Optical Fiber-Type Sugar Chip Using Localized Surface Plasmon

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Optical fiber-type Sugar Chip using localized surface plasmon resonance Masahiro Wakao, Shogo Watanabe, Yoshie Kurahashi, Takahide Matsuo, Makoto Takeuchi, Tomohisa Ogawa, Keigo Suzuki, Takeshi Yumino, Tohru Myogadani, Atsushi Saito, Ken-ichi Muta, Mitsunori Kimura, Kotaro Kajikawa, and Yasuo Suda Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02380 • Publication Date (Web): 19 Dec 2016 Downloaded from http://pubs.acs.org on December 26, 2016

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Analytical Chemistry

Optical fiber-type Sugar Chip using localized surface plasmon resonance Masahiro Wakao,a* Shogo Watanabe,a Yoshie Kurahashi,a Takahide Matsuo,a Makoto Takeuchi,a Tomohisa Ogawa,b Keigo Suzuki,b Takeshi Yumino,b Tohru Myogadani,b Atsushi Saito,b Ken-ichi Muta,b Mitsunori Kimura,c Kotaro Kajikawa,c Yasuo Sudaa,d

a

Department of Chemistry, Biotechnology, and Chemical Engineering, Graduate School of Science and Engineering, Kagoshima University, 1-21-40 Kohrimoto, Kagoshima 890-0065, Japan b

Moritex Corporation, 1-3-3 Azamino-minami, Aobaku, Yokohama 225-0012, Japan c

Interdisciplinary Graduate School of Science and Engineering, Tokyo Institute of Technology, Midoriku, Yokohama 226-8502, Japan d

SUDx-Biotec corporation, 1-42-1, Shiroyama, Kagoshima 890-0013, Japan

*CORRESPONDING AUTHOR EMAIL ADDRESS [email protected] *CORRESPONDING AUTHOR PHONE&FAX (+81)99-285-7843 (+81)99-285-7856

1 ACS Paragon Plus Environment


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ABSTRACT: Optical fiber-type Sugar Chips were developed using localized surface plasmon resonance (LSPR) of gold (Au) nanoparticles. The endface of an optical fiber was first aminosilylated and then condensed with α-lipoic acid containing a dithiol group. Second, gold nanoparticles were immobilized onto the endface via an Au-S covalent bond. Finally, sugar moieties were attached to the gold nanoparticle using our original sugar chain-ligand conjugates to obtain fiber type Sugar Chips, by which the sugar moiety-protein interaction was analyzed. The specificity, sensitivity, and quantitative binding potency against carbohydrate-binding protein were found to be identical to that of a conventional SPR sensor. In this analysis, only a small sample volume (approximately 10 µL) was required compared with conventional SPR sensor (100 µL), suggesting that the fiber-type Sugar Chip and LSPR are applicable for non-pure small masses of proteins. KEYWORDS: Sugar Chips, localized surface plasmon resonance, optical fiber, sugar moiety, sugar moiety-protein interaction

Introduction Carbohydrates or sugar moieties play crucial roles in many biological processes, such as proliferation, differentiation, cell-cell communication, and infection, among others.1–5 Their fundamental biochemical processes include binding to specific molecules such as proteins, carbohydrates (sugar moieties), nucleic acids, and lipids. To understand the functions of carbohydrates, various biosensors for analyzing carbohydrates have been developed.6–19 Surface plasmon resonance (SPR) biosensors are powerful tools for interaction analysis and can detect specific binding interactions between biological molecules on a real-time scale without labeling.10–23 Many studies have investigated carbohydrate immobilization onto gold-coated SPR sensor chips.10–19 Our group developed a new immobilization technique using a linker compound containing thioctic acid and successfully prepared a “Sugar Chip”, in which sugar moiety or sugar chains are immobilized onto the gold-coated sensor chip.19,24 Using the Sugar Chips and SPR, the binding interactions among various proteins were conventionally analyzed. However, the developed system


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using a standard SPR apparatus requires a relatively large amount of sample (several hundred microliters). This can be problematic, particularly for proteins that are difficult to purify. Recently, biosensors based on localized SPR (LSPR) and related technologies have attracted attention.25–35 The LSPR sensing system is divided into two category, which is based on optical absorption and light scattering analysis. The former is constructed in transmission geometry to obtain the absorption spectrum and sometimes needs a complicated optical system to measure .27-30,32,33 On the other hand, the later can be prepared using a simple optical system, and the sensor chip can be constructed even at the endface of a micrometer-sized optical fiber, offering many advantages compared to absorption-based LSPR sensors and conventional SPR biosensors, such as miniaturization of the instrument and smaller amounts of sample.31,34,35 In this study, we fabricated optical fiber-type Sugar Chips for the LSPR sensor and evaluated the lectin-carbohydrate binding interaction. We found that the specificity and sensitivity of the LSPR sensor was identical to that of a conventional SPR sensor using a larger-size Sugar Chip, suggesting that the LSPR is practical for evaluating sugar moiety-protein interactions on a small scale. Experimental section Instrumentation The light source was a red light emitting diode (LED, λmax = 623 nm, λFWHM = 15 nm, 104 mcd, TLSH180P, Toshiba, Tokyo, Japan). The light was coupled to a multimode fiber coupler (2 × 1 50/50, F-CPL-M12855, Newport Corp., Irvine, CA, USA). The end was connected to the fiber chip via a splicer (ULTRAsplicer US-128, Siemon Company, Watertown, CT, USA). The returned light from the endface of the fiber chip was detected using an avalanche photodiode (APD). The LED and APD were controlled by a digital multimeter (AD7461A, Advantest, Tokyo, Japan). A multimode optical fiber (G50/125 3002, Fujikura Ltd., Tokyo, Japan) was used for the fiber-type Sugar Chip. The optical fiber was cleaved using a fiber cutter (high-precision cleaver CT-30, Fujikura). An image of the endface of the fiber was obtained using a field emission scanning electron microscope (FE-SEM, S-4100H, Hitachi High-Technologies Corp., Tokyo, Japan). Reagents



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The endface of the optical fiber was modified with the following agents: N-(2-aminoethyl)-3-aminopropyltrimethoxysilane (Aldrich, St. Louis, MO, USA), 4,4'-dithiobutyric acid (Aldrich), α-lipoic acid (thioctic acid, Nacalai Tesque, Kyoto, Japan), anhydrous N,N-dimethylformamide (DMF, Kanto Chemical Co., Inc., Tokyo, Japan),

1-ethyl-3-(3-dimethylaminopropyl)carbodimide

hydrochloride

(EDC•HCl,

Peptide Institute, Osaka, Japan), 3H-1,2,3-triazolo[4,5-b]pyridine-3-ol (HOAt, TCI, Tokyo, Japan), diisopropylethylamine (DIEA, Nacalai Tesque), and gold nanoparticle solution in H2O (φ ~40 nm stabilized with citrate, TANAKA Chemical Corporation, Tokyo, Japan). Ligand conjugates19 were used to immobilize sugar moieties onto gold nanoparticles immobilized onto the endface of the optical fiber. Concanavalin A (Con A, Aldrich) and RCA120 (Vector Laboratories, Burlingame, CA, USA) were used for interaction analysis. Ultrapure water was used in all experiments. Other chemicals were of commercial grade unless noted. Fabrication of optical fiber-type Sugar Chips The fiber-type Sugar Chips were fabricated as previously described with modifications.31 The endface of a freshly cleaved optical fiber was immersed in ethanol solution containing N-(2-aminoethyl)-3-amino-propyltrimethoxysilane (50 mM) and 5% v/v acetic acid at room temperature for 10 min. The fiber was then rinsed with ethanol and placed in an oven at 120°C for silanization (the fiber obtained from this step was abbreviated as DA-fiber). The obtained fiber was treated with a coupling cocktail containing of DIEA (39 µmol), HOAt (19 µmol), EDC•HCl (19 µmol), and 4,4'-dithiobutyric acid (19 µmol, BA-fiber) or α-lipoic acid (19 µmol, abbreviated as TA-fiber) in DMF (0.8 mL) for 12 h. The fiber was then rinsed with ethanol, immersed in 50 mM aqueous NaBH4 for 2 h, rinsed with water, and dried in an ambient atmosphere. Subsequently, the fiber was immersed in aqueous Au colloid solution while stirring for 30 min, rinsed with H2O, immersed in the solution of the appropriate ligand conjugate (200 µM) for 1 h, and rinsed with H2O. The gold nanoparticles immobilized onto the endface of fiber were functionalized with a sugar moiety. Interaction analysis with lectins The interaction between the optical fiber-type Sugar Chips and sugar-binding protein (lectins) was measured as follows. First, the fiber was immersed in PBS containing 0.05% Tween 20 (PBS/T) (0.5 mL). After 100 s, protein solution (0.5 mL) in PBS/T was


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added, and binding behavior was observed for an additional 200 s. The fiber was then placed in PBS/T solution (1.0 mL) for 100 s. Dissociation was observed during this step. Finally, the fiber was treated with sodium dodecyl sulfate solution (1.0 mL) to remove adsorbed protein and then immersed in PBS/T solution (1.0 mL). In the case of the interaction analysis with 10 µL of protein sample, hand-made glass capilar (φ 1 mm, length: 10 mm) was used. Typically, the fiber was immersed in PBS containing 0.05% Tween 20 (PBS/T) (10 µL). After 100 s, the PBS/T was switched to protein solution (10 µL) in PBS/T, and binding behavior was observed for an additional 200 s. After removal of protein solution, PBS/T solution (10 µL) was immediately added and dissociation was observed for another 100 s. Finally, the fiber was treated with sodium dodecyl sulfate solution (10 µL)) to remove adsorbed protein and then immersed in PBS/T solution (10 µL). Results and Discussion The optical setup was prepared as previously described with modifications (Figure 1).31 A 623-nm LED was chosen as a light source because larger signal changes are observed when using the light ranging from 550 to 680 nm. The light source was coupled to a multimode fiber coupler. The end of the fiber was connected to the fiber-type Sugar Chip via a splicer. The incident light was scattered by gold nanoparticles immobilized to the endface. The return light from the endface of the fiber was detected with APD, and signal changes were monitored using a PC. The optical fiber-type Sugar Chip was prepared as shown in Scheme 1. A standard multimode optical fiber (core diameter 62.5 µm) was used in this study. The fiber was first cleaved by a fiber cutter. The section of the fiber was chemically modified to the surface possessing an amino group by soaking in a solution of aminosilylating agent in ethanol, and heated at 120°C to generate the DA-fiber (1). The resulting amino groups on the DA-fiber were converted into thiol groups by coupling to 4,4'-dithiobutyric acid or α-lipoic acid to afford the BA-fiber (2) or TA-fiber (3) possessing a thiol group, respectively. The TA-fiber was further treated with NaBH4. Further modification of the fiber endface was carried out as shown in Scheme 2. The TA-fiber was immersed in aqueous gold nanoparticles (φ ~40 nm, stabilized with citric acid) to immobilize gold nanoparticles onto the fiber endface via Au-S covalent bonds of thiol groups. The immobilized gold nanoparticles were then functionalized with a sugar-chain ligand conjugate based on our previous study.19 The obtained fiber was used



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for optical fiber-type Sugar Chips. DA and BA-fibers were also treated using the same procedure as that used for the TA-fiber to give the corresponding fiber-type Sugar Chips. It was assumed that gold nanoparticles were electrostatically immobilized in the case of the DA-fiber. The surfaces of all three fibers were characterized by scanning electron microscopy (SEM). Gold nanoparticles were found on the endface of all fibers, although the quantity and density of gold nanoparticles were remarkably different (Figure 2). To investigate the sensitivity, specificity, and reproducibility of the three prepared types of fiber-type Sugar Chips, we measured the sugar moiety-protein interaction. First, Mal-mono ligand conjugate (possessing an α-glucose terminus) was used to functionalize the Sugar Chip, and concanavalin A (Con A, glucose binding protein) was selected for the analyzed lectin (Figure 3). When 5.0 µM of Con A was applied, the relative signal intensity on the DA-fiber (Figure 3A) and BA-fiber (Figure 3B) did not return to the baseline and decreased as the sample solution was replaced, suggesting the detachment and/or aggregation of gold nanoparticles on the endface of the optical fiber. In contrast, the relative signal intensity of the TA-fiber was nearly identical between the beginning and end of the measurement at different time points (Figure 3C), suggesting that gold nanoparticles were stably immobilized to the endface. Although slight baseline shifts among fibers were observed, which were attributed by inadequate equilibration of the fibers, measurements after sufficient washing and equilibration of the fiber afforded reproducible results. Next, various concentrations of proteins (Con A on Mal-mono functionalized Sugar chip in Figure 4A, RCA120 on Lac-mono functionalized chip in Figure 4B) were investigated, and their equilibrium binding constants were evaluated based on the binding curves (Figure 4C and 4D). The KD values of Con A to glucose and RCA120 to galactose were estimated to be 1.4 µM and 140 nM, respectively (see SI). The reverse experiments, in which Con A was applied to the Lac-mono functionalized chip (Figure 4E) and RCA120 was applied to the Mal-mono functionalized chip (Figure 4F), were also performed. However, no increase in intensity was observed in both cases, indicating that the binding phenomena observed in our system was specific based on the properties of lectins. The KD value of Con A to glucose was 3-fold higher than that obtained using a conventional SPR sensor (KD = 480 nM obtained with SPR-670M, Moritex, San Jose, CA, USA, unpublished data). The KD value of RCA120 to galactose was nearly identical to that obtained using the conventional SPR sensor (KD = 200 nM


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obtained with SPR-670M, Moritex, unpublished data). The discrepancies in KD values reported previously15,18,36 particularly in the case of Con A, may be related to the instability of Con A. In the binding interaction analysis with 10 µL of RCA120 sample, signal changes were almost the same as that in Figure 4B (see SI, Figure S1). The experiments were performed using 10 µL of sample solution, suggesting that our LSPR system with a fiber-type Sugar Chip performs well compared to a conventional SPR system and can be used when samples quantities are limited. Conclusion We analyzed sugar moiety-protein interactions using an LSPR biosensor with optical fiber-type Sugar Chips. Optical fiber-type Sugar Chips were easily prepared in four steps via aminosilylation, modification of the thiol group, immobilization of gold nanoparticles, and functionalization of the sugar moiety using our original sugar-chain ligand conjugate. The di-thiol group derived from the thioctic acyl group on the endface of the fiber afforded good properties for immobilizing gold nanoparticles via Au-S covalent bonding and allowed for successful measurements with good signal stability and reproducibility. The LSPR system can be used to analyze sugar moiety-protein binding interactions in a similar manner to conventional SPR systems. Additionally, small quantities of sample (~10 µL) can be used with this system because the fiber probe can be fabricated on a micrometer-sized fiber endface. Acknowledgements The present work was financially supported in parts by grants from the Japan Science and Technology Agency and the Japan Ministry of Health, Labor and Welfare. Supporting Information Calculation of KD and binding analysis using small quantities of sample by LSPR system. This material is available free of charge via the Internet at http://pubs.acs.org. References 1)

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for TOC only LED

coupler APD

computer

core

splicer

hν

optical fiber

= Au nanoparticle

clad

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sample

= carbohydrate moiety

sensing moiety


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Legends to Figures and Schemes

coupler

LED APD

computer

core

splicer

hν

optical fiber

= Au nanoparticle

clad

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sample sensing moiety

= carbohydrate moiety

Figure 1. Localized surface plasmon resonance (LSPR) experimental setup with optical fiber-type Sugar Chips.

A

B

C

Figure 2. Scanning electron microscope (SEM) image for the optical fiber endface. (A) Au nanoparticles on DA-fiber. (B) Au nanoparticles on BA-fiber. (C) Au nanoparticles on TA-fiber. Scale bar is 500 nm.



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Figure 3. Real-time response of LSPR sensor with (A) DA-fiber, (B) BA-fiber, and (C) TA-fiber when 10 µM of Con A (final concentration of Con A was 5.0 µM) was added to the cuvette. The numbered arrows on the graph show (i) injected Con A sample solution, (ii) switch to PBS/T buffer, (iii) washing with 0.5% sodium dodecyl sulfate solution, and (iv) switching and stabilization with PBS/T buffer. Data were collected using freshly prepared fiber (solid line: as-prepared), fiber after drying (gray solid line: after drying), and fiber after 1 day (dashed line: 1 day after).


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Figure 4. (A) Binding of Con A to Mal-mono on the TA-fiber sugar chip. (B) Binding of RCA120 to Lac-mono on the TA-fiber sugar chip. (C) Equilibrium binding data of Con A to Mal-mono chip. (D) Equilibrium binding data of RCA120 to Lac-mono chip. (E) Binding of Con A to Lac-mono on the TA-fiber sugar chip. (F) Binding of RCA120 to Mal-mono on the TA-fiber sugar chip.



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OH OH OH

(MeO)3Si

N H

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NH2 O O Si O

AcOH, EtOH

Fiber endface

NH2

N H

DA fiber (1) O

1)

S

HO

O O Si O

2

EDC•HCl, HOAt, DIEA in DMF

1

H N

N O

2) NaBH4 in H2O (50 mM)

SH

SH O

SH SH

BA fiber (2) O O O Si O

1) HO S S EDC•HCl, HOAt, DIEA in DMF

N

SH SH SH SH

O

O

1

SH SH

H N

2) NaBH4 in H 2O (50 mM)

TA fiber (3)

SH SH

Scheme 1. Preparation of optical fiber with amino or thio group at the endface.

SH SH SH SH

S S in H2O

S S

Mal-mono or Lac-mono

S S

in H2O

S S

Carbhydrate-protein interaction analysis

OH O OH

TA fiber (3) =

Au nanoparticles (40 nm, stabilized with citrate)

OH OH OH O

HO OH

O N H

OH

N H

S

S

S

S

=

(Mal-mono)

or OH HO O OH OH

O

OH OH OH

O N H

OH

N H

(Lac-mono)

Scheme 2. Immobilization of Au nanoparticle onto the fiber endface and functionalization of Au nanoparticles with sugar moiety.


Optical Fiber-Type Sugar Chip Using Localized Surface Plasmon

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