Development of Electrochemical DNA Biosensor for Equine Hindgut Acidosis Detection

The pH drop in the hindgut of the horse is caused by lactic acid-producing bacteria which are abundant when a horse’s feeding regime is excessively carbohydrate rich. This drop in pH below six causes hindgut acidosis and may lead to laminitis. Lactic acid-producing bacteria Streptococcus equinus and Mitsuokella jalaludinii have been found to produce high amounts of L-lactate and D-lactate, respectively. Early detection of increased levels of these bacteria could allow the horse owner to tailor the horse’s diet to avoid hindgut acidosis and subsequent laminitis. Therefore, 16s ribosomal ribonucleic acid (rRNA) sequences were identified and modified to obtain target single stranded deoxyribonucleic acid (DNA) from these bacteria. Complementary single stranded DNAs were designed from the modified target sequences to form capture probes. Binding between capture probe and target single stranded deoxyribonucleic acid (ssDNA) in solution has been studied by gel electrophoresis. Among pairs of different capture probes and target single stranded DNA, hybridization of Streptococcus equinus capture probe 1 (SECP1) and Streptococcus equinus target 1 (SET1) was portrayed as gel electrophoresis. Adsorptive stripping voltammetry was utilized to study the binding of thiol modified SECP1 over gold on glass substrates and these studies showed a consistent binding signal of thiol modified SECP1 and their hybridization with SET1 over the gold working electrode. Cyclic voltammetry and electrochemical impedance spectroscopy were employed to examine the binding of thiol modified SECP1 on the gold working electrode and hybridization of thiol modified SECP1 with the target single stranded DNA. Both demonstrated the gold working electrode surface was modified with a capture probe layer and hybridization of the thiol bound ssDNA probe with target DNA was indicated. Therefore, the proposed electrochemical biosensor has the potential to be used for the detection of the non-synthetic bacterial DNA target responsible for equine hindgut acidosis.

[1]  M. Rizwan,et al.  A highly sensitive electrochemical detection of human chorionic gonadotropin on a carbon nano-onions/gold nanoparticles/polyethylene glycol nanocomposite modified glassy carbon electrode , 2019, Journal of Electroanalytical Chemistry.

[2]  M. Minero,et al.  Circulating miR-23b-3p, miR-145-5p and miR-200b-3p are potential biomarkers to monitor acute pain associated with laminitis in horses. , 2017, Animal : an international journal of animal bioscience.

[3]  T. G. Drummond,et al.  Electrochemical DNA sensors , 2003, Nature Biotechnology.

[4]  M. Rizwan,et al.  Combining a gold nanoparticle-polyethylene glycol nanocomposite and carbon nanofiber electrodes to develop a highly sensitive salivary secretory immunoglobulin A immunosensor , 2018 .

[5]  Ilka Schmueser,et al.  Impedimetric measurement of DNA-DNA hybridisation using microelectrodes with different radii for detection of methicillin resistant Staphylococcus aureus (MRSA). , 2017, The Analyst.

[6]  M. Rizwan,et al.  AuNPs/CNOs/SWCNTs/chitosan-nanocomposite modified electrochemical sensor for the label-free detection of carcinoembryonic antigen. , 2018, Biosensors & bioelectronics.

[7]  Lukas W. Snyman,et al.  Micro optical sensors based on avalanching silicon light-emitting devices monolithically integrated on chips , 2019, Optical Materials Express.

[8]  V. Zucolotto,et al.  Label-free electrochemical DNA biosensor for zika virus identification. , 2019, Biosensors & bioelectronics.

[9]  Ailin Liu,et al.  Development of electrochemical DNA biosensors , 2012 .

[10]  G. Carmichael,et al.  Analysis of single- and double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[11]  N. Buckley,et al.  Electrochemical impedance spectroscopy biosensor for detection of active botulinum neurotoxin , 2014 .

[12]  B Merchant,et al.  Gold, the noble metal and the paradoxes of its toxicology. , 1998, Biologicals : journal of the International Association of Biological Standardization.

[13]  J. Justin Gooding,et al.  Characterisation of gold electrodes modified with self-assembled monolayers of l-cysteine for the adsorptive stripping analysis of copper , 2001 .

[14]  Wei Cheng,et al.  A sensitive electrochemical DNA biosensor for specific detection of Enterobacteriaceae bacteria by Exonuclease III-assisted signal amplification. , 2013, Biosensors & bioelectronics.

[15]  Fred Lisdat,et al.  A label-free DNA sensor based on impedance spectroscopy , 2008 .

[16]  Chang-Woo Lee,et al.  Self-Assembled Monolayer of l-Cysteine on Au(111): Hydrogen Exchange between Zwitterionic l-Cysteine and Physisorbed Water , 2003 .

[17]  Christopher Gwenin,et al.  Development of Solid-Phase RPA on a Lateral Flow Device for the Detection of Pathogens Related to Sepsis , 2020, Sensors.

[18]  U. Rant,et al.  Dissimilar kinetic behavior of electrically manipulated single- and double-stranded DNA tethered to a gold surface. , 2006, Biophysical journal.

[19]  Ilaria Palchetti,et al.  Nucleic acid biosensors for environmental pollution monitoring. , 2008, The Analyst.

[20]  L. Lerman The structure of the DNA-acridine complex. , 1963, Proceedings of the National Academy of Sciences of the United States of America.

[21]  J. Crain,et al.  Label- and amplification-free electrochemical detection of bacterial ribosomal RNA. , 2016, Biosensors & bioelectronics.

[22]  N. Buckley,et al.  Botulinum Neurotoxin Serotypes Detected by Electrochemical Impedance Spectroscopy , 2015, Toxins.

[23]  R. Amann,et al.  Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations , 1990, Applied and environmental microbiology.

[24]  M. V. D. Berg,et al.  Fecal pH and Microbial Populations in Thoroughbred Horses During Transition from Pasture to Concentrate Feeding , 2013 .

[25]  Raj Mutharasan,et al.  A cantilever biosensor-based assay for toxin-producing cyanobacteria Microcystis aeruginosa using 16S rRNA. , 2013, Environmental science & technology.

[26]  C. Gwenin,et al.  A Label Free Colorimetric Assay for the Detection of Active Botulinum Neurotoxin Type A by SNAP-25 Conjugated Colloidal Gold , 2013, Toxins.

[27]  Ying Xu,et al.  Indicator Free DNA Hybridization Detection by Impedance Measurement Based on the DNA‐Doped Conducting Polymer Film Formed on the Carbon Nanotube Modified Electrode , 2003 .

[28]  M. Yeh,et al.  Salmonella detection using 16S ribosomal DNA/RNA probe-gold nanoparticles and lateral flow immunoassay. , 2013, Food chemistry.

[29]  A. V. van Eps,et al.  Fluorescence in situ hybridization analysis of hindgut bacteria associated with the development of equine laminitis. , 2007, Environmental microbiology.

[30]  S. Nadeem,et al.  Entropy generation and temperature-dependent viscosity in the study of SWCNT–MWCNT hybrid nanofluid , 2020, Applied Nanoscience.

[31]  J. Justin Gooding,et al.  Electrochemical DNA Hybridization Biosensors , 2002 .

[32]  Joseph Wang Electrochemical nucleic acid biosensors , 2002 .

[33]  Marc Tornow,et al.  Structural properties of oligonucleotide monolayers on gold surfaces probed by fluorescence investigations. , 2004, Langmuir : the ACS journal of surfaces and colloids.

[34]  L. Lerman,et al.  Structural considerations in the interaction of DNA and acridines. , 1961, Journal of molecular biology.

[35]  R. Hoffmann,et al.  Sulfur-Gold Orbital Interactions which Determine the Structure of Alkanethiolate/Au(111) Self-Assembled Monolayer Systems , 2002 .

[36]  R. Juste,et al.  Detection of latent forms of Mycobacterium avium subsp. paratuberculosis infection using host biomarker-based ELISAs greatly improves paratuberculosis diagnostic sensitivity , 2020, PloS one.

[37]  Jin-Young Park,et al.  DNA Hybridization Sensors Based on Electrochemical Impedance Spectroscopy as a Detection Tool , 2009, Sensors.

[38]  F. Kasten Cytochemical studies with acridine orange and the influence of dye contaminants in the staining of nucleic acids. , 1967, International review of cytology.

[39]  J. Nocek Bovine acidosis: implications on laminitis. , 1997, Journal of dairy science.

[40]  María Marazuela,et al.  Fiber-optic biosensors – an overview , 2002, Analytical and bioanalytical chemistry.

[41]  C. Pollitt,et al.  The genetic diversity of lactic acid producing bacteria in the equine gastrointestinal tract. , 2005, FEMS microbiology letters.

[42]  A. V. van Eps,et al.  Changes in equine hindgut bacterial populations during oligofructose-induced laminitis. , 2006, Environmental microbiology.

[43]  G. Zeng,et al.  Electrochemical detection of Pseudomonas aeruginosa 16S rRNA using a biosensor based on immobilized stem-loop structured probe. , 2011, Enzyme and microbial technology.

[44]  Huaping Peng,et al.  Label-free electrochemical DNA biosensor for rapid detection of mutidrug resistance gene based on Au nanoparticles/toluidine blue–graphene oxide nanocomposites , 2015 .

[45]  Bing Wang,et al.  Development of a multiplex real-time PCR assay using two thermocycling platforms for detection of major bacterial pathogens associated with bovine respiratory disease complex from clinical samples , 2018, Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc.

[46]  S. Satija,et al.  Using Self-Assembly To Control the Structure of DNA Monolayers on Gold: A Neutron Reflectivity Study , 1998 .

[47]  D. Corrigan,et al.  SAM Composition and Electrode Roughness Affect Performance of a DNA Biosensor for Antibiotic Resistance , 2019, Biosensors.

[48]  C. J. Newbold,et al.  Aberystwyth University Identification of a Core Bacterial Community within the Large Intestine of the Horse , 2013 .