Enhancing On-Surface Mismatch Discrimination Capability of PNA

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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 Langmuir, 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: [email protected]) 1

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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, sizedependent 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 2


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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 nanoparticlebased 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 nonionic, 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 3


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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, 4


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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 2mercaptoethylamine (MEA) or cysteamine (Sigma-Aldrich) / 1, 4 - benzenedithiol (SigmaAldrich) 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), 6mercaptohexanol (Sigma-Aldrich), on the on-surface PNA-DNA hybridization, the PNAmodified 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 + (A1A2)/(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 7


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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 AuNP10Au(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, 1.34x1010 and 9.94x1010, respectively]. High-resolution transmission electron microscopy (HRTEM): Carbon-coated copper grids were treated with nanoparticle solution. The HRTEM images were obtained using JEOL-TEM2011. 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 10


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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 rootmean-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 12


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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 13


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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 AuNP10modified 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 DNADNA 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 15


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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 noncomplementary 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 noncomplementary 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 nonspecific 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, 417


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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, 4benzenedithiol treatment and NaCl concentration of 2 mM. Better performance of 1, 4 benzenedithiol, compared to cysteamine, could be related to formation of more stable AuNPAu(111) assembly due to the stronger gold-thiol interactions involved, compared to the goldamine 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) 18


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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 Hbond 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 19


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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) /6mercaptohexanol (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 βME 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 AuNPbound 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 20


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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/cm2 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 PNADNA 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 surfaceattached 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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(a)

(b)

(c)

(d)

(e)

(f)

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].


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100

PNA−DNA DNA−DNA

100

80 Temperature (°C)

Temperature (°C)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

60 40 20 0

20

fully matched singly mismatched mismatch discrimination

80

PNA match PNA mismatch PNA SNP I J K

60 40

40 60 80 20 Nanoparticle size (nm)

100

(a) Figure 2: The plots of 0 melting 20 temperature 40 60[Tm] 80 vs. AuNP diameter for fully matched andsize singly Nanoparticle (nm) 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.


100

Langmuir

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Sensor Probe AuNP linker

Cy3-DNA Before hybridization

After hybridization

Gold(111) on mica

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).


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Langmuir

(a) Table 1: Nucleic acid sequences applied in the present study. The mismatch sites are underlined. DNA/PNA

Sequence

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′


Langmuir

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Table 2: Surface roughness analysis of AuNP-modified Au(111) surface and bare Au(111) surface. Surface

Root-mean-square surface roughness (Rq)

Average surface roughness (Ra)

AuNP10treated Au(111)

10.73 nm

8.89 nm


10.35 nm

8.53 nm


10.20 nm

8.43 nm

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

42.5 °C

29.8 °C

22.3 °C

22.8 °C

ΔTm (PNA) 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

31.0 °C

25.1°C

18.9 °C

18.3 °C

ΔTm (DNA)


Langmuir

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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 AuNP10modified 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 (mM)

Tm

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 78.2 °C

1% TFA 60.8 °C

1% TFA+10% ACN 60.4 °C

1% Acetic acid

1% Formic acid

10% DMF

60.0 °C

58.3 °C

49.2 °C


Langmuir

For TOC use Only

80

Cy3-Target DNA Sensor Probe

Temperature (°C)

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PNA−DNA DNA−DNA mismatch discrimination

70 60 50 40 30 20 10

AuNP

0

linker

0

20

40

60

80

Nanoparticle size (nm)

Gold(111)


100

Enhancing On-Surface Mismatch Discrimination Capability of PNA

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