Analytical Methods (v.9, #38)

Front cover (5561-5562).

Contents list (5563-5569).

Fluorescent sensors for sodium ions by Ge Gao; Yingli Cao; Wei Liu; Dai Li; Wenhu Zhou; Juewen Liu (5570-5579).
Sodium is a highly important metal in biological systems and its concentration in vivo is tightly regulated. However, the fluctuation of sodium levels is still common and problematic and is responsible for several physiological disorders and illnesses, including hypertension and cardiovascular disease. Analytical methods for measuring sodium in biological samples are of great importance for disease diagnosis and for fundamental physiological studies. In this article, various types of fluorescent probes for Na+ based on small molecule chelators, proteins, and nucleic acids are reviewed, and each type has its advantages and disadvantages. While most efforts were made in developing small molecule based fluorescent sensors, recent developments in DNA-based sensing have also been quite promising with excellent sensitivity and selectivity. In particular, a few RNA-cleaving DNAzymes and an aptamer were reported with excellent selectivity for Na+ over K+ and Li+. Currently, the best DNAzyme-based sensors have a detection limit of ∼0.1 mM Na+, and the dynamic range can cover its physiological concentration of ∼140 mM in blood. The limitations of each type of probe are also discussed, and future research opportunities are speculated at the end.

Compound Danshen Dripping Pills (CDDPs) are a Chinese herbal medicinal preparation composed of Salviae miltiorrhizae Radix and two other Chinese medicines, Notoginseng Radix and Borneolum. The main components of CDDPs are protocatechuic aldehyde, danshensu, salvianolic acid A, rosmarinic acid, ginsenoside Rg1, ginsenoside Rb1, l-(−)-borneol and isoborneol. The complex composition of CDDPs, which contains phenolic acids, ginseng saponins and borneols, makes quantitative analysis by traditional methods difficult. Therefore, a fast and accurate 1H qNMR method was established that can simultaneously determine the eight main components of CDDPs. The qNMR method was well validated and successfully applied in the determination of ten sample batches. The vector cosine angle method was used for further analysis of the results to evaluate the sample quality. The results indicate that the qNMR method is suitable for the quality control of CDDPs and has potential as an easy alternative for the analysis of other complex Traditional Chinese Medicines (TCMs) that require two or more traditional instruments for comprehensive quantitative analysis.

A novel and facile method for the fabrication of an indium tin oxide (ITO) photoelectrode modified with eosin Y–rhodamine B duo-dye sensitized Au nanoparticles is presented. After mixing with HAuCl4, a precipitate emerged in the duo-dye solution of eosin Y and rhodamine B. The precipitate was collected, dispersed in ethanol and spin-coated onto the ITO electrode. Different techniques were used to characterize the precipitate, including scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), ultraviolet-visible spectroscopy (UV-Vis), infrared spectroscopy (IR) and photoluminescence (PL). Results of the characterizations showed that HAuCl4 was reduced to Au nanoparticles in situ, which were transformed by rhodamine B, while eosin Y retained its original molecular structure, forming an eosin Y–rhodamine B–Au nanocomposite. Compared with a solution containing only HAuCl4 and rhodamine B, the solution containing all three components produced more eosin Y–rhodamine B–Au nanocomposite precipitate, and exhibited an appreciable photocurrent response in the photoelectrochemical system. Furthermore, the modified ITO photoelectrode gave a specific response in the presence of Hg2+, which offers potential in the fields of environmental monitoring and food safety.

Development of biosensors for the detection of oversulfated chondroitin sulfate (OSCS), the key contaminant in heparin (Hep), is a greatly demanding but challenging task. In this work, a FRET system for the detection of OSCS was successfully constructed by using supercharged green fluorescent protein (ScGFP) as the energy donor and dye labelled Hep (Hep-RF1) as the energy acceptor. With heparinase treatment, Hep-RF1 was hydrolyzed into small fragments, resulting in quenching of the FRET signal. On the other hand, since OSCS is an inhibitor for heparinase, the presence of OSCS would enable the effective FRET from ScGFP to Hep-RF1 even after heparinase treatment. With this ScGFP based FRET sensing platform, as low as 0.001% (w/w) OSCS in Hep has been successfully detected.

A simple, rapid and sensitive colorimetric method has been developed for the detection of Cd2+ using 2,6-dimercaptopurine-modified gold nanoparticles. In the presence of Cd2+, gold nanoparticles functionalized with 2,6-dimercaptopurine were aggregated via chelation between the metal ion and the ligand, accompanied by a color change from red to blue observed with the naked eye. Further, the solution of 2,6-dimercaptopurine-modified gold nanoparticles was monitored using a UV-Vis spectrometer. Under optimized conditions, good linearity varying from 0.75 to 3.0 μM was obtained with a correlation coefficient (R) of 0.996 along with a limit of detection of 32.7 nM. The recoveries were 88.0–112.0% in real samples, and the results agree well with those acquired by atomic absorption spectroscopy with a graphite furnace. The new approach was successfully applied to the determination of Cd2+ in milk, honey, lake water, serum, and urine samples.

Selective separation of xanthones and saponins from the rhizomes of Anemarrhena asphodeloides by modulating the density of surface charges in C18-bonded stationary phases by Jianfeng Cai; Huaxia Xin; Lingping Cheng; Zhimou Guo; Jiatao Feng; Qing Fu; Yu Jin; Xinmiao Liang (5604-5610).
A rapid and robust separation method based on the positively charged reversed-phase (PGRP) stationary phase was developed for selective separation of saponins and xanthones from the rhizomes of Anemarrhena asphodeloides (A. asphodeloides). In this work, the chromatographic performances of three PGRP stationary phases with different surface positive charge densities were systematically evaluated by studying hydrophobicity, effects of pH and buffer concentration of the mobile phase, ion-exchange capacity, etc. The PGRP stationary phase exhibited reversed-phase/anion-exchange mixed-mode properties. Then the retention behaviors of xanthones were investigated. A good retention of xanthones was obtained at high pH and xanthones were easily eluted at low pH. The pH is like an on–off switch on the PGRP stationary phase that controls the retention of xanthones. Finally, this PGRP material was successfully applied to the selective separation of saponins and xanthones from A. asphodeloides. The result demonstrated that neutral and ionic compounds, such as xanthones and saponins from the rhizomes of A. asphodeloides, were separated selectively by modulating both the density of surface charges in the PGRP stationary phase and the pH of the mobile phase.

Dual-modal fluorescence and light-scattering sensor based on water-soluble carbon dots for silver ions detection by Guoliang Liu; Chenglei Xuan; Da-Qian Feng; Dongliang Hua; Tianhao Liu; Gang Qi; Wei Wang (5611-5617).
A facile fluorometric and light-scattering dual-modal nanosensor for silver(i) ions detection was established based on target-responsive aggregation of carbon dots/cysteine system. The synthesized water-soluble fluorescent carbon dots (CDs) by the one-pot hydrothermal method exhibited emissions peak at 441 nm under excitation at 352 nm. CDs fluorescence was quenched by Ag+via electron transfer, while the light-scattering intensity of CDs was significantly enhanced, providing a dual-modal means of analyte Ag+ detection. The relative fluorescence intensity is directly proportional to the concentration of Ag+ between 0.1 μM and 265 μM, and the detection limit is 50 nM. Moreover, the relative light-scattering intensity is proportional to the concentration of Ag+ from 10 to 4000 nM with the detection limit 2 nM. Common compounds including different types of metal ions had no significant interference in the detection mode. The new nanosensor was simple, rapid and sensitive and was applied to the detection of Ag+ in real water samples with satisfactory results. Moreover, the present CDs-based dual-modal sensor provided a promising platform for environmental sensing applications.

A glassy carbon electrode decorated with Fe-OSA was successfully fabricated and its application in electrochemical detection of heavy ions was investigated. Fe-OSA was characterized by scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), X-ray diffraction (XRD), and Fourier transform infrared (FTIR) spectroscopy. The properties of glassy carbon electrode (GCE) before and after modification were analyzed by cyclic voltammetry (CV), and the swelling, reproducibility, and thermal stability of Fe-OSA were studied. In the case of Pb(ii), electrochemical detection was conducted by varying different parameters such as electrolyte, pH and deposition time. The electrochemical behavior of three heavy metal ions (Cd(ii), Cu(ii), and Pb(ii)) on Fe-OSA-modified GCE was investigated under optimum conditions by differential pulse voltammetry (DPV). Using DPV, it was found that Fe-OSA-modified GCE could be used to effectively detect Cd(ii), Cu(ii) and Pb(ii) individually. According to the obtained parameters (correlation coefficients and limit of detection) from the linear relationship between current and concentration, it was revealed that metal ions in low concentrations could also be detected simultaneously with high sensitivity.

Determination of cause of death using paper-based microfluidic device as a colorimetric probe by N. Ansari; A. Lodha; A. Pandya; S. K. Menon (5632-5639).
In recent years, the use of benzodiazepines has increased in the commission of crime, homicide, suicide, drugs overdoses, robbery, and drug-facilitated sexual assault. Assessment of drug exposure is also costly and resource-intensive because the analysis typically requires sophisticated instrumental techniques. Hence, driven by the need to detect alprazolam (ALP), we report a novel, simple, portable, disposable, and rapid inexpensive microfluidic paper-based analytical device (μPAD) for measuring ALP from vitreous humor and blood. This μPAD is embedded with silver nanoparticles for colorimetric detection and rapid quantification of alprazolam in different biological fluids using a smart phone. Under optimal conditions, the limit of detection is found to be 0.8 ng mL−1via a UV-vis spectrophotometer, whereas the limit of μPAD-based smart phone-enabled ALP detection is 10 ng mL−1. This μPAD was successfully applied to drug-exposed real samples of blood and vitreous humor. The proposed method provides mix and measure detection platform using a test paper and smart phone, which offers a new alternative for on-site detection especially in field situations of drugs in forensic cases and as point of care (POC) device for clinical diagnosis for toxicological drug screening. The device formed herein meets the criteria of the present-generation analytical devices in terms of portability, selectivity, sensitivity at the ng mL−1 level, cost effectiveness, simplicity, data transmission facility, and rapid on-site investigation.

Analysis of deferiprone in exhaled breath condensate using silver nanoparticle-enhanced terbium fluorescence by Esmail Mohamadian; Ali Shayanfar; Maryam Khoubnasabjafari; Vahid Jouyban-Gharamaleki; Saba Ghaffary; Abolghasem Jouyban (5640-5645).
Exhaled breath condensate (EBC) has been proposed as an alternative non-invasive biological sample for therapeutic drug monitoring. In this study, a sensitive method for the quantification of deferiprone (DEF) in EBC samples was developed using a silver nanoparticle (AgNP)-enhanced terbium (Tb3+) fluorescence method. Fluorescence properties of the Tb3+–DEF complex were studied in the presence of AgNPs in EBC. Effects of various parameters such as AgNPs and Tb3+ concentrations, pH, and buffer concentration were studied. A calibration curve was obtained with a coefficient of determination (R2) of 0.999, and linearity was observed in the concentration range of 0.06–1.50 mg L−1. The method was successfully applied for the quantification of DEF in sixteen patients with very low concentrations (0.06–0.17 mg L−1) in the EBC samples.

Metal–organic framework enhanced electromembrane extraction – a conceptual study using basic drugs as model substances by Ali Reza Fakhari; Sajad Asadi; Hasan Mohammadi Kosalar; Ali Sahragard; Alireza Hashemzadeh; Mostafa M. Amini (5646-5652).
This work aims to use an appealing metal–organic framework (MOF), MIL-101(Cr), for the enhancement of electromembrane extraction efficiency. The use of MIL-101(Cr) in the supported liquid membrane (SLM) enhanced the analyte partition coefficient within the membrane, resulting in the enhancement of analyte transport. The optimization of the different variables affecting the proposed method was carried out in order to achieve the best extraction efficiency. Three narcotic drugs, including methadone, methamphetamine, and tramadol, were selected as model basic drugs. Optimal extractions were obtained with 2-nitrophenyl octyl ether, containing 2.0 mg mL−1 MIL-101(Cr) as the SLM and 125 V as the driving force with pHs of 4.0 and 1.0 for donor and acceptor solutions, respectively. Equilibrium extraction conditions were obtained after 25 min of operation with a stirring rate of 1250 rpm. Under the optimized extraction conditions, the proposed technique provided good linearity (>0.997), repeatability (RSD = 6.7–9.6%, n = 5), low limits of detection (0.30–0.91 ng mL−1), excellent preconcentration (132–190) and high recoveries (66–95%). Finally, the developed method coupled with CE-UV was successfully employed for the determination of the three narcotic basic drugs in biological samples, including plasma and urine.

A rapid and sensitive turn-on fluorescent probe for ascorbic acid detection based on carbon dots–MnO2 nanocomposites by Yalei Hu; Lin Zhang; Xin Geng; Jia Ge; Haifang Liu; Zhaohui Li (5653-5658).
Ascorbic acid (AA) is a significant small biomolecule and plays a key role in many biochemical processes. In this work, a rapid turn-on fluorescent probe was developed for sensitive and selective sensing of AA based on carbon dots–MnO2 nanocomposites. The nanocomposites were constructed by in situ synthesis of MnO2 nanosheets in carbon dot (C-dot) solution, and the fluorescence of C-dots was quenched with the formation of the nanocomposites due to the fluorescence resonance energy transfer (FRET) from C-dots to the generated MnO2 nanosheets. Once AA was introduced into the system, MnO2 nanosheets were reduced to Mn2+ and the fluorescence of C-dots would be recovered. Under the optimized conditions, this method shows rapid and sensitive response toward AA, the assay time is 2 min and the limit of detection is 68 nM. Moreover, the carbon dots–MnO2 nanocomposite based fluorescence sensing system was successfully employed to determine AA in commercial tablets with satisfactory results. Taking full advantage of C-dots and MnO2 nanosheets, this probe shows remarkable properties including easy operation, low cost and good biocompatibility, and has great potential to be used in biological and clinical diagnostic applications.

Herein, an efficient carrier assisted electromembrane extraction coupled with capillary electrophoresis has been proposed for the quantitation of basic drugs (pseudoephedrine and tramadol) in urine samples. Nonionic lipophilic surfactants in the supported liquid membrane act as carriers and promote the migration of ionic analytes toward the acceptor phase. The volumes of the sample and acceptor solutions were 4000 and 20 μL, respectively. All variables affecting the extraction efficiency were investigated for the proposed method. Optimal extractions were accomplished with 2-nitrophenyl octyl ether, containing 0.25 mM Span 80 as the supported liquid membrane, with 200 V as the driving force and with pH 5.0 in donor and pH 1 in acceptor solutions. Extractions were obtained after 30 min of operation with the whole assembly agitated at 1000 rpm. Under the optimum experimental conditions, the linear range was 10–2000 and 5–2000 ng mL−1 for pseudoephedrine and tramadol, respectively (R2≥ 0.99). The limits of detection were calculated to be 3.03 and 1.51 ng mL−1 for pseudoephedrine and tramadol, respectively. The relative standard deviation was lower than 4.1%. The enrichment factors were in the range of 178 to 188. The method was successfully applied for the analysis of these drugs in urine samples.

In this work, we demonstrated a facile green method for the preparation of fluorescent copper nanoclusters (Cu NCs) with a high quantum yield of 8.6%, using glutathione as the stabilizing agent and ascorbic acid as the reductant. The as-prepared Cu NCs had good water solubility and ultrafine sizes. Further investigation revealed that the fluorescence of Cu NCs was linearly quenched by nitrite ions over the concentration range of 10–225 μM, with the detection limit of 3.4 μM. The proposed method has been successfully applied to determine nitrite ions in real water samples.

Paper test card for detection of adulterated milk by Jamie L. Luther; Valentine Henry de Frahan; Marya Lieberman (5674-5683).
In developing countries, milk is adulterated to meet demand and increase profit. Often the milk is diluted with water, and sugar, urea, and starch are added to mimic the color, consistency, and nitrogen content of the real thing. Since laboratory tests for detecting these adulterants are difficult to perform in a field setting, we developed an inexpensive, paper analytical device (MilkPAD) to detect these analytes in milk. All of the reagents, buffers, and enzymes needed to perform each test are stored in dry form in the paper. The user just needs to pipette several drops of the milk onto each reaction area, wait 40 minutes, and take a cell phone photo of the test card. The test card can colorimetrically detect a variety of starches (0.005% w/v), urea (in excess of 70 mg dL−1), and the sugars glucose and sucrose (in excess of 0.1 mM). The sensitivity and specificity of the MilkPAD is greater than 90% for detection of any of these adulterants at concentrations that are typically found in adulterated milk.

Thrombin is an important serine protease in blood and a therapeutic biomarker. The aptamer-based assays for thrombin take advantage of unique features of nucleic acid aptamers in selection, preparation, stability, and modification of functional groups. Aptamer affinity capillary electrophoresis coupled with laser induced fluorescence (CE-LIF) analysis for thrombin uses a fluorescently labeled aptamer probe and relies on the change of electrophoretic mobility of the aptamer probe caused by protein–aptamer binding. A high-affinity 29-nt DNA aptamer (Apt29) has been used as an affinity probe in CE-LIF analysis of thrombin. In this work, we made a further detailed investigation of the effects of metal ions (e.g. Na+, K+, and Mg2+) in sample buffer on the CE-LIF analysis of thrombin using a dye labeled 29-mer DNA aptamer probe and achieved sensitive detection of thrombin. A complex of thrombin and the Apt29 probe was well separated from the unbound probe in CE separation. We found that the addition of K+ in sample buffer was not preferred in CE-LIF analysis of thrombin using dye-labeled Apt29, and it caused significant decrease of complex peaks of thrombin–Apt29. The use of both Na+ and Mg2+ in sample buffer was favorable for the sensitive detection of thrombin because a larger peak area of complex peaks of thrombin and the aptamer probe was obtained. Under the optimized conditions, as low as 0.1 nM thrombin was successfully detected with good specificity. This work shows that metal ions in sample buffer have a large effect on aptamer affinity CE-LIF analysis of thrombin using an Apt29 probe.

A facile, rapid method based on a liquid–liquid extraction system for the purification of graphene quantum dots is reported in this study. Nitrogen-doped graphene quantum dots (N-GQDs) were synthesized by a simple hydrothermal route for cutting graphene oxide sheets. Then, through the liquid–liquid extraction system of tetrahydrofuran–(NH4)2SO4–water, N-GQDs have been successfully purified. The as-prepared N-GQDs have an average size of 6 nm and exhibit optimum excitation and emission wavelengths of 330 and 435 nm, respectively. N-GQDs were demonstrated as a sensitive fluorescent sensing probe for Fe3+. The sensor can detect Fe3+ (R2 = 0.992) in the concentration range of 0.2–30 μM, with a detection limit of 0.06 μM. This method of quantitative analysis of Fe3+ has good linearity and reliable reproducibility.

Amino acid profiling from fingerprints, a novel methodology using UPLC-MS by Ward van Helmond; Chris-Jan Kuijpers; Elise van Diejen; Jincey Spiering; Brent Maagdelijn; Marcel de Puit (5697-5702).
Fingermark evidence is extensively used in criminal investigations. Hence, there have been many investigations into the chemical compounds present in fingerprint deposits. In this technical note we describe the analysis of non-derivatised amino acid profiles obtained from fingerprints. We used UPLC with an amide stationary phase and subsequent detection using a triple quadrupole MS/MS and TOF-MS detector. The linearity (R2) was satisfactory for both MS detectors (>0.98 for all amino acids in the case of the triple quadrupole MS/MS and >0.96 in the case of the TOF-MS). Although the triple quadrupole had a higher sensitivity for most amino acids, both mass spectrometers were able to retrieve the amino acid profiles of fingerprints from 19 donors. Between these profiles, only minor differences were observed between the separate analyses on the different mass analyzers, mainly in l-proline, l-lysine and l-phenylalanine abundances. Surprisingly, the mean RSD in amino acid profiles from duplicate fingerprints turned out to be lower for the TOF-MS (18.6% ± 6.6% vs. 13.2% ± 3.8%), as did the mean RSD of the intraday reproducibility (8.22% ± 1.94% vs. 9.54% ± 3.07%).

On-line clean-up and LC-MS analysis of primary metabolites in cell culture supernatants by Teresa Mairinger; Julia Tröndle; Michael Hanscho; Stephan Hann (5703-5710).
Current approaches to modern metabolic profiling of biotechnological samples are facing several challenges, e.g. the wide concentration ranges and the complexity of the matrix. While the information on concentration levels of extracellular metabolites is much in demand, especially for flux balance analysis, the measurement is hampered by a highly complex matrix. This affects the general performance of an analytical method over time. In the present study, an automated clean-up procedure, aiming at matrix separation for the analysis of primary metabolites via liquid chromatography coupled with time-of-flight mass spectrometry, was tested and optimized for supernatants of Chinese hamster ovary cultures. A uniformly 13C labeled yeast cell extract was employed for assessing recovery. The optimized on-line clean-up, comprising filtration and solid phase extraction for matrix removal, showed recoveries ranging from 45.0% to 138.7%, with an average of 11.6% relative standard deviation. Additionally, a non-targeted analytical approach by accurate mass spectrometry for the differential assessment of the matrix separation was performed.

Back cover (5711-5712).