Analytical Methods (v.10, #22)

Front cover (2539-2540).

Contents list (2541-2545).

We have rationally constructed a new two-photon fluorescent hypochlorous acid probe (TP-HA). The probe TP-HA displayed a fluorescence enhancement (∼16-fold) in response to HOCl with high sensitivity and selectivity. The new probe was membrane-permeable and suitable for visualization of the changes of HOCl in living cells and tissues.

A novel EI-GC/MS method for the accurate quantification of anti-aging compound oleoylethanolamine in C. elegans by Andrea Annibal; Özlem Karalay; Christian Latza; Adam Antebi (2551-2559).
Endocannabinoids and related N-acyl ethanolamine-derived lipids affect a diverse array of physiological processes and pathological conditions. In the roundworm C. elegans, several endocannabinoid-like molecules have been implicated in regulating axon regeneration, energy balance and food intake as well as aging. One such molecule oleoylethanolamine (OEA) has been shown to promote life extension through nuclear receptor signal transduction, and its accurate quantitation therefore is of high interest. Using a combination of electron impact ionization (EI) and collision induced dissociation coupled to gas chromatography (GC), we found unique fragmentation ions of OEA and designed a specific MRM method for its accurate quantification. Our method should provide a reproducible and robust way to measure OEA dynamics under different genetic, pharmacologic and environmental perturbations.

Determination of Cu2+ and biothiols by novel red fluorescent hybrid nanoparticles by Ling Zhang; Zizhun Wang; Jiaze Hou; Lulu Lei; Jiao Li; Juan Bai; Hui Huang; Yongxin Li (2560-2566).
Red fluorescent hybrid nanoparticles (NPG@PFBD) have been designed and synthesized successfully. The NPG@PFBD nanoparticles are composed of a fluorescent semiconducting polymer (PFBD) and n-propyl gallate (NPG). Because of the coordination between the gallate and Cu2+, Cu2+ could attach to the hybrid nanoparticles and quench their fluorescence through photoinduced electron transfer (PET). In addition, in the presence of biothiols, such as glutathione (GSH), Cu2+ would bind to the thiol. The PET between the hybrid nanoparticles and Cu2+ was weakened, and the fluorescence signal of the nanoparticles was recovered. A facile detection method for Cu2+ and biothiols was therefore developed. The detection limit for Cu2+ and GSH was 40 nM and 90 nM, respectively. The proposed fluorescent hybrid nanoparticles have great potential application for Cu2+ and GSH detection in related biological fields.

Direct and ultrasensitive fluorescence detection of PETN vapor based on a fuorene-dimer probe via a synergic backbone and side-chain tuning by Ao Liu; Huan Liu; Xin Peng; Jinlan Jia; Yanyan Fu; Qingguo He; Huimin Cao; Jiangong Cheng (2567-2574).
Pentaerythritol tetranitrate (PETN), a major component in plastic explosives, poses a major threat to public safety and has extremely low saturated vapor pressure (7.17 ppt) and can hardly be detected directly. Two novel fluorescent sensing materials (6TPA-2FT and 6Py-2FT) were synthesized via Suzuki and Click reactions for a direct detection of PETN explosive vapor. The high binding affinity between the nitro group of PETN and the sensing film of 6TPA-2FT results in rapid and dramatic quenching of its fluorescence. The detection limit of 6TPA-2FT was as low as 0.056 ppt. The sensing mechanism was verified by various experimental characterization, including photophysical properties, SEM, IR, and quantum chemical calculations. For practical application, the sensing film of 6TPA-2FT was successfully used to detect PETN hidden in a bag using only naked eye detection under UV excitation without using any other complicated instrument, indicating its potential application for the detection of PETN vapors.

Evaluation of colorimetric methods for quantification of citrus flavonoids to avoid misuse by Rui Huang; Wenyan Wu; Shuyu Shen; Jiawen Fan; Yue Chang; Shiguo Chen; Xingqian Ye (2575-2587).
The content of flavonoids is important for evaluating the quality of citrus-related products. However, due to the different characteristics of the structures of flavonoids and the lack of a universal method, many methods for quantification have been misused. Here, we used twenty-one citrus flavonoid standards to evaluate four widely used colorimetric methods, and seven species of citrus as natural samples were used to verify the evaluation. The results indicated that NaNO2–Al(NO3)3–NaOH and AlCl3 methods could only detect some of the flavonoids and were inadequate to estimate the total flavonoid content, and the 2,4-dinitrophenylhydrazine (2,4-DNPH) method might be affected by other carbonyl compounds in extracts. The Davis method was modified with naringin as the standard at a measuring wavelength of 420 nm for grapefruit and pomelo, and with hesperidin as the standard at a measuring wavelength of 360 nm for other species. It was proved to be the most reliable method for the quantification of citrus flavonoids and was also recommended for laboratories and factories where needed.

Two methods have been developed and validated for simultaneous determination of amoxicillin sodium and diclofenac sodium in their pure and combined pharmaceutical dosage forms. The first method was HPLC-DAD; the chromatographic separation and resolution has been carried out using a reversed phase BDS Hypersil C18 column with a mobile phase consisting of methanol : acetonitrile : water : orthophosphoric acid (60 : 30 : 9 : 1 by volume), at a flow rate of 1 mL minute−1 and UV detection at 250 nm. The retention times were found to be 3.906 and 6.997 minutes for amoxicillin and diclofenac respectively. The second method was continuous wavelet transform (CWT), which is based on derivative calculation of spectrophotometric spectral data of both drugs in their binary mixture, and the zero crossing point for amoxicillin and diclofenac was found to be at 233 and 247 nm, respectively. The results obtained were statistically compared to reference methods and there were no significant differences between the proposed methods and the reference methods regarding the accuracy and precision. The method was validated according to ICH guidelines and the results were satisfactory.

A comparative study of laser-induced breakdown spectroscopy (LIBS) with spatially resolved single- and double-pulse configurations is performed to investigate the capability of simultaneous multi-element detection in aluminum-based alloys. The spatially resolved technique is used to obtain the LIBS spectra at 6 different positions along the plume expansion (axial) direction. The experimental parameters, including delay time, inter-pulse delay time and gate width, are optimized to achieve sensitive elemental detection. Under these optimized conditions, the limits of detection for Fe, Cu, Mg, Mn, Zn, Sn, Pb, Ni, Ti, Cr, Sr and Ca are obtained with the single- and double-pulse configurations. It is observed that an improvement of the detection capability is achieved in the spatially resolved double-pulse experiments. Moreover, the dependence of the limits of detection on the selection of atomic and ionic lines is discussed.

A method combining a kit with the Bionic e-Eye for rapid on site detection of diarrhetic shellfish poisoning by Xianxin Qiu; Longjie Zhong; Ying Gan; Kaiqi Su; Shihui Hu; Ping Wang (2604-2613).
Okadaic acid is a kind of fat-soluble diarrhetic shellfish poison existing extensively in shellfish. The rapid on site detection of shellfish contaminated by the toxin is a serious problem. Herein, a new method combining a kit with the Bionic e-Eye was developed for the rapid detection of okadaic acid on site. After optimization the kit reached a very low limit of detection, 0.19 μg L−1, and the linear detection range was 0.2–10 μg L−1. The cross reaction rate with dinophysistoxins 1 was 23.6%. The kit had a small cross reaction with other kinds of toxin like saxitoxin, gynotoxin 2 & 3 and pectenotoxins 2-b. The recovery range was about 75.78–102.56%, and the CVs were lower than 20%. A correlation analysis was conducted between the Bionic e-Eye and a microplate reader in PBS and shellfish backgrounds. The detection results of the real shellfish samples by the Bionic e-Eye exhibited great consistency with high performance liquid chromatography. The total time taken to perform the detection was less than 90 minutes. Above all, the method integrated kit with the Bionic e-Eye was very suitable for the rapid detection of okadaic acid on site.

Despite the distinct features of polythymine (T)-templated copper nanoparticles (polyT-Cu NPs) as fluorescent probes for various biosensors, most of the reported methods involve labeling with an appropriate fluorescence quencher, or the addition of enzyme to digest the DNA-template. In this paper, the phenomenon of photo induced electron transfer (PET) between the polyT-Cu NPs and Guanine quadruplex (G4)–K+–hemin (horseradish peroxidase (HRP)–DNAzyme) complexes was found, and was applied for the fabrication of a “turn-on” target modulated fluorescence system for the sensitive determination of the HBV gene, without any labeling process or the addition of enzyme. Using optical and electrical experiments, the PET mechanism was investigated. In the detection process, the PET led to the quenching of the fluorescence of the polyT-Cu NPs, thus lowering the background; then in the presence of target DNA, a conformational change occurred, the PET was blocked and the fluorescence could be recovered. The developed strategy was highly sensitive for the detection of the HBV gene in the concentration range from 0.1 to 80 nM, with a low detection limit of 33.4 pM (S/N = 3). In addition, the novel method for specific DNA sequence detection was highly selective. Finally, this work was successfully utilized for the sensitive and label free fluorescence determination of the HBV gene in blood plasma samples.

A facile colorimetric method for highly sensitive ascorbic acid detection by using CoOOH nanosheets by Qiuyu Nie; Qiyong Cai; Huihui Xu; Zhun Qiao; Zhaohui Li (2623-2628).
In this work, based on a fast redox reaction between CoOOH nanosheets and AA, a very simple and highly sensitive colorimetric assay was developed to detect AA in buffer and real samples. In the detection system, CoOOH nanosheets exhibit a strong ultraviolet-visible (UV-vis) absorption peak at around 430 nm with an observable yellowish-brown color. The presence of AA reduces the CoOOH nanosheets to Co2+ ions and subsequently weakens the color of the solution, which results in a dramatic decrease of the absorption signal. The assay showed a good linear range from 0.25 μM to 60 μM with a detection limit of 57 nM for AA. Meanwhile, the detection system also exhibited high selectivity against other electrolytes and biomolecules. More importantly, this procedure is very simple, and the entire procedure can be finished within 15 min. This method has also been successfully applied to the detection of AA in vitamin C tablets and beverages with reliable results.

A single-chain variable fragment (scFv)-alkaline phosphatase (AP) fusion protein for glycocholic acid (GCA) was produced and characterized. The scFv gene with a 218 linker was generated by splicing by overlap extension (SOE)-polymerase chain reaction (PCR) and sequentially inserted into the expression vector pecan45 containing AP gene to express the scFv-AP fusion protein in Escherichia coli (E. coli). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses revealed that the fusion protein showed the expected molecular weight of about 80 kDa. Both the antibody binding capacity and AP enzyme activity of the scFv-AP fusion protein were validated by colorimetric analysis. One-step competitive direct enzyme-linked immunosorbent assay (ELISA) based on the scFv-AP fusion protein indicated that the average concentration required for 50% inhibition of binding (IC50) and limit of detection (LOD) for GCA were 216 ng mL−1 and 37.0 ng mL−1, respectively, and the linear response range extended from 71.0 to 657 ng mL−1. The cross-reactivity (CR) of the scFv-AP fusion protein was similar to those of its parental scFv antibody. The scFv-AP fusion protein was bifunctional, retaining both antibody binding specificity and AP enzyme activity. This work indicates that the production of the scFv-AP fusion protein in E. coli strain BL21(DE3)pLysS is feasible and suggests that it could be further used as convenient one-step detection probes for GCA.

We describe a methodology for comparing and contrasting a large number of dissolved organic matter samples based on their electrospray ionization mass spectral characteristics. The analytical process and especially the electrospray process give rise to unwanted variation, which needs to be either mitigated, compensated for or monitored, and until now only few examples of multivariate comparison of large sets of dissolved organic matter mass spectral fingerprints where also unwanted variation is monitored have been published. The methodology comprises a tier of processes: (I) establishing a sample set including facilitator and quality control samples; (II) establishing pre-concentration/dilution factors to put samples to the same concentration domain; (III) chemical analysis by direct injection electrospray ionisation time-of-flight mass spectrometry; (IV) data pre-processing – normalisation and variable weighting – to emphasize variation relevant to the objective of the investigation; and (V) multivariate comparison and differentiation of a large number of samples by principal component analysis. The implementation of the methodology was demonstrated on two sample sets: terrestrial and oceanic dissolved organic matter from North America and Antarctica (set 1), and dissolved organic matter from Danish groundwaters (set 2). Samples were re-constituted from dry matter (set 1) or worked up directly (set 2). Artefacts originating from concentration effects were attempted removed by pre-concentration/dilution prior to chemical analysis, pre-concentration/dilution factors determined by either UV-(set 1) or negative mode ESI-MS screening (set 2). Normalisation factors and variable weights were established by use of facilitator sample sets. Artefacts related to unwanted variation induced by remaining concentration effects, sampling-, storage- and analytical processes are described and were monitored by independent quality control sample sets.

Measuring the relative concentration of particle populations using differential centrifugal sedimentation by Alexander G. Shard; Katia Sparnacci; Aneta Sikora; Louise Wright; Dorota Bartczak; Heidi Goenaga-Infante; Caterina Minelli (2647-2657).
The factors that affect the accuracy and precision of differential centrifugal sedimentation (DCS) for the analysis of nanoparticle concentration are described. Particles are separated by their sedimentation rate and detected using light absorption. In principle, the relative concentration of particles in different populations can be found, but the uncertainty in such measurements is unclear. We show that the most appropriate measurement of particle concentration using this technique is the mass concentration, rather than the number concentration. The relative mass concentration of two discrete populations can be measured with reasonable precision, usually without resorting to complicated data analysis. We provide practical approaches to find the relative mass concentrations for two cases: spherical particles of different materials and agglomerated particles of the same material. For spherical particles made of different materials, naïve analysis of the results can provide relative mass concentrations that are many orders of magnitude in error. Correction factors can be calculated that reduce the error to less than 50%. In the case of agglomerated particles we show that errors of less than 20% are possible and demonstrate, in the case of gold particles, that a combination of UV-visible spectroscopy and DCS enable practical values of mass and number based particle concentrations to be obtained.

Back cover (2659-2660).