Biotechnol. J.

2009, 4, 15131529

DOI 10.1002/biot.200900162

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Review

Chemical strategies for immobilization of oligonucleotides

Dalip Sethi1, R. P. Gandhi1, Pradeep Kumar1 and Kailash Chand Gupta1,2
1 Institute
2 Indian

of Genomics and Integrative Biology, Delhi University Campus, Delhi, India

Institute of Toxicology Research, Lucknow, India

The development of oligonucleotide-based microarrays (biochips) is a major thrust area in the rapidly growing biotechnology industry, which encompasses a diverse range of research areas including genomics, proteomics, computational biology, and pharmaceuticals, among other activities. Microarray experiments have proved to be unique in offering cost-effective and efficient analysis at the genomic level. In the last few years, biochips have gained increasing acceptance in the
study of genetic and cellular processes. As the increase in experimental throughput has posed
many challenges to the research community, considerable progress has been made in the advancement of microarray technology. In this review, chemical strategies for immobilization of
oligonucleotides have been highlighted with special emphasis on post-synthetic immobilization
of oligonucleotides on glass surface. The major objective of this article is to make the researchers
acquainted with some most recent advances in this area.

Received 15 July 2009

Revised 1 September 2009
Accepted 7 September 2009

Keywords: Covalent attachment Hybridization Immobilization Oligonucleotides Photoactivation

1 Introduction
The advent of microarray technology for detection
of biomolecular interactions (involving nucleic
acids, peptides/proteins or enzyme-substrate, etc.)
has had an impact on research in diagnostics in the
last decade since it has provided researchers/industries with a tool for rapid sample screenings. In
the beginning, the microarrays (also called biochips) were mostly exploited for monitoring mRNA
expression [13], but the interest soon spread to
other important applications such as single nucleotide polymorphism (SNP) detection, mutation
analysis, genomic or transcriptomic variations,
genotyping of individuals, protein-DNA interaction

Correspondence: Dr. Kailash Chand Gupta, Institute of Genomics and

Integrative Biology, Mall Road, Delhi University Campus, Delhi 110 007,
India
E-mail: kcgupta@igib.res.in
Fax: +91-11-27667471
Abbreviations: NHS, N-hydroxysuccinimide; SAM, self-assembled monolayers; SNP, single nucleotide polymorphism; TFP, tetrafluorophenyl

2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

analysis, etc. [410]. This technology has provided

an alternative analytical device that allows parallel
and simultaneous detection of several thousands of
targets within one sample. The basic concept of
specific base-pair interactions within the DNA or
DNA-RNA hybrids with labeled complementary
strands makes the technology a powerful analytical
tool for monitoring whole genomes. Typically, a
DNA chip (or biochip) consists of a solid surface,
which is functionalized with an array of probe sequences. The principle of its operation involves
three main steps, functionalization (i.e., the attachment of different probes onto different positions),
hybridization (i.e., exposure of the probes to sample solution consisting of targets), and imaging/
readout (i.e., scanning to detect biomolecular interactions). Over the last few years, tremendous improvements in biochip technology have addressed
the problems of low hybridization efficiency, poor
sequence discrimination, long process time and tedious protocols that were inherent in the previous
technologies [1114]. Figure 1 illustrates a flow diagram for construction and use of biochips.
In principle, the efficiency of an oligonucleotide
array depends mainly upon the type of surface, the
sequence of capture probes, size of the arrayed

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Figure 1. Schematic
representation of steps
involved in construction
of biochips.

physical attributes of the surface of choice. Considering the importance of the surface material used
for the fabrication of oligonucleotide-based biochips, a number of surface materials, i.e., nylon, nitrocellulose, glass, polyacrylonitrile, polypropylene,
polystyrene, silicon, teflon, gold, polypyrrole, polyurethane, polymethylmethacrylate, polyethylene
glycol grafted silica surfaces and PMMA, have been
tested [15, 1719]. Of these, glass is the most commonly used material in the form of microscopic
slides owing to its unique advantages [20]: (i) it can
easily be modified via a silanization reaction to
generate desired functional groups (amino, mercapto, carboxyl, epoxy, aldehyde, etc.), (ii) it has excellent chemical resistance against solvents and
can sustain high temperatures and stringent washings, (iii) its flat and non-porous characteristics
help in keeping the hybridization volume to a minimum, and (iv) its low background fluorescence
does not interfere during the imaging/scanning
process, and, therefore, it can be subjected to laser
scanning for visualizing the spots on the surface.

2
probes, immobilization reaction (tethering to the
surface), and the methods of hybridization and detection. Alternatively, the technique involves covalent attachment of the known oligonucleotide sequences at discrete locations on the surface of
choice [15]. Likewise, in expression profiling microarrays, several discrete DNA sequences are
spotted onto a polymeric surface and subsequently
hybridized to fluorophore-labeled targets. Among
various fluorophores, the most commonly used are:
Cy3, Cy5, HEX, TAMRA, TET, etc. Recently, semiconductor nanocrystals (quantum dots, QDs) have
been introduced as sensitive alternatives to organic dyes due to their nanometric dimensions [16].
These represent a new type of fluorescent label
having some distinct advantages over the classical
fluorescent dyes, i.e., high extinction coefficient,
high quantum yields and resistance to photobleaching, which, in turn, increase the sensitivity of
detection. In addition, these can be excited at a single wavelength (emission at different wavelengths)
depending upon the size of the dots. Choice of a
surface material also plays an important role in
manufacturing of biochips. Homogeneity, stability
upon storage and reactivity toward modified/unmodified oligonucleotides significantly affect the
overall performance of a biochip. Consequently, hybridization of nucleic acids targets to immobilized
probes depends on a number of chemical and

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Fabrication of biochips

In general, fabrication of oligonucleotide-based

biochips can be divided into two broad categories:
on-chip synthesis and spotting off-chip synthesized oligonucleotides (i.e., bulk synthesis followed
by deposition on the chip).The first one is based on
in situ synthesis of oligonucleotides following conventional or photolithographic technique at the
pre-determined sites using conventional or photolabile protection groups at 5-end of nucleosides
[2024]. The second method relies on the post-synthesis immobilization of oligonucleotides. Figures
2a and b differentiate two methods of in situ generation of oligonucleotide-based biochips. Conventional oligonucleotide synthesis in conjunction
with ink-jet printing [19] or microfluidic channeling techniques [25] has also been tried.

2.1 In situ synthesis of oligonucleotides

In a simple approach following conventional
oligonucleotide synthesis, glass microslides were
treated with a solution of 3-glycidyloxypropyltrimethoxysilane to generate epoxy functions on the
surface of the substrate (Fig. 2a), which reacted
with hexaethylene glycol linker to provide hydroxyl groups on the surface [20]. Oligonucleotide synthesis was carried out at the desired locations using a conventional phosphoramidite approach and
deprotection was achieved following a standard
method. The strategy resulted in the oligonu-

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cleotide arrays associated with the truncated sequences, which might affect the performance of the
chip during the evaluation process.
Around the same time in 1994, another approach towards synthesis of oligonucleotides on
glass surfaces was developed, which was based on
an in situ photolithographic technique (Fig. 2c). In
this technique, linker molecules modified with
photochemically removable protecting groups
were attached to a glass substrate and light was al-

Figure 2. In situ synthesis of oligonucleotides by (a) conventional method.

(b) photolithographic technique, and (c) fabrication of oligonucleotide
microarrays using photolithographic method.

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lowed to pass through a photolithographic mask

with a high spatial resolution to specific sites on the
surface to produce localized photodeprotection.
The first of a series of chemical building blocks, 5protected deoxyribonucleosides, was incubated
with the surface, and the chemical coupling occurred at those sites that were irradiated in the preceding step. In the next step, light was directed to
different regions of the substrate with a new mask
to generate a second pattern of hydroxyl groups,
and the chemical cycle was repeated. Likewise, patterns were generated and the oligomer chains extended. To make this method more attractive, a
number of photolabile-protecting groups were introduced, which could be removed cleanly and rapidly from the 5_-hydroxyl functionality [2628]
(Fig. 3). Basically, the lability of a photocleavable
group depends on the wavelength and polarity of
the solvent employed. In addition, the success of
this method relies upon three factors: the accuracy
in alignment of the masks, the efficiency of the removal of photo-protecting groups and the yields of
the phosphoramidite coupling step. This method is
of advantage in generating high density (106 sequences/cm2) microarrays without preliminary efforts in synthesis and maintenance of a vast library
of modified oligodeoxynucleotides. An added feature of this method is the construction of small arrays. The smaller the size of the array, the smaller
the volume needed for hybridization. The method
has a disadvantage in that the coupling yields
(~95%) are lower than in the conventional methodology (>99%). Thus, the yield of a 20-mer will be
about 36% as compared to that achieved in conventional synthesis (>80%). In addition, this does not
allow quality control and purification of generated
arrays. Other limitations of the method are the re-

Figure 3. Some of the commonly used photolabile protecting groups for

protection of 5-hydroxyl group in deoxyribonucleosides. NPPOC: 2-(2-nitrophenyl)-propyloxycarbonyl; MeNPPOC: 2-(3,4-methylenedioxy-2-nitrophenyl)-propyloxycarbonyl; MeNPOC: 1-(3,4-methylenedioxy)-6-nitrophenyl ethyloxycarbonyl; NVOC: 6-nitroveratryloxycarbonyl.

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quirement of expensive photomasks, sophisticated

instrumentation, and contamination of desired
length oligonucleotides with truncated sequences.
In an alternative approach, light-assisted synthesis
of high density microarrays in a 5-3 direction has
also been demonstrated [29]. It allows parallel
genotyping and sequencing on the surface itself, as
3-end is available for enzymatic reactions. However, the yields of this 5-3 oligonucleotide synthesis
is lower than that of the 3-5 directed synthesis.
Recently, Phillips et al. [30] reported light-directed in situ synthesis of oligonucleotide arrays on
carbon-based substrates and compared their stability with respect to temperature, basic conditions
concerning serial hybridization and dehybridization efficiency with glass-based microarrays. The
protocol involves UV-light induced functionalization of glassy carbon plates with 9-decene-1-ol to
generate free hydroxyl groups on the surface 3 followed by synthesis using the photolithographic
technique (Fig. 4). The stability of the resulting
oligonucleotide arrays 4 was found to be of much
higher order than in those prepared on glass 2.The
protocol demonstrated that the constructed arrays
could be used repeatedly in applications involving
high temperatures and extremes in pH.

2.2 Post-synthesis immobilization of

oligonucleotides
An alternative way of constructing a microarray
(biochip) is based on the deposition of a small
amount of pre-synthesized oligonucleotides by microdispensing. Unlike on-chip synthesis, this
method not only provides a possibility of cleaning
up the oligonucleotides prior to deposition but can
also be used to generate PCR products- or com-

Figure 4. Functionalized glass and carbon substrates for light-directed

DNA synthesis.

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Biotechnol. J. 2009, 4, 15131529

plete genome-based arrays. Therefore, patterning

of oligonucleotides on the surface of choice via this
method is preferred for many research applications, where low-to-medium density arrays are required [3141]. In this protocol, the construction of
biochips depends on a number of parameters: (i)
choice of appropriate substrate and its functionalization, (ii) synthesis of appropriately modified/unmodified oligonucleotides, (iii) chemistry of attachment, (iv) the thermal and chemical reactivity, (v)
optical and chemical properties of the substrate
material, (vi) binding capacity of the substrate, and
(vii) hybridization conditions and detection techniques.
As most of the features mentioned above have
already been reviewed in varying detail previously
[4249], the present review mainly focuses on recent developments and strategies involving immobilization of oligonucleotides for fabrication of
biochips. The development of efficient chemistries
for manufacturing spatially resolved microarrays
on a glass surface is essential for the realization of
potential of DNA biochips. Besides polymer matrix
used for the construction of biochips, the reliability and integrity of the hybridization reactions are
highly dependent on a number of factors such as
attachment chemistry, nature and length of linkers
tethering oligonucleotides to the substrate surface,
and the hybridization and washing conditions. In
developing a useful and reliable chemistry for producing DNA arrays, the accessibility and functionality of the surface-bound DNA, the density of attachment, the stability (thermal and pH) of the array, the reproducibility of the attachment chemistry, and the conformity of the immobilized sequences are all critical. Considering the importance of these parameters, this section focuses on
various types of interfacial designs that permit the
attachment of pre-synthesized oligonucleotides
onto the glass surface. As mentioned earlier, for
patterning of oligomers, glass is the surface of
choice.Therefore, the main emphasis here is on the
chemistry used for the attachment of oligonucleotides involving glass as the solid surface for
fabrication of biochips.
Currently, two different techniques are being
used for spotting pre-synthesized oligonucleotides
onto glass substrates: contact and non-contact
printing. In the first one, pins or needles are dipped
into the sample solution and brought in contact
with the substrate surface [50]. A small amount
(~0.21.0 nL) is delivered onto the surface.After the
printing process, the pins/needles have to be
cleaned to avoid carry-over. The method, although
widely used, suffers from certain drawbacks including low reproducibility, random morphology of

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the spot and deformation in the shape of pins/needles during repeated contacts to the surface. To
overcome these limitations, the non-contact printing was evolved, which relies on the Piezo effect
[19]. In this technique, a microfluidic nozzle is activated to deliver droplets of a size less than 1 nL
from a certain distance. A reservoir holds a large
volume of the probe solution so that there is no
need to refill after each dispensing.
The immobilization of pre-synthesized oligonucleotides on the substrate can be achieved in the
following two ways: (i) via non-covalent interactions between the oligonucleotides and the surface,
and (ii) by covalent attachment of oligonucleotides
onto the surface of the substrate.

2.2.1 Attachment of oligonucleotides through noncovalent interactions

Non-covalent attachments were mostly explored in
early development of arraying [51]. Li et al. [52]
were the first to study the interaction of biotin with
streptavidin or other avidin species. Subsequently,
many research groups applied the simple concept
of electrostatic interactions between a positively
charged poly-lysine surface and negatively charged oligonucleotides for immobilization, thus eliminating the chemical manipulations [53] (Fig. 5).
These arrays, after drying, were subjected to capping steps, first with acetic anhydride and then
with succinic anhydride to produce a slightly anionic surface making it unsuitable for nonspecific
adsorption. In this protocol, the interactions take
place at several points along the oligonucleotide
backbone, so that the probe sequence is attached to
the glass at several points [54] (Fig. 5).Table 1 summarizes the most cited protocols for the construction of oligonucleotide microarrays via a non-covalent method of attachment. The projected strategy
is relatively simple; however, it suffers from lower
hybridization efficiency, as ionic interactions influence the probes to lie flat on the surface, i.e., they
are less accessible to the hybridization assay. Furthermore, the high background (signal-to-noise ratio) as a consequence of nonspecific adsorption in
this method ultimately paved the way to alternative
covalent linkages for immobilization of oligonucleotides.This protocol is now the preferred one for
selectively linking probes onto a surface of choice.

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These covalently attached probes have been found

to be more stable and result in improved and reproducible hybridization performance.

2.2.2 Covalent mode of attachment of oligonucleotides

This section focuses on the efficient chemistries
developed recently for the attachment of oligonucleotides onto the glass surface via thermochemical as well as photochemical reactions.

Thermochemical methods
In this strategy, chemical functionalities are introduced in the oligomers sequence during the conventional automated synthesis, which form covalent bond under thermal conditions with the complementary groups generated on the surface in the
presence/absence of a coupling agent. Generally,
linkers bearing reactive groups are added either at
the 3-end, internal positions, or at 5-end of the
oligomers. Literature records a number of attachment methods involving organic-organic and inorganic-organic interactions, which vary widely in
chemical mechanism, ease of use, probe density
and stability. A distinguishing feature of the thermochemical method is a single point of attachment
that allows the entire oligonucleotide sequence to
be accessible for hybridization, to withstand high
temperature and salt concentrations, often required during the stringent washing conditions in
subsequent steps of microarray processing. In addition, the methodology allows attachment of the
probes in a regioselective manner with a good surface coverage as well as retention of full activity of
the probes.
Some of the well-studied methods include the
activation of the substrate with standard bifunctional reagents, such as phenylene diisothiocyanate (PDITC) for capturing amine- or thiolmodified oligonucleotides [55], cyanuric chloride
for reaction with aminoalkylated probes [56], succinimidyl 4-(maleimidophenyl)butyrate (SMPB)
for mercaptoalkylated oligonucleotides [57], N(2-trifluoroethanesulfonatoethyl)-N-(methyl)-triethoxysilylpropyl-3-amine (NTMTA) for aminoand mercaptoalkylated oligonucleotides [58], etc.
Choithani et al. [59] reported the synthesis of a
glass-specific heterobifunctional reagent, N-(3-triTable 1. Construction of biochips following non-covalent interactions

Figure 5. Immobilization of negatively charged oligonucleotides onto positively charged glass slide via electrostatic interactions.

2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Functionality
on glass surface

Functionality on
oligonucleotides

Chemistry

Reference

Streptavidin
Poly-L-lysine-glass
Aminopropyl-glass

Biotin
Phosphodiester
Phosphodiester

Non-covalent
Electrostatic
Electrostatic

[52]
[53]
[54]

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Figure 6. Immobilization of modified oligonucleotides using TPMH.

ethoxysilylpropyl)-6-(N-maleimido)-hexanamide
(TPMH) 5, for the preparation of oligonucleotidebased biochips (Fig. 6). The triethoxysilyl function
is specifc toward virgin glass surfaces and the
maleimide function undergoes conjugate addition
to 3- or 5-mercaptoalkyl-modified oligonucleotides. Immobilization of oligonucleotides has been
demonstrated via two routes: through surface activation and through triethoxysilyl-oligonucleotide
conjugate formation, under microwaves. Subsequently, the fabricated chips 6 were successfully
employed in detection of match/mismatches in the
targets based on variation in the fluorescence intensity. The perfect matched duplex gave the highest fluorescence intensity, while the single and
double mismatched duplexes exhibited fluorescence in decreasing order.
In another report, self-quenched DNA hairpin
probes were immobilized on a glass surface [60]
using N-(3-triethoxysilylpropyl)-4-(isothiocyanatomethyl)cyclohexane-1-carboxamide
(TPICC)
(Fig. 7). An advantage of the protocol is that in the
closed state (i.e., hairpin conformation), fluorescence intensity gets quenched due to presence of
guanosine residues in close vicinity of the fluorophore, while on hybridization with a perfectly
matched complementary target, fluorescence gets
restored. The report includes comprehensive studies, i.e., thermal and pH stability, and evaluation of
the performance of the constructed microarrays by
detecting single nucleotide variations.
Recently,Wong and Krull [61] demonstrated improved selectivity of covalently immobilized mixed
films of oligonucleotides and oligomer components
on glass and silicon substrates for detection of multiple base-pair mismatches. In this, the oligomers,

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Figure 7. Immobilization of DNA hairpin probes onto glass surface using

a heterobifunctional reagent, TPICC.

attached through one end, surround the immobilized oligonucleotide probes in such a way that the
probes are isolated from one another and the surface. The probes were tethered to the surface utilizing the reagent, succinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate (sulfo-SMCC).
The strategy significantly reduced nonspecific adsorption, allowed reusability and improved the selectivity of hybridization.
Other efficient protocols comprise the surface
activation with maleimide 7, providing activated
double bonds that are reactive towards thiolated
oligonucleotides 8 [62], and the substitution of a
thiolated substrate 9 with an acrylamide-capped
oligomers 10 (or vice versa) [63] (Fig. 8). Other elegant options include the use of combinations, such
as organic-inorganic interactions involving thiolated oligonucleotides on a gold surface [64], coordination complexes employing phosphorylated
oligonucleotides 13 on a zirconylated surface 14
(Fig. 9), etc. [65]. Lane et al. [66] revealed that oligonucleotide probes bearing a poly(dG) spacer, immobilized on a zirconium phosphonate surface, led
to a higher target capture, compared to probes with
either no spacer or a different polynucleotide
(polyA, poly C and polyT) spacer, during hybridization with complementary targets. The higher probe
coverage, due to formation of G-quadruplexes, resulted in improved affinity of the probes for the zirconium surface.

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Figure 9. Organic/inorganic surfaces for oligonucleotide arrays, attachment of phosphorylated probes via coordinate covalent bond to a zirconylated surface.

Figure 8. Immobilization of modified oligonucleotides onto functionalized

surfaces.

Activation strategies involving coupling or condensing reagents have also been used for covalent
binding of probes to the surface. However, these
strategies often result in an irreproducible and unreliable immobilization performance. A simple and
direct chemistry, which does not require an additional activation/condensing reagent, is always
preferred.Therefore, the chemistry used for immobilizing oligonucleotides is derived, in many cases,
from rather straightforward organic reactions. In
one of the previous studies, epoxy functions were
generated on the glass microslide by reaction with
a 3% solution of 3-glycidyloxypropyltrimethoxysilane 16 [67], which were subsequently used for the
immobilization of the amine-modified oligonucleotides 12 in 0.1 M KOH.
The epoxylated surface generated above was
used for amine-modified oligonucleotides and subsequently employed as a universal type of support
(Fig. 10) [6771], where nucleophilic and electrophilic group bearing oligonucleotides, i.e., mercaptoalkyl- 8, aminooxyalkyl- 17, phosphoryl- 18
and thiophosphorylated 19 oligonucleotides were
immobilized with higher immobilization and hybridization efficiency. In a recent variant of the
method, a facile and efficient attachment of oligonucleotides modified with phosphates at their 3or 5-end to an epoxylated glass slide was demonstrated. The immobilization was effected under
both microwaves and thermal conditions. The immobilization and hybridization efficiency of the
method were found to be ~23% and ~36%, respectively. The constructed microarrays were then successfully employed for discrimination of nucleotide
mismatches. Recently, Gupta and associates [70, 71]

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reported the attachment of aminooxyalkyl- and

mercaptoalkyl-oligonucleotides onto an epoxy surface with high immobilization and hybridization
efficiencies. Aminooxyalkyl-modified oligonucleotides were immobilized at pH 6 and 9 with immobilization efficiency of ~18% and ~22%, respectively (with hybridization efficiency of ~32% at
pH 6). A major advantage of the chemistry is that,
at pH 6, the aminooxyalkylated oligonucleotides
can be immobilized selectively onto the epoxylated
surface in the presence of aminoalkylated probes.
In the same way, mercaptoalkyl-modified oligonucleotides were attached to the epoxy surface under
the influence of microwaves in just 12 min with 24%
immobilization efficiency. The immobilized probes
were found to be stable over a wide range of pH.
Stabilization of the immobilized probes on glass
surface is also an important factor for the generation of reliable biochips. The modified probes immobilized following the projected strategy were
found to be heat stable and displayed stability in
the order phosphoryl < aminooxyalkyl < mercaptoalkyl.
In an alternative approach, Sethi et al. [72] unveiled a facile protocol for the construction of an
oligonucleotide microarray involving immobilization of oligonucleotide-trimethoxysilyl conjugates
20 onto virgin glass microslides 21 (Fig. 9). In this
protocol, aminoalkyl, mercaptoalkyl and phosphorylated oligonucleotides were reacted first with
glycidoxypropyltriethoxysilane (GOPTS) and the
resulting conjugates were directly immobilized
onto the virgin glass surface by making use of
silanization chemistry. The projected strategy reflected high immobilization efficiency (~3640%)
and signal-to-noise ratio (~98), and hybridization
efficiency (~3235%). The constructed microarrays
were then used for detection of base mismatches
on the basis of variation in fluorescence intensity.

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Figure 10. Covalent attachment

of aminoalkyl, aminooxyalkyl,
mercaptoalkyl, phosphoryl and thiophosphorylated oligonucleotides to
virgin/epoxylated glass surface.

The single-step reaction for the formation of conjugates with the commercially available reagent
(GOPTS), omission of capping step and surface
modification, and efficient immobilization of oligonucleotides onto the virgin glass surface are
some of the key advantages of the projected strategy.
Recently, Rozkiewicz et al. [73] reported a
straightforward method to immobilize acetylenemodified oligonucleotides 22 on azide-terminated
glass substrate 23 by click chemistry via microcontact printing (Fig. 11). The immobilization chemistry is irreversible and covalent, resulting in a stable attachment of oligonucleotides 24, which were
subsequently used for hybridization with fulllength complementary targets. The strategy involves a single-step covalent immobilization that
proceeds in high yield without producing any byproduct and without the need of a catalyst. Moreover, the resulting triazole link is biocompatible,
stable and does not affect bioactivity.
In another approach, Pack et al. [74] explored
the reactivity of the oxanine moiety 25, which has
an O-acylisourea moiety, with amine-modified
glass substrate 26 for the fabrication of biochips 27
(Fig. 12). The strategy involves the introduction of

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oxa-nucleotide at the 5-end of the desired oligonucleotide sequences, with or without alkyl spacers,
which were subsequently spotted onto aminefunctionalized glass microslides and the spotted
slides were incubated at 80C for 1 h in a humid
chamber.The resulting microarrays were subjected
to hybridization with the target, and the performance was evaluated in terms of immobilization and
hybridization efficiency, which were found to be
much superior to that observed in existing methods. The results further displayed higher efficiency

Figure 11. Immobilization of alkyne-modified oligonucleotides onto

azidoalkylated glass surface via click chemistry.

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Figure 12. Covalent immobilization reaction of oxa-oligonucleotides with

amine-functionalized glass surface.

of the target recognition by the oligonucleotides

bearing a longer spacer (C6 vs. C2 linker). It is inferred from this study that immobilization of oxanucleotide bearing oligonucleotides onto the
amine-functionalized surface perhaps represents
an advanced method for generating DNA-conjugated solid surfaces for various applications.
In another elegant approach, amine-modified
oligonucleotides 12 were tethered to tetrafluorophenyl (TFP)-activated self-assembled monolayers (SAMs) 28 on gold-coated glass slides [75].
TFP ester was used as an alternative to N-hydroxysuccinimide (NHS) ester 29 because of greater
stability of the former under basic coupling conditions (Fig. 13). In this methodology, the coupling reactions of amine-modified oligonucleotides with
aldehyde, NHS- and TFP-activated SAMs, and the
thiol-modified oligonucleotides with maleimideterminated SAMs were evaluated as a function of
pH. The thiol-maleimide coupling was found to be
pH independent, while aldehyde-, NHS- and TFPactivated SAMs exhibited increasing degree of immobilization of oligonucleotide with increasing pH;
however, at pH 10, NHS-activated SAMs showed a
decrease in coupling efficiency, which might be due
to base-catalyzed hydrolysis of NHS-SAMs that
significantly decreased the coupling reaction. In
addition, the TFP surface displayed lower background fluorescence and smaller spot radii at each
pH. The smaller spot size could be attributed to
the hydrophobic nature of the TFP surface. The
stability of oligonucleotide arrays was demonstrated by hybridization/dehybridization cycles. There
was no significant loss in signal intensity, indicating a high degree of stability of the immobilized
probes.
In a protocol involving reversal of sequential
steps in the conventional methodology for fabrication of biochips, Razumovitch et al. [76] immobilized solution-hybridized amine-modified oligonucleotides 12 onto an aldehyde surface 30 via microcontact printing. Here, complementary oligonucleotides were first hybridized in solution prior to
immobilization onto modified silicon surface
(Fig. 14). In this manner, the immobilization of the

2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Figure 13. Immobilization of amine-functionalized oligonucleotides onto

tetrafluorophenyl (TFP, top) and N-succinimidyl (bottom) ester terminated SAMs on gold-coated glass surface.

hybridized sequences could prevent interactions

between amino groups on the nucleic bases and the
aldehydes on the chip surface. Nevertheless, immobilization occurred through one of the two complementary oligonucleotides bearing 5-aminoalkyl group. Subsequently, the performance of the
constructed array 31 was evaluated by sequential
dehybridization and rehybridization of oligonucleotides labeled with fluorescent dyes under laser
scanning microscopy. This protocol had the advantage of eliminating the hindrances generally encountered during hybridization with the target nucleic acids due to high density of surface-attached
oligonucleotides.
Limitation of sensitivity has been a major concern for realization of the full potential of biochip
technology. A general drawback of glass slides is
the limited density of the surface silanol groups
used for introduction of modifications due to its
non-porous nature. This limits the number of reactive groups that can participate in the binding of
oligonucleotides, resulting in the poor surface coverage with the probes. A simple way to improve the
sensitivity of an array is to increase the surface
density to anchor probes or linker molecules. Considering this, Hackler et al. [77] developed six different surfaces with acrylic 32 and epoxy reactive
groups 33, 34 for immobilization of oligonucleotides (Fig. 15). The surface density of the reac-

Figure 14. Schematic representation of the immobilization of hybridized

amine-modified oligonucleotides onto aldehydic surface.

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Figure 15. Modified high capacity glass surfaces for immobilization of

amine-modified oligonucleotides.

tive functionalities on glass was found to be increased by addition of branched structures. Following this strategy, enhanced immobilization efficiency was achieved. This methodology was subsequently modified by Benters et al. [78], who developed chemically activated glass surfaces 35 for
production of microarrays of oligonucleotides, proteins and low molecular weight ligands. The method was based on the attachment of a dendritic
linker (PAMAM with 64 amino groups in its outer
sphere) onto the aminated glass surface, which was
subsequently activated with PDITC or disuccinimidylglutarate (DSG) to generate a chemically reactive polymeric film 36, covalently affixed to glass
slide. The resulting surface was used for the immobilization of amine-modified oligonucleotides
(Fig. 16). The microarrays prepared were evaluated
in terms of hybridization with the labeled targets.
The results indicated an almost twofold greater
surface coverage with the captured oligonucleotides than that with the conventional microarrays bearing linear linkers, as the dendrimer coating permits the immobilization of many more
probes because of numerous functionalized groups
presence in the structure. Further, the surfaces
were found to be resistant to repeated alkaline regeneration procedures, which is likely due to the
cross-linked polymeric structure of the dendrimer
film. The higher stability of the surface allowed
multiple hybridization experiments without significant loss of signal intensity. Also, a dendrimer coat
acts as a spacer between probe and the substrate
surface, which facilitates efficient binding and accessibility of the immobilized probe to target. In a
diverse approach, Lim et al. [79] developed an improved protocol for the fabrication of a DNA
biochip with PAMAM dendrimers peripherally
modified with biotin and avidin. Biotinyl-oligonu-

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2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Figure 16. Schematic representation of the dendritic-linker bearing glass

surface for immobilization of amine-modified oligonucleotides.

cleotides were tethered to the surface and the

biochips were evaluated with respect to various parameters commonly employed in the microarray
experiments. The results showed significantly improved sensitivity, specificity and stability of the
biochip. A fourfold increase in fluorescence intensity of the spots, and excellent specificity for discriminating SNP with detection limit in the range
of 1 pM to 1 nM were some of the distinctive features of the protocol.
Recently, an exquisite nanoscale-controlled
surface was reported by Hong et al. [80]; this not
only ensured proper spacing between immobilized
biomolecules, but also revealed good discrimination efficiency over a wide temperature range
(3750C) (Fig. 17). In this strategy, a cone-shaped
dendron 37 ensures adequate spacing between immobilized oligonucleotides on surface, which ultimately provides sufficient accessibility for the incoming target DNA, similar to solution-like conditions. Hybridization efficiency was scored in the
range of 80100% (vs. ~2035% usually obtained in
other recorded protocols).
In another protocol, to provide high-capacity
surfaces, glass slides coated with chitosan, an
amine-rich polysaccharide, were used for tethering
biomolecules including synthetic oligonucleotides.

Biotechnol. J. 2009, 4, 15131529

www.biotechnology-journal.com

Figure 17. Dendron-modified surface for the immobilization of oligonucleotides.

The surface amino groups were first activated with

PDITC to generate reactive isothiocyanato functions on the surface, which were subsequently reacted with amine-modified oligonucleotides [81].
The resulting surface exhibited high loading capacity for oligonucleotide immobilization and good
binding efficiency, and enabled excellent availability of the surface-bound probes for hybridization
with the targets and effective detection of SNPs
with low background. Surface-attached oligonucleotide probes revealed highly specific hybridization properties.
Recently, Wu et al. [82] compared the immobilization of unmodified oligonucleotides (2670
mer) and 5-amine-modified oligonucleotides onto
3-D aminoalkylated glass slides modified with
acrylic acid-co-acrylamide copolymer 38 under
the influence of UV light and in the presence of
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide
(EDC)/NHS, respectively (Fig. 18). The investigators observed no significant difference in the hybridization efficiency of the immobilized aminemodified and unmodified oligonucleotides on the

dendritic surface. The results indicate that unmodified oligonucleotides, being cost effective, can be
used for immobilization on the modified glass surface.
More recently, in an elegant approach, modified
analogs, hexitol (HNA) 39 and altritol nucleic acids
(ANA) 40 with high affinity towards DNA and RNA
targets, were employed for the preparation of
biochips [83]. Immobilization of these chimeric nucleic acids was effected by reaction of diene-modified oligonucleotides with maleimidoalkyl-activated glass slides 7 (Fig. 19). The distinguishing features of the strategy are the higher match- and mismatch discrimination of ANA and HNA arrays,
which might be due to improved affinity of the
probes and higher stability of the resulting duplexes (particularly with respect to RNA). The features
accrue from a preorganization (entropy factor) and
enzymatic stability, and the constructed biochips
can be stored for a longer duration and reused.
In another approach, Dendane et al. [84, 85]
have reported an efficient surface patterning of
oligonucleotides onto the inner wall of fused silica
capillary tubes as well as on the surface of glass microslides through oxime bond formation. One of
their strategies involved the preparation of
aminooxy silane protected with 2-(2-nitrophenyl)
propyloxycarbonyl (NPPOC), which was used for
functionalization of glass surfaces 41 (Fig. 20). On
exposure to UV light, NPPOC group was removed
and aldehyde-modified oligonucleotides 42 reacted with the unmasked aminooxyalkylated glass
surface 41 through stable oxime bond formation
43, obviating the need for chemical reduction with

38
39

40

12

Figure 18. Immobilization of 5-amine-modified oligonucleotides onto

poly(acrylic acid-co-acrylamide) copolymer film on glass microslide.

2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Figure 19. Immobilization of diene-modified ANA and HNA onto maleimidoalkyl-glass surface.

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Figure 20. Immobilization of aldehydemodified oligonucleotides onto

aminooxy-glass surface.

sodium cyanoborohydride. The projected approach

offers an alternative methodology for the immobilization of bioactive molecules in the microchannels of labs on chip devices.

Photochemical methods
Covalent attachment of ligands via photoexcitable
moieties constitutes another class of immobilization methods that offers an interesting possibility
of selective activation. In principle, the methodology is simple and offers a specific one-step process
for immobilization of bioactive molecules on a wide
variety of C-H containing surfaces including modified glass surfaces. A number of reports describe
photochemical methods of immobilization triggered by UV irradiation. The method involves the
covalent linking of the oligonucleotides to the surface during irradiation via radical formation.Traditionally, psoralens, benzophenone, azides and carbene precursors have been used for photochemical
immobilization reactions; however, these photoprobes suffer from several inherent drawbacks
[15]. Psoralen-bearing ligands react with the polymeric surfaces that have exposed double bonds in
a 2+2 cycloaddition. Benzophenones have nearly
overlapping absorption spectra with the biological
materials and hence cause severe photoinduced
damages to biomolecules. Nitrenes and carbenes,
which are the most reactive species, however, produce a number of side products leading to lower reaction yields. A Danish research group [86] recently reported anthraquinones as a new class of photoprobes, which possess interesting properties for
ligand immobilization. In its excited state, anthraquinone can react with almost any C-H containing substrate. Further, it has an absorption coefficient above 350 nm, compared to most azides,
diazirines and benzophenones and in contrast to
ketones, the quinoid structure significantly reduces
the reactivity of the carbonyl groups in the ground
state. They presented a facile, orientation-specific,
one-step technique for photochemical immobilization of oligonucleotides using anthraquinone.
However, the oligonucleotide-anthraquinone conjugates involve the time-consuming preparation of
phosphoramidite derivatives and some of these derivatives are not very stable.

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To overcome the limitations of the previous

method, Kumar et al. [87] reported a new heterobifunctional reagent, N-(3-trifluoroethanesulfonyloxypropyl)-anthraquinone-2-carboxamide (NTPAC) for making bioconjugates and immobilization
of biomolecules, i.e., oligonucleotides, peptides,
proteins, etc., on a variety of carbon-containing solid surfaces including modified glass slides. The trifluoroethanesulfonate ester group of the reagent
reacts with aminoalkyl or mercaptoalkyl functions
present in the biomolecules, and the anthraquinone moiety reacts with a variety of carbon-containing polymers under UV irradiation (365 nm).
The reagent, NTPAC, was used in two ways: (i) it
was first brought in contact with the modified glass
slide and exposed to long-wavelength UV light
(365 nm), thereby generating active trifluoroethanesulfonate ester functions on the support, which
subsequently reacted with mercaptoalkyl- or
aminoalkyl-modified oligonucleotides to fix them
on the support; or (ii) the reagent was allowed to react first with 3- or 5-aminoalkyl- or mercaptoalkyl-modified oligonucleotides to form the oligonucleotide-anthraquinone conjugate, which was
then brought in contact with a modified glass surface and exposed to long-wavelength UV light
(365 nm), resulting in immobilization of the conjugate on the support. Both routes work satisfactorily. On the parallel lines, Patnaik et al. [88] recently
reported the synthesis and application of a new
heterobifunctional reagent, N-(iodoacetyl)-N-(anthraquinon-2-oyl)-ethylenediamine (IAED) 44, for
immobilization (Fig. 21). Mercaptoalkyl- and thiophosphoryl-modified oligonucleotides were immobilized on the modified glass microslides under the
influence of UV light and microwaves.The thermochemical reaction between mercaptoalkyl group
and iodoacetyl functionality was carried out under
microwaves and the photochemical reaction involving anthraquinone and modified glass slide
was performed as described above. The immobilized probes 45 were found to be thermally stable
and the constructed microarrays using the projected strategy were successfully employed for the detection of base mismatches.
More recently, another heterobifunctional reagent, 1-N-(maleimidohexanoyl)-6-N-(anthraqui-

Biotechnol. J. 2009, 4, 15131529

Figure 21. Immobilization of modified oligonucleotides onto virgin glass

surface using IAED.

non-2-oyl) hexanediamine (MHAHD) 46, possessing a photoactive anthraquinone moiety on one end
and an electrophilic maleimide group on the other,
was demonstrated to affect efficient immobilization of thiolated oligonucleotides on a modified
glass surface [89]. The addition of thiolated oligonucleotides to maleimidoalkyl group was accelerated by performing reaction under microwaves
(Fig. 22).
Such reactions are generally fast and clean,
since they avoid post-immobilization processing
[90]. In one such report, Kimura et al. [91] employed a different strategy that permits the immo-

www.biotechnology-journal.com

bilization of non-modified probes on a poly-carbodiimide coated glass surface by UV irradiation.

The methodology offers an economical and rapid
route for preparation of biochips as compared to
the existing protocols.
An overview of the attachment chemistries employing covalent linkage (thermochemical and
photochemical) for patterning of arrays is given in
Table 2.
As discussed above, a variety of surface chemistries have been developed for making oligonucleotide-based biochips on glass surfaces. The production and optimal performance of these biochips
also depend on some ancillary factors. One of these
is the length of a linker required to create a suitable
distance between the surface and the probe sequence that is to be used for hybridization experiments. The distance minimizes steric hindrance
and provides accessibility to the incoming molecules. In some cases, polyethylene glycol [92] and
homo-oligomers [93] have been employed, which
not only act as efficient spacers but also reduce
surface inhomogeneities after the silanization step
and nonspecific adsorption of DNA. Other factors
include physical and chemical properties of the
glass surface, derivatization methods used to introduce suitable functional groups on the surface, incorporation of suitable reactive functional groups
on oligonucleotides, density of oligonucleotides on
the surface, delivery of tiny volumes of spotting solution, the blocking of residual functional groups
on the surface, length and type of the target DNA
molecules, hybridization and washing conditions,
etc. Another problem, arising due to uniform distri-

Figure 22. Immobilization of modified

oligonucleotides onto virgin glass
surface using MHAHD.

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Table 2. Some commonly used chemistries for immobilization of pre-fabricated oligonucleotides on glass surface via covalent method

Functional group on glass

Maleimide
Psoralen
4-Nitrophenyl-3-diazopyruvate
-(CH2 )n
-CONHNH2
-CHO
Maleimide
-SH
-SH
-I
-NH2
Gold surface
PAMAM dendrimer
Zirconium
Epoxy

-NCS
-CHO
-OSO2CH2CF3

Functional group on oligomer

Chemical method

Reference

Cinnamide
Probe Sequence
-NH2
Anthraquinone
-CHO
-NH2, -SH
-SH
-S-SR
-SH
-SH
-PO4
Lipoic acid
Amine
-PO4
-NH2, -OPO22, -ONH2, -SH
Trimethoxysilyl-oligomer conjugate
-NH2
-ONH2, NH2
-SH, -NH2
Triethoxysilyl-oligomer conjugate

Photochemical
Photochemical
Photochemical
Photochemical
Thermochemical
Thermochemical
Thermochemical
Thermochemical
Thermochemical
Thermochemical
Thermochemical
Thermochemical
Thermochemical
Thermochemical
Thermochemical
Thermochemical
Thermochemical
Thermochemical
Thermochemical
Thermochemical

[15]
[15]
[15]
[86]
[15]
[15]
[15]
[15]
[15]
[15]
[15]
[18]
[18]
[65]
[6771]
[72]
[78]
[84, 85]
[87]
[88]

bution of spotted oligonucleotides, has been addressed by mixing a suitable co-solvent with such
properties as good wettability and low evaporation
rate; betaine and dimethylsulfoxide are the most
commonly used reagents for this purpose [94].
Similarly, to achieve stronger signals and a lower
detection limit for the probe-target interaction,
Preiniger et al. [95] reported high capacity ARChip
epoxy as the reactive chip substrate obtained after
coating the glass surface with 1% epoxy resin for
immobilization of amine- and thiol-modified oligonucleotides. They investigated the role of buffer
composition on the spot quality and size, and found
that buffers of high viscosity produced more compact spots, which resulted in more reliable data. It
was also shown that too high a level of humidity
caused merger of the spots. These chips generated
stronger hybridization signals (~threefold compared to normal epoxy glass); however, problems
relating to reproducibility of the coating process
and enhanced background signals still have to be
overcome.

Concluding remarks

We have summarized various methods for immobilization of oligonucleotides onto glass surface that
can be utilized as analytical tools in a wide range of
applications. Covalent fixing to surfaces is the preferred approach over physiosorption. The progress
made in the last few years has brought the biochip

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technology to the forefront of rapid diagnostics and

medical research. With the approval of the US FDA
to use DNA chips in the clinical field, concerted efforts are afoot to improve the reliability of the results to satisfy doctors and clinicians. This area has
also opened new avenues towards the development
of sophisticated, safe and reliable analytical tools
for molecular recognition. Likewise, the introduction into the filed of medical diagnostics will certainly boost the microarray industry and help in
promoting human health.
Authors gratefully acknowledge financial support
from the Department of Biotechnology (DBT) and
CSIR Task Force Project (NWP 013). R.P. Gandhi is
an Indian National Science Academy (INSA) Honorary Scientist.
The authors have declared no conflict of interest.

References

[1] Valk, P. J., Verhaak, R. G., Beijen, M. A., Erpelinck, C. A. et al.,

Prognostically useful gene-expression profiles in acute
myeloid leukemia. N. Engl. J. Med. 2004, 350, 16171650.
[2] Heller, M. J., DNA microarray technology: Devices, systems,
and applications. Annu. Rev. Biomed. Eng. 2002, 4, 129153.
[3] Schena, M., Shalon, D., Davis, R. W., Brown, P. O., Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 1995, 270, 467470.
[4] Altshuler, D., Daly, M., Guilt beyond a reasonable doubt. Nat.
Genet. 2007, 39, 813815.

Biotechnol. J. 2009, 4, 15131529

[5] Shai, R. M., Microarray tools for deciphering complex diseases. Front. Biosci. 2006, 11, 14141424.
[6] Bulyk, M. L., Gentalen, E., Lockhart, D. J., Church, G. M.,
Quantifying DNA-protein interactions by double-stranded
DNA arrays. Nat. Biotechnol. 1999, 17, 573577.
[7] Warren, C. L., Kratochvil, N. C. S., Hauschild, K. E., Foister, S.
et al., Defining the sequence-recognition profile of DNAbinding molecules. Proc. Natl. Acad. Sci. USA 2000, 103, 867
872.
[8] Wang, L., Luhm, R., Lei, M., SNP and mutation analysis. Adv.
Exp. Med. Biol. 2007, 593, 105116.
[9] Bulyk, M. L., DNA microarray technologies for measuring
protein-DNA interactions. Curr. Opin. Biotechnol. 2006, 17,
422430.
[10] Mintseris, J., Eisen, M. B., Design of a combinatorial DNA
microarray for protein-DNA interaction studies. BMC
Bioinformatics 2006, 7, 429.
[11] Oh, S. J., Hong, B. J., Choi, K. Y., Park, J. W., Surface modification for DNA and protein microarrays. Omics 2006, 10,
327343.
[12] Halperin, A., Buhot, A., Zhulina, E. B., Sensitivity, specificity, and the hybridization isotherms of DNA chips. Biophys. J.
2004, 86, 718730.
[13] Bao, P., Frutos, A.G., Greef, C., Lahiri, J. et al., High-sensitivity detection of DNA hybridization on microarrays using
resonance light scattering. Anal. Chem. 2002, 74, 17921797.
[14] McQuain, M. K., Seale, K., Peck, J., Levy, S., Haselton, F. R.,
Effects of relative humidity and buffer additives on the contact printing of microarrays by quill pins. Anal. Biochem.
2003, 320, 281291.
[15] Vaijayanthi, B., Kumar, P., Ghosh, P. K., Gupta, K. C., Recent
advances in oligonucleotide synthesis and their applications. Indian J. Biochem. Biophys. 2003, 40, 377391.
[16] Resch-Genger, U., Grabolle, M., Cavaliere-Jaricot, S., Nitschke R., Nann, T., Quantum dots versus organic dyes as fluorescent labels. Nat. Methods 2008, 5, 763775.
[17] Beaucage, S. L., Strategies in the preparation of DNA oligonucleotide arrays for diagnostic applications. Curr. Med.
Chem. 2001, 8, 12131244.
[18] Seliger, H., Hinz, M., Happ, E., Arrays of immobilized oligonucleotides Contributions to nucleic acids technology.
Curr. Pharm. Biotechnol. 2003, 4, 379395.
[19] Pirrung, M. C., How to make a DNA chip. Angew. Chem. Int.
Ed. 2002, 41, 12761289.
[20] Dufva, M., Fabrication of high quality microarrays. Biomol.
Eng. 2005, 22, 173184.
[21] Elder, J. K., Johnson, M., Milner, N., Mir, K. U., Sohail, M.,
Southern, E. M., Antisense oligonucleotide scanning arrays.
in: Schena, M. (Ed.), DNA microarrays: A practical approach,
Oxford Press, New York 1999, pp. 77100.
[22] Fodor, S. P. A., Read, J., Pirrung, M. C., Stryer, L. et al., Lightdirected, spatially addressable parallel chemical synthesis.
Science 1991, 251, 767773.
[23] Pease, A. C., Solas, D., Sullivan, E. J., Cronin, M.T. et al., Lightgenerated oligonucleotide arrays for rapid DNA-sequence
analysis. Proc. Natl. Acad. Sci. USA 1994, 91, 50225026.
[24] Lipshutz, R. J., Fodor, S. P. A., Gingeras, T. R., Lockhart, D. J.,
High density synthetic oligonucleotide arrays. Nat. Genet.
1999, 21, 2024.
[25] Kong, D. S., Carr, P. A., Chen, L., Zhang, S., Jacobson, J. M.,
Parallel gene synthesis in a microfluidic device. Nucleic
Acids Res. 2007, 35, e61.

2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.biotechnology-journal.com

Kailash C. Gupta was born in 1952 and

studied at the University of Rajasthan,
Jaipur, India from where he graduated
in Chemistry in 1975. After completing
his doctoral work at University of
Jodhpur, India in 1978, he joined the
Institute of Genomics and Integrative
Biology (formerly known as Centre for
Biochemicals), Delhi, India. He did his
post-doctoral research in the area of
nucleic acids research in Prof. H. Seligers research group in the Polymer Section, University of Ulm, Germany from 1982 to 1984. He has
had more than 34 years of research experience in the same field. He
published more than 80 research papers in various national and international journals of high repute and contributed several invited review
articles and book chapters. Currently, his research focuses on two
aspects: development of biochips for diagnostic purposes and nanoparticle-aided delivery of bioactive molecules. He is a Fellow of the National Academy of Sciences, India and has recently taken over as a
Director of Indian Institute of Toxicology Research, Lucknow, India.

[26] Beier, M., Hoheisel, J. D., Production by quantitative photolithographic synthesis of individually quality checked
DNA microarrays. Nucleic Acids Res. 2000, 28, e11.
[27] McGall. G., Labadie, J., Brock, P., Wallraff, G. et al., Light-directed synthesis of high-density oligonucleotide arrays using semiconductor photoresists. Proc. Natl. Acad. Sci. USA
1996, 93, 1355512560.
[28] Pirrung, M. C., Lee, Y. R., Park, K., Springer, J. B., Pentadienylnitrobenzyl and pentadienylnitropiperonyl photochemically removable protecting groups. J. Org. Chem. 1999, 64,
50425047.
[29] Albert, T. J., Norton, J., Ott, M., Richmond, T. et al., Light-directed 5-3 synthesis of complex oligonucleotide microarrays. Nucleic Acids Res. 2003, 31, e35.
[30] Phillips, M. F., Lockett, M. R., Rodesch, M. J., Shortreed, M. R.
et al., In situ oligonucleotide synthesis on carbon materials:
Stable substrates for microarray fabrication. Nucleic Acids
Res. 2008, 36, e7.
[31] Salo, H., Virta, P., Hakala, H., Prakash, T. P. et al., Aminooxy
functionalized oligonucleotides: Preparation, on-support
derivatization, and post-synthetic attachment to polymer
support. Bioconjug. Chem. 1999, 10, 815823.
[32] Cheung, V. G., Morley, M., Aguilar, F., Massimi, A. et al., Making and reading microarrays. Nat. Genet. 1999, 21, 1519.
[33] Beier, M., Hoheisel, J. D., Versatile derivatization of solid
support media for covalent bonding on DNA-microchips.
Nucleic Acids Res. 1999, 27, 19701977.
[34] Guo, Z., Guilfoyle, R. A., Thiel, A. J., Wang, R., Smith, L. M.,
Direct fluorescence analysis of genetic polymorphisms by
hybridization with oligonucleotide arrays on glass supports.
Nucleic Acids Res. 1994, 22, 54565465.
[35] Cohen, G., Deutsch, J., Fineberg, J., Levine, A., Covalent attachment of DNA oligonucleotides to glass. Nucleic Acids
Res. 1997, 25, 911912.
[36] Rogers, Y. H., Jiang-Baucom, P., Huang, Z. J., Bogdanov, V. et
al., Immobilization of oligonucleotides onto a glass support

1527

Biotechnology
Journal

[37]

[38]

[39]

[40]

[41]
[42]

[43]

[44]

[45]

[46]
[47]
[48]
[49]
[50]

[51]

[52]

[53]

[54]

[55]

[56]

1528

via disulfide bonds: A method for preparation of DNA microarrays. Anal. Biochem. 1999, 266, 2330.
Chrisey, L.A., Lee, G. U., OFerrall, C. E., Covalent attachment
of synthetic DNA to self-assembled monolayer films. Nucleic Acids Res. 1996, 24, 30313039.
Proudnikov, D., Timofeev, E., Mirzabekov, A., Immobilization
of DNA in polyacrylamide gel for the manufacture of DNA
and DNA-oligonucleotide microchips. Anal. Biochem. 1998,
259, 3441.
Rasmussen, S. R., Larsen, M. R., Rasmussen, S. E., Covalent
immobilization of DNA onto polystyrene microwells: The
molecules are only bound at the 5 end. Anal. Biochem. 1991,
198, 138142.
Morozov, V. N., Morozova, T., Electrospray deposition as a
method for mass fabrication of mono- and multicomponent
microarrays of biological and biologically active substances.
Anal. Chem. 1999, 71, 31103117.
Kumar, P., Gupta, K. C., A rapid method for the construction
of oligonucleotide arrays. Bioconjug. Chem. 2003, 14, 50712.
Tang, J., Xiao, P., Polymerizing immobilization of acrylamide-modified nucleic acids and its application. Biosens.
Bioelectron. 2009, 24, 18171824.
Saluz, H. P., Iqbal, J., Limmon, G. V., Ruryk, A., Wu, Z., Fundamentals of DNA-chip/array technology for comparative
gene-expression analysis. Curr. Sci. 2002, 83, 829833.
Zhao, X., Pan, F., Lu, J.R., Interfacial immobilization of DNA
molecules. Annu. Rep. Prog. Chem., Sect. C, 2007, 103, 261
286.
Oh, S. J., Hong, B. J., Choi, K. Y., Park, J. W., Surface modification for DNA and protein microarrays. Omics J. Int. Biol.
2006, 10, 327343.
Dufva, M., Fabrication of high quality microarrays. Biomol.
Eng. 2005, 22, 173184.
Dufva, M. (Ed.), DNA microarrays for biomedical research:
Methods and Protocols, Humana Press, Totowa 2009.
Matson, R. S. (Ed.), Microarray, methods and protocols, CRC
Press, NY 2009.
Rampal, J. B. (Ed.), Microarrays: Synthesis methods, Humana
Press, Totowa 2007.
Lange, S. A., Benes, V., Kern, D. P., Horber, J. K. H., Bernard,
A., Microcontact printing of DNA molecules. Anal. Chem.
2004, 76, 16411647.
Nikiforov, T. T., Rogers, Y. H., et al., The use of 96-well polystyrene plates for DNA hybridization-based assays: An
evaluation of different approaches to oligonucleotide immobilization. Anal. Biochem. 1995, 227, 201209.
Li, J., Tan, W., Wang, K., Xiao, D., et al., Ultrasensitive optical
DNA biosensor based on surface immobilization of molecular beacon by a bridge structure. Anal. Sci. 2001, 17, 1149
1153.
Wu, Q., Ma, W., Shi, R., Zhang, B., et al., An activated GOPSpoly-L-lysine-coated glass surface for the immobilization of
60mer oligonucleotides. Eng. Life Sci. 2005, 5, 466470.
Belosludstev, Y., Iverson, B., Lemeshko, S., Eggers, R. et al.,
DNA microarrays based on non-covalent oligonucleotide
attachment and hybridization in two dimensions. Anal.
Biochem. 2001, 292, 250256.
Walsh, M. K., Wang, X., Weimer, B. C. Optimizing the immobilization of single-stranded DNA onto glass beads. J.
Biochem. Biophys. Methods 2001, 47, 221231.
Ness, J. V., Kalbfleisch, S., Petne, C. R., Reed, M. W., A versatile solid support system for oligodeoxynucleotide probebased hybridization assays. Nucleic Acids Res. 1991, 19,
33453350.

2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Biotechnol. J. 2009, 4, 15131529

[57] Chrisey, L.A., Lee, G. U., OFerrall, C. E., Covalent attachment

of synthetic DNA to self-assembled monolayer films. Nucleic Acids Res. 1996, 24, 30313039.
[58] Kumar, P., Choithani, J., Gupta, K.C., Construction of
oligonucleotide arrays on a glass surface using a heterobifunctional reagent, N-(2-trifluoroethanesulfonatoethyl)-N(methyl)-triethoxysilylpropyl-3-amine (NTMTA). Nucleic
Acids Res. 2004, 32, e80.
[59] Choithani, J., Kumar, P., Gupta, K.C., N-(3-Triethoxysilylpropyl)-6-(N-maleimido)-hexanamide:An efficient heterobifunctional reagent for the construction of oligonucleotide
microarrays. Anal. Biochem. 2006, 357, 240248.
[60] Misra,A., Shahid, M., Immobilization of self-quenched DNA
hairpin probe with a heterobifunctional reagent on a glass
surface for sensitive detection of oligonucleotides. Bioorg.
Med. Chem. 2009, 17, 58265833.
[61] Wong, A. K. Y., Krull, U. J., Surfaces for tuning of oligonucleotide biosensing selectivity based on surface-initiated
atom transfer radical polymerization on glass and silicon
surfaces. Anal. Chim. Acta 2009, 639, 112.
[62] Strother, T., Cai, W., Zhao, X., Hamers, R.J., Smith, L. M., Synthesis and characterization of DNA-modified silicon (111)
surfaces. J. Am. Chem. Soc. 2000, 122, 12051209.
[63] Wittmann, C. (Ed.), Topics in current chemistry: Immobilization of DNA on chips II, vol. 261, Springer, Berlin 2005.
[64] Dandy, D. S., Wu, P., Grainger, D. W., Array feature size influences nucleic acid surface capture in DNA microarrays.
Proc. Natl. Acad. Sci. USA 2007, 104, 82238228.
[65] Nonglaton, G., Benitez, I. O., Guisle, I., Pipelier, M. et al., New
approach to oligonucleotide microarrays using zirconium
phosphonate-modified surfaces. J. Am. Chem. Soc. 2004, 126,
14971502.
[66] Lane, S. M., Monot, J., Petit, M., Tellier, C., et al., Poly(dG)
spacers lead to increased surface coverage of DNA probes:
An XPS study of oligonucleotide binding to zirconium phosphonate modified surfaces. Langmuir 2008, 24, 73947299.
[67] Lumture, J. B., Beattie, K. L., Burke, B. E., Eggers, M. D. et al.,
Direct detection of nucleic acid hybridization on the surface
of a charge coupled device. Nucleic Acids Res. 1994, 22,
21212125.
[68] Mahajan, S., Kumar, P., Gupta, K. C., An efficient and versatile approach for the construction of oligonucleotide microarrays. BioMed. Chem. Lett. 2006, 16, 56545658.
[69] Mahajan, S., Kumar, P., Gupta, K. C., Oligonucleotide microarrays: Immobilization of phosphorylated oligonucleotides on epoxylated surface. Bioconjug. Chem. 2006, 17,
11841189.
[70] Sethi, D., Patnaik, S., Kumar, A., Gandhi, R. P., et al., Polymer
supported synthesis of aminooxyalkylated oligonucleotides, and some applications in the fabrication of microarrays. Bioorg. Med. Chem. 2009, 17, 5442 5450.
[71] Mahajan, J., Sethi, D., Seth, S., Kumar, A., Kumar, P., Gupta,
K. C., Construction of oligonucleotide microarrays (biochips) via thioether linkage for the detection of bacterial
meningitis. Bioconjug. Chem. 2009, 20, 17031710.
[72] Sethi, D., Kumar, A., Gupta, K. C., Kumar, P., A facile method
for the construction of oligonucleotide microarrays. Bioconjug. Chem. 2008, 19, 21362143.
[73] Rozkiewicz, D. I., Gierlich, J., Burley, G. A., Gutsmiedl, K. et
al., Transfer printing of DNA by click chemistry. ChemBiochem 2007, 8, 19972002.
[74] Pack, S. P., Kamisetty, N. K., Nonogawa, M., Devarayapalli, K.
C. et al., Direct immobilization of DNA oligomers onto the
amine-functionalized glass surface for DNA microarray

Biotechnol. J. 2009, 4, 15131529

[75]

[76]

[77]

[78]

[79]

[80]

[81]

[82]

[83]

[84]

[85]

fabrication through the activation-free reaction of oxanine.

Nucleic Acids Res. 2007, 35, e110.
Lockett, M. R., Phillips, M. F., Jarecki, J. L., Peelen, D., Smith,
L. M., A tetrafluorophenylactivated ester self-assembled
monolayer for the immobilization of amine-modified
oligonucleotides. Langmuir 2008, 24, 6975.
Razumovitch, J., Meier, W., Vebert, C. A., A microcontact
printing approach to the immobilization of oligonucleotide
brushes. Biophys. Chem. 2009, 139, 7074.
Hackler, L. Jr., Dorman, G., Kele, Z., Urge, L. et al., Development of chemically modified glass surfaces for nucleic acid,
protein and small molecule microarrays. Mol. Divers. 2003,
7, 2536.
Benters, R., Niemeyer, C. M., Drutschmann, D., Blohm, D.,
Wohrle, D., DNA microarrays with PAMAM dendritic linker
systems. Nucleic Acids Res. 2002, 30, e10.
Lim, S. B., Kim, K.-W., Lee, C.-W., Kim, H.-S. et al., Improved
DNA chip with poly(aminoamine) dendrimers peripherally
modified with biotin and avidin. Biotechnol. Bioproc. Eng.
2008, 13, 683689.
Hong, B. J., Sunkara, V., Park, J. W., DNA microarrays on
nanoscale-controlled surface. Nucleic Acids. Res. 2005, 33,
e106.
Consolandi, C., Severgnini, M., Castiglioni, B., Bordoni, R. et
al., A structured chitosan-based platform for biomolecule
attachment to solid surfaces: Application to DNA microarray preparation. Bioconjug. Chem. 2006, 17, 371377.
Wu, O., Ma,W., Zhu, L., Guo, Q. et al., Oligo-microarray based
on oligonucleotide immobilization on glass surface modified with activated acrylic acid-co-acrylamide copolymer. J.
Biomed. Materi. Res. B Appl. Biomater. 2008, 87, 6974.
Abramov, M., Schepers, G., Aerschot, A. V., Hummelen, P. V.,
Herdewin, P., HNA and ANA high-affinity arrays for detections of DNA and RNA single-base mismatches. Biosens.
Bioelectron. 2008, 23, 17281732.
Dendane, N., Hoang, A., Renaudet, O., Vinet, F. et al., Surface
patterning of (bio)molecules onto the inner wall of fusedsilica capillary tubes. Lab Chip 2008, 8, 21612163.
Dendane, N., Hoang, A., Defrancq, E., Vinet, F., Dumy, P., Use
of gamma-aminopropyl-coated glass surface for the patterning of oligonucleotides through oxime bond formation.
BioMed. Chem. Lett. 2008, 18, 25402543.

2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.biotechnology-journal.com

[86] Koch, T., Jacobsen, N., Fensholdt, J., Boas, U. et al., Photochemical immobilization of anthraquinone conjugated
oligonucleotides and PCR amplicons on solid surfaces. Bioconjug. Chem. 2000, 11, 474483.
[87] Kumar, P., Agarwal, S. K., Gupta, K. C., N-(3-Trifluoroethanesulfonyloxy-propyl)anthraquinone-2-carboxamide: A new heterobifunctional reagent for immobilization of
biomolecules on a variety of polymer surfaces. Bioconjug.
Chem. 2004, 15, 711.
[88] Patnaik, S., Swami, A., Sethi, D., Pathak, A. et al., N(Iodoacetyl)-N-(anthraquinon-2-oyl)-ethylenediamine
(IAED): A new heterobifunctional reagent for the preparation of biochips. Bioconjug. Chem. 2007, 18, 812.
[89] Choithani, J., Sethi, D., Kumar, P., Gupta, K. C., Preparation
of oligonucleotide microarrays on modified glass using a
photoreactive heterobifunctional reagent, 1-N-(maleimidohexanoyl)-6-N-(anthraquinon-2-oyl)
hexanediamine
(MHAHD). Surf. Sci. 2008, 602, 23892394.
[90] Elghanian, R., Xu,Y., McGowen, J., Seithoff, M. et al., The use
and evaluation of 2 + 2 photoaddition in immobilization of
oligonucleotides on a three dimensional hydrogel matrix.
Nucleos. Nucleot. Nucleic Acids 2001, 20, 13711375.
[91] Kimura, N., Oda, R., Inaki, Y., Suzuki, O., Attachment of
oligonucleotide probes to poly carbodiimide-coated glass
for microarray applications. Nucleic Acids Res. 2004, 32, e68.
[92] Schlapak, R., Pammer, P., Armitage, D., Zhu, R. et al., Glass
surfaces grafted with high density poly(ethylene glycol) as
substrates for DNA oligonucleotide microarrays. Langmuir
2006, 22, 277285.
[93] Zhang, L., Hurek, T., Reinhold-Hurek, B., Position of the
fluorescent label is a crucial factor determine signal intensity in microarray hybridization. Nucleic Acids Res. 2005, 33,
e166.
[94] Diehl, F., Grahlmann, S., Beier, M., Hoheisel, J. D., Manufacturing DNA microarrays of high spot homogeneity and reduced background signal. Nucleic Acids Res. 2001, 29, e38.
[95] Preininger, C., Sauer, U., Dayteg, J., Pichler, R., Optimizing
processing parameters for signal enhancement of oligonucleotide and protein arrays on ARChip epoxy. Bioelectrochem. 2005, 67, 155162.

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