Development of Sphere-Polymer Brush Hierarchical Nanostructure

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Development of sphere-polymer brush hierarchically nanostructure substrates for fabricating microarrays with high performance Xia Liu, Rongrong Tian, Dianjun Liu, and Zhenxin Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b09505 • Publication Date (Web): 09 Oct 2017 Downloaded from http://pubs.acs.org on October 13, 2017

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ACS Applied Materials & Interfaces

4

Development of Sphere-Polymer Brush Hierarchically Nanostructure Substrates for Fabricating Microarrays with High Performance

5

Xia Liua, Rongrong Tiana,b, Dianjun Liua and Zhenxin Wanga,*

1 2 3

6

a

State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of

7

Applied Chemistry, Chinese Academy of Sciences, 5625 Renmin Street, Changchun

8

130022, P. R. China,

9

b

University of Chinese Academy of Sciences, Beijing 100049, P. R. China.

10

11

12

ABSTRACT: In this work, a sphere-polymer brush hierarchically nanostructure

13

modified glass slide has been developed for fabricating high-performance microarray.

14

The substrate consists of a uniform 160 nm silica particle self-assembled monolayer

15

on a glass slide with post-coated poly(glycidyl methacrylate) (PGMA) brush layer

16

(termed as PGMA@3D(160) substrate), which can provide three-dimensional (3D)

1

ACS Paragon Plus Environment


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polymer brushes containing abundant epoxy groups for directly immobilizing various

18

biomolecules. As a typical example, the interactions of three monosaccharides (Gal-β,

19

Glc-β and Man-α) with two lectins (biotin-RCA 120 and biotin-Con A) have been

20

assessed by PGMA@3D(160) substrate-based carbohydrate microarrays. The

21

carbohydrate microarrays show good selectivity, strong multivalent interaction and

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low limit of detection (LOD) in the picomolar range without any signal amplification.

23

Furthermore, the proposed sphere-polymer brush hierarchically nanostructure

24

substrates can easily be extended to fabricate other types of microarrays for DNA and

25

protein detection. PGMA@3D(160) substrate-based microarrays exhibit higher

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reaction efficiencies and lower LODs (by at least one order of magnitude) in

27

comparison with two-dimensional (2D) microarrays which are fabricated on the

28

planar epoxy substrates, making it a promising platform for bioanalytical and

29

biomedical applications.

30

KEYWORDS:

31

carbohydrate-protein binding, carbohydrate microarray, glycoprotein microarray,

32

antibody microarray, DNA microarray.

33

INTRODUCTION

Silica

particle,

poly(glycidyl

methacrylate)

brush,

34

Microarray, as a powerful high-throughput screening tool, can be used to rapidly

35

analyze biological information including genomic, proteomic and glycomic studies,

36

and plays an important role in the fundamental research and development of new 2


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diagnostic and therapeutic methods.1-6 Among these microarray applications,

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carbohydrate microarrays have been widely employed to study carbohydrate-protein

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interactions and received much attention in the field of biological research in recent

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years.7-11 In carbohydrate microarray-based assays, a large number of different

41

carbohydrates with a spatially arrayed and high-density format are immobilized on the

42

surface of support platform and analyzed simultaneously, which greatly enhance our

43

understanding of the interactions of glycans with other biomolecules. However, the

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binding affinity between single carbohydrate molecule and protein on a planar surface

45

is relative low, which limits sensitivity, dynamic range and selectivity of carbohydrate

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microarray-based analysis methods.12-13 Inspired by the multivalent interactions on

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cell surface, the limitation can be overcome through changing the presentation form

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of ligands on the surface.14-19 For instance, the analytical performance of

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microarray-based assays can be improved by density and orientation of immobilized

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carbohydrates, which are directly defined by surface properties of microarray

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substrate.12-13, 16, 20 Therefore, a specifically designed substrate plays a fundamental

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role in the high-throughput microarray-based assay for studying carbohydrate-protein

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interactions.

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Comparing with small molecule functionalized two-dimensional (2D) substrates,

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the three-dimensional (3D) substrates provide higher probe loading capacity and

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larger spacing for adjusting biomolecular distribution.21-23 Up to now, various 3D 3



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substrates have been applied for fabricating carbohydrate microarrays. Rubina’s group

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developed a 3D hydrogel glycan microarray through immobilizing carbohydrates

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inside a porous polymer gel.24 Yan and co-workers proposed a new microarray by

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conjugating carbohydrate ligands on the surface of silica nanoparticles.15 These works

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efficiently improve molecular loading capability of substrates through increasing

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surface areas of substrate materials. Polymer brushes containing carbohydrate

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residues (or DNA) exhibit good binding specificity since polymer brushes, as flexible

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polymer chains, can efficiently decrease steric hindrance between substrate surface

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and

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nanostructured substrate has been employed as platform for highly sensitive detection

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of cancer biomarkers in human serum for the first time.28 Through combining 3D

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surface and densely packed polymer brushes, the substrate can further increase the

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loading amount of biomolecules and provide good accessibility to analytes. In

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comparison with the interactions of antibodies with antigens, more efforts should be

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made to strengthen carbohydrate-protein interactions though adjusting architectural

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presentation of carbohydrates on the surface of the substrate. However, few examples

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of hierarchical 3D substrate-based carbohydrate microarrays have been reported.

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target

molecules.25-27

A

ZnO

nanorod-polymer

brush

hierarchically

Herein, we reported a sphere-polymer brush hierarchical nanostructure (termed as,

75

PGMA@3D(160))

substrate-based

carbohydrate

microarray

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carbohydrate-protein interactions. A slide with uniform silica particle assembled 4


for

studying

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monolayer is used as a 3D backbone to grow brush-like polymer chains. Combining

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globular structure and large contact surface area of silica particle, densely grafted, 3D

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polymer brushes can be obtained, which can efficiently increase loading amount of

80

immobilized biomolecules and reduce steric effect during biomolecular recognition

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reactions. In the proof-of-concept experiments, we demonstrate that binding affinity

82

between carbohydrate with lectin can be greatly enhanced on the PGMA@3D(160)

83

substrate and the PGMA@3D(160) substrate-based carbohydrate microarray is

84

possible to evaluate the dynamic parameters of the interactions (e.g., KD,surf).

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EXPERIMENTAL SECTION

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Materials and reagents. Tetraethyl orthosilicate (TEOS, 99.999%), glycidyl

87

methacrylate (GMA, ≥97%), 2-bromoisobutyryl bromide (BIB), copper (I) bromide

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(CuBr, 98%), triethylamine (TEA, >99.0%), amino-modified monosaccharides

89

(4-aminophenyl

90

β-D-galactopyranoside (Gal-β) and 4-aminophenyl β-D-glucopyranoside (Glc-β)) and

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methoxypolyethylene glycolamine (PEG-NH2) (MW of 750 Da) were purchased from

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Sigma-Aldrich Co. (St Louis, USA). (3-Aminopropyl)triethoxysilane (APTES, 98%)

93

was purchased from Aladdin Co., Ltd. (Shanghai, China). 2, 2’-Bipyridine (Bipy,

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>99%) was purchased from Alfa Aesar (Ward Hill, USA). Human IgG, goat

95

anti-human IgG/Cy5 (anti-IgG-Cy5), and Cy5 labeled streptavidin (streptavidin-Cy5)

96

were obtained from Biosynthesis Biotechnology Co., Ltd. (Beijing, China).

α-D-mannopyranoside

(Man-α),

5


4-aminophenyl


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Asialofetuin (Asf) was purchased from GALAB Technologies GmbH, (Geesthacht,

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Germany). Biotinylated ricinus communis agglutinin 120 (biotin-RCA 120) and

99

biotinylated concanavalin A from Canavalia ensiformis (biotin-Con A) were obtained

100

from Vector Laboratory Ltd. (Burlingame, CA). Synthetic DNA oligomers (as shown

101

in Table S1 for details) were purchased from Sangon Ltd. (Shanghai, China).

102

Ammonium hydroxide (NH3·H2O, American Chemical Society reagent grade,

103

28.0-30.0% NH3 by weight), 1-butanol, H2O2 (34.5% by weight) were obtained from

104

Beijing Chemical Reagents Company (Beijing, China). The plannar glass slides,

105

epoxy-modified glass microscope slides (named as 2D epoxy substrates) and

106

polytetrafluoroethylene (PTFE) grids were obtained from CapitalBio Ltd. (Beijing,

107

China). Other chemicals were analytical grade and obtained from Sinopharm

108

Chemical Reagent Co., Ltd. (Shanghai, China). Milli-Q water (18.2 MΩ·cm) was

109

used in all experiments.

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Characterization. The scanning electron microscope (SEM) micrographs were

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observed by a XL30 ESEM FEG system (FEI, USA) at an accelerating voltage of 10

112

kV. The transmission electron microscopy (TEM) micrographs were performed on a

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JEM 2000FX (JEOL Ltd, Japan) microscope operated at an accelerating voltage of

114

120 kV. X-ray photoelectron spectra (XPS) were recorded with a VG ESCALAB

115

MKII spectrometer (VG Scientific Ltd., UK). Dynamic light scattering (DLS)

116

measurements were carried out on Malvern Zetasizer Nano ZS (Malvern Instruments 6


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Ltd, UK). Thermogravimetric analysis (TGA) measurements were performed on a

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Perkin-Elmer TGA-2 thermogravimetric analyzer under nitrogen from room

119

temperature to 900 °C at a rate of 10 °C min−1. Fourier transform infrared (FTIR)

120

spectroscopic analysis was carried out on a Bruker Vertex 70 spectrometer.

121

Preparation of PGMA brush-modified 3D slides. The PGMA brush-modified 3D

122

slides were fabricated through self-assembling of silica particles on the water surface

123

and surface initiated atom transfer radical polymerization (SI-ATRP) method.28-30

124

Firstly, monodispersed silica particles with average diameters (50±3 nm, 160±8 nm,

125

528±20 nm, 802±30 nm) were synthesized using the Stöber method and redispersed

126

in 1-butanol by centrifugation (see supporting information for details).31

127

Subsequently, the solution containing silica particles were added dropwise to Milli-Q

128

water to form self-assemble monolayers on the water surfaces. The silica particle

129

monolayers were quickly transferred to the surfaces of glass slides by placing clean

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glass slides on the water surfaces. After annealed at 500 °C for 1 hour, the stable

131

silica particle assembled monolayer-modified slides were obtained (termed as, 3D

132

slides). Finally, the planar glass slides and 3D slides were coated by PGMA brushes

133

using a typical SI-ATRP method (see supporting information for details). The PGMA

134

brush-modified slides were referred to as PGMA@2D substrate and PGMA@3D(n)

135

substrate, respectively, where n indicates diameter of used silica particle.

7



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Microarray fabrication and application. The microarrays were fabricated by a

137

SmartArrayer 136 system (Capitalbio Ltd., Beijing, China) under contact printing

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mode as previously reported strategies. 30, 32-33 The details of microarray fabrication

139

and correspondent biorecognition reactions were shown in the supporting information.

140

Generally, amino-modified monosaccharides (Gal-β, Glc-β and Man-α) were printed

141

on PGMA@3D(n) substrate and PGMA@2D substrate for fabricating carbohydrate

142

microarrays,32 and glycoprotein (Asf) was printed on PGMA@3D(160) substrate and

143

2D epoxy substrate for fabricating glycoprotein microarrays,32 respectively. All of the

144

microarrays can be separated into 10 or 12 independent subarrays using PTFE grid.

145

And then the carbohydrate/glycoprotein microarrays were incubated with biotinylated

146

lectins (biotin-RCA 120 and biotin-Con A), and labeled by streptavidin-Cy5,

147

respectively. Human IgG was printed on PGMA@3D(160) substrate and 2D epoxy

148

substrate for studying the interaction of antigen with antibody.30 The Human IgG

149

microarrays were incubated with anti-IgG-Cy5 with the desire concentrations at 37 °C

150

for 2 h. Probe ssDNA (Pm) was printed on the PGMA@3D(160) substrate and 2D

151

epoxy substrate.33 Then, the Pm microarrays were hybridized with target ssDNA (Tm)

152

at the desire concentrations at 55 °C for 1 h, and labeled by Cy5 modified ssDNA

153

(Pf).

154

RESULTS AND DISCUSSIONS

8


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Scheme 1. Fabrication of PGMA@3D(n) substrate. Here, n indicates diameter of used

157

silica particle.

158

Preparation and characterization of the PGMA@3D(n) substrate. The strategy

159

of preparing the PGMA@3D(n) substrates is shown in scheme 1. Firstly, silica

160

particle self-assembled monolayers on glass slides were fabricated by the previously

161

reported method.29 In this case, silica particles are chosen to construct 3D slides

162

because of their controlled size, good monodispersion, similar surface morphology,

163

easy synthesis and facile surface modification, which are very important for

164

generating uniform nanostructure surface. After annealed at 500 °C for 1 h, stable

165

silica particle assembled monolayer-modified slides were obtained. As shown in

166

Figure 1a and 1b, the SEM micrographs show that a uniform and stable silica particle 9



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monolayer has been successfully immobilized on the surface of the glass slide. The

168

surface with globular nanostructure can provide a 3D backbone for immobilizing

169

densely packed polymer brushes, which can significantly increase the efficiency of

170

biomolecular interaction on the solid/liquid interface. After aminosilanization by

171

APTES and initiator immobilization, brush-like polymer chains could grow on the

172

globular

173

polymerization (SI-ATRP). The epoxy groups of PGMA brushes can be used as

174

activate groups for covalently immobilizing various biomolecules through reacting

175

with amino groups in mild conditions. The successful growth of PGMA brushes on

176

the silica particle surface was confirmed by TEM, ATR-FTIR and XPS analysis. After

177

PGMA-grafting, it is clear to observe grey zones of low contrast around the spherical

178

particles that are not shown on the image of silica particles before PGMA-grafting

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and some polymer brush coatings on neighboring particles stick together (as shown in

180

Figure 1c and 1d). The result indicates that silica particles are coated by polymer

181

layers. The ATR-FTIR spectrum of PGMA-grafted silica particles shows an

182

absorption band at 1727 cm-1 which is derived from C=O stretching vibration in the

183

ester of GMA (as shown in Figure S1).30 The XPS spectra of the 3D slide and the

184

PGMA@3D(160) modified slide are shown in the Figure S2. Compared with the 3D

185

slide, the PGMA@3D(160) modified slide shows the signal of N1s (400.0 eV) and

186

Br3d (69.0 eV), which are resulted from aminosilanization and initiator immobilization

nanostructure

surface

by

surface-initiated

10


atom

transfer

radical

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process, respectively.30, 34 The C1s peak of the PGMA@3D(160) modified slide can be

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split into five peaks, which correspond to the C-C/C-H peak at 284.5 eV, the C-C=O

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peak at 285.5 eV, the C-O peak at 286.4 eV, the C-O-C peak at 287.0 eV and the C=O

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peak at 288.6 eV, respectively.35-37 The O1s peak has three new peaks at 531.8 eV,

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533.1 eV and 533.9 eV, which represent the oxygen atoms from O-C=O, C-O-C and

192

O-C=O components of PGMA brushes, respectively.37-38 The ATR-FTIR and XPS

193

results confirm the successful immobilization of PGMA brushes on the surfaces of

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silica particles.

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Figure 1. SEM micrographs (a, b) of 3D slide and TEM micrographs (c, d) of silica

197

particles before (a, c) and after (b, d) PGMA-grafting. 11



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Effect of silica particle size on the performance of PGMA@3D(n) substrate.

199

Silica particles with different diameters (50±3 nm, 160±8 nm, 528±20 nm, 802±

200

30 nm) were employed to immobilize PGMA brushes through SI-ATRP method (as

201

shown in Figure 2). Dynamic light scattering analysis demonstrated that PGMA layer

202

had been formed since the hydrodynamic diameters of silica particles were

203

significantly increased after polymer-grafting (as shown in Table S2). In order to

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compare loading capacities of these substrates, streptavidin-Cy5 was used as a model

205

to calculate surface concentrations of biomolecules binding on these substrates.

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Streptavidin-Cy5 with concentrations from 100 ng/mL to 10 µg/mL were printed onto

207

the substrates and read by LuxScan-10K fluorescence microarray scanner. The

208

volume of printing solution was approximately 0.6 nL. The obtained fluorescence

209

intensity increases linearly with the logarithm of the moles of streptavidin-Cy5

210

printed on these substrates (as shown in Figure S3). After incubating at dark for 12 h,

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these slides were serially washed with 50 mM PB buffer containing 0.15 M NaCl and

212

0.1% (v/v) Tween-20, 50 mM PB buffer containing 0.15 M NaCl, and water. After

213

dried by centrifugation, these slides were read again by the microarray scanner. The

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moles of streptavidin-Cy5 bound on the surface of these substrates can be obtained

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through the standard curves before washing. Each spot in the array is around 0.12 mm

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in diameter. The surface concentrations of streptavidin-Cy5 that bound to the surface

217

after washing can be calculated. Surface concentrations of streptavidin-Cy5 on these

12


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substrates are 58, 186, 214 and 225 molecules/µm2, respectively, when the

219

concentration of streptavidin-Cy5 in printing solution 1 is 10 µg/mL. The high surface

220

concentration should be due to relative high grating density (PGMA@3D(802)＞

221

PGMA@3D(528)＞PGMA@3D(160)＞PGMA@3D(50)) (as shown in Table S2).39

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The binding reaction of monosaccharide Gal-β and lectin biotin-RCA 120 was

223

arbitrary selected to address assay performance of PGMA@3D(n) substrates. The

224

amino-modified monosaccharides were directly immobilized on the PGMA@3D(n)

225

substrates and the PGMA@2D substrate for generating carbohydrate microarrays.

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After recognition of immobilized monosaccharides with biotinylated lectins,

227

streptavidin-Cy5 was used to label these recognition events by avidin-biotin reaction.

228

The resulting fluorescence images from different substrates are shown in Figure 2.

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The fluorescence intensity is increased with increasing the size of silica particles.

230

However, the background signal stemming from the substrate is also increased with

231

increasing the size of silica particles because large silica particles have relatively high

232

light scattering abilities. High signal-to-background ratio is obtained on the

233

PGMA@3D(160) substrate. In addition, two hundred identical recognition reactions

234

of Gal-β and biotin-RCA 120 were performed on the PGMA@3D(160) and

235

PGMA@3D(528) substrates, respectively. The corresponding signal-to-noise ratio

236

(S/N) and quality factors (Z’) were calculated (as shown in Figure S4). The S/N and

237

Z’ values (S/N > 50 and Z’ > 0.8) of the PGMA@3D(160) substrate are much higher

13



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238

than those of the PGMA@3D(528) substrate, which further confirm that the

239

high-throughput screening performance of the PGMA@3D(160) substrate-based

240

microarray is more excellent than that of the PGMA@3D(528) substrate-based

241

microarray.40 In addition, 3D(160) slides with various lengths of PGMA brushes were

242

prepared by varying the ATRP time (i.e., 3 h, 9 h, and 15 h), respectively. As shown

243

in Figure S5, the sensitivities (the slopes of the lines) and standard deviations of

244

biotin-RCA 120 detection are increased with increasing the polymerization time.

245

Taking into account the sensitivity and accuracy of detection assay, nine-hour ATRP

246

was selected in our experiment. Therefore, the optimized PGMA@3D(160) substrate

247

was used in the follow-up experiments.

248 14


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249

Figure 2. SEM (a to e) and TEM (insets of a to e) measurements on PGMA@2D (a),

250

PGMA@3D(50)

251

PGMA@3D(802) (e) substrates, and corresponding fluorescence images (inset of f)

252

and data analysis of Gal-β and biotin-RCA 120 binding assays (f). The concentration

253

of Gal-β is 10 mM in printing solution 1, and the concentration of biotin-RCA 120 is

254

4 µg/mL in binding solution 1. The error bars mean standard deviations (**p＜0.01

255

by ANOVA with Tukey's post-test, n = 6).

(b),

PGMA@3D(160)

(c),

PGMA@3D(528)

(d)

and

256

Detection of carbohydrate-protein interactions on PGMA@3D(160) substrates.

257

In order to confirm the practical applicability of the PGMA@3D(160) substrate as

258

microarray substrate, the interactions of three kinds of monosaccharides (Gal-β, Glc-β

259

and Man-α) and two lectins (biotin-RCA 120 and biotin-Con A) were tested. Figure

260

3a and 3b are the fluorescence images of carbohydrate microarray after reacted with

261

biotin-RCA 120 and biotin-Con A on the PGMA@3D(160) substrate, respectively.

262

For biotin-RCA 120, Gal-β spots show the highest fluorescence signal, while Glc-β

263

spots and Man-α spots exhibit negligible fluorescence signals. For biotin-Con A, the

264

fluorescence intensity of Man-α spots is stronger than that of Glc-β spots, while

265

fluorescence intensity of Gal-β spots is relatively poor. These results are consistent

266

with the previous literature reports, which suggest that the PGMA@3D(160)

267

substrate-based carbohydrate microarray has good selectivity and can be used to

268

discriminate the binding affinities of lectins with saccharides.32, 41 15



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269 270

Figure 3. Fluorescence images (inset) and corresponding data analysis of

271

carbohydrate microarray reacted with biotin-RCA 120 (a) and biotin-Con A (b) on the

272

PGMA@3D(160) substrate. The concentrations of monosaccharides in printing

273

solution 1 and lectins in binding solution 1 are 10 mM and 25 µg/mL, respectively.

274

The biorecognition reactions of monosaccharides with lectins were labeled by 10

275

µg/mL streptavidin-Cy5.

276

A series of experiments were designed to evaluate the sensitivity of

277

PGMA@3D(160) substrate-based carbohydrate microarrays. Various concentrations

278

of amino-modified monosaccharides (Gal-β and Man-α) in printing solution 1 were

279

firstly immobilized on PGMA@3D(160) substrates, and then incubated in

280

corresponding lectin solution. As shown in Figure S6, the LODs (estimated as 3 times

281

of the standard deviation of fluorescence signals of control samples) are 100 µM for

282

Gal-β and 50 µM for Man-α. The fluorescence signals are linearly increased with

283

increasing the concentrations of Gal-β and Man-α in the ranges of 0.1 mM to 3 mM

284

and 0.05 mM to 0.5 mM, respectively, while the concentrations of lectins are kept as 16


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285

constants. For detecting lectins in solutions, different concentrations of biotin-RCA

286

120 and biotin-Con A were applied to the carbohydrate microarrays when 10 mM

287

Gal-β and Man-α in printing solution 1 were spotted on PGMA@3D(160) substrates,

288

respectively. As shown in Figure S7, the fluorescence intensities of microarrays are

289

linearly increased with increasing the concentrations of lectins, which indicates that

290

the linear ranges are 1 ng/mL-0.8 µg/mL (8.3 pM-6.7 nM) for biotin-RCA 120

291

(MW=120 kDa) and 1 ng/mL-4 µg/mL (9.6 pM-38.5 nM) for biotin-Con A (MW=104

292

kDa). The specific binding events could be detected at 1 ng/mL (8.3 pM) for

293

biotin-RCA 120 and 1 ng/mL (9.6 pM) for biotin-Con A, which suggest that the

294

detection limits of lectins are in the picomolar range without any signal amplification.

295

These results are equal to and/or better than those of the previously reported methods

296

(e.g., gold nanoparticle labeled microarray-based plasmon resonance light scattering

297

assay, aminooxyacetyl functionalized glass surface-based oligosaccharide microarrays,

298

microarray-based metal-enhanced fluorescence assay).41-43

299

17



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

300

Figure 4. Fluorescence images (inset) and binding curves of Gal-β and biotin-RCA

301

120 (a), and Man-α and biotin-Con A (b). 10 mM Gal-β and Man-α in printing

302

solution 1 were spotted on PGMA@3D(160) substrates. The biorecognition reactions

303

of monosaccharides with lectins were labeled by 10 µg/mL streptavidin-Cy5. The

304

KD,surf values are obtained by fitting the curves to the equation (see supporting

305

information for details).

306

In order to investigate the binding strength of monosaccharides and lectins on

307

PGMA@3D(160) substrates, the equilibrium dissociation constant (KD,surf) values of

308

the specific carbohydrate-protein interactions were measured. In this case, different

309

concentrations of biotin-RCA 120 and biotin-Con A were applied to Gal-β and Man-α

310

microarrays, respectively. The fluorescence images and the binding curves of two

311

tested carbohydrate-lectin pairs were illustrated in Figure 4. The fluorescence

312

intensities are increased with increasing the concentrations of lectins, and tend to

313

saturate above 20 µg/mL biotin-RCA 120 and biotin-Con A in reaction solutions. The

314

KD,surf values of lectins are 0.73 µg/mL (6.08 nM) for the binding of biotin-RCA 120

315

with Gal-β and 3.44 µg/mL (33.08 nM) for the binding of biotin-Con A with Man-α,

316

which are in the order of nanomolar for the two binding reactions. The binding

317

affinities are obviously higher than those of monovalent binding events reported by

318

previous literatures (typically in the millimolar range), which indicate strong

18


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319

multivalent interactions of monosaccharides and lectins on PGMA@3D(160)

320

substrates.44-47

321

Molecular reaction capability of PGMA@3D(160) substrates. To test universal

322

property of sphere-polymer brush hierarchically nanostructure-based microarray

323

substrates, PGMA@3D(160) substrates were directly used to immobilize different

324

biomolecules (e.g., DNA, antibody and glycoprotein). As shown in Figure 5, S8-S9,

325

the fluorescence intensities of these PGMA@3D(160) substrate-based DNA, antibody

326

and glycoprotein microarrays are sharply increased with increasing concentration of

327

target DNA Tm, anti-IgG-Cy5 and biotin-RCA 120, respectively. In the presence of 1

328

µM Tm, 10 µg/mL anti-IgG-Cy5 and 4 µg/mL biotin-RCA 120, the fluorescence

329

intensities

330

PGMA@3D(160) substrate are 38.7 times, 2.7 times and 2.6 times higher than those

331

of 2D DNA, antibody and glycoprotein microarrays, respectively. Furthermore, the

332

KD,surf value of Asf with biotin-RCA 120 on the PGMA@3D(160) substrate is much

333

lower than that of Asf with biotin-RCA 120 on 2D epoxy substrate. These

334

phenomenons

335

PGMA@3D(160) substrates is much better than those of biomolecules on 2D epoxy

336

substrate.

337

PGMA@3D(160) substrate-based microarrays show higher sensitivities and lower

338

detection limits (as shown in Table S3), which are comparable to our previous

of

the

DNA,

suggest

Compared

that

with

antibody

the

2D

and

reaction

epoxy

glycoprotein

capabilities

microarrays

of

substrate-based

19


on

biomolecules

microarrays,

the

on

the


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

339

reports.30, 33 The results indicate that the PGMA@3D(160) modified slide can be used

340

as a general substrate to fabricate high-performance microarrays for high-throughput

341

screening of a range of disease-related molecular targets.

342 343

Figure 5. Fluorescent images (top) and dose responses of biotin-RCA 120 on Asf

344

microarrays. 500 µg/mL Asf in printing solution 2 were spotted on 2D epoxy

345

substrate and the PGMA@3D(160) substrate. The biorecognition reaction of Asf with

346

lectin was labeled by 10 µg/mL streptavidin-Cy5. The inset shows the assay

347

performance at the low concentrations of biotin-RCA 120.

348

CONCLUSIONS

349

In summary, a sphere-polymer brush hierarchically nanostructure-based microarray

350

substrate has been developed for studying biomolecular interactions. Combining

351

geometrical characterizations of spherical particles and three-dimensional architecture 20


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352

of polymer brushes, the optimized substrate (i.e., PGMA@3D(160) modified slide)

353

can provide large molecular loading amount and good accessibility to the targeted

354

analytes. Compared to 2D epoxy substrates, the obtained carbohydrate, DNA,

355

glycoprotein and antibody microarrays on PGMA@3D(160) substrates exhibit

356

excellent analytical performance for detecting corresponding molecular interactions.

357

The results suggest that our approach provides a new platform for the design of

358

excellent microarrays for further bioanalytical and biomedical applications including

359

disease-related biomarker discovery and biomolecular interaction study.

360

ASSOCIATED CONTENT

361

Supporting Information. Details of fabricating the PGMA@3D(n) substrate and

362

preparing carbohydrate, glycoprotein, antibody and DNA microarrays. Tables

363

including sequences used in the experiment, comparison of analyte detection on the

364

PGMA@3D(160) substrates and 2D epoxy substrates. Figures including ATR-FTIR

365

spectra of silica particles and PGMA-grafted silica particles, XPS characterization of

366

3D slide and PGMA@3D(160) modified slide, the curves obtained by the function of

367

logarithm of printing moles of streptavidin-Cy5 and fluorescence intensities on

368

different substrates, assay performance evaluation of the PGMA@3D(160) substrate

369

and the PGMA@3D(528) substrate, the effects of polymer brush length on the

370

sensitivity of biotin-RCA 120 detection assay, detection assays of monosaccharides

371

and lectins on the PGMA@3D(160) substrates, dose response assays of target DNA 21



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

372

and antibody on the PGMA@3D(160) substrates and 2D epoxy substrates. This

373

material is available free of charge via the Internet at http://pubs.acs.org.

374

AUTHOR INFORMATION

375

Corresponding Author

376

*E-mails: [email protected]. Tel.: +86 431 85262243.

377

Author Contributions

378

The manuscript was written through contributions of all authors. All authors have

379

given approval to the final version of the manuscript.

380

Notes

381

The authors declare no competing financial interest.

382

ACKNOWLEDGMENT

383

The authors would like to thank the National Natural Science Foundation of China

384

(Grant no. 21475126 and YZ201561) for financial support.

385

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31



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Table of Contents/Abstract Graphic

542

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Development of Sphere-Polymer Brush Hierarchical Nanostructure

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