Analytical Methods (v.11, #7)

Front cover (863-864).

Contents list (865-870).

Electrochemical biosensors for autoantibodies in autoimmune and cancer diseases by Susana Campuzano; María Pedrero; Araceli González-Cortés; Paloma Yáñez-Sedeño; José M. Pingarrón (871-887).
Autoantibodies (AAbs) are antibodies produced against our own cells or tissues either providing a first defense against infections or indicating the presence of pathological processes. They are not only able to inform on the evolution of diseases but also to predict some illnesses well in advance. Currently, the evaluation of the number and type of formed AAbs is employed to assess the risk, rate, severity and progression of autoimmune and cancer diseases, and to help find therapies to prevent or mitigate the impact of these illnesses. Conventional methods for the determination of AAbs generally suffer from low sensitivity, time-consuming and laborious methodologies, and need specialized technicians and well-equipped labs. Consequently, seeking new methodologies for the rapid and low-cost screening of AAbs is of great interest for global health because it would shorten the delay between sample collection and diagnosis and improve the introduction of modern diagnostics into the developing world. Electrochemical biosensors are considered as a promising alternative to conventional techniques for the determination of clinical biomarkers due to their simplicity of use, low cost, high sensitivity, multiplexing abilities or compatibility with microfabrication and point of care testing. This review is focused on the critical discussion of selected electrochemical biosensors described to date for the determination of AAbs related to autoimmune diseases and several types of cancer. An overview pointing out future directions in this field is also provided.

Emerging patterns in the global distribution of dissolved organic matter fluorescence by Urban J. Wünsch; Rasmus Bro; Colin A. Stedmon; Philip Wenig; Kathleen R. Murphy (888-893).
The spectra responsible for natural dissolved organic matter (DOM) fluorescence in 90 peer-reviewed studies have been compared using new similarity metrics. Numerous spectra cluster in specific wavelength regions. The emerging patterns suggest that most fluorescence spectra are not tied to biogeochemical origin, but exist across a wide range of different environments.

In environmental trace analysis there is often a need to enrich the compounds of interest from a large sample volume, where the use of solid phase extraction (SPE) is more or less the standard technique. The presence of the sample matrix can cause clogging of the SPE-column, especially at the end of a sample load. Swedish surface waters are often humic rich making the use of traditional sample loading by a vacuum manifold very limited. This obstacle forced the development of a different sample loading technique, based on compressed air and sand as an in-line-filter, designed to load larger sample volumes as needed in trace level analysis of hormones in surface water. The developed technique, combined with a UPLC MS/MS method, showed promising reproducibility and accuracy, and enabled increased sensitivity for the analysis of hormones in humic rich surface water.

A fluorescent fingerprint recording strategy for complex chemical solution by Qiannan Duan; Jianchao Lee; Chaoqun Zheng; Yunyun Zheng; Han Chen (897-900).
A fluorescent fingerprint recording strategy for complex chemical solution (CCS) analysis was proposed. Using ink-jet printing technology, the feature information of the CCS was expressed by fluorescence imaging method. Obtained fluorescent fingerprint images (named PEL-image) might provide some possible interlinks among different samples by the digital image analysis.

An efficient “Ratiometric” fluorescent chemosensor for the selective detection of Hg2+ ions based on phosphonates: its live cell imaging and molecular keypad lock applications by Gujuluva Gangatharan Vinoth Kumar; Ramaraj Sayee Kannan; Thomas Chung-Kuang Yang; Jegathalaprathaban Rajesh; Gandhi Sivaraman (901-916).
Herein, we have reported the ability of smart chemosensors (L1 and L2) to selectively detect g2+ ions over other metal ions and examined this phenomenon by various spectroscopy techniques. Particularly, the proposed chemosensors exhibited notable color and spectra changes with the addition of mercury ions. Moreover, colorimetry measurements and fluorescence could be reversed with the addition of EDTA into the L1-Hg2+ and L2-Hg2+ solutions. Based on the excellent reversibility of this process, we designed a molecular-scale sequential information processing circuit and proposed the “Writing-Reading-Erasing-Reading” and “Multi-write” behaviors in the form of binary logic. Furthermore, L1 and L2 mimic a molecular keypad lock and molecular logic gates with the successive addition of Hg2+ and EDTA. The binding ability of Hg2+ ion with L1 and L2 was further demonstrated by density functional theory (DFT) studies. The biocompatibility of these chemosensors was successfully used for live cell imaging of Hg2+ in HeLa cells under the experimental conditions. Furthermore, the sensors L1 and L2 were useful in the determination of the concentration of Hg2+ ions in real water samples; this indicated their potential for practical applications.

Highly sensitive optical biosensing of Staphylococcus aureus with an antibody/metal–organic framework bioconjugate by Neha Bhardwaj; Sanjeev K. Bhardwaj; Deepanshu Bhatt; Satish K. Tuteja; Ki-Hyun Kim; Akash Deep (917-923).
In this research, a new luminescent bioprobe was developed for the detection of S. aureus based on bio-conjugation of an amine functionalized metal–organic framework (NH2-MIL-53(Fe)) with an anti-S. aureus antibody (Ab). The formation of the desired bioprobe (Ab/NH2-MIL-53), in its liquid phase, has been verified with several spectroscopic and structural characterization techniques. The bioprobe was incubated with varying concentrations of S. aureus bacteria. The resulting antibody conjugated bioprobe (Ab/NH2-MIL-53) maintained a strong inverse correlation in which decreases in the fluorescence intensities were accompanied by an increase in the bacterial count. Thus, the potential of the herein developed probe has been successfully demonstrated for the detection of S. aureus with a low limit of detection (85 CFU mL−1) over a wide concentration range (4 × 102–4 × 108 CFU mL−1). It was further found to be reliable with regard to inter-/intra- precision assays and long-term stability. The feasibility of the method was further supported through the detection of S. aureus spiked in environmental samples (e.g., river water and cream pastry).

The field of point-of-need (PON) screening is currently lacking a rapid, sensitive and cost-effective method for bacterial detection. Such a method would prove invaluable for the efficient identification of pathogenic bacteria that result in a host of human infections and plague the healthcare sector. Surface-enhanced Raman spectroscopy (SERS) has increasingly been put forward as a viable candidate for a bacterial detection and identification platform due to its excellent sensitivity and the corresponding rich vibrational signature. Recently, there has been significant debate within the SERS community as to the origin of the observed SERS signals for cultured bacterial samples. This uncertainty stems from the remarkable similarity among many of the observed SERS signals for a vast array of bacteria, suggesting the spectral features may have a common origin. Electrochemical surface-enhanced Raman spectroscopy (EC-SERS) has recently been shown to largely improve upon the sensitivity of SERS for detection of bacteria. In this report, we will highlight the importance of conducting control studies using filtered bacterial samples. Using a 0.2 μm syringe filter to remove all bacterial cells from the examined sample, which is confirmed via scanning electron microscopy, this study demonstrates an excellent control for determining the origin of the bacterial signal in EC-SERS measurements. Our findings strongly support the argument that SERS spectra observed for bacterial cultures originate from small molecule metabolites released by the bacteria in response to environmental stressors.

Determination of sample stability for whole blood parameters using formal experimental design by Lara P. Murray; Keith R. Baillargeon; Jordan R. Bricknell; Charles R. Mace (930-935).
Biological samples have dynamic properties that can be affected by several preanalytical factors including collection, handling, and storage. When developing diagnostic assays that use biological samples as the input, these factors must be identified and managed to ensure that sample degradation is not jeopardizing the performance of the assay. In this work, we use formal experimental design to identify the factors that influence the stability and integrity of a sample of whole blood with respect to two parameters: the hematocrit and the extent of hemolysis. We found that storage time is the only preanalytical factor with a statistically significant impact on the hematocrit for both the reference centrifugation method and the response of a paper-based hematocrit device, with hematocrit increasing as a function of increased storage time. We attributed this trend to changes in red blood cell morphology that occur with an increased storage time. For this reason, we determined it to be critical to develop the paper-based device and hematocrit assay using whole blood within 48 hours of receipt to ensure that it is not introducing bias to the diagnostic output. Additionally, our results confirmed that our paper-based hematocrit device operates according to our established principle—that the transport distance is determined by the ratio of plasma volume to total volume of whole blood. The approach described in this manuscript can be applied to other parameters or biological samples to develop diagnostic assays with confidence that the selected parameters are consistent with the intended end-user input.

Papain-stabilized silver nanoclusters: dual emission and ratiometric fluorescent sensing of ferrous ions by Qinchao Mo; Minna Jia; Peifeng Zhuang; Simin Yang; Wanting Su; Yixuan Zhu; Na Shao; Meiping Zhao (936-941).
Herein, a dual emissive fluorescent probe based on papain-stabilized Ag nanoclusters was developed. The as-prepared papain–Ag NCs showed a ratiometric fluorescent response to Fe2+, while other cations and anions had no effects on the fluorescence of the papain–Ag NCs. The emissions at 454 nm and 647 nm both decreased and the ratio of the fluorescence intensity at the two wavelengths (F454/F647) had a good linear relationship with the concentration of Fe2+. This probe could be used for detecting Fe2+ in the range from 1.5 to 120.0 μmol L−1 with a detection limit of 500 nmol L−1, which was comparable to or better than those of some other fluorescence approaches for Fe2+ sensing. The responding mechanism was attributed to the chelation of glycine (Gly) residue in the papain ligand with Fe2+, which led to the fluorescence quenching of the papain ligand at 454 nm due to the static quenching effect and reduced the charge transfer from the papain ligand to Ag NCs resulting in the fluorescence quenching of Ag NCs at 647 nm. Moreover, the papain–Ag NC probe was extended to the detection of Fe2+ in a real sample of ferrous lactate oral solution with satisfactory results compared with the standard 1,10-phenanthroline method.

Nowadays, consumption of chicken nuggets as a tasty fast food has become remarkable. Therefore, the safety and quality of this type of food is critical. Heterocyclic aromatic amines (HAAs) are a type of toxic compound formed during the cooking process for meat-based fast food such as nuggets. These substances have detrimental effects on human health and are known to cause chronic diseases such as cancer. In this study, the analysis of 4 types of important HAAs, namely, 2-amino-3‚8-dimethylimidazo[4‚5-f]quinolone (MeIQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-1-methyl-6-phenylimidazo[4‚5-b] pyridine (PhIP) and 2-amino-3-methyl imidazo[4,5-f]quinolone (IQ), were performed with an efficient microwave-assisted extraction-low-density solvent based on a dispersive liquid–liquid microextraction (MAE-LDS-DLLME), followed by reverse phase high-performance liquid chromatography (RP-HPLC). The main parameters affecting microextraction step were selected. Central composite design (CCD) was established to achieve the optimal value for each parameter. The concentration range of 5–500 ng g−1 for HAAs was obtained with a coefficient determination higher than 0.991. The relative standard deviation (RSD%) was between 5.9 and 7.9%. The limit of detection (LOD) was between 2.9 and 4.0 ng g−1. The recovery range was 90–97% and the enrichment factor (EF) was obtained in the range of 165 and 210. The proposed technique is highly sensitive, selective, quick, and easy, and has high capability for removing sample matrix interferences. Different values of HAAs from 284 to 6416 ng g−1 were determined in several real samples. The highest and the lowest average concentration of HAAs (∑4 HAAs) were 8714 ng g−1 and 1657 ng g−1, respectively. PhIP was detected in all samples in the range of 284–1882 ng g−1.

Homing peptide-based ELISA-like method for the selective and sensitive determination of fibrin by Yinghong Zhang; Yuanfu Zhang; Tingting Hou; Rui Li; Qingwang Xue; Shuhao Wang (950-954).
A novel, homing peptide-based ELISA-like method for the determination of fibrin is presented. Compared to the antibody, homing peptides have lower antigenicity, better biodegradability, and higher penetration ability. In this study, fibrin was coated on the surface of a 96-well plate. Then, the homing peptide (CREKA), which was labeled by horseradish peroxidase (HRP), was added and allowed to react with fibrin. After washing, the TMB–H2O2 colorimetric system was utilized for the quantitative analysis of fibrin. The assay showed a linear response toward fibrin concentration in the range of 0.1–10 nM with a correlation coefficient of 0.9976. The limit of detection for fibrin was experimentally determined to be 0.04 nM, based on a signal-to-noise ratio (S/N) of 3. The sensing system has been used for the determination of fibrin in human serum samples. Relative to the conventional methods, this method offers the advantages of high-throughput, stability, simplicity, sensitivity, low cost and selectivity, showing great potential for applications in clinical diagnosis.

Construction of salicylaldehyde analogues as turn-on fluorescence probes and their electronic effect on sensitive and selective detection of As(v) in groundwater by Lina Wang; Yong Li; Xike Tian; Chao Yang; Liqiang Lu; Zhaoxin Zhou; Yunjie Huang; Yulun Nie (955-964).
Arsenic contamination in groundwater is a severe and major environmental problem that endangers public health. As such, a sensitive and selective methodology for the detection of this toxic metalloid must be urgently developed. Aldimine condensation reactions of p-phenylenediamine with salicylaldehyde derivatives yielded “turn-on” fluorescence probes (HBHP, HBBP and HMBP) for the detection of As(v) with high sensitivity. Among these, as a typical AIEgen, HBHP exhibits the highest detection sensitivity. The detection limit of HBHP for As(v) was calculated to be 0.88 ppb, which is due to its higher ability to form intermolecular hydrogen bonds to convert twisted intramolecular charge transfer (TICT) into planar intramolecular charge transfer (PICT) and its higher molecular planarity triggered by specific electronic effects. Job's plots and DFT theoretical studies indicate that HBHP binds to As(v) with a stoichiometry of 1 : 2, and the binding constant was determined using the Benesi–Hildebrand equation to be 4.26 × 104 M2. Commonly existing anions and cations in groundwater exhibited no interference with the selective detection of As(v). Spiking and recovery tests of HBHP in the presence of As(v) in actual water samples gave satisfactory experimental results. Our research would provide novel principles of molecular construction and materials for the detection of As(v) in groundwater.

Spin probe method of electron paramagnetic resonance spectroscopy – a qualitative test for measuring the evolution of dry eye syndrome under treatment by Mihaela Monica Constantin; Catalina Gabriela Corbu; Cristiana Tanase; Elena Codrici; Simona Mihai; Ionela Daniela Popescu; Ana-Maria Enciu; Sorin Mocanu; Iulia Matei; Gabriela Ionita (965-972).
Tear composition is a source of information on the health state of the eye. Dry eye syndrome represents nowadays a common disorder that can be associated with other eye diseases, in particular keratoconus. As such, we focussed on the correlation of selected protein levels with ophthalmic parameters and, as a novelty, we explored the possible correlation of these data, classically used in examination, with electron paramagnetic resonance (EPR) spectral parameters of spin probes. Three groups of patients with (i) dry eye syndrome, (ii) dry eye syndrome and keratoconus and (iii) healthy eyes were considered. Tears were collected from the subjects to determine the levels of total protein, lactoferrin, lysozyme and human serum albumin, and to record the EPR spectra of spin probes with fatty acid-like structures: 5-doxyl stearic acid (5-DSA) and 4-(N,N′-dimethyl-N-hexadecyl)ammonium-2,2′,6,6′-tetramethylpiperidine-1-oxyl iodide (CAT16). In tear secretion, the EPR spectra of spin probes showed two components, one with slow dynamics and another one revealing a quasi-isotropic, rapid motion. We analysed in depth the changes observed in the EPR spectra of 5-DSA for the reason that the differences in dynamics between the 5-DSA components are more evident. The challenge of this study was to provide an alternative to biochemical analysis in monitoring the evolution of the health state of patients diagnosed with dry eye syndrome or keratoconus using the information provided by EPR spectra of spin probes. The results indicate a decreased contribution of the EPR component with rapid motion following treatment, and a significant correlation with the Schirmer and/or BUT values.

Detection of ppb-level NO2 gas using a portable gas-sensing system with a Fe2O3/MWCNTs/WO3 sensor using a pulsed-UV-LED by Pi-Guey Su; Jia-Hao Yu; I-Cherng Chen; Hong-Ci Syu; Shih-Wen Chiu; Ting-I Chou (973-979).
A portable gas-sensing system which is periodically irradiated with light from a pulsed ultraviolet light emitting diode (UV-LED) was fabricated for sensing ppb-level NO2 gas. The portable NO2 gas-sensing system includes a device manager coupled to a pulsed-UV-LED sensing module, a liquid-crystal display module and a data capture module. The sensor made of a composite iron oxide/multi-walled carbon nanotubes/tungsten oxide (Fe2O3/MWCNTs/WO3) material was produced by a one-pot polyol method followed by metal organic decomposition (MOD). Scanning electron microscopy (SEM), energy dispersive spectrometry (EDS) and X-ray diffraction (XRD) were used to analyze the structure and morphology of the fabricated films. The effects of the wavelength and duration of the irradiation on the response of the portable gas-sensing system for sensing NO2 gas were studied. This portable gas-sensing system in periodically pulsed-UV-LED operation mode exhibited high sensitivity, good linearity, good stability, high reproducibility, a low detection limit and high selectivity in the sensing of NO2 gas.

Enzymatic reaction modulation of G-quadruplex formation for the sensitive homogeneous fluorescence sensing of cholinesterase and organophosphate pesticides by Xin Yuan; Sujuan Chen; Shan Li; Qiuyun Liu; Mengqian Kou; Ting Xu; Hong Luo; Ke Huang; Mei Zhang (980-988).
In this work, we present the reported T–Hg2+–T hairpin structure and enzymatic reaction-modulated formation of the G-quadruplex, which is further employed for the fluorescence assays of butyrylcholinesterase (BChE) and organophosphate pesticides (OPs, parathion) in real samples. BChE can catalyze the reaction of acetylthiocholine (ATCh) to produce thiocholine (TCH), which has a thio-ligand to combine with mercury ions (Hg2+) in T–Hg2+–T. The released single-stranded DNA can associate with N-methyl mesoporphyrin IX (NMM) and K+ to form a G-quadruplex, producing a strong fluorescence signal. Therefore, the concentration of the released probe DNA was proportional to the amount of BChE, enabling the conversion of the target reaction events into a detectable signal from the G-quadruplex–NMM. Parathion, as an inhibitor of BChE activity, can be used to prevent the generation of thiocholine and therefore leads to less or no release of free probe DNA. As a result, most of the probe DNA maintains its T–Hg2+–T hairpin structure in the reaction solution, which leads to a significant fluorescence signal decrease. Therefore, by using the “signal-off” mode, the simple and homogeneous fluorescence detection of OPs is readily achieved. Under optimal conditions, the detection limits of BChE and OPs are 0.15 ng mL−1 and 0.025 ng mL−1 respectively. Besides, BChE in serum samples has been detected with satisfactory results. The proposed method retains several unique advantages, including being simple, cost-effective, highly sensitive and selective for label-free analysis.

Back cover (989-990).