Analytical Methods (v.9, #23)

Front cover (3391-3392).

Contents list (3393-3399).

C-Reactive protein: a major inflammatory biomarker by Mukesh Digambar Sonawane; Satish Balasaheb Nimse (3400-3413).
Inflammatory biomarkers are released during tumour growth, and they are highly associated with stages of cancer progression. C-Reactive protein is one of the widely studied marker proteins, which is associated with inflammation. CRP levels have been used to predict survival in patients with different cancers. The assimilation of CRP into prognostic models for cancers improves their predictive accuracy. The analysis of changes in CRP concentration over time can allow for the prediction of tumour aggressiveness and treatment efficacy. Therefore CRP can be an important biomarker for cancer diagnosis and prognosis, and the monitoring of treatment outcomes. This critical review focuses on the role of C-reactive protein in different types of cancers and its relation to the different stages of cancer. This article also focuses on recently reported methods for the detection of CRP.

In this communication, we report phenylboronic acid-functionalized gold nanoclusters (APBA–Lys–AuNCs) using lysozyme as a reducing reagent and capping ligand. Fluorescence quenching was performed for the selective detection of dopamine (DA) in human blood samples with satisfactory results.

The metabolism of ethanol to acetaldehyde has been visualized in living lung epithelial cells using a hydrazinyl naphthalimide fluorescent probe. Utilizing a condensation reaction between carbonyls and a hydrazine moiety, we demonstrate that the fluorescent probe (aldehydefluor-1) AF1 reacts with a range of reactive carbonyl species including formaldehyde, acetaldehyde, glyoxylic acid, and methyl glyoxal. With AF1, it is possible to directly visualize endogenous carbonyl metabolites. Here, we have applied it towards the visualization of acetaldehyde generated from alcohol dehydrogenase mediated ethanol metabolism, validating it as a useful tool to study the roles of alcohol in respiratory disease and other pathological mechanisms.

Ultrafiltration liquid chromatography-mass spectrometry (UF-LC-MS), affinity-guided isolation, and molecular docking were integrated into one strategy for screening of enzyme inhibitors from functional foods. UF-LC-MS facilitated the rapid characterization of ligands that could bind to an enzyme; affinity-guided isolation was helpful for the efficient preparation of ligands for further bioactivity validation; and molecular docking contributed to studying ligand–protein interactions. The strategy was employed to screen pancreatic lipase inhibitors from Monascus-fermented rice (MFR). Finally, three active compounds, namely monascin, monasfluore B, and ankaflavin were discovered by UF-LC-MS and then purified by semipreparative HPLC. Their lipase-inhibitory activities were verified using an enzymatic functional assay. The Lineweaver–Burk plots showed that they inhibited lipase in a non-competitive manner, and the potential mechanisms were preliminarily explored by molecular docking. The results suggest that the strategy is effective in discovering bioactive compounds from complex systems.

Functionalization of gold nanoparticles with nanobodies through physical adsorption by Julie Goossens; Hla Sein; Shaohong Lu; Magdalena Radwanska; Serge Muyldermans; Yann G.-J. Sterckx; Stefan Magez (3430-3440).
Nanobodies (Nbs) are single-domain antibodies with highly desirable properties employed as versatile tools in structural biology, biochemistry and biomedical sciences. Nbs also have a significant potential for the development of highly specific and user-friendly diagnostic assays. These immuno-assays often employ gold nanoparticles (AuNPs) as a detection label. Despite great advances in Nb technology and the immuno-sensor field, the functionalization of AuNPs with Nbs mostly remains unchartered territory. In this paper, we present a study of the functionalization of AuNPs with Nbs through physical adsorption. We show that Nb pI, solution pH, additives and protein engineering strategies have profound effects on the generation of stable Nb–AuNPs. Our results further demonstrate that physical adsorption can be used to produce functional Nb–AuNPs that could be employed in a wide array of applications.

A new approach is described to prepare particle standards of the explosive 1,3,5-trinitroperhydro-1,3,5-triazine (RDX) for use in quantitative wipe-sampling studies. Screening activities to detect traces of explosives are heavily focused on wipe collection from surfaces such as fabrics, metals, and plastics with speed and efficacy as the goals. Studies to improve the sampling protocols, including development of improved wipe materials and selection of best surfaces, depend upon the availability of test materials that mimic particles in fingerprints made after handling explosives. RDX particle arrays were prepared by drop-on-demand (DOD) inkjet printing onto polytetrafluoroethylene (PTFE) substrates and then transferred by rubbing onto target surfaces. The particle size distributions in the transferred deposits approximately match the distributions measured from fingerprints (size range of 1 to 40 μm) for equivalent total masses of RDX, and the transfer is better than 95% efficient to three realistic surfaces; woven nylon, synthetic leather, and brushed steel. DOD inkjet printing can be tailored to produce the desired particle sizes by changing the total deposit mass at each point in the array. Deposits will not transfer efficiently to surfaces smoother than the PTFE transfer substrate. Wipe-sampling studies conducted with the new test materials at a fixed force and for a specific sampling wipe show that the sampling efficiency from woven nylon fabric (e.g., luggage) is 17 ± 4% whereas the efficiency from acrylonitrile butadiene styrene (ABS) plastic (e.g., luggage handles) under the same conditions is 37 ± 6%.

Enabling lateral transport of genomic DNA through porous membranes for point-of-care applications by Samantha A. Byrnes; Joshua D. Bishop; Paul Yager (3450-3463).
Paper-based nucleic acid diagnostics have the potential to translate laboratory assays to simple-to-use, point-of-care devices, but many prototypes of these systems still lack the ability to process realistic samples due to the inability of genomic-sized DNA to move through membranes with small pores. For applications involving pathogen or human gene identification, the ability to fragment and transport DNA would provide more options for device design and broaden the range of applications. To address this challenge, we have developed and characterized a method that combines cell lysis with DNA fragmentation to allow for lateral transport of genomic DNA through commonly-used porous membranes. Additionally, we demonstrate that varying heating time and temperatures allows for control of both lysis and fragmentation based on genome size. These data align with previously published models that describe both DNA denaturation and thermal scission. This level of control allows semi-selective transport of pathogenic DNA, which can reduce the amount of interference from non-target human DNA in downstream applications. This method can be easily automated and is rapid, occurring in less than 10 minutes with one user step.

Biomonitoring method for the analysis of chromium and cobalt in human whole blood using inductively coupled plasma-kinetic energy discrimination-mass spectrometry (ICP-KED-MS) by Joaudimir Castro Georgi; Yuliya L. Sommer; Cynthia D. Ward; Po-Yung Cheng; Robert L. Jones; Kathleen L. Caldwell (3464-3476).
The Centers for Disease Control and Prevention developed a biomonitoring method to rapidly and accurately quantify chromium and cobalt in human whole blood by ICP-MS. Many metal-on-metal hip implants which contain significant amounts of chromium and cobalt are susceptible to metal degradation. This method is used to gather population data about chromium and cobalt exposure of the U.S. population that does not include people that have metal-on-metal hip implants so that reference value can be established for a baseline level in blood. We evaluated parameters such as; helium gas flow rate, choice and composition of the diluent solution for sample preparation, and sample rinse time to determine the optimal conditions for analysis. The limits of detection for chromium and cobalt in blood were determined to be 0.41 and 0.06 μg L−1, respectively. Method precision, accuracy, and recovery for this method were determined using quality control material created in-house and historical proficiency testing samples. We conducted experiments to determine if quantitative changes in the method parameters affect the results obtained by changing four parameters while analyzing human whole blood spiked with National Institute of Standard and Technology traceable materials: the dilution factor used during sample preparation, sample rinse time, diluent composition, and kinetic energy discrimination gas flow rate. The results at the increased and decreased levels for each parameter were statistically compared to the results obtained at the optimized parameters. We assessed the degree of reproducibility obtained under a variety of conditions and evaluated the method's robustness by analyzing the same set of proficiency testing samples by different analysts, on different instruments, with different reagents, and on different days. The short-term stability of chromium and cobalt in human blood samples stored at room temperature was monitored over a time period of 64 hours by diluting and analyzing samples at different time intervals. The stability of chromium and cobalt post-dilution was also evaluated over a period of 48 hours and at two storage temperatures (room temperature and refrigerated at 4 °C). The results obtained during the stability studies showed that chromium and cobalt are stable in human blood for a period of 64 hours.

The research on neurodegenerative diseases is challenged by accurate disease diagnosis and evaluation of treatment response, thus there is a need for indicators at the molecular level, specifically biomarkers, that reliably provide disease diagnostic, progression, and prognostic information. The parenchyma of the brain is immersed in the interstitial fluid (ISF), which is in equilibrium with the cerebrospinal fluid (CSF) allowing for ex vivo sample analysis of potential endogenous in vivo biomarkers. Details on the kinetic parameters of these analytes may provide a more sensitive, specific and accurate measure of disease pathology, biological physiology, and pharmacodynamic response of therapeutics. To study the differential changes in the de novo biosynthesis as well as the clearance of proteinaceous/peptidic biomarkers, a mass spectrometric detectable label is introduced via stable isotope labeling in mammals (SILAM) methodology. Proteolytic processing of the proteins generates surrogate proteotypic peptides. The mass isotopomer distribution analysis (MIDA) of these proxy reporters via high resolution accurate mass spectrometry (HRMS) provides kinetic profiles of the proteins. Presented here is the conceptual methodology of applying LC-HRMS analysis to obtain MIDA data of CSF proteins which were modified by SILAM methodology utilizing D-labeled water (2H2O, D2O).

Integrating qualitative and quantitative assessments of Yougui pill, an effective traditional Chinese medicine, by HPLC-LTQ-Orbitrap-MSn and UPLC-QqQ-MS/MS by Haolong Liu; Feng Qiu; Baolin Bian; Xinwei Yang; Haiyan Zou; Hui Zhao; Zhenzhen Chen; Qingsong Wang; Haiyu Zhao; Lei Wang (3485-3496).
Yougui pill (YGP) is a well-known Chinese medicine formula with definite clinical effects on kidney-yang deficiencies such as infertility. However, Aconitum carmichaeli var. carmichaeli, one of the herbs contained in YGP, shows several side effects. Therefore, it is important to carry out strict and systematic quality evaluation on YGP to ensure its effects and safety. In the present study, qualitative and quantitative analyses were performed on YGP. First, an HPLC-LTQ-Orbitrap-MSn method was applied to validate the chemical profile of YGP. Eighty-three compounds were identified, including iridoids, flavones and alkaloids. Second, an UPLC-QqQ-MS/MS method was used for the simultaneous quantitation of 16 targeted compounds in YGP. The method showed a good linear correlation (R > 0.995) over a wide range and a high sensitivity, as established by LOD (0.04–0.40 ng mL−1) and LOQ (0.10–1.00 ng mL−1). The RSD values of intra- and inter-day precisions were less than 10%. The RSD values of repeatability and stability were less than 5%, and recovery assays were between 85.8% and 96.2%. The cluster analysis indicated that the qualities of YGP from two manufacturers were very different. The present integrated qualitative and quantitative assessment of YGP provides a good example of quality evaluation strategy to assess Chinese medicine formulas.

Multielement determination in medicinal plants using electrothermal vaporization coupled to ICP OES by C. M. M. Santos; M. A. G. Nunes; A. B. Costa; D. Pozebon; F. A. Duarte; V. L. Dressler (3497-3504).
Electrothermal vaporization (ETV)-inductively coupled plasma optical emission spectrometry (ICP OES) is proposed for multielement determination in powdered medicinal plants. Simultaneous determination of As, Cd, Cr, Cu, Mn, Ni, Pb and Zn was performed in 10 herbs consumed in Brazil. Heating of the ETV system and the influence of carrier gas flow rate, modifier gas (Freon R12, CCl2F2) flow rate and sample mass were evaluated and adjusted to obtain the best signal/noise ratio. Temperatures of 300 °C and 1800 °C were used for pyrolysis and vaporization, respectively. Freon has a strong influence on the pyrolysis and vaporization temperatures and the sensitivity. Although the partial elimination of the sample matrix and Freon can act to minimize matrix effects, standard addition calibration was necessary for direct element determination in solid samples. The accuracy of ETV-ICP OES measurements was evaluated by analysing two certified reference materials (NIST 1515 – Apple Leaves and NIST 1574 – Peach Leaves), and the comparison of results obtained by solution nebulization ICP OES (NEB-ICP OES) for acid decomposed samples. The results were in good agreement (t-test at the 95% confidence level) with certified values and those obtained by NEB-ICP OES. Precision was better than 20%, considering the standard deviation of five replicates, and limits of detection and quantification varied from 0.01 to 0.48 and 0.03 to 1.60 μg g−1, respectively. The proposed method allows the simultaneous determination of As, Cd, Cr, Cu, Mn, Ni, Pb and Zn in medicinal plants in less than 5 min, including the sample preparation step.

Design of a ratiometric fluorescent probe for naked eye detection of dopamine by Mohammad Amin Farahmand Nejad; M. Reza Hormozi-Nezhad (3505-3512).
A simple and effective ratiometric fluorescence sensor for selective detection of dopamine (DA) in alkaline media has been developed by simply mixing thioglycolic acid (TGA) functionalized orange fluorescent cadmium telluride (CdTe) quantum dots (QDs) with amino-functionalized blue fluorescent carbon nanodots (CDs). Under a single excitation wavelength of 365 nm, the sensor exhibits dual-emissions centered at 445 and 603 nm. The fluorescence of CdTe QDs is selectively quenched by DA, whereas the fluorescence of CDs is insensitive to the analyte. In the presence of different amounts of DA, the variations in the dual emission intensity ratios exhibit a continuous color change from pink to purple and to blue, which can be clearly observed with the naked eye. The ratiometric fluorescent probe has been successfully applied for the detection of DA in water and urine with a detection limit of 1.3 μM. To facilitate their practical use, ratiometric fluorescent probe–agarose hydrogels were prepared as instrument-free and simple platforms for visual detection of DA.

A pencil drawn microelectrode on paper and its application in two-electrode electrochemical sensors by Ezzaldeen Younes Jomma; Ning Bao; Shou-Nian Ding (3513-3518).
A simple approach for fabricating an inexpensive, disposable pencil drawn microelectrode on paper for a two-electrode electrochemical system is developed. The morphology of the fabricated microelectrode was evaluated by optical microscopy and scanning electron microscopy. The electrochemical behaviors of the pencil drawn microelectrode have been investigated by cyclic voltammetry using ferrocyanide/ferricyanide as redox probes. Moreover, Prussian blue was successfully electrochemically deposited onto the pencil drawn microelectrode to detect hydrogen peroxide with a low detection limit of 0.05 μM and a wide linear range from 1.0 μM to 1300 μM (R = 0.997). This cheap and effective microelectrode fabrication method may offer a wide channel for developing lab-on-paper applications through an appropriate combination of graphite pencil traces and paper properties.

Enhancement of the peroxidase-like activity of cerium-doped ferrite nanoparticles for colorimetric detection of H2O2 and glucose by Morteza Hosseini; Fereshte Sadat Sabet; Hossein Khabbaz; Mustafa Aghazadeh; Farhang Mizani; Mohammad Reza Ganjali (3519-3524).
In this report, cerium doped-ferrite nanoparticles (Ce–Fe3O4 NPs), a novel ceria nanostructure, were proposed to have intrinsic peroxidase-like activity toward a classic peroxidase substrate in the presence of H2O2. The peroxidase-like activity of ceria originated from its facile redox behavior (Ce3+/Ce4+) as a catalytic center, thereby explaining the high performance of ferrite–ceria as an artificial enzyme mimicking peroxidase. The as-prepared Ce–Fe3O4 NPs were first demonstrated to possess intrinsic peroxidase-like activity, enhancing the oxidation of the substrate 3,3′,5,5′-tetramethylbenzidine (TMB) in the presence of either added H2O2 or H2O2 produced by the enzymatic oxidation of glucose, producing a blue product which could be seen by the naked eye. Based on this, we have successfully established a novel platform for the colorimetric detection of H2O2 with a detection limit of 0.6 μM. Furthermore, the Ce–Fe3O4 NP-based platform can also be used for highly sensitive and selective detection of glucose with a linear range of 5.0–150.0 μM and a detection limit of 1.2 μM.

The fluorescent graphene quantum dots (GQDs) provide an amusing nanoplatform, which integrates unique optical properties of quantum dots and prominent electronic properties of carbon materials. However, rational design of dual functional GQDs has been rarely explored. In this study, we constructed an enzyme-free fluorescent sensing method for catechins in green tea, using bifunctional GQDs, which were employed as the peroxidase-like catalyst and a fluorescence probe. In the presence of dissolved oxygen, GQDs with intrinsic peroxidase-like catalytic activity could catalyze the oxidation of catechins, and the oxidation product could efficiently quench GQDs' fluorescence. Based on this finding, an enzyme-free fluorescent nanoplatform was proposed for the sensing of catechins, and the detection limit of 5.0 nM was found. The integration between the excellent fluorescence performance of GQDs and their high catalytic activity made this sensor a simple and sensitive device for the detection of catechins.

A new method was developed for the purification of Nd in geological samples using a single column containing TODGA extraction resin and the precise determination of Nd isotope ratios by multicollector inductively coupled plasma-mass spectrometry (MC-ICPMS). The samples were digested with HF–HNO3 and then transferred to a solution of 3 M HNO3. After loading the solutions onto the TODGA resin column, the column was first eluted with 12 ml of 2.3 M HCl, followed by 8 ml of 1.2 M HCl. The Nd isotope ratios of the latter eluate were measured by MC-ICPMS. This separation procedure was validated by measuring the 143Nd/144Nd ratios (mean ± 2σ) of certified reference materials, BHVO-2, BCR-2 and AGV-2, which were determined to be 0.512989 ± 0.000012, 0.512635 ± 0.000012 and 0.512790 ± 0.000018, respectively, in good agreement with the certified and literature values.

A stable isotope labelling assisted LC-MS method for the determination of polyamines in micro-tissues of rice by Qiu-Yi Wang; Tiantian Ye; Shu-Jian Zheng; Er-Cui Ye; Ren-Qi Wang; Yu-Qi Feng (3541-3548).
A stable isotope labelling assisted LC-MS method was developed for highly sensitive quantitation of polyamines in roots and leaves of rice plants under cadmium stress. Successful quantitation of polyamines in minute amounts of plant extracts allowed the detection of spatial and temporal changes in an organ/tissue-specific manner. Accumulations of putrescine under cadmium stress were observed in the tips of both roots and leaves. However, different regulations of spermine contents in the two regions were observed. Meanwhile, the influence of cadmium stress from the medium was found to be persistent towards the growth of roots after cultivation for 6 days, whereas the leaves gradually reached homeostasis after 3 days. Moreover, our micro-regional analysis on leaf tissues has for the first time revealed that the influences of cadmium stress towards the contents of polyamines gradually translocated directionally on the leaves. Typically, the influences towards the contents of major polyamines shifted from the sheath and central zone towards the tip and peripheral zone.

Development and optimisation of home-made stir bar sorptive extraction for analysis of plastic additives: application in human urine by Aurélie Cortéjade; Audrey Buleté; Laura Prouteau; Saber Chatti; Cécile Cren; Emmanuelle Vulliet (3549-3560).
The overall objective of this study was to develop an analytical method to contribute to the assessment of the exposure of humans to plastic additives based on the use of a new polydimethylsiloxane-based stir bar extraction method. The method focuses on a list of 21 contaminants, including 18 plastic additives and 3 degradation products. They are analysed by ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) after a stir bar sorptive extraction from urine using a home-made stir bar. Thus, the paper focuses on the optimisation of the extraction method. The limits of quantification are variable from one substance to another, but they fall between 0.1 and 305 ng mL−1. The sample preparations lead to recoveries of up to 90% in water and 80% in urine. The method was successfully applied to 15 human urine samples. Among the targeted plastic additives and their degradation products, 7 compounds were quantified in samples at concentrations ranging from 0.3 ng mL−1 to 42.7 ng mL−1.

We reported the use of polydopamine/Au nanoparticles/DNA (PDA/Au NPs/DNA) as a stationary phase for the separation of tryptophan enantiomers by microchip capillary electrophoresis (MCE). The stationary phase was formed by sequentially immobilizing PDA/Au NPs and DNA to the glass microfluidic channel surface using layer-by-layer assembly. The formation of PDA/Au NPs as a functional film involved the use of HAuCl4 as an oxidant to trigger DA self-polymerization. DA was then used as a reductant to reduce HAuCl4 to Au NPs. All these Au NPs were simultaneously deposited on the microchip surface by the adhesive PDA, and the thiolated DNA was strongly adsorbed onto the glass microfluidic channel surface through covalent gold–sulfur bonding. The functional film of the PDA/Au NPs/DNA was characterized by scanning electron microscopy, UV-vis spectra and ATR-FT-IR. The influence of separation voltage and the concentration and pH value of the running buffer were found to significantly influence separation. Successful baseline separation of tryptophan enantiomers was achieved within 65 s utilizing an effective separation length of 33 mm, and coupling with laser-induced fluorescence detection resulted in a resolution factor of 2.95. All these evaluation parameters, including stability and reproducibility, were considered satisfactory. The method outlined here, using the PDA/Au NPs/DNA-coated microfluidic channel as a versatile platform, may offer a processing strategy to prepare a functional surface on microfluidic chips.

Back cover (3569-3570).