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Article

Enhancing On-Surface Mismatch Discrimination Capabilityof PNA Probes by AuNP Modification of Gold(111) Surface

Srabani Ghosh, Sourav Mishra, and Rupa MukhopadhyayLangmuir, Just Accepted Manuscript • DOI: 10.1021/la4019579 • Publication Date (Web): 26 Aug 2013

Downloaded from http://pubs.acs.org on September 8, 2013

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Enhancing On-Surface Mismatch Discrimination Capability of PNA Probes

by AuNP Modification of Gold(111) Surface

Srabani Ghosh, Sourav Mishra and Rupa Mukhopadhyay*

Department of Biological Chemistry, Indian Association for the Cultivation of Science,

Jadavpur, Kolkata-700 032, India

Abstract

Unambiguous identification of single base mismatches in nucleic acid sequences is of great

importance in nucleic acid detection assays. However, ambiguities are often encountered with,

and therefore, a strategy for attaining substantially large enhancement of mismatch

discrimination has been worked upon in this study. Short single-stranded peptide nucleic acid

(PNA) and deoxyribonucleic acid (DNA) sensor probes that are immobilized onto gold

nanoparticle (AuNP) modified Au(111) surface have been applied for target DNA detection. It

will be shown that while both PNA and the analogous DNA probes exhibit generally better target

detection abilities on the AuNP-modified Au(111) surfaces (elicited from fluorescence-based

measurement of on-surface Tm values), compared to the bare Au(111) surface, PNA supersedes

DNA, for all sizes of AuNPs (10, 50 and 90 nm) applied - the difference being quite drastic in

case of the smallest 10 nm AuNP. It is found that while the AuNP curvature plays a pivotal role

in target detection abilities of the PNA probes, the changes in the surface roughness caused by

AuNP treatment do not exert any significant influence. This study also presents a means for

preparing PNA-AuNP hybrids without altering PNA functionality and without AuNP

aggregation by working with the surface-affixed AuNPs.

*Corresponding author: Dr. R. Mukhopadhyay (Telephone: +91 33 2473 4971 Extn. 1506;

Fax: +91 33 2473 2805; E-mail: bcrm@iacs.res.in)

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Introduction

In the last one decade, nanoparticles functionalized with oligonucleotide sequences have been

used as probes in myriads of DNA detection technologies [1–9]. Importantly, it has been shown

that nanoparticle-based assays can be useful in differentiating fully matched targets from those

having single-base mismatches, whereas the analogous assays (devoid of nanoparticles) that

involve the use of fluorophore probes, e.g., Cy3, Fluorescein etc., do not offer such selectivity

[10]. Amongst different material types of the nanoparticles, gold nanoparticles (AuNPs) have

proved to be especially attractive because of ease of preparation, well-defined shapes/sizes, size-

dependent control of the nanoparticle properties, long shelf life (6 months to one year, depending

upon the nature of NPs and storage condition), relatively non-toxic nature, suitability for optical

detection, and straightforward functionalization with oligonucleotide probes [11−13]. Not

surprisingly, there have been a number of reports that describe DNA detection schemes based on

hybridization of target DNA molecules to oligonucleotides immobilized onto gold nanoparticles

[5, 14–19].

Explicit identification of single base mismatches in nucleic acid sequences is of paramount

importance for reliably recognizing genetic variations present amongst individuals that determine

how an individual develops response to external agents like drugs, pathogens, chemicals etc. and

an individual‟s propensity toward development of a specific disease. Herein, we report a

straightforward strategy for amplifying mismatch discrimination capability of PNA and DNA

sensor probes, by immobilizing the probes onto AuNP-modified Au(111) surface (a scheme for

the basic assay setup is shown in Scheme 1). It was presumed that application of differently sized

nanoparticles would assist in generating surfaces of different roughnesses/surface areas/local

curvatures. Such differences could result in different sensor probe densities that are expected to

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influence on-surface target hybridization to different extents since it has been shown earlier that

surface probe density can strongly influence target hybridization [20]. Based on these

presumptions, we aimed at controlling the target hybridization capacity of PNA and DNA sensor

probes immobilized onto AuNP-modified Au(111) surface, and especially, at enhancing the

mismatch discrimination competence of the surface-anchored sensor probes. We employed

Au(111) surface as the substrate in our study, since it has been recently reported that mismatch

discrimination by PNA/DNA sensor probes can generally be improved on Au(111) surface, in

comparison to the solution phase [21, 22]. Also, Au(111) surface is widely used in biosensor

applications [23, 24], especially where immobilization of the sensor molecules via gold-thiol

bond formation [25] is exploited. We applied thiolated 12-mer PNA/DNA sequences having a –

(CH2)6SH at the N-terminal (in case of PNA probes) and at the 5′ end (in case of DNA probes) as

the sensor probes.

Despite the positive attributes of PNA, i.e., PNA probes can bind to DNA oligomers in a

sequence-specific manner with higher affinity compared to the DNA probes obeying Watson−

Crick hydrogen bonding rule [26−29] and that PNA is not susceptible to hydrolytic (enzymatic)

cleavage [30], for being applied as sensor probes, it has not superseded DNA in nanoparticle-

based strategies since PNA attachment to AuNPs has been proven difficult [31, 32]. It has been

observed that when thiolated PNA is added to a citrate-stabilized AuNP dispersion, the particles

are rapidly agglomerated and precipitated out from the solution [32]. It is likely that the non-

ionic, thiolated PNA strands displace citrate anions from gold surface, allowing the bare areas of

AuNPs to bind irreversibly to other exposed AuNPs, making the dispersion unstable. So far,

successful attachment of PNA probes to AuNPs has involved the use of negatively charged PNA

strands, where the negative charges were introduced either by incorporating amino acid residues

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at the tethered end [32, 33], or through construction of PNA–DNA chimeras [32]. Although such

modifications have allowed formation of the PNA-AuNP conjugates, where the colloid stability

has remained intact, PNA functionality had to be altered. Therefore, the present study also offers

a means for constructing PNA (as is) - AuNP hybrids, without getting PNA aggregated, as the

AuNPs are pre-attached onto a solid surface. It will be shown that the ssPNA probes

immobilized onto the AuNP-modified Au(111) surface can efficiently detect complementary

DNA probes and are even capable of single base mismatch discrimination. Importantly, we

report that by reducing the size of the AuNP to as small as 10 nm, mismatch discrimination by

PNA sensor probes can be amplified to a degree, which far supersedes the capacities of the

analogous DNA sensor probes.

Materials and Methods

Preparation of PNA sensor probe solutions: The 12-mer ssPNA sensor probes [PNA 1, PNA

2, PNA 3 and Cy3-PNA 1, see Table 1 for the sequences] (Panagene, Korea), all having a hexyl

thiol [–(CH2)6SH] group at N-ter position, were dissolved in filtered autoclaved Milli-Q water of

resistivity 18.2 MΩcm or other solvents, i.e., sodium phosphate buffer (20 mM sodium

phosphate, 100 mM sodium chloride, pH 7.00), 1% TFA+10% ACN, 10% DMF, 1% TFA, 1%

Acetic acid, and 1% Formic acid, as required. PNA 1 and PNA 2 were the pairs of sequences

having a single base difference at the centre of the sequences [see the underlined residue in Table

1], while PNA 3 was the completely mismatched sequence that was used for the control

experiments. The exact concentrations of PNA solutions were determined by UV-visible

spectrophotometry at room temperature (24±1 ºC), using absorbance values at 260 nm [ε260

(L/(mol × cm) values for PNA 1, PNA 2, PNA 3 and Cy3-PNA 1 taken as 116700, 123800,

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116700 and 122200, respectively - all the ε values applied here or later were obtained from the

manufacturer-provided data sheets].

Preparation of DNA sensor probe solutions: The 12-mer ssDNA sensor probe samples [DNA

1, DNA 2 and DNA 3, see Table 1 for the sequences] (Alpha DNA, Canada), all having a hexyl

thiol [–(CH2)6SH] group at the 5′ position, were dissolved in sodium phosphate buffer (20 mM

sodium phosphate, 100/1000 mM sodium chloride, pH 7.00). DNA 1 and DNA 2 were the pairs

of sequences having a single base difference at the centre of the sequences [see the underlined

residue in Table 1], while the DNA 3 was the completely mismatched sequence that served as

sample for control experiments. The exact concentrations of the DNA solutions were determined

by UV-visible spectrophotometry at room temperature, using absorbance value at 260 nm [ε260

(L/(mol × cm) for DNA 1, DNA 2 and DNA 3 taken as 123020, 131350 and 123020,

respectively].

Preparation of Cy3 labeled DNA target probe solutions: The Cy3 labeled target DNA probes

[Cy3-DNA 1 and Cy3-DNAnc, see Table 1 for the sequences] (IDT, Canada) was taken in

sodium phosphate buffer (20 mM sodium phosphate, 2/100 mM sodium chloride, pH 7.00). The

exact concentrations of the DNA solutions were determined by UV-visible spectrophotometry at

room temperature, using absorbance value at 260 nm [ε260 (L/(mol × cm) for Cy3-DNA 1 and

Cy3-DNAnc taken as 124400 and 116000, respectively].

Immobilization of PNA/DNA sensor probes onto AuNP-modified Au(111) surface: Gold on

mica substrate (Phasis, Switzerland) having a 200 nm thick gold layer was flame annealed until a

reddish glow appeared. This procedure was repeated 7−8 times and after a short period (1−2 s) of

cooling in air, modification with the respective solution was carried out. Generation of clean and

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triangular terraces of high quality Au(111) surface1 due to flame annealing was checked by AFM

imaging at ambient condition (Fig. 1a). The annealed Au substrates were modified with 2-

mercaptoethylamine (MEA) or cysteamine (Sigma-Aldrich) / 1, 4 - benzenedithiol (Sigma-

Aldrich) from a 1.0 mM ethanolic solution for a time period of 30 min. Then the substrate was

washed with 2 ml ethanol and 1 ml Milli-Q water. Immobilization of the 10/50/90 nm AuNPs

(AuNP10/50/90) onto amine-modified (MEA-coated) surfaces (see Figs. 1b and c and Fig. S1 in the

supporting information) was performed from aqueous sols of the AuNPs (concentration 0.17 nM,

which was determined by UV-Visible spectrophotometry) at room temperature. A similar

procedure was applied for AuNP immobilization onto 1, 4 - benzenedithiol modified surface.

The modified gold substrate was washed with 2 ml Milli-Q water. The thiolated PNA/DNA

sequences were immobilized onto the AuNP-modified Au(111) surface (Fig. 1d) via gold-thiol

bond formation [25] by incubating the gold pieces in the nucleic acid solutions of 0.5 µM

concentration for 4 h incubation time in fully immersed condition at room temperature or by

incubation at 60 ºC, as the case may be. After incubation was complete, the modified surface of

the gold piece was washed with 2 mL filtered autoclaved Milli-Q water and dried with soft

nitrogen jet, followed by AFM or fluorescence imaging.

In order to test the effect of a spacer, e.g., β-mercaptoethanol (Sigma-Aldrich), 6-

mercaptohexanol (Sigma-Aldrich), on the on-surface PNA-DNA hybridization, the PNA-

modified Au(111) substrate was treated with 1.0 mM of the spacer molecules for 1 h at room

temperature. After incubation was complete, the substrate was washed with 2 mL filtered

autoclaved Milli-Q water, dried with soft nitrogen jet, and fluorescence images were obtained.

1 The Au(111) has been the preferred choice over the polycrystalline gold since a large number of atomically flat

(111) crystal planes, which offers 3-fold hollow sites, the energetically most favorable thiol-binding sites [34], are

available in case of the former.

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On-surface melting experiments: To investigate the melting behavior of the nucleic acid

duplexes on Au(111) surface and AuNP-modified Au(111) surface, 20 µL droplet of the Cy3

labeled DNA target probe solution of 1.0 µM concentration was deposited on the thiolated

PNA/DNA modified surface and incubated in a humidity chamber for 1 h at room temperature.

The gold pieces were then washed with 4 mL of sodium phosphate buffer (20 mM sodium

phosphate, 2/20/100 mM sodium chloride, pH 7.00). The gold pieces were dried with soft

nitrogen jet and the AFM and the fluorescence images of the resulting surface were captured (see

Fig. 1e for an AFM topographic view of the surface after hybridization and Fig. S2 in Supporting

Information for a representative fluorescence image). For melting the duplexes, the samples were

placed in 600 µL of sodium phosphate buffer and heated to desired temperatures for 15 min. The

samples were taken out, washed with 2 mL (500 µL×4) of the sodium phosphate buffer, dried

with soft nitrogen jet and the fluorescence images were obtained. Heating of a sample was

always performed by starting from the lower temperatures and in steps that could be as small as

1.0 °C (near the anticipated melting temperature value) or as high as 5.0 °C (away from the

melting temperature value) till the target probes were removed from the surface (see Fig S2 in

Supporting Information and Fig. 1f for an AFM topograph of the restored PNA-modified

surface). To calculate the melting temperature from the experimental data, a sigmoidal fit was

carried out employing Boltzman function using the data evaluation software Origin 8 (Origin

Lab Cooperation, Northampton, MA, USA). The equation used for fitting was y = A2 + (A1-

A2)/(1+exp((x-x0)/dx)), where A1 = Initial y value, A2 = Final y value and x0 = centre i.e., the

value of x at (A1+A2)/2, dx = time constant where the constraint is dx! = 0. The melting

temperatures were calculated from the inflection point of the fit function as reported earlier [35].

The standard error of melting temperature measurement was calculated using the standard

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procedure to calculate the standard error measurement of melting temperature2 [36] and was

found to be ± 0.2 °C. The coefficient of variation, which is the ratio of the standard deviation and

the mean, was calculated as per standard procedure [36] and was estimated to be 0.003.

For working with the pre-formed PNA−DNA duplexes (applying fully matched combination

only, i.e., PNA 1 − Cy3 DNA 1), the duplexes were prepared by mixing the two components

(each of 0.5 µM concentration) in equal volumes and standing the solution for 1 h. A freshly

annealed gold piece was immersed in the resulting solution and kept for 4 h. After removing the

gold piece from the solution, it was washed with 2 mL filtered autoclaved Milli-Q water.

Subsequently, it was checked for duplex immobilization by fluorescence imaging. For melting of

the surface-confined duplexes, the gold piece was placed in 600 µL of sodium phosphate buffer

(20 mM sodium phosphate, 2 mM NaCl, pH 7.0) and heated to desired temperatures for 15 min.

Then it was taken out, washed with 2 mL (500 µL×4) of the sodium phosphate buffer, and dried

with soft nitrogen jet. The complete removal of the target DNA strands was checked by

fluorescence imaging. Then the resulting surface containing a PNA layer was treated with the

Cy3 DNA 1 (with or without use of a co-adsorbate) and the melting steps were applied as

described above.

For assessing the ability of the PNA sensor probe modified Au(111) surfaces to retain the

hybridization efficiency after the first use, the PNA DNA duplexes were first formed on

Au(111) surface in sodium phosphate buffer (20 mM sodium phosphate, 100 mM sodium

2 Step 1: Calculation of the mean value; Step 2: Calculation of each measurement's deviation from the mean; Step 3:

Squaring each deviation from the mean. Step 4: Addition of the squared deviations. Step 5: Division of the sum by

the number of measurement. Step 6: Calculation of standard deviation by taking the square root of the number

obtained from step 5. Step 7: Calculation of standard error by dividing the standard deviation by the square root of

the number of measurement, which is 3 in the present case.

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chloride, pH 7.00) by usual procedure, and then dehybridized by heating the respective sample in

sodium phosphate buffer. The morphology of the surface was checked by AFM imaging (Fig.

1f). The fluorescence images of all the re-hybridized samples were obtained at room

temperature.

Estimation of the ‘on-surface’ PNA probe density: The Cy3-PNA 1 probe molecules were

immobilized onto Au(111) or AuNP-modified Au(111) surface using the protocol described

above. The modified substrate was immersed in 12 mM 2-mercaptoethanol for 20 h for removal

of the PNA probes from surface. After removing the gold piece from 2-mercaptoethanol

solution, equal volume of Milli-Q water was added to dilute the solution, the fluorescence

intensity was measured using a Perkin Elmer PTP Fluorescence Peltier system, and the probe

density values were estimated from the fluorescence intensity values.

Control experiments: In order to test whether non-specific adsorption of the DNA target probes

occurs onto the sensor probe modified AuNP-Au(111) surfaces, the PNA 3 modified AuNP10-

Au(111) surface was incubated with 20 µL of 1.0 µM Cy3-DNAnc (fully non-complementary

sequence) in the humidity chamber for 1 h. The sample was then washed with 2 mL (500 µL X

4) phosphate buffer (20 mM sodium phosphate buffer, 100 mM sodium chloride, pH 7.00), dried

under soft nitrogen jet and fluorescence images were obtained.

In order to find out whether heating the substrates for duplex melting could lead to desorption

of the thiol-PNA probes, the AuNP10-modified Au(111) substrate was treated with Cy3-PNA 1,

and fluorescence measurements were performed at 25 °C, 50 °C and 85 °C.

UV-Vis absorbance measurement of the AuNP solutions: The exact concentrations of the

AuNP solutions [Sigma-Aldrich and Nanopartz, Inc. (Loveland CO, USA)] were determined by

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UV-visible spectrophotometry at room temperature. [εAuNP (M-1

cm-1

) for 10 nm, 50 nm and 90

nm particles taken as 1.01x108, 1.34x10

10 and 9.94x10

10, respectively].

High-resolution transmission electron microscopy (HRTEM): Carbon-coated copper grids

were treated with nanoparticle solution. The HRTEM images were obtained using JEOL-TEM-

2011. The TEM sample grids were prepared by depositing 5 µL of the AuNP solution onto the

grid and vacuum-drying overnight.

AFM data acquisition: All the images were recorded in ambient condition at room temperature.

AFM experiments were performed with a PicoLE AFM equipment of Agilent Corp. (USA) using

a 10 micron scanner. Imaging was carried out in the intermittent contact mode (using acoustic

alternating current or AAC signal), to minimize sample damage. The cantilevers (µmasch,

Estonia) having back side coated with Al, and frequencies within 208–232 kHz and force

constant values 3.5–12.5 N/m were used for all the imaging experiments. The probes were

cleaned in a UV–ozone cleaner (Bioforce, Nanosciences) for 10-25 min immediately before

imaging. The tip was engaged in feedback at zero scan range condition to avoid contaminating

the tip during the engage step. The amplitude set point was 90% of the free oscillation amplitude

(8.0 V). Scan speed was typically 0.5–2.4 lines/s. The AFM images were taken at least from

minimum four different areas of each sample to check for reproducibility of the features

observed. All the images presented herein are topographic, and are raw data except for minimum

processing limited to third order flattening. The AFM images were obtained for measuring AuNP

surface coverage and for surface roughness analysis.

Fluorescence data acquisition: The fluorescence images were obtained from an Olympus BX61

fluorescence microscope. All the images were recorded considering λexc= ~550 nm and λem=

~570 nm. The exposure time was kept fixed for all the experiments. All the fluorescence

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experiments were carried out in dark condition. The fluorescence intensity was measured by the

“Image pro plus” software, which is provided with the Olympus IX61 fluorescence microscope.

Results and Discussion

In this work, we tested a strategy for immobilizing thiolated ssPNA probes onto the AuNPs,

which are pre-affixed onto Au(111) surface, thereby preventing AuNP aggregation in presence

of the thiolated PNA. Second, we aimed for enhancing single base mismatch discrimination

capacity of the AuNP-anchored PNA probes. The impetus behind the second objective was that

the local curvatures/ surface roughness/ total surface area could change due to nanoparticle

treatment, thereby altering the total number of immobilized probes and the probe density as well,

which might exert a net beneficial effect on the hybridization efficiency of the nucleic acid

sensor layer.

The Au(111) surface, which is widely used in biosensor applications [23, 24], has been the

substrate of choice in this work, since strong gold-sulfur (thiol) interactions [25] could be

exploited to effectively immobilize the bi-functional linker molecules, i.e., 2-

mercaptoethylamine (MEA), also called cysteamine (thiol-amine terminated), or 1, 4- benzene

dithiol (thiol-thiol terminated), onto the substrate surface. The AuNPs were attached onto the

linker-modified Au(111) surface by means of strong interactions built between the gold surface

of the AuNP and the nitrogen atom of the amine group of cysteamine (or sulfur atom of thiol

group of 1, 4- benzene dithiol). The AuNP-modified Au(111) surface was subsequently

functionalized with the ssPNA probes (or ssDNA probes, as required for obtaining a comparative

view).

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Thiolated 12-mer ssPNA (and thiolated 12-mer ssDNA) sequences having a hexyl spacer

group [–(CH2)6SH] at the N-terminal (for PNA sequences) and 5′ position (for DNA)

respectively, have been employed as the sensor probes. The hexyl spacer [–(CH2)6–] is one of the

standard spacer group, which is widely used for keeping the nucleic acid part away from the gold

surface so that non-specific adsorption via nucleobases can be avoided and the sequence can

remain exposed for target binding in a biosensor experiment. The target Cy3 labeled DNA

probes were chosen considering hybridization to the sensor probes in antiparallel fashion, since it

has been previously reported that the duplexes formed in antiparallel fashion are more stable than

the duplexes formed in parallel fashion [37].

For topographic characterization of the surface at different stages of preparation and

application, atomic force microscopy (AFM) was used (Fig. 1). The sizes of the AuNPs and the

presence of Au(111) crystal planes were confirmed by performing high-resolution transmission

electron microscopy (HRTEM) imaging experiments (Figs. S3 and S4, respectively, in

Supporting Information). For Tm measurement on Au(111) surface, the fluorescence intensity of

the fluorophore-labeled target oligonucleotide (5′-Cy3 modified) probes was monitored by

fluorescence measurements (see Fig. S2 in Supporting Information).

Characteristics of the AuNP-Au(111) Assembly: Since total surface area of a substance can be

increased or decreased by suitably altering the surface roughness, we modified the Au(111)

surface with spherical AuNPs of different sizes, i.e., 10 nm, 50 nm and 90 nm, each at a time,

using the bi-functional linker molecules. The surface roughness of the AuNP10/50/90-modified

Au(111) surface was found to be significantly higher than the bare Au(111) surface - the root-

mean-square roughness (Rq) of AuNP-modified Au(111) surface was about 5 times greater

compared to the planar Au(111) surface (Table 2). Although there was little change in surface

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roughness as the NP size was varied, a consistent rise in the roughness value could be observed

as the NP size was reduced (Table 2). High-resolution transmission electron microscopy

(HRTEM) was used to determine size distribution of the NPs (Fig. S3 in Supporting

Information) and for confirming the presence of the Au(111) planes on the AuNP surface (Fig.

S4 in Supporting Information). The latter observation is especially important in view of the fact

that the Au(111) planes are ideally suited for thiol adsorption because of presence of the

energetically favorable three-fold hollow sites [38].

An evidence for adsorption of the linker molecules onto the Au(111) surface preferentially via

the thiol ends, and not via the amine end, could be obtained from reflection absorption infra red

spectroscopy (RAIRS), since no S-H stretching vibration could be identified, whereas clear

bands for N–H stretching and N–H bending could be observed (see Fig. S5 in Supporting

Information). This is not unexpected though, since gold-thiol interactions (~ 40 Kcal/mol) are

generally stronger than the gold-nitrogen interactions (~ 8 Kcal/mol) [38-40]. Given that the

linkers bind to Au(111) surface via the thiol ends, the amine groups should remain free to bind to

the AuNPs. This is a suitable situation to meet our objective since the amine-functionalized

surfaces have been reported to support strong adsorption of AuNPs by virtue of availability of an

electron lone pair on the amine nitrogen, as well as via ionic interactions with the negatively

charged AuNP particles [41].

Smaller the size of AuNP, better is the mismatch discrimination capacity of the PNA/DNA

sensor probe modified surface: In order to monitor the on-surface Tm of the nucleic acid

duplexes, first the thiolated PNA/ DNA oligomers (Table 1) were immobilized on the Au(111)

surface as per previously optimized protocols [42, 43] or on the AuNP-modified Au(111)

surface, followed by exposing the surface to the Cy3 labeled target DNA probes (Cy3-DNA 1) of

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1.0 µM concentration for hybridization, and fluorescence images were captured. To determine

the Tm values, the gold pieces were heated to desired temperatures by starting from the lower

temperatures going to the higher temperatures. The samples were thoroughly washed after each

heating step, which ensures proper removal of the dehybridized Cy3-DNA 1 strands from the

surface. The fluorescence intensity obtained after each heating step could be directly related to

the remaining portion of the duplexes on the surface. With increasing temperature, the

fluorescence intensity was found to reduce and finally the fluorescence was non-detectable after

reaching a particular temperature. The fluorescence intensity values were plotted against the

temperature in each case and from the denaturation profile the Tm values were determined (see

Figs. S6 and S7 in Supporting Information for the Tm measurement plots).

It is revealed from the measured Tm values that generally an increase in Tm took place in case

of the fully matched duplexes immobilized onto the AuNP-modified Au(111) surfaces whereas

the Tm value of the singly mismatched duplexes remained almost the same, compared to the

respective Tm values obtained on bare Au(111) surface (Table 3). Importantly, the fully matched

duplexes exhibited an increasing order of the Tm values as the size of the NP‟s reduced (Table 3).

Although the Tm values for the duplexes on AuNP90-modified Au(111) surface was found to be

the same (in case of PNA) or almost the same (in case of DNA) as that in case of bare gold

surface [the melting temperature data for the bare Au(111) surface comes from the authors‟

previous report [21]], a drastic increase in the Tm value was observed in case of AuNP10-

modified surface (Table 3). Since the Tm value of the singly mismatched duplexes remained

almost the same for all the NP sizes, the single base mismatch discrimination could be improved

as the NP size was reduced - very significantly in case of AuNP10 [Fig. 2].

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The solution melting temperatures of the fully matched and the singly mismatched PNA/DNA

duplexes were found to be 52.2 °C and 39.1 °C, respectively, and those for DNA/DNA duplexes

were found to be 38.6 °C and 28.7 °C, respectively, which has been reported earlier [21]. A

significant enhancement in the Tm values of both the fully matched PNA−DNA and the DNA-

DNA duplexes therefore took place on the Au(111) surface and the AuNP-modified Au(111)

surfaces, compared to the Tm values obtained in solution – such enhancement being 26.0 °C and

21.3 °C, for PNA-PNA and DNA-DNA duplexes respectively, onto the AuNP10-modified

Au(111) surface. Interestingly, the variation in Tm of the singly mismatched duplexes was found

to be insignificant on the AuNP-modified surface compared to the solution Tm values. Therefore,

the mismatch discrimination by both PNA and DNA probes were found to be much improved on

the AuNP-modified surfaces, in comparison to that in the solution phase, such improvement

being the maximum on the AuNP10-modified surface. The reasons for relatively better

performance of the PNA and DNA sensor probes on surface compared to in solution could be

due to the higher surface probe density values compared to the solution probe density values as

discussed earlier [21].

The increase in the Tm value of the fully matched PNA-DNA duplexes on AuNP10-modified

Au(111) surface [The melting temperature data for the bare Au(111) surface comes from the

author‟s previous report [21]], compared to that obtained on bare Au(111) surface, is found to be

16.4 °C, whereas such increase in case of the DNA-DNA duplexes is 12.1 °C. The larger

increase in case of PNA could be related to the non-ionic nature of PNA backbone since this

could allow close positioning of the PNA strands, leading to a relatively greater increase in the

probe density of PNA on the AuNP-modified surface, than on bare Au(111) surface, compared to

that in case of the negatively charged DNA probes. From Table 3, it can be clearly seen that the

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PNA probes performed considerably better than the DNA probes, in terms of mismatch

discrimination as well. In fact, our attempts to improve the performance of the DNA probes, in

order to match that of the PNA probes, were not generally successful. The Tm value of the fully

matched DNA−DNA duplexes adsorbed onto AuNP10-modified Au(111) surface could be

improved only by few degrees, i.e., from 59.9 °C to 63.1 °C, when a considerably higher salt

concentration of 1000 mM was applied in the immobilization buffer (20 mM Na-phosphate,

1000 mM NaCl, pH 7.00) in an anticipation of increased loading of the DNA probes onto the

AuNP surface as a result of more effective screening of the negative charge of the DNA

backbone. An acidic pH of the immobilization buffer (20 mM Na-phosphate, 100 mM NaCl, pH

6.00), which is thought to facilitate DNA immobilization [20], could improve the Tm value of the

fully matched DNA−DNA duplexes adsorbed onto the AuNP10-modified Au(111) surface, only

by 2.3 °C, compared to that obtained in case of neutral pH.

For the control experiments, the thiolated PNA 3 sensor strands, which are fully non-

complementary sequences, were immobilized on AuNP10-modified Au(111) surface keeping all

the sample preparation conditions same as in case of the fully matched/singly mismatched

PNA−DNA duplexes. The modified gold substrate was then treated with the fully non-

complementary Cy3-DNAnc strands. No fluorescence signal could be detected, meaning that

non-specific attachment of the DNA target probes onto the PNA sensor probe modified

gold(111) surface was negligible (Fig. S8a in Supporting Information). In order to check whether

the thiolated sensor probes are desorbed from the surface due to heating, Cy3-PNA 1 probes

were immobilized on AuNP10-modified Au(111) surface and the fluorescence images were taken

at room temperature, 50 °C and 85 °C. No noticeable loss in the fluorescence intensity values

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could be observed upon heating (Fig. S8b-d in Supporting Information), indicating no significant

level of desorption of the thiol-PNA probes occurred due to heating.

While the absolute Tm values as presented in this study might not be the accurate ones, i.e.,

representative of the dehybridization events alone, since non-specific factors, e.g., non-specific

binding of the target DNA strands (via gold-nucleobase nitrogen interactions) on bare gold

regions of the sensor probe modified surface and their subsequent removal during the heating

steps, might be present, the errors could be small since a convincing evidence of such non-

specific effects could not be obtained (see Fig. S8a). Moreover, the errors would be largely

cancelled out when the Tm values were compared to obtain a measure of the mismatch

discrimination capacity.

The effectiveness of the PNA/DNA sensor probe layers retained upon storage (or

regeneration), after first use, was checked by assessing the hybridization efficiency for a second

time detection that was carried out on the same day, and for a third time detection after a week,

after using the sample. The efficiency was generally reduced compared to the original

hybridization efficiency (Table S1 in Supporting Information) although the morphology of the

dehybridized surface remained almost unchanged [Fig. 1f] compared to the starting surface [Fig.

1d].

Thiol-thiol terminations of the bi-functional linker results in improved performance,

compared to the thiol-amine terminations: In order to study the effect of the terminations of

the linker molecule on the Tm value of the PNA-DNA duplexes, we applied 1, 4-benzenedithiol,

which has thiol groups at both the ends, and compared the results with those obtained using

cysteamine, which has thiol group at one end and an amine group at the other end. The Tm value

of the fully matched PNA-DNA duplexes was found to be marginally increased in case of 1, 4-

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benzenedithiol application as the linker, compared to cysteamine application (see the Tm values

for 100 mM NaCl concentration in Table 4 and Fig. S9 in Supporting Information for the Tm

measurement plots). However, when the salt concentration was reduced to 2 mM [motivated by

one of our previous observation that the Tm value for the fully matched PNA-DNA duplexes,

which are immobilized on Au(111) surface, increases as the salt concentration is reduced [21]],

the Tm values for the fully matched PNA-DNA duplexes increased by 3 to 4 °C (Table 4, Fig.

S9).

Single base mismatch discrimination could be enhanced the maximum in case of 1, 4-

benzenedithiol treatment and NaCl concentration of 2 mM. Better performance of 1, 4

benzenedithiol, compared to cysteamine, could be related to formation of more stable AuNP-

Au(111) assembly due to the stronger gold-thiol interactions involved, compared to the gold-

amine interactions (as in case of cysteamine application). Moreover, since 1, 4-benzenedithiol

contains more rigid backbone than cysteamine due to presence of a planar aromatic ring system

and has therefore relatively restricted conformational flexibility compared to cysteamine, most

likely it could support the AuNP layer onto Au(111) surface in a relatively stable disposition.

Effect of variation in immobilization condition on the performance of the PNA probes:

(a) Effect of variation in solvent: In order to monitor the effects of solvent on PNA

immobilization onto AuNP-modified Au(111) surfaces, we applied a series of mixed solvents

using small amounts (1% to the maximum of 10%) of different polar protic solvents

(trifluoroacetic acid or TFA/water/acetic acid/formic acid) and polar aprotic solvents

(dimethylformamide or DMF/acetonitrile or ACN) mixed in ultrapure water (Milli-Q water). The

thiolated ssPNA probes were taken in water or water having 1% TFA/1% TFA+10% ACN/1%

acetic acid/1% formic acid/10% DMF and immobilized onto AuNP10-modified Au(111)

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surfaces. Since PNA exhibits poor water solubility, mostly due to its non-ionic nature, a greater

solubility was sought for by applying different solvent combinations. If the solubility could be

improved by a suitable change in solvent, an obstruction to the chemisorption of PNA probes

(via thiol groups) caused by the non-specifically attached PNA molecules or PNA aggregates,

could be lessened, since the non-specifically adsorbed PNA molecules would tend to travel back

into solution rather than remaining attached on the surface. Since dielectric constant is generally

considered to be directly proportional to solvent polarity, the descending order of solvent polarity

for the present set of solvents, is expected to be water, followed by water mixed with formic

acid, ACN, DMF, TFA and acetic acid, as the dielectric constants of water, formic acid, ACN,

DMF, TFA and acetic acid are 78, 58.5, 37, 36.7, 8.6, and 6.2, respectively. However, as

indicated in Table 5, no clear order of the Tm values could be observed that could be directly

correlated to the solvent polarity factor, and application of pure water proved to be the most

effective compared to all the other solvent combinations.

The Tm value of the PNA-DNA duplexes was the least in case of application of the 10% DMF

solvent, which is an aprotic solvent - not a H-bond donor. Since DMF is a H-bond acceptor due

to presence of the -CO group, it could make strong H-bond with the water molecules, and disrupt

the water arrangement around the PNA molecules, thereby introducing alterations in the PNA

conformation that could be detrimental to the formation of a bioactive self-assembled PNA film.

Application of ACN, which is also an aprotic solvent, also reduced the Tm value (see Table 5 and

compare between the two situations, one using 1% TFA, and the other using 1% TFA+10%

ACN), most likely due to a similar reason as in case of DMF, since -CN group is also a strong H-

bond acceptor. In case of the other solvent combinations, where protic solvents (the H-bond

donors) were used, the decrease in the Tm values could be related to alterations in PNA

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conformation due to replacement of PNA-bound water molecules by the stronger H-bond donors

(the -OH group of acetic acid, formic acid and TFA is a stronger H-bond donor than the -OH

group of water) and/or changes in water structure rearrangement due to H-bonding formed

between the water molecules and the protic solvents.

(b) Effects of application of spacers: In order to improve the performance of the PNA probes,

we tested introduction of small spacer molecules like β-mercaptoethanol (β-ME) /6-

mercaptohexanol (6-MCH) in the PNA film, so that non-specific adsorption of the target probes

could be avoided on the uncovered part (if any) of the AuNP surface. The PNA probes were

immobilized on the AuNP10-modified Au(111) surface using the standard procedure as applied in

the earlier experiments in this study, followed by application of β-ME/6-MCH. We observed that

the Tm values of the fully matched PNA-DNA duplexes were reduced to 53.2 °C and 57.3 °C,

when β-ME and 6-MCH, respectively, were applied. Based on prior information that thiolated

ssPNA probes form a densely ordered self-assembled monolayer on Au(111) surface [42, 44,

45], it could be that the spacer molecules displaced some of the PNA probes from the surface,

thereby introducing defects in the ordered arrangement of the sensor probe assembly. Since β-

ME could bind to Au(111) surface more strongly than 6-MCH, the damage caused to the PNA

film could be more by β-ME, which is probably why the reduction in Tm was greater in case of

β-ME.

Role of AuNP curvature:

Since the spherical AuNP surfaces offer relatively high curvature (the highest for AuNP10,

followed by AuNP50, and the least by AuNP90), compared to the planar gold surface, the AuNP-

bound duplexes could be subjected to less steric and electrostatic interactions due to an angular

(diverging) orientation with respect to each other, compared to the duplexes immobilized onto

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bare Au(111) surface. The resulting difference in the environment compared to that in case of

planar gold proved to be beneficial for the NP sizes 10 and 50 nm, though not for 90 nm3 (see

Table 3). Such environmental differences were probably directly reflected in the probe density

(i.e., no. of probes within a unit area on the surface) values, since the PNA probe density on the

AuNP10, AuNP50, AuNP90 and planar gold surfaces were estimated to be 2.55×1014

, 1.21×1014

,

5.16×1013

, and 3.80×1013

strands/cm2, respectively, using cysteamine as the linker (the PNA

probe density on the AuNP10 was found to be 3.01×1014 strands/cm

2 using 1, 4- benzenedithiol as

the linker). Clearly, the probe densities in case of the AuNP10 and AuNP50 were an order of

magnitude greater than those in case of AuNP90 and planar gold surfaces. Liu et al. reported

earlier that the DNA probe density on AuNP surfaces could be made much higher than that on

the bare gold surface [19]. Hurst et al. also reported that the DNA probe density could be

controlled by varying the nanoparticle size [47]. For the same probe density value, the steric

repulsion between the adjacent sensor probes could be the least in case of the smallest AuNP,

since the deflection angle between the adjacent probes would be the maximum for the highest

surface curvature offered by the smallest NP. This is expected to result in the highest probe

density in case of the AuNP10 and the lowest probe density in case of the AuNP90. This

presumption has actually found to be true as reflected in the PNA probe density values for

different NP sizes (see above).

A theoretical investigation reported by Schmitt et al. [48] reveals that in case of DNA,

hybridization between the fully matched sequences could be enhanced on surface. This was

primarily attributed to a greater sensor probe density achievable on AuNP surface. In the present

3 It was previously reported that once AuNPs reach a diameter of approximately 60 nm, the local surface

experienced by the oligonucleotides is highly similar to that of a planar surface [46].

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case, the PNA probe densities on the AuNP-modified Au(111) surfaces were generally greater

than that on the bare gold surface. This could result in a difference between the number of PNA-

DNA duplexes formed on the AuNP-modified surface and on Au(111) surface - the

corresponding situation was reflected in the Tm values of the fully matched duplexes4. In case of

the singly mismatched duplexes, since hybridization is largely inhibited due to lack of

complementarity, especially since the mismatch is located at the centre of the sequence, the

probe density factor became less influential that led to almost similar Tm value in case of all the

AuNP. Effectively, the mismatch discrimination could be controlled by changing the AuNP size.

Role of surface roughness:

The Tm (and the ΔTm) values in case of bare Au(111) and AuNP90-modified surface were found

to be almost the same (see Table 3), even if there is a drastic change in surface roughness in case

of the AuNP-modified surface compared to the bare Au(111) surface (Table 2). Also, even if

there was little change in the surface roughness when compared amongst the AuNP10, AuNP50

and AuNP90 modified surfaces, a noticeable change in the Tm (and the ΔTm) values could be

observed as the NP size was reduced from 90 nm to 50 nm, and a drastic change in case of 10 nm

NP (see Table 3). These observation indicate little role of surface roughness in influencing the

Tm values. Since the total surface area would be directly proportional to the surface roughness,

we do not expect any significant role of total surface area either.

4 The PNA probe density reported herein seems to be offering a desirable area per probe, which is sufficiently large

for target entry, and at the same time being sufficiently small for preventing non-specific adsorption of the target

strands. In fact, when we tried to optimize the area per probe by other approaches, e.g., by first immobilizing the

thiolated PNA-DNA duplex (onto AuNP10-modified Au(111) substrate), followed by dehybridizing the duplex by

heating and removing the DNA strands, leaving a „PNA only‟ layer on the gold substrate, the Tm value of the fully

matched PNA-DNA duplex subsequently formed onto the „PNA only‟ layer was found to be 67.7 °C (without use of

any co-adsorbate, e.g., 6-MCH) and 58.9 °C (using 6-MCH as the co-adsorbate), which is much less than the value

observed, i.e., 78.2 °C, in case of direct immobilization of the thiolated ssPNA probes onto the AuNP-modified

Au(111) substrate.

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Conclusions

In conclusion, the mismatch discrimination ability of both PNA and DNA sensor probes could be

enhanced onto AuNP-modified Au(111) surface, compared to bare gold surface, especially using

the smallest AuNP10 and the PNA probes. The simple strategy for formation of the surface-

attached AuNP-PNA construct appears to be beneficial not only because the difficulty in

attaching PNA probes onto AuNPs, without AuNPs getting aggregated, can be overcome, but

importantly, because this allows an increase in the sensor probe density and therefore increasing

the hybridization probability. The AuNP curvature appears to play a decisive role, while the

surface roughness does not. What is left to be found out is whether this AuNP-based simple and

straightforward strategy could be extended to detection of other types of nucleic acids, e.g.,

RNA. It would particularly be useful to find out whether this strategy could be utilized in

multiplexed high-throughput applications where gold-coated sensor arrays are used, e.g., in

label-free methods like surface plasmon resonance (SPR), nanomechanical cantilever sensor etc.

Acknowledgements

We gratefully acknowledge the financial support (Grant No. BT/ PR-11765/MED/32/107/2009)

from Department of Biotechnology, Govt. of India, and the fellowships of S.G. and S.M. from

Indian Association for the Cultivation of Science, Kolkata and Council of Scientific and

Industrial Research, Govt. of India, respectively.

Supporting Information

The AFM topograph and HRTEM images of AuNPs, fluorescence images of PNA-DNA

duplexes on AuNP10 modified Au(111) surface at different heating stages, RAIR spectra of

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MEA-coated Au(111), Tm measurement plots, results of control experiments, and information on

rehybridization efficiency of the PNA film and the DNA film on AuNP10 and gold(111) surface.

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Au Films: Particle Size Dependence. J. Phys. Chem. B 1999, 103, 5826–5831.

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Assembled Peptide Nucleic Acid Structures on Gold(111) Surface. J. Colloid Interface Sci.

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(43) Mishra, S.; Ghosh, S.; Mukhopadhyay, R. Ordered Self-assembled Locked Nucleic Acid

(LNA) Structures on Gold(111) Surface with Enhanced Single Base Mismatch Recognition

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Figure 1: AFM topograph images of (a) flame

annealed bare Au(111) surface; (b) Cysteamine

modified Au(111) surface; (c) AuNP10 coating

on cysteamine-modified Au(111) surface; (d)

PNA layer formed onto the AuNP-coated

surface using thiolated ssPNA probes; (e) after

hybridization with target ssDNA probes; and (f)

after heat-induced dehybridization, the PNA

layer is restored [Scale bar for (a – f) is 200 nm

and Z-range for (a) 0–0.75 nm, (b) 0–1.09 nm,

(c) 0–10.2 nm, (d) 0–1.56 nm, (e) 0–2.39 nm and

(f) 0–1.56 nm].

(a)

(c)

(b)

(d)

(e) (f)

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(a)

Figure 2: The plots of melting temperature [Tm]

vs. AuNP diameter for fully matched and singly

mismatched (along with mismatch discrimination

profiles) PNA-DNA and DNA-DNA duplexes. It is

clearly elicited from these plots that with decrease

in the AuNP size, the mismatch discrimination

capacity of both PNA and DNA sensor probes

continuously increases, mostly due to increase in

Tm value of the fully matched duplexes. Though the

PNA probes outperform the DNA probes for all the

AuNP sizes, its superior performance is the most

prominent for the smallest NP.

0 20 40 60 80 100

20

40

60

80

100T

emp

erat

ure

(°C

)

Nanoparticle size (nm)

PNA−DNA fully matched DNA−DNA singly mismatched

mismatch discrimination

0 20 40 60 80 100

20

40

60

80

100

Tem

pera

ture

(°C

)

Nanoparticle size (nm)

PNA match

PNA mismatch

PNA SNP

I

J

K

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Scheme 1: Basic scheme of fluorescence-based on-surface detection of nucleic acid duplex

formation on AuNP-modified Au(111) surface (the drawings of the nucleic acid probes/

linkers are not representative of their actual molecular structures).

linker

AuNP

Sensor Probe

Gold(111) on mica

Cy3-DNA

Before hybridization

After hybridization

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(a)

Table 1: Nucleic acid sequences applied in the present study.

The mismatch sites are underlined.

PNA 1 N-ter-HS-C6-CTA-TGT-CAG-CAC-CONH2-C-ter

PNA 2 N-ter-HS-C6-CTA-TGT-AAG-CAC-CONH2-C-ter

PNA 3 N-ter-HS-C6-CGA-TCT-GCT-AAC-CONH2-C-ter

Cy3-PNA 1 N-ter-HS-C6-CTA-TGT-CAG-CAC-CONH-Lys-Cy3-C-ter

DNA 1 5′-HS-C6-CTA-TGT-CAG-CAC-3′

DNA 2 5′-HS-C6-CTA-TGT-AAG-CAC-3′

DNA 3 5′-HS-C6-CGA-TCT-GCT-AAC-3′

Cy3-DNA 1 5′-Cy3-GTG-CTG-ACA-TAG-3′

Cy3-DNAnc 5′-Cy3-CGA-TCT-GCT-AAC-3′

DNA/PNA Sequence

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Table 2: Surface roughness analysis of AuNP-modified

Au(111) surface and bare Au(111) surface.

Surface Root-mean-square Average surface

surface roughness roughness

(Rq) (Ra)

AuNP10- 10.73 nm 8.89 nm

treated Au(111)

AuNP50- 10.35 nm 8.53 nm

treated Au(111)

AuNP90- 10.20 nm 8.43 nm

treated Au(111)

Bare Au(111) 2.113 nm 1.32 nm

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Table 3: The Tm values of PNA-DNA/DNA-DNA duplexes on differently sized AuNP-modified Au(111)

surface and on bare Au(111) surface using cysteamine linker and Na-phosphate hybridization buffer (20

mM Na-phosphate, 100 mM NaCl, pH 7.0). Differences in melting temperatures between fully matched and

singly mismatched situations are shown as ΔTm.

Surface

Nucleic acid duplexes

AuNP10-treated AuNP50-treated AuNP90-treated Bare Au(111)

PNA 1 Cy3-DNA 1 78.2 °C 68.4 °C 61.8 °C 61.8 °C

PNA 2 Cy3-DNA 1 35.7 °C 38.6 °C 38.8 °C 39.0 °C

ΔTm (PNA) 42.5 °C 29.8 °C 22.3 °C 22.8 °C

DNA 1 Cy3-DNA 1 59.9 °C 54.1 °C 48.8 °C 47.8 °C

DNA 2 Cy3-DNA 1 28.9 °C 29.0 °C 29.9 °C 29.5 °C

ΔTm (DNA) 31.0 °C 25.1°C 18.9 °C 18.3 °C

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Table 4: A comparison between the effectiveness of two linkers – cysteamine (thiol-amine terminated) and 1,

4-benzenedithiol (thiol-thiol terminated), as reflected in the Tm values of PNA-DNA duplexes on AuNP10-

modified Au(111) surface using Na-phosphate hybridization buffers (20 mM Na-phosphate, pH 7.00) having

moderate and low concentrations of NaCl. Differences in the Tm values for fully matched and singly

mismatched situations are shown as ΔTm.

Nature of duplex Linker molecule NaCl concentration Tm

(mM)

PNA 1−Cy3-DNA 1 1, 4-Benzenedithiol 100 78.9 °C PNA 2−Cy3-DNA 1 1, 4-Benzenedithiol 100 36.1 °C ΔTm 42.8 °C PNA 1−Cy3-DNA 1 1, 4-Benzenedithiol 2 82.1 °C PNA 2−Cy3-DNA 1 1, 4-Benzenedithiol 2 37.6 °C ΔTm 44.5 °C PNA 1−Cy3-DNA 1 Cysteamine 100 78.2 °C PNA 2−Cy3-DNA 1 Cysteamine 100 35.7 °C ΔTm 42.5 °C PNA 1−Cy3-DNA 1 Cysteamine 2 81.7 °C PNA 2−Cy3-DNA 1 Cysteamine 2 39.9 °C ΔTm 41.8 °C

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Table 5: Influence of variation in immobilization medium on melting temperature of fully matched

PNA-DNA duplexes (tested for AuNP10-modified surface only).

Water 1% TFA 1% TFA+10% ACN 1% Acetic acid 1% Formic acid 10% DMF

78.2 °C 60.8 °C 60.4 °C 60.0 °C 58.3 °C 49.2 °C

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For TOC use Only

Cy3-Target DNA

linker

AuNP

Gold(111)

Sensor Probe

0 20 40 60 80 1000

10

20

30

40

50

60

70

80

PNA−DNA

DNA−DNA

mismatch discrimination

Tem

per

atu

re (

°C)

Nanoparticle size (nm)

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