The history from bio-imaging to live single-cell mass spectrometry (MS) is herein reviewed. The limitation of the current bio-imaging method is probing only known molecules, and a method for finding new molecules is needed for cells which, however, show individual behaviors even in the same incubation dish. Single-cell MALDI-TOF/MS has been developed, but it can detect only molecules that can be easily ionized, and not be exhaustive. Recently, the contents of a single cell have been sucked out by a nano-electro spray tip, and directly introduced into MS by nano-spray ionization. Thousands of molecular peaks have been successfully and exhaustively detected, and an extraction method for key molecules was also developed. This new method is now being widely applied to explore site- or state-specific molecules in various aspects of cell dynamisms.
D-Amino acids, the enantiomers of L-amino acids, are candidates to be novel physiologically active substances and the biomarkers in mammals. However, the amounts of D-amino acids in the tissues and physiological fluids are extremely small in most cases, and sensitive and selective analytical methods are needed for their determination. In the present manuscript, we review the analytical technologies including our recent advances for the determinations of small amounts of D-amino acids in mammals and the applications to clarify their physiological meanings.
Biomolecules, especially large polymeric molecules such as enzymes and antibodies, mediate various biological functions, including biochemical reactions and molecular recognition, with high reactivity, efficiency, selectivity and accuracy. Many researchers have investigated methods to take advantage of these characteristics in analytical devices. One way to accomplish this is to immobilize biomolecules in the devices. For example, biomolecules have been immobilized by means of silica sol-gel technology and used for basic research in the food and pharmaceutical industries. Proteins encapsulated by this method retain their structure and biological activity for a prolonged period. This review describes methodologies for immobilization of biomolecules and the applications of sol-gel technology to analytical devices, especially flow-through systems.
High-speed analyses of proteins by microchip isoelectric focusing (MCIEF) were investigated on straight channel chips. By employing a commercially available microchip electrophoresis instrument equipped with a linear-imaging UV detector, sample proteins focused in a separation channel could be detected without a mobilization step. Typically, standard proteins were focused within 210 s at different positions in a separation channel with its total length of 38.8 mm on the basis of their pI values. The linear relationship between the focused position of each protein and its pI value was observed in the pH range from 6 to 10. The formation process of a pH gradient in the microchannel and the effects of the applied voltage and the channel length on the MCIEF separation were also investigated.
Ion mobility-mass spectrometry (IM-MS) was applied to analyses of isomeric oligosaccharides linked by α1-4, α1-6, β1-3, and β1-4 glycosyl linkages. Negative-ion electrospray ionization (ESI) quadrupole IM time-of-flight mass spectrometry allowed the combination of IM separation and collision-induced dissociation (CID) MS/MS product ion analysis. Multimer formations of hexa-saccharide linkage isomers differ from each other, and their molecular shapes were analyzed by ion mobility spectrometry (IMS). The product ion spectra of the oligosaccharide isomers were measured by negative-ion CID-MS/MS for each IM separated peak. The spectrum for each isomer was distinct, and their corresponding linkage structures were identified by MS/MS analysis.
A candidate reference measurement procedure involving isotope dilution coupled with gas chromatography-mass spectrometry (GC-MS) has been developed and critically evaluated. An isotopically labeled internal standard, cortisol-d2, was added to a serum sample. After equilibration, solid-phase extractions (SPE) for sample preparation and derivatization with heptafluorobutyric anhydride (HFBA) were performed for GC-MS analysis. The limit of detection (LOD) and the limit of quantification (LOQ) were 5 and 20 ng g−1, respectively. The recovery of the added cortisol ranged from 99.8 to 101.0%. Excellent precision was obtained with a within-day variation (RSD) of 0.7% for GC-MS analysis. The accuracy of the measurement was evaluated by comparing of results of this reference measurement procedure on lyophilized human serum reference materials for cortisol (European Reference Materials (ERM)-DA 192) as Certified Reference Materials (CRMs). The results of this method for total cortisol agreed with the certified values within some uncertainty. This method, which demonstrates simply, easy, good accuracy, high precision, and is free from interferences from structural analogues, qualifies as a reference measurement procedure.
A new class of DNA probes having a mechanically detectable tag is reported. The DNA probe, which consists of a single-stranded recognition sequence and a double-stranded circular DNA entity, was prepared by polymerase reaction. M13mp18 single strand and a 32mer oligodeoxynucleotide whose 5′-end is decorated with the recognition sequence were used in combination as template and primer, respectively. We have successfully demonstrated that the DNA probe is useful for bioanalytical purposes: by deliberately attaching target DNA molecules onto Au(111) substrates and by mechanically reading out the tag-entity using a high-resolution microscopy including atomic force microscopy, visualization/detection of the individual target/probe DNA conjugate was possible simply yet straightforwardly. The present DNA probe can be characterized as a 100%-nucleic acid product material. It is simply available by one-pod synthesis. A surface topology parameter, image roughness, has witnessed its importance as a quantitative analysis index with particular usability in the present visualization/detection method.
A rapid, sensitive immunoassay based on a surface plasmon resonance sensor in a flow system for the determination of alkylphenol polyethoxylate (APEO) is described. The method is based on an indirect competitive reaction between an anti-APEO antibody in the sample solution and APEO immobilized on a sensor chip and APEO in the same sample solution. A sensor chip was prepared by immobilizing an APEO-horseradish peroxidase (APEO-HRP) conjugate on the thin gold film of the sensor chip. The adsorption constants for the APEO-HRP conjugate on the sensor chip and the surface density of the APEO-HRP adsorbed on the sensor chip at the saturated state were estimated to be 4.7 × 105 M−1 and 5.0 × 10−14 mol/mm2, respectively, using a Langmuir adsorption isotherm equation and results from the adsorption experiments. The affinity constants for the immunocomplexes of the anti-APEO antibody with the APEO conjugate on the sensor chip and for APEO in the sample solution were estimated to 2.0 × 106 and 5.1 × 106 M−1, respectively. A typical sigmoid calibration curve for APEO was obtained in the concentration range from 1 ppb to 1000 ppb. The detection limit, defined as the concentration of APEO, at which 85% of the sensor signal was observed, was ca. 10 ppb. The assay was applied to the determination of APEO in tap water in conjunction with a solid phase extraction pretreatment; APEO levels of approximately 50 ppt were successfully determined.
A simple method for determining short-chain fatty acids (SCFAs) in rat and human feces was developed using high-performance liquid chromatography with electrochemical detection (HPLC-ECD). A two-channel HPLC-ECD system was fabricated using an ion exclusion column and an electrochemical detector with a glassy carbon working electrode. Aqueous solutions of 0.1 mM HClO4 and of ethanol containing 2-methyl-1,4-naphthoquinone served as a mobile phase and a quinone solution, respectively. Peak areas for lactic, acetic, propionic, butyric, isovaleric, and valeric acids at a detection potential of −0.9 V vs. an Ag/AgCl electrode showed a linear relationship with the acid amount in the range 0.1 to 40 nmol. Standard acids at 4 nmol were determined ten times with relative standard deviations (RSD) of less than 2.0%. The analytical results of healthy human feces were measured within 35 min. RSD (n = 5) in all SCFAs were less than 2.7%, and recoveries of SCFAs were more than 92%. The present method was characterized by reproducibility with the simple and rapid procedure without derivatization of analytes, and it has the potential for clinical and biomedical applications.
A uric acid biosensor based on the direct electron transfer of a hemoglobin-encapsulated chitosan-modified glassy carbon electrode was developed for a highly sensitive and selective analysis in urine samples. The modified electrode was prepared by the encapsulation of hemoglobin and uricase in a chitosan matrix. The hydrogen peroxide produced from the catalytic oxidation of uric acid by uricase was reduced electrocatalytically by immobilized hemoglobin and used to obtain a sensitive amperometric response to uric acid. The linear response of the uric acid concentrations ranged from 2.00 to 30.0 μM with a correlation of 0.9982, the detection limit of uric acid was estimated to be 0.85 μM at a signal/noise ratio of 3. The uric acid biosensor can efficiently exclude the interference of commonly coexisted ascorbic acid, dopamine, epinephrine, etc. The relative standard deviation was under 2.56% (n = 5) for the determination of real samples. This biosensor is satisfactory for the determination of human urine samples compared with the HPLC-UV method.
The interaction between double-strand calf thymus gland DNA (ds-DNA) and Al(III) was studied by using differential pulse voltammetry (DPV) at a hanging mercury drop electrode (HMDE), Raman spectrometry and circular dichroism (CD) spectra. It was shown that at neutral pH ds-DNA did not produce any cathodic peak at the HMDE in the potential window from −550 to −2000 mV vs. SCE. However, in the presence of Al(III), a cathodic peak was generated at about −1660 mV, which is ascribed to a reduction of adenine and cytosine residues of single denatured DNA (sd-DNA). It was concluded that ds-DNA was completely denatured to sd-DNA by Al(III) at a neutral pH. The apparent denaturing kinetic velocity constants of ds-DNA by Al(III) were derived from linear increases of the cathodic peak currents with time. When [Al(III)]×[OH−]3 ≥ 2 × 10−26, the precipitation of Al(OH)3 was observed and identified by the Raman spectrum, and inductively coupled plasma atomic emission spectrometry (ICP-AES). CD spectra showed that the B-type of structure conformations of ds-DNA and related sd-DNA did not change with the increment of Al(III) from 5.0 × 10−7 to 1.0 × 10−5 M, but the corresponding absorption strengths increased. The related physiological significances and possible applications of the observations were considered.
A simple capillary enzymatic biosensor was developed. This was prepared by simply coating a dissolvable membrane containing enzyme/s on the inner wall of a square glass capillary. An easy measurement was carried out by capillary force sample introduction with concurrent enzyme release and a reaction with a certain substrate. Enzyme-release capillary (ERC) biosensors showed long-term storage stability of at least two weeks for a β-galactoside derivative and glucose. Moreover, this could be integrated on a capillary-assembled microchip (CAs-CHIP) to broaden its multiple analyte sensing potential for clinical diagnostic applications.
A reporter gene assay (RGA) that uses a mouse liver recombinant Hepa1c1c7 containing the firefly luciferase gene was developed to screen for dioxins in human plasma. For a high-sensitivity method, the addition of cycloheximide to the culture medium brought about a fivefold increase in the sensitivity. The detection limit was 0.1 pg/μL/well. Aryl hydrocarbon receptor (AhR) binding affinity factors (AhR-BAF), calculated from the effect concentration 50 (EC50) value, showed approximately the same values as those in WHO-TEF (2006). A significant correlation between RGA and the conventional gas chromatography/mass spectrometry (GC/MS) method was obtained.
A simple and highly sensitive method that involves miniaturized hollow fiber assisted liquid-phase microextraction (HF-LPME) with in situ acyl derivatization and GC-MS was developed for the determination of benzophenone (BP) and related compounds in human urine samples. The limits of detection (S/N = 3) and quantification (S/N > 10) of BPs in human urine samples are 0.01 to 0.05 and 0.05 to 0.2 ng ml−1, respectively. The average recoveries of BPs (n = 5) in human urine samples spiked with 10 and 50 ng ml−1 BPs are 93.1 to 106.7% (RSD: 1.5 to 8.4%) and 96.3 to 101.5% (RSD: 3.0 to 7.7%), respectively. When the proposed method was applied to human urine samples, BPs were detected at the sub ng ml−1 level.
Sugar analysis by postcolumn labeling using phenylhydrazine with high-performance liquid chromatography is herein introduced. Phenylhydrazine reacts not only with the reducing end of oligosaccharides, but also with mono-sugar components with degraded glycosyl bonds. N-Acethylhexosamine is highly sensitive at a sub-pico molecular range, and this method is useful for detecting glycans on glycoproteins. The detection limit of fetuin, a protein possessing 3 N-glycans and 3 O-glycans, was 0.17 pmol by infusion analysis, and chromatograms of glycopeptides separated on an ODS column were obtained. These results suggest that this method is useful for the preparation of glycopeptides for glyco-proteomics research.
The thermally controlling fluorescence intensity of a molecular probe for cellular imaging has been investigated. A reversible temperature-induced phase transition of N-isopropylacrylamide/fluorescein copolymers [poly(NIPAAm-co-FL)] was used as a molecular switch to control the fluorescence intensity of the imaging probe. The copolymer displayed environmentally sensitive fluorescence properties, in which the fluorescence intensity changed with the response to both the temperature and the pH. Utilizing these features, we monitored the thermal-aggregation process of BSA by the fluorescence resonance energy transfer (FRET) method. Additionally, the cellular uptake in RAW264.7 cells of poly(NIPAAm-co-FL) conjugated with lipid was studied using a confocal laser scanning microscope.