Efficient self-assembly of DNA-functionalized fluorophores and gold nanoparticles with DNA functionalized silicon surfaces: the effect of oligomer spacers.

2.7 MB·English

by  Milton, James A.

Published online 29 January 2013 Nucleic Acids Research, 2013, Vol. 41, No. 7 e80
doi:10.1093/nar/gkt031
Efficient self-assembly of DNA-functionalized
fluorophores and gold nanoparticles with DNA
functionalized silicon surfaces: the effect of
oligomer spacers
James A. Milton1, Samson Patole2, Huabing Yin2, Qiang Xiao3, Tom Brown3 and
Tracy Melvin2,4,*
1National Oceanography Centre, University of Southampton, Southampton, Hampshire, SO14 3ZH, 2School of
Electronics and Computer Science, University of Southampton, Southampton, Hampshire, SO17 1BJ, 3School
of Chemistry, University of Southampton, Southampton, Hampshire, SO17 1BJ and 4Optoelectronics Research
Centre, University of Southampton, Southampton SO17 1BJ, Hampshire, UK
Received October 15, 2012; Revised December 5, 2012; Accepted January 4, 2013
ABSTRACT useful for application with nanosensors, the
self-assembly ofnanoelectronic devices andtheat-
Although strategies for the immobilization of DNA
tachment of nanoparticles to biomolecules for
oligonucleotides onto surfaces for bioanalytical
single-molecule biophysical studies.
and top-down bio-inspired nanobiofabrication ap-
proaches are well developed, the effect of intro-
INTRODUCTION
ducing spacer molecules between the surface and
the DNA oligonucleotide for the hybridization of Silicon is used ubiquitously by the semiconductor
nanoparticle–DNA conjugates has not been previ- industry, and nanofabrication approaches have been
ously assessed in a quantitative manner. The both implemented and are being developed for ever
smaller devices. The uniform chemical reactivity of crys-
hybridization efficiency of DNA oligonucleotides
talline silicon makes it an ideal substrate for the direct
end-labelled with gold nanoparticles (1.4 or 10nm
immobilization of biomolecules, so permitting the use of
diameter) with DNA sequences conjugated to
silicon structures for biosensors and for the self-assembly
silicon surfaces via hexaethylene glycol phosphate
DNA/nanomaterial conjugates as building blocks in the
diester oligomer spacers (0, 1, 2, 6 oligomers) was
creation of nanostructures by the so-called ‘bottom-up’
found to be independent of spacer length. To methods for the next generation of nanoelectronic
quantify both the density of DNA strands attached devices. During the past decade, a number of approaches
to the surfaces and hybridization with the havebeendevelopedforthecovalentattachmentofDNA
surface-attached DNA, new methodologies have oligonucleotides onto crystalline silicon surfaces (1–5),
been developed. Firstly, a simple approach based and sub-micron motifs of surface-attached DNA oligo-
on fluorescence has been developed for determin- nucleotides in high densities on silicon can be achieved
(5). Although approaches for the creation of hybrids of
ation of the immobilization density of DNA oligo-
DNA-conjugated nanosized particles to surface-attached
nucleotides. Secondly, an approach using mass
DNA has been reported (6,7), there has not been a sys-
spectrometry has been created to establish (i) the
tematic study of the hybridization efficiency of DNA
mean number of DNA oligonucleotides attached to
oligonucleotide–nanoparticle conjugates, and this infor-
the gold nanoparticles and (ii) the hybridization
mation is crucial for the creation of next-generation
density of nanoparticle–oligonucleotide conjugates nanobiosensors and bio-inspired self-assembly of nanoe-
with the silicon surface–attached complementary lectronic devices. This is especially important for the so-
sequence. These methods and results will be called ‘bottom-up’–based nanofabrication methods (8,9).
*To whom correspondence should be addressed. Tel:[phone]; Fax:[phone]; Email: [email protected]
Present address:
Huabing Yin, School of Engineering, James Watt South Building, University of Glasgow, Glasgow G12 9QQ, UK.
The authors wish it to be known that, in their opinion, the first three authors should be regarded as joint First Authors.
(cid:2)TheAuthor(s)2013.PublishedbyOxfordUniversityPress.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionNon-CommercialLicense(http://creativecommons.org/licenses/
by-nc/3.0/),whichpermitsunrestrictednon-commercialuse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
e80 NucleicAcidsResearch,2013,Vol.41,No.7 PAGE2OF12
In comparison with the case of solution-phase DNA the approach taken here is to first attach a monolayer of
hybridization, the requirements for effective DNA N-succinimide ester functionalized undecyl alkane chains
hybridization to conjugated DNA sequences on surfaces on the surface in a similar approach to Peeters et al. (25)
is not so well understood. It has been shown that the and then conjugate these to DNA oligonucleotides with a
density of immobilized DNA, or probe, on a substrate ‘spacer’ of hexaethylene glycol phosphate diester oligo-
strongly affects the hybridization yield of unlabelled or mers between the DNA 30-end and a hexylamine linker
fluorophore-labelled DNA as well as the kinetics of for reaction with the succinimide group at the surface.
hybridization such that high efficiency of hybridization This strategy was used to provide both a high and the
is usually achieved in low-density regimes (10–12). same immobilized density of DNA conjugated to the
Although low densities may be preferable for some surface (irrespective of the spacer length).
bioanalysis applications, where the size of the sensor or The density for hybridization of DNA to silicon
devicesurfaceareareachesthenanoscale,thenthenumber surfaces(withoutaspacer)haspreviouslybeenestablished
of conjugated DNA strands is limited by the size and by us to be 7(cid:2)1012 DNA oligonucleotides per square
sometimes infinitesimally small. So for nanoscale sensors centimetre (5). This is slightly higher than for covalently
and devices, understanding the conditions for achieving attached DNA on glass surfaces (2.3(cid:2)1012 DNA oligo-
high hybridization efficiencies and densities of DNA con- nucleotides per square centimetre) (2). The presence of
jugates to complementary DNA strand surfaces is oligomers of hexaethylene glycol phosphate diester
important. spacers between the oligonucleotide and the linker on
The steric hindrance, the accessibility and surface the surface is anticipated to facilitate the hybridization
charge of surface-immobilized DNA probe as well as the of DNA oligonucleotides conjugated to gold nanopar-
ionicstrengthtowardshybridizationwithanunlabelledor ticles (DNA–GNP). Using relatively high densities of
fluorophore-labelled complementary sequence has been immobilized DNAsequences onthesurface isanticipated
the subject of a number of studies (10,11,13–20). The to reduce the probability of the DNA sequences being
most common approach to improve the hybridization positioned within the surface-immobilized ethylene
efficiencyofcomplementarystrandswithsurface-attached glycol oligomers at the surface (21,22), and thus allow
DNAprobesequencesinvolvestheuseof‘spacers’,which access of the bulky GNP–DNA (26,27).
are conjugated between the surface and the probe (21). Theimpactofspacersontheconjugationefficiencyand
These spacers have a number of effects including (i) hybridizationofDNAtoDNA–GNPhasbeenthesubject
‘raising’ the DNA probe from the surface (ii) impacting of a number of studies (9,27–30), It is the disassembly of
on the local electrostatic and hydrophilic environment of DNA–GNPs with complementary DNA–GNPs that has
theDNAprobe,(iii)influencingthe‘steric’accessibilityof providedthemostinterest,wherecooperativethermalde-
the DNA probe to hybridization and (iv) producing naturationorenzymaticcleavageresultinacolourchange
‘brush-like’ surfaces (10,11,13–15,22). In addition, some (31,32).ThekineticsfortheassemblyofDNA–GNPwith
spacer molecules, for instance oligomers of ethylene complementaryDNA–GNPisoftenslowandhasbeenthe
glycol, can reduce non-specific association of DNA com- subject of a number of recent studies (33–35), notably Oh
plementary sequences with the surface (16,17). et al., who demonstrated that the use of spacers of poly-
Spacers that are based on oligomers of ethylene glycol nucleotides of adenine or thymine between the oligo-
are considered to facilitate the hybridization of nucleotide and the GNP impacts on the hybridization
fluorophore-labelled or unlabelled DNA oligonucleotides kinetics; a spacer of 15 nucleotides was considered
with surface-attached complementary sequences optimal, and additional nucleotides in the space did not
(16,17,23,24). Perhaps the most well-known study in this improve the hybridization rate (35). Given the wealth of
field is that of Southern et al. (17), where the effect of literature for the assembly of DNA–GNP with comple-
surface density of the attached sites and of ethylene mentary DNA–GNPs, there are few examples of
glycololigomerswithdifferentlengthsandcontainingdif- nanoparticles conjugated to oligonucleotides that have
ferentchargedgroupshasbeeninvestigated.Thesestudies been hybridized with oligonucleotides attached to planar
suggestthattheoptimalspacerisonewithalownegative- surfaces (6,7,36); optimized conjugation for highly effi-
charge density that is amphiphilic and has a chain length cient assembly has not been studied in detail before now.
of 30–60 atoms. Longer or shorter spacers were found to In this article, we investigate the influence of the
bedetrimentaltothehybridizationyieldwitholigonucleo- oligomer spacer length on the hybridization performance
tides. More recently, Peeters et al. have investigated the ofimmobilizedDNA.Toachievethis, wehavedeveloped
effectofhexylandundecylalkanespacerslinkedtoagold novel methods to quantitatively assess the density of (i)
surface and then linked via triethylene glycol and immobilized DNA oligonucleotide probes on the silicon
hexaethylene glycol phosphate diester groups to DNA surface, (ii) DNA oligonucleotides attached to GNPs and
oligonucleotides (25). It is proposed that the use of the (iii) hybridized targets, including DNA–GNP conjugates.
undecyl groups provides DNA films in which the mol-
ecules are oriented perpendicular to the surface.
Unfortunatelynoconclusionscouldbededucedregarding MATERIALS AND METHODS
the effect of ethylene glycol oligomer size on the
Reagents
hybridization efficiency from that study.
Rather than synthesize oligonucleotides and linkers All chemicals were obtained from the Aldrich Chemical
directly on surfaces as reported by Southern et al. (17), Companyunlessotherwisenotedandusedwithoutfurther
PAGE3OF12 NucleicAcidsResearch,2013,Vol.41,No.7 e80
Table 1. DNA oligonucleotide sequences
Reference Sequencea
A-HEG 30-NH -(CH) CH(CH OH)CH-TGA CGA TAG ATA GAC GT-50
0 2 24 2 2
A-HEG 30-NH -(CH) CH(CH OH)CH-(HEG)-TGA CGA TAG ATA GAC GT-50
1 2 24 2 2 1
A-HEG 30-NH -(CH) CH(CH OH)CH-(HEG)-TGA CGA TAG ATA GAC GT-50
2 2 24 2 2 2
A-HEG 30-NH -(CH) CH(CH OH)CH-(HEG)-TGA CGA TAG ATA GAC GT-50
6 2 24 2 2 6
A-EG 30-NH -(CH) CH(CH OH)CH-PEG4000-TGA CGA TAG ATA GAC GT-50
91 2 24 2 2
B-HEG 30-NH -(CH) CH(CH OH)CH-TGA CGA TAG ATA GAC GTPT-FAM-50
0 2 24 2 2
B-HEG 30-NH -(CH) CH(CH OH)CH-(HEG)-TGA CGA TAG ATA GAC GTPT-FAM-50
1 2 24 2 2 1
B-HEG 30-NH -(CH) CH(CH OH)CH-(HEG)-TGA CGA TAG ATA GAC GTPT-FAM-50
2 2 24 2 2 2
B-HEG 30-NH -(CH) CH(CH OH)CH-(HEG)-TGA CGA TAG ATA GAC GTPT-FAM-50
6 2 24 2 2 6
A0-com 30-ACG TCT ATC TAT CGT C-FAM-50
A01-mis 30-ACG TCT ATC TTT CGT C-FAM-50
A0-non 30-CTT ATG CTA GCC ATT C-FAM-50
C-com 30-FAM-ACG TCT ATC TAT CGT C-50
D-HEG 30-NH -(CH) CH(CH OH)CH-ACG TCT ATC TAT CGT C-50
0 2 24 2 2
E-HEG 30-HS-(CH) -ACG TCT ATC TAT CGT C-50
0 26
E-HEG 30-HS-(CH) -(HEG) -ACG TCT ATC TAT CGT C-50
6 26 6
aHEG, hexaethylene glycol diphosphate ester; P, photolabile linker; T, mismatch nucleotide.
purification. Deionized (DI) water (ELGA genetics removal of the lithographic resist in acetone and
system) was used for the preparation of all the aqueous thorough cleaning. Diced pieces (2(cid:2)2 cm2) of single-
solutions. Undecylenic acid N-hydroxysuccinimide ester side polished silicon <100> wafer were precleaned
(UANHS) was synthesized as described elsewhere (4). (RCA clean), etched in 2% hydrofluoric acid for 2 min,
The oligonucleotides used for this study (Table 1) were washed thoroughly with water, dried under pressurized
synthesized in-house using conventional phosphoramidite nitrogen and immediately spin-coated with 40ml of 1%
chemistry and purified by reverse-phase high pressure (or UANHS solution in CH Cl at 2000r.p.m. for 50s (4).
2 2
high performance) liquid chromatography (HPLC). The The wafer was exposed through a quartz mask in
A-series oligonucleotides for attachment to the UANHS contact (containing parallel lines of 10m wide with 30m
linker functionalized silicon surface weresynthesized with spacing over a total area of 18 mm2) under nitrogen pro-
an aminomodifier C7 CpG (Glen Research) at the 30-end, tectionusinga500-WHgXelamp(OrielInstrument)fitted
and hexaethylene glycol diphosphate ester (HEG) spacers withaninterferencefilter(253.7±10nm,MellesGriot)at
were synthesized by inserting one, two or six 17-O-(4,40- 1 mW/cm2 for 5 min. After photoreaction, the wafer was
dimethoxytrityl)-hexaethyleneglycol, 1-[(2-cyanoethyl)- thoroughly washed with dichloromethane.
(N,N-diisopropyl)]-phosphoramidite units (Link
Technologies) and oligonucleotides with 92 ethylene Immobilization of DNA oligonucleotides onto the
glycol oligomers by addition of the PEG4000 NHS ester succinimidyl ester functionalized silicon surface
(Creative PEGWorks) to the aminohexyl group on the
The A-series of oligonucleotides used for immobilization
30-end of the oligonucleotide [identical to A-(HEG) ]
0 weredissolvedina0.1Msodiumbicarbonatesolutiontoa
following the solid-phase oligonucleotide synthesis.
finalconcentrationof50mM(pH=8.3).Eightmicrolitre
The B-series of oligonucletides prepared to establish
of the oligonucleotide solution was deposited on the
the surface-attachment density were synthesized by
UANHS alkene-patterned silicon wafer and covered
incorporationofthephotolabilegroup,3-(4,40-dimethoxy- with a cover slip (2(cid:2)2 cm2). The wafer was incubated
trityl)-1-(2-nitrophenyl)-propane-1,3-diol-[2-cyanoethyl-
overnight at room temperature in a humid chamber.
(N, N-diisopropyl)]-phosphoramidite (Link Technologies)
After incubation, the wafer was washed thoroughly with
between the DNA sequence and 6-carboxyfluorescein
DI water. Samples immobilized with the oligonucleotides
(FAM), which allowed the photolytic release of the were kept in DI water at 4(cid:3)C before characterization or
fluorophoregroupsfromtheoligonucleotidesonultravio- hybridization.
let (UV) irradiation (37). For the preparation of DNA–
GNP conjugates, 30-aminomodified oligonucleotides Preparation of DNA oligonucleotide–gold nanoparticle
(the D-series) and 30-thiolmodified oligonucleotides (the conjugates
E-series), all with a complementary sequence to the
(i) Gold nanoparticles (1.4nm diameter) with a single
A-series, were synthesized.
DNA oligonucleotide linked to the surface were prepared
as follows: mono-N-succinimide functionalized 1.4nm
Photopatterning of silicon surfaces with undecylenic acid
GNPs (Mono-sulfo-NHS-Nanogold, Nanoprobes) at a
N-hydroxysuccinimide
concentration of 15mM in 10mM sodium phosphate
Masks were prepared on UV-grade fused silica (Suprasil) buffer at pH 8.0 were mixed with two equivalents of the
by chrome evaporation followed by patterning by photo- D-series oligonucleotides at 4(cid:3)C overnight, and then
lithography and removal of the revealed chrome areas purified by ultracentrifugation (67000g) and triple
using the wet etching method. This was followed by washing of the pellet with 10mM phosphate buffer,
e80 NucleicAcidsResearch,2013,Vol.41,No.7 PAGE4OF12
0.3M NaCl, pH 7.0, to yield the 30-GNP end-labelled was illuminated through a band pass filter (345(cid:4)395nm)
DNA conjugate (HEG -GNP 1.4nm). with a 500-W HgXe lamp at various intensities and
0
(ii) Ten-nanometre diameter colloidal GNPs were exposure times. The photolysis products were analysed
conjugated with multiple DNA oligonucleotides per by HPLC to determine the percentage of oligonucleotide
particle using a similar procedure to that previously re- chains cleaved from the FAM group in comparison with
ported (28). The E-series oligonucleotides were treated the starting intact oligonucleotide (i.e. the photolysis
with 0.1M dithiothreitol (DTT) in 0.1M NaHCO at yield). The optimum conditions were found to be 30-min
3
room temperature for 1h to activate the thiol terminal illumination at 25–30 mW/cm2. The B-series of oligo-
groups. After the extra DTT was extracted with ethyl nucleotides were attached to the silicon surface in
acetate, the oligonucleotides were desalted using a patterns by the same method used for the A-series oligo-
Nap-10 column (Pharmacia) and freeze dried. The nucleotide attachment and described above. The wafer
activated thiol oligonucleotides were incubated with with the B-series oligonucleotide patterns was photolysed
10nmdiameterGNPs(BBInternational)at4(cid:3)Covernight using the optimal conditions determined above, and then
typically in a ratio of 0.009mmole gold nanoparticles to placedin800mlofwaterat90(cid:3)Cfor15mintodissolvethe
1.8mmole oligonucleotides. The DNA–GNP (10nm cleaved FAM groups. The wafer was removed, rinsed
diameter) conjugates were gradually exposed to 10mM twice with 100ml water and evaluated by fluorescence mi-
phosphate buffer, 0.1M NaCl, pH 7.0 for 16h for croscopy to make sure all the fluorophores had been
‘aging’, and then purified by ultracentrifugation removed. The collected solution (1000ml) was freeze
(26000g) and triple washing of the pellet with 10mM dried. The resulting material was re-dissolved in 70ml of
phosphate buffer, 0.1M NaCl, pH 7.0. The final product 0.05M Tris buffer (pH 8.7) and the fluorescence yield
was re-suspended and stored in a 10mM phosphate measured in a 50ml cuvette by fluorimeter (Cary Ellipse,
buffer, 0.3M NaCl, pH 7.0 solution. The concentration Varian Inc., (cid:2) =488nm, (cid:2) =525nm). The concen-
exc em
of the final DNA–GNP concentrations was estimated by tration of cleaved fluorophore groups present was
UV-visiblespectroscopy.Theextinctioncoefficientsofthe determined by reference to a standardization curve
characteristic adsorption peaks ofgold nanoparticles [420 produced by illumination [30 min illumination at 25–30
nm for 1.4nm (Nanoprobe Inc.) and 520nm for 10nm mW/cm2 (345(cid:4)395nm)] of a series of solutions of
gold particles] did not change significantly as a result of known concentration of the oligonucleotides containing
the oligonucleotide attachment (28). the photolabile and FAM groups in DI water.
Hybridization and denaturation of fluorophore and gold Hybridization density with single-stranded DNA
nanoparticle DNA conjugates from surface-attached DNA The hybridization density was quantified using our previ-
ously reported method (5). Briefly, A-series oligonucleo-
FAM-labelled A0-series or C-series oligonucleotides
tides were patterned on silicon surfaces using a
(20mM) were dissolved in phosphate-buffered saline
photolithographicmask.Complementaryfluorescentoligo-
(10mM phosphate buffer, 0.3M NaCl, pH 7.0) for
nucleotides (C-com) were hybridized on to the A-series
hybridization. A wafer patterned with an A-series oligo-
oligonucleotidepatternsandwashedthoroughlywithphos-
nucleotide was pre-soaked in phosphate-buffered saline
phatebuffer. Fluorescent images were acquired to confirm
for 20 min, and then excess buffer was removed.
hybridization and calculate the surface area of the oligo-
Immediately afterwards, 8ml of the oligonucleotide
nucleotidepatterns.Thewaferwasthenplacedin800mlof
hybridization solution was deposited on the wafer and
water at 90(cid:3)C for 15 min to allow for the denaturation of
covered with a cover slip. The wafer was incubated in a
the duplex. The wafer was removed and rinsed twice with
humid chamber for 4h at room temperature. After incu-
200ml of water and the wash collected. The wafer was
bation,thewaferwasrinsedthoroughlyandthenstoredin
examined by fluorescence microscopy to make sure all the
the phosphate buffer.
C-com had been removed. The water was removed in a
TheGNPconjugates(either1.4or10nm)withD-series
freeze drier and then the residue carefully dissolved in
or E-series oligonucleotides ((cid:4)10nM) were dissolved in
70ml of 0.05M Tris buffer (pH 8.7). The fluorescence
phosphate-buffered saline (10mM phosphate buffer,
yield from the sample was measured in a 50ml cuvette in
0.3M NaCl, pH 7.0) for hybridization. A pre-soaked
a fluorimeter (Cary Ellipse, Varian Inc., (cid:2) =488nm,
wafer patterned with an A-series oligonucleotide was exc
(cid:2) =525nm); at least four separate samples were
treated with 20ml of the oligonucleotide–GNP em
determined. The concentration of DNA strands present in
hybridization solution and incubated in a humid
the sample was determined by reference to a standardiza-
chamber for 8h at room temperature. After incubation,
tion curve prepared from a series of fluorescent-labelled
the wafer was rinsed thoroughly and then stored in the
DNA oligonucleotide samples of known concentration.
phosphate buffer.
Quantification of DNA–gold nanoparticle conjugates
Quantification of the surface-immobilization density and
hybridized to the DNA functionalized surfaces
hybridization efficiency
The conjugates hybridized on to A-series oligonucleotide
Surface-immobilization density patterns attached on the surface of the silicon were
Photolysis conditions for cleaving the photolabile group released by heating in water at 90(cid:3)C for 15 min, and the
from the surface-attached B-series oligonucleotides were collected DNA–GNP conjugate solutions were too dilute
optimized. Typically, the sample dissolved in DI water toestablishtheconcentrationbyabsorptionspectroscopy,
PAGE5OF12 NucleicAcidsResearch,2013,Vol.41,No.7 e80
soamassspectrometrymethodwasdevelopedtoestablish 7.0)onaglassslidetowardsEpiplanNeofluar10(cid:2)or20(cid:2)
thehybridizationdensity,andthisisachievedbymeasure- objective lenses.
ment of the gold concentration following chemical diges-
tion as follows: it is assumed that the density of spherical
RESULTS AND DISCUSSION
gold nanoparticles is equivalent to that of bulk gold
(=19.30g/cm3), so the average particle mass was Overview: formation of oligonucleotide patterns on silicon
calculated to be 2.77(cid:2)10(cid:5)20g for 1.4nm gold and
1.01(cid:2)10(cid:5)17g for 10nm gold nanoparticles. By establish- Our previously reported two-step method forcovalent at-
tachment of oligonucleotides onto crystalline silicon
ing the mass of gold in a DNA–GNP conjugate solution
surfaces in patterns has been exploited for attachment of
ascomparedwiththetheoreticalnanoparticlemassallows
DNA sequences to the silicon surface (Figure 1) (4). The
for the number of GNPs to be determined. The samples
subsequent coupling of the N-hydroxysuccinimide ester
were prepared for inductively coupled plasma–mass spec-
terminated surface with an amino-modified oligonucleo-
troscopy (ICP–MS) analysis by digestion of the collected
tides[SeriesA(seeTable1)]inanaqueoussolution,ledto
DNA–GNPconjugatesinwater(1ml)with50mlof0.1M
its covalent attachment of the single-stranded DNA
KCN and 1mM K Fe(CN) for 1h to dissolve the gold
3 6 (ssDNA) sequences (See Figure 2a for the schematic
nanoparticles. This solution was then diluted with 9ml of
showing the composition of the linker and spacer). In
5% HCl and analysed by ICP–MS following the method
this study, DNA oligonucleotides are attached to silicon
describedinPitcairnetal.(38).Controlsamplesprepared
surfaces via the linker with a chain of 18 atoms and a
in the same manner without DNA–GNP but with similar
spacermadeofoligomersofone,twoandsixhexaethylene
amountsofoligonucleotideswereusedasbackgroundref-
glycolphosphatediestergroupswithmonomerseachwith
erence samples. All measurements were repeated four
a chain of 20 atoms in the monomer. These chains
times.
provided combined linker and spacer lengths with 18,
38, 58 and 138 atoms long, covering a full range of
Quantification of the number of oligonucleotides attached
chain lengths; Southern et al. suggested that chains of
to the 10-nm diameter gold nanoparticles
30–60 atoms between surfaces and DNA sequences are
ThesurfacecoverageofDNAstrandson10-nmGNPswas
optimal for hybridization (17), and thus the chain
determined using a combination of ICP–MS and induct-
lengths chosen for the studies reported here cover this
ively coupled plasma–optical emission spectroscopy (ICP–
range.
OES) methods. The number of DNA strands was
ThepatternsofattachedDNAsequenceswererevealed
calculated from the phosphorus concentration determined
by epifluorescence microscopy after hybridization with
byICP–OESusingthefollowingmethod.Toavoidadven-
FAM-labelled complementary DNA sequences [A0-com
titious phosphate contamination, the DNA–GNP conju-
(see Table 1)], and a fluorescence signal of >95% of the
gates were aged in 0.1N NaCl solution (pH (cid:4)7.0). 150ml
desired pattern was achieved. Shown in Figure 3 are
of the DNA–GNP conjugate solution was weighed and
typicalepifluorescentimagesofthesamples(afterincuba-
transferred to a polytetrafluoroethylene high-pressure
tion and washing with buffer) with the 50-end FAM-
microwave digestion vessel and then spiked with an
labelled complementary sequence (A0-com), a sequence
yttrium as an internal standard. Following the addition of
containing a mis-matched thymine residue in the
1ml of sub-boiled concentrated HNO , the DNA–GNP
3 sequence(A01-mis)andasequencethatisnotcomplemen-
samples were digested for 30 min using a microwave
tarytotheprobesequence(A0-non).Thereislimitedtono
digestor (CEM, MDS2000). The digested samples were
levels of fluorescence revealed in the samples treated with
transferred to polyethylene containers and then diluted
thesequencecontainingamis-matchedthymineresiduein
with 10ml ultrapure water. The concentration of phos-
the sequence (A01-mis) and the sequence that is not com-
phorus was determined using ICP–OES calibrated with
plementary to the probe sequence (A0-non). The level of
synthetic reference standards. The number of moles of
fluorescence detected for the sample prepared with the
phosphorus was calculated from the determined mass
sequenceA-EG andhybridizedwiththecomplementary
divided by the atomic mass of phosphorus (30.9736). The 91
sequence A0-com is far lower than for any of the other
number of moles of the target oligonucleotide was
samples hybridized with A0-com.
calculated by dividing the number of phosphate groups in
the oligonucleotides (E-HEG )=17 and (E-HEG )=23.
0 6 Quantification of density of surface-immobilized probe
ThenumberoftargetDNAsequencesattachedpergold
nanoparticle was established by the determination of the Quantification of the surface-immobilized probe DNA
gold nanoparticle numbers in a comparable solution, but density is necessary, as this has been demonstrated to in-
using the ICP–MS method as described above. fluence the efficiency for hybridization (11). The quantifi-
cation of the probe density of DNA conjugated onto
surfaces has been achieved using radio-labelled oligo-
Epifluorescence microscopy
nucleotides (14,17,39,40). Radioactive-labelled oligo-
Thefluorescein-labelledoligonucleotide-patternedsurfaces nucleotides have a short shelf-life, are hazardous and are
werevisualizedusingaZeissAxiovert200invertedmicro- onlyaccessibletoresearchlaboratorieswiththeappropri-
scope with epifluorescence illumination fitted with a Zeiss ate infrastructure. Although an approach using alkaline
Filterset09.Allsampleswereplacedfacedownonadrop phosphatasedigestion hasbeenreportedforthequantita-
of phosphate buffer (10mM phosphate, 0.3M NaCl, pH tiveevaluationofthesurfacecoverageofoligonucleotides
e80 NucleicAcidsResearch,2013,Vol.41,No.7 PAGE6OF12
Figure 1. Schematic showing the steps for immobilization of oligonucleotides onto silicon surfaces.
Figure 2. Schematicshowingthechemicalgroupsusedforconjugationtosurfaces.(a)TheDNAoligonucleotidelinkagetothesiliconsurface.The
figureshowstheundecylamide‘linker’tothesiliconsurfaceattachedviaahexylchain(termedthe‘linker’)tothehexaetheneglycolphosphatediester
chain oligomer (termed the ‘spacer’), where n=0, 1, 2, 6. The spacer is conjugated to the 30-end of the DNA oligonucleotide. (b) The DNA
oligonucleotideconjugationusedtoestablishtheimmobilization densityatthesiliconsurface.Thesurfaceattachmentisthesameasin(a)butthe
oligonucleotideis50-endlabelledwithaphotolabilegroup[1-(2-nitrophenyl)-propane-1,3-phosphatediester]attachedtoFAMviaathymineresidue.
(c)TheDNAoligonucleotidelinkagetothegoldnanoparticle(10nm).Thegoldnanoparticle(10nm)linkedviaathiollinkagetoahexylchainand
then to the spacer, where n=0, 6 (for E-HEG -GNP and E-HEG -GNP, respectively). The spacer is linked to the 30-end of the DNA
0 6
oligonucleotide.
at relatively low densities ((cid:4)109 oligonucleotides\cm2) hexyl amino group used to attach to the N-hydroxysuc-
(41), polyethylene glycol is well known to interact with cinimide ester surface (Figure 2b). The nitrobenzyl group
proteins (42). An alternative approach to establish the offersahighlyefficientphotocleavablegroupandiseasily
immobilized density of DNA oligonucleotides with incorporated into DNA oligonucleotides during DNA
ethylene glycol oligomer spacers was required. Here we synthesis by the use of commercially available phospho-
have developed a simpler safer fluorescence-based tech- ramidite derivatives (43). Here the DNA sequence (Series
nique for the determination of the surface-attachment A, Table 1) is attached to the silicon surface via attach-
density of DNA oligonucleotides based on the work of ment to the N-hydroxysuccinimide ester surface and only
Ju et al. (37). In short, a photolabile linker based on a the FAM released by UV illumination. The photore-
2-nitrobenzyl group is attached between the 50-end of the action, as reported, results in FAM-photolabile group
oligonucleotide and the FAM label, to the 30-end of the release leaving behind the DNA oligonucleotide still
oligonucleotide is the ‘spacer’ group that is linked to a attached to the surface (37). The FAM-containing
PAGE7OF12 NucleicAcidsResearch,2013,Vol.41,No.7 e80
Figure 3. Epifluorescent images of the patterns of A-series of oligonucleotides (A-HEG , A-HEG , A-HEG , A-HEG and A-EG ) on silicon
0 1 2 6 91
surfacesafterhybridizationwithfluorescein-labelled(a)non-complementarysequences(A0-non),(b)sequencescontainingasinglemismatch(A0-1mis)
and (c) complementary sequence (A0-com). The sequences used are identified in Table 1.
moleculesarecollectedandtheDNAimmobilizeddensity ShowninTable2istheimmobilizeddensityofB-series
established as detailed in the ‘Materials and Methods’ oligonucleotides covalently attached on silicon surfaces.
section. High densities of 5.4–9.6(cid:2)1012strands/cm2 for oligo-
A study was carried out by illumination of each of the nucleotides with and without hexaethylene glycol phos-
B-series oligonucleotides in aqueous solution using the phate diester oligomers have been achieved.
band pass filtered UV irradiation (375±25nm) of a
HgXe lamp at a range of illumination intensities from The effect of spacer on the surface-attached DNA
10 to 50 mW/cm2, and a range of illumination periods oligonucleotides on the hybridization density for
from 10 to 120 min. The UV absorption peak of DNA fluorescently labelled and gold-nanoparticle-labelled
(260nm) and the fluorescence quantum yield of FAM oligonucleotides
((cid:2) =488nm) were monitored to establish the optimal
exc Fluorophore-labelled DNA oligonucleotide
photolysisconditionstoreleasetheFAMwithoutdamage
The immobilized densities for the DNA sequences
to the DNA oligonucleotide. No obvious change was
A-HEG , A-HEG , A-HEG and A-HEG on silicon are
observed at a UV absorbance of 260nm after photolysis 0 1 2 6
listedinTable2.TheoligonucleotideusedisC-com,which
underanyoftheconditionsstudied,indicatinganegligible isthe30-FAMend-labelledcomplementaryDNAsequence
quantity of DNA photolysis products, such as thymine toprovideconsistencywiththe30-GNPend-labelledcom-
dimers. A decrease in the fluorescence quantum yield of
plementaryDNAsequenceused(seestudiesbelow).There
thesolutionwasobservedafterphotolysis.Thisvariability
is a small variation in the immobilized density for the se-
in the reduction in fluorescence yield with filtered UV il-
quences A-HEG , A-HEG , A-HEG and A-HEG on
0 1 2 6
lumination, for example, repeat individual experiments of silicon as well as a small difference in the hybridization
oligonucleotide B-HEG6 solutions (at 1(cid:2)10(cid:5)8M after densitywithC-com.Totakeintoaccounttheminordiffer-
photolysis at 20(cid:4)25mW/cm2 for 30 min) showed a ence in the immobilized density, the hybridization effi-
decrease from initial values by 53±1% for three ciency was calculated (Table 2). For all cases, a high
samples. Standardization curves prepared from a series efficiencyofhybridizationwasobtained,ofalmostfulloc-
of FAM-photolabile group-oligonucleotide solutions of cupancyof theimmobilized complementary sequences. In
known concentration after the photolysis showed a short, one DNA hybrid is formed on average for every
linear relationship between oligonucleotide concentration (cid:4)20–40 nm2, which is almost at the density of the
and fluorescence intensity. These results demonstrate that immobilized complementary sequence. Some of the
the quantification of immobilized oligonucleotides on earlierreportshaveshownthatthehybridizationyield in-
silicon surfaces can be achieved by reference to a creases where the hexaethylene glycol phosphate diester
standard curve of the same type of oligonucleotide chain length increases (10,11). For the system studied
under the same photolysis conditions. The photolysis here, with both a surface linker and a spacer, the length
yield, as determined by HPLC, approached 100% at illu- of the hexaethylene glycol phosphate diester oligomer
minationintensities >25mW/cm2and>30minexposure. spacer has an effect, if minor, on the hybridization effi-
Therefore, an illumination intensity of (cid:4)28mW/cm2 for ciency for oligonucleotide C-com. The hybridization effi-
30 min was used for the photolysis of immobilized oligo- ciency for the surface-immobilized sequences A-HEG ,
1
nucleotides on silicon surfaces, and for the calibration A-HEG and A-HEG is slightly higher than with
2 6
standards. surface-immobilized A-HEG (Table 2). Although
0
e80 NucleicAcidsResearch,2013,Vol.41,No.7 PAGE8OF12
Table 2. Densities of the immobilized oligonucleotides on silicon surfaces and the complementary ssDNA strands hybridized onto them
Immobilized DNA Number of atoms Immobilized density Hybridization density Hybridization efficiencya
between DNA and silicon surface number/cm2 ((cid:2)1012) number/cm2 ((cid:2)1012)
A-HEG 18 5.4±0.8 2.3±0.3 42%
0
A-HEG 38 9.6±2.1 5.9±0.7 62%
1
A-HEG 58 7.3±1.9 4.8±0.9 67%
2
A-HEG 138 5.4±0.7 3.8±0.7 70%
6
aHybridization efficiency, determined from the immobilized density versus the hybridization density.
A-HEG hasthelowestsurface-immobilizeddensity,there chemicalstructureofthelinkageisshowninFigure2c.So
0
isarelativelysmallincreaseinthehybridizationefficiency far a direct method for the quantification of
withlengthofhexaethyleneglycolphosphatediesterspacer thiol-terminated oligonucleotides coupled onto GNPs
fromA-HEG ,A-HEG toA-HEG . has not been reported. The feed ratio of thiol-terminated
1 2 6
oligonucleotides to GNPs used in the synthesis does not
GNP of 1.4nm conjugated with a single complementary provide the number of strands per DNA–GNP because
strand (HEG -GNP 1.4nm)
0 theyieldofattachmentisinfluencedbysurfaceadsorption
The hybridization of immobilized oligonucleotides on
and the equilibrium, oligonucleotide length, nucleotide
silicon surfaces with DNA–GNP conjugates was
bases and even washing steps during the preparation
investigated. Two types of DNA–GNP conjugates were
(13,28,44). Existing methods for the determination of
used:a1.4nmGNPconjugatedwithasinglecomplemen-
surfacecoverageofoligonucleotidesonGNPsarefluores-
tary strand (HEG -GNP 1.4nm) and a 10-nm gold
0 cence based (13). However, accurate data for this existing
nanoparticle labelled with multiple complementary
approach is dependent on the successful removal of the
strands (HEG -GNP 10nm), described below.
0 GNPsfromthefluorescentlabels,soanewmethodwhere
A new method was developed for the determination of
the elemental composition is quantified has been de-
the number of DNA–GNPs hybridized to surface-at-
veloped here.
tached complementary sequences. In brief, DNA–GNP
Phosphorus exists stoichiometrically in DNA and can
samples were hybridized with the DNA-functionalized
be quantified using ICP–OES with an accuracy of (cid:4)1%
silicon surfaces, washed and then released by heating the
(45).QuantificationoftheamountofphosphorusbyICP–
substrate at 90(cid:3)C in an aqueous solution. The collected
OES and gold by ICP–MS in the same sample provides a
solution containing the released DNA–GNP conjugates
new approach to deduce the ratio of phosphorus to gold
from the DNA sequences attached to surface were
and hence the number of oligonucleotides to GNPs. This
evaluated by ICP–MS following the method described in
hasbeenappliedinthisstudy.Inallcases,theactualratio
Pitcairn et al. (38) (see ‘Materials and Methods’ section).
of oligonucleotides bound to GNPs is less than the feed
Figure4containsplotsofthehybridizationdensitiesof
ratio.TheaveragesurfacecoverageofHEG oligonucleo-
DNA–GNP conjugates (HEG -GNP 1.4nm), and these 0
0 tidesonGNPsatafeedratioof200is124±8strandsper
can be compared directly on the same plot with the
nanoparticle (corresponding to 66±4pmol/cm2,
hybridization densities of the fluorescently labelled DNA
3.9(cid:2)1013 strands/cm2). This coverage is consistent to
(C-com) onto immobilized A-HEG , A-HEG , A-HEG
0 1 2 that reported for DNA–GNP conjugates prepared in a
and A-HEG oligonucleotide patterns on silicon. There is
6 similar manner (42pmol/cm2) (13).
no significant variation in the hybridization density for
Figure4containsaplotofthehybridizationdensitiesof
1.4nm DNA–GNP conjugates as a function of the
HEG -GNP 10nm, in comparison with those of the
spacer length (A-HEG , A-HEG , A-HEG and 0
0 1 2 ssDNA (C-com) and HEG -GNP 1.4 nm onto immo-
A-HEG ) of the sequence attached to the silicon surface. 0
6 bilized A-HEG , A-HEG , A-HEG and A-HEG oligo-
The hybridization density of 1.4nm DNA–GNP conju- 0 1 2 6
nucleotide patterns on silicon. The hybridization density
gates is approximately four times lower than for
of the HEG -GNP 10nm with the A-HEG , A-HEG ,
fluorescently labelled DNA (C-com), but this still repre- 0 0 1
A-HEG and A-HEG oligonucleotides attached on the
sents a hybridization efficiency of between 10 and 20%— 2 6
silicon are not significantly different and is (cid:4)3(cid:2)1010
which is good, providing a hybridization density of
(cid:4)1(cid:2)1012 DNA–GNP (1.4nm)/cm2—or one complemen- hybrids/cm2 or (cid:4)1 hybrid per (cid:4)3000 nm2. In fact, the
tary sequence per (cid:4)100 nm2. The lack of variation in the hybridization density of A-HEG0-GNP 10nm conjugates
is lower by a factor of 30 as compared with the A-HEG -
hybridization density suggests that hexaethylene glycol 0
GNP 1.4nm conjugates and yet again is not determined
phosphate diester spacers between the immobilized oligo-
simply by the length of the spacer for the oligonucleotide
nucleotides and the silicon surface have a minor effect on
attached to a planar silicon surface. Thus, the size of the
their hybridization with HEG -GNP (1.4nm) conjugates.
0
GNP in the DNA–GNP conjugates appears to be an im-
GNP of 10nm conjugated with multiple complementary portant factor affecting hybridization at surfaces and the
strands (HEG -GNP 10nm) yield of hybridization. An increased DNA–GNP conju-
0
The 30-thiol end-labelled DNA oligonucleotide (Table 1, gate dimension would occupy a higher exclusive cross-
E-HEG) was conjugated to 10nm gold nanoparticles; the section area, and cause the hybridization density to be
PAGE9OF12 NucleicAcidsResearch,2013,Vol.41,No.7 e80
8e+12 nanoparticle). Of note, a previous report indicates that
the insertion of 20-dA or 20-dT spacers leads to a lower
6e+12
surface coverage of oligonucleotides on GNPs (13). The
4e+12 small improvement in the surface coverage of GNPs with
2m) theoligonucleotideE-HEG ascomparedwithDNAwith
er/c 2e+12 20-dAor20-dTspacerscou6ldbearesultoftwofactors:(i)
b
m
u a transition from an amorphous coil to a brush-like state
n
y ( occurring as the packing density of oligonucleotides
nsit 4e+10 increase, which is a well known scenario for polyethylene
e
D glycol layers of higher molecular weight (Mw > 1000)
3e+10
(47,48); and (ii) the cross-section of an EG unit in its
2e+10 idealizedhelical form isonly0.21 nm2(49)incomparison
with that of ssDNA at 0.9 nm2 (the cross-section of
1e+10
0 2 4 6 ssDNA is 1.07nm). So, more DNA strands containing
number of hexaethyleye glycol phosphate diester units hexaethylene glycol phosphate diester oligomer spacers
could be accommodated onto the 10-nm nanoparticle
Figure 4. Plot of the hybridization density of fluorescently labelled
DNA (C-com), DNA–GNP conjugates (E-HEG -Au 1.4nm and surface as a result of spatial reasons.
0
E-HEG-Au 10nm) onto oligonucleotide patterns (A-HEG , The hybridization density of the E-HEG -GNP 10nm
0 0 6
A-HEG1, A-HEG2 and A-HEG6) on silicon surfaces versus number conjugates onto the immobilized complementary strands
of hexaethylene glycol phosphate diester oligomers. Hybridization A-HEG (Figure 5a) is 1.26±0.24(cid:2)1011 particles/cm2,
density of the DNA-modified silicon surfaces after hybridization with 0
which is more than four times higher than that of the
the FAM-labelled complementary stands (dash line), GNP (1.4nm)–
ssDNA conjugates (solid line) and GNP (10nm)–DNA conjugates E-HEG -GNP (10nm) onto immobilized complementary
0
(dash-dot line). Error bars shown are standard error of the mean. strands A-HEG (Figure 5b) (3.1±0.8(cid:2)1010 particles/
0
cm2),showingasignificantenhancementforhybridization
of E-HEG -GNP 10nm due to the presence of
6
lower than for the A-HEG -GNP 1.4nm or C-com. In hexaethylene glycol phosphate diester oligomer spacer
0
solution, the hydrodynamic radius of a short DNA on the GNP surface. The hybridization density of
chain of <20 bases has been determined to be 1nm (46) E-HEG -GNP 10nm onto silicon surface functionalized
0
and 8.7 for a 10-nm GNP labelled with multiple 12 bp with A-HEG (Figure 5c) is 2.1±0.5(cid:2)1010 particles/
6
DNA oligonucleotides prepared in a similar manner cm2. The hybridization density of E-HEG -GNP 10nm
6
(16). Using published data (46), the approximate ratio of conjugates onto the immobilized complementary strands
exclusive cross-section areas between an HEG0-GNP A-HEG0(Figure5a)issixtimeshigherthanforE-HEG0-
10nm conjugate and an ssDNA (A-HEG0, 17 base) GNP 10nm conjugates onto A-HEG6 (Figure 5c). These
strand is estimated to be (cid:4)75. This difference, however, results suggest that the presence of the spacer of
is too small to account for the observed difference in oligomeric hexaethylene glycol phosphate diester has the
hybridizationdensity.Thissuggeststhatthereducedflexi- biggest impact when present on the 10-nm GNP–DNA
bility of a DNA strand after attachment to the 10nm conjugate. Incorporating the hexaethylene glycol phos-
GNP and steric hindrance between neighbouring strands phate diester oligomer spacer on the GNP surface will
on the DNA–GNP 10nm conjugates are the major deter- result in the following: (i) a greater distance of the DNA
minants impacting on the hybridization efficiency. from theGNP surfaceand thus higher accessibility ofthe
Additional factors that may influence the hybridization conjugatedDNAstrands;and(ii)agreatercross-sectional
efficiency could include electrostatic factors resulting area of the DNA–GNP conjugate as a result of the
from the surface charge of the GNP or the high density increased spacer size. Thus, on collision with the DNA
of DNA attached; however, under the saline buffer con- functionalized silicon surface, the E-HEG6-GNP will
ditions used, these effects are anticipated to be minor. occupy a higher coverage (by up to (cid:4)50nm diameter) as
comparedwithE-HEG -GNP(byupto(cid:4)23nmdiameter)
0
Ten nanometre GNP conjugated with multiple (see Figure 5). DNA hybridization occurs via a two-step
complementary strands containing a hexaethylene glycol process:(i)DNAnucleation,followedby(ii)rapidzipper-
phosphate diester oligomer (HEG -GNP 10nm) ing to yield the duplex (35). It is thus possible that there
6
A hexaethylene glycol phosphate diester oligmer spacer are both a higher probability of nucleation due to
group between the gold nanoparticle (10nm) surface and increased flexibility of the linker-spacer chain of
theattachedsingle-strandedoligonucleotideswastestedto E-HEG -GNP and coverage of the underlying silicon-
6
establishtheeffectontheyieldofhybridization.Theratio DNA surface on collision (Figure 5).
of oligonucleotides per nanoparticle was determined by Given the cross-sectional area of the DNA–GNP
ICP–OES and ICP–MS methods, as detailed above. At a (10nm) conjugates, taking into account the size of the
feed ratio of 200 oligonucleotides per GNP, 144±3 attached DNA oligonucleotides and spacers, the density
strands of E-HEG oligonucleotides per nanoparticle of hybridized DNA–GNP conjugates is high and is typic-
6
were determined, resulting in a higher packing density ally more than one DNA–GNP (10nm) per (cid:4)3000 nm2.
(76±1pmol/cm2, 4.6(cid:2)1013 strands/cm2) in comparison Thus the method represents an efficient approach for the
with the oligonucleotides E-HEG (124±8 strands per self-assemblyofGNPsonsiliconsurfaces.Itisanticipated
0
e80 NucleicAcidsResearch,2013,Vol.41,No.7 PAGE10OF12
Figure 5. Schematic showing silicon surfaces functionalized with DNA oligonucleotides with (a) no spacer linkage (A-HEG ) and hybridized with
0
DNAwithahexaethyleneglycolphosphatediesteroligomerchainSpacerlinkedtoagoldnanoparticle(10nm)(E-HEG -GNP10nm).Thefigureis
6
notdrawntoscale,theGNPis10nmdiameter,theDNAstrand(shownbythewavyline)is(cid:4)5nmlongandthespacerHEG is(cid:4)15nmlong(fully
6
extended). (b) No spacer linkage (A-HEG ) and hybridized with DNA with no spacer, but linked to a gold nanoparticle (10nm) (E-HEG -GNP
0 0
10nm). The linker between the GNP and DNA is (cid:4)1nm long and the linker between the silicon and DNA is 2.4nm. (c) a hexaethylene glycol
phosphatediesteroligomerchainspacer(A-HEG )hybridizedwithDNAsequenceslinkedtogoldnanoparticles(10nmdiameter)withoutaspacer
6
(E-HEG -GNP10nm).Thefigureisnotdrawntoscale,thelinkerattachedtothesiliconsurfaceis(cid:4)2.4nmandthespacer,HEG is(cid:4)15nmlong
0 6
(fully extended).
that other nanoparticles and surfaces could be A-HEG -DNA attached to the silicon surface provided a
0
self-assembled in a similar manner. 6-fold increase in the hybridization density as compared
with E-HEG -DNA with the A-HEG -DNA attached to
0 36
the silicon surface. These results suggest that the attach-
CONCLUSIONS mentlinkerandspacerlengthoftheDNAattachedtothe
gold nanoparticle of 10nm has a more significant impact
Ourapproachinperformingthisstudyistodevelopquan-
thantheattachment linkerand spacer lengthofthe DNA
titative approaches to provide an accurate evaluation of
attachedtothesiliconsurface.Thisismostlikelyafunction
(i) the immobilization density of DNA on the surface
the DNA molecule attached on the gold nanoparticle via
of silicon and on the surface of gold nanoparticles and
a hexaethylene glycol phosphate diester linker being
to then evaluate (ii) the hybridization efficiency of sur-
more free to diffuse around on the silicon surface during
face-attachedDNAasafunctionoftheattachmentchem-
collisionwiththeDNAfunctionalizedsiliconsurface.
istry. For our case, we have developed DNA-attachment
TheDNAconjugationmethodreportedhereprovidesa
chemistry to provide high hybridization efficiencies for
simple and reliable way to provide controllable interfaces
fluorophore-labelled DNA with the surface-attached
betweensiliconsurfacesandgoldnanoparticles.Although
DNA. The silicon surfaces, as studied, provide efficient
thesizeofthegoldnanoparticlehasamajorimpactonthe
hybridization of fluorophore-labelled DNA hybridization
hybridization yield at the surface, the approach reported
with efficiencies of between 42 and 70%. Even with the
here provides high yields of hybridization of fluorophore
high hybridization efficiencies, there is a small trend
and1.4-nmgoldnanoparticle-labelledDNAwithcomple-
showing improved efficiencies as a function of the spacer
mentarysequencesonasiliconsurface.Applicationofthe
length. In contrast, tuning the spacer length for the DNA
method is likely to offer potential for the creation of
oligonucleotide attached to the silicon surface does not
bioanalytical tools and nanobioelectronic structure
have a major effect on the yield of DNA–GNP
fabrication.
hybridization, whether for DNA–GNP (1.4nm) or
DNA–GNP (10 nm). The hybridization efficiencies of
DNA–GNP were proportional to the nanoparticle size. FUNDING
The DNA–GNP (1.4nm) hybridization efficiencies were
We are grateful for the financial support of the
onlymarginallylessefficientthanthefluorophore-labelled
Biotechnology and Biological Sciences Research Council
DNA hybridization efficiency, but independent of the
(ReferencesE16611and51/E18926)andtheRoyalSociety
spacer size for the DNA attached to the surface. The
(Biomolecule Patterns on Silicon). Funding for open
density for hybridization for the E-HEG -DNA (10nm),
0 access charge: A partial waiver of fees is gratefully
even taking into account the cross-section of the hybrid,
acknowledged, the remaining fees are funded from
was poor. The incorporation of a hexaethylene glycol
personal research funds.
phosphatediesteroligomer betweenthegoldnanoparticle
and the DNA oligonucleotide (E-HEG -DNA) with the Conflict of interest statement. None declared.
36