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Chemcomm Acceptedchemical Communications Manuscript View Article Online View Journal ChemComm AcceptedChemical Communications Manuscript This article can be cited before page numbers have been issued, to do this please use: B. Kaur, K. Malecka, C. S. Chay, D. A. Cristaldi, I. Mames, H. Radecka, J. Radecki and E. Stulz, Chem. Commun., 2018, DOI: 10.1039/C8CC05362F. Volume 52 Number 1 4 January 2016 Pages 1–216 This is an Accepted Manuscript, which has been through the Royal Society of Chemistry peer review process and has been ChemComm accepted for publication. Chemical Communications Accepted Manuscripts are published online shortly after www.rsc.org/chemcomm acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available. You can find more information about Accepted Manuscripts in the author guidelines. Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s ISSN 1359-7345 standard Terms & Conditions and the ethical guidelines, outlined in our author and reviewer resource centre, still apply. In no COMMUNICATION Marilyn M. Olmstead, Alan L. Balch, Josep M. Poblet, Luis Echegoyen et al. Reactivity diff erences of Sc3N@C2n (2n = 68 and 80). Synthesis of the fi rst methanofullerene derivatives of Sc N@D -C 3 5h 80 event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains. rsc.li/chemcomm Page 1 of 5 Please doChemComm not adjust margins View Article Online DOI: 10.1039/C8CC05362F ChemComm COMMUNICATION Approaching single DNA molecule detection with an ultrasensitive electrochemical genosensor based on gold nanoparticles and Received 00th January 20xx, Cobalt-porphyrin DNA conjugates Accepted 00th January 20xx †a †a †b †b †b DOI: 10.1039/x0xx00000x Balwinder Kaur, Kamila Malecka, Domenico A. Cristaldi, Clarissa S. Chay, Iwona Mames, Hanna Radecka,a Jerzy Radecki*a and Eugen Stulz*b www.rsc.org/ We describe an ultrasensitive electrochemical genosensor based markers, such as methylene blue or ferrocene, are often called on gold nanoparticles and cobalt-porphyrin functionalised ssDNA signal-on or signal-off DNA sensors, depending on their probes. The sensitivity at the attomolar concentration level arises design.2b, 2d from an increased density of redox labels on the electrode surface Improving the sensitivity and detection limit are two of the compared to sensors without NP modification. The electrode major challenges in the preparation of genosensors, and detects as few as 23 DNA molecules, approaching single molecule particularly the amount of immobilised ssDNA probe on the detection. biosensor interface further determines its recognition Manuscript capability towards complementary ssDNA. In order to increase There is currently a great interest in highly sensitive DNA the immobilisation amount of the ssDNA probe and improve biosensors due to increased risks and threats from pathogens, its hybridisation efficiency, different nanomaterials have been but also for early diagnostics of gene associated diseases such investigated for electrode surface modification to increase 1 as cancer. The genosensors find widespread use in clinical their surface areas.6 In recent years, DNA genosensors based diagnosis, pathogen detection, DNA sequencing and mapping, on metal nanoparticles (Pt, Au and Ag) for signal amplification 2 and in forensics. In particular, the detection of single have been developed with substantial improvement in nucleotide polymorphism (SNP) is of high importance in sensitivity.7 DNA-gold nanoparticle conjugates have been specific allele analysis, which can indicate susceptibility for explored as one of the most attractive nanomaterials because 3 Accepted specific diseases. of their unique chemical and physical properties and ease of Generally, in genosensors the hybridisation process between formation.7d, 7e, 8 The reported sensors with nanoparticle the ssDNA probe and its complementary target is converted modification generally exhibit detection limits (LODs) in the into a readable signal by appropriate transducers like range of 10-9 to 10-15 M;7e, 8a, 8b, 9 one report describes a LOD of Published on 06 August 2018. Downloaded by University of Southampton 8/6/2018 11:57:25 AM. electrochemical, optical or mass-sensitive elements. Among 1.7 × 10−18 M where a AuNP was attached to the end of a the different sensing methods, electrochemical detection 3−/4− hairpin loop and acts as mediator for the [Fe(CN)6] redox offers one of the best options for DNA sensing due to its couple detection.10 simplicity, fast response, low cost, ease of miniaturization and Here we report a new strategy to prepare DNA biosensors 4 potential for multiplexing in microfluidic chips. To date a based on gold nanoparticles (AuNPs) combined with cobalt- variety of electrochemical genosensors for specific ssDNA porphyrin (CoP) labelled ssDNA probes,11 which are 5 sequence detection have been reported in the literature. immobilized on the gold electrode surface. The CoP acts as These genosensors are based either on direct oxidation or redox marker for the sensitive detection of the target ssDNA reduction of nucleic acids, on electrodes modified with label- derived from Avian Influenza Virus (AIV); this marker has ChemComm free ssDNA probe and diffusional redox markers such as previously shown to be very sensitive and selective, allowing 3-/4- 3+ Fe[(CN)6] , or [Ru(NH3)6] in the sample solution. The efficiently discrimination of single base pair mismatches in the sensors which incorporate a ssDNA probe labelled with redox femtomolar range and does not require an externally added redox mediator, which is a great advantage.11a AIV, especially the type H5N1, is a highly contagious, poultry threatening pathogen which can also infect humans. Thus, there is a requirement for highly sensitive, accurate and rapid tests for diagnosis of AIV infection which would allow early antiviral therapy.12 In recent years, our laboratories have developed DNA biosensors towards the detection of H5N1.2b, 11a, 13 Using This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 1 Please do not adjust margins Please doChemComm not adjust margins Page 2 of 5 View Article Online DOI: 10.1039/C8CC05362F COMMUNICATION Journal Name AuNPs, an increase of ssDNA probe molecules and with it the in air, and representative images are shown in Fig. 1a. The number of redox centres responsible for the analytical signal bare gold surface shows a high degree of structural generation was achieved on an electrode, resulting in a homogeneity with well-defined terraces and a root mean genosensor with greatly improved sensitivity. A detailed study squared (RMS) roughness of 3.27 ± 0.17 nm, which was to determine the effect of AuNPs surface functionalization determined over an area of 0.50 µm2. The topography of the with different amounts of CoP-ssDNA on the detection of DNA AuNP modified gold surface is shown in Fig. 1b, and well targets is presented. separated NPs are visible. The RMS roughness, determined The steps in the genosensor fabrication and the working over the same area as before, was equal to 7.49 ± 0.45 nm. principle are depicted in Scheme 1 (details of the procedures The size of the observed AuNPs was found to be 46.6 ± 1.7 nm, can be found in the electronic supporting information). The which is larger than the AuNPs themselves (13 nm). Similar size first step involves modification of the gold electrode surface increase in AuNP-DNA conjugates with a comparable DNA with a thiol-DNA capture strand, which is complementary to length (from 17 nm NP to 69.8 nm NP-DNA conjugate with an the CoP-ssDNA probe; the capture strand is embedded in a 18 bp DNA) have been reported,14 therefore the increase can self-assembled monolayer (SAM) of mercaptohexanol (MCH). be accounted for by the ssDNA layer. The dispersion of the NPs In a separate step, AuNPs (13 nm, Fig. S4) were functionalised on the gold surface also indicates that no aggregation occurs, with CoP-DNA (probe strand)11b through covalent Au-thiol which would show substantially larger sizes.14 bonding; as in our previous sensor, three dithiol serinol units at the 5’-end were used to ensure stability of the conjugate.11a Different ratios of AuNPs to ssDNA at 1:10, 1:100 and 1:200 were used (by virtue of concentration); no MCH filler was used on the NPs. The AuNP–CoP-ssDNA conjugates were then attached to the gold electrode surface via hybridisation of the two complementary strands. In this respect, some of the ssDNA probe is used to bind the NPs to the gold surface and Manuscript burying this amount of the porphyrin markers; however, since Figure 1. AFM images of: (a) Au(111) thin film on mica substrate and (b) Au(111) not all probe strands are used in this process it leaves enough substrate modified with SH-ssDNA+MCH/AuNPs-CoP-ssDNA (1:200). CoP-ssDNA strands for capturing the target strands in solution. The functional electrode was next fully characterised using electrochemical methods. The presence of the redox active probe (CoP) on the electrode surface was confirmed by cyclic voltammetry (CV), Osteryoung square wave voltammetry (OSWV) and differential pulse voltammetry (DPV). Representative voltammograms for all systems are shown in Fig. S6 (supporting information). The gold electrodes, which Accepted were modified with AuNPs-CoP–ssDNA using 1:10, 1:100 and 1:200 ratio, all exhibited well defined quasi-reversible Co(II)/Co(III) redox peaks. The electrochemical parameters Published on 06 August 2018. Downloaded by University of Southampton 8/6/2018 11:57:25 AM.
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