Analytical Methods (v.11, #17)

Front cover (2233-2234).

Contents list (2235-2239).

Carbon quantum dots: synthesis, properties, and sensing applications as a potential clinical analytical method by Saipeng Huang; Wenshuai Li; Pu Han; Xin Zhou; Jiewei Cheng; Huiyun Wen; Weiming Xue (2240-2258).
Carbon quantum dots (CDs) are a novel class of fluorescent carbon nanoparticles and nanomaterials with fascinating properties, which have attracted worldwide attention and have been applied in different fields during the last decade. In this article, we have reviewed the recent progresses made in the field of bioanalysis and biosensor, focusing on their synthesis methods, use of novel precursors, particle size control, construction methodologies and modification strategies, quantum yields, photoluminescence properties and luminescence mechanisms, linear relationships and detection limits, biocompatibility and water solubility, functionalization, and technical applications, particularly focusing on their applications in biosensing different bioactive ingredients along with some discussions on the challenges and perspectives in this promising field. This review can arouse research interest in the fabrication of biosensors by using CDs, including detection in major biomarkers, significant responses, and crucial enzymes in the pathological process, which will provide a vital tool and important bioinformatics for the early prevention, diagnosis, and treatment of diseases.

Identification of specific pre-analytical quality control markers in plasma and serum samples by Luz Ruiz-Godoy; Virginia Enríquez-Cárcamo; Lourdes Suárez-Roa; María Lourdes Lopez-Castro; Abel Santamaría; Mario Orozco-Morales; Ana Laura Colín-González (2259-2271).
Blood is a liquid connective tissue with a complex composition that supplies oxygen and nutrients to cells. It is minimally invasive to obtain and can be used in “omics” technologies. Current research regarding blood-based biomarkers has gained considerable attention as a useful tool for diagnostic, therapeutic, and drug development; however, several research groups have reported controversial results despite using the same analytical platform. Such discrepancy can be caused by errors made during the pre-analytical phase. Therefore, quality control of pre-analytical variables is an important factor that could affect the reproducibility and credibility of results. This review discusses the challenge to identify blood quality control markers and describes several molecules that could be potential candidates.

Alkaline phosphatase (ALP) is a membrane-bound glycoprotein widely distributed in humans. It can catalyze the hydrolysis of phosphate monoesters under alkaline conditions. Since the variation of ALP activity is associated with various diseases, it is crucial to develop sensitive and accurate methods for monitoring ALP activity. We report here a fluorescence assay for testing ALP activity and inhibition based on fluorescent hydroxyapatite nanoparticles (HAP-NPs). The fluorescence intensity of the HAP-NPs can be initially quenched by Cu(ii) and then recovered by inorganic pyrophosphate (PPi), a substrate of ALP. So ALP can regulate the HAP-NP fluorescence intensity by hydrolyzing PPi into phosphate ions (Pi). According to the test results, ALP can be quantitatively detected in the range from 1 U L−1 to 625 U L−1. When applied for real sample analysis, the level of ALP activity in human serum samples detected by our assay was in good agreement with the clinical test results. In addition, the assay was utilized for screening ALP inhibitors. These data indicate that the assay has good potential for clinical and pharmaceutical applications.

A methodology for the fast identification and monitoring of microplastics in environmental samples using random decision forest classifiers by Benedikt Hufnagl; Dieter Steiner; Elisabeth Renner; Martin G. J. Löder; Christian Laforsch; Hans Lohninger (2277-2285).
A new yet little understood threat to our ecosystems is microplastics. These microscopic particles accumulate in our oceans and in the end may find their way into the food chain. Even though their origin and the laws governing their formation have become ever more clear fast and reliable methodologies for their analysis and identification are still lacking or at an early stage of development. The first automatic approaches to analyze μFTIR images of microplastics which have been enriched on membrane filters are promising and provide the impetus to put further effort into their development. In this paper we present a methodology which allows discrimination between different polymer types and measurement of their abundance and their size distributions with high accuracy. In particular we apply random decision forest classifiers and compute a multiclass model for the polymers polyethylene, polypropylene, poly(methyl methacrylate), polyacrylonitrile and polystyrene. Further classification results of the analyzed μFTIR images are given for comparability. The study also briefly discusses common issues that can arise in classification such as the curse of dimensionality and label noise.

High-resolution colorimetric detection of lipase activity based on enzyme-controlled reshaping of gold nanorods by Hao Zhang; Shengnan Wu; Linghua Zhang; Ling Jiang; Fengwei Huo; Danbi Tian (2286-2291).
We developed a novel enzyme-controlling colorimetric assay to measure lipase activity. The method relied on enzymatic reaction-assisted gold deposition on gold nanorods (AuNRs) to generate a significant color change, which was strongly dependent on lipase activity. The H2O2 produced by autoxidation of the ethylene oxide subunits in Tween 80 reduced AuCl4 ions to Au atoms, which were coated onto of the surface of the AuNRs. In addition, lipase catalyzed the hydrolysis of the carboxyl ester bond in Tween 80, which controlled the rate of reshaping of AuNRs and tailored the localized surface plasmon resonance of the AuNRs. A linear response ranging from 0 to 4.5 mg mL−1 and a detection limit of 0.017 mg mL−1 for lipase were obtained. The methodology provided a sensitive, convenient, and rapid assay to monitor enzyme activities by judging different colors using both naked-eye detection and colorimetric instruments.

Highly sensitive enzyme-free amperometric sensing of hydrogen peroxide in real samples based on Co3O4 nanocolumn structures by Palanisamy Kannan; Thandavarayan Maiyalagan; Alagarsamy Pandikumar; Longhua Guo; Pitchaimani Veerakumar; Perumal Rameshkumar (2292-2302).
Developing a non-noble metal nanostructure based highly active nanocatalyst for sensitive detection of small molecules is of great interest due to its large active surface area and rapid catalytic activity towards analytes. In this work, we demonstrate the synthesis of porous cobalt oxide nanocolumn arrays (Co3O4 NCs) and explore them as an efficient catalyst material for electrochemical determination of hydrogen peroxide (H2O2) in real samples (river water and antiseptic solutions). The morphology and crystalline properties of the as-synthesized Co3O4 NC structures are characterized by using scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), and electrochemical methods. Unlike Co3O4 nanoparticles (Co3O4 NPs), the Co3O4 NCs show significantly higher sensing performance towards the electrochemical reduction of H2O2 in 0.1 M NaOH solution. The presence of nanoporous structures in Co3O4 NCs could provide a large specific surface area, higher conductivity and rapid analyte diffusion, which makes them a promising candidate for detection of H2O2. The amperometric analysis shows that Co3O4 NCs exhibited a faster electrocatalytic response towards a wide concentration range of H2O2 (100 to 2000 μM) with the lowest limit of detection (LOD) of 0.28 μM (S/N = 3). The Co3O4 NC electrode is highly selective for detecting H2O2 in the presence of major important biomolecules such as glucose, ascorbic acid, uric acid, dopamine, and other anionic interfering compounds. These results demonstrate that Co3O4 NCs serve as a potential sensing platform towards the electrochemical detection of small molecules.

Refractive index (RI) sensing in microfluidics has the advantage of universal detection, capable of sensing all species from simple monoatomic ions to complex proteins without external labels or additional contrast agents. Various forms of interferometry have been developed for RI sensing in microfluidics. In particular, backscatter interferometry (BSI) is easily implemented and well-suited for miniaturization. This is important for future applications in point-of-care or point-of-interest measurements, where the total analysis system needs to be easily deployed. The optical arrangement in BSI is similar to that used in optical pickup heads (OPHs), found in CD and DVD drives. This offers intriguing possibilities for repurposing OPHs for miniaturized RI detection in microfluidics. To explore the feasibility of this approach, commercially available OPHs are modified for RI detection in 75 μm i.d. (363 μm o.d.) fused silica capillaries. BSI interference patterns measured using a modified OPH positioned near the capillary are compared with simulations as a function of wavelength. Once characterized, the modified OPH is used to measure refractive index changes as sucrose solutions are injected through the 75 μm i.d. capillary. Signal level changes were recorded following the introduction of solutions ranging in concentration from 67 μM to 19.3 mM and the resulting calibration plot (67 μM to 4.8 mM) exhibited good linearity (R2 = 0.9993). Finally, a modified OPH was used to detect the electrophoretic separation of Na+ and Li+ using RI detection. While the measurements reported here used modified OPHs that bypassed the built-in photodiode detector, eventually all on-board components could be utilized for a completely self-contained, inexpensive, universal detector for field deployable microfluidic applications.

A switch-on fluorophore using water molecules via hydrogen bonding and its application for bio-imaging of formaldehyde in living cells by Yile Wang; Yifan Chen; Yan Huang; Qi Zhang; Yucang Zhang; Jianwei Li; Chunman Jia (2311-2319).
Water molecules always quench fluorescence, limiting the practical application of fluorophores in bio-imaging. Probes developed from the well-established Aggregation Induced Emission (AIE) effects seem to be able to overcome this challenge. However, the background interference issue has hampered the development of the field for a long time. Herein, we report a type of fluorophore whose emission could be switched on using water molecules. The fluorophores were derived with aldehyde groups from a natural compound chromene and synthesized in just one-step. The triggering of fluorescence enhancement was not related to the well-known AIE mechanism or covalent reaction, but was further elucidated to be due to the non-covalent hydrogen bonding between the fluorophores and water molecules. Subsequently we optimized structures of the chromene derived fluorophores and chose a fluorophore for further application as a fluorescent probe. The probe showed high sensitivity and selectivity for formaldehyde. Moreover the excellent pH-, redox-, and photo-stability and low cytotoxicity guaranteed its successful imaging of formaldehyde in living cells. Our findings not only provide an example of fluorophores whose emission mechanism is complementary to the AIE mechanism, but also introduced a new type of probe for biosensing, bio-imaging and potential precise diagnosis in the future.

Development of a biomimetic enzyme-linked immunosorbent assay based on a molecularly imprinted polymer for the detection of cortisol in human saliva by Giulia Spano; Simone Cavalera; Fabio Di Nardo; Cristina Giovannoli; Laura Anfossi; Claudio Baggiani (2320-2326).
The development of reliable molecularly imprinted sorbent assays is ensured by in-depth knowledge of the binding between the tracer, conventionally based on a template analogue conjugated to an enzyme, and the imprinted polymer used as a recognition element. To this end, the binding properties of a cortisol-3-(O-carboxymethyl)oxime-horseradish peroxidase conjugate to cortisol-imprinted microparticles previously adsorbed at the bottom of microplates were assessed. The effect of different blocking agents as well as of different percentages of Tween 20 in the working buffer were investigated in order to minimise the non-specific binding of the enzyme tracer to the adsorbed imprinted microparticles. The capability of the enzyme tracer to bind the imprinted solid phase and to compete with free cortisol was assessed by measuring the apparent equilibrium dissociation constant, KD (39.7 ± 13.5 pmol L−1), and the apparent binding site concentration, Bmax (21.7 ± 4.3 nmol L−1), whereas the IC50 value for the cortisol competition curve was found to be 5.32 ± 1.15 ng mL−1. Moreover, binding selectivity measured for several cortisol-related steroids confirmed the experimental results previously published for cortisol-imprinted polymers. A competitive assay for the determination of cortisol in human saliva was developed with a limit of detection of 1.02 ng mL−1, providing quantitative results comparable to those of a commercial ELISA.

As the merit of ratiometric assay is impregnable due to potentially interfering processes, a ratiometric method for pesticide detection was developed. By adjusting glutathione : HAuCl4 to an appropriate ratio, dual-emission luminescent ultra-small gold nanoparticles (AuNPs) with a high emission at 800 nm and a low emission at 600 nm were synthesized. Interestingly, the sulfhydryl-containing compounds were found to result in completely opposite changes to strengthen the 600 nm emission and weaken the 800 nm emission. Therefore, dual-emitted AuNPs were engaged to develop a ratiometric pesticide biosensing strategy. In the presence of acetylcholinesterase (AChE), acetylthiocholine can be hydrolyzed into thiocholine, whose newly generated sulfhydryl can interact with AuNPs, resulting in the opposite change of the dual emissions. While adding pesticide as an AChE inhibitor, the catalytic activity of AChE is inhibited and less thiocholine was produced. The biosensing system shows an obvious sensitivity to the pesticide with a limit of detection (LOD) of 0.2 nM for aldicarb and 0.07 nM for chlorpyrifos. Therefore, this simple assay is suitable for AChE activity and pesticide detection, even in vegetable samples.

A multiclass method for the determination of pharmaceuticals in drinking water by solid phase extraction and ultra-high performance liquid chromatography-tandem mass spectrometry by Júlia A. de Oliveira; Lenise J. P. Izeppi; Roberta F. Loose; Daniela K. Muenchen; Osmar D. Prestes; Renato Zanella (2333-2340).
In this work, a simple method for the determination of 20 pharmaceuticals of different classes in drinking water using solid phase extraction (SPE) and ultra-high performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) was developed and validated. Different sorbents, solvents and elution volumes were evaluated. The optimized conditions of the extraction step were the Strata™-X cartridge, samples without pH adjustment but with addition of 0.1% (w/v) of EDTA, eluting solvent 1% (v/v) acetic acid in acetonitrile/methanol 1 : 1 (v/v) and an elution volume of 1 mL. Recovery results were adequate ranging from 70 to 119%, with relative standard deviation (RSD) ≤ 19%. The limits of quantification and detection of the method were from 0.01 to 0.1 μg L−1 and from 0.003 to 0.03 μg L−1, respectively, considered suitable for monitoring of these compounds in drinking water. The proposed method was successfully applied in real samples. Atenolol, carbamazepine and paracetamol were found in some samples at concentrations ≤ 0.027 μg L−1.

Development of fast-response turn-on phosphorescent probes for biothiols based on ruthenium(ii) complexes by Ze-Bao Zheng; Ji-Chun Cui; Yin-Feng Han; Yan-Qing Ge; Jian Zuo; Wen-Xin Hao (2341-2350).
A novel ruthenium(ii) complex of [Ru(bpy)2(BIPP-DNBS)](ClO4)2 (Ru-BIPP-DNBS) {bpy = 2,2′-bipyridine, BIPP-DNBS = 4-bromo-2-(1H-imidazo[4,5-f][1,10]phenanthrolin-2-yl)phenyl 2,4-dinitrobenzenesulfonate} has been designed and synthesized as a fast-response turn-on phosphorescent probe for the detection of biothiols. The ruthenium(ii) polypyridine complex was used as a phosphorescence reporter and 2,4-dinitrobenzenesulfonate (DNBS) was used as both a recognition unit and phosphorescence quencher. Ru-BIPP-DNBS was weakly phosphorescent owing to the effectual photoinduced electron transfer (PET) from the ruthenium(ii) luminophore to the sensing group DNBS. Upon treatment with biothiols, the 2,4-dinitrobenzenesulfonyl group of Ru-BIPP-DNBS was cleaved, and the phosphorescence was “turned on”, as a result of the formation of Ru-BIPP. The favorable steric interactions between the 1H-imidazo[4,5-f][1,10]phenanthroline group and ortho-2,4-dinitrobenzenesulfonate highly ameliorated the response rate of Ru-BIPP-DNBS toward biothiols, resulting in a rapid thiol-induced SNAr substitution reaction. This probe also exhibited good linearity ranges with detection limits of 0.47 μM for cysteine and 0.27 μM for glutathione. Furthermore, application of Ru-BIPP-DNBS for the imaging of endogenous biothiols in glioma cell samples was successfully demonstrated.

Molecularly imprinted polymers (MIPs) have been attracting increasing attention and used in various applications due to their tailor-made and adjustable selectivity. By means of innovation of the imprinting methods, such as using winnowing templates based on the analytical targets, the selectivity of MIPs can either be narrowed down to one particular molecule (high specificity), or broadened up to one group of analogues (wide selectivity). Based on the recent finding that polyhedral oligomeric silsesquioxanes (POSS) can enhance the absorption capacity of separation media, a baicalin-imprinted POSS hybridized monolith was synthesized and systematically investigated in terms of the preparation conditions and running parameters when using the MIP monolith as a capture column in on-line in-tube SPME-HPLC. The resulting MIP could be utilized to enrich baicalin out of a complex matrix of Scutellaria baicalensis for facile quantitative analysis. Furthermore, by means of off-line LC-MS/MS, wide selectivity for capturing analogues of baicalin was also qualitatively confirmed. This work provides a new perspective and method for controlling the wide selectivity of MIPs, and also a facile 2-dimensional HPLC system for the analysis of traditional Chinese medicine (TCM).

Correction for ‘Application of a novel electromembrane extraction and microextraction method followed by gas chromatography-mass spectrometry to determine biogenic amines in canned fish’ by Marzieh Kamankesh et al., Anal. Methods, 2019, 11, 1898–1907.

Back cover (2363-2364).