A renewable, chemoselective, and quantitative ligand density microarray for the study of biospecific interactions

Article abstract of DOI:10.1039/c0cc01509a

Novel renewable microarray technology has been developed to immobilize and release carbohydrates and proteins from self-assembled monolayers (SAMs) of electroactive quinone-terminated alkanethiolates on gold surfaces. This method may be applied to a variety of research fields for use in biosensor technology and the generation of renewable and tailored microarrays for biospecific cell-based assays.

Full text of DOI:10.1039/c0cc01509a

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A renewable, chemoselective, and quantitative ligand density microarray
for the study of biospecific interactionswz
Abigail Pulsipher and Muhammad N. Yousaf*
Received 21st May 2010, Accepted 12th October 2010
DOI: 10.1039/c0cc01509a
Novel renewable microarray technology has been developed to
immobilize and release carbohydrates and proteins from self-
assembled monolayers (SAMs) of electroactive quinone-
terminated alkanethiolates on gold surfaces. This method may
be applied to a variety of research fields for use in biosensor
technology and the generation of renewable and tailored
microarrays for biospecific cell-based assays.
Herein, we present a renewable microarray technology to
immobilize and release carbohydrates and proteins through
control of electrochemical potential and pH. Monosaccharides
were synthesized to incorporate oxyamine functionality for
chemoselective conjugation to the ketone in electroactive
quinone-terminated SAMs on gold. Sugar immobilization
and release was monitored, and the SAM and ligand density
were precisely controlled and quantified by cyclic voltammetry.
In addition to the formation of mixed hydroquinone- and
tetra(ethylene glycol)-terminated SAMs that provide
resistance to nonspecific lectin adhesion, microarray
technology allows for control over the orientation and
spatial distribution of SAM molecules and ligands, criteria
that are both essential for studying the biospecific interactions
Microarray-based technologies serve as high-throughput
analytical tools for the evaluation of
a variety of
biomolecular interactions, providing a platform for rapid
analysis and automation, and requiring low analyte and
1
reagent volumes. Such technologies have incorporated the
use of functionalized self-assembled monolayers (SAMs) of
alkanethiolates on gold surfaces for the preparation of DNA,
peptide, and carbohydrate microarrays to conduct studies in
3
,8–11
between molecules.
Different microarray densities of
quinone-terminated SAMs were generated, and a number of
oxyamine-containing sugars were immobilized. The resultant
oxime linkages were characterized by electrochemistry, X-ray
photoelectron spectroscopy, and contact angle, and lectin-
carbohydrate binding interactions were observed by
fluorescence microscopy. This method may be used in
conjunction with the tailoring of other biomolecules such as
DNA, peptides, and phospholipids for studies in genomics,
transcriptomics, and proteomics.
2
genomics, proteomics, and glycomics, respectively. SAMs can
also be specifically tailored to present a number of derivatized
ligands from the surface through covalent modification,
3
affording a well-defined, biocompatible system. Current
chemoselective conjugation strategies include Staudinger
8
4
5
6
7
ligation, Click chemistry, maleimide, amide, Diels–Alder
9
and oxime chemistry. One methodology in particular that uses
electroactive, hydroquinone-terminated SAMs, has proven to
be a powerful approach toward the immobilization and release
The general strategy for developing a chemoselective,
renewable, and quantitative, ligand density microarray for
biological analysis is described in Fig. 1. We have chosen to
1
0
of ligands. This strategy enables the quantification of the
amount of attached ligand, as well as provides the ability to
monitor the reaction progression in real-time and calculate
use different ratios of hydroquinone-(H
Q, 5, supporting
SH,
2
1
1
kinetic rates.
information, Scheme S2) and tetra(ethylene glycol)-(EG
4
Carbohydrates represent the most abundant class of organic
compounds found in living organisms and are known to
mediate several biological processes, including neural
development, signal transduction, viral invasion, and cancer
4, supporting information, Scheme S2) terminated
alkanethiolates to construct our microarrays. Alkanethiolate
solutions are transferred to a bare gold substrate using an
automated microarray method, and microspots of SAMs are
rapidly formed. The remaining unpatterned surface is then
1
2
metastasis. Microarray technologies have been designed to
perform high-throughput screenings of various vital events
including enzymatic glycosylation, sugar identification, and
1
3
bacterial detection. A method based on a renewable and
quantitative platform that can carry out similar analyses and
also prove to be compatible with numerous biological systems,
including carbohydrates, would greatly benefit a number of
industries and tremendously cut down costs, materials,
and time.
Fig. 1 Strategy to develop a renewable and quantitative, ligand
density microarray for biological analysis. (1) Different mixtures of
alkanethiolates are transferred from a 96-well microplate to a bare gold
substrate via automated array technology, and microspots of SAMs
University of North Carolina at Chapel Hill, Department of
Chemistry, Chapel Hill NC, USA. E-mail: mnyousaf@email.unc.edu;
Fax: 91 9962 2388; Tel: 91 9843 3203
w Electronic supplementary information (ESI) available: Experimental
details, carbohydrate synthesis, XPS, and contact angle data. See DOI:
4
are formed. (2) The substrate is backfilled with EG SH. (3) Biological
ligands are then immobilized to the array. (4) The corresponding
bioassay is performed, (5) analyzed, and (6) the original microarray
is regenerated for a subsequent cycle of ligand immobilization and
analysis.
10.1039/c0cc01509a
zC hT ehmi sCa or mt i cml e. is part of the ‘Emerging Investigators’ themed issue for
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Chem. Commun., 2011, 47, 523–525 523

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2
trace, Fig. 2B), indicating full oxime (H Qox/Qox) bond
formation. It is also important to note that the sugar-Q
reaction was also monitored at various time points to
determine the percentage of ligand bound (supporting
information, Fig. S2), as well as the approximate rate of
product conversion (data not shown). To release the ligand
2
and regenerate the original H Q/Q-terminated SAM (blue,
Fig. 2C), potential was applied for 60 cyclic scans, or 15 min,
under reducing conditions (PBS, pH = 7). Approximately
one-third of the carbohydrate ligand was released following
2
0 scans, as shown in the green trace in Fig. 2C, with the
presence of H Q/Q production.
2
Additional surface characterization was conducted to
confirm that consistent SAMs of H Q/Q were being formed
2
and that oxyamine-functionalized carbohydrates were being
immobilized after reaction. The static contact angle of water
(
10 mL) was measured on bare gold substrates, as well as on
surfaces with SAMs of Q-, H Q-, and glucose-presenting
2
groups (supporting information, Fig. S1B). There was a
significant decrease in the contact angle, from 64.191 to
33.321, for unreacted Q-terminated SAMs and Q-containing
Fig.
distinctive redox peaks (blue) of a H
30 mV and [H
Q] = ꢀ24 mV. Once an oxyamine-containing
carbohydrate is immobilized, (B) the redox peaks (green) shift to
ox] = 610 mV and [H ox] = 283 mV, indicating the presence of
2
Electrochemical characterization. (A) This CV shows
2
Q/Q-terminated SAM at [Q] =
6
2
2
SAMs reacted with glc-ONH (90 mM in MeOH, 4 h),
[
Q
2
Q
respectively. The large difference in contact angle is
accounted for in the increase of wettability and cohesive
forces between the water and hydrophilic hydroxyl groups,
an oxime (ox) bond. (C) The bound carbohydrate (green) can be
released by electrochemical reduction (20 scans), and the original
2
H Q/Q SAM (blue, 60 scans) can be regenerated. The two blue
indicating
there
was
successful
attachment
of
traces in A and C show the same peaks, which correspond to H
before carbohydrate immobilization and after its release.
2
Q/Q
carbohydrate–oxyamine ligands. X-ray photoelectron
spectroscopy (XPS) was also performed to confirm oxyamine
conjugation to substrates bearing Q-terminated SAMs. The 1s
nitrogen peak observed at 398 eV, corresponds to the presence
4
backfilled with EG SH, oxidized to reveal Q, and biological
ligands are then reversibly conjugated to the microarray.
Depending on the particular biomolecule presented from the
substrate, the corresponding bioassay can be performed and
analyzed. The original microarray can then be regenerated for
a subsequent round of immobilization, analysis, and release.
The surface chemistry and renewable nature of this microarray
technology is illustrated in and characterized by CV (Fig. 2).
Mixed SAMs of H Q/EG SH are electrochemically oxidized
of glc-ONH
2
immobilized to the surface (green trace,
supporting information, Fig. S1A), while there is no nitrogen
present on substrates with Q-terminated SAMs, as shown by
the black trace. SAM and ligand density were also quantified
by CV (supporting information, Fig. S2).
Although our renewable and quantitative ligand microarray
strategy is designed to be extended toward a number of
biological systems, we chose to represent the biospecific
interactions between carbohydrate ligands and lectins. Fig. 3
displays three cycles of ligand immobilization and release.
Automated microarray technology was utilized to generate a
2
4
(
4
1 M HClO , pH = 0, scan rate of 100 mV/s) to Q-terminated
groups which can then chemoselectively react with oxyamine-
functionalized compounds. In this particular study, our
ligands of interest include galactose-, glucose-, and mannose-
oxyamine (Gal-ONH
2
, 1; Glc-ONH
2
, 2; and Man-ONH
2
, 3,
24 x 24 microarray of 100 mm in diameter spot-arrays of H
Eg SH-containing alkanethiolates (1 : 10, 1 mM in EtOH) on a
bare gold substrate. Glc- and gal-ONH (10 mL, 90 mM in
2
Q/
respectively, supporting information Scheme S1 and S2).
4
After the monosaccharide is covalently bound to the surface
through an oxime linkage, its corresponding lectin, or sugar-
binding protein, is added. The protein then adheres to and
interacts with its recognition signal and is able to be
characterized by fluorescence microscopy. To regenerate the
surface for another subsequent cycle of ligand immobilization,
sugars and lectins are released by applying a constant potential
under reducing conditions (PBS, pH = 7, 60 cyclic scans,
2
MeOH) were transferred to the microarray of SAMs at
random using the same program. The surface was then
backfilled with EG SH (1 mM in EtOH, 16 h), and a mixture
4
containing fluorescently-conjugated Concanavalin A (ConA,
1 mg/mL in H O, 2 h) and Peanut Agglutinin (PNA, 1 mg/mL
2
in H O, 2 h) was allowed to bind. ConA and PNA are lectins,
2
known to specifically recognize and bind to glucose and
galactose residues, respectively. Carbohydrate ligands and
lectins were then released from the substrate after 60 cyclic
scans of electric potential (PBS, pH = 7, 15 min), renewing the
1
5 min). The result consists of a reduced alcohol and renewed
Q-terminated SAM; this cycle may be performed again. As
H
2
previously mentioned, CV was employed to characterize ligand
immobilization and release from Q-terminated SAMs on gold
original mixed H
which was also determined by electrochemistry. Glc- and
gal-ONH were again randomly delivered to the SAM
2 4
Q/EG SH-terminated SAM microarray,
(
Fig. 2). Distinctive H
2
Q/Q redox peaks were observed at ꢀ24
and 630 mV, respectively (blue trace, Fig. 2A). Following four
hours of sugar–oxyamine conjugation (90 mM in MeOH),
peaks were completely shifted to 283 and 610 mV (green
2
microarray, followed by reaction with ConA and PNA for
two more cycles. Carbohydrate–protein binding was then
5
24 Chem. Commun., 2011, 47, 523–525
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and galactose) were synthesized to incorporate oxyamine
functionality and tethered to the surface. The oxime linkages
were confirmed and characterized by CV, XPS, and contact
angle. By applying constant potential (15 min) under reducing
conditions (PBS, pH = 7), sugar ligands were released,
renewing the original H Q-terminated array for another round
2
of immobilization and release. The extent of the molecule
reaction, as well as its release and amount bound was
monitored and quantified by CV. Fluorescently-conjugated,
carbohydrate-binding proteins ConA and PNA were added to
2
the microarray following man-, glc-, and gal-ONH reaction,
and substrates were visualized by fluorescence microscopy.
The images displayed distinctly colored spots, corresponding
to specific signal (sugar) recognition, indicating successful
observation of ligand–receptor interactions on our platform.
This method may be applied to a variety of scientific fields for
use in biosensor technology and the generation of renewable
and tailored microarrays for biospecific ligand–receptor
interaction assays.
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2
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4
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8
2
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2
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2 4
used to transfer different mixtures of H Q- and EG SH-
terminated alkanethiols to a bare gold substrate, resulting in
the tailoring of SAM microspots (100 mm diameter). Following
electrochemical oxidation of H Q to the corresponding Q,
2
1
oxyamine compounds were reacted and immobilized to the
microarray. A number of monosaccharides (mannose, glucose,
This journal is c The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 523–525 525