Hierarchical-Multiplex DNA Patterns Mediated by Polymer Brush

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Hierarchical-multiplex DNA patterns mediated by polymer brush nanocone arrays which possess the potential application for specific DNA sensing Wendong Liu, Xueyao Liu, Peng Ge, Liping Fang, Siyuan Xiang, Xiaohuan Zhao, Huaizhong Shen, and Bai Yang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 26 Oct 2015 Downloaded from http://pubs.acs.org on October 26, 2015

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ACS Applied Materials & Interfaces

Hierarchical-multiplex DNA patterns mediated by polymer brush nanocone arrays which possess the potential application for specific DNA sensing Wendong Liu, Xueyao Liu, Peng Ge, Liping Fang, Siyuan Xiang, Xiaohuan Zhao, Huaizhong Shen, and Bai Yang* State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun 130012, People’s Republic of China. Fax: +86 431 85193423; Tel: +86 431 85168478; E-mail: [email protected]. KEYWORDS: polymer brush, DNA patterns, nanocone arrays, sensing, hierarchicalmultiplex ABSTRACT: This paper provide a facile and cost-efficient method to prepare single strand DNA nanocone arrays and hierarchical DNA patterns which were mediated by PHEMA brush. The PHEMA brush nanocone arrays with different morphology and period were fabricated via colloidal lithography. The hierarchical structure was prepared through the combination of colloidal lithography and traditional photolithography. The DNA patterns were easily achieved via grafting the amino group modified single strand DNA (ssDNA) onto the side chain of polymer brush and the anchored DNA maintained their reactivity. The as-prepared single strand DNA nanocone arrays can be applied for target DNA sensing with the detection limit reaching 1.65 nM. Besides, with the help of introducing microfluidic ideology, the hierarchical-multiplex DNA patterns on the same substrate could be easily achieved with each kind of pattern possess one kind of ssDNA, which are promising surfaces for the preparation of rapid, visible, and multiplex DNA sensors. 1 ACS Paragon Plus Environment


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1. INTRODUCTION Deoxyribonucleic acid (DNA) is representative polymer exist naturally with the history dating from the origins of life forms, and it attracts the researchers’ attention since it carries genetic information and performs as the foundational matter for biological heredity.

[1, 2]

Meanwhile,

as a interdisciplinary of chemistry, materials science and bioscience, the science of DNA not only has been focused by life sciences, but also become the foundation of current technology research, which is due to its specific double helix structure. The specific structure of DNA chains endows them substantial recognition capabilities for it can exist in various secondary structures which depending on their sequences and environment conditions. And it makes DNA can specifically recognized various kinds of target molecules, such as DNA, RNA, protein, drugs, organic and inorganic molecules, via the interactions including classical Watson-Crick base pairing interactions, π-π stacking, Van der Waals interactions, hydrophobic interactions and other non-covalent interactions. [3, 4, 5] Thus, DNA based sensing plays a very important role in many healthcare related fields, such as clinical diagnostics, gene therapy, and so on. In recent years, many techniques have been exploited to prepare DNA based sensing system, such as integrating DNA chains with electrochemical electrode, [6, 7, 8, 9]

14]

field-effect transistor,

[10, 11, 12]

surface-enhanced Raman Scattering (SERS) substrate,

metal substrate with plasmonic effect,

[15, 16]

photonic crystals,

[17, 18, 19, 20, 21]

[13,

and polymer

substrate. [22, 23, 24] Though many DNA sensing systems possess a low detection limit, it is still a challenging to prepare a visible integrated DNA sensor with simplified approach and less input at the same time, especially for the specific platforms which contain both micro and nanoscale patterns. During these years, DNA patterns are proposed to have great potential in preparing visible sensor and have attracted great interests of researchers in many fields. Series of DNA patterns have been achieved with the help of photolithography, lithography,

[28]

and dip-pen nanolithography.

[29, 30]

[25, 26]

inkjet printing,

[27]

e-beam

Additionally, patterns with the feature

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structure size equals 100 nm have been achieved by nanoimprinting lithography,

[31, 32, 33]

and

functionalized µ-CP (microcontact printing). [34] Nevertheless, these methods are either costly, time-consuming to prepare nanostructures with large area or the feature sizes of the structures are too large. Therefore colloidal lithography is proposed as a easy-operated, cost-efficient, and high throughput way for the preparation of microscale or nanoscale structures. It have several preponderances in contrast with the techniques described retrospect above. Firstly, the colloidal crystals which are used as etching masks can easily be obtained with the diameters finely regulated. Secondly, the structure parameters can be well controlled through regulating the size of the colloidal microspheres and the etching condition. Thirdly, complex 3D nanoscale structures which possess specific symmetry can be obtained as well. Based on these superiorities, microscale or nanoscale structures obtained by colloidal lithography have been broadly applied in biosensing and biomolecule patterning. In the past decade, ordered polymer brush nanocone arrays with the sizes equal to submicrometer and nanometer have been proposed having great potential for the fabrication of sensors since they need a smaller reagent dose and have more reactive sites than the unordered one, leading to an improved sensitivity which need a small dosage of analytes, and modified kinetics.

[35]

Since, few works have aimed at investigating the polymer brush

nanocone arrays to prepare polymer/DNA arrays for specific DNA sequences sensing, it is greatly desired to exploit a facile method to fabricate DNA patterns for biological and technological research. In this paper, we provide a facile and cost-efficient approach to prepare single strand DNA (ssDNA) nanocone arrays and hierarchical DNA patterns which were mediated by PHEMA (poly (2-hydroxyethyl methacrylate)) brushes. PHEMA brush nanocone arrays of various morphologies and periods were obtained by colloidal lithography with the sample area equals 4 cm2 while the hierarchical PHEMA brush patterns were prepared via the combination of colloidal lithography and traditional photolithography. The 3 ACS Paragon Plus Environment


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single strand DNA nanocone arrays with feature morphologies and kinds of hierarchical DNA patterns were easily achieved by grafting the amino group modified single strand DNA onto the side chain of polymer brush. In addition, different with other flat platforms, the specific structure of the nanocone arrays which is quasi-three dimensional, which can effectively enhance the specific-surface-area and raise the grafting efficient of the complementary target DNA to hybrid with the ssDNA nanocone patterns, thus the as-prepared ssDNA nanocone arrays can be used for target DNA sensing with the detection limit reaching 1.65 nM. Besides, with the help of the introducing microfluidic ideology, this work provide a new method to prepare hierarchical-multiplex structures for the sensing application, and the hierarchicalmultiplex DNA patterns are achieved which are regarded as promising surfaces for the fabrication of rapid, visible, and multiplex DNA sensors. 2. EXPERIMENTAL SECTION Materials: Silicon wafers (100) were cut into pieces with the size equal to 2.0 cm × 2.0 cm and boiled in a mixture of H2SO4 and 30% H2O2 (the volumetric mixture ratio equals to 7:3) for 30 min to modify the silicon substrate to be hydrophilic, and then cleaned with deionized water, and dried by nitrogen gas (N2) flow. [36] Phosphate buffer saline (PBS) was provided by Sigma. PMDETA (N, N, N’, N’’, N’’-pentamethyldiethylenetriamine) was provided by TCI. 2-Hydroxyethyl methacrylate (HEMA) monomer, 3-Aminopropyl trimethoxysilane (APTMS), 2-Bromoisobutyryl bromide, DMAP (4-Dimethylaminopyridine), DSC (N, N’-disuccinimidyl carbonate), CuCl (Copper (I) chloride) were purchased from Aldrich. The single strand DNAs used in this work which were modified at the terminal group were provided by Sangon Biotech (Shanghai) Co., Ltd. while the sequences of the single strand DNAs are as following: NH2-Probe DNA A (NH2-5’-CCG TAC AAG CAT GGA ACG GCA AAT GCA ACT-3’), FITC-Target DNA A (FITC-5’-AGT TGC ATT TGC CGT TCC ATG CTT GTA CGG-3’), NH2-Probe DNA B (NH2-5’-TAC TCA GTA GCG ACA CAT GGC CAA CCG TAA-3’), 4 ACS Paragon Plus Environment

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FITC-Target DNA B (FITC-5’-TTA CGG TTG GCC ATG TGT CGC TAC TGA GTA-3’), NH2-Probe DNA C (NH2-5’-TCA AGG CTC AGT TCG AAT GCG CGC CCG GCC-3’), FITC-Target DNA C (FITC-5’-GGC CGG GCG CGC ATT CGA ACT GAG CCT TGA-3’), NH2-Probe DNA D (NH2-5’-AAA TAT TAT TAA ATT ATA TTG CGC GGC CAG-3’), FITC-Target DNA D (FITC-5’-CTG GCC GCG CAA TAT AAT TTA ATA ATA TTT-3’), NH2-Probe DNA HIV (NH2-5’-TGC ATC CAG GTC ATG TTA TTC CAA ATA TCT TCT3’), FITC-Target DNA HIV (5’-AGA AGA TAT TTG GAA TAA CAT GAC CTG GAT GCA-3’)，NH2-Probe DNA H (NH2-5’-GCT ATA CAT TCT TAC TAT TT-3’)，Target DNA H (5’-AAT GGA TTA AAT AAA ATA GTA AGA ATG TAT AGC-3’), Dye DNA H (5’-TAT TTA ATC CAT T-3’-FITC). PDMS (Polydimethylsiloxane elastomer kits (Sylard 184)) was provided by Dow Corning (Midland, MI). The polystyrene microspheres with the diameter of 490 nm, 620 nm, and 1 µm were purchased from Wuhan Sphere Scientific Co., Ltd., and washed by water and 50 % ethanol for several times prior to use. Sodium Hydroxide (NaOH), 40% hydrofluoric acid (HF), nitric acid (HNO3), ammonium fluoride (NH4F), dichloromethane, toluene, triethylamine, absolute ethanol, sodium dodecyl sulfate (SDS), N, N-Dimethylformamide (DMF) and the photoresist were used directly after received; The masks used for photolithography were machined by Institute of Microelectronics of Chinese Academy of Sciences. The TJ-1A-Micro Flow Syringe Pump was purchased from Baoding Longer Precision Pump Co., Ltd. The water used in the experiments of this work was deionized before using. Preparation of polymer brush film via surface-initiated atom transfer radical polymerization (SI-ATRP): In this article, HEMA was chosen to synthesis polymer brush film through the SIATRP approach as demonstrated in our previous work.

[36]

Firstly, silicon substrates were

modified with amino groups via grafting APTMS onto the hydroxylated silicon wafers by chemical vapor deposition method. Then the ATRP initiator (2-bromoisobutyrylbromide) was covalently bonded to the amino group modified surfaces via immersing the modified wafers 5 ACS Paragon Plus Environment


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in a mixture of 10 mL anhydrous dichoromethane, 140 µL triethylamine, and 100 µL 2bromoisobutyrylbromide at 0℃ for 1 h, and then the reaction system were placed in the atomsphere for 15 h. After that the substrates were cleaned by dichoromethane and ethanol, repectively, and dried with N2 flow. As soon as the ATRP initiator grafted wafers achieved, the polymerization was conducted by immersing the wafers into a homogeneous dark blue solution which contains 46 µL PMDETA (0.22 mmol), 7.24 mg (0.0724 mmol) CuCl, and 4 mL monomer aqueous solution (the mixture ratio of HEMA/H2O equals 3:1) and maintained for 8 h under ultrapure N2 atmosphere at room temperature. Then the polymer brush films were achieved after washing the samples by ethanol and DMF. Fabrication of PHEMA brush nanocone arrays and hierarchical patterns: The PHEMA nanocone arrays were fabricated by colloidal lithography method using two dimensional assembled polystyrene (PS) colloidal crystal as a mask during the reactive ion etching approach.

[37]

The size of the PS microspheres we used was 490 nm, 620 nm and 1 µm,

respectively. First, the PS microspheres were self assembled on PHEMA brush film to form hcp (hexagonal-close-packed) two dimensional colloidal crystal by interfacial modified method.

[38]

Then the PHEMA brush nanocone arrays were obtained via etching the PS

microspheres experiencing a ncp (non-close packed state). At the same time, the exposed PHEMA brush film was also etched by the oxygen plasma, the RIE (reactive ion etching) process was conducted on Plasmalab 80 Plus (Oxford Instrument). In this part, the RIE was conducted at the chamber pressure equals 10 mTorr, the flow rate of oxygen gas equals 50 SCCM, and RF power and ICP power reaching 30 W and 100 W, respectively, was excecuted out ranging from 360 s to 420 s with interval of 20 s. After RIE, the maintained PS was dissolved by absolute ethanol and DMF under ultrasonication, and the samples were dried by N2 stream. [36]


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The hierarchical patterns of PHEMA brush were achieved through the combination of colloidal lithography and traditional photolithography. The substrate with PHEMA brush nanocone arrays was covered with a layer of positive photoresist by the spin-coating method. And then the photoresist were patterned by traditional photolithography techniques with the help of the masks that possess one to four patterns. After washing off the photoresist that exposed to UV light and removing the PHEMA brush on the substrate without covered by photoresist with oxygen plasma, the hierarchical patterns of PHEMA brush were achieved by rinsed the substrates with absolute acetone and ethanol to wash off the remained photoresist and dried with N2 stream. Preparation of ssDNA-polymer conjugates: from nanocone arrays to hierarchical-multiplex patterns: The single strand DNA-polymer conjugates were obtained with the help of the hydroxy group on the side chain of PHEMA and the amino group modified on the terminal of single strand DNA’s coupling which was mediated by DSC. Thus, the PHEMA brush patterns were firstly functionalized with DSC as presented in previous work.

[36]

The patterned

polymer brush substrates were soaked in a mixed solution of 0.1 M DSC and 0.1 M DMAP in anhydrous dimethylformamide (DMF) which were deoxygenated for 24 h through filling the system with ultrapure N2 stream. And then the substrates were cleaned with DMF, dichloromethane, absolute ethanol and dried by N2 stream. In order to conjugate one type of single strand DNA (ssDNA) to the polymer brush to prepare ssDNA nanocone arrays and hierarchical patterns, the functionalized samples were immersed in 1.5 mL phosphate buffer saline (PBS, pH=7.4) solution of NH2-Probe DNA A with the concentration of 0.02 µM for 1 h under atomsphere. Afterwards, the samples were cleaned by PBS for to remove the ssDNAs that absorbed on the surface of the substrates physically. For the purpose of confirming the proper incubation time for the target DNA grafting, the samples with ssDNA nanocone arrays were immersed in 1.5 mL PBS solution of FITC-Target DNA A (complementary to NH2-Probe DNA A) with the concentration of 0.02 µM for 0.5h, 1 7 ACS Paragon Plus Environment


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h and 2h, respectively. And then rinsed with PBS solution and treated with ultrasonication. To evaluate the reactivity of the ssDNA anchored on the polymer brush patterns, the wafers with ssDNA nanocone arrays and hierarchical ssDNA patterns were immersed in 1.5 mL PBS solution of FITC-Target DNA A (complementary to NH2-Probe DNA A) with the concentration of 0.02 µM for 1 h, and then the samples were dealed with PBS and ultrasonic to remove the non-specifically absorbed ssDNA. For the conjugation of four types of single strand DNAs to the different polymer brush patterns that integrated on the same sample, which were used to prepare hierarchicalmultiplex ssDNA patterns, an as-prepared PDMS mask (with the cavum depth of nearly 240 µm) which possess four isolated cavum on one side were introduced. The fabrication of PDMS mask was as follows, glass molds are fabricated by glass microlithography. [39] In brief, the layout of the network with the width of the cross inside equals 100 µm was transferred onto the resist-precoated glass plate by UV exposure with a PET mask aligner. After developing, the network was etched into the substrate by bathing in a HF−HNO3−NH4F solution for a predefined time. To prepare the PDMS mask, a prepared glass mold was casted with the PDMS prepolymer; then the casted mold was placed in a drying oven at 60 °C for 3 h to cure the PDMS prepolymer, the PDMS mask achieved after the PDMS was peeled off from the glass mold, and the microcavums were achieved on one side of the PDMS mask while the inlet and outlet of each cavum were fabricated with the help of a puncher.

[40]

After that, the

PDMS mask was compressed on the surface of the hierarchical polymer brush patterns with the cavum side adhered to the sample, while each cavum is corresponded to one kind of pattern with the help of optical microscopy. Then conduits were anchored to the inlet that intercommunicate the cavum with the outside, while the other terminal was integrated with the micro flow syringe pump. Afterwards, NH2-Probe DNA A, NH2-Probe DNA B, NH2Probe DNA C, NH2-Probe DNA D solution with the concentration of 30 µM in PBS were injected into the four cavums respectively. In the injection process, the flow rate was set as 1 8 ACS Paragon Plus Environment

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µL/min which was controlled by the syringe pump. These four kinds of Probe DNA solution were injected sequentially, and the injection time of each cavum went on 1.5 min after the solution filled and drained out from the outlet of each cavum. After the four cavums were all filled with Probe DNA solution, the reaction system was placed under room temperature for 1 h to make the Probe DNA completely react with polymer brushes. Finally, PBS solution was injected into each cavum from the consistent inlet for 5 min to clean out the unreacted probe DNA in each cavum which prevented the cross reaction between cavums, then the PDMS mask was removed from the substrate followed by rinsing the substrate with PBS for several times and dried with N2 stream. To confirm that different probe DNA has been grafted onto corresponding pattern specifically, the multiple ssDNA grafted substrate were reacted with 1.5 mL PBS solution of FITC-Target DNA A, FITC-Target DNA B, FITC-Target DNA C, and FITC-Target DNA D, with the concentration of 0.02 µM in PBS for 1 h sequentially. After each reaction process, the substrate was rinsed with PBS to remove the unreacted dye conjugate target ssDNA, dried with N2 stream, and characterized by fluorescent microscopy. Qualitative experiment for DNA sensing: In this part, the functionalized PHEMA brush nanocone arrays were grafted with NH2-Probe DNA HIV through immersing the PHEMA brush nanocone arrays in 1.5 mL PBS solution of NH2-Probe DNA HIV with the concentration of 0.02 µM for 1 h, and then washed by PBS. After that, the NH2-Probe DNA HIV grafted substrates were immersed in 1.5 mL PBS solution of FITC-Target DNA HIV (tDNA) with the concentration of 1.65 nM, 4.95 nM, 9.90 nM, 16.5 nM, and 49.5 nM for 1 h. Afterwards, the samples were dealed under ultrasonic bath for 30 s, and cleaned with PBS to remove the ssDNAs that absorbed on the surface nonspecifically. As a concept for practical application, we changed the probe DNA into a shorter strand which possess 20 bases (NH2Probe DNA H) complementary with 20 bases on one terminal of the target DNA, and then use this substrate to react with the unmodified target DNA H (33 bases, equal to FITC-Target DNA HIV) with the concentration of 9.90 nM for 1 h, then the hybrid DNA pattern was used 9 ACS Paragon Plus Environment


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to react with a FITC modified single strand reporter DNA (Dye DNA H) which have 13 bases (complementary with another terminal of the target DNA) to make the single strand terminal of the target hybrid with the reporter DNA. After that, the samples were characterized with fluorescent microscopy. Characterization: SEM images were taken under the help of a JEOL FESEM 6700F electron microscope with the primary electron energy equals 3 kV. The substrates were sputtered with a thin layer of Pt prior to taking images. X-ray photoelectron spectroscopy (XPS) was evaluated by ESCALAB 250 spectrometer with a mono X-Ray source Al Ka excitation (1486.6 eV). The atomic force microscopy (AFM) images were recorded in tapping mode with a Nanoscope Ⅲa scanning probe microscope from Digital Instruments under ambient conditions. The optical microscope images and fluorescent microscopy images were taken by the microscopy OLYMPUS BX51. Fluorescence spectroscopy was performed with a Shimadzu RF-5310 PC spectrophotometer. The depth of the PDMS cavum was determined using a Dektak 150 surface profiler (Veeco). The average photoluminescence (PL) intensity of the fluorescent image was calculated by a scientific soft (Image-Pro Plus 6.0). Optical photographs were taken by a digital camera (Canon PowerShot G9, Japan). 3. RESULTS AND DISCUSSIONS 3.1. Preparation and modulation of PHEMA brush nanocone arrays through colloidal lithography. PHEMA brush nanocone arrays are prepared via colloidal lithography techniques. Figure 1 presents the schematic illustration of the basic process. Two dimensional hcp (hexagonalclose-packed) polystyrene colloidal crystals with the periods of 490 nm, 620 nm, and 1 µm were assembled on the surface of the PHEMA brush film grafted from the silicon substrate through the interfacial modified method as mentioned in the experimental section. After that, the two dimensional polystyrene colloidal crystals were used as the mask during the RIE (reactive ion etching) treatment. And the PHEMA brush nanocone arrays were obtained after 10 ACS Paragon Plus Environment

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the exposed PHEMA brushes were etched off along with the hcp two dimensional colloidal crystals changed into a ncp (non-close-packed) one. Figure 2a presents the hcp structure of the PS mask with the period of 620 nm. The area of the well ordered hcp PS microspheres can reach 150×150 µm2. Figure S1 presents the optical microscopy image of the self assembled PS microsphere that lifted up to the substrate with the area equals 4 cm2, which contains some linear and point defects, meanwhile, more than 99% of the area were occupied by PS microspheres, which pledged the quality of the PHEMA brush nanocone arrays obtained later. With the partially etching of PS microsphere, the hcp structure turned into ncp one. The PHEMA brush film underneath the PS microsphere was etched off simultaneously, when the polymer brush film between the spheres was exposed to the oxygen plasma while the PS microspheres turning small. Finally, the PHEMA brush nanocone arrays were obtained after removing the remained PS microspheres. Figure 2b presents the AFM image of the PHEMA brush nanocone arrays with the period of 620 nm, which exhibits hexagonal ncp state and homogeneous arrangements over a large area that can be larger than 225 µm2. Figure 2c presents the 3D AFM image of the polymer brush nanocone arrays which performs cone morphology and maintains the ordering of the mask. It indicates that the polymer brush nanocone arrays have been prepared successfully. Moreover, it provides a suitable approach to prepare micro-building blocks which can be used for the fabrication of complex patterns of polymer. Taking advantage of SI-ATRP, complex patterns that constructed by different functional polymers can be realized. The fabrication process was greatly relied on the RIE conditions. Therefore, we regulated the etching time to find an optimised operation condition to achieve available PHEMA brush nanocone arrays. Figure S2 presents the AFM images and section analysis of the PHEMA brush nanocone arrays with the period of 620 nm that obtained via treating with oxygen plasma for 360s, 380, 400s, and 420s respectively. It is obvious that it experienced different specific states before the isolated PHEMA brush nanocone formed. The diameter of the 11 ACS Paragon Plus Environment


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PHEMA structures reduces while the spaces between the nanostructures become larger with increasing the etching time. For the sample being etched for 360s (Figure S2a), the cone morphology formed, but the substrate are still unexposed to atmosphere for the bottom of the nanocones attaching to each other and the height of the cones is lower than the original polymer film. When the etching time increased to 380 s (Figure S2b), the diameter of the nanocones was further reduced with the silicon substrate exposed to the atmosphere. However, this etching time is still not long enough to form a polymer brush nanocone that performs a completely isolated state, but the height of the nanocones is equal to that of the polymer film, which proves the formation of nanocones. When the etching time reaches 400s (Figure S2c), each structure displays cone morphology with the height equal to the polymer film. And the distances between each other were large enough to show completed morphology. But when the etching time reaches 420s (Figure S2d), the nanocones performs greater degree of separation while the height greatly reduces. Therefore, 400s was chosen as the etching time to prepare PHEMA brush nanocone arrays. Besides, take the advantage of this fabrication method, PHEMA brush nanocone arrays with different periods can be achieved simply via regulating the diameter of the microspheres that used as masks. Figure 3a-c demonstrates the SEM images of polystyrene microspheres with the diameter of 504 nm, 620 nm, and 1 µm, respectively. And all these colloidal crystals perform hexagonal close-packed structures over a large area. Figure 3d-f present the AFM images of the polymer brush nanocone arrays that possess distinct periods, which were consistent with the diameters of PS microspheres showed in Figure 3a-c, and all these PHEMA brush nanocone arrays inherited the ordering of the masks. Hence, it is suitable to fabricate polymer brush nanocone arrays by colloidal lithography, and this nanofabrication technique can be broadening the applicaiton of other functional polymers. 3.2. Fabrication of hierarchical PHEMA brush patterns combined colloidal lithography with photolithography. 12 ACS Paragon Plus Environment

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Hierarchical polymer patterns are important substrates for antifouling surfaces, sensors and bio-adhesion surfaces. In this work, hierarchical PHEMA brush patterns are fabricated by combining colloidal lithography with traditional photolithography. The schematic procedure is shown in Figure 4a. Firstly, a positive photoresist layer was spin coated on the substrate which possesses the as-prepared PHEMA brush nanocone arrays followed by conventional UV light exposure and development, then a photoresist pattern was formed with the PHEMA brush nanocone arrays exposed in the uncovered area. After that, the photoresist pattern was used as the mask during the RIE process for etching off the exposed PHEMA nanocone arrays. Finally, the hierarchical PHEMA brush nanocone arrays were obtained after removing the remained photoresist with organic solvent. The achieved hierarchical PHEMA brush patterns perform homogeneous arrangements over wafer scale while the borderlines between the areas with and without PHEMA brush nanocones are distinct. Figure 4b, c show the optical microscopy images of the hierarchical polymer brush ring and disk patterns fabricated on the silicon substrates, which are uniformly over a large area. The dark areas constructed by PHEMA brush nanocones while the bright areas are the silicon matrix. To address the detailed morphology of the hierarchical polymer brush patterns, we charactrized their morphologies with the help of AFM. The insets in Figure 4b, c present that these microscale ring and disk structures are composed by polymer brush nanocones which keep well ordering. The feature perimeters of the polymer brush nanocone arrays can be well controlled by regulating the colloidal lithography conditions. The patterns of masks for photolithography can be prefabricated arbitrarily, which make it possible to fabricate integrated hierarchical polymer brush patterns on one chip as shown in Figure S3, and hierarchical polymer brush patterns will provide a novel path to prepare complex and multiscale biomarcomolecules patterns for practical applications. 3.3. Monocomponent single strand DNA (ssDNA) patterns.



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The reaction process of the preparation of ssDNA patterns are presented in Figure 5 via covalently grafting ssDNA chains to the PHEMA brush patterns. The substrates with asprepared PHEMA brush patterns were soaked in the mixture of 0.1 M N, N’-disuccinimidyl carbonate (DSC) and 4-Dimethylaminopyridine (DMAP) in anhydrous diemthylformadime (DMF) to functionalize the PHEMA brush with succinimidyl groups. Then the ssDNA patterns are facilely obtained by soaking the functionalized PHEMA patterns in amino modified ssDNA (NH2-Probe DNA A) solution, owing to the post-grafted succinimidyl group’s highly activity to react with the primary amino group covalently.

[36]

Figure 6 shows

the high-resolution XPS spectrum of the ssDNA nanocone arrays. The existence of the characteristic peak of phosphorus indicates that the oligonuecleotide has been successfully grafted onto the polymer brush. The detailed information about the binding energy peak position of each element was presented in Table S1. In order to give an intuitive proof that the ssDNA patterns are achieved and maintained their reactivity, the fluorescein isothiocyanate conjugated Target DNA A (FITC-Target DNA A) which possess the complementary sequences to Probe DNA A was introduced to hybridize with the ssDNA pattern as mentioned in the experimental section. The fluorescence was charactrized by fluorescent microscopy. Samples were illuminated by blue light. Figure S4 presents the fluorescent images obtained after the ssDNA nanocones reacted with FITC-conjugate complementary oligonucleotide for 0.5 h, 1 h and 2 h. Through the comparation of these three images, it performs that the PL intensity is increase with the incubate time increase before it reach an equilibrium time (1 h), after the equilibrium time reached, the PL intensity will not change with the incubation time increase. Thus, 1 h was choose as the incubation time for the hybridization of probe DNA and target DNA. Figure 7 shows the fluorescent images of the hybridized DNA nanocone arrays with the periods of 504 nm (Figure 7a), 620 nm (Figure 7b), and 1 µm (Figure 7c), respectively. These results demonstrate that the arrangement of DNA nanocone arrays are homogeneous in a large area which maintain the ordering of the PHEMA brush nanocone 14 ACS Paragon Plus Environment

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arrays and the fluoresence intensity is almost equal over the whole area that the microscopy took, which certified that the DNA nanocone arrays are well fabricated and the reactivity preserved. With the comparison of Figure 7a, b, and c, we can see that the period of 504 nm is not large enough to reach the resolution of the fluorescent microscopy, and it does not perform detailed patterns which make it not suitable to be used for the fabrication of distinguished patterns. For the period of 620 nm and 1 µm, the sizes are suitable, while the fluorescence intensity of Figure 7c is lower than that of Figure 7b. It is because the ratio of height to the bottom diameter of nanocones is reduced with the increasing of period, which decreases the specific surface area and the hybridization efficiency of the FITC-Target DNA A. As a result, the period of 620 nm was chosen in this work to fabricate PHEMA and DNA nanocone arrays. Besides the characterization methods of above, the atomic force microscopy (AFM) was used to track the height changes after modifying the polymer brush nanocone arrays with succinimidyl group and grafted with ssDNA and double strand DNA (dsDNA). Figure 8a shows the height changes of the nanocone arrays after modifing the polymer chains with succinimide (PHEMA + Succinimide), grafting of the amino-modified single strand probe DNA (PHEMA + ssDNA) and binding the FITC-conjugate complementary oligonucleotide (PHEMA + dsDNA). The height of polymer nanocone arrays used for DNA patterning is about 93 nm. After the modification of succinimidyl group and grafting of ssDNA, it increases to about 100 nm and 132 nm which proves that the polymer chains have been successfully modified and the ssDNA pattern achieved. After the hybridization with FITC conjugate complementary ssDNA, this value greatly reduced to about 111 nm, which is further evidence that the ssDNA have been grafted to polymer chains. Because with the hybridization of Probe ssDNA and complementary Target ssDNA, the length of DNA reduced since it changed from a flexible chain state into rigid double helix state as shown in Figure 8b. This futher proved that the ssDNA have been grafted to polymer chains. Besides that, many hierarchical DNA patterns mediated by polrymer brush patterns can be fabricated. As a 15 ACS Paragon Plus Environment


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proof-of-concept experiment, miocrostripe (Figure 9a), microdisk (Figure 9b), microring (Figure 9c), and integrated microword (Figure 9d) patterns of monocomponent DNA have been fabricated and the DNA maintained their reactivity. Figure 9e shows the enlarged image of the DNA micropatterns. It presents that each unit consists of DNA nanocone arrays with the period of 620 nm which possess several line and point defects, the DNA nanocone can be seen clearly and exhibit hexagonal non-close-packed structure. These nanocone and hierarchical patterns of ssDNA have distinct advantages. Firstly, DNA performs three dimensional distributions on the polymer brush since the hydroxyl groups of the PHEMA brushes are distributed in three dimensional. Secondly, the DNA is robustly anchored on the polymer brush mediated by the covalent bond between the DNA and the polymer brush. Thirdly, the specific molecular recognition capability and site-specific modification property of DNA. At last not least, the specific property of structure changing from ssDNA to dsDNA endowed the ssDNA patterns their own features that few molecules can compare with it. With the benefit above, they have great potentials in mediating cell adhesion, preparing responsive devices and sensors, and so on. 3.4. Qualitative target ssDNA sensing based on ssDNA nanocone arrays. After the monocomponent DNA patterns were prepared, we compared the efficiency of the complementary target ssDNA grafting to the ssDNA nanocone arrays and the flat PHEMA brush film anchored with single strand DNA that prepared via the same coupling reaction. Figure S5 presents the atom percentage of the elements (N, O, P, C) after grafting the FITCTarget DNA onto the ssDNA naoncone arrays and ssDNA anchored polymer brush film, the detailed values were shown in Table S2 which were characterized by high-resolution XPS. It clearly demonstrates that the carbon element content comes from DNA on the nanocone arrays is about 2 to 3 times for that on the flat polymer brush film anchored with ssDNA, which means that the ssDNA nanocone arrays are more beneficial for the target DNA to hybridize than the flat film. The reason is that once a DNA hybridized with the probe DNA 16 ACS Paragon Plus Environment

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anchored on the polymer brush film and the rigid double helix structure formed, it increases the space occupancy compared with the flexible single strand DNA, reducing the opportunity for the target DNA to hybridize with the neighboring probe DNA and the grafting efficiency of target DNA. But for the nanocone one, the probe DNA grafting on the polymer brush are distributed on a curved surface which decreased the steric hindrance for the target DNA to hybridize with the neighboring probe DNA after the rigid double helix formed. Thus it improved the grafting efficiency of the target DNA. Besides the reason mentioned above, the curvature due to probes grafted onto the nanocones may be another important reason, because the interaction between chains can greatly influence the grafting of next step, thus when the length of the single strand probe DNA is not so long, the probe DNA may be perform a straight state during the hybridization process which is greatly helpful for the target DNA grafting.These results also provide us a positive basis to make an attempt on target DNA sensing. For the qualitative sensing experiment, a FITC conjugated partial sequence of the HIV-1 gag gene, 5’-AGA AGA TAT TTG GAA TAA CAT GAC CTG GAT GCA-3’,

[41]

we named

FITC-Target DNA HIV was chosen as the target, while a thirty three bases ssDNA with the amino group modified at the 5’ terminal named NH2-Probe DNA HIV with the sequence of NH2-5’-TGC ATC CAG GTC ATG TTA TT CCA ATA TCT TCT-3’) which complementary with the target DNA was grafted to the PHEMA brush nanocone arrays to capture the target DNA. The detail process of this part is mentioned in the experimental section. In detail, the functionalized PHEMA brush nanocone arrays were grafted with NH2-Probe DNA HIV through immersing the PHEMA brush nanocone arrays in solution of NH2-Probe DNA HIV with the same concentration to prepare the DNA nanocone arrays with the same features. Afterwards, the Probe DNA HIV nanocone arrays reacted with the FITC-Target DNA HIV solution with the concentration of 1.65 nM, 4.95 nM, 9.90 nM, 16.5 nM, and 49.5 nM for 1 h to capture the single strand target DNA onto the probe DNA patterns. The samples were 17 ACS Paragon Plus Environment


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characterized with fluorescent microscopy. Figure 10 shows the fluorescent images of the samples after the probe DNA naonocone arrays reacted with different concentration of FITCTarget DNA (a, f: C(tDNA) = 1.65 nM; b, g: C(tDNA) =4.95 nM; c, h: C(tDNA) =9.90 nM; d, i: C(tDNA) =16.5 nM; e, j: C(tDNA) =49.5 nM). The integration time of taking the fluorescent images (a-d, f-i ) is 3 s while the for the images (e, j) is 0.8 s. Figure 10a-e demonstrate that with the concentration of target DNA increasing, the fluorescent naoncone arrays gradually revealed and the fluorescent intensity turned strong. From the enlarged fluorescent images Figure 10f-j, we can get the information that the quantity of the target DNA hybridized onto each nanocone gradually increased with the increasing of target DNA concentration until it reach a threshold value. Meanwhile, the central fluorescence of each nanocone becomes brighter with the boundary between each nanocones become clear. When the concentration of target DNA exceeded the threshold value, the nanocone were fully covered with target DNA, the fluorescence were so strong that it makes the whole naonocone demonstrate homogeneous fluorescent, and the boundary presents an obscure view. Figure S6 shows the fluorescence images of the Probe DNA HIV grafted substrate (Figure S6a) and after the Probe DNA HIV grafted substrate reacted with FITC-Target DNA A which is uncomplementary to the probe (Figure S6b). This images presents none ordering fluorescence patterns except some fluorescence spots which is caused by the nonspecific absorption of the uncomplementary DNA strands. And it demonstrate the selectivity of this platform since the organization of the target is based on the base paring rule, and this ssDNA patterns can provide a low noise for the fluorescent images. After we got the fluorescent images, the scientific soft named ImagePro Plus was used to calculate the average PL intensity of each image achieved from a specific concentration of target DNA. The relationship between the average PL intensity and the concentration of the target DNA was shown in Figure S7a, it present the information that the PL intensity increase with the concentration raising when the fluorescent images were took under the same conditions, and the PL intensity were similar when the fluoresence of 18 ACS Paragon Plus Environment

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image is looks the same. Besides this calculation by soft, we also measured the fluorescence spectra of the FITC conjugate target DNA solution with different concentrations before and after the reaction with the probe DNA patterns (Figure S7b), through calculating the difference value of the maximum PL intensity of each concentration before and after reaction, the relationship between the PL intensity and the concentration of target DNA is also given indirectly (Figure S7c), which is consistent with the fluorescent images obtained. In addition, as concept for the practical applications, Figure S8 shows the fluorescent images achieved from the experiment as described in the qualitative experiment part. The fluorescence of these two images are similar with the improved experiment result little dark than the other. These result demonstrate that the detection ability doesn’t reduce too much, thus our platform can be applied in practical use, since our motivation is fabricate a sensing platform for the predetection of some specific markers, which is service for the accurately detection of Medizin. These results prove that this kind of ssDNA patterns possess the potential to be used for sensing. It is worth to mention that when the concentration of target DNA is as low as 1.65 nM, just few fluorescent nanocone appear, showing that the target DNA have been successfully captured on the probe DNA nanocone, which means that the detection limit of DNA sensing using this method can reach 1.65 nM visually. Though this detection limit cannot reach the value of the other works reported which are based on the methods of HyperRayleigh Scattering,

[41]

electrochemistry,

[42, 43]

surface plasmon resonance.

[44, 45]

The

fabrication process presented here provide a facile and time-efficient method to prepare single strand DNA nanocone arrays, this special DNA nanocone arrays have the potential to provide a platform for quick semi-quantitative detection of some specific marker of underlying disease before the accurate inspection needed. 3.5. Hierarchical-multiplex ssDNAs patterns which available for rapid, trace, and visible DNA sensor.



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As mentioned above, the DNA nanocone arrays possess the potential for the application of multiple DNA sensing, then it demands us to integrate multiplex ssDNA on the same chip to achieve its practical application. Therefore, we compressed a PDMS mask (with the cavum depth equal 240 µm) on the integrated hierarchical PHEMA brush patterns to realize the fabrication of hierarchical-multiplex ssDNAs patterns that are available for rapid, trace DNA sensor as mentioned in the experimental section. Figure S9 shows the basic process of preparing the PDMS mask which possesses four microcavums while each cavum has an inlet and an outlet. After that, the PDMS mask was compressed on the surface of the functionalized hierarchical polymer brush patterns with the cavum side adhere to the sample as illustrated in Figure S10, while each cavum is corresponding to one kind of pattern with the help of optical microscopy. Then, NH2-Probe DNA A, NH2-Probe DNA B, NH2-Probe DNA C, NH2-Probe DNA D solution was injected into the four cavums with help of a micro flow syringe pump, respectively. After the four cavums were all filled with Probe DNA solution, the reaction system was placed under room temperature for 1 h to make the Probe DNA completely react with polymer brushes, and the hierarchical-multiplex ssDNA patterns were achieved after removing the PDMS mask and rinsed the sample with PBS. To confirm different probe DNA has been grafted onto corresponding pattern specifically, the multiple ssDNA grafted substrate reacted with 1.5 mL PBS solution of FITC-Target DNA A, FITC-Target DNA B, FITC-Target DNA C, and FITC-Target DNA D, with the concentration of 0.02 µM in PBS for 1 h sequentially, and after each reaction process, the substrate was rinsed with PBS to remove the unreacted dye conjugate target ssDNA, and dried with N2 stream, and characterized. Figure 11a presents the optical photograph of the integrated hierarchical polymer brush patterns on silicon substrate that we used, the sample performs vivid structural color which is nearly the same as the two dimensional colloidal crystal of polystyrene which proves that the polymer brush nanocone arrays that construct the microscale patterns finely maintained the ordering of the RIE mask. Figure 11b shows the optical photograph of the 20 ACS Paragon Plus Environment

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integrated PDMS mask on the patterned substrate, which separated the substrate into four parts. Figure 11c shows the optical microscopy image of this integrated system which present that each part separated by the PDMS mask possesses one kind of pattern. To evaluate the practical utility of this method to prepare multiplex ssDNA patterns that without cross interaction between different parts before using the ssDNA solution to fill cavums. This system was tested via filling the cavums with PBS solution sequently. After one cavum is filled with PBS solution, maintain that state for 30 min before filling the next cavum to see whether the PDMS can successfully prevent the liquid flow into the neighboring cavum. Figure S11a presents the optical photograph of the system we used which contains the syringe pump. Figure S11b-f show the optical photograph of the assembled system before (Figure S11b) and after the injected the PBS solution into different cavums sequentially (Figure S11cf). These photographs demonstrate that after compressed the PDMS mask onto the patterned substrate, the cross-shaped PDMS that separate the mask into four cavums has successfully contact with the substrate which sealed the space for liquid flow through. Thus, after the PBS solution filled each cavum, the color changed and then with the liquid wetting the polymer brush structures, the structural color cannot be seen, and even stay for 30 min after the cavum filled with PBS solution, the neighboring cavum showed no changes and the substrate was not wet. These results prove that this home-made system can be used for the preparation of hierarchical-multiplex ssDNA patterns. Based on these results, the multiplex DNA grafting was proceeded and characterized after each cycle as mentioned above. Figure 11d shows the fluorescent image of the hierarchical-multiplex DNA patterns after each section were bonded with specific FITC-conjugate complementary oligonucleotides while the regions covered by the cross-shaped PDMS as shown in Figure 11c were no fluorescent patterns appeared. It proves that a simple hierarchical-multiplex DNA sensor is obtained since each pattern was constructed by ssDNA nanocone arrays. The words “STATE”, “KEY”, “LAB”, and “JLU” were grafted with single strand DNA : Probe DNA A, Probe DNA B, Probe DNA C, and 21 ACS Paragon Plus Environment


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Probe DNA D, respectively, and each pattern was displayed after the coupling with the corresponding complementary single strand DNA modified with FITC. Figure S12 shows the fluorescent images achieved after the hierarchical-multiplex DNA patterns react with different FITC-conjugate complementary DNA stepwise. The fluorescent patterns of different parts appeared step by step, while the center of the sample forms a cross-shaped dark region which proved that there was no probe DNA grafted. In each image, there were no patterns formed in the regions without reacting with corresponding ssDNA except some fluorescent spots which might be caused by the nonspecifically absorbed FITC conjugated target DNA. These results indicate that the integrated system presented in our work possesses the ability to prepare hierarchical-multiplex single strand DNA patterns and these patterns can be used as rapid, trace, and visible DNA sensor for it maintained the properties of the ssDNA nanocone arrays. 4. CONCLUSION In conclusion, a facile and cost-efficient method to prepare single strand DNA nanocone arrays and hierarchical DNA patterns which used PHEMA brush arrays as medium is presented. The polymer brush nanocone arrays with various morpholoies and periods were prepared via colloidal lithography. The hierarchical one was prepared through the combination of colloidal lithography and traditional photolithography. The DNA nanocone arrays with feature morphologies and kinds of hierarchical patterns were easily achieved via grafting the amino group modified single strand DNA onto the side chain of polymer brush. Meanwhile the detailed process was tracked with the help of AFM. We also presented a universal method to graft the DNA chains onto substrate via covalent bonding. In addition, the as-prepared ssDNA nanocone arrays can be used for target DNA sensing with the detection limit of 1.65 nM. Besides, with the help of the introducing of microfluidic ideology, the hierarchical-multiplex DNA patterns and other macromolecule patterns, which are promising surfaces for the fabrication of rapid, visible, and multiplex DNA sensors can be easily achieved. Moreover, taking advantage of the easily fabricated and modulated structures, 22 ACS Paragon Plus Environment

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the facile functionality of the polymer brush and the covalently grafting of DNA chains which maintained the specificity of the structural changes, this DNA pattern has potential applications in the fields of sensing, biology interfaces, responsive interfaces, genetic screening and recombination.



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Figure 1. Schematic illustration of fabricating PHEMA brush nanoarrays via colloidal lithography.


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Figure 2. (a) SEM image of the close-packed polystyrene microsphere with the diameter of 620 nm which were used as the RIE mask; (b) AFM image of PHEMA nanocone arrays of 620 nm period over large area and (c) 3D AFM images of PHEMA nanocone arrays, Z scale is 200 nm, size is 15 µm×15 µm.



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Figure 3. (a-c) SEM images of the close-packed polystyrene microspheres with the diameter of 504 nm (a), 620 nm (b), and 1 µm (c), respectively; (d-f) AFM images of the PHEMA nanocone arrays with the period of 504 nm (d), 620 nm (e), and 1 µm (f) which were consistent with the diameter of the microspheres used as mask, Z scale is 200 nm, sizes are 10 µm × 10 µm.


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Figure 4. (a) Schematic procedures of fabricating the hierarchical polymer brush patterns combined colloidal lithography and photolithography. (b-c) Optical microscopy images of the hierarchical polymer brush disk (b) and ring (c) patterns, the insets demonstrate the AFM images of the PHEMA brush nanocone arrays which constituted the patterns of microscale.



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Figure 5. Basic process of grafting biomolecules onto polymer chains.


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Figure 6. (a) High-resolution XPS spectrum of the DNA nanocone arrays achieved through grafting the amino-modified oligonucleotide onto the polymer nanocone arrays; (b) Highresolution P2p XPS spectrum of the DNA nanocone arrays.



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Figure 7. Fluorescent images of the DNA nanocone arrays with the period of 504 nm (a), 620 nm (b), and 1 µm (c) after binding the FITC-conjugate complementary oligonucleotide.


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Figure 8. (a) Height changes of the nanocone arrays after modified the polymer chains with succinimide, amino-modified single strand probe DNA and FITC-conjugate complementary oligonucleotide; (b) Simulation of the DNA chains structure transition.



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Figure 9. (a-d) Fluorescent images of the different hierarchical DNA patterns after binding FITC-conjugate complementary oligonucleotide; (e) The enlarged fluorescent photograph of the DNA pattern in image d.


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Figure 10. Fluorescent and enlarged images of the samples after the Probe-DNA nanocone arrays react with different concentration of Target-DNA (a, f: C(tDNA)= 1.65 nM; b, g: C(tDNA)= 4.95 nM; c, h: C(tDNA)= 9.90 nM; d, i: C(tDNA)= 16.5 nM; e, j: C(tDNA)= 49.5 nM), the integration time of taking the fluorescent images (a-d, f-i ) are 3 s while the for the images (e, j) are 0.8 s.



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Figure 11. Optical photograph of the hierarchical polymer brush pattern on silicon substrate (a), the assembled reaction system (b), and the optical microscopy image after the PDMS channel covered onto the silicon substrate which possess hierarchical polymer brush patterns (c); (d) Fluorescent image of the hierarchical-multiplex DNA patterns after each section were bond with specific FITC-conjugate complementary oligonucleotides.


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ASSOCAITED CONTENT Supporting Information. The binding energy peak position and atom percentage of each element (N, O, P, C). Optical microscopy, AFM images of self assembled 2D PS microspheres, operating system, PHEMA nanocone arrays, integrated hierarchical PHEMA brush patterns. Fabrication of PDMS mask. Fluorescent image of negative control, time regulation and the steply formation of hierarchical-multiplex DNA patterns. Relationship between PL intensity and concentration. Fluorescence spectra. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author The author to whom correspondence should be addressed. E-mail: [email protected], Fax: +86 431 85193423. Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS This work was financially supported by the National Science Foundation of China (NSFC) under Grand Nos. 91123031, 21221063, 51373065, and the National Basic Research Program of China (973 program) under Grant No. 2012CB933800. REFERENCES (1) Wu L.; Xiong E.; Zhang X.; Zhang X.; Chen J. Nanomaterials as Signal Amplification Elements in DNA-Based Electrochemical Sensing. Nano Today 2014, 9, 197-211. (2) Tjong V.; Tang L.; Zauscher S.; Chilkoti A. ‘‘Smart’’ DNA Interfaces. Chem. Soc. Rev. 2014, 43, 1612-1626. 35 ACS Paragon Plus Environment


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Hierarchical-multiplex DNA patterns with each microscale unit construted by nanocone arrays.


Hierarchical-Multiplex DNA Patterns Mediated by Polymer Brush

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