Analytica Chimica Acta (v.889, #C)

A review on enzyme and ultrasound: A controversial but fruitful relationship by M.M. Delgado-Povedano; M.D. Luque de Castro (1-21).
A critical review on the effect of ultrasound (US) on enzymes and their biocatalytic action is presented here. Discussion on the information users of US acquire before utilizing the different devices, and the importance they give to US frequency is constant along the review. The authors have gone into the different areas in which the US–enzyme binomial has been applied. The lack of enough information on the US–enzyme-working conditions under which each piece of research has been developed, and the necessity to provide complete information on the data and metadata to give enough light on each piece of research (and thus on the potential comparison of results from different studies) are critically exposed. With this aim, the study has been divided into the positive effect of US on enzymes to favor the production of metabolites, polymers or proteins; and the degradation, inhibition or activation of the biocatalyst under US application. Also the effect of US on enzyme production and the main fields of application of the US–enzyme binomial are discussed.Common commercial ultrasound devices.Display Omitted
Keywords: Enzymes; Ultrasound; Metabolites; Proteins; Polymers; Enzymatic activation–inhibition;

Nanoparticle-based assays in automated flow systems: A review by Marieta L.C. Passos; Paula C.A.G. Pinto; João L.M. Santos; M. Lúcia M.F.S. Saraiva; André R.T.S. Araujo (22-34).
Nanoparticles (NPs) exhibit a number of distinctive and entrancing properties that explain their ever increasing application in analytical chemistry, mainly as chemosensors, signaling tags, catalysts, analytical signal enhancers, reactive species generators, analyte recognition and scavenging/separation entities.The prospect of associating NPs with automated flow-based analytical is undoubtedly a challenging perspective as it would permit confined, cost-effective and reliable analysis, within a shorter timeframe, while exploiting the features of NPs.This article aims at examining state-of-the-art on continuous flow analysis and microfluidic approaches involving NPs such as noble metals (gold and silver), magnetic materials, carbon, silica or quantum dots. Emphasis is devoted to NP format, main practical achievements and fields of application. In this context, the functionalization of NPs with distinct chemical species and ligands is debated in what concerns the motivations and strengths of developed approaches. The utilization of NPs to improve detector's performance in electrochemical application is out of the scope of this review.The works discussed in this review were published in the period of time comprised between the years 2000 and 2013.Display Omitted
Keywords: Nanoparticle; Automation; Flow analysis; Microfluidics;

Effect of temperature on acid–base equilibria in separation techniques. A review by Leonardo G. Gagliardi; Marcos Tascon; Cecilia B. Castells (35-57).
Studies on the theoretical principles of acid–base equilibria are reviewed and the influence of temperature on secondary chemical equilibria within the context of separation techniques, in water and also in aqueous-organic solvent mixtures, is discussed. In order to define the relationships between the retention in liquid chromatography or the migration velocity in capillary electrophoresis and temperature, the main properties of acid–base equilibria have to be taken into account for both, the analytes and the conjugate pairs chosen to control the solution pH. The focus of this review is based on liquid–liquid extraction (LLE), liquid chromatography (LC) and capillary electrophoresis (CE), with emphasis on the use of temperature as a useful variable to modify selectivity on a predictable basis. Simplified models were evaluated to achieve practical optimizations involving pH and temperature (in LLE and CE) as well as solvent composition in reversed-phase LC.Display Omitted
Keywords: Acid–base equilibria; Temperature; Buffer properties; Selectivity; Separation techniques;

Post column derivatisation analyses review. Is post-column derivatisation incompatible with modern HPLC columns? by Andrew Jones; Sercan Pravadali-Cekic; Gary R. Dennis; R. Andrew Shalliker (58-70).
Post Column derivatisation (PCD) coupled with high performance liquid chromatography or ultra-high performance liquid chromatography is a powerful tool in the modern analytical laboratory, or at least it should be. One drawback with PCD techniques is the extra post-column dead volume due to reaction coils used to enable adequate reaction time and the mixing of reagents which causes peak broadening, hence a loss of separation power. This loss of efficiency is counter-productive to modern HPLC technologies, -such as UHPLC. We reviewed 87 PCD methods published from 2009 to 2014. We restricted our review to methods published between 2009 and 2014, because we were interested in the uptake of PCD methods in UHPLC environments. Our review focused on a range of system parameters including: column dimensions, stationary phase and particle size, as well as the geometry of the reaction loop. The most commonly used column in the methods investigated was not in fact a modern UHPLC version with sub-2-micron, (or even sub-3-micron) particles, but rather, work-house columns, such as, 250 × 4.6 mm i.d. columns packed with 5 μm C18 particles. Reaction loops were varied, even within the same type of analysis, but the majority of methods employed loop systems with volumes greater than 500 μL.A second part of this review illustrated briefly the effect of dead volume on column performance. The experiment evaluated the change in resolution and separation efficiency of some weak to moderately retained solutes on a 250 × 4.6 mm i.d. column packed with 5 μm particles. The data showed that reaction loops beyond 100 μL resulted in a very serious loss of performance.Our study concluded that practitioners of PCD methods largely avoid the use of UHPLC-type column formats, so yes, very much, PCD is incompatible with the modern HPLC column.Display Omitted
Keywords: Post column derivatisation; HPLC; Chromatography; Amino Acids; Antioxidants;

A new resin- Diphonix® in Diffusive Gradients in Thin Films (DGT) technique for the determination of uranium was investigated and compared with previously used binding phases for uranium, Chelex®-100 and Metsorb™. The DGT gel preparation and the elution procedure were optimized for the new resin. The U uptake on Diphonix® resin gel was 97.4 ± 1.5% (batch method; [U] = 20 μg L−1; 0.01 M NaNO3; pH = 7.0 ± 0.2). The optimal eluent was found to be 1 M 1-hydroxyethane-1, 1-diphosphonic acid (HEDPA) with an elution efficiency of 80 ± 4.2%. Laboratory DGT study on U accumulation using a DGT samplers with Diphonix® resin showed a very good performance across a wide range of pH (3–9) and ionic strength (0.001–0.7 M NaNO3). Diffusion coefficients of uranium at different pH were determined using both, a diffusion cell and the DGT time-series, demonstrating the necessity of the implementation of the effective diffusion coefficients into U-DGT calculations. Diphonix® resin gel exhibits greater U capacity than Chelex®-100 and Metsorb™ binding phase gels (a Diphonix® gel disc is not saturated, even with loading of 10.5 μmol U). Possible interferences with Ca2+ (up to 1.33 × 10−2 M), PO 4 3 − (up to 1.72 × 10−4 M), SO 4 2 − (up to 4.44 × 10−3 M) and HCO 3 − (up to 8.20 × 10−3 M) on U-DGT uptake ([U] = 20 μg L−1) were investigated. No effect or minor effect of Ca2+, PO 4 3 − , SO 4 2 − , and HCO 3 − on the quantitative measurement of U by Diphonix®-DGT was observed. The results of this study demonstrated the DGT technique with Diphonix® resin is a reliable and robust method for the measurement of labile uranium species under laboratory conditions.Display Omitted
Keywords: Uranium species; Diffusive Gradients in Thin Films; Uranium diffusion coefficients; Chelex®-100; Metsorb™; Diphonix®;

A novel electrochemiluminescent immunosensor based on the quenching effect of aminated graphene on nitrogen-doped carbon quantum dots by Jing Zhou; Tongqian Han; Hongmin Ma; Tao Yan; Xuehui Pang; Yueyun Li; Qin Wei (82-89).
Nitrogen-doped carbon quantum dots (N-CQDs) with an average diameter of 2 nm were synthesized by carbonization of diethylene triamine pentacetate acid (DTPA). The simple prepared N-CQDs showed excellent electrochemiluminescence (ECL) property and were used as luminophors to fabricate a sandwich-type ECL immunosensor. Aminated graphene (NH2-G) was also synthesized and used as a label of secondary antibody. The labeled NH2-G could effectively quench the ECL of N-CQDs modified on electrodes due to ECL resonance energy transfer (ERET). Immunological recognition which induced ECL quenching enabled the quantitative determination of biomarkers. Alpha fetoprotein (AFP) was selected as a model analyte to investigate the analytical performance of the proposed immunosensor. Under optimal conditions, a good linear relationship between ECL intensity and the logarithm of AFP concentration was obtained in the range of 0.01–100 ng mL−1 with the detection limit of 3.3 pg mL−1. The proposed ECL immunosensor showed good stability, acceptable selectivity and reproducibility.Display Omitted
Keywords: Resonance energy transfer; Nitrogen-doped carbon quantum dots; Alpha fetoprotein; Aminated graphene;

Coupling a universal DNA circuit with graphene sheets/polyaniline/AuNPs nanocomposites for the detection of BCR/ABL fusion gene by Xueping Chen; Li Wang; Shangchun Sheng; Teng Wang; Juan Yang; Guoming Xie; Wenli Feng (90-97).
This article described a novel method by coupling a universal DNA circuit with graphene sheets/polyaniline/AuNPs nanocomposites (GS/PANI/AuNPs) for highly sensitive and specific detection of BCR/ABL fusion gene (bcr/abl) in chronic myeloid leukemia (CML). DNA circuit known as catalyzed hairpin assembly (CHA) is enzyme-free and can be simply operated to achieve exponential amplification, which has been widely employed in biosensing. However, application of CHA has been hindered by the need of specially redesigned sequences for each single-stranded DNA input. Herein, a transducer hairpin (HP) was designed to obtain a universal DNA circuit with favorable signal-to-background ratio. To further improve signal amplification, GS/PANI/AuNPs with excellent conductivity and enlarged effective area were introduced into this DNA circuit. Consequently, by combining the advantages of CHA and GS/PANI/AuNPs, bcr/abl could be detected in a linear range from 10 pM to 20 nM with a detection limit of 1.05 pM. Moreover, this protocol showed excellent specificity, good stability and was successfully applied for the detection of real sample, which demonstrated its great potential in clinical application.Display Omitted
Keywords: BCR/ABL fusion gene; Universal DNA circuit; Catalyzed hairpin assembly; Transducer hairpin; Electrochemical DNA biosensor;

A single glass capillary nanopore-based sensing platform for rapid and selective detection of cupric ions is demonstrated by utilizing polyglutamic acid (PGA) as a non-immobilized probe. The detection is based on the significant decrease of ionic current through nanopore and the reversal of ion current rectification responses induced by the chelated cupric ions on the probes when in the presence of cupric ions. PGA shows high selectivity for detecting cupric ions rather than other metal ions. The sensitivity of the sensing platform can be improved about 1–2 orders of magnitude by employing asymmetric salt gradients during the measurements. And the PGA-based nanopore sensing platform shows excellent regenerability for Cu2+ sensing applications. In addition, the method is found effective and reliable for the detection of cupric ions in real samples with small volume down to 20 μL. This nanopore-based sensing platform will find promising practical applications for the detection of cupric ions.Display Omitted
Keywords: Nanopore; Polyglutamic acid; Cupric ions; Selectivity; Asymmetric salt gradients; Regenerability;

A label free aptasensor for Ochratoxin A detection in cocoa beans: An application to chocolate industries by Rupesh K. Mishra; Akhtar Hayat; Gaëlle Catanante; Cristina Ocaña; Jean-Louis Marty (106-112).
Contamination of food by mycotoxin occurs in minute/trace quantities. Nearly 92.5% of the cocoa samples present Ochratoxin A (OTA) levels at trace quantity. Hence, there is a necessity for a highly sensitive and selective device that can detect and quantify these organic toxins in various matrices such as cocoa beans. This work reports for the first time, a facile and label-free electrochemical impedimetric aptasensor for rapid detection and quantitation of OTA in cocoa beans. The developed aptasensor was constructed based on the diazonium-coupling reaction mechanism for the immobilization of anti-OTA-aptamer on screen printed carbon electrodes (SPCEs). The aptasensor exhibited a very good limit of detection (LOD) as low as 0.15 ng/mL, with added advantages of good selectivity and reproducibility. The increase in electron transfer resistance was linearly proportional to the OTA concentration in the range 0.15–2.5 ng/mL, with an acceptable recovery percentage (91–95%, RSD = 4.8%) obtained in cocoa samples. This work can facilitate a general model for the detection of OTA in cocoa beans based on the impedimetric aptasensor. The analysis can be performed onsite with pre-constructed and aptamer modified electrodes employing a portable EIS set up.Display Omitted
Keywords: Aptasensor; Cocoa beans; Label free; Impedimetric; Ochratoxin; Screen printed electrodes;

Catalytic amplification based on hole-transporting materials as efficient metal-free electrocatalysts for non-enzymatic glucose sensing by Yue Gu; Rongrong Yuan; Xiaoyi Yan; Cong Li; Weilu Liu; Ruixue Chen; Liu Tang; Bo Zheng; Yaru Li; Zhiquan Zhang; Ming Yang (113-122).
Hole-transporting materials with tunable structures and properties are mainly applied in organic light-emitting diodes as transport layer. But their catalytic properties as signal amplifiers in biological assays are seldom reported. In this paper, a starburst molecule, 4,4,4″-tri(N-carbazolyl)-triphenylamine (TCT), containing a triphenylamine as the central core and three carbazoles as the peripheral functional groups was designed and synthesized. Subsequently, the hole-transporting material based on the TCT polymer, poly(TCT) (PTCT), was achieved via a low-cost electrochemical method and exploited as an efficient metal-free electrocatalyst for non-enzymatic glucose detection. Here, this hole-transporting material served three purposes: electrochemical recognition (owing to hydrogen bonding interaction and the biomimetic microenvironment created by the polymer), electrocatalysis (owing to the hole-transporting capability of triphenylamine and the catalytic property of carbazole), and signal amplification (owing to energy migration along the conductive polymer backbone). The electrocatalytic and sensing performances of the sensor based on PTCT were evaluated in detail. Results revealed that the PTCT film could efficiently catalyze the oxidation of glucose at a less-positive potential (+0.20 V) in the absence of any enzymes. The response to glucose was linear in the concentration range of 1.0–6000 μM, and the detection limit was 0.20 μM. With good stability and selectivity, the proposed sensor could be feasibly applied to detect glucose in practical samples. The encouraging sensing performances suggest that the hole-transporting material is one of the promising biomimetic catalysts for electrocatalysis and relevant fields.Display Omitted
Keywords: Non-enzymatic biosensor; Glucose; Signal amplification; Electrocatalysis;

For the first time, a simple and efficient in situ emulsification microextraction method using a dicationic ionic liquid followed by magnetic assisted physisorption was presented to determine trace amounts of lead. In this method, 400 μL of 1.0 mol L−1 lithium bis (trifluoromethylsulfonyl) imide aqueous solution, Li[NTf2], was added into the sample solution containing 100 μL of 1.0 mol L−1 1,3-(propyl-1,3-diyl) bis (3-methylimidazolium) chloride, [pbmim]Cl2, to form a water immiscible ionic liquid, [pbmim][NTf2]2. This new in situ formed dicationic ionic liquid was applied as the acceptor phase to extract the lead-ammonium pyrrolidinedithiocarbamate (Pb-APDC) complexes from the sample solution. Subsequently, 30 mg of Fe3O4 magnetic nanoparticles (MNPs) were added into the sample solution to collect the fine droplets of [pbmim][NTf2]2, physisorptively. Finally, MNPs were eluted by acetonitrile, separated by an external magnetic field and the obtained eluent was subjected to micro-sampling flame atomic absorption spectrometry (FAAS) for further analysis. Comparing with other microextraction methods, no special devices and centrifugation step are required. Parameters influencing the extraction efficiency such as extraction time, pH, concentration of chelating agent, amount of MNPs and coexisting interferences were studied. Under the optimized conditions, this method showed high extraction recovery of 93% with low LOD of 0.7 μg L−1. Good linearity was obtained in the range of 2.5–150 μg L−1 with determination coefficient (r2) of 0.9921. Relative standard deviation (RSD%) for seven repeated measurements at the concentration of 10 μg L−1 was 4.1%. Finally, this method was successfully applied for determination of lead in some water and plant samples.Display Omitted
Keywords: Dicationic ionic liquid; In situ emulsification; Microextraction; Magnetic physicosorption; Lead;

The microextraction of diclofenac and mefenamic acid from water samples was performed by using rotating disk sorptive extraction (RDSE) with molecularly imprinted polymer (MIP) as the sorptive phase. The MIP was synthesized from the monomer 1-vinylimidazol (VI) together with the cross-linker divinylbenzene (DVB) using diphenylamine as the template molecule. Scanning electron microscopy (SEM) analyses of the MIP revealed clusters of spherical particles having a narrow size distribution, with diameters of approximately 1 μm.The optimized extraction conditions involved a disk rotation velocity of 3000 rpm, an extraction time of 120 min, a sample volume of 50 mL, and a sample pH of 2 as well as 25 mg of MIP immobilized in the disk. Desorption of the extracted analytes was performed with 5 mL of methanol for 10 min. Analysis by gas chromatography-mass spectrometry (GC–MS) was carried out after derivatization of the analytes with N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA).Nonmolecularly imprinted polymer (NIP) was also synthesized for comparison. It was observed that under the same conditions, MIP extracted significantly more NSAIDs containing diphenylamine (or part of this molecule) in their structure than NIP. Higher significant differences between MIP and NIP were observed for diclofenac, mefenamic acid and paracetamol, clearly indicating the effect of the template on the extraction.Recoveries of the method were between 100 and 112%, with relative standard deviations of 5–6%. The limits of detection were between 60 and 223 ng L−1. Water samples from a wastewater treatment plant (WWTP) of Santiago de Chile, were found to contain concentrations of these acidic drugs between 1.6 and 4.3 μg L−1 and between 1.4 and 3.3 μg L−1 in the influent and effluent, respectively.Display Omitted
Keywords: Rotating-disk sorptive extraction; Molecularly imprinted polymer; Non steroidal anti-inflammatory drugs; Water; Gas chromatography-mass spectrometry;

In the present study, humic acids (HAs) were applied as both a matrix for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and an adsorbent of magnetic solid phase extraction (MSPE) for the first time. As natural macromolecule compounds, HAs are inherently highly functionalized and contain laser energy absorbing–transferring aromatic structures. This special molecular structure made HAs a good candidate for use as a MALDI matrix in small molecule analysis. At the same time, due to its good adsorption ability, HAs was prepared as MSPE adsorbent via a simple co-mixing method, in which the commercially available HAs were directly mixed with Fe3O4 magnetic nanoparticles (MNPs) in a mortar and grinded evenly and completely. In this process, MNPs were physically wrapped and adhered to tiny HAs leading to the formation of magnetic HAs (MHAs). To verify the bi-function of the MHAs, Rhodamine B (RdB) was chosen as model compound. Our results show that the combination of MHAs-based MSPE and MALDI-TOF-MS can provide a rapid and sensitive method for the determination of RdB in chili oil. The whole analytical procedure could be completed within 30 min for simultaneous determination of more than 20 samples, and the limit of quantitation for RdB was found to be 0.02 μg/g. The recoveries in chili oil were in the range 73.8–81.5% with the RSDs less than 21.3% (intraday) and 20.3% (interday). The proposed strategy has potential applications for high-throughput analysis of small molecules in complex samples.Display Omitted
Keywords: Humic acids; Matrix; Magnetic solid phase extraction; Rhodamine B;

Polyethoxylated tallow amine (POEA) surfactants have been used in many glyphosate-based herbicide formulations for agricultural, industrial and residential weed control. The potential for release of these compounds into the environment is of increasing concern due to their toxicity towards aquatic organisms. Current methods for analysis of POEA surfactants require significant time and effort to achieve limits of quantification that are often higher than the concentrations at which biological effects have been observed (as low as 2 ng mL−1). We have developed a rapid and robust method for quantifying the POEA surfactant mixture MON 0818 at biologically relevant concentrations in fresh water, sea water and lake sediment using reversed phase high-performance liquid chromatography and electrospray ionization-tandem mass spectrometry. Water samples preserved by 1:1 v/v dilution with methanol are analyzed directly following centrifugation. Sediment samples undergo accelerated solvent extraction in aqueous methanol prior to analysis. Large volume (100 μL) sample injection and multiple reaction monitoring of a subset of the most abundant POEA homologs provide limits of quantification of 0.5 and 2.9 ng mL−1 for MON 0818 in fresh water and sea water, respectively, and 2.5 ng g−1 for total MON 0818 in lake sediment. Average recoveries of 93 and 75% were achieved for samples of water and sediment, respectively spiked with known amounts of MON 0818. Precision and accuracy for the analysis of water and sediment samples were within 10 and 16%, respectively based upon replicate analyses of calibration standards and representative samples. Results demonstrate the utility of the method for quantifying undegraded MON 0818 in water and sediment, although a more comprehensive method may be needed to identify and determine other POEA mixtures and degradation profiles that might occur in the environment.Display Omitted
Keywords: Polyethoxylated tallow amines; Glyphosate surfactants; Water; Sediment; High-performance liquid chromatography–tandem mass spectrometry; Large volume injection;

This paper reports on the optimisation, characterisation, validation and applicability of gas chromatography coupled to triple quadrupole mass spectrometry in its tandem operation mode (GC-QqQ(MS/MS) for the quantification of polychlorinated dibenzo-p-dioxins and furans (PCDD/Fs, dioxins) and dioxin-like polychlorinated biphenyls (DL-PCBs) in environmental and food matrices. MS/MS parameters were selected to achieve the high sensitivity and selectivity required for the analysis of this type of compounds and samples. Good repeatability for areas (RSD = 1–10%, for PCDD/Fs and DL-PCBs) and for ion transition ratios (RSD = 0.3–10%, for PCDD/Fs, and 0.2–15%, for DL-PCBs) and low instrumental limits of detection, 0.07–0.75 pg μL−1 (for dioxins) and 0.05–0.63 pg μL−1 (for DL-PCBs), were obtained. A comparative study of the congener specific determination using both GC-QqQ(MS/MS) and gas chromatography-high resolution mass spectrometry (GC-HRMS) was also performed by analysing several fortified samples and certified reference materials (CRMs) with low (feed and foodstuffs), median (sewage sludge) and high (fly ash) toxic equivalency (TEQ) concentration levels, i.e. 0.60, 1.83, 72.9 and 3609 pg WHO-TEQ(PCDD/Fs) g−1. The agreement between the results obtained for the total TEQs (dioxins) on GC-QqQ(MS/MS) and GC-HRMS in all the investigated samples were within the range of ±4%, and that of DL-PCBs at concentration levels of 0.84 pg WHO-TEQs (DL-PCBs) g−1, in the case of feedstuffs, was 0.11%. Both instrumental methods have similar and comparable linearity, precision and accuracy. The GC-QqQ(MS/MS) sensitivity, lower than that of GC-HRMS, is good enough (iLODs in the down to low pg levels) to detect the normal concentrations of these compounds in food and environmental samples. These results make GC-QqQ(MS/MS) suitable for the quantitative analysis of dioxins and DL-PCBs and a real alternative tool to the reference sector HRMS instruments.Display Omitted
Keywords: Gas chromatography; Triple quadrupole mass spectrometry; Polychlorinated dibenzo-p-dioxins; Dioxin-like polychlorinated biphenyls; Foodstuffs; Environmental samples;

This study developed an affinity-gradient nano-stationary phase (AG-NSP) for protein analysis using nanofluidic capillary electrochromatography (nano-CEC) conjugated with matrix assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS). The AG-NSP can be used for protein pre-separation in nano-CEC and as a matrix carrier for protein analysis in MALDI-TOF-MS. A hydrophobicity gradient in AG-NSP was photochemically formed by grafting 4-azidoaniline hydrochloride on vertically arrayed multi-wall carbon nanotubes (MWCNTs) through gray-level exposure to UV light. The reversed-phase gradient stationary phase in AG-NSP was tailored according to the properties of the mobile phase gradient in capillary electrochromatography. As a result, the operation of the system is easily automated using a single buffer solution without the need for multiple solvents for elution. The use of nano-CEC with AG-NSP demonstrated excellent separation efficiency and high resolution for various types of DNA/protein/peptide. MALDI-TOF-MS analysis was then performed directly on the separated proteins and peptides on the chip. The proposed system was then used for the detection of three types of proteins with different molecular weights and PI values, including Cytochrome c (12,360, pI = 10), Lysozyme (14,300, pI = 11), and BSA (86,000, pI = 5)), and digested IgG fragments. The proposed system provided resolution of 1000 Da for the proteins in this study and the separation of digested IgG fragments at a low concentration of 1.2 pmol μL−1.Display Omitted
Keywords: Nanofluidic capillary electrochromatography; Affinity-gradient nano-stationary phase; Matrix carrier; MWCNTs nanostructured chromatographic supports;

We reported here for the first time on the use of cotton thread combined with novel gold nanoparticle trimer reporter probe for low-cost, sensitive and rapid detection of a lung cancer related biomarker, human ferritin. A model system comprising ferritin as an analyte and a pair of monoclonal antibodies was used to demonstrate the proof-of-concept on the dry-reagent natural cotton thread immunoassay device. Results indicated that the using of novel gold nanoparticle trimer reporter probe greatly improved the sensitivity comparing with traditional gold nanoparticle reporter probe on the cotton thread immunoassay device. The assay avoids multiple incubation and washing steps performed in most conventional protein analyses. Although qualitative tests are realized by observing the color change of the test zone, quantitative data are obtained by recording the optical responses of the test zone with a commercial scanner and corresponding analysis software. Under optimal conditions, the cotton thread immunoassay device was capable of measuring 10 ng/mL human ferritin under room temperature which is sensitive enough for clinical diagnosis. Moreover, the sample solution employed in the assays is just 8 μL, which is much less than traditional lateral flow strip based biosensors.Display Omitted
Keywords: Gold nanoparticle trimer reporter probe; Cotton thread immunoassay device; Point-of-care diagnosis; Human ferritin detection;

DNA aptazymes are allosteric DNAzymes activated by the targets of DNA aptamers. They take the advantages of both aptamers and DNAzymes, which can recognize specific targets with high selectivity and catalyze multiple-turnover reactions for signal amplification, respectively, and have shown their great promise in many analytical applications. So far, however, the available examples of DNA aptazyme sensors are still limited in utilizing only several DNAzymes and DNA aptamers, most likely due to the lack of a general and simple approach for rational design. Herein, we have developed such a general approach for designing fluorescent DNA aptazyme sensors. In this approach, aptamers and DNAzymes are connected at the ends to avoid any change in their original sequences, therefore enabling the general use of different aptamers and DNAzymes in the design. Upon activation of the aptazymes by the targets of interest, the rate of fluorescence enhancement via the cleavage of a dually labeled substrate by the active aptazymes is then monitored for target quantification. Two DNAzymes and two aptamers are used as examples for the design of three fluorescent aptazyme sensors, and they all show high selectivity and sensitivity for the detection of their targets. More DNA aptazyme sensors for a broader range of targets could be developed by this general approach as long as suitable DNAzymes and aptamers are used.A simple and general approach for rational design of fluorescent DNA aptazyme sensors based on the target-induced unfolding of DNA hairpins.Display Omitted
Keywords: Aptamer; DNAzyme; DNA aptazyme; Functional DNA;

3-D microarray and its microfabrication-free fluidic immunoassay device by Yingshuai Liu; Yuanyuan Zhang; Zhisong Lu; Chang Ming Li (187-193).
Conventional 2-D microarray is known to have high-throughput detection capability; however, the sensing spots density is significantly hindered by the spot-to-spot distance (gap) requirement for eliminating cross-talks between adjacent spots. Herein a new conceptual 3-D microarray device is proposed to significantly improve the spots density. To demonstrate advantages of the 3-D array, a microfabrication-free fluidic immunoassay device is further made by simply coupling an antibodies-arrayed glass cuboid into a circular glass tube. Rapid, sensitive and high-throughput flow-through immunoassays were accomplished with the 3-D array-based device for detection limits of 10–100 pg mL−1 and wide dynamic range over 4–5 orders of magnitude in human serum with cancer biomarkers α-fetoprotein (AFP) and carcinoembryonic antigen (CEA) as model targets, holding great promise for practical clinical applications. The 3-D microarray device not only significantly increases the density of sensing spots, but also greatly enhances the mass transport for rapid immunoassay when using in a flow-through device.Display Omitted
Keywords: 3-Dimensional microarray; High-throughput; Microfabrication-free; Flow-through immunoassay; Cancer biomarkers;

We present a novel electrochemical glucose sensor employing an interdigitated array (IDA) of 1:1 aspect ratio carbon nanoelectrodes for the electrochemical-enzymatic redox cycling of redox species (ferricyanide/ferrocyanide) between glucose oxidase (GOx) and the two comb-shaped nanoelectrodes of the IDA. The carbon nanoelectrodes were fabricated using a simple, cost-effective, reproducible microfabrication technology known as the carbon-microelectromechanical-systems (C-MEMS) process. One comb (comb 1) of the IDA was selectively modified with GOx via the electrochemical reduction of an aryl diazonium salt, while the other comb (comb 2) remained unmodified; this facilitates electrochemically more active surface of comb 2, resulting in sensitive glucose detection. Ferricyanide is reduced to ferrocyanide by the GOx in the presence of glucose, and ferrocyanide diffuses to both combs of the IDA where it is oxidized. The limited electrochemical current collection at the surface-modified comb 1 is counterbalanced by the efficient redox cycling between the enzyme sites at comb 1 and the bare carbon surface of comb 2. Reducing the electrode-to-electrode gap between the two combs (gap = 1.9 μm) increases the diffusion flux of redox species at comb 2 hence, enhanced the sensitivity and limit of detection of the glucose sensor by ∼2.3 and ∼295 times, respectively at comb 2 compared to comb 1. The developed IDA-based glucose sensor demonstrated good amperometric response to glucose, affording two linear ranges from 0.001 to 1 mM and from 1 to 10 mM, with limits of detection of 0.4 and 61 μM and sensitivities of 823.2 and 70.0 μA mM−1 cm−2, respectively.Display Omitted
Keywords: Glucose sensor; Interdigitated array nanoelectrodes; Electrochemical-enzymatic redox cycling; Diazonium salt; Glucose oxidase;

An immunochemical strategy based on peptidoglycan synthetic peptide epitopes to diagnose Staphylococcus aureus infections by Carme Pastells; Gerardo Acosta; Nuria Pascual; Fernando Albericio; Miriam Royo; M.-Pilar Marco (203-211).
The characteristic pentaglycyl cross-bridge of the Staphylococcus aureus peptidoglycan (PG) cell wall component is an attractive epitope to raise specific antibodies against this microorganism. Based on this approach, we report here for the first time a competitive ELISA able to detect S. aureus down to 104 CFU mL−1, without pre-enrichment on cell culture. The antibodies were raised against peptide-protein bioconjugates prepared by covalently coupling peptide haptens (PSau6 and PSau8) designed and synthesized taking into consideration the complex tridimensional structure in the PG polymer. Deglycosylation of the PG under acidic conditions has found to increase assay detectability. Assay performance has been evaluated in clinical samples such as bronchoalveolar lavage (BAL) and bronchoalveolar endotracheal aspirates (BAS) showing promising results for further implementation of this immunoassay as a daily routine diagnostic tool. Cross-reactivity studies have demonstrated that the immunoassay is specific for S. aureus.Display Omitted
Keywords: Staphylococcus aureus; Peptidoglycan; Immunoassay; Diagnostic; Trifluoromethanesulfonic acid; Antibodies;