{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2024,7,14]],"date-time":"2024-07-14T08:04:36Z","timestamp":1720944276930},"reference-count":57,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2015,5,22]],"date-time":"2015-05-22T00:00:00Z","timestamp":1432252800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"U.S. Department of Defence","award":["N00024-13-P-4543"]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Sensors"],"abstract":"In this paper, we report the ultra-sensitive indirect electrochemical detection of E. coli O157:H7 using antibody functionalized primary (magnetic) beads for capture and polyguanine (polyG) oligonucleotide functionalized secondary (polystyrene) beads as an electrochemical tag. Vacuum filtration in combination with E. coli O157:H7 specific antibody modified magnetic beads were used for extraction of E. coli O157:H7 from 100 mL samples. The magnetic bead conjugated E. coli O157:H7 cells were then attached to polyG functionalized secondary beads to form a sandwich complex (magnetic bead\/E. coli secondary bead). While the use of magnetic beads for immuno-based capture is well characterized, the use of oligonucleotide functionalized secondary beads helps combine amplification and potential multiplexing into the system. The antibody functionalized secondary beads can be easily modified with a different antibody to detect other pathogens from the same sample and enable potential multiplexing. The polyGs on the secondary beads enable signal amplification up to 10\\(^{8}\\) guanine tags per secondary bead (\\(7.5\\times10^{6}\\) biotin-FITC per secondary bead, 20 guanines per oligonucleotide) bound to the target (E. coli). A single-stranded DNA probe functionalized reduced graphene oxide modified glassy carbon electrode was used to bind the polyGs on the secondary beads. Fluorescent imaging was performed to confirm the hybridization of the complex to the electrode surface. Differential pulse voltammetry (DPV) was used to quantify the amount of polyG involved in the hybridization event with tris(2,2'-bipyridine)ruthenium(II) (Ru(bpy)\\(_{3}^{2+}\\)) as the mediator. The amount of polyG signal can be correlated to the amount of E. coli O157:H7 in the sample. The method was able to detect concentrations of E. coli O157:H7 down to 3 CFU\/100 mL, which is 67 times lower than the most sensitive technique reported in literature. The signal to noise ratio for this work was 3. We also demonstrate the use of the protocol for detection of E. coli O157:H7 seeded in waste water effluent samples.<\/jats:p>","DOI":"10.3390\/s150512034","type":"journal-article","created":{"date-parts":[[2015,5,26]],"date-time":"2015-05-26T08:16:36Z","timestamp":1432628196000},"page":"12034-12052","source":"Crossref","is-referenced-by-count":43,"title":["Highly Sensitive Bacteria Quantification Using Immunomagnetic Separation and Electrochemical Detection of Guanine-Labeled Secondary Beads"],"prefix":"10.3390","volume":"15","author":[{"given":"Harikrishnan","family":"Jayamohan","sequence":"first","affiliation":[{"name":"Department of Mechanical Engineering, University of Utah, Salt Lake City, UT 84112, USA"}]},{"given":"Bruce","family":"Gale","sequence":"additional","affiliation":[{"name":"Department of Mechanical Engineering, University of Utah, Salt Lake City, UT 84112, USA"},{"name":"Espira Inc., 825 N 300 W Suite N-223, Salt Lake City, UT 84103, USA"}]},{"given":"Bj","family":"Minson","sequence":"additional","affiliation":[{"name":"Espira Inc., 825 N 300 W Suite N-223, Salt Lake City, UT 84103, USA"}]},{"given":"Christopher","family":"Lambert","sequence":"additional","affiliation":[{"name":"Department of Bioengineering, University of Utah, Salt Lake City, UT 84112, USA"}]},{"given":"Neil","family":"Gordon","sequence":"additional","affiliation":[{"name":"Guanine Inc., Salt Lake City, UT 84103, USA"}]},{"given":"Himanshu","family":"Sant","sequence":"additional","affiliation":[{"name":"Department of Mechanical Engineering, University of Utah, Salt Lake City, UT 84112, USA"},{"name":"Espira Inc., 825 N 300 W Suite N-223, Salt Lake City, UT 84103, USA"}]}],"member":"1968","published-online":{"date-parts":[[2015,5,22]]},"reference":[{"key":"ref_1","unstructured":"World Health Organization (2014). Water-Related Diseases, WHO."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"394","DOI":"10.1039\/B510888H","article-title":"Simultaneous detection of Escherichia coli O157:H7 and Salmonella Typhimurium using quantum dots as fluorescence labels","volume":"131","author":"Yang","year":"2006","journal-title":"Analyst"},{"key":"ref_3","first-page":"102","article-title":"Enumeration of Escherichia coli and the coliform bacteria","volume":"8","author":"Feng","year":"2002","journal-title":"Bacteriol. Anal. Man."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"174","DOI":"10.1016\/j.bios.2013.01.009","article-title":"Highly sensitive detection of pathogen Escherichia coli O157:H7 by electrochemical impedance spectroscopy","volume":"45","author":"Agusil","year":"2013","journal-title":"Biosens. Bioelectron."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1657","DOI":"10.1111\/j.1365-2672.2007.03405.x","article-title":"Enumeration of Salmonella and Escherichia coli O157:H7 in ground beef, cattle carcass, hide and faecal samples using direct plating methods","volume":"103","author":"Arthur","year":"2007","journal-title":"J. Appl. Microbiol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"3388","DOI":"10.1039\/c3an00056g","article-title":"Solid-state voltammetry-based electrochemical immunosensor for Escherichia coli using graphene oxide\u2013Ag nanoparticle composites as labels","volume":"138","author":"Jiang","year":"2013","journal-title":"Analyst"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"19207","DOI":"10.1073\/pnas.1008768107","article-title":"Electrical detection of pathogenic bacteria via immobilized antimicrobial peptides","volume":"107","author":"Mannoor","year":"2010","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"761","DOI":"10.1016\/j.talanta.2013.06.034","article-title":"Real-time and sensitive detection of Salmonella Typhimurium using an automated quartz crystal microbalance (QCM) instrument with nanoparticles amplification","volume":"115","author":"Salam","year":"2013","journal-title":"Talanta"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"998","DOI":"10.1016\/j.bios.2005.03.007","article-title":"A mixed self-assembled monolayer-based surface plasmon immunosensor for detection of E. coli O157:H7","volume":"21","author":"Subramanian","year":"2006","journal-title":"Biosens. Bioelectron."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"613","DOI":"10.1016\/j.snb.2010.01.007","article-title":"Detection of low levels of Escherichia coli in fresh spinach by surface plasmon resonance spectroscopy with a TMB-based enzymatic signal enhancement method","volume":"145","author":"Linman","year":"2010","journal-title":"Sens. Actuators B Chem."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"22072","DOI":"10.1039\/c2jm33667g","article-title":"Detection of bacteria with organic electrochemical transistors","volume":"22","author":"He","year":"2012","journal-title":"J. Mater. Chem."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"315","DOI":"10.1016\/j.talanta.2012.03.049","article-title":"Electrochemical immunosensor for multiplexed detection of food-borne pathogens using nanocrystal bioconjugates and MWCNT screen-printed electrode","volume":"94","author":"Viswanathan","year":"2012","journal-title":"Talanta"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"3461","DOI":"10.1016\/j.bios.2009.04.042","article-title":"Highly sensitive amperometric immunosensor for the detection of Escherichia coli","volume":"24","author":"Ashkenazi","year":"2009","journal-title":"Biosens. Bioelectron."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"4427","DOI":"10.1039\/c3an00438d","article-title":"Advances in nano-scaled biosensors for biomedical applications","volume":"138","author":"Wang","year":"2013","journal-title":"Analyst"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"4804","DOI":"10.3390\/s90604804","article-title":"Microfluidic systems for pathogen sensing: A review","volume":"9","author":"Mairhofer","year":"2009","journal-title":"Sensors"},{"key":"ref_16","unstructured":"(2014). Beaches and Coasts, Questions and Answers, EPA."},{"key":"ref_17","unstructured":"(2014). CWA Table IA.\u2014List of Approved Biological Test Procedures, American Water Works Association."},{"key":"ref_18","unstructured":"IDEXX Corporation (2014). A Comparison of IDEXX Coliform and E. coli Tests, IDEXX."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"337","DOI":"10.1016\/j.bios.2011.09.029","article-title":"Detection of E. coli O157:H7 by immunomagnetic separation coupled with fluorescence immunoassay","volume":"30","author":"Zhu","year":"2011","journal-title":"Biosens. Bioelectron."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"31","DOI":"10.1017\/S0950268800051438","article-title":"Immunomagnetic separation as a sensitive method for isolating Escherichia coli O157 from food samples","volume":"113","author":"Wright","year":"1994","journal-title":"Epidemiol. Infect."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"99","DOI":"10.1016\/j.snb.2007.03.045","article-title":"A label-free, microfluidics and interdigitated array microelectrode-based impedance biosensor in combination with nanoparticles immunoseparation for detection of Escherichia coli O157:H7 in food samples","volume":"128","author":"Varshney","year":"2007","journal-title":"Sens. Actuators B Chem."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"787","DOI":"10.1016\/j.talanta.2009.05.003","article-title":"Immunomagnetic separation and rapid detection of bacteria using bioluminescence and microfluidics","volume":"79","author":"Qiu","year":"2009","journal-title":"Talanta"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"3011","DOI":"10.1093\/nar\/28.16.3011","article-title":"Survey and summary from DNA biosensors to gene chips","volume":"28","author":"Wang","year":"2000","journal-title":"Nucleic Acids Res."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1957","DOI":"10.1039\/b912653h","article-title":"Electrochemical techniques on sequence-specific PCR amplicon detection for point-of-care applications","volume":"134","author":"Luo","year":"2009","journal-title":"Analyst"},{"key":"ref_25","unstructured":"Jayamohan, H., Sant, H.J., and Gale, B.K. (2013). Microfluidic Diagnostics, Springer."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"15","DOI":"10.3390\/bios2010015","article-title":"Electrochemical biosensor for rapid and sensitive detection of magnetically extracted bacterial pathogens","volume":"2","author":"Setterington","year":"2012","journal-title":"Biosensors"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1884","DOI":"10.1126\/science.1088755","article-title":"Nanoparticle-based bio-bar codes for the ultrasensitive detection of proteins","volume":"301","author":"Nam","year":"2003","journal-title":"Science"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"5932","DOI":"10.1021\/ja049384+","article-title":"Bio-bar-code-based DNA detection with PCR-like sensitivity","volume":"126","author":"Nam","year":"2004","journal-title":"J. Am. Chem. Soc."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"83","DOI":"10.1016\/S1388-2481(02)00542-8","article-title":"Electrochemical detection of DNA hybridization based on DNA-templated assembly of silver cluster","volume":"5","author":"Wang","year":"2003","journal-title":"Electrochem. Commun."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"2158","DOI":"10.1002\/anie.200453832","article-title":"DNA-Based Amplified Bioelectronic Detection and Coding of Proteins","volume":"43","author":"Wang","year":"2004","journal-title":"Angew. Chem. Int. Ed."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"6974","DOI":"10.1021\/ac060809f","article-title":"Sensitive immunoassay of a biomarker tumor necrosis factor-\u03b1 based on poly (guanine)-functionalized silica nanoparticle label","volume":"78","author":"Wang","year":"2006","journal-title":"Anal. Chem."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"2434","DOI":"10.1016\/j.bios.2008.12.023","article-title":"An electrochemical immunoassay for protein based on bio bar code method","volume":"24","author":"Ding","year":"2009","journal-title":"Biosens. Bioelectron."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1736","DOI":"10.1016\/j.bios.2010.08.012","article-title":"A multiplex nanoparticle-based bio-barcoded DNA sensor for the simultaneous detection of multiple pathogens","volume":"26","author":"Zhang","year":"2010","journal-title":"Biosens. Bioelectron."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"5011","DOI":"10.1039\/c3an00606a","article-title":"Electrochemical immunoassay for Salmonella Typhimurium based on magnetically collected Ag-enhanced DNA biobarcode labels","volume":"138","author":"Pratiwi","year":"2013","journal-title":"Analyst"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"267","DOI":"10.1016\/j.snb.2012.01.050","article-title":"Dual amplified, sensitive electrochemical detection of pathogenic sequences based on biobarcode labels and functional graphene modified electrode","volume":"163","author":"Wang","year":"2012","journal-title":"Sens. Actuators B Chem."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"3381","DOI":"10.1002\/ange.200600124","article-title":"Multiplexed DNA detection with biobarcoded nanoparticle probes","volume":"118","author":"Stoeva","year":"2006","journal-title":"Angew. Chem."},{"key":"ref_37","unstructured":"Gordon, N. (2014). Ultra-Sensitive Detection of Extremely Low Level Biological Analytes Using Electrochemical Signal Amplification and Biosensor. USPTO 14\/173,064."},{"key":"ref_38","unstructured":"ATCC (2014). ATCC Bacterial Culture Guide, ATCC."},{"key":"ref_39","unstructured":"Life Technologies (2014). Dynabeads MAX E. Coli O157 Kit, Life Technologies."},{"key":"ref_40","unstructured":"KPL Inc. (2014). Biotin-Labeled BacTrace Anti-E. coli O157:H7 Antibody Datasheet, KPL."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"133","DOI":"10.1016\/j.elecom.2010.11.033","article-title":"Direct electrodeposition of reduced graphene oxide on glassy carbon electrode and its electrochemical application","volume":"13","author":"Chen","year":"2011","journal-title":"Electrochem. Commun."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"370","DOI":"10.1016\/j.snb.2013.05.018","article-title":"Microfluidic integrated multi-walled carbon nanotube (MWCNT) sensor for electrochemical nucleic acid concentration measurement","volume":"185","author":"Kim","year":"2013","journal-title":"Sens. Actuators B Chem."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"14725","DOI":"10.1039\/c1jm12028j","article-title":"Graphene oxide for electrochemical sensing applications","volume":"21","author":"Roy","year":"2011","journal-title":"J. Mater. Chem."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"2989","DOI":"10.1021\/ac100036p","article-title":"Sensitive immunosensor for cancer biomarker based on dual signal amplification strategy of graphene sheets and multienzyme functionalized carbon nanospheres","volume":"82","author":"Du","year":"2010","journal-title":"Anal. Chem."},{"key":"ref_45","unstructured":"Wang, J. (2006). Analytical Electrochemistry, John Wiley & Sons."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"3249","DOI":"10.1039\/c2lc40630f","article-title":"Microfluidic designs and techniques using lab-on-a-chip devices for pathogen detection for point-of-care diagnostics","volume":"12","author":"Foudeh","year":"2012","journal-title":"Lab Chip"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"2768","DOI":"10.1039\/c0an00590h","article-title":"Graphene electrochemistry: An overview of potential applications","volume":"135","author":"Brownson","year":"2010","journal-title":"Analyst"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"272","DOI":"10.1002\/elan.201000425","article-title":"Electrochemical Monitoring of Nucleic Acid Hybridization by Single-Use Graphene Oxide-Based Sensor","volume":"23","author":"Muti","year":"2011","journal-title":"Electroanalysis"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"543","DOI":"10.1016\/j.colsurfb.2010.10.014","article-title":"Novel electrochemical sensor based on functionalized graphene for simultaneous determination of adenine and guanine in DNA","volume":"82","author":"Huang","year":"2011","journal-title":"Colloids Surf. B Biointerfaces"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"1668","DOI":"10.1002\/chem.201102850","article-title":"Nucleic acid functionalized graphene for biosensing","volume":"18","author":"Bonanni","year":"2012","journal-title":"Chem. A Eur. J."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"10763","DOI":"10.1002\/chem.201101117","article-title":"Electrochemistry at chemically modified graphenes","volume":"17","author":"Ambrosi","year":"2011","journal-title":"Chem. A Eur. J."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"1339","DOI":"10.1021\/ja01539a017","article-title":"Preparation of graphitic oxide","volume":"80","author":"Offeman","year":"1958","journal-title":"J. Am. Chem. Soc."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"8933","DOI":"10.1021\/ja00140a006","article-title":"Electrochemical measurement of the solvent accessibility of nucleobases using electron transfer between DNA and metal complexes","volume":"117","author":"Johnston","year":"1995","journal-title":"J. Am. Chem. Soc."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"4044","DOI":"10.1021\/ac020221i","article-title":"Square wave voltammetric detection of chemical DNA damage with catalytic poly (4-vinylpyridine)-Ru (bpy) 22+ films","volume":"74","author":"Mugweru","year":"2002","journal-title":"Anal. Chem."},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"597","DOI":"10.1021\/nl0340677","article-title":"Carbon nanotube nanoelectrode array for ultrasensitive DNA detection","volume":"3","author":"Li","year":"2003","journal-title":"Nano Lett."},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"1239","DOI":"10.1088\/0957-4484\/14\/12\/001","article-title":"Ultrasensitive label-free DNA analysis using an electronic chip based on carbon nanotube nanoelectrode arrays","volume":"14","author":"Koehne","year":"2003","journal-title":"Nanotechnology"},{"key":"ref_57","unstructured":"Thorp, H.H. (2004). Long-Range Charge Transfer in DNA II, Springer."}],"container-title":["Sensors"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1424-8220\/15\/5\/12034\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,6,3]],"date-time":"2024-06-03T10:35:52Z","timestamp":1717410952000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1424-8220\/15\/5\/12034"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2015,5,22]]},"references-count":57,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2015,5]]}},"alternative-id":["s150512034"],"URL":"https:\/\/doi.org\/10.3390\/s150512034","relation":{},"ISSN":["1424-8220"],"issn-type":[{"value":"1424-8220","type":"electronic"}],"subject":[],"published":{"date-parts":[[2015,5,22]]}}}