Harnessing aptamers for electrochemical detection of endotoxin.

Lipopolysaccharide (LPS), also known as endotoxin, triggers a fatal septic shock; therefore, fast and accurate detection of LPS from a complex milieu is of primary importance. Several LPS affinity binders have been reported so far but few of them have proved their efficacy in developing electrochemical sensors capable of selectively detecting LPS from crude biological liquors. In this study, we identified 10 different single-stranded DNA aptamers showing specific affinity to LPS with dissociation constants (K(d)) in the nanomolar range using a NECEEM-based non-SELEX method. Based on the sequence and secondary structure analysis of the LPS binding aptamers, an aptamer exhibiting the highest affinity to LPS (i.e., B2) was selected to construct an impedance biosensor on a gold surface. The developed electrochemical aptasensor showed excellent sensitivity and specificity in the linear detection range from 0.01 to 1 ng/mL of LPS with significantly reduced detection time compared with the traditional Limulus amoebocyte lysate (LAL) assay.

[1]  Byung-Gee Kim,et al.  Screening of LPS-specific peptides from a phage display library using epoxy beads. , 2005, Biochemical and biophysical research communications.

[2]  J. Szostak,et al.  In vitro selection of RNA molecules that bind specific ligands , 1990, Nature.

[3]  L. Gold,et al.  Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. , 1990, Science.

[4]  J. Bruno,et al.  In Vitro antibacterial effects of antilipopolysaccharide DNA aptamer-C1qrs complexes , 2008, Folia Microbiologica.

[5]  A. Ellington,et al.  Dissecting protein:protein interactions between transcription factors with an RNA aptamer. , 1995, RNA.

[6]  Jian-Ding Qiu,et al.  A label-free amperometric immunosensor based on biocompatible conductive redox chitosan-ferrocene/gold nanoparticles matrix. , 2009, Biosensors & bioelectronics.

[7]  Michael Musheev,et al.  Nonequilibrium capillary electrophoresis of equilibrium mixtures: a universal tool for development of aptamers. , 2005, Journal of the American Chemical Society.

[8]  Sergey N Krylov,et al.  Non-SELEX: selection of aptamers without intermediate amplification of candidate oligonucleotides , 2006, Nature Protocols.

[9]  L. Gold,et al.  The mathematics of SELEX against complex targets. , 1998, Journal of molecular biology.

[10]  Qing-Wu Yang,et al.  A novel lipopolysaccharide-antagonizing aptamer protects mice against endotoxemia. , 2009, Biochemical and biophysical research communications.

[11]  Bansi D. Malhotra,et al.  Electrochemical DNA sensor for Neisseria meningitidis detection. , 2010, Biosensors & bioelectronics.

[12]  T. Tosa,et al.  Characteristics of immobilized histamine for pyrogen adsorption. , 1983, Journal of chromatography.

[13]  T. M. Herne,et al.  Characterization of DNA Probes Immobilized on Gold Surfaces , 1997 .

[14]  R. Ulevitch,et al.  A new method for reduction of endotoxin contamination from protein solutions. , 1987, Journal of immunological methods.

[15]  F. B. Anspach,et al.  Removal of endotoxins by affinity sorbents. , 1995, Journal of chromatography. A.

[16]  J. Nolan,et al.  Endotoxin Binding by Charged and Uncharged Resins 1 , 1975, Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine.

[17]  Michael Zuker,et al.  Mfold web server for nucleic acid folding and hybridization prediction , 2003, Nucleic Acids Res..

[18]  Woochang Lee,et al.  Immobilization of antibody fragment for immunosensor application based on surface plasmon resonance. , 2005, Colloids and surfaces. B, Biointerfaces.

[19]  M. Apicella,et al.  The lipooligosaccharides of pathogenic gram-negative bacteria. , 1996, Critical reviews in microbiology.

[20]  M. Berezovski,et al.  Kinetic capillary electrophoresis (KCE): a conceptual platform for kinetic homogeneous affinity methods. , 2005, Journal of the American Chemical Society.

[21]  R. Malone,et al.  Improved method for the removal of endotoxin from DNA. , 1996, Journal of biotechnology.

[22]  B. Ho,et al.  Single-Stranded DNA Oligoaptamers: Molecular Recognition and LPS Antagonism Are Length- and Secondary Structure-Dependent , 2008, Journal of Innate Immunity.

[23]  Victor Okhonin,et al.  Selection of smart aptamers by methods of kinetic capillary electrophoresis. , 2006, Analytical chemistry.

[24]  L. Gold,et al.  Selective enrichment of RNA species for tight binding to Escherichia coli rho factor , 1993, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[25]  M. Famulok,et al.  Nucleic acid aptamers-from selection in vitro to applications in vivo. , 2000, Accounts of chemical research.

[26]  A. Issekutz.  Removal of gram-negative endotoxin from solutions by affinity chromatography. , 1983, Journal of immunological methods.

[27]  J. Szostak,et al.  In vitro selection of functional nucleic acids. , 1999, Annual review of biochemistry.

[28]  M. Pumera,et al.  New materials for electrochemical sensing VI: Carbon nanotubes , 2005 .

[29]  I-Ming Hsing,et al.  Label-free protein recognition using an aptamer-based impedance measurement assay , 2006 .

[30]  P. He,et al.  An Aptamer-Based Protein Biosensor by Detecting the Amplified Impedance Signal , 2006 .

[31]  Michael Musheev,et al.  Non-SELEX selection of aptamers. , 2006, Journal of the American Chemical Society.

[32]  M. Shin,et al.  Monolith-based immobilized metal affinity chromatography increases production efficiency for plasmid DNA purification. , 2011, Journal of chromatography. A.

[33]  Youngkwan Lee,et al.  Synthesis, characterization and self-assembled film of poly(3-((2,3-dihydrothieno[3,4-b][1,4]dioxin-2-yl)methoxy)propane-1-thiol) (PEDTMSHA) , 2010 .

[34]  G. Marrazza,et al.  Carbon and gold electrodes as electrochemical transducers for DNA hybridisation sensors. , 2004, Biosensors & bioelectronics.

[35]  L. Gold,et al.  Selection of high-affinity RNA ligands to reverse transcriptase: inhibition of cDNA synthesis and RNase H activity. , 1994, Biochemistry.

[36]  V. Lakshminarayanan,et al.  Cyclic voltammetric and electrochemical impedance studies on the structure, adsorption kinetics, and barrier properties of some organic dithiol self-assembled monolayers on gold. , 2003, Journal of colloid and interface science.

[37]  R. Ulevitch,et al.  Receptor-dependent mechanisms of cell stimulation by bacterial endotoxin. , 1995, Annual review of immunology.

[38]  F. Battaglini,et al.  Electronic tongue for simultaneous detection of endotoxins and other contaminants of microbiological origin. , 2010, Biosensors & bioelectronics.

[39]  I. Wicks,et al.  Bacterial lipopolysaccharide copurifies with plasmid DNA: implications for animal models and human gene therapy. , 1995, Human gene therapy.