A novel rapid method based on a background fluorescence quenching immunochromatographic assay (bFQICA) was established to achieve simultaneously the quantitative detection of 3-methyl-quinoxaline-2-carboxylic acid (MQCA) and quinoxaline-2-carboxylic acid (QCA), which were efficiently extracted and enriched 4 times using immunomagnetic beads from pork. The analysis of field pork samples by bFQICA was in accordance with that of LC-MS/MS; especially, the proposed bFQICA exhibited great advantages in convenience and efficiency, which only takes 30 min for the detection of MQCA and QCA.

In this reported work, we achieved high-throughput, highly sensitive fluorescent analysis using an enzyme-linked immunosorbent assay (ELISA) that employed a metallic nanoparticle (NP)-immobilized 96-well plate. The immobilization of metallic NPs on a 96-well plate effectively amplified fluorescent signals of the assay. The silver (Ag) NP-immobilized plate showed the best fluorescent enhancement effect of all plates immobilized by metal NPs. Our results demonstrate the potential of applying Ag NPs to enhance the efficiency of direct and indirect ELISA by the labeling of targets.

Extracellular vesicles (EVs) play an important role in cell-to-cell communication by carrying molecular messages that reflect physiological and pathological conditions of the parent cells. EVs have been identified in all body fluids; and among them, urine stands out as a sample that is easy and inexpensive to obtain and can be collected over time to monitor changes. Various protocols have been established to study urinary extracellular vesicles (UEVs) and they have shown great potential as a biomarker source for clinical applications, not only for urological, but also non-urological diseases. Due to the high variability and low reproducibility of pre-analytical and analytical methods for UEVs, establishing a standardized protocol remains a challenge in the field of diagnosis. Here, we review UEV studies and present the techniques that are most commonly used, those that have been applied as new developments, and those that have the most potential for future applications. The workflow procedures from the sampling step to the qualitative and quantitative analysis steps are summarized along with advantages and disadvantages of the methodologies, in order to give consideration for choosing the most promising and suitable method to analyze human UEVs.

Immunosensors are used to provide simple, quick and effective ways to detect analytes for the diagnosis of a number of diseases. This work reports on a novel optical immunosensor based on thyroxine antibody labeled gold nanoparticles (anti-T4/C-AuNPs) for the sensitive detection of the thyroxine (T4) hormone. T4 hormone is a reliable diagnostic biomarker present in the human blood serum that is necessary for normal neural development and cellular metabolism. The free T4 hormone, a major key player of the total biologically active T4 hormone, in serum regulates the patient’s thyroid function. The immunosensor was synthesized by using amine functionalized cysteamine capped gold nanoparticles (C-AuNPs) of size 36 nm approximately. The T4 specific antibody (anti-T4) was covalently attached to C-AuNP by EDC-NHS (1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide) cross-linking chemistry. This anti-T4/C-AuNP bioconjugate was used for the analysis of the free thyroxine hormone in samples. The proposed method exhibited high performance immunosensors having a linear range of 0.52 to 65.1 pg/mL and the detection limit of 9.11 pg/mL.

A novel polymeric membrane sensor was developed by using 2-hydroxyethyl acrylate, trimethylolpropane triacrylate, (3-mercaptopropyl)trimethoxysilane, and poly(ethylene glycol)diacrylate for As(III) determination. Various parameters, like the pH, response time, the foreign ions, and concentration effects were investigated for deciding the optimum working conditions of the polymeric sensor. As a result of this investigation, the optimum pH was found to be 2, and the response time was found to be 30 s. The linear range of the sensor was 6.65 × 10⁻⁹ – 3.99 × 10⁻⁸ mol L⁻¹ (0.50 – 2.99 μg L⁻¹) with a detection limit of 2.33 × 10⁻⁹ mol L⁻¹ (0.18 μg L⁻¹). Soy flour and well-water samples were successfully analyzed with the developed sensor. The sensor can be used at least 100 times after regeneration. It could be reused by washing with purified water and showed good stability for 6 months.

A very simple and non-extractive spectrofluorometric method for the swift determination of aluminum at nano-trace levels using 2′,3,4′,5,7-pentahydroxyflavone (morin) has been developed. Morin reacts in a slightly acidic (0.005 – 0.025 M H₂SO₄) solution with aluminum in 20% ethanol to produce a highly fluorescent complex in aqueous solution, which has excitation and emission wavelengths of λ_ex = 270 and λ_em = 565 nm, respectively. Linear calibration graphs were obtained for 0.01 – 800 μg L⁻¹ of Al, providing a detection limit of 1 ng L⁻¹. The limit of quantification of the reaction system was 10 ng L⁻¹. The stoichiometric composition of chelate is 3:2 (Al:morin). The developed method was successfully used in the determination of aluminum in several Standard Reference Materials (SRM) as well as in some water, biological, hemodialysis solutions, food, pharmaceutical, soil sample, and complex synthetic mixtures. The results of the proposed method for biological and food analysis were found to be in excellent agreement with those obtained by AAS. The results of the proposed method for hemodialysis solutions were analogous with those obtained using the method described in British Pharmacopoeia within 95% confidence limits.

High-fat diet (HFD)-induced obesity is a primary risk factor for serious health problems. Although much research has been performed at the genomic level, lipidomic studies were limited. In this study, we aim to obtain a comprehensive profile of circulating plasma lipids, which are altered in rodent rat obesity by untargeted liquid chromatography–mass spectrometry. Rats fed with HFD for 8 weeks had increased body weight, liver and adipose tissue weight. The analysis results revealed that polyunsaturated fatty acids (PUFAs) and their corresponding phosphatidylcholine, phosphatidylinositol, and phosphatidylserine were significantly decreased in rats fed with HFD. In contrast, less unsaturated and ether type phosphatidylglycerols were increased. The triacylglycerides (TAGs) having saturated FA were increased in the HFD condition, whereas TAGs having PUFA were decreased. The levels of many plasma lipids were altered, and interestingly PUFA derived lipids were negatively associated with obesity. This signifies the importance of a PUFAs enriched diet to overwhelm obesity associated diseases.

A substrate of 4-nitrophenyl phosphate was enzymatically hydrolyzed by alkaline phosphatase (ALP) in a capillary tube, while an injected zone of the substrate was electrophoretically migrating in the separation buffer containing the enzyme by capillary electrophoresis (CE). During CE migration of the substrate from the start time of the electrophoresis to the detection time of the substrate, the substrate was continuously hydrolyzed by ALP to form a product of 4-nitrophenolate, and a plateau signal of 4-nitrophenolate was detected as a result of the zero-order kinetic reaction. The height of the plateau signal was directly related to the reaction rate, and it was used for the determination of a Michaelis–Menten constant through Lineweaver–Burk plots. Since the plateau signal is attributed to the dynamic formation of the product by the enzymatic reaction in CE, this analysis method is named as capillary electrophoresis/dynamic frontal analysis (CE/DFA). In CE/DFA, the CE separation is included on detecting the plateau signal, and the hydrolysis product before the sample injection is resolved from the dynamically and continuously formed product. The inhibition of the enzyme with the product is also eliminated in CE/DFA by the CE separation.

Sensitive and specific detection of nucleic acids or proteins, which act as biomarkers, is of great importance in disease diagnosis. By combing the concept and operation of an endonuclease-assisted target-responsive amplification method and peroxidase-mimic DNAzyme generated by terminal deoxynucleotidyl transferase (TdT), a novel and facile colorimetric biosensor was developed for DNA and protein. Target DNA and thrombin were chosen as representative biomolecules. The production of cleavage fragments can only be triggered by specific target binding and the following nicking process, which do not occur spontaneously. In the signal collection part, numerous guanine-rich DNA were produced through the prolongation of cleavage fragments by TdT and formed highly effective DNAzyme with hemin. In this novel amplification method, we succeeded in realizing sensitive and specific detection of target DNA and thrombin. Under optimal conditions, target DNA can be detected as low as 1 pM, and thrombin with a detection limit of 100 pM. The method also proves the potential versatility and feasibility of TdT-generated DNAzyme in various bio-analyses.

Chemical oxygen demand (COD) is important for water quality assessment as it represents the level of reductive organic pollution from eutrophication in aquatic systems. For surface water quality monitoring, permanganate is usually applied as an oxidizing reagent, and the routine COD_Mn determination is mostly achieved by titration method. However, this titration method is tedious and time consuming, and the results suffer from environmental temperature fluctuations and complicated operation techniques. In this study, a novel COD_Mn determination method was developed using gas-phase molecular absorption spectrometry equipped with an online automated digestion device for the first time. The effects of digestion temperature, digestion time and sulfuric acid content were thoroughly studied. This method exhibited good linearity (0.35 to 12 mg/L), a low detection limit (0.12 mg/L), and good RSD from various water samples (0.71 – 2.37%). When used for COD_Mn determination in routine water quality monitoring, this automated GPMAS can considerably improve analysis speed, efficiency, accuracy and stability compared to the traditional titration method.

Batchwise studies have demonstrated that the retention of acetylacetone (Haa) from an aqueous phase in non-endcapped octadecylsilyl (ODS) silica with silanol intentionally left (NEC) is stronger than the retention in endcapped ODS silica under ambient pressure, due to the enhanced wettability and the contribution of adsorption to silanol (–SiOH) as well as of the distribution into the ODS phase. Equilibrium analysis on solid phase extraction (SPE) of copper ion with Haa to NEC has indicated the adsorption of 1:1 species by the proton replacement reaction with silanol to give [–SiOCu(aa)] at relatively low Haa concentrations and the adsorption of [Cu(aa)₂] to silanol, as well as the distribution of [Cu(aa)₂] into the ODS phase. These adsorbed and distributed species of the same composition could be differentiated by ESR spectroscopy. The advantages of residual silanol in SPE of metal ions are discussed.

A parallel facing electrode (PFE) structure for adherent cell monitoring by electrochemical impedance spectroscopy (EIS) was developed, and its characteristics were investigated by both computer simulation and experiment. The PFE model consists of two facing gold electrode strips separated by 40 μm, and the area of its intersection is 500 × 500 μm. Computer simulation of EIS with adherent cells showed a distinct difference in solution resistance for different cell coverage, which was confirmed by experimental results using latex beads suspension. A well-defined relationship between solution resistance and cell coverage in our PFE is promising for quantitative evaluation of cell density, morphology and fatality.

The quantum size effect of CdTe quantum dots (QDs) has been one of the targets of extensive research concerning the optical spectroscopy of semiconductors, but little is known about their effects on electrochemiluminescence (ECL) behavior, especially in the anodic potential range. In this present study, water-soluble CdTe QDs with different sizes were synthesized with a microwave-assisted hydrothermal method. Upon electrochemical oxidation of the CdTe QD in the presence of tri-n-propylamine (TPrA) as a coreactant, two ECL signals, called ECL₁ and ECL₂, were observed at potentials corresponding to the oxidation of TPrA (at +0.8 V) and CdTe QDs (at +1.2 V), respectively. The relative intensity of ECL₁ significantly increased with increasing the particle size of CdTe QDs, and disappeared when the particle size was less than 2.4 nm. Upon an anodic potential of +0.8 V, TPrA is oxidized at the electrode surface, where the intermediate radical species like TPrA^•+ radical cation and CdTe QDs^•− radical anion are supposed to be formed to give the excited chemical species of CdTe QDs*. Possible ECL mechanisms are proposed from a view point of thermodynamics.

Melanin-related compounds in paraffin-embedded tissue specimens of basal cell carcinoma (BCC), seborrheic keratosis (SK), malignant melanoma (MM), and nevus pigmentosus (NP) were nondestructively investigated using permeability measurements (light penetration into pigmented lesion), X-band (9.4 GHz) electron paramagnetic resonance (EPR), and EPR imaging (EPRI). The paramagnetic species in BCC, SK, MM, and NP specimens were analyzed using intensity, linewidth, spectral pattern, and X-band EPRI. The EPR spectra of BCC, SK, and NP showed a single line pattern. The EPR signal intensities of the BCC, SK, and NP samples corresponded to the permeability values that are directly related to pigment color tone, except for MM. The correlation coefficient between EPR and permeability was supported by the high degree of linear relation in the range. We further analyzed MM and speculated that MM contains an additional signal of the pheomelanin radical. In MM and NP samples, two-dimensional (2D) EPRI revealed paramagnetic species distribution and different magnitudes. The paramagnetic (radical) species are directly related to the pigmented lesion site. To conclude, spectroscopic analyses suggest that pheomelanin-related compounds may exist in malignant melanoma.

The production of antibody-drug conjugates (ADCs) has been in great demand in the field of cancer therapeutics. Although cysteine-based conjugation is the most common and well known process for producing ADCs, multiple analytical methods are required for accurate drug-antibody ratio (DAR) determination due to the heterogeneity of the ADCs. Here we report various analytical methods for DAR analysis of traditional cysteine-based ADCs; additionally, apply a good manufacturing practice (GMP) strategy to produce a four hundred milligram ADC batch for use in good laboratory practice (GLP) studies. The work described herein not only evaluates several analytical performances but also provides guidance for future phase appropriate ADC production while establishing a unique analytical strategy.

The deuteration of N²-ethyl-2′-deoxyguanosine (Et-dG), which is a DNA adduct generated from acetaldehyde, was studied by the addition reaction of acetaldehyde-d₄ to 2′-deoxyguanosine (dG) in deuterium oxide (D₂O), with the aim to obtain an isotope internal standard for the liquid chromatography/tandem mass spectrometry (LC/MS/MS) quantitation of Et-dG. The replacement of the dG C-8 hydrogen atom by a deuteron atom took place at 50°C in D₂O and afforded a mixture of Et-dG-d₄ and Et-dG-d₅. Et-dG-d₄, which was stable in aqueous solutions, was prepared by incubating the mixture in H₂O at 60°C for 48 h. The calibration curve was obtained by multiple reaction monitoring (MRM) measurements using a hydrophilic interaction chromatography–electrospray ionization–tandem mass spectrometric (HILIC/ESI-MS/MS) system between the Et-dG concentration, ranging from 1.0 × 10⁻¹⁰ to 4.0 × 10⁻⁹ M in the sample solutions, and the relative peak areas of Et-dG (m/z: 296.1 → 180.1) to the value of Et-dG-d₄ (m/z: 300.2 → 184.2), with an internal standard showing good linearity (R² = 0.995, n = 5).

¹H/¹⁹F NMR-based screening methods were applied to a human serum albumin-fleroxacin complex. Fleroxacin contains three fluorine atoms in a molecule, which is suitable as a model fluorinated compound for NMR analysis with ¹H and ¹⁹F detection. The ¹⁹F{¹H} and ¹H{¹H} saturation transfer difference spectra were acquired and the ¹H/¹⁹F spin-lattice relaxation rates were measured with and without any selective irradiation of protein resonance to identify the binding epitopes of fleroxacin. Because several ¹H signals of fleroxacin resonated close to water, its precise signal intensities were unavailable. The ¹⁹F NMR-based screening methods successfully provide complementary information, indicating its importance in the analysis of fluorinated compounds.

A practical computation of fast Fourier transformation (FFT) based generalized two-dimensional (2D) correlation spectroscopy is described. Using simple sinusoids, we tested and confirmed that the method served effectively and properly, invariant to the changes of the number of data points of the time profiles. This computation is applicable to any type of waveforms in a versatile manner.

In this paper, we proposed a high-throughput microwave digestion system based on multi-batch reactors (three quartz test tubes inside commercial PTFE vessels). This original configuration was validated by ICP-MS analysis of several elements in biological certified reference materials (fish tissues and plankton). The proposed system was proved to be free from contamination showing very low LODs. The improved hardware configuration is therefore highly beneficial for the detection of trace elements in microsamples from the marine food web.