Analytical Methods (v.8, #13)

Front cover (2709-2710).

Contents list (2711-2717).

We present herein a simple, sensitive and specific method for CK-MB detection for the early diagnosis of myocardial infarction through magnetic separation integrated with chemiluminescence. FITC and ABEI labelled anti-CK-MB antibodies and magnetic beads functionalized with anti-FITC antibodies are used to make a CK-MB complex after magnetic separation and the intensity of the chemiluminescence is used as a readout signal. The limit of detection is as low as 0.296 ng mL−1 in serum and other common substances in serum do not cause interference. We test 120 real clinical samples using our method and the results are consistent with the existing commercial platform, which confirms the practical promise of our proposed method.

Hydrothermal synthesis of blue-emitting silicon quantum dots for fluorescent detection of hypochlorite in tap water by Yongming Guo; Lianfeng Zhang; Fengpu Cao; Lianghong Mang; Xiaoling Lei; Shengjuan Cheng; Jintong Song (2723-2728).
A facile, sensitive and selective method for the fluorescent detection of hypochlorite in tap water has been developed by utilization of blue-emissive silicon quantum dots (SiQDs) with a quantum yield of 10.6%. The SiQDs are prepared via a simple hydrothermal treatment of (3-aminopropyl)trimethoxysilane and sodium citrate, and they display excellent stability in high salt conditions. The proposed preparation method for SiQDs does not require complicated synthesis and does not need the protection of an inert gas. It is attractive that the as-prepared SiQDs can be utilized to detect hypochlorite in tap water with relatively good selectivity and sensitivity, and as low as 0.01 μM hypochlorite could be successfully determined.

The use of 3-naphthyl-1-phenyl-5-(4-carboxyphenyl)-2-pyrazoline as a pre-column derivatization agent for the HPLC analysis of biomolecules containing an –NH2 group is proposed in this article. The derivatization conditions to obtain quantitative reaction were optimized by considering different parameters (time, temperature, pH and reagent concentration) using seven amino acids as model analytes. The resulting derivatives were well separated on a reversed-phase column with an isocratic elution profile and detected fluorimetrically at excitation and emission wavelengths of 370 and 480 nm, respectively. The proposed method was validated for accuracy, precision, sample stability, linearity, and detection and quantification limits and were all found to be satisfactory. Through this study, we are forecasting the emergence of pyrazoline based heterocyclic dyes as organic fluorescent markers for the labelling of diagnostically important compounds, for their determination and quantification of practical interest.

Simultaneous preparation and characterization of three high-purity type B fumonisins from maize culture by Wenbo Guo; Zheng Han; Junhua Yang; Qinxiong Rao; Zhihui Zhao (2737-2742).
A reliable and large-scale preparative method was developed for the simultaneous preparation of high-purity fumonisin B1 (FB1), fumonisin B2 (FB2) and fumonisin B3 (FB3) from maize culture inoculated with Fusarium moniliforme in this study. The targeted B-series fumonisins (FBs) were firstly extracted with acetonitrile/water (50 : 50, v/v) and then purified using MAX cartridges. The subsequent chromatographic preparative procedures on a Unitary C18 column (250 mm × 10 mm, 5 μm) and a SB-CN column (250 mm × 9.4 mm, 5 μm) were performed to effectively eliminate the other interferents to obtain the high-purity compounds. The prepared compounds were clearly identified to be FB1, FB2 and FB3 using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR). The purities were 96.25 ± 0.18%, 98.07 ± 0.18% and 97.36 ± 0.74% for FB1, FB2 and FB3, respectively, which were determined by the external standard LC-MS/MS method. Within 72 h, large amounts of high-purity FB1 (80 mg), FB2 (16 mg) and FB3 (16 mg) could be simultaneously prepared by the established approach.

Cholesterol is a major modifiable risk factor in cardiovascular disease and can be effectively managed by a combination of medication and monitoring. There continues to be a need for new point-of-care diagnostics to measure lipid panels, including total cholesterol. Enzyme assays based on the generation of hydrogen peroxide have been very effective in this regard. This work demonstrates the application of printed electrochemical sensors to the measurement of total cholesterol in serum. The assay uses the surfactant Triton X-100 to provide electrocatalytic enhancement of a silver paste screen-printed electrode to hydrogen peroxide, while also achieving effective solubilisation of total cholesterol from lipoprotein. The resulting biosensors employed 0.5% (v/v) Triton X-100 in PBS with 156 U mL−1 cholesterol esterase and 60 U mL−1 cholesterol oxidase. Measurement of total cholesterol in serum in the range of 0 to 10 mM had a sensitivity of 2.24 × 10−8 A mM−1, with coefficient of determination of 0.984, detection limit of at least 2 mM and average relative standard deviation of 10.8% (n = 3).

Digital droplet LAMP as a microfluidic app on standard laboratory devices by Friedrich Schuler; Clara Siber; Sebastian Hin; Simon Wadle; Nils Paust; Roland Zengerle; Felix von Stetten (2750-2755).
Digital nucleic acid amplification methods are a growing research field that allows for absolute quantification of DNA making the need of standard curves redundant. However, most of the existing digital amplification systems require specialized laboratory devices and costly investments. The required disposable cartridges are device specific and not interchangeable. Here, we present digital droplet loop-mediated isothermal amplification (ddLAMP) as a microfluidic app on standard laboratory devices. ddLAMP is implemented on a disposable polymer chip (DropChip) in the format of a standard microscope slide. After off-chip DNA denaturation, the reaction mix is emulsified in the DropChip in a mini centrifuge for 6 minutes. The DropChip is transferred to an in situ thermal cycler for 1 hour of incubation. Afterwards, a fluorescence scan in a microarray scanner is performed. The DropChip allows for absolute quantification with a dynamic range of 15–1500 DNA copies per μl. Assay conditions were optimized for ddLAMP and comparison of ddPCR and ddLAMP for genomic E. coli DNA reveals very good concordance.

A novel switchable hydrophobic–hydrophilic transition dispersive solid–liquid microextraction (SHT-DSLME) method has been used for the first time as a dispersive/extractive method for the removal of total selenium from real samples. Tetraethylenepentamine modified multi-walled carbon nanotubes (MWCNTs-TEPA) were synthesized and employed as an adsorbent for the removal and analysis of total inorganic selenium by SHT-DSLME coupled with graphite furnace atomic absorption spectrometry (GFAAS). In this study, the hydrophobic–hydrophilic transition of MWCNTs-TEPA was achieved by exposing them to a green, inexpensive, non-hazardous and non-accumulating antisolvent trigger: CO2. The switching phenomena of functionalized MWCNTs from hydrophobic to hydrophilic were confirmed using FTIR spectrophotometry and SEM measurements. Under optimized experimental conditions, the enhancement factor (EF) and limit of detection (LOD) were found to be 72 and 0.015 μg L−1, respectively for selenium. Validation of the developed method was carried out by selenium determination using water for trace elements (TM-28.3) as a certified reference material and the results were found to be in good agreement with the certified values. The proposed novel method was then successfully applied for the determination of selenium in real water samples.

Development of a facile and sensitive fluorimetric derivatization reagent for detecting formaldehyde by Chao Liu; An-Wei Cheng; Xue-Kui Xia; Yu-Fa Liu; Sheng-Wen He; Xu Guo; Jin-Yue Sun (2764-2770).
Formaldehyde is one of the main environmental pollutants and has significant impacts on human health. Herein, we developed a derivatization reagent for detecting formaldehyde rapidly, facilely and sensitively. Upon the introduction of formaldehyde, the reagent exhibited significant enhancement in fluorescence spectra at room temperature within 9 minutes, and the enhanced green fluorescence could be easily observed by the naked eye. Compared with other relevant analytes such as H2S, CO, NO and benzene, only formaldehyde could elicit the obvious increase in fluorescence. Furthermore, we prepared fluorescent test papers with the reagent and demonstrated their application for the detection of formaldehyde qualitatively.

Rapid detection of Dam methyltransferase activity based on the exonuclease III-assisted isothermal amplification cycle by Xue Li; Zhipeng Xie; Wei Wang; Yunlei Zhou; Huanshun Yin; Zhiqing Yang; Shiyun Ai (2771-2777).
The DNA isothermal amplification cycle is an effective expansion strategy, which can overcome the inherent limitation of the target to signal ratio of 1 : 1. In this work, we fabricated an electrochemical biosensor for the rapid detection of Dam methyltransferase activity based on the exonuclease III (Exo III) assisted isothermal amplification cycle. First, double stranded DNA (ds-DNA) containing the symmetric sequence of 5′-GATC-3′ was immobilized on the magnetic bead surface. After the ds DNA was methylated by Dam MTase, it could be further cleaved by DpnI and formed a blunt end, which could lead to the digestion of a DNA strand by Exo III and release of a target single stranded DNA (ts-DNA). Then, the ts-DNA can further hybridize with DNA probes (DNA3) immobilized on the AuNPs/Au electrode surface and form a blunt end DNA–DNA duplex. Subsequently, the DNA3 in the DNA–DNA duplex can be digested by Exo III and release ts-DNA. The released ts-DNA can further hybridize with the unhybridized single stranded DNA (ss-DNA) and an isothermal amplification cycle is achieved. The method showed high sensitivity with a low detection limit of 0.031 unit per mL. Moreover, inhibition investigation indicates that Dam MTase activity can be inhibited by using 5-fluorouracil with an IC50 value of 131.22 μM.

A new method was developed for the isolation and preconcentration of gatifloxacin (GTFX) and prulifloxacin (PUFX) based on magnetic mixed hemimicelle solid-phase extraction (MMHSPE) using sodium dodecyl sulphate (SDS) coated Fe2−xAlxO3 (x = 0.4) MNPs as the sorbent. The Fe2−xAlxO3 MNPs not only prevent dissolution of the Fe3O4 MNPs in acidic solutions but also extend their application without sacrificing their unique magnetic characteristics. Due to the high surface area of these new sorbents and the excellent adsorption capacity after surface modification by SDS, satisfactory preconcentration factors and extraction recoveries were produced with only 8 mg of Fe2−xAlxO3 MNPs. It is important to note that the largest adsorption capacity of the as-synthesized Fe2−xAlxO3 nanoparticles occurs at x = 0.4. Several parameters that affected the extraction efficiency were investigated, including the type and volume of desorption solvent, extraction and desorption time, pH of the solution, addition of NaCl and the sorbent amount. Under the optimized extraction conditions, a linear response was observed in a concentration range of 0.4 to 120 ng mL−1 for GTFX and 2 to 240 ng mL−1 for PUFX, and the limits of detection were 0.030 and 0.070 ng mL−1, respectively. The proposed procedure was applied successfully for the detection of the investigated drugs in their pharmaceutical dosage forms, plasma and urine.

Algorithmic modeling of spectroscopic data to quantify binary mixtures of vinegars of different botanical origins by José S. Torrecilla; Regina Aroca-Santos; John C. Cancilla; Gemma Matute (2786-2793).
Multiple binary mixtures of different kinds of vinegars have been analyzed through UV-Vis absorption. Two types of mathematical models (multiple linear regression (MLR) and artificial neural networks (ANNs)) have been employed to identify and quantify the components of such blends. Six different vinegars were used to prepare these mixtures, each one with a particular botanical origin: white wine, red wine, apple cider, apple, molasses, and rice. The best results have been obtained with ANN based models, offering mean estimation error value averages of 1% (v/v) and mean correlation coefficients (R2) over 0.99. This model is adequate to perform the estimation and achieve an accurate and reliable tool. Nevertheless, although the MLR models provide worse results (0.88 in terms of R2 and 5% v/v error), they can be used depending on the application and required accuracy.

The authentication and identification of different edible oils have become a focus of attention in the food safety field. In this work, we propose a method for distinction of edible oils by using a terahertz (THz) spectrum combined with genetic algorithm (GA) and partial least squares discriminant analysis (PLS-DA). To evaluate the robustness of the model, we also employ full spectra PLS (fsPLS), interval PLS (iPLS), and backward interval (biPLS) algorithms to verify the classification performance through variable selection. The results demonstrate that the GA-PLS-DA model has a smaller root mean square error of prediction (RESEP), a larger correlation coefficient of prediction (Rp), and higher classification accuracy than other models. In conclusion, the THz spectrum coupled with chemometrics is an effective method for differentiating various types of edible oils.

Nano-crystal ZSM-5 was fabricated through the reflux crystallization method under atmospheric pressure and the crystallization evolutionary process was investigated utilizing Diffuse Reflectance Infrared Fourier Transform (DRIFT) spectroscopy. The control of the crystallization process and understanding of the growth mechanism of nano-crystals are considerable in developing technological applications. The aim was to compare the outcome of the proposed approach as an alternative with that of XRD. Multivariate curve resolution-alternating least squares (MCR-ALS) was employed to resolve the DRIFT spectral data set providing the evolution trend of the crystallization process using Evolving Factor Analysis (EFA) as an initial estimate approach of concentration profiles. The non-parametric Wilcoxon signed rank test (t-test) was applied to evaluate the obtained results which confirmed the reasonable agreement between two methods.

Reorganization energy and Stokes shift calculations from spectral data as new efficient approaches in distinguishing the end point of micellization/aggregation by Mintu Halder; Shubhashis Datta; Priyanka Bolel; Niharendu Mahapatra; Sudipta Panja; Harsh Vardhan; Saurav Kayal; Deb Kumar Khatua; Ishita Das (2805-2811).
Critical micellar concentration (CMC) is an important parameter which indicates the matured associated state of amphiphilic molecules. The present work demonstrates a new, accurate, and generalized method for the determination of the critical micellar concentration (CMC) based on the estimation of the reorganization energy (RE) and Stokes shift of fluorescent probes placed inside micellar aggregates. With increasing concentration of surfactant, the fluorophore is well portioned in the micellar environment. The change in the magnitude of the Stokes shift and reorganization energy with respect to bulk water is a result of the formation of aggregates from surfactant monomers and there is a substantial change of RE in the vicinity of the CMC like what is observed with various other physical parameters during micellization. CMC values determined from both Stokes shift calculation and reorganization energy estimations are comparable with those previously reported in the literature. However, we demonstrate that reorganization energy calculation provides improved consistency and is more user-friendly than that estimated from Stokes shift measurements.

Characterization of poloxamers by reversed-phase liquid chromatography by Bing Guan; Amr Ali; Haofan Peng; Weiwei Hu; Lam Raga Markely; Scott Estes; Shashi Prajapati (2812-2819).
Poloxamer 188 plays a critical role in protecting cells during cell culture bioprocessing. To better understand the properties of poloxamers, a reversed-phase liquid chromatography (RPLC) method was developed to characterize the ingredients in poloxamers. A Waters SunFire C18 column gave good separation of ten poloxamer ingredients, which were monitored by using an ultraviolet (UV) detector at 200 nm and an evaporative light scattering detector (ELSD). UV detection of less hydrophobic ingredients showed good linearity and precision of the selected poloxamer ingredients. Furthermore, nine fractions were isolated from a single poloxamer 188 lot, and the molecular weights of all the fractionated ingredients were evaluated by size exclusion chromatography (SEC). This RPLC method can detect the variation not only among different types of poloxamers, but also among different lots of the same poloxamer. In addition, this RPLC method can be used to measure the stability of some poloxamer ingredients under various conditions.

Assessment of organosilane-functionalized nano-carbon black for interference-free on-line Pb(ii) ion enrichment in water, herbal medicines and environmental samples by Daniel Morais Nanicuacua; Mariana Gava Segatelli; Marcela Zanetti Corazza; César Ricardo Teixeira Tarley (2820-2830).
In the present work, commercial nano-carbon black (CB) particles were functionalized with 3-mercaptopropyltrimethoxysilane (3-MPTMS) through a silanization reaction to improve their adsorption capacity towards Pb(ii) ions and hence extend their application as an alternative and low-cost nanocarbonaceous material in the field of separation science. The material was characterized by FT-IR, scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), Raman spectroscopy, as well as by thermogravimetric analysis and textural data. After the characterization of the material (3-MPTMS/CB) a FIA-FAAS preconcentration method for Pb(ii) ions was developed, which in turn was performed by loading 20.0 mL of a 200 μg L−1 Pb(ii) solution at pH 4.76 (buffered with 0.0358 mol L−1 acetate buffer) through 100 mg of 3-MPTMS/CB packed into a mini-column at a flow rate of 4.0 mL min−1. The optimized conditions of the proposed method have been achieved through a 25-1 fractional factorial and Doehlert matrix design. The improvements in the adsorption capacity of CB towards Pb(ii) after chemical modification with 3-MPTMS were noticed by comparing the sensitivity of the analytical curve built with CB and 3-MPTMS/CB. Very satisfactory figures of merit including a limit of detection (LOD) of 1.33 μg L−1, limit of quantification (LOQ) of 4.45 μg L−1, preconcentration factor of 28.0, consumptive index of 0.714 mL, preconcentration efficiency of 5.6 min−1 and sample throughput of 12 h−1 were obtained. The precision of the method was assessed as relative standard deviation (RSD) (%) for 10 measures of 10.0 and 160.0 μg L−1 Pb(ii) solutions yielding values of 3.0 and 2.3%, respectively. The preconcentration method was successfully applied to the Pb(ii) ion determination in different kinds of water and Ginkgo biloba samples with satisfactory recovery values (91–108%). In addition, the accuracy of the proposed method was also checked by analysis of a certified marine sediment reference material (MESS-3).

Dephosphorylation of DNA by DNA 3′-phosphatases plays significant regulatory roles in DNA damage repair, replication, and recombination. Here, we present a novel, highly sensitive and label-free fluorescent assay for T4 polynucleotide kinase/phosphatase (T4 PNKP) activity and its inhibitors on the basis of nicking enzyme-assisted signal amplification and poly(thymine)-templated fluorescent copper nanoparticles (CuNPs) as a fluorescent indicator. It was designed such that an A-rich hairpin primer with a 3′-phosphoryl end can serve as the substrate for T4 PNKP. Once the phosphorylated hairpin primer was hydrolyzed by T4 PNKP, the resulting hairpin primer with a 3′-hydroxyl end was immediately elongated to form a long double-strand product using DNA polymerase as well as a double-stranded recognition site for nicking enzyme Nb.BbvCI. With the help of nicking enzyme Nb.BbvCI, the double-strand product could be cleaved to release a single-stranded poly-(thymine) (poly T) DNA, which could be used as a template for the formation of fluorescent CuNPs. Subsequently, the remaining probe could be reacted as the primer to trigger the next cycle of polymerization and scission process, thus significantly improving the sensitivity of the sensor. Furthermore, the application of the proposed strategy for screening T4 PNKP inhibitors also demonstrated satisfactory results. With the advantages mentioned above, this simple, cost-effective, and highly sensitive strategy has potential for the study of DNA damage repair mechanisms.

A simple, fast and green nanofibers mat-based disk solid-phase extraction technique for chrysoidine analysis in soybean products by Feifei Qi; Xiaoqing Li; Jingjing Liu; Kaiping Huang; Fei Rong; Qian Xu (2837-2844).
A simple, fast and green sample preparation method followed by HPLC-DAD was developed for the determination of chrysoidine in soybean products. A polypyrrole (PPy) functionalized nanofibers mat (PPy-NFsM mat) was positioned in a home-made filter assembled to a nanofibers mat-based disk solid-phase extraction (NFsM-disk SPE) device for sample cleanup and preconcentration. After a simple extraction with alkaline ethanol–water solution, the sample extractants were diluted and directly purified by the technique. Then, the eluent was analyzed forthrightly by HPLC without an evaporation–concentration step. Compared to present methods, it was a faster sample preparation (10 min per sample) method with remarkably reduced organic solvent (15.7 mL). The developed method was validated by the analysis of six different kinds of spiked soybean products, resulting in satisfactory recoveries (78.2–106.5%; RSD ≤ 9.7%), and limit of quantitation (0.05 mg kg−1), which demonstrate the feasibility of the proposed method. In addition, the PPy NFsM mat could be reused three times excluding matrix interference.

Certified reference materials (CRMs) for the determination of the water content of solids have been used to calibrate Karl Fischer titrators and to validate the measurement method for the accuracy and consistency of results. In the present study, CRMs of sodium tartrate dihydrate and potassium citrate monohydrate for the determination of water content were investigated and certified. The stability of water content against humidity, the thermal stability and the composition of substances evaporated on drying of the CRMs candidates were characterized. A mass balance approach based on mass loss on drying was investigated to measure their water content. Karl Fischer coulometric and volumetric titrations were improved, especially the sampling method, and were utilized to certify the two CRMs. The certified water content and its expanded uncertainty for the two CRMs were found to be 156.3 mg g−1 (1.3 mg g−1, k = 2) and 55.8 mg g−1 (0.6 mg g−1, k = 2), respectively.

Back cover (2853-2854).