Facile fabrication of branched-chain carbohydrate chips for studying carbohydrate-protein interactions by QCM biosensor

Article abstract of DOI:10.1016/j.cclet.2017.08.003

A novel approach for fabricating branched-chain (BC) carbohydrate chips to study carbohydrate-protein interactions using Quartz Crystal Microbalance (QCM) biosensor was developed. This approach utilizes functional alkynyl-branch molecule modified chip surfaces, which through the terminal alkynyl group for covalent linking of unprotected azide-carbohydrates. The unprotected azide-carbohydrates were syhthesized in one-step using 2-azido-1,3-dimethyl-imidazolinium as catalyst, avoiding complex chemical modifications. Additionally, the branch surface modified with the carbohydrates not only supplies more specific binding site but also reveals significant cluster effect. To exemplify the sugar cluster effect on BC carbohydrate chips, BC Galactose and Mannose chips prepared in this work were used to determine carbohydrate-lectin interactions using QCM biosensor. The results clearly showed that BC chip significantly improves the detection sensitivity compared with the straight-chain (SC) chip. More importantly, the BC galactose chip sensitivity was enhanced 40% compared with the SC galactose chip.

Full text of DOI:10.1016/j.cclet.2017.08.003

Accepted Manuscript
Title: Facile fabrication of branched-chain carbohydrate chips
for studying carbohydrate-protein interactions by QCM
biosensor
Authors: Yuchao Lu, Siyu Song, Chenxi Hou, Shuang Pang,
Xueming Li, Xiaowen Wu, Chen Shao, Yuxin Pei, Zhichao Pei
PII:
S1001-8417(17)30279-6
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.cclet.2017.08.003
CCLET 4152
To appear in:
Chinese Chemical Letters
Received date:
Revised date:
Accepted date:
4-5-2017
1-7-2017
2-8-2017
Please cite this article as: Yuchao Lu, Siyu Song, Chenxi Hou, Shuang Pang, Xueming
Li, Xiaowen Wu, Chen Shao, Yuxin Pei, Zhichao Pei, Facile fabrication of branched-
chain carbohydrate chips for studying carbohydrate-protein interactions by QCM
biosensor, Chinese Chemical Lettershttp://dx.doi.org/10.1016/j.cclet.2017.08.003
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Communication
Facile fabrication of branched-chain carbohydrate chips for studying carbohydrate-
protein interactions by QCM biosensor
Yuchao Lua,+, Siyu Songa,+, Chenxi Houa, Shuang Panga, Xueming Lia, Xiaowen Wua, Chen Shaoa, Yuxin
Peia, Zhichao Peia,b,

aShaanxi Key Laboratory of Natural Products & Chemical Biology, College of Chemistry and Pharmacy, Northwest A&F University,
Yangling 712100, China
bAttana, SE-11419, Stockholm, Sweden
Graphical Abstract
A novel approach for fabricating branched-chain (BC) carbohydrate chips to study carbohydrate-protein interactions using
Quartz Crystal Microbalance (QCM) biosensor was developed. This approach utilizes functional alkynyl-branch molecule
modified chip surfaces, which is functionalized with terminal alkynyl group for covalent linking of unprotected azide-
carbohydrates via click chemistry.
ABSTRACT
A novel approach for fabricating branched-chain (BC) carbohydrate chips to study carbohydrate-protein interactions using Quartz Crystal
Microbalance (QCM) biosensor was developed. This approach utilizes functional alkynyl-branch molecule modified chip surfaces, which through the
terminal alkynyl group for covalent linking of unprotected azide-carbohydrates. The unprotected azide-carbohydrates were syhthesized in one-step
using 2-azido-1,3-dimethyl-imidazolinium as catalyst, avoiding complex chemical modifications. Additionally, the branch surface modified with the
carbohydrates not only supplies more specific binding site but also reveals significant cluster effect. To exemplify the sugar cluster effect on BC
carbohydrate chips, BC Galactose and Mannose chips prepared in this work were used to determine carbohydrate-lectin interactions using QCM
biosensor. The results clearly showed that BC chip significantly improves the detection sensitivity compared with the straight-chain (SC) chip. More
importantly, the BC galactose chip sensitivity was enhanced 40% compared with the SC galactose chip.
Keywords:
Multivalent
Brach carbohydrate chip
QCM
Carbohydrate-protein interactions
———
 Corresponding author.
E-mail address: peizc@nwafu.edu.cn ( Z. Pei) ;
+these authors contributed equally to this work.

Carbohydrate-receptor interactions, present at the surface of nearly every cell in the biological activities such as recognition
markers, cell adhesion and development, fertilization, immune response, signal transduction and viral/bacterial infections [1-4].
Efficient analysis and study of these important biological activities are, however, largely hampered by the weak affinities which often
associated with carbohydrate-protein interactions [5]. To overcome this issue, multivalent carbohydrate-protein interactions are utilized
to achieve high affinity due to the glycoside multivalent effect [6]. The glycoside multivalent effect has prompted the development of
synthetic multivalent glycoconjugates with the ability to interact with target lectins, such as glycoclusters [7-9], glycopolymers [10-13],
glycodendrimers [14-18] and glyconanoparticles [7,19-23]. Remarkably, branched molecules which were used as templates for
synthesizing multivalent glycoconjugates have attracted our attention.
In another aspect, carbohydrate chips have received considerable attentions as useful high throughput analytical tools for the rapid
analysis of carbohydrate–protein interactions [5, 24-28]. Recent efforts have led to the development of carbohydrate chips via
multivalent glycoconjugate modification strategy [29] in surface plasmon resonance (SPR) [7,30], carbohydrate microarray [31-33].
However, QCM sensors as a real-time and label-free technology has been widely employed in carbohydrate-protein interaction studies,
where QCM carbohydrate chip based on multivalent glycoconjugate has not yet been reported. To the best of our knowledge, QCM
carbohydrate chips are normally prepared through straight-chain, which are chemically modified by a uniform monolayer of linear
molecules with functional groups. For example, Ramström et al. explored the thiol-ene/-yne reactions as surface functionalization
strategies for the fabrication of carbohydrate-presenting surfaces. The resulting portion of bifurcate carbohydrate-presenting surfaces
showed higher binding capacity [34].
Herein, we present a facile method of branch alkynyl surfaces for the site-directed immobilization of unprotected azide-
carbohydrates on QCM biosensor chip surface for studying carbohydrate-protein interactions. The unprotected azide-carbohydrates
were covalent linked to branch alkynyl-functionalized surfaces in aqueous solution [35-37]. It has consisted of three steps:
Photochemical insertion of perfluoro-phenyl azides into the polystyrene, amide coupling to produce a branch alkynyl surface, and the
last step, Cu-catalyzed azide-alkyne cycloaddition (click chemistry) linked unmodified azide carbohydrates (Scheme 1). And this
protocol was based on the assumption that the higher functionalized surface of branch alkynyl molecules attached to a solid support
should help to maximize the immobilization capacity for azide-carbohydrates and in turn provide the multivalence (i.e., the cluster
effect) required for carbohydrate-protein interaction. Furthermore, the last step of the protocol varies between the productions of
different carbohydrate-functionalized surfaces, allowing for large scale production of the intermediate branched-chain alkyne surfaces,
which can be stored for long time after production before usage. The details of compounds synthesis and carbohydrate chips
fabrication can be found in Supporting information.
In order to test carbohydrate-protein interactions in real-time, the prepared BC carbohydrate chips were docked into the A100
QCM instrument and equilibrated under a continuous flow (25 μL/min) of running buffer. The measurements were initiated when the
resonant frequency stabilized (baseline drift < 0.2 Hz/min). The interactions between lectins and BC carbohydrate chips were evaluated
by injecting lectins into the running buffer over the surfaces. Surface regeneration was performed between the interaction tests. The
binding responses of the three replicate injections of Con A were reproducible (Fig. 1a). This result confirmed that the regeneration of
the BC carbohydrate surfaces performed between three successive injections was effective and enabled the reproducible evaluation of
several binding events on the same surface of the BC chip. Furthermore, a microscopic evaluation of the above-mentioned interaction
was performed before and after the injection of the lectin. Both the surface (non-fluorescent, shown in black in Fig. 1b) and lectins
(FITC-Con A, shown in green in Fig. 1b) were observed before and after injection of FITC-Con A. The fluorescent evaluation
performed after the interaction between the lectin and the carbohydrate, monitored with the biosensor, showed a mainly peripheral
localization of the lectin (in green, Fig. 2b). These observations indicated that the frequency shift monitored with QCM specifically
reflected the interactions occurring at the surface between mannose and Con A.
———

To evaluate whether the BC carbohydrate chip surface has more site-glycosyl and cluster effect than SC chip, lectins were
injected over SC and BC carbohydrate chip surfaces, where the low pH buffer (pH 1.5) was used as the regeneration solution. The
responses were simultaneously monitored using the Attana A100 QCM biosensor and the maximal frequency shifts obtained for each
lectin were recorded and summarized in Fig. 2. The resulting binding analyses for SC and BC chips were presented in Fig. 2a, where
the maximum frequency corresponded to the binding of the lectin. As can be seen in Fig. 2a, the different frequency shifts were
produced from the interactions between lectins and different carbohydrate chips due to the level of glycosylation on the chips surfaces,
where the BC chips yielded a higher binding of lectin, resulting from the cluster effect. To further exemplify the cluster effect, the
binding studies of different concentration of PNA to SC and BC galactose chip were performed using QCM biosensor, respectively.
With the increase of PNA concentration, the BC chips were sensitivity significantly enhanced compared to SC chips (Fig. 2b).
Especially, the BC galactose chip signal was enhanced more than 40% compared to the SC galactose chip at PNA concentration 0.5
mg/mL. These results confirmed that BC galactose chips have significantly improved glycosylation level on the chips’ surfaces, and
increased carbohydrate-protein binding capacity.
The specific binding study was further performed by measuring the different lectins interaction with BC mannose chip. Typical
binding curves for the two lectins used were presented in Fig. 3. As can be seen for the α-D-mannopyranose specific lectin Con A, the
frequency dropped immediately upon injection as a consequence of the association of the lectin to the surface. As the injection plug
came to an end, dissociation took place where the lectin was washed away from the surface. In comparison, WGA showed low binding,
which was in agreement with the specificities for mannose.
In summary, a facile method for preparing BC carbohydrate chips has been established and demonstrated to be able to improve the
glycosylation level of the QCM carbohydrate chip surfaces. The key feature of the method is the use of branch alkynyl molecule
modified surfaces to achieve higher binding of lectin than the linear surfaces, where the branch surfaces not only supplies more specific
bingding site but also reveals significant cluster effect. Simultaneously, the azide-carbohydrates modified branch alkynyl surfaces were
evaluated using QCM biosensor, allowing for real-time and label-free analysis of the association and dissociation phases of
biomolecular interactions. Remarkably, the BC carbohydrate chips showed stronger binding capacity with lectins, and exhibited
significant stability during the subsequent interaction detection and regeneration steps. This work provides a good example for
preparing the QCM BC carbohydrate chips, which have great potential applications in glycobiology and biomedicine.
Acknowledgment
We thank the National Natural Science Foundation of China (Nos. 31270861 and 21572181) for financial support.

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400
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Regeneration
Regeneration
Regeneration
b)
a)
FITC-ConA
Con A
Con A
5000 6000
Con A
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1000
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3000
4000
200
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Time (s)
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Time (s)
Fig. 1. a) Frequency shift recorded during the binding and regeneration of Con A (200 μg/mL) to immobilized BC mannose chips; b)
Specificity of the binding of FITC-Con A (200 μg/mL) to BC-Mannose chip. Fluorescent images of both BC-Mannose chip (in black) and
FITC-Con A (in green) were taken before (left hand side) and after (right hand side) injection of FITC-Con A. (Olympus BX53); The scale
bars stand for 20 μm.
80
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PNA Concentration
Fig. 2. a) The binding of PNA to SC (black line) and BC (red line) galactose chip; Con A to SC (blue line) and BC (green line) mannose chip.
Lectins at 0.2 mg/mL were introduced onto the chip surfaces and the interactions were monitored in a period of 85 s for association and 315 s
for dissociation; b) Comparison of the binding of different concentration of PNA to SC and BC galactose chip. PNA at 0.05, 0.2, 0.5 mg/mL
was injected to the SC and BC galactose chip, respectively; All experiments were performed at a flow rate of 25 μL/min using PBS running
buffer (pH 7.4) under 25 oC. The maximal frequency shift observed at the end of the injection phase was recorded.
70
WGA (0.2 mg/mL)
Con A (0.2 mg/mL)
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Fig. 3. General binding curve of Con A and WGA to BC mannose functionalized surface. Con A, which recognizes and binds to mannose
specifically, showed a high binding to the BC mannose chip. As comparison, WGA, which recognizes and binds to N-acetylglucosamine
specifically, showed a very low binding to the BC mannose chip.

Scheme 1 General surface functionalization procedure. The polymeric surfaces were spin-coated with a solution of NHS-activated compound
2 and irradiated with UV-light (254 nm) for 5 min. The NHS-activated ester surfaces were linked through an amidation process with the
deprotection branch alkynyl-terminated 8 to produce the alkyne functionalized surfaces. The alkyne surfaces were then differentiated with
azide-functionalized carbohydrates (11, 13) through click chemistry.