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Analytica Chimica Acta (v.578, #1)
A galactose polyacrylate-based hydrogel scaffold for the detection of cholera toxin and staphylococcal enterotoxin B in a sandwich immunoassay format by Paul T. Charles; Freddie Velez; Carissa M. Soto; Ellen R. Goldman; Brett D. Martin; Richard I. Ray; Chris R. Taitt (pp. 2-10).
A galactoside-based polyacrylate hydrogel was used as a scaffold to immobilize antibodies for the development of a sandwich immunoassay to detect cholera toxin (CT) and staphylococcal enterotoxin B (SEB). The hydrogel possesses large pores and simulates a solution-like environment allowing easy penetration of large biomolecules. Highly crosslinked hydrogels containing pendant amine or carboxyl functionalities were polymerized through a free-radical polymerization process. Covalent crosslinking of the antibodies on hydrogel films was accomplished using a homobifunctional crosslinker or carbodiimide chemistry. Utilizing the two different crosslinking methodologies, our results demonstrated the effectiveness of repetitive additions of crosslinker reactant into a single location on the gel surface. This approach in fact increased the amount of immobilized antibody. Patterned arrays of the immobilized antibodies for sandwich immunoassay development were achieved using a PDMS template containing micro-channels. This template provided a suitable means for applying reagents in multiple cycles. Fluorescence and three-dimensional (3D) imaging by confocal microscopy and laser scanning confocal microscopy of Cy3-labeled anti-CT and/or Cy3-anti-SEB tracer molecules provided qualitative and quantitative measurements on the efficiency of protein immobilization, detection sensitivity and signal-to-noise ratios. As a result of using the galactose polyacrylate-base hydrogel as a platform for immunoassay development, we have successfully been able to achieve low limits of detection for SEB and cholera toxins (1.0ngmL−1). Repetitive additions (>3 cycles) of the crosslinker and antibody have also shown a dramatic increase in the immobilization of antibody resulting in improved immunoassay sensitivity. Fluorescence signal-to-noise ratios using the hydrogel-based immunoassays have been observed as high a 40:1.
Keywords: Hydrogel; Toxin; Fluorescence; Antibody; Protein; Array
A galactose polyacrylate-based hydrogel scaffold for the detection of cholera toxin and staphylococcal enterotoxin B in a sandwich immunoassay format by Paul T. Charles; Freddie Velez; Carissa M. Soto; Ellen R. Goldman; Brett D. Martin; Richard I. Ray; Chris R. Taitt (pp. 2-10).
A galactoside-based polyacrylate hydrogel was used as a scaffold to immobilize antibodies for the development of a sandwich immunoassay to detect cholera toxin (CT) and staphylococcal enterotoxin B (SEB). The hydrogel possesses large pores and simulates a solution-like environment allowing easy penetration of large biomolecules. Highly crosslinked hydrogels containing pendant amine or carboxyl functionalities were polymerized through a free-radical polymerization process. Covalent crosslinking of the antibodies on hydrogel films was accomplished using a homobifunctional crosslinker or carbodiimide chemistry. Utilizing the two different crosslinking methodologies, our results demonstrated the effectiveness of repetitive additions of crosslinker reactant into a single location on the gel surface. This approach in fact increased the amount of immobilized antibody. Patterned arrays of the immobilized antibodies for sandwich immunoassay development were achieved using a PDMS template containing micro-channels. This template provided a suitable means for applying reagents in multiple cycles. Fluorescence and three-dimensional (3D) imaging by confocal microscopy and laser scanning confocal microscopy of Cy3-labeled anti-CT and/or Cy3-anti-SEB tracer molecules provided qualitative and quantitative measurements on the efficiency of protein immobilization, detection sensitivity and signal-to-noise ratios. As a result of using the galactose polyacrylate-base hydrogel as a platform for immunoassay development, we have successfully been able to achieve low limits of detection for SEB and cholera toxins (1.0ngmL−1). Repetitive additions (>3 cycles) of the crosslinker and antibody have also shown a dramatic increase in the immobilization of antibody resulting in improved immunoassay sensitivity. Fluorescence signal-to-noise ratios using the hydrogel-based immunoassays have been observed as high a 40:1.
Keywords: Hydrogel; Toxin; Fluorescence; Antibody; Protein; Array
Colorimetric multiplexed immunoassay using specific aggregation of antigenic peptide-modified luminous nanoparticles by Toshihiro Ihara; Yasunori Mori; Takaaki Imamura; Motoko Mukae; Shojiro Tanaka; Akinori Jyo (pp. 11-18).
A rapid immunoassay capable of detecting specific antibodies in one-step procedure is described. Antigenic peptides with cationic (KKKKC) or anionic (DDDDC) pentamer tail were immobilized on luminous nanospheres of 40nm diameter (Ø) through cystamine and bifunctional linker molecules under various conditions. The numbers of each peptide anchored to a sphere were 5.0×102 and 0.8–3.8×103, respectively. A mixture of the antigenic peptides of FAK and c-Myc was immobilized to the spheres with red emission, while that of c-Myc and α-catenin was likewise to green spheres. Multiplexed immunoassay was easily achieved by adding the antibodies to a mixed dispersed solution of these spheres under appropriate conditions. Anti-FAK and anti-α-catenin antibodies formed aggregates with red and green emissions, respectively. On the other hand, the anti-c-Myc antibody formed aggregates emitting a yellow light. This system enabled us to differentiate three antibodies in one vessel from the definite differences in aggregate color.
Keywords: Multiplexed immunoassay; Oligopeptides-modified nanosphere; Fluorescence microscopy; Specific aggregation; Crosslinking; Cystamine
Colorimetric multiplexed immunoassay using specific aggregation of antigenic peptide-modified luminous nanoparticles by Toshihiro Ihara; Yasunori Mori; Takaaki Imamura; Motoko Mukae; Shojiro Tanaka; Akinori Jyo (pp. 11-18).
A rapid immunoassay capable of detecting specific antibodies in one-step procedure is described. Antigenic peptides with cationic (KKKKC) or anionic (DDDDC) pentamer tail were immobilized on luminous nanospheres of 40nm diameter (Ø) through cystamine and bifunctional linker molecules under various conditions. The numbers of each peptide anchored to a sphere were 5.0×102 and 0.8–3.8×103, respectively. A mixture of the antigenic peptides of FAK and c-Myc was immobilized to the spheres with red emission, while that of c-Myc and α-catenin was likewise to green spheres. Multiplexed immunoassay was easily achieved by adding the antibodies to a mixed dispersed solution of these spheres under appropriate conditions. Anti-FAK and anti-α-catenin antibodies formed aggregates with red and green emissions, respectively. On the other hand, the anti-c-Myc antibody formed aggregates emitting a yellow light. This system enabled us to differentiate three antibodies in one vessel from the definite differences in aggregate color.
Keywords: Multiplexed immunoassay; Oligopeptides-modified nanosphere; Fluorescence microscopy; Specific aggregation; Crosslinking; Cystamine
Development of a chemiluminescent immunosensor for chloramphenicol by In-Seon Park; Namsoo Kim (pp. 19-24).
A direct competitive chemiluminescent immunosensor system that exploits the competition between chloramphenicol (CAP) as an analyte and CAP–horseradish peroxidase conjugate as a tracer for binding to an anti-CAP antibody on a solid support was devised by installing a flow-through cell which was connected to an injector and a peristaltic pump inside a dark box, followed by positioning a photomultiplier tube as light detector in front of it. The anti-CAP antibody was immobilized onto positively charged Biodyne B membrane pieces by a dipping procedure. The operating conditions for the immunosensor were selected with respect to substrate composition (0.25, 13.3 and 0.66mM for luminol, H2O2 and p-iodophenol, respectively), injection volume of the substrate solution (200μL) and the concentrations of antibody for immobilization (0.10mgmL−1) and tracer (0.030mgmL−1). At these conditions, sensor response according to analyte concentration was well fitted to a linear equation when plotted in semi-logarithmic scale, with the limit of detection for CAP of 10−8M. By using the immunosensor, CAP measurement in the model samples prepared from five food materials was conducted.
Keywords: Direct competitive chemiluminescent immunosensor; Chloramphenicol detection; System optimization; Model sample analysis
Development of a chemiluminescent immunosensor for chloramphenicol by In-Seon Park; Namsoo Kim (pp. 19-24).
A direct competitive chemiluminescent immunosensor system that exploits the competition between chloramphenicol (CAP) as an analyte and CAP–horseradish peroxidase conjugate as a tracer for binding to an anti-CAP antibody on a solid support was devised by installing a flow-through cell which was connected to an injector and a peristaltic pump inside a dark box, followed by positioning a photomultiplier tube as light detector in front of it. The anti-CAP antibody was immobilized onto positively charged Biodyne B membrane pieces by a dipping procedure. The operating conditions for the immunosensor were selected with respect to substrate composition (0.25, 13.3 and 0.66mM for luminol, H2O2 and p-iodophenol, respectively), injection volume of the substrate solution (200μL) and the concentrations of antibody for immobilization (0.10mgmL−1) and tracer (0.030mgmL−1). At these conditions, sensor response according to analyte concentration was well fitted to a linear equation when plotted in semi-logarithmic scale, with the limit of detection for CAP of 10−8M. By using the immunosensor, CAP measurement in the model samples prepared from five food materials was conducted.
Keywords: Direct competitive chemiluminescent immunosensor; Chloramphenicol detection; System optimization; Model sample analysis
Identification of P-glycoprotein substrates using open tubular chromatography on an immobilized P-glycoprotein column: Comparison of chromatographic results with Caco-2 permeability by Ruin Moaddel; Rachid Hamid; Sharvil Patel; Peter L. Bullock; Irving W. Wainer (pp. 25-30).
The Caco-2 cell monolayer model was used to classify 13 compounds as P-glycoprotein ( Pgp) substrates or non-substrates. The apparent permeability coefficients (Papp) in the basal-to-apical direction (PappB–A) and in the apical-to-basal direction (PappA–B) were determined for each compound and a compound was designated as a Pgp substrate if PappB–A/PappA–B, the permeability ratio, exceeded 2.0. The same compounds were chromatographed on open tubular glass columns containing membranes from cell lines that either expressed Pgp ( Pgp(+)-OT column) or did not express Pgp ( Pgp(−)-OT column). The differential retentions in min, Δ t values, of the compounds were determined using the following relationship Δ t= t( Pgp(+)-OT)− t( Pgp(−)-OT). A statistically significant correlation was observed between the Δ t values and the permeability ratios, r2=0.7749 ( p=0.0063), indicating that the differential chromatography approach could be used to quantitatively assess permeability ratios. The results also indicated that a Δ t value ≥0.5min was a reliable measure of a permeability ratios >2 and could be used as a rapid qualitative determination of whether a test compound was a Pgp substrate. The chromatographic study took 1h to complete and a single pair of columns could be used to screen at least 150 compounds a week and 600 compounds during the 4-week lifetime of the columns.
Keywords: Online screening; Drug absorption; Affinity chromatography
Identification of P-glycoprotein substrates using open tubular chromatography on an immobilized P-glycoprotein column: Comparison of chromatographic results with Caco-2 permeability by Ruin Moaddel; Rachid Hamid; Sharvil Patel; Peter L. Bullock; Irving W. Wainer (pp. 25-30).
The Caco-2 cell monolayer model was used to classify 13 compounds as P-glycoprotein ( Pgp) substrates or non-substrates. The apparent permeability coefficients (Papp) in the basal-to-apical direction (PappB–A) and in the apical-to-basal direction (PappA–B) were determined for each compound and a compound was designated as a Pgp substrate if PappB–A/PappA–B, the permeability ratio, exceeded 2.0. The same compounds were chromatographed on open tubular glass columns containing membranes from cell lines that either expressed Pgp ( Pgp(+)-OT column) or did not express Pgp ( Pgp(−)-OT column). The differential retentions in min, Δ t values, of the compounds were determined using the following relationship Δ t= t( Pgp(+)-OT)− t( Pgp(−)-OT). A statistically significant correlation was observed between the Δ t values and the permeability ratios, r2=0.7749 ( p=0.0063), indicating that the differential chromatography approach could be used to quantitatively assess permeability ratios. The results also indicated that a Δ t value ≥0.5min was a reliable measure of a permeability ratios >2 and could be used as a rapid qualitative determination of whether a test compound was a Pgp substrate. The chromatographic study took 1h to complete and a single pair of columns could be used to screen at least 150 compounds a week and 600 compounds during the 4-week lifetime of the columns.
Keywords: Online screening; Drug absorption; Affinity chromatography
Capillary electrophoresis for capture and concentrating of target nucleic acids by affinity gels modified to contain single-stranded nucleic acid probes by Andrew Chan; Ulrich J. Krull (pp. 31-42).
Selective capture and pre-concentration of target nucleic acids from relatively complicated samples may provide a method to facilitate introduction to a microfluidic-based detection system to improve detection limits. An acrylamide polymer gel modified with Acrydite™ that contained 20mer oligonucleotide probe was prepared and loaded into a capillary column. The results indicated that the amount of probe DNA that was captured into the acrylamide was about 40% of the starting monomer, and different quantities of probe could therefore be coupled into the gel. The gel was passivated by pre-treatment with non-complementary DNA oligonucleotide to block non-selective adsorption sites, and the gel was determined to be stable for multiple cycles of use. The probe could hybridize with target sequences that were introduced by electrokinetic injection from a sample solution. The target could be freed from the polymer gel by use of a combination of heating, chaotropic salt and voltage conditions. Target capture efficiency was up to 90% when using samples that did not saturate probe sites in the columns, and recovery of target from the gel could be as high as 95%.
Keywords: Acrylamide; Capillary electrophoresis; Selective capture; Oligonucleotides
Capillary electrophoresis for capture and concentrating of target nucleic acids by affinity gels modified to contain single-stranded nucleic acid probes by Andrew Chan; Ulrich J. Krull (pp. 31-42).
Selective capture and pre-concentration of target nucleic acids from relatively complicated samples may provide a method to facilitate introduction to a microfluidic-based detection system to improve detection limits. An acrylamide polymer gel modified with Acrydite™ that contained 20mer oligonucleotide probe was prepared and loaded into a capillary column. The results indicated that the amount of probe DNA that was captured into the acrylamide was about 40% of the starting monomer, and different quantities of probe could therefore be coupled into the gel. The gel was passivated by pre-treatment with non-complementary DNA oligonucleotide to block non-selective adsorption sites, and the gel was determined to be stable for multiple cycles of use. The probe could hybridize with target sequences that were introduced by electrokinetic injection from a sample solution. The target could be freed from the polymer gel by use of a combination of heating, chaotropic salt and voltage conditions. Target capture efficiency was up to 90% when using samples that did not saturate probe sites in the columns, and recovery of target from the gel could be as high as 95%.
Keywords: Acrylamide; Capillary electrophoresis; Selective capture; Oligonucleotides
High sensitivity detection of bisphenol A using liposome chromatography by Xue-Ying Liu; Chikashi Nakamura; Itsuro Tanimoto; Shiro Miyake; Noriyuki Nakamura; Takashi Hirano; Jun Miyake (pp. 43-49).
An antibody column in tandem with a fluorescent dye entrapped liposome column was developed for highly sensitive detection of an endocrine disruptor, bisphenol A (BPA). Anti-BPA antibody was immobilized in a protein G column with orientation control. A derivative of BPA was conjugated to phospholipase A2 (PLA2). BPA sample solutions mixed with the BPA-PLA2 conjugates were injected on to the anti-BPA antibody column and competitive binding occurred in the antibody column. The amount of the free conjugate was proportional to the concentration of the BPA sample. The eluted conjugates were injected on to the second column gel on which calcein-entrapped liposomes were immobilized and the PLA2-catalyzed hydrolysis of liposomal phospholipids causing fluorescent dye leakage as a signal amplification. In this system, the mixture of BPA and BPA-PLA2 conjugate were incubated for 60min in the anti-BPA column, and then the collected solution was applied to the liposome column. The BPA detection range of 0.02–140ngmL−1 was wider than 0.03–6.6ngmL−1 obtained by the method of competitive ELISA using the same antibody. Moreover, this system could be adapted to an HPLC system resulting in almost the same detection limit in online detection. The method could be applied to environmental samples, river water and soil extracts. The BPA concentration of 0.1ngmL−1 and 10ngg−1 was detectable in water and soil extract, respectively.
Keywords: Abbreviations; BPA; bisphenol A; calcein; 3,3-bis(; N; ,; N; -di(carboxymethyl)aminomethyl) fluorescein; DMSO; dimethyl sulfoxide; EPC; egg yolk phosphatidylcholine; HEPES; 2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid; ILC; immobilized liposome chromatography; PLA; 2; phospholipase A; 2Bisphenol A detection; Fluorescent; Liposome column; Antibody column; Phospholipase A; 2; Conjugate
High sensitivity detection of bisphenol A using liposome chromatography by Xue-Ying Liu; Chikashi Nakamura; Itsuro Tanimoto; Shiro Miyake; Noriyuki Nakamura; Takashi Hirano; Jun Miyake (pp. 43-49).
An antibody column in tandem with a fluorescent dye entrapped liposome column was developed for highly sensitive detection of an endocrine disruptor, bisphenol A (BPA). Anti-BPA antibody was immobilized in a protein G column with orientation control. A derivative of BPA was conjugated to phospholipase A2 (PLA2). BPA sample solutions mixed with the BPA-PLA2 conjugates were injected on to the anti-BPA antibody column and competitive binding occurred in the antibody column. The amount of the free conjugate was proportional to the concentration of the BPA sample. The eluted conjugates were injected on to the second column gel on which calcein-entrapped liposomes were immobilized and the PLA2-catalyzed hydrolysis of liposomal phospholipids causing fluorescent dye leakage as a signal amplification. In this system, the mixture of BPA and BPA-PLA2 conjugate were incubated for 60min in the anti-BPA column, and then the collected solution was applied to the liposome column. The BPA detection range of 0.02–140ngmL−1 was wider than 0.03–6.6ngmL−1 obtained by the method of competitive ELISA using the same antibody. Moreover, this system could be adapted to an HPLC system resulting in almost the same detection limit in online detection. The method could be applied to environmental samples, river water and soil extracts. The BPA concentration of 0.1ngmL−1 and 10ngg−1 was detectable in water and soil extract, respectively.
Keywords: Abbreviations; BPA; bisphenol A; calcein; 3,3-bis(; N; ,; N; -di(carboxymethyl)aminomethyl) fluorescein; DMSO; dimethyl sulfoxide; EPC; egg yolk phosphatidylcholine; HEPES; 2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid; ILC; immobilized liposome chromatography; PLA; 2; phospholipase A; 2Bisphenol A detection; Fluorescent; Liposome column; Antibody column; Phospholipase A; 2; Conjugate
Capturing molecules with templated materials—Analysis and rational design of molecularly imprinted polymers by Shuting Wei; Michael Jakusch; Boris Mizaikoff (pp. 50-58).
The creation of synthetic tailor-made receptors capable of recognizing desired molecular targets with high affinity and selectivity is a persistent long-term goal for researchers in the fields of chemical, biological, and pharmaceutical research. Compared to biomacromolecular receptors, these synthetic receptors promise simplified production and processing, less costs, and more robust receptor architectures. During recent decades, molecularly imprinted polymers (MIPs) are widely considered mimics of natural molecular receptors suitable for a diversity of applications ranging from biomimetic sensors, to separations and biocatalysis.A remaining challenge for the next generation of MIPs is the synthesis of deliberately designed and highly efficient receptor architectures suitable for recognizing biologically relevant molecules, for which natural receptors are either not prevalent, or difficult to isolate and utilize. Hence, this review discusses recent advances in synthetic receptor technology for biomolecules (e.g. drugs, amino acids, steroids, proteins, entire cells, etc.) via molecular imprinting techniques. Surface imprinting methods and epitope imprinting approaches have been introduced for protein recognition at imprinted surfaces. Imprinting techniques in aqueous solution or organic-water co-solvents have been introduced avoiding denaturation of biomolecules during MIP synthesis. In addition, improved bioreactivity of entire enzyme or active site mimics generated by molecular imprinting will be highlighted. Finally, the emerging importance of molecular modeling and molecular dynamics studies detailing the intermolecular interactions between the template species, the porogenic solvent molecules, and the involved monomer and cross-linker in the pre-polymerization solution will be addressed yielding a rational approach toward next-generation MIP technology.
Keywords: Molecularly imprinted polymers (MIPs); Biomolecular recognition; Biomimetic receptors; Protein recognition; Synthetic enzymes; Rational design; Molecular modeling
Capturing molecules with templated materials—Analysis and rational design of molecularly imprinted polymers by Shuting Wei; Michael Jakusch; Boris Mizaikoff (pp. 50-58).
The creation of synthetic tailor-made receptors capable of recognizing desired molecular targets with high affinity and selectivity is a persistent long-term goal for researchers in the fields of chemical, biological, and pharmaceutical research. Compared to biomacromolecular receptors, these synthetic receptors promise simplified production and processing, less costs, and more robust receptor architectures. During recent decades, molecularly imprinted polymers (MIPs) are widely considered mimics of natural molecular receptors suitable for a diversity of applications ranging from biomimetic sensors, to separations and biocatalysis.A remaining challenge for the next generation of MIPs is the synthesis of deliberately designed and highly efficient receptor architectures suitable for recognizing biologically relevant molecules, for which natural receptors are either not prevalent, or difficult to isolate and utilize. Hence, this review discusses recent advances in synthetic receptor technology for biomolecules (e.g. drugs, amino acids, steroids, proteins, entire cells, etc.) via molecular imprinting techniques. Surface imprinting methods and epitope imprinting approaches have been introduced for protein recognition at imprinted surfaces. Imprinting techniques in aqueous solution or organic-water co-solvents have been introduced avoiding denaturation of biomolecules during MIP synthesis. In addition, improved bioreactivity of entire enzyme or active site mimics generated by molecular imprinting will be highlighted. Finally, the emerging importance of molecular modeling and molecular dynamics studies detailing the intermolecular interactions between the template species, the porogenic solvent molecules, and the involved monomer and cross-linker in the pre-polymerization solution will be addressed yielding a rational approach toward next-generation MIP technology.
Keywords: Molecularly imprinted polymers (MIPs); Biomolecular recognition; Biomimetic receptors; Protein recognition; Synthetic enzymes; Rational design; Molecular modeling
Prospects of conducting polymers in biosensors by Bansi D. Malhotra; Asha Chaubey; S.P. Singh (pp. 59-74).
Applications of conducting polymers to biosensors have recently aroused much interest. This is because these molecular electronic materials offer control of different parameters such as polymer layer thickness, electrical properties and bio-reagent loading, etc. Moreover, conducting polymer based biosensors are likely to cater to the pressing requirements such as biocompatibility, possibility of in vivo sensing, continuous monitoring of drugs or metabolites, multi-parametric assays, miniaturization and high information density. This paper deals with the emerging trends in conducting polymer based biosensors during the last about 5 years.
Keywords: Conducting polymer; Biosesnor; Soliton; Polaron; Bipolaron; Enzyme; Glucose oxidase; Urease; Cholesterol oxidase; Deoxyribonucleic acid (DNA); Peptide nucleic acid (PNA); Microorganism; Amperometric; Potentiometric; Immunosensor
Prospects of conducting polymers in biosensors by Bansi D. Malhotra; Asha Chaubey; S.P. Singh (pp. 59-74).
Applications of conducting polymers to biosensors have recently aroused much interest. This is because these molecular electronic materials offer control of different parameters such as polymer layer thickness, electrical properties and bio-reagent loading, etc. Moreover, conducting polymer based biosensors are likely to cater to the pressing requirements such as biocompatibility, possibility of in vivo sensing, continuous monitoring of drugs or metabolites, multi-parametric assays, miniaturization and high information density. This paper deals with the emerging trends in conducting polymer based biosensors during the last about 5 years.
Keywords: Conducting polymer; Biosesnor; Soliton; Polaron; Bipolaron; Enzyme; Glucose oxidase; Urease; Cholesterol oxidase; Deoxyribonucleic acid (DNA); Peptide nucleic acid (PNA); Microorganism; Amperometric; Potentiometric; Immunosensor
Electronic deoxyribonucleic acid (DNA) microarray detection of viable pathogenic Escherichia coli, Vibrio cholerae, and Salmonella typhi by Yanming Liu; Zhilong Gong; Nicole Morin; Odell Pui; Michael Cheung; Hai Zhang; Xing-Fang Li (pp. 75-81).
An electronic deoxyribonucleic acid (DNA) microarray technique was developed for detection and identification of viable Escherichia coli O157:H7, Vibrio cholerae O1, and Salmonella typhi. Four unique genes, the E. coli O157 lipopolysaccharide (LPS) gene ( rfbE) and H7 flagellin gene ( fliC), the V. cholerae O1 LPS gene ( rfbE), and the S. typhi LPS gene ( tyv), were chosen as the targets for detection. These targets were selectively amplified from mRNA of viable cells using reverse transcription polymerase chain reaction (RT-PCR) and detected using the electronic DNA microarray technique. Specific captures and reporters were designed and examined for selective detection and correct identification of the target pathogens. The technique was able to detect as few as 2–150 cells of E. coli O157:H7. The co-presence of six other common bacteria and a parasite at 10- and 1000-fold higher concentrations than the target E. coli O157:H7 did not interfere with the specific detection. Comparative analysis of live and heat-killed E. coli O157:H7 cells showed that the technique only responded to the viable cells and not to the dead cells. Thus, the integration of RT-PCR of specific mRNA with the electronic DNA microarray technique enables specific and sensitive detection of viable target cells. This technique is potentially useful for high throughput screening of multiple pathogenic bacteria in different samples.
Keywords: Deoxyribonucleic acid (DNA) microarray; Escherichia coli; O157:H7; Viability; Pathogen; Detection
Electronic deoxyribonucleic acid (DNA) microarray detection of viable pathogenic Escherichia coli, Vibrio cholerae, and Salmonella typhi by Yanming Liu; Zhilong Gong; Nicole Morin; Odell Pui; Michael Cheung; Hai Zhang; Xing-Fang Li (pp. 75-81).
An electronic deoxyribonucleic acid (DNA) microarray technique was developed for detection and identification of viable Escherichia coli O157:H7, Vibrio cholerae O1, and Salmonella typhi. Four unique genes, the E. coli O157 lipopolysaccharide (LPS) gene ( rfbE) and H7 flagellin gene ( fliC), the V. cholerae O1 LPS gene ( rfbE), and the S. typhi LPS gene ( tyv), were chosen as the targets for detection. These targets were selectively amplified from mRNA of viable cells using reverse transcription polymerase chain reaction (RT-PCR) and detected using the electronic DNA microarray technique. Specific captures and reporters were designed and examined for selective detection and correct identification of the target pathogens. The technique was able to detect as few as 2–150 cells of E. coli O157:H7. The co-presence of six other common bacteria and a parasite at 10- and 1000-fold higher concentrations than the target E. coli O157:H7 did not interfere with the specific detection. Comparative analysis of live and heat-killed E. coli O157:H7 cells showed that the technique only responded to the viable cells and not to the dead cells. Thus, the integration of RT-PCR of specific mRNA with the electronic DNA microarray technique enables specific and sensitive detection of viable target cells. This technique is potentially useful for high throughput screening of multiple pathogenic bacteria in different samples.
Keywords: Deoxyribonucleic acid (DNA) microarray; Escherichia coli; O157:H7; Viability; Pathogen; Detection
Ferrocenylnaphthalene diimide-based electrochemical detection of methylated gene by Shinobu Sato; Koji Hokazono; Tatsuya Irie; Takashi Ueki; Michinori Waki; Takahiko Nojima; Hiroki Kondo; Shigeori Takenaka (pp. 82-87).
Ferrocenylnaphthalene diimide (FND)-based electrochemical hybridization assay was applied to the detection of methylated cytosine of DNA using the products obtained after treatment with bisulfite followed by polymerase chain reaction (PCR), where unmethylated cytosine is converted to thymine and methylated one to cytosine. Twenty-meric DNA probes for the methylated (cytosine) and unmethylated (thymine) types of the part of the promoter region of cyclin D-dependent protein kinase inhibitor, p16, gene (p16Ink4a) were used to be immobilized on the electrochemical array (ECA) chip. Using 1μL of 10ng/μL of methylated sample obtained from the methylation-specific PCR of methylated genome containing 10-times excess of unmethylated one, the methylated PCR sample could be detected by the identical electrochemical signals from the two DNA probes under the settled optimum hybridization conditions.
Keywords: Methylation analysis; Ferrocenylnaphthalene diimide (FND); Electrochemical hybridization assay; Methylation-specific PCR (MSP)
Ferrocenylnaphthalene diimide-based electrochemical detection of methylated gene by Shinobu Sato; Koji Hokazono; Tatsuya Irie; Takashi Ueki; Michinori Waki; Takahiko Nojima; Hiroki Kondo; Shigeori Takenaka (pp. 82-87).
Ferrocenylnaphthalene diimide (FND)-based electrochemical hybridization assay was applied to the detection of methylated cytosine of DNA using the products obtained after treatment with bisulfite followed by polymerase chain reaction (PCR), where unmethylated cytosine is converted to thymine and methylated one to cytosine. Twenty-meric DNA probes for the methylated (cytosine) and unmethylated (thymine) types of the part of the promoter region of cyclin D-dependent protein kinase inhibitor, p16, gene (p16Ink4a) were used to be immobilized on the electrochemical array (ECA) chip. Using 1μL of 10ng/μL of methylated sample obtained from the methylation-specific PCR of methylated genome containing 10-times excess of unmethylated one, the methylated PCR sample could be detected by the identical electrochemical signals from the two DNA probes under the settled optimum hybridization conditions.
Keywords: Methylation analysis; Ferrocenylnaphthalene diimide (FND); Electrochemical hybridization assay; Methylation-specific PCR (MSP)
Tetrakis-acridinyl peptide: Distance dependence of photoinduced electron transfer in deoxyribonucleic acid assemblies by Keiji Mizuki; Takahiko Nojima; Bernard Juskowiak; Shigeori Takenaka (pp. 88-92).
The distance dependence of photoinduced electron transfer in deoxyribonucleic acid (DNA) duplex was investigated using the “TAP cassette? systems of the general formula (AT)6A nXA9− n (X denote guanine (G) or cytosine (C)). The tetrakis-9-acridinyl peptide (TAP) binds tightly with (AT)6 duplex region showing strong fluorescence that was not quenched by the A nXA9− n single-stranded region. Quenching was observed after duplex formation with the complementary T9− nXT n strand (G-C pairing), showing clear dependence on the distance between the TAP and a guanine. An extremely low β value of 0.22 was obtained in our electron transfer (ET) system that suggests exceptional good mediation of ET process. Experiments with G-mismatches showed negligible quenching for systems with guanine separated by more than one AT base pair that indicated rather inefficient ET process for duplexes containing disrupted π-electronic system.
Keywords: Electron transfer; Deoxyribonucleic acid duplex; Hopping mechanism; Tetrakis-acridinyl peptide
Tetrakis-acridinyl peptide: Distance dependence of photoinduced electron transfer in deoxyribonucleic acid assemblies by Keiji Mizuki; Takahiko Nojima; Bernard Juskowiak; Shigeori Takenaka (pp. 88-92).
The distance dependence of photoinduced electron transfer in deoxyribonucleic acid (DNA) duplex was investigated using the “TAP cassette” systems of the general formula (AT)6A nXA9− n (X denote guanine (G) or cytosine (C)). The tetrakis-9-acridinyl peptide (TAP) binds tightly with (AT)6 duplex region showing strong fluorescence that was not quenched by the A nXA9− n single-stranded region. Quenching was observed after duplex formation with the complementary T9− nXT n strand (G-C pairing), showing clear dependence on the distance between the TAP and a guanine. An extremely low β value of 0.22 was obtained in our electron transfer (ET) system that suggests exceptional good mediation of ET process. Experiments with G-mismatches showed negligible quenching for systems with guanine separated by more than one AT base pair that indicated rather inefficient ET process for duplexes containing disrupted π-electronic system.
Keywords: Electron transfer; Deoxyribonucleic acid duplex; Hopping mechanism; Tetrakis-acridinyl peptide
Photoactive, covalent attachment of deoxyribonucleic acid on gold with double-strand specificity using self-assembled monolayers containing psoralen by Koji Nakano; Hideshi Matsunaga; Keisuke Sai; Nobuaki Soh; Toshihiko Imato (pp. 93-99).
Taking advantages of psoralen photochemistry, we have developed a new method of immobilizing DNA on gold substrate surfaces. A psoralen derivative having an alkylamine function was synthesized, and was self-assembled on gold substrate surfaces in a combined use of a thiol-derivatized molecule, 3,3′-dithiobis(succinimidyl propionate) forming amide bonds on the surface. We found that by irradiating with long wavelength ultraviolet light (320–400nm), DNA molecules added in the solution phase were covalently immobilized on the monolayer surface through the photoadduct formation of the psoralen molecules with the DNA nucleobases. The present method has its advantage that is applicable to native DNAs, no chemically modifying DNAs, in spite of its covalent immobilization principle. We have examined 12 mer synthetic oligonucleotide immobilizations and have found that the surface concentration thus attained was to be 20pmolcm−2, which is consistent with saturated surface coverage. Interestingly, the immobilization occurred double-stranded-DNA-preferentially; no immobilization for single-stranded DNAs. Characterization of the immobilization chemistry has been achieved using atomic force microscopic imaging, infrared absorption, X-ray photoelectron spectroscopy, electrochemistry, and quartz-crystal microbalance and their results were described.
Keywords: DNA; Immobilization; Self-assembly; Psoralen; Photoreaction; Double-stranded DNA specificity
Photoactive, covalent attachment of deoxyribonucleic acid on gold with double-strand specificity using self-assembled monolayers containing psoralen by Koji Nakano; Hideshi Matsunaga; Keisuke Sai; Nobuaki Soh; Toshihiko Imato (pp. 93-99).
Taking advantages of psoralen photochemistry, we have developed a new method of immobilizing DNA on gold substrate surfaces. A psoralen derivative having an alkylamine function was synthesized, and was self-assembled on gold substrate surfaces in a combined use of a thiol-derivatized molecule, 3,3′-dithiobis(succinimidyl propionate) forming amide bonds on the surface. We found that by irradiating with long wavelength ultraviolet light (320–400nm), DNA molecules added in the solution phase were covalently immobilized on the monolayer surface through the photoadduct formation of the psoralen molecules with the DNA nucleobases. The present method has its advantage that is applicable to native DNAs, no chemically modifying DNAs, in spite of its covalent immobilization principle. We have examined 12 mer synthetic oligonucleotide immobilizations and have found that the surface concentration thus attained was to be 20pmolcm−2, which is consistent with saturated surface coverage. Interestingly, the immobilization occurred double-stranded-DNA-preferentially; no immobilization for single-stranded DNAs. Characterization of the immobilization chemistry has been achieved using atomic force microscopic imaging, infrared absorption, X-ray photoelectron spectroscopy, electrochemistry, and quartz-crystal microbalance and their results were described.
Keywords: DNA; Immobilization; Self-assembly; Psoralen; Photoreaction; Double-stranded DNA specificity
Spatiotemporal control of cell adhesion on a self-assembled monolayer having a photocleavable protecting group by Jun Nakanishi; Yukiko Kikuchi; Tohru Takarada; Hidekazu Nakayama; Kazuo Yamaguchi; Mizuo Maeda (pp. 100-104).
Control of cell adhesion is a key technology for cell-based drug screening and for analyses of cellular processes. We developed a method to spatiotemporally control cell adhesion using a photochemical reaction. We prepared a cell-culturing substrate by modifying the surface of a glass coverslip with a self-assembled monolayer of an alkylsiloxane having a photocleavable 2-nitrobenzyl group. Bovine serum albumin (BSA) was adsorbed onto the substrate to make the surface inert to cell adhesion. When exposed to UV light, the alkylsiloxane underwent a photocleavage reaction, leading to the release of BSA from the surface. Fibronectin, a protein promoting cell adhesion, was added to cover the irradiated regions and made them cell-adhesive. Seeding of cells on this substrate resulted in their selective adhesion to the illuminated regions. By controlling the sizes of the illuminated regions, we formed cell-adhesive spots smaller than single cells and located focal adhesions of the cells. Moreover, by subsequently illuminating the region alongside the cells patterned on the substrate in advance, we released their geometrical confinements and induced migration and proliferation. These manipulations were conducted under a conventional fluorescence microscope without any additional instruments. The present method of cell manipulation will be useful for cell biological studies as well as for the formation of cell arrays.
Keywords: Cell adhesion; Cell migration; Cell proliferation; Self-assembled monolayer; Focal adhesion
Spatiotemporal control of cell adhesion on a self-assembled monolayer having a photocleavable protecting group by Jun Nakanishi; Yukiko Kikuchi; Tohru Takarada; Hidekazu Nakayama; Kazuo Yamaguchi; Mizuo Maeda (pp. 100-104).
Control of cell adhesion is a key technology for cell-based drug screening and for analyses of cellular processes. We developed a method to spatiotemporally control cell adhesion using a photochemical reaction. We prepared a cell-culturing substrate by modifying the surface of a glass coverslip with a self-assembled monolayer of an alkylsiloxane having a photocleavable 2-nitrobenzyl group. Bovine serum albumin (BSA) was adsorbed onto the substrate to make the surface inert to cell adhesion. When exposed to UV light, the alkylsiloxane underwent a photocleavage reaction, leading to the release of BSA from the surface. Fibronectin, a protein promoting cell adhesion, was added to cover the irradiated regions and made them cell-adhesive. Seeding of cells on this substrate resulted in their selective adhesion to the illuminated regions. By controlling the sizes of the illuminated regions, we formed cell-adhesive spots smaller than single cells and located focal adhesions of the cells. Moreover, by subsequently illuminating the region alongside the cells patterned on the substrate in advance, we released their geometrical confinements and induced migration and proliferation. These manipulations were conducted under a conventional fluorescence microscope without any additional instruments. The present method of cell manipulation will be useful for cell biological studies as well as for the formation of cell arrays.
Keywords: Cell adhesion; Cell migration; Cell proliferation; Self-assembled monolayer; Focal adhesion