Highly Transparent Cyclic Olefin Copolymer Film with a Nanotextured

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Biological and Medical Applications of Materials and Interfaces

Highly Transparent Cyclic Olefin Copolymer Film with Nano-Textured Surface Prepared by One-Step Photografting for High Density DNA Immobilization Yuan Qi, Yindian Wang, Changwen Zhao, Yuhong Ma, and Wantai Yang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b09662 • Publication Date (Web): 19 Jul 2019 Downloaded from pubs.acs.org on July 21, 2019

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ACS Applied Materials & Interfaces

Highly Transparent Cyclic Olefin Copolymer Film with Nano-Textured Surface Prepared by One-Step Photografting for High Density DNA Immobilization Yuan Qi,a,b Yindian Wang,a,b Changwen Zhao,*,a,b,d Yuhong Ma,e Wantai Yang*,a,b,c,d

aState

Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical

Technology, Beijing, 100029, China bBeijing

Laboratory of Biomedical Materials, Beijing University of Chemical Technology, Beijing,

100029, China cBeijing

Advanced Innovation Centre for Soft Matter Science and Engineering, Beijing University

of Chemical Technology, Beijing, 100029, China dKey

Laboratory of Biomedical Materials of Natural Macromolecules, Ministry of Education

Beijing, Beijing University of Chemical Technology, 100029, China eKey

Laboratory of Carbon Fiber and Functional Polymers Ministry of Education, Beijing

University of Chemical Technology, Beijing, 100029, China

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ABSTRACT: Compared with conventional glass slides and two-dimensional (2D) planar microarrays, polymer-based support materials and three-dimensional (3D) surface structures have attracted increasing attention in the field of biochips due to their good processability in microfabrication and low cost in mass production, as well as their improved sensitivity and specificity for the detection of biomolecules. In the present study, UV-induced emulsion graft polymerization was carried out on a cyclic olefin copolymer (COC) surface to generate 3D nano-textures composed of loosely stacked nanoparticles with a diameter of approximately 50 nm. The introduction of a hierarchical nano-structure on a COC surface only resulted in a 5% decrease in its transparency at a wavelength of 550 nm but significantly increased the surface area, which markedly improved immobilization density and efficiency of an oligonucleotide probe compared with functional group and polymer brush modified substrates. The highest immobilization efficiency of the probes reached 93%, and a limit of detection of 75 pM could be obtained. The hybridization experiment demonstrated that the 3D gene chip exhibited excellent sensitivity for target DNA detection and single-nucleotide polymorphism (SNP) discrimination. This one-step approach to the construction of nano-textured surfaces on COC has promising applications in the fields of biochip and immunoassays. KEYWORDS: surface modification, photografting, hierarchical surface structure, microarray, biochip 1. INTRODUCTION Biochips, which capture a high density of biomolecule microarrays on their surfaces, have been one of the most powerful tools in various bio-related fields, including disease diagnostics, drug screening, genomics, and proteomics, due to their fast, selective, sensitive and high-throughput detection of target molecules.1-5 Compared with the most commonly used glass substrates in biochip technology, many polymer materials also possess satisfactory optical transparency and suitable bulk rigidity in slide form for standard measurement. Moreover, thermoplastic polymers commonly have excellent processability and, thus, are amenable to large-scale


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production and can be micro-fabricated in a cost effective manner for further integration with lab-on-a-chip devices.6-9 Recently, some polymers have been intensively investigated as promising supports for preparation of biomolecule microarrays.10-15 Among them, the cyclic olefin copolymer (COC) has attracted special interest because of its unique features of high transparency, low autofluorescence, good chemical resistance and low water uptake under moist conditions.16-18 However, the surface of COC mainly consists of C-H bonds, which lack reactive sites for further covalent immobilization of biomolecules. Several techniques have been explored to overcome the surface inertness of COC by introducing functional groups on its surface, including plasma treatment,19 UV/ozone oxidation,20,21 adsorption of functional polymers by a hydrophobic interaction,22 plasma enhanced chemical vapour deposition (PECVD),23 UV-photografting24 and catalytic chemical oxidation.25,26 Although these methods can effectively activate the COC surface with functional groups (e.g., COOH, OH, NH2) or a reactive polymer brush, they focus little on improving the sensitivity of the biochips. On a flat substrate, the thickness of functional groups (one dimension) and polymer brush (two dimensions) are in the range of angstroms to tens of nanometers, which provides a limited density of anchor sites for the immobilization of biomolecules. However, low probe-loading capacity always results in poor sensitivity for target molecules. It is necessary to develop suitable surface modification strategies that can endow COC substrates with a higher density of binding sites to capture an adequate amount of probes. Comparing with one- or two-dimensionally modified surfaces, substrates decorated with a three-dimensional (3D) surface texture have enlarged surface area and higher thickness of functional layer, which is more suitable for fabricating microarrays with a high loading capacity.27,28 The surface 3D structure on COCs can be constructed by direct creation of complex topography. Nanoimprint lithography,29 electron-beam lithography30 and plasma treatment31 are the commonly used techniques to alter the surface morphology of polymer materials. However, nanoimprint lithography can regulate the surface texture but is not able to introduce functional groups.32 The



complicated process of electron-beam lithography makes it less than ideal for largescale fabrication. Under appropriate conditions, plasma treatment can etch a large area of the polymer surface and generate nanostructures carrying polar functional groups.33 In contrast, the surface functional groups generated by plasma modification are unstable due to the rearrangement of polymer chains, which results in the surface returning to its pristine unmodified form over a period of days.34,35 Introducing nano/microparticles is an alternative way of fabricating a surface 3D structure on substrates. Various inorganic and organic particles, including silica nanospheres,36 ZnO nanorods37 and polystyrene (PS) microspheres,38,39 have been utilized to prepare high-performance biomolecules microarrays for sensitive biomedical assays. The drawback of these strategies is that most of them need a complex procedure to fix particles on the surface and then functionalize them. Photografting is an effective technique for tailoring the surface properties of polymers due to the advantages of its fast reaction rate, easy operation and utilization of low-cost equipment, moderate modification depth and spatial/temporal controllability.40 This technique commonly focuses on introducing a surface-tethered polymer brush, while paying little attention to fixing nano/microparticles on the surface. Emulsion polymerization is a widely used method for preparing polymer nanoparticles in both the factory and laboratory. In our previous work, we have found that direct conducting UV-initiated emulsion surface graft polymerization could produce a coarse surface fixed with colloid particles.41 Based on this work, a facile strategy to introduce a 3D nano-texture with functional groups on the COC surface was developed. Compared with the methods for fixing nanoparticles on a substrate from suspension, this strategy only needs one step to introduce a 3D texture composed of nanoparticles carrying reactive epoxide groups for the following oligonucleotide conjugation. Moreover, unlike many nanoparticle coatings showing poor transmittance in the visible light range, the 3D nano-structure on COC fabricated by our strategy only slightly compromise the optical transparency of the COC substrate. The surface 3D structure provided a higher specific surface area and greater density of functional groups, which


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markedly improved immobilization density and efficiency of the DNA probe compared with functional group (one dimension, 1D) and polymer brush (two dimension, 2D) modified substrate. It was demonstrated that the 3D DNA chip exhibited excellent performance for target DNA hybridization and discrimination of single-nucleotide polymorphism (SNP). This one-step approach to construct a nano-textured surface on COC has promising applications in the fields of biochip and immunoassays. 2. MATERIALS AND METHODS 2.1. Materials. COC was purchased from Polyplastics (TOPAS 5013L, Japan) and was dissolved in toluene (10 wt%) then casted on a glass culture dish to form a slide with a thickness of 100 µm after evaporation of the toluene. Biaxially oriented polypropylene (BOPP) film with a thickness of 30 µm was used after extraction with acetone for 36 h to remove the additives and impurities, and then dried in vacuum at 25 °C. Methyl methacrylate (MMA, from Alfa-Aesar, Tianjin, China) and glycidyl methacrylate (GMA, from TCI, Shanghai, China) were distilled under reduced pressure before use. Benzophenone (BP), ethylene glycol dimethacrylate (EGDMA) and Tween 20 were purchased from Alfa-Aesar (Tianjin, China). Cetyl trimethylammonium bromide (CTAB), saline sodium citrate (SSC), formamide, acetone and methanol were purchased from Beijing Chemical Regents Company and used as received. Bovine serum albumin (BSA) was obtained from Sigma-Aldrich (Shanghai, China). All of the oligonucleotides were purchased from Takara Bio Inc. (Dalian, China), and the sequences are listed in Table 1. All the probes contained a spacer tail of 15 thymines at the 5′ end to physically separate the sequence from the surface, avoiding steric interferences.42 Table 1. Oligonucleotide sequences of probes and target. Name

Sequence (5´- 3´)

5´end

3´end

Probe A

(T)15 CAT GTT CAT GGT GCT GTC CAC G

NH2

Cy3

Probe B

(T)15 CAT GTT CAT GGT GCT GTC CAC G

NH2

none

Probe C

(T)15CAT GTT CAT GGA GCT GTC CAC G

NH2

none

Probe D

(T)15CAT GTT CAT GAA GCT GTC CAC G

NH2

none

Probe E

(T)15CAT GTT CAT ATT GCT GTA CAC G

NH2

none



Target A

CGT GGA CAG CAC CAT GAA CAT G

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Cy5

none

2.2. Characterization. Contact angle measurements were carried out with an OCA 20 from Dataphysics. X-ray photoelectron spectroscopy (XPS, ESCALAB 250 from Thermo-Fisher Scientific Co., USA) was used to probe elementary composition on the surface with a monochromator. Atomic force microscopy (AFM, CPII from VEECO Co., USA) was used to observe the surface morphology of the substrate. Scanning electron microscopy (SEM) measurements were obtained using a JEOL JSM7500F (Japan Electronics Co., Ltd, Japan). A UV–Vis Spectrophotometer (U3900H, HITACHI, Japan) was used to determine the difference in transmittance before and after photografting emulsion particles. Personal Array 16 (CapitalBio Corporation, China) was used to print oligonucleotides by using the contact dispensing mode. The fluorescent images and intensity of the microarrays was collected by a LuxScan-10K/A (CapitalBio Corporation, China) scanner. 2.3. Emulsion Photografting Polymerization on a COC Surface. First, a predetermined amount of monomer containing MMA, GMA and EGDMA (molar ratio, 15:15:1) was added to an aqueous solution of CTAB (40 wt% concentration) with vigorous stirring at room temperature. The system was further stirred for 30 min and a stable emulsion was formed. For the photografting polymerization, 10 wt% BP solution in acetone was first coated evenly between a COC film (4  4 cm2) and a BOPP film, then the solvent was evaporated. In the second step, 20 μL of the emulsion was injected between the two films and spread evenly to form a sandwich structure. Then, the sandwich structure with the COC on the bottom was placed between two quartz plates and irradiated with UV light (high-pressure mercury lamp, 9000 μW/cm2 at a wavelength of 254 nm) at room temperature for 2 min. After irradiation, the BOPP film was peeled off, and the modified COC film was extracted with acetone to remove the unreacted photoinitiator and ungrafted particles. It was further rinsed with excess deionized water to wash away the absorbed emulsifier followed by washing with acetone. Finally, the COC film thus obtained was dried at room temperature to constant weight.


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2.4. Oligonucleotide Immobilization. The oligonucleotide probe was diluted in 0.5 M Na2CO3 / NaHCO3 buffer (pH = 9.0) containing 0.04 wt% Tween 20 to give a final concentration between 0.25 and 5 μM and was printed on modified COC by using the contact-dispensing mode. After 2 h incubation at 80 °C, the film was scanned using a microarray scanner to determine the fluorescence intensity. The microarray was then blocked by a BSA buffer (10 g · L-1 BSA, 5 × SSC and 0.1% SDS) for 60 min, followed by rinsing with deionized water for 10 min and drying at room temperature. BSA has many amino groups that can react with the unreacted epoxy groups on the surface to avoid non-specific absorption of the target nucleotide.43 The fluorescence intensity of the microarray after blocking was recorded to calculate the immobilization efficiency, which was defined as the ratio of fluorescence intensity before and after blocking. The immobilization density of the oligonucleotide microarray was established from the corresponding calibration curves in Figure S1. 2.5. DNA Hybridization. A certain amount of Cy5-labelled complementary Target A was dissolved in a hybridization buffer (4SSC, 0.1 wt% SDS). The solutions was then directly dropped onto the microarray chip and then covered by a coverslip. The hybridization assembly was placed into a hybridization chamber and incubated at 45 °C for 45 min. After the hybridization reaction, the film was washed 5 times with 1  SSC containing 0.1 wt% SDS, rinsed with deionized water and then dried at room temperature. Finally, the hybridized film was scanned to obtain the fluorescence intensity. The density of hybridization was calculated with the standard curve in Figure S2. 3. RESULTS AND DISCUSSION 3.1. Photografting Functional Nano-Texture onto a COC Film. Generally, the monomer solution used in photografting is a homogeneous solution that is transparent to UV light penetration so as to initiate polymerization. When an emulsion is used, the transmittance of this heterogeneous system to UV light should be seriously considered because it is directly associated with the success of photografting polymerization. In this work, the emulsion was confined between two films (COC and BOPP) to form a



thin layer with a thickness of several micrometres. This reduced the blocking effect of the emulsion on UV transmittance and greatly improved the efficiency of photografting polymerization. The preparation procedure of a functional nano-texture on COC film is shown in Scheme 1. First, a photosensitizer (BP) was coated onto the COC surface, and then the emulsion was added to the surface and covered by a BOPP film to form a thin layer under appropriate pressure. Because BP is hydrophobic, it cannot diffuse into the aqueous phase thus the photoreaction only occurs on the surface of the COC. When the system was irradiated by UV light, BP was excited and could abstract the surface hydrogen of COC, generating surface radicals that initiated the graft polymerization of monomers (MMA and GMA). In the presence of a crosslinker (EGDMA), the grafted polymer chains were further crosslinked to form 3D networks. Combined with the stabilization effect of the emulsifier, surface-tethered nanoparticles were obtained by this unique photoinduced emulsion graft polymerization. As this graft polymerization proceeded, nanoparticles stacked together to form a uniformly covered layer on the COC. Moreover, the PGMA part in the nanoparticle layer contained reactive epoxide groups and could serve as a binding site to immobilize biomolecule probes. Compared with other methods, the emulsion photografting modification could be easily achieved in one step and within several minutes, which has great potential as a platform for the construction of complex surface textures.


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Scheme 1. Schematic illustration of preparation of functional nano-texture on cyclic olefin copolymer substrate.

The surface morphology of modified COC was first characterized by SEM and AFM, as shown in Figure 1. It was observed that the surface of COC became coarse after emulsion photografting polymerization. Successive layers composed of randomly stacked nanoparticles appeared on modified COC, verifying the feasibility of using our strategy to construct a 3D surface nano-texture. The surface topographies of modified COC varied with changing monomer concentration in the emulsion. A loosely stacked nanoparticle layer (Figure 1a, a', b) was obtained when a monomer concentration of 15 wt% was used. However, the graft layer was not continuously connected by adhered nanoparticles and there were still many large defective regions. As the monomer concentration of emulsion increased to 18 wt%, the coverage of the graft layer on COC became uniform (Figure 1c, c', d). The introduced layer was constituted of loosely stacked nanoparticles with a diameter of approximately 50 nm, and the nanoparticles that adhered together showed a shish-kebab structure. It could be observed that there were many spaces between the shish-kebabs of nanoparticles, giving rise to a porous structure which increased the specific surface area of the 3D graft layer. The surface roughness analysis by AFM measurement also confirmed the surface morphology



change. The surface of pristine COC showed a root-mean-square (RMS) roughness of 5.32 nm, indicating a smooth surface. In contrast, the average RMS roughness of the modified COC (18 wt% monomer concentration) increased to 35.67 nm, which was coarse and in good accordance with the SEM observation. A further increase in the monomer concentration to 22 wt% generated a densely stacked nanoparticle layer with some local cracks (Figure 1 e, e', f), and the size of the nanoparticles increased (70 nm). The nanoparticles packed tightly together with little space between them, possibly lowering the surface area of the nano-texture. Overall, all the modified COC exhibited unique hierarchical surface 3D structures constituted by nanoparticles, and it is expected that this increased surface area could provide more attachment sites for the subsequent immobilization of DNA probes.

Figure 1. SEM images of the modified COC prepared with a monomer concentration in emulsion of 15 wt% (a, a´), 18 wt% (c, c´) and 22 wt% (e, e´). Figures (a’, c’, e’) are partially enlarged images of figures (a, c, e), respectively. The corresponding AFM images of surface-modified COC


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at a monomer concentration of 15 wt% (b), 18 wt% (d) and 22 wt% (f).

The surface chemical composition of COC at different stages was analysed by using XPS. As shown in Figure 2a, the curve-fitted C 1s core-level spectrum of pristine COC showed a predominance of C−C/C-H bonds (284.6 eV). An additional small peak at 286.2 eV should be attributed to thermal oxidation of the surface during the processing. After the emulsion graft polymerization, the C 1s envelope could be curvefitted into three components (Figure 2b) at binding energies of 284.6 eV, 286.2 eV and 288.4 eV, attributed to the C-C/C-H, C-O, C=O species derived from polymethacrylate and epoxide structures in the nanoparticle layer.44,45 This finding further confirmed that the hierarchical 3D layer carrying functional groups was introduced on the surface. The epoxide groups on surface-modified COC can react with the amino end-group of the oligonucleotide probe (Table 1), thus it is easy to covalently attach probes to the surface. When comparing Figure 2c to Figure 2d, the typical P 2p signal appeared on the surface-modified COC spotted with oligonucleotides, indicating that the DNA probes had been successfully immobilized. The change in the surface element composition is also consistent with the above results (Table S1).



Figure 2. Core level XPS C 1s spectra of (a) pristine COC and (b) COC modified with a stacked nanoparticle layer. Core level XPS P 2p spectra of COC modified with a stacked nanoparticle layer before (c) and after (d) immobilizing oligonucleotides.

The excellent optical transparency of COC is a crucial property when it is used as a substrate for biochips. Therefore, the transmittance change of COC after constructing a nano-texture on its surface was investigated, and the results are shown in Figure 3. The transmittance of pristine COC was very high in the visible light range (400-700 nm), and it reached 85% at 550 nm. Grafting the nanoparticle layer slightly reduced the transparency of COC, but it only showed a 5% decrease and is still approximately 80% at 550 nm. Increasing the concentration of the monomer in the emulsion from 15 to 22 wt% led to little decrease of transmittance in 550 nm, indicating the grafted nanoparticle layer had negligible adverse effect on the clarity of the COC. This result suggests that our modification strategy for COC is suitable for the fluorescent detection of labelled oligonucleotides.


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Figure 3. UV-vis spectra of original COC and COC modified with a stacked nanoparticle layer with different emulsion concentrations.

3.2. Oligonucleotides Immobilization. To obtain a higher immobilization efficiency of DNA probes on the nano-textured substrate, a hydrophilic surface is required to ensure the effective permeation of the spotted solution into hierarchical structures. The pristine COC has a hydrophobic surface with a water contact angle (WCA) of 99° (Figure S3). After emulsion graft polymerization, the surface hydrophilicity of the COC film was significantly improved, and the WCA decreased to 58°, which is more suitable for probe immobilization. Because the grafted nanoparticle layer contained many functional epoxide groups, oligonucleotides (Probe A, Table 1) with amino groups were immobilized on the surface-modified COC through a nucleophilic ring-opening reaction.46,47 Here, we utilized Cy3 as a fluorescence label of the oligonucleotide probe to evaluate their immobilization properties on substrate. The probe solutions with concentrations of 0.25 μM, 0.5 μM, 1.0 μM, 2.0 μM and 5.0 μM were spotted on the surface-modified COC. As shown in Figure 4, all the spots in the microarrays are of a regular circle shape, and their fluorescence signals decreased as the probe concentration dispensed on the surface decreased. The fluorescence intensity of the microarrays was in the range of 10,000 a.u. to 56,000 a.u. After blocking and washing treatment, a slight decline in the fluorescence intensity was observed, indicating some absorbed oligonucleotides were removed. The immobilization density



of the probes could be calculated by a calibration curve (Figure S1) from the fluorescence signal, which reached 1.8 pmol/cm2 when the concentration of Probe A was 5 μM. Concerning the uniformity of the DNA array (5 μM of Probe A), the spotto-spot coefficient of variation (CV) was calculated with respect to spot size and fluorescence intensity. The low variation in the spot radius (72 µm, CV = 3.21%) and fluorescence intensity (CV = 8.32%) indicated that a uniform microarray could be prepared on the 3D substrate after blocking and washing.

Figure 4. Fluorescence images of the oligonucleotide microarray before (a) and after (a') blocking treatment (probe concentration from top to bottom: 5.0 μM, 2.0 μM, 1.0 μM, 0.5 μM and 0.25 μM). (b) Fluorescence intensity of the oligonucleotide microarray before (b1) and after (b2) blocking treatment, and the immobilized density (b3) of Probe A after blocking treatment.

The immobilization efficiency of the oligonucleotide microarray on the nanotextured COC with different surface morphologies and spotting concentrations was determined from the ratios of fluorescence intensity before and after blocking treatment. The results are shown in Figure 5. It was found that the surface-modified COC prepared with 15 wt% of monomer in emulsion had a relatively low immobilization efficiency (44%-71%) as the spotting concentration of Probe A increased from 0.25 μM to 2 μM. This should be attributed to its loosely stacked nanoparticle layer and large cracks (Figure 1a, a'), which reduced the coverage as well as the availability of functional epoxide groups. The COC modified with a higher concentration of emulsion (18 wt% and 22 wt%) showed improved immobilization efficiency from 64% to 93% as the probe concentration increased from 0.25 μM to 5 μM, which is higher than that of DNA immobilized on a 2D surface6 (30% - 50% for a probe concentration from 2.5 μM to 25


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μM) and 3D substrates.48,49 According to Ref. 48, the DNA immobilized on 3D hydrogel had 87% immobilization efficiency for 44 µM of probe and only 25% for 3 µM of probe. The immobilization efficiency in Ref. 49 for in situ prepared 3D DNA gel drops is only 50% within the DNA concentration range of 0.0013-13.3 mM.

Figure 5. Immobilized efficiency of Probe A on COC modified with a stacked nanoparticle layer with spotting concentrations of Probe A ranging from 0.25 μM to 5 μM.

To further confirm the superiority of our strategy under the same experimental conditions, 1D and 2D substrates were prepared for comparison. As shown in Figure 6, oligonucleotide microarrays could also be fabricated on poly(glycidyl methacrylate) (PGMA) brush-grafted COC (COC-PGMA, 2D substrate) or a (3-glycidoxypropyl) trimethoxysilane-modified glass surface (Glass-epoxy, 1D substrate), although smaller spots were obtained due to the hydrophobic characteristic of PGMA-modified COC (Figure S3c). However, the fluorescence intensity of microarray on the 3D nanotextured surface was 2-4 times higher than that on the 1D and 2D surfaces (Figure 6), indicating a higher density of immobilized probes on the 3D surface. In addition, the immobilization efficiencies of the 1D and 2D surfaces were also lower than those of the 3D surface at the same probe concentration, only in the range of 30% to 70%. The stacked nanoparticle structure on COC provides an increased surface area and higher density of epoxide groups, which significantly increases the capacity of the substrate to capture DNA probes.



Figure 6. Immobilized efficiency of Probe A (a) on the COC-nanoparticle layer surface, (b) on the COC-PGMA brush surface and (c) on the Glass-epoxy. Fluorescence intensity of Probe A on (d) COC-nanoparticle layer surface, (e) on the COC-PGMA brush surface and (f) on the Glass-epoxy with spotting concentrations of Probe A ranging from 0.25 to 5.0 μM.

3.3. DNA Hybridization Assays. Cy3-labelled probes (Probe A) were printed on the modified COC to prepare the DNA microarray. After deactivating the remaining active sites with blocking buffer, the microarray was incubated with Cy5-labelled complementary targets (Target A) at 45 °C for 45 min to facilitate the hybridization. The appearance of Cy5 fluorescence spots in Figure 7a suggested that Target A was selectively attached on the surface-modified COC. The merged fluorescence images of the Cy5 and Cy3 (Figure 7b) channels in Figure 7c shows a yellow colour due to the combination of red and green light, which further demonstrated that the hybridization between immobilized probes and complementary targets was successfully achieved on the COC modified with a stacked nanoparticle layer.

Figure 7. Fluorescence images of microarrays on surface-modified COC with (a) the Cy5 channel,


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(b) the Cy3 channel and (c) merged (a) and (b). The surface-modified COC was prepared with a monomer concentration in emulsion of 18 wt%.

The densities of hybridized Target A at different concentrations (0.125 nM to 500 nM) were determined by using a calibration curve (Figure S2) according to the fluorescence density of hybridized microarrays with different probe concentrations (0.25 μM to 5 μM), and the results are shown in Figure 8. The hybridization density increased with the concentration of the Target A solution up to a concentration of 100 nM. Further increasing the concentration of Target A to 500 nM did not result in a significant increase in their attachment, and the obtained maximum DNA hybridization density was 0.43 pmol/cm2 (5 μM of probe and 500 nM of target). It was noted that the hybridization density increased a small amount when the concentration of the probe increased from 2 μM to 5 μM at high Target A concentrations, which might be caused by the repulsive electrostatic and steric interactions between the probes and targets at high DNA immobilization density.50,51 At low Target A concentrations (0.125 nM) and high probe dispensing concentration (5 μM), the hybridization density could still reach 0.12 pmol/cm2, corresponding to 15000 a.u. of fluorescence intensity (Figure S2) with a signal-to-noise ratio (S/N) (Figure S5(a)) close to 30. As shown in Figure S6, the fluorescence intensity increased exponentially as the concentration of Target A increased to 1 nM. We further investigated the hybridization behaviour of a microarray prepared by a 5 µM probe A at lower Target A concentration and the result is shown in Figure S5(b). It was found that the hybridization fluorescence intensity increased linearly with the concentration of Target A, ranging from 50 pM to 50 nM. The limit of detection (LOD) of Target A was determined when the signal-to-background ratio was equal or greater than 3.52 Accordingly, the LOD of Target A corresponds to a concentration of 75 pM (S/N = 3.2). These results demonstrated that the sensitivity of biochips prepared by our strategy is comparable or even superior to the reported strategies.53 Under the same conditions (5 μM probe and 0.125 nM target), the hybridized density in Ref. 53 (below 0.2 pmol/cm2) is lower than that of our method 0.43 pmol/cm2). On the chip functionalized



with dendrimers, a minimal concentration of oligonucleotides probes of 5 μM is needed for detectable hybridization, while in our work, the microarray prepared from probe A at 1.25 µM still exhibited a wide detection range for Target A.54 The LOD (75 pM) in this work, which is on the same order as those of most fluorescence detection methods,55 is similar to that of the plasma-treated PS (50 pM) with a 3D surface texture56 but is lower than the DNA microarray fabricated on glass slides bearing shrink-wrapped 3D structures (280 pM).57

Figure 8. Hybridization density of microarrays at different concentrations of Target A.

The identification of DNA variation plays a key role in the prevention and diagnosis of various inherited diseases and cancer.58 The sensitivity of the microarrays printed on surface-modified COC was further evaluated by hybridization with different oligonucleotide probes containing mismatched sequences towards Target A. None fluorescence labelled probes with a fully complementary sequence (probe B) or with one, two or three base-mismatched sequences (probes C, D and E, respectively) were immobilized on the 3D COC substrates. The stringent condition that using buffers consisted of lower ionic strength (0.9 × SSC) and a high concentration of formamide is an effective way to improve the discrimination efficiency in SNP detection.59 The hybridization results in Figure S7 show that when the concentration of formamide in the hybridization buffer was 30%, the fluorescence intensity ratio (discrimination ratio) of the full complementary sequence to one-, two- and three-base mismatched sequences


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were 1.5, 1.7 and 7.1, respectively. This means under that condition it is not possible to discriminate the one- and two-base mismatched sequence. However, a satisfactory discrimination ratio of 10.0 for SNP identification could be obtained in a buffer containing 50% formamide, indicating a good capability for SNP detection. 4. CONCLUSION In summary, a facile strategy to introduce 3D architecture onto COC by UVinduced emulsion graft polymerization was developed. By tuning the monomer concentration, a loosely stacked layer of nanoparticles that uniformly covered the COC could be obtained. The introduction of hierarchical surface 3D structure only slightly compromised the optical transparency of the COC substrate. The epoxide groups in the 3D structure and the increased surface area allow the modified COC to be used as an excellent support for oligonucleotide immobilization. The highest immobilization efficiency of a probe was 93%, which is extremely high compared with other substrates with 3D structures. The hybridization experiment suggested that the 3D biochip showed high sensitivity in the hybridization between the probe and its complementary target as well as in SNP detection. This one-step approach to construct a hierarchical 3D texture on the COC surface has promising applications in the fields of biochip and immunoassays. ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website Calibration curve, water contact angle images and other additional details (PDF) AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] (C. Z.) [email protected] (W. Y.) ORCID Changwen Zhao: 0000-0002-9929-1826 Wantai Yang: 0000-0002-7763-4947 Notes



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Highly Transparent Cyclic Olefin Copolymer Film with a Nanotextured

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