Single-mismatched DNA Research Articles

Label-Free and Ultrasensitive Electrochemical DNA Biosensor Based on Urchinlike Carbon Nanotube-Gold Nanoparticle Nanoclusters

Nanomaterials have been extensively utilized in biosensing systems for highly sensitive and selective detection of a variety of biotargets. In this work, a facile, label-free, and ultrasensitive electrochemical DNA biosensor has been developed, based on "urchinlike" carbon nanotube-gold nanoparticle (CNT-AuNP) nanoclusters, for signal amplification. Specifically, electrochemical polymerization of dopamine (DA) was employed to modify a gold electrode for immobilization of DNA probes through the Schiff base reaction. Upon sensing the target nucleic acid, the dual-DNA (reporter and linker) functionalized AuNPs were introduced into the sensing system via DNA hybridization. Afterward, the end-modified single-wall carbon nanotubes with DNA (SWCNT-DNA) were attached to the surface of the AuNPs through linker-DNA hybridization that formed 3D radial nanoclusters, which generated a remarkable electrochemical response. Because of the larger contact surface area and super electronic conductivity of CNT-AuNP clusters, this novel designed 3D radial nanostructure exhibits an ultrasensitive detection of DNA, with a detection limit of 5.2 fM (a linear range of from 0.1 pM to 10 nM), as well as a high selectivity that discriminates single-mismatched DNA from fully matched target DNA under optimal conditions. This biosensor, which combines the synergistic properties of both CNTs and AuNPs, represents a promising signal amplification strategy for achieving a sensitive biosensor for DNA detection and diagnostic applications.

An enzyme-free electrochemical sandwich DNA assay based on the use of hybridization chain reaction and gold nanoparticles: application to the determination of the DNA of Helicobacter pylori.

An ultrasensitive enzyme-free electrochemical sandwich DNA biosensor is described for the detection of ssDNA oligonucleotides. A DNA sequence derived from the genom of Helicobacter pylori was selected as a model target DNA. The DNA assay was realized through catching target DNA on capture DNA immobilized gold electrode; then labeling the target DNA with reporter DNA (rpDNA) and initiator DNA (iDNA) co-modified gold nanoparticles (AuNPs). The high density of iDNAs serves as one of the amplification strategies. The iDNA triggers hybridization chain reaction (HCR) between two hairpins. This leads to the formation of a long dsDNA concatamer strand and represents one amplification strategy. The electrochemical probe [Ru(NH3)5L]2+, where L stands for 3-(2-phenanthren-9-ylvinyl)pyridine, intercalated into dsDNA chain. Multiple probe molecules intercalate into one dsDNA chain, serving as one amplification strategy. The electrode was subjected to differential pulse voltammetry for signal acquisition, and the oxidation peak current at -0.28V was recorded. On each AuNP, 240 iDNA and 25 rpDNA molecules were immobilized. Successful execution of HCR at the DNA-modified AuNPs was confirmed by gel electrophoresis and hydrodynamic diameter measurements. Introduction of HCR significantly enhances the DNA detection signal intensity. The assay has two linear ranges of different slopes, one from 0.01 fM to 0.5 fM; and one from 1 fM to 100 fM. The detection limit is as low as 0.68 aM. Single mismatch DNA can be differentiated from the fully complementary DNA. Conceivably, this highly sensitive and selective assay provides a general method for detection of various kinds of DNA. Graphical abstractSchematic representation of the detection and the amplification principles of the electrochemical sandwich DNA assay. Purple curl: Captured DNA; Green curl: Reporter DNA; Orange curl: HCR initiator DNA; Yellow solid-circle: Gold nanoparticle; H1 and H2: Two hairpin DNA; [Ru(NH3)5L]2+: Signal probe.

The detection of a mismatched DNA by using hairpin DNA-templated silver nanoclusters

Fluorescence assays have been developed to detect biomolecules using DNA-templated silver nanoclusters (DNA-AgNCs) utilizing their unique physical and optical properties. This study was designed to detect single-mismatched DNA by the hybridization of target DNA to template DNA either before or after DNA-AgNCs synthesis. The results showed that the detection specificity of a single-mismatched DNA was clearly enhanced when the target DNA (cDNA) was hybridized to template DNA prior to DNA-AgNCs synthesis compared with cDNA hybridization subsequent to DNA-AgNCs synthesis.

Neutralized chimeric DNA probe for detection of single nucleotide polymorphism on surface plasmon resonance biosensor

An implementation of neutralized chimeric DNA oligomer as a probe for sensitive detection of single nucleotide polymorphisms (SNPs) on a surface plasmon resonance imaging sensor is investigated. The chimeric DNA oligomer was synthesized in a conventional DNA synthesizer, containing neutral nucleotides with a methylated phosphate group. The secondary structures and melting points of the chimeric DNA fragment and its complexes with perfect-matched and single-mismatched complementary DNA molecules were examined by using circular dichroism and UV–vis spectroscopy in comparison with the native probe DNA counterpart. The results indicate that the chimeric DNA complexes can form a B-form structure and exhibit high thermostability. Moreover, the hybridization and discrimination efficiency of the chimeric probe DNA for the SNP genotyping were verified by using the SPRi sensor under different experimental conditions. The data reveal the effects of the ionic strength and operation temperature on the selectivity of the chimeric probe DNA for the SNP detection. The hybridization condition with a low ionic strength and high temperature allows the chimeric probe DNA distinguishing perfect-matched and single-mismatched target DNA molecules to the best extent, likely due to the reduced electrostatic repulsive force and presence of the additional methyl group on the backbone. Consequently, the direct and label-free detection with the SPR technique and neutralized chimeric probe DNA can be realized for the SNP genotyping by optimizing the operation condition and sequence design.

Ultrasensitive chemiluminescence assay for the lung cancer biomarker cytokeratin 21-1 via a dual amplification scheme based on the use of encoded gold nanoparticles and a toehold-mediated strand displacement reaction

A catalytic circuit is described in which the addition of a single input DNA strand leads to the release of more than one output strand. The system consists of (a) a three-stranded substrate complex, (b) fuel DNA, and (c) trigger DNA. The whole network is activated by the trigger DNA in the catalytic circuit, which regenerates the trigger to catalyze another new circuit and releases two kinds of bio-barcoded gold nanoparticles (AuNPs) after toehold-mediated strand displacement. The AuNPs are then dissolved to form Au (III) ions which catalyze the luminol-H2O2 chemiluminescence. Cytokeratin 21-1 (CYFRA21-1), associated with so-called non-small cell lung cancer, was used as the model target. By taking advantage of the above dual amplification scheme, this gene assay works in the 20 f. to 8.0 nM CYFRA21-1 concentration range with a 6 f. detection limit. The method exhibits excellent selectivity even over single-mismatched DNA.

Ionic strength-controlled hybridization and stability of hybrids of KRAS DNA single-nucleotides: A surface plasmon resonance study

The discrimination of a fully matched, unlabeled KRAS wild-type (WT) (C-G) target sample with respect to three of the most frequent KRAS codon mutations (G12 S (C-A), G12 R (C-C), G12C (C-T)) was investigated using an optimized detection strategy involving surface plasmon resonance (SPR), based on optimized probe-surface density and ionic strength control. The changes observed in the SPR signal were always larger for WT compared with the single-mismatch target DNA oligonucleotides, and were aligned with the theoretical energy differences between the base pair C-G, C-T, C-A, C-C. Hybridization rates of ∼106M−1s−1 were detected without the introduction of high temperature and labels, usually needed in conventional hybridization methods. One hundred percent mutation discrimination of the matched KRAS wild-type (C-G) sequence with respect to three mismatched G12C (C-T), G12 S (C-A), G12 R (C-C) target sequences was achieved.

Polymer–DNA Molecular Net for Selective Transportation of Target Biomolecules and Inhibition of Tumor Growth

We demonstrate a new concept of a molecular net for capturing, release, and transportation of target molecules by utilizing polymer-based DNA molecular net (PD-mn). Target biomolecules like DNA, ATP, and VEGF were successfully captured from a heterogeneous mixture of biomolecules and released by a stimulus without nonspecific degradation by PD-mn prepared by polymerization of acrylamide and acrydite-modified DNA. Furthermore, by introducing fluorescence-labeled competitor DNA, single-mismatched DNA was specifically detected. As a concept of artificial transporter, PD-mn selectively captured target biomolecules from a heterogeneous mixture of molecules into a specific enzymatic reaction solution and induced enzyme-mediated reaction. Finally, effective in vivo antitumor effect was demonstrated utilizing sheet-shaped PD-mn which has improved capture ability against VEGF as a target biomolecule by amplified DNA VEGF aptamer. This PD-mn could be used as not only a selective molecular adsorbent or artificial tr...

Immobilization-free electrochemical DNA detection with anthraquinone-labeled pyrrolidinyl peptide nucleic acid probe

Electrochemical detection provides a simple, rapid, sensitive and inexpensive method for DNA detection. In traditional electrochemical DNA biosensors, the probe is immobilized onto the electrode. Hybridization with the DNA target causes a change in electrochemical signal, either from the intrinsic signal of the probe/target or through a label or a redox indicator. The major drawback of this approach is the requirement for probe immobilization in a controlled fashion. In this research, we take the advantage of different electrostatic properties between PNA and DNA to develop an immobilization-free approach for highly sequence-specific electrochemical DNA sensing on a screen-printed carbon electrode (SPCE) using a square-wave voltammetric (SWV) technique. Anthraquinone-labeled pyrrolidinyl peptide nucleic acid (AQ-PNA) was employed as a probe together with an SPCE that was modified with a positively-charged polymer (poly quaternized-(dimethylamino-ethyl)methacrylate, PQDMAEMA). The electrostatic attraction between the negatively-charged PNA–DNA duplex and the positively-charged modified SPCE attributes to the higher signal of PNA–DNA duplex than that of the electrostatically neutral PNA probe, resulting in a signal change. The calibration curve of this proposed method exhibited a linear range between 0.35 and 50nM of DNA target with a limit of detection of 0.13nM (3SDblank/Slope). The sub-nanomolar detection limit together with a small sample volume required (20μL) allowed detection of <10fmol (<1ng) of DNA. With the high specificity of the pyrrolidinyl PNA probe used, excellent discrimination between complementary and various single-mismatched DNA targets was obtained. An application of this new platform for a sensitive and specific detection of isothermally-amplified shrimp's white spot syndrome virus (WSSV) DNA was successfully demonstrated.

Rapid nucleic acid melting analyses using a microfabricated electrochemical platform

Microfabrication methods have been used to fabricate a new microscale platform that integrates thermal control and multi-electrode components to enable rapid, temperature-dependent electrochemical measurements on small-volume fluid samples. A wide range of biochemical phenomena can be characterized with the device, for example, when monitoring interactions at the working electrode between probe and target species which include an electroactive moiety. Employing square wave voltammetry, we have demonstrated the utility and reproducibility of the microplatform in melting studies on full-match, single-mismatch, and double-mismatch DNA structures of relevance to single-nucleotide polymorphism (SNP) discrimination. As shown, the small size of the reported device, low volume for the samples it can interrogate (∼10μL), individual addressing of platform components and fast localized heating (settling times ∼5s) combine to allow for efficient sample analyses. In addition, a straight-forward route exists, involving replication into array formats and integration with microfluidics, for extending the technology toward eventual high throughput work on drug discovery and medical diagnostics.

Trinucleotide duplex formation inside a confined nanospace under supercooled conditions

Self-assembly of nucleotides of fewer than three base pairs is often found in protein-nucleotide conjugations, despite their energetic instability, and is regarded as the potential starting point for the creation of artificial hydrogen-bonded supramolecular complexes. Here we report duplex formation of 3-mer DNA fragments confined within silica mesopores modified with a positively charged trimethyl aminopropyl monolayer, and their further stabilization under supercooled conditions (T<273 K). We load 3-mer DNA fragments with donor- or acceptor-dye into modified silica mesopores and examine their hybridization behaviours using FRET measurements. The FRET results clearly reveal that efficient duplex formation through at least two A-T base pairs can be achieved at 233 K. Enthalpy changes for duplex formation are found to be nearly equal between complementary and single-mismatched 3-mer DNA duplexes. These results confirm confined mesoscale cavities to be a novel low-temperature reaction space for hydrogen-bonded supramolecular complexes.

A gold nanorods-based fluorescent biosensor for the detection of hepatitis B virus DNA based on fluorescence resonance energy transfer

In this study, we designed a fluorescence resonance energy transfer system containing gold nanorods (AuNRs) and fluorescein (FAM) for the detection of hepatitis B virus DNA sequences. AuNRs were synthesized according to the seed-mediated surfactant-directed approach, and the surface of the AuNRs was wrapped with a thin layer of cetyltrimethylammonium bromide (CTAB), resulting in the AuNRs being positively charged. When FAM-tagged single-stranded DNA (FAM-ssDNA) was added into the AuNRs suspension, it was adsorbed onto the surface of the positively charged AuNRs and formed a FAM-ssDNA-CTAB-AuNRs ternary complex, the resulting structure led to a fluorescence resonance energy transfer (FRET) process from FAM to AuNRs and the fluorescence intensity of FAM was consequently quenched. When complementary target DNA was added to the FAM-ssDNA-CTAB-AuNRs complex solution, a further decrease in fluorescence intensity was observed because of an increased FRET efficiency. Under optimal conditions, the decline of the fluorescence intensity of FAM (ΔF) was linear with the concentration of the complementary DNA from 0.045 to 6.0 nmol L(-1) and the detection limit was as low as 15 pmol L(-1) (signal/noise ratio of 3). When this fluorescent DNA sensor was used to detect the polymerase chain reaction product of hepatitis B virus gene extracted from a positive real sample, a positive response was obtained. Impressively, the biosensor exhibits good selectivity, even for single-mismatched DNA detection.

Fabrication of a Sensitive Electrochemical Biosensor for Detection of DNA Hybridization Based on Gold Nanoparticles/CuO Nanospindles Modified Glassy Carbon Electrode

AbstractIn this work, a sensitive electrochemical DNA biosensor for the detection of sequence‐specific target DNA was reported. Firstly, CuO nanospindles (CuO NS) were immobilized on the surface of a glassy carbon electrode (GCE). Subsequently, gold nanoparticles (Au NPs) were introduced to the surface of CuO NS by the electrochemical deposition mode. Probe DNA with SH (HS‐DNA) at the 5′‐phosphate end was covalently immobilized on the surface of the Au NPs through AuS bond. Scanning electron microscopy (SEM) was used to elucidate the morphology of the assembled film, and electrochemical impedance spectroscopy technique (EIS) was used to investigate the DNA sensor assembly process. Hybridization detection of DNA was performed with differential pulse voltammetry (DPV) and the methylene blue (MB) was hybridization indicator. Under the optimal conditions, the decline of reduction peak current of MB (ΔI) was linear with the logarithm of the concentration of complementary DNA from 1.0×10−13 to 1.0×10−6 mol·L−1 with a detection limit of 3.5×10−14 mol·L−1 (S/N=3). In addition, this DNA biosensor has good selectivity, and even can distinguish single‐mismatched target DNA.

Ultrasensitive electrochemiluminescence detection of DNA based on nanoporous gold electrode and PdCu@carbon nanocrystal composites as labels

A sensitive electrochemiluminescence (ECL) DNA biosensor based on nanoporous gold (NPG) electrode and PdCu@carbon nanocrystals (CNCs) composites is developed. The CNCs were obtained simply by electrooxidation with abundant carboxyl groups at their surfaces. The NPG can be easily prepared by a selective dissolution of silver from silver-gold alloy in nitric acid, which has free-standing noble metal membranes with controllable three-dimensional (3D) porosity. The PdCu bimetallic nanocomposites with hierarchically hollow structures were fabricated through a simple replacement reaction using dealloyed nanoporous copper (NPC) as both a template and reducing agent. Structure characterization was obtained by means of transmission electron microscope (TEM) and scanning electron microscope (SEM) images. The PdCu@CNCs composites exhibit 6 times higher ECL intensity than the pure CNC-labeled reporter DNA. Taking advantage of dual-amplification effects of the developed probe, a limit of detection as low as 18 aM can be achieved and the assay exhibits excellent selectivity for single-mismatched DNA detection even in human serum. The proposed ECL based method should have wide applications in diagnosis of genetic diseases due to its simplicity, low cost, and high sensitivity at extremely low concentrations.

Gold-based optical biosensor for single-mismatched DNA detection using salt-induced hybridization

In this study, a gold nanoparticle (Au-NP)-based detection method for sensitive and specific DNA-based diagnostic applications is described. A sandwich format consisting of Au-NPs/DNA/PMP (Streptavidin-coated MagnetSphere Para-Magnetic Particles) was fabricated. PMPs captured and separated target DNA while Au-NPs modified with oligonucleotide detection sequences played a role in recognition and signal production. Due to the much lower stability of mismatched DNA strands caused by unstable duplex structures in solutions of relatively low salt concentration, hybridization efficiency in the presence of different buffers was well investigated, and thus, the optimized salt concentration allowed for discrimination of single-mismatched DNA (MMT) from perfectly matched DNA (PMT). Therefore, quantitative information concerning the target analyte was translated into a colorimetric signal, which could easily and quantitatively measured by low-cost UV–vis spectrophotometric analysis. The results indicated this to be a very simple and economic strategy for detection of single-mismatched DNA strands.

3-Aminopyrrolidine-4-carboxylic acid as versatile handle for internal labeling of pyrrolidinyl PNA

Conformationally restricted pyrrolidinyl PNAs with an α/β-dipeptide backbone consisting of a nucleobase-modified proline and a cyclic five-membered amino acid spacer such as (1S,2S)-2-aminocyclopentanecarboxylic acid (ACPC) (acpcPNA) can form very stable hybrids with DNA with high Watson-Crick base pairing specificity. This work aims to explore the effect of incorporating 3-aminopyrrolidine-4-carboxylic acid (APC), which is isosteric to the ACPC spacer, into the acpcPNA. It is expected that the modification should not negatively affect the DNA binding properties, and that the additional nitrogen atom in the APC should provide a handle for internal modification. Orthogonally-protected (N(3)-Fmoc/N(1)-Boc and N(3)-Fmoc/N(1)-Tfa) APC monomers have been successfully synthesized and incorporated into the acpcPNA by Fmoc-solid-phase peptide synthesis. T(m), UV and CD spectroscopy confirmed that the (3R,4S)-APC could substitute the (1S,2S)-ACPC spacer in the acpcPNA with only slightly decreasing the stability of the hybrids formed between the modified acpc/apcPNA and DNA. In contrast, the (3S,4R) enantiomer of APC caused substantial destabilization of the hybrids. Furthermore, a successful on-solid-support internal labeling of the acpc/apcPNA via amide bond formation between pyrene-1-carboxylic acid or 4-(pyrene-1-yl) butyric acid and the pyrrolidine nitrogen atom of the APC spacer has been demonstrated. Fluorescence properties of the pyrene-labeled acpc/apcPNAs are sensitive to their hybridization states and can readily distinguish between complementary and single-mismatched DNA targets.

A sensitive fluorimetric biosensor for detection of DNA hybridization based on Fe/Au core/shell nanoparticles

In this study, we reported a sensitive fluorescent biosensor for detection of DNA hybridization based on Fe/Au core/shell (Fe@Au) nanoparticles (NPs). First, Fe@Au NPs were synthesized using a reverse micelle method, with gold as the shell and iron as the core. The nanoparticle size was confirmed by transmission electron microscopy (TEM). Scanning electron microscopy (SEM) was performed in order to elucidate the morphology of the Fe@Au NPs. Then probe DNA with -SH at the 5'-phosphate end was covalently immobilized onto the surface of the Fe@Au NPs. The DNA hybridization event can be detected by a fluorescent method and methylene blue (MB) as the fluorescent probe. The decline of the fluorescence intensity of MB (ΔF) was linear with the concentration of the complementary DNA from 3.0 × 10(-13) to 1.0 × 10(-9) M with a detection limit of 1.0 × 10(-13) M (S/N = 3). In addition, this approach of DNA detection exhibited excellent selectivity, even for single-mismatched DNA detection.

A Single‐Electrode, Dual‐Potential Ferrocene–PNA Biosensor for the Detection of DNA

A Fc-PNA biosensor (Fc: ferrocenyl, C(10)H(9)Fe) was designed by using two electrochemically distinguishable recognition elements with different molecular information at a single electrode. Two Fc-PNA capture probes were therefore synthesized by N-terminal labeling different dodecamer PNA sequences with different ferrocene derivatives by click chemistry. Each of the two strands was thereby tethered with one specific ferrocene derivative. The two capture probes revealed quasi-reversible redox processes of the Fc(0/+) redox couple with a significant difference in their electrochemical half-wave potentials of Delta E(1/2)=160 mV. A carefully designed biosensor interface, consisting of a ternary self-assembled monolayer (SAM) of the two C-terminal cysteine-tethered Fc-PNA capture probes and 6-mercaptohexanol, was electrochemically investigated by square wave (SWV) and cyclic voltammetry (CV). The biosensor properties of this interface were analyzed by studying the interaction with DNA sequences that were complementary to either of the two capture probes by SWV. Based on distinct changes in both peak current and potential, a parallel identification of these two DNA sequences was successful with one interface design. Moreover, the primary electrochemical response could be converted by a simple mathematical analysis into a clear-cut electrochemical signal about the hybridization event. The discrimination of single-nucleotide polymorphism (SNP) was proven with a chosen single-mismatch DNA sequence. Furthermore, experiments with crude bacterial RNA confirm the principal suitability of this dual-potential sensor under real-life conditions.

Electrochemiluminescence Biobarcode Method Based on Cysteamine−Gold Nanoparticle Conjugates

The recently developed DNA-gold nanoparticle (DNA-GNP) biobarcode assay provides polymerase chain reaction (PCR)-like sensitivity for nucleic acid and protein targets without a need for enzymatic amplification. However, application of the conventional assay is challenged by its complex, expensive, time-consuming, and labor-intense procedure. Herein, we present a new electrochemiluminescence (ECL) biobarcode method based on cysteamine-GNP conjugates. In this strategy, an ECL nanoprobe is fabricated that relies on GNP that is modified with tris-(2,2'-bipyridyl) ruthenium (TBR) labeled cysteamine to boost ECL signals and single strand DNA for target recognition. Specifically, a sandwich complex that consists of a biotin labeled capture probe, target DNA, and cysteamine-GNP conjugate is captured by magnetic microparticles (MMPs) and subsequently identified by the ECL signals from loaded TBR. With the use of the developed probe, a limit of detection as low as 100 fM can be achieved and the assay exhibits excellent selectivity for single-mismatched DNA detection even in human serum. The proposed ECL based method should have wide applications in diagnosis of genetic diseases due to its high sensitivity, simplicity, and low cost.

A sensitive DNA biosensor fabricated from gold nanoparticles, carbon nanotubes, and zinc oxide nanowires on a glassy carbon electrode

We outline here the fabrication of a sensitive electrochemical DNA biosensor for the detection of sequence-specific target DNA. Zinc oxide nanowires (ZnONWs) were first immobilized on the surface of a glassy carbon electrode. Multi-walled carbon nanotubes (MWCNTs) with carboxyl groups were then dropped onto the surface of the ZnONWs. Gold nanoparticles (AuNPs) were subsequently introduced to the surface of the MWNTs/ZnONWs by electrochemical deposition. A single-stranded DNA probe with a thiol group at the end (HS–ssDNA) was covalently immobilized on the surface of the AuNPs by forming an Au–S bond. Scanning electron microscopy (SEM) and cyclic voltammetry (CV) were used to investigate the film assembly process. Differential pulse voltammetry (DPV) was used to monitor DNA hybridization by measuring the electrochemical signals of [Ru(NH 3 ) 6 ] 3+ bounding to double-stranded DNA (dsDNA). The incorporation of ZnONWs and MWCNTs in this sensor design significantly enhances the sensitivity and the selectivity. This DNA biosensor can detect the target DNA quantitatively in the range of 1.0 × 10 −13 to 1.0 × 10 −7 M, with a detection limit of 3.5 × 10 −14 M (S/N = 3). In addition, the DNA biosensor exhibits excellent selectivity, even for single-mismatched DNA detection.

Affinity and selectivity of C2‐ and C5‐substituted “chiral‐box” PNA in solution and on microarrays

Two peptide nucleic acids (PNAs) containing three adjacent modified chiral monomers (chiral box) were synthesized. The chiral monomers contained either a C2- or a C5-modified backbone, synthesized starting from D- and L-arginine, respectively (2D- and 5L-PNA). The C2-modified chiral PNA was synthesized using a submonomeric strategy to avoid epimerization during solid-phase synthesis, whereas for the C5-derivative, the monomers were first obtained and then used in solid-phase synthesis. The melting temperature of these PNA duplexes formed with the full-match or with single-mismatch DNA were measured both by UV and by CD spectroscopy and compared with the unmodified PNA. The 5L-chiral-box-PNA showed the highest T(m) with full-match DNA, whereas the 2D-chiral-box-PNA showed the highest sequence selectivity. The PNA were spotted on microarray slides and then hybridized with Cy5-labeled full match and mismatched oligonucleotides. The results obtained showed a signal intensity in the order achiral >2D-chiral box >5L-chiral box, whereas the full-match/mismatch selectivity was higher for the 2D chiral box PNA.