taken at an angle of 90°, defined as the angle between the collection axis of photoelectron analyzer and sample plane. All data were collected and analyzed using software provided by the manufacturer. 2.6. Fabrication of NFM Chip. First, we placed a piece of glass slide as the collector on the cathode, and the ES mats would deposit on the glass slide; second, we put a special plastic grid (http:// www.capitalbio.com/zh-hans/node/417, also schematically showed in Figure S1 in the Supporting Information) on the ES mats and pressed (about 50 N) the grid for about 5 min when the grid adhered to the uncured PDMS ES mats, and ES mats also adhered to the glass slide; third, we heated the samples in an oven to 70 °C for 2 h, and at this time, PDMS prepolymer reacted with the curing agent and formed 3D network.

a Formed linker is the molecular chains that link the PDMS prepolymers after curing.

2.7. Protein Microarray. The protocol mainly included the following four steps. First, antigens (rabbit IgG, serial diluents) were robotically printed on chip (20 nL/dot) by the use of a microarray spotter (BIODOT AD1500) under room temperature and 60% constant humidity, yielding spots about 300 µm in diameter. Second, 5% casein solution was used as blocking solution to incubate the antigen microarray for 2 h to decrease the nonspecific adsorption, followed by the use of phosphate buffer saline (PBS, pH 7.4) to wash the NFM chip. Third, the chip was incubated with fluorescence-labeled antibody (goat-antirabbit IgG, 5 µg/mL) for 2 h, when antibody would specifically bind to immobilized antigen. Last, after using PBS with Tween 20 (PBST) solution to rinse the chip three times and drying the chip, we used a microarray scanner (CapitalBio LuxScanTM10K) to read the fluorescent signals.

3. Results and Discussion Direct ES of PDMS failed (data not shown) because there were insufficient chain entanglements due to the relatively low molecular weight of the prepolymer (estimated to be a few thousands). The ES of PDMS was realized, however, by first using a macromolecule as the carrier polymer and then curing the blend nanofibers to form a stable 3D network via covalent bonds (Scheme 1A). Compared with previous demonstrations of ES of small molecules (e.g., phospholipids), ES of PDMS shows special advantage: the phospholipid nanofibers are not stable because these nanofibers are stabilized via van der Waals force, but differently, covalent bonds between PDMS prepolymer chains render 3D network and make PDMS nanofibers stable. We found that the choice of carrier polymer and solvent were crucial for the success of ES of PDMS. PMMA (M.W. 350 000) was chosen as the carrier polymer because PMMA could be

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Table 1. XPS Analysis of PMMA and PDMS/PMMA Nanofibrous Membrane atom %a b

PMMA

c

PDMS/PMMA

calculated measured calculated measured

C

O

Si

71.4 73.7 ( 0.5 60.0 55.7 ( 0.4

28.6 26.3 ( 0.2 26.7 28.6 ( 0.3

0 0 13.3 15.7 ( 0.1

a Atom % was based on the survey scan of Si 2p, C 1s, and O 1s. Data averaged from 9 points, ( standard error. b Atom % was calculated using (O2C5) and (SiOC2) as the repeat unit for PMMA and PDMS, respectively. The ratio of PDMS: PMMA was 2:1. c Atom % was calculated using (O2C5) and (SiOC2) as the repeat unit for PMMA and PDMS, respectively. The ratio of PDMS: PMMA was 2:1.

Figure 1. Electrospun PDMS/PMMA nanofibers. (A) Macroscopic image. The unit of the ruler is centimeters. (B) Typical SEM image of PDMS/PMMA nanofibers. (C) XPS of PMMA and PDMS/PMMA electrospun nanofibers confirmed the incorporation of PDMS into the nanofibers. Experimental parameters are: PDMS/PMMA 2:1, PMMA concentration 10%, voltage 26 kV, work distance 13 cm, ES time 5 min.

electrospun into nanofibers easily, and PMMA also had very low autofluorescence.29,32 As for the PDMS/PMMA composite, the solvents must satisfy two prerequisites: compatible with both PDMS and PMMA and suitable for ES. We chose a mixture of N,N-dimethylformamide (DMF) and tetrahydrofuran (THF) as the solvent for ES of PDMS/PMMA. This decision was made by employing solubility parameter δ to evaluate the compatibility between PDMS/PMMA and solvents. THF was a good solvent for PMMA because δ of THF (9.5) was close to that of PMMA (9.2 to 9.4). However, THF alone was not compatible for ES of PMMA because THF is so volatile that the orifice of the needle could be easily blocked by concentrated solutions. DMF (δ 12.1) with high boiling point was often added as cosolvent. THF was also a good solvent for swelling PDMS: THF and DMF ranked 7th and 27th in swelling PDMS, respectively.33 The optimized weight ratio of DMF to THF was found to be 1:1 (data not shown). We first prepared pure PMMA solution with a desired concentration (e.g., 10%, by weight) to yield a transparent solution and then added calculated PDMS prepolymer together with curing agent (10:1) to the solution to achieve a desired PDMS/PMMA weight ratio. Note that the ratio was PDMS/neat PMMA. The fabrication process was demonstrated in Scheme 1. We found that the stability of the ES process was crucial for the macroscopic morphology of ES mats and finally determined the uniformity of the thickness of ES mats. If the process was stable, then the ES mats formed a round area; otherwise, it was irregular. There are two observable indications to prove the stability of the ES process of our current system. First, during ES, the “Taylor cone” at the orifice of the needle was of conical shape and stable, and the ES mats deposited on the collector continuously and uniformly, as observed by the naked eye. Second, the digital camera picture (Figure 1A) shows that ES mats deposited on aluminum foil formed a round area (white part) with a diameter of ∼8 cm. After ES, the PDMS/PMMA ES nanofibers were finally assembled into a uniform and integrated membrane, satisfying the prerequisites for further applications. The detailed SEM image (Figure 1B) shows that

the ES mats comprise smooth and continuous nanofibers and the diameters of nanofibers are uniform. At this step, the ES nanofibers contained PMMA, PDMS prepolymer, curing agent, and residual solvent. Note that the prepolymer has not cured at this time. Finally, we placed ES mats in an oven at 70 °C for 2 h, where covalent cross-linking formed that resulted in stable 3D PDMS networks. Obvious observation proved that PDMS prepolymer indeed cured after the curing process: before curing ES mats, were sticky, and after curing, they were no longer sticky. The morphology (both macro- and microscales) of ES mats did not change after curing (data not shown). To eliminate the residual solvent, we dried the ES mats in a vacuum oven at 40 °C for 5 h. The composition was similar between the feed (i.e., bulk solution) and the surface of the resultant PDMS/PMMA nanofibers, as confirmed by XPS. Compared with pure PMMA nanofibers, survey scan of PDMS/PMMA showed a unique Si 2P peak at 102 eV, which proved that PDMS presented at the surface of composite nanofibers (Figure 1C). The calculated and measured atomic concentration (atom %) agreed well for both PMMA and PDMS/PMMA for C, O, and Si (Table 1). For pure PMMA nanofibers, the atom % was reasonably close to the calculated value (e.g., for C atom, 71 vs 74). For PDMS/PMMA composite nanofibers, the measured atom % values of C, O, and Si also agreed well with the calculated values of composite nanofibers with a PDMS/PMMA ratio of 2:1. By tailoring the solution ingredient, we could control the morphology of ES mats, which was found to be critical for protein microarrays (see below). As showed in Figure 2A-F, when the PDMS/PMMA ratio varied from 0:1 to 4:1 with PDMS content gradually increasing, different morphologies were observed. Pure PMMA yielded uniform and smooth fibers (Figure 2A). After the addition of PDMS to PMMA solution, we still could obtain continuous, uniform, and smooth fibers (Figure 2B-E). It was interesting that junctions or solders existed at the crossing of every two fibers in PDMS/PMMA ES mats (Figure 2B-E), and we reasoned that before curing, the viscous PDMS prepolymer PDMS/PMMA nanofibers tended to stick together, and after curing, junctions formed at the crossing sites. Junctions made the ES mats to form a stable 3D network at the fiber level. With the increase in PDMS content, the diameter of fibers increased (Figure 2G and Table 2), which was attributed to the increment of the viscosity of PDMS/ PMMA solutions (Table 2). When the PDMS/PMMA ratio was over 4:1, ES fibers adhered together and could not keep the shape of individual fibers (Figure 2F). Furthermore, for solutions with a given PDMS/PMMA ratio, we could control the diameter of PDMS/PMMA composite nanofibers by changing the concentration of PMMA solutions (Figure 2H). With the decrease in PMMA concentration, the diameter of ES fibers decreased

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Figure 2. (A-F) Electrospun PDMS/PMMA nanofibers with different PDMS/PMMA ratios: (A) pure PMMA, (B) 1:2, (C) 1:1, (D) 2:1, (E) 3:1, (F) 4:1. A-E have the same scale bar. Experimental parameters: PMMA concentration 10%, work distance 13 cm, voltage 22 kV. (G) Plot of diameter versus PDMS/PMMA ratio measured from A-E. (H) Plot of diameter versus PMMA concentration. Experimental parameters are: PDMS/PMMA ) 2:1, work distance 13 cm, voltage 26 kV. Table 2. Correlation of PDMS/PMMA Ratio, Viscosity of Solutions, and Diameter of ES Fibers PDMS/PMMA

viscosity (mPa · s)

diameter (nm)

0:1 1:2 1:1 2:1 3:1

67.6 81.5 115.2 193.2 342.2

465 ( 32 564 ( 124 632 ( 107 1064 ( 59 1571 ( 436

because of the decrease in the viscosity of the solutions. This trend will increase the specific surface areas; therefore, such adjusting is pursued for protein microarray. We next applied PDMS/PMMA NFM as a substrate for protein microarrays. A practical problem that needs to be solved first for using PDMS/PMMA NFM as the microarray substrate is how to keep the shape of PDMS/PMMA NFM during operation. We developed a standard protocol to solve this problem. First, we placed a piece of glass slide as the collector on the cathode. Therefore, the ES mats were deposited on the glass slide. Second, we put a special rubber cover grid (schematically shown in Figure S1 of the Supporting Information) on the ES mats and pressed (about 50 N) the cover grid for about 5 min. Third, we heated the samples in an oven at 70 °C for 2 h to ensure that the PDMS prepolymer cross-linked and PDMS/PMMA NFM adhered tightly with the glass slide. After taking this integrated slide (abbreviated as PDMS/PMMA NFM chip thereafter) out from the oven, the PDMS/PMMA NFM chip was ready for protein microarray application. The fibrous structure at the middle of PDMS/PMMA NFM was

unaffected (data not shown) because only the peripheral part contacting the grid was pressed. Next, we evaluated the performance of the PDMS/PMMA NFM chip as a microarray substrate by conducting a model immunoassay. The protocol included the following four steps (Figure S1 of the Supporting Information). First, antigens (rabbit IgG, serial diluents) were robotically printed on the PDMS/ PMMA NFM surface (20 nL/dot) by using a microarray spotter (BIODOT AD1500) under room temperature and 60% humidity, yielding spots about 300 µm in diameter. Second, 5% casein solution was used as the blocking solution and incubated for 2 h to decrease the nonspecific adsorption, followed by copious washing with PBS solutions. Third, the chip was incubated with fluorescein-labeled antibody (Goat-antirabbit IgG, 5 µg/mL) for 2 h. After rinsing with PBST, we used a microarray scanner (CapitalBio LuxScanTM10K) to read the fluorescent signals. Representative images of protein microarrays are shown in Figure S2 of the Supporting Information. The fibers in the PDMS/PMMA NFM substrate had a mean diameter of 686 nm. The dots in the same row had the same concentration as that indicated on the left, and eight dilutions were tested. After processing, SEM tests indicated that the morphology of PDMS/ PMMA NFM did not change (data not shown). In Figure S2 of the Supporting Information, we can observe four important performances of PDMS/PMMA NFM chip: (i) the topography of protein microarrays was regular, which meant that PDMS/ PMMA NFM maintained its shape during operation. One would see disordered microarrays if the membrane was deformed; (ii)

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Figure 3. (A) Comparison of microarrays on PDMS/PMMA NFM (686 nm) and NC. (B) Plot of SNR versus antigen concentration on different substrates.

the morphology and intensity of every dot in the same row were almost uniform, which indicated that the PDMS/PMMA NFM chip was a uniform membrane; (iii) with the decrease in concentration, the intensity decreased gradually, which ensured the experiment for finding LOD; (iv) the background or the noise at nonspotting areas was nearly zero, which indicated the low autofluorescence and limited nonspecific adsorption. So we believed that the performance of PDMS/PMMA NFM satisfied the basic requirements for protein microarrays. Diameters of PDMS nanofibers determine the specific surface areas of PDMS/PMMA NFM and finally influence the performance of protein microarrays. As discussed above, we could control the diameter of nanofibers by adjusting the concentration of solutions; that is, 6, 8, and 10% yielded nanofibers with mean diameters of 686, 926, and 1327 nm, respectively. With the decrease in the diameter, the signal-to-noise ratio (SNR) increased (Figure 3B, taking 40 µg/mL antigen as example, SNR is 18, 23, and 42 for 1327, 926, and 686 nm, respectively), which was attributed to the increase in the specific surface areas. We also compared the performance and determined LOD of PDMS/ PMMA NFM chip against a commonly used commercial NC membrane. Microarrays on NC showed high-fluorescence background due to the high autofluorescence of NC membrane (Figure 3A, right image): intensity of fluorescence signals on NC membrane and PDMS/PMMA NFM 686 nm is 4997 and 450, respectively. The intensity of fluorescence and SNR of PDMS/PMMA NFM chip were much higher than those of the NC membrane (Figure 3B, taking 20 µg/mL antigen as example, SNR is 27 and 3 for PDMS/PMMA NFM 686 nm and NC membrane, respectively). LOD refers to the lowest quantity of fluorescence signal that the microarray scanner can distinguish, and the microarray scanner can identify signals with SNR >2, so SNR ) 2 is set as LOD. On NC membrane, LOD was 20 µg/mL, and on PDMS/PMMA NFM, LOD was 0.625 µg/mL, indicating that LOD of PDMS/PMMA NFM was 32 times lower than that of NC membrane. In comparison with featureless PDMS slab, PDMS/PMMA NFM also allowed the detection at

lower concentration due to the relatively large surface areas and 3D structures. Under the same condition (antigen concentration 40 µg/mL), the fluorescence intensity on PDMS/PMMA NFM was about 52 times stronger than that on PDMS membrane (Figure S3 of the Supporting Information). As a control, we also printed protein microarrays on pure PMMA nanofibrous membrane (PMMA NFM), but with no satisfactory results. When spotting proteins on PMMA NFM, we observed that proteins spread out on the PMMA NFM, resulting in irregularly shaped spots with diameter of ∼600 µm (Figure S4 of the Supporting Information, 300 µm for PDMS/ PMMA NFM). We attributed the poor performance of PMMA NFM partially to its loose nature and wettability (Figure S5 of the Supporting Information). It also supported the idea that PDMS was exclusively responsible for the excellent performance of PDMS/PMMA NFM. Therefore, PMMA NFM was not included in the LOD experiments. In these preliminary experiments, LOD of NC was in the range of micrograms per milliliter, which was higher than the reported results, such as picograms per milliliter in Chikoti group.19 This difference was attributed to the different choice of antigen-antibody pair as well as the detection system. Under the same experimental conditions, however, these immunoassay results showed that PDMS/PMMA NFM exhibited superior performance to flat PDMS and commercial NC membrane. Furthermore, current results indicated that minimizing the diameter of nanofibers was effective to lower LOD.

4. Conclusions In conclusion, we reported the first case that fabricated PDMS/PMMA nanofibers via ES, which extended the scope of ES materials from thermoplastic polymers to thermosetting materials. The successful fabrication of PDMS/PMMA nanofibers should ascribe to the presence of carrier polymers in that carrier polymers provide sufficient chain entanglements to make up for the deficiency of chain entanglements in the PDMS

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prepolymer. We believe this protocol will also be applicable for other thermosetting materials such as epoxy. Preliminary results indicated that the PDMS/PMMA NFM has the potential of being a novel solid supporting material for protein microarray. The improved sensitivity will potentially benefit proteomics and clinical diagnostics using protein microarrays such as detection of infectious diseases. Given the simplicity, low cost, and high efficiency of ES technology, we believe that PDMS/PMMA NFM is a promising 3D microarray substrate. Acknowledgment. This work was supported by SINANO startup funds, National Science Foundation of China (20904037, 50773001, 90813032), the Ministry of Science and Technology (2009CB320305, 2009CB930001) and Natural Science Foundation of Jiangsu Province (BK2009141). We thank the Public Center for Characterization and Test at SINANO for the SEM support, and Mr. Yuanzi Wu and Professor Jinglin Xie for the help of XPS. Supporting Information Available. Protocol of immunoassay, microarrays on PDMS/PMMA NFM chip, comparison of immunoassays between PDMS and PDMS/PMMA NFM, microarrays on PMMA NFM, and wettability of protein solutions on PMMA NFM and PDMS/PMMA NFM. This material is available free of charge via the Internet at http://pubs.acs.org.

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