Analytical Methods (v.8, #39)

Front cover (6989-6990).

Contents list (6991-7001).

Recent advances in microscale western blotting by Brittany J. Sanders; Daniel C. Kim; Robert C. Dunn (7002-7013).
Western blotting is a ubiquitous tool used extensively in the clinical and research settings to identify proteins and characterize their levels. It has rapidly become a mainstay in research laboratories due to its specificity, low cost, and ease of use. The specificity arises from the orthogonal processes used to identify proteins. Samples are first separated based on size and then probed with antibodies specific for the protein of interest. This confirmatory approach helps avoid pitfalls associated with antibody cross-reactivity and specificity issues. While the technique has evolved since its inception, the last decade has witnessed a paradigm shift in western blotting technology. The introduction of capillary and microfluidic platforms has significantly decreased time and sample requirements while enabling high-throughput capabilities. These advances have enabled western analysis down to the single cell level in highly parallel formats, opening vast new opportunities for studying cellular heterogeneity. Recent innovations in microscale western blotting are surveyed, and the potential for enhancing detection using advances in label-free biosensing is briefly discussed.

Ozone induced chemiluminescence is one of the most viable detection techniques for the chromatographic determination of ultra-trace sulfur compounds in difficult matrices. First reported and commercialized in the early 90s, the sulfur chemiluminescence detector has undergone substantial transformation in terms of critical component design, performance, and reliability making it a near ideal detector for sulfur detection especially in sample matrices that become increasingly complex. In this article, the development of the detector for chromatography from its inception is presented. In addition to key applications that have been reported in the literature, recent new techniques developed with the most advanced version of the detector, like the use of the detector for total sulfur analysis without chromatographic separation is also reported.

We developed an electrochemical cell based on all graphite pencil electrodes (GPEs) for the detection of capsaicin. The cell produced a larger detection range of 0.1–100 μM compared to other reported studies, which is necessary for real sample testing. In addition, the cell was adopted for the analysis of capsaicinoid contents in chili powder and sauces bought from the supermarket. Through data processing by principal component analysis (PCA) and k-means clustering, it is possible to differentiate between the different brands of chili sauce. The low-cost and small construction of the cell may lead to a portable electronic tongue for on-site analysis of chili products.

Monitoring metabolite change in ice stored ghol fish (Protonibea diacanthus) by using a 1H NMR technique: identification of pyruvate as a spoilage marker by Ratish R. Nair; Nidhi Joshi; Vinod P. Boricha; Soumya Haldar; Pabitra B. Chatterjee (7030-7033).
Proton nuclear magnetic resonance (1H NMR) spectroscopy based metabolic analysis of ghol fish (Protonibea diacanthus) tissue extracts was carried out to acquire information on quality changes in fish samples during their prolonged storage in ice soon after sea-catch. This study revealed a wide distribution of metabolic signatures with the post-mortem age of ice stored fish. Up to 4 days (D-4) of ice storage, there were little changes in the concentration of a few selected metabolites such as trimethylammonium oxide (TMAO), creatine/phosphocreatine, and pyruvate; however, the concentration of the latter increased significantly afterwards.

Most electrochemical biosensors for microRNA (miRNA) detection need the immobilization of DNA on the electrode; first, the process is tedious and the DNA/RNA hybridization occurs on the electrode/solution interface, resulting in low efficiency. In this study, a homogeneous electrochemical biosensor has been proposed for sequence-specific miRNA in pancreatic cancer cells and lung adenocarcinoma cells, which combines the advantages of the simplicity of immobilization free and high efficiency of duplex-specific nuclease (DSN) assisted target recycling amplification. A DNA probe modified with a methylene blue group at the 3′ terminal (eMB) contains negative charges on its backbone, which cannot diffuse easily to the surface of the negatively charged indium tin oxide (ITO) electrode because of electrostatic repulsion, so a low electrochemical signal was detected. In the presence of miRNA, eMB can recognize and hybridize with miRNA to form a DNA–RNA heteroduplex. The eMB can be hydrolysed by DSN, so miRNA is retained. miRNA can cause a new cycle of hybridization–cleavage–releasing, and this recycling process generates numerous short MB-labelled oligonucleotide fragments (MB-OFs). The MB-OFs with less negative charges can diffuse easily to the surface of the ITO electrode, so an enhanced electrochemical signal was detected. Under the optimal conditions, the differential pulse voltammetric (DPV) response of the system had a linear relationship with the logarithm of the target miRNA concentration in the range of 0.1 pM to 10 nM. The proposed biosensor has been successfully applied to detect miRNA in real samples.

A label-free DNAzyme-based nanopore biosensor for highly sensitive and selective lead ion detection by Guangchao Liu; Ling Zhang; Duo Dong; Yang Liu; Jinghong Li (7040-7046).
As the lead ion (Pb2+) can severely induce environmental pollution and harm human health, it is important to develop a simple, highly sensitive and selective sensor for Pb2+ detection. Herein, a label-free DNAzyme-based nanopore biosensor for the sensitive and selective quantification of Pb2+ has been developed. Pb2+ can trigger the cleavage of a hairpin substrate in the presence of DNAzyme and cause the increase of single-stranded DNA (ssDNA) products, which could generate current blockade signals distinguished from that of the hairpin substrate when tested with an α-hemolysin nanopore. The presence of Pb2+ can be detected at ∼3.48 nM or higher and the sensor is highly selective to Pb2+. Furthermore, the nanopore-based sensing strategy can be extended to other analytes which can trigger similar DNAzyme cleavage reactions.

A new method for the determination of total furanic aldehyde compounds in Brazilian cachaça samples using liquid–liquid extraction and UV detection by Erika M. G. Melo; Julys P. A. Fernandes; Francisco A. S. Cunha; Jéssica O. B. Lira; Ricardo A. C. Lima; Kássio M. G. Lima; Luciano F. Almeida (7047-7053).
This work proposes a simple, rapid and low-cost method for the determination of total furanic aldehyde (TFA, sum of furfural and hydroxymethylfurfural) compounds in Brazilian cachaça samples, based on liquid–liquid extraction followed by UV-vis spectrophotometry. For this, 5.0 mL of chloroform and the sample are mixed and the mixture is then stirred for 2 minutes to form an emulsion, which rests for 1 minute to complete the phase separation. An aliquot of the decanted chloroform phase is then analyzed in a quartz cell (10 mm optical path) for absorbance at 279 nm. Effects of solvent, pH, temperature, possible interferences and fraction extracted were investigated. This method is not only sensitive and accurate, with an LOQ of 0.0203 mg 100 mL−1 and recoveries in the range 101–109%, but also simple and rapid (15 h−1). The new method for TFA compounds can be a potential reference method in routine safety testing for Brazilian cachaça samples or other beverages.

A label-free, exonuclease III (Exo III) assisted and cost-effective Hg2+ fluorometric assay based on a strand displacement amplification strategy was developed with high sensitivity and selectivity. Thymine–thymine (T–T) mismatches between the toehold domains in hairpin DNA and assistant DNA can serve as specific recognition elements for Hg2+ binding with the help of T–Hg2+–T base pairs to initiate a toehold-mediated strand displacement reaction. Exo III provides a reduced background signal, and the method is label-free using an intercalator, Sybr green I (SG). The number of T–T mismatches, the concentration of SG and Exo III, the incubation temperature and time were all optimized. Under the optimized condition, the limit of detection for Hg2+ can be as low as 9.42 pM with a linear range from 0.01 to 1 nM. Moreover, this proposed strategy shows excellent selectivity, and was successfully applied for the detection of Hg2+ in laboratory tap water and Yanjing lake water samples in our university.

Inkjet printing on transparency films for reagent storage with polyester–toner microdevices by Shannon T. Krauss; Thomas P. Remcho; Ehsan Monazami; Brandon L. Thompson; Petra Reinke; Matthew R. Begley; James P. Landers (7061-7068).
The use of overhead transparencies as substrates for the fabrication of microfluidic devices brings new capabilities in the creation of fully-integrated systems. Here, we describe a reagent storage method through inkjet printing that is complementary to the simple print, cut and laminate (PCL) fabrication technique, which already incorporates printing toner onto commercial off-the-shelf materials for fabrication of polyester–toner microdevices. With the surface of the polyester film dramatically different than paper, substrate optimization was required to increase printability without incorporating additional substrate-coating steps. Functionality for chemical sensing devices, operated centrifugally here, was demonstrated through standard human serum albumin analysis. Quantitative results were obtained through image analysis using a desktop scanner to determine color change in the presence of various total protein and albumin concentrations. Printed devices exhibited a shelf life of at least two months with no loss in activity, resulting in a relative standard deviation of 0.652% for total protein analysis.

Determination of fourteen UV filters in bathing water by headspace solid-phase microextraction and gas chromatography-tandem mass spectrometry by Marlene Vila; Maria Celeiro; J. Pablo Lamas; Thierry Dagnac; Maria Llompart; Carmen Garcia-Jares (7069-7079).
A methodology based on solid-phase-microextraction (SPME) followed by gas chromatography-tandem mass spectrometry (GC-MS/MS) has been developed for the simultaneous analysis of 14 UV filters of different chemical nature in water. The extraction parameters (the extraction mode and temperature, the fibre coating, and the addition of salt) were optimised by means of experimental designs in order to select the best extraction conditions. The final proposed conditions were 10 mL of water sample with 35% NaCl added, extracted in the headspace mode with a polyacrylate (PA) fibre at 100 °C for 20 minutes. The SPME-GC-MS/MS method was validated in terms of linearity (R2≥ 0.9937), accuracy and precision, obtaining LODs in the range of 0.068–12 ng L−1. The validated methodology was then applied to the analysis of different bathing water samples including sea, river, spa, swimming pool, and aquapark water, allowing the detection of 10 of the 14 target compounds; some of them were found at concentrations up to 692 ng mL−1.

A new sample collection method for the assessment of the percentage of water absorbed in frozen and quick-frozen poultry cuts (chicken breasts with skin) by Leonardo Rodrigo Fonseca Tigre Maia; Sergio Borges Mano; Eliane Teixeira Mársico; Carlos Adam Conte Júnior; Carlos Frederico Marques Guimarães; Danielli Vacari Brum (7080-7086).
The determination of the mean physiological water/protein ratio of samples obtained from homogenized poultry cuts using a powerful mincer is an efficient way of determining their total water content. However, this standard method demands expensive equipment and takes unnecessary time. The proposed new system to obtain samples uses a drill and a metal funnel to perform the collection, and it has the same accuracy, a faster performance and a less costly process. Comparing the traditional method to the method proposed in this current work to calculate the average water and protein content and water/protein ratio measured in 22 samples of breast meat cuts, the results analysis has shown that all of the tests performed on the 22 samples gave very similar data (paired t-test p > 0.05). Therefore, these positive outcomes indicate the high potential of using this new sample collection method to determine the total water content of poultry cuts (chicken breasts with skin).

An electrochemical immunosensor based on a silver nanoparticles (Ag NPs)-doped polyaniline (PANI) modified glassy carbon electrode (GCE) transducer, wherein polyclonal anti-polychlorinated biphenyl (PCB) antibody (Ab) was immobilized by a covalent linkage with glutaraldehyde (GA), was developed. The optimum conditions for the fabrication of the immunosensor were immersion, 30 min incubation in 1.0% GA. Electrochemical measurements of PCB 28 were done using the square wave voltammetry (SWV) technique. The optimum methodology conditions were a 20 mV s−1 scan rate, sweep potential range of −1 to 1 V and PCB incubation period of 2 h. The electrochemical response obtained under these optimum conditions was linear within 0.2 and 1.2 ng mL−1 with limit of detection (LOD) and limit of quantitation (LOQ) values of 0.063 ng mL−1 and 0.209 ng mL−1, respectively. The specificity of the developed sensor towards PCB 28 against benzyl chloride (BnCl) and PCB 180 was poor due to structural similarities. However, the PCB 180 results correspond to total PCB, thus indicating the sensor's applicability to regular total PCB determination. Cations and anions tested had a minimum effect on the sensor. Recoveries in water and guava juice ranged from 90% to 102%. Thus, proving the immunosensor's selectivity to PCBs and its possible application in the detection and monitoring of PCBs in food, water bodies and general environmental samples.

An archeological Egyptian cartonnage dating back to the Greco-Roman period around the third century BC was elementally analyzed via the sample-friendly technique, Laser-Induced Breakdown Spectroscopy (LIBS). The unique characteristics of LIBS as being a simple and fast technique, has been exploited to obtain surface elemental distribution and depth profiles for the investigated sample. An Nd:YAG laser operating at the fundamental wavelength of 1064 nm has been used as the excitation source along with an echelle spectrometer coupled to an ICCD detector for spectral dispersion and detection. Under the experimental conditions, four distinct regions were studied as ten elements were analyzed for spatial distribution along the surface and also for their depth profiles. Rapid and comprehensive visualization of the investigated regions are provided. The most relevant elements i.e. carbon, calcium, silicon, oxygen and iron suggest that the cartonnage was manufactured via the traditional method, mainly dependent on natural pigments such as metal oxides and calcium carbonate (calcite) which is consistent with the previous results of other conventional techniques used in the field of Archaeology. Supporting studies obtained by both scanning electron microscopy with energy dispersive X-ray micro-analysis (SEM-EDX) and X-ray diffraction (XRD) have been presented for comparison purposes.

Attenuated total reflection Fourier transform-infrared (ATR-FTIR) spectroscopy as a new technology for discrimination between Cryptococcus neoformans and Cryptococcus gattii by Fernanda S. L. Costa; Priscila P. Silva; Camilo L. M. Morais; Thales D. Arantes; Eveline Pipolo Milan; Raquel C. Theodoro; Kássio M. G. Lima (7107-7115).
Systemic fungal infections are among the most difficult diseases to manage in humans, especially when the recognition of the correct species is required for a precise and successful treatment. This is the case for Cryptococcus species and its genotypes, which are the main cause of meningitides in immunocompromised patients. Attenuated total reflection Fourier transform-infrared (ATR-FTIR) spectroscopy with discriminant analysis was employed to distinguish between the pathogenic fungal species Cryptococcus neoformans and Cryptococcus gattii by determining which wavenumber–absorbance/intensity relationships might reveal biochemical differences. Cryptococcus inactivated colonies were applied to an ATR crystal, and vibrational spectra were obtained in the ATR mode. Twenty-eight Cryptococcus isolates, fourteen C. neoformans and fourteen C. gattii were investigated. Spectral categories were analyzed using principal component analysis (PCA), successive projection algorithm (SPA) and genetic algorithm (GA) followed by linear discriminant analysis (LDA) and quadratic discriminant analysis (QDA). Multivariate classification accuracy results were estimated based on sensitivity, specificity, positive (or precision) and negative predictive values, Youden index, and positive and negative likelihood ratios. Sensitivity for C. neoformans and C. gattii categories were 84.4% and 89.3%, respectively, using a QDA-LDA model with 17 wavenumbers with respect to their “fingerprints”. Compared to classical methods for differentiation of Cryptococcus species, this new technology could represent an alternative and innovative tool for faster and cheaper fungal identification for routine diagnostic laboratories.

The high sensitivity of proton induced γ-ray emission technique to light elements is exploited to determine F, Na, Mg, Al and P in several Indian tea powders. Measurements are performed on black as well as green tea powders before (i.e. made tea) and after their infusion (brewing) in hot aqueous medium. Quantification against a Standard Reference Material (SRM) 1571 (orchard leaves) reveals that made tea, in general, contains these elements in a concentration range of a few tens to several hundred mg kg−1. Although made black or green tea does not have any characteristic elemental profile, green tea, in general, contains Al at elevated levels. The elements are leached from made tea into aqueous medium (liquor) on infusion, as ascertained by analyzing the infused tea powders. The extent of leaching is highest for F and least for Al. These measurements can be used to estimate the concentrations of the elements in liquors. Statistical analysis of the results on solid samples shows that the standard deviation of the measurements is about 4%, while their combined standard uncertainty is 5–20%. The uncertainty in elemental concentrations of SRM is the main contributor to the combined standard uncertainty of the measurements.

A nanocomposite containing ferrocenoyl glutaric acid hydrazone and multiwalled carbon nanotubes (MWCNTs) has been synthesized and characterized for its structural, morphological and electrochemical properties. Characterization has been done using scanning electron microscopy (SEM), transmission electron microscopy (TEM), thermogravimetric analysis (TGA), infrared (IR), X-ray photoelectron spectroscopy (XPS), and UV-visible studies. The electrochemical behavior and stability of the modified electrode have been investigated using cyclic voltammetry (CV) and differential pulse voltammetry (DPV). A self-standing film of this electroactive and homogeneous composite has been obtained by solution casting method. It is observed that the ferrocenoyl glutaric acid hydrazone (L)/MWCNTs/Nafion composite has better electrochemistry, electrical properties and firm adhesion of the material at the electrode surface. The modified electrode showed an electro-catalytic response to the oxidation of azide ions at the potential of 0.30 V in 0.1 M phosphate buffer solution (pH 7.1). The linear range and detection limit for the azide ion were found to be 0.02 mM to 20 mM and 0.312 μM, respectively.

Natto is a typical fermented food, and bears various health benefits. Its production involves complex biochemical processes and requires versatile monitoring techniques. In this work, LF-NMR and MRI techniques were introduced to monitor a typical fermentation process of natto. Upon a multi-exponential fitting of NMR data obtained by using a Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence, spin–spin relaxation time (T2) spectra display the presence of four populations of water molecules during the fermentation: bound water (T2,1), immobilized water (T2,2), moderately immobilized water (T2,3) and free water (T2,4). The concomitant increase in apparent physicochemical properties and the T2,3 water populations can be correlated with the extended fermentation time. The chemometric method of principal component analysis (PCA) based on the CPMG data further reveals that the fermentation is characteristic of a process with three main phases. On the other hand, the proton density weighted images, derived from MRI, reveal the spatial distribution of water molecules within the fermented products and project a full hydration process inside natto through a gradual penetration of water molecules from the edge to the center of the granules. Therefore, LF-NMR and MRI combined with chemometrics provide unique insights into the monitoring of natto fermentation.

Analysis of Ni, Cr, Cu, Pb and Cd in marine bioindicators using mixed-micelles with microwave assisted micellar extraction and GF-AAS by A. M. Hernández-Martínez; C. Padrón-Sanz; M. E. Torres-Padrón; Z. Sosa-Ferrera; J. J. Santana-Rodríguez (7141-7149).
A new method has been developed for the extraction and determination of the heavy metals Ni, Cu, Cr, Pb, and Cd in mussels (Mytilus galloprovincialis) and polychaetes (Nereis diversicolor), using biodegradable mixed-micelles (SDS and Triton X-100) as the extractant, and GF-AAS analysis. This method proves to be effective in the extraction of metals from this type of matrix by employing only mixed-micelles as the extractant, without the addition of chelating agents or any other reagents to enhance the efficiency of the method. The proposed method has shown satisfactory recovery percentages for all the metals under study in both cases: 83–110% for Mytilus galloprovincialis and 72–106% for Nereis diversicolor, good linearity and reproducibility (with RSDs lower than 4.6% and 3.24%, respectively), as well as detection limits ranging from 0.02 to 0.05 μg g−1 for Mytilus galloprovincialis and from 0.003 to 0.04 μg g−1 for Nereis diversicolor. The results obtained for the optimized method were compared with those obtained using MAE/GF-AAS (ISO 11047:1998) analysis. This method has been applied to the Certified Reference Material ERM-CE 278K, as well as to other species of bivalves (Donax trunculus and Cerastoderma edule) and polychaetes (Lumbrineris latreilli and Marphysa sanguinea), with satisfactory results.

Ultrasound-assisted micellar extraction of phenolic antioxidants from spices has been developed for the first time. The effect of the surfactant nature and its concentration on the extraction efficiency has been evaluated. Electrogenerated hexacyanoferrate(iii) ions have been used as coulometric titrants owing to their selectivity towards phenolic antioxidants. The best recovery of phenolic antioxidants has been obtained using nonionic 0.1 mol L−1 Brij® 35 micellar medium by single extraction for 10 min with ultrasound treatment. The spice/extractant ratio that provided maximum recovery of phenolic antioxidants was found for 20 spices. Comparisons with ethanolic and methanolic extracts have been performed. Micellar extracts show significantly higher recovery of phenolic antioxidants. Reactivities of the individual phenolic antioxidants of the spices with electrogenerated hexacyanoferrate(iii) ions, bromine and Ce(iv) in Brij® 35 micellar medium have been found. The antioxidant properties of the spices' micellar extracts have been estimated using total antioxidant capacity (TAC), ferric reducing power (FRP) and ceric reducing/antioxidant capacity (CRAC) based on their reactions with coulometric titrants (the electrogenerated bromine, hexacyanoferrate(iii) ions and Ce(iv), respectively). Positive correlations of these parameters with antioxidant activity towards 2,2-diphenyl-1-picrylhydrazyl (DPPH˙) (r = 0.8676, 0.9502 and 0.8855 for TAC, FRP and CRAC, respectively) have been obtained.

Electrochemical sensing of nitro-aromatic explosive compounds using silver nanoparticles modified electrochips by S. Singh; V. K. Meena; B. Mizaikoff; S. P. Singh; C. R. Suri (7158-7169).
In the present study an electrochemical deposition method for glycine-stabilized silver nanoparticles (Glyc-AgNPs) at screen printed electrodes is shown in a single step with minimal sample preparation. The advantage of this method is the direct assembly of Glyc-AgNPs at the electrode surface without external modification. The Glyc-AgNPs were characterized for their size, crystallinity and redox activity. The nanoparticles were further used for electrochemical sensing of 2,4,6-tri-nitro toluene (TNT) and di-nitro benzene (DNB), i.e. nitro-aromatic explosive compounds using differential pulse voltammetry (DPV). The Glyc-AgNP modified electrochip revealed significantly more sensitivity towards TNT as compared to DNB, as evident from the current intensity obtained due to the electrochemical reduction process. Earlier studies have suggested that tri-nitro compounds are more easily reduced as compared to mono- and di-nitro compounds, which is hypothesized as the main reason for the observed sensitivity for TNT. A linear response was observed in the range from 1 × 10−10 to 0.1 M for TNT, and 1 × 10−7 to 0.1 M for DNB. The results obtained with spiked samples provided solid grounds for future field trials with the developed sensing device.

Establishment and validation of quantitative analysis of multi-components by a single marker for quality control of polygoni multiflori radix by Dan Gao; Yu-meng Li; Na Wang; Chun-yu Li; Cong-en Zhang; Zhao-fang Bai; Xiao-he Xiao; Wei-wei Gao; Jia-bo Wang (7170-7176).
Quality control of Traditional Chinese Medicine (TCM) is one of the biggest challenges facing its clinical practice. The quality of Polygoni Multiflori Radix (PM) is mainly attributed to the content of stilbenes and anthraquinones. Herein, a sensitive, precise, and specific high-performance liquid chromatography (HPLC) method is established for the quantification of the six major components in PM, which include cis-2,3,5,4′-tetrahydroxystilbene-2-O-β-d-glucoside (cis-TSG), trans-2,3,5,4′-tetrahydroxystilbene-2-O-β-d-glucoside (trans-TSG), emodin-8-O-β-d-glucoside, physcion-8-O-β-d-glucoside, emodin and physcion. Based on this, using trans-TSG as the internal reference, quantitative analysis of multi-components by a single marker (QAMS) was carried out, and relative correction factors (RCFs) were calculated to determine the contents of the other five components. Tested by a robustness experiment under certain changeable conditions, the RCFs proved to be stable with RSDs below 5.0%. Moreover, on comparing the contents of the target components determined by an external standard method (EMS) and QAMS, there were no significant differences observed between the two methods with absolute relative errors of <5%. The abovementioned results indicate that QAMS possesses high convenience, accuracy, and feasibility and can be successfully applied to PM and its preparations for multi-component quality control.

Quantification of Mn in glass matrices using laser induced breakdown spectroscopy (LIBS) combined with chemometric approaches by Praveen Devangad; V. K. Unnikrishnan; M. M. Tamboli; K. M. Muhammed Shameem; Rajesh Nayak; K. S. Choudhari; C. Santhosh (7177-7184).
A Q-switched solid state Nd:YAG laser operating at a third harmonic (355 nm) wavelength and an echelle spectrograph coupled with an ICCD system were used to study the plasma on a glass target. The present work is mainly focused on the investigation of multivariate calibration methods like principal component regression (PCR) and partial least squares regression (PLSR) for the analysis of Mn in complex matrices like glass. The glass studied has Mn as an analyte of interest whose doping concentration in the matrix varies from 0.77% to 11.61%. The performance of univariate and multivariate methods has been presented in this paper through their figures of merit. Improved prediction accuracy, limit of detection (LOD) and regression coefficients (R2) have been reported when the data were analyzed using PCR and PLSR. The calibration curves of six emission lines of Mn have been analyzed using a univariate method that resulted in R2 values varying from 0.85 to 0.98. This method resulted in a correlation uncertainty of 10% and a LOD of 0.20 wt%. R2 values of 0.98 to 0.99 have been obtained for the multivariate calibration curves of Mn analyzed in three selected regions of the LIBS spectrum. The optimum LOD and root mean square error of prediction (RMSEP) using PCR and PLSR were found to be 0.02 wt% and 0.54 wt%, respectively. The significant improvement in the analytical performances of multivariate calibration methods for the investigation of LIBS data is evident from the aforementioned results. Finally, the results of PCR and PLSR were confirmed by PCA classification.

Homocysteine was distinguished from cysteine and assayed by colorimetry with gold nanocomposites composed of 13 nm gold nanoparticles conjugated with thermoresponsive copolymers, poly(N-isopropylacrylamide-co-2-(dimethylamino)-ethylmethacrylate). The colorimetric assay of homocysteine was based on the disassembly of the assembled gold nanoparticles due to conjugation with the thermoresponsive copolymers. Heating followed by cooling a solution of the gold nanoparticles induced the phase transition of the thermoresponsive copolymers conjugated with gold nanoparticles to cause the disassembly, implying that the color of the solution changed from bluish purple to red. Homocysteine inhibited the thermally induced disassembly, whereas cysteine did not. The a* value in the L*a*b* color coordinates quantified the colorimetric change in the gold nanoparticle solution. The a* value decreased monotonically as the homocysteine concentration was increased to 7 × 10−6 mol L−1, and the detection limit, defined as 3σ of the blank, was estimated to be 0.35 × 10−6 mol L−1. The colorimetric assay was used to determine the homocysteine concentration in bovine serum.

High-throughput sialylation measurement using lectins on an Octet platform for clone screening by Kanvasri N. Jonnalagadda; Lam Raga A. Markely; Bing Guan; Christina Alves; Shashi Prajapati (7193-7198).
A high-throughput method to quantify sialic acids was developed on a ForteBio Octet QK384 platform to support cell-line development. The high-throughput sialylation analysis method using the ForteBio Octet (HTFBS) is based on the binding rate of Maackia amurensis lectin II (MALII) to (α-2,3)-linked sialic acids of proteins bound to Protein-A sensors. The HTFBS method can specifically quantify the sialylation levels of secreted proteins in crude cell culture samples and does not require purified samples. Using this method, thirty crude cell culture samples can be analyzed for titers and sialylation in 60 minutes. In a comparison study between the HTFBS method and HPLC method, a linear correlation was observed between the MALII binding rate obtained by using the ForteBio Octet QK384 and the total sialic acid content obtained by HPLC for samples in the sialic acid content range of 5–24 mol mol−1. The MALII binding rate was consistent for samples with titers between 6.25 and 100 μg mL−1. The HTFBS method is robust and the effect of protein concentration is negligible on the MALII binding rate for samples between 6.25 and 100 μg mL−1. The HTFBS method was successfully applied to the cell-line development platform for screening highly sialylated clones. The current HTFBS method can quantify sialylation levels in crude samples and can readily be used to accelerate cell line development for producing therapeutic proteins with desired product quality.

An aptasensor based on cobalt oxyhydroxide nanosheets for the detection of thrombin by Yuan Yang; Yao Cen; Wen-Jing Deng; Ru-Qin Yu; Ting-Ting Chen; Xia Chu (7199-7203).
An aptasensor based on a fluorophore-labelled aptamer and cobalt oxyhydroxide (CoOOH) nanosheets was developed for determining the concentration of thrombin. We first found that the CoOOH nanosheets had the ability to distinguish a G-quadruplex from single-stranded DNA (ssDNA) due to the different abilities of the nanosheet to absorb these two DNA forms. The absorption of 6-carboxyfluorescein (6-FAM) labelled aptamer (ssDNA) on the surface of the CoOOH nanosheets resulted in the quenching of the fluorescence of the FAM through fluorescence resonance energy transfer (FRET) between the FAM and the CoOOH nanosheets. However, the binding of the aptamer to its target resulted in the formation of an antiparallel G-quadruplex complex, for which the CoOOH nanosheets had weak affinity, resulting in a recovery of fluorescence. We applied this strategy to the detection of thrombin. The intensity of the fluorescence recovery was found to be linear with the logarithm of the thrombin concentration in the range of 0.5 to 100 nM, and the limit of detection (LOD) was determined to be 0.5 nM. Because of the high selectivity of the aptamer and strong quenching ability of the CoOOH nanosheets, similar aptasensors but with different specific aptamers may potentially serve as platforms to detect a wide range of analytes, and may hence have promising applications in clinical diagnosis.

Discrimination of adulterants in UHT milk samples by NIRS coupled with supervision discrimination techniques by Aderval S. Luna; Jéssica S. A. Pinho; Luciana C. Machado (7204-7208).
A methodology was developed for distinguishing different ultra-high temperature (UHT) milk adulterants (water, urea, and formaldehyde) at various levels using NIR spectroscopy (NIRS) coupled with supervision discrimination techniques (SIMCA, SVM-DA, and PLS-DA). The figures of merit in discrimination were established based on the results given by the best preprocessing for each technique. Discrimination of formaldehyde was 100% independent of the discrimination technique used. For water discrimination SVM-DA furnished better results as compared to PLS-DA and SIMCA. The same occurred with urea discrimination as well.

Characterization of non-intentionally added substances (NIAS) and zinc oxide nanoparticle release from evaluation of new antimicrobial food contact materials by both LC-QTOF-MS, GC-QTOF-MS and ICP-MS by M. J. Martínez-Bueno; S. Cimmino; C. Silvestre; J. L. Tadeo; A. I. Garcia-Valcárcel; A. R. Fernández-Alba; M. D. Hernando (7209-7216).
The migration of non-intentionally added substances (NIAS; namely organic chemicals) and added metal oxide nanoparticles from new active nanomaterials intended for use as food contact materials (FCMs) has been determined by a mass spectrometry-based methodology. The FCMs consist of polypropylene (PP)-based nanocomposites functionalized with zinc oxide nanoparticles (ZnO NPs) as the antimicrobial agent. This work presents a multi-analytical approach incorporating ICP-MS (inductively coupled plasma mass spectrometry), GC-QTOF-MS (gas chromatography coupled to quadrupole-time-of-flight mass spectrometry) and LC-QTOF-MS (liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry) platforms. The screening approach for investigating both the GC- and LC-amenable NIAS consisted of initial peak detection and structural characterization based on accurate mass acquisition of both the full-scan and fragmentation spectra. The workflow for the unknown screening comprised retrospective analysis and data processing using both a mass spectral library and databases. Migration testing, based on standard operating conditions established by EU legislation, provided tentative identification of both LC-amenable NIAS: 2,4,6 triamino-1,3,5-triazine; azepan-2-one; (2E)-3-phenylprop-2-enal, and GC-amenable NIAS: diethyl phthalate (DEP). To determine the optimal antimicrobial FCM performance, we evaluated the release of ZnO NPs from different PPR-based nanocomposites. Out of the nanopolymers evaluated, a film consisting of PP, polylimonene (PL) and ZnO NPs, in wt% (92/5/3), proved to be a suitable contact material with improved functionality.

ISVR modeling of an interferon gamma (IFN-γ) biosensor based on graphene by Elnaz Akbari; Zolkafle Buntat; Mehrbakhsh Nilashi; Abdolkarim Afroozeh; Yousef Farhang; Alireza Zeinalinezhad (7217-7224).
Nowadays the detection of proteins plays a crucial role for the early diagnosis of diseases. The combination of biosensor application with nanotechnology has offered new alternatives for clinical diagnostic techniques. One of the major public health problems in many developing countries is tuberculosis (TB) susceptibility and Interferon-gamma (IFN-γ) can be used in the diagnosis of this infectious disease. In this study, a prototype graphene based FET structure was employed as a biosensor. Additionally, a PDMS layer was deployed beneath the graphene as a dielectric layer. As a result of the changeability of Ids (drain–source current), the carrier concentration would change when the IFN-γ molecules attach to the surface of graphene. To acquire another pattern for the I–V (current–voltage characteristic), the Incremental Support Vector Regression (ISVR) algorithm was also employed. The comparative study based on the outcomes of the ISVR and pre-existing analytical models with experimental data found that there was acceptable agreement, which was able to substantiate the proposed models. Moreover, the ISVR showed that the proposed method remarkably improved the accuracy of prediction.

Representative subset selection and outlier detection via isolation forest by Wo-Ruo Chen; Yong-Huan Yun; Ming Wen; Hong-Mei Lu; Zhi-Min Zhang; Yi-Zeng Liang (7225-7231).
In order to a build robust and predictive model, all outliers should be eliminated and representative samples should be selected. In this study, Isolation forest Outlier detection and Subset selection (IOS) has been proposed, which can detect outliers and select representative subsets simultaneously. IOS is different from the classical subset selection method, which is cluster-based and has a uniform design. A comparative study among the IOS, Kennard–Stone (KS), sample set partitioning based on joint x–y distances (SPXY) and random sampling (RS) methods was conducted. The performances of these algorithms were benchmarked with four datasets, including two normal NIR datasets, which are free of outliers: soil and diesel fuel, and two datasets with outliers: milk NIR dataset and solubility QSAR dataset (LogS). Results show that IOS can detect outliers and select representative subsets of samples simultaneously, which reduces prediction errors significantly compared with the KS, SPXY and RS methods. IOS can eliminate outliers and select representative samples without y values. Hence, the proposed method may be an advantageous alternative to the other three strategies. IOS is implemented in MATLAB language and is available at

A colorimetric probe for the detection of aluminum ions based on 11-mercaptoundecanoic acid functionalized gold nanoparticles by Ruiqi Zhu; Jinping Song; Qi Ma; Ying Zhou; Jun Yang; Shaomin Shuang; Chuan Dong (7232-7236).
An easy, simple and selective colorimetric method for aluminum ions has been developed using 11-mercaptoundecanoic acid functionalized gold nanoparticles (MUA–AuNPs). The MUA–AuNPs could be induced to aggregate in the presence of Al3+ ions according to the chelation effect between the carboxyl group and Al3+, and a color change from red to purple with increasing Al3+ ion concentration was observed. The plasmon absorbance of the MUA–AuNPs allows the quantitative detection of Al3+ ions. A sensitive linear correlation existed between the ratio of absorbance at 650 nm to that at 520 nm and the Al3+ ion concentration ranging from 10−6 M to 10−4 M. The detection limit was 5.7 × 10−7 M, which is below the maximum recommended contamination level of the World Health Organization (WHO) for the aluminum content (7.4 μM) in drinking water. The MUA–AuNPs chelated with Al3+ showed excellent selectivity in the presence of EDTA compared to other metal ions (Ag+, Ba2+, Ca2+, Cd2+, Cr3+, Cu2+, Fe2+, Hg2+, Mg2+, Mn2+, Ni2+, Pb2+ and Zn2+). The proposed method can offer the additional advantage of the efficient determination of Al3+ ions in drinking water, and the recovery was in the range of 98.5–101.6% with a relative standard deviation below 2%.

Bimetallic gold–silver nanocluster fluorescent probes for Cr(iii) and Cr(vi) by Yuanlian Yang; Yongfang Sun; Shuzhen Liao; Zhaoyang Wu; Ruqin Yu (7237-7241).
Bimetallic gold–silver nanoclusters (AuAgNCs) have been successfully synthesized using 11-mercaptoundecanoic acid (11-MUA) as a reducing agent and capping ligand. It was found that the fluorescence of AuAgNCs was quenched selectively by Cr(iii) when using ethylenediaminetetraacetic acid (EDTA) as the masking agent and there was a good detection linear range for Cr(iii) from 0.08 μM to 6 μM with high sensitivity (LOD = 0.05 μM, S/N = 3). At the same time, the experimental results show that the AuAgNCs were not sensitive to the ascorbic acid (AA) reductant and they can thus be utilized to detect Cr(vi) indirectly by employing an additional reducing step. Linear quenching of fluorescence intensity related to Cr(vi) concentration was over the range of 0.6 μM to 10 μM with a LOD of 0.3 μM (S/N = 3). In addition, the recovery experiments further demonstrated that this AuAgNCs based method can achieve the chemical speciation and quantitative determination of Cr(iii) and Cr(vi) in real water samples.

Graphene quantum dots as a fluorescence-quenching probe for quantitative analysis of Ponceau 4R solution by Jianpo Zhang; Lihua Na; Yunxia Jiang; Dawei Lou; Li Jin (7242-7246).
Ponceau 4R is widely used as a colorant in China. However Ponceau 4R poses potential risks to human health, especially when consumed in excess amounts. In this paper, quantitative analysis of Ponceau 4R in solution was achieved based on the fluorescence quenching of graphene quantum dots. UV absorption spectra and calculations based on a modified Stern–Volmer curve were employed to study the fluorescence quenching mechanism of this system. All results indicate that it is a static quenching process. Furthermore, the effect of pH, temperature and reaction times on the fluorescence intensity of GQDs with Ponceau 4R was studied. Under the optimal conditions, the linear response of the fluorescence intensity of GQDS to the concentration of Ponceau 4R allowed the quantitative analysis of Ponceau 4R in a range of 5–150 μg mL−1, and the limit of detection was 2.57 μg mL−1. And the low detection limit could reach 8.32 ng mL−1 by using more diluted GQDs. Particularly, this method was used to quantitatively analyze real samples, indicating that this method could be more widely applied to similar samples.

A digoxin electrochemical aptasensor using Ag nanoparticle decorated graphene oxide by Mohammad Hossein Mashhadizadeh; Niloofar Naseri; Masoud A. Mehrgardi (7247-7253).
In this work, a highly sensitive aptasensor for digoxin determination in biological samples has been introduced. The surface of a gold screen-printed electrode was modified by using electrodeposited gold nanoparticles (GNPs). A monolayer of 3-mercaptopropionic acid (MPA) was then self-assembled on the GNP surfaces. Subsequently, an amino-labeled digoxin specific aptamer was covalently bonded to the carboxylic groups of MPA on the GNPs through imide bond formation. The silver nanoparticle decorated graphene oxide (AgNPs–GO) interacted with the immobilized aptamer viaπ–π interaction and the oxidation signal of AgNPs was monitored. In the presence of digoxin, the hybrid left the electrode surface due to the specific interaction between the aptamer and digoxin, and the oxidation signal decreased. The proposed aptasensor delivered a linear dynamic range of 1 pM to 0.1 μM and a detection limit of 0.3 pM and was successfully utilized for digoxin determination in biological samples with good reliability.

A new hybrid nanomaterial based on single-walled carbon nanotubes incorporating nanocrystalline carbon quantum dots (SWCNT/C-dots) was developed and characterized by means of high resolution transmission electron microscopy, atomic force microscopy, Raman spectroscopy and electrochemical techniques. A glassy carbon electrode modified with SWCNT/C-dots exhibited an enhanced electrocatalytic response for 17α-ethynylestradiol, reflecting an increase of the active area of the hybrid material, promoted by the C-dots. However, the best activity was observed for laccase immobilized on CGE/SWCNT/C-dots, suggesting that the nanocrystalline C-dots improve the electron transport between the substrate, SWCNTs and the copper ions in the enzyme active sites. Such an association provided a very efficient bioelectrochemical sensor for the 17α-ethynylestradiol endocrine interfering agent, with a detection limit of 4.0 nmol L−1 in real samples.

Designing a biostable L-DNAzyme for lead(ii) ion detection in practical samples by Hao Liang; Sitao Xie; Liang Cui; Cuichen Wu; Xiaobing Zhang (7260-7264).
A promising biosensor for effective lead(ii) ion detection in practical applications was developed by constructing a Pb2+-specific L-DNAzyme, the enantiomer of the natural nucleic acid-constructed D-DNAzyme. This fluorescence sensor contains the L-enzyme strand with a quencher at the 3′ end, and the L-substrate strand with a fluorophore at the 5′ and a quencher at the 3′ ends that formed a complex. In the presence of Pb2+, the L-substrate is cut into two fragments, leading to the recovery of fluorescence. The sensor shows high sensitivity and selectivity for Pb2+ detection with a linear response in the range of 5–100 nM and a detection limit of 3 nM in aqueous solution. Importantly, based on the fact that L-DNAzyme consists of non-natural nucleic acids, which are insensitive to nuclease digestion, protein adsorption and D-DNA hybridization, our sensor shows a specific response to Pb2+ in practical water and serum samples. Therefore, it is expected that our L-DNAzyme-based strategy may offer a new method for developing simple, rapid and sensitive sensors in complex systems.

A camelid VHH-based fluorescence polarization immunoassay for the detection of tetrabromobisphenol A in water by Kai Wang; Zhiping Liu; Pei Ji; Jianfeng Liu; Sergei A. Eremin; Qing X. Li; Ji Li; Ting Xu (7265-7271).
Tetrabromobisphenol A (TBBPA) is a widely used flame retardant and is classified as an endocrine disruptor. A convenient and sensitive fluorescence polarization immunoassay (FPIA) for TBBPA was developed with a camelid variable domain of heavy chain antibody (VHH) being specific for TBBPA. 3-(2,6-Dibromo-4-(2-(3,5-dibromo-4-hydroxyphenyl)propan-2-yl))propanoic acid (hapten T1) was conjugated with ethylenediamine fluorescein thiocarbamoyl (EDF) to form a tracer that binds with anti-TBBPA VHH. The limit of detection and half-maximum inhibition concentration of TBBPA by FPIA were 5 ng mL−1 and 90 ng mL−1, respectively. This FPIA was used to detect TBBPA in water samples without any pre-treatment and each analysis was performed within 20–30 min. The recovery of TBBPA from spiked water samples ranged from 73% to 102%, with coefficients of variation ranging from 7% to 14%. The FPIA exhibited high potential for rapid and accurate determination of TBBPA in real water samples.

Gold nanoclusters-catalyzed rhodamine 6G–K3Fe(CN)6 chemiluminescence and its application by Dongqin Yang; Yanyan He; Yanyan Sui; Funan Chen (7272-7278).
An ultra-facile flow injection chemiluminescence (CL) strategy was designed for bisphenol A (BPA) detection. It was based on the inhibition of rhodamine 6G chemiluminescence by BPA on the gold nanoclusters (Au NCs) enhanced rhodamine 6G–K3Fe(CN)6 system in an alkaline medium for the first time. Furthermore, BSA-stabilized Au NCs were synthesized via a simple method with desirable reproducibility and stability. Under optimized conditions, the relative CL intensity was found to be proportional to the BPA concentration in the range of 2.0 × 10−7 to 1.0 × 10−5 mol L−1, with a detection limit of 7.0 × 10−8 mol L−1 (S/N = 3). The effects of interfering substances on the detection of 1.0 × 10−6 mol L−1 BPA were analyzed for confirming good selectivity. A brief discussion on the possible CL reaction mechanism was presented through the results of CL spectra, fluorescent spectra, UV-visible spectroscopy and radical scavenger reactions. Finally, this suggested method was used successfully to detect BPA in real samples with acceptable recovery values of 93.0–106.2%.

Back cover (7279-7280).