Analytical and Bioanalytical Chemistry (v.391, #8)
Nanotechnologies in the biosciences by Manabu Tokeshi; Yoshinobu Baba (2693-2694).
is Associate Professor of the Department of Applied Chemistry at Nagoya University. He is also Researcher at MEXT Innovative Research Center for Preventive Medical Engineering at Nagoya University and Adjunct Researcher in the Micro Chemistry Group at Kanagawa Academy of Science and Technology. His research interests are microfluidics, nanofluidics, micro/nano biosystems, and miniaturization of diagnosis systems. is Professor of the Department of Applied Chemistry and the Plasma Nanotechnology Research Center at Nagoya University. He is also Group Leader of the MEXT Innovative Research Center for Preventive Medical Engineering at Nagoya University, Vice Director of Nanobio Research, Health Technology Research Center at the National Institute of Advanced Industrial Science and Technology, and Visiting Professor at the Institute for Molecular Science at the National Institute of Natural Sciences. His research interests are nanobioscience and nanobiotechnology for genomics, proteomics, glycomics, systems biology, single-molecule manipulation, and medical applications.
Microtechnologies for membrane protein studies by Hiroaki Suzuki; Shoji Takeuchi (2695-2702).
Despite the rapid and enormous progress in biotechnologies, the biochemical analysis of membrane proteins is still a difficult task. The presence of the large hydrophobic region buried in the lipid bilayer membrane (transmembrane domain) makes it difficult to analyze membrane proteins in standard assays developed for water-soluble proteins. To handle membrane proteins, the lipid bilayer membrane may be used as a platform to sustain their functionalities. Relatively slow progress in developing micro total analysis systems (μTAS) for membrane protein analysis directly reflects the difficulty of handling lipid membranes, which is a common problem in bulk measurement technologies. Nonetheless, researchers are continuing to develop efficient and sensitive analytical microsystems for the study of membrane proteins. Here, we review the latest developments, which enable detection of events caused by membrane proteins, such as ion channel current, membrane transport, and receptor/ligand interaction, by utilizing microfabricated structures. High-throughput and highly sensitive detection systems for membrane proteins are now becoming a realistic goal. Although most of these systems are still in the early stages of development, we believe this field will become one of the most important applications of μTAS for pharmaceutical and clinical screenings as well as for basic biochemical research.
Keywords: Microtechnology; Membrane protein; Lipid bilayer; Ion channel; Transporter
Incubation type Si-based planar ion channel biosensor by Tsuneo Urisu; Toshifumi Asano; Zhenlong Zhang; Hidetaka Uno; Ryugo Tero; Han Junkyu; Isoda Hiroko; Yusuke Arima; Hiroo Iwata; Koji Shibasaki; Makoto Tominaga (2703-2709).
A new planar-type ion channel biosensor with the function of cell culture has been fabricated using silicon on an insulator substrate as the sensor chip material. Coating of the sensor chip with fibronectin was essentially important for cell incubation on the chip. Although the seal resistance was quite low (∼7 MΩ) compared with the pipette patch-clamp gigaohm seal, the whole-cell channel current of the transient receptor potential vanilloid type 1 (TRPV1) channel expressing HEK293 cells was successfully observed, with a good signal-to-noise ratio, using capsaicin as a ligand molecule. Figure A new planer type ion channel biosensor with function of cell culture is fabricated using the silicon on insulator substrate as the sensor chip material. The coating of the sensor chip by the fibronectin was essentially important for the cell incubation on the chip. Whole cell channel current of TRPV1 channel was successfully observed using capsaicin as a ligand molecule with good signal to noise.
Keywords: Ion channel biosensor; SOI; Extracellular matrix; Fibronectin; Nystatin
Integration of an electrochemical-based biolithography technique into an AFM system by Soichiro Sekine; Hirokazu Kaji; Matsuhiko Nishizawa (2711-2716).
An ordinary atomic force microscopy (AFM) was functionalized and applied to electrochemically draw micropatterns of biomolecules. To fabricate an electrochemical AFM probe having an electrode at the tip, a metal-coated AFM probe was first insulated with Parylene C, and then the apex of the tip was ground mechanically to expose the electrode. The effective electrode diameter was estimated to be ca. 500 nm. The electrode probe was positioned close to a heparin-coated antibiofouling substrate and used to locally generate hypobromous acid from a dilute Br− solution to render the substrate surface protein-adhesive. In situ topographical imaging after the electrochemical treatment suggested the heparin layer became detached to allow the adsorption of proteins, in this case fibronectin. The diameter of the drawn fibronectin pattern was 2 μm, which is one order of magnitude smaller than we achieved previously using a microdisk electrode (tip diameter 10 μm). Figure AFM configuration integrated with the electrochemical-based surface modification and resultant micropatterns of fluorescence-labeled fibronectin
Keywords: AFM; Biolithography; Electrochemistry; Fibronectin; Surface modification; Parylene; Protein patterning
An artificial virus-like nano carrier system: enhanced endosomal escape of nanoparticles via synergistic action of pH-sensitive fusogenic peptide derivatives by Kentaro Sasaki; Kentaro Kogure; Shinji Chaki; Yoshio Nakamura; Rumiko Moriguchi; Hirofumi Hamada; Radostin Danev; Kuniaki Nagayama; Shiroh Futaki; Hideyoshi Harashima (2717-2727).
We previously reported that transferrin (Tf)-modified liposomes (Tf-L) additionally modified with a cholesterylated pH-sensitive fusogenic peptide (Chol-GALA) can release an encapsulated aqueous phase marker to cytosol via endosomal membrane fusion. However, further obstacles need to be overcome to bring the Tf-L to the level of a viral-like gene delivery system. In this study, we developed a novel packaging method to encapsulate condensed plasmid DNA into PEgylated Tf-L (Tf-PEG-L) to form a core–shell-type nanoparticle. The most difficult challenge was to provide a mechanism of escape for the condensed core from endosome to cytosol in the presence of polyethylene glycol (PEG). We hypothesized that a membrane-introduced Chol-GALA and a PEgylated GALA would interact synergistically to induce membrane fusion between liposome and endosome. By simultaneously incorporating Chol-GALA into the membrane of Tf-PEG-L and GALA at tips of PEG chains, a condensed core was released into cytosol, and transfection acitivty increased 100-fold. We concluded that topological control was responsible for the synergistic effect of GALA derivatives introduced on Tf-PEG-L.
Keywords: Artificial virus; Multifunctional envelope-type nanodevice; Polyethyleneglycol; Transferrin; pH-sensitive fusogenic peptide GALA; Synergistic action
On-chip fabrication of mutifunctional envelope-type nanodevices for gene delivery by Hiroshi Kuramoto; Yeon-Su Park; Noritada Kaji; Manabu Tokeshi; Kentaro Kogure; Yasuo Shinohara; Hideyoshi Harashima; Yoshinobu Baba (2729-2733).
Microfluidic devices may be highly beneficial to the rapid fabrication of small quantities of various nonviral vectors with different functionalities, which is indispensable for effective order-made gene therapy. We adapted a microfluidic chip-based approach for fabricating small quantities of nonviral vectors in a short time in preparation for order-made gene therapy applications. This approach permitted us to fabricate multifunctional envelope-type nanodevices (MENDs), composed of a compacted (or condensed) DNA core and a lipid bilayer membrane shell, which are considered as promising nonviral vectors for gene therapy applications. The on-chip fabrication of the MEND was very simple, rapid, convenient, and cost-effective compared with conventional methods. The size of the MEND showed strong dependence on the concentration and flow rate of the reaction precursors and could be controlled to be much smaller than that achievable by conventional methods. This, together with abovementioned merits, makes our microfluidic chip-based approach very attractive for the fabrication of MENDs for effective application to order-made gene therapy.
Keywords: On-chip fabrication; Multifunctional envelope-type nanodevice (MEND); Nonviral vector; Gene delivery; Gene therapy; Microfluidic device
DNA molecule manipulation by motor proteins for analysis at the single-molecule level by Ryuji Yokokawa; Junichi Miwa; Mehmet Cagatay Tarhan; Hiroyuki Fujita; Masahiro Kasahara (2735-2743).
Massively parallel and individual DNA manipulation for analysis has been demonstrated by designing a fully self-assembled molecular system using motor proteins. DNA molecules were immobilized by trapping in a polyacrylamide gel replica, and were digested by a restriction enzyme, XhoI, for DNA analysis. One end of the λDNA was modified with biotin and the other end was modified with digoxin molecules by fragment labeling and ligation methods. The digoxin-functionalized end was immobilized on a glass surface coated with anti-digoxigenin antibody. The biotinylated end was freely suspended and experienced Brownian motion in a buffer solution. The free end was attached to a biotinylated microtubule via avidin–biotin biding and the DNA was stretched by a kinesin-based gliding assay. A stretched DNA molecule was fixed between the gel and coverslip to observe the cleavage of the DNA by the enzyme, which was supplied through the gel network structure. This simple process flow from DNA manipulation to analysis offers a new method of performing molecular surgery at the single-molecule scale. Figure DNA molecule manipulation by motor proteins for analysis at the single-molecule level
Keywords: DNA; Molecular surgery; Motor protein; Nanomanipulation
Development of a pressure-driven nanofluidic control system and its application to an enzymatic reaction by Takehiko Tsukahara; Kazuma Mawatari; Akihide Hibara; Takehiko Kitamori (2745-2752).
A novel air-pressure-based nanofluidic control system was developed and its performance was examined. We found that the flow in a 100 nm scale nanochannel on a chip (called an extended nanospace channel) could be controlled within the pressure range of 0.003–0.4 MPa, flow rate range of 0.16–21.2 pL/min, and residence time range of 24 ms–32.4 s by using the developed nanofluidic control system. Furthermore, we successfully demonstrated an enzyme reaction in which the fluorogenic substrate TokyoGreen-β-galactoside (TG-β-gal) was hydrolyzed to the fluorescein derivative TokyoGreen (TG) and β-galactose by the action of β-galactosidase enzyme as a calalyst in a Y-shaped extended nanospace channel. The parameters for the reaction kinetics, such as K m, V max and k cat, were estimated for the nanofluidic reaction, and these values were compared with the results of bulk and microfluidic reactions. A comparison showed that the enzyme reaction rate in the Y-shaped extended nanospace channel increased by a factor of about two compared with the rates in the bulk and micro spaces. We thought that this nanospatial property resulted from the activated protons of water molecules in the extended nanospace. This assumption was supported by the result that the pH dependence of the maximum enzyme activity in the Y-shaped extended nanospace channel was slightly different from that in the bulk and micro spaces.
Keywords: Extended nanospace; Nanofluidic; Pressure-driven; Enzyme reaction; Size-confinement effect
High-throughput evaluation of quiescent hematopoietic progenitor cells using a micro-multiwell plate by Satoshi Fujita; Yutaka Morita; Hiroo Iwata (2753-2758).
Conventional assays for hematopoietic progenitor cells (HPCs) require long-term culturing, a labor-intensive procedure, and technique proficiency. We aimed to develop a high-throughput method to determine frequency of quiescent primitive HPCs by a combination of the micro-multiwell plate and 5-fluorouracil (5-FU) treatment. The micro-multiwell plate was made of a silicone sheet with a 6 × 6 array of 1-mm diameter holes and a glass substrate. To enrich primitive HPCs in a CD34 population, CD34 cells and stromal cells were applied to micro-multiwells and cultured in the presence of 5-FU for 2 days. The quiescent primitive HPCs that survived after 5-FU treatment were then expanded with cytokines in the absence of 5-FU for a further 10 days. After culturing, cells were immunostained and the number of primitive HPCs in inoculated CD34 cells was estimated from fluorescent intensity for each well under a stereoscopic fluorescent microscope. The frequencies of primitive HPCs correlated well with frequencies of cobblestone area-forming cells for two CD34 cell lots. Our method allows high-throughput screening for primitive HPCs in CD34 cells. Figure Representative image of a micro-multiwell plate fordetecting primitive hematopoietic stem cells
Keywords: High-throughput screening; Hematopoietic progenitor cell; 5-Fluorouracil; Microwell
Electroactive chitosan nanoparticles for the detection of single-nucleotide polymorphisms using peptide nucleic acids by Kagan Kerman; Masato Saito; Eiichi Tamiya (2759-2767).
Here we report an electrochemical biosensor that would allow for simple and rapid analysis of nucleic acids in combination with nuclease activity on nucleic acids and electroactive bionanoparticles. The detection of single-nucleotide polymorphisms (SNPs) using PNA probes takes advantage of the significant structural and physicochemical differences between the full hybrids and SNPs in PNA/DNA and DNA/DNA duplexes. Ferrocene-conjugated chitosan nanoparticles (Chi-Fc) were used as the electroactive indicator of hybridization. Chi-Fc had no affinity towards the neutral PNA probe immobilized on a gold electrode (AuE) surface. When the PNA probe on the electrode surface hybridized with a full-complementary target DNA, Chi-Fc electrostatically attached to the negatively-charged phosphate backbone of DNA on the surface and gave rise to a high electrochemical oxidation signal from ferrocene at ∼0.30 V. Exposing the surface to a single-stranded DNA specific nuclease, Nuclease S1, was found to be very effective for removing the nonspecifically adsorbed SNP DNA. An SNP in the target DNA to PNA made it susceptible to the enzymatic digestion. After the enzymatic digestion and subsequent exposure to Chi-Fc, the presence of SNPs was determined by monitoring the changes in the electrical current response of Chi-Fc. The method provided a detection limit of 1 fM (S/N = 3) for the target DNA oligonucleotide. Additionally, asymmetric PCR was employed to detect the presence of genetically modified organism (GMO) in standard Roundup Ready soybean samples. PNA-mediated PCR amplification of real DNA samples was performed to detect SNPs related to alcolohol dehydrogenase (ALDH). Chitosan nanoparticles are promising biometarials for various analytical and pharmaceutical applications. Figure The electrochemical method for SNP detection using PNA probes and chitosan nanoparticles takes advantage of the significant structural and physicochemical differences between PNA/DNA and DNA/DNA duplexes. Single-stranded DNA specific enzymes selectively choose these SNP sites and hydrolyze the DNA molecules on gold electrode (AuE) surface.
Keywords: Electrochemical DNA biosensor; Chitosan nanoparticles; Peptide nucleic acids; Single-nucleotide polymorphism detection; Nuclease S1
Metabolomics approach for determining growth-specific metabolites based on Fourier transform ion cyclotron resonance mass spectrometry by Hiroki Takahashi; Kosuke Kai; Yoko Shinbo; Kenichi Tanaka; Daisaku Ohta; Taku Oshima; Md. Altaf-Ul-Amin; Ken Kurokawa; Naotake Ogasawara; Shigehiko Kanaya (2769-2782).
Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR/MS) is the best MS technology for obtaining exact mass measurements owing to its great resolution and accuracy, and several outstanding FT-ICR/MS-based metabolomics approaches have been reported. A reliable annotation scheme is needed to deal with direct-infusion FT-ICR/MS metabolic profiling. Correlation analyses can help us not only uncover relations between the ions but also annotate the ions originated from identical metabolites (metabolite derivative ions). In the present study, we propose a procedure for metabolite annotation on direct-infusion FT-ICR/MS by taking into consideration the classification of metabolite-derived ions using correlation analyses. Integrated analysis based on information of isotope relations, fragmentation patterns by MS/MS analysis, co-occurring metabolites, and database searches (KNApSAcK and KEGG) can make it possible to annotate ions as metabolites and estimate cellular conditions based on metabolite composition. A total of 220 detected ions were classified into 174 metabolite derivative groups and 72 ions were assigned to candidate metabolites in the present work. Finally, metabolic profiling has been able to distinguish between the growth stages with the aid of PCA. The constructed model using PLS regression for OD600 values as a function of metabolic profiles is very useful for identifying to what degree the ions contribute to the growth stages. Ten phospholipids which largely influence the constructed model are highly abundant in the cells. Our analyses reveal that global modification of those phospholipids occurs as E. coli enters the stationary phase. Thus, the integrated approach involving correlation analyses, metabolic profiling, and database searching is efficient for high-throughput metabolomics.
Keywords: Fourier transform ion cyclotron resonance mass spectrometry; Metabolomics; Metabolite annotation; Cellular conditions; Correlation network
Biosensor incorporating cell barrier architectures on ion selective electrodes for early screening of cancer by Gargi Ghosh; Leonidas G. Bachas; Kimberly W. Anderson (2783-2791).
Angiogenesis occurs during the early phase of cancer. Recruitment of new blood vessels by existing cancer cells leads to the release of higher concentrations of cytokines as compared to cells in healthy individuals. Some of the common cytokines observed at higher concentrations, such as vascular endothelial growth factor, basic fibroblast growth factor, hepatocyte growth factor and tumor necrosis factor-alpha, are also known to induce increased permeability across an endothelial cell monolayer. A whole-cell-based biosensor has been developed that can detect the presence of small quantities of the abovementioned cytokines individually and in different combinations. It was observed that the biosensor could differentiate between the cytokine concentrations observed in the sera of healthy individuals and cancer patients. The biosensor was also evaluated by exposing it to actual serum. These results demonstrated that the sensor can distinguish between healthy individuals and cancer patients and that the corresponding biosensor responses correlate with the stages of cancer.
Keywords: Screening cancer; Angiogenesis; Cytokines; Cell-based biosensor; Ion-selective electrode
Interaction of native α-cyclodextrin, β-cyclodextrin and γ-cyclodextrin and their hydroxypropyl derivatives with selected organic low molecular mass compounds at elevated and subambient temperature under RP-HPLC conditions by P. K. Zarzycki; H. Ohta; Y. Saito; K. Jinno (2793-2801).
The main focus of this study was to explore the capability of native α-cyclodextrin, β-cyclodextrin and γ-cyclodextrin and their hydroxypropyl derivatives for host-guest interaction with 7,8-dimethoxyflavone, selected steroids (estetrol, estriol, estradiol, estrone, testosterone, cortisone, hydrocortisone, progesterone and 17α-hydroxyprogesterone) and polycyclic aromatic hydrocarbons (toluene, naphthalene, 1,8-dimethylnaphthalene, 1-acenaphthenol, acenaphthylene and acenaphthene) under reversed-phase liquid-chromatography conditions. The study revealed that native cyclodextrins interact more efficiently with the analytes investigated than do their hydroxypropyl counterparts. In the low-temperature region, enormously high ratios were observed for polycyclic aromatic hydrocarbons, particularly 1,8-dimethylnaphthalene, acenaphthene and acenaphthylene chromatographed on a β-cyclodextrin-modified mobile phase. In such a case, the retention times of the polycyclic aromatic hydrocarbons were strongly reduced (e.g. from 127 to 1.2 min for 1,8-dimethylnaphthalene) and were close to the hold-up time of the high-performance liquid chromatography (HPLC) system (0.7 min). Moreover, chiral separation of 1-acenaphthenol optical isomers was observed and the elution order of the enantiomers was determined. Within the steroids group, strong interaction was observed for estradiol and testosterone. The results of cluster analysis indicate that β-cyclodextrin as well as γ-cyclodextrin and its hydroxypropyl derivative can be most effective mobile-phase additives under reversed-phase HPLC conditions for 3D-shape-recognition-driven separation, performed at subambient and elevated temperatures, respectively.
Keywords: Cyclodextrins; Polycyclic aromatic hydrocarbons; Steroids; Enantiomers; Temperature; High-performance liquid chromatography; Cluster analysis
Combining chip-ESI with APLI (cESILI) as a multimode source for analysis of complex mixtures with ultrahigh-resolution mass spectrometry by Philippe Schmitt-Kopplin; Matthias Englmann; Ramon Rossello-Mora; Ralf Schiewek; Klaus J. Brockmann; Thorsten Benter; Oliver J. Schmitz (2803-2809).
Recently we have established atmospheric-pressure laser ionisation (APLI) as a method for coupling time-of-flight mass spectrometric detectors (TOF MS) with chromatographic systems (HPLC and GC) to allow two-photon ionisation of non-polar aromatic compounds. Here we demonstrate that APLI can be combined with chip-electrospray ionisation (cESI) coupled to Fourier-transform-ion cyclotron resonance mass spectrometry (FT-ICR MS) for ultrahigh-resolution analysis of complex samples. With the laser turned off, the analytes are ionised only by ESI, whereas when the laser is switched on non-polar aromatic substances also are ionised. In combination with the extremely high mass resolution of an FT-ICR MS, simultaneous qualitative analysis of polar and non-polar analytes is possible in both positive and negative modes, as is exemplified with a crude oil sample. Nevertheless, ion suppression was observed (up to ca. 70% for D10-pyrene) and thus sample preparation with chromatographic or electrophoretic pre-separation is necessary for quantitative analysis of targets. In addition, for the first time, the dopant-assisted APLI method in combination with cESI (DA-cESILI) was used for determination of 1-nitrocoronene.
Keywords: APLI; FT-ICR MS; Oil; Mass spectrometry; ESILI
Simultaneous analysis of vitamins and caffeine in energy drinks by surfactant-mediated matrix-assisted laser desorption/ionization by David C. Grant; Robert J. Helleur (2811-2818).
As a new approach to rapid small-molecule analysis, surfactant-mediated matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF-MS) was successfully used in the analysis of caffeine and the vitamins riboflavin, nicotinamide, and pyridoxine in various energy drinks. Of five common MALDI matrices tested (α-cyano-4-hydroxycinnamic acid, sinapinic acid, 2,5-dihydroxybenzoic acid, dithranol, and 2′,4′,6′-trihydroxyacetophenone), α-cyano-4-hydroxycinnamic acid was found to be most suitable for analysis of high-sugar-containing energy drinks. Cetyltrimethylammonium bromide (CTAB) surfactant was used as a matrix-ion suppressor, at a matrix:surfactant mole ratio of approximately 500:1. The resulting mass spectra show very few matrix-related ions, while analyte signals were clearly observed. For comparative purposes the same analytes were identified and quantified in energy drinks by LC–ESI–MS with UV detection. Quantitatively the calibration curves of all four analytes showed a marked improvement when the surfactant-mediated method was used compared with traditional MALDI–TOF-MS; correlation coefficients of 0.989 (nicotinamide), 0.991 (pyridoxine), 0.983(caffeine) and 0.987 (riboflavin) were obtained. It was found that in quantitation of the energy drink analytes the surfactant-mediated MALDI–TOF-MS results were comparable with those from LC analysis. In reproducibility experiments RSD values ranged from 9.7 to 18.1%. The work has demonstrated that this mass spectrometric approach can be used as a rapid screening technique for fortified drinks.
Keywords: MALDI; Surfactants; Energy drinks; Mass spectrometry; Vitamins; Caffeine
Study on molecular interactions between proteins on live cell membranes using quantum dot-based fluorescence resonance energy transfer by Tian-Cai Liu; Hai-Li Zhang; Jian-Hao Wang; Hai-Qiao Wang; Zhi-Hong Zhang; Xiao-Feng Hua; Yuan-Cheng Cao; Qing-Ming Luo; Yuan-Di Zhao (2819-2824).
Mouse anti-human CD71 monoclonal antibody (anti-CD71) was conjugated with red quantum dots (QDs; 5.3 nm, emission wavelength λ em = 614 nm) and used to label HeLa cells successfully. Then green QD-labeled goat anti-mouse immunoglobulin G (IgG; the size of the green QDs was 2.2 nm; λ em = 544 nm) was added to bind the red-QD-conjugated anti-CD71 on the cell surface by immunoreactions. Such interaction between anti-CD71 and IgG lasted 4 min and was observed from the fluorescence spectra: the fluorescence intensity of the “red” peak at 614 nm increased by 32%; meanwhile that of the “green” one at 544 nm decreased by 55%. The ratio of the fluorescence intensities (I 544 nm/I 614 nm) decreased from 0.5 to 0.2. The fluorescence spectra as well as cell imaging showed that fluorescence resonance energy transfer took place between these two kinds of QDs on the HeLa cells through interactions between the primary antibody and the secondary antibody.
Keywords: Fluorescence resonance energy transfer; Molecular interaction; Quantum dot; Bioprobe
Immunofunctionalisation of gold transducers for bacterial detection by physisorption by Olivier Laczka; Eva Baldrich; F. Javier del Campo; Francesc Xavier Muñoz (2825-2835).
Functionalisation of the sensing surface is a key factor in immunosensor fabrication as it allows target-selective capture and prevents nonspecific adsorption of undesired components. Gold immunofunctionalisation using self-assembled monolayers (SAM) has been widely exploited to this end for the detection of small targets. However, we recently demonstrated that this strategy fails when detecting whole bacteria cells (Baldrich et al., Anal Bioanal Chem 390:1557–1562, 2008). We now investigate different physisorption-based alternatives using E. coli as the target organism. Our results demonstrate that physisorption generates the appropriate substrate for the specific detection of bacteria on gold surfaces, providing detection limits down to 105 cells mL−1 in an ELISA-type colorimetric assay. Additionally, surface coverage is highly reproducible when assayed by impedance spectroscopy and the inter- and intra-assay coefficients of variation are below 10–15% in all cases. These surfaces were stable, retained functionality and did not suffer from significant biomolecule desorption after 10 days storage in PBS at 37 °C, hence confirming physisorption as a cheap, simple and efficient strategy for the detection of bacteria.
Keywords: Bacteria detection; Immunosensor; Gold immunofunctionalisation; Antibody conjugation; ELISA assay
Analytical prospect of compact disk technology in immunosensing by Sergi Morais; Jesús Tamarit-López; Javier Carrascosa; Rosa Puchades; Ángel Maquieira (2837-2844).
A sensitive and versatile methodology involving recordable compact disks as molecular screening surfaces and a standard optical CD/DVD drive as detector, is reported. Quantitative immunoanalysis, in microarray format, of a cancer marker (alpha-fetoprotein, AFP) and a selective herbicide (atrazine) on four types of audio-video disc was conducted. Enzyme or gold nanoparticle-labeled antibodies were used as tracers, forming a precipitate on the sensing disk surface. The principle of disk reading is based on capture of analog signals with the disk drive that were proportional to the darkness of the immunoreaction product. Detection limits for AFP (8.0 μg L−1) and for atrazine (0.04 μg L−1) were under the threshold needed to detect nonseminomatous testicular cancer, and below the maximum E.U. residue limit for drinking water, respectively. The described methodology improves the previous developments using CDs and highlights the enormous potential of immunoassay methods using standard audio–video disk surfaces in combination with the CD/DVD drive for clinical analysis, drug discovery, or high-throughput multiresidue screening applications. Figure Eye-catching image The analytical potential of commercial audio–video discs as molecular screening surfaces in combination with use of a standard CD/DVD drive as detector for quantitative immunoanalysis of a cancer marker and agrochemical residues is demonstrated.
Keywords: Microimmunoassay; Compact disk; Cancer markers; Agrochemicals; Screening
Identification of in vitro differential cell secretions due to cigarette smoke condensate exposure using nanoflow capillary liquid chromatography and high-resolution mass spectrometry by Jason W. Flora; Jeff Edmiston; Rebecca Secrist; Guoya Li; Gaurav S. J. B. Rana; Timothy B. Langston; Willie McKinney (2845-2856).
Secreted proteins, the secretome, can be isolated from biological fluids (e.g., blood) and are often responsible for the regulation of biological processes such as cell signaling, growth, and apoptosis. The identification of secreted proteins can lead to an understanding of disease mechanisms and they can serve as early candidate biomarkers of disease and exposure. However, it is time-consuming and costly to conduct in vivo interrogations of the human secretome. The purpose of this article is to provide a detailed description of a rapid in vitro technique for the analysis of differential protein secretion due to exposure to smoking-machine-generated cigarette smoke (CS) condensate (total particulate matter, TPM). Endothelial cells were exposed to CS-TPM, the supernatant was collected, and the secretome was elucidated by nano liquid chromatography coupled with high-resolution mass spectrometry. A total of 1,677 unique peptides were identified in the cell culture supernatants. Several proteins were differentially expressed following CS-TPM exposure that relate to several biological processes, such as metabolism, development, communication, response to stimulus, and response to stress.
Keywords: Fourier transform ion cyclotron resonance mass spectrometry; LTQ FT mass spectrometer; Nanoflow capillary liquid chromatography; Secreted proteins; Secretome; Endothelial cells; Cigarette smoke total particulate matter
Determination of phenanthrene by antibody-coated competitive real-time immuno-PCR assay by Chun Zhou; Qiong-E Wang; Hui-Sheng Zhuang (2857-2863).
A reliable selective and sensitive antibody-coated competitive real-time immuno-PCR (RT-IPCR) assay for the determination of phenanthrene (PH) was developed. Phenanthrene butanoic acid (γ-oxo-PHA) was synthesized as the hapten of PH. An active ester method was used to couple the PHA to bovine serum albumin to form an artificial immune antigen. Male New Zealand white rabbits were immunized with immune antigen to obtain polyclonal antibodies, with which a novel RT-IPCR assay for determination of PH was developed. Under the optimized assay conditions, PH can be determined in the concentration range from 10 fg/mL to 100 pg/mL with a detection limit of 5 fg/mL. The cross-reactivities of the anti-PH antibody to seven structurally related compounds were below 12.5%. Some environmental water samples were analyzed with satisfactory results, which showed good accuracy and suitability to analyze PH in environmental water. Compared with high-performance liquid chromatography, the recovery was lower or higher with agitation but would still be acceptable for use in an on-site field test to provide rapid, semiquantitative, and reliable test results for making environmental decisions.
Keywords: Phenanthrene; Antigen; Polyclonal antibody; Real-time immuno-PCR assay
Short-end injection capillary electrophoresis for quantification of plasma chondroitin sulfate isomer disaccharides by Angelo Zinellu; Salvatore Sotgia; Maria Franca Usai; Elisabetta Zinellu; Antonio J. Lepedda; Luca Deiana; Marilena Formato; Ciriaco Carru (2865-2868).
We describe a new ultra-rapid capillary electrophoresis method with UV detection for analysis of the disaccharides obtained after enzymatic depolymerization of plasma chondroitin sulfates. The free reducing groups of the released carbohydrate molecules are derivatized with 2-aminoacridone by reductive amination in the presence of cyanoborohydride. The fluorotagged products can be separated by short-end injection capillary electrophoresis in a capillary with an effective length of 10.2 cm. The migration times of Δdi-0S and Δdi-4S were 0.95 and 1.81 min, respectively. We compared the proposed method with UV detection to a reference CE-LIF assay by measuring plasma chondroitin sulfate in 94 subjects. The described assay for total plasma CS measurement may, owing to the high throughput and the fast analytical times, be a good tool for routine studies both in research and in clinical applications.
Keywords: Short-end injection; Glycosaminoglycan; Chondroitin sulfate; 2-Aminoacridone
An impedance immunosensor for the detection of the phytohormone abscisic acid by Yu-Wei Li; Kai Xia; Ruo-Zhong Wang; Jian-Hui Jiang; Lang-Tao Xiao (2869-2874).
The phytohormone abscisic acid (ABA) is the major player in mediating the adaptation of plants to stress. Previously developed phytohormonal biosensors usually employed indirect detection of the products of conjugated oxidase reactions. A label-free electrochemical impedance immunosensor for ABA detection was developed using an anti-ABA antibody adsorbed directly on a porous nanogold film. The film was produced electrochemically on a glassy carbon electrode in 0.008 mol/L hydrogen tetrachloroaurate solution containing 0.004 mol/L lead acetate with an applied potential of −0.5 V (versus Ag/AgCl) for 50 s. The anti-ABA antibody was immobilized onto the porous nanogold through electrostatic adsorption and covalent conjugation. Electrochemical impedance spectroscopy was used to characterize the successful construction of the porous nanogold film and the stepwise modification of the glassy carbon electrode. The concentration increase of the antigen brought about a decrease of the interfacial electron transfer, which also meant an increase of the impedance signal. The experimental parameters pH, antibody incubation time, and antibody concentration were optimized. The results showed significant linearity R = 0.9942, with the content of ABA in the range 0.5–5,000 ng/mL with a detection limit at about 0.1 ng/mL.
Keywords: Impedance immunosensor; Abscisic acid; Immunoassay; Porous nanogold
Application of a quartz crystal nanobalance and principal component analysis for the detection and determination of histidine by Maryam Shojaei; Abdolreza Mirmohseni; Maryam Farbodi (2875-2880).
The aim of the present investigation was to develop a biosensor based on a quartz crystal nanobalance (QCN) for the detection of histidine (His). A thin layer of nickel was electrochemically deposited over the gold crystal electrode and exposed to H2O2 to form nickel oxide. The composite electrode was then used to determine His. The frequency shifts were linear with respect to the concentration of His in solution. His can be measured in the range of 100–2000 mg L−1. A lower limit of detection of 48 mg L−1 and a sensitivity factor of 0.0307 Hz/mg L−1 was obtained. Some possible interferences were checked for, and the performance of the sensor was found to be unaffected by any interference except for those from arginine, cysteine and NaH2PO4. Principal component analysis (PCA) was used to process the frequency response data of the single piezoelectric crystal at various times, considering the different adsorption–desorption dynamics of His and the interfering compounds. Over 85% of the variance in the data was explained by two principal components. A score plot of the data for the first two PCs showed that the modified QCN yields favorable identification and quantification performances for His and the interfering compounds.
Keywords: Histidine; Quartz crystal nanobalance; Nickel; Principal component analysis
GC-MS with ethyl chloroformate derivatization for comprehensive analysis of metabolites in serum and its application to human uremia by Xiumei Tao; Yumin Liu; Yihuang Wang; Yunping Qiu; Jingchao Lin; Aihua Zhao; Mingming Su; Wei Jia (2881-2889).
An optimized method based on GC-MS with ethyl chloroformate derivatization has been developed for the comprehensive analysis of endogenous metabolites in serum. Twenty-two reference standards and serum samples were used to validate the proposed method. The correlation coefficient was higher than 0.9900 for each of the standards, and the LOD varied from 125 to 300 pg on-column. The analytical equipment exhibited good repeatability (RSD<10%) for all of the standards. Both the repeatability and the within-48-h stability of the analytical method were satisfactory (RSD<10%) for the 18 metabolites identified in the serum samples. Mean recovery was acceptable for the 18 metabolites, ranging from 70% to 120% with RSDs of less than 10%. Using the optimized protocol and a subsequent multivariate statistical technique, complete differentiation was achieved between the metabolic profile of uremic patients and that of age- and sex-matched normal subjects. Significantly decreased levels of valine, leucine, and isoleucine and increased levels of myristic acid and linoleic acid were observed in the patient group. This work demonstrated that this method is suitable for serum-based metabolic profiling studies.
Keywords: Metabolic profiling; GC-MS; Ethyl chloroformate; Serum; Multivariate statistical analysis; Metabonomics; GC; Bioanalytical methods; Clinical/biomedical analysis
Violet and greenish photoprotein obelin mutants for reporter applications in dual-color assay by Ludmila A. Frank; Vasilisa V. Borisova; Svetlana V. Markova; Natalia P. Malikova; Galina A. Stepanyuk; Eugene S. Vysotski (2891-2896).
Two kinds of Ca2+-regulated photoprotein obelin with altered color of bioluminescence were obtained by active-center amino acid substitution. The mutant W92F-H22E emits violet light (λmax = 390 nm) and the mutant Y139F emits greenish light (λ max = 498 nm), with small spectral overlap, both display high activity and stability and thus may be used as reporters. For demonstration, the mutants were applied in dual-color simultaneous immunoassay of two gonadotropic hormones—follicle-stimulating hormone and luteinizing hormone. Bioluminescence of the reporters was simultaneously triggered by single injection of Ca2+ solution, divided using band-pass optical filters and measured with a two-channel photometer. The sensitivity of simultaneous bioluminescence assay was close to that of a separate radioimmunoassay. Figure Two kinds of Ca2+-regulated photoprotein obelin with altered color of bioluminescence were obtained and applied in dual-color simultaneous immunoassay of two gonadotropic hormones.
Keywords: Ca2+-regulated photoprotein; Bioluminescence; Dual-color assay
Real-life application of a QCM-based e-nose: quantitative characterization of different plant-degradation processes by Peter A. Lieberzeit; Abdul Rehman; Bita Najafi; Franz L. Dickert (2897-2903).
Continuous surveillance of composting processes would enable a feedback loop to be obtained for both analysis and process control. For this purpose, we designed e-noses based on a six-electrode quartz-crystal microbalance (QCM) array coated with affinity materials and molecularly imprinted polymers (MIP). They enable quantitative monitoring of volatile organic compounds (VOCs) emitted directly in a compost bin and are highly suitable tools for achieving on-line characterization of the degradation processes occurring. During grass and pine composting (duration 14 days and 40 days, respectively), we observed concentrations of up to 250 ppm of esters, 700 ppm of alcohols, 250 ppm of terpenes, and 90% relative humidity directly on-line with such a system and could validate the data off-line by GC-MS. The sensor also gave direct insight into the differences between the two composting batch types. Besides duration, during grass composting larger amounts of alcohols are emitted whereas relative amount of terpenes is twice as high for pine composting. Detailed correlation of the sensor and the GC-MS data allows approximate estimation of the sensitivity of the sensor materials towards analyte classes such as, e.g., aliphatic alcohols or terpenes. Figure Mass sensitive sensor arrays coated with different molecularly imprinted and affinity materials are a highly suitable tool for quantitatively monitoring solvent patterns during composting procedures on-line in a composter headspace.
Keywords: Sensor array; QCM; Composting; Molecular imprinting; GC-MS
Development of a selective molecularly imprinted polymer-based solid-phase extraction for indomethacin from water samples by Tao Yang; Ya-Hui Li; Shuang Wei; Yuan Li; Anping Deng (2905-2914).
A selective molecularly imprinted solid-phase extraction (MISPE) for indomethacin (IDM) from water samples was developed. Using IDM as template molecule, acrylamide (AM) or methacrylic acid (MAA) as functional monomer, ethylene dimethacrylate (EDMA) as crosslinker, and bulk or suspension polymerization as the synthetic method, three molecularly imprinted polymers (MIPs) were synthesized and characterized with a rebinding experiment. It was found that the MIP of AM-EDMA produced by bulk polymerization showed the highest binding capacity for IDM, and so it was chosen for subsequent experiments, such as those testing the selectivity and recognition binding sites. Scatchard analysis revealed that at least two kinds of binding sites formed in the MIP, with the dissociation constants of 7.8 μmol L−1 and 127.2 μmol L−1, respectively. Besides IDM, three structurally related compounds — acemetacin, oxaprozin and ibuprofen — were employed for selectivity tests. It was observed that the MIP exhibited the highest selective rebinding to IDM. Accordingly, the MIP was used as a solid-phase extraction sorbent for the extraction and enrichment of IDM in water samples. The extraction conditions of the MISPE column for IDM were optimized to be: chloroform or water as loading solvent, chloroform with 20% acetonitrile as washing solution, and methanol as eluting solvent. Water samples with or without spiking were extracted by the MISPE column and analyzed by HPLC. No detectable IDM was observed in tap water and the content of IDM in a river water sample was found to be 1.8 ng mL−1. The extraction efficiencies of the MISPE column for IDM in spiked tap and river water were acceptable (87.2% and 83.5%, respectively), demonstrating the feasibility of the prepared MIP for IDM extraction. Figure Molecularly imprinted polymer-based solid-phase extraction for indomethacin
Keywords: Molecularly imprinted polymer (MIP); Indomethacin (IDM); Solid-phase extraction (SPE); Water sample
Rapid determination of amide herbicides in environmental water samples with dispersive liquid–liquid microextraction prior to gas chromatography–mass spectrometry by Ru-Song Zhao; Chun-Peng Diao; Xia Wang; Ting Jiang; Jin-Peng Yuan (2915-2921).
This paper describes a novel, simple and environmentally friendly method for rapid determination of the amide herbicides metoalchlor, acetochlor, and butachlor. It is based on dispersive liquid-liquid microextraction and gas chromatography–mass spectrometry. Factors that may influence the enrichment efficiency, such as type and volume of extraction solvent, type and volume of dispersive solvent, extraction time, and content of NaCl, were investigated and optimized in detail. Under the optimum conditions, the limits of detection of metoalchlor, acetochlor, and butachlor were 0.02, 0.04, and 0.003 μg L−1, respectively. The experimental results indicated that there was linearity over the range 0.1–50 μg L−1 and good reproducibility with relative standard deviations over the range 1.6–3.0% (n = 5). The proposed method has been applied for the analysis of real-world water samples, and satisfactory results were achieved. Average recoveries of spiked herbicides were in the range 80.3–108.8%. All of these indicated that the developed method would be an efficient method for simultaneous determination of the three herbicides in environmental water samples.
Keywords: Dispersive liquid-liquid microextraction; Gas chromatography-mass spectrometry; Metoalchlor; Acetochlor; Butachlor
Reactive matrix clean-up of naturally occurring perylene in young lignite by Torsten Spitzer (2923-2929).
Young lignite from two locations in Sendai City, Japan, were analyzed for polynuclear aromatic compounds (PACs). Lignite samples were extracted with toluene and PACs were isolated by a compound-class-selective, reactive matrix clean-up. This clean-up separates all compounds of polynuclear aromatic structure, for example hydrocarbons and ketones and their primary metabolites, from interfering organic compounds. The result of this isolation procedure is, therefore, a group of a large number of polycyclic compounds with different functional groups. Further analysis is done by glass capillary gas chromatography. Perylene was identified as the only compound obtained by the reactive matrix clean-up of lignite samples from both locations. Concentrations were in the range of 1–10 mg kg−1. Conversely, PACs resulting from particulate emissions from fossil fuel combustion always contain isomeric PAHs (polynuclear aromatic hydrocarbons) with some polycyclic aromatic ketones and thousands of primary and secondary metabolites at very low concentrations. This was demonstrated for a sample of urban air particulate matter, which is the source of PAC contamination of surface soil. The absence of accompanying PAHs and polynuclear aromatic ketones in the lignite samples confirms that perylene did not originate from a combustion process. It is assumed that the high concentrations of perylene are the result of a reduction of perylene quinone. Thus, the high perylene content of the lignite samples investigated is of biogenic origin.
Keywords: Reactive matrix clean-up; Perylene; Lignite; PAHs; PACs; Particulate matter; Mean capacity factor
A green flow-based procedure for fluorimetric determination of acid-dissociable cyanide in natural waters exploiting multicommutation by Carlos M. C. Infante; Jorge C. Masini; Fábio R. P. Rocha (2931-2936).
A flow system designed with solenoid valves is proposed for determination of weak acid dissociable cyanide, based on the reaction with o-phthalaldehyde (OPA) and glycine yielding a highly fluorescent isoindole derivative. The proposed procedure minimizes the main drawbacks related to the reference batch procedure, based on reaction with barbituric acid and pyridine followed by spectrophotometric detection, i.e., use of toxic reagents, high reagent consumption and waste generation, low sampling rate, and poor sensitivity. Retention of the sample zone was exploited to increase the conversion rate of the analyte with minimized sample dispersion. Linear response (r = 0.999) was observed for cyanide concentrations in the range 1–200 µg L−1, with a detection limit (99.7% confidence level) of 0.5 µg L−1 (19 nmol L−1). The sampling rate and coefficient of variation (n = 10) were estimated as 22 measurements per hour and 1.4%, respectively. The results of determination of weak acid dissociable cyanide in natural water samples were in agreement with those achieved by the batch reference procedure at the 95% confidence level. Additionally to the improvement in the analytical features in comparison with those of the flow system with continuous reagent addition (sensitivity and sampling rate 90 and 83% higher, respectively), the consumption of OPA was 230-fold lower.
Keywords: Flow analysis; Green analytical chemistry; Multicommutation; Cyanide; Fluorimetry; Waters
Monitoring of substrate and product concentrations in acetic fermentation processes for onion vinegar production by NIR spectroscopy: value addition to worthless onions by J. M. González-Sáiz; I. Esteban-Díez; C. Sánchez-Gallardo; C. Pizarro (2937-2947).
Wastes and by-products of the onion-processing industry pose an increasing disposal and environmental problem and represent a loss of valuable sources of nutrients. The present study focused on the production of vinegar from worthless onions as a potential valorisation route which could provide a viable solution to multiple disposal and environmental problems, simultaneously offering the possibility of converting waste materials into a useful food-grade product and of exploiting the unique properties and health benefits of onions. This study deals specifically with the second and definitive step of the onion vinegar production process: the efficient production of vinegar from onion waste by transforming onion ethanol, previously produced by alcoholic fermentation, into acetic acid via acetic fermentation. Near-infrared spectroscopy (NIRS), coupled with multivariate calibration methods, has been used to monitor the concentrations of both substrates and products in acetic fermentation. Separate partial least squares (PLS) regression models, correlating NIR spectral data of fermentation samples with each kinetic parameter studied, were developed. Wavelength selection was also performed applying the iterative predictor weighting–PLS (IPW-PLS) method in order to only consider significant spectral features in each model development to improve the quality of the final models constructed. Biomass, substrate (ethanol) and product (acetic acid) concentration were predicted in the acetic fermentation of onion alcohol with high accuracy using IPW-PLS models with a root-mean-square error of the residuals in external prediction (RMSEP) lower than 2.5% for both ethanol and acetic acid, and an RMSEP of 6.1% for total biomass concentration (a very satisfactory result considering the relatively low precision and accuracy associated with the reference method used for determining the latter). Thus, the simple and reliable calibration models proposed in this study suggest that they could be implemented in routine applications to monitor and predict the key species involved in the acetic fermentation of onion alcohol, allowing the onion vinegar production process to be controlled in real time.
Keywords: Acetic fermentation; Onion vinegar; Valorisation; NIR spectroscopy; Multivariate calibration; Variable selection
A multivariate approach for the simultaneous determination of losartan potassium and hydrochlorothiazide in a combined pharmaceutical tablet formulation by Rubén M. Maggio; Patricia M. Castellano; Teodoro S. Kaufman (2949-2955).
A convenient new method for the simultaneous determination of losartan potassium and hydrochlorothiazide, with minimum sample pretreatment, is described. The procedure, based on the multivariate analysis of spectral data in the 220−274 nm region by the partial least squares algorithm, is linear in the concentration range 1.06−5.70 mg L−1 for hydrochlorothiazide and 4.0−22.2 mg L−1 for losartan. It is simple, rapid and robust, allowing accurate and precise results, with drug recovery rates of 99.3 and 100.4% and relative standard deviations of 1.7 and 1.0% obtained for hydrochlorothiazide and losartan, respectively. The method was applied to the simultaneous determination of both analytes in tablets, and it provided good results which were in statistical agreement with those provided by independent HPLC analyses of the samples. The method has also been successfully applied for the construction of drug dissolution profiles of a commercial pharmaceutical preparation containing both analytes. Figure A UV-PLS method for the simultaneous determination of losartan potassium and hydrochlorothiazide in pharmaceutical tablet formulations has been developed and validated
Keywords: Chemometric method; Drug content; Dissolution profiles; Simultaneous determination; Losartan potassium; Hydrochlorothiazide