Analytica Chimica Acta (v.969, #C)

Rapid imaging and detection of circulating tumor cells using a wide-field fluorescence imaging system by Tomoko Yoshino; Kaori Takai; Ryo Negishi; Tatsuya Saeki; Hisashige Kanbara; Yoshihito Kikuhara; Tadashi Matsunaga; Tsuyoshi Tanaka (1-7).
Circulating tumor cells (CTCs) provide potentially accessible in vivo sources of metastatic cancer cells. As such, considerable focus has been placed on analyzing the genetics of single-CTCs. Prior to these analyses, however, CTCs must first be detected within the blood samples of cancer patients. Current methods for detection of CTCs by fluorescence microscopy require the analysis of hundreds of images per blood sample, making this a time-consuming process that creates a bottleneck in CTC analysis. In this study, we therefore developed a wide-field fluorescence imaging system for rapid CTC detection. For these analyses, CTCs were first isolated using the microcavity array (MCA), a micro-sized filter for CTC recovery that separates cells based on differences in cell size and deformability. Notably, the proposed imaging system enabled rapid (∼10 s) visualization of all stained cells within the 6 mm × 6 mm MCA field via one-shot imaging. Furthermore, the morphology of the cells in the resulting images accurately reflected that observed by conventional microscopy. In total, isolation and detection of CTCs using the MCA combined with our novel wide-field fluorescence imaging system was achieved within 1 h. Thus, our proposed system will provide rapid CTC detection system.Display Omitted
Keywords: Circulating tumor cells; Microcavity array; One shot imaging; Wide-field imaging; Fluorescence; CMOS sensor;

Novel hybridization proximity-regulated catalytic DNA hairpin assembly strategy has been proposed for electrochemical immunoassay based on in situ DNA template-synthesized Pd nanoparticles as signal label. The DNA template-synthesized Pd nanoparticles were characterized with atomic force microscopic and X-ray photoelectron spectroscopy. The highly efficient electrocatalysis by DNA template synthesized Pd nanoparticles for NaBH4 oxidation produced an intense detection signal. The label-free electrochemical method achieved the detection of carcinoembryonic antigen (CEA) with a linear range from 10−15 to 10−11 g mL−1 and a detection limit of 0.43 × 10−15 g mL−1. Through introducing a supersandwich reaction to increase the DNA length, the electrochemical signal was further amplified, leading to a detection limit of 0.52 × 10−16 g mL−1. And it rendered satisfactory analytical performance for the determination of CEA in serum samples. Furthermore, it exhibited good reproducibility and stability; meanwhile, it also showed excellent specificity due to the specific recognition of antigen by antibody. Therefore, the DNA template synthesized Pd nanoparticles based signal amplification approach has great potential in clinical applications and is also suitable for quantification of biomarkers at ultralow level.A novel label-free and enzyme-free electrochemical immunoassay based on proximity hybridization-regulated catalytic DNA hairpin assemblies for recycling of the CEA.Display Omitted
Keywords: Palladium nanoparticles; Proximity hybridization; Sensors; Immunoassay;

Quantitative determination of polysulfide in albumins, plasma proteins and biological fluid samples using a novel combined assays approach by Mayumi Ikeda; Yu Ishima; Akitomo Shibata; Victor T.G. Chuang; Tomohiro Sawa; Hideshi Ihara; Hiroshi Watanabe; Ming Xian; Yuya Ouchi; Taro Shimizu; Hidenori Ando; Masami Ukawa; Tatsuhiro Ishida; Takaaki Akaike; Masaki Otagiri; Toru Maruyama (18-25).
Hydrogen sulfide (H2S) signaling involves polysulfide (RSSnSR′) formation on various proteins. However, the current lack of sensitive polysulfide detection assays poses methodological challenges for understanding sulfane sulfur homeostasis and signaling. We developed a novel combined assay by modifying Sulfide Antioxidant Buffer (SAOB) to produce an “Elimination Method of Sulfide from Polysulfide” (EMSP) treatment solution that liberates sulfide, followed with methylene blue (MB) sulfide detection assay. The combined EMSP-MB sulfide detection assay performed on low molecular weight sulfur species showed that sulfide was produced from trisulfide compounds such as glutathione trisulfide and diallyl trisulfide, but not from the thiol compounds such as cysteine, cystine and glutathione. In the case of plasma proteins, this novel combined detection assay revealed that approximately 14.7, 1.7, 3.9, 3.7 sulfide mol/mol released from human serum albumin, α1-anti-trypsin, α1-acid glycoprotein and ovalbumin, respectively, suggesting that serum albumin is a major pool of polysulfide in human blood circulation. Taken together with the results of albumins of different species, the liberated sulfide has a good correlation with cysteine instead of methionine, indicating the site of incorporation of polysulfide is cysteine. With this novel sulfide detention assay, approximately 8,000, 120 and 1100 μM of polysulfide concentrations was quantitated in human healthy plasma, saliva and tear, respectively. Our promising polysulfide specific detection assay can be a very important tool because quantitative determination of polysulfide sheds light on the functional consequence of protein-bound cysteine polysulfide and expands the research area of reactive oxygen to reactive polysulfide species.Display Omitted
Keywords: Polysulfide; Sulfide; Human serum albumin; Reactive oxygen species; Reactive polysulfide species; Redox signaling;

The comprehensive description of complex mixtures such as bio-oils is required to understand and improve the different processes involved during biological, environmental or industrial operation. In this context, we have to consider how different ionization sources can improve a non-targeted approach. Thus, the Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) has been coupled to electrospray ionization (ESI), laser desorption ionization (LDI) and atmospheric pressure photoionization (APPI) to characterize an oak pyrolysis bio-oil. Close to 90% of the all 4500 compound formulae has been attributed to CxHyOz with similar oxygen class compound distribution. Nevertheless, their relative abundance in respect with their double bound equivalent (DBE) value has evidenced significant differences depending on the ion source used. ESI has allowed compounds with low DBE but more oxygen atoms to be ionized. APPI has demonstrated the efficient ionization of less polar compounds (high DBE values and less oxygen atoms). The LDI behavior of bio-oils has been considered intermediate in terms of DBE and oxygen amounts but it has also been demonstrated that a significant part of the features are specifically detected by this ionization method. Thus, the complementarity of three different ionization sources has been successfully demonstrated for the exhaustive characterization by petroleomic approach of a complex mixture.Display Omitted
Keywords: Non-targeted analysis; Bio-oils; Petroleomic approach; Ultra-high resolution mass spectrometry;

Accurate and sensitive determination of molar fractions of 13C-Labeled intracellular metabolites in cell cultures grown in the presence of isotopically-labeled glucose by Mario Fernández-Fernández; Pablo Rodríguez-González; David Hevia Sánchez; Pedro González-Menéndez; Rosa M. Sainz Menéndez; J. Ignacio García Alonso (35-48).
This work describes a methodology based on multiple linear regression and GC-MS for the determination of molar fractions of isotopically-labeled intracellular metabolites in cell cultures. Novel aspects of this work are: i) the calculation of theoretical isotopic distributions of the different isotopologues from an experimentally measured value of % 13C enrichment of the labeled precursor ii) the calculation of the contribution of lack of mass resolution of the mass spectrometer and different fragmentation mechanism such as the loss or gain of hydrogen atoms in the EI source to measure the purity of the selected cluster for each metabolite and iii) the validation of the methodology not only by the analysis of gravimetrically prepared mixtures of isotopologues but also by the comparison of the obtained molar fractions with experimental values obtained by GC-Combustion-IRMS based on 13C/12C isotope ratio measurements. The method is able to measure molar fractions for twenty-eight intracellular metabolites derived from glucose metabolism in cell cultures grown in the presence of 13C-labeled Glucose. The validation strategies demonstrate a satisfactory accuracy and precision of the proposed procedure. Also, our results show that the minimum value of 13C incorporation that can be accurately quantified is significantly influenced by the calculation of the spectral purity of the measured cluster and the number of 13C atoms of the labeled precursor. The proposed procedure was able to accurately quantify gravimetrically prepared mixtures of natural and labeled glucose molar fractions of 0.07% and mixtures of natural and labeled glycine at molar fractions down to 0.7%. The method was applied to initial studies of glucose metabolism of different prostate cancer cell lines.Display Omitted
Keywords: Glucose metabolism; 13C-labeled compounds; Multiple linear regression; Cell cultures; Prostate cancer;

Fluorescence probe for hypochlorous acid in water and its applications for highly lysosome-targetable live cell imaging by Xiaojie Jiao; Chang Liu; Qing Wang; Kun Huang; Song He; Liancheng Zhao; Xianshun Zeng (49-56).
Hypochloric acid (HOCl) plays important roles in cell signaling and homeostasis, such as anti-inflammation and immune regulation, pathogen response and so on. Accordingly, direct detection of HOCl at the organelle level is important for investigation of the complex contributions of HOCl to human health. In the present study, a water soluble lysosome-targeting fluorescent probe Lyso-1 bearing a hydrazone moiety as a HOCl-responsive site and a morpholine unit as a lysosomal-targeting group has been synthesized and evaluated for its ability to image lysosomal HOCl. The probe Lyso-1, based on a novel HOCl-promoted hydrazone oxidation strategy, showed a highly selective fluorescent off/on response to HOCl with the various reactive oxygen species in water. With increasing amount of ClO from 0.5 to 2.5 μM, a linear correlation between the fluorescence intensity (570 nm) of Lyso-1 and [ClO] was found, and the regression equation was y = 96.65 + 110.2068[ClO] with a linear coefficient R of 0.9920. The detection limit is determined to be 60 nM. Lyso-1 demonstrated a perfect lysosomal targetable ability, and was successfully applied to image of exogenous, endogenous produced lysosomal HOCl in live L929 cells. The success of subcellular imaging indicated that the lysosome-targetable probe Lyso-1 could be used in further applications for the investigation of biological functions and pathological roles of HClO at organelle levels.Display Omitted
Keywords: Fluorescent probe; Rhodamine; Hypochlorous acid; Lysosome; Imaging;

This paper describes a low-cost, sensitive, visual and rapid immunochromatographic assay method on cotton thread for carcinoembryonic antigen (CEA) detection by using novel carbon nanotube/gold nanoparticles (CNT/GNPs) nanocomposite reporter probe. CEA, a lung cancer protein biomarker, was used as analyte to demonstrate the principle of the immunochromatographic assay on cotton thread biosensor. In the presence of target CEA, the decreasing aggregation amount of CNT/GNPs nanocomposite reporter probes on the test zone induced directly readout by naked eye. Meanwhile, quantitative detection could be performed conveniently with a commercial available scanner. The performance with respect to sensitivity of the method was greatly improved by 2–3 magnitudes comparing with traditional gold nanoparticles (GNPs) or carbon nanotubes (CNTs) as reporter probe. Under optimal conditions, the biosensor was capable of detecting 2.32 ng/mL CEA (S/N ≥ 3) which is sensitive enough for clinical diagnosis. These results indicated the novel CNT/GNPs nanocomposite reporter probe based immunochromatographic assay on cotton thread is particularly suitable for point-of-care (POC) diagnostics in resource-limited regions.Display Omitted
Keywords: Carbon nanotube/Gold nanoparticles nanocomposite probe; Cotton thread; Point-of-care diagnosis; Immunochromatographic assay;

Escherichia coli adhesive coating as a chiral stationary phase for open tubular capillary electrochromatography enantioseparation by Qifeng Fu; Kailian Zhang; Die Gao; Lujun Wang; Fengqing Yang; Yao Liu; Zhining Xia (63-71).
Bacteria, the microorganism with intrinsic chirality, have numerous fascinating chiral phenomena such as various chirality-triggered biological processes and behaviors. Herein, bacteria were firstly explored as novel chiral stationary phases in open-tubular capillary electrochromatography (OT-CEC) for enantioseparation of fluoroquinolone enantiomers and simultaneous separation of six fluoroquinolone antibiotics. The model strain, i.e. non-pathogenic Escherichia coli (E. coli) DH5α, was adhered onto the inner surface of positively charged polyethyleneimine (PEI) modified capillaries based on the bacterial adhesion characteristics and strong electrostatic interaction. The morphology and thickness of the bacteria adhesive coatings in the capillary were characterized by field emission scanning electron microscopy (FESEM) and atomic force microscopy (AFM). Baseline separation of ofloxacin and partial separation of lomefloxacin enantiomers could be achieved by the E. coli coated columns. The preparation parameters including the coating time and concentration of bacteria that affecting the chiral resolution were intensively investigated. The electrophoretic parameters, including pH, buffer concentration and applied voltage, were also optimized. The developed method was validated (linearity, LOD, LOQ, intra-day, inter-day and column-to-column repeatability and recovery) and successfully utilized for the quantitative analysis of ofloxacin enantiomers in the ofloxacin tablets. Moreover, only a slight decrease in the separation efficiency was observed after 90 consecutive runs on the E. coli@capillary. These results demonstrated that bacteria are promising stationary phases for chiral separation in CEC.Display Omitted
Keywords: Bacteria; Stationary phase; Chiral separation; Open-tubular capillary; Electrochromatography;

An ultra-specific and sensitive sandwich ELISA for imatinib using two anti-imatinib antibodies by Tetsuya Saita; Yuta Yamamoto; Kazuhisa Hosoya; Yutaro Yamamoto; Sakiko Kimura; Yutaka Narisawa; Masashi Shin (72-78).
The development of an immunoassay for a low-molecular-weight drug first requires the identification of specific antibodies that do not cross-react with the drug's metabolites. If two antibodies can simultaneously recognize the entire structure of the drug, we can then utilize them to establish an ultra-specific sandwich ELISA, free from interference due to the metabolic products of the drug. This paper reports an ultra-specific and sensitive sandwich ELISA for determination of the tyrosine kinase inhibitor imatinib using two anti-imatinib antibodies. The anti-imatinib antibodies were obtained by two partial structures of imatinib as haptens (2-(5-amino-2-methylanilino)-4-(3-pyridyl)pyrimidine and 4-{(4-methyl-1-piperazinyl)-methyl}-benzoate). Under optimized conditions, this sandwich ELISA shows a linear detection range from 64 pg mL−1 to 8 ng mL−1, and a limit of detection of approximately 64 pg mL−1 for 100-μL samples. The ELISA is specific to imatinib and while there was no cross-reactivity with the major metabolite N-desmethyl-imatinib, slight cross-reactivity was found with metabolite pyridine-N-oxide-imatinib. This assay demonstrated significantly lower cross-reactivity with metabolites than competitive ELISAs. Using this assay, drug levels were easily measured in rat blood after oral administration of imatinib via a single dose of 30 mg kg−1 or 100 mg kg−1. The levels in rat serum measured by this ELISA were comparable with those measured by HPLC, and there was a strong correlation between the values determined by the two methods (y = 0.983x + 0.081, R2 = 0.948). Thus, we have successfully developed the first specific and sensitive sandwich ELISA for imatinib using two anti-imatinib antibodies. This sandwich ELISA will be a valuable tool for therapeutic drug monitoring and pharmacokinetic studies of imatinib.Display Omitted
Keywords: Imatinib; Sandwich enzyme-linked immunosorbent assay; Low molecular weight drug; Competitive enzyme-linked immunosorbent assay; Therapeutic drug monitoring;