Analytica Chimica Acta (v.916, #C)

The monitoring of microRNA (miRNA) expression levels is of great importance in cancer diagnosis. In the present work, based on two cascaded toehold-mediated strand displacement reactions (TSDRs), we have developed a label- and enzyme-free target recycling signal amplification approach for sensitive electronic detection of miRNA-21 from human breast cancer cells. The junction probes containing the locked G-quadruplex forming sequences are self-assembled on the senor surface. The presence of the target miRNA-21 initiates the first TSDR and results in the disassembly of the junction probes and the release of the active G-quadruplex forming sequences. Subsequently, the DNA fuel strand triggers the second TSDR and leads to cyclic reuse of the target miRNA-21. The cascaded TSDRs thus generate many active G-quadruplex forming sequences on the sensor surface, which associate with hemin to produce significantly amplified current response for sensitive detection of miRNA-21 at 1.15 fM. The sensor is also selective and can be employed to monitor miRNA-21 from human breast cancer cells.Display Omitted
Keywords: Strand displacement; Target recycling; Electronic sensor; microRNA detection; Cancer cells;

Prediction of retention time in reversed-phase liquid chromatography as a tool for steroid identification by Giuseppe Marco Randazzo; David Tonoli; Stephanie Hambye; Davy Guillarme; Fabienne Jeanneret; Alessandra Nurisso; Laura Goracci; Julien Boccard; Serge Rudaz (8-16).
The untargeted profiling of steroids constitutes a growing research field because of their importance as biomarkers of endocrine disruption. New technologies in analytical chemistry, such as ultra high-pressure liquid chromatography coupled with mass spectrometry (MS), offer the possibility of a fast and sensitive analysis. Nevertheless, difficulties regarding steroid identification are encountered when considering isotopomeric steroids. Thus, the use of retention times is of great help for the unambiguous identification of steroids. In this context, starting from the linear solvent strength (LSS) theory, quantitative structure retention relationship (QSRR) models, based on a dataset composed of 91 endogenous steroids and VolSurf + descriptors combined with a new dedicated molecular fingerprint, were developed to predict retention times of steroid structures in any gradient mode conditions. Satisfactory performance was obtained during nested cross-validation with a predictive ability (Q2) of 0.92. The generalisation ability of the model was further confirmed by an average error of 4.4% in external prediction. This allowed the list of candidates associated with identical monoisotopic masses to be strongly reduced, facilitating definitive steroid identification.Display Omitted
Keywords: Retention time prediction; Steroids; Isotopomers identification; LSS theory; Reversed-phase liquid chromatography; Quantitative structure–retention relationships;

New approaches to antimony film screen-printed electrodes using carbon-based nanomaterials substrates by Clara Pérez-Ràfols; Núria Serrano; José Manuel Díaz-Cruz; Cristina Ariño; Miquel Esteban (17-23).
Three different commercial carbon nanomaterial-modified screen-printed electrodes based on graphene, carbon nanotubes and carbon nanofibers were pioneeringly tested as electrode platforms for the plating with Sb film. They were microscopically and analytically compared to each other and to the most conventional unmodified carbon screen-printed electrode (SPCE). The obtained detection and quantification limits suggest that the in-situ antimony film electrode prepared from carbon nanofibers modified screen-printed electrode (SbSPCE-CNF) produces a better analytical performance as compared to the classical SPCE modified with antimony for Pb(II) and Cd(II) determination, approving its appropriateness for measuring low μg L−1 levels of the considered metals. In-situ SbSPCE-CNF was successfully used for the simultaneous determination of Pb(II) and Cd(II) ions, by means of differential pulse anodic stripping voltammetry, in a certified reference estuarine water sample with a very high reproducibility and good trueness.Display Omitted
Keywords: Antimony film electrodes; Screen-printed electrodes; Anodic stripping voltammetry; Heavy metal ions;

A high-throughput solid-phase extraction microchip combined with inductively coupled plasma-mass spectrometry for rapid determination of trace heavy metals in natural water by Tsung-Ting Shih; Cheng-Chuan Hsieh; Yu-Ting Luo; Yi-An Su; Ping-Hung Chen; Yu-Chen Chuang; Yuh-Chang Sun (24-32).
Herein, a hyphenated system combining a high-throughput solid-phase extraction (htSPE) microchip with inductively coupled plasma-mass spectrometry (ICP-MS) for rapid determination of trace heavy metals was developed. Rather than performing multiple analyses in parallel for the enhancement of analytical throughput, we improved the processing speed for individual samples by increasing the operation flow rate during SPE procedures. To this end, an innovative device combining a micromixer and a multi-channeled extraction unit was designed. Furthermore, a programmable valve manifold was used to interface the developed microchip and ICP-MS instrumentation in order to fully automate the system, leading to a dramatic reduction in operation time and human error. Under the optimized operation conditions for the established system, detection limits of 1.64–42.54 ng L−1 for the analyte ions were achieved. Validation procedures demonstrated that the developed method could be satisfactorily applied to the determination of trace heavy metals in natural water. Each analysis could be readily accomplished within just 186 s using the established system. This represents, to the best of our knowledge, an unprecedented speed for the analysis of trace heavy metal ions.Display Omitted
Keywords: High-throughput solid phase extraction; Inductively coupled plasma-mass spectrometry; Micromixer; Multi-channeled; Automation;

In this study, a new polymeric ionic liquid-based adsorbent was prepared and used as the extraction medium of stir cake sorptive extraction (SCSE) of three organic acid preservatives, namely, p-hydroxybenzoic acid, sorbic acid and cinnamic acid. The adsorbent was synthesized by the copolymerization of 1-ally-3-vinylimidazolium chloride (AV) and divinylbenzene (DVB) in the presence of a porogen solvent containing 1-propanol and 1,4-butanediol. The effect of the content of monomer and the porogen solvent in the polymerization mixture on the extraction performance was investigated thoroughly. The adsorbent was characterized by infrared spectroscopy, elemental analysis, scanning electron microscopy and mercury intrusion porosimetry. To obtain the optimal extraction conditions of SCSE/AVDVB for target analytes, key parameters including desorption solvent, adsorption and desorption time, ionic strength and pH value in sample matrix were studied in detail. The results showed that under the optimized conditions, the SCSE/AVDVB could extract the preservatives effectively through multiply interactions. At the same time, a simple and sensitive method by combining SCSE/AVDVB and high-performance liquid chromatography with diode array detection was developed for the simultaneous analysis of the target preservatives in orange juices and tea drinks. Low limits of detection (S/N = 3) and quantification limits (S/N = 10) of the proposed method for the target analytes were achieved within the range of 0.012–0.23 μg/L and 0.039–0.42 μg/L, respectively. The precision of the proposed method was evaluated in terms of intra- and inter-assay variability calculated as relative standard deviation (RSD), and it was found that the values were all below 10%. Finally, the proposed method was used to detect preservatives in different orange juice and tea drink samples successfully. The recoveries were in the range of 71.9–116%, and the RSDs were below 10% in the all cases.Display Omitted
Keywords: Stir cake sorptive extraction; Polymeric ionic liquid; Adsorbent; High-performance liquid chromatography; Preservatives;

Quantitation of a PEGylated protein in monkey serum by UHPLC-HRMS using a surrogate disulfide-containing peptide: A new approach to bioanalysis and in vivo stability evaluation of disulfide-rich protein therapeutics by Naiyu Zheng; Jianing Zeng; Amy Manney; Lakenya Williams; Anne-Françoise Aubry; Kimberly Voronin; Adela Buzescu; Yan J. Zhang; Alban Allentoff; Carrie Xu; Hongwu Shen; William Warner; Mark E. Arnold (42-51).
To quantify a therapeutic PEGylated protein in monkey serum as well as to monitor its potential in vivo instability and methionine oxidation, a novel ultra high performance liquid chromatography-high resolution mass spectrometric (UHPLC-HRMS) assay was developed using a surrogate disulfide-containing peptide, DCP(SS), and a confirmatory peptide, CP, a disulfide-free peptide. DCP(SS) was obtained by eliminating the step of reduction/alkylation before trypsin digestion. It contains an intact disulfide linkage between two peptide sequences that are essential for drug function but susceptible to potential in vivo cleavages. HRMS-based single ion monitoring (SIM) on a Q Exactive™ mass spectrometer was employed to improve assay specificity and sensitivity for DCP(SS) due to its poor fragmentation and low sensitivity with SRM detection. The assay has been validated for the protein drug in monkey serum using both surrogate peptides with excellent accuracy (within ±4.4%Dev) and precision (within 7.5%CV) with a lower limit of quantitation (LLOQ) at 10 ng mL−1. The protein concentrations in monkey serum obtained from the DCP(SS)-based assay not only provided important pharmacokinetic parameters, but also confirmed in vivo stability of the peptide regions of interest by comparing drug concentrations with those obtained from the CP-based assay or from a ligand-binding assay (LBA). Furthermore, UHPLC-HRMS allowed simultaneous monitoring of the oxidized forms of both surrogate peptides to evaluate potential ex vivo/in vivo oxidation of one methionine present in each of both surrogate peptides. To the best of our knowledge, this is the first report of using a surrogate disulfide-containing peptide for LC-MS bioanalysis of a therapeutic protein.Display Omitted
Keywords: UHPLC-HRMS; PEGylated protein; Disulfide-containing peptide; Protein quantitation; In vivo stability; Methionine oxidation;

Matrix-assisted ionization vacuum for protein detection, fragmentation and PTM analysis on a high resolution linear ion trap-orbitrap platform by Bingming Chen; Christopher B. Lietz; Chuanzi OuYang; Xuefei Zhong; Meng Xu; Lingjun Li (52-59).
Matrix-assisted ionization vacuum (MAIV) is a novel ionization technique that generates multiply charged ions in vacuum without the use of laser ablation or high voltage. MAIV can be achieved in intermediate-vacuum and high-vacuum matrix-assisted laser desorption/ionization (MALDI) sources and electrospray ionization (ESI) sources without instrument modification. Herein, we adapt MAIV onto the MALDI-LTQ-Orbitrap XL platform for biomolecule analysis. As an attractive alternative to MALDI for in solution and in situ analysis of biomolecules, MAIV coupling to high resolution and accurate mass (HRAM) MS instrument has successfully expanded the mass detection range and improved the fragmentation efficiency due to the generation of multiply charged ions. Additionally, the softness of MAIV enables potential application in labile post-translational modification (PTM) analysis. In this study, proteins as large as 18.7 kDa were detected with up to 18 charges; intact peptides with labile PTM were well preserved during the ionization process and characterized MS/MS; peptides and proteins in complex tissue samples were detected and identified both in liquid extracts and in situ. Moreover, we demonstrated that this method facilitates MS/MS analysis with improved fragmentation efficiency compared to MALDI-MS/MS.Display Omitted
Keywords: Mass spectrometry; Matrix-assisted ionization vacuum; High resolution and accurate mass; Post-translational modification; Multiply charged ions; Protein analysis;

The possibility of the absolute quantitation of GMO events by digital PCR was recently reported. However, most absolute quantitation methods based on the digital PCR required pretreatment steps. Meanwhile, singleplex detection could not meet the demand of the absolute quantitation of GMO events that is based on the ratio of foreign fragments and reference genes. Thus, to promote the absolute quantitative detection of different GMO events by digital PCR, we developed a quantitative detection method based on duplex digital PCR without pretreatment. Moreover, we tested 7 GMO events in our study to evaluate the fitness of our method. The optimized combination of foreign and reference primers, limit of quantitation (LOQ), limit of detection (LOD) and specificity were validated. The results showed that the LOQ of our method for different GMO events was 0.5%, while the LOD is 0.1%. Additionally, we found that duplex digital PCR could achieve the detection results with lower RSD compared with singleplex digital PCR. In summary, the duplex digital PCR detection system is a simple and stable way to achieve the absolute quantitation of different GMO events. Moreover, the LOQ and LOD indicated that this method is suitable for the daily detection and quantitation of GMO events.Display Omitted
Keywords: Absolute quantitation; Digital polymerase chain reaction; Duplex detection; Pretreatment-free;

Versatile microfluidic complement fixation test for disease biomarker detection by Man Li; ZhuanZhuan Shi; Can Fang; AnXiu Gao; Chang Ming Li; Ling Yu (67-76).
The complement fixation test (CFT) is a serological test that can be used to detect the presence of specific antibodies or antigens to diagnose infections, particularly diseases caused by microbes that are not easily detected by standard culture methods. We report here, for the first time, a poly(dimethylsiloxane) (PDMS)/glass slide hybrid microfluidic device that was used to manipulate the solution compartment and communication within the microchannel to establish sampler and indicator systems of CFT. Two types of on-chip CFT, solution-based and solid phase agar-based assays, were successfully demonstrated for biomarker carcinoembryonic antigen (CEA) and recombinant avian influenza A (rH7N9) virus protein detection. In addition, the feasibility of the on-chip CFT in assaying real biopsy was successfully demonstrated by specifically detecting rH7N9 and CEA in human serum. The results demonstrated that the miniaturized assay format significantly reduced the assay time and sample consumption. Exemption from protein immobilization, blocking, complicated washing steps and expensive enzyme/fluorescein conjugates highlights the merits of on-chip CFT over ELISA. Most attractively, the on-chip agar-based CFT results can be imaged and analysed by smartphone, strengthening its point-of-care application potential. We anticipate that the on-chip CFT reported herein will be a useful supplemental or back-up tool for on-chip immunoassays such as ELISA for disease diagnosis and food inspection.For the first time, complement fixation test (CFT) was demonstrated on microfluidic devices for disease biomarker detection. Exempting from protein immobilization, blocking, complicated washing steps and expensive enzyme/fluorescein conjugates strengthens its point-of-care application potential.Display Omitted
Keywords: Microfluidic; Complement fixation test; Serological test; Point-of-care test;

We present a facile fabrication of layer-by-layer (LbL) microarrays of quantum dots (QDs) and acetylcholinesterase enzyme (AChE). The resulting arrays had several unique properties, such as low cost, high integration and excellent flexibility and time–saving. The presence of organophosphorous pesticides (OPs) can inhibit the AChE activity and thus changes the fluorescent intensity of QDs/AChE microscopic dot arrays. Therefore, the QDs/AChE microscopic dot arrays were used for the sensitive visual detection of OPs. Linear calibration for parathion and paraoxon was obtained in the range of 5–100 μg L−1 under the optimized conditions with the limit of detection (LOD) of 10 μg L−1. The arrays have been successfully used for detection of OPs in fruits and water real samples. The new array was validated by comparison with conventional high performance liquid chromatography-mass spectrometry (HPLC-MS).A fluorimetric assay for high-throughput screening of organophosphorous pesticides was developed based on the CdTe QDs/AChE microarrays via inkjet-assisted LbL printing techniques.Display Omitted
Keywords: Quantum dot; Acetylcholinesterase; Biosensor; Organophosphorus pesticide; Layer-by-layer assembly; Inkjet-printing;

As a popular detection model, the fluorescence “turn-off” sensor based on quantum dots (QDs) has already been successfully employed in the detections of many materials, especially in the researches on the interactions between pesticides. However, the previous studies are mainly focused on simple single track or the comparison based on similar concentration of drugs. In this work, a new detection method based on the fluorescence “turn-off” model with water-soluble ZnCdSe and CdSe QDs simultaneously as the fluorescent probes is established to detect various pesticides. The fluorescence of the two QDs can be quenched by different pesticides with varying degrees, which leads to the differences in positions and intensities of two peaks. By combining with chemometrics methods, all the pesticides can be qualitative and quantitative respectively even in real samples with the limit of detection was 2 × 10−8 mol L−1 and a recognition rate of 100%. This work is, to the best of our knowledge, the first report on the detection of pesticides based on the fluorescence quenching phenomenon of double quantum dots combined with chemometrics methods. What's more, the excellent selectivity of the system has been verified in different mediums such as mixed ion disruption, waste water, tea and water extraction liquid drugs.Display Omitted
Keywords: Double quantum dots; Turn-off; Chemometrics methods; Qualitative and quantitative analysis of various pesticides;

Multiplex electrochemiluminescence DNA sensor for determination of hepatitis B virus and hepatitis C virus based on multicolor quantum dots and Au nanoparticles by Linlin Liu; Xinyan Wang; Qiang Ma; Zihan Lin; Shufan Chen; Yang Li; Lehui Lu; Hongping Qu; Xingguang Su (92-101).
In this work, a novel multiplex electrochemiluminescence (ECL) DNA sensor has been developed for determination of hepatitis B virus (HBV) and hepatitis C virus (HCV) based on multicolor CdTe quantum dots (CdTe QDs) and Au nanoparticles (Au NPs). The electrochemically synthesized graphene nanosheets (GNs) were selected as conducting bridge to anchor CdTe QDs551-capture DNAHBV and CdTe QDs607-capture DNAHCV on the glassy carbon electrode (GCE). Then, different concentrations of target DNAHBV and target DNAHCV were introduced to hybrid with complementary CdTe QDs-capture DNA. Au NPs-probe DNAHBV and Au NPs-probe DNAHCV were modified to the above composite film via hybrid with the unreacted complementary CdTe QDs-capture DNA. Au NPs could quench the electrochemiluminescence (ECL) intensity of CdTe QDs due to the inner filter effect. Therefore, the determination of target DNAHBV and target DNAHCV could be achieved by monitoring the ECL DNA sensor based on Au NPs-probe DNA/target DNA/CdTe QDs-capture DNA/GNs/GCE composite film. Under the optimum conditions, the ECL intensity of CdTe QDs551 and CdTe QDs607 and the concentration of target DNAHBV and target DNAHCV have good linear relationship in the range of 0.0005–0.5 nmol L−1 and 0.001–1.0 nmol L−1 respectively, and the limit of detection were 0.082 pmol L−1 and 0.34 pmol L−1 respectively (S/N = 3). The DNA sensor showed good sensitivity, selectivity, reproducibility and acceptable stability. The proposed DNA sensor has been employed for the determination of target DNAHBV and target DNAHCV in human serum samples with satisfactory results.Display Omitted
Keywords: Multiplex electrochemiluminescence; DNA sensor; Graphene nanosheets; Multicolor quantum dots; Au nanoparticles;

Investigations into the separation behaviour of perfluorinated C8 and undecanoic acid modified silica hydride stationary phases by Chadin Kulsing; Yuanzhong Yang; Roshanak Sepehrifar; Michael Lim; Joshua Toppete; Maria T. Matyska; Joseph J. Pesek; Reinhard I. Boysen; Milton T.W. Hearn (102-111).
In this study, the surface charge properties of perfluorinated C8 (PerfluoroC8) and undecanoic acid (UDA) modified silica hydride stationary phases have been investigated. The zeta potential values of these stationary phases were measured in aqueous/acetonitrile mobile phases of different pH, buffer concentrations and acetonitrile contents. The retention behaviour of several basic, acidic and neutral compounds were then examined with these two stationary phases, with U-shaped retention dependencies evident with regard to the organic solvent content of the mobile phase. Plots of the logarithmic retention factor versus buffer concentration revealed slopes ≥ −0.41 for both stationary phases, indicating the involvement of mixed mode retention mechanisms with contributions from both ionic and non-ionic interactions. Using a linear solvation energy relationship approach, the origins of these interactions under different mobile phase conditions were differentiated and quantified. The PerfluoroC8 stationary phase exhibited stronger retention for basic compounds under high acetonitrile content mobile phase conditions, whilst stronger retention was observed for all compounds with the UDA stationary phase under high aqueous content mobile phase conditions. The more negative zeta potentials of the UDA stationary phase correlated with higher total charge density, surface charge density and charge density at the beta plane (the outer plane of the double layer) compared to the PerfluoroC8 stationary phase. With mobile phases of low buffer concentrations, more negative zeta potential values were unexpectedly observed for the PerfluoroC8 stationary phase with slight increases in the C descriptor value, reflecting also the greater accessibility of the analytes to the stationary phase surface. Comparison of the retention behaviours on these phases with other types of silica hydride stationary phases has revealed different patterns of selectivity.Display Omitted
Keywords: Silica hydride stationary phases; Aqueous normal-phase behaviour; Linear solvation energy relationships; Zeta potential measurements;

Bioanalysis of bevacizumab and infliximab by high-temperature reversed-phase liquid chromatography with fluorescence detection after immunoaffinity magnetic purification by Kenichiro Todoroki; Tatsuki Nakano; Yasuhiro Eda; Kaname Ohyama; Hideki Hayashi; Daiki Tsuji; Jun Zhe Min; Koichi Inoue; Naoki Iwamoto; Atsushi Kawakami; Yukitaka Ueki; Kunihiko Itoh; Toshimasa Toyo'oka (112-119).
This study presents two simple and rapid methods for the quantification of therapeutic mAbs based on LC. Two mAbs (bevacizumab and infliximab) in plasma samples were purified using magnetic beads immobilized with a commercially-available idiotype antibody for each mAb. Purified mAbs were separated with HT-RPLC and detected with their native fluorescence. Using immunoaffinity beads, each mAb was selectively purified and detected as a single peak in the chromatogram. The HT-RPLC achieved good separation for the mAbs with sharp peaks within 20 min. The calibration curves of the two mAbs ranged from 1 to 20 μg mL−1 (bevacizumab) and 1–10 μg mL−1 (infliximab), and they had strong correlation coefficients (r2 > 0.998). The LOD of bevacizumab and infliximab was 0.07 and 0.15 μg mL−1, and the LLOQ of bevacizumab and infliximab was 0.12 and 0.25 μg mL−1, respectively. Thus, the sensitivities were sufficient for clinical analysis. Immunoaffinity purification with HT-RPLC produced a selective and accurate bioanalysis without an LC-MS/MS instrument. Both methods could become general-purpose analytical methods and complement the results obtained with conventional LBA.Display Omitted
Keywords: Therapeutic antibodies; High-temperature reversed-phase liquid chromatography; Immunoaffinity purification; Bevacizumab; Infliximab;