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Analytica Chimica Acta (v.776, #)

Editorial Board (pp. iii).

Effect of quality characteristics of single sample preparation steps in the precision and coverage of proteomic studies—A review by Thomas Krüger; Thomas Lehmann; Heidrun Rhode (pp. 1-10).

Display Omitted► In contrast to usual analytics exorbitant numbers of steps are combined in proteomics. ► Overall precision and yield are virtually impossible to be determined experimentally. ► We estimated overall bias using published values of various singular procedures. ► Unexpectedly, calculated overall bias is in the range of aberrations of many candidate biomarkers. ► Proposals to improve precision and yield include hyphenation, parallelization and automation.Proteomic profiling and biomarker search are analytical tools as many other. Nevertheless, in the proteomic discovery phase considerable sample fractionation is inevitable before readout. Since these procedures are of notable complexity, proteomic tools need in particular analytical quality validation standards as prevail for other analytical methods. With acceptance of the rule of error propagation the values of imprecision and yield of each preparation step determine overall reproducibility and therewith information harvest of a propagated method series. Thereto, we examined recent proteomic reports with reproducibility data and with parallelization, and automation approaches. Based on the data available from literature it is highly probable, that at least a part of current proteomic platforms actually suffer from high technical variance.The general need for quantification and the impact of precision and recovery levels with each step of typical multi-step workflows are illustrated. All sample preparation approaches that maximize yields (percent recoveries) and minimize overall imprecision should be suitable to improve reliability of biomarker search. This has to be realized by technical innovations that (a) minimize the imprecision of each serial sample preparation step, that (b) minimize the number of serial sample preparation steps, and that (c) parallelize all procedures with a maximum of automation.

Keywords: Abbreviations; AEC; anion exchange chromatography; ConA; concanavalin A; CV; coefficient of variation; =; relative standard deviation; EIC; extracted ion chromatogram; FFE; free flow electrophoresis; HP; high-performance; LAC; lectin affinity chromatography; MARS; multiple affinity removal system; MCE; multichannel electrolyte; PF2D; protein fractionation by 2D liquid chromatography; RP; reversed phase; SOP; standard operation procedure; SXC; strong cation exchange chromatography; WGA; wheat germ agglutininSample preparation; Imprecision; Recovery; Parallelization; Automation; Error propagation; Validation parameters

A sensitive and label-free impedimetric biosensor based on an adjunct probe by Xi Yuan Zhang; Long Yin Zhou; Hong Qun Luo; Nian Bing Li (pp. 11-16).

Using an adjunct probe for highly sensitive and label-free detection of DNA sequence based on electrochemical impedance spectroscopy was achieved.•A label-free impedimetric sensor is constructed for DNA sequences detection.•The key point is adjunct probes immobilized nearby capture probes.•The biosensor achieves simple and highly selective detection for DNA sequence.•The sensor is readily extended for other biomolecules, proteins and mRNAs detection.A highly sensitive and label-free impedimetric biosensor is achieved based on an adjunct probe attached nearby the capture probe. In this work, the adjunct probe was co-assembled on the surface of gold electrode with the capture probe hybridized with the reporter probe, and then 6-mercapto-1-hexanol was employed to block the nonspecific binding sites. When target DNA was added, the adjunct probe functioned as a fixer to immobilize the element of reporter probe displaced by the target DNA sequences and made the reporter probe approach the electrode surface, leading to effective inhibition of charge transfer. The increase in charge transfer resistance is related to the quantity of the target DNA in a wide range. The linear range for target DNA with specific sequences was from 0.1nM to 0.5μM with a good linearity ( R=0.9988) and a low detection limit of 6.3pM. This impedimetric biosensor has the advantages of simplicity, sensitivity, good selectivity, and large dynamic range.

Keywords: Label-free biosensor; DNA sequence; Adjunct probe; Electrochemical impedance spectroscopy; Charge transfer

Fabrication of glucose biosensor for whole blood based on Au/hyperbranched polyester nanoparticles multilayers by antibiofouling and self-assembly technique by Chong Sun; Xiaohan Chen; Qiaorong Han; Min Zhou; Chun Mao; Qinshu Zhu; Jian Shen (pp. 17-23).

Display Omitted•A novel method for detection of glucose in whole blood has been developed.•The method based on antibiofouling and self-assembly technology was investigated.•The antibiofouling technique utilized for sensor is significant for diagnostics.Acknowledging the benefits of hyperbranched polymers and their nanoparticles, herein we report the design and synthesis of sulfonic acid group functionalized hydroxyl-terminated hyperbranched polyester (H30-SO₃H) nanoparticles and their biomedical application. The H30-SO₃H nanoparticles were characterized by transmission electron microscopy (TEM), Fourier transform infrared (FTIR) spectroscopy and proton nuclear magnetic resonance spectroscopy (¹H NMR). The good hemocompatibility of H30-SO₃H nanoparticles was also investigated by coagulation tests, complement activation and platelet activation. The novel glucose biosensor was fabricated by immobilizing the positively charged Au nanoparticles, H30-SO₃H nanoparticles and glucose oxidase (GOx) onto the surface of glassy carbon electrode (GCE). It can be applied in whole blood directly, which was based on the good hemocompatibility and antibiofouling property of H30-SO₃H nanoparticles. The biosensor had good electrocatalytic activity toward glucose with a wide linear range (0.2–20mM), a low detection limit 1.2×10⁻⁵M in whole blood and good anti-interference property. The development of materials science will offer a novel platform for application to substance detection in whole blood.

Keywords: Hyperbranched polyester nanoparticles; Antibiofouling; Self-assembly technology; Glucose nano-biosensors; Whole blood

Polyphosphonate induced coacervation of chitosan: Encapsulation of proteins/enzymes and their biosensing by Hailing Liu; Yanyun Cui; Pan Li; Yiming Zhou; Yu Chen; Yawen Tang; Tianhong Lu (pp. 24-30).

Based on the coacervation of chitosan via the ionotropic crosslinking interaction, proteins/enzymes can be encapsulated in situ into chitosan matrix.•The ionotropic crosslinking interactions result in the coacervation of chitosan.•A phosphonate-assisted encapsulation of proteins in chitosan matrix is introduced.•The encapsulated proteins retain their bioactivity.•The encapsulation method can be used to fabricate various chitosan-based biosensors.Based on the polyphosphonate-assisted coacervation of chitosan, a simple and versatile procedure for the encapsulation of proteins/enzymes in chitosan–carbon nanotubes (CNTs) composites matrix was developed. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), energy dispersive spectrum (EDS) mapping demonstrated the hemoglobin (Hb) uniformly distributed into chitosan–CNTs composites matrix. Raman measurements indicated the CNTs in composites matrix retained the electronic and structural integrities of the pristine CNTs. Fourier transform infrared (FT-IR), ultraviolet–visible (UV–vis) and circular dichroism (CD) spectroscopy displayed the encapsulated Hb preserved their near-native structure, indicating the polyphosphonate–chitosan–CNTs composites possessed excellent biocompatibility for the encapsulation of proteins/enzymes. Electrochemical measurements indicated the encapsulated Hb could directly exchange electron with the substrate electrode. Moreover, the modified electrode showed excellent bioelectrocatalytic activity for the reduction of hydrogen peroxide. Under optimum experimental conditions, the fabricated electrochemical sensor displayed the fast response (less than 3s), wide linear range (7.0×10⁻⁷ to 2.0×10⁻³M) and low detection limit (4.0×10⁻⁷M) for the determination of hydrogen peroxide. This newly developed protocol was simple and mild and would certainly find extensive applications in biocatalysis, biosensors, bioelectronics and biofuel cells.

Keywords: Phosphonic acid; Chitosan; Carbon nanotubes; Hemoglobin; Direct electrochemistry

Simultaneous assay of pigments, carbohydrates, proteins and lipids in microalgae by Yimin Chen; Seetharaman Vaidyanathan (pp. 31-40).

•Simultaneous assay of major biochemical components with a unified method.•New formulae for the assay of pigments with higher sensitivities.•Standardised pretreatment of samples for the assay of carbohydrates and proteins.•Conservation of sample, time, chemicals, cost and energy using the unified assay.Biochemical compositional analysis of microbial biomass is a useful tool that can provide insight into the behaviour of an organism and its adaptational response to changes in its environment. To some extent, it reflects the physiological and metabolic status of the organism. Conventional methods to estimate biochemical composition often employ different sample pretreatment strategies and analytical steps for analysing each major component, such as total proteins, carbohydrates, and lipids, making it labour-, time- and sample-intensive. Such analyses when carried out individually can also result in uncertainties of estimates as different pre-treatment or extraction conditions are employed for each of the component estimations and these are not necessarily standardised for the organism, resulting in observations that are not easy to compare within the experimental set-up or between laboratories. We recently reported a method to estimate total lipids in microalgae (Chen, Vaidyanathan, Anal. Chim. Acta, 724, 67–72). Here, we propose a unified method for the simultaneous estimation of the principal biological components, proteins, carbohydrates, lipids, chlorophyll and carotenoids, in a single microalgae culture sample that incorporates the earlier published lipid assay. The proposed methodology adopts an alternative strategy for pigment assay that has a high sensitivity. The unified assay is shown to conserve sample (by 79%), time (67%), chemicals (34%) and energy (58%) when compared to the corresponding assay for each component, carried out individually on different samples. The method can also be applied to other microorganisms, especially those with recalcitrant cell walls.

Keywords: Biochemical composition; Pigments; Carbohydrates; Proteins; Lipids

Automated SPME–GC–MS monitoring of headspace metabolomic responses of E. coli to biologically active components extracted by the coating by S.M. Zakir Hossain; Barbara Bojko; Janusz Pawliszyn (pp. 41-49).

Display Omitted•In vivo HS-SPME was used for monitoring of Escherichia coli metabolic profile changes.•For the first time SPME fiber coating was used for simultaneous delivery of the antibacterial agent.•Feasibility of automation of this process was demonstrated.Monitoring extracellular metabolites of bacteria is very useful for not only metabolomics research but also for assessment of the effects of various chemicals, including antimicrobial agents and drugs. Herein, we describe the automated headspace solid-phase microextraction (HS-SPME) method coupled with gas chromatography–mass spectrometry (GC–MS) for the qualitative as well as semi-quantitative determination of metabolic responses of Escherichia coli to an antimicrobial agent, cinnamaldehyde. The minimum inhibitory concentration of cinnamaldehyde was calculated to be 2gL⁻¹. We found that cinnamaldehyde was an important factor influencing the metabolic profile and growth process. A higher number of metabolites were observed during the mid-logarithmic growth phase. The metabolite variations (types and concentrations) induced by cinnamaldehyde were dependent on both cell density and the dose of cinnamaldehyde. Simultaneously, 25 different metabolites were separated and detected (e.g., indole, alkane, alcohol, organic acids, esters, etc.) in headspace of complex biological samples due to intermittent addition of high dose of cinnamaldehyde. The study was done using an automated system, thereby minimizing manual workup and indicating the potential of the method for high-throughput analysis. These findings enhanced the understanding of the metabolic responses of E. coli to cinnamaldehyde shock effect and demonstrated the effectiveness of the SPME–GC–MS based metabolomics approach to study such a complex biological system.

Keywords: Sampling; Sample preparation; Solid Phase Microextraction (SPME); In vivo; Microbiology; Drug discovery

Preparation and examination of monolithic in-needle extraction (MINE) device for the direct analysis of liquid samples by Monika Pietrzyńska; Adam Voelkel; Katarzyna Bielicka-Daszkiewicz (pp. 50-56).

•MINE device for isolation of analytes from water samples.•Nine polymer poly(styrene-divinylbenzene) monoliths prepared in stainless steel needles.•High efficiency of in-needle extraction systems based on monolithic materials.•New possibilities in sample preparation area.Combination of extraction and chromatographic techniques opens NEW possibilities in sample preparation area. Macroporous poly(styrene-divinylbenzene) (PS-DVB) monoliths were prepared by in situ polymerization in stainless steel needles. The surface of stainless steel needle was modified earlier by the silane coupling agent. Monolithic materials located inside needles were used as the in-needle extraction device. Scanning electron microscope (SEM) images were obtained for nine monoliths. Spectra of prepared materials were also performed with the use of two techniques: Attenuated Total Reflectance (ATR) and Fourier Transform Infrared Spectroscopy (FTIR). The new monolithic in-needle extraction (MINE) devices were used in the preparation of a series of test water samples for chromatographic analysis. The extraction of phenolic compounds from water samples was carried out by pumping liquid samples through the MINE device. Obtained results indicate a high efficiency of in-needle extraction systems based on monolithic materials. Breakthrough volume and the sorption efficiency of prepared monolithic in-needle extraction devices were determined experimentally. The achieved recovery was close to 90%, and determined LOQ values varied between 0.4 and 6μg.

Keywords: Monolithic materials; In-needle extraction; Needle trap device; Poly(styrene-divinylbenzene)

Proteomic characterization of human platelet-derived microparticles by Anna Laura Capriotti; Giuseppe Caruso; Chiara Cavaliere; Susy Piovesana; Roberto Samperi; Aldo Laganà (pp. 57-63).

•Shotgun proteomic study of platelet-derived microparticles.•Low molecular weight protein enrichment by hydrogel nanoparticles.•NanoHPLC-MS/MS analysis of in-solution tryptic digests of microparticle proteins.Microparticles (MPs) are small fragments of apoptotic or activated cells that may contribute to pathological processes in many diseases. Platelet-derived MPs (PMPs) are the most abundant type of MPs in human blood. To characterize the proteins in PMPs we used a shotgun proteomics approach by nanoHPLC separation followed by MS analysis on an LTQ Orbitrap XL. PMPs were produced from isolated platelets stimulated with adenosine diphosphate (ADP). We developed an analytical platform constituted by two different steps: in the first one we used a standard shotgun strategy; in the second one, to improve low-molecular weight, low-abundance-proteins identification, the samples were fractionated using hydrogel nanoparticles, an enrichment system based on a mixed mechanism of dimensional exclusion and colorant affinity. This was chosen to tackle a common issue with shotgun approaches, in which the low-abundance proteins are not detected when surveys are on a broad scale. By means of the entire analytical platform, we identified 603 proteins, 243 of which were not previously identified. A simple and straightforward procedure for the study of PMPs was provided, producing a tool for further understanding their biological and pathological roles, and a baseline for future studies aimed at discovering biomarkers involved in several diseases.

Keywords: Abbreviations; MP; microparticles; PMP; platelet microparticle; ADP; adenosine diphosphate; RBC; red-blood cell; HN; hydrogel nanoparticles; RP; reversed-phase; LMW; low-molecular weight; Poly(NIPAm/CB) core (VSA); poly(N-isopropylacrilamide/cibacron blue) core (vinylsulfonic acid); SPE; solid phase extraction; LAP; low abundance protein; MW; molecular weight; HMW; high molecular weight; FDR; false discovery rate; GO; gene ontology; PF4; platelet factor 4Microparticles; Platelet; Shotgun proteomics; Hydrogel nanoparticles

Collection method for chemical particulates on surfaces with detection using thermal desorption-ion trap mass spectrometry by K.J. Ewing; D. Gibson; J. Sanghera; F. Miklos (pp. 64-68).

•We developed a sticky screen sampler for contact sampling of surfaces.•We investigated the ability of the sticky screen sampler for collecting chemical particulates from a surface.•We demonstrated that the sticky screen was capable of collecting chemical particulates from a surface.•We demonstrated that chemical particulates collected by the sticky screen could be analyzed using thermal desorption-ion trap mass spectrometry.•We demonstrated nanogram (ng) level detection limits for DMMP immobilized into silica gel.Successful analysis of particulate/low vapor pressure analytes such as explosives and toxic chemicals, and commercial pesticides require new sampling tools that enable detection of these analytes using current vapor phase detection instruments. We describe a sampling approach that uses stainless steel screens coated with a sticky polydimethyl siloxane (PDMS) coating to capture particulates from surfaces. Preliminary results for the collection of dimethyl methylphosphonate (DMMP) sorbed onto silica gel (SG) particulates (DMMP/SG) from a surface with subsequent analysis by thermal desorption-cylindrical ion trap mass spectrometry (TD-CITMS) are reported.

Keywords: Chemical particulates; Collection; Surfaces; Screens

A “turn-on” fluorescent chemosensor for zinc ion with facile synthesis and application in live cell imaging by Kai Li; Xiaoyan Wang; Aijun Tong (pp. 69-73).

•A “turn-on” fluorescent chemosensor for the detection of Zn(II) was facilely synthesized.•Detection of Zn(II) can be performed in water at neutral pH with high sensitivity.•The chemosensor exhibits remarkably selective to Zn(II) over other metal ions.•The chemosensor could be efficiently delivered to live cells for bioimaging of Zn(II).Compound1 was facilely synthesized through a one step reaction from commercially available materials. As a sensitive and selective “turn-on” fluorescent chemosensor for Zn(II),1 exhibits a 40-fold fluorescence enhancement response to Zn(II) over other physiological relevant metal ions in aqueous solution at neutral pH. Furthermore,1 could be efficiently delivered to live cells for bioimaging of Zn(II).

Keywords: Zinc ion; Fluorescence; Chemosensor; Cell imaging

Well-oriented ZZ–PS-tag with high Fc-binding onto polystyrene surface for controlled immobilization of capture antibodies by Jin-Bao Tang; Xi-Feng Sun; Hong-Ming Yang; Bao-Gang Zhang; Zhi-Jian Li; Zhi-Juan Lin; Zhi-Qin Gao (pp. 74-78).

•A versatile platform for immobilizing functionally intact IgG is proposed.•The mechanism relies on properly oriented ZZ–PS-tag onto a hydrophilic PS surface.•The oriented ZZ–PS-tag presents ~fivefold higher IgG-binding activity.•The platform shows tenfold higher sensitivity and a wider linear range in ELISA.The site specificity and bioactivity retention of antibodies immobilized on a solid substrate are crucial requirements for solid phase immunoassays. A fusion protein between an immunoglobulin G (IgG)-binding protein (ZZ protein) and a polystyrene-binding peptide (PS-tag) was constructed, and then used to develop a simple method for the oriented immobilization of the ZZ protein onto a PS support by the specific attachment of the PS-tag onto a hydrophilic PS. The orientation of intact IgG was achieved via the interaction of the ZZ protein and the constant fragment (Fc), thereby displayed the Fab fragment for binding antigen. The interaction between rabbit IgG anti-horseradish peroxidase (anti-HRP) and its binding partner HRP was analyzed. Results showed that the oriented ZZ–PS-tag yielded an IgG-binding activity that is fivefold higher than that produced by the passive immobilization of the ZZ protein. The advantage of the proposed immunoassay strategy was demonstrated through an enzyme-linked immunosorbent assay, in which monoclonal mouse anti-goat IgG and HRP-conjugated rabbit F(ab′)₂ anti-goat IgG were used to detect goat IgG. The ZZ–PS-tag presented a tenfold higher sensitivity and a wider linear range than did the passively immobilized ZZ protein. The proposed approach may be an attractive strategy for a broad range of applications involving the oriented immobilization of intact IgGs onto PS supports, in which only one type of phi-PS (ZZ–PS-tag) surface is used.

Keywords: Abbreviations; IgG; immunoglobulin G; PS; polystyrene; phi-PS; hydrophilic polystyrene; Pho-PS; hydrophobic PS; PS-tag; polystyrene-binding peptide; HRP; horseradish peroxidase; ELISA; enzyme-linked immunosorbent assay; BSA; bovine serum albuminEnzyme-linked immunosorbent assay; Immunoglobulin G; Hydrophilic polystyrene plate; Oriented immobilization; Polystyrene-binding peptide; ZZ–PS-tag fusion protein

Irregular-shaped platinum nanoparticles as peroxidase mimics for highly efficient colorimetric immunoassay by Zhuangqiang Gao; Mingdi Xu; Li Hou; Guonan Chen; Dianping Tang (pp. 79-86).

•We report a new colorimetric immunoassay of rabbit IgG.•Irregular-shaped platinum nanostructures were used as peroxidase mimics.•The assay was implemented based on nanocatalysts.Enzyme-linked immunosorbent assay (ELISA) methods based on natural enzyme-labeled probes have been applied in the immunoassays, but most have some inevitable limitations (e.g. harsh preparation, purification and storage) and are unsuitable for routine use. Herein we synthesized a new class of irregular-shaped platinum nanoparticles (ISPtNP) with a mean length of 7.0nm and a narrowing width from 2.0 to 5.0nm along the longitudinal axes, which were utilized as peroxidase-like mimics for the development of colorimetric immunoassays. Compared with bioactive horseradish peroxidase (HRP), the synthesized ISPtNP exhibited a low K_m value (~0.12mM) and a high K_cat value (~2.27×10⁴s⁻¹) for 3,3′,5,5′-tetramethylbenzidine (TMB) with strong thermal stability and pH tolerance. The catalytic mechanism of the ISPtNP toward TMB/H₂O₂ was for the first time discussed and deliberated in this work. Based on a sandwich-type assay format, two types of colorimetric immunoassay protocols were designed and developed for the detection of rabbit IgG (RIgG, as a model) by using the synthesized ISPtNP and conventional HRP as the labeling of detection antibodies, respectively. Similar detection limits (LODs) of 2.5ngmL⁻¹ vs. 1.0ngmL⁻¹ were obtained toward RIgG with the ISPtNP labeling compared to HRP format. Intra- and inter-assay coefficients of variation were less than 13%. Importantly, the ISPtNP-based assay system could be suitable for use in a mass production of miniaturized lab-on-a-chip devices and open new opportunities for protein diagnostics and biosecurity.

Keywords: Colorimetric immunoassay; Irregular-shaped platinum nanoparticles; Peroxidase mimics; Rabbit IgG

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Analytica Chimica Acta (v.776, #)