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Exposure to mycotoxins, which may contaminate food and feed commodities, represents a serious health risk for consumers. Ochratoxin A (OTA) is one of the most abundant and toxic mycotoxins, thus specific regulations for fixing its maximum admissible levels in foodstuff have been established. Lateral Flow ImmunoAssay (LFIA)-based devices have been proposed as screening tools to avoid OTA contamination along the whole food chain. We report a portable, user-friendly smartphone-based biosensor for the detection and quantification of OTA in wine and instant coffee, which combines the LFIA approach with chemiluminescence (CL) detection. The device employs the smartphone camera as a light detector and uses low-cost, disposable analytical cartridges containing the LFIA strip and all the necessary reagents. The analysis can be carried out at the point of need by non-specialized operators through simple manual operations. The biosensor allows OTA quantitative detection in wine and coffee samples up to 25 μg L−1 and with limits of detection of 0.3 and 0.1 μg L−1, respectively, which are below the European law-fixed limits. These results demonstrate that the developed device can be used for routine monitoring of OTA contamination, enabling rapid and reliable identification of positive samples requiring confirmatory analysis.

ATP-driven bioluminescence relying on the D-luciferin-luciferase reaction is widely employed for several biosensing applications where bacterial ATP detection allows to verify microbial contamination for hygiene monitoring in hospitals, food processing and in general for cell viability studies. Several ATP kit assays are already commercially available but an user-friendly ATP biosensor characterized by low-cost, portability, and adequate sensitivity would be highly valuable for rapid and facile on site screening. Thanks to an innovative freeze-drying procedure, we developed a user-friendly, ready-to-use and stable ATP sensing paper biosensor that can be combined with smartphone detection. The ATP sensing paper includes a lyophilized “nano-lantern” with reaction components being rapidly reconstituted by 10 μL sample addition, enabling detection of 10−14 mol of ATP within 10 min. We analysed urinary microbial ATP as a biomarker of urinary tract infection (UTI), confirming the capability of the ATP sensing paper to detect the threshold for positivity corresponding to 105 colony-forming units of bacteria per mL of urin, This research was sponsored in part by PRIN 2015 “Multifunctional nanotools for advanced cancer diagnostics” (Prot. 2015TWP83Z) and the NATO Science for Peace and Security Programme under Grant No. 985042. The ICN2 is funded by the CERCA Programme/Generalitat de Catalunya and also supported by the Severo Ochoa program of the Spanish Ministry of Economy, Industry and Competitiveness (MINECO, grants No. SEV-2017-0706 and MAT2017-87202-P).

High polyphenols levels are one of the main quality characters of Extra Virgin Olive Oil (EVOO), leading to high antioxidant activity and contributing to the EVOO health beneficial effects. Analytical methods for the rapid assessment of EVOO polyphenols content, possibly directly at the place of production or distribution, are therefore important for the commercial enhancement of this product. Unfortunately, most analytical methods for quantification of polyphenols in EVOO require laboratory instrumentation and preliminary sample extraction procedures. In this paper we describe a paper-based chemosensor based on the well-known Folin-Ciocalteu colorimetric assay for the rapid (assay time 15 min) quantitative detection of polyphenols in EVOO without any preliminary sample extraction. Use of n-propanol for diluting the EVOO sample allows its direct analysis on paper supports loaded with the Folin-Ciocalteu reagent. The color change of the paper supports is measured using a smartphone camera. A disposable analytical cartridge containing all necessary reagents, including calibration standards, and accessories for performing the assay using a Samsung S8 smartphone have been developed to perform on-site analysis. Measurement of total polyphenol content of EVOO samples gave results in good agreement with the conventional Folin-Ciocalteu assay, and the limit of detection was 30 μg gallic acid equivalents g−1 EVOO. The same approach can be employed to measure polyphenols in other oils of vegetal origin.

A simple and versatile continuous air-segmented flow sensor using immobilized luciferase was designed as a general miniaturized platform based on sensitive biochemiluminescence detection. The device uses miniaturized microperistaltic pumps to deliver flows and compact sensitive light imaging detectors based on BI-CMOS (smartphone camera) or CCD technology. The low-cost components and power supply make it suitable as out-lab device at point of need to monitor kinetic-related processes or ex vivo dynamic events. A nylon6 flat spiral carrying immobilized luciferase was placed in front of the detector in lensless mode using a fiber optic tapered faceplate. ATP was measured in samples collected by microdialysis from rat brain with detecting levels as low as 0.4 fmoles. The same immobilized luciferase was also used for the evaluation of bile salt hydrolase (BSH) activity in intestinal microbiota. An aminoluciferin was conjugatated with chenodeoxycholic acid forming the amide derivative aLuc-CDCA. The hydrolysis of the aLuc-CDCA probe by BSH releases free uncaged aminoluciferin which is the active substrate for luciferase leading to light emission. This method can detect as low as 0.5 mM of aLuc-CDCA, so it can be used on real faecal human samples to study BSH activity and its modulation by diseases and drugs.

Increasingly, smartphones are used as portable personal computers, revolutionizing communication styles and entire lifestyles. Using 3D-printing technology we have made a disposable minicartridge that can be easily prototyped to turn any kind of smartphone or tablet into a portable luminometer to detect chemiluminescence derived from enzyme-coupled reactions. As proof-of-principle, lactate oxidase was coupled with horseradish peroxidase for lactate determination in oral fluid and sweat. Lactate can be quantified in less than five minutes with detection limits of 0.5 mmol L(-1) (corresponding to 4.5 mg dL(-1)) and 0.1 mmol L(-1) (corresponding to 0.9 mg dL(-1)) in oral fluid and sweat, respectively. A smartphone-based device shows adequate analytical performance to offer a cost-effective alternative for non-invasive lactate measurement. It could be used to evaluate lactate variation in relation to the anaerobic threshold in endurance sport and for monitoring lactic acidosis in critical-care patients.

The development of user-friendly devices for on site analysis and characterization of painting materials is one of the most challenging objectives in the diagnostics for cultural heritage. Thanks to the specificity of antigen–antibody reactions, immunological methods have been already successfully applied for the detection of proteins and for their localization within painting stratigraphies. Moreover, by combining the advantages of the immunological techniques with the high detectability offered by chemiluminescence detection, it has been possible to achieve good analytical performance and very low detection limits. This work was aimed at developing a portable analytical device for the detection of chicken ovalbumin (a protein found in egg tempera and in egg-based protective varnishes) in painting samples employing ready-to-use analytical cartridges and a thermoelectrically-cooled CCD camera as a chemiluminescence detector. The protein was extracted from using a simple procedure and revealed by a non-competitive immunoassay with chemiluminescence contact imaging detection. The assay was simple, fast and suitable for the detection of ovalbumin in small samples. This analytical system provided positive identification of ovalbumin in samples obtained from both fresh or artificially aged paint reconstructions and historical paintings. It might be easily employed in different contexts (such as small museums, restoration laboratories or even on site) by restorers to obtain prompt information during restoration actions, such as cleaning operations. In perspective this device could be also employed for the detection of other proteinaceous and organic painting components.

Bile acids (BAs) are endogenous steroids involved in the transport of lipids in bile, acting also as molecular signaling hormones. Primary BAs synthesized in the liver undergo several metabolic pathways in the intestine by gut microbiota to produce secondary BAs. Together with secondary BAs, other metabolites have been recovered from human faeces, including many oxo-BA analogues produced in the colon through oxidation of BA hydroxy groups. However, the complete oxo-BA characterization in biospecimens (particularly intestinal content and faeces) has not been reported yet, hampering the assessment of their potential physiological role. Herein, we have developed and validated a new RP-HPLC-ESI-MS/MS method in negative ionization mode for the simultaneous analysis of 21 oxo-BAs and their 7 metabolic BAs precursors in human faeces. The elution was performed in gradient mode and 28 compounds, including primary, secondary BAs, and their oxo-derivatives, were separated within 50 min at 40 °C column temperature. The method is accurate (bias%

The present work aimed at optimizing a two-step enzymatic plus solvent-based process for the recovery of bioactive compounds from white grape (Vitis vinifera L., mix of Trebbiano and Verdicchio cultivars) pomace, the winemaking primary by-product. Phenolic compounds solubilised by water enzyme-assisted and ethanol-based extractions of wet (WP) and dried (DP) pomace were characterised for composition and tested for antioxidant, anti-tyrosinase and anti-inflammatory bioactivities. Ethanol treatment led to higher phenol yields than water extraction, while DP samples showed the highest capacity of releasing polyphenols, most probably as a positive consequence of the pomace drying process. Different compositions and bioactivities were observed between water and ethanol extracts and among different treatments and for the first time the anti-tyrosinase activity of V. vinifera pomace extracts, was here reported. Enzymatic treatments did not significantly improve the total amount of solubilised compounds; Celluclast in DP led to the recovery of extracts enriched in specific compounds, when compared to control. The best extracts (enzymatic plus ethanol treatment total levels) were obtained from DP showing significantly higher amounts of polyphenols, flavonoids, flavanols and tannins and exerted higher antioxidant and anti-tyrosinase activities than WP total extracts. Conversely, anti-inflammatory capacity was only detected in water (with and without enzyme) extracts, with WP samples showing on average a higher activity than DP. The present findings demonstrate that white grape pomace constitute a sustainable source for the extraction of phytochemicals that might be exploited as functional ingredients in the food, nutraceutical, pharmaceutical or cosmetic industries.

Electronic nose and capillary electrophoresis were applied in quality control of green tea samples subjected to long-term storage. Twelve representative green teas were considered, available as an "aged" (tea leaves stored during a long-term period of two years) and/or "not aged" (fresh products) samples. Their infusions were analyzed by an electronic nose, equipped with an array of six metal oxide semiconductor (MOS) sensors to obtain olfactive fingerprints of the volatile compounds in the infusions headspace. Upon training and chemometric analysis of acquired data (linear discriminant analysis), the electronic nose was found to be able in correctly classifying unknown samples as "aged" or "not aged". Concomitantly, the infusion samples were analyzed by Cyclodextrin-modified Micellar Electrokinetic Chromatography (CD-MEKC) for determination of catechins. The analysis of seven most represented catechins and the methylxanthines theobromine and caffeine revealed a general loss of the polyphenols in each of the considered aged samples (up to 45%, w/w). In addition, the applied enantioselective method based on (2-hydroxypropyl)-beta-cyclodextrin (HP-beta CD) as chiral selector, was exploited for the estimation of (+)-Gallocatechin in the presence of (-)-Gallocatechin; the latter, as the non-native enantiomer, can be associated to the epimerisation of (-)-Epigallocatechin and was assumed as a marker occurring in case of uncorrected storage conditions of tea leaves. Interestingly, it was observed that epimerization did not significantly occur during aging. The application of CD-MEKC and electronic nose allowed for a fast characterization of green teas taking into account that the aroma is a decisive parameter for the acceptance of the product, whereas the catechins content is associated to the biological value.

Berberine (1) is an alkaloid used widely in the treatment of several diseases. However, its physicochemical properties, pharmacokinetics, and metabolism remain unclear, and conflicting data have been reported. In this study, the main physicochemical properties of 1 and its metabolites were evaluated, including lipophilicity, solubility, pKa, and albumin binding. A sensitive HPLC-ESIMS/MS method was developed and validated to identify 1 and its main metabolites in human plasma. This method was used to quantify their levels in the plasma of healthy volunteers and hypercholesterolemic patients following a single dose and chronic administration, respectively. In both cases, berberrubine (2) was found to be the main metabolite. Surprisingly, 2 is more lipophilic than 1, which suggests that this compound tautomerizes to a highly conjugated, electroneutral quinoid structure. This was confirmed by NMR studies. These results indicate that the higher plasma concentration of 2 was a consequence of a more efficient intestinal absorption, suggesting that berberrubine is potentially more pharmacologically active than berberine.

In this paper, we report, for the first time, the use of a smartphone to image and quantify biochemiluminescence coupled biospecific enzymatic reactions to detect analytes in biological fluids. Using low-cost three-dimensional (3D) printing technology, we fabricated a smartphone accessory and a minicartridge for hosting biospecific reactions. As a proof-of-principle, we report two assays: a bioluminescence assay for total bile acids using 3α-hydroxyl steroid dehydrogenase coimmobilized with bacterial luciferase system and a chemiluminescence assay for total cholesterol using cholesterol esterase/cholesterol oxidase coupled with the luminol-H2O2-horseradish peroxidase system. These assays can be performed within 3 min in a very straightforward manner and provided adequate analytical performance for the analysis of total cholesterol in serum (limit of detection (LOD) = 20 mg/dL) and total bile acid in serum and oral fluid (LOD = 0.5 μmol/L) with a reasonable accuracy and precision. Smartphone-based biochemiluminescence detection could be thus applied to a variety of clinical chemistry assays.

The qualitative identification of proteinaceous substances, as well as their location within a complex paint stratigraphy, is one of the most challenging issues in the characterization of painting materials. Nevertheless, information on paint components represent a crucial task for studies concerning both the ancient painting techniques adopted and the state of conservation, being fundamental investigations for the selection of appropriate conservation actions. The present research was aimed at developing a new detection approach for the immunochemical localization of ovalbumin in paint cross-sections based on the use of scanning electrochemical microscopy (SECM). The immunochemical analyses were performed using an anti-ovalbumin primary antibody and a secondary antibody labelled with horseradish peroxidase (HRP). SECM measurements were performed in feedback mode using benzoquinone (BQ)/hydroquinone (H2Q) redox couple. In presence of hydrogen peroxide (H2O2), HRP catalyzes the re-oxidation of H2Q to BQ and the increment of BQ concentration in correspondence of the target protein was detected by SECM through the electrochemical reduction of the regenerated BQ at the microelectrode. Indeed, the localization of ovalbumin was possible thanks to a clear discrimination of SECM currents, achieved by the comparison of the measurements recorded before and after H2O2 administration, based on the HRP on/off approach. The method was evaluated both on samples from standard mocks-up and on a historical sample, collected from a Renaissance wood painting. The obtained results were promising, foreseeing a wider application of SECM on cultural heritage researches.

A new HPLC-ESI-MS/MS method for the determination of glucosamine (2-amino-2-deoxy-d-glucose) in rabbit cartilage was developed and optimized. Glucosamine was extracted from cartilage by cryogenic grinding followed by protein precipitation with trichloroacetic acid. The HPLC separation was achieved with a polymer-based amino column using a mobile phase composed of 10mM ammonium acetate (pH 7.5)-acetonitrile (20:80%, v/v) at 0.3 mL min flow rate. d-[1-(13)C]Glucosamine was used as internal standard. Selective detection was performed by tandem mass spectrometry with electrospray source, operating in positive ionization mode and in multiple reaction monitoring acquisition (m/z 180→72 and 181→73 for glucosamine and internal standard, respectively). Limit of quantification was 0.045 ng injected, corresponding to 0.25 μg g-1 in cartilage. Linearity was obtained up to 20 μg g-1 (R(2)>0.991). Precision values (%R.S.D.) were

New cell sorting methodologies, which are simple, fast, non-invasive, and able to isolate homogeneous cell populations, are needed for applications ranging from gene expression analysis to cell-based therapy. In particular, in the forefront of stem cell isolation, progenitor cells have to be separated under mild experimental conditions from complex heterogeneous mixtures prepared from human tissues. Most of the methodologies now employed make use of immunological markers. However, it is widely acknowledged that specific markers for pluripotent stem cells are not as yet available, and cell labelling may interfere with the differentiation process. This work presents for the first time gravitational field-flow fractionation (GrFFF), as a tool for tag-less, direct selection of human hematopoietic stem and progenitor cells from cell samples obtained by peripheral blood aphaeresis. These cells are responsible to repopulate the hemopoietic system and they are used in transplantation therapies. Blood aphaeresis sample were injected into a GrFFF system and collected fractions were characterized by flow cytometry for CD34 and CD45 expression, and then tested for viability and multi-differentiation potential. The developed GrFFF method allowed obtaining high enrichment levels of viable, multi-potent hematopoietic stem cells in specific fraction and it showed to fulfil major requirements of analytical performance, such as selectivity and reproducibility of the fractionation process and high sample recovery.

The identification of serum biomarkers for the diagnosis of inflammatory bowel diseases able to reduce the need for invasive tests represents a major goal in their therapy and follow-up. We report here a methodological approach for the evaluation of specific changes in the serum peptides abundance in healthy (H) and Crohn's disease (CD) subjects, based on a label-free LC ESI/Q-TOF differential mass spectrometry (MS) approach combined with targeted MS/MS analysis. The low molecular weight serum proteins were separated by RP nano-LC ESI/Q-TOF MS and the resulting datasets were aligned with msInspect software. The differently abundant peptides, evaluated using Proteios Software Environment, were identified by MS/MS analysis and database search. The identification of clusters of peptides resulting from proteins (such as fibrinogen-alpha) commonly involved in physiological processes lead to the evaluation of a possible role in CD of specific serum exoproteases. An assay based on synthetic peptides spiked into H, CD and ulcerative colitis (UC) serum samples as substrate, followed by MALDI MS and chemometric analysis of the metabolite patterns has been developed achieving a 100% discrimination between CD, UC and H subjects. The results are promising for the application of this approach as a simple tool for diagnostic aims and biomarker discovery in CD.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a useful technique for the identification of bacteria on the basis of their characteristic protein mass spectrum fingerprint. Highly standardized instrumental analytical performance and bacterial culture conditions are required to achieve useful information. A chemometric approach based on multivariate analysis techniques was developed for the analysis of MALDI data of different bacteria to allow their identification from their fingerprint. Principal component analysis, linear discriminant analysis (LDA) and soft independent modelling of class analogy (SIMCA) were applied to the analysis of the MALDI MS mass spectra of two pathogenic bacteria, Escherichia coli O157:H7 and Yersinia enterocolitica, and the non-pathogenic E. coli MC1061. Spectra variability was assessed by growing bacteria in different media and analysing them at different culture growth times. After selection of the relevant variables, which allows the evaluation of an m/z value pattern with high discriminant power, the identification of bacteria by LDA and SIMCA was performed independently of the experimental conditions used. In order to better evaluate the analytical performance of the approach used, the ability to correctly classify different bacteria, six wild-type strains of E. coli O157:H7, was also studied and a combination of different chemometric techniques with a severe validation was developed. The analysis of spiked bovine meat samples and the agreement with an independent chemiluminescent enzyme immunoassay demonstrated the applicability of the method developed for the detection of bacteria in real samples. The easy automation of the MALDI method and the ability of multivariate techniques to reduce interlaboratory variability associated with bacterial growth time and conditions suggest the usefulness of the proposed MALDI MS approach for rapid routine food safety checks.

Hollow-fiber flow field-flow fractionation is here applied to untreated, whole human blood serum. Matrix-assisted, laser desorption/ionization time-of-flight-mass spectrometry (MALDI-TOF-MS) of serum fractions shows mass signals in the M r range where low-abundance, serum protein components are known to be present, though a membrane of nominal 30 000 Da cutoff was employed for the fractionation device. Using diluted sera spiked with low amounts (0.06–0.1%, w/w) of an artificial mixture constituted the human adrenocorticotropic hormone fragments 18–39 ( M r = 2465.7) and 7–38 ( M r = 3659.2), and of bovine insulin ( M r = 5734), horse cytochrome c ( M r = 12 384) and chicken lysozyme ( M r = 14 388), a hybrid fractionation/microfiltration mechanism shows to govern the separation of the low- M r components.

A new high pressure liquid chromatography-electrospray ionization-tandem mass spectrometry method for the simultaneous determination of glucosinolates, as glucoraphanin and glucoerucin, and the corresponding isothiocyanates, as sulforaphane and erucin, was developed and applied to quantify these compounds in Eruca sativa defatted seed meals and enriched functional foods. The method involved solvent extraction, separation was achieved in gradient mode using water with 0.5% formic acid and acetonitrile with 0.5% formic acid and using a reverse phase C18 column. The electrospray ion source operated in negative and positive mode for the detection of glucosinolates and isothiocyanates, respectively, and the multiple reaction monitoring (MRM) was selected as acquisition mode. The method was validated following the ICH guidelines. Replicate experiments demonstrated a good accuracy (bias%

A multiplex chemiluminescent biosensor for simple, rapid and ultrasensitive on-site quantification of aflatoxin B1 and type B-fumonisins in maize samples has been developed. The biosensor integrates a multiplex indirect competitive lateral flow immunoassay (LFIA) based on enzyme-catalyzed chemiluminescence detection and a highly sensitive portable charge-coupled device (CCD) camera, employed in a lensless "contact" imaging configuration. The developed assay requires a simple extraction of the analytes from maize flour samples followed by their detection with a 30 min assay time. The use of chemiluminescence detection allowed accurate and objective analytes quantification, enabling simultaneous detection of type B-fumonisins and aflatoxin B1 down to 6 mu g kg(-1) and 1.5 mu g kg(-1), respectively, thus fulfilling the standards imposed by the legislation of European Union. Maize flour samples spiked with both analytes were subjected to multiplex analysis obtaining recoveries ranging from 80 to 115% and the coefficient of variation below 20%. Finally, analysis of naturally contaminated maize samples resulted in a good agreement between CL-LFIA and a validated confirmatory HPLC-UV and commercial ELISA kit, obtaining recoveries in the range 88-120%. The proposed CL-LFIA protocol is rapid, inexpensive, easy-to-use, and fit for the purpose of rapid screening of mycotoxins in maize flour.

A fast and ultrasensitive chemiluminescent enzyme immunoassay for aflatoxin M(1) in milk samples has been developed and validated. The method is an indirect competitive type format involving the immobilization of an aflatoxin M(1)-bovine serum albumin conjugate on 384 well black polystyrene microtiter plates and the use of a secondary antibody labeled with horseradish peroxidase detected with a luminol-based substrate. Aflatoxin M(1) standard solutions were prepared in milk-based buffer, and milk samples were analyzed without any cleanup procedure. The limit of quantification was 1 ppt, the coefficient of variation was below 9% for both intra- and interassay precision, and the recovery ranged from 96 to 122%. The method is specific, and other aflatoxins do not significantly cross-react with the antibody. Twenty-four milk samples were analyzed, and a good correlation was observed (y = 0.98x + 1.71, r(2) = 0.98, n = 24) when the data were compared with a reference high-performance liquid chromatography method with a fluorescent detector. The developed method is suitable for an accurate, sensitive, and high-throughput screening of aflatoxin M(1) in milk samples with a reduction of costs and increased detectability, as compared with previously developed immunoassays.

Capabilities of mass spectrometry for the analysis of intact proteins can be increased through separation methods. Flow field-flow fractionation (FlFFF) is characterized by the particularly "soft" separation mechanism, which is ideally suited to maintain the native structure of intact proteins. This work describes the original on-line coupling between hollow-fiber FlFFF (HF FlFFF), the microcolumn variant of FlFFF, and electrospray ionization/time-of-flight mass spectrometry (ESI/TOFMS) for the analysis and characterization of intact proteins. The results show that the native (or pseudonative) structure of horse heart myoglobin and horseradish peroxidase is maintained. Sample desalting is also observed for horse heart myoglobin. Correlation between the molar mass values independently measured by HF FlFFF retention and ESI/TOFMS allows us to confirm the protein aggregation features of bovine serum albumin and to indicate possible changes in the quaternary structure of human hemoglobin.

We describe a rapid and sensitive high-performance liquid chromatography/electrospray tandem mass spectrometry (HPLC/ESI-MS/MS) method for simultaneous determination of the most relevant metabolites of benzene and toluene, t,t-muconic acid (t,t-MA), S-phenylmercapturic acid (S-PMA), and S-benzylmercapturic acid (S-BMA). Urine samples were purified before analysis by solid-phase microextraction (SPE) on SAX cartridges with 50 mg sorbent mass. The developed method fulfils all the standard requirements of precision and accuracy. Calibration curves were linear within the concentration range of the standards (0-80 microg/L(urine) for t,t-MA, and 0-25 microg/L(urine) for S-PMA and S-BMA), and had correlation coefficients > or =0.997. Limits of detection were 6.0 microg/L for t,t-MA, 0.3 microg/L for S-PMA, and 0.4 microg/L for S-BMA. The method was used to determine t,t-MA, S-PMA and S-BMA levels in urine of 31 gasoline-station workers, with personal monitoring data obtained from radial symmetry passive diffusive samplers. In the context of mean work-shift exposures of 75.9 microg/m(3) (range 9.4-220.2) for benzene and 331.9 microg/m(3) (78.2-932.1) for toluene, metabolite concentrations in end-of-shift urine samples ranged from 23.5-275.3 microg/g(creatinine) for t,t-MA, non-detectable to 0.9 microg/g(creatinine) for S-PMA, and 3.8-74.8 microg/g(creatinine) for S-BMA. No significant correlation was found between the environmental concentrations and urinary metabolites (p > 0.05 for all cases); the ratios of benzene metabolites could be influenced by exposure levels and co-exposure to xylenes and toluene. The high throughput of this procedure should facilitate exploration of the metabolic effects of benzene-related co-exposure to toluene and alkylbenzenes in large populations of subjects exposed to gasoline.

On-line, continuous chemiluminescence detection has been recently applied to gravitational field-flow fractionation as a new method suitable for flow-assisted assays based on the separation between the analyte in solution and the analyte bound to micrometer-sized particles. In this work, flow field-flow fractionation coupled with off-line chemiluminescence detection is exploited for the development of flow-assisted, multianalyte assays. Micrometer-sized, polystyrene beads coated with enzymes suitable for chemiluminescence detection, such as horseradish peroxidase and alkaline phosphatase, are used as model samples. The high size-based selectivity of flow field-flow fractionation and specificity of chemiluminescence detection make possible, in a single run with very short analysis time, the simultaneous, high sensitivity detection of the different enzymes linked to beads of different size.

This Chapter deals with applications of CL and BL in topics of significant relevance, which are not described in the previous Chapters of this book. In particular, principle and limitations of the well-known luminol test, based on the catalysis of the luminol CL reaction by blood traces and utilised by forensic scientists in investigations involving violent crime for more than 40 years, are reviewed. Afterwards, the various CL-based methods employed for the rapid and sensitive detection of explosives, from portable explosive analyzers to gas chromatographic and HPLC techniques with CL detection and immunoassays, are described. Finally, examples of application of CL and BL in the study and conservation of cultural heritage are reported, including detection of biodeteriogen agents (bacteria, fungi, yeast, algae and lichens) on artworks by BL imaging of adenosine triphosphate (ATP) and immunolocalization of protein binders in painting cross-sections by CL imaging microscopy.

This work describes a rapid and sensitive solid-phase microextraction (SPME) method for the isolation and analysis of methyl tert-butyl ether in water samples. Methyl tert-butyl ether was extracted from aqueous solutions using SPME fibre coated with Divinylbenzene/Carboxen™/polydimethylsiloxane (30 μm film thickness) and analysed by GC–MS with a Hewlett Packard 6890/5973 system equipped with a capillary column coated with Vocol™ (30 m ×0.25 mm ,1.5 μm film thickness). Extraction parameters and chromatographic separation conditions were optimised. The developed method showed good analytical performance in terms of precision (RSD between 2% and 8%) and accuracy (mean recovery from 96% to 104%) with a detection limit of 14 ppt. Finally the method was applied to surface, tap and commercial mineral water samples, as well as snow samples collected along a busy road of Bologna town area. The median concentration of methyl tert-butyl ether in all these samples (0.05–0.4 ppb) was well below the maximum aqueous contamination levels in water adopted in the United States (13 ppb).

We used a luminescence-based biosensor to determine mercury in urine samples of subjects with or without dental amalgam restorations. The system utilizes Escherichia coli as a host organism and firefly luciferase luc gene as a reporter under the control of the mercury-inducible promoter from the mer operon from transposon Tn21. The presence of mercury induces the specific gene sequence with consequent synthesis of the luciferase enzyme, and luciferin/luciferase-mediated light output occurs. The method showed a linear relationship between the light signal intensity and the Hg2+ concentration in the range of 1.67 × 10−13–1.67 × 10−7 M with a detection limit of 1.67 × 10−13 M, corresponding to 4.0 × 10−18 mol/tube. The system proved precise, accurate, and highly selective for mercury and methylmercury, with no light emission induced by other heavy metals; the matrix effect caused by urine components was minimal. The analysis of urine specimens showed that urinary mercury levels in subjects with amalgam filli...

A fast and sensitive chemiluminescent (CL) enzyme immunoassay for clenbuterol (CLB) analysis in bovine urine has been developed. Clenbuterol (CLB) specific polyclonal antibodies were raised in rabbit using a CLB azo derivative conjugated with ovalbumin. Horseradish peroxidase (HRP) was used as label and conjugated with the same derivative. In the developed competitive method, antibodies were immobilized on 384-wells black polystyrene microtiter plates; the sample volume was 20 μl and HRP-labeled CLB activity was immediately measured, using different CL substrates, after 10 min incubation time. Emitted light was recorded using a sensitive back-illuminated, cooled CCD camera or a conventional, photomultiplier-based micrtotiter plate reader. The developed method fulfills all the requirements of precision (CV below 10%) and accuracy (mean recovery from 96 to 110%) with a detection limit of 0.08 ppb in urine matrix. The use of 384-wells microtiter plate allows a 5-fold reduction in reagent quantity and the CL detection improves the detectability of the HRP-labeled tracer, thus reducing analysis time. The developed method is therefore suitable for high-throughput screening of CLB in urine samples, with reduced costs as compared with conventional colorimetric enzyme immunoassays, thanks to the possibility to optimize the system in non-equilibrium immunological conditions and with a very fast chemiluminescence detection of the HRP-label activity.

Analytical chemiluminescence and bioluminescence represent a versatile, ultrasensitive tool with a wide range of applications in diverse fields such as biotechnology, pharmacology, molecular biology, clinical and environmental chemistry. Enzyme activities and enzyme substrates and inhibitors can be efficiently determined when directly involved in luminescent reactions, and also when they take part in a reaction suitable for coupling to a final light-emitting reaction. Chemiluminescence detection has been exploited in the fields of flow-injection analysis and column-liquid chromatographic and capillary-electrophoretic separative systems, due to its high sensitivity when compared with colorimetric detection. It has widely been used as an indicator of reactive oxygen species formation in cells and whole organs, thus allowing the study of a number of pathophysiological conditions related to oxidative stress. Chemiluminescence represents a sensitive and rapid alternative to radioactivity as a detection principle in immunoassays for the determination of a wide range of molecules (hormones, food additives, environmental pollutants) and in filter membrane biospecific reactions (Southern, Northern, Western, dot blot) for the determination of nucleic acids and proteins. Chemiluminescence has also been used for the sensitive and specific localization and quantitation of target analytes in tissue sections and single cells by immunohistochemistry and in situ hybridization techniques. A relatively recent application regards the use of luminescent reporter genes for the development of bioassays based on genetically engineered microorganisms or mammalian cells able to emit visible light in response to specific inorganic and organic compounds. Finally, the high detectability and rapidity of bio- and chemiluminescent detection make it suitable for the development of microarray-based high throughput screening assays, in which simultaneous, multianalyte detection is performed on multiple samples.

trans-4-Hydroxy-2-nonenal (HNE) is an end-product of lipid peroxidation in biological systems which produces a variety of powerful biological effects. A method based on electrospray mass spectrometry was developed for the determination of 4-HNE at cellular levels. Quantification was carried out by using HNE-d(11) as internal standard; the mass chromatograms were acquired in the single ion monitoring mode (SIM) on the [M + H](+) monoisotopic species for HNE and HNE-d(11). With this approach a higher precision and lower detection limit and biological sample size than those typical of the methods so far employed are achieved. Furthermore the determination of the analyte from the cell extract is directly performed without the need of any HNE derivatization. As a first application the method was used to identify and quantify HNE in human T cell leukemia extracts.

The analysis of bile salts in biological samples has remained a difficult task, due to the complex nature of the salts and also to their low concentration in common sample fluids such as plasma and urine. Given their importance, the development of accurate and sensitive methods of instrumental analysis has been the subject of intensive research, and recent advances have eliminated or lessened some of the difficulties. Currently available techniques are the following: thin-layer chromatography, gas chromatography, high-performance liquid chromatography, supercritical fluid chromatography, gas chromatography-mass spectrometry and capillary electrophoresis. Liquid chromatography coupled with mass spectrometry (thermospray, fast atom bombardment, electrospray and ionspray), a method undergoing continuous improvement, is also being applied to bile salts analysis. In this paper, these various techniques, which differ greatly in specificity, accuracy and simplicity, are reviewed and discussed, in terms of analytical performance, applicability to a given sample fluid, major limitations, ability to identify uncommon bile salts, including unsaturated oxo derivatives, glucuronides, sulfates, glycosides and bile alcohols.

Bothrops jararaca snake venom extract has been analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electrospray mass spectrometry (ESI-MS) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) in order to obtain a rapid and accurate characterization. By the SDS-PAGE and MALDI-MS techniques about fourteen components are evidenced; with the latter technique, faster analysis and a more accurate molecular weight determination have been achieved. By ESI-MS the data obtained strongly depend on the operating conditions employed. © 1997 John Wiley & Sons, Ltd.

Proteins immobilized on polymer supports are extensively used to develop bioanalytical methods and the determination of the protein/polymer molar ratio is important both for quantifying the catalytic activity and calculating the diffusion rate of the substrates. In this paper a method, based on electrospray mass spectrometry, for characterizing subtilisin and lipase immobilized on chemically modified poly(ethylene glycol)-5000 is described. The results obtained show that this technique is particularly effective. The information provided by it can be used to optimize the synthesis of these polymer–protein conjugates a well as to evaluate the efficiency of their separation and purification. © 1997 John Wiley & Sons, Ltd.

A miniaturized multiplex biosensor exploiting a microfluidic oligonucleotide array and chemiluminescence (CL) lensless imaging detection has been developed for parvovirus B19 genotyping. The portable device consists of a reaction chip, comprising a glass slide arrayed with three B19 genotype-specific probes and coupled with a polydimethylsiloxane microfluidic layer, and a charge-coupled device camera modified for lensless CL imaging. Immobilized probes were used in DNA hybridization reactions with biotin-labeled targets, and then hybrids were measured by means of an avidin-horseradish peroxidase (HRP) conjugate and CL detection. All hybridization assay procedures have been optimized to be performed at room temperature through the microfluidic elements of the reaction chip, with sample and reagents delivery via capillary force exploiting adsorbent pads to drive fluids along the microchannels. The biosensor enabled multiplex detection of all B19 genotypes, with detectability down to 80 pmol L(-1) for all B19 genotype oligonucleotides and 650 pmol L(-1) for the amplified product of B19 genotype 1, which is comparable with that obtained in traditional PCR-ELISA formats and with notably shorter assay time (30 min vs. 2 h). The specificity of the assay has been evaluated by performing DNA-DNA hybridization reactions among sequences with different degrees of homology, and no cross hybridizations among B19 genotypes have been observed. The clinical applicability has been demonstrated by assaying amplified products obtained from B19 reference serum samples, with results completely consistent with the reference PCR-ELISA method. The next crucial step will be integration in the biosensor of a miniaturized PCR system for DNA amplification and for heat treatment of amplified products.

A non-invasive test for oro-ileal transit time (OITT) evaluation was developed, based on the measurement of tauroursodeoxycholic acid (TUDCA) oral fluid concentration profile after its oral administration. Exploiting the fact that TUDCA is actively absorbed only in the ileum, OITT is measured as the time corresponding to TUDCA maximum oral fluid concentration ( t max ). To measure oral fluid TUDCA concentration in a point-of-care setting, an ultrasensitive portable immunosensor was developed, based on a competitive chemiluminescent enzyme immunoassay (CL-EIA), using immobilized anti-TUDCA antibody and an ursodeoxycholic acid (UDCA)-peroxidase conjugate as tracer, detected by enhanced chemiluminescence employing a portable charge-coupled device (CCD)-based device. The test was validated in 24 healthy subjects before and after treatment with Loperamide, a drug that increases OITT. The developed CL-EIA was accurate and precise, with a LLOQ of 50 pmol L −1 . The measured OITT for healthy subjects (291 ± 50 min) was fairly well correlated with OITT values obtained by measuring TUDCA in serum ( r = 0.89). An increased OITT was observed in all the studied subjects after Loperamide treatment. The CL immunosensor can be employed directly in gastroenterology and paediatric units and it can thus represent a new non-invasive simple test for OITT evaluation in a point-of-care setting, with improved diagnostic utility.

A highly sensitive and rapid chemiluminescent assay for the determination of the activity of xanthine oxidase (XOD) was developed. The chemiluminescent signal was obtained from the catalyzed oxidation of hypoxanthine, accelerated and amplified using a Fe-EDTA complex and perborate, which acts on luminol. The same luminescent mixture was previously used as detection system for immunoassays. Two different mixtures were used, which differ in their luminol and perborate content, with (CLMrho) or without (CLMb) addition of 0.1 μM rhodamine fluorophor. The response obtained from XOD standard solutions in buffer was linear from 5 to 500 U L−1 and from 0.7 to 250 U L−1 for CLMrho and CLMb respectively, at 25°C. 5 and 0.7 U L−1 were the detection limits at 1 standard deviation level. The intra- and inter-assay relative standard deviations ranged from 6 to 12 % for both CLM. Measurements were made using the high performance, low-light level imaging Berthold luminograph LB-980 which allows simultaneous dete...

Some commercial preparations of common natural conjugated bile salts contain impurities (e.g., amines, lipids, and calcium) that are likely to affect their physicochemical properties. A method was developed for purifying commercial preparations of sodium salts of glycine- and taurine-conjugated bile acids. The method consists of passage of a dilute aqueous solution of the sodium bile salt through three columns in sequence: graphitized carbon, a hydrophobic bonded octadecylsilane (C18) cartridge, and a calcium-chelating resin. The final solution was extracted with chloroform, and the purified bile salt was then isolated by freeze-drying, with a yield of 65-75%. Each bile salt purified by this method was compared with the corresponding bile salt purified by conventional adsorption chromatography on a silicic acid column, using a mixture of methanol and chloroform as eluant. Purity was assessed by visible spectra, by surface tension measurements (using the maximum bubble-pressure method and a Wilhelmy wire method), by chloroform extractability of impurities in the conjugated bile acid, by liposome solubilization, and by chemical analysis of the calcium content. Both purification methods removed colored and surface-active impurities, but the new method was always as or more effective than silicic acid column chromatography. Calcium ion, present in commercial bile salts in concentrations up to 16 mmol/mol bile salt, was removed completely by the three-column method, but not by silicic acid chromatography. The new method is thus a simple, rapid, and efficient procedure for purification of the sodium salts of glycine- and taurine-conjugated bile acids for physicochemical measurements, in which elimination of surface-active impurities and polyvalent cations is desired.

The critical micellar concentration (cmc) values of some mixed systems containing two bile salts were determined by a maximum pressure bubble method and compared with those derived from a theoretical model developed for nonionic surfactants to assess the applicability of this model to such systems. Some assumptions on which the presumed validity of this model was based are discussed. The following binary mixtures were investigated: sodium chenodeoxycholate with cholate, ursocholate and ursodeoxycholate, either unconjugated or conjugated with taurine and glycine at different mole fractions (0, 0.25, 0.5, 0.75, 1) in 0.15 M NaCl. For these mixtures, experimentally determined data were in good agreement with values predicted by the theoretical model: both the cmc and the surface tension at this concentration of the mixtures were intermediate between those of the two pure bile salts; also, as the total bile salt concentration increased, the mixed micelles became enriched with the bile salt having the highest cmc, whereas the total monomer activity, determined by a potentiometric method employing a bile salt-selective electrode, increased only slightly. To test this model in an in vitro system, surface tension was also measured in ox bile samples that were enriched by 50% with sodium ursodeoxycholate, chenodeoxycholate, or their taurine amidates. The cmc and the surface tension at this concentration of the artificial bile increased when enriched with a bile salt with a cmc higher than that of endogenous salts (e.g. ursodeoxycholate versus taurocholate), whereas the reverse occurred for mixtures enriched with a bile salt with a lower cmc, such as chenodeoxycholate.(ABSTRACT TRUNCATED AT 250 WORDS)

The present work describes the development of HPLC-mass spectrometric systems equipped with an electrospray interface for the quantitative analysis of bile acids. Good separation of free as well as glycine- and taurine-conjugated bile acids was achieved with a C18 reversed-phase column (3 microns particle size, 70 x 4.6 mm I.D.) employing methanol-15 mM ammonium acetate as the mobile phase for both isocratic and gradient mode, at a flow-rate of 0.3 ml/min. This system permits post-column splitting of the eluate for analysis by two different detectors: (1) electrospray-mass spectrometer with a flow-rate of 18 microliters/min; and (2) a complementary evaporative light scattering mass detector. When bile salts were ionized in the electrospray interface operating in the negative-ion mode, only [M-H]- molecular ions were generated; the detection limit was 15 pg injected for all bile acids studied. In the second system, a semi-micro pre-column splitting apparatus (Acurate, LC Packings) was utilized: with this device the flow-rate from the HPLC pump was reduced to 1.4 microliters/min and bile acids were separated with a micro-bore C18 column (3 microns particle size, 150 x 0.30 I.D.), using the same mobile phase as above. With this latter system, a head-column enrichment technique can be used: the amount injected can be increased from 60 to 200 nl, permitting an improvement in the detection limit to 5 pg injected. Application of the HPLC-electrospray-mass spectrometric method to bile and serum bile acid analysis is described; preliminary data on the ability of the first system to determine the 13C/12C isotope ratio in 13C-labeled bile acid enriched serum is also critically discussed.

A sensitive chemiluminescent sandwich-type enzyme immunoassay for hen egg lysozyme was developed. The assay was performed on polystyrene microtitre plates using immobilized specific polyclonal rabbit antibody against lysozyme, a peroxidase conjugate and the H2O2/luminol-enhanced chemiluminescence detection reagent. The chemiluminescent signal was detected using either a microplate luminometer, or photographic film in a camera luminometer. The detection limit for lysozyme was 0.3 ng/mL, and this was three times lower than that obtained using a colorimetric method with H2O2 and o-phenylendiamine as substrates. Recovery of the assay was 97-112% and the relative standard deviation ranged from 3.6% to 10.3%. The immunoassay overcame interference from the food sample matrix when lysozyme, used as a bacteriostatic agent, was measured.

A “Point-Of-Care-Testing” (POCT) system relies on portable and simply operated self-standing analytical devices. To fulfill diagnostic requirements, the POCT system should provide highly sensitive simultaneous detection of several biomarkers of the pathology of interest (multiplexing) in a short assay time. One of the main unsolved issues in POCT devices development is the integration of pre-analytical sample preparation procedures in the miniaturized device. In this work, an integrated POCT system based on gravitational field-flow fractionation (GrFFF) and chemiluminescence (CL) detection is presented for the on-line sample pre-analytical treatment and/or clean-up and analysis of biological fluids. As a proof of principle for the new GrFFF-CL POCT system, the automatic on-line analysis of plasma alkaline phosphatase activity, a biomarker of obstructive liver diseases and bone disorders, starting from whole blood sample was developed. The GrFFF-CL POCT system was able to give quantitative results on blood samples from control and patients with low sample volume (0.5 microL) and reagents consumption, short analysis time (10 minutes), high reproducibility and with a linear range of 50-1400 IU/L.The system can be easily applied to on-line prepare plasma from whole blood for other clinical biomarkers and for other assay formats, based on immunoassay or DNA hybridization.

A compact portable chemiluminescent biosensor for simple, rapid, and ultrasensitive on-site quantification of fumonisins (fumonisin B1 + fumonisin B2) in maize has been developed. The biosensor integrates a competitive lateral flow immunoassay based on enzyme-catalyzed chemiluminescence detection and a highly sensitive portable charge-coupled device (CCD) camera, employed in a contact imaging configuration. The use of chemiluminescence detection allowed accurate and objective analyte quantification, rather than qualitative or semi-quantitative information usually obtained employing conventional lateral flow immunoassays based on colloidal gold labeling. A limit of detection of 2.5 μg L −1 for fumonisins was achieved, with an analytical working range of 2.5–500 μg L −1 (corresponding to 25–5000 μg kg −1 in maize flour samples, according to the extraction procedure). Total assay time was 25 min, including sample preparation. A simple and convenient extraction procedure, performed by suspending the sample in a buffered solution and rapidly heating to eliminate endogenous peroxidase enzyme activity was employed for maize flour samples analysis, obtaining recoveries in the range 90–115%, when compared with LC–MS/MS analysis. The chemiluminescence immunochromatography-based biosensor is a rapid, low cost portable test suitable for point-of-use applications.

[EN] Simple, rapid and highly sensitive assays, possibly allowing on-site analysis, are required in the security and forensic fields or to obtain early signs of environmental pollution. Several bioanalytical methods and biosensors based on portable devices have been developed for this purpose. Among them, Lateral Flow ImmunoAssays (LFIAs) offer the advantages of rapidity and ease of use and, thanks to the high specificity of antigen-antibody binding, allow greatly simplifying and reducing sample pre-analytical treatments. However, LFIAs usually employ colloidal gold or latex beads as labels and they rely on the formation of colored bands visible by the naked eye. With this assay format, only qualitative or semi-quantitative information can be obtained and low sensitivity is achieved. Recently, the use of enzyme-catalyzed chemiluminescence detection in LFIA has been proposed to overcome these problems. In this work, we describe the development of a quantitative CL-LFIA assay for the detection of 2,4,6-trinitrotoluene (TNT) in real samples. Thanks to the use of a portable imaging device for CL signal measurement based on a thermoelectrically cooled CCD camera, the analysis could be performed directly on-field. A limit of detection of 0.2¿gmL -1 TNT was obtained, which is five times lower than that obtained with a previously described colloidal gold-based LFIA developed employing the same immunoreagents. The dynamic range of the assay extended up to 5¿gmL -1 TNT and recoveries ranging from 97% to 111% were obtained in the analysis of real samples (post blast residues obtained from controlled explosion). © 2012 Elsevier B.V., This work was supported by grants from the Italian Ministry of Instruction, University and Research (PRIN 2007AWK85F "Development of integrated analytical instruments based on luminescence principles for multiplexed analysis in POCT (Point-Of-Care Testing) applications" and PRIN 2006033429 "New Analytical Tools for Security and Criminal Investigations: Trace Detection and Identification of Explosives and Related Compounds") and from the University of Bologna ("Fundamental Oriented Research").

A chemiluminescence-based flow method for the determination of some organophorus and carbamate pesticides based on inhibition of acetylcholinesterase was developed. Acetylcholinesterase in solution or immobilized on methacrylate beads (Eupergit C) was coupled to choline oxidase and peroxidase immobilized on Eupergit C. In this system choline formed by acetylcholinesterase was oxidized by choline oxidase and the H2O2 produced was determined via the luminol/peroxidase luminescent reaction. The detection limits (3 σ) for Paraoxon and Aldicarb were 0.75 μg 1−1 and 4 μg 1−1, respectively, when soluble acetylcholinesterase was used under the following optimized experimental conditions: 56 μM luminol in working solution, sample volume 60 μl, flow-rate 0.3 ml min−1 and 60 min incubation time. The flow sensor device using all the three enzymes in the immobilized form had a higher detection limit of 125 μg 1−1 for Paraoxon. The mid-range relative standard deviation (n=10) using 1 mM standard substrate solution was 3.7%. The recovery from contaminated samples (soil, vegetables) varied from 81 to 108%. The results obtained by the developed methods were in good agreement with those obtained by a commonly used colorimetric test.

A new enteric-coated formulation of sodium ursodeoxycholate was prepared and administered to man. The barrier film disintegrates and releases the drug only at pH > or = 5.5. The sodium salt of glycoursodeoxycholate was also prepared and encapsulated like ursodeoxycholate. Serum levels of ursodeoxycholate and glycoursodeoxycholate were measured by specific enzyme immunoassay after oral administration of their sodium salts in an enteric-coated formulation at equimolar doses of 475 and 540 mg. The same subjects also received in separate experiments ursodeoxycholic acid, sodium ursodeoxycholate, and glycoursodeoxycholic acid in gelatin capsules. The mean area under the curve (mumol/L.hr) following administration of enteric-coated sodium ursodeoxycholate (45 +/- 8) was significantly higher than that of either ursodeoxycholic acid (26 +/- 5; P < 0.01) or sodium ursodeoxycholate (25 +/- 6; P < 0.001) administered in a conventional gelatin capsule. No differences were found when glycoursodeoxycholic acid was administered as an enteric-coated sodium salt or in acid form in gelatin capsules. Ursodeoxycholic was administered at a dose of 10 mumol/min/kg over 1 hr to bile fistula rats both intraduodenally (i.d.) and intravenously (i.v.). The experiment included administration of the sodium salt in solution and the acid as a suspension. A similar experiment was performed with glycoursodeoxycholic acid. The ratio of the amount recovered from bile in the i.d. to that in the i.v. experiment is almost 1 for the sodium salt of ursodeoxycholate in solution, while it drops to 0.55 for ursodeoxycholic acid. No differences were found between i.v. and i.d. administration when glycoursodeoxycholic acid was administered in acid form and as a soluble sodium salt.(ABSTRACT TRUNCATED AT 250 WORDS)

A highly sensitive and rapid chemiluminescent assay for the determination of the activity of alkaline phosphatase (ALP) in serum was developed by monitoring the chemiluminescent signal obtained from an ALP triggerable dioxetane, adamantyl-1,2-dioxetane phenyl phosphate (PPD) used as substrate for the enzyme. The response was linear from 0.1 to 500 U 1−1 at 25 °C for standard (buffered) ALP solutions and from 10 to 1000 U 1−1 at 25 °C for standard ALP added to serum. The intra- and inter-assay relative standard deviations were less than 6% and recoveries ranged from 91 to 106%. Results agreed well with those obtained with a spectrophotometric method for the determination of total ALP activity in serum and for liver- and bone-type ALP isoenzymes activity.

The analytical performance of a low-light imaging luminograph for quantitative luminescence analysis was evaluated in terms of sensitivity, spatial resolution, accuracy, precision, and sample geometry, at the macrolevel and in combination with optical microscopy. The system allows for the detection of 400 amol of enzymes such as alkaline phosphatase and horseradish peroxidase using 1,2-dioxetanes and luminol/p-iodophenol or acridancarboxylate esters, respectively, as chemiluminescent substrates. Enzymatic activity and spatial distribution of nylon net immobilized-alkaline phosphatase was studied; the system permits the quantification of the immobilized enzyme with a spatial resolution as low as 1 μm. Other applications, such as the alkaline phosphatase localization in 8 μm intestinal mucosa cryosections, quantitative immunocytochemistry, and dot blot DNA hybridization reactions, were studied and optimized. The system was also employed for in situ hybridization assay of cytomegalovirus DNA in infected human fibroblasts. The presence of a viral genome was revealed with digoxigenin-labeled probes and alkaline phosphatase-labeled anti-digoxigenin antibody, using chemiluminescent substrate for this enzyme. The luminescent signal was intense and stable, and the probe was imaged and quantified within single cells with higher intensity in the nuclei, with a spatial resolution as low as 1 μm and very low background. The results show that this technique is an ultrasensitive and potent analytical tool to localize and quantify biomolecules at microscopic level, and it is suitable for many bioanalytical applications.

A simple and versatile analytical device designed to perform, even simultaneously, different types of bioassays has been developed and optimized. A transparent microfluidics-based reaction chip, where analytes were quantitatively detected by means of biospecific reactions and chemiluminescence detection, was placed in contact with a thermoelectrically cooled CCD sensor through a fiber optic taper. Such a lensless contact imaging configuration combined adequate spatial resolution and high light collection efficiency within a small size portable device. The miniaturization of the reaction chamber ensured short analysis times (in the minutes range), while the use of chemiluminescence detection provided wide signal dynamic range and high detectability, down to attomole levels of protein and femtomole levels of nucleic acid analytes. A model hybrid panel test was realized by combining an enzyme assay for alkaline phosphatase activity, a nucleic acid hybridization assay for Parvovirus B19 DNA, and an immunoassay for horseradish peroxidase as a model antigen. The successful simultaneous quantification of the three targets demonstrated that a range of analytes, from enzymes to antigens, antibodies, and nucleic acids, can be measured in a single run, thus enabling the realization of a complete, personalized diagnostic panel test for early diagnosis of a given disease and patient follow-up.

Whole-cell bioluminescent (BL) bioreporter technology is a useful analytical tool for developing biosensors for environmental toxicology and preclinical studies. However, when applied to real samples, several methodological problems prevent it from being widely used. Here, we propose a methodological approach for improving its analytical performance with complex matrix. We developed bioluminescent Escherichia coli and Saccharomyces cerevisiae bioreporters for copper ion detection. In the same cell, we introduced two firefly luciferases requiring the same luciferin substrate emitting at different wavelengths. The expression of one was copper ion specific. The other, constitutively expressed, was used as a cell viability internal control. Engineered BL cells were characterized using the noninvasive gravitational field-flow fractionation (GrFFF) technique. Homogeneous cell population was isolated. Cells were then immobilized in a polymeric matrix improving cell responsiveness. The bioassay was performed in 384-well black polystyrene microtiter plates directly on the sample. After 2 h of incubation at 37 °C and the addition of the luciferin, we measured the emitted light. These dual-color bioreporters showed more robustness and a wider dynamic range than bioassays based on the same strains with a single reporter gene and that uses a separate cell strain as BL control. The internal correction allowed to accurately evaluate the copper content even in simulated toxic samples, where reduced cell viability was observed. Homogenous cells isolated by GrFFF showed improvement in method reproducibility, particularly for yeast cells. The applicability of these bioreporters to real samples was demonstrated in tap water and wastewater treatment plant effluent samples spiked with copper and other metal ions.

Liquid chromatography-mass spectrometry (LC-MS) has become an important analytical tool for quantitative proteomics and biomarker discovery. In the label-free differential LC-MS approach computational methods are required for an accurate alignment of peaks extrapolated from the experimental raw data accounting for retention time and m/z signals intensity, which are strongly affected by sample matrix and instrumental performance. A novel procedure "MassUntangler" for pairwise alignment has been developed, relying on a pattern-based matching algorithm integrated with filtering algorithms in a multi-step approach. The procedure has been optimized employing a two-step approach. Firstly, low-complexity LC-MS data derived from the enzymatic digestion of two standard proteins have been analyzed. Then, the algorithm's performance has been evaluated by comparing the results with other achieved using state-of-the-art alignment tools. In the second step, our algorithm has been used for the alignment of high-complexity LC-MS data consisting of peptides obtained by an Escherichia coil lysate available from a public repository previously used for the comparison of other alignment tools. MassUntangler gave excellent results in terms of precision scores (from 80% to 93%) and recall scores (from 68% to 89%), showing performances similar and even better than the previous developed tools. Considering the mass spectrometry sensitivity and accuracy, this approach allows the identification and quantification of peptides present in a biological sample at femtomole level with high confidence. The procedure's capability of aligning LC-MS data previously corrected for distortion in retention time has been studied through a hybrid approach, in which MassUntangler was interfaced with the Open MS TOPP tool MapAligner. The hybrid aligner yielded better results, showing that an integration of different bioinformatic approaches for accurate label-free LC-MS data alignment should be used. (C) 2011 Elsevier B.V. All rights reserved.

2-Bromoacetyl-6-methoxynaphthalene was used as a pre-chromatographic fluorescent labelling reagent for the high-performance liquid chromatographic (HPLC) analysis of bile acids. The derivatization reaction was performed in an aqueous medium in the presence of tetrahexylammonium bromide by ultrasonication at 40 degrees C to give fluorescent esters which were separated by reversed-phase HPLC and detected fluorimetrically (lambda ex = 300 nm, lambda em = 460 nm). Applications to the determination of ursodeoxycholic acid (UDCA) and chenodeoxycholic acid (CDCA) in their pharmaceutical formulations are described. The method was also applied to the determination of free and conjugated bile acids in human bile samples.