- About this Journal ·
- Abstracting and Indexing ·
- Advance Access ·
- Aims and Scope ·
- Annual Issues ·
- Article Processing Charges ·
- Articles in Press ·
- Author Guidelines ·
- Bibliographic Information ·
- Citations to this Journal ·
- Contact Information ·
- Editorial Board ·
- Editorial Workflow ·
- Free eTOC Alerts ·
- Publication Ethics ·
- Reviewers Acknowledgment ·
- Submit a Manuscript ·
- Subscription Information ·
- Table of Contents
BioMed Research International
Volume 2013 (2013), Article ID 640163, 6 pages
An Internalin A Probe-Based Genosensor for Listeria monocytogenes Detection and Differentiation
Department of Biomedical Sciences, Section of Microbiology, University of Cagliari, via Porcell 4, 09124 Cagliari, Italy
Received 19 December 2012; Revised 13 February 2013; Accepted 22 February 2013
Academic Editor: Dimitrios Karpouzas
Copyright © 2013 Laura Bifulco et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Internalin A (InlA), a protein required for Listeria monocytogenes virulence, is encoded by the inlA gene, which is only found in pathogenic strains of this genus. One of the best ways to detect and confirm the pathogenicity of the strain is the detection of one of the virulence factors produced by the microorganism. This paper focuses on the design of an electrochemical genosensor used to detect the inlA gene in Listeria strains without labelling the target DNA. The electrochemical sensor was obtained by immobilising an inlA gene probe (single-stranded oligonucleotide) on the surfaces of screen-printed gold electrodes (Au-SPEs) by means of a mercaptan-activated self-assembled monolayer (SAM). The hybridisation reaction occurring on the electrode surface was electrochemically transduced by differential pulse voltammetry (DPV) using methylene blue (MB) as an indicator. The covalently immobilised single-stranded DNA was able to selectively hybridise to its complementary DNA sequences in solution to form double-stranded DNA on the gold surface. A significant decrease of the peak current of the voltammogram (DPV) upon hybridisation of immobilised ssDNA was recorded. Whole DNA samples of L. monocytogenes strains could be discriminated from other nonpathogenic Listeria species DNA with the inlA gene DNA probe genosensor.
Listeria monocytogenes is a Gram-positive, aerobic, rod-shaped, foodborne pathogenic bacterium inducing listeriosis, an illness characterized by encephalitis, septicaemia, and meningitis [1–4]. It is the only pathogenic species of Listeria in humans and has been the cause of several well-documented food poisoning outbreaks [1, 5–9]. It can also cause gastroenteritis in otherwise healthy individuals and more severe invasive diseases in immunocompromised patients, pregnant women, newborns, and elderly people [10–15].
L. monocytogenes enters mammalian cells by inducing its own phagocytosis. Internalin A (InlA) is an 80 kDa surface protein which allows Listeria to enter the cells. It is a complex key virulence factor protein encoded by the inlA gene and is specific only for L. monocytogenes and not for other listerial species or for other genera. It mediates the attachment of Listeria to, and the invasion of, hepatocytes, epithelial, and endothelial cells. The bacterial adhesion and invasion of human intestinal epithelial cells is also mediated through specific interaction with its host cell receptor E-cadherin [16–19].
Conventionally, the detection and identification of bacteria mainly rely on specific microbiological and biochemical identification methods, which require at least 3 and as many as 7 days to yield results. Genetic characterisation methods are more rapid than the classical identification methods and lead to unequivocal species identification [20, 21]. Among these, polymerase chain reaction (PCR), followed by hybridisation of the PCR amplified target with a labelled single-stranded oligonucleotide probe is an effective method of sequence-specific DNA detection [16, 17].
Rapid and reliable detection methods of this pathogenic, toxin-producing bacterium are required since it is able to survive and grow at low temperatures  and because the mortality rate for infected individuals is much higher than for other common foodborne pathogens [23–27].
Although there are many DNA hybridisation assays currently suitable for diagnosis, faster, cheaper, miniaturised, multianalyte, easier to use, and more sensitive approaches are highly desirable, especially in the case of decentralised analysis. In this context, electrochemical detection of DNA hybridisation events offer innovative routes [28–37].
An effective and sensitive biosensor requires a probe that can be immobilized on a sensing platform. An ideal probe should be able to achieve sensitive and specific detection of the target analyte. It must also be easy to produce and withstand environmental stresses, such as changes in temperature and pH. The proposed methodology aims at the detection of L. monocytogenes incidence in either environmental or clinical samples, based on the detection of the inlA gene in DNA extracts from isolated strains. For achieving our goal, we have embedded an inlA-specific probe on the surface of a SPE using several signal enhancing protocols and measured hybridization events in DNA extracts and control templates, based on generated electrochemical signals [29–31, 38–41].
At the best of our knowledge, this is the only genosensor so far described that allows the discrimination of L. monocytogenes from different nonpathogenic Listeria strains based on the detection of a specific listerial pathogenic factor such as internalin A. It proved to allow a definite and significant differentiation of pathogenic from nonpathogenic listerial species.
2. Materials and Methods
2.1. Apparatus, Chemicals, and Probe
Differential pulse voltammetry (DPV), for measurements, and cyclic voltammetry (CV), for electrode cleaning, were carried out using an AUTOLAB PGSTAT 30 electrochemical analysis system and a GPES 4.8 software package (Eco Chemie, The Netherlands). Electrodes: screen-printed gold electrodes (Au-SPEs) were obtained from Ecobioservices & Researches s.r.l. (Florence, Italy). The two 59-base oligonucleotide sequences, the L. monocytogenes internalin inlA gene probe (A) (Gen Bank M67471.1) originally designed by Ingianni et al. , and its complementary sequence target (B) were obtained from Invitrogen.
The sequences were as follows : DNA probe (59-base sequence A): 5_-CCATTAGCTAATTTAACAACACTAGAACGACTAGATATTTCAAGTAATAAGGTGTCAGA- 3_; DNA target (59-base sequence B): 5_-TCTGACACCTTATTACTTGAAATATCTAGTCGTTCTAGTGTTGTTAAATTAGCTAATGG- 3_.
2.1.1. Reagents, Buffers, and Solutions
Methylene blue (MB) was purchased from Difco. The Methylene blue solution was prepared with 20 M MB and 20 mM NaCl in 20 mM Tris-HCl buffer (pH 7.00). 3-mercaptopropionic acid (MPA), N-hydroxysulfosuccinimide (NHS), and -ethylcarbodiimide hydrochloride (EDC) were obtained from Sigma-Aldrich (Steinheim, Germany). All chemicals were of an analytical reagent grade. In-house distilled and sterilised water was used for the preparation of all buffers and solutions. 50 mM solution was used for electrochemical cleaning of the electrodes.
2.1.2. Microbial Strains and Conditions
6 listerial strains from foods (Lys 1: L. innocua, Lys 2: L. monocytogenes, Lys 3: L. monocytogenes, Lys 4: L. monocytogenes, Lys 5: L. monocytogenes, and Lys 6: L. ivanovii) and 4 listerial collection strains (Lys 7: C 315 L. innocua, Lys 8: C 276 L. innocua, Lys 9: C 383 L. monocytogenes, and Lys 10: C 483 L. monocytogenes) taken from our Institute’s collection and representing important species of the genus Listeria were used in this work. DNA samples were prepared as described. The strains were grown on BHI plates and reidentified by metabolic tests according to Ingianni et al.  and use of the API Listeria galleries (bioMèrieux Italia, Milan, Italy).
2.1.3. DNA Extracts
DNA samples were prepared as follows: the bacteria strains were incubated overnight in BHI broth and washed twice in PBS before DNA extraction using an Easy-DNA kit (Invitrogen, Carlsbad, Ca, USA) following the manufacturer’s protocol. DNA concentration and purity were determined by UV light absorbance measured by an Ultrospec III spectrophotometer (Pharmacia LKB).
2.1.4. PCR Performance
PCR was performed as described by Ingianni et al. . DNA oligonucleotide stock solutions (100 mg/L) and Listeria DNA extracts (dsDNA 100 mg/L) were prepared with TE solution (10 mM Tris-HCl, 1 mM EDTA, and pH 8.00) and kept frozen. Working DNA solutions were prepared with either 500 mM acetate buffer (pH 4.80) or 20 mM Tris-HCl buffer (pH 7.00), according to the hybridisation protocol .
2.2. SAM Preparation and Electrode Modification
The SAM modification of Au-SPEs was performed following a protocol described by Gooding et al.  and Kerman et al.  for gold rod electrodes. This protocol was adjusted for screen-printed electrode modification. The gold surfaces of the working electrodes were prepared by electrochemical cleaning before modification. The electrodes were cleaned by cycling between the 0 V and +1.5 V potentials in a 50 mM solution at a scan rate of 100 mV/s for approximately 15 min. until reproducible scans were recorded. The electrodes were rinsed with sterile distilled water before SAM modification.
SAMs were prepared by covering the surfaces of the clean Au-SPEs with a freshly prepared 75 : 25 (v/v) ethanol:water solution containing 20 mM MPA. Au-SPEs were incubated in this ethanolic solution overnight for approximately 15 h. The Au-SPEs/SAM were rinsed with 75 : 25 (v/v) ethanol:water and then with water, prior to covalent activation by immersion in the 50 mM phosphate buffer solution (pH 7.40) containing 2 mM EDC and 5 mM NHS for 1 h. Then, the Au-SPEs/SAM/Linker surfaces were rinsed with the 50 mM phosphate buffer solution (pH 7.40).
Next, DNA immobilisation was performed on the working electrode surfaces. 20 L of 500 mM acetate buffer solution (pH 4.80) containing 100 ppm probe were pipetted onto the surface of each Au-SPEs/SAM/Linker. The probe droplets were left to air-dry overnight. Sensors were then soaked in water for 2 h and rinsed again with water to remove unbound DNA. Thus, inlA probe-modified Au-SPEs were obtained. Three inlA probe-modified electrodes were utilised as a control (inlA probe) for each experiment. The cost of each electrode was about 1.5 euros, and it was found to be stable for at least 1 week, when kept in the refrigerator.
20 l of 20 mM Tris buffer solution (pH 7.00) containing 100 ppm target (complementary sequences or whole Listeria DNAs) were pipetted onto the inlA probe-modified Au-SPE surfaces. Whole Listeria DNA samples were prepared immediately before hybridisation by high temperature denaturation (94°C) for 10 min. to obtain ssDNA. The target droplets were air-dried for 30 min. This allowed hybrid-modified Au-SPEs to be obtained. Each test required about 50–60 min of work by a technician.
2.4. MB Binding
MB was accumulated on the surface of either the modified or the hybridised electrodes, by pipetting 20 L of 20 mM Tris-HCl buffer (pH 7.00) containing 20 mM MB with 20 mM NaCl, which was then left for 5 min. without applying any potential. After MB accumulation, the electrodes were rinsed with 20 mM Tris-HCl buffer (pH 7.00) for a few seconds.
2.5. Voltammetric Transduction
The reduction signal of the accumulated MB was measured by using differential pulse voltammetry (DPV) with an amplitude of 10 mV and scan rate of 20 mV/s. Experiments were carried out in 20 mM Tris-HCl buffer (pH 7.00). Each experiment was carried out in triplicate.
3. Results and Discussion
The genosensors relied on the electrochemical transduction of the hybridisation between the immobilised ssDNA probe and its unlabelled complementary sequences. By following the modified protocol, we could form the SAMs on the surfaces of the screen-printed gold electrodes and activate them. Then, the original inlA probe was covalently linked onto the gold-working electrodes. The sensors were optimised for use with the complementary oligonucleotide and then tested on samples of Listeria culture DNA extracts. Hybridisation detection was accomplished by measuring the MB reduction signal. Electroactivity of this label could discriminate the hybrid from the probe. The decrease in the magnitude of the MB voltammetric reduction signals, thus, reflected the extent of hybrid formation. Probe specificity and probe-method sensitivity were further tested using PCR products of inlA gene targets as templates.
3.2. Probe Immobilisation
To understand probe coverage and surface organization at the Au-SPEs/SAM, the Au-SPEs/SAM/activated, and the Au-SPEs/SAM/probe-modified electrodes, we recorded peak current magnitudes at the respective electrodes after incubation in MB solutions.
Measurements of MB reduction were carried out at the bare electrodes (Figure 1), at the MPA-SAM modified electrodes, at the EDC/NHS-activated SAM electrodes and at the SAM/ssDNA inlA probe-modified electrodes. The voltammetric signal of MB reduction at the bare electrodes decreased after SAM modification and activation and increased again after inlA probe linking. The MPA-SAM restricted MB access to the electrode but still allowed significant electrochemistry to occur at the underlying electrode (MPA-SAM in Figure 1). The activation of this carboxylic acid terminated SAM with EDC/NHS further restricted MB access to the electrode without completely passivising it (activated SAM in Figure 1). Immobilisation of the probe on the SAM-modified electrodes resulted in an increase in MB peak currents (inlA probe in Figure 1) due to the affinity of MB for the free guanine bases of the DNA as previous reported [30, 42]. The values of inlA probe against bare, SAM, and activated SAM were found to be significant: .
3.3. Detection of the InlA Complementary DNA Sequence
The sensors were studied for hybridisation detection using the complementary sequence of the immobilised probe. The genosensors were usable for one shot only. Therefore, we compared the data obtained from series of 3 to 5 genosensors produced during each experiment. Data shown are the average of each experiment. Figure 2 shows the DP voltammograms for the MB reduction signal at the inlA probe-immobilised Au-SPEs (blue) and after hybridisation with the target (red). The shown voltammetric curves are the average of 5 electrodes. The highest MB reduction signal was observed with the ssDNA probe on the electrode alone (Figure 2, blue), because MB has a strong affinity for the free guanine bases; hence, the greatest amount of MB accumulation occurs on this surface. An obvious decrease in the voltammetric peak was observed for the indicator after double-strand formation (Figure 2, red), since the interaction between MB and the guanine residues of the probe was prevented by hybrid formation on the electrode surface.
The sensors were tested with different concentrations of complementary oligonucleotide. A voltammetric signal was still observed (Figures 3 and 4), even when all the DNA probe was completely hybridised to a duplex, because MB can also act as an intercalator. However, the rapid decrease of the MB signal after hybridisation, as shown in the calibration curve, indicates that the voltammetric signal due to intercalation is small compared to the signal from direct interaction with the guanine bases.
3.4. Detection of L. monocytogenes Strains and Discrimination from Different Listeria Strains
Genosensors were tested on whole DNA samples of different L. monocytogenes strains. The differences recorded in the reduction signals indicate a different grade of hybridization between L. monocytogenes strain DNAs. It could be due to the presence of a different number of copies of the inlA gene; thus, the voltammogram peaks of L. monocytogenes DNAs showed a minimum high when a low number of copies are present, while L. monocytogenes peaks were higher for a larger number of copies of the gene.
Genosensors were then tested on whole DNA samples of different Listeria strains. Figure 4 shows the comparison of MB reduction peaks after hybridisation with two DNA samples of L. monocytogenes strains, one from L. ivanovii and two DNA samples of L. innocua strains. The inlA gene sequences are only present in the DNA of L. monocytogenes.
However, the L. monocytogenes voltammogram current was always lower than Listeria non-monocytogenes voltammograms (the difference between the media of L. monocytogenes and the media of other Listeria species was found to be highly statistically significant: ).
MB reduction peak data of the six L. monocytogenes strains were compared with the voltammograms of the other strains. Figure 5 shows that the L. innocua voltammograms are comparable to the inlA probe signals, while L. monocytogenes voltammograms are comparable to the complementary oligonucleotide signals.
Experiments were carried out to investigate genosensor stability. They were stored at 4°C after preparation, and measurements were performed after 24, 48, 72, and 96 hours. When kept in a freezer at −20°C the genosensors presented the same responses for at least 6 weeks, and at −80°C, they were still efficient after 3 months.
In this work, we investigated the possibility of an Internalin A (inlA) probe application for the construction of a genosensor for the identification of the pathogenic bacterium L. monocytogenes. The inlA probe utilised was previously designed in our laboratory. Due to the formation of the alkanethiol SAMs adsorbed on Au-SPE surfaces, the inlA probe could be attached using covalent linkers such as EDC and NHS. These genosensors were used for the detection of hybridisation on the Au-SPE surfaces by means of MB as the electroactive reporter. The inlA probe-modified Au-SPEs were shown to transduce hybridisation with complementary and, more interestingly, with whole DNA samples of L. monocytogenes that contained the inlA gene. Furthermore, discrimination between different pathogenic and nonpathogenic Listeria species was recorded. The test is quite inexpensive, requires less than 60 min of technical work, when DNA extracts are available, and can be useful for assaying DNA extracted from clinical isolates, as well as from environmental and food strains, especially in the case of decentralised analysis.
The authors wish to thank Dr. Ilaria Palchetti from the “Ugo Schiff” Dipartimento di Chimica, Florence, Italy, for the stimulating discussions and critical suggestions. The authors also acknowledge the financial support from the Consorzio per le Ricerche e lo Sviluppo delle Biotecnologie Biotecne, Cagliari, Italy, and the Fondazione Banco di Sardegna, Sassari, Italy (Project 2010-11).
- C. G. Clark, J. Farber, F. Pagotto et al., “Surveillance for Listeria monocytogenes and listeriosis, 1995–2004,” Epidemiology and Infection, vol. 138, no. 4, pp. 559–572, 2010.
- R. J. Meldrum, P. W. Ellis, P. T. Mannion, D. Halstead, and J. Garside, “Prevalence of Listeria monocytogenes in ready-to-eat foods sampled from the point of sale in wales, United Kingdom,” Journal of Food Protection, vol. 73, no. 8, pp. 1515–1518, 2010.
- D. Nucera, S. Lomonaco, D. M. Bianchi et al., “A five year surveillance report on PFGE types of Listeria monocytogenes isolated in Italy from food and food related environments,” International Journal of Food Microbiology, vol. 140, no. 2-3, pp. 271–276, 2010.
- L. O'Connor, M. O'Leary, N. Leonard et al., “The characterization of Listeria spp. isolated from food products and the food-processing environment,” Letters in Applied Microbiology, vol. 51, no. 5, pp. 490–498, 2010.
- Centers for Disease Control and Prevention (CDC), “Surveillance for foodborne disease outbreaks: United States, 2007,” Morbidity and Mortality Weekly Report, vol. 59, no. 31, pp. 973–979, 2010.
- Centers for Disease Control and Prevention (CDC), “Surveillance for foodborne disease outbreaks—United States, 2006,” Morbidity and Mortality Weekly Report, vol. 58, pp. 609–615, 2006.
- A. di Pinto, L. Novelleo, F. Montemurro, E. Bonerba, and G. Tantillo, “Occurrence of Listeria monocytogenes in ready-to-eat foods from supermarkets in Southern Italy,” New Microbiologica, vol. 33, no. 3, pp. 249–252, 2010.
- C. W. Donnelly, “Listeria monocytogenes: a continuing challenge,” Nutrition Reviews, vol. 59, no. 6, pp. 183–194, 2001.
- J. Koch, R. Dworak, R. Prager et al., “Large listeriosis outbreak linked to cheese made from pasteurized milk, Germany, 2006-2007,” Foodborne Pathogens and Disease, vol. 7, no. 12, pp. 1581–1584, 2010.
- S. B. Barbuddhe and T. Chakraborty, “Listeria as an enteroinvasive gastrointestinal pathogen,” Current Topics in Microbiology and Immunology, vol. 337, no. 1, pp. 173–195, 2009.
- I. A. Gillespie, P. Mook, C. L. Little, K. Grant, and G. K. Adak, “Listeria monocytogenes infection in the over-60s in England between 2005 and 2008: a retrospective case-control study utilizing market research panel data,” Foodborne Pathogens and Disease, vol. 7, no. 11, pp. 1373–1379, 2010.
- H. Hof, “History and epidemiology of listeriosis,” FEMS Immunology and Medical Microbiology, vol. 35, no. 3, pp. 199–202, 2003.
- K. A. Jackson, M. Iwamoto, and D. Swerdlow, “Pregnancy-associated listeriosis,” Epidemiology and Infection, vol. 138, no. 10, pp. 1503–1509, 2010.
- J. McLauchlin, “Human listeriosis in Britain, 1967–85, a summary of 722 cases: 1. Listeriosis during pregnancy and in the newborn,” Epidemiology and Infection, vol. 104, no. 2, pp. 181–189, 1990.
- P. Mook, K. A. Grant, C. L. Little, G. Kafatos, and I. A. Gillespie, “Emergence of pregnancy-related listeriosis amongst ethnic minorities in England and Wales,” Eurosurveillance, vol. 15, no. 27, pp. 17–23, 2010.
- A. Ingianni, M. Floris, P. Palomba, M. A. Madeddu, M. Quartuccio, and R. Pompei, “Rapid detection of Listeria monocytogenes in foods, by a combination of PCR and DNA probe,” Molecular and Cellular Probes, vol. 15, no. 5, pp. 275–280, 2001.
- R. H. Orsi, H. C. D. Bakker, and M. Wiedmann, “Listeria monocytogenes lineages: genomics, evolution, ecology, and phenotypic characteristics,” International Journal of Medical Microbiology, vol. 301, no. 2, pp. 79–96, 2011.
- A. J. Roberts, S. K. Williams, M. Wiedmann, and K. K. Nightingale, “Some Listeria monocytogenes outbreak strains demonstrate significantly reduced invasion, inlA transcript levels, and swarming motility in vitro,” Applied and Environmental Microbiology, vol. 75, no. 17, pp. 5647–5658, 2009.
- W. D. Schubert, C. Urbanke, T. Ziehm et al., “Structure of internalin, a major invasion protein of Listeria monocytogenes, in complex with its human receptor E-cadherin,” Cell, vol. 111, no. 6, pp. 825–836, 2002.
- D. Elhanafi, V. Utta, and S. Kathariou, “Genetic characterization of plasmid-associated benzalkonium chloride resistance determinants in a Listeria monocytogenes train from the 1998-1999 outbreak,” Applied and Environmental Microbiology, vol. 76, no. 24, pp. 8231–8238, 2010.
- C. Mammina, A. Aleo, C. Romani et al., “Characterization of Listeria monocytogenes isolates from human listeriosis cases in Italy,” Journal of Clinical Microbiology, vol. 47, no. 9, pp. 2925–2930, 2009.
- R. O. Azizoglu, J. Osborne, S. Wilson, and S. Kathariou, “Role of growth temperature in freeze-thaw tolerance of Listeria spp.,” Applied and Environmental Microbiology, vol. 75, no. 16, pp. 5315–5320, 2009.
- P. P. Banada, S. Guo, B. Bayraktar et al., “Optical forward-scattering for detection of Listeria monocytogenes and other Listeria species,” Biosensors and Bioelectronics, vol. 22, no. 8, pp. 1664–1671, 2007.
- P. Banerjee and A. K. Bhunia, “Cell-based biosensor for rapid screening of pathogens and toxins,” Biosensors and Bioelectronics, vol. 26, pp. 99–106, 2010.
- T. Curtis, R. M. Z. G. Naal, C. Batt, J. Tabb, and D. Holowka, “Development of a mast cell-based biosensor,” Biosensors and Bioelectronics, vol. 23, no. 7, pp. 1024–1031, 2008.
- V. Nanduri, A. K. Bhunia, S. I. Tu, G. C. Paoli, and J. D. Brewster, “SPR biosensor for the detection of L. monocytogenes using phage-displayed antibody,” Biosensors and Bioelectronics, vol. 23, no. 2, pp. 248–252, 2007.
- E. Tully, S. P. Higson, and R. O'Kennedy, “The development of a “labeless” immunosensor for the detection of Listeria monocytogenes cell surface protein, Internalin B,” Biosensors and Bioelectronics, vol. 23, no. 6, pp. 906–912, 2008.
- F. Farabullini, F. Lucarelli, I. Palchetti, G. Marrazza, and M. Mascini, “Disposable electrochemical genosensor for the simultaneous analysis of different bacterial food contaminants,” Biosensors and Bioelectronics, vol. 22, no. 7, pp. 1544–1549, 2007.
- Y. Jin, X. Yao, Q. Liu, and J. Li, “Hairpin DNA probe based electrochemical biosensor using methylene blue as hybridization indicator,” Biosensors and Bioelectronics, vol. 22, no. 6, pp. 1126–1130, 2007.
- P. Kara, K. Kerman, D. Ozkan et al., “Electrochemical genosensor for the detection of interaction between methylene blue and DNA,” Electrochemistry Communications, vol. 4, no. 9, pp. 705–709, 2002.
- K. Kerman, M. Kobayashi, and E. Tamiya, “Recent trends in electrochemical DNA biosensor technology,” Measurement Science and Technology, vol. 15, pp. R1–R11, 2004.
- F. Lucarelli, A. Kicela, I. Palchetti, G. Marrazza, and M. Mascini, “Electrochemical DNA biosensor for analysis of wastewater samples,” Bioelectrochemistry, vol. 58, no. 1, pp. 113–118, 2002.
- F. Lucarelli, G. Marrazza, A. P. F. Turner, and M. Mascini, “Carbon and gold electrodes as electrochemical transducers for DNA hybridisation sensors,” Biosensors and Bioelectronics, vol. 19, no. 6, pp. 515–530, 2004.
- F. Lucarelli, S. Tombelli, M. Minunni, G. Marrazza, and M. Mascini, “Electrochemical and piezoelectric DNA biosensors for hybridisation detection,” Analytica Chimica Acta, vol. 609, no. 2, pp. 139–159, 2008.
- B. Meric, K. Kerman, G. Marrazza, I. Palchetti, M. Mascini, and M. Ozsoz, “Disposable genosensor, a new tool for the detection of NOS-terminator, a genetic element present in GMOs,” Food Control, vol. 15, no. 8, pp. 621–626, 2004.
- O. Pänke, A. Kirbs, and F. Lisdat, “Voltammetric detection of single base-pair mismatches and quantification of label-free target ssDNA using a competitive binding assay,” Biosensors and Bioelectronics, vol. 22, no. 11, pp. 2656–2662, 2007.
- J. P. Tosar, K. Keel, and J. Laíz, “Two independent label-free detection methods in one electrochemical DNA sensor,” Biosensors and Bioelectronics, vol. 24, no. 10, pp. 3036–3042, 2009.
- J. J. Gooding, P. Erokhin, and D. B. Hibbert, “Parameters important in tuning the response of monolayer enzyme electrodes fabricated using self-assembled monolayers of alkanethiols,” Biosensors and Bioelectronics, vol. 15, no. 5-6, pp. 229–239, 2000.
- K. Kerman, D. Ozkan, P. Kara, B. Meric, J. J. Gooding, and M. Ozsoz, “Voltammetric determination of DNA hybridization using methylene blue and self-assembled alkanethiol monolayer on gold electrodes,” Analytica Chimica Acta, vol. 462, no. 1, pp. 39–47, 2002.
- B. Meric, K. Kerman, D. Ozkan et al., “Electrochemical DNA biosensor for the detection of TT and hepatitis B virus from PCR amplified real samples by using,” Talanta, vol. 56, no. 5, pp. 837–846, 2002.
- D. Ozkan, P. Kara, K. Kerman et al., “DNA and PNA sensing on mercury and carbon electrodes by using methylene blue as an electrochemical label,” Bioelectrochemistry, vol. 58, no. 1, pp. 119–126, 2002.
- W. Yang, J. J. Gooding, and D. B. Hibbert, “Characterisation of gold electrodes modified with self-assembled monolayers of L-cysteine for the adsorptive stripping analysis of copper,” Journal of Electroanalytical Chemistry, vol. 516, no. 1-2, pp. 10–16, 2001.