Analytical Methods (v.11, #19)

Front cover (2505-2506).

Contents list (2507-2512).

In this paper, a novel fluorescent probe DHOA based on excited-state intramolecular proton transfer (ESIPT) principle was designed, prepared, and then applied to quantitative measurement and cell imaging of cysteine (Cys). The probe had high selectivity and large Stokes shift, and could quickly and sensitively detect the content of Cys with a detection limit of 55 nM. With its low cytotoxicity and good membrane permeability, the probe was successfully applied to fluorescence imaging of endogenous and exogenous Cys in HepG2 cells.

Label-free characterization of biochemical changes within human cells under parasite attack using synchrotron based micro-FTIR by Hany M. Elsheikha; Nashwa A. Elsaied; K. L. Andrew Chan; Chris Brignell; Mohammad S. R. Harun; Katia Wehbe; G. Cinquee (2518-2530).
The protozoan Toxoplasma gondii is responsible for severe, potentially life-threatening, infection in immunocompromised individuals and when acquired during pregnancy. In the meantime, there is no available vaccine and the anti-T. gondii drug arsenal is limited. An important challenge to improve antiparasitic therapy is to understand chemical changes that occur during infection. Here, we used Fourier transform infrared spectroscopy (FTIR) to investigate the effect of T. gondii infection on the chemical composition of human brain microvascular endothelial cells (hBMECs) at 3, 6, 24 and 48 hours postinfection (hpi). Principal component analysis (PCA) showed that the best separation and largest difference between infected and uninfected hBMECs was detected at 24 hpi and within the 3400–2800 cm−1 region. At 48 hpi, although the difference between samples was obvious within the 3400–2800 cm−1 region, more differences were detected in the fingerprint region. These findings indicate that infected and control cells can be easily distinguished. Although differences between the spectra varied, the separation was most clear at 24 hpi. T. gondii increased signals for lipids (2853 cm−1) and nucleic acids (976 cm−1, 1097 cm−1 and 1245 cm−1), and decreased signals for proteins (3289 cm−1, 2963 cm−1, 2875 cm−1) in infected cells compared to controls. These results, supported by amino acid levels in culture media, and global metabolomic and gene expression analyses of hBMECs, suggest that T. gondii parasite exploits a wide range of host-derived chemical compounds and signaling pathways for its own survival and proliferation within host cells. Our data demonstrate that FTIR combined with chemometric analysis is a valuable approach to elucidate the temporal, infection-specific, chemical alterations in host cells at a single cell resolution.

A sequential injection analysis system with sensitive chemiluminescence detection using a flow-batch detection cell was developed for the real time monitoring of kinetic profiles of antioxidant activity of phenolic substances in a luminol–Fe(iii)–hydrogen peroxide system during a 60 s stop–flow period. Selected substances including coumaric acid, caffeic acid, hesperidin, ascorbic acid, resveratrol, rutin, chlorogenic acid, quercitrin, epicatechin, phloridzin, phloretin, and Trolox as a reference standard were tested with respect to different chemiluminescence signal kinetics. The evaluation of antioxidant activity was expressed as the quenching efficacy index Q. Our method has a wide working concentration range of 4–250 μmol L−1 for Trolox antioxidant activity. The real time kinetic profiles based on the chemiluminescence quenching half-life of both quick and slow antioxidants were also determined. A sample throughput of 30 h−1 was demonstrated. The antioxidant activity of apple and black elder fruit extracts was evaluated.

A rapid room-temperature DNA amplification and detection strategy based on nicking endonuclease and catalyzed hairpin assembly by Guixiu Dong; Jianyuan Dai; Limin Jin; Hongli Shi; Fang Wang; Cuisong Zhou; Baozhan Zheng; Yong Guo; Dan Xiao (2537-2541).
A novel and rapid room-temperature DNA amplification strategy to simultaneously amplify and detect target HIV DNA was developed by coupling catalyzed hairpin assembly (CHA) with a “non-sequence dependent” nicking endonuclease. In this system, a Nt.BsmAI nicking endonuclease recognition sequence and HIV target DNA sequence were integrated into hairpin probes H1 and H3, respectively. The HIV target DNA was repeatedly used to trigger a CHA reaction, forming numerous H1–H2 duplexes. Then the newly formed sticky ends of H1–H2 caused the hairpin structure of H3 to open. As a result, the double-stranded nicking enzyme recognition site was reunited, which was recognized and cleaved by the nicking enzyme. Finally, the target DNA replica with a sequence matching that of the HIV target DNA was obtained. The above reactions, namely, the CHA reaction, hairpin opening and enzyme cleavage, can be continuously repeated, leading to rapid room-temperature DNA amplification. In addition, this proposed rapid DNA amplification strategy enables picomolar detection of HIV DNA target within 10 min, providing a potential clinical application at point-of-care.

An integrated homogeneous SPARCL™ immunoassay for rapid biomarker detection on a chip by Natalia Sandetskaya; Nicole Isserstedt-John; Andreas Kölsch; Sebastian Schattschneider; Dirk Kuhlmeier (2542-2550).
We developed an integrated version of the homogeneous SPARCL™ (Spatial Proximity Analyte Reagent Capture Luminescence) immunoassay for rapid measurement of biomarkers on a chip. Our development is based on a simple microfluidic design with on-chip preserved dry reagents. The assay requires no washing steps and can be performed in a “mix-and-read” format. A syringe pump and a luminometer supported the detection. Only one manual pipetting step is necessary to load the sample onto the chip, and the entire incubation and measurement can be accomplished in approximately 35 min. We have demonstrated the application of the SPARCL™ on a chip to the quantitative detection of the tissue inhibitor of metalloproteinases 1 (TIMP-1) in spiked mock samples and a limited number of gingival crevicular fluid specimens. The integrated SPARCL™ assay could detect as little as 49.3 pg ml−1 (1.76 pM) TIMP1. To our knowledge, this is the first demonstration of the fully integrated homogeneous SPARCL™ immunoassay in a lab-on-a-chip. Further, we discuss the optimization of the chip and the assay in order to improve its analytical performance.

Characterisation and optimisation of ion discrimination in a mini ion funnel for a miniature mass spectrometer by Xiaohua Zhang; Xinming Huo; Fei Tang; Zhuoyue Zha; Fengbo Yang; Yafan Ni; Yanjun Wang; Mingfei Zhou; Xiaohao Wang (2551-2558).
Ion funnels have been miniaturised and integrated into miniature ion trap mass spectrometers to improve instrument sensitivity. However, the ion discrimination effect of the ion funnel constrains its application. Previous research has generally reported that the ion discrimination is related mainly to the mass of the ions. In this paper, the Runge–Kutta algorithm was used to characterise the trajectories of ions in the funnel under collisions with gas molecules. It is theoretically and empirically proved that, in a continuous atmospheric-pressure-interfaced miniature mass spectrometer, ion discrimination in the funnel shows two aspects: mass discrimination and collision cross-section discrimination. It was found that DC gradient, RF amplitude and operating pressure of the ion funnel will affect ion transmission. Among these parameters, RF amplitude is the key parameter controlling ion discrimination. To weaken the ion discrimination, RF amplitude scanning method was introduced in the injection section of the ion trap to improve ion transmission efficiency over the entire mass range. After optimisation of the ion funnel, the dynamic range of the instrument concentration reached four orders of magnitude. The detection limit of roxithromycin reached a low level of 1 ng mL−1. The results of this study increase the theoretical understanding of the ion funnel, providing guidance for applying ion funnels in improving the sensitivity of miniature mass spectrometers and expanding their actual applicability.

A fluorescent probe for Cd2+ detection based on the aggregation-induced emission enhancement of aqueous Zn–Ag–In–S quantum dots by Chang Wei; Xian Wei; Zhe Hu; Dan Yang; Shiliang Mei; Guilin Zhang; Danlu Su; Wanlu Zhang; Ruiqian Guo (2559-2564).
A sensitive and selective fluorescent probe for the cadmium ion (Cd2+) was developed based on the aggregation-induced emission enhancement (AIEE) of Zn–Ag–In–S quantum dots (ZAIS QDs). The QDs were synthesized by a convenient hydrothermal method with l-cysteine as ligands, with the average size of 3.2 nm and the emission peak at 520 nm. The interference study indicated the good selectivity of the probe. It has a broad detection range from 25 μM to 2 mM and the detection limit is 1.56 μM. AIEE is triggered by the interaction between Cd2+ and thiol groups on the surface of QDs, which causes weaker electrostatic repulsion between QDs and the passivation of surface defects. To the best of our knowledge, this is the first report on the AIEE of quaternary QDs. The method was successfully applied to the determination of Cd2+ in water samples with recoveries between 98.1% and 103.3%. This work shows its potential as a candidate for Cd2+ detection and provides a new understanding of AIEE.

A novel rhodamine-based fluorescent probe was designed and synthesized. Its metal ion selectivity was illuminated by the highly specific recognition of the probe towards Fe3+ over other metal ions; moreover, the color change in the recognition process of Fe3+ could be used for its “naked-eye” detection in an aqueous environment. In addition, a theoretical calculation was performed to reveal the possible reaction mechanism between the probe and Fe3+. The fluorescent imaging of Fe3+ in living cells further suggested future applications of the probe for instant Fe3+ detection in a clinical diagnosis and dynamic tracking of Fe3+ in biological systems.

A novel extraction method for simultaneous determination of neonicotinoid insecticides and their metabolites in human urine by Shiming Song; Yuan He; Bo Zhang; Mingwei Gui; Jiping Ouyang; Tao Zhang (2571-2578).
In this study, a novel method (i.e., liquid–liquid micro-extraction, LLME) coupled to liquid chromatography tandem mass spectrometry is proposed for the simultaneous determination of urinary levels of six neonicotinoid insecticides (NEOs), namely, imidacloprid, clothianidin, acetamiprid, thiacloprid, thiamethoxam, and dinotefuran, and four metabolites, including 1-methyl-3-(tetrahydro-3-furylmethyl) guanidine, 1-methyl-3-(tetrahydro-3-furylmethyl) urea, 5-hydroxy-imidacloprid, and N-demethyl-acetamiprid has been developed. Ethyl acetate was used as the extraction solvent, and the supernatant was concentrated and dissolved by methanol. The method was validated for repeatability, reproducibility, linearity, and percent recovery. Satisfactory recovery of the NEOs and their metabolites (71–107%) was observed with intraday relative standard deviations (RSDs, n = 3) of 2–11% and inter-day RSDs (n = 9) of 3–17%. Good linearity (R2 > 0.99) was obtained for all analytes. Limits of quantification and detection for all target analytes ranged from 0.01 ng mL−1 to 0.1 ng mL−1 and from 0.003 ng mL−1 to 0.03 ng mL−1, respectively. Trace levels of NEOs and their metabolites were found in human urine samples (mean: 0.06–1.99 ng mL−1). To the best of our knowledge, this study is the first to apply the proposed LLME method for simultaneous identification and quantification of NEOs and their metabolites in urine samples.

Modulation of ICT and PET processes in boranil derivatives: a ratiometric fluorescent probe for imaging of cysteine by Dongjian Zhu; Xiaowei Yan; Aishan Ren; Wen Cai; Zhenhua Duan; Yanghe Luo (2579-2584).
A novel ratiometric fluorescent probe 1 based on boranil derivatives modulated by ICT and PET processes was designed and synthesized for the first time. Upon addition of Cys, the emission peak of 1 changed from 461 nm to 474 nm, accompanied by a fluorescence color change from dark blue to blue under illumination with a 365 nm UV lamp. 1 exhibited a fast response (within 9 min), high sensitivity (detection limit of 95.1 nM) and excellent selectivity toward Cys over Hcy and GSH owing to a specific conjugate addition–cyclization reaction between Cys and the acrylate moiety. Furthermore, 1 was successfully implemented for monitoring Cys in living cells.

Herein, an indole-attached fluorescein dye (FI-1) was designed and synthesized as an excellent sensor for fluoride ions. The probe FI-1 strikingly changed from colorless to yellow and exhibited a turn-on fluorescence response towards fluoride with superior sensitivity and selectivity in an aqueous medium; the fluoride ions facilitated the ring opening of the fluorescein spiro ring; this caused changes in the absorbance and fluorescence, supported by the results of 1H-NMR titrations. The observed UV-vis and fluorescence changes are supported by the density functional theory calculations. Thus, the proposed probe can be applied to detect fluoride ions with the lowest range of 31.8 nM; moreover, the probe can be applied for the imaging of fluoride ions in living cells using fluorescence microscopy.

A ‘‘naked-eye’’ colorimetric and ratiometric fluorescence probe for trace hydrazine by Cuiyan Wu; Hai Xu; Yaqian Li; Ruihua Xie; Peijuan Li; Xiao Pang; Zile Zhou; Haitao Li; Youyu Zhang (2591-2596).
Hydrazine (N2H4) is widely used in industry but also very toxic. Herein, we describe the preparation of a ‘‘naked-eye’’ colorimetric and ratiometric fluorescent probe (DH), bearing α,β-unsaturation carbonyl group as a recognition site, and employ it for the excited-state intramolecular proton transfer based (ESIPT-based) detection of N2H4. The probe can detect N2H4 by colorimetric and ratiometric fluorescence dual signals with high sensitivity and selectivity; the detection limit of N2H4 was 0.063 μM (2.01 ppb), which was far below the safety level (10 ppb) stated by the U.S. Environmental Protection Agency (EPA). It enables “naked-eye” detection for hydrazine determination in aqueous solution. More importantly, we successfully applied DH to detect N2H4 in real water samples, indicating its potential utility for N2H4 sensing in environmental samples.

A simple and portable potentiometric immunoassay was designed for the quantitative detection of matrix metalloproteinase-7 (MMP7) in acute kidney injury and renal cancer on a copper ion-selective electrode (Cu-ISE). To construct such an assay system, a sandwich-type immunoreaction was initially carried out on monoclonal anti-MMP7 capture antibody-coated microplates by using an initiator DNA strand-labeled anti-MMP7 secondary antibody. Thereafter, a hybridization chain reaction (HCR) was used for the signal amplification between copper nanocluster-labeled two hairpin DNA probes. Accompanying the formation of the sandwiched immunocomplex, the labeled DNA initiator strand on the secondary antibody triggered the HCR between two alternating DNA hairpins to form a nicked double-helix with copper nanoclusters. The subsequent potentiometric measurement of copper ions released from the concatenated copper nanoclusters under acidic conditions was conducted on a portable handheld potentiometer. The immuno-HCR assay combined an easy-operation potentiometric assay with signal amplification by using an HCR-induced copper nanocluster concatemer. Under optimum conditions, the developed immuno-HCR assay exhibited a dynamic linear range of 0.01–100 ng mL−1 with a detection limit of 5.3 pg mL−1 and good reproducibility. Also, the immuno-HCR assay gave good anti-interfering capacity toward other biomarkers. The analysis for clinical human serum specimens revealed good accordance with the results obtained by an enzyme-linked immunosorbent assay. Importantly, our strategy is promising for enzyme-free and cost-effective analysis of low-abundance cancer biomarkers.

A type I Bi2S3@ZnS core–shell structured photocatalyst for the selective photoelectrochemical sensing of Cu2+ by Jiajia Xi; Hao Wang; Bihong Zhang; Xiaopeng Hu; Faqiong Zhao; Baizhao Zeng (2605-2610).
A type I core/shell structured Bi2S3@ZnS photocatalyst was synthesized by an electrostatic interaction mechanism and used for the photoelectrochemical detection of Cu2+. The Bi2S3@ZnS composite showed excellent photoelectrochemical activity and stability due to the high photoelectrochemical activity of Bi2S3 and the anti-photocorrosion function of the ZnS shell. In addition, the ZnS shell also provided a transfer platform for the photogenerated holes through its acceptor states (i.e. VZn and IS) and suppressed the recombination of photogenerated carriers. The photocurrent signal of the obtained Bi2S3@ZnS/ITO electrode was much higher than that of pure Bi2S3 under visible-light irradiation. When it was used for the photoelectrochemical sensing of Cu2+, a wide detection range (0.003–30.0 μM) and a low detection limit (0.001 μM) were observed. The sensor also displayed high stability and acceptable selectivity. It was applied to detect real samples and good results were achieved.

The detection of petroleum contaminants in soil based on multiphoton electron extraction spectroscopy by Chenwei Zhu; Ran Zhou; Youjian Zhang; Ji Chen; Shisong Tang; Xiangyou Li; Xiaoyan Zeng (2611-2616).
The fast detection of petroleum contaminants in soil is very important and urgent for environmental monitoring. Generally, the current methods for the detection of these kinds of contaminants require complex sample pre-treatment with the excessive use of toxic and volatile organic solvents or the analytical performances are not satisfactory enough for field-based applications. A method based on photocurrent measurements, named multiphoton electron extraction spectroscopy (MEES), was proposed to detect petroleum contaminants in a soil matrix in this study. The results showed that the resonance-excited wavelength of 326 nm had the highest selectivity for petroleum determination after a wide range of wavelengths was scanned with an optical parametric oscillator (OPO) laser under ambient conditions. Linear regression was performed to fit the intensities and areas of the photocurrent curves to the petroleum concentration via a least squares method. A LoD of 0.0091 wt% was achieved with an R2 value of 0.9912 and a RMSECV value of 0.013 wt% via the peak area approach, whose sensitivity and accuracy were slightly higher compared to the peak intensity method (LoD of 0.011 wt%, an R2 value of 0.9315 and a RMSECV value of 0.020 wt%). This research provided us with a new method for the rapid determination of petroleum contaminants in soil samples with high accuracy.

Back cover (2617-2618).